Kỹ Thuật Xét Nghiệm Huyết Học Và Truyền Máu Ứng Dụng Trong Lâm Sàng

VIEN HUYET HOC - TRUYEN MAU TRUNG JONG
Chu bien: GS.TSKH. D6 Trung Phan

Cao Dang Y te f’liu Tlio - Thu vi£»
K M .0 0 7 7 1 4
KY THUAT XET NGHIEM
LfhG DUNG TROMG LAN ÂNG
■ » ♦
NHA XUAT BAN Y HOC

V I E N H U Y E T HOC - T R U Y E N M A U T R U N G 1/dNG
Chu bien: GS. TS K H . Oo Trung Phan
K? THUAT
■ X£T NGHl£M
■
HUYET HOC VA TRUYEN MAU

IJMG DUNG TRONG LAM SANG
■
( T d i b a n Ia n t h i i n h a t c o sU a c h i i a va b o s u n g )
NHA X U A T BAN Y HOC
HA NQI - 2009
Chu bien
GS.TSKH. DO TRUNG PHAN
Tham gia bien soan
TS. BUI THI MAI AN
TS. TRUONG CONG DUAN
ThS. PHAM TUAN DUONG
ThS. NGUYEN TH| Y LANG
TS. NGUYEN TH( NLT
GS. TSKH. DO TRUNG PHAN
PGS.TS. THAI GUY
BSCKII. TRAN THI HONG THUY
BSCKI1. DO MANH TUAN
BSCKI. NGUYEN CHI TUY^N
PGS.TS. CUNG TH( TY
TS. NGUYEN TRlEU VAN
PGS.TS. PHAM QUANG VINH
Thu* ky bien soan
TS. NGUY§N TRlEU VAN
PGS.TS. PHAM QUANG VINH

1
LOI NOI 0AU
Trong 10 nam gan day, difdc sii quan tarn cua Bo Y te ve sU phat trien khoa
hoc ky thuat, chifdng trinh ky thuat cao da h6 trd cho svf phat trien cua nhieu
chuyen nganh, do do chat lifdng chan doan va dieu tri benh difdc nang len ro ret.
Cung vdi sif phat trien chung do, nganh Huyet hoc - Truyen mau co nhieu doi
mdi ve trang thiet bi va ky thuat, chat lifdng chan doan va dieu tri benh mau, cung
nhif an toan truyen mau va sii dung mau da co bifclc phat trien dang ke gop phan
khong nho vao sif nghiep cham soc siic khoe nhan dan. De tiep tuc ho trd cho cong
tac dao tao cua cac TrUdng Dai hoc va Trung hoc y te, ho trd cho cac thay thuoc lam
dieu tri hieu ro hdn cac trang bi, ky thuat m6 i va sii dung tot hdn cac ket qua xet
nghiem ve Huyet hoc - Truyen mau, chung toi bien soan cuon sach “Ky thuat xet
nghiem Huyet hoc va Truyen mau ting dung trong lam sang” , do cac giao sif, tien
si. thac si, bac si lau nam co kinh nghiem thuoc chuyen nganh Huyet hoc - Truyen
mau bien soan. Cuon sach bao gom 10 chifdng:
Chifdng 1 : Te bao - To chtic hoc cd quan tao mau
Chifdng 2: Dong mau - Cam mau
Chifdng 3: Di truyen huyet hoc
Chifdng 4: Sinh hoa huyet hoc
Chifdng 5: M ien dich huyet hoc
Chifdng 6 : An toan truyen mau
Chifdng 7: San xuat, bao quan va phan phoi mau cac san pham mau, sU
dung mau d cac benh vien.
Chifdng 8 : Mot so’ van de cd ban trong xet nghiem Huyet hoc - Truyen mau
Chifdng 9: Dam bao chat lifdng xet nghiem huyet hoc va truyen mau.
Chifdng 1 0 : Cac gia tri sinh hoc ve huyet hoc va mien dich huyet hoc.
Tai ban lan nay chung toi co siia chufa va bo sung mot so van de va ky thuat
cap nhat.
3
Hy vong cuon sach tai ban kv nay se lam hai long cac can bo. ky thuat vien
Huyet hoc - Truyen mau. cac thav thuoc lam cong tac dao tao d cac Trtfdng Dai
hoc va Trung hoc y te cung nhii cac bac si dieu tri d tat ca cac benh vien trong
toan quoc. Tuy nhien. trong qua trinh bien soan khong tranh khoi con thieu sot.
Rat mong dude ban doc gop y.
Xin tran tron g cam On
THAY MAT TAP THf; BI^N SOAN
Chu bien
GS. TSKH. D d TRUNG PHAN

UA
MUC LUC
Lai no I dau 3
C h i/d n g 1: TE BAO - TO CHLfC HOC CO QUAN TAO MAU

TS. Trwang Cong Duan; BSCK II. Tran Thi Hong Thuy
Cach lay va bao quan benh pham xet nghiem te bao 9
Phifcfng phap lam tieu ban xet nghiem 11
Dem so li/dng hong cau 14
Dem so liidng bach cau 16
Dem so li/ong tieu cau 18
Dem hong cau li/di 20
Omh li/cng huyet sîc to 21
Do the tich khoi hong cau 23

Do toe do mau lîng 24
Cong thirc bach cau 25
Ky thuat tap trung bach cau 27
May dem te bao. Nguyen tSc hoat dong va phirong phap sif dung 28
Huyet do 33
Tuy do 34
Hoa hoc te bao 37
Sinh thiet tuy xi/ong 44
Hach do 49
Sinh thiet hach 50
Lach do 51
Xet nghiem tim te bao Hargrave 52
Tim ky sinh trung sot ret 53
Tim au trung giun chi 58
Xet nghiem te bao trong dich nao tuy 59
Xet nghiem te bao trong dich mang phoi 61
Xet nghiem te bao trong dich mang bung 62
Xet nghiem te bao trong dich khdp va bao khdp 63
Xet nghiem te bao trong nude tieu 64
Xet nghiem can Addis 67
D
C hi/dng 2: D O N G MAU - CAM MAU
PGS. TS. Cung Thi Ty; TS. NguySn Thi NCf
Nguyen tac lay benh pham xet nghiem dong mau 70
Dau hieu day that ’ 71
Thai gian mau chay 72
Thdi gian mau dong 73
Co cuc mau 75
Thdi gian Howell (Phuc hoi calci) 76
Thai gian prothrom bin (PT, Q uick) 77
Thai gian throm boplastin tC/ng phan hoat hoa (APTT - Cephalin - Kaolin) 79
Thai gian throm bin (TT) 80
Nghiem phap Von-Kaulla (Thai gian tieu sdi huyet) 81
Thai gian tieu Euglobulin huyet ti/ong 83
Nghiem phap Ethanol hoSc protam insulphat de phat hien fibrin m onom er 84
Phudng phap xac djnh fibrinogen 85
Ojnh lifang cac yeu to difa tren ca sa xet nghiem thai gian prothrom bin 87
DLnh lirang cac yeu to di/a tren co sd thai gian throm boplastin ti/ng phan 89
Djnh lifang yeu to XIII 91
Djnh li/ang san pham thoai hoa fibrinogen va fibrin 92
Hoat ti'nh yeu to 3 tieu cau 96
Xet nghiem do do ngi/ng tap tieu cau 97
Xet nghiem xac djnh sl/ co mat cua cac chat khang dong Il/ u hanh 99
Xet nghiem cac yeu to khang dong mau (anti - coaglulation): protein C,
protein S, antithrom bin III. 101
C hi/dng 3: Dl TR UYEN HUYET HOC

PGS. TS. Pham Quang Vinh
Cay mau ngoai vi phan tich nhiem s§c the 103
Lam tieu ban va phan tich nhi§m sac the te bao tuy xudng 106
Cach mo ta cong there nhiem sac the 108
Cac ky thuat nhuom nhi§m sac the 110
Nhuom phan biet nhiem sSc tir chj em 119
X et nghiem vat the Barr 121
Ky thuat sinh hoc phan tif Crng dung trong y hoc 123
• Ky thuat tach chiet ADN 123
• Ky thuat tham ADN va lai vdi m lu “do" 127
• Ky thuat PCR (Polym erase chain reaction) 132
6
C hi/dng 4: SINH HOA H U Y ^T HOC
BSCK I. N g u y in Chi TuySn
Sat huyet thanh 136

Ferritin huyet thanh 138
SCrc ben hong cau 140
Dien di huyet sac to 141
Huyet sac to khang kiem 143
Djnh li/ong m ethem oglobin reductase 145
Xet nghiem si/ thieu hut men G 6 PD hong cau bang phi/ong phap soi den tif ngoai 148
Dinh li/ong men G 6 PD trong dung djch hong cau bang phi/ong phap quang pho 149
Djnh li/ong haptoglobin trong huyet thanh 151
C hi/dng 5: M I§N D|CH HUYET HOC

TS. NguySn Trieu Van; GS. TSKH. D6 Trung Phan
Mot so khai niem co ban ve ky thuat mien djch Crng dung trong
Huyet hoc - Truyen mau 155
Ky thuat man cam dong vat 158
Ky thuat phan lap te bao mi§n dich 170
Ky thuat ket tua 179
Ky thuat ngirng ket 197
Ky thuat do hoat tinh bo the 206
Ky thuat phat hien khang the khang nhan 208
Ky thuat dien df mi§n djch 212
Ky thuat xac dinh khang nguyen biet hoa tren be mat te bao mau 213
Ky thuat chuyen dang lym pho 218
Ky thuat gay doc bach cau trong ong nghiem 220
Ky thuat tim khang the khang tieu cau 221
Ky thuat dinh nhom khang nguyen bach cau (HLA) 225
Ky thuat cay cum te bao (Colony form ing culture) 228
Phirong phap tao quang dung huyet phat hien te bao lach san xuat khang the 230
Ky thuat thi/c bao (Phagocytosis) 232
7
Chi/dng 6: AN TOAN TRUYEN MAU
PGS. TS. Thai Guy; BSCK II Dd Manh Tuan; TS Bui Thi Mai An:
ThS. Nguyen Thi Y Lang; GS. TSKH. Dd Trung Phan
Van dong hien mau nhan dao 235

Tuyen chon ngi/di cho mau 238
Ky thuat thu gom mau 243
Ky thuat sang loc cac benh nhiem trung truyen qua di/ong truyen mau 249
Ky thuat djnh nhom mau ABO va phat mau an toan 269
Ky thuat dinh nhom mau he Rh 277
Ky thuat djnh nhom mau cac he khac 281
Phan ting cheo (phan ting hoa hop) 282
Phi/ong phap hieu gia khang the mien djch 283
Phirong phap tach khang nguyen 286
Chi/dng 7: SAN XUAT, BAO QUAN VA PHAN PHOI MAU, CAC SAN PHAM MAU
ThS. Pham Tuan Duong; ThS. Nguyen Thi Y Lang; GS. TSKH. Dd Trung Phan
Cach san xuat hong cau mau va bao quan 288

Ky thuat dieu che khoi hong cau 289
Ky thuat dieu che khoi hong cau ngheo bach cau 291
Ky thuat dieu che khoi hong cau rtia 294
Ky thuat dieu che khoi tieu cau 296
Ky thuat dieu che huyet ti/ong 300
Ky thuat dieu che tua lanh giau yeu to VIII 301
Ky thuat lifu trti, bao quan va phan phoi mau 303
Chi/dng 8: MOT SO VAN BE CO BAN TRONG XET NGHIEM HUYET HOC - TRUYEN MAU
BSCK II. Tran Thi Hong Thuy
Phi/ong phap lam sach va khti trung dung cu lam xet nghiem 309
Pha cac hoa chat, dung dich sti dung trong xet nghiem huyet hoc 311
Cach sti dung va bao quan kinh hien vi 315
Chuung 9: DAM BAO CHAT LUONG XET NGHIEM HUYET HOC VA TRUYEN MAU
PGS TS Pham Quang Vinh 321
Chudng 10: CAC GIA TRj SINH HOC VE HUYET HOC VA MI^N D(CH HUYET HOC
GS TSKH. Do Trung Phan va CS 329
Tai lieu tham khao 343
Chifdng 1
TE BAO - TO CHLfC HOC

■ CO QUAN TAO
■ MAU
CACH LAY VA BAO QUAN BENH PHAM XET NGHIEM TE BAO
Xet nghiem huyet hoc ngay cang difdc sif dung nhieu d cac cd sd y te nham
gop phan tich cuc vao viec chan doan. dieu tri. nghien cifu khoa hoc cung nhif
cham soc sifc khoe ban dau. Viec chuan hoa cac ky thuat xet nghiem la can thiet.
Trong do viec lay va bao quan benh pham la het siic quan trong. Xet nghiem
huyet hoc gom xet nghiem te bao, xet nghiem dong - cam mau, xet nghiem mien
dich va xet nghiem di truyen. Benh pham xet nghiem te bao co the la mau. nifdc
tieu va cac dich: nao tuy. mang phoi. mang tim. mang bung. khdp... de cac xet
nghiem nay co ket qua va co gia tri cho chan doan, lay mau benh pham xet
nghiem chiem 50% ket qua xet nghiem. Vi vay phai co mot so nguyen tac ma
ngu'di lay benh pham phai tuyet doi tuan thu:
- Dung ten benh nhan
- Lay benh pham dung quy cach
- Thuc hien dung quy trinh bao quan va van chuyen
1. M au
1.1. Ten xet nghiem
Ten xet nghiem Viet t i t
1. So lifdng hong cau, bach cau, cong thifc bach cau goi chung CTM
la cong thifc mau
2. So lifdng tieu cau TC
3. The tich khoi hong cau hay hematocrit Hct
4. Lifdng huyet s ic to hay hemoglobin Hb
5. Toe do mau lang ML
6 . Ky sinh trung sot ret KSTSR
7. Hong cau lifdi HC lifdi
8 . Huyet do (gom dac diem hinh thai va so lifdng cua hong cau, HD
bach cau, tieu cau; the tich khoi HC, huyet s ic to, hong cau
lifdi, cong thifc bach cau).
9. Tap trung bach cau TTBC
10. Te bao Hargraves
Cac loai xet nghiem tif 1 den 8 co the lay mau vao cung mot ong chong
dong kho. the tich = lml.
Xet nghiem tap trung bach cau lay vao ba ong chong dong kho co s&n. moi
ong = lm l mau.
9
Xet nghiem te bao Hargraves lay 3ml vao ong chong dong = Heparin
Mau da difdc chong dong, neu mua he (nhiet do tren 30°C) co the de ong mau
da nut kin vao ngan mat cua tu lanh nhtfng cung phai diidc xet nghiem trong
vong 6 gid ke tCr khi lay mau, neu qua thdi gian do, tinh chat ly hoa cua mau thay
doi lam anh htfdng tdi ket qua xet nghiem.
1.2. Phircmg phap lay mau

1.2.1. Truce khi li'y m au
Doi chieu giay xet nghiem vdi ho ten, tuoi, so' gitfdng, can benh cua benh nhan.
Thtfdng lay mau vao buoi sang, benh nhan chtfa an gi va cd the nghi ngdi
thoai mai.
a. Mau mao mach: Chu yeu dtfdc thiic hien tai labo hoac do khong lay difdc
mau tinh mach (Hien nay it dung vi dem bang may).
- Dung cu: bong, con sat trung 70°, kim chich, dung cu lay mau x 6 t nghiem.
- Tien hanh: sat khuan dau ngon tay deo nhan (d ngUdi 16n), d ngon chan
cai hoac got chan (d tre nho). Dung kim chich sau dung c3 kim co san cho mau
chay ta nhien. Khong lay mau d ndi bi phu ne, co u hoac nhutng ndi nghi t i c mach.
Tranh nan bop nhieu.
b. M au tinh mach: chu yeu do y ta benh phong lay.
- Dung cu: bong, con sat trung 70°, garo, kim lay mau, ong nghiem da co
san chat chong dong kho, co nut.
- Tien hanh: Ga ro phia tren vi tri dinh choc.
Sat khuan vi tri dinh choc.
Chich kim vao tinh mach hut mau ra.
Rut kim va md garo.
Ep bong vao vi tri choc.
- Ndi lay mau thtfdng la tinh mach khuyu tay, mu ban tay, khi can co the lay d
tinh mach ben hoac boc lo tinh mach.Tre em co the lay d tinh mach co hoac dau.
- Mau lay vao ong chong dong phai diidc lac nhe, tron deu va nut kin.
1.2.2. Sau khi lay m au: ghi len nhan ong mau ho ten, tuoi, so" gitldng, phong benh
dung nhtf trgn giay xet nghiem cua benh nhan.
Chu y:
- Khong lay mau qua lau, mau de bi dong day.
- Tuy so ltfdng mau can dung ma chon ong nghiem phu hdp.
- Mau lac tron it, khong deu de gay dong.
- Mau lay dat yeu cau la: mau lay du so liidng, khong dong, khong v3
hong c&u.
10
2. Nirdc djch
Gom nifdc tieu, nUdc nao tuy, dich mang tim, mang phoi, mang bung, dich
khdp, dich kyste phoi, th&n...
Ong dung benh ph^m phai kho sach, benh pham phai difdc xet nghiem trong
vong 6 gid ke tut khi l&y.
• Can Addis
Buoi sang benh nhan di tieu het.
Sau do uong 300ml nifdc soi de nguoi, n&m nghi. Trong 3 gid, di tieu binh
thifdng vao mot cai bo sach va do difdc bao nhieu m ililit thi ghi vao giay xet
nghiem. L&c deu toan bo nifdc tieu va lay 10ml dem den phong xet nghiem .
• M ot so nguyen tac: Ngoai cac yeu c&u chung cho benh nhan thi viec lay va
bao quan benh pham xet nghiem te bao c£n thilc hien mot so" yeu c£u sau:
- Khong lam thay doi m&t do te bao.
- GiQ dude tinh nguyen ven cua te bao.
De dat difdc nhQng yeu cau tren, can lifu y:
- Dung cu (ong nghiem) phai kho, sach.
- Neu dung chat chong dong, phai la chat chong dong kho.
- Lay mau dung thdi diem (thUdng lay vao buoi sang trUdc khi an).
- Lay dung so" ltfdng (theo ty le chong dong).
- Ong nghiem phai dtfdc day nut, de nhiet do phong xet nghiem.
- Thdi gian tCf luc lay mau den khi lam xet nghiem khong qua 6 gid.
- Neu can van chuyen m lu, phai nhe nhang tranh lam vd te bao.
PHUOTNG PHAP LAM TIEU BAN XET NGHIEM
1. Lam tieu ban mau ngoai vi

1.1. Tieu ban mau dan
Nho mot giot mau cach dau lam kmh 1 cm, lam nay diidc giQ ngang bang
mot tay. Tay kia cam lam keo, nghieng 45°, tiep xuc canh ng^n vao giot mau de
cho mau lan toa theo canh nay, khi giot mau da diidc chia deu, day nhanh va deu
lam keo theo chieu dai cua lam nSm ngang.
a! /s * • ,
De kho tu nhien trong phong xet nghiem (kho va mat), danh dau tieu ban
b^ng but chi.
Tieu ban tot la tieu ban mau difdc dan deu, khong mong qua, khong day
qua, cach hai dau lam kinh khoang 1 cm, phan duoi tao thanh hinh “lifdi meo” .
(H inh 1.1).
11
Yeu cau:
- Tieu ban phai sach.
- Khong dUdc keo hai lan tren cung mot giot mau.
I 2mm
1cm 1cm
Hinh 1.1: Tieu ban mau dan
1.2. Tieu ban giot dac
Nho hai giot mau len hai dau cua mot lam ki'nh. Dung que thuy tinh hoac
goc cua mot lam ki'nh khac di nhe len giot mau theo vong Iron dong tarn tu trong
ra ngoai. den khi dudng ki'nh giot mau khoang 1 - 1 ,5 cm. De kho tu nhien tren
mot mat phang ngang. Danh dau tieu ban. nhuom ngay. khong can co djnh.
1.3. Tieu ban soi tuoi

Nho mot giot mau len gitia lam ki'nh. Dung mot lamen (la ki'nh) mong dat
len giot mau. de mau trail ra khap lamen. Dua len ki'nh hien vi soi ngay.
ThUdng dung de quan sat au trung giun chi con cii dong hoac hien tUdng
hong cau chuoi tien.
2. Lam tieu ban djch tu y xU dng

Nho 0.2 - 0.3 ml dich hut tuy xUdng da dUdc tron deu len mot lam ki'nh kho
va sach. Lam 6 - 1 0 tieu ban giong nhu tieu ban "mau dan". De kho tu nhien. co
dinh. nhuom Giemsa hai thi.
Trong trUdng hop can thiet co the lam them tieu ban ap: de dich tuy lang
tren lam ki'nh 2 - 3 phut, nghieng lam kinh nhe nhang va dung mot mieng bong
tham d mep tha'p. lay can con lai. Ap nhe lam kfnh sach len can dich tuy xUdng.
de kho tu nhien. co dinh. nhuom Giemsa hai thi.
3. Lam tieu ban nUdc djch
Dich long (mang b u ng, mang tim. mang phoi. nao tuv....) dUdc de lang tu
nhien hoac lv tam nhe ( 1 0 0 0 vong/phut trong 1 0 phut), lay can nho mot giot len
lam kinh sach. Dung que thuy tinh dau nhan det di giot benh pham theo hinh
tron dong tam. dUdng ki'nh 2.0 - 2.5 cm. De kho tu nhien. co dinh. nhuom Giemsa
mot thi.
Ltiu y: Tuy theo do day cua tieu ban de dat thdi gian nhuom nhUng bao gid
cung ngan hdn thdi gian nhuom mot tieu ban mau dan. Rua tieu ban phai het sue
nhe n h a n g de tranh bi troi b en h ph am .
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Trong mot so" tnidng hdp, dich dac va quanh c ln dvfdc lam tieu ban cang sdrn
cang tot sau khi lay ra khoi cd the. Dung tam bong ia"y chat dich cho len lam kinh.
Dung lam keo dan tieu ban nhu lam tieu ban mau dan hoac dung tam bong di nhe
nhu lam tieu ban giot dac. De kho, danh dau, co" dinh va nhuom.
4. Lam tieu ban mo

4.1. Tieu ban tuyxiromg (phtrang phap paraphin)
4.1.1. XCf ly manh sinh thiet
Co" dinh trong dung dich Helly: 5-6 gid

Rtia dutdi voi nUdc chay: 1 gid
Khijt calci bang dung dich Custer: 1 2 gid
ROfa dudi voi nude chay: lgid
Loai nUdc bang con: Con 90° 30 phut
Con tuyet doi I 1 gid
Con tuyet doi II 1 gid
Con tuyet doi III 3 gid
Loai con bang xylen: Xylen I 30 phut
Xylen II 90 phut
Xylen III 90 phut
Vui trong paraphin d 60°C 1 2 gid
Due khuon.
Cat tieu ban chieu day 2 -2 ,5^m.
4.1.2. Nhuom H &E
- Tay nen bang xylen 3 lan: 5 phut 1 lan

- Chuyen qua con 100°, 95°, 80°: 5 phut 1 lan
- R ia diidi voi ntfdc chay: 1 0 phut
- Phu tieu ban bang dung dich lugol: 10 phut
- R ia dudi voi nifdc chay: 10 phut
- Phu tieu ban bang dung dich N a 2 S2 0 3: 5 phut
- Riia dtfdi voi nifcic chay: 10 phut
- Nhuom trong dung dich Hemalun de M ayer (hoac hematoxylin): 3-5 phut
- Rua dtfdi voi nildc chay: 10 phut
- Nhung tieu ban trong dung dich con-HCl 1%: vai giay
- Phu tieu ban bang dung dich N a 2 C 0 3 1%: 1 phut
13
- Ruta difdi voi nifdc chay: 10 phut
- Nhuom trong dung dich erythrocin B (hoac eosin ) 1 %: 1 phut
- Rifa nhanh trong nUdc
- Tay tieu ban bang con tuyet doi, lam trong bang xylen, g in lamen (la kinh)
4.2. Tieu ban mo mem ( Lach, hach,...)

Cac bifdc xtf ly benh pham sinh th iet giong nhif doi v 6 i tieu ban tuy
xifdng. Co dinh benh pham bang form ol 10% trong 24-48 gid tuy kich thifdc
benh pham, khong can khijf calci cho nhGng benh pham nay. Quy trinh nhuom
H & E nhif sau:
- Tay nen bang xylen 3 lan: 5 phut 1 lln
- Chuyen qua con 100°, 95°, 80°: 5 phut 1 lln
- Rufa difdi voi nifdc chay: 10 phut
- Nhuom trong dung dich Hemalun de M ayer (hoac hematoxylin): 3-5 phut
- Rita difdi voi ntfdc chay: 10 phut
- Nhung tieu ban trong dung dich con-HCl 1 %: vai giay
- Phu tieu ban bang dung dich N a 2 C 0 3 1 %: 1 phut
- RtJa dtfdi voi nude chay: 10 phut
- Nhuom trong dung dich erythrocin B (hoac eosin) 1%: 1 phut
- Rijfa nhanh trong nildc
- Tay tieu ban bang con tuyet doi, lam trong bang xylen, gsin lamen
DEM SO LlfflNG HONG CAU

(Bang kinh hiSn vi quang hoc)
1. Nguyen tac
Dem so lifdng hong clu cua mau toan phan trong mot the tich da biet vdi mot ty
le pha loang nhat dinh de tinh ra so" lifdng hong cau trong mot lit mau toan phan.
2. Mau xet nghiem, hoa chat va dung cu

- Mau tinh mach hoac mao mach difdc chong dong bang ED TA kho l , 5 mg/ml.
- Dung cu la y mau mao mach (dau ngon tay) va tinh mach.

- Dung dich Marcano.
- Qua bop cao su.
14
- Ong pha loang (potain) hong c£u, loai pha loang 2 0 0 lin .
- Buong dem, lamen.
- Kinh hien vi quang hoc.
3. Ky thuat
3.1. Pha loang, nho len buong dem
L ie ky ong mau, neu chich d£u ngon tay thi nhd th&'m bo giot d£u. Dung
potain hut mau den vach 0,5. Lau sach d£u ong hut, chu y khong de mau trong
ong hut bi thieu do dong tac nay. Sau do hut tiep dung dich Marcano den vach
1 0 1 d phia tren bû (do pha loang 1 /2 0 0 ), bo qua bop ra, dung ngon tay cai va tro
(hoac giuta) bit chat hai dau potain, lie theo chieu doc potain khoang 1 - 2 phut.
Chu y khong de co bot khi hut mau va dung dich pha loang, khong hut mau qua
vach chuan, khong difdc de mat mau khi hut dung dich pha loang.
Trifdng hdp benh nhan thieu mau, co the hut mau den vach 1 , sau do hut
dung dich pha loang den vach 1 0 1 (do pha loang 1 /1 0 0 ).
G&n lamen len buong dem kho, sach. Sau khi lSc ky potain, bo vai giot dau,
de dau difdi potain sat canh lamen, nho mot giot h 6 n dich sao cho lan toa khSp
buong dem nhifng khong tran ra ngoai, ddi 5 phut. Dung vat kinh x 10 kiem tra
xem hong cau co trai deu tren buong dem hay khong.
3.2. DS'm so I iron g hong cau
Lifa chon khu vtfc dem hong c iu tren buong dem. Co nhieu loai buong dem
khac nhau nhif Goriaep, Thoma, Neubauer va Smic, nhifng co dac diem chung khi
dem hong cau la deu dem 5 khu viic (5 o vuong 16n), trong do 4 khu vifc d bon goc
va mot khu viic d giula buong dem. Moi khu vifc dem gom 16 o vuong nho. M oi o
vuong nho co the tich la 1/4000 mm3. BSng vat kinh x 10, dem tat ca hong cau
nam gon trong khu vxfc o vuong 16n va cac hong cau nam tren hai canh cua moi o
vuong 16n.
3.3. Tinh ket qua
K et qua difdc chap nhan khi so' lifdng hong cau d moi khu vile chenh lech
nhau khong qua 1 0 . Goi n la tong so hong cau tren 5 khu v\fc dem, do pha loang
la 1/200, N la so lift'ng hong cau trong mot lit mau, thi:
N = n x 200 x 4000 /80 x 106 = n/100 x 10 12 / lit
4. Nguyen nhan sai so

- Mau xet nghiem khong dung quy cach: dong day, mau mao mach bi pha
loang bcii dich gian bao do nan bop dû ngon tay nhieu khi chich mau, ong mau de
lau khong day nut,...
- Dung cu khong du tieu chuan: ban, ifdt, stit me ...
- Gan lamen khong khit.
15
- Pha loang khong chu&n: hut mau khong dung vach quy dinh tren potain,
dung dich pha loang thieu hoac thifa.
- Dung dich pha loang nhieu can hoac v&n due.
- Khong tron deu.

- Nho tren buong dem khong dung ky thuat: co bot khi, hong c£u phan bo
khong deu...
- Dem hoac tinh ket qua sai.
DEM SOi LITCTNG BACH CAU

(Bang kinh hien vi quang hoc)
1. Nguyen tac
Dem so" lifdng bach cau cua mau toan phan trong mot the tich da biet vdi
mot ty le pha loang nhat dinh de tinh ra so lifdng bach c&u trong mot lit mau
toan phan.

- Mau tinh mach hoac mao mach chUa kip dong hay da difdc chong dong
bang EDTA kho l,5mg/ml.
- Dung cu lay mau mao mach (dau ngon tay) va tinh mach.
- Dung dich dem bach cau: co the dung dung dich Lazarus, dung dich
Hayem hoac dung dich xanh acetic.
- Qua bop cao su.
- Ong pha loang (potain) bach cau, loai pha loang 2 0 lan.
- Buong dem. lamen.
3. Ky thuat
3.1. Pha loang, nho len buong dem
Lac ky ong mau, neu chich dau ngon tay thi nhd tham bo giot dau. Dung
potain hut mau den vach 0.5. Lau sach dau ong hut, chu y khong de mau trong
ong hut bi thieu do dong tac nay. Sau do hut tiep dung dich pha loang bach cau
den vach 1 1 d phia tren bau (do pha loang 1 /2 0 ), bo qua bop ra, dung ngon tay
eai va tro (hoac giiJa) bit chat hai dau potain. lac theo chieu doc potain khoang 2 -
3 phut den khi mau cua mau trong potain chuyen sang mau sam. Chu y khong de
co bot khi hut mau va dung dich pha loang, khong hut mau qua vach chuan.
khong dude de mat mau khi hut dung dich pha loang.
16
GSn lamen len buong dem kho, sach. Sau khi lac ky, bo giot dau, de dau
difdi potain sat canh lamen, nho mot giot hon dich vao sao cho lan toa khap buong
dem nhifng khong tran ra ngoai, ddi 5 phut. Dung vat kinh x 10 kiem tra xem
bach cau co trai deu tren buong dem hay khong.
3.2. Dem so Iupng bach cau va tinh ket qua

3.2.1. Buong dem Goriaep
Dem bach cau trong 25 khu viic (moi khu viic co 4 o vuong). Goi n la tong so"
bach cau dem dUdc trong 25 khu vUc, do pha loang 1/20 thi:
So" lifdng bach cau = n x 2 0 x 10/4 x 1 0 6 = 50 n x 1 0 9/lit
Trong do: 10 la chieu cao buong dem.
4 la 4 mm2(dien tich cua 25 khu vUc dem).
3.2.2. Buong dem N eubauer va Smic

Dem bach cau tro n g 4 k h u vUc (moi k h u vUc co 16 o v u on g). Goi n la tong so"
b ach cau dem dUdc tro n g 4 k h u vUc, do p h a lo a n g 1/20 thi:
So" lifdng bach cau = n x 2 0 x l 0 / 4 x 106 = 50 n x 1 0 9/lit

Trong do: 10 la chieu cao buong dem.
4 la 4 mm 2 (dien tich cua 4 k h u vUc dem).
3.2.3. Buong dem Thoma

Dem bach cau trong 16 khu vUc (moi khu vUc co 16 o vuong) co dien tich la
1 mm2. Goi n la tong so" bach cau dem dUdc trong 16 khu vUc, do pha loang 1/20 thi:
So" lifdng bach cau = n x 20 x 1 0 x 1 0 6= 2 0 0 n x 109/ lit
3.2.4. Buong dem M alassez

Dem bach cau trong 100 khu viic (moi khu viic co 20 o vuong) co dien tich la
1 mm2. Goi n la tong so"bach cau dem dtidc trong 1 0 0 khu vttc, do pha loang 1/20 thi:
So" lifdng bach cau = n x 2 0 x 1 0 x 1 0 6 = 2 0 0 nx 1 0 9/lit

loang bdi dich gian bao do nan bop dau ngon tay nhieu khi chich mau, ong mau de
lau khong day nut,...
- Dung cu khong du tieu chuan: ban, ifclt, sift me ...

- Gan lamen khong khit.
TRUON
- Pha loang khong chuan: hut mau khong dung vach quy <tiRl£)ta% pqtam,
Y TE'
- Dung dich pha loang nhieu can hoac van due. fH U Tf?Q [
THU VIEN
'w & L 17
c
- Khong tron deu.
- Nho tren buong dem khong diing ky thuat: co bot khi, bach cau phan bo
khong deu,...
DEM SO LINING TIEU CAU

(B£ng ki'nh hien vi quang hoc)
1. Nguyen tic
Dem so liidng tieu c&u cua mau toan ph&n trong mot the tich da biet vdi
mot ty le pha loang nhat dinh de tinh ra so ltfdng tieu cau trong mot lit mau
toan phan.

- Mau tinh mach hoac mao mach chUa kip dong hay da dUdc chong dong
bang ED TA kho l,5mg/ml (tot nhat khi benh nhan chUa an sang).
- Dung cu lay mau mao mach (d&u ngon tay) va tinh mach.
- Dung dich amoni oxalat hoac dung dich Marcano.
- Qua bop cao su.
- Ong pha loang (potain) hong cau, loai pha loang 2 0 0 lan hoac potain bach
cau loai pha loang 2 0 lan.
- Buong dem, lamen.
- Kinh hien vi doi pha hoac kinh hien vi quang hoc.
3. Ky thuat dem true tiep

3.1. Pha loang tieu cau bang dung djch Marcano
Lac ky ong mau, neu chich dau ngon tay thi nhd tham bo giot d&u. Dung
potain hong cau hut mau den vach 0,5. Lau sach dau ong hut, chu y khong de
mau trong ong hut bi thieu do dong tac nay. Sau do hut tiep dung dich Marcano
den vach 1 0 1 d phia tren bau (do pha loang 1 /2 0 0 ), bo qua bop ra, dung ngon tay
cai va tro (hoac gi€ta) bit chat hai dau potain. lac theo chieu doc potain khoang 3-
5 phut. Chu y khong de co bot khi hut mau va dung dich pha loang. khong hut
mau qua vach chuan, khong dUdc de mat mau khi hut dung dich pha loang.
TrUdng hdp benh nhan giam tieu cau. co the hut mau den vach 1, sau do hut
dung dich pha loang den vach 1 0 1 (do pha loang 1 /1 0 0 ).
3.2. Pha loang tieu cau b$ng dung djch amoni oxalat
Lac ky ong mau. neu chich dau ngon tay thi nhd tham bo giot dau. Dung
potain bach cau hut mau den vach 0.5. Lau sach dau ong hut, chu y khong de
18
mau trong ong hut bj thieu do dong tao nay. Sau do hut tiep dung dich amoni
oxalat den vach l i d phia tren b lu (do pha loang 1 /2 0 ), bo qua bop ra, dung ngon
tay cai va tro (hoac gifla) bit chat hai d&u potain, lac theo chieu doc potain
khoang 3-5 phut de hong cau tan het. Chu y khong de co bot khi hut mau va dung
dich pha loang, khong hut mau qua vach chuan, khong dUdc de mlit mau khi hut
dung dich pha loang.
TrUdng hdp benh nhan giam tieu cau, co the hut mau den vach 1 , sau do hut
dung dich pha loang den vach 1 1 (do pha loang 1 /1 0 ).
3.3. Dem s o lu a n g tieu cau
Gan lamen len buong dem kho, sach. Sau khi lac ky, bo mot vai giot d&u, de
dau dudi potain sat canh lamen, nho mot giot hdn dich vao sao cho lan toa khap
buong dem nhUng khong tran ra ngoai, ddi 10 phut. Dung vat ki'nh x 10 dem so"
lUdng tieu c&u.
Khu vUc dem tieu cau chi'nh la khu vUc dem hong c&u tren buong dem. Co
nhieu loai buong dem khac nhau nhu Goriaep, Malassez, Thoma, Neubauer va
Smic, nhUng co dac diem chung khi dem tieu cau la deu dem 5 khu vUc (5 o vuong
ldn), trong do 4 khu vUc d bon goc va 1 khu vUc d gifla buong dem. Moi khu vUc
dem gom 16 o vuong nho. Moi o vuong nho co the tich la 1/4000 mm3. Bang vat
kinh x 1 0 , dem tat ca tieu cau (cham sang nho bang 1 / 1 0 hong c&u, c ln phan biet
vdi cac hat md) nam gon trong khu vUc o vuong ldn va cac tieu cau nam tren hai
canh cua moi o vuong ldn.
3.4. Tinh ket qua
Goi n la ton g so" ltfdng tieu c&u tren 5 k h u vUc dem, do p h a lo a n g la 1/200.
N la so" liidng tieu cau tro n g mot lit m au, thi:
N = n x 200 x 40 00 /80 x 105 = n x 10 9/lit
Neu do pha loang la 1/20 thi:
N = n x 20 x 40 00/80 x 105 = n/10 x 109/lit
4. Ky thuat gian tiep

Dem so" ltfdng tieu cau thong qua ty le phan bo" bach cau/tieu cau tren tieu
ban nhuom Giemsa va so lifdng bach cau cua chinh mau mau dd.
PhUdng phap nay tuy co sai so" ldn nhUng no cd vai trd bo sung hieu qua cho
phUdng phap dem so" liidng tieu cau trUc tiep, ngay ca khi dem tieu cau b in g may
dem te bao.

loang bdi dich gian bao do nan bop dau ngon tay nhieu khi chich mau, ong mau de
lau khong day nut....
- Dung cu khong du tieu chuan: ban, Udt, sift me...
19
- G£n lamen khong khit.
- Pha loang khong chuan: hut mau khong dung vach quy dinh tren potain,
- Dung dich pha loang co nhieu can hoac v&n due.
- Khong tron deu.

- Nho tren buong dem khong dung ky thuat: co bot khi, hong cau phan bo
khong deu,...
DEM HONG CAU LlfOfl

(Phi/ong phap nhuom xanh cresyl)
1. Nguyen tac
H o n g cau ludi la giai doan t r u n g g ia n g i d a h o n g c£u co n h a n v a h o n g c l u
trUdng thanh, dUdc dac trU ng bdi A R N con lai tro n g b ao tUdng. N g U d i ta co the
n h an biet dac diem n a y nhd cac thuoc n h u om la m tua A R N t r o n g h o n g c&u.
2. Dung cu, hoa chat

- Mau tinh mach chong dong bang ED TA kho.
- Ong nghiem kho sach co nut.
- Lam kinh, lam keo kho sach.
- Pipette Pasteur.
- Xanh cresyl bao hoa.
- Tu am hoac Bain marrie.
3. Ky thuat
Cho vao ong nghiem hai giot mau xet nghiem va hai giot xanh cresyl bao
hoa, lac deu, day nut, u d 37°C/20 phut. Lac deu, lam tieu ban mau dan (keo tieu
ban that mong), de kho tu nhien. Doc ket qua tren kinh hien vi vdi vat kinh dku.
Tinh ty le phan tram hong cau ludi trong 1000 hong cau trUdng thanh.
4. Nguyen nhan sai sot thirdng gap

- Lac khong deu: khi lay mau de u. khi lam tieu ban.
- Thuoc nhuom kern chat lUdng. hoac can.
- Doc nham the vui hoac bach cau
20
D|NH LINING HUYET SAC TO
(Phirong phap quang pho ke)
1. Nguyen tac
Do mat do quang hoc cua mau nghiem sau khi da chuyen huyet sac to" thanh
methemoglobin - mot dan xuat ben vutng ma sU hap phu tot nhat d budc song
540nm. Do mat do quang ty le thuan vdi nong do cua huyet sac to nen de dang
tinh ra dUdc lUdng huyet sac to" cua m lu nghiem.
2. Mau xet nghiem, thuoc thur va dung cu

. - Mau tinh mach hoac mao mach chUa kip dong hay da dUdc chong dong
bang E D TA kho l,5mg/ml.
- Dung dich drabkin sif dung.
- Hemo-trol chuan.
- Pipette vach 20 (j.1 (0,02 ml).
- Quang pho ke co budc song 540 nm.
3. Ky thuat
3.1. Thiet lap bieu do mau
Lam bang dung dich chuan ma hieu gia cyanmethemoglobin (ben vUng
trong dieu kien bao quan d 4°C) da dUdc xac dinh chinh xac va ghi ro tren mdi lo
mau chuan, thUdng khoang 60 mg/100 ml, nghla la mat do quang hoc tUdng Ung
vdi mot m lu mau chUa lUdng huyet sac to" 15 mg/100 ml khi dUdc pha loang trong
dung dich drabkin (20|il/5ml). De ve dudng thang mau, ngUdi ta thUc hien tren
mot loat ong nghiem tUdng Ung vdi hieu gia cua hemo-trol da ghi chu:
Bang 1.1: TUdng quan giufa hieu gia hem o-trol va mat do quang (OD)
So ong nghiem 1 2 3 4 5 Chufng

Drabkin sif dung 0 ml 1 ml 2 ml 3 ml 4 ml 5 ml
Hemo-trol 5 ml 4 ml 3 ml 2 ml 1 ml 0 ml
O
Q
Mat do quang OD, od2 od3 od5 od6

Huyet sac to (g/l) 5n/20 4n/20 3n/20 2 n/20 1 n/20 0
Do mat do quang hoc cac ong nghiem d budc song 540nm, ghi tren giay ke o
vuong m ilim et cac mat do quang hoc doc dUdc (ODi, OD2,..., OD6) len true tung va
lUdng huyet skc to tUdng Ung tinh dUdc len true hoanh (ddn vi g/1).
21
Cach tinh lifdng huyet s ic to tUdng U n g tU m at do q u a n g do dUdc n h u sau:
Vi du, neu ty le huyet s ic to cua hemo-trol chuan la 58 mg/lOOml = 580g/l. thi:
Ong 1= 580 x 5: 20 = 145g/l.
Ong 5= 580 x 1: 20 = 29g/l.
Ong chUng = 0.
Nhd co bieu do m£u dUdc thiet lap (mot dudng thang), doc huyet sac to se
ddn gian bang cach ghi mat do quang hoc d true tung va suy ra lUdng huyet sac to
d true hoanh tUdng Ung.
Trong dieu kien kho khan, khong thiet lap dUdc bieu do mau hoac vi moi
trUdng phong xet nghiem khong dam bao (khong du kin, khong co dieu hoa, khong
co hut am) nen do cac mau nghiem cung vdi mot mau hemo-trol chuan sau dd tinh
ra lUdng huyet sac to" cho mau nghiem.
3.2. Do mau xet nghiem
Chuan bi nhUng ong nghiem dung 5ml dung dich drabkin sU dung. Cho vao
moi ong 0,02 ml mau chUa kip dong hoac da dUdc chong dong kho, \ic that deu,
de khoang 5 phut cho tan hoan toan hong cau. Do vao cong cua quang ke dung
dich drabkin sif dung, dieu chinh mat do quang d budc song 540 nm ve so 0 (trU
tr^ng). Do dung dich can do vao cong, do mat do quang hoc d budc song 540nm.
Sau dd doi chieu vdi bieu do mau hoac dung dich chuan (hem o-trol) de cd ty le
huyet sac to" g/1 .
4. Nguyen nhan sai so thi/dng gap

- Do pha loang khong chuan: hut mau hoac dung dich drabkin khong dung
so lUdng quy dinh.
- Hong cau chUa tan het trong dung dich drabkin: lac khong deu, thdi gian
qua ngan.
- Huyet tUdng due.
- Bach cau qua cao.
- Dien nguon khong on dinh hoac qua thap.
- Kinh loc md do hdi nude hoac cu.
- Dung dich drabkin sU dung khong dung nong do hoac bi hong.
5. Cac phirong phap khac

5.1. Phi/ong phap may dem te bao: Nguyen tac do huvet sac to cua may dem
te bao cung giong nhu phUdng phap do bang quang pho ke ndi tren. Tu y nhien,
phudng phap may dem te bao (ke ca may tu dong va may ban tu dong) tien ldi
va chinh xac hdn nhieu. Xet nghiem vien khong can chuan bi dung dich chuan,
khong can thiet lap bieu dd mau. khong can pha loang mau trong dung dich
drabkin.
5.2. Phuong phap do tri/c tiep b&ng mau toan phan: Dtfa tren stf chenh l$ch
cudng do anh sang phan xa khi chieu vao giot mau toan phan so vdi chuan trang
cua may do. Phtfdng phap nay gon nhe ve trang thiet bi, ddn gian ve ky thuat,
thich hdp cho cong tac thUc dia nhat la cac vung xa, vung sau; nhtfng ket qua k 6 m
on dinh so vdi phtidng phap quang pho’ ke.
5.3. Phuong phap so mau b£ng mat tren huyet sdc ke Sahli: Phudng phap nay
kern chinh xac. Hien nay khong nen ap dung.
DO THE TICH KHOI HONG CAU

(Phuong phap microhematocrit)
1. Nguyen tac
The tich khoi hong cau la the tich ma khoi hong cau chiem cho so vdi ltfdng
mau toan phan da biet, khi mau dUdc chong dong va dung ktc ly tam lam hong
cau lfing xuo'ng thanh mot khoi. Bieu thi bang 1/1.
2. Mau xet nghiem, chong dong va dung cu

- Mau tinh mach hoac mao mach chtfa kip dong hay da dtfdc chong dong
bang E D TA kho l,5mg/ml.
- Heparin trang ong vi the tich (neu mau chUa dtfdc chong dong).
- M ay ly tam vi the tich toe do lO.OOOg/phut.
- Thudc do kem theo mdy.
- Ong ly tam vi the tich chuan.
- Matis.
3. Ky thuat
Neu lay mau ma khong cho vao ong chong dong thi phai trang ong vi the
tich bang heparin va tham bo giot mau dau tien sau khi chich dau ngon tay, neu
mau tinh mach la'y vao ong chong dong thi phai tron deu.
Nhung dau ong vi the tich vao mau, nghieng 45°-60°, de mau mao d in den
khoang 3/4 chieu dai ong vi the tich. Lau sach mau d dau ong vi the tich va gan
dau ong bang matis.
De cac ong vi the tich tren khay cua ly tam vi the tich, dau gan matis quay
ve phia ngoai. Neu phai ddi lam hang loat thi de ong mao d in thang dvtng tren
khay matis theo so thti tu.
Sau khi day nap ly tam can than, ly tam 5 phut tdc do 10.000 g/ phut.
23
Doc ket qua ngay sau khi may ly tam dUng, bang cach dieu chinh miic tren
va dudi cua ong vi the tich vdi vach chuan cua thudc do.
4. Nguyen nhan sai so thtfdng gap

- Lay mau: Lay ca giot dau khi chich mau dau ngon tay, garo lau qua 1 phut.
- Nong do ED TA khong thich hdp hoac heparin bi hong do bao quan.
- Mau de qua 6 gid.
- Lac mau khong deu trudc khi mao din.
- Thdi gian va toe do cua ly tam khong dam bao.
- Hong cau tu ngUng ket, v5 hong cau.
- Huyet tUdng bay hdi trong thdi gian ly tam do may nong qua hoac ly tam
xong nhUng chUa doc ngay.
- G a n matis chUa du chat.
5. Cac phtfdng phap khac

5.1. May dem te bao
Tat ca may dem te bao tU 5 thong so” trd len deu do dUdc hematocrit. Nguyen
tac cd ban la tinh hematocrit tU tong kich cd cac xung dien dUdc tao ra bdi hong
cau. Day la phudng phap cho ket qua nhanh, on dinh va chinh xac nhat hien nay.
Tuy nhien, khi hong cau tu ngUng ket thi phan ldn cac trUdng hdp may cho ket
qua hematocrit khong chinh xac.
5.2. Phutyng phap macrohematocrit

Su dung ong Wintrobe, quay tren may ly tam thong thUdng. PhUdng phap
nay ton mau, sai so" ldn, hien nay rat it dUdc sU dung.
DO TOC DO MAU LANG
1. Nguyen ly
Mau toan phan lay ra khoi cd the. chong dong, cho vao ong thuy tinh de
thang dUng. Sau mot thdi gian te bao mau se lang xuong de lai cot huyet tUdng d
phia tren.

- Mau chong dong b a n g ED TA kho (lay mau k hi chUa an ), hoac ph ud n g
tien lay mau mao mach.
- Gia va ong mau lang (Pachenkow hoac W estergreen).

- Dong ho.
- Dung cu pha loang: ong nghiem. dung dich natricitrat 3.8%.
24
3. Ky thuat
3.1. Phuong phap Westergreen
Pha loang 1 , 6 ml mau vdi 0,4 ml dung dich natricitrat 3,8% (ty le 1/5). Lac
deu nhe nhang. Dung ong W estergreen hut mau da pha loang den vach 0. Lau
sach xung quanh ong mau lSng. Cam thang dting ong mau lang len gia
W estergreen. Doc chieu cao cot huyet ttfdng bang thudc vach co san tren ong
W estergreen sau 1 gid va 2 gid.
3.2. Phuong phap Pachenkow
Trang ong Pachenkow b ln g dung dich chong dong. Hut dung dich
natricitrat 3,8% den vach P (50), thoi vao ong nghiem nho, kho, sach. Hut hai lan
mau den vach K (0) thoi vao ong nghiem da co dung dich natricitrat. Lac deu nhe
nhang. Sau cung, hut mau da pha loang chong dong (1/5) vao ong Pachenkow den
vach K. Cam thang dting ong mau lang len gia. Doc ket qua sau 1 gid va 2 gid.
4. Nguyen nhan sai sot thUdng gap

- Mau lay khong du hoac dong day.
- Ong mau lSng khong sach, licit, suit me...
- T y le pha loang khong chinh xac.
- Lac tron mau khong deu.
- Co bot khong khi trong ong mau l&ng.
- Ong mau lang khong thang dting.
- Doc ket qua khong dung thdi gian hoac khong dung cach thijfc quy dinh.
5. PhiTdng phap khac

Hien nay co nhieu loai may xet nghiem mau lang, bao gom:
- Loai may chi ddn thuan doc ket qua mau lang.
- M ay co chifc nang hut mau nhUng khong tti rifa ong mau lang.
- M ay tu dong hoan toan.
Cac may do toe do mau l&ng deu dUa tren nguyen the W estergreen.
CONG THUrC BACH CAU
1. Nguyen ly
Dua vao hinh thai va dac diem bat mau thuoc nhuom cua cac loai bach cau
tren tieu ban mau ngoai vi de tinh ty le phan tram cua moi loai.
25
- Ki'nh hien vi quang hoc
- Dau soi
- Dung cu dem
- Kim chich dau ngon tay
- Lam ki'nh kho sach
- Con tuyet doi
- Giemsa
- PhUdng tien sat khuan tai cho
3. Quy trinh ky thuat

3.1. Benh pham
- Mau tinh mach co chong dong
- Mau mao mach: chich d dau ngon tay
3.2. Lam tieu ban mau dan (xem muc 1.1 bai Phudng phap lam tieu ban xet nghiem)
Hinh 1.2: Tieu ban mau dan
3.3. Lap cong th u t bach cau
Dung vat kinh x 10 quan sat toan bo tieu ban, tieu ban phai dat cac veu
cau: sach. dan deu. bat mau deu.
Chuyen sang vat kinh dau x 1 0 0
Nho 1 giot dau len 1/3 gicta cua tieu ban
Quan sat va phan loai it nhat 100 bach cau. xem moi loai co bao nhieu va
ghi ty le % moi loai.
26
4. Nhan xet ket qua
4.1. So sdnh vdi ch i so binh thtf&ng tinh theo tuoi: (bang 1.2)
Bang 1.2: Chi so bach cau binh thi/dng tinh theo tuoi
So li/dng te bao TrUdng

Tre mdi de 9 thang 3 tuoi 10 tuoi
trong 1 mm3 thanh
SLBC(G/I)
5 - 25 4 - 15 4-11 5- 10 4-11
CTBC ( % ) ^ ^ ^ ^ ^ ^
Bach cau doan trung tinh 54-86 25-40 35-50 45-60 55-75
Bach cau lia acid (toan) 0 - 2 1 -2 1 -4 1 -4 4 -8
Bach cau Ua base (kiem) 0 0,4 0,2 0,1 0,1
Lymphocyt 10-38 50-70 45-60 40-59 25-35

Monocyt 0 -7 8 6 6 1 -4
Bach cau dua 6 6 6 4 1 -4
Cac te bao non dong tuy 2 2 1 0 0
4.2. C a s a de phan loai bach cau

- Hinh thai va kich thudc cua bach clu.
- Nhan: hinh thai va ty le nhan tren bao tUdng.
- Bao tufdng: mau s&c, cac hat, hoc trong bao tUdng.
4.3. Co the tinh so luong tuyet doi cua moi loai BC theo cong th u t
Ty le phan tram x so" lifdng bach cau chung.
5. NhCrng sai sot thi/dng gap

- Quy trinh khong dung: tieu ban qua day hoac qua mong, co" dinh khong
tot (tieu ban chifa that kho, do am cao), thdi gian nhuom sai.
- Hoa chat: con, thuoc nhuom khong dam bao chat lifdng.
- Nhan dinh sai hinh thai bach cau, dem khong du so" liidng te" bao can thiet.
KY THUAT TAP TRUNG BACH CAU
1. Nguyen ly
Trong cac trUdng hdp co ft te" bao bat thUdng d mau ngoai vi, dac biet d benh
nhan co so" lUdng bach cau thap, khong the phat hien dUdc bSng tieu ban mau
dan. Tap trung bach cau la mot ky thuat lam tang so" lUdng va mat do te" bao co
nhan tren tieu ban. giup khac phuc kho khan tren.
27
- Pipette Pasteur.
- Bo dung cu lam tieu ban mau dan.
- May ly tam.
- Chat chong dong (ED TA, natricitrat hoac heparin).Thuoc nhuom Giemsa.

- Lay 3-5 ml mau tinh mach chong dong.
- De ong mau th&ng dUng cf nhiet do phong 30 phut, hut lay phan huyet
tUdng cho tdi qua ranh gidi vdi hong cau khoang 0,5 mm.
- Ly tam 1000 vong/phut trong 15 phut, hut bo phan huyet tUdng phia tren
chi de lai 0,5 ml can.
- LSc deu, lam tieu ban, de kho, co dinh, nhuom Giemsa nhu tieu ban mau dim.
4. Ket qua
- Quan sat tieu ban bang vat kinh x 10: danh gia mat do va dac diem phan
bo te bao co nhan, tim kiem cac te bao kich thudc ldn. Tieu ban dat yeu cau phai
co > 10 te bao cd nhan trong moi vi trUdng x 100, te bao cd nhan khong bi nat.
- Quan sat tieu ban d vat kinh x 100: danh gia hinh thai, phan ldai theo ty le
phan tram te bao cd nhan. Chu y phat hien te bao la, dac biet la “blast” .
- Tra ldi ket qua b&ng mot ban cong thUc te bao cd nhan tren tieu ban, co
the kern theo cac nhan xet neu can thiet.
MAY DEM TE DAO

NGUYEN TAC HOAT DONG VA PHUONG PHAP SUf DUNG
1. Mo dau
Nam 1642, Leeuwenhook phat hien ra te bao mau. Cho den naml934,
Moldavan mdi de xuat mot phudng phap dem sc" lUdng hdng cau da dUdc pha
loang san dua tren nguyen tac dien tu. day la nen mong dau tien cho ky thuat
may dem te bao phat trien d nhUng nam sau nay. Nam 1845, mot phUdng phap
dem te bao hong cau khac dUdc mo ta dua tren nguyen tac quang hoc hoan toan.
Bang viec xac dinh mat do quang cua hdn dich hong cau so vdi chu&n de tinh ra so
lUdng hong cau trong mau benh pham. Den dau nhUng nam 1950, ky sU Wallace
Coulter da nghien cUu thanh cong va bat dau trien khai phudng phap tu dong
dem va phan loai kich thudc cac te bao mau. Tti dd nguyen tac nay dUdc biet den
28
vdi ten goi la " Nguyen tac Coulter". Nam 1953, vdi sU ra ddi cua may dem te bao
Coulter model "A", nguyen t&c dem te bao tu dong cua Coulter dUdc pho bien gan
nhu kh&p the gidi. Nam 1968, hang Coulter che tao chiec may dem tu dong dau
tien "Coulter model S" vdi 7 chi so". Phai chd 10 nam sau, nam 1978, cung vdi
nhtfng tien bo cua nganh dien tvf, may "Coulter model S plus" ra ddi mdi cd kha
nang dem so" lUdng va tinh dUdc kich thudc tieu cau.
Cang ngay, cac the" he may dem te" bad cang hoan thien ve tinh nang va do
tin cay. Nguyen ly cd ban cua may dem te" bao theo dong la sU bien doi dien trd
khi te" bao di qua dien trUdng tao thanh cac xung dien. Nguyen ly nay giup phan
tich sU khac biet ve kich thudc cac loai te" bao khac nhau. Han che" cua loai may
nay la khong cd kha nang nhan dien chinh xac te" bao bach c&u de phan loai. De
khac phuc nhUdc diem nay, ngUdi ta sti dung cac xung dien cong hUdng da chieu
va tia laser, giup boc lo cac khac biet ve hinh thai va cau true nhan, nen cd the lap
dUdc cong thUc bach cau mot cach chinh xac. Cho den nay, tren the" gidi, cd nam
hang dUdc coi la ldn nhat va cung la cac hang cd uy tin cad tren thi trUdng qudc te"
la Cdulter, Abbott, T O A (Sysmex), Technicon va Roche. Cac may dem te bao hien
dang dUdc sti dung cd the chia lam hai loai:
- Cac may dem te" bao nguyen ly trd khang: Bao gom may dem te" bao ban tu
dong va tu dong hoan toan. NhUdc diem cua the" he may nay la ket qua phan loai bach
cau chi chinh xac trong cac trUdng hdp hinh thai bach cau hoan toan binh thUdng.
- Cac may the he sau: iTu diem hdn han cua the" he may nay la toe do cao
va phan loai bach cau chinh xac. Vdi nhting may san xuat theo cac model trUdc
nam 1996 nhu Technicon H I, Cell Dyn 3500, M AXM , Helios, SE 9000, sai so"
trong phan loai bach ciu , ndi chung, khoang 1 0 %. Cac model gan day, vdi viec ap
dung tong hdp cac cd che" trd khang, xung dien da chieu, laser, te" bad bach cau can
phan loai dUdc dat trong mot khong gian phan tich ba chieu, do dd kha nang nhan
dien te" bao dUdc nang len den khoang 95%, ke ca viec nhan dien cac te" bao blast
trong leukemia cap. M ot so"hang con ap dung them cd che" nhuom men peroxydase
de tang cUdng cho kha nang nhan dien bach cau hat. Mot so" may nhu Cell Dyn
4000 cua hang Abbott, SE-Avante cua Sysmex con dUdc tang cUdng hieu qua bdi
kha nang dem trUc tiep hong cau ludi trong cung mot m lu mau.
Khong chi diing d mUc sti dung cac may dem te" bad ddn le, mdt s6 " trung tam
xet nghiem hien dai da trien khai mdt day chuyen xet nghiem tu ddng. Cac thanh
phan cd ban bad gdm mot may dem te bao ddng vai tro may chu va mot so" may
khac nhu may dem hong cau ludi, may lam tieu ban, may nhuom tieu ban. Nhu
vay, con ngUdi chi can lay mau benh nhan va phan tich mot so" (thUdng la rat It)
cac tieu ban Giemsa cd bat thUdng ngoai kha nang cua may.
29
2. Nguyen tic hoat dong cua may dem te bao
Nguyen tac cd ban la trd khang:
Hinh 1.3: Nguyen tac hoat dong cua m ay dem te bao theo nguyen ly trd khang
Cac may hien dai cd them:

- Cd che laser scatter va nhuom men peroxydase: tao ra cd che khong gian
ba chieu de nhan dang bach c&u, ke ca te bao blast.
- Nhuom A R N cho dem hong cau ludi.
3. Phuong phap surdung may dem te bao

3.1. Cac thong so
Tam thong so" cd ban cho da so" cac may dem te bao hien nay dang lUu hanh
tren thi trUdng la:
- So lUdng hong cau (Red blood cell - RBC),
- So lUdng bach cau (W hite blood cell - W BC),
- LUdng huyet sac to" (Hemoglobine - HGB),
- Hematocrit (Het)
- The tich trung binh hong c&u (Mean corpuscular volume - M CV),
- LUdng huyet sac to trung binh hong cau (M ean corpuscular Hemoglobine
- MCH),
- Nong do huyet sac to" trung binh hong cau (M ean corpuscular
hemoglobine concentration - M CH C)
- So' lUdng tieu cau (Platelet - PLT).
Cac may the he trd khang cd the cd them: s6 lUdng va ty 1§ p h d n tram cua
cac loai bach cau, the tich trung binh tieu cau (M ean platelet volume- M PV ). dac
30
biet la do dao dong dudng kinh hong cau (Red distribution wide - RD W ) va tieu
cAu (Platelet Distribution W ide;PD W ). Cac thong so ve thanh phan bach cau chi
chinh xac khi hinh thai va kich thudc bach c&u binh thUdng, muon ch&c chan nen
kiem tra tren tieu ban nhuom Giemsa. Cac may the he laser va hoa te bao co kha
nang phan loai bach cau vdi do chinh xac cao; ngoai ra, may con co kha nang phan
loai dUdc hong cau non, te bao blast va dem dUdc hong cau ludi.
3.2. Chuan may

3.2.1. Yeu cau
- M au chuan: d a m b ao chat lUdng, cd du b a mUc thap, b in h thUdng, cad.
- N hiet do phong xet nghiem.
3.2.2. Quy trinh chuan may: Dung may de dem cac mau mau chuan. Doi chieu ket
qua vdi cac thong so" cua bo mau chuan. Tinh he so" chenh lech giUa ket qua thu
dude va cac thong so" cua mau chuan, dieu chinh he so" chuan cua may. Kiem tra lai
mau chuan. Chi ket thuc qua trinh chuan may khi nao tat ca ket qua thu dUdc d ca
ba mUc nam trong khoang dao dong cho phep. Dac biet lUu y cac thong so" MCV,
MCH va MCHC. Mot may dem te bao dUdc goi la chuan khi ba thong so" nay nam
trung hoac gan trung gia tri giUa khoang dao dong cho phep cua mau chuan. Cung
cd the sU dung quy trinh chuan tu dong cua may (neu cd).
3.3. Mot so yeu cau khi surdung may

- Ndi dat may sach, can nhat la khong bui, kho va mat.
- Benh pham dung quy cach, sU dung ong nghiem cd nut va chat chong
<tong kho, tot nhat la ong nhua cd ED TA kho.
- Lac mau can than: tot nhat la cd may lac chuyen dung.
- ThUc hien nghiem tuc cac quy dinh bao quan va van hanh may:
+ Phai cd day dat chong nhieu dung quy cach.
+ Dung dich cua may nao chi sii dung cho may ay.
+ Han che sU dung on ap.

+ Bao dudng ngay, tu ln, thang theo quy dinh.
3.4. Phan tich ket qua
3.4.1. Cac thong so hong cau:
- So lUdng h o n g cau: b in h thUdng luon ton tai moi lien q u a n q u y lu at gitia

m a u sac ong m a u vdi so" lUdng h o n g cau. T h o n g thUdng so lUdng h o n g cau ty le
t h u a n vdi m au sac o n g m au. T r U d n g hdp so lUdng ho n g cau cao k h o n g tUdng xU n g
vdi m a u sac o n g m au . can xem n g a y cac chi so" MCV, M CH va M CH C xem cd p h ai
h o n g cau nho. nhudc sac khong? N e u so" lUdng hong cau th a p k h o n g tUdng xU n g
vdi m a u sac ong m au . can xem cd p h ai m a u bi do ng day. lac k h o n g deu, m a u tu
n g U n g ket h a y hut k h o n g du m a u (do m a u q u a n h hoac hut kim)?
31
- LUdng huyet sic to': neu hu y et s ic to' cao k h o n g tUdng xting voi so lUdng
hong cau can xem cd phai do huyet tUdng due h a y bach c l u q u a cad khdng? Neu
huyet sic to' thap b a t thUdng, can xem ky cac b a d ddn g cu a m ay tren b a n in ket
qua, cd the do loi cua m ay hoac l i e k h o n g deu, d o n g day, m a u n g U n g ket hoac hut
khong du mau.
- Hematocrit: cac sai lam son g h a n h vdi s6' lUdng h o n g c l u v a huyet s i c to.
- Nong do huyet sic to' trung binh trong mot lit hong clu: binh thUdng
MCHC 320-360 g/1, neu > 380 g/1 phai tim nguyen nhan gay sai so'.
3.4.2. Cac dau hieu bat thudng trong ban ket qua
- Ky thuat: tuy loai m ay m a cd he tho'ng q u y Udc b a d d d n g r i e n g khi cac

yeu to ky thu at an h hU dng den ket qu a , vi du d ie n the n g u d n th a p, nhieu. thieu
thuoc thii...
- Benh ly: khi cac thong so' trong ket qua n lm ngoai khoang binh thudng
(do cai dat) may se bao gia tri cao hay thap. Ngoai ra, tuy tting loai may ma cd the
cd bao dong mot so' tinh trang, vi du tieu cau von, hong cau lan vao tieu cau,...
3.4. 3. M dt so diem can lUu y
- Doi chieu cac thong so' cua may vdi tieu ban nhuom Giemsa.
- Hong cau ngUng ket: cac thong so' cao thap khong cd quy luat, khdng
tUdng xUng vdi mau ong mau, dac biet M CH C thudng rat cao. Can kiem tra ky
ong mau de khang dinh.
- Tieu cau von: trong moi trUdng hdp doi vdi may trd khang, can kiem tra
mat do phan bo' va do tap trung tieu cau tren tieu ban nhuom Giemsa. Neu tieu
cau von, so lUdng se khdng tUdng xUng vdi mat do, do tap trung tieu cau.
- Bach cau cao: do lam tang mat do quang nen gay tang gia tao huyet sic
to, MCHC rat cao.
- Tang sUc ben hong cau: vi du trUdng hdp bilirubin mau cad, dung dich pha
vd hong cau vdi nong do va thdi gian binh thudng cua may tu dong khong du lam tan
het hong cau trUdng thanh, gay ra tang gia tad so' lUdng bach cau trong ket qua xet
nghiem. Chi bang cach kiem tra tren tieu ban Giemsa mdi phat hien dUdc.
- Huyet tUdng due: lam tang gia tao huyet sac to'.
- Tang do nhdt huyet tUdng: vdi thdi gian va ap lUc hut thdng thudng cua
may dem te bao cd the gay ra giam ba ddng ngoai vi gia tao do hut khdng du mau.
- Mau bui ban: lam tang tieu cau gia tao.
Tom la i: May dem te bao la mot trong nhiing Ung dung hieu qua tien bo khoa
hoc ky thuat vao y hoc, md ra nhieu kha nang quan trong ma phUdng phap thu cong
khong lam dude nhung ro rang khdng the thay the hoan toan dUdc Cdn ngUdi.
32
HUYET DO
1. Khai niem ve huyet do

Nhieu tinh trang sinh ly va benh ly cua cd the dUdc phan anh trUc tiep hcac
gian tiep qua so lUdng, hinh thai cung nhif thanh phan cac te bac mau. Huyet do
la ban tong ket cd binh luan cac bieu hien dd.
2. Dung cu
Bo dung cu lay mau tinh mach, lam tieu ban, nhuom Giemsa va hong cau ludi.
May dem te bao hoac cac thiet bi thay the (quang pho ke, ly tam vi the tich, potain).
Kinh hien vi quang hoc.
3. Cac thong so can thiet

- So' lifdng hong cau, so lifdng bach cau, so' lifdng tieu cau, lifdng huyet sac
to', hematocrit, the tich trung binh hong cau, lifdng huyet sac to' trung binh hong
cau, nong do huyet sac to' trung binh hong cau, ty le phan tram hong cau lifdi.
- Cong thifc bach cau, dac diem hinh thai te bao hong cau, bach cau, tieu
clu , do tap trung tieu cau va nhUng bat thifdng tren tieu ban.
4. PhUdng phap phan tich ket qua

4.1. N guyen tac
- Quan sat ky cac dac diem hinh thai tren tieu ban mau nhudm Giemsa,
doi chieu cac thong so' do, dem dUdc (so' lifdng te bao cac loai, huyet sfic to',...) vdi
thifc te tren tieu ban, neu khong phu hdp phai tim nguyen nhan: do ky thuat hay
bat thifdng benh ly.
- So sanh cac thong so', cac dac diem quan sat vdi gia tri tham chieu tifdng
iifng cua ngifdi khoe manh. can lifu y den tuoi, gidi cua benh nhan.
- Doi chieu lam sang bao gom benh sU, cac trieu chUng thuc the dac biet la
tinh trang nhiem trung, xuat huyet, gan to, lach to, hach to; tien sU tiep xuc, benh
tat va dung thuoc cua benh nhan.
4.2. Cach thu thap dOr lieu
- Hong cau: so lifdng binh thifdng, tang hay giam; mUc do tang, giam? Dac
diem phan bo' tren tieu ban (binh thifdng, ngUng ket, chuoi tien)? Kich thudc dong
deu hay khdng, neu khdng dong deu thi hong cau to hay nho chiem Uu the? Hong
c&u binh sac hay nhudc sac? Hinh thai cd binh thudng khdng, neu khdng binh
thudng thi phai mo ta cac hinh thai quan sat dUdc va mUc do nhieu it cua tifng
33
loai (hinh bia b£n, hinh giot nifdc, hinh liem,...). Cac the bat thudng trong hong
C£tu: the Jolly, vong Cabot,... Co hong cau non hay khong, loai nao la chu yeu?
Hong cau ludi binh thifdng, tang hay giam?
- Bach cau: so lifdng bach cau binh thifdng, tang hay giam; mtic do tang,
giam? Nhan xet tifng loai bach c&u quan sat dUdc tren tieu ban mau ve so lifdng
va hinh thai, dac biet lUu y cac bach d i u cd hinh thai bat thudng: phai co' g in g xac
dinh mile do biet hoa, cd phai blast khdng, thuoc loai bach cau nao (nhuom hoa
hoc te bao khi c&n thiet).
- Tieu c&u: so' lifdng va do tap trung tieu cau cd binh thudng khdng. tang
hay giam? Neu tieu ban lam tif mau chong ddng thi thUdng khdng danh gia dtfdc
do tap trung tieu cau. Kich thifdc tieu cau binh thifdng, to hay nho? Cd tieu cau
khong lo hay khdng, nhieu hay l't? Trong trifdng hdp nghi ngd sinh mau ngoai tuy
can tim xem cd mau tieu cau d mau hay khdng?
- Cac bat thifdng khac: ky sinh trung sot ret, au trung giun chi, ung thu
di can...
4.3. Tra leri ket qua
4.3.1. So lieu: Ghi day du cac thong so' can thiet (muc 3).
4.3.2. Nhan xet
- Doi vdi cac duf kien binh thifdng: k h d n g n h a t thiet p h ai liet ke d&y du, tuy
theo tifng trUdng hdp cu the ma ghi n h iin g dac diem c&n thiet.
- Ddi vdi cac du( kien na m ngoai m ien gia tri b in h thifdng thi ph ai ghi d&y
du va cang lUdng hoa chinh xac ca n g tot.
4.3.3. Ket luan: Chi ket luan khang dinh khi cd cac yeu to chac chan (vi du tim
thay ky sinh trung sot ret). Da so trifdng hdp ket qua phan tich huyet do chi cho
phep gdi y dinh hifdng chan doan (vl du nghi ngd leukemia cap) hoac khu tru van
de do loai trif difdc mot hay nhieu kha nang.
4.3.4. De nghi: Cd the de xuat yeu cau can thiet cho chan doan benh (vl du de nghi
dien di huyet sac to khi huyet do gdi y mot tan mau bam sinh).
TUY DO
1. Khai niem
, ^ nghiem p h a n tich so lifdng va h in h thai cac te b a d tuy xUdng

de tham dd chiic n a n g tao m au cu n g n h u gdi y cac n g u y e n n h a n g a y rdi loan chtic
n a n g tao m au tai tuy xUdng.
34
Bo dung cu sat trung tai cho: Con iod 5%, con 70°, bong,...
Bdm tiem 5 - 1 0 ml.
Kim choc tuy.
Ong nghiem co ED TA (K 2 hoac K 3) kho.

V at lieu c&m mau.
Bo dung cu lam tieu ban tuy, nhuom Giemsa va hong cau ltfdi.
M ay dem te bao hoac cac thiet bi thay the (quang pho ke, ly tam vi the
tich, potain).
Kinh hien vi quang hoc.
Xylocain 2 %.
Thuoc nhuom Giemsa va hong cau liicii.

3.1. Chuan bi
Phong thu thuat phai sach, dung cu phai vo trung: kim choc tuy, kim lay
mau, gac tham mau...
Benh nhan phai dtfdc chuan bi tinh th in trudc khi lam thu thuat, giai thich
ve sii can thiet cua thu thuat.
Thii test thuoc te.
3.2. Vj tri choc
- Gai chau sau tren: Benh nhan n lm sap thoai mai, ke noi hai mao chau
vdi nhau, ke tiep mot dtfdng noi dinh xUdng cut vdi mad chau d dtfdng nach giiia,
lam thanh mot tam giac xUdng cut-chau-cdt song. Diem choc n im chinh giiia
dtfdng phan giac cua tam giac tren ke tii goc cot sohg-chau. Ddn gian hdn, sd tay
vao vung gai chau sau tren (diem lom khi benh nhan n4m sap) se thay mot diem
go len, dd chinh la diem choc.
- Xtfdng tic: Khoang lien siidn 2-3 tren dtfdng chinh giGa.
- Ngdai ra, cd the choc d gai chau trtfdc tren, phan tren trong cua dau tren
xtfdng chay, phia trong giiia xtfdng got.
3.3. Thu thuat

- Xac dinh vi tri choc.
- Sat trung da theo hinh xoay oc tii diem moc ra xung quanh ban kinh 5 cm
b&ng con iod, sau dd bang con 70°.
- Gay te tai cho tiing I6 p b in g lm l xylocain, bat dau tii diidi da den mang
xiidng, chd 2-3 phut.
35
- Cam kim choc tuy bang hai ngon cai va tro, long ban tay ty len doc kim.
Dua kim qua da dung vao diem gay te bang cach xoay nhe nhang nghieng 45° so
vdi mat da, sau do dung dtfng kim choc qua phan cd. khoan nhe tren mang xtfdng.
neu benh nhan khdng dau tiep tuc khoan qua ban xtfdng cting. khi cd cam giac
xop hdn. khoan tiep 3-5mm, lay nhe kim neu thay ch ic thi dtfng tai dd.
- Rut nong thong de vao hop vo trung.
/ . ■> . A
- Lap bdm tiem can than.
- Hut gon. ap ltfc vtfa phai (0,5 ml) benh nhan cam thay hdi dau. khi gan
du so ltfdng 0 . 5 ml dich tuy. ndi long bdm tiem hut tiep cho du so' ltfdng.
- Rut bdm tiem. lap nong thong lai va rut kim.
Bdm 0,3 ml dich tuy vao ong nghiem cd chong ddng kho, lac nhe de dem so
ltfdng te bao tuy va u hong cau ludi; 0 , 2 ml len mot lam ki'nh, keo 8 tieu ban. Cung
cd the lam them tieu ban ap neu can thiet.
- De tieu ban kho tu nhien trong phong xet nghiem (mat, kho), nhuom
Giemsa hai thi.
4. Phan tich ket qua

4.1. Nguyen tac
- Ve cd ban gio'ng nhtf nguyen tac phan tich huyet do.
- Can doi chieu cac dti lieu thu thap dtfdc gitfa mau ngoai vi va dich hut tuy
xtfdng cua benh nhan trong cung thdi diem.
4.2. Quan sat toan bo tieu ban nhuom Giemsa b&ng vat kinh x 10
- Danh gia mat do te bao cd nhan va dac diem phan bo cua te bao ke ca
hong cau trUdng thanh.
- Tim kiem mau tieu cau va cac te bao kich thtfdc ldn (ung thtf di can).
- Ltfa chon cach thtic tinh ty le phan tram cac te bao cd nhan.
4.3. Quan sat bang vat kinh dau x 100
- Xem xet ky khu vtfc dau. duoi. trung tam va hai canh tieu ban nhuom
Giemsa de rut ra nhtfng nhan dinh ve dac diem so ltfdng. hinh thai te bao va tinh
trang biet hoa cua mdi ddng te bao cung nhtf ttfdng quan phat trien cua cac ddng
te bao.
Tim hinh the bat thtfdng: ung thtf di can. ky sinh trung... Neu cd blast,
phai can etf vao hinh thai va hoa hoc te bao de xac dinh xem blast thudc ddng nao
(ddng hat, lympho. mono,...).
- Lap cong thtfc tuy ttf 100-500 te bao cd nhan tuy thed muc dich chan doan
ha\ nghien ctiu. Tinh chi so trtfdng thanh cua ddng hat, dong hong cau va ty le
nguyen hong cau/bach cau hat.
- Lap cong thtfc mau tieu cau ttf 1 0 0 mau tieu cau neu benh nhan cd giam
tieu cau ngoai vi.
36
4.4. Tra l&i kS't qua
4.4.1. Ban tra Idi ket qua: ngoai cac so' lieu cu the phai neu nhan xet ve so' ltfdng va
hinh thai te bao tuy va cac bat thtfdng (neu cd).
- Ch&'t ltfdng cua tieu ban.

- Mtfc do giau - ngheo cua tuy xtfdng.
- Phan tich sd' ltfdng va hinh thai cac ddng te bao binh thtfdng.
- Cac bat thtfdng neu cd.
4.4.2. Ket luan

o
- Khang dinh chan doan.
- Khu tru pham vi hoac dinh htfdng tim kiem chan doan.
- Loai trtf mot hay nhieu kha nang.
- M ot so' it trtfdng hdp khdng ket luan dtfdc.
4.4.3. De nghj: Trong mot so' trtfdng hdp cd the ghi yeu c lu can thiet cho chan
doan benh neu ket qua tuy do khdng khang dinh dtfdc chan doan.
HOA HOC TE BAO
Hoa hoc te bao la phtfdng phap khao sat mot so' thanh phan co chtia trong
cac khdng bao n&m trong bao ttfdng cua te bao, dtfdi kinh hien vi quang hoc sau
khi lam hien mau bang cac thuoc nhuom hoac cac cd chat thich hdp.
1. Nguyen tac chung

1.1. Tat ca cac phuong phap nhuom hoa hoc te bao deu bao gom ba giai doan
1.1.1. Co dinh: Ngoai muc dich cd' dinh te bao tren tieu ban, do'i vdi moi phtfdng
phap nhuom phai ltfa chon hoa chat co' dinh hdp ly de bad ton toi da thanh phan
can khao sat: vi du cac khdng bad md trong nhuom sudan den, men peroxydase
trong nhuom peroxydase,...
1.1.2. N huom : Dung cac thuoc nhuom (sudan den) hay cac cd chat (benzidin,
oc naphtdl AS acetat,...) de trtfc tiep hoac gian tiep lam hien mau cac thanh phan
can khao sat.
1.1.3. Tao nen: Dung mot thuoc nhuom lam hien mau hinh thai te bao (nhan va
bao ttfdng) tren tieu ban. Mau nen phai cd do ttfdng phan can thiet de giup quan
sat thuan ldi cac hat dtfdng tinh trong mdi phtfdng phap nhuom.
37
1.2. Phai loai bo het chat nhuom hay co djnh va 6 4 khd tieu ban truoc khi
chuyen sang budc tiep theo
1.3. Doc ket qua

1.3.1. Danh gia mire do duong tinh
- Do 0: Khong co hat b it mau hoa hoc te bao la (-)

- Do 1: Cac hat b it mau chiem khoang < 1/3 bao ttfdng te bao la (+)
- Do 2 : Cac hat b it mau chiem khoang >173 den < 3/4 bao ttfdng te bao la (++)
- Do 3: Cac hat b it mau chiem het bao ttfdng te bao la (+++)

- Do 4 : Cac hat b it mau chiem het bao ttfdng va de len ca nhan te bao la
(++++)
1.3.2. Tinh diem (score): Danh gia mtfc do dtfdng tinh hoa hoc te bao cua 100 te
bao can nghien ctiu. Gia stf ty le phan tram te bao d cac dd 0, 1, 2, 3, 4 ttfdng ting
vdi a, b, c, d, e thi cong thtfc tinh diem nhtf sau:
Score = (a x 0 ) + (b x 1 ) + (c x 2 ) + (d x 3) + (e x 4)
2. Gia tri■ cua hoa hoc

• te bao
- Ho trd phtfdng phap hinh thai hoc de xac dinh ddng te bao, mtfc do biet
hoa te bao trong phan loai Id xe mi cap, hoi chting roi loan sinh tuy va cac b&t
thtfdng khac.
- Trong mot so trtfdng hdp dac biet, hoa hoc te bao giup xac dinh mot quin
the te bao cd phai blast hay khdng: vi du cac microblast ddng hat tren tieu ban
Giemsa giohg nhtf lympho roi loan hinh thai.
3. Bieu hien hoa hoc te bao cua mot so dong te' bao mau
Bang 1.3: Phan ting hoa te bao cua cac te bao mau
Te bao
Dong hat Ddng mono Dong hong cau Ddng lympho
Phtfdng phap ^
Peroxydase +++ ± -> ++ - -
Sudan den +++ + -> ++ - -
Esterase dSc hieu +++ - - -
Esterase khdng dSc hieu ++ +++ - - -> ±
Esterase khdng
+± -» ++ - -> ± - - -> ±
dac hieu cd NaF
PAS + ± + . _ > ++♦
LAP +++ - - -
Peris - - - -> + J
38
4. Mot so phuong phap nhuom hoa hpc te bao
4.1. Nhuom peroxydase (Phtfdng phap cai tien cua Nguyen Van Tinh)
4.1.1. Co che: Dtfdi tac d u n g cua peroxydase, h y d ro g e n peroxyd (H 2 0 2) se giai

p h o n g ra mot oxy n g u y e n tut, oxy hoa cd chat (b e n z id in ) tao chat tua m a u tUdng
lin g tro n g bao tUdng bach cau:
Peroxydase Cd chat
H 20 2 ......... -> H 20 + 0 -------- -------- ► Oxy hoa

Chat mau
4.1.2. Pha dung djch benzidin 0,1%
- Benzidin: lOOmg
- Con tuyet doi: 1 0 ml
- Nude cat vtfa d u 100ml
- Oxy gia (30 the tich): vai giot
Lac deu, bao quan d nhiet do phong xet nghiem. Lac trtfdc khi stf dung.
4.1.3. Q uy trinh nhuom

- Ngam tieu ban trong con formol 10%: 10 phut.
- Riia dudi vdi ntfdc chay 30 giay, de kho ttf nhien.
- Nhuom benzidin 0,1%: 2 - 3 phut.
- Rua dudi vdi ntfdc chay 1 phut, de kho ttf nhien.
- Nhudm Giemsa 1/10: 10-12 phut.
- Riia dudi vdi ntfdc chay 1 phut, de kho.
4.1.4. D oc ke t qua
Phtfdng phap nhuom nay cho cac hat dtfdng tinh mau vang xin (gi s&t) trong
bao ttfdng te bao. Tinh so' ltfdng te bad dtfdng tinh trong tong so' 100 te bao can
xem xet. Doi vdi leukemia cap thi dd la 100 te bad blast. Ngtfdi ta chi quan sat de
cd nhan xet chung ve mtfc do dtfdng tinh chtf khdng can thiet tinh scdre trong
phtfdng phap nhudm peroxydase.
4.2. Nhuom sudan den
4.2.1. CO che: Chat mau sudan den cd thudc tinh hoa tan trong lipid, ngtfdi ta ldi
dung dac diem nay de phat hien thanh phan lipid cd trong te bao.
4.2.2. Pha dung dich sudan
( 1 ): Sudan den B: 0 ,lg

Con 96°: 30ml
39
(2): Dung dich phenol:
Dung dich phenol: 2,96ml
N a 2 H P 0 4. 12 H 2 0: 0,06g
Con 96°: 6 ml
Nude cat: 20ml
Tron (1) va (2)
4.2.3. Quy trinh nhuom

- Co dinh tieu ban bang hdi formol: 10 phut.
- ROfa dtfdi vdi ntfdc chay 30 giay, de kho ttf nhien.
- Nhuom sudan: 25 phut.
- Nhung con 70°: 1/ 2-1 phut.
- Riia dtfdi vdi ntfdc chay 30 giay, de kho ttf nhien.
- Nhuom Giemsa 1 /1 0 : 12 phut.
- Riia dtfdi vdi ntfdc chay 1 phut, de kho.
4.2.4. Doc ket qua
Cac te bao dtfdng tinh chtfa cac hat, cue mau den trong bao ttfdng va thtfdng
chdm len nhan, cd khi che km ca te bad. Cach doc ket qua giong nhtf phtfdng phap
nhuom peroxydase.
4.3. Nhuom PAS (Periodic Acid Schiff)
4.3.1.Co che: Dtfdi tac dong cua periodic (H I 0 V2H 2 0 ), nhom chtfc rtfdu cua
glycogen dtfdc chuyen thanh aldehyd, se tac dung vdi thuoc thtf schiff, cho hdp
chat mau hong ttfdi trong bao ttfdng te bao.
HIO,. 2H20 O
R - CH2O H ........... -> H 20 + 2 R - C^ + a. Fucsin sulfurd —> Chat mau hong ttfdi
H (thuoc thtf schiff) (mau hoa mtfdi gid)
4.3.2. Pha dung dich nhuom
a. Dung dich periodic 1%

- Periodic: lg
- Ntfdc cat: 1 0 0 ml
b. Dung dich schiff
- Fuchsin basic: 0 ,5 g
- Ntfdc cat: 1 0 0 ml / 40-50°C.
- Lac deu cho tan het, de nguoi, loc.
40
- Them 0,9 ml acid HC1 nguyen cM't
- Them 0,5 g N a 2 S2 0 5
De trong tu toi 24 gid, dung dich chuyen sang mau vang nhat.
Bao quan trong lo mau cf 4°C de dung din.
c. D u ng dich hematoxylin:
Alun de kali: 25g
1 . Nude cat 350ml (dun soi cho tan het)
Them 0,5g hematoxylin
Iodat kali ( K I 0 3): 0 ,lg
2.
Nude c£t: 50ml
Khi nao (1) da nguoi, tron ( 1 ) va (2), sau do cho them 100ml glycerin
nguyen chat.
- Ngam tieu ban trong formol 10%: 10 phut.

- RiJta dtfdi voi ntfdc chay 30 giay, de kho ttf nhien.
- Nhuom periodic 1%: 10-15 phut.
- Rtia dtfdi voi ntfdc chay 30 giay, de kho ttf nhien.
- Nhuom schiff: 10-12 phut.
- Riia dtfdi voi ntfdc chay 30 giay, de kho ttf nhien.
- Nhuom hematoxylin: 20 phut.

4.3.4. Doc ket qua
Cac te bao dtfdng tinh co cac hat, cuc mau do ttfdi (mau hoa mtfdi gid) trong
bao ttfdng. Cac te bao dtfdng tinh lan toa (bao ttfdng co mau do ttfdi min, khong co
hat) khong co gia tri. Doc ket qua giong nhtf phtfdng phap nhuom peroxydase.
4.4. Nhuom esterase khong dac hieu
4.4.1. Cd che: Trong dieu kien pH va nhiet do thich hdp, naphtol ttf do dtfdc giai
phong ttf cd chat (vl du °c Naphtol ASD acetate) dtfdi tac dung cua men esterase
cua oacn cau liav mono G3 ket hdp vdi muoi diazo (tan, khong mau) de tao
thanh mot chat tua va co mau (azo).
Diazo
Cd chat -» Naphtol ttf do -> I Oxy hoa (men esterase)
Azo (mau, tua)
41
Tuy nhien, co stf khac nhau gitfa hoat tinh men esterase trong bach ciu
mono va bach clu hat khi them N aF vao dung dich nhuom: men esterase trong
bach clu mono bi mat hoat tinh (tfc che) g in nhtf hoan toln , ngtfdc lai, hoat tinh
men esterase trong bach clu hat h lu nhtf khong thay doi. Chinh vi d&c diem n&y
ma ngtfdi ta sif dung hai phtfdng phap nhuom esterase khong dac hieu la tic che
va khong tfc che de phan dinh dong hat va mono.
4.4.2. Pha dung dich nhuom :
1 . Dem Tris: -Tris: 2,43g

- Ntfdc cat: 25ml
- Acid HC1 IN : 18,5ml
- Ntfdc cat vtfa du 100ml
2 . Dung dich cd chat: - oc Napthol -AS acetate: lOmg
- Propylen glycol: 0,3ml
Tron 20ml (1) vdi ( 2 ) lac deu, them mot it muoi fast blue (»30m g). Dung dich
co mau vang chanh, sau do dem nhuom ngay.
a. Khong tic che
- Co dinh b ln g hdi formol 40%: 15 phut.
- U trong dung dich nhuom d 37°C: 1 gid.

- Rijfa dtfdi voi ntfdc chay 30 giay, de kho ttf nhien.
- Nhuom Kernetrot: 1 2 phut
- Rtfa dtfdi voi ntfdc chay 30 giay, de kho ttf nhien.
- Chu y: tieu ban phai dtfdc co dinh ngay, cang sdm cang tot ke ttf khi lay
benh phlm ra khoi cd the.
b. lie che bang N a F
- Co dinh bang hdi formol 40%: 15 phut.
- U trong dung dich nhuom d 37°C' 1 gid.
fed them N a F vao dung dich nhuom: 5mg I m l)

- Nhuom Kernetrot: 12 phut.
- Rtfa dtfdi vdi ntfdc chay 30 giay, de kho ttf nhien.
42
4.4.4. Doc ket qu£
Cac te bô dtfdng tinh co hat mau xanh th lm trong bao ttfdng. Trong phan
loai leukemia cflfp, khi doc ket qua phai tinh score.
4.5. Nhugm phosphatase kiem bach cau

4.5.1. Co che
Muoi phosphat cua oc naphtol dtf6 i tac dong cua men phosphatase ki€m bach
cau trong moi trtfdng co ion magie se giai phong ra oc naphtol. oc naphtol ket hdp
vdi muoi diazo (fast blue) tao thanh mot san pham azo tua, co mau.
4.5.2. Pha dung djch nhuom

- Dung dich dem
+ TRIS: 12,5g.
+ Ntfdc cat: 12,5 ml.
+ Acid HC1 IN : 41ml.
+ Ntfdc cat vtfa du 500 ml, pH = 8,07.
- Dung dich cd chat
+ 40 mg oc naphtol phosphat.
+ 1 , 2 ml N -N dim ethyl formamid.
+ Lac deu cho tan het.
+ Them vao 200 ml dung dich dem TRIS.
+ Khi nhuom cho them vao hon dich tren mot it muoi fast blue sao cho hon
dich co mau xanh hdi nhat.
- Dung dich Kernetrot:
+ Nuclear fast red: 0,2g.
+ A12 (S 0 4) 3: 5 g.
+ Ntfdc cat: 100ml
Dun nong de A12 (S 0 4) 3 tan het trong ntfdc cat, sau do cho nuclear fast red
vao de 5 phut, de nguoi, them vao vai hat thymol. Loc trtfdc khi sii dung.
4.5.3. Ky thuat
- Co" dinh tieu ban mau va tuy bang con formol 10%: 15 phut.
- Rtia sach, de kho.
- U trong dung dich nhuom 1 gid, 37°C.
- Rtfa dtfdi voi ntfdc chay, de kho.
- Nhuom nen bang dung dich Kernetrot.
- Chu y: tieu ban phai dtfdc co dinh ngay, cang s6 m cang tot ke ttf khi lay
benh pham ra khoi cd the.
43
4.5.4. Doc ket qua
Cac te bao dtfdng tinh co hat mau xam den trong bao ttfdng. Tinh score
trong 1 0 0 te bao can xem xet.
4.6. Nhudm Peris
4.6.1. Co che
Trong moi trtfdng acid, ion s it cua ferritin (F e+++) se tac dung vdi ferrocyanid
tao ra ferric ferrocyanid co mau xanh pho (xanh cobalt dam).
4.6.2. Pha dung djch nhuom
Tron lan hai dung dich sau theo ty le 1/2:

- Acid HC1 2%: 0 1 the tich.
- Dung dich Potasium Ferrocyanid (K 4 FeC N 6 . 3H 2 0 ) 2%: 02 the tich.
4.6.3. Ky thuat
- Tieu ban mau, tuy co' dinh bang con metylic: 10 phut
- Riia tieu ban, de kho

- Nhuom trong dung dich nhuom: 30 phut
- Rtfa tieu ban
- Nhung trong acid HC1 1 %.
- Rtfa tieu ban
- Nhuom nhan b&ng dung dich Kernetrot: 12 phut
- Rtfa tieu ban
- Tieu ban mau, tuy de kho va doc ket qua
4.6.4. Doc ket qua
Cac te bao dtfdng tinh co hat hoac cuc mau xanh pho trong bao ttfdng. Tinh
score trong tong so 1 0 0 te bao hong cau trtfdng thanh hoac nguyen hong cau.
Ngoai ra, can tinh ty le phan tram te bao nguyen hong cau sat vong.
SINH THIET TUY XlfflNG
1. Nguyen ly
Bang ky thuat cat lat, co dinh va nhuom to chtic hoc, sinh thiet tuy xtfdng
cho phep khao sat:
44
- C&'u true mo benh hoc cua tuy sinh mau.
- So' ltfdng, hinh thai, c&'u true, thanh phan va ca vi tri nguyen uy cua te
bao trong vi moi trtfdng sinh mau.

- Dung cu va vat lieu sat trung tai cho: bong, con iod 5%, con 70°.
- Microtom.
- V at lieu cam mau.
- Bdm tiem 5ml
- Kim sinh thiet tuy xtfdng: thtfdng dung kim Jamshidi.
- Lo thuy tinh 60 ml, cd to.
- Be nhuom.
- Phien kinh, la kinh va chat gan (Bom Canada).
- Thuoc gay te tai cho (thtfdng dung: xylocain 2%)
- Cac dung dich xtf ly manh sinh thiet: Dung dich co' dinh H elly
Dung dich khtf calci Custer.
Con 90°, con tuyet ddi.
Xylen.
- Paraphin diem nong chay 58° C.
- Cac dung dich nhuom manh sinh thiet.
3. Pha dung djch

3.1. Dung djch Helly
- Dung dich Zenker:
+ K 2 Cr 2 0 7: 2,5g
+ H gC l2: 5g
+ N a 2 SO.,: lg
+ Ntfdc cat: 100ml
- Cach pha khi dung:
+ C H 3 COOH: 5ml
+ H CH O trung tinh: 5ml
+ Dung dich Zenker: 100ml
3.2. Dung djch Custer
- Acid formic 85- 90%: 50ml
45
- Niidc cat: 35ml
- Citrat sodium: 17g/85ml niidc cat am (40° C)
3.3. Dung djch Hemalun de mayer

- Alun de kali: 5g
- Hematein: 0,6g
- Acid acetic: 5ml

4.1. Thu thuat
- Xac dinh diem choc (xem bai tuy do).
- Sat trung vung gai chau sau tren bang con iod va con 70°.
- Gay te tifng ldp, dac biet can than mang xUdng gai chau sau tren.
- Cho 2 phut.
- Dung hai dau ngon tay (ngon giiia va tro trai) co dinh diem moc choc.
- Tay phai cam kim sinh thiet bang ngon cai va tro, long ban tay ty len doc
nong kim sinh thiet.
- Dua kim qua da nhe nhang bang lUc an xoay, kim nghieng 45° so vdi mat da.
- Sau khi qua da, dung kim thang ddng khoan nhe nhang qua ldp cd.
- Xac dinh lai diem moc bang dau ngon tro trai: dich chuyen dong thdi da
va kim sang mot phia.
- Khoan nhe kim tren mang xUdng, neu benh nhan khong dau tiep tuc
khoan xoay tii tii qua mang xUdng. goc quay kim khoang 10-15°.
- Khi co cam giac vao phan xop, tay trai giu! co dinh phan vo kim, tay phai
rut nong kim.
- Dem mot mieng gac vo trung len doc kim, tay phai tiep tuc khoan xoay
nhe nhang 1-1,5 cm.
- Lac nhe kim de cat.
- Xoay kim tai cho theo mot chieu 2-3 vong.
- Rut kim bang cach xoay, cho den het phan xUdng.
- Nghieng kim. day dong thdi ca kim va phan da sang phai 0 ,5 -lcm bang tay
phai va hai ngon (tro va giiia) trai. an chat ngon giiia trai vao ho sinh thiet.
- Rut kim qua da, cam mau. dan bang.
- Tha manh sinh thiet vao dung dich co din h.'
4.2. X irly manh sinh thiet

Co dinh trong dung dich Helly: 5-6 gid.
Rua dudi voi nude chay: l gio.
46
KhOf calci bang dung dich Custer: 12 gid.
Rtia dtfdi voi ntfdc chay: lgid-
Loai nticic bang con: Con 90° 30 phut
Con tuyet doi I 1 gid
Con tuyet doi II 1 gid
Con tuyet doi III 3 gid
Loai con bang xylen: Xylen I 30 phut
Xylen II 90 phut
Xylen II I 90 phut
Vui benh pham trong paraphin d 60 °C 1 2 gid.
Due khuon.
Cat tieu ban chieu day 2-2,5^m.
4.3. Nhuom sinh thiet (phuong phap H&E)
- Tay nen bang xylen 3 lan: 5 phut 1 lan

- Chuyen qua con 100°, 95°, 80°: 5 phut 1 lan
- Rufa dtfdi voi ntfdc chay: 10 phut
- Phu tieu ban bang dung dich lugol: 10 phut
- Rifa dudi voi ritfdc chay: 1 0 phut
- Phu tieu ban bang dung dich N a 2 S 2 0 3: 5 phut
- Rtfa dudi voi nude chay: 10 phut
- Nhuom trong dung dich hemalun de mayer: 3-5 phut
- Rtfa dudi voi ntfdc chay: 10 phut
- Nhung tieu ban trong dung dich con-HCl 1 %: vai giay
- Phu tieu ban bang dung dich N a 2 C 0 3 1 %: 1 phut
- Rtfa dudi voi ntfdc chay: 10 phut
- Nhuom trong dung dich eosin 1%: 1 phut
- Riia ntfdc nhanh
- Tay tieu ban bang con tuyet doi, lam trong bang xylen, gan la kinh
5. Phi/dng phap phan tich ket qua

5.1. Tieu chuan tieu ban sinh thiet
Dai 1-1,5 cm.
Co it nhat 1 0 khoang sinh mau.
M ot ldp te bao.
Gitf nguyen ven cau true mo benh hoc tuy xUdng.
The hien dtfOc dac diem te bao va mo sinh mau.
5.2. Nguyen tdc

- Quan sat tong the (bang vat ki'nh x 4 hoac x 10) cau true khoang tao mau
va cac thanh phan co kich thudc ldn nhu mau tieu cau, di can ung thtf trtfdc khi
quan sat chi tiet hinh thai te bao bang vat ki'nh x 40.
- So sanh cac dtf lieu thu thap dtfdc vdi ngtfdi binh thtfdng cung tuoi, gidi.
- Trong nhieu trtfdng hdp, can doi chieu vdi lam sang va ket qua huyet tuy
do (neu co).
5.3. Cach thu thap ddrheu

5.3.1. Danh gia chung (vat kinh x 4)
- Ki'ch thtfdc manh sinh thiet co du de danh gia tinh trang benh ly cu the
hay khong.
- Co sai sot ky thuat nhtf ep tuy qua manh, cham mach mau, hay xe rach
khoang tao mau khong?
- Co nhting thay doi cau true ldn nhtf di can ung thtf, hoac u lympho hay khong?
5.3.2. Danh gia chi tiet (vat kinh x 40)
- Cau true khoang tao mau, bao gom:

+ Ki'ch thtfdc va dac diem cua be xtfdng: Co the bi r o n g di (ro xtfOng) hoac
day len. Binh thtfdng tao cot bao n am trong cac be xtfdng, h a u nhtf k h o n g ga p huy
cot bao.
+ Mat do va phan bo te bao: Danh gia sd bo ty le te bao tao mau va te bao
dem (xd, md) tren toan bo te bao tuy. Binh thtfdng khu vtfc te bao tao mau chiem
khoang 60-70% dien tich sinh mau, cang ldn tuoi to chtfc md cang phat trien.
- Ty le dong hat/dong hong cau: Binh thtfdng 2/1 - 4/1.
- Mtfc do trtfdng thanh cua dong hat va dong hong cau: Ty le trtfdng thanh
cua dong hat va dong hong cau binh thtfdng khoang 1 /4 .
- Dong mû tieu cau: Binh thtfdng co 1-4 MTC/ vi trtfdng x40. Khi so ltfdng
mau tieu cau gap 3-4 lan binh thtfdng mdi dtfdc coi la tang mau tieu cau.
- Lymphocyt: Binh thtfdng chiem khoang 5 % te bao tuy, nam rai rac trong
khoang tao mau khong theo quy luat. 0 ngtfdi tren 40 tuoi, lymphocyt chiem
khoang 10-15%.
- Plasmocyt: dtfdi 5% te bao tuy. thtfdng nam canh thanh mach mau.
- Dai thtfc bao: Ty le rat it. bao ttfdng md nen kho phat hien tren cac tieu ban
sinh thiet. Khi quan sat thay dai thtfc bao la chtfng to tang dai thtfc bao.
- Dtf trtf sat: Nhuom Peris.
48
- Sdi lien vong: Nhuom Gomogi va Van Gieson.
- Xam nh&p cua cac te bao ngoai tuy xtfdng (lanh tinh hoac ac tinh).
5.4. Tra l&i ket qua

La mot ban mo ta kem theo nh&n xet dtfa tren cd scf phan tich khach quan
nhtfng chi tiet cu the, co the minh hoa b ln g so" lieu gom:
- V i tri sinh thiet, cac diem dac biet khi lam thu thuat (xtfdng xop, cting,...)
va dac trtfng cd ban ve tieu chuan cua tieu ban.
- Dac diem cau true va thanh phan khoang tao mau (kich thtfdc be xtfdng
va khoang sinh mau, mat do va dac diem phan bo' te bao...).
- Dac diem ve hinh thai hoc va ttfdng quan so" ltfdng cua cac dong te bao,
bao gom ca xd, md, huy cot bao va tao cot bao.
- Tinh trang xam nhap cua cac te bao ngoai tuy, dac biet la di can ung thtf.
HACH DO
1. Nguyen ly
Hach do la mot xet nghiem te bao hoc can thiet trong phan ldn cac trtfdng
hdp hach to, nhat la doi vdi cac trtfdng hdp nghi ngd ac tinh hoac viem dac hieu.
Thong qua viec choc hut va lam tieu ban, co the danh gia dtfdc thanh phan va ty
le te bao trong hach. La xet nghiem tham do trtfc tiep, hach do rat co gia tri cho
ch in doan xac dinh.

- Bo dung cu sat khuan va cam mau tai cho.
- Bdm tiem 5- 1 0 ml, kim tiem loai 20 G x 1 V2 hoac ldn hdn.
- Phien ki'nh lam tieu ban.
- Bo dung cu nhuom Giemsa.
- Ki'nh hien vi quang hoc.

- Ltfa chon hach: tot nhat la cac hach ngoai bien nam phia tren cd hoanh,
trtfdng hdp dac biet mdi choc hach dtfdi cd hoanh. Phai can nhlc rat ky khi choc cac
hach d sau (nguy cd chay mau cao) nhtf hach o bung, trung that.
- Sat kh u ln tai cho.
- Co dinh hach b ln g ngon tro va ngon cai tay trai. Tay phai cam bdm tiem
lip s in kim, choc nhe nhang qua da, khi choc qua vo hach se co cam giac mat do
49
chic hdn. Tuy theo kich thtfdc hach to hay nho ma quyet dinh do sau cua kim. N 6 i
chung, nen choc vao hach 0,5-1 cm hoac den vung trung tam hach. Hut manh 4-5
lain (ap ltfc - 5 den - 8 ml), neu hach co mat do ch ic thi co the gia bdm tiem d ap ltfc
am quay bdm tiem 2-3 vong dong tam. Tha ttf ttf pit tong cua bdm tiem cho den
khi het ap ltfc am. Rut bdm tiem va kim tiem. Bdm nhe chat hach len phien ki'nh,
dan tieu ban.
- De kho tieu ban ttf nhien, co' dinh nhuom Giemsa.
- Kem theo hach do, ngtfdi ta thtfdng lam hai tieu ban mau ngoai vi de so
sanh khi can thiet.
4. Doc ket qua

- Dung vat ki'nh x 4 va x 10 quan sat toan bo tieu ban. Nen xem nhieu tieu
ban choc hut. Chon cac tieu ban giau te bao, cac tieu ban nghi ngd de quan sat ky.
- Quan sat chi tiet te bao trong cac tieu ban da ltfa chon b in g vat ki'nh x
40, trUdng hdp dac biet mdi can dung vat ki'nh x 100.
- Thanh phan te bao cua hach binh thtfdng nhtf sau:
Te bao lympho: 90 - 99%, trong do 90 - 98% la lymphocyt.
Te bao co the gap nhtfng ty le rat thap la lien vong, monocyt, plasmocyt, to
chtic bao, mastocyt.
SINH THIET HACH
1. Nguyen ly
Trong mot so trtfdng hdp benh ly, hach do khong du dtf kien thuyet phuc chin
doan, can thiet lam sinh thiet hach. La mot xet nghiem mo benh hoc, sinh thiet hach
cho phep quan sat cau true hach, hinh thai va phan bo te bao trong hach.

- Bo dung cu sat trung da.
- Bo dung cu tieu phiu.
- Bo dung cu cam mau tai cho, bao gom ca kim va chi khau.
- Bo dung cu chuyen due, c it va nhuom tieu ban mo.

3.1. Lay hach
- Ltfa chon hach: chon hach ngoai bien nong, kich thtfdc v i a hoac nho
(dtfdng kinh dtfdi 2 cm), tot nhat d phia tren cd hoanh.
- Thong thtfdng lay ca hach. chi c it mot phan khi hach qua to.
50
3.2. XCrly hach va lam tieu ban
- C it hach theo chieu doc thanh 2-3 manh tuy kich thudc hach.
- Co' dinh, chuyen, due, cat, nhuom theo quy trinh ky thuat lam tieu ban to
chile mo mem.
4. Doc ket qua

- Quan sat tieu ban 0 vat kinh nho (x 4 hoac x 1 0 ) de co nh^n xet tong the
ve cau true hach va dac diem phan bo' te bao.
- Quan sat tieu ban 0 vat kinh x 40 de xem xet thanh p h in va hinh thai
te bao.
LACH DO
1. Nguyen ly
BSng viec choc hut va lam tieu ban co the quan sat dUdc thanh phan te bao
trong lach, giup cho chan doan mot so' benh, nhat la d benh nhan co lach to.

- Bo dung cu sat khuan va cam mau tai cho.
- Bdm tiem 5- 1 0 ml, kim tiem loai 20 G x lV 2 hoac 16n hdn.
- Lam kinh lam tieu ban.
- Bo dung cu nhuom Giemsa.

- Chu&n bi benh nhan: Trong trUdng hdp benh nhan co cac xet nghiem ve
dong mau khong binh thudng, can can nhSc ky chi dinh choc lach, phai lUdng
trUcfc bien chting chay mau trong o bung sau khi tien hanh thu thuat, nhat la d
benh nhan lach to da lau ngay.
- Benh nhan n&m n g ia , thoai mai.
- Chon vi tri choc: Thudng choc vao mom lach tren diidng nach trUcfc.
- Sat trung vi tri choc.
- Choc nhe nhang kim da l&p sSn bdm tiem den het chieu day thanh bung.
Cho benh nhan hit sau va nin thd, chdc that nhanh vao lach, hut 1-3 lan v 6 i ap
luc tif -5ml den - 8 ml, tha pit tong cho het ap lilc am, rut bdm tiem cung kim
tiem. Cho benh nhan thd binh thudng.
51
- Bdm chat hut dtfdc ra lam ki'nh, lam tieu ban nhtf ky thu^t mau din.
nhuom Giemsa.
- Benh nhan can nam bat dong 2 gid. Ban giao cho bac si dieu tri theo doi
benh nhan.
- Kem theo lach do, ngtfdi ta thtfdng lam hai tieu ban mau ngoai vi de so
sanh khi c&n thiet.
4. Doc ket qua

- Dung vat ki'nh x 4 va x 1 0 quan sat toan bo tieu ban. Nen xem nhieu tieu
ban choc hut. Chon cac tieu ban giau te bao, cac tieu ban nghi ngd de quan sat ky.
- Quan sat chi tiet te bao trong cac tieu ban da ltfa chon bang vat ki'nh x
40, trtfdng hdp dac biet mdi can dung vat ki'nh xlOO.
- Thanh phan te bao cua lach binh thtfdng nhtf sau:
Te bao lympho: 50 - 60%.
Bach cau doan: 20 - 30%.
Te bao lien vong: 10 -15%
Ngoai ra, co the gap te bao to chtic lach, te bao bieu mo, te bao thtfc bao
vdi ty le r&'t It.
XET NGHIEM TiM TE BAO HARGRAVE
1. Nguyen ly
Khang the ttf sinh trong huyet thanh ket hdp vdi khang nguyen ttfdng tfng
cua mang nhan te bao, dtfdi tac dong cua bo the, lam ton thtfdng mang nhan, tao
thanh khoi thuan nhat. Bach cau doan trung tinh thtfc bao phtfc hdp nay tao ra
“te bao Hargrave” .

- Bdm tiem 3 ml.
- Ong nghiem 5 ml.

- May lac ong nghiem.
- Bi nhtfa nhd.
- Bo dung cu lam tieu ban mau dan.
- Bo dung cu va hoa chat nhuom Giemsa.
52
- 3 ml mau tinh mach chong dong b&ng heparin (20 ddn vi / ml).
- Tron deu, de bdm tiem thSng d in g quay kim len tren trong 60 phut.
- Be cong kim, bom nhe nhang mot ntfa phan huyet ttfdng ra mot ong
nghiem kho va sach (ong 1). Ph&n lfing gifla khoi hong ciu va huyet ttfdng dtfdc
bdm ra mot ong nghiem khac, tron,deu va dtfdc chia thanh hai ph&n: mot ntfa de
nguyen (ong 2), con mot ntfa dem lac bi trong 15 phut (o'ng 3).
- Hoa lSn ong 1 , ong 2 , ong 3. L ie tron deu nhe nhang, u 37°C trong 60 phut.
- L ie deu, ly tam nhe lay can keo 4 tieu ban va nhuom Giemsa nhtf tieu
ban mau dan.
4. Ket qua
Te bao H argrave dtfdc tao ra do bach cau doan trung tmh thtfc bao mot nhan
dang thoai hoa. Nhan thoai hoa la mot khoi thuan nhat tron, to, mau gach non
tren tieu ban nhuom Giemsa. Cac doan nhan (2-4 doan) cua bach cau thtfc bao bi
day dat ra xung quanh khoi thuan nhat tao nen hinh "hoa hong". Bao ttfdng cua
bach cau doan trung tinh thtfc bao rat md nhat, kho quan sat tren tieu ban
nhuom Giemsa. Phai doc it nhat 2 tieu ban (xem toan bo tieu ban) mdi du ket
luan. Cach tra ldi ket qua tot nhat la co hay khong thay te bao Hargrave? Neu co
thi moi tieu ban co bao nhieu te bao Hargrave (tinh trung binh).
TIM KY SINH TRUNG SOT RET
I. CAN CLf DE CHAN DOAN SOT RET

1. Djch te
Benh nhan co d vung sot ret ltfu hanh hay khong ?
2. Trieu chumg lam sang

- Benh nhan co:
+ Sot cao
+ Nhtic dau
+ Ret run
+ CJn lanh
+ Va mo hoi
+ Dau toan than
53
- Cdn sot ret dien hinh thtfdng gap d ngtfdi chtfa co m iln dich hoac mien
dich yeu
- Cdn sot ret khong dien hinh thtfdng gap d ngtfdi co m iln dich hoac la dan
song d vung sot ret ltfu hanh
3. Xet nghiem mau tim ky sinh trung sot ret (KSTSR)

La yeu to quan trong nhat. Khi KSTSR dtfqJc ngtfdi xet nghiem tim tha'y
trong mau, luc do mdi khang dinh la benh nhan mac sot ret
- l/u diem:
+ Nhanh, chinh xac, phan loai dtfdc KST
+ Ddn gian, re tien
- Nhtfdc diem:
+ Benh nhan it KSTSR thi kho phat hien
+ Benh nhan da dieu tri sot ret nen khong dien hinh
+ Phu thuoc kha nang ch&n doan cua ngtfdi xet nghiem
4. Cac ky thuat mien djch

Khac phuc dtfdc nhtfdc diem tren nhtfng doi hoi nhtfng trang thiet bi hien
dai, dat tien.
II. KY THUAT TIM KY SINH TRUNG SOT RET TRONG MAU

1. Phuong tien dung cu
- Lam kinh kho, sach.
- But chi ghi tieu ban.
- Kim chich mau.
- Dung cu sat khuan: bong, con.
- Gia de tieu ban.
- Dau soi kinh.
- Giemsa, ong dong, dua thuy tinh. pipette nho dung dich.
- Con tuyet doi.
- Ntfdc cat pha dung dich Giemsa pH trung tinh.

2.1. Co hai loai tieu ban mau de xet nghiem ky sinh trung sot ret
- Tieu ban giot day (giot dac ): gom nhieu ldp hong cau nen K S T co mat se
tap trung tren mot dien nho hdn va nhtf vay se tim thay K S T nhanh hdn qua kinh
hien vi.
54
- Tieu ban mau dan (giot mong): thtfdng gom mot 16p hong cau, sau khi da tim
tha'y KST sot r6 t d giot d&y thi xem tieu ban giot dan de phan loai KST SR.
Co the lam ca giot d£y va mau dan tren cung mot tieu ban, hai gi<?t cach
nhau khoang 0,5 cm.
2.2. C£c bu’dc ky thuat: Co the lay ttf ong mau co chong dong hoac lay trtfc tiep
mau mao mach:
- Sat khuan dau ngon tay (thtfdng lay mau d ngon nhan, tre nho co the lay
6 got chan hoac ngon chan cai).
- Dung kim chich sau dung cd quy dinh.
- Bop nhe cho mau chay roi cham len phien kinh 1 giot khoang 3 - 5 fo.1, lam
tieu ban mau dan.
- Cham mot giot mau len mot phien kinh khac lam tieu ban giot dac.
- De tieu ban kho ttf nhien. Ghi ten benh nhan vao dau tieu ban.
- Co' dinh tieu ban mau dan bang con tuyet doi. Khong co dinh tieu ban
giot dac.
- De kho ttf nhien roi nhuom Giemsa 3 % - 4% trong 45 phut.

- ROfa sach bang ntfdc thtfdng. Chu y: de nguyen Giemsa, cho ntfdc chay nhe
vao goc tren tieu ban de khong lam bong mau. Dtfng tieu ban len gia.
- De kho ttf nhien.
* Co the nhuom nhanh bang cach pha dung dich Giemsa 10% de trong 1 0 -
15 phut. R ia sach va lam kho nhanh bang may sa'y toe hoac den.
3. Doc ket qua

Dtfa vao hinh thai cua ky sinh trung sot ret trong hong cau (neu co) de ket luan.
3.1. Hinh thai cac giai doan phat trien cua KSTSR (xem hinh 1.4; 1.5; 1.6)
3.2. Phan loai KSTSR: cd 4 loai
- Plasm odium fa lcip a ru m : Thtfdng gap nha't cf V iet Nam
- Plasm odium vivax : Thtfdng gap
- Plasm odium m alariae : ft gap
- Plasm odium ovale : Hiem gap

Sau day la mot so' dac diem cua hai the K STSR thtfdng gap nha't d ntfcic ta
(fa lcip a riu m )
55
B ang 1.4: Mot so dac diem ky sinh trung P. falciparum va P. vivax:
The P. falciparum P. vivax

1. Ttf dtfdng Hinh n hin, dau phay, cham than Hinh nhan, meo mo, da dang
Nhan manh, nho, co the co 2 nhan Nhan thd, to
Nguyen sinh chat dong deu Nguyen sinh chat day, dtit doan
2. Hoa thj It g3p d mau ngoai vi Hay gSp d mau ngoai vi
3. Giao bao Hinh qua chuoi, hinh liem Hinh to tron
Nhieu s ic to hinh que den hoac Co nhieu sac to vang phu len
tang den NSC mau xanh
4. Hong cau vat chu Khong thay doi, so it co nho Trtfdng to
Co hat Maurer nho min Co hat Schuffner thd, to
5. Sac to Hinh que, den Hinh trtfng, nau vang
■»
^ *
e
* C* *
} — ►The ti/duQng non (Trophozoites)
O ^
C
_ O
i
}' • *
j •
— * The ti/dufing gia
* * * * *
V w
Id i * \
’%r
H inh 1.4. Hinh thai cac giai doan phat trien cua Plasmodium falciparum
tren tieu ban giot dan, giot day
3.3. So bo danh gia ket qua
- (+): co 1 - 1 0 KST SR/100 vi trUdng d tieu ban giot dac
- (++): 1 1 - 1 0 0 KST SR/ 1 0 0 vi trtfdng d tieu ban giot dac

(+++): 1 - 1 0 KST SR / 1 vi trtfdng d tieu ban giot dac
- (++++): tren 10 KST SR / 1 vi trtfdng d tieu ban giot dac
56
0 ; #v • 9* t
8 * •' ' — * The ti/di/ang non (Trophozoites)
• 9 I ® « '< } „ ‘l
The tu dudng gia

O '-i <3
§ § » * * *
The phan Net (Schizonts)
« « <
------ Giao bao (Gametocytes)

• w “ 2 ? •$$■. •
Hinh 1.5: Hinh thai cac giai doan phat trien cua Plasmodium vivax
i £!S?*
• t »-> r ^
—* ThetUdU0ng non (Trophozoites)
The tu du0ng gia

• # # — •
V ^ —
The phan Met (Schizonts)
& p.* »,/..• ^wTf-
> o — *• Giao bao (Gametocytes)
Hinh 1.6: Hinh thai cac giai doan phat trien cua Plasmodium ovale
57
4. Nhumg sai sot thifdng gap
- Phien ki'nh khong sach, con md, hoac bi moc.
- Phien ki'nh cu, bi sU6 c nhieu.
- Tieu ban bi ban.
- Tieu ban chtfa kho da co dinh, nhuom.
- Pha va nhuom Giemsa khong dung ti le, thdi gian.
- Rtfa tieu ban khong dung quy cach de lai can Giemsa.
- Nhtfng hinh gia co the nham vdi sot ret: bach diu nat d i nham vdi the
hoa thi, tieu cau, can Giemsa, nam tren tieu ban.
- Kh 6 nhan biet vi KSTSR bi bien dang sau dieu tri thuoc hoac KSTSR
khang thuoc, d ngtfdi song lau trong vung sot r 6 t.
* C hu y: Chi ket luan la am tinh khi da soi 100 vi trtfdng d tieu ban giot dfic
ma khong tim thay KSTSR.
5. Ky thuat tap trung hong cau mang KSTSR

La ky thuat do W ardlaw va Levine de xuat nh&m phat hien nhving trtfdng
hdp it KST trong mau.
Nguyen ly: Dung ong mao dan co thuoc nhuom Acridin mau vang cam de l£y
mau benh nhan. Sau khi lay du mau thi day nap va gan orig vao phao chat deo rat
nho. Dem quay ly tam se thay chia thanh 10 ldp. Ldp hong cau dtfdc tap trung cao
va hong cau co KST dtfdc nhuom b in g Acridin. Dtfdi ki'nh hien vi huynh quang se
thay dtfdc KSTSR trong hong cau mot cach de dang.
Phtfdng phap nay nhay hdn, dac biet la cac trtfdng hdp co K S T m&t do thap
nhtfng doi hoi phai co kinh hien vi huynh quang nen kho pho bien d cac cd sd.
TIM AU TRUNG GIUN CHI
Hiejn nay, ky thuat phat hien au trung giun chi trong mau dtfdc s i dung
rong rai de ch&n doan benh giun chi bach huyet.
1. Nguyen tic
Giun chi Brugia malayi va Wuchereria bancrofti trtfdng thanh song d he thong
bach huyet va sinh ra au trung. Au trung chi di chuyen ra mau ngoai vi vao ban dem.
Do do phai lay mau ngoai vi vao ban dem de xet nghiem, thdi gian tot nhat la ttf 20
gid tdi 2 gid sang. Trtfdc khi lay mau, benh nhan phai nam nghi ttf 1 - 2 gid.
2. Phuong tien va dung cu

- Kinh hien vi
58
- Bong, con sat trung
- Kim chich mau
- Phien kinh kho sach
- Giemsa, con co" dinh

- Benh pham la mau tinh mach co chong dong.
- Co the la mau mao mach lay cf dau ngon tay. Mua lanh, nhung tay vao
chau ntfdc nong co the lam tang ltfdng au trung trong mau. Sat khuan d iu ngon
tay b in g con 70°. Dung kim chich cho mau chay ra. Cham 3 giot cach deu nhau
len phien kinh. Dung goc cua mot phien kinh khac, danh 3 giot mau tren thanh 3
giot dac, tron, dtfdng kinh khoang 1,5 cm. Khoang cach gitfa cac giot khong xa qua
va cung khong dinh vao nhau. De tieu ban kho ttf nhien, khong nen hd nong hoac
phdi n&ng (se lam au trung co lai hoac bien dang hinh the au trung ).
Tieu ban co the soi ttfdi tren kinh hien vi bang vat kinh nho x 10 hoac nhuom
Giemsa 1 0 % trong 1 0 phut, riia nhe nhang bang ntfdc thtfdng. De kho ttf nhien.
Soi bang vat kinh dau x 100.
A u t r u n g b a t m a u tim tren nen m a u h o n g nhat, cac h a t nh iem sac v a n h a n
bat m a u ro.
XET NGHIEM TE BAO TRONG DjCH NAO TUY
1. Nguyen ly
Binh thtfdng dich nao tuy trong suot, co it te bao. So" ltfdng va thanh phan te
bao trong dich nao tuy tang len d mot so" benh. Khi do xet nghiem te" bao dich nao
tuy co the co gia tri trong chan doan.

- Buong dem.
- Phien kinh kho sach.
- Pipette Pasteur.
- Dung dich Giemsa.
- Dung dich pha vd hong cau gom:
+ Acid acetic : 5 ml
+ Ntfdc cat viia du:100 ml
- Dau soi kinh.
- Dung cu de lap cong thtfc te bao.
59
Lac nhe benh pham (dich nao tuy), cho vao 2 ong nghiem, moi ong 1 - 2 ml.
3.1. Dem so luung bach cau

- Nho vao ong thtf nhat 2 - 3 giot dung dich pha v5 hong ciu , lac deu. Sau
do, lay mot giot cho vao buong dem. de lang 5 phut roi dem so ltfdng bach c&u tren
ki'nh hien vi.
- Vdi buong dem Nageotte: loai buong dem de dem te bao trong dich nao
tuy co the tich chung 50 mm3. Chia lam 40 bang ke theo chieu ngang buong dem,
moi bang co the tich = 1,25 mm3. Dem so bach cau tren 4 bang dtfdc bao nhieu
chia cho 5 de co so ltfdng bach cau trong 1 mm3.
- Vdi buong dem Goriacep: dem so ltfdng bach cau trong cac khu vtfc dung
dem bach c&u, dtfdc bao nhieu nhan vdi 62,5 roi chia cho 25, se co so ltfdng bach
cau trong 1 mm3.
- Dem bang buong dem Neubauer: dem so bach ciu d khu vtfc dung de dem
bach cau dtfdc bao nhieu nhan 10 chia 4 , dtfdc so bach cau trong 1 mm3.
- Ket qua:
+ Binh thtfdng trong ntfdc nao tuy co it te bao (khong qua 1 te bao / 1 mm1)
+ Tang it: co 3 - 10 bach c&u /I mm 3
+ Tang vtfa: tren 10 bach cau / 1 mm 3
+ Tang cao: tren 100 bach cau / 1 mm 3
3.2. Phan loai bach cau: Khi co tren 10 bach cau /mm3
Ly tam ong nghiem thtf 2 toe do 200 vong/phut trong 30 phut. Do ntfdc
trong d tren, lay can lam tieu ban giot day, dtfdng kinh khoang 2 cm. De kho, co
dinh bang con tuyet doi, de kho roi nhuom Giemsa 1 / 1 2 trong 1 0 phut, rtfa bang
ntfdc thtfdng (chu y khong doi trtfc tiep vao phan benh pham tranh lam bong). De
kho va doc ket qua bang vat kinh dau, dem 1 0 0 te bao de phan loai.
4. Nhan xet ket qua

- Ty le bach cau hat tang cao (thtfdng tren 75%) gap trong viem mang nao
mu do tu cau, phe cau. nao mo cau.
- Ty le lymphocyt tang cao (thtfdng tren 75%) gap trong viem mang nao do
lao. virus, giang mai..
- Khi thay nhieu hong cau thi co the do xuat huyet nao, chan thtfdng so nao..
- Ngoai ra con co the gap nhting te bao tham ngam nhtf te bao cua cac
benh mau ac tinh, mot so te bao bieu mo. nam. hoac mot so' tinh the.
Li/u y: Khi dich nao tuy co hong cau thi co the do chay mau hoac do thu
thuat choc cham mach mau. trong ket qua xet nghiem can ghi r 5 neu co hong cau.
Dich nao tuy rat de bi nhiem khuan nen ong benh pham phai day nut kin va
dtfa den phong xet nghiem soi ngay.
60
5. Cac yeu to £nh htfdng
- N h im benh phikm, n h im giay x 6 t nghiem
- Ntfdc nao tuy sau khi choc khong giSfi ngay di xet nghiem
- Khong lac deu benh pham trtfdc khi dem so' ltfdng
- Lam tieu ban khong dung ky thuat: qua day hoac qua mong, rvta manh
qua lam bong benh phim , nhuom qua d im hoac qua nhat nen khong nhan dinh
dung te bao.
XET NGHIEM TE BAO TRONG DjCH MANG PHOI
1. Nguyen ly
Dich mang phoi xuat hien trong cac trUdng hdp benh ly nhtf: viem phoi,
viem mang phoi, viem gan, ung thtf phoi hoac do nguyen nhan cd hoc trong cac
benh tim, gan, th in . M oi benh, dac diem te bao trong dich co khac nhau. Xet
nghiem te bao trong dich mang phoi giup cho viec chsLn doan benh.

- M ay ly tam.
- Ong nghiem.
- Lam kmh kho sach.
- Pipette Pasteur.
- Giemsa, con co' dinh.

Ly tam 6 'ng benh pham 3000 vong/phut trong 30 phut. Lay can, lam tieu ban
giot day, dtfdng kinh khoang 2 cm. De kho ttf nhien, co' dinh bang con tuyet doi.
De kho. Nhuom Giemsa pha ty le 1/10, de 5 - 7 phut. Rtfa sach hang ntfdc thtfdng,
rtfa nhe nhang vi tieu ban ra't de bong. De kho ttf nhien. Doc tieu ban tren kinh
hien vi, dau tien quan sat bang vat kinh nho (xlO), sau chuyen sang vat kinh dau
de xem chi tiet.
4. Nhan djnh ket qua

4.1. Djch thanh ta huyet (mau vang chanh)
4.1.1. Tran djch c£p tinh
- Do lao, giang mai, benh tim: thay nhieu lymphocyt, te bao bieu mo va mot
it hong cau.
61
- Do benh tim, than: thay nhieu te bao bieu mo, mot it te b io lymphocyt v i
hong ciu.
- Do viem mang phoi: thay nhieu bach ciu doan trung tinh chtfa thoai hoa.
- Do lao mang phoi c&p, viem m&ng phoi thtf phat cua giang mai, th&'p
khdp cap, gap nhieu bach cau doan via acid.
4.4.2. Tran djch man tinh

- Do lao mang phoi tiep theo ton thtfdng phoi: th&y nhieu lymphocyt v i t£
bao bieu mo.
- Do ung thtf phoi va mang phoi thay nhieu te bao bieu mo, hong c iu va te
bao ung thtf.
- Do benh tim, phoi: thay nhieu te bao bieu mo, it hong ciu , it te bao
lymphocyt.
4.2. Djch m u (mau due): gap trong benh viem mang phoi do vi khuin, trong lao
phoi, co nhieu bach ciu trung tinh thoai hoa.
4.3. Djch m au (mau do hong) thtfdng gap trong cac benh sau:
- Chan thtfdng phoi, mang phoi.
- Lao hoac cac benh gay xuat huyet.
- Ung thtf phoi hoac mang phoi.
4.4. Dich dtfdng chap (mau trang due)

- Do vd ong ngtfc.
- Do thoai hoa cac te bao trong moi trtfdng khong bi nhiem khuin.
Tren tieu ban thay nhieu hat md va cac manh te bao.
XET NGHIEM TE BAO TRONG DjCH MANG BUNG
1. Nguyen ly
Dich mang bung con goi la ntfdc bang, chi gap trong benh ly, trong do mot so
trtfdng hdp co xuat hien te bao. V i vay xet nghiem te bao dich mang bung cd the
giup cho chan doan.

Giong nhtf lam xet nghiem dich mang phoi.

Giong nhtf lam xet nghiem dich mang pho’i.
62
Co hai loai:
- Neu co it te bao: thifdng 1& dich tM'm.
- Neu nhieu te bao: thifdng la dich tiet do viem, nhilm kh u in (gap trong
lao phuc mac, ung thtf...).
+ Nhieu bach cau doan trung tinh gap trong o ap xe vd, viem phuc mac do
viem khuan.
+ Nhieu hong cau va te bao bieu mo, co the co te bao ung thu gap trong ung
thif mot so" tang.
* C h u y:
- Co the xet nghiem nhieu l&n de so sanh doi chieu ket qua (khi c in thiet).
- Phan biet vdi nhtfng te bao trong dich u nang buong trtfng: te bao hinh
tru, ki'ch trtfdc to, dau co long.
- Khi choc hut ntfdc mang bung can lifu y co the gay nhiem khu&n thtf phat
va chay mau, dich tham se trd thanh dich tiet, ket qua x 6 t nghiem se thay doi. Ong
dtfng benh pham phai difdc nut kin va mang di xdt nghiem ngay.
XET NGHIEM TE BAO TRONG DICH KHflfP VA BAO KHITP
1. Nguyen tac
Dich sau khi choc hut phai dtfdc nut kin va dtf a den ngay phong xet nghiem.
2. Phtfdng tien - dung cu

Nhtf lam xet nghiem cac dich mang bung, mang p h o i...
3. Tien hanh
Nhtf lam xet nghiem cac dich mang bung, mang phoi ...
4. Nhan xet ket qua

4.1. Djch thanh soi huyet
- Trong thap khdp: gap nhieu bach cau doan trung tinh, mot so" monocyt, te"
bao bieu mo, te" bao lympho, te" bao qua nho (ragocyte).
- Trong lao phoi: thay nhieu bach cau lym phocyt va te bao trung tinh
thoai hoa.
- Trong giang mai: thay nhieu te" bao bieu mo, mot so" bach cau doan trung tinh.
63
4.2. Djch mu (mau tr&ng due)
- Do lao: th&y nhieu bach ciu doan trung tinh va te bao lymphocyt.
- Do nhiem khuan nao mo ciu: gap nhieu bach cau doan trung tinh thoai hoa.
4.3. Djch m4u (mau do hong)

- Do chan thtfdng gay xuat huyet trong khdp va bao khdp. Ngoai hong chu
con thSfy bach ciu va te bao bieu mo.
- Dich dudng chap (mau trang nhtf ntfdc gao): thtfdng do giun chi gay ra,
c&n kiem tra them x 6 t nghiem mau.
XET NGHIEM TE BAO TRONG NtfttC TIEU
1. Nguyen ly
Ntfdc tieu binh thtfdng chi cd mot so" te bao bieu mo, tinh the. Khi th&y xuit
hien nhieu hong ciu, bach cau, tru la benh ly.

- Kinh hien vi.
- May ly tam.
- Pipette Pasteur.
- Lam kinh, la kinh.

- Thtfdng lay ntfdc tieu vao buoi sang sau khi bo phan dau.
- Lac deu ong ntfdc tieu, cho vao ong khoang 5 ml, ly tam 2000 vong/phut
trong 15 phut. Neu khdng ly tam thi de lin g 1 - 2 gid.
- Do phan tren, hut mot giot can, nhd len lam kinh. Day la kinh len giot
can cho lan deu.
- Dat len kinh hien vi doc b in g vat kinh x 10 hoac x 40.
- Kiem tra khoang 2 0 - 30 vi trtfdng. So vi trtfdng kiem tra khdng dtfdc it
hdn 1 0 .
4. Nhan xet ket qua

4.1. Te bao
- Hong cau
ft : dtfdi 5 HC / vi trtfdng
64
+ : 5- 1 0 HC/ vi trtidng
++ : 10 - 20 HC /vi trUdng
+++: tren 2 0 HC /vi trtfdng
- Bach cau
It: dudi 10 BC / vi trUdng
+: 10 - 20 BC/ vi trUdng
++: tren 20 BC/ vi trUdng

+++: tren 50 BC/ vi trUdng
- Tinh trung
- Te bao bieu md nieu dao: te bao to hinh da dien, nhan rd.
- Te bao bieu md bang quang: te bao to hinh vcft, nhan ro.
- Te bao bieu md than: te bao to trung binh, hinh bau due, nhan tron ro.
4.2. Trichomonas
4.3. Tru nieu: Cau tao bdi c h a t nh ay , te b a o cu a m a u k h i q u a o n g t h a n , d o n g

la i v a m a n g k h u o n c u a o n g th a n . Dua v a o t h a n h p h i n c a u tao n g U di ta ch ia
h a i lo ai tr u
4.3.1. Tru khong cd te bao

- Tru trong: cdn goi la tru thau quang, hinh dai, bd nhan, trong suot.
Nude tieu binh thudng thai ra 3000 tru trong vong 12 gid. Tru nay tang khi lao
dong nang, sot, sau gay me b&ng ether. Gap nhieu cd the nghi do viem than.
- Tru sap (tru keo): ng&n va to hdn tru trong, ong anh do chiet quang
nhieu, mau xam, thudng cd vet ntit. NgUdi ta cho rang do n&m lau trong ong than
nen bi kho va tao thanh tru sap.
- Tru xd: mau vang nhat, trong nhu cd nhieu sdi ghep lai va keo dai,
thudng gap trong viem than cap.
- Tru md: do bao tUdng te bao thoai hoa, hoac do md trong mau bai tiet ra
tao thanh. Cac hat md hien ro tren than tru, thudng gap trong than nhiem md.
4.3.2. Tru cd te bao: thudng gap trong viem cau than
- Tru hat: giong nhu tru trong nhUng tren mat cd nhulng hat to nhd bam
len, do cac te bao hoac cac hat cholesterol cua cac te bao thoai hoa tao thanh.
Thudng gap trong viem than cap.
- Tru bieu mo: cdn goi la tru lien bao gom nhUng te bao d ong than tao thanh.
- Tru mu hay tru bach cau: do bach cau hat thoai hoa tao thanh, thudng
dUt thanh doan ngan.
- Tru hong cau cdn goi la tru mau, do hong cau ket tu, bd tru thudng 1dm
chdm khdng deu.
- Tru vi khuan ( it gap ): do vi khu&n tao nen.
65
Khi doc b in g v i t kinh x 10, tru diidc danh gia nhif sau:
- Khong co tru
+ 1 tru / 1 0 0 vi trtfdng
++ 1 tru / 1 vi trtfdng
+++ 1 0 tru / 1 vi trtfdng
++++ 1 0 0 tru / 1 vi trtfdng
4.4. Cdn tinh t h i

- Sulfat calci: hinh kim dai, hoa thi, khong mau.
- Oxalat calci: hinh phong bi, banh quy, kich thudc 10 - 20 p.m, hinh cu lac
khoang 50 |im, chiet quang.
- Carbonat calci: hinh cau, tan trong acid.
- Can phosphat: hinh chii nhat, la difdng xi, hinh sao. Kich thtfdc 30 - 150nm.
Khong mau, chiet quang.
- Acid uric: hinh thoi, mui giao, hoa thi canh nhon, hinh ngoi sao, mau
vang hay nau do.
- Amoni urat: hinh c&u gai, xtfdng rong, hinh bo kim, kich thtfdc 20 ^m,
mau vang, chiet quang.
- Phosphat tricalci: hat nho, khong co hinh thu nhat dinh, tan trong acid.
- Sulfa diazin: hinh bo ma, hinh choi.
- Sulfa thiazon: hinh luc lang.
- Sulfa pyridin: hinh la du du.
- Cholesterol: la nhiing ban mong manh, khong mau trong suot, chong
len nhau.
- Phosfat dicalci: hinh tam giac, goc nhon, chum thanh hinh hoa thi.
4.5. Cac tap chat khac

- Do lay ntfdc tieu khong dung ky thuat.
- Dung cu ban, tieu ban ban.
- Sdi chat nhay.
- Long, sdi bong, bot khi.
5. Nhumg dieu can chu y
- Phai ghi ro rang loai mau, cach lay mau: mau lay ttf nhien, mau lay qua dat
sonde, mau lay dau, giiia hay cuoi bai, mau lay sau khi phau thuat dtfdng tiet nieu...
- Ve sinh sach se trtfdc khi lay m&u ntfdc tieu.
- Dung cu lay m iu phai dam bao kho, sach.
66
- Mliu x 6 t nghiem phai dtfdc dtfa ngay den labo sau khi liy , neu de lau
hong ciu , bach ciu , te bao bieu mo v i tru se giam trong khi vi kh u in va men
tang. Tot nh&'t la trong vong 2 gid.
- Luon luon nhd dieu tra ve kinh nguyet vi co the gay sai sot nghiem trong
do hong ciu , bach ciu , te bao bieu mo va vi kh u in co the anh htf3ng den ket qua
xet nghiem trong va sau hanh kinh vai ngay.
- Ong ntfdc tieu phai d iy nut kin de tranh do vai va lin them tap khuin.
XET NGHIEM CAN ADDIS
1. Nguyen ly
Dinh ltfdng ntfdc tieu trong mot thdi gian nha't dinh va tien hanh dem te bao
cho phep xac dinh so' ltfdng te bao ntfdc tieu trong mot phut giup cho chan doan,
theo doi va dieu tri mot so' benh he tiet nieu
2. Cach lay benh pham

Buoi sang benh nhan day sdm di tieu het, sau do uo'ng 300 ml ntfdc dun soi
de nguoi va n im nghi. Trong 3 gid di tieu binh thtfdng vao mot cai bo sach va do
dtfdc bao nhieu m ililit thi ghi vao giay xet nghiem. L ie deu toan bo ntfdc tieu va
lay 1 0 ml dem den phong xet nghiem.

- M ay ly tam
- Buong dem Nageotte
- Pipette Pasteur
- Kinh hien vi

Dem 1 0 ml ntfdc tieu ly tam 1500 - 2 0 0 0 vong/ phut trong 5 phut. Bo 9 ml d
phan tren. Lay 1 ml can lac deu va nho len buong dem Nageotte.
Dem so' ltfdng te bao d cac o quy dinh, ttf do tm h ra so' ltfdng te bao hong
cau va bach cau trong mot phut.
T h i du:
Trong 3 gid (180 phut) benh nhan di tieu dtfdc 360 ml .
Trong 1 mm 3 dem dtfdc 5 bach cau.
Cach tinh nhtf sau:
N x a x 1000/ 10 hay N x a x 100 = 5 x 360/180 x 1000 / 10 = 1000
67
Trong do: - N la so' ltfdng te bao dem dtfdc trong 1 m m 3.
- a = V / 1 . V la ltfdng ntfdc tieu thu dtfdc
. t la thdi gia n 180 ph ut
- 1 0 la 10 ml ntfdc tieu dem ly tam

- 1 0 0 0 la 1 0 0 0 mm 3 = 1 ml
Nhtf vay trong mot phut co 1000 bach cau trong ntfdc tieu
5. Nhan xet ket qua

Phtfdng phap nay co gia tri ve chin doan va dieu tri dac biet trong viem than:
- Neu hong ciu dtfdi 3000 giai doan on dinh
bach cau dtfdi 2500
- Neu hong ciu tren 3000
giai doan tien trien
bach cau tren 2500
- Neu hong cau va bach cau tren 100 000: nghi soi than
- Neu hong cau tang nhieu, bach cau tang it nghi viem be than ho§c
bang quang.
6. Cac yeu to anh hi/Ong

- Cach lay benh pham khong dung
- Do ltfdng ntfdc tieu khong chmh xac
- Khong lac deu ntfdc tieu trtfdc khi lay lam xet nghiem
- Tinh toan sai.
68
TE BAO TINH THE
Tinh the
Tru trong Hong cau carbonatcalci Tinh the Sulfadiazin
Tru bach cau

111® Bach cau
^ ^ T rm h th e
< E > ^ ^ A m o n im a g ie phossphat
Tinh the
Sulfapyridin
ooo BJ f
ooo i^ t
Tinh the Tinh the
Tru mfl 0 0 0 Hat m3 K Dicalci phosphat Sulfapyridin
o o OQ&
O04 0 □
QJOOOq
% V o o£ o Tinh the
Tru hat » Tricalci phosphat
Te bao bieu mo than
Te bao t a g BI,0
Tinh the m s / m .Tinh the
bieu mo
bang quang Bc Oxalatcalci Acetyl sulfanilamid
Tru xd
,X 6 c r
Te bao C '1 j $
<s< bieu mo Tinh the > f O c Tmh the
Tinh trung nieu dao Iff f Calci sulfat s— J Amoni urat
Hinh 1.7. Hinh anh te bao nirdc tieu

Chifdng 2
DONG MAU - CAM MAU
NGUYlN TiC LAY 6ENH PHAM XET NGHIEM B&NG MAU
Ket qua cua cac xet nghiem dong mau phu thuoc rat nhieu vao cach lay
benh pham. De tien hanh tot cac ky thuat xet nghiem dong mau, can tuan thu
mot so" nguyen tac cd ban sau day trong khau lay benh pham:
- Trtfdc khi lay mau, can nam dtfdc chinh xac tien svi gia dinh, tien sti ban
than benh nhan ve chay mau. Hien tai hoac trong thdi gian g in day nhat co dtfdc
dieu tri cac thuoc co anh htfdng den he thong dong mau nhtf aspirin, heparin, din
xuat coumarin v.v... hay khong ?
- Benh nhan khong dtfdc an chat beo trong vong 12 gid trtfdc khi l£y mau
nham tranh sai lac ket qua xet nghiem do huyet ttfdng bi due.
- Trtf nhtfng trtfdng hdp cap ctiu, benh nhan nen dtfdc nghi ngdi trtfdc khi
lay mau vi moi stf van dong manh deu gay tinh trang tang nong do cac yeu to
dong mau trong huyet ttfdng. Doi vdi phu ntf, nen tranh xet nghiem dong mau
trong nhtfng ngay dang hanh kinh.
- Tot nhat la lay mau tinh mach, chi lay mau dong mach, mao mach trong
nhtfng trtfdng hdp khong the lay dtfdc mau tinh mach.
- Lay mau phai dam bao han che den mtfc toi da stf khdi dong dtfdng dong
mau noi sinh: c in sti dung bdm tiem nhtfa hoac trang silicon. Tranh khdi dong
dtfdng dong mau ngoai sinh: dung kim s ic cd 2 1 , choc trung tinh mach, khong
dtfdc choc vao ndi co tu mau hoac vet choc trtfdc.
- Mau lay ra cho vao ong nhtfa co chtfa chong dong (loai chong dong, ty le
chong dong tuy theo ttfng xet nghiem) tranh tao bot, day nut nhtfa hoac bit b in g
giay paraphin, tron deu mau va chong dong mot cach nhe nhang, khong lie ngang
tranh tan mau.
- Cac xet nghiem can dtfdc tien hanh trong khoang thdi gian 4 gid ke ttf khi
lay mau. Bao quan m iu mau d 4°C (trtfdng hdp m iu xet nghiem la huyet ttfdng
giau tieu c iu thi bao quan d 2 2 °C).
- Noi chung, ket qua cua cac xet nghiem dong mau khong phan anh chinh
xac ltfdng cac yeu to dong mau trong mau kiem tra ma chi la ty le p h in tram so
vdi m iu chtfng (binh thtfdng). Trong khi do nong do cac yeu to dong mau d ngtfdi
binh thtfdng dao dong trong mot khoang kha rong (vl du yeu to V III: 50-200°o). Vi
70
v i y mSu mau chtfng phai la m lu mau tron cua nhieu ngtfdi (pool), mot pool tot
nha't la la'y ttf 15 ngtfdi binh thtfdng va toi thieu cung phai ttf 5 ngtfdi binh
thtfdng. M iu chtfng luon phai dam bao ia'y cung thdi diem vdi m iu benh hoac
dtfdc dong kho va bao quan trong nhtfng dieu kien chugLn. M lu chtfng va m iu
benh luon dtfdc tien hanh cung ky thuat, cung dieu kien va cung do mot ngtfdi
thtfc hien.
DAU HIEU DAY THAT
1. Nguyen ly
Danh gia stfc ben mao mach qua cac not xua't huyet dtfdi da sau khi tang ap
ltfc cua mau trong mao mach bang cach tao ra mot stf tf dong tinh mach.

- M ay do huyet ap.
- Dong ho.
3. Tien hanh ky thuat

- Kiem tra not xuat huyet tren tay benh nhan (neu co, can ghi r5 de phan
biet vdi not xuat huyet mdi xuat hien sau khi tien hanh ky th u a t).
- Do huyet ap benh nhan.
- Duy tri ap ltfc bang may do huyet ap d tri so' trung binh gitfa huyet ap toi
da va toi thieu trong 1 0 phut.
- Thao nhanh may do huyet ap, gid cao tay benh nhan de mau ltfu thong
binh thtfdng.
- Doc ket qua bang cach dem cac not xua't huyet mdi d vung dtfdi dai quan
do huyet ap.
4. Ket qua
- Binh thtfdng khong co not xuat huyet mdi. Khi co hdn 10 not xua't huyet
mdi tren dien tich 1 0 cm2, dau hieu day that dtfdc goi la dtfdng tinh va tuy theo so'
not xua't huyet xua't hien ma ket qua dtfdc bieu thi +, ++, +++.
Dau hieu day that dtfdng tinh the hien tinh trang stfc ben thanh mach kem,
thtfdng gap trong cac benh ve thanh mach: thieu vitam in C, viem mao mach, benh
ve so' ltfdng va chat ltfdng tieu cau.
5. Cac yeu to anh htfdng

- N h lm vdi not xuat huyet cu, do khong kiem tra hoac kiem tra khong ky.
- Tao ap ltfc qua cao hoac qua thap.
- Khong dam bao thdi gian tang ap ltfc.

THOI GIAN MAU CHAY
Co hai phtfdng phap thong dung hien nay: phtfdng phap Duke va phtfdng
phap Ivy.
1. Thdi gian mau chay (Phi/dng phap Duke)

1.1. Nguyen ly
Do thdi gian ttf luc tao mot vet chich nam ngang d vung gitfa dai tai den khi
mau ngtfng chay.
1.2. Dung cu, hod chat

- Kim chich (blood lancet)
- Dong ho bam giay, giay tham
- Bong tham, dung dich sat trung (ether, con)
1.3. Tien hanh ky thuat

- Dung ether sat trung nhe nhang vung dai tai. Ddi 1-2 phut cho ether
bay hdi.
- Dung kim chich choc dtft khoat vao vung gitfa dai tai tao mot vet thtfdng
dai 5mm sau 2mm. Khdi dong dong ho bam giay.
- Ctf 30 giay mot lan, dung giay tham cac giot mau chay ra ttf vet chich cho
den khi mau ngtfng chay, bam dong ho ngtfng lai.
1.4. Ket qua
- Thdi gian mau chay thtfdng dtfdi 5 phut. Tri so nay co the thay doi tuy
phong xet nghiem.
- Khi thdi gian mau chay keo dai tren 5 phut, nen kiem tra d tai doi dien
hoac tot nhat nen tien hanh theo phtfdng phap Ivy.
- Thdi gian mau chay keo dai trong cac benh ly ve thanh mach (thieu
vitam in C ...), benh ly ve so ltfdng, chat ltfdng tieu cau (xuat huyet giam tieu cau,
Glanzmann...).
1.5. Nguyen nhan sai lam
- Kich thtfdc vet chich khong dat tieu chuan: qua nong hoac qua sau.
- Dong tac tham mau ttf vet chich qua manh gay bong nut tieu cau vtfa mdi
hinh thanh.
- Co bat thtfdng mach mau vung dai tai.
72
2. Thdi gian mau chay (Phtfdng phap Ivy)
2.1. Nguyen ly
Dtfdi ap ltfc dtfdng, mau ttf chay qua vet thtfdng thanh mach cho den khi tîo
thanh nut elm mau. Dtfa vao hien ttfdng nay, phtfdng phap xet nghiem thdi gian
may chay Ivy tien hanh d vung cang tay.
2.2. Dung cy, hod chat

- M ay do huyet ap.
- Kim chich, bong con hoac ether.
- Dong ho bam giay (3 chiec ).
2.3. Ti§'n hanh ky thuat

- Dung may do huyet ap bdm d ap ltfc 40mmHg va gitf on dinh.
- Chon mot vung d mat trtfdc cang tay khong co long, khong nhin thay
mach mau, sat trung bfing ether.
- Ddi 1-2 phut cho ether bay hdi, tao 3 vet chich co kich thtfdc nhtf nhau
cach nhau khoang 2cm va co do sau khoang 3mm. Khcli dong dong ho ngay khi tao
cac vet thtfdng. Ctf 30 giay mot lan, dung giay tham, tham nhe nhang mau ri ra
ttf cac vet thtfdng ttfdng ttf nhtf phtfdng phap Duke. Ghi thdi gian mau chay cua
ttfng vet thtfdng.
2.4. Ket qua

Thdi gian mau chay trong trtfdng hdp nay la thdi gian trung binh cua ca 3
vet thtfdng. Tuy nhien cung co phong xet nghiem ghi rieng re thdi gian mau
chay cua ttfng vet thtfdng. Thdi gian mau chay theo phtfdng phap Iv y thtfdng
dtfdi 5 phut.

Nhtfng nguyen nhan gay nen sai lam trong phtfdng phap Duke cung lam sai
lac ket qua thdi gian mau chay theo phtfdng phap Ivy. Ngoai ra can ltfu y tranh
choc phai mot mach mau n im kha sau ê lam cho thdi gian mau chay keo dai.
Trtfdng hdp nay dtfdc phat hien neu thdi gian mau chay cua hai vet thtfdng kia
trong thdi han binh thtfdng.
THQTI GIAN MAU DONG

(Phuung phap Lee - White)
1. Nguyen ly
Thdi gian ttf khi mau tiep xuc vdi be mat ong nghiem den khi hinh thanh
cuc dong la thdi gian dong mau.
73
- Bdm tiem nhtfa loai 5ml kim cd 21.
- Ong nghiem sach, kho, kich thtfdc 75 x 9,5mm.
- Binh cach thuy 37°C.

- Dong ho bam giay.

- Chu&n bi 2 ong nghiem cho moi benh nhan, ghi ten, tuoi, khoa, phong.
- Dung bdm tiem nhtfa lay 2-3ml mau tinh mach (yeu c lu dong tac nhanh,
gon, choc chinh xac, khong luon lach kim, khong choc di choc lai).
- Phan phoi deu vao 2 ong nghiem da chuan bi. Moi ong 1 -1,5ml, ltfu y
tranh lam noi bot khi cho mau vao ong nghiem. Khcli dong dong ho bi'm giay ngay
khi mau tiep xuc vdi thanh ong nghiem.
- De 2 ong mau vao binh cach thuy 37°C.
- Sau 3 phut, ctf 30 giay nghieng nhe nhang ong 1 de kiem tra cho den khi
mau dong.
- Tiep tuc kiem tra ong thtf hai nhtf ong thtf nh&'t. B&'m dong ho dtfng lai
khi mau d ong thtf hai dong chat. Thdi gian dong mau la thdi gian den khi mau d
ong thtf hai dong.
4. Ket qua
- Binh thtfdng thdi gian dong cua ong 1 ttf 6 - 8 phut va cua ong 2 ttf 8-10
phut. Stf chenh lech thdi gian dong gitfa ong 2 va ong 1 ttf 2 phut - 2 phut 30 giay.
Tuy nhien tri so' binh thtfdng cua thdi gian mau dong phu thuoc nhieu vao ttfng
dieu kien ky thuat, dong tac cua ky thuat vien, vi vay moi phong xet nghiem nen
co mot tri so' rieng cua minh.
- Thdi gian mau dong keo dai thtfdng gap trong cac trtfdng hdp roi loan
dtfdng dong mau noi sinh nhtf hemophilia, dieu tri heparin v.v...
- Day la mot xet nghiem tho, ddn gian nen do chinh xac bi han che ngay ca
khi da tuan thu nghiem ngat cac dieu kien ky thuat. V i vay thdi gian mau dong
binh thtfdng khong co nghla la he thong dong mau binh thtfdng.
- Thdi gian mau dong co the v ln binh thtfdng d benh nhan co so' ltfdng tieu
cau giam nang va ngay ca d benh nhan hemophilia.
5. Nguyen nhan sai lam

- Ong nghiem khong sach, kich thtfdc khong dam bao (qua ldn hoac qua nho).
- Nhiet do d binh cach thuy khong dam bao 37°C.
- Ky thuat lay mau sai: kim khong sac, kim qua to hoac phai choc nhieu
lan mdi lay dtfdc mau, choc vao ndi tu mau cu v.v...
74
- Lifdng mau l&y qua nhieu hoac qua it.
- K y thu^t nghieng kiem tra khong dam bao, khoang cach giiia cac l&n
nghieng khong dat 30 giay.
CO eye MAU
(Phuong phap Budtz-Olsen)
1. Nguyen ly
Qua trinh dong mau la qua trinh hinh thanh sdi huyet. Sau do sdi huyet se
co lai, tao nen stf co cua cue dong. Mtfc do co cua cue dong phu thuoc vao vai tro
cua tieu cau va sdi huyet.
2. Dung cu, thuoc thur

A/
- Ong nghiem sach kich thudc 75 x 9,5mm.
- Bdm kim tiem nhtfa, bong, con sat trung.

- Lay 3ml mau tinh mach, phan phoi deu vao 2 ong nghiem (co the sif dung
luon 2 ong mau dong lam theo phiidng phap Lee - W hite ).
- De vao binh cach thuy 37°C.
- Sau 2-4 gid doc ket qua.
4. Ket qua
Co hai cach de danh gia ket qua:
- Dtfa vao mtfc do co cua cuc dong (co hoan toan, co khong hoan toan,
khong co, cuc dong bi tan).
+ Cue mau co hoan toan: tao cuc mau bd ro rang, phan huyet thanh con lai
chiem khoang 50 den 65% the tich mau ban dau, khong cd hong cau tii do.
+ Cue mau co khong hoan toan: tao cue mau, phan huyet thanh con lai It
(dudi 40% the tich mau ban dau) hoac con hong cau tii do.
+ Cue m4u khong co: khong tao rieng phan huyet thanh.
+ Cue mau bi nat: hau het hong c lu tii do trong huyet thanh.
- Hoac dtfa vao ltfdng hong cau tii do con lai dtfdi day ong sau khi co va chia
lam cac mtfc do: +, ++, +++.
Tuy theo mtfc do benh ly so' ltfdng, chat ltfdng tieu cau hoac sdi huyet se gay
nen tinh tr?n«? cuc mau khong co, co khong hoan toan hoac bi tan.
70
- Ltfdng mau lay qua nhieu hoac qua it.
- Ong nghiem ban, kich thtfdc khong dam bao.
- Nhiet do binh cach thuy.

- Nhtfng trtfdng hdp da hong cau hoac thieu mau deu anh htfdng den ket
qua co cuc mau.
THQTI GIAN HOWELL

(Thdi gian phuc hoi calci)
1. Nguyen ly
Chong dong b in g natri citrat se lam ngtfng qua trinh dong mau d giai doan
can ion calci. Khi hoi phuc calci, qua trinh dong mau se tiep tuc. Dtfa vao dac tinh
nay, ngtfdi ta khao sat thdi gian dong cua huyet ttfdng sau khi cho thtfa calci de
v 9 .^ v
danh gia dtfdng dong mau noi sinh vdi stf cd mat cua tieu cau.

- CaCl2 M/40.
- Ong nghiem kich thtfdc 75 x 9,5mm.
- Pipette.
- Dong ho bam giay.

- Mau chtfng va mau benh nhan chong dong bang natri citrat 3,8% hoac
3.2% vdi ty le 9 the tich mau 1 the tich chong dong. Thu huyet ttfdng giau tieu
cau bang li tam nhe (500 vong/phut trong 5 phut) hoac de ttf lang.
- Phan phoi 0,2ml huyet ttfdng giau tieu cau cua benh nhan va mau chtfng
vao moi ong nghiem.
- De vao binh cach thuy 37°C trong 5 phut.
- Cho them 0 ,2 ml CaCl2 M/40. Tron deu, khdi dong dong ho ngay khi cho
calci vao. De yen d binh cach thuy 37°C.
- Ttf 1 phut 15 giay bat dau nghieng nhe kiem tra dong ctf 15 giay mot lan
cho den khi dong chat. Bam dong ho dtfng lai va ghi thdi gian dong.
Lap lai ttfdng ttf vdi ong thtf hai cua mau kiem tra.
Tien hanh ttfdng ttf vdi ong mau chtfng (mot ngay co the chi lam mot lan).
4. Ket qua
- Thdi gian Howell 1& ket qua trung binh cua 2 ong.
- Binh thtfdng ttf 1 phut 15 giay den 2 phut 30 giay. T ri so' nay co the thay
doi tuy ttfng phong x 6 t nghiem.
- Neu thdi gian Howell cua ong chtfng nSm ngoai gia tri tren thi phai kiem
tra lai thuoc thOf, ky thuat va mau chtfng.
- Thdi gian H owell keo dai trong cac trtfdng hdp benh ly co roi loan dtfdng
dong mau noi sinh do thieu hut cac yeu to' dong mau (benh hemophilia) hoac do co
khang dong ltfu hanh, do dieu tri chong dong dang heparin.
- Thdi gian H owell la mot xet nghiem ddn gian de danh gia dtfdng dong
mau noi sinh nen do chinh xac khong cao vi vay hien nay nhieu phong xet nghiem
da ap dung xet nghiem A P T T thay cho thdi gian Howell.

A/ 9
- Ong nghiem ban, khong dung kich thtfdc.
- N hiet do binh cach thuy khong dam bao 37°C.
- Chat ltfdng CaCl2 M/40.
- M&u huyet ttfdng kiem tra dtfdc chuan bi sai quy cach: khong dung chat
chong dong, ty le chong dong, ly tam manh lam mat tieu cau.
THdl GIAN PROTHROMBIN

(Thdi gian Quick)
1. Nguyen ly
Mau chong dong bling natri citrat se dtfdc phat dong qua trinh dong mau
theo con dtfdng ngoai sinh khi hoi phuc calci va co mat thromboplastin. Dtfa vao
dac tinh nay, ngtfdi ta khao sat thdi gian dong cua huyet ttfdng sau khi cho thtfa
thromboplastin calci de danh gia cac yeu to' dong mau dtfdng ngoai sinh (phtfc he
prothombin: II, V, V II, X).

- Dong ho bam giay.
- Pipette.
- CaCl2 M/40.
77
- Thromboplastin calci.
- Trtfdng hdp sti dung thromboplastin calci dang dong kho: pha ntfdc cflft
theo chi d in d nhan lo, nghieng nhe nhang cho tan het (tranh tao bot), sau 30
phut tien hanh ky thuat.
- Trtfdng hdp sii dung thromboplastin bot: ctf 50mg thromboplastin bot, cho
vao lm l NaCl 0 ,9 %, tron deu va u d binh cach thuy 37°C trong 15 phut. Sau d6
tron vdi CaCl2 M/40 ty le 1 /1 , de lin g, gan lay p h in trong cl tren. Day chinh l i
thromboplastin calci san sang de lam x 6 t nghiem.

- Mau chtfng va benh chong dong bang citrat natri 3,2% hoac 3,8% theo ty
le 9 the tich mau 1 the tich chong dong. Ly tam 2000 vong trong 10 phut, tach lfiy
huyet ttfdng lam xet nghiem.
- M&u huyet ttfdng dtfdc bao quan d 4°C hoac nhiet do phong va tien hanh x6 t
nghiem trong vong 4 gid ke ttf khi lay mau. Trtfdng hdp c ln k 6 o dai thdi gian bao
quan, m iu huyet ttfdng can phai dtfdc de d -2 0 °C hoac lanh hdn.
- Phan phoi 0,1 ml huyet ttfdng cua mau can kiem tra vao ong nghiem.
d binh cach thuy 37° C trong 5 phut.
- Cho vao 0,2 ml thromboplastin calci. Khdi dong dong ho ngay. Tron deu d
binh cach thuy 37°C trong 9 giay.
- Sau 9 giay, bat dau dao nhe va quan sat, den khi xuat hien mang dong,
bam dtfng dong ho lai.
- Lap lai ttfdng ttf vdi ong thtf 2 cua mSu kiem tra va ket qua dtfdc tinh la
tri so' trung binh cua 2 lan nay.
- Hang ngay trtfdc khi tien hanh xet nghiem, phai tien hanh ky thuat vdi mau
chtfng trtfdc de lay thong so chuan va kiem tra hoa chat, sinh vat pham.
4. Ket qua
- Tuy theo loai thromboplastin si’l dung ma moi phong xet nghiem co tri so
binh thtfdng khac nhau. Thdi gian Quick binh thtfdng khi stf dung thromboplastin
cd hoat tinh day du thtfdng ttf 11 den 13 giay.
- Ket qua cua xet nghiem nay cd the bieu thi bang thdi gian (giay) hoac
bang phan tram. N gay nay, de tranh nhtfng sai sot ket qua gay ra do stf dung cac
loai thromboplastin khac nhau, U y ban chuan hoa quoc te cua To chtfc Y te the
gidi yeu cau moi loai thromboplastin phai ghi ro I.S.I (chi so' do nhay quoc te).
- Cach tinh ty le prothrombin ttf thdi gian Quick:
+ Trtfdng hdp co bang tinh san kem theo vdi lo thromboplastin stf dung:
viec tinh se ddn gian sau khi xac dinh thdi gian Quick cua chtfng va benh.
+ Trtfdng hdp khong co bang kem theo: sii dung cong thtfc:
(T ”+ T ’ )x l0 0 / {T benh- [ T ’-( T ” -T’ )]}= % prothrombin
Trong do: T ” : thdi gian dong cua chtfng 50%
T ’: thdi gian dong cua chtfng 100%
- Cach tinh IN R (International Norm alized Ratio):
/ P T benh \l-S.I
IN R = P T R = I ---------------
y P T chtfng /
- Thdi gian Quick keo dai trong cac trtfdng hdp roi loan dtfdng dong mau ngoai
sinh (giam nong do cac yeu to phtfc he prothrombin do: suy chtfc nang gan hoac thieu
vitamin K, dieu tri chong dong bang dan xuat coumarin).
- Xet nghiem nay nhay nhat vdi stf thieu hut prothrombin.

- Do mau huyet ttfdng kiem tra: dong day, sai ty le chong dong
- Do ky thuat: tien hanh ky thuat sau 4 gid ke'ttf khi lay mau vdi mau m?u
dtfdc bao quan d nhiet do phong.
- Do mau huyet ttfdng chtfng: khong lay pool hoac lay pool ttf mot ltfdng ft
hdn 5 ngtfdi.
- Do chat ltfdng thromboplastin khong dam bao hoac sti dung thromboplastin
da bao quan lau sau khi chuan bi.
THOI GIAN THROMBOPLASTIN TUTNG PHAN HOAT HOA

(APTT: activated partial thromboplastin time)
(Thdi gian Cephalin - Kaolin)
1. Nguyen ly
Thdi gian phuc hoi calci cua huyet ttfdng citrat hoa sau khi u vdi mot ltfdng
thtfa kaolin (hoat hoa yeu to" tiep xuc) va cephalin (thay the yeu to 3 tieu cau)
giup danh gia chinh xac cac yeu to" khac cua dtfdng dong mau noi sinh. Vdi xet
nghiem nay, dieu kien hoat hoa yeu to" tiep xuc cung nhtf so" ltfdng, chat ltfdng tieu
cau trong mau kiem tra khong anh htfSng den ket qua xet nghiem.

- CaClo M/40.
- Kaolin-cephalin thtfdng pham dong kho: pha theo chi dan, nghieng nhe
den khi tan het, 2 0 phut sau stf dung lam xet nghiem.
- Kaolin-cephalin ttf san xua't: pha kaolin vdi NaC l 0,9% nong do 5mg/ml,
pha cephalin nong do thich hdp (theo chi dan cua ndi san xua't ). Tron hon dich
kaolin - cephalin theo ty le 1 /1 .
79
- Lay mau va tach huyet tUdng ngheo tieu c lu cua chtfng va benh nhan
nhtf xet nghiem thdi gian Quick.
- Phan phoi 0,1ml huyet ttfdng ngheo tieu c£u c in kiem tra vao ong
nghiem, de vao binh cach thuy 37°C.
- Them vao 0 , 1 ml hon dich kaolin - cephalin. Tron deu, u d binh cach thuy
37°C trong 3 phut. Trong thdi gian u, ctf 15 giay lac tron deu mot lan.
- Cho vao 0 , 1 ml CaCl2 M/40, khdi dong dong ho va theo doi den khi xu&'t
hien mang dong, bam dong ho dtfng lai. Ghi thdi gian dong.
- Moi mau huyet ttfdng can dtfdc tien hanh 2 lan va ket qua chinh la thdi
gian trung binh cua 2 lan kiem tra nay.
Tien hanh ttfdng ttf vdi mau chtfng.
4. Ket qua
Thdi gian thromboplastin ttfng phan hoat hoa cua huyet ttfdng binh thtfdng
thay doi ttf 30-35 giay tuy loai cephalin-kaolin, tuy ky thuat, tuy dieu kien ky
thuat ma ttfng phong xet nghiem sti dung.
Khi ket qua keo dai tren 8 giay so vdi mau chtfng dtfdc goi la thdi gian
thromboplastin ttfng phan hoat hoa keo dai. Thtfdng gap trong tinh trang roi loan
dtfdng dong mau noi sinh do thieu hut yeu to" dong mau (hemophilia...) hoac do
chong dong ltfu hanh (benh leukemia cap, dieu tri heparin...).

- Khong tuan thu thdi gian u kaolin-cephalin vdi huyet ttfdng hoac thdi
gian u cua chtfng va benh khong giong nhau.
- Khong tron deu hon dich kaolin-cephalin trtfdc khi cho vao huyet ttfdng vi
kaolin rat de lang xuong day ong nghiem.
- M iu huyet ttfdng kiem tra khong dtfdc bao quan dung quy dinh.
THOI GIAN THROMBIN
1. Nguyen ly
Do thdi gian dong cua huyet ttfdng khi cho thrombin vao. Xet nghiem nay
danh gia giai doan chuyen fibrinogen thanh fibrin.
2. Dung cu, thuoc thur, hoa chat

- Ong nghiem.
80
- Dong ho bam giay.
- Pipette.
- Thrombin pha loang (bang NaCl 0,9%) va bao quan b ntfdc da dang tan.
Nong do pha loang tuy theo loai thrombin. Thtfdng vdi nong do thrombin 25 ddn
vi/ml se cho thdi gian thrombin ct m iu huyet ttfdng chtfng 15-20 giay.

- Lay mau va tach huyet ttfdng cua m iu chtfng va benh nhan nhtf ky thuat
thdi gian Quick.
- Phan phoi 0,1ml huyet ttfdng can kiem tra vao ong nghiem.
- Cho them vao 0 , 1 ml NaC l 0,9%, tron deu, de d binh cach thuy 37°C trong
30 phut.
- Cho them 0 , 1 ml thrombin d nong do thich hdp da xac dinh. Khdi dong
dong ho.
- Quan sat stf xuat hien cuc dong. Bam dong ho dtfng lai. Moi mau kiem tra
2 lan va thdi gian dong trung binh cua hai lln nay chinh la thdi gian thrombin.
- Tien hanh ttfdng ttf vdi m iu chtfng.
4. Ket qua
Thdi gian thrombin cua mau kiem tra dtfdc coi la keo dai khi dai hdn thdi
gian thrombin cua mau chtfng tren 5 giay.
Thdi gian thrombin keo dai gap trong cac trtfdng hdp thieu hut fibrinogen
(dtfdi lg/1), bat thtfdng ve cau true phan tii fibrinogen, cd mat cac chat tfc che
thrombin (heparin) hoac chat tfc che trung phan fibrin (PDF, mot so protein khac
thtfdng thay trong benh da u tuy).

- Sii dung thrombin pha loang vdi nong do khong thich hdp ( thdi gian
thrombin cua huyet ttfdng chtfng ng&n hdn 15 giay) hoac dieu kien bao quan
khong dam bao vi thrombin sau khi pha loang khong ben vtfng.
- Pipette, ong nghiem v.v... cd heparin.
NGHIEM PHAP VON - KAULLA
1. Nguyen ly
Khi huyet ttfdng dtfdc pha loang va acid hoa se lam tua fibrinogen va cac yeu
to hoat hoa he thong tieu sdi huyet (plasminogen hoat hoa, plasminogen). Cac
chat tfc che tieu sdi huyet va yeu to V II cd trong tua rat it. Do dd khi tua nay dtfdc
81
lam dong, thi cuc dong se bi tieu nhanh hdn nhieu lln so vdi binh thtfdng. Dtfa
vao tinh c h it nay, ngtfdi ta tien hanh theo doi thdi gian tieu cua tua dtfdc l&m
dong de danh gia mtfc do hoat dong cua he thong tieu sdi huyet.
2. Hoa chat, thuoc thur

- Dem Michaelis pH 7,35
- CaCl2 M / 10
- Acid acetic 2 %
- Ch&'t chong dong natri citrat 3,8%

- Lay mau va tach huyet ttfdng: mau tinh mach chong dong bang citrat
natri 3,8% vdi ty le: 1 the tich ch£t chong dong/ 9 the tich mau. De ttf lang hoic ly
tam 1500 vong/phut trong 5 phut, lay huyet ttfdng.
- Trong ong nghiem chtfa 0,3 ml huyet ttfdng cua m iu chtfng ho&c cua benh
nhan, cho vao 3ml ntfdc cat.
- Cho them vao moi ong 1 giot acid acetic 2%, kiem tra de cd pH 5,2
- Ly tam 3000 vong/phut trong 15 phut. Gan bo ntfdc trong, gitf lai phan tua.
- Dung giay tham, tham kho thanh ong.
- Cho vao moi ong 0,3 ml dung dinh dem Michaelis pH 7,35 da pha loang
1/4 trong dung dich N aC l 0,9 %, dung que danh tan tua.
- Cho them vao moi ong 1 giot CaCl 2 M/1 0 , dat ong nghiem vao binh cach
thuy 37° C, chd dong. Bam dong ho theo doi thdi gian tan hoan toan.
4. Ket qua
Tan hoan toan: Trtfdc 15 phut: tieu sdi huyet toi cap
1 5 -3 0 phut: tieu sdi huyet cap
30 - 45 phut: tieu sdi huyet ban cap
45 - 60 phut: tieu sdi huyet tiem tang
Tren 60 phut: binh thtfdng

- Khong tao dtfdc cuc dong do: tien hanh sai ky thuat, ltfdng acid acetic cho
khong dung, tham ong nghiem khong kho.
- Chu y: neu ltfdng fibrinogen cua huyet ttfdng qua it, cung khong tao dtfdc
cuc dong, ltfc dd phai lam lai xet nghiem bang cach bo sung vao huyet ttfdng ngtfdi
benh mot ltfdng fibrinogen.
82
THOI GIAN TIEU EUGLOBULIN HUYET TlTONG
1. Nguyen ly
Dtfa vao dac tinh tua trong moi trUdng acid cua cac euglobulin huyet tUdng,
ngtfdi ta acid hoa huyet tUdng de thu nhtfng chat nay va loai bo cac ch&t tfc che
tieu sdi huyet. Sau dd lam dong euglobulin huyet ttfdng va theo doi thdi gian tieu
dong, de danh gia nhanh mtfc do hoat dong cua he thong tieu sdi huyet.
2. Hoa chat va thuoc thur

- Huyet ttfdng citrat chtfng va benh
- CaCl2 M / 40
- Acid acetic 1 ,6 % pha loang th&nh 1/100 trong ntfdc cat
- Dung dich dem borat pH 7,6

- Trong ong nghiem kich thtfdc 75 x 12 mm cho 0,5 ml huyet ttfdng ngheo
tieu cau, cho them 9,5 ml acid acetic da dtfdc pha 1/100 trong ntfdc C£Lt.
- Dao nhe ong tron deu
- De vao tu lanh 4°C trong 20 phut.
- Lay ra chia deu sang 2 ong ( moi ong 5 ml)
- L y tam 3000 vong trong 10 phut
- Lay ra gan bo phan ntfdc trong gitf lai tua, dung giay loc tham kho thanh ong.
- Cho vao moi ong 0,25 ml dem borat va hoa tan tua.
- Cho them vao moi ong 0,25 ml CaCl 2 M / 40 dat vao binh cach thuy 37°C,
theo doi tdi luc dong va bam dong ho theo doi thdi gian tan.
4. Ket qua
- Tan trtfdc 10 phut: tieu sdi huyet toi cap, nang
- Tan 1 0 -3 0 phut: tieu sdi huyet cap
- Tan 30 - 60 phut: tieu sdi huyet ban cap
- Tan ttf 60 - 120 phut: tieu sdi huyet tiem tang
- Tan sau 120 phut: binh thtfdng

- Trtfdng hdp tieu sdi huyet cap, nang va tieu sdi huyet nghiem trong, dung
dich se khong dong dtfdc, can cung cap them fibrinogen cho moi ong de dung dich
co the dong va theo doi tan.
- Can phan biet giam sdi huyet va tieu sdi huyet khi theo doi bien doi cua
cuc dong.
83
NGHIEM PHAP ETHANOL HOftC PROTAMIN SULPHAT
DE PHAT HIEN FIDRIN MONOMER
1. Nguyen ly
Khi thrombin tac dong len fibrinogen tao fibrin, mot it fibrin khong dtfdc
polyme hoa se hinh thanh phtfc he hoa tan vdi fibrinogen va FDP. Nhtfng phtfc he
nay co the difdc phat hien in vitro bdi ethanol hoac protamin sulphat.
2. Thuoc thur, hoa chat

- Huyet tUdng citrat
- Protamin sulphat 1% hoac ethanol 50% trong nUdc cat.
- Chtfng dtfdng: them 0,1 ml thrombin ( 0,2 ddn vi N IH /ml ) vao 0,9 ml
huyet ttfdng citrat vh u vao binh cach thuy 37°C trong 30 phut. Nhtfng sdi fibrin
se dtfdc hinh thanh trong thdi gian u va se dtfdc nh&n th&y khi quay ly tam.
3. Tien hanh Ky thuat

3 .1. Nghiem phap protamin sulphat
- Them 0,05 ml protamin sulphat vao ong nghiem chtfa 1 ml huyet ttfdng
benh va ong chtfng dtfdng.
- De ong dtfng trong binh cach thuy 3 phut.
Ket qua dtfdng tinh khi thay hinh thanh ltfdi hoac sdi fibrin. Neu khong
thay la ket qua am tinh.
3.2. Nghiem phap ethanol
- Trong ong nghiem cd 0,45 ml huyet ttfdng citrat giau tieu cau cho them
0,15 ml ethanol 50%, day nut kin, dat d nhiet do 4°C trong 1 0 phut.
- Lay ong nghiem ra xem. Neu cd stf hinh thanh gel hoac dong la ket qua
dtfdng tinh. Neu khong cd tua v ln trong la am tinh.

- Dung cu lay mau khong dung quy cach (ong dtfng benh pham phai la
nhtfa hoac thuy tinh cd trang silicon).
- Mau mau xet nghiem bi dong
- Dung cu lam xet nghiem khong sach
84
0
Cac xet nghiem dtfdc thuc hien vdi dung dich pha loang: (a) trong ntfdc muoi,
(b) trong nUdc muoi cd chtfa epsilon aminocaproic acid (E A C A ) de ngan ngtfa tieu
sdi huyet va (c) trong ntfdc muoi cd chtfa protamin sulphat de ngan ngtfa tac dung
tfc che cua san pham thoai hoa fibrinogen va fibrin neu cd. Trong nhtfng trtfdng
hdp cap ctfu chi nhtfng nong do pha loang vdi ntfdc muoi cd the da cho nhtfng
thong tin c&n thiet.
2.2. Thuoc thu, hoa chat

- Huyet ttfdng citrat cua benh nhan va ngtfdi binh thtfdng.
- Thrombin: pha 50 ddn vi N IH trong 2 ml dung dich N aC l 0,9% ngay trtfdc
khi dung.
- EACA: 1 mg/ ml dung dich NaC l 0,9%.
- Chuan bi 3 bo, moi bo 7 ong nghiem de xet nghiem huyet ttfdng binh
thtfdng va 3 bo khac ttfdng ttf de xet nghiem huyet ttfdng benh nhan.
- Moi ong cua bo thtf nhat cho 0,5 ml ntfdc muoi.
- Moi ong cua bo thtf hai cho 0,5 ml E AC A pha trong ntfdc muoi.
- Trong ong mot cua bo thtf ba cho 0,5 ml protamin sulphat pha trong ntfdc
muoi va 6 ong con lai cho 0,5 ml ntfdc muoi.
- Them 0,5 ml huyet ttfdng binh thtfdng hoac mau xet nghiem vao ong dau
cua moi bo, tron deu va chuyen 0,5 ml sang ong 2 va tiep tuc ttfdng ttf sang ong 3,
4...nhtf vay ta se cd nong do huyet ttfdng ttf 1/2 den 1/128.
- Them 0,1 ml thrombin vao moi ong va tron deu.
- Dat cac ong vao binh cach thuy 37°C va de yen 15 phut, sau dd kiem tra
xem cd fibrin hay khong (cd dong hay khong) ?
2.4. Ket qua
- Binh thtfdng fibrin se cd d trong tat ca cac nong do pha loang huyet ttfdng
ttf 1 den 1/128. Trong giam fibrinogen nang thi khong thay cuc dong d bat ky nong
do nao, neu giam nhe thi se cd cuc dong fibrin d 1 - 3 ong dau.
- Neu thay cuc dong d cac ong cd E A C A pha loang hdn so vdi ong chi cd
ntfdc muoi thi chtfng to tieu sdi huyet nang.
- Neu thay cuc dong d ong cd protamin sulphat nong do pha loang cao hdn
so vdi ong chi cd ntfdc muoi la cd san ph&m thoai hoa fibrinogen va fibrin trong
huyet ttfdng.
- Cac ong nghiem cua bo huyet ttfdng binh thtfdng dung lam chtfng dtfdng.
86
DjNH LUONG CAC YEU TO DI/A TREN CO SOI
XET NGHIEM THQfl GIAN PROTHOMDIN
(Prothrombin, yS'u to V, VII va X)
1. Nguyen ly chung
- Thdi gian prothrombin cua huyet ttfdng bieu hien nong do cac yeu to V, VII,
X, fibrinogen va prothrombin. Muon dinh ltfdng mot trong cac yeu to", ngtfdi ta theo
doi thdi gian prothrombin cua huyet ttfdng vdi thuoc thii cd dtf cac yeu to khac.
- Thuoc thti dd la cac mau dong kho cua huyet ttfdng ngtfdi khong cd
prothrombin hoac yeu to" V hoac yeu to" V II dtfdc ban sin. Nhtfng san pham nay
chtfa hoat tinh yeu to" c&n dinh ltfdng khong qua 1 % nhtfng cd du cac yeu to" khac
can thiet cho xet nghiem thdi gian prothrombin.
- Dinh ltfdng dtfa tren stf so sanh mtfc do dieu chinh thdi gian prothrombin
cua huyet ttfdng thieu hut yeu to" da biet sau khi them vao cac nong do pha loang
cua huyet ttfdng benh va sau khi them vao cac nong do pha loang cua huyet ttfdng
chtfng.
- Thdi gian prothrombin dtfdc danh gia b ln g cach dung thromboplastin ttf
oc dong vat (tho) vdi CaCl2. '
2. Djnh ltfdng yeu to II, V, VII

2.1. Thuoc thur, hoa chat ’
- Huyet ttfdng thieu hut yeu to" dinh kiem tra.
- Dem muoi barbiton
- Thromboplastin ttf oc tho hoac oc ngtfdi.
- CaCl2 M / 40 ( 0,025 mol/1).
- Huyet ttfdng chtfng.
- Pha loang huyet ttfdng chtfng va huyet ttfdng c ln kiem tra d 3 nong do:
1/10, 1/50, 1/100 trong dem.
- Lay 3 ong nghiem, trong moi ong cho:
+ 0 ,1 ml huyet ttfdng chtfng da pha d cac nong do tren
+ 0,1 ml huyet ttfdng khong cd yeu to" kiem tra (II hoac V hoac V II)
+ 0 ,1 ml thromboplastin
- De vao binh cach thuy 30 giay;
87
- Sau do them vao moi ong 0 ,1 ml CaCl2, bam dong ho theo doi thdi gian dong.
- Ke dtfdng bieu d iin gitfa thdi gian dong vdi do pha loang tren giay
logarit kep.
- Tiep tuc lam nhtf the vdi huyet ttfdng benh roi doc ket qua tren do thi.
G hi chu: cd the chuan bi cac huyet ttfdng thieu cac yeu to nhtf sau:
- Huyet ttfdng khong cd yeu to V cd the dtfdc chuan bi bang cach lay huyet
ttfdng oxalat u d 37°C trong 3 ngay trong dieu kien vo trung (mau chong ddng ty
le 9: 1 vdi oxalat natri 14 g/1), bao quan d - 2 0 °C.
- Huyet ttfdng khong cd yeu to V II •co the chuan bi ttf huyet ttfdng benh
nhan dung W arfarin sau 48 gid hoac huyet ttfdng cua mot loai cho cd thieu hut
yeu to" V II bam sinh.
- Noc ran Viper Dai Loan (oxyuranus scutellatus) cd kha nang trtfc tiep
bien prothrombin thanh thrombin trong stf vang mat cua nhtfng yeu to ddng mau
da biet nao dd. V i the cd the dtfng no nhtf mot thuoc thii dac hieu de dinh ltfdng
yeu to" II mot thi.
- Bach Quoc Tuyen va cong stf da san xuat thuoc thii de dinh ltfdng yeu to
II ttf huyet thanh oxalat hoa va huyet ttfdng hut vdi ty le 1: 2.
- Thuoc thtf de dinh ltfdng yeu to V II dtfdc san xuat ttf huyet ttfdng ctfu chong
ddng bin g wintrobe, hut bang bot silicagen G hoac tren mang loc amiang 2 0 %.
3. Djnh li/dng yeu to X

3.1. Nguyen ly
- Thay the thromboplastin trong thdi gian Quick bang noc ran ho’ mang
Russell pha loang ta se cd thdi gian dong dai hdn. Noc ran hoat hoa ra't manh yeu
to" X ma khong can stf tham gia cua yeu to" V II.
- De dinh ltfdng yeu to" X, ngtfdi ta dung mot thuoc thtf dd la huyet ttfdng bo
loc qua than hoat. Thuoc thuf nay thieu hut ca hai yeu to" V II va X. Nhtfng neu
thay thromboplastin bang noc ran Russell de xet nghiem thdi gian prothrombin,
yeu to" V II thieu hut se khong anh htfdng den ket qua.
- So sanh thdi gian prothrombin vdi noc ran Russell cua thuoc thtf nhd yeu to
X cua huyet ttfdng chtfng va benh de biet nong do yeu to" X cua benh nhan.
3.2. Thuoc thu, hoa chat

- Huyet ttfdng bd loc amiang ( khong cd yeu to" X )
- Dung dich noc ran Russell 1/150000 pha vdi cephalin.
- Cephalin pha vdi dem muoi pH 7,4 de cd nong do thich hdp.
- Huyet ttfdng citrat cua chtfng va benh.
- CaCl2M/40
- Ong nghiem, pipette, thung cach thuy. dong ho ba"m giay.
88
3.3. Ti£n hanh ky thuat
- Pha loang huyet ttfdng chtfng va benh 1/10, 1/100, 1/1000 trong dem muoi
pH 7,4.
- Cho vao moi ong nghiem 0,1 ml huyet ttfdng bd khong cd yeu to"X, 0,1 ml
huyet ttfdng chtfng va benh pha loang d cac nong do va 0 , 1 ml thuoc thii gom noc
rSn vdi cephalin.
- De vao thung cach thuy.
- Sau dung 30 giay cho them 0,1 ml CaCl 2 M/40, bam dong ho theo ddi thdi
gian dong. Lam moi ong cua moi nong do lap lai 2 lan.
3.4. Ket qua
- Thdi gian ddng cua cac nong do huyet ttfdng chtfng se dtfdc ve thanh
dtfdng th in g tren giay logarit k 6 p.
- Hoat tinh yeu to" X cua benh cd the doc tren do thi theo ket qua thdi gian
dong cua ong cd huyet ttfdng benh pha loang 1 /1 0 .

(cua dinh ltfdng cac yeu to" dtfa tren cd sd thdi gian prothrombin).
Ngoai nhtfng nguyen nhan chung nhtf lay benh pham khdng chinh xac, dung
cu khdng sach, d day can chu y den chat ltfdng cua thuoc thti: neu trong dd con
nhieu yeu to" ma ta can loai trtf, ket qua se khdng chinh xac.
D|NH LUDNG CAC YEU TO DUA TREN CO SOI

THOI GIAN THROMDOPLASTIN TUNG PHAN
(Yeu to VIII, IX, X I va XII)
1. Nguyen ly
Thdi gian thromboplastin ttfng phan cua huyet ttfdng ngtfdi hay huyet ttfdng
bd khdng cd mot trong cac yeu to" V III, IX, X I va X II se bi keo dai. Thdi gian nay
se dtfdc dieu chinh khi bo’ sung huyet ttfdng cd yeu to" thieu hut dd. Mtfc do dieu
chinh phu thuoc nong do yeu to" thieu hut trong huyet ttfdng bo’ sung. Dtfa vao dd,
ngtfdi ta pha loang huyet ttfdng can xet nghiem ra cac nong do khac nhau va tron
vdi huyet ttfdng khdng cd yeu to" can khao sat (huyet ttfdng thii) de theo ddi mtfc
do dieu chinh va tinh ra nong do yeu to" dong mau dd.
CJ day chung toi md ta phtfdng phap dinh ltfdng yeu to" V III C mot thi con
phtfdng phap dinh ltfdng cac yeu to" IX, XI, X II cung ttfdng ttf.
89
2. Ky thuat
2.1. Djnh hrpng yeu to VIII C
2.1.1. Dung cu, hoa chat

- H u yet ttfdng thut: la huyet ttfdng khong cd yeu to' V I I I C, cd the dung
loai dong kho ban sin , cd the dung huyet tUdng cua benh nhan hem ophilia A
cd hoat tinh yeu to' V I I I C dtfdi 1 % hoac cd thdi gian sinh throm boplastin ttfng
phan hoat hoa tren 150 giay va khdng cd khang the khang yeu to V I I I C.
_ Cephalin: cd the dung loai ddng kho ban s in hoac ttf san xua't tai phong
thi nghiem.
. CaCl2 M/40.
_ Kaolin: 5 g/1 trong dem.
- Huyet ttfdng chtfng: cd the dung pool huyet ttfdng cua ngtfdi binh thtfdng,
cd the dung huyet ttfdng da biet trtfdc hoat tinh yeu to' V III C.
2.1.2. Quy trinh ky thuat

- Lay mau benh nhan tach huyet ttfdng nhtf xet nghiem thdi gian Howell.
- Pha loang huyet ttfdng
+ Pha loang huyet ttfdng chtfng theo ty le: 1/10, 1/20, 1/40, 1/80 trong dem
Michaelis pH 7,3.
+ Pha loang huyet ttfdng benh nhan theo ty le 1/10 (neu benh pham la m iu
yeu to 'V III cd dac thi can pha loang hdn).
- Tien hanh xet nghiem:
+ Xay dtfng do thi chu&n: trong mot day ong nghiem:
Moi ong cho 0,1 ml huyet ttfdng chtfng d moi nong do pha loang,
0 ,1 ml huyet ttfdng thtf (khdng cd yeu to' V III C),
0 ,1 ml hdn dich cephalin-kaolin
U d binh cach thuy 37°C trong 2 phut.
Sau dd them 0 ,1 ml CaCl 2 M/40 .
Bam dong ho, theo ddi thdi gian ddng.
Dtfa vao thdi gian ddng d cac do pha loang, ta dtfng mot do thi trong dd
true tung la thdi gian ddng cua cac do pha loang, true hoanh la hoat tinh
cua yeu to 'V III C. Do thi se la mot dtfdng th in g.
+ Xet nghiem mau kiem tra: cac btfdc tien hanh nhtf ddi vdi huyet ttfdng chtfng.
Dtfa vao do thi ta tmh dtfdc nong do yeu to' V II I C.
_ Hoat tinh yeu to V IIIC cua ngtfdi binh thtfdng la 50 - 200%.
2.1.3. Nguyen nhan sai lam
_ Huyet ttfdng thtf chtfa dung tieu chu&n (cdn hoat tinh V III C tren l% ).
90
_ K et qua thdi gian ddng cua cac nong do pha loang huyet ttfdng chtfng
khdng chinh xac nen do thi khdng phai la mot dtfdng thing.
_ Mot so" nguyen nhan chung: dung cu lam xet nghiem khdng sach, lay
benh ph^m khdng chu^n.
Litu y: Ngoai sti dung huyet ttfdng chtfng, ngtfdi ta cdn dung huyet ttfdng
chu^n de kiem tra, quy trinh giong nhtf doi vdi huyet ttfdng chtfng va do thi la
mot dtfdng thang song song vdi dtfdng thang ctia huyet ttfdng chtfng.
2.2. Djnh twang cac y§'u to khdc
Cac btfdc tien hanh ttfdng ttf dinh ltfdng yeu to" V III C, 6 day chi trinh bay
cach chuan bi huyet ttfdng thti.
2.2.1. Djnh lifang yeu to IX
Huyet thanh khdng cd yeu to" IX:

- Lay 3 ml mau benh nhan bi benh hemophilia B cho vao ong nghiem nho
- Cho them vao 3 giot thromboplastin da pha loang 1/10 trong dung dich
NaCl 0,9% hoac dung dich dem Michelis pH 7,3.
_ De ddng d thung cach thuy 37°C trong 4 gid cho cue mau co hoan toan,
quay ly tam tach huyet thanh.
2.2.2. Djnh lifting yeu to X I

Huyet ttfdng khdng cd yeu to" XI:
- Lay mau chong ddng bang citrat
- L y tam manh tach lay huyet ttfdng ngheo tieu cau
- Hoat hoa yeu to" X II bang Celit
- De cf binh cach thuy 37°C trong 6 gid de tieu thu het yeu to" XI.
- Thti lai thdi gian A P T T , chi sti dung lam huyet ttfdng thti khi thdi gian
A P T T ttf 150 giay trci len.
L liu y:
Mau ctia benh nhan hemophilia dung san xua"t thuoc thti phai khdng cd
khang the khang yeu to" dd ( IX, X I ).
DINH LUDNG YEU TO XIII
1. Nguyen ly
Yeu to X III dtfdc hoat hoa trong qua trinh dong mau nhd ion calci va
thrombin. Yeu to 'X III hoat hoa lam o’n dinh cuc fibrin.
91
Khi co du yeu to" X III cuc fibrin se khong h o i tan trong dung dich ure
5mol/l, con cuc fibrin dtfdc tao ttf huyet ttfdng thieu yeu to X III se bi tan.
2. Thuoc thur, hoa chat

- Huyet ttfdng citrat cua benh va cua chtfng.
- CaCl2 M/40
- Ure 5 mol/1 (300 g/1)

- Chuan bi 2 ong nghiem thuy tinh kich thtfdc 75 x l 2 mm
- Cho 0 ,2 ml huyet ttfdng chtfng hoac benh vao moi ong
- Cho them 0 ,2 ml CaCl2M/40 vao ttfng ong
- U d thung cach thuy 37°C trong 30 phut, sau dd them 3 ml dung dich ure
5 mol/1.
- Lac cuc ddng trong dung dich ure. De ong nghiem cf nhiet do phong qua
dem va kiem tra cuc ddng vao hom sau.
4. Ket qua
- Neu yeu to" X III cd trong huyet ttfdng thi cuc ddng v ln cdn.
- Phtfdng phap nay thtfdng chi dung de chan doan thieu yeu to X III bam
sinh va nhtfng trtfdng hdp yeu to" X III < 1 %.
LiCu y:
- De biet kha nang lam tan cuc fibrin cua dung dich ure, ngtfdi ta tien
hanh lam xet nghiem vdi ong chtfng dtfdng nhtf sau: trong ong nghiem cho 0 , 2
ml huyet ttfdng citrat, 0 ,2 ml thrombin, sau dd cho dung dich ure nhtf tren. Cue
ddng se tan trong ure vi yeu to" X III khdng hoat hoa do thieu Ca++.
- Cd the thay dung dich ure 5 moi /I bang acid monochloracetic 2%.
DjNH LI/ONG SAN PHAM THOAI HOA FIBRINOGEN VA FIBRIN

(Fibrinogen and fibrin degradation products - FDP)
- FD P cd mat nhanh chong trong mau cf ca hai trtfdng hdp: tieu sdi huyet
toan the va tieu sdi huyet cuc bo ket hdp vdi stf lin g dong fibrin trong long mach
(cd ddng mau trong long mach).
- Trong mau ngtfdi binh thtfdng cd the cd F D P dtfdi dang vet, dd la ket qua
cua tieu sdi huyet sinh ly. FD P cao hdn trong thdi ky kinh nguyet, dac biet d
ngtfdi rong kinh. FD P cung tang d ngtfdi xd gan, tac mach, nhoi mau phoi va trong
benh nhan ghep than. 0 benh nhan ghep than, tang FD P trong ntfdc tieu la bieu
hien cua tinh trang thai ghep.
92
- FDP can trd phan ting thrombin - fibrinogen, lam k6 o dai thcfi gian thrombin
huyet ttfdng. Huyet thanh cd FDP se lam tang nhanh stf ket tu cua tieu clu.
- Phtfdng phap m iln dich la phtfdng phap nhay nh&'t de xac dinh FDP.
1. Djnh lifdng FDP difa vao phifdng phap mien djch ure che ngifng ket
hong cau cua Merskey
1.1 Nguyen ly
FD P cd tinh khang nguyen, se phan tfng vdi khang the khang fibrinogen
ngtfdi. Khi cho vao huyet thanh cd FDP, khang the khang fibrinogen ngtfdi se bi
trung hoa va do dd giam kha nang ngtfng ket hong cau dtfdc gSn fibrinogen (tfc
che ngtfng ket hong cau).
1.2. Chuan bi thuoc thur, hod chat va dung cu
1.2.1. Thuoc thCr

- Dung dich dem pH 7,2
+ K H 2 P 0 4 0,15 M 23,9 ml
+ N a 2 H P 0 4 0,15 M 76 ml
+ NaC l 0,85% 100 ml
- Dung dich dem phosphat citrat pH 6,4
+ 0,35 the tich N a 2 H P 0 4 0,15 M
+ 0,65 the tich K H 2 P 0 4 0,25 M
+ 1 the tich natri citrat 0,1 M
1.2.2. Huyet thanh ngudi binh thudng va huyet thanh benh

- Lay 3 ml mau khdng chong ddng vao oiig nghiem cd san 10 mg chUit tfc
che: acid amino caproic (hoac chat tfc che Kuniet).
- De d thung cach thuy 37°C trong 3 gid.
- Quay ly tam 3000 vong/phut trong 1 0 phut, tach lay huyet thanh.
1.2.3. Huyet ti/ong chimg
- Tron huyet ttfdng citrat cua nhtfng ngtfdi binh thtfdng cung nhom hong
cau he ABO.
- Ly tam manh 3000-5000 vong/phut trong 20 phut. Bo can, dinh ltfdng
fibrinogen trong huyet ttfdng dd.
1.2.4. Huyet thanh chong fibrinogen ngudi
- Tiem fibrinogen ngtfdi cho tho tao khang the. Khi cd hieu gia cao dat yeu
cau thi lay mau tho khdng chong ddng, thu huyet thanh, kiem tra b 4 ng dien di
mien dich va hieu gia khang the trtfdc khi ltfu trtf.
93
1.2.5. H6ng cau ngudi nhom O ho$c hdng cau cCru
- L&y 20ml mau, chong dong b&ng 3 ml ACD ho&c citrat, mau de ltfu trtf 2
ngay trtfdc khi cam nhiem.
- XOf ly hong clu vdi acid tanic 1/200.000:
+ Rtia hong clu 7 lln bang dem pH 7,2.
+ Pha hong clu 5% trong dem pH 7,2
+ Xti ly vdi acid tanic: 1 the tich hong c iu 5 % vdi 1 the tich acid tanic
1 /2 0 0 .0 0 0 , de cf nhiet do phong (co the trong gidi han 2 2 - 37°C) trong 2 0 phut.
- ROta hong c iu 3 lan b&ng dem pH 7,2 va cuoi cung pha th&nh hong clu
5 % trong dem.
1.2.6. Dung cu vi ngtfng ket, khay nhi/a nhiSu hang 16

1.3.1. Hieu gia khang thS
- Cam nhilm hong cau

+ Hong cau 5% tiep xuc vdi huyet tUdng ngtfdi pha loang 1/250 trong dung
dich dem pH 7,2.
+ Hong cau 5% tiep xuc vdi huyet thanh pha loang 1/250
(1the tich hong clu , 1 the tich huyet ttfdng hoac huyet thanh pha loang
1/250 u cl 37°C trong 30 phut).
+ Rtia hong cau 3 lan roi pha lai 5%.
- Chon nong do khang huyet thanh
+ Pha loang khang huyet thanh b ln g dung dich dem pH 7,2 cd chtfa 1%
gelatin tren hai hang lo.
+ Hang lo 1 se cd them hong cau 5% da tiep xuc v 6 i huyet ttfdng pha 1/250
+ Hang lo 2 cd them hong cau da tiep xuc vdi huyet thanh pha 1/250
+ De cf nhiet do phong, sau 1-2 gid doc ngtfng ket
+ Nong do khang huyet thanh dtfdc dung la nong do ldn hdn 2 do pha loang
so vdi do pha loang cuoi cung cdn ngtfng ket.
1.3.2. Djnh li/ong FDP
- Pha loang huyet ttfdng chuan (Pool da dtfdc dinh ltfdng fibrinogen) trong
dem pH 7,2 cd 1 % gelatin vdi nong do: 1/30, 1/90, 1/270, 1/540.
(Vdi cac m iu kiem tra va huyet thanh binh thtfdng (cd chat tfc che) thi
pha 1/3, 1/9, 1/27...).
- Cho 0,05 ml huyet thanh cf hieu gia da chon vao moi lo u 10-30 phut d
4°C, them vao mdi lo 0,05 ml hong cau 5% da cam n hilm de nhiet do phong 2 gid.
94
1.4. K4t qua
- Chting dtfdng \k h&ng cd huyet ttfdng chultn.

- Chtfng am Ik h&ng cd huyet thanh ngtfdi binh thtfdng.
- Mtfc do ngtfng ket cl do pha loang cua m iu c ln kiem tra dtfdc so sanh vdi
huyet ttfdng chu^n.
Thi du; huyet ttfdng chu^n cd chtfa 300mg/l, fibrinogen tfc che do ngtfng ket
b 1/600 thi moi do pha loang se 1 &:
1 0 0 0 1
300 x ------ x ------ = 0,5pg/ml

1 0 0 6000
Neu lie che ngtfng ket xay ra b 1/80 cua hang m iu c ln kiem tra thi trong
m iu se cd 0,5îg/ml x 80/1 =40jag/ml FDP.
Binh thtfdng trong huyet thanh cd ttf 0-5ng/ml.

- Nong do acid tanic khong thich hdp de xvL ly hong clu
- Khang huyet thanh khdng dam bao chat ltfdng.
2. Djnh ltfdng FDP b£ng phtfdng phap ngtfng ket latex

2.1. Nguyen ly
Khi tron huyet thanh hoac ntfdc tieu vdi dich treo cd hat latex da g in khang
the dac hieu chong FDP, neu cd hien ttfdng ngtfng ket, nghia la trong huyet thanh
hoac ntfdc tieu cd FDP. Phtfdng phap ban dinh ltfdng dtfdc tien hanh b ln g cach
pha loang m iu xet nghiem b cac nong do khac nhau.
2.2 Thuoc thur, hod c h it

Dung thrombo-wellcotest kit (Wellcome reagents LTD . Beckenham, Kent).
2.3. Tii'n hanh ky thuat

- Tach huyet thanh:
+ Lay 2ml mau vao mot ong nghiem chuyen dung cho xet nghiem FD P (cd
chtfa thrombin va chat tfc che tieu dam de mau dong nhanh va ngan ngtfa tieu sdi
huyet in vitro).
+ De ong mau vao thung cach thuy 37°C cho den khi cuc mau b it dau co,
quay ly tam tach lay huyet thanh de lam xet nghiem.
- Thtfc hien phan tfng:
+ Pha loang huyet thanh 1/5, 1/20 vdi dem glycin cd trong bo kit.
+ Lay mot giot hon dich d mdi do pha loang cua huyet thanh nho vao phien
kinh vdi mot giot dich treo latex. L ie phien kinh nhe nhang cho den khi nhin thay
ngtfng ket cac hat latex b ln g m lt thtfdng (thdi gian khong qua 2 phut).
95
- Neu chat xet nghiem la ntfdc tieu cung lam ttfdng ttf nhtf huy£t thanh: cho
2 ml ntfdc tieu vao ong chuyen dung, dat vao thung cach thuy 37°C khoang 30
phut, sau dd loc ntfdc tieu qua giay loc hinh dla (Whatman GF/B). Thtfc hien phan
tfng vdi ntfdc tieu khdng pha loang va pha loang 1/5.
2.4. Ket qua

- Huyet thanh: neu xuat hien ngtfng ket d do pha loang 1/5 thi ltfdng FDP
trong huyet thanh khdng qua 1 0 pg/ml, neu ngtfng ket cf do pha loang 1 / 2 0 thi
ltfdng FD P trong huyet thanh khdng qua 40 jig/ml.
- Ntfdc tieu: Neu ngtfng ket d ntfdc tieu khdng pha loang thi FD P khdng qua 2
jag/ml, neu ngtfng ket d do pha loang 1/5 thi FDP khdng qua 10 mg/ml ntfdc tieu.
- Ket qua cua phtfdng phap dung latex ttfdng ttf vdi phtfdng phap dung
hong cau xuf ly bang acid tanic nhtfng no cho ket qua nhanh va tien cho nhtfng
phong xet nghiem khdng lam thtfdng xuyen.
HOAT TINH YEU TO 3 TIEU CAU

(Phi/ong phap Rabiner va Hrodek)
1. Nguyen ly
Hoat tinh yeu to' 3 tieu cau xac dinh bSng stf khac nhau cua thdi gian dtfdc
phuc hoi calci cua huyet ttfdng sau khi dtfdc hoat hoa bdi kaolin trtfdc va sau khi
loai bo tieu cau. Ban dinh ltfdng dtfdc danh gia qua dtfdng bieu dien thdi gian
ddng cua huyet ttfdng binh thtfdng vdi so' ltfdng tieu cau 50 x 109 /1, 25 x 109/1, 12,5
X 1 0 9/ 1 .
2. Thuoc thu, hoa chat

- Mau chong ddng bang citrat natri 3,8% ti le 1/10 trong ong nghiem nhtfa
hoac thuy tinh trang silicon. Quay li tam de cd huyet ttfdng giau tieu cau va
ngheo tieu cau.
- Dung huyet ttfdng ngheo tieu cau de pha loang huyet ttfdng giau tieu cau,
de cd 50 x 109 va 12,5 x 109/ 1, cd the dung podl huyet ttfdng ngheo tieu cau cua
ngtfdi binh thtfdng nhtfng de lau.
- Dung pool huyet ttfdng ngheo tieu cau binh thtfdng pha huyet ttfdng giau
tieu cau de cd so' ltfdng tieu cau 50 x 109, 25 x 109va 12,5 x 109 / 1.
- CaCl2 M/40
- Kaolin 5mg/ml hoac celit 15mg/ml.
- Dem Michalis pH 7,3
96
- Trong cac ong nghiem chtfa 0,1ml huyet ttfdng cd chtfa so' ltfdng tieu clu khac
nhau: 50 x 109, 25 x 109 va 12,5 x 109/1 va ngheo tieu clu chtfng, 50 x 109, 25 x 109; va
ngheo tieu clu benh.
- Dat vao thung cach thuy 37°C, cho them 0 , 1 ml hdn dich kaolin 5mg/ml
hay celit 15mg/ml lac deu. Dung 2 phut sau cho them 0,1ml CaCl M/40 b&'m dong
ho theo doi thdi gian ddng.
4. Ket qua
- Stf khac nhau gitfa thdi gian ddng cua ong huyet ttfdng giau va ngheo tieu
cau ndi len hoat tmh yeu to' 3 tieu cau.
- Qua thdi gian ddng cua cac ong huyet ttfdng chtfng, ve dtfdng thang th in g
chuan tren giay logarit kep vdi true tung la thdi gian ddng, true hoanh la cac nong
do tieu cau (50 x 109/1 ttfdng dtfdng 100%, 25 x 109/1 ttfdng dtfdng 50%...). Ttf do thi
nay tinh dtfdc hoat tinh yeu to' 3 tieu clu cua benh nhan qua thdi gian ddng.

- Mau lay de lau qua 3 gid mdi lam xet nghiem
- Pha loang tieu c lu khdng chinh xac
XET NGHIEM DO 00 NGUNG TAP TIEU CAU
Co hai phtfdng phap chinh dtfdc sti dung de danh gia do ngtfng tap tieu clu:
Do do ngtfng tap tieu c lu ttf phat va do do sau khi cho chat kich tap vao.
1. Nguyen ly: K y thuat do ngtfng tap tieu c lu b&ng may do ngtfng tap tieu c lu ttf
dong khi cho chat kich tap ttf ngoai vao:
Khi cho chat kich tap (ADP, collagen...), tieu clu se ngtfng tap khdng hoi phuc
va lam thay doi mat do quang hoac do trd khang cua ddng dien. Mtfc do thay doi
ngtfng tap dtfdc phan anh qua mtfc do thay doi mat do quang hoac do trd khang.
2. Hoa chat, phtfdng tien, dung cu

- M ay do ngtfng tap tieu c lu ttf dong
- Chat kich tap: AD P, collagen..., vdi nong do thich hdp
- Mau mau: mau tinh mach, lay bang bdm tiem nhtfa, ong nhtfa, chong
ddng bang citrat natri 3,8% (hoac 3,4%) theo ty le 1 the tich chong ddng tron vdi 9
the tich mau. Ly tam thu huyet ttfdng giau tieu c lu va huyet ttfdng ngheo tieu
clu. Trong mot so trtfdng hdp, cd the sti dung mau toan p h ln de lam xet nghiem.
97
— Phan phoi huyet ttfdng giau tieu cau va ngheo tieu c lu vao cac cdng
(ltfdng huyet ttfdng tuy theo ttfng may) va dat vao cac vi tri quy dinh tren may.
- Tien hanh cac btfdc theo tu ln ttf quy dinh (phu thuoc ttfng loai may).
4. Ket qua va danh gia

Ket qua ngtfng tap tieu clu dtfdc the hien bang: do ngtfng tap toi da, do doc
cua dtfdng cong ngtfng tap, thdi gian xuat hien ngtfng tap, thdi gian ke ttf khi xua't
hien ngtfng tap den khi dat ngtfng tap toi da. K et qua ngtfng tap tieu c lu the hien
bang do ngtfng tap toi da thtfdng dtfdc sti dung nhat.
Tri so' binh thtfdng cua ngtfng tap tieu clu tuy thuoc vao ttfng loai may, tuy
thuoc vao loai, nong do chat kich tap. Mdi phong xet nghiem nen cd tri so' binh
thtfdng ctia rieng minh.
Thdi gian
Hinh 2.1: Dtfdng bieu d iin ngtfng tap tieu cau vdi ADP 6 ngtfdi binh thtfdng
Ngtfng tap tieu c lu giam trong cac benh ly giam chtfc nang tieu c lu ba’ m
sinh hoac m ic phai. Tuy ttfng loai benh ma ngtfng tap tieu c lu giam vdi cac cha't
kich tap khac nhau.
98
Thai gian
Hinh 2.2: Dtfdng bieu dien ngtfng tap tieu cau vdi ADP d ngudi do ngung tap tieu cau giam
N g U n g tap tieu cau tan g trong n h U ng trUdng hdp t a n g hoat hoa tieu cau:
phu nU co thai, p h a u thuat, mot so b en h ly tim mach.
5. Nhurng lifu y khi tien hanh ky thuat

- Doi vdi nhtfng trUdng hdp sii dung may do do ngUng tap b a n g nguyen ly thay
doi mat do quang. benh nhan can nhin an trong vong 12 gid trUdc khi lay mau.
- Ket qua ngung tap tieu cau d nhUng mau huyet tUdng giau tieu cau cd so"
ltfdng tieu cau < 200.1071 hoac > 400.109/1 it cd gia tri trong danh gia chat ltfdng
tieu cau.
- Doi vdi nhtfng trUdng hdp sii dung mau toan phan de do ngung tap tieu
cau: ket qua bi anh htfdng bdi nhieu yeu to", dac biet la hematocrit.
XET NGHIEM XAC DjNH Sif CO MAT CUA CAC CHAT

KHANG DONG Ll/U HANH
1. Nguyen ly
Thdi gian mot xet nghiem dong mau keo dai do: hoac thieu hut mot hay
nhieu yeu to ddng mau hoac stf cd mat cua chat khang ddng lUu hanh. Nguyen ly
phat hien khang ddng ltfu hanh dtfa vao kha nang bu cua cac yeu to' ddng mau.
Thdi gian ddng cua hdn hdp 1 the tich huyet tUdng binh thtfdng va 1 the tich
huyet tUdng cua benh nhan thieu hut 1 hay nhieu yeu to" ddng mau se gan bang
thdi gian ddng cua huyet ttfdng binh thtfdng. Trai lai, thdi gian ddng cua hon hdp
99
1 the tich huyet ttfdng binh thtfdng va 1 the tich huyet ttfdng cua benh nhan cd
chat khang ddng ltfu hanh se keo dai gan bang thdi gian ddng cua huyet ttfdng
benh nhan.

- O n g nghiem ki'ch thtfdc 75x9.5mm.
- Dong ho bam giay.

- Huyet tUdng chtfng.
- Tuy theo loai xet nghiem can tien hanh (thdi gian prothrombin hay thdi
gian kaolin-cephalin hoat hoa v.v...) ma chuan bi thudc thtf.

- Lay mau tach huyet ttfdng: nhtf xet nghiem thdi gian Howell.
- Tien hanh xet nghiem ddng thdi vdi 3 mau huyet ttfdng trong cung dieu kien:
+ Mau huyet ttfdng chtfng.
+ Mau huyet ttfdng benh.
+ Mau huyet ttfdng hdn hdp chtfng va benh theo ty le 1/1.
- Ghi thdi gian ddng cua ca 3 mau.
Trong mot so trtfdng hdp, tien hanh lai xet nghiem vdi ca 3 mau sau khi u 2
gid d binh cach thuy 37°C.
4. Ket qua
Thdi gian ddng cua mau huvet ttfdng hdn hdp chtfng va benh ty le 1/1 keo
dai gan vdi thdi gian ddng cua huyet ttfdng benh chtfng to stf cd mat cua khang
ddng ltfu hanh. Can tien hanh xac dinh ban chat cua khang ddng ltfu hanh nay.
Thdi gian Thdi gian Thdi gian Chat khang ddng

cephalin - kaolin prothrombin thrombin ltfu hanh
Keo dai Binh thudng Binh thifdng XII, IX, XI, VIII
Binh thudng Keo dai Binh thudng VI la- thromboplastin to chifc.
Keo dai Keo dai Binh thtfdng X, V, II
Keo dai Keo dai Keo dai Thrombin
Chi so Rosner:
A P T T (benh + chtfng) - A P T T (chtfng) x 100
A P T T (benh)
Ket qua chi so:
< 12: Khang ddng noi sinh (-)
>12 - <15: Khang ddng noi sinh nghi ngd (±)
> 15: Khang ddng noi sinh (+)
100
XET NGHIlM CAC YEU TO KHANG DONG MAU (ANTI • C0A6UBATI0N):
PROTEIN C, PROTEIN S, ANTITHROMBIN III
Cac chat khang dong sinh ly bao gom: Protein C (Pc), protein S (Ps) va
antithrombin III (A T III), khi bi thieu hut (gap trong cac benh nhan bi benh di
truyen bam sinh) hoac giam sut ve ham ltfdng va hoat tinh (gap trong cac benh
nhan bj roi loan dong mau theo htfdng tang dong) co nguy cd dan den huyet khoi
va t ic mach. Cho nen viec xet nghiem cac yeu to nay rat can cho chan doan va
tien ltfdng benh.
1. Xet nghiem protein C va protein S
0 cac benh nhan thieu hut Pc, Ps cac xet nghiem A PTT , PT binh thtfdng, vi
vay viec xac dinh nong do va hoat tinh cua Pc, Ps d cac benh nhan nay co gia tri
chan doan cao.
- Protein C: Nguyen ly ky thuat: vi Pc la 1 khang nguyen, nen xu htfdng
dung phtfdng phap mien dich nhtf dien di mien dich, phong xa mien dich, mien
dich gam men (E L IS A ) dung khang the ddn dong dac hieu Pc dang dtfdc chu y
phat trien, ky thuat quang hoc so mau dung kit STA - Staclot - Pc cung dtfdc ap
dung. 0 ngtfdi ldn mtfc do Pc > 70%, d tre em thap hdn.
- Protein S: Do ham ltfdng protein s phtfc tap hdn vi co 2 thanh phan:
Protein S ttf do va Ps ket hdp gan vdi C4b. Thieu hut Ps co the phat hien qua 2
dang: giam Ps toan ph ln va giam Ps ttf do. Thong thtfdng cac ky thuat mien dich
htfdng phat hien Ps tu do, ttfdng ttf nhtf ky thuat E LIS A phat hien Pc. Cung co
the dung ky thuat dien di mien dich cheo (Crossed Immunophoresis) phat hien co
tinh chat dinh tinh Ps ttf do va protein s lien hdp.
2. Xet nghiem Anti - thrombin III (AT III)

A T III la chat khang dong quan trong, cung vdi Pc, Ps dong vai tro la cac
yeu to nguy cd gay huyet khoi, tac mach. 6 lam sang co the gap hai the: bam sinh
di truyen va mac phai, ca hai deu co the gay hau roi loan dong mau theo htfdng
tang dong gay huyet khoi (Thrombosis).
Nguyen ly ky thuat xac dinh A T III: A T III la 1 protein chung la 1 khang

nguyen, nen phtfdng phap mien dich dtfdc coi la phtfdng phap tfu tien phat trien.
Tuy nhien. cung nhtf Pc. Ps viec chiet tach Pc, Ps, A T III tao khang the ddn dong
la cong viec phtfc tap. Co hai nhom ky thuat: ky thuat theo dtfdng chtfc nang va
ky thuat theo dtfdng mien dich.
Cac ky thuat mien dich dtfdc sii dung de xac dinh A T III, cac ky thuat nay
bao gom:
- Dien di mien dich (Immunophorosis)
101
- Mien dich khuech tan (Immunodiffusion)
- Phong xa mien dich (Radio - Immunoassay)
- Mien dich gan men (Enzym linted Immunoassay = E L IS A )
Trong do ky thuat E LIS A sii dung khang the ddn dong (monoclonal
antibodies) dac hieu A T III dtfdc quan tam phat trien nhat.
O ntfdc ta, cac xet nghiem ve cac chat khang dong sinh ly tren tdi nay chtfa
dtfdc phat trien rong trong chan doan va tien ltfdng, cac yeu to gay huyet khoi, tic
mach. Nhtfng trong thdi gian khong xa do stf gia tang cac tai bien ve tim mach.
san khoa v.v... doi hoi ap dung cac ky thuat dinh htfdng cac yeu to khang dong
gop phan chan doan va tien ltfdng huyet khoi va tac mach do vai tro cua Pc. Ps.
A T III hoac bam sinh hoac mac phai gay nen.
102
C hiidng 3
DI TRUYEN HUYET HOC ■
CAY MAU NGUAIVI PHAN TfCH NHliM SAC THE
1. Nguyen ly
Cac te bao lympho mau ngoai vi khi tiep xuc vdi chat gay phan bao (mitogene)
co kha nang chuyen dang thanh te bao non va phan chia. Ldi dung kha nang do,
ngtfdi ta nuoi cay mau ngoai vi trong moi trUdng co c h it kich thich phan bao va
oau do lam ngUng phan bao d ky gitfa - giai doan co hinh dang nhilm s ic the
(N ST) dien hinh de lam tieu ban quan sat NST. Phan tich N S T te bao mau ngoai
vi cho phep ch in doan cac hoi chting di truyen do bat thtfdng NST, xac dinh'N ST
gidi cua ca the hoac phat hien mot so ton thtfdng NST.

2.1. Hod chat
1 . Heparin 5000 UI/ ml. Lo 5 ml.

2. Moi trtfdng nuoi cay te bao: TC 199, R P M I hoac moi trtfdng MEM.
3. Chat kich thich phan bao (P H A = Phytohemagglutinine).
4. Huyet thanh nhom mau AB.
5. Dung dich colcemid 0,004 ° / 00 (4 pg/ml)
6 . Chuan bi dung dich nhtfdc trtfdng
Hoac dung dich KC1 0,075M
Hoac dung dich huyet thanh A B pha loang 1/6 trong ntfdc cat
7. Chuan bi dung dich co" dinh
- Dung dich Carnoy I
+ Con ethylic 99° : 6 the tich.
+ Acid acetic dac : 1 the tich.
+ Chorofoor : 3 the tich.
- Dung dich Carnoy II
- Con ethylic 99° : 3 the tich.
+ Acid acetic : 1 thie tich.
103
2.2. Chuan bi dung cu
- Bdm tiem vo khu&n.

- Lo c£y vo trung co dung tich 15 - 30 ml.
- Pipette cac loai.
- Ong ly tam nhon day dung tich 10 - 15ml.
- Lam kinh sach.
- Tu am 37°C.
- Tu lanh.
- M ay ly tam quay ngang.
- Buong cay vo khu&n.
- Kinh hien vi.

3 .1. Nuoi cay
- Lay mau tinh mach vao bdm tiem co trang heparin, ltfdng mau l£Ly ttf l-2ml.
- Trong moi lo cay cho: 6,5 ml dung dich nuSi c&y.

1 ml huyet thanh AB.
0,1 ml PHA.
0,4 ml mau.
Lac nhe lo cay, de tu am 37°C co 5% C 0 2 (co the day nut lo cay va de tu am
thtfdng 37°C) trong thdi gian 72 gid, hang ngay lac nhe 1 - 2 lan.
Sau 72 gid u d tu am, chd vao m 6 i lo cay 0 ,1 ml dung dich colcemid 0,004°l00
lfic deu, dat lai tu am tiep 2 gid.
3.2. Lam tieu ban NST
3.2.1. Nhitoc tri/ong

- Chuyen toan bo hon dich d lo cay sau khi u 2 gid vdi colcemid vao ong ly
tam nhon day, ly tam d may ly tam ngang 5 phut x 1000 vong/ phut.
- Sau ly tam, hut bo phan dich trong d tren, de lai can te bao (chi hut den
cach mat tren can te bao khoang 3 mm).
- Cho them vao ong ly tam 8 ml dung dich nhtfdc trtfdng da de am 37°C
trtfdc, 0,1ml dung dich E D TA 40 mg/ml, tron deu va dat lai vao tu am 37°C trong
thdi gian 1 0 - 1 2 phut.
3.2.2. Co dmh
- Lan 1 : Sau khi u vdi dung dich nhtfdc trtfdng. lay ong ly tam ra, cho them
vao moi ong 0,2ml dung dich Carnoy II, dung pipette Pasteur tron deu, roi lai ly
104
tam l&y can nhtf tren, sau khi hut bo dung dich trong d phia tren, cho them vao
moi ong 5-7 ml dung dich Carnoy I, tron deu, de b nhiet do phong trong thdi gian
15 phut.
- iJin 2: Sau 15 phut lai ly tam va hut bo phan tren, them vao moi ong 5-7ml
dung dich Carnoy II, dung pipette Pasteur tron deu, de b nhiet do phong 10 phut.
3.2.3. Nhd ti&u bSn

- Cac phien kinh sach, ruta qua vdi ntfdc c£t, de tren gia lam va gia lam
dtfdc dat trong ngan da tu lanh khoang 10-15 phut, sau do lay ra de ph&ng tren
giay tham.
- Cac ong ly tam sau 1 0 phut u vdi dung dich Carnoy II, dtfdc ly tam 1000
vong/phut trong 5 phut, lay ra hut bo dung dich trong tren. Dung pipette
Pasteur tron deu can te bao va lay hon dich can nay nho len cac phien kinh. Chu
y khi nho tieu ban, de dau pipette cao hdn mat phien ki'nh ttf 1 0 - 2 0 cm.
3.2.4. De tieu ban kho tu nhien va tien hanh nhudm Giemsa ho$c cac ky thuat nhudm khac
Nhuom Giemsa:
- Pha Giemsa ty le 1/10 trong dem co pH = 6 ,8 .
- Danh dau tieu ban va dat vao be nhuom 10 phut
- Rtia b ln g ntfdc may va de kho, doc ket qua.
3.3. Phan tich ket qua

- Soi tieu ban sau khi dtfdc nhuom vdi cac ky thuat nhuom Giemsa hoac
nhuom bang dtfdi kinh hien vi quang hoc b vat kinh 10 de tim cum N S T dan deu,
chuyen sang vat kinh x 1 0 0 de phan tich chi tiet.
- Dem so' ltfdng ttfng cum NST.
- Tam xep cong thtfc NST.
- Chup anh, lam anh NST.
- Cat N S T rdi ttfng chiec va xep theo danh phap quoc te .
- Danh gia cac bat thtfdng neu co: can phan tich du so ltfdng cum N S T vi
nhieu khi benh nhan co dang kham NST.
4. Cac yeu to anh hiring

4.1. Cay khong ket qua do te bao khong phan chia
- Tu am khong dam bao nhiet do
- Moi trtfdng nuoi ca'y: pH qua kiem hay acid, n h ilm trung, qua han
- P H A khong dam bao chat ltfdng
4.2. TS'bao phan chia nhung hinh anh cum N S Txau
- pH cua moi trtfdng khong phu hdp
105
Nhiet do tu am khong on dinh
Cac dung dich sti dung khi chu^n bi tieu ban khong dung quy cach.
Tien hanh sai ky thuat.
LAM TltU BAN VA PHAN TfCH NHlEM SAC THE

Tt BAD TUY XUUNG
1. Nguyen ly
Binh thtfdng 0 tuy xtfdng, cac te bao tao mau lien tuc tang sinh va trtfdng
thanh do vay qua trinh phan chia dien ra mot cach ttf nhien. Dtfa vao tinh ch&'t
nay co the lam tieu ban N S T te bao tuy xtfdng trtfc tiep hoac sau khi nuoi cfty
trong moi trtfdng nhan tao de phan tich NST.

2.1. Hod chat
- Heparin 5000 Ul/ml.
- Moi trtfdng nuoi cay: R P M I 1640, Parker 199...
- Huyet thanh AB.
- Dung dich colcemid 0,0040/oo (4 pg/ml).
- Cac dung dich nhtfdc trtfdng va dung dich co dinh giong nhtf chu^n bi cho
cay mau ngoai vi.
- Dung dich dem bach cau.
2.2. Dung cu
- Giong nhtf chuan bi dung cu cay mau ngoai vi.
- Ngoai ra them bo dung cu dem bach cau.
- Chuan bi ong nghiem vo trung co nut kin, dtfng khoang 2 ml moi trtfdng
de am 37°C khoang 15 phut de lay tuy.

3.1. Lam tieu ban NST true tiep
3.1.1. Lay tup va Cfc che phan bao
- Lay 0,5 ml dich tuy vao bdm tiem vo trung co trang heparin 5000 Ul/ml
lSc nhe.
- Bdm ngay dich tuy vao ong nghiem co chtfa khoang 2 ml moi trtfdng nuoi
cay da de am trtfdc, tron deu, dem so" ltfdng te bao tuy trong hon dich bang buong
dem bach cau.
106
- Trong ong ly tam nhon day cho:
+ 8 ml moi trtfdng da de a'm 37° C.
+ 0 ,1 ml dung dich colcemid 0,004°/00-
+ Ltfdng hon dich tuy chtfa 4- 8.106 te bao.
- U d tu a'm 37° C trong thdi gian 1 gid.
3.1.2. Ldm tieu b£n

Sau khi u 1 gid, tien hanh cac btfdc nhtf lam tieu ban N S T mau ngoai vi.
3.2. C£y tuy ng&n han khdng dung chSt kich thich phan bao
3.2.1. Lify tup xitong va nu6i c£y
- L^y 0,5 ml dich tuy vao bdm tiem da trang heparin 5000 UI/ ml va cho
vao ong nghiem vo trung co chtfa 2 ml moi trUdng nuoi c&y, tron deu va dem te
bao tuy trong hon dich.
- Trong moi lo nuoi c&y cho:
+ 6 ml moi trUdng nuoi cay.
+ 1 ,6 ml huyet thanh AB.
+ Ltfdng hon dich tuy chtfa ttf 4- 6 . 1 0 6 te bao. L ie nhe lo ca'y va de tu a'm
37°C trong 24 gid.
- Sau 24 gid, cho vao moi lo 0 ,1 ml dung dich colcemid 0,004°/oo (4 pg/ml) de
lai tu am 1 gid.
3.3.2. Lam tieu ban
Tu^n ttf cac btfdc nhtfdc trtfdng, co dinh va nho tieu ban giong nhtf lam tieu
ban N ST mau ngoai vi.

Phan tich ket qua N S T te bao tuy xtfdng ttfdng ttf vdi mau ngoai vi. Song can
ltfu y la phtfdng phap nay dtfdc sti dung chu yeu de nghien ctfu benh ac tinh cd
quan tao mau, tren cung mot tieu ban co the vtfa co cum N S T bat thtfdng, vtfa co
cum N ST binh thtfdng nen can phan tich chi tiet ca cac cum N S T co hinh anh
khong dep vi cac bat thtfdng N S T thtfdng thay d loai “cum” nay.
Sau khi chup anh cac cum N S T dien hinh, cat va xep N S T theo danh phap
quoic te. Cac bat thtfdng ve so" ltfdng va cau true dtfdc danh gia:
- Thtf a NST: thay d toi thieu 2 cum NST.
- Thieu NST: thay d toi thieu 3 cum N S T .
- Bat thtfdng cau true: d toi thieu 2 cum N S T .
5. Cac yeu to anh hirdng

5.1. Khdng cd cum phan bao
- Moi trtfdng nuoi cay bi hong.
107
- Choc khong dung tuy.
- Tuy xtfdng bi suy, khong con te bao phan chia.
5.2. Cum phan bao xau

- Moi trtfdng kem chat ltfdng.
- K y thuat tien hanh chtfa dat.
CACH MO TA CONG THtfc NHllM SAC THE
1. Danh phap quoc te ve te bao di truyen ngi/di nSm 1995

Trong te bao than cua ngtfdi binh thtfdng co 46 N S T chtfa toan bo thong tin
di truyen cua ca the. Phtfdng phap dung cac ky thu&t de quan sat va nghien ctfu
N S T goi la phtfdng phap te bao di truyen hoc.
Ttf nam 1960 cac nha te bao di truyen tren the gidi da hop tai Denver de
thong nhat ten goi va cach trinh bay N S T binh thtfdng cung nhtf cac bat thtfdng
(Danh phap NST). K et qua s ip xep bo N S T cua te bao goi la cong thtfc N S T hay
cong thtfc nhan (karyotype). Nhd stf tien bo cua ky thu&t, ngtfdi ta co the phan
tich chi tiet hdn cac bat thtfdng N S T nen doi hoi phai bo sung danh phap thtfdng
xuyen. Da co nhieu hoi nghi ve danh phap dtfdc to chtfc. Hoi nghi g in day n hit
nam 1994 tai Memphis va 1 nam sau do, U y ban danh phap cua Hoi nghi da cong
bo" danh phap quoc te ve te bao di truyen ngtfdi 1995 (An International System for
Human Cytogenetic Nomenclature 1995) ma noi dung cd ban dtfa tren cac quy tic
cong bo' trtfdc, co bo sung.
1.1. Soluvng va hinh dang NST
Trong cong thtfc NST, cac N S T thtfdng dtfdc danh so' ttf 1 - 2 2 theo thtf ttf
giam dan cua chieu dai. Can ctf vao chieu dai va vi tri tam cua N S T ngtfdi ta chia
cac N S T ra 7 nhom (A - G).
- Nhom A (gom N S T 1 - 3) la cac N S T ldn tam gitfa.
- Nhom B (gom N S T 4 - 5) la cac N S T 16n tam gan gitfa.
- Nhom C (gom N S T 6 - 1 2 ) N S T kich thtfdc trung binh, tam gitfa hoac gin
gitfa, N S T X gan giong N S T ldn trong nhom nay.
- Nhom D (gom cac N S T 13 - 15) cac N S T tam cuoi, kich thtfdc trung binh
co the co ve tinh.
- Nhom E (gom cac N S T 16 - 18): N S T kha nho, tam gitfa hoac g in gitfa.
- Nhom F (gom cac N S T 19-20): N S T nho tam gitfa.
- Nhom G (gom cac N S T 2 1 -2 2 ): N S T nho, tam cuo'i, co the co ve tinh.
108
1.2. Vung, bdng vd dudi bdng cua NST
Cac ky thuat nhuom bang (bang G, bang R, bang Q) tao nen hinh anh cac
bang dam nhat xen ke va lien tiep theo chieu doc cua NST. Cac bang nay dac hieu
cho ttfng NST, dtfa vko do ngtfdi ta chia N S T ra nhieu vung, moi vung co cac bang,
moi bang lai chia thanh dtfdi bang.
Ky hieu p va q la chi canh ngan va canh dai cua NST. So' cua vung, bang,
dtfdi bang dtfdc danh so' ttf phia tam ra, bat dau la so' 1 . De xac dinh mot bang
nha't dinh ngtfdi ta dung 4 ky ttf lien tuc khong co da'u ngan cach: so' NST, ky hieu
canh, so' cua vung, so' cua bang trong vung; V i du: lp31 chi N S T so' 1, canh ngan,
vung 3, bang 1 . Khi phan tich chi tiet. them cac dtfdi bang cung dtfdc danh so ttf
phia tam ra ngoai va dtfdc the hien sau ky ttf chi bang, co ngan cach bang dau
cham (.). V i du bang lp31 chia thanh 3 dtfdi bang lp31.1, lp31.2, lp31.3 thi dtfdi
bang lp31.1 d phia g in tam nha't va lp31.3 xa tam nhat. Neu lai chia nho tiep thi
them ky ttf nhtfng khong can da'u.
2. Md ta cong thurc NST

2.1. Nguyen tac chung
De trinh bay cong thtfc NST, dau tien la viet tong so' N S T (gom ca N S T gidi)
tiep la da'u (,) roi den N S T gidi, sau do la cac ba't thtfdng neu co. Nhtf vay, cong
thtfc N ST binh thtfdng cua ntf la 46, X X -, cua nam la 46,X Y .
2.2. Md ta cac bat thudng

Sau N S T gidi la da'u phay roi den cac ky hieu va N S T ba't thtfdng. Neu cd
nhieu bat thtfdng thi bat thtfdng cua N S T gidi viet trtfdc roi den N S T thtfdng, theo
thtf ttf so' ttf be den ldn. Moi ba't thtfdng cach mot da'u phay. Khi ba't thtfdng cau
true chi lien quan den 1 N S T thi so' N S T di ngay sau ky hieu chi ba't thtfdng va de
trong ngoac ddn. Neu cd ttf 2 N S T tham gia bat thtfdng thi dung dau cham phay
(;) de ngan cach gitfa cac NST.
2.2.1. Bat thudng so luong

Dau cong (+) hay da'u trtf (-) de trtfdc N S T ndi len them hoac mat NST, trtf
ba't thtfdng bam sinh N S T gidi.
V i du 47,XX, + 21 ttfc la: cd 3 N S T 21
Bat thtfdng bam sinh N S T gidi viet theo nguyen t ic chung, V i du: 47,XXX
ttfc la thtf a mot N S T X bam sinh. Ba't thtfdng mac phai N S T gidi dtfdc mo ta nhtf
NST thtfdng.
2.2.2. B it thudng cau true
- Dau cong (+) hay trtf (-) de sau ky hieu canh N S T ndi len them doan hay
mat doan canh do.
- add (addition): Them doan N S T nhtfng khong biet nguon go'c doan them.
V i du 46,XX, add (19) (p l3 ) ttfc la them mot p h in vat lieu di truyen vao
canh ngan N S T 19 ttf doan pl3.
109
- del (deletion): M at doan.
- der (derivative): N ST bat thtfdng diidc hinh th&nh do ton thiidng tii 2 NST
hoac hdn.
- die (dicentric): N S T hai tam.

- dup (duplication): nhan doan NST: mot doan NST nhan len hai hay nhieu lAn.
- ins (insertion): xen doan NST. Khi co b i t thtfdng xen doan lien quan den
2 N S T thi N S T nhan doan viet trtfdc.
V i du: 46,XX, ins (5;2) (p l4 ; q32 q22)
- inv (invertions): dao doan: co the dao doan ngoai tam (khi 2 diem gay d tren
mot canh NST) va dao doan quanh tam (khi hai diem g ly d tren hai canh NST).
- i (isochromosome): d in g NST: N S T cd hai canh hoan toan giong nhau

V i du: 46,XX, i(17) (qlO)
6 day N S T co hai canh deu la canh dai.

- mar (m arker chromosome): N S T danh dau la N S T cd b it thtfdng c&'u true
ma khong biet nguon goc.
- r (ring chromosome) N S T vong.
- t (translocation) chuyen doan: khi trinh bay thi N S T gidi hoac N S T co so
nho viet trtfdc. Trong trtfdng hdp 3 hay 4 N S T tham gia chuyen doan thi: dau tien
la N S T gidi hay N S T co so' nho, tiep den la N S T nhan doan cua N S T d iu , roi cuoi
cung la N S T cho doan N S T dau.
2.3. Dang kham NST
Khi phan tich cong thtfc N S T thay cd 2 hay nhieu dong te bao cd cong thtfc
khac nhau thi phan cach b in g dau gach cheo (/) va so' te bao phan tich dtfdc d moi
dong de trong dau [ ].
V i du: 46.X X [13]/45.X [16]
Cd nghia: da phan tich dtfdc 29 cum NST, trong dd 13 cum cd cong thtfc NST
ntf binh thtfdng, 16 cum cd 45NST va chi co mot N S T gidi la X.
CAC KY THUAT NHUOM NHliM SAC THE
1. Ky thuat nhuom Giemsa thong thirdng

1.1. Nguyen ly
Nhiem sac the (N S T ) la phan bat mau ro nha't cua te bao vdi thuoc nhuom
Giemsa nen sau khi nhuom, N S T cd mau dong nhat (Hinh 3.1).
110
1.2. Chutfn bj thudc nhu$m va dung djch d?m
- Dung dich Giemsa m§: Pha thong thtfdng va de san.
- Dung dich dem pha s&n:

+ Dung dich 1 : N a H P 0 4 0,07 M.
+ Dung dich 2: K H 2 P 0 4 0,07 M.
- Dung dich dem sti dung: Pha tron dung dich 1 va dung dich 2 vdi ty le 1:1 ve
the tich.
1.3 Quy trinh ky thuat

- Pha Giemsa trong dem vdi ty le 3,5%, cho vao coic nhuom, dung giay
tham, th&'m hot d be mat.
- Tieu ban N S T da chuan bi de kho, nhung qua nUdc cat 3- 5 giay.
- De dting tieu ban 10 phut trong coc nhuom, cho dau co chu! len tren.
- Rtia b S n g nUdc m ay, t r a n g lai bling nUdc cat, de kho v a doc k et qua.
Nhuom Giemsa thong thtfdng cho hinh anh N S T ro, dtfdc sti dung de danh
gia so ltfdng va tim cac ton thtfdng nhtf dtft, gay hoac cac ton thtfdng c&'u true
khac. Nhtfng N S T bat mau dong nhat nen r£t kho khan trong viec xac dinh cu the
ttfng N ST cung nhtf nhtfng thay doi nho nhtf chuyen doan, dao doan. V i v£y ngtfdi
ta sti dung ky thuat nhuom bang NST.
1 *
r 0 * f | * S-
* d *■ b ^
X * 8
Hinh 3.1: Hinh anh NST nhuom Giemsa
2. Cac ky thuat nhuom b5ng NST

Hien nay nhieu ky thuat nhuom bang N S T dtfdc sti dung: bang Q, bang G,
bang R, bang C, bang T. Viec nhuom bang nao la tuy thuoc muc dich xet nghiem
va kha nang cua ttfng phong xet nghiem.
Ill
2 . 1 Nguyen ly
Xti ly tieu ban N S T bang men tieu protein hoac trong moi trtfdng muoi 6
nhiet do cao. Do cau true N S T co ttfng doan A D N giau A ,T (adenin va thymidin)
dac trtfng cho ttfng cap NST, vi vay phan bo' cac loai protein cung dac trtfng doc
theo chieu dai cua moi NST. Mtfc do tac dong cua men tieu protein va nhiet len
cac doan N S T se khac nhau, do do sau khi xti ly va nhuom se co hinh anh ttfng
doan dam- nhat xen ke nhau doc theo chieu dai NST. Do dai, vi tri, so' ltfdng cac
doan dam nhat vi vay cung dac trtfng cho ttfng doi NST, qua do co the xac dinh
chinh xac ttfng N S T va phat hien dtfdc cac ton thtfdng ma bang ky thuat nhuom
thong thtfdng khong phat hien dtfdc.
Moi phong xet nghiem te bao di truyen co the ltfa chon va tfng dung cho
rieng minh mot ky thuat, cac ky thuat co the thay doi d mot so btfde. Chung toi
gidi thieu d day cac btfdc cd ban cua cac ky thuat dtfdc sti dung rong rai hien nay.
2.2. Ky thuat nhudm bdng G
N S T dtfdc xti ly vdi men trypsin hoac xti ly nhiet do trong muoi citrat, sau do
nhuom Giemsa, cac bang dam ttfdng tfng vdi nhtfng doan chtfa nhieu A va T.
2.2. 1. Being G x ti ly nhiet: AS G (Acetic-Saline- Giemsa)

Summer va cong stf sti dung dau tien vao nam 1971, nay da co nhieu thay doi.
a. Chuan bi tieu ban: Cac tieu ban N S T sau khi chuan bi, de khoang 1 tu&n
d nhiet do phong hoac 3 ngay d nhiet do 37°C.
b. Chuan bi hod chat va dung cu
- Dung dich SSC x2: N aC l 0,3 M.
N atricitrat 0,03 M.
- Dung dich Giemsa, dung dich dem phosphat nhtf nhuom thong thtfdng.
- Be am de nhiet do 60°C.
- Coc nhuom.
c. Tien hanh ky thuat
- Cho dung dich SSC x 2 vao coc nhuom, de vao be a'm d 60°C trong 15 phut
- U tieu ban N S T trong SSC x 2 d 60°C trong 1 gid 30 phut.
- Rtia ngay bang ntfdc cat.
. - Nhuom 30 phut d dung dich Giemsa 2% trong dem phosphat.
- De kho va doc ket qua.
2.2.2. Nhuom b&ng G x ti ly vdi men trypsin
a. Hod chat, dung cu cdn thiet

- Trypsin: lo trypsin Diffco 1: 250.
- Huyet thanh thai be.
112
- Dung dich dem phosphat pH = 6,8 (nhtf tren).
- Dung dich d in g trtfdng (ntfdc muoi sinh ly).
- Ntfdc cat khii ion.
- Coc nhuom.
b. Pha hod chat
- Dung dich trypsin (0,025%):
+ Trypsin (1: 250) Diffco 0,05g.
+ Ntfdc muoi dang trtfdng 200ml.
- Dung dich huyet thanh thai be:
+ Huyet thanh thai be 4ml.
+ Ntfdc muoi sinh ly 200ml.
- Dung dich nhuom
+ Dung dich Giemsa me 4ml.
+ Dem phosphat 200ml.
- Ntfdc muoi sinh ly 2 coc x 200 ml.
- Dung dich dem phosphat 2 coc x 200 ml.
- Ntfdc cat khii ion 2 coc x 200 ml.
c. Quy trinh ky thuat
- U tieu ban trong dung djch trypsin 0,025%.

- Thdi gian u tuy tuoi (so ngay ttf khi co dinh) cua tieu ban va tuy loai NST.
N S T mau, to chtfc u khoang 12 - 18 giay cho tieu ban da lam 1 tuan.
N S T ntfdc 01 12 - 16 giay.
N S T tuy 4 - 6 giay.
Tieu ban mdi chuan bi thdi gian u phai lau hdn.
Thdi gian nay can dieu chinh theo ttfng phong xet nghiem
- Cho tieu ban vao coc dtfng dung dich huyet thanh thai be hai lan, moi lan
5-10 giay.
- Cho tieu ban vao coc dtfng ntfdc muoi sinh ly 2 lan, moi lan 5 - 10 giay
- Nhuom 2- 3 phut trong dung dich Giemsa. Thdi gian co the thay doi tuy
ttfng phong xet nghiem.
- Cho tieu ban vao dung dich dem phosphat 2 lan, moi lan 5-10 giay.
- Rtfa tieu ban bang cach nhung vao coc ntfdc cat 2-3 lan.
- De kho d 50"C trong 15 phut va doc ket qua, chup anh, xep cong thtfc
(Hinh 3.2).
* »
\ 4
* A i t
u
1 •
s * * ( v n C "
1 2 3 4 ■i
1 1 ! 1
• • • •
S i 1 1 1 1
6 7 8 9 10 n 12
« 1
4 • * #* u
i t » *»
13 14 15 16 17 18
V V U * » f •
r* -'
t *
19 20 2! 22 • XY
H in h 3.2: Hinh anh NST nhuom bang G
2.2.3 Danh gia ket qua: Dtfa vao hinh anh mau cua bang G de phan tich.
2.2.4 Cac yeu to anh htfdng: nhuom thanh cong khi co cac bang dam nhat xen ke
nhau doc chieu dai NST.
a. Ky thuat xit ly nhiet

- Tieu ban mdi lam hoac de qua lau thtfdng ket qua kho dep, tieu ban dtfdc
lam ttf 1 tuan - 2 thang thtfdng cho ket qua tot.
- Nhiet do be am qua cao hoac qua thap.
- Thdi gian u tieu ban trong dung dich SSC x 2 qua lau hay qua ngan deu
cho ket qua kem.
b. Ky thuat dung men trypsin
- Tieu ban mdi lam thtfdng kho dep.
- Nong do cua men khi pha cao hoac thap qua.
- Thoi gian u men ngan hay dai qua deu cd the lam hong ket qua.
2.3. Ky thuat nhuom bang R
Ky thuat nhuom tao nen cac bang dam nhat ngtfdc lai vdi bang G ttfc la vung
dam d bang G se nhat d bang R va ngtfdc lai nen goi la bang ngtfdc (Reverse band).
Co nhieu phtfdng phap nhuom bang R, phtfdng phap dau tien la dung moi
trtfdng muoi xtf ly tieu ban N ST d nhiet do cao sau dd nhuom Giemsa (R bands by
heat using Giemsa = RHG). Ngoai ra cac phtfdng phap khac cd the dung acndin hoac
chat nhuom mau fluor nhtf chromomycin hay dung chat thay the thymidin (BrdU).
114
Chung toi trinh bay d day phtfdng phap nhuom nong bang R (R H G ) do
Dutrillaux va Lejeune mo ta 1971, co chinh ly.
2.3.1. Dung cu va hoa chat
- Be am co the de nhiet do 87°C.

- Coc nhuom va cac coc dtfng ntfdc cat.
- Dem phosphat (nhtf phan tren).
- Dung dich nhuom: Giemsa 3,5% trong dem phosphat.
- Dung dich Earle.

+ NaCl 6800 mg
+ KC1 400 mg
+ N a H 2P 0 4 . H 20 140 mg
+ M g S 0 4 . 7H20 200 mg
+ Glucose 1000 mg
+ CaCl2 100 mg
-i- Ntfdc cat vtfa du 1000 ml.
Dung dich Earle nay cd the pha dac 10 lan (dung dich me) va gitf d 4°C, khi
sii dung pha loang vdi ntfdc cat. Chuan bi 2 coc dung dich Earle sii dung, moi coc
khoang 100ml.
+ Coc 1: dung N a 2H P 0 4 dieu chinh cho pH » 5,3.
+ Coc 2: dung N a 2H P 0 4 dieu chinh cho pH « 6,5.
- Tieu ban N S T da chuan bi de 2 - 3 ngay d nhiet do phong hoac qua mot

dem d 37°C.
- Nhung tieu ban vao ntfdc cat 1 - 2 phut. •
- Dat tieu ban trong dung dich Earle co pH = 5,3 d 87°C

(De coc dtfng Earle vao be am 87°C trtfdc 15 phut).
Thdi gian khoang 45 phut.
- Chuyen tieu ban sang coc dtfng Earle cd pH = 6,5 cung d 87°C trong thdi
gian 45 phut. (Thdi gian u tieu ban d Earle cd pH = 5,3 va 6,5 la bang nhau va tuy
thuoc tuoi cua tieu ban, neu tieu ban da chuan bi tren 1 tuan thi thdi gian nay
ngan hdn).
- Chuyen tieu ban vao coc dtfng ntfdc cat d nhiet do phong va trang rtfa tieu
ban 3- 4 lan.
- Nhuom trong dung dich Giemsa trong 8 phut.
115
qua, chon cac cum ky giiia N S T ro chup anh, xep cong thtfc N S T va phan tich ket
qua (Hinh 3.3).
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Hinh 3.3: Hinh anh NST nhuom bang R
2.3.3. Cac yeu to anh hudng

- Tieu ban N S T khong dep, N S T bi nat.
- Nhiet do be am qua cao hoac khong du.
- pH cua dung dich Earle khong dung.
- Thdi gian u d Earle nhat la dung dich Earle pH= 5,3 khong phu hdp.
- Rtia tieu ban trtfdc khi nhuom khong ky.
2.4. Ky thuat nhuom bang C
Khac vdi cac ky thuat nhuom bang khac. nhuom bang C la ky thuat nhuom
cac vung di nhiem sac, cac vung nay thtfdng nam d phan tam cua N ST va canh dai
cua N S T Y. Ngtfdi ta xtf ly tieu ban trong dung dich kiem: NaOH hay B a(O H )2 sau
do xtf ly tiep trong moi trtfdng muoi d nhiet do cao va nhuom vdi Giemsa. Chi cd
vung di nhiem sac bat mau dam con lai mau nhat. Chung toi gidi thieu kv thuat
do Summer dtfa ra 1972, co chinh ly.
2.4.1. Hoa chat va dung cu
a. Dung cu
- Cac loai coc nhuom.
- Be am, cd the de nhiet do 65°C.
116
b. Hod chat
- Dung dich bari hydroxid 5%
B a(O H )2 = 5g
Nude ca't du 100ml
Hoa tan B a(O H )2 b^ng may khu&y tii, loc, nen pha dung dich n&y trUdc
khi dung (trong ngay).
- Dung dich SSC x 2
+ NaCl 17,5 g
+ Natricitrat ngam 2 phan tii nUdc
(N a 3C6H 50 7.2 H 20 ) 8,8 g
+ Nude cat du 100ml.
- Dung dich HC1 0,2N.
- Dung dich nhuom Giemsa 2%.
+ Dem phosphat pH = 6,8 = 98 ml
+ Giemsa 2 ml
- Con tuyet doi, con 80 do, con 70 do.
- Ngam tieu ban 1 gid trong dung dich HC1 0,2 N.

- Rtfa bang niidc khii ion va de kho.
- U tieu ban 45-60 phut trong B a(O H )2 5% d nhiet do phong.
- Rifa tieu ban bang cach nhung vao nhieu coc dUng nude khu ion; sau dd
chuyen tieu ban vao cac coc dung con lan lUdt 70 do, 80 do, tuyet doi, thdi gian
nhung tieu ban d moi coc khoang 15 - 20 giay. De kho tieu ban.
- U tieu ban 1 gid 30 phut trong dung dich 2 x SSC d 65°C.

- Rifa tieu ban bang nUdc khii ion va de kho.
- Nhuom tieu ban vdi thuoc nhuom 2% (de phang tieu ban va phu thuoc
nhuom len tieu ban) trong thdi gian 20- 25 phut. Rtia tieu ban bang nUdc khif ion,
trang bang nUdc cat, de kho va doc ket qua.
2.4.3. Cac yeu to anh hudng

- Thdi gian ngam tieu ban trong HC1 0,2N va Ba(OH)2 qua ngan hoac qua lau.
- Thdi gian u tieu ban trong dung dich 2 x SSC d 65°C khong dam bao.
- Nhuom Giemsa dam qua (se lam mat bang).
2.4.4. Danh gia ket qua: Nhuom bang C se cho hinh anh dam d phan tam cua N ST nhat
la cac NST 1, 9, 16 va canh dai NST Y, cac phan cdn lai NST nhat mau (hinh 3.4).
117
4 -5
8 -X -12
JUAftAA ** A* * *
13-15 16 17-18
XXXM A fl y\ * m
19-20 21-22 Y
Hinh 3.4: Hinh anh NST nhuom bang C
2.5. Ky thuat nhuqm bang Q
Bang Q la bang diidc sti dung sdm nhat, nhilng do phai dung quinacrin, kinh
hien vi huynh quang va can doc sdm sau khi nhuom nen khong diidc dung hang
ngay. Tuy vay cung can thiet de lam can cti doi chieu hoac cho cac nghien ctiu ve
phan di nhiem s ic ctia NST.
2.5.1. Hoa chat
a. Dem M cllva ine (p H = 5,4)

- Acid citric (H 3C6H 50 7) 2 ,lg
- N atri hydro phosphat (N a 2H P 0 4) 3,9g
- Nude cat 500 ml.
b. D ung dich nhuom quinacrin
- Atabrin 1 vien hoac 100 mg
- Dem M cllvaine (pH = 5,4) 200 ml

Hoa tan va loc, de d 2- 5°C.
- Nhuom cac tieu ban 10- 15 phut trong dung dich nhuom quinacrin. trong
buong toi hoac hoc gieky den coc nhuom.
118
- Rvia difdi ntfdc may, loai h£t quinacrin thtfa.
- D&t tieu ban 1 phut trong dem Mcllvaine.
- Phu dem M cllvaine len tieu ban v& th&'m bdt dem thtfa.
- Soi dtfdi kinh hien vi huynh quang, dung btfdc song ttf 450- 500 nm.
2.5.3. Danh gia ket qua
Cac N S T se the hien cac doan sang, toi dtfdi kinh hien vi huynh quang, vung
sang ttfdng tfng vdi phan N S T chtfa A D N giau A va T. Chon cac cum N S T ro, chup
anh de phan tich ket qua dtfa tren phan bo" bang chu^n.
Cac vung d&m tren N S T khi nhuom bang G, vung nhat khi nhuom bang R
ttfdng tfng vdi vung sang cua bang Q.
Ltfu y ky thuat nhuom bang Q vdi quinacrin phai doc va chup anh ngay sau
khi nhuom, de lau se khong con ro cac vung bang.
NHUOM PHAN BIET NHliM SAC TUT CM EM
1. Nguyen ly
T e bao phan chia dtfdc la nhd stf nhan doi NST, do do can den base nitd.
Ngtfdi ta dung chat ttfdng ttf thymidin (Bromodesoxy uridine = BrdU) de thay the
base nitd nay trong hai chu ky phan bao lien tiep, se tao ra dtfdc mot N S T con
(nhi§m s&c tii) co ca hai mach A D N deu chtfa BrdU. Khi nhuom vdi fluorochrom
hay Giemsa, nhiem sac tii nay co mau khac nhiem sac tti con lai. Qua do co the
quan sat dtfdc cac hinh anh trao doi gitfa hai nhiem sSc tuf - trao doi nhilm s^c tit
chi em (Sister chromatrid exchanges: SCEs.)
Nhtf vay ky thuat nay dtfdc tien hanh qua hai btfdc:
- Btfdc 1: Gfin BrdU vao A D N d hai chu ky phan bao lien tiep.
- Btfdc 2: Nhuom phan biet hai nhiem sac tut.

2.1. Gan BrdU vao nhiem sac the
2.1.1. Hoa chat va dung cu can thiet
Ngoai cac dung cu, phtfdng tien hoa chjit can cho cay mau ngoai vi, can them
dung dich BrdU.
BrdU: 10 mg
Ntfdc cat: 10 ml.
119
2.1.2. Cac budc tien hanh
- Cay lymphocyt binh thtfdng, u d 37°C trong 42 gid.
- Sau 42 gid them vao moi lo cay dung dich BrdU de nong do cuoi cung la
10 pg/ml, u tiep 30 gid d 37°C .
- Sau 30 gid, tfc che bang colcemid va lam tieu ban binh thtfdng.
2.2. Nhuom phan biet hai nhiem sac t il

- Dung dich Hoechst 33258
Hoechst 33258: 1,5 mg
Ntfdc cat 10 ml
Dung dich nay gitf trong toi, de dong lanh, co the dung vai thang.
- Dung dich 2 x SCC
NaCl: 17,5 g.
N atri citrat ngam 2 phan tti ntfdc
(N a 3C6H 50 7.2H20 ): 8,8 g.
Ntfdc cat vtfa du 1000 ml
Dieu chinh pH 7,0 bang NaO H IN hay HC1 IN .
- Dem Sorensen (pH 7.0):
K H 2P 0 4: 5,26 g
N a 3H P 0 4: 8,65 g
Ntfdc cat: 1000 ml.
- Dung dich nhuom Giemsa 2%:
Dem Sorensen: 49 ml
Giemsa: 1 ml.
- Be am, cac coc nhuom, coc thuy tinh.
- Den ctfc tim: 1 bong den ctfc tim.
- Kinh hien vi chup anh.
- Nho dung dich Hoechst 33258 len tieu ban, day tieu ban bang dia thuy
tinh (dia petri) trong 10-15 phut d nhiet do phong.
- Rtfa tieu ban bang ntfdc cat 3 lan, de kho.
- Phu len tieu ban mot ldp dung dich 2 x SCC (pH 7.0) va dat dtfdi den ctfc
tim trong 1- 2 gid. (Khoang cach ttf den den tieu ban khoang 20 cm).
- Rtfa lai tieu ban bang ntfdc cat, de kho va nhuom Giemsa 2% trong 15- 20 phut.
- Doc ket qua dtfdi kinh hien vi quang hoc, chup anh cac cum N S T va
phan tich.
120
Sau khi nhuom phan biet, nhiem sac til co ca hai sdi A D N mdi dtfdc tong
hdp se bat mau khac nhiem sac tti con lai. Trong qua trinh phan chia co the co
nhtfng trao doi gitfa cac doan cua hai nhiem sac ttf chi em- dtfa vao do ngtfdi ta
co the danh gia cac mtfc do thay doi. K y thuat nay dtfdc dtfng de danh gia tac
nhan gay dot bien NST.
4. Nhufng yeu to anh hirdng

- Dung dich pha khong dam bao.
- Thdi gian xti ly dtfdi den ctfc tim hay trong dung dich qua lau hoac chtfa dti.
XET NGHIEM VAT THE BARR
Trong thtfc te nhieu khi can xac dinh gidi nhanh, ngtfdi ta co the sti dung ky
thuat xet nghiem vat the gi6i. Tuy nhien day cung chi la x6t nghiem sd bo, muon
chfic chfin can phai phan tich NST.
1. Nguyen ly
Te bao cf giai doan gian ky, chi co mot N S T X hoat dong, khi nhuom Orcein
hay carbofuchsin, chat nhilm sfic b N S T X con lai bSt mau d&m tao nen wkt the
Barr, bam vao mang nhan. Co the chon mot trong hai ky thuat sau.

2.1. Ky thuat nhuom vdi orcein
2.1.1. Dung cu va hoa chat
- Phien kinh sach, de ltfdi, be am co lSc.

- Dung dich Aceto- orcein 2%:
+ Cho 40g orcein tong hdp vao 200 ml acid acetic, l&c deu trong be am co
may l&c.
+ Them ntfdc cat du 2 lit, loc 3 lan b&ng giay loc W hatman so" 1, de ch6 toi
hay hoc giay den.
2.1.2. Tien hanh
- Phet tieu ban: Dung de ltfdi lay te bao niem mac ma, phet len phien kinh,
thtfdng moi ben l&'y mot tieu ban. De kho tieu ban d nhiet do phong.
- Nho 3- 4 giot dung dich Aceto- orcein 2% len tieu ban, day bang dia petri
trong khoang 15 phut.
121
- Do dung dich orcein thtfa, th&"m kho, doc kcst qua ngay dtfdi kinh hien vi,
doc toi thieu 100 te bao va tinh ty le ph£n tram cac te b&o c6 v&t the Barr.
2.2. Ky thuat nhuom carbofuchsin r

- Chuan bi dung dich carbofuchsin III:
+ Dung dich I: Fuchsin basic 3 g
Con 70° : 100 ml
+ Dung dich II: Dung dich I: 100 ml
Phenol 5%: 900 ml
+ Dung dich III: Dung dich II: 120 ml
Acid acetic: 120 ml
Formaldehyd 34%: 120 ml
- Con 96°- 98°.
- Dung dich HC1 nong do 5 N.
- Con methanol.
2.2.2. Tien hanh

- Phet tieu ban: Dung de ltfdi lay te bao niem mac ma phet len phien kinh,
de kho.
- Co" dinh ngay sau khi tieu ban kho vao con 98° trong 30 phut sau do de
kho d nhiet do phong.
- Nhung tieu ban vao dung dich HC1 5N trong 5 - 6 phut.
- Trang tieu ban b&ng ntfdc may,de kho.
- Nhuom tieu ban vdi carbofuchsin II I trong 20 phut.
- Rifa bo fuchsin thtfa bang methanol, de kho.
Sau do soi dtfdi kinh hien vi, tim vat the Barr tren 100 te bao.
3. Danh gia ket qua

Vat the Barr bat mau sam, hinh thoi, tam giac hoac bau due nam bam vao
mang nhan te bao. Theo ly thuyet so" ltfdng vat the Barr trong te" bao bang so
ltfdng N S T X trtf 1. Nhtf vay d nam gidi binh thtfdng khong co vat the Barr, d ntf
binh thtfdng co mot vat the Barr, ngtfdi co 3 N S T X (XXX) se co 2 vat the Barr.
Trong thtfc te" khi xet nghiem te" bao niem mac ma cua ntf co khoang 10 -
80% te bao co vat the Barr tuy theo giai doan phat trien va chu ky kinh nguyet. d
nam binh thtfdng cung co the co vat the Barr (nhtfng dtfdi 5%).
4. Cac sai so hay gap

- M ieng benh nhan qua ban,lan nhieu vi khu&n va vet b^n kho doc ket qua.
122
- Nhuom qua lau, dung dich nhuom qua dac khong phan biet dtfdc vSt the
Barr vjk p h ln chat nhan con lai.
- Doc it t£ b&o chtfa phat hien dtfdc.
KY THUAT SINH HOC PHAN TUT UTNG DUNG TRONG Y HOC
Cd scl di truyen cua cd the n&m trong cac gen tren N S T ma ban chat cua no
la acid desoxy ribonucleic (A D N ). Cau true A D N la chuoi xo&n kep cac nucleotid
ma moi m&t xich la mot base nitd (Adenin - A, Thym idin - T, Guanin - G, Cytosin
- C). Hai sdi cua chuoi xo&n k6p lien ket vdi nhau mot cach khong phai ng&u
nhien ma co tinh dac trtfng theo ttfng cap base nitd gitfa sdi nay vdi sdi kia theo
cach A di vdi T, G di vdi C...
Khi phan chia te bao, co qua trinh nhan doi AD N, luc nay cac sdi tach nhau
ra va moi sdi se lam khuon de tong hdp nen mot sdi bo sung theo nguyen t ic cap
doi(cd che ntia bao ton) tao nen hai chuoi A D N hoan toan giong nhau va giong
chuoi ban dau.
Ngtfdi khoe manh co bo gen binh thtfdng, ttfc la trinh ttf cac base nitd trong
cac gen (doan A D N ) binh thtfdng. Cho den nay ngtfdi ta th&y toan bo cac gen da
biet chtfc nang chi chiem 10% ltfdng A D N cua te bao, con lai 90% la nhtfng doan
A D N co trinh ttf nucleotid lap lai ma nay ngtfdi ta chtfa biet chtfc nang. Trtf hai
anh chi em sinh doi cung trtfng, that kho tim dtfdc hai ca the co bo gen giong
nhau, cho nen xac dinh gen noi len dtfdc tinh chat dac trtfng cua ca the va nguon
goic bo' me (mot ntia bo gen co nguon goc ttf bo" va mot ntia ttf me).
Trinh ttf cac base nitd cua gen bi thay doi co the gay nen benh di truyen.
Ngtfdi ta co the dung ky thuat sinh hoc phan tti de xac dinh nhtfng thay doi do va
xac dinh nguyen nhan gay benh. Ngoai ra, cung co the sti dung ky thuat nay de
xac dinh sdm stf cd mat cua cac virus, vi khuan gay benh.
KY THUAT TACH CHIET ADN

•
Tuy theo muc dich ctia xet nghiem va dieu kien thtfc te, co the tach A D N ttf
cac nguon khac nhau: bach cau mau ngoai vi, ttf to chtfc, ttf chan toe, ttf benh
pham nghi cd tac nhan gay benh (chat tiet, huyet thanh....). CJ day chung toi gidi
thieu ky thuat chiet tach A D N ttf bach cau mau ngoai vi. Do de lay m iu nen ky
thuat nay thuan ldi cho cac xet nghiem tim gen hay tim dac trtfng ca the.
1. Nguyen ly
A D N d trong nhan te bao cung vdi cac protein, A R N ... nen qua trinh tach va
thu hoach A D N dtfa tren cac btfdc: pha mang te bao va mang nhan, loai bo A R N
va protein, tap trung AD N.
123
2. Hoa chat va dung cu can thiet
2.1. Dung cu
- May ly tam thtfdng va may ly tam lanh co rotor dung ly tam ong Eppendorf.
- M ay 1Ic ong nghiem.
- M ay dien di.
- Tu elm, be am.
- Den U ltraviolet (U V )
- M ay chup anh (U V )
- Pipette man (Micro pipette), ong Eppendorf.
- Cac thiet bi lanh -30°C, tu lanh - 80 °C.
2.2. Hoa chat
- Dem PBS: pha s in de 4°C

- Dem T ris lM : pha s in de 4°C
- E D TA 0, 5M: pha s in de 4°C
- NaC l 5M: pha s in de 4°C
- SDS 10%
- Con tuyet doi va con 70°
- Dem T.E
- N atri acetat 3M
- AR Nase 10 mg/ml de - 30°C
- Proteinase 10 mg/ml de - 30°C
- Phenol bao hoa
- Chlorofoor
- Isoamyl alcohol
- Bromo phenol blue
- Dung dich Ethidium bromic 1%; 50/qo
- Dem TBE x 10
+ Tris base 108g
+ Acid boric 55g
+ ED TA pH = 8 0,5M
+ Ntfdc cat du 1 lit
Dung dich nay sau pha de san va khi dung pha loang 10 lln .
124
1. Lay 0,5 ml mau tinh mach chong dong b&ng heparin (trang heparin vao
bdm tiem trtfdc khi lay), cho vao ong ly tam Eppendorf vo trtfng.
2. Rtfa protein huyet ttfdng
- Tron PBS va mau ti le 1: 1 lac deu, li tam 15 phut, 6000 vong/phut d
nhiet do phong.
- Rut bo phan trong tren (de lai khoang 500 pi)
3. Tay pha mang
Bo sung Buffer cho du 1 ml (bo sung 500 pi)
- Tris 1M: lOpl
- EU TA 0,5M: 20pl
- NaCl 5M: 20pl
- SDS 10%: 200pl
- Ntfdc cat khii ion du: 500pl
(Cac thanh phan cd the cho rieng vao ong chtfa can te bao cung cd the pha
san roi tron vao).
4. Loai bo A R N
Cho 5 pi A R N ase lOmg/ml vao hon dich tren, lac deu, u am 37 °C trong 1 gid.
5. Loai protein:
- Sau 1 gid u vdi ARNase, lay ra.
- Bo sung 24pl proteinase lOmg/ml, lac deu.
- U 3 gid d 56 °C
(De thuan ldi nen cam ong nghiem Eppendorf vao tam bot xop mong tha
trong ntfdc am 56 °C).
- Sau u 3 gid. lay ra va de nguoi: goi la ong 1
* Cho phenol bao hoa vao ong 1 vdi ty le 1:1
+ Lac bang may.
+ Li tam 10000vong/ phut trong 15 phut d 24°C (N h iet do phong)
+ Hut lay phan tren cho sang ong Eppendorf 2 (Chu y hut ttf tren mat
xuong bo lai mang trang gitfa va phan den d dtfdi).
* Cho tiep phenol bao hoa vao ong 2 vdi ty le 1:1, lac tron. li tam. hut phan
tren cho vao ong Eppendorf 3.
* Cho vao ong 3 hon hdp phenol/chlorofoor 50/50 vdi ty le 1:1 (1 the tich
phenol, 1 chlorofoor. 2 hon dich).
125
+ Tron, lie , li tam 10 000 vong/ phut trong 15 phut d 24°C (C6 th§ dung
chlorofoor da cd san 1/25 isoamyl alcohol).
+ Hut phan tren cho vao ong 4.
• Cho vao ong 4 hon hdp nhtf ong 3, lie, li tam, hut cho sang ong Eppendorf 5
• Cho vao orig 5: 24 the tich chlorofoor + 1 the tich isoamyl alcohol + 25 the
tich chat tach, lie, tron, li tam vdi toe do 14 000 vong/phut trong 15 phut d 4°C.
Hut lay p h ln tren cho vao ong 6 (Hut se dtfdc khoang 700|il).
6. Tua A D N
- Cho them vao ong 6 da cd san 700jil dich tach:
+ N atri acetat 3M: 100fil
+ Con tuyet doi: 1 ml
- Tron deu b ln g each lie ong nghiem va xem tua (A D N tua thanh “sdi”
trang Id lting).
- Ly tam 15 phut, 14000 vong/phut d 4°C.
- Lay ong ra do bo phan tren (can than de khong do mat A D N - nen do vao
mot ong nghiem khac va cat di).
- Them vao ong 1,5ml con 70° va lie nhe. Nhtf vay da dtfdc A D N tinh sach,
A D N nay cd the gitf d - 20 °C, - 80 °C hoac tien hanh cac xet nghiem khac.
7. Dien di A D N
Sau khi chiet tach A D N can dien di de kiem tra co thu hoach dtfdc ADN
hay khong.
• Chuan bi A D N
- Ly tam ong 6 (d tren) 14 000 vong/ phut trong 15 phut d 4°C
- Do bo phan con 70°.
- Say kho A D N trong buong hut vo trung (H ote vo trung) thdi gian 1 gid.
- Sau 1 gid cho 50|il TE vao can kho va lac.
- Lay 7|al hon dich A D N trong TE tron vdi 3(il bromophenol blue lam chat
lieu chay dien di.
• Chuan bi gel (thach Agarose 1% trong T B E )
- Pha agarose 0,8% vao coc thuy tinh roi dun soi vdi bep thtfdng, hoac pha
1% roi dun b lo g lo vi song; de nguoi ttf nhien den khoang 50 - 60 °C.
- Dung ltfdc tao gieng cai vao gia gel (gia thach).
- Do ttf ttf thach agarose len gia da cai ltfdc (tfdc ltfdng de “gieng” co do sau
khoang 2 mm).
• Tien hanh dien di:
- Cho dung dich A D N da tron vdi thuoc nhuom mau bromophenol blue vao
moi gieng thach 10 fil .
126
- DSt thach (gel) v&o buong dien di.
- Do TBE ng&p gel, den vach quy dinh.
- Chay dien di vdi dong dien 50mA, 80V trong thdi gian 30 phut
• N huom A D N
- Sau dien di dat gel thach vao dia vuong
- Do dung dich Ethidium bromic 5 °/00 trong TBE vao dia
- Lie nhe khoang 5 phut, lay gel thach ra
- Soi: dat len gia soi tti ngoai va chup anh.
Cd the nhuom b ln g cach pha san thuoc nhuom 5°/00 v ô khi agarose dang d
60 °C, tron deu va do gel. Sau khi chay dien di co the soi ngay va chup anh.
4. Danh gia ket qua

A D N tren dien di se the hien vach phat sang va mau trang tren anh chup.
5. Luu y
Vi mau xet nghiem ft, ky thuat tien hanh qua nhieu budc nen yeu cau thao
tac het sUc can than, tranh nhiem trung va tranh hut hoac do mat AD N.
KY THUAT THAM ADN VA LAI V 6 l MAU “DO”
Ky thuat tham A D N va lai la ky thuat sti dung m iu do “probe” dtfdc danh

dau de phat hien vi tri doan A D N ttfdng tfng tren dien di sau khi A D N dtfdc cat
b ln g enzym han che, qua dd xac dinh dtfdc stf cd mat cac dot bien trong ADN.
Hien nay cac ky thuat cd the cd khac nhau d cac chi tiet song deu dtfa tren nguyen
tac do Edward M. Southern dtfa ra nam 1975.
1. Nguyen ly
- Dtfa vao dac diem cua cac enzym han che la cat A D N d cac vi tri ltfa chon
dac hieu, do dd mot enzym se cat A D N bo gen ngtfdi ra nhieu doan co do dai khac
nhau ttf vai doi base den doan cd hang tram kilo base (kb). Nhtfng doan nay sau
khi dien di tren gel agarose se d nhtfng vi tri nhat dinh theo do dai cua chung.
- Mot dac diem ntfa la A D N se bi bien tinh (hai sdi tach nhau ra) dtfdi tac dung
cua dung dich kiem (NaOH) sau do chuyen len mang ran vdi cac vi tri ttfdng tfng.
- Dtfa tren nguyen tac bo sung cua hai sdi A D N sau khi bien tinh va trung
hoa trd lai, ngtfdi ta dung cac doan A D N dd (probe) co trinh ttf A D N biet trtfdc de
cho ket hdp “bo sung” vdi A D N tren mang, qua do phan tich va tim ra dot bien.
Hinh 3.5 trinh bay mot dot bien do tai to hdp lam mat doan A D N gitfa doan gen A
va doan gen B:
127
- Binh thifdng enzym han che c it d vi tri X tao ra hai doan AD N:
+ Doan 3.7 kb cd chtfa phan gen A
+ Doan 9.0 kb co chtfa phan gen B
- Khi dot bien (do tai to hdp hay chuyen doan, mat doan) enzym nav cat va
tao nen doan 7 kb chtfa ca A va B (Hinh 3.5c).
- Sau khi dien di, cac doan 3,7 kb, 9kb,7kb d cac vi tri khac nhau. Dung
mau do (probe) A. hoac probe B se xac dinh dtfdc .
Gen A Gen B
Vdi mi u v a mi l .
IIIII IIIII --------- A D N do A d03
X X X
----------
3, 7 kb — -------------------------------9 kb -----------► 37 kb
a: So do doan AND chira gen A,

gen B va cac diem c it (X ) cua
enzym han che. 9 <ti
------ Vach
xoat phai
b: Sau dien di va lai
Gs n A GenB
Vdi mau Vdi mau
lllll urn ADN dd A ac B
X X
« ------------------------------ 7 k : ---------------------------------- ►
c: Doan AND bi dot bien mat doan 7 kb --------- 7k:

giCfa A va B. mat ca 1 vi tri c it
cua enzym han che
------ ------ Vac*

»jct :ftat
d: Sau dien di va lai

vdi m iu do A. m iu do B
Hinh 3.5: Dung mau do (probe) phat hien dot bien mat doan ADN
a, b : d ngudi binh thi/dng; c, d : d ngUdi mat doan gen
128
2. Hoa chat, dung cu
2.1. ADN: Do tach chi«*t A D N
2.2. Hod ch£t
- Enzym (men) han che: EC0 RI; Hind III; Pst I... Da so la cac enzym
hexacutter (nhan biet doan 6 base nitd), m6i enzym co mot dem pha thich hdp,
thtfdng hoat dong d 37°C.
- HCl 0,5M
- Agarose 1 % trong dem TBE
- NaOH 0,5 M; NaOH 0,25 M
- NaCl 1,5 M; NaCl 0,5 M
- Dung dich SSC x 20; Dung dich SSC x 2
- Dem TE (Tris 10 mM, ED TA 1 mM pH 7,6)
- Mang lai (mang nitrocellulose hay mang nilon)
- Dich lai va cac sinh pham kem theo
- Mau do
- Dich danh dau
- Glutaraldehyd
- Cac chat hien phim
- Ntfdc cat
2.3. Dung cu
- Hop lai thtfdng la loai ECL
- M ang lai
- Giay s ic ky, giay whatman
- Giay tham
- Tuam
- Tu lam da
- Be am
- Dung cu dien di
3. Cac birdc tien hanh

3.1. Cat ADN bang enzym gidi han
- Trong ong nghiem Eppendorf cho:
+ 10 pg A D N trong dem TE
+ 1 0 -2 0 ddn vi enzym han che (co the cho 50 ddn vi) trong dem ttfdng tfng
+ Ntf6c cat du 50 pi
129
- U qua dem d 37°C
- Lay ra mot p h ln cho chay dien di tren gel agarose 1 %
- Nhuom va chup anh kiem tra xem A D N da dtfdc c it phu hdp chtfa
3.2. Tham (Southern Blot)

3.2.1. Dien di
- Do gel agarose 1 % tao gieng nhtf da noi d tren

- Chay dien di A D N da cat vdi dong dien IV / 1cm chieu dai gel, thdi gian
10 - 12 gid (qua dem).
3.2.2. XCfly gel (lam bien tinh gel)
- Trong hop thuy tinh cho 300 ml HC1 0,5 M

- Dat gel vao va lie khoang 15 phut (chu y quan sat khi mau cua dung dich
trong hop chuyen sang mau nau la dtfdc).
Muc dich cua btfdc nay la lam mat purin (depurination) de cat dtft nhtfng
doan A D N qua dai thanh nhieu manh (nhtfng v ln n lm yen mot cho) tao dieu kien
de tham.
3.2.3. Lam bien tinh ADN (Denaturation)
- Cho gel agarose vao hop dtfng dung dich I (N aC l 1,5 M; N aO H 0,5 M)
- L ie nhe trong 25 phut, (chu y quan sat khi dich chuyen ttf mau nau
thanh mau xanh la dtfdc).
Muc dich btfdc nay la lam hai sdi trong chuoi xoan kep A D N tach ra
3.2.4. Trung hoa (Neutrolisation)
Nhac gel ra va cho vao hop dtfng dung dich II (N aC l 1,5 M ;NaOH 0,2 M),
lac nhe trong 15 phut.
3.2.5. Tham ADN len mang (mang nitrocellulose hay mang nilon)
- C it mang, giay whatman, giay loc dung b ln g kich thtfdc gel
- Dung be tham, cd the thay b ln g mot khay nhtfa hay khay thuy tinh, dat
tam ngang tren thanh khay.
- Cho dung dich SSC x 20 vao trong be (khay)
- Dat thtf ttf tren tam chan ngang ttf dtfdi len:
1. Cau giay tham
2. Ba ldp giay whatman
3. Gel da dien di (de up)
130
4. Mang
5. Ba ldp gi§fy whatman
6. Nhieu ldp giay tha'm
7. Vat nang de len
- De tu tha'm qua dem (khoang 10 gid) d nhiet do phong
3.2.6. Co djnh ADN len mang

- Sau khi tham, lay mang ra, rOfa nhe bang dung dich SSC x 2
- Tham kho dich tren mang
- Dat mang dudi den U V thdi gian 5 phut (hoac de mang gitfa hai ldp giay
tham 3 - 4 gid) d nhiet do phong.
3.3. Lai ADN

3.3.1. Lam tien lai
- Pha dich lai: chuan bi 0,25 ml dich lai/ 1 cm2 mang gom dich lai, NaC l du
0,5 M, va 5% sinh pham di kem.
- Cho dich lai vao hop kin
- Dat mang vao ttf ttf, lac nhe
- Day km hop cho vao be a'm 42°C khoang 1 gid
3.3.2. Danh dau m iu dd (probe)

- Cho 100 ng m iu do A D N vao ong Eppendorf
- Dun trong ntfdc soi 5 phut (can than tranh bat nut)
- Lay ra cho ngay vao lanh (da vun)
- Cho dich danh dau (labelling reagent) vdi the tich b ln g the tich m iu do,
tron deu.
- Them glutaraldehyd vdi the tich b ln g the tich dich danh da'u
- De d 37°C ttf 13 - 15 phut
3.3.3. Tien hanh lai
- Dtfa mau do da danh dau ttf ttf vao hop da cd san dich tien lai va mang,
tranh do th in g len mang.
- Tron deu, de qua dem d 42°C
3.4. Phat hien
3.4.1. RCfa mang lai: sau 12 den 24 gid u d 42°C
- Lay ra ngam mang lai hai lan x 10 phut trong dung dich I
(SDS 0.4 % va SSC x 5) d 55°C
131
- Rtia tiep mang lai hai lan x 5 phut trong dung dich II (SSC x 2) d nhiet do
phong (moi lan chuyen dung dich dung giay tham kho mang).
3.4.2. Hien mang

- Pha tron thuoc hien 1 va 2 (ban san) vdi ti le 1: 1 ltfdng thuoc pha du
0.125 ml / cm2 mang.
- Trang len mang
- Tham kho va up phim X quang len mang, u 1 • 2 gid
- Rtia va hien phim
4. Danh gia ket qua

Dtfa vao thang chuan do dai AD N , so sanh vi tri co probe de biet co dot bien
hay khong.
5. Luu y
M iu benh pham It, ky thuat tien hanh qua nhieu btfdc nen yeu cau het stfc
c ln than trong cac thao tac.
6. Cac phtfdng phap khac

Nguyen t ic cua “ lai” la dung cac m iu do dac hieu de tim doan A D N co
trinh ttf nucleotid ttfdng dong nen cd the dung trtfc tiep len N S T hay stf dung
phtfdng phap lai Dot Blot la cho A D N len mang, co dinh khong qua dien di sau
do dung mau do gan, rtfa va phat hien ndi cd mau do. N goai ra cd the stf dung ky
thuat lai tai cho (F IS H - Fluorescent Insitu H ybridization) de phat hien cac bat
thtfdng gen.
KY THUAT PCR (POLYMERASE CHAIN REACTION)
Ky thuat PCR dtfdc Kary M ullis phat minh nam 1980 nhd cac thanh ttfu:
1. Phat hien men tong hdp A D N (A D N polymerase) cd dac diem: tong hdp
tiep tuc doan A D N dang “dang dd” tren khuon cd sin.
2. Biet dtfdc trinh ttf cac nucleotid d nhieu doan gen ngtfdi. Tong hdp dtfdc
mot doan A D N bang phtfdng phap hoa hoc (oligo polynucleotid) lam moi (primer).
3. Ung dung tinh chat bo sung cua sdi kep A D N va cd the cat cac lien ket
hydro gitfa hai sdi b ln g nhiet do cao.
1. Nguyen ly
Ngtfdi ta dung nhiet lam bien ti'nh hai sdi cua chuoi xoan kep A D N cho
chung tach ra, tao nen hai sdi ttf do cd kha nang gan vdi doan A D N co trinh ttf
base nitd bo sung, sau do lay doan moi (prim er) la doan A D N ngan cho gan vao
132
A D N phan lap diidc d vung co trinh tii bazd nitd ttfdng dong, roi dieu chinh nhiet
do thich hdp cho hoat dong cua enzym (vdi stf cd mat cua cac nucleotid hoat hoa,
doan moi da g in len A D N khuon thi d nhiet do thich hdp A D N polymerase se to’ng
hdp tiep A D N ). B ln g nhieu lan nhtf vay, ngtfdi ta da to’ng hdp dtfdc khoi ltfdng ldn
AD N tren khuon co san.
De nhan len mot doan gen, ngtfdi ta dung hai moi (cap moi) cd trinh ttf
nucleotid ttfdng tfng vdi hai d lu cua doan gen nay.
Vi qua trinh tong hdp A D N luon luon dtfdc tien hanh theo chieu ttf d lu 5’
den dau 3’, va vdi 2 moi cd trinh ttf nucleotid bo’ trd cho 2 sdi A D N d 2 dau (phia 3’
cua moi sdi), thi d chu ky mot se tao dtfdc 2 doan A D N mdi dai hdn doan can tim
nhtfng moi doan cd mot dau (dau 5’) bi gidi han bdi moi. Den chu ky 2 se tao dtfdc
2 doan A D N co gidi han hai dau d vi tri ma moi “gan” (Hinh 3.6).
A B
i 1
soi 1 5' i mm mm 1 3' \
| Chu&i xoin ADN
Stf 2 3' I I V /m ........................................................... - -------- 1 5' J
mm nnnnnii **** (pnmer)
5- C
Stf 1b <£ S
Chu ky 1
I 5'
Soi 2b
Soi 1b WM 5'
Soi 1c 5 'd e e m : 3"
DCmg
Chu ky 2
Soi 2b
Soi 2c - z m s'
DCmg
DOrng
Soi 1c 5’ ME
S tf 2c
3'
3 -m s: ! 5’
I Chu ky 3
s’lrnmr >3'
t
Dflmg
Hinh 3.6: Sd do phan ting PCR tong hop doan ADN ttf A -> B
133
N g u y e n ly h o a t d o n g c u a m a y P C R :
Thtfc cM't cua PCR la phan ting enzym trong ong nghiem difdi tac dung cua
nhiet. M ay PC R la may dieu khien nhiet do theo chtfdng trinh de dam bao ba che
do nhiet:
- Nhiet do 94 - 95°C de tach sdi A D N kep ra hai sdi A D N ddn
- N hiet do 30- 65°C (tuy theo yeu c lu cua doan moi va tinh chat cua doan
gen) co tac dung “g in ” moi vao vi tri co trinh ttf base nitd tUdng dong cua doan
gen (AD N).
- Nhiet do 70-73°C dam bao hoat dong tot cho enzym tong hdp A D N tiep ttf
doan moi.
Do dac diem cua men taq A D N polymerase co the chiu nhiet va m&'t tac
dung sau mot thdi gian nen ngtfdi ta thiet ke cac may co thdi gian thay doi nhiet
do gitfa cac che do cang ngan cang tot
2. Hoa chat, dung cu

1. A D N khuon: ttf tach chiet A D N
2. A D N moi (prim er) va cac dung dich di kem
3. D N T P (desoxy ribonucleotid hoat hoa)
4. Enzym (taq A D N polymerase)
5. M ay PCR, may va dung cu hoa chat dien di
M ay va cac dung cu chup anh UV, micropipette

1. Trong ong Eppendorf
- Pha hon hdp dung moi va moi
- Tron moi, men, nucleotid
- A D N khuon
- Cho d lu paraphin neu can (tuy ttfng loai may, neu co bo phan chong mat
hdi ntfdc co the khong can dau).
2. Chuan bi chtfdng trinh nhiet cho may
3. De may chay theo chtfdng trinh da dat, thdi gian khoang 30 phut
Thong thtfdng mot chu ky khoang 1 phut, sau khoang 30 phut thi men hoat
dong kem va ltfdng A D N cung da nhieu. Ttf 1 A D N ban dau sau khi chay se co 2"
sdi, n la so' chu ky.
4. Sau khi may ngtfng chay, de ve nhiet do 10°C
- Lay ra hut bd dau tren (neu cd)
134
- Trong ong Eppendorf cho:
+ 8 fxl dich A D N vtfa tong hdp
+ 2 ^1 thuoc nhuom
Tron deu va cho chay dien di tren gel vdi dong dien 80 V, 45 mA, thdi gian
khoang 45 phut. Nhuom, soi va chup U V se th&y r5 1 vach (neu dung mot cap moi).

PCR la phan tfng rat nhay, de dtfdng tinh gia neu de lin A D N ngoai lai, nen
moi thao tac d cac btfdc phai vo trung va ngtfdi ta thtfdng chay them mot chtfng
am (khong cho A D N khuon).
5. Cac tfng dung cua PCR

- Dung de nhan len A D N sau do lai, phat hien b ln g m iu do dac hieu
- Dung cap moi dac hieu, cd the 2 hoac 3 moi de phat hien cac dot bien hay
tmh dac trtfng A D N cho loai, ca the...
135
Chifdng 4
SINH HOA HUYET HOC ■
SAT HUYET THANH

(Phuong phap spectrophotometric ferrozine)
1. Nguyen ly
Transferrin g in vao s it ferric (s it III). Trong huyet thanh, dtfdc giai phong
va bien doi thanh ferrous (sat II) bdi hydroxylamin. Sat II (ferrous) phan tfng vdi
ferozin thanh phtfc hdp mau dtfdc dinh ltfdng bang quang ke.

2.1. Hoa chat
- Bon thuoc thii A: 48 ml
+ Guanidinum chlorid : 1,0 mol/L
+ Hydroxylamin : 0,6 mol/L, pH = 4,0
- Bon thuoc thii B: 2ml: Ferrozin 40 mmol/L
- Sat chuan: 5ml: Sat 200 |ig/L
(Ion standard)
Chuan bi thuoc th ti xet nghiem :
(Cho hai ong nghiem/cho 1 mau thtf khi tien hanh)

Chuyen cac loai thuoc thtf trong thuoc B vao 1 lo thuoc thii A (chu y 1). L ie
deu va on dinh dtfdc 6 thang d 2 - 8°C.
Chuan bi dung dich xet nghiem nhtf sau: Thuoc thtf B: lm l
Thuoc thOf A: 24 ml
2.2. Dung cu
Pipette, ong nghiem, quang ke co btfdc song 560 nm (550 - 570nm).
2.3. Benh pham
H u yet thanh hoac huyet ttfdng chong dong bang heparin, on dinh 7 ngay
d 2 - 8°C.
136
3. Tien hanh
3.1. Mot ong thu (one tube/sample)
a. Dung pipette cho vao ong nghiem theo thtf ttf sau:
Ong trang Ong chuan (ST) Thur (S)
1 Ntfdc cat 200 |il 0 0

2 Sat chuan 0 200 til 0
3 M iu thLf 0 0 200 ill
4 Thuoc thif A 1,0ml 1,0 ml 1,0 ml
b. L ie tron deu, roi doc mat do quang ( A l) cua ong chuan (S T) va ong thtf (S)
d btfdc song 560nm ddi chieu vdi dng trang.
c. Dung pipette cho vao cac ong nghiem thuoc thii B
5 Thudc thir B 40nl 40jal 40|jl
d. Lac tron, de yen d nhiet do trong phong trong 5 phut, doc mat do quang
(A2) cua ong chuan va dng thuf vdi btfdc song 560nm doi chieu vdi dng trang
3.2. Hai ong thufTwo tubes/sample)

a. Dung pipette cho vao ong nghiem theo thil ttf sau:
Ong Thuoc
Ong trang chuan thtf trang Thtf (S)
(ST) (SB)
1 Ntfdc cat 200 |il 0 0 0

2 Sat chuan 0 200 |il 0 0
3 Mau thif 0 0 200 [i\ 200 nl
4 Thudc thir A 0 0 1,0ml 0
5 Thudc thif dung hang ngay (Working 1,0ml 1,0ml 0 1,0ml
Reagent)
b. Lac deu, de yen 5 phut tai phong 20°C
c. Doc mat do quang (A) cua ong thti trang d 560 nm doi chieu vdi ong ntfdc cat.
d. Doc mat quang cua ong chuan va thii tai 560 nm doi chieu vdi dng trln g.
4. Cach tinh ket qua

4.1. Mot ong th ir Tinh theo cong thtfc sau:
A 2-A! (m iu thtt) S
I ;---------- x 200 (ig/dl s it
A jj-A, (mau chuan) ST
137
4.2. Hai dng thu
As'AgB
------------ x 200 îg/dl sat
A St
Ddn vi quoc te (SI unit) = ng/dL s it x 0,179 = nmol/LsIt

Gia tri binh thtfdng
Nam : 70 - 155 |J.g/dL = 12,5 - 27,7 nmol/1
Ntf : 55 - 140 îg/dL = 9,84 - 25,1 ^xmol/1
Moi phong xet nghiem can co gia tri binh trung binh rieng cua minh.

- Khong dung huyet thanh vd hong c lu se gay sai ket qua
- Ong nghiem thtfdng dung nen nia b ln g acid hay dung ong nghiem nhtfa.
FERRITIN HUYET THANH

(Phucmg phap do 6$ due latex)
1. Nguyen ly
Ferritin huyet thanh bi ngtfng ket bdi hat latex dtfdc g in vao khang huyet
thanh ngtfdi co khang the ferritin. Stf ngtfng ket cua hat latex ty le thu&n vdi
nong do cua ferritin, va co the do dtfdc b ln g do due.

2.1. Cach thOe ddng gdi va ma so
Latex: COD 31926 : 1 x 5 ml
COD 31026 : 2 x 5 ml
Diluent: COD 31926 : 1 x 45 ml
(Dung dich pha loang) COD 31026 : 2 x 45ml
(Standard) Chuan: COD 31926 : 1 x 3 ml
COD 31026 : 4 x 3 ml
- Dung dich treo (Surpension) cua hat latex dtfdc g in vao huyet thanh
ngtfdi khang the ferritin. N atri azit + bao quan nong do 0,95 g/1.
- Dung dich pha loang (Diluent). Dung dich dem chlorid ammonium 0,2 mol/1,
co natriazit 0,95 g/1 pH = 8,2.
- Ferritin chuan: Hoa tan vdi 3ml ntfdc cat, on dinh 1 thang d 2 - 8°C
138
2.2. Dyng cu
- Noi cach thuy 37°C

- Quang ke hay quang ke co cach thuy 37°C co btfclc song 540 nm (530 - 550nm)
2.3. B?nh phim

Huyet thanh on dinh 7 ngay cf 2 - 8°C, huyet thanh co hong c lu bi vd va co
nhieu md khong thich hdp cho xet nghiem, gay sai so.

1. Thuoc thtf va quang ke de 37°C
2. Dieu chinh quang ke ve vi tri “0” v6i ntfdc ceTt.
3. Trong cong (Cuvetle) cho:
SoTT Thuoc thur Cong (Cuvetle)

1 Dung djch hoa loang 0,90ml
2 Chuan hay mau xet nghiem 0 ,1 0 ml
3 Latex 0 ,1 0 ml
4. Tron deu, lie ngang cong roi cho vao may d 37°C
5. Doc mat do quay d 540 nm (sau 10 giay goi la A j) va sau 8 phut sau khi
cho them m lu thtf vao (A 2)
Cach tmh ket qua
(Az-Aj) (m iu thtf)
-------------------- ------- x nong do chuan = u.g/1 ferritin
(A 2-A j) chuan
4. Ket qua: binh thtfdng

Nam : 30 - 220 ng/L
Ntf :2 0 -1 1 0 *ig / L
Theo tai lieu cua Biosystems Clinical Chemistry
C O STA - B R A V A 30 - 4
08030 Barcelona - spain
ISO - 9001

- Hong cau vd, huyet thanh khong dam bao, gay sai so
- Trtfdc khi doc phai de cach thuy 37°C
139
sure BEN HtfNG c a u
1. Nguyen ly
Hong clu dtfdc cho vao nhiing dung dich dem muoi nhtfdc trtfdng c6 pH = 7,4
de d nhiet phong thi nghiem 22°C. Sau mot thdi gian nh£ft dinh de quan sat xem
mtfc do tan cua hong clu.

Dung dich me co ap ltfc tham thau ttfdng dtfdng vdi dung dich natri chlorua
10% (bao quan dtfdc hang thang) bao gom cac muoi sau day:
- N atri chlorua : 180 g
- N atri dihydrophosphat (N a H 2P 0 42H20 ) : 4,86 g
- Di natri hydro phosphat (N a 2H PO J : 27,31 g
- Ntfdc cat vtfa du : 2000 ml
Dung dich nay khong trong thi phai loc. Ttf dung dich me 10% pha th&nh
cac dung dich 1%, 2%, 2,5%, 2,75%...6%, 7%, 8%. Ttf dung dich co nong do 2,75%
den 6% ctf moi nong do cach nhau 0,25%.
Ttf cac dung dich ph ln tram nhtf tren pha thanh cac dung dich p h ln nghin
ttfdng tfng 196o, 2%o, 2,5%o, 2,75%o, 8%o (Ttf nong do 2,75%o den 6%o ctf moi nong do
cach nhau 0,25%o).

3.1. Cach lay benh pham
Lay 3ml hay 4 ml mau tinh mach cd chong dong b ln g heparin hay natri
oxalat hoac natri citrat.
3.2. Tien hanh

Chuan bi 18 dng nghiem deu mau loai ong nghiem ly tam dtfdng kinh 1cm x
6cm. Danh so ttf 1 den 18. Cho vao mdi ong nghiem 5 ml cac dung dich dem da
pha loang ra phan nghin nhtf tren theo nong do giam d in (ong so" 1 nong do 8°/oo---
ong so" 18 nong do l°/oo).
- Cho vao mdi dng nghiem 2 - 3 giot hong cau (sau khi ong mau da gan bo
bdt phan huyet ttfdng).
- Lac nhe 2 - 3 lan cho deu hong cau trong cac ong nghiem.
- De d nhiet do phong xet nghiem 22°C ttf 30 - 60 phut thi doc ket qua (cd
the quay ly tam roi doc ket qua va tinh ty le phan tram huyet tan.
140
3.3. C4ch dqc k it qua
- B it d lu tan: P h ln tren co m&u hong
- Tan ho^n toan: Toan bo dung dich co mau do trong suot.
4. Ket qua
4.1. Binh thudng
- Bat d lu ttf 4,5%o —> 5%o
- Tan hoan toan ttf 3%o -> 3,5%o
4.2. Benh ly
- Stfc ben hong cau giam: (D i tan d nong do cao) trong thieu mau huyet
tan, trong thieu mau Minkowski Chauffard.
- Stfc ben hong cau tang: (Tan d nong do thap) trong nhtfng benh ve huyet
sic to noi chung, ngoai ra con tang sau c it lach, thieu mau va mot so'benh ve gan.
5. Cac yeu to anh hudng

- Dung dich nhtfdc trtfdng phai chinh xac nong do, sau khi pha phai kiem
tra bang cach tien hanh xet nghiem vdi 1 - 2 hong cau cua ngtfdi binh thtfdng xem
ket qua cd nam trong gidi han binh thtfdng khong roi mdi sii dung cho benh nhan.
Neu khdng chinh xac phai pha lai.

- Khdng lam xet nghiem nay khi benh nhan truyen mau chtfa dtfdc hai tuan.
DIEN DI HUYET SAC TO
1. Nguyen ly
Phtfdng phap dien di la phtfdng phap ly hoa dung ddng dien mot chieu lam
di chuyen cac phan ttf mang dien. Muc dich cua phtfdng phap nay dtfa tren stf tich
dien cua cac phan ttf, va toe do di chuyen khac nhau de tach cac thanh p h ln ma
ta mong muon. Ttf dd cd the dinh tinh va dinh ltfdng.

2.1. Bang acetat - cellulose co kich thudc: 4 x 17 cm ; 2,5 cm x 17 cm
2.2. Chuan bi dung djch huyet sac to
hay mau tinh mach cd chong ddng b ln g natrioxalat, natricitrat. Tot nha't
dung heparin (1 giot loai 25.000 ddn vi cho 5 ml mau).
- Ly tam 2500 vong/phut/ 5 phut.
- Loai bo huyet ttfdng - ROta 4 lln b ln g ntfdc moi sinh ly 9%o.
- Lam vd hong cau b ln g ntfdc ctft v& chlorofoor
+ 1 the tich hong c lu
+ 1 the tich ntfdc cat
+ 1 the tich chlorofoor
- Lac manh trong 5 phut. De tu lanh + 4°C trong mot dem. Sau dd ly tam
5.000 vong/phut trong 20 phut. Hut phan tren cung b ln g pipette Pasteur sang
mot ong nghiem khac vd trung, ly tam 5000 vong/ phut/ 20 phut. Sau dd loc lai de
cd dung dich huyet s ic to that trong.
- Ty le huyet sac to cua dung dich tren dtfdc xac dinh bang phtfdng phap so
mau thtfdng dung dich nay chtfa khoang 10% huyet s ic to.
2.3. Dung dich dem

2.3.1. Dem Veronal pH = 8,6
- Veronal sodique (natri veronal) : 10,3 g
- Veronal acid : 1,34 g
Dieu chinh de pH = 8,6
2.3.2. Dung djch dem phosphat p H = 6,5

- K 2H P 0 4 : 3,05 g
- K H 2P 0 4 : 2,40 g
Dieu chinh de pH = 6,5
2.4. Dung dich nhuom mau

- Den amino (amino noir) : 0,5 g
- Methanol : 50 ml
- Acid acetic : 10 ml
- Ntfdc cat : 40 ml
2.5. Dung djch tay mau

- Methanol 500 ml
- Acid acetic 50 ml
- Ntfdc cat 450 ml
2.6. Dung djch lam trong bang cellulogen

- Acid acetic glacial 7 ml
- Methanol 35 ml
- Ntfdc cat 50 ml
142
- Nhung nhiing bang cellulogen vao dung dich dem 15 phut.
- Tha'm nhiing bang cellulogen vao giiia hai td giay loc de loai bo dung dich thifa.
- Dat nhiing bang nay tren gia dien di va nho vao phia am ctfc tren bang
mot giot huyet s ic to' ttfdng dtfdng 5 jal.
- Lam clu no'i cho cac bang chay. Cho chay vdi ctfdng do 4mA cho mdi bang
va thdi gian chay ttf 1 gid 30 phut -> 2 gid (mdi may phai ttf dieu chinh thdi gian
chay cho thich hdp).
- TSt may, lay cac bang dien di ra nhung trtfc tiep nhtfng bang nay vao
dung dich nhuom mau trong 5 phut.
- ROia bang dung dich tay mau den khi trang hoan toan.
- Sau khi rtfa, se lam trong cac bang bang dung dich lam trong trong 10 phut.
4. Ket qua
Dtfa vao hinh anh cua bang mau ctfc dtfdng
HUYET SAC TO KHANG KIEM
1. Nguyen ly
Dtfdi tac dung cua chat kiem, cac huyet sac to' bi pha huy dtfdi 1 phut. Rieng
huyet sac to' F khdng bi pha huy.
143
2.1. Hod chat
- N atri hydroxyd N/12 + NaO H IN : 5ml
+ Ntfdc c^t hai lln : 55 ml
- Dung dich ngtfng ket
+ Amoni sulfat [(N H 4) 2S 0 4] : 76 g
+ Acid chlorhydric N : 0,5ml
+ Ntfdc cat : 200 ml
- Dung dich huyet sac to' (Xem phan ky thu&t dien di huyet sac to)
2.2. Dung cu: Pipette, quang ke cd btfdc song 500 - 540 nm

3.1. Dung djch A
Mdi benh pham chuln bi hai ong nghiem sach
- Mot ong: 1,6ml dd NaO H N/12
- Mot ong: 3,4 ml (N H 4) 2S 0 4.
Dung micropipette hut 0,1ml dung dich huyet sac to' cho vao dng dtfng
NaOH N/12. Bam dong ho ngay. Chd dung 1 phut thi do’ ong nghiem chtfa 3,4 ml
dung dich amoni sulfat sang, lac deu roi loc qua giay loc. Ntfdc loc la dung dich A.
3.2. Dung djch B

Mdi benh pham mot ong nghiem de ten va danh dau chfl B. Cho vao ong 4ml
ntfdc cat va 0,02 ml dung dich huyet sic to', lie deu. Dd la dung dich B. Moi lan lam
xet nghiem nay phai lam song song vdi mot huyet sac to' cua ngtfdi ldn binh thtfdng.
Cung cd the lam chtfng dtfdng tinh b ln g huyet sic to' cua mau rau thai nhi.
Dung dich A va B dtfdc doc d btfdc song 540 nm.
4. Ket qua
Doc va tinh ket qua theo cong thtfc:
M D Q H A x 25
F = ----------------------- = % Hb
MDQHB
MDQH: Mat do quang hoc
Phan tram huyet s ic to' F d day la so sanh vdi huyet s ic to' toan phln.
Tri so' binh thtfdng = Ngtfdi ldn : 0,2 - 2%
Sd sinh : 60 - 80%
3 thang : 20%
7 thang 1 - 5%
144
- Dung dich NaOH N/12 pha sai, dung dich A khong trong, due, mat do
quang hoc cao gay ra so dung dich NaOH N/12 lam dung dich huyet sac to' ttf do
sang nau, pha lau phai kiem tra lai.
- Tat ca cac dung dich phai dtfdc pha mot cach chinh xac, hemolysat phai
lam chinh xac.
DjNH LI/ONG METHEMOGLOBIN REDUCTASE
1. Nguyen ly
Methemoglobin (M et-Hb) dtfdc tao ra dtfdi tac dung cua K 3F e(C N )6, khi co
mat cua N A D H 2 nhd tac dung cua Met-Hb-Reductase co trong hemolysat cua mau
xet nghiem se bi khii thanh Hb khii. Hb khii nay se ket hdp vdi 0 2 hoa tan trong
ntfdc tao H b 0 2 va tuy theo stf tang mat do quang hoc d btfdc song 575 nm va tinh
ra hoat tinh Met-Hb-Reductase.
Glucose
NADP O
li
G6PD Glucose 6 (p ) C ---------
I
HC - O H
I
6 phosphogluconolacten HO-CH O
I
Met Hb redutase HC-OH
NADP
NADPH,
I
HC
l
G-SH GS-SG Met-Hb Hb CH-OPO- —
Coleglobin
HbHO, HbO, + HjO (khong chuyen
lai thanh Hb di/dc
va ket tinh thanh
Tac nhan oxy hoa the Heinz
Thudc th ir
Feri
1. 2Fe3Hb + NaDH -► 2Fe2+ + NAD* + H"
Reductase
2. Dung djch EDTA. 0.01M
So do 4.2: Chu trinh tao m ethem oglobin (M eth-Hb)
145
2. D u n g c u , ho a c h a t
2.1. Dung dich EDTA 0,01 M
ED TA (C 10H 14O N 22H20 ) : 0,186 g

Nude cat : 50ml
2.2. Dem citrat 0,05M pH - 4,7
Acid citric (C 6H 80 7H 20 ) : 1,05 g

Nude cat vtfa du : 100 ml
Dung NaO H I N dieu chinh pH ve 4,7 roi them nUdc c£t vUa du 100ml.
2.3. Dung djch kaliferricyanua K3Fe(CN)s : 0,5nm
Kaliferricyanua : 16,5mg
Nude cat : 100 ml
2.4. NADH2kho
Thudng dung loai muoi co 2Na

C21H 27N 70 14P 2N a 2 (M W 709,4) : 9 mg
Nude cat : 1 ml
2.5. DEAE cellulose dang bot, sol (0,1 - 0,25mm)
2.6. Dung djch huyet sac to (M W 64450) da loai bo Met-Hb-Reductase, nong do

1,16 g%
Cach lam :
- M a u chong dong, h e p a r in hoac citrat, ly tam 2000 vong/phut/10 p h u t loai
bo huyet tUOng.
- Rtfa hong cau hai lan b ln g nUdc muoi 9°/00 ty le 1/10
- Ly tam lay khoi hong cau 3000 vong/phut/10 phut.

- CU 10ml khoi h o n g c i u cho them 60 ml nUdc cat h a i la n v a 1.6 g DEAE
cellulose kho.
- Khuay deu de 10 phut, thinh thoang lac.

- Ly tam va lay dich trong phia tren roi lai cho mot lUOng D EAE vao lie
deu them 10 phut.
- Pha loang dung dich nay bang nUdc cat den nong do Hb cuoi cung la 1.2 g%.
Day la dung dich Hb khong co Met-Hb-Reductase.
Kiem tra tren may quang ke.
- Chia thanh lo nho de bao quan (2 thang). Cac thuoc thu de dinh lu<ftig
huyet sac to benh nhan nen dung phUOng phap cyan met hemoglobin.
146
3. Q u y trin h k y th u a t
Trade het chuln bi hon dich dem co chtfa Met-Hb. Cho vao ong nghiem 10 ml.
0,5 ml dung dich ED TA (1)
1 ml dem citrat (2)
1,8 ml dung dich K 3F e(C N )6 (3)
1 ml dung dich HB (6)
- Lay mau benh nhan: lay 6 dau ngon tay benh nhan hoac l£y co chong dong.
Hoac khoi hong cau neu la mau toan phan thi lay 0,04ml thoi ngay vao hon dich tren,
trang pipette nhieu lan, dong thdi lay mau de dinh ltfdng huyet sac to ngay.
- Sau khi cho mau benh nhan vao roi de them ntfdc den 9,9 ml de mot the
tich con lai cho them N A D H 2.
- Tron deu roi de > 1 gid d nhiet do phong, neu 37°C thi 20 phut.
- Ly tam loai bo can.
- Lay nUdc trong phia tren de dinh ltfdng men.
- Dinh ltfdng men. Cho vao 2 cong (1 cm)
Chtfng ( 1 ) Thur (2)

Ni/dc trong 2,97ml 2,97ml
Nifdc cat 0,03 ml 0
nadh2 0 0,03 ml
Tron deu
Doc lai stf tang mat do quang hoc d budc song 575 nm trong 10 phut (ghi
ttfng phut).
- Dinh lifdng huyet sac to" trong mau mau xet nghiem.
4. K et qua
Hoat tinh M et-Hb-Reductase bieu thi bang fimol Hb bi khii sau 1 phut cho
1 gram Hb xet nghiem. Trtfdc het tinh A E/1 phut. Thtfdng lay phut thtf 2, 3 va 4
roi chia 3.
A E 575nm/l’
Hoat tinh men = --------------------- x 595
Hbg%
Ket qua: Vdi ky thuat nay hoat tinh men d ngtfdi cho mau la 3,2 ddn vi (jimol
H b 0 2/l’gHb (dao dong ttf 2,2 - 3,8 ddn vi).
0 ngtfdi thieu men hoat tinh men se giam hay khong con.
5. C a c y e u to a n h h ird n g
- Cac dung dich phai pha chinh xac.
147
- Dung dich huyet sic to phai chung va chuln chinh xac
- Quang ke phai chuan de cho mat do quang hoc chinh xac
Cac dieu kien tren khong dam bao se dan den nguyen nhan sai lam.
XET NGHIEM SIT THIEU HUT MEN G6PD HONG CAU

BANG PHUONG PHAP SOI DEN TUT NGOAI
Trang 161 - Pratical Hematology Sixth Eidtion. Sir John V. Dace - S.M Lewis
Phtfdng phap cai tien cua Beutler va Mitchell.
1. N g u y e n ly
Dtfa vao tinh chat phat quang dac biet cua NADP-H d vung anh sang ttf ngoai.
2. D u n g cu , ho a c h a t
2.1. Hoa chat

- D-glucose 6 phosphat: 10 mmol/1. Hoa tan 305 mg muoi dinatri hoac
dikali vdi so" ltfdng ttfdng ttf trong 100 ml ntfdc cat.
- NADP* (C2iH27N70 17P3Na) = 7,5 mmol/1. Hoa tan 60mg N A D P dinatriphosphat
10ml ntfdc cat.
- Saponin 750mmol/l (10 g/1)
- Dung dich dem TRIS-HC1: pH = 7,8
Tris (hydroxymethyl) aminomethan (24,23 g/1): 250 ml
Oxyd glutathion (glutathione oxy hoa) (GSSG), 8 mmol/1. Hoa tan 49 mg
GSSG trong 10 ml ntfdc cat tron thuoc thut theo cach sau:
Hai the tich G6PD, 1 the tich N A D P +, 2 the tich saponin, 3 the tich dung
dich dem 1 the tich dung dich GSSG, 2 the tich ntfdc. Dung dich nay on dinh 2
nam hay hdn d nhiet do -20°C, 2 thang neu d 4°C, azid co the cho them vao de
phong nam phat trien hoac cd the chia ra ong nghiem nhd, moi ong 100 pi, gitf am
20°C moi khi dung mot ong nghiem.
2.2. Dung cu
- Khay nhtfa co lo
- Giay tham whatman N:1
- Dong ho bam giay
- Den tii ngoai.
3. Q u y trin h ky th u a t
Lay 10 fo.1 mau toan phan chong dong bang heparin hay EDTA. cho 100 |il
dung dich tron de nhiet do 15 - 25°C. Lav 10 fil mau da phan tfng vdi thuoc thtf
148
tren vao giay tham whatman so' 1 tai thdi diem b it dau phan ting va sau do 0 - 1
phut, 4 - 6 - 8 - 1 0 phut neu muon nhanh hdn co the de giay d nhiet do 25 - 30°C.
Cho vao lo md khay nhtfa: 10 pi mau toan phan chong dong b ln g heparin
hay EITTA. Cho 100 pi dung dich cd chat, bam dong ho vao tinh la phut 0.
Dung que thuy tinh tron deu va cham ra giay tham whatman tai cac thdi
diem: O’ - 1’ - 4’ - 6’ - 8’ - 10’ ’
De kho d nhiet do phong (khoang 15 phut) dem soi dtfdi den ttf ngoai, danh
gia mtfc do phat quang.
4. D o c k e t qua
- Phat quang mau xanh da trdi ro: Hoat tinh men binh thtfdng goi la phan
tfng am tinh (-)
- Khong phat quang (thieu men r5) goi la phan tfng dtfdng tinh (+)
- Phat quang yeu (thieu men mtfc do vtfa phai) phan tfng (±)
5. C ac y e u to a n h h u £ n g
- Dung cu ban
- Lay mau chong dong khong tot se d in den sai ket qua
DINH LUANG MEN G6PD TRONG DUNG DjCH HONG CAU

BANG PHITONG PHAP QUANG PHO
1. Nguyen ly
Phtfdng phap nay cho phep xac dinh so' ltfdng hoat tinh men G6PD dtfa tren
viec do btfdc song ttf ngoai 340nm - to'c do tao thanh N A D P khii (N A D P H trong he
thong chtfa mot ltfdng thtfa G6PD va N A D P). Hoat tinh men trong dung dich tan
hong cau la yeu to' quyet dinh to'c do bieu dien phan ting.

2.1. Dem TRIS-HCI
- pH = 8.0 0,1 M
- TR IS : 1,21 M
- HC1: 0.1 N 70 ml
- Ntfdc cat vtfa du 100 ml
- Chinh pH bang HC1 0,1 N
149
2.2. G6P: 20mM
- N a 2G6P 3H20 : 7,2mg

- Ntfdc cat : lm l
2.3. NADP: lOmM trong ntfdc
NADP : 7,7 mg
Ntfdc cat :1 m l
2.4. Dung djch hoat hoa MgCI2 0,1 M
M gC l2 6H20 : 20,3 g
Ntfdc cat : 100 ml
2.5. May quang pho ke
Dong ho bam giay

Dung dich Prabkin
3. Q u y trin h k y th u a t
3 . 1. Rua hong cau
Rtfa 3 lan b in g NaCl 9°/00 lanh chu y gan bach cau va tranh lam vd ldp hong
cau be mat tren.
3.2. Chuan bi dung djch huyet sac to
0,1 ml hong cau rtfa

4,9 ml ntfdc cat
Lac can than trong 3 phut, ly tam d 4°C 3000 vong/phut trong 30 phut. Hut
phan ntfdc trong d tren de do hoat tinh men.
3.3. Do hoat tinh men
Trtfdc khi cho vao cong de tien hanh phan tfng ngtfdi ta cho them 0,1 ml GGP
Cho vao cong: 0,3 ml dd dem TR IS
0,3 ml dd hoat hoa
2,0 ml ntfdc cat
0,1 ml dd N A D P
0,2 ml dd huyet sac to
Tron deu, do mat do quang hoc d btfdc song 340 nm phan tfng bat dau khi
cho them 0,1 ml dd G6PD tron deu roi do stf tang mat do quang hoc ttfng phut
trong 10 phut.
150
H oat ti'nh men diidc tinh theo cong thtfc
AE/1’ x 3,0ml x 100

Men Ul/g Hb =
6,22 x Hb (g) x 0,2ml
Chu thich:
- AE/1’ thay doi mat do quang hoc trong 1 phut
- 6,22 he so" hap thu phan tut cua N A D P
- 3,0 ml the tich toan bo dung dich phan tfng.
- 0,2 ml the tich dung dich huyet sac to"
- Hb (g) so’ gram hemoglobin trong 100 ml (dung dich huyet sac to do trtfdc
khi tien hanh phan tfng).
Thong thtfdng ngtfdi ta do hoat tinh G6PD d nhiet do 30°C trong cong co
dieu nhiet.
Neu do d nhiet do phong thi ket qua phai nhan vdi he so" hieu chinh
K = [1 - (t° do - 30°C) x 0,06]
4. Ket qua
4.1. Hoat tmh men binh thifdng vdi dieu kien
+ Hong cau ltfdi 0,2 - 1,2 %
+ N hiet do do 30°C
Se bang 3,7 - 6.8 ddn vi quoc te/gHb
4.2. Thieu hdn hoat tinh men

0,0 - 0,4 ddn vi quoc te /gHb
4.3. Thieu vCra

1,0 - 3,5 ddn vi quoc te / g Hb

- Cac loai dung dich pha khong chuan
- Quang pho ke co btfdc song 340 nm khong chinh xac
- Dung dich huyet sac to' khong chinh xac
DJNH LI/ONG HAPTOGLOBIN TRONG HOTET THANH
Hatoglobin la mot a mucoprotein, no giam nang khi vd hong cau trong hoac
ngoai huyet quan.
Haptoglobin la mot protein lam nhiem vu van chuyen huyet sac to ttf do
trong huvet ttfdng - huyet thanh ve gan theo ty le 2 phan ttf hemoglobin (Hb) cho
1 phan ttf haptoglobin.
151
1. Nguyen ly
Mot lifdng hemoglobin biet trade cho vao huyet thanh. Phtic hdp Hb -
Haptoglobin dUdc tach ra b ln g phudng phap dien di tren giay cellulose acetat.
Moi quan he cua so" lUdng xac dinh va huyet s ic to' tu do dUdc tinh ra sau khi
nhuom cac bang dien di b ln g Densitometer d budc song 450mm vdi dudng c it
ngang 0,3mm.
Nong do haptoglobin dUdc tinh ra b ln g mg phtic hdp hemoglobin trong 1 lit
huyet thanh.
2. Thuoc thur
2.1. Dung dich dem: pH = 7,0 Ion luc 0,05
N a 2H P 0 4: 7,lg/l,2 the tich
N a 2P 0 4 - H 20: 6,9g/l,l the tich
Bao quan d +4°C
2.2. Hemoglysat
Mau dUdc lay vao ong nghiem vo trung co chat chong dong la ACD
Rtia 3 lan b ln g nUdc muoi sinh ly 0,9%. Sau do cho vao khoi hong cau da rtia
2 the tich nUdc cat va 1 the tich Toluene hoac Carbon Tetrachloride (CC14), cac
loai dung dich tren rat doc phai het stic can than.
Dung dich dUdc khuay deu va lie b ln g may lie , sau do ly tam 3000 vong
(1200g) trong 30 phut. Sau do dung pipette hut dung dich Hb. Loai bo Hb va
globin tu do. Tot nhat cho vao dung dich hemolysat 2 the tich nUdc (khong co
Toluene) va ly tam 20000 vong (18000g) d 4°C trong 20 phut. Co the bao quan d
4°C dUdc 1-2 ngay, neu d -20°C dUdc vai tuln.
2.3. Dung djch nhuom
Hoa tan 0,5g O-dianisidin (3,3’ - dimethoxybenzidin) trong 70ml cua ethanol 95%.
TrUdc khi dung cho them 10ml dung dich dem acetat, pH = 4,7 (Sodium
acetate 2,92g, acid acetic glacial lm l va cho nUdc cat vUa du 1000 ml). 2,5 ml cua
3% (H 20 210VOL) sau do cho nUdc cat vUa du 100ml.
2.4. Dung djch lam ro
Acid acetic 25ml + 75ml ethanol 95%

2.5. Dung djch rifa
Acid acetic 50ml + nUdc vUa du 1000ml
3. Phi/ong phap
3.1. Chuan bi mau: Huyet thanh lay vao ong nghiem, roi u trong chau nude am
(37°C) cho tiet huyet thanh. huyet thanh dUdc bao quan d -20°C cho den khi su dung.
152
Lay 01 the tich Hem olysat (da chuan bi nhu tren) cong vdi 09 the tich
huyet thanh (da chuan bi nhu tren) de trong phong d nhiet do 20°C-22°C trong
vong 10 phut.
3.2. Tien hanh chay dien di

Nhung giay cellulose acetat trong dung dich dem (kich thudc giay chay dien
di la 12 x 2,5cm). Sau do vdi giay cellulose acetat de tren giay tham cho se kho.
Dat khoang 0,75ml dung dich m iu da chuan bi tren d vach xua't phat. Chay d dien
the 150 V chieu dai bang chay khoang 5-7 cm trong vong 30 phut.
Vi du nhu sau:
A: Chi co hemoglobin (0,4g/l). Bang xuat hien d vi tri P globulin.
B: Huyet thanh binh thudng cong vdi huyet sac to', bang xuat hien d ca p
globulin (Hb) va vung a 2 globulin (phUc hdp hemoglobin va haptoglobin).
C: LUdng haptoglobin giam nhe. Bang d vi tri p globulin dam hdn bang d vi
tri a 2 globulin. j
D: Huyet thanh cua mot trUdng hdp thieu mau tan mau. Haptoglobin khong
co. Hemoglobin xua't hien d vi tri P globulin.
DUdng xuat phat dUdc chi b ln g mui ten.
Sau khi chay 30 phut la'y cac bang ra cho vao dung dich nhuom O-dianisidin
trong 10 phut. Sau do rtia vdi nUdc va dung dich rtia trong 5 phut, cho vao dung
dich co dinh ethanol 95% chinh xac trong 1 phut. Roi cho vao dung dich lam ro
trong 30 giay. Chuyen bang giay cellulose acetat tren lam thuy tinh va say d
100°C trong 10 phut.
153
Sau khi de nguoi tien hanh do ket qua b ln g ty trong ke d budc song 540nm
vdi dudng c it ngang la 0,3mm.
4. Tinh ket qua

Haptoglobin (g/1) = Haptoglobin Faction x Nong do huyet s ic to' (g/1)
Trong huyet thanh ngUdi binh thudng haptoglobin lien ket co tU 0.3-2.0g cua
huyet sac to'/h't gia tri trung binh la l,lg .
Nam cao hdn nU
Bat thitdng:
Tang: Khi co thai, nhiem trung man, cac benh ac tinh huy hoai to chUc, Hodgkin,
lupus ban do.
Giam: Thieu mau hong cau to (megaloblastic anemia)
Tang tan mau va theo doi chay mau d cac to chUc.
154
Chtfdng 5
M l i N D IC
■
H H U Y E T H O■ C
MOT SO KHAI NIEM CO BAN Vt KY THUAT M liN DjCH

UNG DUNG TRONG HUYlT HOC - TRUYEN MAU
Mien dich huyet hoc la mot ph ln cua m iln dich hoc noi chung. De co the
n lm bfit dUdc cac ky thuat va ting dung trong Huyet hoc-Truyen mau, chung toi
xin nhfic lai mot so" khai niem cd ban ve m iln dich hoc nhif: khang nguyen (K N ),
khang the (K T), phan ting ket hdp khang nguyen - khang the (K N -K T ).
1. Khang nguyen
1.1. Dinh nghia: Khang nguyen la chat la doi vdi cd the, co kha nang kich thich cd
the sinh ra K T dac hieu chong lai chat do.
1.2. Dac tinh cua khdng nguyen

Dac tmh cua K N phu thuoc vao nhieu yeu to" nhu dac tinh cua vat chu, tinh
la c u a K N , ban chat KN...’.
1.2.1. Tinh la cua KN: Neu K N la mot chat cang khac xa vdi vat chu ve mat tien
hoa bao nhieu thi kha nang sinh K T cang manh bay nhieu. Dua tren tinh chat la
it hay nhieu cua K N ma ngUdi ta co cac khai niem: di KN, dong loai KN, dong gen
KN, tu KN.
1.2.2. Trong lifting phan tCf cua KN: Thudng nhting chat co trong lUdng phan tti cao
thi tinh K N manh. Nhu vay, protein la nhting chat co tinh K N manh nhat. DUa
tren tinh chat nay, ngUdi ta co khai niem: K N hoan toan, K N khong hoan toan.
1.2.3. Cac nhom quyet djnh KN: Cac nhom quyet dinh K N la p h in quan trong
trong viec hinh thanh K T dac hieu chong lai chung.
1.2.4. Ban chat di truyen cua vat chu: M ot cd the chi co the mien dich chong lai
mot K N ma cd the do khong co.
1.2.5. So phan KN trong co the
K N du vao cd the b ln g bat ky dUdng nao thi sau 1 0 - 1 5 phut cung xuat
hien trong mau. Sau do K N khuech tan ra khoang gian bao lam cho nong do K N
trong mau ha nhanh. Sau 3 - 4 ngay, K N dot ngot bien mat. Do chinh la do cd the
155
bat dau xuat hien KT. K N khi vao cd the bi dai thtfc bao an va trinh dien cac sieu
K N cho cac te bao co tham quyen mien dich de cac te bao nay san xuat K T theo
khuon cua KN.
1.2.6. NhCmg yeu to anh hudng den tinh sinh mien djch cua KN
Ngoai tinh phan ting cua cd the vat chu, tinh sinh mien dich cua K N con
phu thuoc vao rat nhieu cac yeu to" nhu: lieu ltfdng KN, dtfdng vao cua KN , vai tro
cua cd the va ta chat,...
2. Khang the
2.1. Dinh nghia: K T la nhtfng chat do cd the sinh ra chong lai cac yeu to co hai
xam nhap cd the. Co hai loai KT: K T dich the va K T te bao. K T dich the la nhtfng
protein huyet thanh nam d khu vtfc gamma globulin. K T te bao la nhtfng K T co
dinh tren be mat te bao mien dich. C) day, chung toi gidi thieu chu yeu ve K T dich
the hay con goi la globulin mien dich (Immunoglobuline - Ig).
2.2. Cac lop globulin mien djch: co 5 ldp globulin mien dich nhtf sau:
Trong ltfdng Nong do trong

Ten Viet Nam
phan ttf huyet thanh
IgG hay y G 150.000 800-1000mg/100 1000 ±160mg/100
IgA hay yA 170.000 100-450mg/100 352 ± 126 mg/100

IgM hay y M 900.000 60-280mg/100 174 ± 31 mg/100
IgD hay y D 155.000 3-140mg/100
IgE hay y E 195.000 0 , 1 - 1 ,4mg/100
2.3. Cau true cua globulin mien djch: Cau true cua 5 loai globulin mien dich
ttfdng doi giong nhau, gom mot hay nhieu ddn vi hinh thanh. Moi ddn vi la mot
phan tti protid co 4 chuoi da peptid: 2 chuoi nang (H ) co trong ltfdng phan ttf la
50.000 va 2 chuoi nhe (L ) co trong ltfdng phan tti la 25.000. Hai chuoi nang nam
ke nhau con hai chuoi nhe n&m d 2 canh ngoai chung dtfdc noi vdi nhau b ln g cau
noi disulfua (SH ) (Hinh 5.1). Cac chuoi nang (H ) cua cac Ig thi khac nhau. thi du
IgG chuoi nang ky hieu la y; Ig A ky hieu la a, IgM ky hieu la |i, IgD ky hieu la 6,
IgE ky hieu la e; con chuoi nhe cua cac Ig chi co 2 typ hoac X hoac K.
156
L
S
I
S
S S
I I
s s
— I-----------------------1------------------ H
KHBT KHBT
KHKN S
I
S
____ L -------------------- L
H inh 5.1: Sd do cau true phan tCr cua IgG
2.4. Dac tmh cua globulin mien djch (xem phan ly thuyet)
3. Phan urng ket hdp khang nguyen - khang the

Phan tfng ket hdp K N -K T la phan ting cd ban ctia qua trinh mien dich.
Trong cd the, phan ting K N -K T la nguyen nhan cua cac hien ttfdng bao ve cung
nhtf hien ttfdng qua man cua qua trinh mien dich. Trong labo, phan tfng K N -K T
la cd sd de phat hien K N , K T hoac ca hai.
3.1. Co che ket hop KN-KT: K N va K T ket hdp dac hieu vdi nhau chu yeu dtfa vao
cac ltfc lien ket. Co 4 loai ltfc ket kdp dtfdc noi den la: (1) Ltfc hap dan phan tti
Van der Walls, (2) Ltfc tinh dien coulomb, (3) Ltfc lien ket gitfa cac cau noi hydro
va (4) Ltfc o" thuy (ltfc lien ket gitfa KN, K T khi khong co phan ttf ntfdc d gitfa).
3.2. Cac yeu to anh hudng den phan ung ket hop KN-KT: Co nhieu yeu to anh
htfdng den qua trinh ket hdp K N -K T bao gom: (1) Nhiet do phan tfng, (2) Do am,
(3) pH cua moi trtfdng, (4) ltfdng KN, K T tham gia phan ting, (5) Nong do cac ion
trong moi trtfdng phan tfng...
3.3. Tac dung sinh hoc cua phan ung ket hop KN-KT: Muc dich cuoi cung cua
ket hop nay la loai trtf K N ra khoi cd the. Viec loai trtf thong qua K T dich the co
trong huyet thanh (M ien dich dich the) va K T te bao (M ien dich qua trung gian
te bao).
Sau day, chung toi xin gi6i thieu mot so" ky thuat chinh cua m iln dich hoc
ting dung trong Huyet hoc - Truyen mau bao gom: (1) K y thuat man cam dong
vat, (2) Ky thuat phan lap te bao, (3) Ky thuat ket tua, (4) K y thuat ngtfng ket va
mot so" ky thuat khac.
157
KY THUAT
■ MAN CAM DONG
» VAT
«
1. Mot so yeu to anh hirdng den qua trinh tao khang the
Su tao thanh khang the chiu anh hUcfng cua nhieu yeu to khac nhau: loai
khang nguyen, lieu lUdng khang nguyen, dudng vao, khoang cach gitia cac lan
m ln cam, sU lUu lai cua khang nguyen trong cd the, loai dong vat, tuoi dong v&t
cung nhu tinh trang chung cua dong vat khi tiep xuc v6i khang nguyen, tinh
khang nguyen... Ban chat, cau true cua khang nguyen cung nhu cac te bao c6
tham quyen mien dich dong mot vai tro rat quan trong, co tinh dac hieu trong qua
trinh tao khang the va tat nhien dUdc lUu y dac biet trong cong tac san xua't cac
khang huyet thanh mien dich. Trong phan nay, chung toi neu len mot so"yeu to vS
phudng dien dong vat thi nghiem: chung loai, tuoi dong vat... va vai tro cua ta
chat trong qua trinh man cam.
1.1. Kha nang dap Ong mien djch cua cac loai dgng vat
1.1.1. Cac loai dong vat
Khi co khang nguyen xam nhap, cd the sinh vat phan Ung lai de loai trti
khang nguyen, trong do bao gom qua trinh tao khang the. N oi nhu the khong phai
tat ca cac loai dong vat va cung khong phai tat ca cac chung trong cung mot loai
dong vat deu phan Ung tao khang the nhu nhau doi v6i mot khang nguyen. Trong
cac dong vat thi tho va ngUa la dong vat dUdc sti dung nhieu de gay m ln cam vk
do do huyet thanh m iln dich cung dUdc xep thanh hai loai chinh: huyet thanh tho
mien dich chtia khang the loai R (rabbit type) va huyet thanh ngUa m iln dich
chtia khang the loai H (horse type).
• Tho la dong vat thong dung nhat trong cong tac dieu che khang huyet
thanh m iln dich. Tho d l nuoi, sach se, hien lanh cho nen rat tien dung trong cac
phong thi nghiem.
• Ngi/a, de la nhting dong vat 16n cho lUdng huyet thanh nhieu. Nhung
trong cong tac thuc nghiem noi chung va dieu che khang huyet thanh noi rieng,
ngUa, de la nhting dong vat tUdng doi hiem va dat, gia thanh huyet thanh cao.
• Chuot lang thi co phan ting tao khang the dich the khong tot nhung lai
thich hdp dac biet cho cac phan ting khang nguyen - khang the te bao. Chuot lang
cung nhu chuot cong trang va chuot nhat, mot mat cho ta lUdng huyet thanh qua
it oi, mat khac phan ting ket tua mien dich cua cac khang huyet thanh nay yeu.
• Ga, chim : Huyet thanh chim can dung dich co ion luc cao (co nong do g£p
10 den 12 lan dung dich dang trUdng) de tao phan ting ket tua trong moi trUdng
long, trong khi do d moi trUdng gel chi can nong do d in g trUdng se tao tua tot.
Ambrosius da tien hanh so sanh kha nang tao ket tua cua huyet thanh mien
dich tho, cho, chuot cong trang, chuot lang, chuot nhat va chuot dong. Khang
158
nguyen difdc sti dung la huyet thanh ldn ba't hoat. Tac gia nhan tha'y kha nang
ket tua cua cac khang huyet thanh khac nhau ro ret: khang huyet thanh tho co
kha nang ket tua cao nha't roi den cho va chuot dong la thap nhat.
Noi tom lai, tho la dong v a t co nhieu Uu diem v a tien dung nhat dung gay
m ln cam dieu che cac khang huyet thanh m iln dich, sau do den ngUa, de.
1.1.2. Tu6i ddng vat.
Tuoi dong vat co anh hudng quan trong do'i vdi qua trinh tao khang th<f.
Phoi va dong vat mdi lot long hau nhu chUa co kha nang tao khang the hoac noi
mot cach chinh xac hdn la qua yeu. Kaliss va cong sU (1962) da tien hanh tiem
mot khang nguyen de gay m ln cam cho tho mdi de, ket qua dem lai la dung nap
mien dich do'i vdi khang nguyen do. Nhiing tre mdi de, ngay tu ln l l dau dUdc m ln
cam mot khang nguyen thi khong co hien tUdng tao khang the trUdc thang thU 2.
Su trUdng thanh cua cac cd quan co tham quyen mien dich g in lien vdi tuoi
cua dong vat, do do dieu che cac khang huyet thanh m iln dich phai tien hanh
tren dong vat trUcfng thanh khoe manh.
Trong cung mot chung va cung mot dieu kien sinh song, co con phan Ung
manh do'i vdi khang nguyen m ln cam tUc la tao dUdc khang the hieu gia cao,
nhUng lai co nhUng con phan Ung yeu, cho khang the hieu gia tha'p. Do do trong
qua trinh gay m ln cam, can chon loc nhflng con co phan Ung tot de th u ln chung.
Ngoai ra yeu to' than kinh, noi tiet, cac thuoc Uc che mien dich cung anh
hudng kha ldn den qua trinh tao khang the. V i vay can lUu y den tinh trang
chung va dieu kien song cua con vat khi tien hanh gay m ln cam.
1.2. Vai tro ta chat
Ta chat la nhUng chat dUdc tron v6i khang nguyen khi gay man cam. Ta
chat co tac dung tang cao kha nang dap Ung mien dich khong dac hieu, qua do ma
tang cUdng san xuat khang the, cho nen co ten la ta cha't m iln dich. Ve cd che tac
dung cua ta cha't, co tac gia cho r in g ta cha't da tang hoat tinh phospholipase A
cua dai thuc bao. Nhd do lexitin chuyen thanh lyzolexitin, co chUc nang cua mot
ta chat noi sinh.
1.3. Chan nuoi dong vat thi nghiem
Trong hoan canh chUa co cd sd chan nuoi 16n de cung cap cac dong vat thuan
chung on dinh, moi ddn vi nghien cUu deu co mot cd sd chan nuoi nho. Dong vat
thi nghiem hau het deu mua d chd hoac cl cac gia dinh chan nuoi rieng le mot cach
tuy tien. Che do nuoi dUdng va khau phan an hoan toan tu do theo kha nang cung
cap cua tUng cd sd. Che do cham soc theo doi sUc khoe dong vat thi nghiem con
yeu. Viec nhap dong vat thi nghiem ra't bi dong va khong chon loc tot. Dong vat
thi nghiem cha't lUdng kem thi do tin cay cua ket qua nghien cUu rat sai lech.
Khong co dong vat th u ln chung tot la mot kho khan ldn cho cong tac thi nghiem
noi chung va cong tac gay man cam dieu che khang huyet thanh m iln dich noi
rieng. Tinh trang con vat co tinh quyet dinh do'i vdi kha nang dap Ung mien dich
159
tao khang the. De khac phuc mot so' kho khan tam thdi tren day, trudc khi cd trai
chan nuoi ldn cung cap dong vat th u ln chung vdi che do nuoi dtfdng vsk kh au phan
thong nhat, can de cao cong tac ltfa chon khi nhftp dong vat thi nghiem.
Trtfdc het, cac dong vat dtfdc chon phai cd stfc khoe tot, khong m lc benh nhtf
cac benh ngoai da: ghe, nam... cd can nang toi tieu tuy ttfng loai dong vat. Tho
phai cd can nang tren 2,5kg, chuot lang phai nang tren 300 gam. Sau khi nhftp
dong vat can phai nuoi trong mot thdi gian it nhat 15 ngay roi mdi tien hanh gay
man cam.
Cd sd chan nuoi can phai cao rao, sach se, thoang, ve mua dong khdng de gid
lua, ve mua he khdng qua nong. Phai cd che do ve sinh chuong trai hang ngay
cung nhtf ve sinh cho dong vat. Dac biet c ln gitf ve sinh tot cho cac khu vtfc tiem
nhtf gan ban chan, tai, bung de tranh hien ttfdng nhilm trung.
Ngtfdi chan nuoi phai ton trong noi quy bao ho lao dong va ve sinh dinh
dtfdng. Khau p h ln an can du ve so' ltfdng va dam bao chat ltfdng, dac biet phai
dam bao ve protein.
2. Dieu che khang huyet thanh mien djch

2.1. Nguyen tac chung
Ndi chung cho den nay chUa cd mot phtfdng phap m ln cam nao thich hdp,
tien ldi nhat doi vdi mdi mot khang nguyen va cho mdi mot loai dong vat. Ngtfdi ta
cung chtfa tim ra dtfdc mot md hinh man cam nao cd tinh van nang. M ot md hinh
man cam tot dtfdc danh gia d cho khang the dtfdc tao ra nhanh, hieu gia cao, tinh
khiet. Tuy nhien, viec dieu che khang huyet thanh m iln dich c ln phai tuan theo
mot so nguyen tac nhat dinh nhtf chat ltfdng khang nguyen, lieu ltfdng khang
nguyen, cach tiem, khoang cach gitfa cac lan tiem...
2.1.1. Chat liftin g khang nguyen
Tinh khang nguyen cua mot chat (kha nang gay dap tfng m iln dich) phu
thuoc vao ban chat, trong ltfdng phan ttf, cau true, tinh la, kha nang chuyen hoa
cua chat dd. Tinh khang nguyen cdn phu thuoc vao tinh ddn gia hay da gia, nghla
la khang nguyen gom nhieu protein hay chi cd mot protein.
Do tinh khiet cua khang nguyen quyet dinh do tinh khiet cua khang huyet
thanh. Can phai dac biet chu y den do tinh khiet cua khang nguyen khi dieu che
khang huyet thanh ddn dac hieu. Bang cac phtfdng phap tua, sac ky, hap phu
mien dich, ngtfdi ta dieu che dtfdc khang nguyen tinh khiet.
160
2.1.2. Lieu luong khang nguyen, khoang cach gidra cac lan tiem
Bang 5.1: Lieu ltfdng khang nguyen va phi/ong phap man cam
Ti&m ldn Ltfdng khdng Tao Thdi gian Ltfdng Xuat hien Tiem nhac Tdc dung Tdc dung
Itfdt nguyen CFA khang thd’ c6 hieu gia khdng the qua m ln lai lin II tren hieu tren phan
(ml) khdng the cham gid khdng tfng qud
cao nhd't the m in cham
Trong da 0,4 ml Nhanh 2-4 tu3n 80mcg ++ 10mg 500mcg Giam ro

KT.N/ml KT.N/ml
25mg RSA RSA
1mg MB (ni/dc muoi)
Gan b^n 0,4 ml Nhanh 2-4 tuan 200mcg ++ 10mg 1000mcg Giam ro
ch3n KT.N/ml KT.N/ml
25mg RSA RSA
1mg MB (ni/dc muoi)
Mang 0,4 ml Cham hdn 6 - 9 tuan 200mcg ± 10mg 400mcg Giam ro
bung KT.N/ml KT.N/ml
25mg RSA RSA
1mg MB (nude muoi)
Di/di da 0,4 ml Cham hdn 6 - 9 tuan 400mcg ± 10mg Thay doi it Giam ro
KT.N/ml
25mg RSA RSA
1mg MB (ni/dc muoi)
Tiem b3p 0,4 ml Cham hdn 6 - 9 tuan 400mcg ± 10mg Thay doi it Giam ro
KT.N/ml
25mg RSA RSA
1mg MB (ni/dc muoi)
TTnh mach 0,4 ml Cham hdn 10mg 600mcg
KT.N/ml
25mg RSA RSA
1mg MB (nirdc muoi)
CFA: Ta chat Freund toan ven

MB: Mycobacterium
RSA: Albumin huyet thanh bo
KT-N: Nitd cua khang the
2.1.3. Dudng tiem
De m ln cam dong vat, ngtfdi ta tien hanh cac cach tiem khac nhau nhtf tiem
trong da, tiem dtfdi da, tiem tmh mach. Ngoai ra con sii dung cach tiem mang
bung va gan ban chan phoi hdp. Tuy theo loai dong vat ma chon vi tri tiem bap,
tiem dtfdi da, tiem tinh mach cho thich hdp. Moi mot cach tiem deu co tac dung
khac nhau. Tiem tinh mach co tac dung nhanh toan than, dac biet gan, lach. Tiem
mang bung cung co tac dung toan than nhtfng trtfdc het la tac dung den cac hach
cua mac treo. Tiem bap, tiem dtfdi da co tac dung tot doi vdi cac hach dia phtfdng.
Tiem trong da va gan ban chan lam khang nguyen tieu cham hdn, keo dai
kich thich cua khang nguyen tai cho. Nhtfng ltfdng khang nguyen cho moi lan
tiem It, do do thtfdng dung doi vdi cac khang nguyen co trong ltfdng phan ttf cao,
hoac khang nguyen khong phai protid nhtf polysaccharid... Nam 1960 Leskowitz
va Waksmann da tien hanh so sanh ket qua qua trinh tao khang the va qua m ln
cham bdi cac cach tiem khac nhau (bang 5.1).
161
2.1.4. Ddng vat thi nghiem : Tho, chuot lang, chuot cong trln g, ga... la nhtfng dong
vat dtfdc sti dung nhieu trong dieu che khang huyet thanh. Dtfdi day chung toi
gidi thieu ky thuat tiem man cam cho mot so dong v&t tren.
a. Tho
• Tiem tinh mach: Vi tri tiem tinh mach tot nh&'t doi v6i tho la tinh mach
tai. Trtfdc khi tiem c ln phai ve sinh ndi tiem nhtf c it sach long va sat trung bln g
con. De tinh mach noi ro de tiem thi sau khi c it long, nen xoa nhe vung tiem bln g
tay. Vi qua trinh gay man cam phai tiem nhieu lan, do do phai tiem lln theo tinh
mach tti d lu tai den goc tai nham tranh hien ttfdng vd mach, tac mach. Nen co
dinh tho b ln g mot hop go, chi de tai tho ra ngoai thi khi tiem se ddn gian, dam
bao chac chan ket qua.
• Tiem ditdi da.Vi tri tiem dtfdi da doi vdi tho la d vung stfdn hoac vung
ltfng. Trtfdc khi tiem cung can phai c it long va sat trung b ln g con. De dam bao
chinh xac khi tiem, lay mot tay cam da len va tay kia luon mui kim vao dung vi
tri dtfdi da.
• Tiem mang bung: Trong ky thuat tiem mang bung, can chu y ttf the tho
khi tiem va kim tiem. Kim tiem khong dtfdc qua sac, dtfdng tiem di chech 30° de
tranh tiem sau qua vao ruot. Trong khi tiem phai doc ngtfdc tho de ruot don het
ve phia tren, tao dieu kien tiem dtfdc chinh xac, mat khac tay trai cam da bung
keo len, tay phai luon kim tiem vdi dudng chech 30°.
• Tiem trong da: Trtfdc khi tiem can c it long that sach va sat trung. Ky thu&t
tiem phai chinh xac tao dtfdc mot not noi san nhtf trong cac thti nghiem noi bi.
b. Chuot lang
• Tiem tinh mach: Tinh mach chan sau la ndi de tiem nhat doi vdi chuot
lang. Cd the tiem vao tinh mach nam gitia ngon chan gitia va ngon chan ngoai; cd
the tiem vao tinh mach cua phan tren dui sau khi boc lo tinh mach. Ngoai ra, cd
the tiem vao tinh mach tai, hoac tinh mach quy dau. Can co dinh chuot lang tren
mot mieng go phang (ban mo chuot) trong khi tiem. Vung tiem cung phai lam
sach long va sat trung can than. Neu boc lo tinh mach sau khi tiem phai khau 1-2
mui va bang sat trung.
• Tiem trong da: Thtfdng tiem trong da khi tien hanh cac thti nghem. Ndi
tiem trong da doi vdi chuot lang la da vung ltfng va da vung bung.
c. Chuot (chuot cong trang, chuot dong).
• Tiem tinh mach: V i tri tiem tinh mach ddi vdi chuot cong trang va chuot
dong la tinh mach duoi. Cd the gay me bang ether trtfdc khi tiem, hoac co dinh
tren ban mo chuot. De tinh mach noi ro de tiem thi trtfdc khi tiem c ln ngam duoi
chuot 5 - 10 phut trong ntfdc am khoang 50°C va lam that sach vung tiem. Hai
tinh mach duoi n lm doc hai ben cua duoi.
• Tiem trong da: V i tri tiem trong da nhtf doi vdi chuot lang.
d. Ga mdi
Gay man cam cho ga cung nhtf cho chim, thtfdng tien hanh tiem tinh mach.
Tinh mach nam d mat trong vung goc canh la ndi de tiem nhat. Can phai co’ dinh
ga that tot c!e dam bao tiem chinh xac.
162
e. T riin g ga: G in day, ngtfdi ta con dung trtfng ga mai de san xuat khang
the. Sau khi m ln cam, ga mai dtfdc nuoi de de trtfng, khang huyet thanh (dtfdc
goi la Ig Y dtfdc chuyen tdi long do trtfng va thu hoach trtfng ga de phan lap khang
the b ln g viec ket hdp phtfdng phap ket tua vdi natri sulphat va/hoac acid caprylic.
Ngtfdi ta da thanh cong trong viec gay m ln cam khang nguyen noc r ln loai
Bothrops hoac loai Crotalus cho ga mai Leghorn de tao ra khang the khang noc
rln , sii dung trong cac trtfdng hdp bi cac loai r ln nay cln.
2.2. Man cam cho dong vat
Tuy theo khang nguyen, tuy theo yeu cau dieu che khang huyet thanh ddn
gia hay da gia ma co the tien hanh theo cac cach sau day:
- M ln cam b ln g dung dich khang nguyen ddn thuan.
- Man cam hon hdp khang nguyen vdi alumin hydroxyd va ta chat Freund.
- Man cam phoi hdp. Lan man cam dau dung hon hdp khang nguyen vdi ta
chat, sau do man cam bang dung dich khang nguyen khong co ta chat.
De tien hanh nghien ctfu giai quyet cac van de thtfc tien de ra, cac nganh
mien dich thuoc y hoc cung nhtf thuoc chan nuoi thu y... phai co cac khang huyet
thanh da gia cung nhtf khang huyet thanh ddn gia. Cac khang huyet thanh da
dtfdc nhieu ntfdc san xuat ban tren thi trtfdng quoc te nhtfng rat dat, va bao quan
kho khan, phai luon luon gitf d nhiet do toi thieu 4°C de hieu gia khang the khong
bi giam. Mot mat de giai quyet kho khan chung, mat khac, de chu dong trong
cong tac nghien ctfu, viec ttf san xuat khang huyet thanh la dieu can thiet. Do vay
chung toi gidi thieu mot so" mo hinh man cam chung nham muc dich giup cac cd sd
co the nghien ctfu tfng dung tuy theo yeu cau va dieu kien cua minh.
2.2.1. Man cam bSng khang nguyen den thuan
a. D o'i vdi khang nguyen hod tan: Cac khang nguyen hoa tan thtfdng dtfdc sii
dung vdi nong do ttf 0,01g% den lg% trong qua trinh gay man cam. Nong do
protein binh thtfdng cua huyet thanh: 6 - 7g%, trong do khoang mot ntfa la
alumin, khoang 1/5 la gamma blobulin va khoang 2% trong so con lai la hon hdp
cua hdn 20 thanh phan protein co dac tinh khang nguyen.
De gay man cam cho tho, Gillert va Eichhorn da sii dung dung dich khang
nguyen 0 ,lg % theo mo hinh ddn gian bang cach tiem tinh mach dtfdi day (Bang 5.2)
Bang 5.2: Mo hinh mln cam cho tho theo Gillert
Ngay tiem The tich tiem

Ngay thir nhat 1 ml
Ngay thCr hai 3ml

Ngay thif ba 3ml
NgaythCr21 3ml
163
Ngay thti 28 l&y mau thti hieu gia khang the, neu dat hieu gia cao mong
muon thi co the giet tho lay toan bo huyet thanh.
De tranh nguy cd soc phan ve do tiem protein la vao tinh mach thi lln dlu
man cam tiem tinh mach, sau do tiem dtfdi da hoac tiem mang bung. M at khac,
trtfdc khi man cam 1 - 2 gid, co the thti phan ting noi bi vdi 0,1 ml dung dich
khang nguyen, hoac cd the dung khang histamin tong hdp.
b. Ddi vdi khdng nguyen khong tan (khdng nguyen vi khuan).

Tiem tinh mach va trong da thi dat ket qua tot hdn so vdi tiem bap va tiem
dtfdi da, mat khac, vi khuan song gav dap ting mien dich tot hdn vi khuan chet.
De bao ve dong vat man cam. nen tang lieu ltfdng dan dan. Vdi hon dich vi khuan
cd 1 ty mam benh trong lm l thi Alfoldy Hegedus gay man cam cho thd. bang cach
tiem tinh mach.
Bang 5.3: Mo hinh man cam cho tho theo Alfoldy
Ngay tiem The tich tiem

Ngay thtf nhat 0 , 1 ml
Ngay thtf 6 0 ,2 ml
Ngay thtf 11 0,5ml

Ngay thtf 16 1 ,0 ml
Ngay thtf 21 2 ,0 ml
Ngay thtf 25 lay mau thti hieu gia khang the, va cd the giet thd lay huyet
thanh neu hieu gia dat yeu cau. Neu hieu gia cdn thap, cd the tiep tuc tiem nhlc
lai mot lan ntfa 2ml, hoac tiem 5 ngay lien moi ngay 2ml.
Dieu che khang huyet thanh m iln dich ma khang nguyen la vi khuan, cho
den nay it sti dung phtfdng phap man cam khang nguyen ddn thuan ma thtfdng sti
dung hdn hdp khang nguyen vdi ta chat. V i khi sti dung them ta chat thi dat dtfdc
hieu gia khang the cao hdn nhieu.
2.2.2. M in cam khang nguyen vdi ta chat
Cd nhieu loai ta chat dung de man cam, d day chung tdi chi gidi thieu ta
chat thong dung nhat, ta chat Freund.
Man cam b ln g hon hdp khang nguyen - ta chat Freund la phtfdng phap dtfdc
ting dung rong rai d cac ntfdc trong cong tac dieu che khang huyet thanh. Hdn hdp
khang nguyen - ta chat Freund da dem lai dtfdc stf dap tfng mien dich tot, tao
dtfdc ltfdng khang the nhieu. Cd hai loai ta chat Freund dtfdc ban tren thi trtfdng.
Loai khdng hoan toan (incomplete): - Arlacel A: 1,5ml
- Bavol F : 8,5ml
Loai hoan toan (complete): - Arlacel A: 1,5ml
- Bayol F : 8,5ml
- Mycobacterium: 5mg
164
(1) Dieu che hon hdp*khang nguyen - ta chat:
Hon hdp khang nguyen - ta cha't Freund chi dieu che trtfdc khi gay m ln cam.
Dung cu cln co:
- Bdm tiem loai 10ml vdi kim tiem to.
- Coc co mo loai 10 - 25ml
Dung cu phai dtfdc rtia sach, say kho, vo trung trtfdc khi dung.
Tien hanh dieu che:
- Dung dich khang nguyen (protein) hoac nhu dich khang nguyen (vi
khuan, virus, te bao): 1 the tich.
- Ta chat Freund loai hoan toan hoac khong hoan toan: 1 the tich
Tron ta chat vdi khang nguyen. Dau tien cho vao coc co mo 1 the tich ta chat
Freund, sau do cho tti tti 1 the tich dung dich khang nguyen vao. Dung bdm tiem
de bdm, hut nhieu lan khoang 30 - 40 phut la dat yeu cau (hon dich da thuan
nha't) cho den khi nao hon dich trln g due nhtf stfa, co do keo dac tot, nghia la nho
mot giot hon dich len mat ntfdc la ma giot do khong bi tan ra.
Co the dung co'i chay stf nho co dung tich khoang 50ml vo trung de nghien
hon hdp khang nguyen - ta chat Freund.
(2) Gay man cam cho tho:
- Vdi dung dich khang nguyen, tong lieu protein cho mot tho: 75 - 100 mg.
Moi tuan tiem mot lan trong 3 - 4 tuan lien. Co the tiem dtfdi da hoac tiem bap.
Lieu ltfdng cho moi lan tiem: 2ml hon hdp khang nguyen - ta chat Freund
(lm l dung dich khang nguyen: 25mg khang nguyen trong lm l ntfdc muoi sinh ly +
lm l ta chat Freund).
Huyet thanh co khang the tang cao keo dai trong qua trinh gay m ln cam ttf
2 - 4 thang sau. Khoang 5 - 6 ngay sau lan tiem cuo'i cung thi lay mau thti.
- Khang nguyen la chat chiet xua't cua phan hoa. Tinh chat phan hoa dtfdc
chiet b ln g dung dich caca hoac phenol lm l dung dich chiet tach (37,25mg/ml)
tron vdi lm l ta chat Freund chia thanh 4 phan tiem vao 4 gan ban chan. Moi tuan
tiem 2 lan, trong 3 tuan.
- Protein ket hdp. Thi du: picryl clorua - gamma globulin bo; P - sulfanilic -
gamma globulin bo: 2,4 dinitrophenylalbumin bo... Co nhieu phtfdng phap gan cac
chat co trong ltfdng phan tti thap (cac hapten) len mot cha't co trong ltfdng phan tti
cao ma chu yeu len protein. 10 mg picryl clorua - gamma globulin bo dtfdc tron vdi
ta chat Freund tiem dtfdi da d vung gay. 7 ngay sau tiem nhac lai. Sau do 1 tuan
thi tiem tiep 3 tuan moi tuan 2 lan, moi lan 2mg picryl clorua - gamma globulin
bo khong co ta chat.
(3) Gay man cam chuot lang.
Tiem mot lan 10 - 20 mg khang nguyen (albumin long trang trtfng. gamma
globulin...) trong lm l ntfdc muoi sinh ly + lm l ta chat Freund. Chia deu lam 2,
tiem dtfdi da hoac tiem bap. Sau 4 tuan lay mau.
165
Co the tiem 3 tuan lien, moi tu4n mot lln tiem b lp lm l (gom 2 - 2.5mg
khang nguyen + ta chat Freund). Mot tuan sau l&n tiem cuoi cung thi lay mau.
Neu m in cam chuot lang b ln g protein ket hdp thi tien hanh nhtf sau: 2mg
picryl clorua - gamma globulin bo vdi ta chat Freund tiem dtfdi da d gan ban chan
sau. 17 ngay sau tiem lai mot lln vdi 3 mg picryl clorua - gamma globulin + ta
chat vao dtfdi da vung gay. Sau 8 ngay lay mau.
(4) Gay man cam chuot cong trang.
lm g khang nguyen trong 0,1ml ntfdc muoi sinh ly + 0,1 ml hon hdp tren
(0,5 mg khang nguyen). Khoang 19 - 20 ngay sau lay mau kiem tra hieu gia
khang the.
2.3. Man cam phoi hop

La phtfdng phap phoi hdp cach sii dung ta chat trong qua trinh gay m ln cam
cho dong vat. Mot qua trinh man cam gom nhieu lan tiem cach nhau, do dd mot
vai lan tiem sii dung hon hdp khang nguyen - ta chat Freund, mot sd l&n tiem sii
dung hdn hdp khang nguyen - alumin hydroxyd, mot sd l&n tiem chi sii dung dung
dich khang nguyen ddn thuln.
Phtfdng phap man cam phoi hdp cd the dung ddi vdi cac khang nguyen nhtf
collgagen gelatin, hoac khang nguyen to chtfc: to chtfc nao, thtfdng than, gan, da
day, ruot...
2.3.1. M6 hinh m in cam phoi hop (theo Steffen, 1968)

Thi du m ln cam cho thd dieu che khang huyet thanh da gia.
Tuan thtf nhat, tiem dtfdi da vung gay: 10 mg protein + ta chat Freund.
Tuan thtf hai, tiem nhac lai mot lan nhtf tuan thtf nhat.
Sau dd mot tuan, tiem tiep 4 tuan vdi hdn hdp protein - alumin hydroxyd.
Tien hanh nhtf sau ( tiem tinh mach):
Bang 5.4: Mo hinh man cam theo Steffen
Tuan le So lan tiem Lieu ltfdng moi lan tiem

Tuan 1 3 1,5mg
Tuan 2 3 2 ,0 mg
Tuan 3 3 2,5mg
Tuan 4 3 3,0mg
Tam ngay sau lan tiem cuoi cung thi lay mau thtf hieu gia khang the.
2.3.2. Md hinh m in cam phoi hop.
Sau day gidi thieu mot vai md hinh man cam phoi hdp da cd ket qua tot.
a. Dieu che khang huyet thanh da gia (huyet thanh tho chong protein huyet
thanh ngiicri,).
Nguyen lieu, dung cu:

- Coic co mo, bom tiem, kim tiem coi chay stf.
- Huyet thanh ngtfdi binh thtfdng (5 - 10 m iu huyet thanh ngtfdi cho mau
binh thtfdng, tron deu) khong dung huyet.
- Ta chat.
Neu khong co ta chat Freund ngoai, co the ttf dieu che ta ch&'t vdi cac
nguyen lieu san co nhtf sau:
- D lu paraphin tinh khiet: 6 ml
- Lanolin: 2 g
- BCG chet 30 mg
Dau tien cho lanolin vdi dau paraphin vao coi chay stf nghien cho that deu
va nhuyen, sau do cho BCG chet vao, lai nghien tiep cho den khi co dtfdc mot hon
dich keo tot.
Dieu che hon hdp khang nguyen (K N )- ta chat:
- Dung dich khang nguyen (huyet thanh ngtfdi binh thtfdng pha loang vdi
NaCl 0,9% theo ty le 1: 1): 1 the tich.
- Hon dich ta chat (d tren): 1 the tich dung bdm tiem vdi kim tiem to hoac
coi chay stf de dieu che nhu dich (xem d tren).
Tien hanh man cam:
B a n g 5.5: Mo hinh man cam phoi hop
Tuan le Djch tiem Lieu ltfdng Cach tiem

Thir nhat Hon hdp KN - ta chat 1,5ml Mang bung
Thtf hai Hon hdp KN - ta chat 1,5ml Mang bung
Thif ba Dung dich KN pha loang 1: 1 1 ml Tiem bap
Thtf ttf Nghi, khong tiem
Thi/ n3m Dung dich KN pha loang 1: 1 1 ml TTnh mach
7 - 8 ngay sau lay mau thti hieu gia khang the. Neu hieu gia con thap thi
tiem lai mot lan: lm l dung dich khang nguyen pha loang (0,5ml huyet thanh +
0,5ml NaCl 0,9%) tiem bap hoac tinh mach; 7 - 8 ngay sau lay mau thti.
De tranh tinh trang soc bdi lan tiem tinh mach, can sti dung huyet thanh ttfdi,
khong vd hong cau, khong co lipid, dong thdi dung cu phai vo trung va tiem cham.
Nhtf da trinh bay d tren, moi mot dong vat, moi mot chung loai deu co stf
dap tfng mien dich khac nhau. V i vay can gay m ln cam, mot ddt cho nhieu tho, it
ra la 4 - 5 con. Qua kinh nghiem, co tho thi cho phan ung tot vdi albumin (dtfdng
tua albumin dam ro) nhtfng co tho lai cho phan tfng tot vdi cac protein khac cua
huyet thanh nhtf IgG hoac IgA, hoac haptoglobin... Neu de rieng huyet thanh
ttfng tho thi khong dtfdc mot huyet thanh da gia tot mong muon, nhtfng neu tron
lai vdi nhau thi se dtfdc mot huyet thanh da gia tot, phan tfng tua dam ro vdi tren
30 thanh phan protein huyet thanh ngtfdi (khang nguyen).
167
b. Dieu che khdng huyet thanh dctn gid.
Dieu che khang huyet thanh ddn gia nhtf: huyet thanh tho khang IgG,
khang IgA, khang IgM , khang alpha - foetoprotein (AFP)... Dtfdi day 1& mo hinh
dieu che huyet thanh tho khang IgG cua ngtfdi.
IgG trong huyet thanh ngtfdi dtfdc chiet tach b ln g phtfdng phap sac ky cot
qua DEAE Sephadex A 50. Dung dich IgG co nong do 3mg/ml.
Dieu che hon dich khang nguyen - ta chat:
- Dung dich IgG (3mg/ml): 1 the tich
- Ta chat Freund: 1 the tich
B a ng 5.6: Mo hinh man cam phoi hop
Tuan le Djch tiem Lieu ltfdng Cach tiem

Thif nhat Hon hdp KN 2 ml Mang bung
Thif hai Hon hdp KN 2 ml Mang bung
Thif ba Dung dich IgG (3mg/ml) 1,5ml Tiem bap
ThCf tu Nghi
Thif nSm Dung dich IgG 1,5ml TTnh mach
7 - 8 ngay sau lay mau thtf hieu gia khang the. Neu hieu gia chtfa cao thi
tiep tuc tiem mot lan ntfa: 1,5ml dung dich khang nguyen tiem bap hoac tinh
mach. Sau do 7 - 8 ngay lay mau thti lai.
2.4. Ky thuat lay mau va tach huyet thanh

2.4.1. Lay mau kiem tra hieu gia khang the
Co the lay mau tinh mach hoac dong mach de kiem tra hieu gia khang the.
K y thuat va vi tri lay mau tinh mach doi vdi ttfng loai dong vat nhtf p h ln tiem
tinh mach d tren. Chu y: ong nghiem va bdm tiem phai sach, kho va vo trung.
Moi lan thti can lay 0,5 - lm l mau tuy loai dong vat. Sau khi lay mau, can nam
ong nghiem trong tay khoang 15 phut roi de d nhiet do phong tti 30 phut den 1 gid
de cuc mau co tot; co the de d tu lanh + 4°C khoang 20 phut. L y tam tach lay
huyet thanh dung de thti hieu gia khang the.
2.4.2. Ky thuat kiem tra hieu gia khang the
Kiem tra hieu gia khang the bang ky thuat khuyech tan Ouchterlony va
dien di mien dich. Xem phan ky thuat ket tua.
2.4.3. Lay mau va huyet thanh toan bo
Sau khi kiem tra thay hieu gia khang the da dat thi giet dong vat. lay mau
toan bo.
Dung cu can: dao keo mo va ong ly tam to.
168
Co dinh tho tren ban mo vdi tii the n lm ngtia. Lam ve sinh vung co: c it
long, sat trung. Boc lo mach canh va luon mot ong thong vao dong mach canh de
l&'y mau.
Cho mau chay vao ong ly tam to. Cho chay tii tii va khi mau chay g in het
thi doic ngtfdc tho de l&y dtfdc toi da.
De cac ong mau vao tu I ’m 37°C khoang 15 phut, sau do de vao tu lanh 4°C
khoang 30 phut den 1 gid. Tach cuc huyet khoi thanh ong thuy tinh bang que
thuy tinh ly tam lay huyet thanh (3000 vong/ phut trong 15 phut). Dung ong hut
Pasteur de hut huyet thanh sang cac ong hoac chai thuy tinh vo trung.
2.5. Tinh khiet khang huyet thanh
Doi vdi cac khang huyet thanh dOn dac hieu, can co do tinh khiet khang the
cao. Co the tien hanh hap phu mien dich de loai trtf cac khang the tap (xem ky
thuat mien dich hap thu).
2.6. Bao quan khang huyet thanh mien dich

Hieu gia khang the cua huyet thanh giam nhanh neu khong dtfdc bao quan
6 dieu kien lanh hoac neu lam tan ra dong lai nhieu lan trong qua trinh sii dung.
Do do c ln phai chia huyet thanh thanh nhieu ong nho, vo trung, moi ong 1 -
2ml de bao quan va hang ngay lay ra mot ltfdng du dung. Ngoai lo phai co nhan
ghi ngay lay va loai khang huyet thanh hieu gia toi tfu, the tich.
Tuy theo dieu kien cho phep co the bao quan theo hai cach dtfdi day:
2.6.1. Bao quan difdi dang long
Sau khi chia huyet thanh vao cac ong nho thi bao quan d do lanh - 20°C.
Hang ngay lay ra sii dung neu khong het thi de vao tu lanh + 4°C va sti dung cho
4 - 5 ngay sau.
2.6.2. Bao quan dudi dang ddng kho
Dong kho la hinh thtfc tot nhat de bao quan huyet thanh. Co the dong kho It
mot b ln g tuyet carbonic ket hdp vdi hut chan khong.
Cd the dong kho bang nito long. Tien ldi nhat la dung may dong kho hien dai.
Sau khi dong kho cung bao quan d dieu kien lanh, hieu gia khang the giti
dtfdc 1 - 2 nam sau. Trtfdc khi sti dung, hoa khdi ltfdng dong kho vdi mot ltfdng
ntfdc muoi sinh ly bang the tich huyet thanh trtfdc khi dong kho.
De chong stf phat trien cua vi khuan, du dang long hay dang dong kho thi
trtfdc khi bao quan can cho them vao huyet thanh mot ltfdng 0,5% phenol hoac
0,01% methiolat, hoac 0,02% Na - azit hoac hon hdp 0,25% phenol + 0,005%
methiolat (neu khdng dung de dinh ltfdng cac protein cd tinh chat men).
169
KY THUAT PHAN LAP T t BAO Ml£N DjCH
Muon nghien ctfu dac diem cac te bao m iln dich thi trUdc het phai tach dtfdc
cac te bao nay ra khoi hon hdp gom nhieu te bao mau khac. Th i du muon c6
lympho tinh khiet ttf mau thi phai tach dtfdc lympho ra khoi hon hdp cac te bao
hong cau, tieu cau, bach cau da nhan va dai thtfc bao. Chi khi co hon dich te bao
tinh khiet mdi co the nghien ctfu cac dac diem cua chung. Cho nen phan lap cac
dong te bao la mot ky thuat khong the thieu dtfdc khi nghien ctfu cac phan tfng
mien dich te bao cung nhtf cac dac diem khac cua te bao mien dich. Ky thuat phan
lap te bao da co ttf lau, ngay cang dtfdc cai tien va hoan thien, do do co nhieu loai
ky thuat khac nhau. Trong pham vi nay, chung toi gidi thieu hai loai ky thu&t:
phan lap lympho va phan lap dai thtfc bao, chu yeu la cac te bao mien dich cua
ngtfdi de phuc vu cho viec nghien ctfu cac ky thuat te bao.
Ky thuat phan lap lympho bao gom:
- Phan lap lympho ttf mau.
- Phan lap lympho ttf hach, lach va cac to chtfc lympho khac.
Ky thuat phan lap dai thtfc bao gom:
- Phan lap dai thtfc bao ttf mau.
- Phan lap dai thtfc bao ttf cac cd quan khac: o bung, cac xoang, phoi...
1. Phan lap lympho

1.1. Phan lap lympho tCrmau
Co nhieu cach phan lap lympho ttf mau nhtf: phan lap b ln g dung dich
dextran, phan lap b ln g gelatin, phan lap b ln g ficoll. 0 day, chung toi gidi thieu
phtfdng phap phan lap lympho thong dung nhat ttf mau la phtfdng phap phan lap
lympho b ln g ficoll, con cac phtfdng phap khac chi neu nguyen ly chung.
1.1.1. Phan lap lympho being dung djch dextran

Nguyen ly: dung dextran trong ltfdng phan tti ldn, cac phan dextran se g in
vao be mat hong cau, nhtfng khong gan vao cac te bao mau khac. Stf lien ket nay
se lam cho hong cau nang them, do do khi lang ttf nhien, hong cau se lang nhanh
hdn cac te bao mau khac. Lang nhanh nhat la cac hong cau, roi den bach clu da
nhan, sau cung la lympho. Sau thdi gian de lang, d 2/3 tren khoi ntfdc mat co tdi
tren 80% la lympho.
1.1.2. Phan lap lympho being dung djch gelatin 3%
Nguyen ly: Giong nhtf dextran, gelatin cung bam vao hong cau lam tang
trong ltfdng cua hong cau, do do khi de lang. hong cau se lang nhanh hdn cac te
bao khac. Gelatin con co tfu diem la lam hong cau lang nhanh hdn va lang nhieu
hdn, nen so' hong cau con sot it hdn.
170
Thifdng dung gelatin 3% trong dung dich Hanks. Dung dich nay dtfdc hap
cach thuy cho gelatin tan hoan toan. Neu can vo trung thi phai loc qua mang loc
0,45 milimicron, sau d6 de d tu a'm 37°C de cho nhiet do cua dung dich trd ve
37°C. Dong thdi mau da chong dong cung de d tu am 37°C. De tranh chd ddi lau,
ngtfdi ta thtfdng chueJn bi dung dich gelatin ttf chieu hom trtfdc, lien tuc de tu am
37°C den hom sau sti dung.
1.1.3. Phan lap lympho bdng dung djch ficoll (Hinh 5.2)
Dung ficoll (Pharm acia) trong ltfdng phan tti 400.000 trong dung dich
uromiro, dung dich nay co ty trong xac dinh; vdi ty trong dd; khi ly tam, cac
lympho khong the lang xuong day qua ldp ficoll, do dd tap trung lai d be mat tiep
xuc gitfa dung dich ficoll va huyet ttfdng. Rieng hong cau va bach cau da nhan
lang xuong day. Phtfdng phap nay cdn goi la ly tam phan ldp theo ty trong.
• Nguyen lieu
/s' v
- Ong ly tam cd 10 x 75 mm, pipette Pasteur dau nhan.
- Pha dung dich ficoll: ficoll (400.000) 9,6 g + uromiro 0,75% 20 ml (1 ong) +
ntfdc cat 130 ml. Hien nay, cd nhieu loai ficoll ban san tren thi trtfdng, khdng c&n
pha che.
Danh cho tan deu, bao quan d nhiet do 4°C cd the dung 2 - 3 thang.
- Dung dich Hanks pH 7,4 de rtia te bao.
- M ay li tam lanh 4°C.
- Kinh hien vi va buong dem te bao bach cau.
• Tien hanh
- 2 ml mau chong ddng bang heparin, lac deu, cd the pha loang them vdi
mot khoi 2 ml dung dich Hanks.
- Dung pipette chia do hut 2 ml mau roi nhe nhang dtfa vao ong nghiem da
cd san 1 ml dung dich ficoll, cho mau chay ttf ttf vao theo thanh ong nghiem xuong
tiep xuc vdi be mat cua dung dich ficoll, tao thanh hai khoi ro ret: ficoll d dtfdi,
mau d ldp tren.
- Nhe nhang dtfa vao may ly tam 1200 vong / 20 phut d nhiet do 4°C.
Lay ra quan sat bang mat thtfdng se thay 4 ldp ro ret: dtfdi cung la hong cau,
cd bach cau da nhan. tiep theo la khoi ficoll, tren be mat ficoll la mot ldp mong te
bao mau trang nhat va tren cung la huyet ttfdng. Cdn goi la ldp “ may” te bao
(Hinh 5.2).
- Dung pipette Pasteur nhe nhang hut lay ldp “ may” te bao, chuyen sang
ong nghiem khac.
- Rtia te bao bang dung dich Hanks. Do day ong nghiem bang dung dich
Hanks, dung pipette Pasteur thuan nhat huyen dich te bao, ly tam 1500 vong
trong 10 phut, rtia ba lln nhtf vay d nhiet do 4°C.
- Pha thanh huyen dich te bao trong dung dich Hanks.
- Xac dinh sd ltfdng te bao, ty le te bao song bang dung dich Hanks.
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A B
Hinh 5.2: Tach lympho bang ky thuat ly tam phan ldp vdi Ficoll
A: Mo hinh ve nguyen ly cua ky thuat
B: Anh sau ly tam tach lympho tao thanh ldp m&ng trong ong ly tam.
Thu hoach ldp “may” nay cd lympho chiem > 95%
Ky thuat nay cho huyen dich te bao tren 90% la lympho, 2 - 5% la bach c£u
da nhan va 5% la hong cau. T y le song cua te bao la tren 95%.
Ap dung: Huyen dich te bao lympho nay kha tinh khiet nen co the dung cho
cac ky thuat sau:
- Tao hoa hong T va B.
- Nuoi cay, chuyen dang lympho in vitro.
- Lam doc te bao lvmpho.
Ky thuat nay tiet kiem dtfdc mau vi te bao lympho khong bi mat nhieu.
1.1.4. Chuan bi huyen djch te bao lym pho hoan toan tinh khiet (khong co bach diu da
nhan, hong clu va tieu cau).
• Nguyen lieu
- Ong nghiem 10 x 75 mm. Pipette Pasteur, pipette chia do.

- Bdm tiem loai 20 ml ,
- Bong nilon, bong thuy tinh (neu khong co ca hai thtf thi co the dung bong
tham ntfdc xe tdi).
- Dung dich ficoll.
- Huyet thanh khang ABH .
- Dung dich Hanks.
• Tien hanh: Co hai cach lam: co the lam ngtfng ket hong cau bang huyet
thanh khang ABH hoac de lang ttf nhien vdi dextran nhtf tren, roi ly tam vdi ficoll.
(1) Dung phan tfng ngtfng ket hong cau bang huyet thanh khang ABH:
- Lay 2 ml mau chong dong cho vao ong nghiem nho (0,5 x 6cm) moi ong 1 ml.
- L y tam 3500 vong trong 5 phut, lay ntfdc mat chuyen sang ong nghiem
khac co 0,3 ml huyet thanh khang AB, hoac khang H (tuy thuoc vao nhom mau).
- Can ngtfng ket dtfdc loai bo b ln g cach ly tam 1000 vong trong 10 giay.
- Lay ntfdc mat chuyen sang ong nghiem khac co 0,3 ml ficoll.
- Ly tam 3000 vdng/3 phut, ta"t ca bach cau da nhan va dam hong cau
ngtfng ket se lin g xuong day, tieu cau n lm trong ntfdc mat co khang ABH, con te
bao lympho nam d be mat tren ldp ficoll. Te bao lympho dtfdc lay ra bang pipette
Pasteur va chuyen sang ong nghiem khac co dung dich Hanks, tron deu bang
pipette Pasteur.
- Ly tam tiep 1000 vong trong 1 phut, loai bo ntfdc mat, trong do co tieu
cau. Con can lympho tinh khiet dtfdc chuyen sang ong nghiem khac, rtfa tiep mot
lan ntfa bang dung dich Hanks.
- Can te bao dtfdc hoa tan trong dung dich Hanks, dem so" ltfdng te bao,
danh gia ty le song chet bang dung dich xanh trypan.
(2) Dung bong nilon
Budc 1: Loai bo sd bo hong clu bang dung dich dextran.
Budc 2: Loai bo bach cau da nhan. Sau khi da lam lang hong cau bang
dextran, lay toan bo ntfdc mat. Chuyen ntfdc mat vao bdm tiem nhtfa 20 ml co sin
bong da xe tdi (dung 600 mg bong tdi cho vao 20 ml huyen dich bach cau). Huyen
dich tham deu vao bong. Bdm tiem dtfdc de dtfng d tu am 37°C trong 30 phut.
Trong thdi gian nay, bach cau da nhan bam vao bong, sau do dung dung dich
Hanks rtia ldp bong cho te bao lympho (te bao khong bam vao bong) theo dung
dich chay xuong, cuoi cung dung loi bdm tiem ep cho sach ntfdc. Ntfdc chay xuong
co chtia te bao lympho va hong cau, con bach cau da nhan dtfdc giti lai d ldp bong.
Bi/dc 3: loai bo hoan toan hong cau: Huyen dich te bao lympho con lln hong
cau dtfdc phan lap tiep tuc b ln g cach ly tam vdi dung dich ficoll . 2 ml huyen dich
te bao dtfdc ly tam vdi 1 ml ficoll. Hong cau se lang hoan toan xuong day ong, te
bao lympho dtfng lai d be mat tiep xuc gitfa ficoll va dich chtfa te bao. Te bao
lympho dtfdc lay ra bang pipette Pasteur, rtia lai ba lan b ln g dung dich Hanks.
Dem so" ltfdng te" bao, xac dinh ty le song chet b ln g xanh trypan 2%. Chuan bi
huyen dich te" bao theo yeu cau cua thi nghiem.
Ap dung: Te bao lympho tach b ln g phtfdng phap nay rat tinh khiet, gan
100% te bao la lympho, khong co bach cau da nhan va hong cau, tieu cau. Thtfdng
chi ap dung cho cac ky thuat sau day:
- Doc te bao, dac biet la doc te bao trong phat hien khang nguyen H LA.
- Nuoi cay te bao lympho hon hdp.
Phtfdng phap nay co nhtfdc diem la ton mau vi tach qua nhieu btfdc nen te"
bao bi mat nhieu.
1.2. Phan lap lympho tCrong nguc
Cac te bao lav tii ong ngtfc co chu yeu la lympho bao, cac te bao khac nhtf
bach cau da nhan va hong cau chiem ty le rat it. Ngtfdi ta thay r in g co tdi 90% cac
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lympho d ong ngtfc co dtfdng ki'nh cua cac lympho nho la 8 pm, mot vai phan tr&m
la cac lympho bao nhtf khi phan lap ttf mau. Co the lam nhtf sau:
- Boc lo ong ngtfc, dung bdm tiem vo trung hut dich bach huyet (con goi la
ky thuat dan ltfu ong ngtfc).
- Bao quan dich bach huyet trong mot binh nho co s§n dung dich bao quan.
Dung dich bao quan gom dung dich Parker, heparin 20 ddn vi/ml, penicillin 50
ddn vi/ml, streptomycin 60|ig/ml. Vdi dung dich nay, te bao co the song 12 gid d
nhiet do 4°C, sau do thi ltfdng te bao chet se tang len. Cung co the bao quan d
nhiet do 4°C trong dung dich nuoi cay co 0,5% dtfdng, 10% huyet thanh cung loai,
co khang sinh, do dam dac cua te bao ttf 10 - 100 x 106 te bao trong 1 ml dung
dich nuoi. Trong trtfdng hdp nay, te bao co the song tren 24 gid vdi ty le te bao
song tren 80%.
1.3. Phan lap lympho tCfhach

Co the lay hach d ngtfdi (hach Hodgkin, hach lympho - sarcom) hoac hach cua
sue vat (thtfdng nhat la hach ben, hach co cua chuot, v.v...). Tien hanh nhtf sau:
(1) Hach lay ra dtfdc bao quan ngay trong dung dich Parker co huyet thanh
cung loai 10% va khang sinh.
(2) Cat loc cac to chtfc xd, md (neu co).
(3) Rtfa lai vai lan bang dung dich Parker.
(4) Dung keo nho, sac, cat thanh manh nho, kich thtfdc 5 x 5 mm. Neu hach
chuot thi chi can cat doi. Ttf btfdc nay co hai cach lam: tach lympho bao ra khoi
hach bang dung dich trypsin, hoac nghien hach qua ltfdi loc.
Phan lap bang dung dich trypsin 0,25%. To chtfc hach dtfdc tieu bang dung
dich trypsin co khuay ttf. moi lan khuay khoang 10 - 15 phut, d nhiet do 37°C.
Khuay nhtf vay 3 - 4 lan, moi lan khuay xong, gan lay ntfdc, ntfdc nay co te bao,
ngay lap ttfc phai bao quan d 4°C de tfc che tac dung cua trypsin. Cach lam nay co
nhtfdc diem la trypsin lam giai phong A D N va nucleoprotein. Cac thanh p h in nay
bam vao be mat te bao, lam anh htfdng den hoat dong binh thtfdng cua cac cam
thu quan be mat te bao. Sau cung. phai rtia te bao 3 lan bang dung dich Hanks de
loai bd trypsin.
Phan lap te bao ttf hach bang cach nghien to chtfc hach. Cac manh to chtfc
cua hach dtfdc nghien nhe nhang qua ltfdi loc te bao bang que thuy tinh. Vtfa
nghien vtfa xoi ntfdc cho te bao da tach ra dtfdc ttf do theo ntfdc, qua mang loc
xuong binh chtfa d dtfdi. Sau khi nghien, te bao dtfdc rtia lai hai lan bang dung
dich Hanks. Bang cach nav, ty le te bao chet cung vao khoang ttf 5 - 10%.
1.4. Phan lap lympho tCflach
Lay lach: Thtfdng lay lach ttf sue vat thi nghiem, hoac co the ttf benh nhan
qua phau thuat. To chtfc lach dtfdc bao quan trong dung dich Parker co heparin
(10 ddn vi/ml), de d nhiet do 4°C.
174
- Tach te bao ra khoi to chtfc lach: tien hanh nhtf da mo ta d phan tren
(phan lap te bao ttf hach).
- Loai bo sd bo te b&o hong clu: hong clu dtfdc lam lin g sd bd b ln g dung
dich dextran 6% hoac gelatin 3% (xem p h ln phan lap lympho ttf mau).
- Loai bo hoan toan bach clu da nhan va hong clu bang cach ly tam vdi
dung dich ficoll. Ntfdc mat sau khi lam lin g hong clu d btfdc tren dtfdc ly tam vdi
dung dich ficoll (xem ph ln phan lap lympho ttf mau).
- Rtia sach te bao 3 lln b ln g dung dich Hanks, moi lln rtia ly tam 1200
vong trong 10 phut d nhiet do 4°C.
- Dem so' ltfdng lympho, xac dinh ty le song va chet cua te bao b ln g dung
dich xanh trypan.
- Pha thanh huyen dich te bao co dam dac theo yeu clu thi nghiem.
1.5. Tach lympho tLramidan

Giong nhtf tach ttf tuyen tfc, nhtfng cln chu y hai diem:
- Rtia sach tuyen bang NaCl 0,9% vi co lan mau trong qua trinh phau thuat.
- Sau khi rtia sach hong clu, c ln md rong hach, rtia cac o mu ben trong,
neu la viem amidan. Dung dich rtia nay c ln co khang sinh, nhtf penicillin 60 ddn
vi, streptomycin 60 pg/ml.
1.6. Tach lympho tirdjch xuat tiet 6 o phuc mac

Ntfdc xuat tiet d o bung thong thtfdng co the co ba loai te bao: dai thtfc bao,
lympho bao, it hong clu.
Phan lap te bao lympho cung nhtf dai thtfc bao trong ntfdc xuat tiet cua o
bung dtfdc ap dung cho cac nghien ctfu ve qua trinh mien dich hoc hoac qua man.
1.6.1. Lay nude xuat tiet

- Choc do o bung, hoac neu la sue vat thi nghiem thi giet chet roi rtia o
bung b ln g dung dich Parker.
- Dung dich chtia ntfdc xuat tiet la dung dich Parker co 1% huyet thanh be,
co heparin chong dong (moi ddn vi heparin cho 1 ml). Dich te bao lay ra dtfdc bao
quan d nhiet do lanh, tot nhat la dat lien tuc trong chau ntfdc da.
1.6.2. Ly tam phan ldp theo trong
Trong ong nghiem 20 ml, co san 5 ml dung dich ficoll, dung pipette Pasteur
nho ttf ttf hon dich te bao vao ldp ficoll (nhtf da mo ta) ly tam nhe 1200 vong trong
30 phut d 4°C. Cac dai thtfc bao va hong clu se lang xuong, con te bao lympho
nam d vung gitfa ficoll va dung dich chtfa te bao nguyen uy. Dung pipette Pasteur
lay ldp te bao nay ra, rtia hai lln bang dung dich Hanks.
Chu y: Neu dich te bao xuat tiet qua nhieu va loang thi co the co dac lai
b ln g cach ly tam 1500 vong trong 10 phut, loai bdt ntfdc mat roi th u ln nhat. Dich
dac nay se dtfdc ly tam vdi ficoll. Lam nhtf vay dd ton ficoll ma van khong anh
htfdng den ket qua.
175
Phan lap b ln g cach nay co the cung mot luc dtfdc hai loai te b&o tinh khi£t:
dai thtfc bao nam 0 phan can, lympho bao n lm 0 p h ln gitfa.
2. Phan lap cach difdi nhom lym pho (T va B lym pho) tir hdn hop te bao lym pho
2.1. Phan lap B lympho

Chuan bi hon hop te bao lympho
- Chuan bi ong te bao:
+ Moi ong cho phep xti ly khoang 100 x 106 te bao
+ Sti dung cac ong nhiia (kich thtfdc: 19,5 cm x 0,6 cm) dtfdc c it dai khoang
12 - 14 cm va han day tao goc nghieng 45°.
- Nhung 0,1 g sdi bong vao dung dich Hanks.
- Dat ong vao dung dich Hanks va nhoi bong deu dan vao long ong den
5 cm, tranh bot khi.
- Nhtfng ong nav co the de d 4°C trong mot dem hoac bao quan d - 20nC,
hai d lu ong dtfdc han lai.
- Cat dau ong de thao dung dich ra khoi ong
- Rtia lai bang 5 ml dung dich Hanks, sau do bang 3 ml R P M I co 5% huyet
thanh bao thai be (SCF = serum calf fetal).
- Dat ong thang dting, de lai mot chut moi trtfdng ngap tren ldp sdi.
- De ong d 37° trong 30 phut trtfdc khi cho te bao vao.
- Phan lap lymphocyt B
+ Dung 50 - 100 x 106 lympho toan phan pha trong 0,5 ml RPM I co 5% SCF
(pha bang pipette Pasteur co trang silicon), cho hon dich te bao vao trong ong.
+ Ddi cho ldp te bao tham het vao ldp sdi, them 0,2 ml moi trtfdng vao ben
tren ldp sdi, dat ong th in g dtfng, u 30 phut d 37°C.
+ Loai bo te bao khong ket dinh (T lympho) b ln g 5 ml dung dich Hanks
37°C (de ong nghieng tren mot ong nghiem loai 5 ml. Lam lai lan thtf hai.
+ T h u te bao ket dinh ( B lv m p h o ) b a n g cach them vao o n g 1 ml d u n g dich
R P M I co 5% S C F v a ep ldp sdi. H o n dich thu dtfdc cho vao o n g F is h e r v a rtfa hai
lan trong d u n g dich H a n k s b l n g cach ly tam 1000 g/1 phut.
2.2. Phan lap T lympho: co hai phtfdng phap

- Dung ky thuat bong: co the dung bong thuy tinh, bong y te. bong nilon, T
lympho khong dinh vao bong, chung se dtfdc thu hoach ttf dung dich rtfa bong
trong ong thi nghiem nhtf mo ta d tren. Te bao T dtfdc tap trung, co dac bang cach
ly tam lin g can te bao. Con co the phan lap T lympho b ln g phtfdng phap tao E
rosette vdi hong cau ctfu.
- Dung ky thuat khang the (K T ) ddn dong dac hieu B lympho: K y thuat
dtfdc tien hanh theo cac btfdc sau day:
176
+ G in K T v&o dia phan ting
+ C huln bi hon hop dich te bao lympho
+ Dtfa hon hop lympho co do dam dac 5 x 106 te bao vao dia phan ting, u am
37°C/30 phut, cac te bao co khang nguyen dac hieu se gan vao K T tren dia (Hinh 5.3).
+ Rtia dia thi nghiem bfing dung dich Hanks, cac te bao khong phan ting vdi
KT se dtfOc thu hoach qua dung dich rtia (Hinh 5.3).
+ Tap trung dung dich rtia, ly tam, co dac, can te bao la T lympho (Hinh 5.3)
Blympho
H5n hop
Tva Blympho
T lympho
Dia phan ong
Hinh 5.3: Nguyen ly ky thuat phan lap T lympho bang KT don dong dac hieu B lympho
KT dac hieu B lympho gan vao dTa phan ifng, B lympho phan tfng v6i KT nay bi giCf lai,
con lympho tu do se thu di/Oc bang cach rtfa dTa bang dich PBS .
3. P h an la p d a i th u c b a o
Ta c o t h e c o d tfO c d a i t h t f c b a o t i n h k h i e t b l n g c a c h p h a n l a p c h t i n g t t f
m a u , ttf n tfd c x u a t t i e t c u a c a c x o a n g n h tf o b u n g , x o a n g m u i, h o a c d ich tie t
d tfd n g h o h a p .
Co the tien hanh phan lap d tfO c ca d ngtfdi va sue vat thi nghiem. Dai thtfc
bao kho nuoi, kho giti d tfO c lau, bdi vi chung khong cd kha nang nhan len nhtf
177
lympho bao. Nhtfng nhieu thi nghiem in vitro rat can c6 huyen djch dai thtfc bao
tinh khiet nhtf: trong nghien ctfu quan he gitfa lympho va dai thtfc bao trong qua
trinh tao khang the, nghien ctfu chi so' thtfc bao dai thtfc bao da dtfdc mien dich,
nghien ctfu yeu to tfc che di tan dai thtfc bao. Do do phan lap dai thtfc bao la mot
ky thuat ttf lau da dtfOc chu y.
3.1. Phan lap dai thifc bao tCrmau

Trong mau, dai thtfc bao co rat it nen phan lap cung rat kho khan. Dtfa vao
kha nang bam kinh cua dai thtfc bao, chung ta co the tien hanh theo cac btfdc
sau day:
Budc 1: Lay 10 ml mau chong dong bang heparin. Pha loang mot khoi mau
vdi mot khoi dung dich Hanks, roi ly tam 800 vong trong 10 phut, loai bo can
hong cau, con ntfdc mat, (co dai thtfc bao, lympho bao va it hong cau) dtfdc de lang
trong binh nuoi cay te bao kieu Leighton (binh day phang va rong). LtfOng dich te
bao chtfa trong binh tuy thuoc vao binh day rong hay hep, thtfdng sau khi dtfa
dich te bao vao sao cho ldp dich do cao khoang 2 - 3 mm la dtfdc va de trong tu am
37°C trong 45 - 60 phut. Trong thdi gian nay, dai thtfc bao va cac te bao khac se
lang xuong day, rieng dai thtfc bao khi lang xuong se bam chat vao day binh.
Budc 2: Rtfa de loai bo te bao khong bam (hong cau va lympho) bang cach
trang nhe day binh 3 - 4 lan bang dung dich Hanks, cuoi cung se co mot ldp te bao
bam vao day binh. Ldp te bao nay chu yeu la dai thtfc bao, cd it lympho B (te bao
B cung cd kha nang bam kinh).
Budc 3: Tach dai thtfc bao ra khdi day binh, rtfa ldp te bao bang dung dich
Hanks cd 0,25% trypsin. Trang nhe nhang, de tu am 37°C trong 10 - 15 phut cho
ldp te bao bong ra khdi day binh. Ngay lap ttfc, dtfa nhe nhang vao binh nuoi cay
bang dung dich Hanks cd 10% huyet thanh be de tfc che tac dung tiep cua trypsin.
Budc 4: Rtia te bao de loai bd trypsin bang dung dich Hanks. Khi rtia ly tam
1200 vong/ 5 phut.
Sau lan rtia cuoi cung, can te bao dtfdc thuan nhat trong dung dich Parker
cd 20% huyet thanh be cd khang sinh penicillin va streptomycin.
Budc 5: Dem so' ltfdng te bao, xac dinh ty le song (dung dung dich xanh
trypan 0,2%).
3.2. Phan lap dai thuc bao tuchat tiet dudng ho hap
Dich tiet d dtfdng ho hap cd mot ltfOng ldn dai thtfc bao. Cd the lay cac dai
thtfc bao ttf dich nay bang cach sau day (thtfdng lam tren sue vat).
Giet thd, mo long ngtfc, lay toan bo phoi va khi quan. Khi phau tich. chu y
khong de ton thtfdng dtfdng ho hap va lam chay mau vao to chtfc phoi.
Dung kep. kep mot mep khi quan. nang len. dung pipette Pasteur lay dung
dich rtfa bdm vao khi quan (khoang 10 - 15 ml). Dung djch rtfa la dung dich
Hanks cd heparin, ctf 1 ml dung dich cd mot don vi heparin. Dung pipette Pasteur
hut len roi lai tia vao thanh khi quan. lam nhtf vay 4 - 5 lan, roi hut dich rtfa dtfa
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v&o ong nghigm ldn (20 ml), tiep tuc rtia lai nhu tren 2 - 3 lan. Dich rtia 6 moi lan
difdc tap trung lai va ly tam lay can, trong can nay co lan cac dich nhay. Pha huy
cha't nh£y bang cach cho tac dung vdi dung dich trypsin 0,25% co khua'y tti trong
15 phut d nhiet do phong.
Sau do bo sung vao dich te bao dung dich Hanks de tic che tac dung cua
trypsin. Rtia te bao hai l£n bang dung dich Hanks. Khi rtia, ly tam 1200 vdng/5
phut d nhiet do 4°C.
Sau lan rtia cuoi cung, te bao dtfdc hoa tan trong dung dich Parker co 20%
huyet thanh be, co khang sinh. Dem so' lifdng te bao, xac dinh ty le song bang
dung dich xanh trypan 0,2%.
Tach dai thtfc bao bang cach nay cung nhan dtfdc mot ltfOng ldn te bao, va ty
le song co the dat tren 85%. Muon co dai thtfc bao tinh khiet va loai bo te bao
chet, co the tiep tuc de lang trong binh nuoi ca'y day rong nhtf da mo ta.
KY THUAT KET TUA
1. K hai niem c d b an v e k e t tu a m ie n djch
Ket tua mien dich la san pham cua phan tfng khang nguyen - khang the.
Noi mot cach dung hon thi ket tua m iln dich la san pham cua mot chuoi phan
ting ke tiep nhau gitia hai phan tti: gitia mot khang nguyen da hoa tri va mot
khang the da hoa tri hoac it nhat la hai hoa tri (khang nguyen va khang the deu d
dang hoa tan). Cung nhtf phan tfng tua trong hoa hoc, de co ket tua thi phai co
chuoi phan tfng xay ra ma chuoi phan tfng muon xay ra thi can co mot phan tfng
lien ket md man ban dau. tao nen mot phtfc hdp nho ban dau con tan trong dung
moi. Phtic hop ban dau nay lam nen de chuoi phan ting tiep tuc xay ra va nhd vay
da tao ra mot phtic hdp ldn khong tan kieu “ mang ltfdi” .
Trong phan nay, chung toi trinh bay mot so' diem c0 ban ve thuyet “mang
ltfdi” nham muc dich phuc vu cho cac ky thuat cua mien dich-huyet hoc ve sau.
1.1. Thuyet mang ludi trong phan ung khang nguyen - khang the
Thuyet “ mang ltfdi” hay la thuyet mot giai doan, dtfdc M arrack gidi thieu lan
dau (1938) sau do Pauling, Pressman, Camphel tiep tuc phat trien.
Khi tron mot dung dich khang nguyen vdi mot dung dich khang the (khang
huyet thanh) dac hieu thi: dau tien phan tti khang nguyen tiep xuc vdi phan tti
khang the nhd ltfc lien ket dac hieu, bieu lo qua tinh hao va ai ltfc cua khang the.
Cac nhom quyet dinh tren hai phan tti khang nguyen va khang the phan
ting lien ket vdi nhau nhd cac ltfc nhtf ltfc tinh dien, ltfc Van der Waals, cau lien
ket hydro tao nen phan tfng dau tien cua qua trinh tua.
KT + KN K T - K N (phan tfng lan thtf nhat)
179
Phan tu khang the cung nhu phan tti khang nguyen trong phtic hdp ban dftu
nay la loai da hoa tri, cho nen con co cac hoa tri ttf do, vi v&y phtic hdp ban d&u
nay lai lien ket v6i phan tti khang the hoac khang nguyen tu do hoSc vdi mot phtic
hop ban dau khac. Do la chuoi phan ting lan thti hai:
KT - KN + KT <-» K T - K N - K T hoac
K T - K N + K T - K N <-> K T - K N - K T - K N (phan ting lan thti hai)
Chuoi p h a n t i n g l i e n ket l a n b a c u n g c o the x a y r a v d i mot l U d n g l d n c a c
p h a n tin g .
K T - K N - K T - K N + K T <-> K T - K N - K T - K N - K T
K T - K N - K T + K T - K N - K T <-» K T - K N - K T
KT - KN - KT
(p h a n ting la n thti b a ) - theo Steffen (1968)
C h u o i p h a n ting la n thti b a n a y x u a t h ie n t r o n g trUOng hop k h a n g the

thtia, n g h ia la k hi co q u a n h ie u p h a n tti k h a n g the thtia, tu do so vdi p h a n tti
k h a n g ngu ye n .
Neu n o n g d o p h a n t t i k h a n g n g u y e n v a k h a n g t h e t U d n g d U O n g n h a u t h i c h i
x a y ra p h a n tin g la n th ti n h a t , roi s a u d o x a y ra p h a n tin g k ie u t r u n g h o p cac
p h t i c h o p dUOc t a o t h a n h 6 p h a n t i n g l a n t h t i n h a t .
T r o n g trUOng hop k h a n g n g u y e n thtia thi k h a n a n g lien ket d p h a n ting l&n

thti hai k em hon n h ieu so vdi k hi k h a n g the thtia. Phtic hop dUdc tao th a n h de bi
huy hoai vi cac moi lien ket k em b e n vting. K h i k h a n g n g u y e n q u a thtia thi xuat
hien cac phtic hop nho hoa tan. N h u v a y thu yet m a n g ludi da co mot ting dung
thuc tien tron g viec giai thich cac p h a n ting tu a c u n g n h u p h a n ting n g U n g ket
m i l n dich. K h a n g the co tti h a i hoa tri trd len mdi co k h a n a n g tao dUdc m a n g ludi
n g U n g ket vdi k h a n g n g u y e n ; k h a n g the mot h o a tri ( K T k h o n g h o a n toa n ) khong
co k h a n a n g tao dUOc m a n g ludi n g U n g ket vdi k h a n g ngu ye n , n g h ia la k h o n g tao
dUdc p h a n ting tu a v a p h a n ting n g U n g ket m ien dich.
1.2. Cac dudng bieu d iin ket tua

1.2.1. Dudng bieu di§n H eidelberger
H e id e lb e r g e r v a K e n d a l la nhting ngUdi d a u tien neu r a p h a n ting ket tua

dinh lUdng v a da d u a r a nhting k hai niem ve dong hoc cua p h a n ting k h a n g nguyen
- k h a n g the, ty le gitia k h a n g n gu yen v a k h a n g the tron g phtic hop m ien dich cung
n h u ve so nhom quyet dinh cua mot p h a n tti k h a n g nguyen. N h tin g hieu biet ve stf
khac n h a u trong q u a trinh p h a n ting dinh ltfdng tua cd mot y n gh ia q u a n trong ve
cac hien ttfdng b en h ly m ien dich. dac biet la q u a m an nhanh.
Khang the cd kha nang ket tua vdi khang nguyen goi la ket tua to', he thong
phan ting lien ket khang nguyen - khang the goi la he thong ket tua.
Ky thuat dinh ltfdng phan ting ket tua nay bao gom:
180
- Cho v& o c a c o n g n g h i e m m o t n o n g d o k h a n g t h e x a c d i n h r o i c h o v a o d d
lt f O n g k h a n g n g u y e n t a n g d £ n l e n , p h a n t f n g k h a n g n g u y e n - k h a n g t h e s e x a y r a .
Tua d tfd c l o a i tr tf b a n g l y t a m r o i t i e n h a n h x e t n g h i e m k h a n g n g u y e n ttf d o c u n g
n h t f k h a n g t h e ttf d o c u a d u n g d i c h t r o n g 0 t r e n .
- 0 o ng n gh iem thtf nh&'t, nong do k h a n g n g u y e n thap, cho nen cdn thay

k h a n g the cdn lai d p h ^ n d u n g dich trong. D o dd goi la o n g n g h iem cd k h a n g the
thtfa, tron g llnh vtfc mien dich thi goi la v u n g k h a n g the thtfa.
- O cac ong n ghiem tiep theo, do t a n g d a n ltfOng k h a n g n g u y e n nen ltfOng

k h ang the ttf do cua d u n g dich tron g d tren lien tuc gia m d a n v a den mot ong
nghiem ma d dd k h a n g n g u y e n v a k h a n g the ttfOng dtfOng nh au , tat ca cac k h a n g
the lien ket vdi cac k h a n g n g u y e n lam cho d u n g dich tron g d tren k h o n g cdn
k ha n g the va k h a n g n g u y e n ttf do, n g h ia la ket tua h o an toan. C h in h d ong
nghiem nay cd ltfOng tu a ldn nhat, phtfc hcfp tua ben vtfng. Ngtfdi ta goi v u n g nay
la v u n g ttfOng dtfOng.
- C) c a c d n g n g h i e m t i e p t h e o ( s a u o n g t t f O n g d t f O n g ) v a n t i e p t u c c h o t a n g
d a n lt f O n g k h a n g n g u y e n l e n t h i t h a y l t f O n g t u a g i a m x u o n g v a p h a n d u n g d ich
t r o n g d t r e n x u a t h i e n k h a n g n g u y e n t t f d o , d tfO c g o i l a v u n g k h a n g n g u y e n t h t f a .
0 d ay n o n g do k h a n g th e k h d n g d u d e lie n k e t vdi ta t ca k h a n g n g u y e n ch o n e n d a
tao n e n ca c p h tfc h o p n h d k h a n g n g u y e n - k h a n g t h e h o a ta n .
Ttf c a c ket q u a tren day c d the bieu dien tron g mot he thong toa do: true
hoanh la nong do k h a n g n g u y e n ta n g d a n ttf trai s a n g phai, true t u n g la ltfdng tua
phat hien. Dtfdng bieu dien n ay dtfO c goi la dtfdng bieu dien tua h a y la dtfdng bieu
dien H e id elb erge r.
N g a n h len cu a dtfdng b ie u dien H e i d e l b e r g e r cho thay ltfOng tua ty le th u an

vdi k h a n g nguyen. D i n h cu a dtfdng b ieu dien la v u n g ttfOng dtfOng k h a n g n g u y e n -
k h ang the. N g a n h x u o n g cua dtfdng bieu dien la ltfdng tua g ia m d a n vdi stf ta n g
nong do k h a n g n g u y e n vi x u a t hien phtfc hop m ien dich hoa tan.
1.2.2. Cac loai dudng bieu d iin ket tua khac

Dtfdng bieu dien tua khac n h a u tuy theo n g u on goc cua hu y et th a n h mien
dich va dtfOc xep t h a n h hai nhom: nhom k h a n g hu y et th a n h thd v a nhom k h a n g
huyet th an h ngtfa.
T r o n g nhom k h a n g thu y et th a n h thd, hien ttfOng tfc che ket tu a chi x ay r a d

vu n g k h a n g n g u y e n thtfa, cdn d v u n g k h a n g the thtfa v a v u n g ttfOng dtfOng thi
hien ttfdng ket tu a x a y r a theo ty le thuan . NgtfOc lai, tro n g nh om k h a n g huyet
thanh ngtfa, hie n ttfOng tfc che ket tu a k h d n g nhtfng chi x a y r a d v u n g k h a n g
nguyen thtfa m a n g a y ca v u n g k h a n g the thtfa. Q u a d a y cho th a y h u y et th a n h
m i ln dich thuoc nh om ngtfa it thich hdp cho hoa m ien dich d in h ltfdng tua. T r o n g
khi sii d u n g cac loai hu y et th a n h m ien dich dac biet, can chu y den dac diem nay
va vdi moi loai can tim v a sii d u n g lieu ltfdng toi tfu tro n g k h u vtfc ttfOng dtfdng.
1.3. Cau tao cua ph ut hop tua

C a u tao cu a phtfc hdp tua p h u thuoc trtfdc het vao p h a n tfng k h a n g n g u y e n -
k h a n g the v a he thong k h a n g n g u y e n - k h a n g the. T r o n g trtfdng hdp k h a n g
181
ngu yen q u a thtia thi x u a t hien phtic hdp nho ho& tan, phtic hdp n a y chi gom hai
p h a n tti k h a n g n g u y e n lien ket vdi mot p h a n tti k h a n g the.
N h u vay, tro n g trUOng hop k h a n g the thtia thi mot so ldn cac nh dm quy£t
dinh cua p h a n tti k h a n g n g u y e n se lien ket vdi cac p h a n tti k h a n g the, trong
trUOng hdp tUdng d u d n g thi con nh ieu nhom quyet din h cu a p h a n tti k h a n g
n g u y e n con tu do (k h o n g lien ket vdi k h a n g the), tron g trUdng hdp k h a n g nguyen
thtia thi hai nhom quyet din h cua hai p h a n tti k h a n g n g u y e n lien k et vdi mot
p h a n tti k h a n g the ( K N - K T - K N ) . T r o n g thuc te, de dat dUdc sii tUdng dUOng
gitia k h a n g n g u y e n v a k h a n g the (ket tua ho an toan), neu cho d a n d i n it mot
k h a n g n g u y e n vao thi tong lUdng k h a n g n g u y e n can thiet it hdn tong lUdng khang
n g u ye n khi ta chi cho vao mot la n (hien tUdng D a n y s z ). H ie n tUdng do dUdc giai
thich la khi cho d a n d a n it mot k h a n g n g u y e n v ao thi da so cac nh om quyet dinh
ctia p h a n tti k h a n g n g u y e n da hoa tri lien ket vdi cac p h a n tti k h a n g the, cho nen
lUdng k h a n g n g u y e n can thiet de ket ttia ho an toan doi hoi it hdn. N g o a i ra, do
cho vao it mot k h a n g n g u y e n n h u vay nen k h o n g co mot so k h a n g n g u y e n bi bao
v ay trong m a n g ludi lien ket n h u khi cho vao mot lan.
D i e u n a y c o m o t y n g h i a q u a n t r o n g d o i v d i p h a n t i n g d o c t o v a k h a n g d o c to.
N eu dem t r o n n g a y m o t l a n m o t l U d n g d o c to' v d i m o t l U d n g k h a n g d o c t o tU O n g
dUO ng t h i k h o n g c o n d oc, n h u n g n e u ta c h ia lU d n g d o c to a y ra n h ie u p h i n n h o va
t r o n d a n v d i k h a n g d o c to' t h i c o n d o c v i c o n d o c to' t u d o c h U a b i l i e n k e t .
Jt.4. Qua trinh phat trien cua ky thuat ket tua

1.4.1 Ket tua d m oi trifdng long
P h a n ting ket tu a m ien dich dUdc K r a u s mo ta n a m 1897 n h u n g luc do chi
tien h a n h d moi trUOng long, ttic la tron d u n g dich k h a n g n g u y e n vdi d u n g dich
k h a n g the t r o n g o n g n g h ie m . P hUOng p h a p tron ddn g i a n n a y da p h a t h ie n dUdc
s a n p h a m p h a n ting m i l n dich m a nien dich vi k h u i n d a d a u tien ting d u n g de
p h a t hien k h a n g the. U h l e n h u t h (1927) d a p h a t t rie n ky t h u a t k et tu a d moi
trUOng lo n g n a y vdi ky t h u a t ket tu a v o n g ( n h a n ) don g ia n . S a u do, n h ie u tac gia
da ting d u n g p h u o n g p h a p n a y vdi n h ie u he t h o n g k h a n g n g u y e n - k h a n g the
k h a c n h au .
1.4.2. Ket tua d m di truong gel

K e t ttia tron g moi trUdng gel h a y la mot tro n g moi trU dng co gia do. la mot
budc p h at trien q u a n tro n g cu a ky t h u a t ket tu a m ien dich. N a m 1905. Bechhold
mo ta mot p h a n ting cd n h ie u dUOng n g U n g k et k hi cho hu y et th a n h de (k h a n g
n g u y e n ) .......t a n v a o m o i t r U d n g g e l d a c d c h t i a k h a n g h u y e t t h a n h d a c h i e u ( h u y e t
th a n h chong huyet t h a n h de toan p h a n ). 40 n a m sau, ky th u at khech tan tua
tron g gel dUOc ho an chinh. C a c th a n h p h a n th a m gia p h a n ting dUdc k h u e c h tan
tron g gel n en goi la k h u e ch tan m ien dich, m at khac s an p h i m cua p h a n ting lai
ket tu a v a g a n chat tro n g gel nen dUdc goi la ket tua mien dich. G e l th ach la loai
thong dung, ngoai r a cdn cd the d u n g gelatin, pectin, po ly acry lam it, silicage... Su
ph at trien cua p h u d n g p h a p tua tron g gel da lam ra ddi mot loat cac ky thuat:
O u d in , O u ch te rlon y, M a n c in i. dien di mien dich, dien di mien dich p h o n g xa. S a u
day la mot so mo h in h ket tua th u d n g dUdc sti d u n g hien n a y ( H i n h 5.4 d e n 5.7).
182
2. M o t s o y e u to a n h h i/d n g va c a c d ie u k ie n q u y d jn h doi vd i q u a trin h
k h u e c h ta n va k e t tu a
2.1. Khuech tan
2.1.1. Dinh luat khuech tan doi vdi khuech tan mien dich
Khuech tan mien dich tuan theo nguyen ly cac dinh luat khuech tan cua cac
chat hoa tan.
Su k h u e c h t a n t r o n g g e l t i n h k h i e t c o to 'c d o g a n g i o n g n h u t r o n g m o i t r U d n g
l o n g v i l u c d o t h a n h p h a n t h a m g i a p h a n U n g ( k h a n g n g u y e n k h a n g t h e ) dUOc c o i
la n h t i n g c a u t t i h i n h t r o n , t r o n g l U O n g p h a n t t i b e , c o n m o i t r U d n g g e l d U O c c o i
n h u m o t m a n g ludi v d i m a t ludi t o .
2.1.2. Nhiet do va thdi gian khuech tan

Khang huyet thanh thd khuech tan va tua tot d 37°C va 4°C. Khang huyet
thanh cua cac chung loai khac (ngUa) chi khuech tan va tua tot d 37°C. O 37°C, toe
do khuech tan tUOng doi nhanh thich hop vdi nhting xet nghiem doi hdi cd ket qua
sdm. NhUng toe do khuech tan nhanh lai anh hudng den qua trinh ket tua. Do do
nhiet do va thdi gian khuech tan quy dinh nhu sau: huyet thanh cd hieu gia
khang the cao thi de 1 gid d 37°C va 24-48 gid d 4°C, huyet thanh co hieu gia
khang the thap thi can keo dai 4-8 ngay d 4°C. Khi c&n keo dai thdi gian thi phai
cho them chat chong vi khuan (methiolat, natriumazid).
2.1.3. M ot so yeu to khac

a. Tinh chat cua khang nguyen vd khdng the.
Hinh 5.4: Mien djch tua-khuech tan hai chieu

Trong phan Ung gom co: Thach long co khang the dac hieu, tren dia phan ifng due 3 lo,
cac lo nay dat khang nguyen dinh tim, Khi khuech tan hai chieu KT va KN gap nhau
se phan ting va tao thanh vach tua tren moi trUdng thach.
A: Phan Ung (+) vdi 1 khang nguyen (anti-1) 2 dudng phan Ung khep kin
B: Phan Ung (+) vdi 3 khang nguyen (anti 1,2,4): Ket qua khong khancj dinh dUdc khang nguyen nao
C: Phan Ung (+) vdi 2 khang nguyen, trUdng hdp nay cung khong khang dinh (anti 1,2).
183
Ky thuat nay kha chuan, co the dung danh gia do tinh khiet cua K N khi c6
K T dOn dac hieu.
A B
Hinh 5.5: Phan ifng tua dang “ R ocket” (Dien di kieu Rocket)
A: So do tua dang Rocket, tren phieu thach co khang the dac hieu.
Trong cac 16 due tren phien thach co chifa KN (Ag), KN se khuech
tan vao moi trifdng thach co KT dac hieu va tao tua mau trying due
nhif hinh qua Rocket do do goi la dien di Rocket.
B: Anh that cua tua Rocket
K T d on dong
d ac hi6u vdi K N
K T da gia phan ifng

voi huyet thanh ngaoi
Hinh 5.6: Phan Crng tua dang dien di mi§n dich

Tren day la hinh anh dien di mien dich: Tren phien thach long co cac lo va ranh dai doc
theo phien. Trong lo co KN, chay dien di de cac thanh phan KN dan trai doc phien thach,
lay bo thach o ranh, sau do cho KT vao ranh. KT khuech tan va phan ting voi KN dac
hieu. Day la ky thuat ket hop gitia dien di va khuech tan.
184
Trong 16 co khang nguyen
1 II. .. lOJi _t1----■■1 1
•% fg lff Vo ng phan
img tua
Thach I6ng co khang th i
CVs/
K hang th i phan ung
1- ■'•
:;
vOi cac do dam dac
5
’'>*. > r< t ■ khac nhau cua K N
B
Hinh 5.7: Phan ting tua dang mien dich khuech tan vong (Mancini test).
Khang the dSc hieu hoa tron trong moi trUdng thach agar, tren do due cac 16 co kich
thudc giong nhau, d3t vao cac 16 KN co do pha loang khac nhau (thi du pha loang
10 lan, 25, 50, 100). DTa phan ting de nhiet do 37°C/24-72 gid. Doc ket qua bang
cach do dudng kinh vong khuech tan, ve do thi chuan ve nong do pha loang KN.
Khi kiem tra m iu khang nguyen bat ky, can sir dung nong do chuan, tti do tinh ra
nong do cua mau kie’ m tra tUOng ting.
A: Hinh anh khuech tan theo nong do khac nhau cua KN

B: Do thi chuan ve nong do KN pha loang, sir dung djnh ltfOng nong do KN hoSc KT.
Dac tmh mien dich, nong do, hieu gia khang the ddng vai trd quan trong doi
vdi qua trinh khuech tan va ket tua. Ngoai ra qua trinh khuech tan cdn bi anh
hudng bdi cac yeu to':
(1) Trong lu 'O n g p h a n tti: Trong l U O n g p h a n t t i c a n g c a o t h i k h u e c h t a n c a n g
c h a m v a ngUO c la i.
(2) Hinh dang phan tti: Phan tti hinh tron khuech tan nhanh hon phan tti
hinh bau due. hinh thoi...
185
b. Tinh chat cua gel.
Gel thach la loai thong dung doi vdi phan ting ket tua.
Do tinh khiet cu a thach. K h u e c h tan cua k h a n g n g u y e n v& k h a n g the cd deu
d a n h a y khdng, trUdc het p h u thuoc vao do tinh khiet cu a thach. T h a c h ( a g a r )
binh th u dn g chtia n h ieu ion, S 0 4” , M g+\ ... la m t a n g tac d u n g dien t h a m v a de
lien ket hoa hoc vdi protein. Do dd cd mot so' trUdng hop q u a trin h k h u e c h tan bi
can trd, p h a n ting ket tua a m tinh bdi vi thach k h o n g dUOc tinh khiet. T a c du n g
nay cua thach h ay ga p doi vdi lipoprotein, c h u n g p h a n ting vdi nh om s u lfo n in dua
den hien tUOng tu a tu p h at l&ng xuong. Do dd hien n a y tren thi trU d n g da cd loai
a g a r o z a la thach da dUOc loai trti M g++, C a ++, r a t tot cho cong tac hoa m ien dich
ket tua, dac biet tot doi vdi dien di m ien dich vi la m g ia m hien tUOng noi th&'m.
Doi vdi thach khac, mot so' tac gia k h u y e n nen xti ly trUdc k hi d u n g b& ng cach cho
nUdc cat chay qua thach mot thdi gian, hoac rtia b&ng ether, hoac ethanol, ho&c
dien phan.
Nong do thach trong dem. Nong do thach thich hop cho khuech tan va ket
tua tti 0,3-2%. Neu nong do thach nhd hon 0,3% thi cac phan tti tham gia phan
ting khuech tan nhanh, khdng gan chat dUOc cac san pham phan ting, do dd khd
khan cho viec danh gia ket qua. Neu nong do thach tren 2% thi khuech tan ch&m,
thdi gian khech tan va tua keo dai, anh hudng den qua trinh hinh thanh tua.
c. Do am vd mat phang khuech tan
- Do &m: Trong qua trinh khuech tan va ket tua phai giti bao hoa hoi nUdc,
nghia la phai giti do am tot, neu khdng thi thach se khd lam cho qua trinh khuech
tan khd khan hoac dinh tre.
- M at phSng khuech tan: M at p h in g khuech tan phai ph&ng va deu, khdng
dUOc go ghe, cho day cho mong anh hudng den qua trinh khuech tan. Do dd trong
khi do thach cung nhu ca thdi gian khuech tan, phai luon luon giti d mat phang
thang bang.
2.2. K it tua
De qua trinh ket tua xay ra tot thi phai chu y den mot so' dieu kien nha't dinh.
2.2.1. pH
Qua trinh ket tua thudng xay ra tot d pH 6,5 - 8,5. Tuy theo he thong nghien
ctiu ma mot so tac gia thay qua trinh ket tua cd the xay ra d pham vi pH tti 4-12,
song lUOng tua giam ro ret khi pH qua thay doi (acid hoac kiem qua).
2.2.2. Dung djch khang the va khang nguyen
Dung dich khang the phai trong, khdng cd lipid. Neu dung dich khang the
van due hoac cd lipid thi phai loai trti b4ng cach ly tam gan bd.
Dung dich khang nguyen phai cd pH trung tinh on dinh.
2.2.3. Nong do muoi
Qua trinh ket tua tot d nong do muoi 0.15M. Nong do N aC l va KC1 cd the
tang den 1,5M vi It anh hudng den qua trinh ket tua. NhUng neu nong do muoi
186
qua cao (N aC l 15%) ket hdp vdi nhiet do tang thi ket tua bi phan ly. Kleinschmidt
nghien ctiu anh htfdng cua cac anion trong cac muoi KC1, KBr, Kl... doi vdi qua
trinh ket tua, th&y ket tua giam ro khi nong do cac ch&'t cao hon 0,15M. Nhiing
dung dich khong co muoi cung lam giam ket tua hoac khong co ket tua mac d&u
pH =7 vk v&n co phan ting khang nguyen - khang the. Khang huyet thanh chim la
loai dac biet ngoai le, ket tua xay ra 0 nong do NaC l 8-10%. 0 nong do 8-10%,
hien ttfdng ket tua chi xay ra doi vdi khang the thuoc IgG, cdn IgM chi ket tua d
nong do muoi 0,15M.
2.2.4. KhCf hoat b 6 thS

Bo’ the lien ket vdi phtic hdp khang nguyen - khang the cd the xay ra trong
qua trinh ket tua hay la sau khi u vdi huyet thanh ttfdi. Bo the lien ket trong
phtic hdp khang nguyen - khang the khdng bi loai trti trong khi rtia, mat khac no
lam roi loan phan ting ket tua dinh tmh cung nhtf dinh ltfdng. Do dd c ln khti hoat
bo’ the trtfdc khi tien hanh thi nghiem (de 30 den 60 phut d 56°C). Khang huyet
thanh cd hieu qua thap thi hieu gia cang giam khi khti hoat bo the, cho nen ngtfdi
ta khuyen nen hap phu bo’ the cua huyet thanh nay vdi mot lUdng ket tua khac
loai (vdi san pham cua mot khang huyet thanh khac khti hoat). K et tua trong mdi
trtfdng cd 0,01 ethylen diamin tetraacetic se han che stf lien ket cua bo the tren
phtfc hop khang nguyen - khang the.
3. Ket tua djnh tinh

3.1. Thu"nghiem chuan bj
Dinh tinh nhfim muc dich danh gia so bo phan tfng khang nguyen - khang
the ma d day la danh gia khang the ket tua trong khang huyet thanh. De danh
gia dtfdc chinh xac, can cd thti nghiem chuan bi trtfdc nhfim sd bo danh gia ltfdng
khang the ket tua trong khang huyet thanh mot cach nhanh chong.
3.1.1. Dung cu, hoa chat. Can cd:
- Ong nghiem nhd.

/s'
- Ong hut chia do.
- Khang nguyen.
B a n g 5.7: Ty le KN /KT trong ky thuat tua dinh tinh
Ong thur Ong chtfng 1 Ong chtfng 2

Khang huyet thanh 0,2-0,5ml 0,2-0,5ml
Khang nguyen 10-15ng (trong 0 ,1 -0 ,2 ml 10-15ng (trong 0,1- 0,2ml
NaCl 0,9%) NaCl 0,9%)
Huyet thanh binh thudng 0 , 1 - 0 ,2 ml 0 , 1 - 0 ,2 ml
ho3c NaCl 0,9%
- Khang huyet thanh.

- H u y e t t h a n h b in h thtfdng.
- N aC l 0.9%) trong ntfdc cat.
187
3.1.2. Tien hanh
Tat ca cac ong de 6 37°C trong 30-60 phut.
3.1.3. Doc ket qua

So sanh vdi ong chtfng
Ong thti co ttia ro: Khang huyet thanh co khang the ket tua vdi khang nguyen.
Tua khdng ro ma dung dich due thi ly tam 2000-3000 vong/phut trong 30
phut roi doc ket qua.
Khdng cd tua, dung dich due khdng ro thi de tiep 0 4°C qua mot dem. Horn
sau lay ra ly tam nhif tren va doc ket qua.
S a u khi ly tam nhif tren m a v a n k h d n g cd tu a (k h d n g cd h ie n ttfOng ket hop
k h a n g n g u ye n - k h a n g the) thi cd the n g h i den hien ttfOng tic che ket tua do
k h a n g n g u ye n thtia hoac hieu gia k h a n g the q u a thap. T r o n g nhting trUdng hop
nay, can tien h a n h thti n g h iem vdi ltfOng k h a n g n g u y e n nhd (2-10 ng k h a n g
ngu yen cho l m l k h a n g hu y et th a n h ), trifdng hop c l n thiet cd the t a n g the tich
k h a n g huyet th a n h len 2-3ml.
Trong thti nghiem chuan bi nay, cd the tien hanh thti nghiem xac dinh hien
ttfOng khang nguyen thtfa, hoac khang the thtfa b in g cach chia p h ln dung dich
trong d tren lam hai phan, mot phan tron vdi 5-10 microgam khang nguyen (trong
0,1ml ntfdc muoi sinh ly), cdn phan kia tron vdi 0,l-0,2ml khang huyet thanh).
Cung de 37°C trong 30-60 phut va ly tam nhtf tren. Neu cd ket tua d ong thti
nhat la cd khang the thtia, neu ket tua d dng thti hai la khang nguyen thtia. Kinh
nghiem cho thay, de doc ket qua chinh xac thi nen soi ong nghiem tren nen den.
3.2. Ket tua vdng don gian (theo Steffen 1968)

3.2.1. Nguyen ly
Phan ting ket tua xuat hien sdm nhat d mat tiep xuc cua dung dich khang
nguyen va dung dich khang the va vdi dien tiep xuc hinh tron se cho mot vdng
ket tua.
3.2.2. Dung cu va nguyen lieu

- Ong nghiem nhd, ong hut Pasteur
- Khang huyet thanh va khang nguyen.
3.2.3. Tien hanh
Cho 0,5ml khang huyet thanh vao ong nghiem. Sau dd dung ong hut
Pasteur cho can than va nhe nhang 0,5ml dung dich khang nguyen (nong do
khang nguyen 0,5-5mg/ml) len tren be mat dung dich khang huyet thanh. khdng
dtfOc xao tron. De d nhiet do cua phong.
3.2.4. Doc ket qua
Doc ket qua sau 30 phut, 60 phut, 120 phut hoac 240 phut. Vung tiep xtic
gitia khang huyet thanh va dung dich khang nguyen xuat hien mot vanh due ro:
phan ting dtfOng tinh. Neu khdng cd vanh due ro: phan tfng am tinh.
188
Khong nen doc cham hdn thdi gian tren vi vdng ngtfng ket se bi xoa nhoa boi
hien ttfdng khuech tan.
Chu y: Roi loan ngang ket do hien ttfgng khang nguyen thtia trong thi
nghiem n&y hoan toan khdng lo ngai. The tich dung dich khang nguyen khdng
dtfOc ldn hOn the tich khang huyet thanh. Khi cho dung dich khang nguyen vao,
nen ty nh<? d lu ong hut Pasteur vao thanh ong nghiem g in mat dung dich khang
huyet thanh va cho chay tti tti nhe nhang theo chu vi ong nghiem. Ket qua vdng
don gian cd the cai tien bang cach tien hanh trong cac ong mao quan.
4. Ket tua djnh ltfdng

4.1. Ban djnh lupng hieu gia khdng the, khang nguyen
Xac dinh hieu gia khang the, khang nguyen dtfa tren hai phuong phap cd ban:
4.1.1. Phi/ong phap alpha (ky thuat Dean va Webb)

Cho vao moi ong nghiem ltfdng khang the bang nhau xac dinh, sau dd
cho lan ltfdt tti dng thti nhat trd di mot the tich khang nguyen cd do pha loang
tang dan.
4.1.2. Phi/ong phap beta (ky thuat Ramon)

Cho vao mdi ong nghiem ltfdng khang nguyen xac dinh bang nhau, sau dd cho
lan ltfOt tti ong thti nhat trd di mot the tich khang the cd do pha loang tang dan.
Hieu gia chinh la do pha loang cua ong cd ket tua rd.
Thi du: Xac dinh hieu gia cua huyet thanh m iln dich (khang mot khang
nguyen nao dd) ta cd the tien hanh nhu sau:
Neu ket tua xuat hien ro d dng so' 9 chang han thi hieu gia cua khang huyet
thanh mien dich nay la 1: 512. Tuy nhien stf ket tua dau tien (ty le khang the/
khang nguyen) cdn phu thuoc vao nong do chung cua hdn hop khang nguyen
khang the. PhtfOng phap tren day cd nhanh chong cho ket qua song cd the bi sai
lac bdi hien ttfOng ket tua khdng dac hieu nhtf phan ting cheo cua cac khang
nguyen doi vdi khang the. V i the, de xac dinh hieu gia cua khang the va khang
nguyen, dong thdi giup phat hien cac phan ting cheo hoac stf tinh khiet cua khang
nguyen, khang the thi ngtfdi ta thtfdng dung phtfdng phap ngtfng ket trong gel (ky
thuat Ouchter lony) se trinh bay dtfdi day.
Bang 5.8: Ty le pha loang KT trong ky thuat tua djnh Itfpng
Ong nghiem 1 2 3 4 5 6 7 8 9 10 11 12
Dung djch khang nguyen 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2 0,2
5mg/ml
0,2ml dung dich khang 11:2 11:4 1:8 1:16 11:32 11:64 1:128 1:256 1:512 1:1024 1:2048 1:4096
the cd do pha loang
4.2. Cac phuong phap ket tua djnh lupng

Ly thuyet ve dtfdng bieu d iln ket tua cua H eidelberger va Kendall da chi ro,
phan ting ket tua xay ra tot nhat d vung ttfdng dtfdng gitia khang nguyen va
189
k h a n g the. Do d o c o m o t so ' t a c g i a t r t f d c k h i d i n h l t f d n g k e t t u a , c o n t i e n h a n h t h t f
n g h i e m cO b a n d e x a c d i n h v u n g t t f O n g d t f d n g c u a k h a n g n g u y e n . k h a n g t h e .
4.2.1. ThCf nghiem chuan bi
De x a c d i n h v u n g t t f O n g d t f O n g , c h o v a o m o i o n g n g h i e m 0,4ml d u n g d i c h
k h a n g n g u y e n co n o n g do ta n g d a n (50, 100, 200, 400 p . g / m l ; c o t h e t a n g d e n 2000-
3000 | i g / m l ) v a d u n g d i c h k h a n g t h e c o n o n g d o c o d i n h (0,4ml).
De tat c a c a c o n g n g h i e m 0 37°C t r o n g 60 p h u t va q u a m o t d e m d 4°C. Cac
o n g n g h i e m d tfO c l y t a m 30 p h u t vdi to 'c d o 2000-3000 v o n g / p h u t , r o i xac d i n h
k h a n g n g u y e n t h t f a va k h a n g t h e t h t f a c u a d u n g d i c h t r o n g n t f d c n o i d t r e n .
O n g n a o co ltfd n g k e t t u a n h ie u n h a 't, k h o n g c o n khang nguyen t h t f a v&

k h a n g t h e t h t fa d p h a n d u n g d ic h d t r e n n tfa , t h i d o la p h a m v i ttfO n g d tfd n g c u a
k h a n g n g u y e n , k h a n g th e.
4.2.2. ThCf nghiem chinh thifc
Dinh ltfdng ltfdng ket tua cua mot he thong phan tfng khang nguyen khang
the doi hoi khang huyet thanh phai co chat ltfdng cao. Tong the tich cua hon hdp
khong qua 3-4ml. Do'i vdi khang huyet thanh co hieu gia thap thi phai keo dai
thdi gian ket tua 6-8 ngay 0 4°C va co the cho tang the tich khang the.
a. Nguyen ly cua phitcmg phap
Ket tua dtfOc tao thanh sau phan tfng khang nguyen - khang the, dtfdc rtfa
vdi dung dich NaCl 0,9% lanh va hoa tan trong NaO H 0,1N roi tien hanh dinh
ltfOng protein cua tua bang phtfdng phap vi Kjeldahl, phan tfng Folin hoac phtfOng
phap do mat do quang 0 btfdc song 280nm.
b. Dung cu, hod chat
- Ong nghiem. ong hut chia do

- Khang huyet thanh, khang nguyen
- NaCl 0.9%, NaOH 0.1N

c. Tien hanh
- Chu&n bi khang huyet thanh va khang nguyen:
+ Khtf hoat bo the cua khang huyet thanh (30 phut d 56°C).
+ Pha cac dung dich khang nguyen 5-10% trong ntfdc muo'i sinh ly (0.9%)
- Phan tfng ket tua:
Ong thtf Ong chtfng
+ Khang huyet thanh khtf hoat 0,4ml 0,4ml
+ Dung dich khang nguyen 0.4ml

+ NaCl 0.9% 0,4ml
190
C6 th i tien h&nh them mot ong chting thti hai: 0,4ml dung dich khang nguyen +
0,4ml NaCl 0,9% de xem co hien ttfdng ket tua lam sai lech ket qua khong.
De ta't ca cac ong d 37°C trong 30 phut va 24-48 gid d 4°C. Trong thdi gian do
thinh thoang phai lie nhe.
4.2.3. RCra va hoa tan tda

Cac ong dtfcfc ly tam 2000 - 3000 vong/phut x 30 phut. Nhe nhang c h it phln
dung dich trong sang mot ong nghiem khac de kiem tra khang nguyen, khang the
thtfa. P h ln can d tfO c rtia 2-3 lln vdi NaCl 0,9% lanh, moi lln hoa can vdi l-2m l
NaCl 0,9% lanh, ly tam va c h it bo p h ln dung dich trong 0 tren.
Hoa can vdi NaO H 0,1N de tien hanh dinh ltfOng. D lu tien cho 0,5ml NaOH
0,1N hoa can, sau do cho them d in de tong the tich khoang 2-3ml.
4.2.4. Djnh liftin g protein ttia

Sau khi hoa tan can, co the tien hanh dinh ltfdng protein cua tua bang cach:
- Dinh ltfdng nitd cua tua bang phtfdng phap vi Kjeldahl. Ttf ltfdng nitd tinh
dtfdc ltfdng protein bang cach nhan vdi 6,25. PhtfOng phap nay chinh xac nhtfng
phien phtfc va mat thdi gian, nen chi de tien hanh vdi chuan.
- Phan tfng Folin, kha nhay nhtfng mat do quang thay doi doi vdi cac
protein khac nhau, khac biet tdi 50%. Do do phai tien hanh song song vdi chuln
khang nguyen, khang the cung loai de dieu chinh ket qua.
- PhtfOng phap do mat do quang d btfdc song 280mm. PhtfOng phap nay don
gian nhtfng he so' mat do quang cung thay doi tuy theo loai protein.
Thi du: E 1% d pH = 7 thi doi vdi albumin d btfdc song 280nm la 5,8, doi vdi
gamma globulin la 13,8... Do mat do quang so vdi ong trln g la NaO H 0,1N.
4.2.5. Kiem tra dung djch trong d tren

Khang nguyen thtia Khang the thtia
Dung dich trong 0,3ml 0,3ml
Khang huyet thanh 0,1ml
Khang nguyen 5-10 ng
(trong 0,1ml N aC l 0,9%)
Neu khong co khang nguyen thtia va khang the thtia d p h ln dung dich trong
la qua trinh dinh ltfdng da tien hanh tot d vung ttfOng dtfOng, ket qua dam bao
chinh xac. Neu co khang nguyen thtia hoac khang the thtia, phai dieu chinh lai.
4.2.6. ifn g dung cua phuong phap

T in h d tfd c ltfO n g k h a n g t h e n g t f n g k e t t r o n g k h a n g h u y e t t h a n h ( l a y t o n g
ltfO n g t u a t r t i d i l t f d n g k h a n g n g u y e n c h o v a o d a x a c d i n h t r tfd c ).
Tinh dtfdc ty le khang nguyen/khang the d vung ttfdng dtfdng hoac vung
khang the thtfa. Dat co sd cho viec dieu che phtfc hdp khang nguyen, khang the co
hoat tinh sinh vat hoc.
191
Phtfdng phap dtfdc ap dung de dinh ltfdng khang doc to va doc to vi khuan,
co the ap dung nghien ctfu benh ly thtfc vat va thu y. (Khang nguyen la cac cha't
chiet xuat, khang the la huyet thanh dong vat dtfdc gay m ln cam bang khang
nguyen ttfdng tfng).
5. Ket tua trong gel

K e t t u a m i l n d i c h t r o n g g e l l a h i e n ttfO n g k h a n g n g u y e n , k h a n g t h e ( h o a c ca
h a i ) k h u e c h t a n v a o m o t l d p g e l v a cl m o t v i t r i x a c d i n h c u a g e l x u a t h i e n d t f d n g
k et tu a h oac v u n g k et tu a cu a k h a n g n g u y e n k h a n g th e.
Stf tao thanh ket tua cung nhtf vi tri ket tua phu thuoc vao nhieu yeu to'.
Trtfdc het, no tuan theo dinh luat khuech tan trong moi trtfdng long cua Fick 2
cung nhtf he so' khuech tan, co nghia la phu thuoc vao tmh cha't cua khang
nguyen, khang the, nong do va trong ltfdng phan tti cua khang nguyen, khang
the. (Dtfdng ket tua nam gan vi tri khang nguyen hdn neu khang nguyen co trong
ltfdng phan tti cao hon trong ltfdng phan tti khang the, va ngtfdc lai). Ngoai ra con
phu thuoc vao pH, ion ltfc, nhiet do, thdi gian.
Uu diem cua phtfdng phap. Vdi phtfdng phap ket tua trong gel, co the tien
hanh nghien ctfu he thong khang nguyen, khang the da gia. Trong moi trtfdng gel
hon hdp, khang nguyen va khang the da gia se cho nhieu dtfdng tua doc lap bdi
dac tinh khuech tan va dien di khac nhau cua chung.
Khuech tan trong gel giup cho stf danh gia mot cach chinh xac do tinh khiet
cua khang nguyen khang the, phat hien dtfdc tinh dong tinh, phan tfng cheo... cua
cac khang nguyen. Cac dtfdng tua dtfdc gan trong gel lam cho viec danh gia ket
qua va ltfu lai dtfdc thuan ldi. Ket tua m iln dich trong gel, ngoai tac dung xac
dinh dinh tinh, con dung dinh ltfdng cung nhtf ban dinh ltfdng ket tua mien dich.
Chung toi chi gidi thieu nhting ky thuat thong dung nha't.
5.1. Khuech tan vdng don gian (ky thuat Mancini)
Ky thuat khuech tan mien dich vong trong gel thach dtfdc Mancini,
Carbonara va Heremans neu ra nam 1965 vdi mot he thong so sanh de xac dinh
dinh ltfdng khang nguyen tti mot ngtfng ket mien dich dac hieu.
5.1.1. Nguyen t i c cua phuong phap
Khang nguyen hoa tan tti mot lo hinh tron khuech tan ra xung quanh mot
cach deu dan vao mot ldp gel thach co do day nhat dinh chtia khang the ttfdng
ting. Stf tiep xuc khang nguyen vdi khang the ttfdng ting dac hieu se tao thanh
mot vung ket tua hinh tron khong tan.
Dien tich hinh tron ty le thuan vdi nong do khang nguyen.
Phtfdng phap Mancini dtfdc sti dung trong hoa mien dich de dinh ltfdng cac
protein trong mot he thong khang huyet thanh don dac hieu vdi khang nguyen
ttfdng tfng. Thi du dinh ltfdng IgG, Ig A vdi khang huyet thanh ddn dac hieu khang
IgG, khang IgA.
192
5.1.2. Thiet bi, nguyen lieu, hoa chat
- B an th a n g bang, buong am ,
- T h u d c k e p d o h o a c k fn h h ie n vi co th u d c do.
- G ia y k e o ly (m m )
- Ong nghiem, o'ng hut vi ltfOng, ong hut chia do

- Dia thuy tinh co dtfclng kinh 8,5cm, hoac cac phien kinh phang 8 x 10cm
- Thach 1,5% trong dem 8,6 vdi ion ltfc 0,1
- Khang huyet thanh don dac hieu, khang nguyen chuan
- Huyet thanh can thtf (huyet thanh benh nhan)
- NaCl 0,9%
- Dung dich nhuom: co the dung dung dich 0,5% Amidoschwarz 10B (trong
methanol: acid acetic ty le 9:1) hoac dung dich 0,2% xanh Coomassie trong ntfdc.
- Dung dich co' dinh: acid acetic 2% trong nUdc hoac acid tricloacetic 12,5%.
- Dung dich rUa: methanol/acid acetic (9/1) hoac ethanol/acid acetic/nUdc (4/1/5)
5.1.3. Tien hanh
- Dieu che thach chUa khang the. Dun soi cach thuy thach 1,5% de thach
tan ra, sau do de vao noi am cach thuy 56°C de nhiet do thach giam xuong gan
56°C. Dong thdi de khang huyet thanh vao noi cach thuy am 56°C.
Cho vao ong nghiem hoac lo penicillin mot the tich thach va khang huyet
thanh theo ty le: khang huyet thanh trong thach la 2-5%, hoac co the 10%. Tat ca
deu tien hanh d 56°C.
- Do thach chUa khang the vao dia thuy tinh co day phang va dat len mat
phang da thang bang, lam sao thach dan deu dan co do day khoang 2mm; de d
nhiet do phong 20-30 phut, sau do de vao tu lanh (4°C) khoang 20 phut cho thach
dong tot.
- Due lo. Dung khuon hoac mot o'ng co dudng ki'nh 2mm due cac lo tren ban
thach due va hut phai can than de lo thach tron, nhan va sach. Khoang each gitfa
cac lo ttf: 7-10mm tuy nong do khang nguyen cua mau thu.
- Cho dung dich khang nguyen vao lo. Bang o'ng hut vi lUdng. cho vao moi
lo 3-5 microlit dung dich khang nguyen (moi lo la mot mau khang nguyen. ttfc la
moi mau huyet thanh benh nhan). Tuy theo yeu cau ma pha loang huyet thanh.
(Thi du: de dinh ltfdng IgG thi huyet thanh benh nhan can pha loang 1/10, dinh
ltfdng IgA thi pha loang 1/2, cdn dinh ltfdng IgM khong pha loang.
Dong thdi cho vao ba lo ba nong do khang nguyen chuan ttfdng tfng da biet
ro nong do de ve dtfdng chuan.
Thdi gian khuech tan: de vao hop co do am cao (bao hoa hdi nUdc) d nhiet do
phong trong 48 gid.
193
Loai bo protein thtia khong ngtfng ket: ngam 2 ng&y trong NaC l 0.9%, h&ng
ngay thay 2 den 3 lan dung dich NaCl 0,9%.
Co' dinh: ngam 30 phut trong dung dich acid acetic 2%.
Nhuom mau: ngam 10 phut trong dung dich 0,5% Amidoschwarz 10B ho&c
Coomassie 0,2%.
Loai bo mau thtfa. Ngam lan ltfOt 6 nhieu chliu methanol: acid acetic (9:1)
hoac ethanol: acid acetic ntfdc (4:1:5) moi l&n khoang 2 phut.
De kho 6 nhiet do phong.
5.1.4. Doc ket qua
Dung thtfdc kep ly met hoac kinh hien vi co chia do de do dtfOng kinh khuech tan.
Ve d t f O n g b i e u d i l n . Can c t f v a o n o n g d o k h a n g n g u y e n c h u & n b i e t t r t f d c \h
d t f d n g k i n h d o d tfO c c u a c a c n o n g d o k h a n g n g u y e n c h u & n , t a v e d t f d c d t f d n g b i e u
d ie n t r e n g ia y k e ly (it n h a t p h a i co 3 d i e m ) . Ttf d o ti'n h r a n o n g d o k h a n g n g u y e n
ttfO n g t fn g c u a m a u t h ti.
Co the ve dtfdng chu&n theo cac cach sau day: Thi du:
- Dtfdng ki'nh khuech tan IgG cua huyet thanh benh nhan A la 5mm.
- Nong do IgG ttfOng tfng tren true hoanh la 30,
- Do pha loang huyet thanh: 40
Vay: nong do IgG cua huyet thanh benh nhan A la: 30 x 40 = 1200mg/100ml.
5 . 1.5. Cac yeu to anh hudng
- Khong de thang b&ng trong qua trinh lam va qua trinh khuech tan nen
ldp thach day mong khac nhau do do khuech tan khdng deu, dien khuech tan meo
mo, loi lom hoac hinh bau due.
- Do thtfa dung dich khang nguyen tran ra ngoai lo hoac nong do khang nguyen
qua thtfa lam cho dien khuech tan md. gidi han khdng ro (do tao phtfc hop tan).
- De d nhiet do qua cao khdng du do £tm nen thach bi khd, anh htfdng den
toe do khuech tan.
- Thdi gian khuech tan de qua lau ma khoang cach gitia cac lo thi be nen co
the xay ra hien ttfdng khuech tan chong len nhau.
- Huyet thanh cd nhieu lipid hoac cd nhieu fibrinogen gay nen stf ket tua
ttf phat.
5.1.6. Gidi han cua phuong phap
Phtfdng phap nay chi phu hdp dac biet doi vdi viec nghien ctfu dinh ltfdng cac
protein trong trang thai sinh ly. benh ly ma co trong ltfdng phan tti khdng qua ldn.
De khac phuc stf khdng deu dan cua ldp thach khi dd, chung tdi da dung hai
mieng kinh phang chong len nhau va lot gitfa hai ki'nh 0 ba canh nhtfng bang nhd
194
cha't deo hoac cao su day do l,5-2mm va co dinh chat bang cap phoi quan ao. Nhif
vSy hai mieng kinh phang da cach nhau khoang 2mm, ba phia da kin, con mot
phia khong lot cao su la khu vtfc de do thach chtia khang the. Sau khi do thach
chtia khang the vao thi de nghieng khoang 30° trong 10 phut 0 nhiet do phong roi
de vao tu lanh 4°C trong 20 phut cho thach chtia khang the dong tot. Sau khi
thach da dong tot thi bo di mot phien kinh bang cach vtia miet nhe nhang phien
kinh do tren ldp thach, vtia keo ra. Ta se dtfOc mot ldp thach chtia khang the day
khoang 2mm nam lai tren phien kinh kia. Tien hanh due lo va cho khang nguyen
vao cac lo nhif phan tren.
5.2. Khuech tan vdng kep

Tti 1949, ky thuat Ouchterlony dtfOc ting dung mot cach pho bien trong
nghien ctiu ket tua mien dich bdi ky thuat kha don gian va cho ket qua nhanh.
5.2.1. Nguyen ly cua ph i/on g phap
Khang nguyen va khang the hoa tan tti hai vi tri khac nhau khuech tan
vong vao trong moi trtfdng gel thach tao nen nhting dtfdng ket tua hinh cung khi
khang nguyen va khang the ttfOng tfng tiep xuc nhau.
Hinh dang do cong cua dtfdng tua phu thuoc vao he so khuech tan cua phan
tti khang nguyen va phan tti khang the.
5.2.2. Thiet bi, dung cu, hoa chat
- Mat phang thang bang, buong am nhtf do'i vdi ky thuat Mancini.
- Hop quan sat vdi nen den, lam kinh, binh non
- Ong hut chia do: 0,1ml, lm l, 5ml, 10ml
- O n g h u t m a o q u a n , k h u o n d u e lo
- Dung dich thach 1,5% trong dem pH 8,6 ion liic 0,05.
- Dung dich khang nguyen: huyet thanh benh nhan hoac cac thanh phan protein.
- Khang the: khang huyet thanh ttfOng tfng.
- NaCl 0,9%
- Dung dich nhuom va dung dich rtia nhtf trong ky thuat Mancini.
5.2.3. Tien hanh

- Chuan bi cac lam kinh. Cac lam kinh dtfOc rtia va lau kho sach, danh so
va dat len tren mat phang thang bang.
- Dun soi cach thuy dung dich thach 1,5% de thach tan deu.
- Dung ong hut dai len moi lam kinh 2,5ml thach lam sao cho ldp thach
dan deu va co do day khoang 3mm. De d nhiet do phong cho thach dong lai, sau dd
de vao tu lanh khoang 15-20 phut cho thach ddng chac.
Neu chtfa kip lam thi nghiem thi de cac lam thach vao buong am.
195
- Due lo. Dung khuon hoac cac o'ng dong de due cac lo tren ban thach.
- Dung o'ng hut mao quan cho khang huyet thanh day lo giiia. con cac lo
xung quanh cho day dung dich khang nguyen co do pha loang khac nhau.
- De cac lam kinh vao buong am tren mot tiet dien bang va 0 nhipt dp
phong trong 24 gid.
5.2.4. Doc ket qua
Dung anh sang tu nhien hoac hop quan sat (hop co bong den. day nen den va
tren la mat kinh) de doc ket qua cac dtfdng ket tua.
De tien hanh dinh ltfdng tiep hoac de gitf lam tai lieu thi loai protein thtfa
khong ngtfng ket (ngam 2 ngay trong dung dich NaCl 0,9%). Sau dd nhuom 10
phut vdi dung dich Amidoschwarz va rtia bang hdn hop ethanol: acid acetic + ntfdc
va de khd nhtf do'i vdi ky thuat Mancini.
5.2.5. ifn g dung cua ky thuat Ouchterlony.
- Kiem tra do tinh khiet cua khang nguyen. De tien hanh kiem tra do tinh
khiet cua mot hdn hdp khang nguyen hoac mot khang nguyen don gia (mot thanh
phan protein) sau khi dtfOc dieu che thi ngtfdi ta cd the sti dung ky thuat
Ouchterlony.
De c d k e t q u a c h i n h x a c , c a n p h a l o a n g k h a n g n g u y e n t h a n h n h i e u n o n g d o khac
n h a u d e c d t h e t a o dtfOc m o t p h a m v i t tf O n g d t f d n g c u a k h a n g n g u y e n , k h a n g t h e .
- So sanh cac khang nguyen. Ky thuat Ouchterlony giup cho viec nghien
ctfu so sanh tinh dong nhat (stf giong nhau) phan tfng ch6o va tinh khac loai cua
hai khang nguyen.
N h om 1: Ca hai khang nguyen hoan toan giong nhau, ttfc la chung cd
cung nhom quyet dinh cua khang nguyen, do dd hai dtfdng ttia khep kin (tinh
dong nhat).
N hom 2: Khdng dong nhat. Hai khang nguyen cd nhting nhom quyet dinh
khac nhau nghia la mdi mot khang nguyen phan ting vdi mot thanh phan khang
the trong khang huyet thanh. Hai dtfdng tua cat nhau.
N hom 3: Dong nha't mot phan thti nha't. Hai khang nguyen cd chung mot
nhom quyet dinh. Ngoai ra khang nguyen I cdn cd mot nhom quyet dinh phan ting
vdi mot thanh phan khang the chtia trong khang huyet thanh ma d khang nguyen
I I khdng cd. Khang the chong lai nhom quyet dinh nay tao thanh mot dtfdng ket
tua di qua khang nguyen I I cho hinh anh mot ctfa ga.
N hom 4: Dong nhat mot phan thtf hai. Hai khang nguyen cd chung mot
nhom quyet dinh. Ddng thdi mdi khang nguyen lai cd mot nhom quyet dinh khac
nhau va phan tfng vdi hai thanh phan khac nhau trong khang huyet thanh va tao
nen hinh anh "ctfa ga kep". Loai dong nhat mot phan nay ttfOng do'i khd phan biet
vdi nhom khdng dong nhat.
5.3. Ky thuat dien di mien djch (xem bai rieng)
196
KY THUAT NGUNG KET
1. Nguyen ly cd ban cua phan tfng ngutig ket

1.1. Djnh nghia
Ngtfng ket la hien ttfdng ket lai thanh dam va ngtfng thanh can (ttf mot hon
dich) cua cac vi khuan, huyet cau hoac cac tieu phan khac.
1.2. Co so ly hoa cua phan ung ngung ket
Mot hon dich bao gom nhtfng tieu phan lo ltfng trong dung moi. Cac tieu
phan gitf trang thai phan tan deu nhtf vay la nhd d tinh trang ion hoa trong dung
moi, cac phan tti ntfdc bam xung quanh cung ion hoa hinh thanh mot ldp bao boc
lam mtfc do ion hoa xung qauanh ldn, nhtfng giam dan cho den khi bang ntfdc
thtfdng cua dung mdi. Mtfc do ai thuy nhieu it quyet dinh do on dinh cua hdn dich.
Mot hdn dich ai thuy it thi qua trinh hydrat hoa tieu phan ndi tren chi tien hanh
tren mat do stfc hut dien ma cd hut ntfdc, cdn hdn dich ai thuy nhieu khi ntfdc di
sau vao cau tao cua tieu phan hinh thanh mot ldp ntfdc dem day bao quanh.
Chinh vi the ma khi cho mot dung dich dien giai vao hdn dich thi hdn dich cang it
ai thuy cang d l ngtfng ket, trai lai hon dich ai thuy nhieu khi khdng ngtfng ket.
Nhtfng neu cho vao day mot chat lay ntfdc di nhtf rtfdu thi ldp ntfdc dem xung
quanh mat va hdn dich ai thuy cung se de dang bi ngtfng ket.
1.3. Nhung yeu to anh hudng den phan ung ngung ket
Co sd ly hoa vtfa ndi tren cho thay ngay phan tfng ngtfng ket chiu anh htfdng
chu yeu vao liic ion va pH cua dung mdi, sau den mot so' yeu to' thti yeu hon nhtf
nhiet do va nhtfng chat khac cho them vao dd.
1.3.1. Dam do ion cua m o i trtfdng

Nhtf tren da ndi, cac tieu phan do cd tai dien, nen da hinh thanh nhting ltfc
day thang ltfc hut lan nhau lam cho tieu phan phan tan deu trong dung mdi. Nay
neu lam giam hieu the be mat xuong thi se lam giam ltfc day va tang stfc hut lan
nhau dan den ket tu. Do dd cho them mot chat dien giai vao trong hdn dich tieu
phan se tao dieu kien thuan ldi cho hien ttfOng ngtfng ket hinh thanh. Dd chinh la
cd sd ly hoa cua cac phan tfng len bong ket tua trong huyet thanh hoc, hay dd la
hien ttfOng ngtfng ket hoa hoc.
1.3.2. pH cua m d i trtfdng

pH cua mdi trtfdng anh htfdng nhieu it den phan tfng ngtfng ket tuy theo do
chenh lech cua no vdi diem dang dien cua tieu phan. Stf khac biet cang it thi ldp
ntfdc dien xung quanh tieu phan cang vting chac va hdn dich cang ben vting; stf
197
khac biet cang xa thi ldp ion quanh tieu phan bi trung hoa va nhtf vay chung de
dang hop lai vdi nhau dtfa den ngtfng ket. Nhtfng trong phan tfng ngtfng ket sinh
hoc, yeu to' pH anh htfdng khong ldn lam vi no v&n co the xay ra trong mot vung
pH rat rong so vdi diem dang dien cua chat sinh hoc.
1.3.3. N hiet do
Nhiet do lam thay ddi chuyen dong Brown, anh htfdng den do phan tan cua
tieu ph&n trong dung mdi. No cung anh htfdng den stfc tai dien. Rieng doi vdi
phan tfng ngtfng ket sinh hoc thi nhiet do qua cao (tren 60°C) se gay thoai hoa va
tua, nhiet do qua thap gay ddng dac dung mdi.
1.3.4. Chat keo khac
Trong phan tfng ngtfng ket, ngoai nhtfng yeu to vtfa ke tren thi neu cho vao
day bat ky mot chat gi cd anh htfdng den nhtfng yeu to dd, deu anh htfdng den
phan tfng. Dac biet trong phan tfng ngtfng ket sinh hoc thi ngtfdi ta thtfdng tien
hanh trong dung mdi keo loai nhtf huyet thanh, dung dich chat cao phan ttf... cho
nen nhtfng chat nay cd anh htfdng nhtf lam cho cac tieu phan de dinh vdi nhau,
lam hien ttfOng ngtfng ket de hinh thanh hon.
1.4. Phan loai phan ung ngung ket

Cd the p h a n biet hai loai p h a n tfng ngtfng ket chinh sau:
- Phan tfng ngtfng ket hoa hoc. phu thuoc chu yeu vao pH va dam do ion
cua mdi trtfdng. Dd la phan tfng hay dung de xac dinh can bang keo loai huyet
thanh trong cac benh gan.
- Phan tfng ngtfng ket sinh hoc hay dac hieu, xay ra khi trong hdn hop dich co
khang nguyen la tieu phan va cd khang the hoa tan. No phu thuoc vao ca 4 yeu to" da
ndi tren, song tuy mdi trtfdng hop cu the, cd mtfc do khac nhau.
2. Phan ung ngung ket dac hieu

2.1. Khai niem ve phan ung ngung ket
Trong m iln dich hoc, phan tfng ket hop khang nguyen - khang the rat hay
xay ra va la co sd cua rat nhieu ky thuat huvet thanh dung de chan doan cung
nhtf de nghien ctfu. Neu dem tron mot khang huyet thanh dac hieu vdi mot khang
nguyen dtfdi hinh thtfc tieu phan (te bao, huyet cau, hat trO cd gan khang nguyen
len tren...) thi cac tieu phan se ket lai thanh dam. ngtfng lai thanh can, tach khdi
pha dich the cua hdn dich.
Phan tfng ngtfng ket dac hieu lan dau tien dtfOc Gruber va Durham phat
hien vao nam 1896 khi hai dng dem tron mot giot hdn dich vi khuan thtfOng han
vdi mot giot huyet thanh benh nhan mac benh a'y thi tha'y vi khua’ n bi ngtfng ket
lai thanh ttfng dam trong giot lo ltfng. N gay nay hien ttfOng nay do cd du moi
tieu chuan dac hieu cua tat ca cac phan tfng mien dich cho nen dtfOc dung de
phat hien mot trong hai yeu to' chinh tham gia vao phan tfng ket hop khang
nguyen-khang the.
198
K h i k h a n g n g u y e n d a b ie t th i co th e tim tr o n g h u y e t t h a n h n h a t d in h n a o do
stf h i e n d i e n c u a k h a n g t h e d a c h i e u tU O ng tin g .
Ngtfdc lai, khi ngtfdi ta co nhting khang huyet thanh dac hieu da biet ro co
chtia nhting khang the dac trtfng thi co the xac dinh tinh chat khang nguyen
ttfOng tfng co trong mot thanh phan nao do chtfa biet.
2.2. Cor c h i cua phan ling ngung ket dac hieu

2.2.1. Gia thuyet cua Bordet
Nam 1899, Bordet da cho rSng phan tfng ngtfng ket mien dich xay ra qua
hai thi:
- Thi ket hop khang nguyen - khang the trong do khang the dtfdc co" dinh
tren mat cac te bao mang khang nguyen; thi nay co the xay ra ngay trong dung
dich ntfdc va hoan toan khong chiu anh htfdng cua cac yeu to" chi phoi phan tfng
ngtfng ket.
- Thi ket tu va ngtfng dong xay ra tiep theo, ddi hdi phai cd dung mdi
dien giai. Tron g thi nay, phan tfng ngtfng ket tuan theo nhting quy luat cua
phan ting ngtfng ket hoa hoc, nghia la thay doi theo ltfc ion, pH, nhiet do...
Nhtfng gia thuyet cua Bordet khdng giai thich dtfOc hien ttfcfng khang
nguyen khang the ket hop trong mot vung pH kha rong, khdng giai thich dtfOc
hien ttfOng khu vtfc.
2.2.2 Gia thuyet m ang ludi cua M arrack va Pauling
Trong cd che ket hop khang nguyen - khang the, hien ttfOng ngtfng ket cung
nhtf hien ttfOng ket tua dtfdc quyet dinh bdi stf ket hdp gitia cac nhom quyet dinh
dac hieu cd tren tieu phan khang nguyen vdi cac nhom ttfOng tfng cd tren phan tti
khang the. Hai tieu phan khang nguyen dtfOc noi vdi nhau bang mot phan tti
khang the cd hai hoa tri va cti nhtf the, cai no mac vdi cai kia hinh thanh mot
mang ltfdi cach quang ctf mot khang nguyen lai den mot khang the. N gay nay,
qua kinh hien vi dien tti, ngtfdi ta thay gia thuyet nay la dung vdi sti that. Qua
khuech dai ldn da thay la nhting cue ngtfng ket gitfa khang the dac hieu va virus,
cho hinh cach quang khang nguyen-khang the trong dd phan tti khang the la nhting
hinh ong cd chieu dai khoang 250 - 270 Angstrom (1 Angstrom = 1.10 'mm). Dieu dd
cho thay la khi ket hdp, hai canh Fab cua khang the da md han ra 180° nhtf trong
phan ly thuyet da ndi.
Nhtf vay cO che cua ngtfng ket cung giong nhtf ket tua chi cd khac la trong
phan tfng nay cd kho cua tieu phan khang nguyen to hdn rat nhieu phan tti khang
the vi chung la nhting te bao. Cung vi the ma tren mat tieu phan khang nguyen
cd rat nhieu nhom quyet dinh khang nguyen khac nhau.
2.2.3. NhCmg yeu td anh hudng den phan Ung ngung ket m iin djch
Qua cd che vtia ndi tren thi cd hai loai ltfc anh htfdng den phan tfng ngtfng
ket dac hieu: cac ltfc anh htfdng den ket hdp khang nguyen - khang the va cac ltfc
anh htfdng den phan tfng ngtfng ket.
199
a. Cac ltfc anh htfdng den ket hap khdng nguyen - khdng the
Ngoai nhiing ltfc nhtf ltfc Van der Walls, lien ket hydro, ltfc dien Coulombs
da noi trong phan ly thuyet cung can phai ke hai yeu to" chinh sau:
Tinh da hoa tri cua khang the. Phai la nhtfng khang the toi thieu co hai hoa
tri thi mdi tao dtfOc phan tfng ngtfng ket trtfc tiep. Neu dung men ma cat mot
phan ttf khang the IgG chang han thanh hai manh Fab, thi chung van co kha
nang ket hop vdi nhom quyet dinh khang nguyen tren mat tieu phan. song khong
tao noi hien ttfOng ngtfng ket. Mandy da thanh cong trong viec cat hai khang the
khac nhau, sau dd lai chap manh Fab cua cai no vao vdi Fab cua cai kia. thu dtfOc
mot phan ttf khang the lai cd hai hoa tri nhtfng khac nhau ve tinh dac hieu. Song
khang the lai ay cung khdng gay ngtfng ket dtfOc.
L ie u ltfOng ttfOng dtfOng va hien ttfOng khu vtfc. N e u tron g mot loat nhieu
ong n ghiem ngtfdi ta cho mot ltfdng n h a t dinh k h a n g n g u y e n (vi du 1, 1/2,
1/4...1/n) thi se thay hien ttfOng ngtfng ket x ay r a ro n h a t 0 mot sd o n g d gitfa, cdn
tron g nhtfng o n g d hai d a u ngtfng ket rat it hoac k h d n g cd. D d la hien ttfOng khu
vtfc. O nhtfng ong n ghiem gitfa, do lieu ltfdng k h a n g n g u y e n va k h a n g the ttfdng
dtfdng vdi n h au , hin h th a n h m a n g ltfdi ro n g k h a p ga y ngtfng ket toan bo k h a n g
n g u y e n (h o n g cau), p h a n d u n g dich cdn lai ben tren g a n nhtf tro n g suot. Con
tron g nhtfng o n g d a u cd chtfa k h a n g the. M o t tieu p h a n k h a n g n g u y e n cd the ket
hdp vdi nh ieu p h a n ttf k h a n g the, h a y ndi mot cach khac mdi p h a n tii k h a n g the
k h d n g sii d u n g het hoa tri cua no, v a n cdn hoa tri thtfa k h d n g h in h t h a n h dtfOc
m a n g ltfdi ldn. k h d n g ket dtfOc th a n h nhtfng d a m du ldn m a ngtfng dong. 0 nhtfng
o'ng sau thi lai thtfa k h a n g n g u y e n va nhtf v a y k h d n g du k h a n g the de nd'i tat ca
cac k h a n g n g u y e n vdi n h a u th a n h m a n g ltfdi. ket q u a c u n g ttfOng ttf nhtf k hi thtfa
k h a n g the (xem them p h a n ky thu at ket tua).
b. Cac ltfc anh htfdng den phan tfng ngtfng ket

Day la nhtfng ltfc da ndi trong phan tfng ngtfng ket khdng dac hieu ttfc dam
do ion, pH cua mdi trtfdng, nhiet do va cac chat cao phan tii.
- D a m do ion r a t can thiet do dam b ao p h a n tfng tien h a n h dtfOc tot. Trtfdc
tien p h a n tfng ngtfng ket thtfdng d u n g cac te b ao cd m a n g k h a n g n g u y e n , cho nen
can cd mot d a m do ion toi tfu de cho te b ao khdi bi htf h a i ( d a n g trtfOng). s a u ntfa
la de cac te bao gitf dtfdc t r a n g thai p h a n tan d o n g deu ttfdng do'i on d in h trtfdc stf
xay ra p h a n tfng ket hop vdi k h a n g the. N h tf the mdi t r a n h dtfOc h ie n ttfdng
ngtfng ket n g a u phat.
- Vai trd cua pH rat ro khi khang the d dam do thap. Neu do'i vdi cac loai
khang huvet thanh cd khang the dam dac thi khdng can chu y den pH. song khi
pha loang nhieu hoac khi dung nhtfng khang huyet thanh ma hoat tinh khang the
veu thi can duy tri phan tfng d mot pH to'i tfu. Do dd ma nhtfng phan tfng nhay ddi
hdi phai dung dung dich dem.
- Nhiet do tang thong thtfdng lam tang chuyen dong Brown va nhtf the lam
cho stf tiep xuc gitfa khang nguyen va khang the dtfdc tot hon, stf ket hop gitfa cac
nhom quyet dinh khang nguyen va khang the nhieu hon, lam phan tfng x ay ra
nhanh hdn. Song cung cd nhtfng ket hop khang nguyen - khang the ddi hdi mot
nhiet do tdi tfu de hinh thanh. vi du nhtf do'i vdi loai khang the nong thi la 37°C,
cdn vdi khang the lanh la 4°C.
200
- Cac cha't cao phan tti ma thong thtfdng la cac protein khdng dac hieu cua
huyet thanh cd trong khang huyet thanh gitf mot vai trd rat quan trong trong phan
tfng ngtfng ket. Cac globulin cd ve lam cho phan tfng ngtfng ket de xay ra hon. Vai
trd cua cac cha't cao phan tti dtfpc thay ro rang nha't khi lam phan tfng vdi cac
khang the khdng hoan toan. Lltn d lu tien Race va W iener (1944) nhan thay khang
the chong Rh v5n bam len hong cau Rh(+) nhtfng khdng gay ngtfng ket neu tien
hanh trong ntfdc muo'i. Ve sau, Diamond va Abelson thay chac chan se ngtfng ket
neu cho xay ra trong huyet thanh AB cua ngtfdi, huyet thanh bd hoac cac chat cao
phan tti tong hop da dtfdc dung de thay the (nhtf dextran, PVP...).
Den nay thi ngtfdi ta da ro la khang the khdng hoan toan thtfdng la loai IgG
qua ngan, khdng choc thung dtfOc mang tai dien bao quanh hong cau, va khdng
lam tron nhiem vu cau no'i gitfa hai tieu phan khang nguyen. Theo Pollack thi cac
cha't cao phan tti, tuy theo kha nang cua mdi thti, se giup pha cai vd boc ion am
quanh hong cau lam giam bdt cac ltfc day gitia chung vdi nhau, giup chung den
gan nhau hon de khang the IgG du stic no'i chung lai. Cd le cac men tieu dam
thtfdng dung de xti ly hong cau trtfdc khi cho ket hop vdi khang the khdng hoan
toan nhtf trypsin, bromelin cung la lam thay doi ltfc tai dien am quanh hong cau.
S o n g tron g cac cha't cao p h a n tti cu n g cd nhting chat ga y tic che p h a n ting
ngtfng ket. V i d u nhtf h u y et th a n h ttfOi. T heo gia thuyet ly hoa thi c h u n g lam ta n g
mtfc tai dien a m q u a n h h o n g cau. C u n g cd y kien cho r i n g hien ttfOng k h u vtfc
chinh la do cac ch at tfc che g a y r a cho nen khi p h a lo a n g la m bdt hoat n a n g cua
chung thi ngtfng ket cd the xay r a dtfOc.
2.2.4. Gia tri cCia phan Cmg ngi/ng ket
Phan tfng ngtfng ket la mot ky thuat ra't thong dung trong mien dich vi
don gian va do nhay cao. Tuy theo he thong sti dung, trong nhting dieu kien toi
tfu thi phan tfng cho phep phat hien nhtfng dam do khoang 0,003 microgam nito
khang the trong 1 ml. Do nhay dd phu thuoc vao nhieu yeu to', dac biet phu
thuoc vao so' ltfdng cac nhom quyet dinh khang nguyen tren be mat tieu phan,
the tich cac tieu phan.
2.3. Cac hinh thifc hoat dong cua phan ung ngung ket
Mac du cd nhieu loai ky thuat khac nhau ap dung cho ttfng hoan canh rieng
biet, nhtfng nguyen tac chung deu ra't ddn gian va chinh nhd tinh chat gian ddn
ay ma ky thuat ngtfng ket trd nen rat pho cap. Phan ting ngtfng ket cd the thtfc
hien dtfdi hinh thtfc dai the hay vi the.
2 .3 . 1. Phan Cmg ngtm g ket dai the
Dtfdc thtfc hien trong nhtfng o'ng nghiem nhd 10 - 100 mm, thtfdng dtfdc
dung trong vi khuan hoc. Cach tien hanh ndi chung da dtfdc md ta sd bo d tren va
ket qua dtfdc doc ngay hoac sau khi u mot thdi gian, hoac sau khi ly tam nhe. Neu
cd ngtfng ket thi khi lac nhe, nhtfng dam ngtfng ket se bong benh noi len trong ldp
ntfdc trong ben tren, trong rat ro. Khi phan tfng khdng xay ra thi lac nhtf vay,
dam ngtfng tu se tan ra va cac tieu phan phan tan deu trong dung mdi thanh mdt
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hon dich nhtf luc chUa la m p h a n ting. Co the sti d u n g mot cai gU dng lorn (gU dn g
K a h n ) de chieu difdi d a y o'ng vdi lu o n g a n h s a n g chech nh in tha'y ra't ro nhting
d am n g U n g tu ket nhd neu cd.
D o i vdi mot so' k h a n g huyet th a n h tao p h a n ting n h a n h ch on g thi cd the l&m

n g a y tren cac la m k in h h a y tren m ien g gach m en t r a n g n h u tro n g viec xac dinh
nhom m au. K h i n ay thi nen lac nhe m ien g kinh, p h a n ting n g U n g ket se nh an h
chong hin h thanh.
2.3.2. Phan ifng ngung ket vi the
H ie n n a y de tiet kiem thdi g ia n cu n g n h u k h a n g h u y e t thanh , n h ieu phan

ting n g U n g ket dUOc thuc hien tron g nhting o'ng rat nhd h a y ndi cho d u n g hon la
trong nhting k h a y b a n g chat deo tron g dd da due s a n h a n g loat nhting g ie n g nhd
cd stic chtia tti 0,5ml den 1,5ml. V iec p h a lo a n g dUOc b a n g nhting d n g h u t mao
q u a n 0,2ml hoac b l n g nhting o'ng nhd tii dong cho k h o a n g 0,025m l h a y It hon cho
mdi l&n nhd. N g u d i ta cdn sti d u n g nhting “b o n g p h a lo a n g ” g io n g n h u chiec khuy
cd can, mdi lan n h u n g x u on g d u n g dich thi p h a n r o n g cua no chtia dUOc du n g
0,025ml. K et q u a n g U n g ket vi the k h d n g the tien h a n h doc n h u vdi p h a n ting dai
the, m a chu yeu nh in tti tren xuong, d a n h gia can d o n g tro n g d a y cac g ie n g cua
khay. K h i k h d n g cd n g U n g ket thi cac tieu p h a n la n g x u o n g d a y th a n h mot hinh
tron kin, deu, sac canh. K h i cd n g u n g ket thi c u n g la n g n h u v a y s o n g canh 1dm
chdm, hoac h in h th a n h v a n h ngan. K h i cd n g U n g ket m a n h thi th a y ro nhting dam
tua dai ro n g r a k h a p d a y gieng.
T r o n g trUdng hop nghi ngd thi nen la'y mot giot d d a y o'ng (cho p h a n ting dai
the) h ay d day g ie n g (cho p h a n ting vi the) d a n len la m k in h m a xem dudi kinh
hien vi.
2.3.3. Phan Ung ngung ket trong gel (gelcard) sCr dung dinh nhom m au A B O va phat
hien khang the bat thudng (xem phan djnh nhom mau, chUdng 6)
2.3.4. Phan Ung ngung ket dinh lUdng
B a n g p h u d n g p h ap p h a lo a n g cd the d a n h gia tUdng do'i lUdng k h a n g the

hoac k h a n g n g u y e n cd tron g mot cha't sinh hoc nao dd. C u n g n h u do'i vdi p h a n ting
ket tua, ngudi ta p h a lo a n g k h a n g the h a i la n mot, tro n g n h ie u o'ng v a de ph an
ting n g U n g ket h in h th a n h vdi mot lUdng k h a n g n g u y e n co' dinh. T r o n g nhting o'ng
d a u cd the p h a n ting rat yeu vi thtia k h a n g the (tien k h u vUc). T ie p den k h u vUc
cd p h a n ting rd r a n g roi p h a n ting lai trd n en k h d n g ro r a n g d d a u kia. N’h u v a y do
p h a lo an g cao n h a t cua k h a n g the m a cdn cho hien tUdng n g U n g ket ro r a n g dUOc
d u n g de bieu thi d a m do p h a k h a n g the. N g u d i ta th u d n g d u n g con so' dao cua do
p h a lo a n g de chi hieu gia k h a n g h u y et thanh . V I d u do p h a lo a n g cao n h a t cdn gay
n g U n g ket ro la 1/n thi h ieu g ia cu a k h a n g h u y et th a n h a'y se la n.
S o n g cu n g p h ai nhd r a n g p h a n ting n g U n g ket k h a c p h a n ting ket tu a la

k h d n g the din h lUdng can tua n h u tron g p h u d n g p h a p H e i d e l b e r g e r K e n d a l l; hon
ntia lieu lUOng tUOng dUOng rat r o n g cho nen p h a n ting n g U n g ket din h lUdng chi
cd tinh cha't tUOng do'i, th u d n g cho phep so s a n h ket q u a cu a h a i thdi d ie m khac
n h a u tren cu n g mot b e n h n h a n hoac so s a n h hai hu y et t h a n h k h a c n h a u . C u n g
202
c ln n h S n m a n h r a n g m u o n n h i i n g k e t q u a t h u d t f d c c o t h e s o s a n h d tfO c t h i c a n
th ie t p h a i tie u c h u ln hoa c a o k y t h u a t c u n g n h t f c a c t h u o c t h t i . Hon n t i a c o n y e u
to' c o n n g t f d i k h i d o c k e t q u a , c h o n e n n g t f d i t a t h t f d n g k h u y e n l a c h i n e n c o i t r o n g
n h t f n g t h a y d o i h i e u g ia ld n hOn h a i l l n p h a lo a n g .
2.3.5. Gia tri cua phan tfng ngtfng ket
Mac du nhting thieu sot vtia ndi tren, phan ting ngtfng ket v ln la mot ky
thuat ra't thong dung trong m iln dich hoc bdi vi no ddn gian va cd do nhay cao.
Tuy theo he tho'ng sti dung, trong nhting dieu kien toi tfu thi phan tfng cho phep
phat hien nhtfng dam do khoang 0,003 microgam nito khang the trong lm l. Do
nhay dd phu thuoc vao nhieu yeu to', dac biet la vao ltfOng cac nhom quyet dinh
khang nguyen cd tren be mat tieu phan, the tich cua tieu phan du no chi lam
nhiem vu mang cac quyet dinh khang nguyen ma thdi.
3. Phan loai phan umg ngung ket

Tuy theo tieu phan mang khang nguyen ma cd the phan biet hai loai phan
ting sau:
- Phan ting ngtfng ket trtfc tiep hay chu dong khi khang nguyen da san cd
tren tieu phan nhtf khang nguyen nhom mau ABO tren hong cau, khang nguyen
ttf nhien cua vi khuan (Hinh 5.8).
- Phan tfng ngtfng ket gian tiep, khi khang nguyen d tfO c gan mot cach
nhan tao tren be mat cua tieu phan trtfdc khi tien hanh phan tfng. Tieu phan khi
ay cd the la te bao it nhieu da d tfO c xti l y hoac la cha't cao phan tti tong hop (nhtf
hat latex).
3.1. Phan ung ngung ket true tiep (hay chu dong)
Phan tfng ngtfng ket chu dong hay trtfc tiep dtfOc dung rong rai trong vi sinh
hoc va huyet hoc. Tren mat cac vi khuan va huyet cau cd mang nhom quyet dinh
khang nguyen, cd the bi ngtfng ket khi gap khang the ttfOng tfng dac hieu. Nhtfng
tren cung mot te bao lai cd nhieu khang nguyen khac nhau, cho nen cung loai te
bao dd cd the bi ngtfng ket bdi nhieu khang huyet thanh, mdi thtf dac hieu cho
mot khang nguyen.
Mat khac, mot khang huyet thanh cd the chtfa nhieu khang the cho nen khi
muon cd mot loai khang huyet thanh ddn dac hieu (ttfc chi phan tfng vdi mot
nhom quyet dinh khang nguyen) thi phai hap phu no, loai bd nhtfng khang the
khdng can thiet.
3.1.1. Phan tfng ngtfng ket tri/c tiep hong cau va vi khuan
Mot sd chung vi khuan gay ngtfng ket hdn dich hong cau, hien ttfdng nay
khdng do cO che m iln dich vi khdng cd ngtfng ket to' ma la do ca'u true dac biet cua
mot sd chung vi khuan. Neu dem tron hdn dich vi khuan ay vdi hdn dich hong clu
thi se cd ngtfng ket. Phan tfng thtfdng dtfOc dung trong nghien ctfu de phan loai
chung vi khuan cd fimbrae hay khdng, nhtf de phan biet Hemophilus aegyptius
vdi Hemophilus influenza.
203
i
A Khang thi IgG di phar

Ung vdi KN trtn M mac
HC tao n«n hat ngung »«
B: Khdng thi th&j (don hoa

tri) c6 mat trSn mat HC
khdng tao hat ngung kit
Nhung khi cho th&n
anti-globulin hat ngung
kit tire khac dupe tao
thanh (net dam la anb-
yglobulin)
C: DTa phan Cmg ngung k it

- Huylt thanh pha loang
1/2,1/4...
- Cac Id chimg (♦), ctiirng (-)
- L6 khong ngung k it
HC ling gon xuong day £
-L6 co ngung ket (♦)
HC lang lan toa loan bd
day Id
H in h 5.8: Mo hinh phan i/ng ngi/ng ket tri/c tiep

A: Phan ifng ngi/ng ket trtfc tiep cua KT typ IgG vdi KN hong cau
B: Phan O'ng ngtfng ket bdi khang globulin (anti-globulin) vdi khang the thieu da phan im g
vdi KN tren be mat hong cau-anti-globulin da lien ket vdi khang the thieu tao thanh cac
hat ngtfng ket (Coom bs true tiep)
C: DTa phan ting ngung ket
204
3.1.2. Phan tfng ngi/ng ket trtfc tiep trong huyet hoc
C ac te bko m a u deu m a n g tren be m a t r a t n h ieu k h a n g n g u y e n dac hieu cho

nen ve n g u y e n tic, p h a n ting ngtfng ke trtfc tiep co the ap d u n g cho ch u n g dtfOc
neu ta co k h a n g huyet th a n h ttfOng tfng. D o i vdi b ac h cau v a tieu cau, do b a n than
ch ung cu n g de ttf nh ien ket d m h vdi n h a u cho nen p h a n tfng ngtfng ket m at tinh
chinh xac v a thtfdng ph ai d u n g ky t h u a t te bao. D o i vdi h o n g cau thi p h a n tfng
nay dtfOc d u n g r a t r o n g rai de p h at hien k h a n g n g u y e n k hi biet k h a n g the hoac
ngtfpc lai. Cac ky t h u a t bao gom:
- Xac dinh nhom mau he ABO (xem phan ky thuat xac dinh nhom mau he
ABO).
- P h a n tfng ngtfng k et cheo (xem p h a n phtfOng p h a p thti p h a n ting cheo
ngtfdi cho va ngtfdi n h an ).
- Xac dinh nhom mau thuoc cac he thong khac (xem phan ky thuat dinh
nhom mau khac cua he hong cau).
- Xac dinh ngtfng ket to' khong hoan toan. Nghiem phap Coombs (xem phan
nghiem phap Coombs).
- Nhting nham lan trong nhan dinh phan ting ngtfng ket dinh nhom mau
Trong viec doc ket qua ngtfng ket cung co the co nham lan. Sau day la mot
so' nham lan co the gap can biet de de phong.
(1) Trtfdng hop co ngtfng ket ma doc nham la khong co. Sai lam nay co may
nguyen nhan chinh sau:
- Doc qua voi vang nen khong chu y c ln than, nhat la khi sti dung nhting
o'ng nghiem nho. Khong lac ky, tron deu nen khi ly tam hong cau lang xuong
trtfdc khi co ket hop khang nguyen - khang the. Cho nen co nOi ngtfdi ta khong ly
tam ma de tu am 37° trong mot gid roi doc ket qua. Ket qua cho chinh xac hon
song mat nhieu thdi gian hon.
- H ie n ttfOng ngtfng ket q u a yeu do k h a n g huyet th a n h yeu, ho n g cau xau,

hay la do ty le gitia h o n g cau v a k h a n g huyet th a n h k h o n g thich hop cho nen xua't
hien hien ttfOng k h u vtfc da noi tren.
(2) Trtfdng hop ngtfng ket gia. Co doi khi phan tfng cho ket qua dai the nhtf
la co ngtfng ket, nhtfng neu nhin qua kmh hien vi thi khong thay hong cau ngtfng
tu vdi nhau ma chi thay chung tap hop dinh chat nhau lam thanh ttfng chuoi nhtf
chuoi tien. Hien ttfOng nay thtfdng thay trong nhtfng trtfdng hdp dung loai khang
huyet thanh mau co dam do protein hoac hieu gia cao, hay khi hong clu dinh thti
bi thay doi (cua benh nhan viem phoi, sot ret, cum, benh da u tuy...). Muon phan
biet, co the pha loang giot mau trong dung dich sinh ly, neu ngtfng ket that thi
dam hong cau se lang xuong dtfdi. Neu la ngtfng ket gia thi hong clu se tron deu
vdi huyet thanh ma cho moi hon dich mau hong.
(3) Hien ttfOng Thomsen hay la ngtfng ket do vi khuan. Hien ttfdng nay xay ra
khi hong c lu mau hay khang huyet thanh mau dung lau bi nhiem khuan. Ngtfng
ket xay ra khong phai do ket hop khang nguyen - khang the dac hieu nhom mau
205
ma la do nhiing thay doi do vi khuln gay ra tren m&t hong c lu va trong huySt
thanh. Cho nen nhiing thii dung nhieu lan trong phan ting phai giti cho dtfdc vo
trung va bao quan trong tu lanh. Khi nghi ngd co nhiem khuln thi phai bo.
(4) Hien tUOng loan ngtfng ket. Co tac gia cho r in g hien ttfdng nay vh hi^n
ttfdng Thomsen la mot song thtfc ra loan ngtfng ket c6 the gap khong phai trong
nhiem trung ma la do dung hong clu trtf lau ngay trong nhiet do cao. Day la hien
ttfOng ngtfng ket lan tran vdi tat ca moi loai khang huyet thanh hdp cung nhtf
khong hdp. Cach de phong la trtf hong c lu trong nhiet do thich hop va thay hong
c lu mdi.
(5) Hien ttfOng ttf ngtfng ket. Doi khi giiia hong c lu va huyet thanh cua cung
mot ngtfdi co phan tfng ngtfng ket. Hien ttfOng nay co the gap d nhiing ngtfdi bi
dau gan, vang da sung huyet hay bi man cam vdi thuoc. Do la benh ttf mien va
c ln phat hien ttf ngtfng ket to' (xem p h ln dtfdi).
(6) Ngtfng ket to lanh. Trong mua lanh, khi nhiet do cua mot so ngtfdi gay
ngtfng ket vdi cac loai hong clu. Nhtfng khi cho phan ting tien hanh trong nhiet
do am thi khong xuat hien. Do la trong huyet thanh co ngtfng ket to' lanh, mot
loai khang the chi hoat dong trong nhiet do thap. Co the khti b ln g cach dun huyet
thanh len 56°C trong mot gid.
(7) Ngtfng ket do phu loai: Cac phu loai cua nhom mau A co the gay nen
nhtfng hien ttfOng ngtfng ket khong ro rang hoac ngtfng ket gia. V I du nhtf ngtfng
ket to' chong A 2 co phan tfng vdi hong c lu loai 0 hoac nhtf hong c lu loai A 2B de
n hlm vdi loai B.
3.2. Phan ung ngung ket gian tiep (thu ddng)

3.2.1. Nguyen ly: Phan tfng nay cd do nhay cao nen dung de phat hien cac khang
the chong lai cac K N hoa tan ma phan tfng tua khong phat hien dtfdc. Ngtfdi ta
gan cac phan tti K N hoa tan len be mat cac tieu p h ln ttf nhien (hong c lu ) hay
nhan tao (latex, bentonit...), cac tieu p h ln co tac dung nhtf gia dd. Phan ting xay
ra khi tron vdi huyet thanh cd K T ttfdng tfng.
3.2.2. Cac phan (mg ngtfng ket thu dong
Phan ting ngtfng ket thu dong dtfOc sti dung nhieu trong vi sinh vat, huyet
hoc de phat hien K T hoa tan. G in day, ngtfdi ta ap dung nguyen ly nay de san
xuat kit S E R O D IA de sang loc H IV trong truyen mau.
KY THUAT DO HOAT TINH BO THE
1. Nguyen ly
Hong c lu khi dtfOc man cam d l y d u vdi khang the tan mau (hem olyzin) se bi
dung giai nhieu hay it tuy theo ltfdng bo the cd mat, nghia la tuy theo hoat tinh
bo the. Nhtf vay day la do hoat tinh tong hdp cua tat ca cac thanh phan bo the.
206
Chi c ln mot trong cac thanh phan cua bo the giam hoac thieu cung du lam hoat
tmh chung bi anh htfdng, vi phan tfng day truyen bi gian doan, te bao khang
nguyen khong bi tan.

- Ly tam
- Ong nghiem
- Hong clu ctfu (mau ctfu chong dong)
- Huyet thanh tan mau
- Huyet thanh can xet nghiem (xet nghiem trong vong 6 gid sau khi lay mau).
- Ntfdc muoi sinh ly 0,9%

- Cam nhiem hong cau: Hong cau ctfu rtfa 3 lan bang NaCl 0,9% (ly tam
1500-2000 vong/phut/10 phut), pha thanh hon dich hong cau 5% vdi huyet thanh
man dang trtfOng. Sau do tron huyet thanh tan mau vdi hong cau ctfu da pha
loang 5%, ty le bang nhau, u 37°C trong mot gid, ctf 30 phut lac mot lan.
- Tien hanh phan tfng: Lay 10 o'ng nghiem nho, dung pipette lan ltfOt dong
cac dung dich (Bang 5.9).
B ang 5.9: Do pha loang huyet thanh trong ky thuat do hoat tinh bd the
So ong 1 2 3 4 5 6 7 8 9 10
Huyet thanh xet 4 4 4 4 4 4 4 4 4 4
nghiem (giot) —> —>
NUdc muoi sinh ly 0 4 4 4 4 4 4 4 4 4

H6 n djch hong cau 1 1 1 1 1 1 1 1 1 1
5% da cam n h ilm
Do pha loang 1 1/2 1/4 1/8 1/16 1/32 1/64 1/128 1/256 1/512
- Trtf o'ng nghiem dau, cac o'ng nghiem cdn lai deu nho 4 giot ntfdc muo'i. C)
o'ng so 10, lay 4 giot dung dich bo di.
- U 0 tu am 37°C/30 phut, sau do quay ly tam 2000 vong/1 phut, doc ket qua.
4. Ket qua
- Doc ket qua bang mat thtfdng, xem hien ttfdng tan mau (khong thay cdn
hong cau lang xuong day o'ng nghiem). Tim o'ng nao d do, ltfdng bo the vtfa du de
lam tan 100% hong cau.
- Vdi huyet thanh ngtfdi binh thtfdng, tan mau d hieu gia 1/16; 1/32 (ong 5,6)
- Hieu gia bo the tang trong mot so benh, mot so trang thai: viem khdp,
nhiem nong, nhiem xa.
207
- H ie u gia bo the gia m do t h a m gia cac p h a n ting ket hop k h a n g n g u y e n -
k h a n g the: b e n h thieu m a u tan m a u do k h a n g the lanh, tan m a u do m ien dich...
hoac bi sti d u n g n h ieu tron g mot so" trUdng hop viem (viem phoi, thtfdng han...).

- Ltfdng h o n g cau k h o n g chu&n dtfdc mot cach ch in h xac
- H u y e t th a n h dtfdc thti p h a lo a n g theo he so" 2, vi v&y k h o p h a t h ie n nhting

hieu gia t r u n g gia n lam tan het h o n g cau.
KY THUAT PHAT HIEN KHANG THE KHANG NHAN
1. Nguyen ly
Trong benh ly ttf mien, cd the til sinh ra khang the chong lai cac khang
nguyen cua ban than. Dtfa tren nguyen ly sii ket hop gitia khang nguyen v&
khang the dac hieu de phat hien cac khang the khang nhan trong cd the. 0 day,
chung toi gidi thieu hai ky thuat de phat hien: K y thuat mien dich huynh quang
va ky thuat ngtfng ket. Co nhieu loai ttf khang the:
- Loai chong deroxyribonucleoprotein, bieu hien b&ng hinh anh huynh
quang dong nhat phu len toan bo nhan te bao. Hieu gia cao rat co gia tri trong
chan doan va tien ltfdng.
- Loai chong phan tti A D N xoan kep bieu hien bang mau phat quang hinh
vong (khang the dac hieu nhat cho benh lupus).
- Loai chong cac thanh phan khac trong nhan bieu hien bang stf phat
quang lom dom, tmh dac hieu khong cao trong lupus, bao gom: khang nguyen
Smith (Sn), khang nguyen protein ribonucleo (co trong nhieu benh: lupus, xd ctfng
bi, nhiem collagen...), khang nguyen Ro (do Robert phat hien) va khang nguyen
Lane (do Lane phat hien).
2. Phat hien khang the khang nhan blng ky thuat mien djch huynh quang
2.1. Dung cu, hoa chat
- Tieu ban gan khang nguyen (D ung cac lam kinh gan s in cac te" bao
nuoi cay nhtf te bao W il-2 hoac te bao Hep-2, hoac dung hong cau ga, hong cau
chim xtf ly trtfdc).
- A nti-IgG -FITC
- Kinh hien vi huynh quang
- Dung dich dem PBS
- Xanh Evans, Glycerin, lamen.
208
- Huyet thanh chting:
+ Chting dtfOng: huyet th a n h ben h n h a n lu p u s giai doan cap.
+ Chting am: Huyet thanh ngtfdi binh thtf Ong, cang tre cang tot.
2.2. Quy trinh ky thuat

- Nho m iu huyet thanh c ln thti, chting dtfdng, chtfng am len tieu ban da
gin san khang nguyen.
- U tieu ban trong toi d nhiet do phong (30 phut)
- Dung dem PBS rtia sach khang the thtia.
- Phu anti-IgG -FITC
- U 30 phut d nhiet do phong
- Rtia lai 3 lln b ln g dem PBS, de kho til nhien
- Nho glycerin, phu lamen, doc ket qua tren kinh hien vi huynh quang
2.3. Nhan dinh ket qua

Xet nghiem dtfdng tinh khi co mot trong cac mau phat quang sau:
- Mau phat quang dong nhat (lan toa hoac day dac): phan anh cac khang the
chong deoxyribonucleoprotein. Neu co hieu gia cao thi thtfdng co benh lupus ban do
he thong hoat dong. Khi benh thoai lui thi hieu gia giam.
- Mau phat quang hinh vong: phan anh cac khang the chong A D N kep
xoan, co tinh dac hieu cao nhat trong benh lupus ban do he thong (de nhan thay
khi dung bach cau ngtfdi lam nen).
- Mau phat quang lom dom: phan anh nhieu loai khang the chong lai nhieu
loai khang nguyen la thanh p h ln cua nhan te bao (khang nguyen: Srri, n-RNP,
Ro, La).
- Ngoai 3 m iu phat quang tren, con co m iu phat quang hat nhan (diem
sang tap trung trong nhan, khong trai deu tren toan bo nhan te bao), day la cac
khang the chong hat nhan.
2.4. Cac yeu to anh hudng
- Do pha loang huyet thanh: Doi vdi khang nguyen la tieu ban te bao Hep-2, do
pha loang huyet thanh benh nhan la 1/80 de tranh dtfdng tinh gia.
- Phat quang gia: C ln luon luon tien hanh chtfng am va chting dtfdng kem
theo de so sanh ket qua.
3. Phat hien khang the khang nhan blng ky thuat ngimg ket
Trong bo kit phat hien khang the khang n-D N A (native deoxyribonucleic
acid) cua hang Human da co du hoa chat va vat lieu de tien hanh phan tfng phat
hien khang the khang nhan. Sau day, chung toi xin tom tat thanh phan kit va
quy trinh tien hanh xet nghiem.
209
3.1. Thanh phan
- Huyen dich hat latex co gkn n-D NA (lo nut trsing)
- Chting dtfOng (lo n u t do)
- Chting am (lo nut xanh)
- 01 tam nhtia co 6 o de tien hanh phan ting
- Que khuay
Bao quan: 2-8°C
M&u thti: Dung huyet thanh benh nhan cho phan ting. Neu huyet thanh l&'y
trong vong 48 gid thi bao quan d 2-8°C.

M ang hoa chat va huyet thanh c&n thti ve nhiet do phong, lac deu lo dvfng
hat latex trtfdc khi sti dung
Tren phien nhtfa phan tfng, dat trong moi o (giot):
Hoa chat o 1 o 2 o 3
Chtfng dtfdng 1
Chtfng am 1
M iu huyet thanh can thti 1
Hat latex gan khang nguyen 1 1 1
D u n g que k h u a y tron deu huyen dich gan k h a n g k h a n g ngu yen va huyet thanh
thti (hoac chting dtfOng, chtfng am), doc ph an tfng ngtfng ket trong vong 2 phut.
3.3. Nhan djnh ket qua

Doc ket qua chtfng trtfdc, chtfng dtfdng co cac hat ngtfng ket thanh ttfng dam
nhd va chting am khong cd cac dam ngtfng ket. So sanh ket qua m iu huyet thanh
can thti va chting dtfdng, chtfng am de ket luan.
3.4. Cac yeu to anh hu&ng

- Tam phan tfng ban, tfdt cd the lam sai ket qua, can rtia sach, say kho
trtfdc khi tien hanh phan ting.
- Huyet thanh benh nhan can thti phai dtfdc tach c&n than, tranh vd hong clu.
4. Phan ting Waaler-Rose

4.1. Nguyen ly
Phan tfng W aaler-Rose la phan tfng ngtfng ket thu dong de phat hien yeu to
dang thap xuat hien trong huyet thanh cua cac benh nhan bi benh viem khdp
dang thap. Yeu to dang thap la mot loai protein cd kha nang ngtfng ket cac hong
cau da dtfdc u vdi globulin hoac khang the. Lay huyet thanh cua benh nhan viem
khdp dang thap tron vdi hong cau da phu IgG ngtfdi. yeu to' dang thap se ket hdp
vdi IgG va keo theo ngtfng ket hong cau.
4.2. Dung cy, hod c h it
- Hong c£u 0 , Rh (-) da rtia sach vdi niidc muoi 0,9%
- Huyet thanh tho khang hong c£u 0
- Ong nghiem
- Pipette Pasteur
- M iu huyet thanh benh nhan can thti.

- Chu^n bi dung dich he thong phat hien:
+ Hong cau 0 rtia 4 lan b in g NaCl 0,9%, pha thanh huyen dich hong clu 5%
+ Pha 1 the tich hong cau 5% vdi 1 the tich huyet thanh tho khang hong
clu 0 (da hieu gia), de d tu am 37°C/1 gid.
- Tien hanh phan ting:
Trong 12 o'ng nghiem dtfOc danh so' tti 1 - 12, nho dung dich theo thti ttf
(Bang 5.10).
B a ng 5.10: D o pha loang huyet thanh trong ky thuat W aaler-R ose
Ong 1 2 3 4 5 6 7 8 9 10 11 12
Huyet thanh 4 4
benh nhan (giot) —>
NaCl 0,9% (giot) 4 4 4 4 4 4 4 4 4 4 4
Do pha loang 1 1/ 1/ 1/ 1/ 1/ 1/ 1/ 1/ 1/ 1/ 1/
2 4 8 16 32 64 128 256 512 1024 2048
Sau khi pha xong, cho vao moi o'ng 2 giot dung dich he thong phat hien da
chuan bi d tren. Lac deu, de vao tu am 37°C/1 gid.
Lay ra x e m h i e n t t f O n g n g t f n g k e t b & n g k i n h l o m ( d o c s o b o k e t q u a l a n 1)
De vao 4°C trong 18 gid, doc ket qua lln 2.

Vdi p h t f O n g p h a p n a y :
- Neu d do pha loang 1/16 trd xuong ngtfng ket coi nhtf phan tfng am tinh
- N e u d do p h a lo a n g 1/32 trd nen co ngtfng ket la p h a n tfng dtfOng tinh
4.5. Cac yeu to anh hudng

- Xet n g h ie m dtfOng tinh ttf 70-90% cac trtfdng hop viem khdp d a n g thap,
nhtfng chi dtfOng tinh > 6 t h a n g s a u k hi khdi p h at b enh. N g o a i ra, xet n g h iem
con dtfOng tinh t r o n g mot sd b e n h khac nhtf xO gan, b e n h phong, g i a n g mai, cac
benh he th o n g (S LE 35%, cting bi 40%...).
- Xet nghiem am tinh trong mot so' benh khdp khac: Thap khdp cap tinh,
htf khdp, viem ctfng khdp song.
211
KY THUAT DIEN DI M liN DjCH
1. Nguyen ly
Trong mot dien trtfdng, cac thanh phan protein se di chuyen den nhtfng vi
tri khac nhau tuy theo stf tich dien cua chung (qua trinh dien di). Sau khi dien di,
neu cho khang huyet thanh ttfOng tfng vao mot ranh doc theo stf di chuyen cua
hon hop protein thi cac thanh phan protein (khang nguyen) va khang huyet
thanh (khang the) se khuyech tan va tao thanh cac dtfOng ket tua dac hieu tai cac
vi tri chung tiep xuc (qua trinh mien dich).

- M ay dien di
- Ban thang b in g
- Dung dich dem Veronal pH = 8,2-8,6, ion ltfc 0,05
- Thach 2% pha trong dem pH = 8,2-8,6
- Khang huyet thanh da dtfcfc m iln dich dat tieu chuan
- Lam kinh trong 2,5-7,5cm
- Dung dich tay
- Dung dich nhuom protein: amidoschwars

- Pha thach trong dung dich dem Veronal pH = 8.2-8.6 trong binh thuy
tinh. Neu thach V iet Nam pha 2%, neu thach ngoai pha 1,5%. Ctf 100 ml thach
cho 0,2ml dung dich methiolat de chong nhiem khuan. Dun thach cho den khi
khong con bot khi la dtfOc. do thach len lam dung ngay hoac chia vao cac ong
nghiem de dung dan.
- Dun cach thuy ong thach cho tan hoan toan roi nho 1 giot len lam, dung
que thuy tinh sach phet thanh 1 ldp mong tren lam roi de kho trong tu say
56°C/15-20 phut lam chan thach.
- Lay lam thach da phet chan ra, dat tren ban thang bang, do moi lam 4 ml
thach pha nhtf tren.
- De thach dong hoan toan khoang 1 gid roi due lo va ranh theo mau
- Nho vao moi lo 20 microlit huvet thanh benh nhan va huyet thanh chtfng
- Dat lam thach vao be dien di. dung giay tham lam cau noi vdi dung dich dem
- Nho eosin de danh dau diem dtfng.
- Chay dien di vdi hieu the 15 Von/1 lam, thdi gian chay tuy khoang ttf 1
gid 30 - 2 gid.
212
- Lfify lam thach ra, hdt bo ranh, nho khang huyet thanh vao day ranh, roi
cho v&o buong am, doc ket qua sau 24 gid.
- Neu c ln ltfu tieu ban, dem s&y kho va nhuom nhtf sau:
+ Sau khi doc ket qua xong, ngam lam thach vao N aC l 0,9% trong 3 ngay,
moi ngay thay NaCl 0,9% 1-2 lln cho sach cac protein thtfa.
+ Vdt lam ra, hut het ntfdc muoi co trong ranh va lo roi do bu thach 1% cho
day ranh va lo. Phu len lam thach mot ldp giay tham, de kho ttf nhien.
+ Boc bo ldp giay tham, rtia sach sOi gi&y tham
+ Nhuom protein: ngam lam thach trong dung dich Amidoschwarz 10 phut.
+ Tay bd thuoc nhuom thtfa b&ng cach ngam lam thach trong dung dich acid
acetic 2% (co 10% glycerin). Nen co 4 - 5 be nhuom lien tiep de ngam lan ltfOt moi
be 2 phut. De kho d nhiet do phong, doc ket qua.

Ky thuat dien di mien dich cd the tach dtfOc tdi 35 thanh phan khac nhau
cua protein huyet thanh.
So sanh gitfa vach tua cua huyet thanh chtfng va vach tua cua huyet thanh
benh nhan can thti de ket luan stf cd mat hay tang, giam cua cac chuoi gamma
globulin (chuoi nang, chuoi nhe).
Dien di mien dich dtfOc sti dung de chan doan cac tinh trang benh ly nhtf
khong co gamma globulin, giam protein huyet thanh, cac benh nhiem khu&n,
benh Kahler...
5. Cac yeu to anh htfdng

- Khang huyet thanh khong tinh khiet hoac qua han sti dung.
- Huyet thanh benh nhan bi vd hong cau
- Cat thach khdng can doi lam dtfdng tua khdng chinh xac
- Dieu chinh dien the va ctfdng do ddng dien khdng dung lam cho qua trinh
dien di chay nhanh qua hoac cham qua dan den dtfdng tua khdng chinh xac.
KY THUAT XAC DjNH KHANG NGUYEN BIET HOA

TREN BE MAT TE BAO MAU
1. Nguyen ly
Khang nguyen biet hoa (cluster differentration antigen) ky hieu CD hieu
theo nghia rong bao gom tat ca cac loai protein xuat hien tren be mat te bao bach
cau trong suot trong qua trinh biet hoa ttf khi dtfOc sinh ra ttf te bao nguon (stem
cell) den khi hoan thanh chtfc nang. Phat hien dtfOc cac khang nguyen biet hoa
213
giup nhan biet dtfOc giai doan phat trien cua ttfng loai te bao cung nhtf qua trinh
benh ly cua chung. Mot trong nhtfng tfng dung cO ban cua ky thuat xac dinh
khang nguyen biet hoa (CD ) la phan loai leukemia va nghien ctfu tinh trang benh
ly co lien quan den roi loan mien dich. Dtfa tren nguyen ly ky thuat mien dich
danh dau: dung mot chat danh dau (chat mau huynh quang, chat phong xa hay
men...) gan vao khang nguyen hoac khang the de phat hien ra khang the hofic
khang nguyen ttfOng tfng 0 mtfc vi the (to chtfc, te bao...) hoac dinh ltfOng chat do 6
mtfc vi ltfOng. K y thuat xac dinh khang nguyen biet hoa dung chat mau huynh
quang de danh dau nen con goi la ky thuat mien dich huynh quang.

C) day, chung toi trinh bay ky thuat mien dich huynh quang trtfc tiep
(M D H Q TT), mot ky thuat pho bien hien nay tfng dung trong phan loai leukemia
va xac dinh tinh trang mien dich cua co the trong mot so benh ly.
- Ki'nh hien vi huynh quang
- M ay ly tam lanh
- M ay dem te bao (dem so ltfdng bach cau )
- Ong nghiem
- Lamen, lam kinh
- Cac loai khang the khang CD - tuy theo muc dich nghien ctfu
- Cac loai hoa chat khac: Dung dich ficoll-Isopaque 1,060, dung dich pha
hong cau, dung dich co dinh te bao, dung dich dem PBS, bo kit chtfng...

3.1. Quy trinh ky thuat MDHQTT phan loai leukemia
3.1.1. Ky thuat phan lap te bao blast trong m iu nghiem tup xuong
- Dieu chinh so' ltfdng te bao co nhan trong dich hut tuy xtfOng 0 mat do 10 -
15 x 109/1
- Chuan bi dung dich ficoll-Isopaque 1.060, dat 2 ml ficoll vao day ong
nghiem 10 x 100mm.
- Dat nhe nhang 4 ml huyen dich te bao len be mat ldp ficoll. tranh tron
lln gitfa hai ldp.
- L y tam 2200 vdng x 20 phut 0 16°C. hut bd dich noi
- Thu hoach ldp te bao mau trang due d gitfa, rtfa 3 lan trong dem PBS
- Kiem tra ty le te bao song chet bang xanh trypan 0,1%: te bao chet se bi
nhuom xanh trypan trong nguyen sinh chat. Dam bao ty le te bao song tren 90%.
Cd the kiem tra ty le te bao blast bang cach huyen dich lai vdi huyet ttfdng cua
chinh benh nhan va dan tieu ban, nhuom Giemsa va doc tieu ban. Tien hanh xet
nghiem M D H Q T T ngay sau khi tach. Neu ty le blast tuy xtfOng cao > 90% te bao
cd nhan thi khdng can tach blast.
214
3.1.2. K y thuat M D H Q TT
- Dieu chinh huyen dich te bao 0 mat do 5 - 10 x 109/1, lac deu
- Trong cac ong nghiem sach, kich thtfdc 10 x 100 mm, danh dau mau
nghiem, dat nhe nhang 0,1ml huyen dich te bao xuong day ong, tranh dinh m iu
nghiem len thanh ong. Dat tiep 0,02ml dung dich anti-CD ttfOng tfng vao day ong,
tron deu.
- U trong toi d nhiet do phong thdi gian 30 - 45 phut, ctf 10 phut lai lac deu 1 lan.
- Pha hong cau b ln g dung dich pha hong cau, trong 7 - 10 phut den khi
mau cua huyen dich trong suot la dtfdc.
- Rtia b ln g dung dich PBS: ly tam 1500 vdng x 15 - 20 phut d 25 - 25°C, hut
bd dich noi. Rtia lien tiep 3 lan.
- Cd" dinh can te bao cdn lai b ln g dung dich co" dinh (0,05ml), bao quan
trong toi 0 nhiet do 2 - 6°C den khi doc ket qua (trong vdng 24 gid).
- Doc ket qua tren kinh hien vi huynh quang, btfdc song 450-492nm, vat
kinh 40:
Anti-CD g in F ITC (CD3, CD10): mau xanh luc
Anti-CD g in PE (CD5 hoac CD7, CD14, CD19, CD16/56, CD33, CD34):
mau vang cam.
Cac kit chtfng deu gan ca FITC lan PE. Doc chtfng am, xac dinh nen te
bao am tinh. Danh gia ty le % blast dtfOng tinh.
3.2. Quy trinh ky thuat MDHQTT trong nghien ciiu phan b o te bao mien djch
- Dem so" ltfdng te" bao cd nhan trong mau nghien ctfu (mau hoac dich hut
tuy xtfOng) b ln g may dem te" bao. Dieu chinh mat do te" bao d mtfc 5 - 10 x 109/1.
- Tron g cac ong nghiem sach kich thtfdc 7x10mm, danh dau mau nghien
ctiu, dat 0,1ml huyen dich te bao xuong day ong nghiem (tranh dinh len thanh
ong), sau dd dat tiep 0,02ml anti-CD ttfOng ting, tron deu nhe nhang.
- Cac btfdc tien hanh tiep theo nhtf ky thuat M D H Q T T phan loai leukemia.

- Chtfng am: de xac dinh tinh trang gan huynh quang khdng dac hieu va
tinh trang ttf phat quang cua te" bao.
- Doc ket qua, dem ttf 200 te bao cd nhan trd len, tinh ty le phan tram te
bao phat quang tren so" te bao cd nhan. Cac te bao se phat mau xanh luc (chat
mau huynh quang F IT C ) (CD3, CD4) va mau vang cam (chat mau huynh quang
PE) (CD 8, CD16/56, CD34).
- Tinh sd" ltfdng tuyet doi: nhan ty le te" bao phat quang ttfng loai vdi so"
ltfOng tuyet doi te bao cd nhan (dem tren may dem te bao).
5. Cac yeu to anh hi/dng

- Dem sd" ltfdng te bao cd nhan khdng chinh xac
- Tinh trang ttf phat quang cua te bao trong benh ly ac tinh cua te bao mau
215
- Qua it te bao
- Phat quang gia
6. Ghi chu
K y thuat M D H Q la ky thuat nhanh nham phat hien khang nguyen dac hi§u
tren be mat te bao hoac to chtfc cat. PhtfOng phap nay bao gom 4 ky thuat:
M D H Q TT dung K T dac hieu gan HQ; M D H Q G T dung anti-y-globulin gan huynh
quang phat hien K T dac hieu; M D H Q T T khuyech dai b ln g bo the; M D H Q phan
tich qua dong chay te bao (FA C S) (hinh 5.9 va 5.10)
H in h 5.9: Mo hinh phan tfng mien djch

huynh quang phat hien KN be m at te bao va to chifc
A: Mo hinh MDHQ tri/c tiep: Dung kit khang the g in huynh quang u trtfc tiep vdi te bao d ich ho3c to
chtfc dich.
B: Mo hinh M DHQ gian tiep: SCf dung anti-y-globulin m ien dich (an ti-lm m u no glo bu lin ) g in huynh quang
phat hien khang the typ IgG da phan dng dac hieu vdi KN te bao dich.
C: MDHQ gian tiep khuyech dai b ln g bd the hoat hoa: Day la phan dng lam tang do nhay cua khang the cd
dinh bo the. Phan dhg KN+KT vdi su co mat cua bd the, bo the bi hoat hoa, tao nhieu thanh phan C3b.
C3b gan vao phan Fc cua IgG va tn/c tiep hap phu tren be mat te bao dich. Sau do, khang bd the dac
hieu C3b gan huynh quang difdc bd sung, phan ifng j+ ) se xuat hien a nhieu vi tri co g in bo the tren be
mat te bpo dich. Nhd vay phan dng khuyech dai bd the lam tang do nhay cua MDHQGT.
216
r
Huynh quang
mau do (-)
Huynh quang
mau xanh (+)
Tia laser
A n h sang chi6u thang 90°

Tia sang phan tich cac hat nho
r,eii© trai r6ng
6ng nghiem
H in h 5.10: Mo hinh MDHQ sCf dung may FACS

(Fluorescence - activated - cell - sorter)
- Bira vao buong dem te bao djch te bao da nhuom vdi khang the huynh quang
- Nhd co he thong “rung" te bao tach ra va chuyen dong theo ddng chay
- Ddng chay te bao chay qua bo phan phan tich bang tia laser, d day moi te bao difpc
tach rieng, di/pc do kich thifdc (size), cac hat nho di/pc xac djnh, mau xanh huynh
quang (+) va mau do (-) di/pc xac djnh va ghi tren bang giay.
- Nhd he thong rung, djch te bao lai di/pc tach ra ti/ng giot, moi giot la 1 te bao. Cac giot
te bao nay chay qua dTa lorn doi dang, hai nhom te bao (+) va (-) di/pc thu hoach vao
ong nghiem , he thong may dirpc rifa lai, tiep tuc kiem tra m iu khac.
- Tren co sd 3 chi tieu thu dirpc: kich th i/dc te bao (s), so li/png te bao (n) va mau huynh
quang (f) tinh ra di/pc sd te bao tham gia phan Cmg.
217
KY THUAT CHUYEN DANG LYMPHO
1. Nguyen ly
Te bao lympho khi nuoi cay vdi stf co mat cua ch&'t kich thich phan bao hoac
khang nguyen dac hieu da man cam so bo cho cac te bao nay se co hien ttfpng thay
doi hinh dang te bao: te bao co kinh thtfdc ldn hon, bao ttfOng rong hon, trong
nhan co nhieu hat nhan, nhan bat mau nhat hdn. Dtfa vao tinh ch&'t tren day,
ngtfdi ta nuoi cay lympho bao de nghien ctfu chtfc nang cua cac te bao nay. Ch&'t
gay phan bao co the la P H A (Phytohem aglutinin) ( Khang nguyen khong dac
hieu) hay b&ng doc to' vi khuan, tuberculin, cac chat hoa hoc... (Khang nguyen
dac hieu).

- Lo nuoi cay te bao say vo trung
- Bdm tiem, pipette vo trung
- Buong nuoi cay vo trung
- M ay ly tam lanh
- Kinh hien vi thtfdng
- Tu nuoi cay C 0 2 co bo phan dieu hoa ap ltfc 0 2 95% va khi C 0 2 5%.
- Moi trtfdng Parker 199 hoac dung dich M E M (M inim al Eagale Medium)
- Dung dich Hanks: pH 7,2 - 7,4
- Chat phan bao: P H A hoac Con-A, PPD hoac cac chat nghien to’ chtfc, tuy
theo muc dich nghien ctfu.
- Huyet thanh AB da khtf bo the 0 56°C /30 phut hoac huyet thanh bao
thai be.
- Cdn co dinh, dung dich nhuom Giemsa.

- Dung bdm tiem trang heparin, lay 4 ml mau tinh mach, cho vao 1 dng
nghiem da khti khuan.
- Phan lap te bao qua ficoll hoac de lang ttf nhien vdi do nghieng 45° trong
20 phut sau do de dtfng 90° trong 5 phut. Lay 2/3 tren ntfdc mat, hdn hop nav co
tren 80% la lympho, lan mot it bach cau da nhan va hong cau. Dem so' ltfdng te
bao lympho va xac dinh ty le song chet cua te bao, chi dung nuoi cay khi ty le te
bao song tren 95%, so' ltfdng te bao trong huyen dich 106 te bao/1 ml.
- Chuan bi hai nhom lo cay (moi nhom can 3 lo):
218
+ Nhom lo (+) gom co:
8 ml moi trtfdng P a r k e r
0,05 ml P H A hoac khang nguyen can kiem tra
0,5 ml dung dich nuoi co 106 te bao lympho, lie deu.
+ Nhom lo (-) gom co:
8 ml moi trtfdng P a r k e r
0,5 ml dung dich nuoi co 106 te bao lympho, \kc deu.
Chu y: Neu dung khang nguyen dsic hieu thi c£n xac dinh lieu khang nguyen
thich hpp cho 106 te bao.
- D&y kin cac lo nuoi cay, de tu am 37° trong 72 gid (neu la khang nguyen
khong dac hieu, lau hon neu la khang nguyen dac hieu), moi ngay lac nhe mot l&n
tat ca cac lo, tot nh&'t la nuoi cay trong tu §"m C 0 2.
- Thu hoach: H et thdi gian nuoi c&'y, lay cac lo ra, dung pipette Pasteur
thuan nhat de lam tan cac dam te bao, chuyen vao ong ly tam, ly tam nhe 1000
vong/phut/10 phut, hut bo ntfdc noi, lac nhe cho te bao bong ra, lay can dan tieu
ban, de kho ttf nhien, co" dinh bang con tuyet doi trong 5 phut, nhuom Giemsa.

- Doi vdi moi lo, doc 1000 te bao de tinh ty le phan tram so" ltfdng te" bao
chuyen dang.
- Binh thtfdng, nhom lo (-) khong co te bao chuyen dang
- Neu chat kich thich phan bao la P H A thi ty le chuyen dang la 80% ± 5, ty
le chuyen dang < 20% la benh ly
- Neu chat kich thich la cac loai khang nguyen khac thi ty le chuyen dang
> 5% la dtfOng tinh
Chu y: Doc ket qua b 3 lo, tinh ty le trung binh.
Tieu chuan de danh gia te" bao chuyen dang: cac te bao lympho chuyen dang
la cac te bao lympho non, dtfdng kinh 10 - 12 (im, nguyen sinh chat bat mau kiem,
trong nhan co 2 - 3 hat nhan, nguyen sinh chat co vai khong bao, ty le gitfa nhan
va nguyen sinh chat lcin. Ta con co the thay cac giai doan khac nhau cua qua
trinh chuyen dang.

- Nuoi cay bi nhiem trung
- Dan tieu ban khong deu hoac khong thuan nhat te bao trtfdc khi lam
tieu ban.
- Dem te bao chuyen dang khong chinh xac, neu chi dem d duoi tieu ban
thi nhieu te bao lympho khong chuyen dang, d ngon tieu ban thi nhieu te bao
lympho chuyen dang, do do nen dem ca hai khu vtfc va lay gia tri trung binh.
219
KY THUAT GAY DOC BACH CAU TRONG ONG NGHIEM
1. Nguyen ly
K h i te b a o dich (te b a o m a n g k h a n g n g u y e n dac h i e u ) p h a n ting vdi k h a n g
h u y e t th a n h dac h ie u c u n g vdi stf co m a t cu a bo the thi te b a o dich se bi doc v&
chet. Phtic hop k h a n g n g u y e n - k h a n g t h e -bo the la m t h a y doi tin h t h a m m a n g te
bao, ton thtfOng m a n g , ntfdc se n g a m vao b a o tUOng la m te b a o trUOng to v a chet.
B S n g cach n h u o m vdi cac ch at m a u ( x a n h t r y p a n hoac eo sin ), cd the xac dinh
dtfOc so ltfOng te bao s o n g chet, tti dd b ie t dtfOc h ie u gia g a y doc te b a o cua
k h a n g h u y e t th an h .

- Ong nghiem, pipette chinh xac, bain-marie, phien nhtfa
- Dung dich ficoll tach lympho
- Dung dich dem PBS
- Dung dich Parker 199
- Dung dich Hanks pH 7,2
- Dung dich nhuom te bao xanh trypan 0,2%
- Bo the tho: thtfdng lay mau tinh mach tai cua 9 - 10 con tho, de rieng ong
mau cua ttfng con mot. De mau ttf dong 30 phut/37°C va 2 gid/4°C. L y tam 3000
vong/phut/10 phut. Tach huyet thanh cua tat ca cac ong mau tho, tron deu. Ly
tam lai 4000 vong/phut/10 phut. Chia deu huyet thanh thu dtfOc vao cac ong nho
dung dan. Bao quan d -80°C hoac tot nhat la bao quan dtfdi dang dong kho. (Chv^
y: tat ca cac btfdc thtic hien d 4°C, dong lanh ngay d -80°C, huyet thanh tho da lay
ra khdi -80°C thi khong dong lai ntia).
- Dau paraphin
- Dung dich dem bach cau
- Khang huyet thanh dac hieu can thti

3.1. Tap trung bach cau lympho
- Lay mau tinh mach, chong dong b ln g heparin (20 don vi heparin cho 1 ml
mau), lac deu trong o'ng nghiem, de l&ng tti 15 - 30 phut.
- L y tam 1000 vong/phut/10 phut, hut lay phan huyet ttfdng, cach ldp hong
cau 2 mm, de tranh hut phai ldp bach cau da nhan va hong clu.
- Cho huyet ttfdng vao ong nghiem da cd san dung dich ficoll, ly tam 1800-
2000 vong/phut/10 phut, lay vong bach cau sat ldp ficoll cho vao ong nghiem khac.
220
- Rtia bach c lu bang dung dich PBS 2 lln (ly tam 1500 vong/phut/10 phut),
hut bo dich ndi, huyen dich lai dung dich bach c lu sao cho so bach cau tti 5x 109/1
trong dung dich Parker.
3.2. Pha loang khdng huyi t thanh: Lay 0,5 ml khang huyet thanh, pha loang nhu
sau: L&y 10 dng nghiem, danh sd tti 1 - 10, cho vao moi dng 0,5 ml dung dich Parker.
Sau do dtfa vao ong so' 10,5 ml huyet thanh, tron deu, chuyen 0,5ml sang o'ng sd 2,
tron deu, chuyen 0,5 sang o'ng so' 3..., ctf nhtf vay, den ong sd 10, bo di 0,5 ml cuoi
cung (chu y: ta't ca cac btfdc dtfOc tien hanh 6 nhiet do ntfdc da dang tan).
3.3. TiS'n hanh phan Ong

- Dung micropipette dtfa vao moi o'ng 5 jil huyen dich te bao (moi dng cd
25.000 te bao), lac deu, u chau ntfdc am 37°C/30 phut.
- La'y ra, rtia lai 2 lan bang dung dich Hanks, loai bo ntfdc mat
- Bo sung vao can te bao 0,2 ml huyet thanh thd ttfoi (bd the) pha loang 1/8
trong dung dich Parker, lac deu, u chau ntfdc am 37°C/45 phut.
- Bo sung vao mdi o'ng 0,3 ml dung dich xanh trypan, lac deu, de trong
chau ntfdc da, doi 5 phut sau doc ket qua.

- Dung pipette Pasteur dau nhan, hut 1 giot nhd vao buong dem bach clu,
dem so' te bao chet dtfdi kinh hien vi, vat kinh 40, cac te bao trtfOng to, bat mau
xanh la te bao chet.
- Dem ttf 200 - 400 te bao, tinh ty le phan tram te bao chet, ttf dd suy ra
hieu gia khang huyet thanh.
5. Cac yeu to anh hi/dng

- So' ltfdng bach cau khdng du de tien hanh phan tfng
- Xanh trypan khdng dung nong do
- Khang huyet thanh can thti khdng cd khang the
KY THUAT TIM KHANG THE KHANG TIEU CAU
1. Tim khang the khang tieu cau blng ky thuat ELISA

1.1. Nguyen ly
Khang nguyen tieu cau khi dtfOc gan vao gieng phan tfng se ket hdp vdi
khang the khang tieu cau dac hieu cd trong huyet thanh benh nhan can thti, sau
khi them anti-IgG-peroxydase se tao nen phtic hop: Tieu cau-KT khang tieu cau-
anti IgG-peroxydase. Khi them co chat OPD (O-phenylene Diamine), phan ting
doi mau, can cti vao mau sac phan ting de nhan dinh ket qua.
221
1.2. Dung cu, hod chat
- M ay ly tam
a' O n
- Ong nhiia tach tieu cau
- Pipette chinh xac
- Phien nhiia gan tieu c lu
- Buong dem tieu c lu hoac may dem te bao
- Dung dich dem PBS
- Dung dich dem PBS-BSA 2%
- Dung dich dem PBS-Tween-20/0,05%
- Dung dich PB S-ED TA N a 2
- Tieu cau lay tii ngiidi cho mau nhom 0
- Dung dich ED TA N a 2
- Dung dich cong hop anti-IgG-peroxydase
- OPD
- Niidc muoi sinh ly
- Dung dich N a N 3 chong nam

1.3.1. Ky thuat g in khang nguyen tiSu c iu
- Lay mau ngudi cho nhom O, tii 5 - 10ml, cua 10 ngudi, chong dong b ln g
ED TA (1 the tich ED TA - 9 the tich mau).
- L y tam 1200 vong/phut/10 phut/22°C de co huyet tUOng giau tieu cau
- T a c h h u y e t tU O ng g i a u t i e u c a u s a n g t u i k h a c ( t r a n h l l n h o n g c l u )
- Ly tam 2500 vong/phut/10 phut de cd khoi tieu clu

- Riia tieu c lu 3 lln bang dem PB S-E D TA N a 2
- Hut bd dich noi, lay can tieu clu, cho them N a N 3 0,1% chong nhiem trung
- Hoa loang khoi tieu c lu b ln g dem PB S-E D TA N a 2 de co huyen dich tieu
c lu mat do tieu c lu 1,5-2 x 109/1.
- Nhd vao moi gieng 0,1ml huyen dich tieu clu, de 12-16 gid 0 4°C (qua dem)
- Lay r a , vay s a c h n U d c t r o n g g i e n g va n h d 0,2ml PBS-BSA 2%, d e n h i e t d o
p h o n g 60 p h u t .
- Riia 3 lln b ln g dem PBS-Tween-20/0,05%

- De 37°C/30 phut (lam kho)
- Bao quan d -20°C trong vdng 6 thang
1.3.2. Ky thuat p h a t hien khang the khang tieu cau
- Rua phien nhtia da gan tieu c lu bang dem PBS Tween-20, tham kho
222
- Nho 0,1ml huyet thanh c ln thti (co chting am va chting d t f O n g kem theo)
- U 37°C/60 phut, rtia 3 lln b&ng dem PBS Tween-20, th&'m kho
- Nho 0,05ml anti-IgG-peroxydase, de 45 phut d nhiet do phong
- Rtia 3 lln biing dem PBS Tween-20, 0 lan rtia sau cung, ngam dung dich
rtia tti 10 - 15 phut. Rtia lai mot lln ntfa, tham kho.
- Nho 0,05ml OPD, de 10 phut d nhiet do phong.
- Doc ket qua bang m&t thtfdng hoac may doc ELISA, btfdc song 492nm.
1.4. Nhan djnh k it qua

- Ket q u a d t f O n g t i n h k h i m a t d o q u a n g d m i u t h t i b l n g h o a c d a m h o n
c h t i n g dtfO n g
- Ket qua am tmh khi mat do quang cua mau thti nhtf mat do quang cua
chtfng am.
1.5. Cac y iu to anh hudng

- Gfin khang nguyen tieu cau vao phien nhtfa khdng dat yeu clu.
- Phien nhtfa g&n tieu cau qua thdi gian sti dung cho ph6p.
- Phan ting dtfOng tinh gia khi rtia khdng sach anti-IgG-peroxydase d btfdc 5
- Cd chat khdng thich hop
2. Tim khang the khang tieu cau blng ky thuat mien djch huynh quang
2.1. Nguyen ly
■) 9 \ s » 9 9 x
Khang the khang tieu cau khi xuat hien trong cO the cd the ton tai trong
huyet thanh hay tren mang tieu cau benh nhan. Khang the nay khi ket hop vdi
anti-Ig-FITC thi cd the phat hien d tfO c tren kmh hien vi huynh quang.
2.2. Dung cu, hoa chat

- Dem PBS, pH = 7,4
- PBS/EDTA/BSA 0,2%
- Dung dich N H 4C1 (tach hong cau ttf huyen dich tieu cau)
- Paraform aldehyd (P F A ) 1% trong PBS (trong ltfdng /the tich)
- A n ti-Ig-FITC
- Glycerol 33,3% trong PBS (the tlch/the tich)
- Phien nhtfa day tron

2.3.1. Phan lap tieu cau
- Lay 10ml mau chong ddng b&ng EDTA, ly tam trong 1500-1800 vong/phut/10
phut, chuyen huyet ttfOng giau tieu cau sang ong nhtfa khac, ly tam 3000-4000
vong/phut/7 phut, hut phln plasma sang mot ong khac.
223
- Nho lm l PBS/EDTA/BSA vao can tieu clu vh lac deu, them dung dich
PBS/EDTA/BSA cho day o'ng va ly tam 0 3000-4000 vong/phut/7 phut.
Chu y: Neu con hong c lu trong huyen dich tieu clu , nho 2,5ml dung dich
N H 4C1 va u 5 phut trong da lanh dang tan.
- Rtia tieu cau 2 lan vdi dung dich PBS/EDTA/BSA va dieu chinh sd ltfdng
tieu cau 3 x 108/ml.
2.3.2. Co djnh tieu cau
Them 2,5ml dung dich paraformaldehyd va dung dich tieu clu , lie deu va u
5 phut 0 nhiet do phong, rtia 2 lan vdi dung dich PBS/EDTA/BSA va dieu chinh
mat do tieu c lu d 3 x 108/ml trong PBS/EDTA/BSA.
2.3.3. Ky thuat mi§n dich huynh quang true tiep
- Dat 0,02ml huyen dich tieu cau benh nhan trong mot lo cua phien nhtfa
day tron
- Them 0,02ml anti-Ig-FITC, lie deu va u 30 phut d nhiet do phong trong
bong toi. Sau khi u, them 0,15ml dung dich PBS/EDTA/BSA, lac deu va ly tam
1500-1800 vdng/phut/5 phut. Rtia them 2 lan nhtf vay. (!) lln rtia sau cung. hut bo
dich noi, them 0,015ml dung dich glycerol 33,3% va can tieu clu , lac deu va nhd
len lam kinh.
- Phu la men len va de 15 phut, doc ket qua tren kinh hien vi huynh quang.
2.3.4. Ky thuat m i§n djch huynh quang gian tiep
- Dat 0,02ml huyen dich tieu cau da co dinh P F A va 0,02ml huyet thanh cln
thti trong mot gieng day tron tron va lac deu, u 30 phut d nhiet do phong.
- Them 0,15ml PBS/EDTS/BSA, rtia 3 lan.
- Cac btfdc tiep theo tien hanh nhtf ky thuat trtfc tiep.

- Phan tfng d t f O n g tinh khi thay hinh anh phat quang mau xanh luc quanh
tieu cau
- Phan tfng am tinh khi khdng thay hinh anh phat quang quanh tieu clu
2.5. Cac yeu to anh huung
- Qua trinh rtia khdng sach gay d t f O n g tinh gia
- Tieu cau qua it do dem khdng chinh xac lam cho khdng gan hoac gan
khdng du khang the khang tieu cau
224
KY THUAT DjNH NHOM KHANG NGUYEN BACH CAU
(ky thuat djnh nhom HLA)
1. Nguyen ly
Khang nguyen bach cau ngtfdi (Human Leucocyte Antigens - H L A ) la he
khang nguyen mang tinh dac hieu loai. He khang nguyen H L A co the sti dung
nhu mot dau an nhan chung hoc va dau an benh ly. Khang nguyen H L A ldp I
(HLA-A, HLA-B, H LA-C ) dtfdc phat hien bang ky thuat vi doc te bao. Khang
nguyen H L A ldp II (H LA- DR, DP, DQ) dtfOc phat hien bang ky thuat PCR
(Polymerase chain reaction).

2.1. Ky thuat vi doc lympho
2.1.1. Dung cu hoa chat
- Khay nhtfa nuoi cay to’ chtfc (Terasaki)
- Bom tiem Hamilton gom 3 cd: 100, 250, 500 microlit
- Pipette Pasteur, kinh hien vi
mf
- Ong ly tam cd lx 10cm
- Dung dich Hanks pH 7,2; ntfdc muoi sinh ly
- Dung dich formalin 35% co pH 7,2
- Dung dich xanh trypan 0,2% (pha trong ntfdc muoi sinh ly)
- Dung dich ficoll tach bach cau.
2 . 1.2. Khang huyet thanh chong lym pho bao
K h a n g h u y e t t h a n h lay ttf cac ca the truy en m a u n h ieu la n hoac m a u cua

cac ba me chtia de n h ieu lan cd b a t d o n g he k h a n g n g u y e n H L A gitfa me v a con
hoac k h a n g h u y et th a n h dtfOc s a n x u a t theo phtfOng p h a p k h a n g the don ddng cua
cac labo H L A tren the gidi da dtfdc thtfdng m ai hoa. K h a n g hu y et t h a n h dtfOc khti
bo the d 56°C/30 phut. B a o q u a n d - 80°C.
2.1.3. Bo the: thtfdng dung bo the thd. Lay mau dong mach thd, tach huyet thanh
bang ly tam 3000 vong/phut/15 phut d 4°C. Huyet thanh thd dtfdc ha'p phu mot
lan vdi hong cau, bach cau, tieu cau cua ngtfdi cd nhom mau AB. Lay mau AB, rtia
3 lan bang N aC l 0.9%, lay can te bao u vdi huyet thanh thd 1 gid d 4°C, the tich
nhtf nhau. Sau khi hap phu, ly tam 3000 vong/phut/15 phtit 0 4°C. Lay ntfdc mat
(huyet thanh thd) bao quan d -80°C.
2.1.4. Tien hanh tach bach cau: nhtf trong ky thuat doc te bao.
225
2.1.5. Tien hanh phan Cmg
Dung phien nhtfa co lo, tien hanh phan ting nhtf sau:
- Nho deu dau paraphin vao moi lo (lp l)
- Cho 1 pi dung dich khang huyet thanh vao moi lo
- Cho 1 pi dung dich bach cau 5x10®, tron deu, sau do de 30-45 phut 6 nhiet do phong.
- Cho them 5 pi bo the tho, u 37°C ttf 60 - 90 phut, lay ra v&y sach dlu
paraphin, cho vao moi lo 2 pi xanh trypan da pha 0 tren, tron deu, de nhiet do
phong 5 phut
- LSc deu, de nhiet do phong 5 phut
- 5 pi formalin de co' dinh te bao.
- Doc ket qua tren kinh hien vi dao ngtfOc
2.1.6. Nhan djnh ket qua

Bach cau chet la nhtfng bach cau ngam doc, trtfOng to, bat mau xanh. Dem
so te bao chet va tinh ty le:
- Ttf 0 - 15% la (-)
- Ttf 15 - 20% la (±)
- Ttf 20 - 30% la (+)
- Ttf 30 - 50% la (++)
- Ttf 50 - 80% la (+++)
- Ttf 80% trd len la (++++)
Do'i chtfng: Gom chtfng dtfOng va chtfng am:
Chtfng dtfOng: dung khang huyet thanh d do pha loang 100% te bao chet
Chtfng am: chi cd lympho, khdng cd khang huyet thanh.
Khi doc ket qua, can doc chtfng trtfdc.
2.1.7. Cac yeu to anh hudng den ket qua
- M at do te bao bach clu
- Hieu gia bo’ the
- Nong do xanh trypan
- N hiet do phan tfng
- Ctfdng do ddng dien dien di
2.2. Ky thuat PCR

2.2.1. Nguyen ly
Nhd cap moi dac hieu (Prim er), mot doan D N A dtfOc tong hop ttf nguon
nguyen lieu la cac nucleotid ttf do vdi stf xuc tac cua D N A polymerase.
226
2.2.2. Dung cu, hoa c h it
- May PCR
- M ay dien di
- Lay ly tam
- Tu a'm (co the dieu chinh nhiet do 37°C va 52°C)
- May lac
- May doc ket qua dien di (co anh sang den U V)
- Cac loai micropipette
- Ong Eppendof (1 ml va 1,5ml)
- Dung dich pha hong cau, bach clu
- Proteinase K, NaCl 0,9%
- Ethanol 99%, Agarose
- Ethidium bromid
- D N A size marker, d NTD
- D N A polymearase
- Prim er (tuy thuoc doan D N A can khuech dai)
- Chiet tach D N A
Co rat nhieu phtfOng phap chiet tach D N A khac nhau nhtfng noi chung
deu theo mot so' btfdc cd ban sau:
+ La'y mau tinh mach, chong dong bang E D TA (3 - 5ml)
+ Pha hong cau, bach cau
+ Xtf ly bach cau da dtfcfc pha bang proteinase K
+ Tua protein bang dung dich NaCl bao hoa
+ Loai bo tua protein bang ethanol 99%
+ Tai hoa tan tua D N A trong dem TE (Tris-E D TA)
- Thtfc hien qua trinh khuech dai
So' chu ky khuech dai, nhiet do va thdi gian cua cac giai doan trong mot chu
ky hoan toan phu thuoc vao ttfng cap moi. Qua trinh nay thtfdng gom cac btfdc cO
ban sau:
- Chu^n bi hdn hop khuech dai: tong the tich 50 - 100 |il (dung eppendorf
chuyen dung), cac thanh phan trong hdn hop bao gom: D NA, D N A polymerase,
dNTP, primer (cd the them mot vai thanh phan khac tuy theo htfdng dan cua nha
san xua't).
- Thtfc hien stf khuech dai tren may chuyen dung: thtfdng cd so' chu ky
khuech dai la 30 -60 (tuy theo ttfng ky thuat).
227
- Moi chu ky gom 3 giai doan: giai doan thao chuoi xoan kep cua D N A tao
chuoi don DNA, giai doan gan cap moi vao chuoi don DNA. giai doan keo dai
chuoi bo ctfu mdi dtfdc tong hop.
- Dien di kiem tra san pham khuech dai
San pham khuech dai dtfdc kiem tra bang dien di tren gel thach agarose, ket
qua dtfdc xem la dtfdng tinh khi thay vet trang due tren dtfdng chay cua san pham
trong gel thach.
KY THUAT CAY CUM TE BAO

(Colony forming units)
1. Nguyen ly
Cac te bao goc tao mau (Hemopoietic Stem cells) khi gap cac yeu to kich thfch
tao mau (trong mdi trtfdng dieu kien hoa) se phan chia va nhan len tao cac cum te
bao khac nhau tuy thuoc vao te bao dich va chat ki'ch thich phat trien. Dtfa vao ti'nh
chat nay. ngtfdi ta nuoi cay tao cum de danh gia kha nang tao mau d mot so benh
mau, hoac cua dich te bao goc stf dung cho ghep tuy, danh gia kha nang moc cua te
bao tuy ghep.

- Ong nghiem vd trung. dia nuoi cay. pipette Pasteur
- Tu am COj, may ly tam lanh. ki'nh hien vi ngtfOc
- Mdi trtfdng MEM. dung dich McCoy’s, thach, huyet thanh bao thai be.
- Te bao lympho ngtfdi khoe manh binh thtfdng

3.1. Lam moi truong dieu kien hoa (tao moi truong kich thich phat trien)
Mau ngoai vi chong ddng bang heparin (5UI/1 ml mau). de lang. lay phan
tren dem roi dieu chinh nong do te bao sao cho cd 7.5xl0,; / ml mdi trtfdng.
- Hdn dich te bao 10 ml gom cd:
- Mdi trtfdng M E M (x 2) 3.0 ml.
- Huyet thanh thai be 1.0 ml.
- Dich te bao (7.5 x 10r7ml) 3.0 ml.
- Thach 1,5% 3.0 ml.
Cho 10 ml nay vao hop petri dtfdng ki'nh 94 mm. De d nhiet do phong 5-10
phut cho thach ddng.
Cho vao tu am 37°C trong 7 ngay trong dieu kien am manh cd 5% CO,.
Thu hoach mdi trtfdng dieu kien hoa: lay dia nuoi cay de 24-48 gid d -i'C cho
thach lang xuong. Hut phan tren (mdi trtfdng dieu kien hoa) bao quan d -20 C. Mdi
trtfdng nav cung cd the san xuat duoc bang cach nuoi cav gai banh rau trong RPM I.
228
3.2. Cay te bao tuy
- Lay tuy: choc tuy va lay tuy toan phan da duoc chong dong giong mau
ngoai vi de lin g roi lay phan tren, dem so' ltfOng te bao va dieu chinh nong do te
bao 6.5x107ml moi trtfdng.
- Chuan bj hon dich te bao: 5 ml gom cd:
Mdi trtfdng Me Coy’s (x 2), MEM (x 2) 1,5 ml
Huyet thanh thai be 1.0 ml
Mdi trtfdng dieu kien hoa 0,6 ml
Thach 0.9% 1,5 ml
Te bao (6.5 x 10'Vml) 0,4 ml
Tron hdn dich te bao. sau dd cho lm l vao dia petri (dtfdng kinh 34mm) x 3
dia. chd thach ddng roi nhe nhang chuyen vao tu nuoi cay 37°C, 5% COj do am
cao. nuoi cay trong 14 ngay lay ra doc ket qua.
4. Ket qua
Dem cac te bao dtfdi kinh hien vi dao ngtfOc do phong dai 20-40 lan. Tap hop
te bao > 20 te bao. dtfOc coi la mot cum (colony) va cay dat yeu cau (hinh 5.11).
5. NhCirng yeu to anh hudng

- Dieu kien vd trung: tu am, tu say...
- Hoa chat: do pH cua mdi trtfdng. chat chong ddng dung ty le (tot nhat la heparin).
- Ky thuat: lam mdi trtfdng dieu kien hoa, tach lymphocyt...
H inh 5.11: Cum (Colony) te bao dUdc tao thanh ttf 1 te bao goc nuoi cay 14 ngay
trong mdi trtfdng thach long (Som isolid medium). Tren day la mot ddn vi tao cum
(Colony form ing unit) cua te bao goc 2 ddng (Bipotent stem cells): bach cau hat/mono.
(Trich dan tilr luan an TSKH cua Do Trung Phan, Budapest 1987).
229
Ghi chu: H i e n n a y t r e n t h t f O n g t r t f d n g c o m o i t r t f d n g c a y c u m t e b a o d t f d c
san x u a t san t i i m e t h y l c e l l u l o s e v d i d u c a c c h a t k i c h t h i c h s i n h s a n v a b i e t h o a . c o
hai d a n g m o i t r U d n g d o n d o n g v a m o i t r t f d n g d a d d n g r a t t h u a n t i e n c h o n g t f d i s i i
d u n g , c h a t ltfd n g t o t. D a y la t i e n b o m d i v e k y t h u a t n u o i c a y t a o c u m stf d u n g c h o
nghien c t f u t e b a o g o c .
PHUDNG PHAP TAO QUANG DUNG HUYET

PHAT HIEN TE BAO LACH SAN XUAT KHANG THE
1. Nguyen ly
Tren dia nuoi cay te bao. bao gom te bao lach da dtfOc man cam va hong cau
ctfu (HCC) la khang nguyen dac hieu, te bao lach san xuat khang the se khuech
tan ra xung quanh va phan tfng vdi HCC vdi stf cd mat cua bo the. cac te bao HCC
se bi ly giai tao mot vanh dai khdng cd HCC xung quanh te bao lach. Hinh anh
nay goi la “quang dung huyet”
2. Nguyen vat lieu

1. Chuot nhat trang da man cam HCC, 21 ngay sau lay lach chuan bi dich te
bao lach (10(’/ml).
2. HCC t u o i r t f a 3 l a n b a n g n t f d c m u o i , c h u a n b i d a m d a c 5,0 x 10 t e b a o / m l
3. Buong phan tfng: chuan bi lam sach va la kinh sach. paraphin ran
4. Bd the: huyet thanh thd tuoi

1. Chuan bi hdn hop te bao va bd the theo thtf ttf sau:
- lm l dich te bao lach (10b te bao)
- lm l dich HCC rtfa (4x10' te bao)
- 0,5ml bd the, huyet thanh thd tuoi
2. Tron deu, nhanh chong dtfa vao buong phan tfng da dtfOc chuan bi trtfdc.
han lai 4 phia bang paraphin nong chay.
3. U am 37°C trong 45 phut thdi gian can thiet de te bao tiet K T ddng thdi
phan tfng vdi K N la HCC + bd the.
4. D o c k e t q u a : q u a n s a t b a n g m a t t h t f d n g b u o n g p h a n t f n g d e m s o l t f O n g
q u a n g d u n g h u v e t v a s o lt f O n g t e b a o b a c h c a u k h d n g t a o q u a n g d u n g h u y e t .
Sau dd tfnh ra sd te bao tao khang the/10” te bao lach (ca te bao tao quang
dung huyet va bach cau khdng san xuat KT).
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4. Chu y
- Vi la ldp ddn te bao cho nen can han ky de tranh xo day te bao khi van
hanh ky thuat.
- Can dem chinh xac so' luong bach cau trong buong phan tfng va can
khAng dinh so ltfdng hon hdp te bao lach da dtfa vao phan tfng.
- Te bao tao quang rat de nhan dinh: Trung tam la bach cau, xung quanh
la vung khong co HCC (Hinh 5.12).
A B
H inh 5.12: Mo hinh tao hoa hong (HH=Rosette)

tu nhien vdi HC ctfu (HCC) cua T lympho
A: T lym pho co thu the dac hieu vdi HCC, khi u lym pho T vdi HCC chung
se g in ket vdi nhau tao hinh hoa hong, goi la E-rosette.
B: Anh tao HH vdi HCC cua T lympho: Trung tam la T lympho, xung quanh la HCC
A B
H inh 5.13: Mo hinh tao quang dung huyet phat hien te bao san xuat khang the
A: Mo ta quy trinh thi nghiem: Hon hdp lym pho vdi HCC theo ty le 1/40, u trong buong kin
vdi su co mat cua bo the. Neu co te bao san xuat khang the quang dung huyet se
dtfdc tao thanh. Neu te bao khong san xuat khang the dac hieu HCC se khong co
quang dung huyet.
B: Quang dung huyet: Trung tam la te bao tiet KT, quang trSng la HCC bi ly giai vdi stf co
mat cua bo the.
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KY THUAT THUC BAO
(Phagocytosis)
1. N h a c lai hien tu d n g th u c b a o (c d c h e th u c b a o )
Thu c bao la hien tUOng sinh ly binh thtfdng cua bach cau t r u n g tinh. dai
thuc bao va te bao duoi gai (den dritic cells), n g a n gon goi c h u n g la thtfc bao
(Phagotocytes). D o la mot tron g cac chtfc n a n g bao ve q u a n tron g cua CO the.
Thieu chtfc n a n g nay cO the de bi nhiem trung. q u a trinh thtfc bao dien bien lien
tuc. bao gom cac khau:
H oa tfng dong: D o chat gay hoa htfdng d o n g (cac chat g a y viem. cac
loai vi trung. cac tinh chat huy hoai. cac cvto k in ...) hoat hoa thtfc
bao. htfdng thtfc bao tdi tran dia b a n g cach xu yen mach. di dong tdi o
viem.
T iep can: Tdi tran dia (o viem ) thuc bao tim cach tiep can vdi ddi
|<D ttfOng thtfc bao.
B ao vay: K hi tiep can dtfOc vdi cac ddi ttfOng thtfc bao. thtfc bao tho
ra doi tay ttf m a n g bao ttfOng. om lay doi ttfOng. T h e n a v goi la
p h ago som e tien than.
N u o ’t doi ttfOng: Doi ttfOng thtfc bao cu n g vdi phian m a n g bao tUOng
lot vao trong n g u y e n sinh chat cua thtfc bao. T h e n a y goi la the
( § ) ph ago som e chinh thtfc.
(g)
T h e p h ago som e bi bao v a y bdi cac hat ly sosom e, the n ay goi la the
ph agolysosom e. D o n g thdi thtfc hien hoa m a n g lysosom e va m an g
bao ttfOng cua p h agosom e, lysosome dtfOc p h o n g thich v a tieu huy
doi ttfOng thtfc bao (vi trun g).
D e n day. ve CO b a n la ket thuc mot q ua trinh thtfc bao v a CO the dtfOc

bao ve.
——
Loi d u n g hien ttfOng nay. ttf 1964 M a c - F a l a n d da x ay dtfng ky thuat
thtfc bao de d a n h gia chtfc n a n g thuc bao cua bach cau t r u n g tinh.
dai thtfc bao.
2. N g u y e n ly ky th u a t th u c b a o
Su' d u n g t u c a u t r a n g k h o n g g a y b e n h h o a c h a t l a t e x l a m d o i t t f O n g t h t f c b a o .
Khi u b a c h c a u tu'Oi v d i d o i t t f O n g t h u c b a o 0 37"C. b a c h c a u t h t f c h i e n q u a t r i n h
thtfc b a o . Ket q u a du'Oc d a n h g i a b a n g 3 c h i t i e u :
- Phan tram te bao co thuc bao (goi tat la phan tram thtfc bao)
232
- Chi so thtfc bao la so trung binh doi t t f O n g t h t f c b a o c d t r o n g 1 t e b a o c o
thUc bao.
- Ty le do'i tUOng thtfc bao bi tieu huy l a s o t u c a u c h e t / t o n g so' t u c a u b i
thtfc bao.
3. C h u a n bj
3.1. Dung cu can thiet

- Ong nghiem cd 10 x 75m/ml
- Ly tam dang o'ng nam ngang
- Tu am 37"C
- Ki'nh hien vi an h s a n g thtfdng
- L a m ki'nh pipette P a s t e u r
3.2. Cac chat lieu can thiet

- Tu cau trang khong gay benh
- Hat latex
- Dung dich dem PBS
- Dung dich dextran 6%
- Dung dich pha hong cau N H 4C1
- Dung dich nhuom AO/EB (Acridin Orange/ Ethidiun bromide)
- Ficol (hypaque) 400.000.
4. Tien hanh
4.1. Chuan bj doi twang thwc bao

- Tu cau trang: Tu cau dtfOc nuoi cay trong moi trtfdng canh thang d 37°C
trtfdc 24 gid. Lay tu cau va rtfa tu cau bang ntfdc muoi sinh ly 3 lan bang cach ly
tam 3000 vong/10 phut. Chuan bi dich tu cau co do dam dac bang 2 x l0 8/ml, dtfOc
xac dinh tren may so mau, btfdc sang... tu cau trong o'ng chuan dtfOc bat hoat va
co dinh bang formalin 5%.
- H a t latex: P h a hat latex dat. dtfOc nong do 2 x lO t'/ml b a n g phtfOng p h ap so
mau nhtf vdi tu cau ndi tren.
4.2. Chuan bi te bao thwc bao (bach cau hat trung tmh)
- 4ml mau tinh mach chong dong bang heparin
- Loai lympho bang dung dich Ficoll (xem ky thuat phan lap te bao mien
dich. chtfo'ng V).
- Loai hong cau qua hai btfdc:
+ Budc 1: Loai bdt hong cau bang dextran 6%: dich te bao da loai lympho
3ml + 3ml PBS + 400f.il dextran 6%, lac deu, de nghieng 45° trong 30 phut d 37°C.
233
loai bo hong cau la n g lay dich te bao d ldp tren, ly tam 1.500vong/5 phut, lay can
te bao. lac nhe dng nghiem cho can te bao tan deu.
+ Budc 2: P h a hong cau: C a n te bao dtfOc hoa tan tron g 2ml d u n g dich
XH,C1. lac deu. khi thay hong cau tan deu. n h a n h chong bo s u n g 4ml d u n g dich
dem. lac deu. ly tam 1.500 vong/5 phut rtfa lai 2 lan b a n g P B S . thu la y can. chuan
dich bach cau cd do dam dac la 2 x l 0 6/ml.
4.3. Tien hanh ky thuat thuc bao

- Trong 1 ong nghiem hemolyse cd 0.5ml dung dich tu cau 2x10* tu cau/ml. 0.5ml
dich te bao bach cau t r u n g tinh 2 x l 0 6/ml, u hdn hop tren 0 37°C/1 gid (ctf 10 phut
lac lai 1 lan). vdi hat latex cun g tien h a n h tUOng ttf.
- Rtfa 3 lan b a n g P B S de loai bd doi ttfOng thtfc b ao k h d n g bi thtfc b ao ban g

cach ly tam 1.500 vong/5 phut, thu can te bao, keo tieu b a n (giot d a n ), nhuom
G ie m sa .
- T r a lai dich te bao la lm l, bo s u n g 5|il A O / E B . lac deu. u 37°C/5 phut lam

tieu ban giot dan. doc ket qua.
4.4. Doc ket qua dudi kinh hien vi (anh minh hoa)
- D e m so' ltfOng te bao cd thtfc bao tren tong so’ te bao b ach cau, dem ttf
100 - 200 bach cau. d u n g kinh hien vi a n h s a n g thtfdng, do p h o n g dai xlOO.
- D e m so’ ltfOng doi ttfOng thtfc b ao cd tron g 1 te b a o cd thtfc bao, dem

k h o a n g 10 - 20 bach cau cd thtfc bao. dtfdi k inh hien vi h u y n h q u a n g , do p h o n g dai
xlOO. dem so ltfOng vi t r u n g bi tieu diet (m a u n a u dd), te b ao son g (b at m a u xanh).
- D e m so’ ltfOng tu cau chet tren tong so tu cau bi thtfc bao. So’ tu cau chet
bat m au vang. dd: tu cau song b a t m au x an h . den k h o a n g 10 - 20 b a c h cau co
thtfc bao.
4.5. Danh gia ket qua: T in h 3 chi so sau day:
So’ te b ao cd thtfc b ao xlOO

1. T y le % thtfc bao =
T o n g so’ te bao dem dtfOc
Tong so vi k h u a n hoac hat latex cd trong so thtfc bao

2. Chi sd thtfc bao =
So ltfOng te bao cd thtfc b a o dem dtfOc
3. T y le diet k h u a n (ty le tu cau bi tieu diet)
T o n g so vi k h u a n chet (m a u dd ca m )
Ty le diet k h u a n =
T o n g so vi k h u ^ n song + chet dem dtfOc
4.6. Chi so thuc bao 6 ngudi binh thudng

- T y le thuc bao : >70%
- Chi sd thtfc bao : >3
- Ty le diet k h u a n : >60%
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Chifdng 6
AN TOAN TRUYEN MAU
VAN DONG HIEN MAU NHAN DAO
1. D ai cU dng
T r u y e n m a u la m o t k h a u t r o n g y e u c u a h e t h o n g c a p c tiu v a d ie u tr i th tfO n g
b e n h b in h , b e n h n h a n v a n a n n h a n . M u o n t r u y e n m a u h ie u q u a v a a n t o a n , d ie u
c a p t h i e t l a p h a i c o d u s o ’ l t f d n g m a u v a d a m b a o d u n g c h a t lt f O n g .
K h o n g t h e c o d tfd c c h a t ltfO n g v a a n t o a n t r o n g t r u y e n m a u n e u k h o n g d u s o
ltfO n g m a u t r u y e n . N g u o n n g t f d i c h o m a u tuf n g u y e n t h i e u t h i k h a u t u y e n c h o n ,
s a n g lo c c a c b e n h l a y l a n q u a d tfd n g m a u s e k h o n g t h e th tfc h i e n m o t c a c h b a i b a n
nhtf q u y c h e t r u y e n m a u .
T r o n g t r u y e n m a u cap ctfu vi k h o n g co m a u dtf trtf ( N g a n h a n g m a u ) nen

phai lay m a u cap ctfu. M a da lay m a u cap ctfu thi k h o n g the d a m b a o dtfdc d a y
du cac q u y che k h a m tu ye n v a s a n g loc nhtf lay m a u thtfdng quy, v a luc n a y
dtfng trtfdc stf s o n g v a cai chet thi ngtfdi ta chon la y cai s o n g trtfdc da. M a u
ngtfdi con p h a i la y ttf ngtfdi. D a la y ttf ngtfdi thi p h a i p h u thuoc v ao lo n g n h a n
dao cua con ngtfdi, vi lo n g vi tha bd tinh ich ki, tii ve ca n h a n , d a u oc loi n h u a n
ma m a n g dao li tot de p “Thtfdng ngtfdi nhtf the thtfdng t h a n ”, m in h vi moi ngtfdi
moi ngtfdi vi m inh.
Theo cong tfdc Q u oc te cua H o i chti th a p do ( C T D ) , t r a n g ltfdi liem dd ( T L L D )

nam 1949 viec hien m a u n h a n dao ( H M N D ) p h ai dtfdc thtfc hien tren p h a m vi
toan cau. M a cac to chtfc C T D v a T L L D cua ttfng ntfdc ttfng quoc g ia cd n h iem vu
dtfng ra to chtfc cuoc v a n d o n g nay.
2. N g u y e n ta c v a n d o n g h ien m a u n h a n d a o
1. C o n g tac t r u y e n t h o n g gia o due can la m cho moi ngtfdi t r o n g co ng d o n g

hieu r5 y n g h ia n h a n v a n , n h a n dao cao ca, dao li n h a n ai de ttf n g u y e n hien
m au n h a n dao vdi tin h t h a n k h d n g chi la t r a c h n h ie m m a la n g h i a v u cao quy
ddi vdi cuoc s o n g d d n g loai t h a n yeu, k h d n g chiu b a t ctf mot stf r a n g bu oc nao,
dieu k ien nao.
2. Ngtfdi tinh n g u y e n hien m a u n h a n dao p h a i dtfdc k h u y e n cao ve moi ngu y

cO tron g viec cho m au . V e stfc khoe v a an toan cua tinh n g u y e n p h a i dtfdc q u a n
tam h a n g dau.
235
3. Hien mau nhan dao khong co mot dong cd n&o ve vu ldi ke ca ngtfdi hi£n
mau tu nguyen den ngtfdi thtfc hien.
4. Khong co mot s\i phan biet doi sti nao trong viec hien mau nhtf:
- Chung toe
- Quoc tich
- Ton giao
- DSng cap
- Giau, ngheo, sang, hen.
5. Phai d t f a v a o q u y c h e q u o c g i a v e : so' l a n h i e n m a u v a k h o i l t f O n g m a u
m o i la n la y m a u phai d t f d c x a c d i n h d t f a t r e n c d sd :
- Gidi tmh
- Trong l t f O n g cO t h e
- Gidi han tuoi tac, cao nhat va thap nhat cua ngtfdi hien mau
6. Phai dtfdc tfu tien lay mau d vung co nguy cO thap nha't de tranh giai
doan ctia so, lay mau phai dtfdc kham xet can than de tim ra dtfdc cac yeu to ba't
thtfdng, de cham soc stfc khoe va ttf van cho ngtfdi hien mau.
7. Cong tac van dong hien mau phai trd thanh mot phong trao thtfdng
xuyen, rong khap, nhtf vet dau loang lan rong khap cong dong, vdi phtfdng cham
“mtfa dam ngam lau, mtfa lau ngam dan” phong trao phai kien tuc ngay mot tang,
tranh dau voi duoi chuot.
8. Phong trao phai ke tiep, nghia la can co truyen tnong giao due trong cac
trtfdng m iu giao, cap co sd, cap II, cap II I de ke tiep cac ldp cha anh lam cho
nguon mau on dinh ngay cang tang.
9. V a n d o n g hien m a u p h ai dtfOc n a n g cao k h o n g chi la m a u toan p h a n ma

p h ai v a n dong ca g a n hu y et ttfOng, g a n te b ao de sti d u n g - can gi t r u y e n na'y,
k h o n g can k h o n g truyen, de thtfc hien theo n g u y e n tac la “la'y m a u to'i thieu, sti
d u n g to'i d a ”.
10. Van dong hien mau phai biet dtfa vao phong trao de nuoi dtfdng va phat
trien manh me phong trao, bang cach dtfa chinh vao nhting ngtfdi hien mau an
toan thanh nhtfng tuyen truyen vien de lam cd sd cho stf song con cua cong tac
van dong hien mau nhan dao.
3. B ien p h a p th irc hien
3.1. Bien phap va to chut
- Phai thanh lap Ban chi dao hien mau nhan dao ttf Trung tfdng den dia
phtfdng va phap che thanh: Uy ban truyen mau quoc gia (UBTM Q G ) theo sd do sau:
236
- Thanh lap lien doan nhiing ngtfdi cho mau nhan dao ttf Trung UOng den
cO scf, de sii dung co hieu qua nhtfng ngtfdi cho mau ttf nguyen an toan.
- To chtfc mot he thong mang ltfdi tuyen truyen vien (T T V ) 0 cac co scf, cO
quan phtfOng xa, trtfdng hoc. Cac tuyen truyen vien nay phai dtfpc dao tao mot
cach chu dao ve cong tac van dong hien mau nhan dao iV D H M N D ).
3.2. Cong tac tuyen truyen giao due (TTGD)

• Cong tac tuyen truyen giao due phai dtfOc tien hanh rong khap tren binh
dien quang dai quan chung lam cho hp hieu sau sac hon phong trao H M N D ve
tam quan trong cua no, nhat la y nghia dao li cua no doi vdi dai bieu Quoc hoi, vi
chi nhtfng ngtfdi nay mdi dtfa ra dtfpc nhtfng luat tren binh dien quoc gia.
• Cac bien phap truyen thong giao due.
- Bao chi:
+ Phai co mot to bao chuyen trach ve H M N D
+ To bao cua chtf thap do
+ Bao stfc khoe cua Bo y te.
- Dai phat thanh ve hoi dap HM ND, trtfdc luc phat thanh co nhac, nhac dac
trtfng cho chtfOng trinh (nghe nhac, ngtfdi nghe co the doan dtfdc chtfdng trinh).
- Dai truyen hinh Trung tfdng:
+ Moi tuan co 10 phut phat hinh noi ve H M N D cua cac cd sd - co nhac dac
trtfng cho chtfdng trinh kieu nhtf truyen thanh.
+ Co nhtfng bai hat, ban nhac do nhtfng nhac si cd tieng sang tac va cac ca
si noi tieng hat nham dong vien nhtfng ngtfdi H M ND , thi chac chan se cd tinh hap
d in va di vao long ngtfdi.
- C a n bien soan tai lieu, giao trin h v a xua't b a n tai lieu sii p h a m dlia viko
chUOng trin h g i a n g d a y tron g cac trtfdng.
- To chtfc cac buoi doi thoai vdi thanh nien, sinh vien, hoc sinh do la tiem
ltfc cho mau rat quan trong.
- Can co nhtfng cuon phim ndi ve moi lien quan den mau, trong do nhac
den nguon goc, cac khd khan, nhu cau, thanh cong va stf c ln thiet phai hien mau
ttf nguyen nhan dao.
Phim nay cung can nhd cac tac gia cd ten tuoi viet kich ban va mot nhom nghe
si quen biet sam vai va dao dien. Viec nay phai dtfOc bao trO do cac cap co uy tin.
- Can cd nhtfng chiec xe tuyen truyen khap dtfdng pho phan phat cac tai
lieu bd tui td roi ve H M ND .
- Dung loa phong thanh de thong bao nhtfng buoi lay mau. Cac trung tam
van hoa, du lich nen cd ap phich, bang-ron, khau hieu ve H M ND .
4. T h a i d o tie p x u c v d i n g u d i c h o m au
- Thai do vui ve niem nd, hoa nha the hien long ton trong va kinh men vdi
ngtfdi H M N D de lam vui long ngtfdi hien mau luc den va luc ra ve.
- Noi tiep ddn phai thuan loi cho viec di lai, nOi gtii xe.
- Ngay gid tiep xuc vao tat ca cac ngay trong tuan (ttf 7 gid 30 den 22 gid).
- Dien thoai thtfdng tru 24/24 gid de vtfa tiep ddn, giai dap, vtfa ttf va'n cho
ngtfdi cho mau.
- Nhan vien lay mau phai an mac lich stf ôn gang dung vdi q u y che
phong xet nghiem lay mau.
- Nhan vien lay mau tre khoe, cd ky thuat tay nghe cao (lay mau la dtfOc
ngay), tranh gay d a u cho ngtfdi hien mau.
- Vdi ngtfdi hien mau la nam gidi thi nen bo" tri ntf ky thuat vien lay mau
va ngtfOc lai neu la ntf thi nen bo’ tri nam ky thuat vinn lay mau.
- Ghe lay mau phai la loai ghe chuyen dung, phong rieng biet ttfng ngtfdi
de dam bao thoai mai va yen tinh. Trtfdc luc lay mau, ky thuat vien nen cd ldi xin
phep “Xin ldi cho phep dtfOc lay mau” . Sau luc la'y mau xong cd ldi cam On vi da
hien nhtfng giot mau minh cho dong loai than yeu, tiep dd mdi an nhe, uo'ng ntfdc
mat va mong gap lai lan sau.
TUYEN CHON NGlAJl CHO MAU
1. Md dau
T u y e n chon ngtfdi cho m a u la mot n h iem v u het stfc q u a n tro n g cu a cac cO sd
t ru y e n m a u tro n g viec d a m b a o c u n g cap m a u mot cach d a y du v a an toan.
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- Ngtfdi cho m au co the cho m au toan ph ln, hoac ttfng thanh p h l n m au cua minh.
- Hien tai d ntfdc ta co ba nhom ngtfdi cho mau chinh sau:

+ Ngtfdi cho mau chuyen nghiep
+ NgUdi nha, ngtfdi than benh nhan cho mau
+ Ngudi cho mau nhan dao, khdng lay tien
Mot nhom ntfa trong ngtfdi cho mau la ngudi cho mau ttf than kh(5ng de cap den
trong bai nay. Muc tieu cua nganh truyen mau la phai sdm nhat tien tdi cd mot he
thong ngudi cho mau thifdng xuyen, tinh nguyen, nhan dao va khdng lay tien.
2. N g u y e n ta c
V i e c t u y e n c h o n n g u d i c h o m a u p h a i d a t d tfO c n h i i n g m u c t i e u v a n g u y e n
tac sau:
- N g tf d i c h o m a u l a n g tfd i h o a n t o a n ttf n g u y e n , k h o e m a n h , k h d n g t h u o c
c a c d o i ttfO n g cd h a n h v i n g u y cd c a o .
- Phai dam bao dtfdc stfc khoe ngudi cho mau, khdng gay bat ky anh htfdng
x&'u tdi stfc khoe hoac cac benh lay nhiem do qua trinh cho mau.
- Cd dtfOc c a c d d n v i m a u c o c h a t l t f d n g d e d a m b a o d t f d c h i e u q u a d i e u t r i t o t ,
d o n g thdi k h d n g g a y ra ca c b e n h la y tr u y e n q u a d u d n g tr u y e n m a u ch o b e n h n h a n .
- Viec tuyen chon ngudi cho mau dtfdc tien hanh dong thdi tai cac cd sd
truyen mau, lay mau lUu dong theo quy dinh chung
- Quan ly ngudi cho mau: ngtfdi cho mau phai dtfdc lap ho so theo ddi can
than vdi noi dung sau:
2.1. Nhan than (ly lich ngudi cho mau): Khi mot ngudi den cho mau ta phai co
dtfdc cac thong tin ve ho mot cach cu the va chinh xac gom:
- Ho va ten
- Gidi
- Ngay thang nam sinh
- Dia chi
- So' dien thoai neu cd
- Nghe nghiep
- Noi cong tac
- Tien sii benh tat
- Tinh trang stfc khoe hien tai
Vdi nhiing ngtfdi mdi, ho den de tham gia vao nhom ngudi cho mau chuyen
nghiep chung ta phai lap so’ ho sd va the ngUdi cho mau, vay can bo’ sung them:
*> N
+ Anh chup gan nhat
+ Dac diem nhan dang
239
Qua hoi ve tien sU benh tat va tinh trang siic khoe hien tai cua ngtfdi cho
mau, mot van de quan trong la xac dinh xem ngtfdi cho mau co thuoc nhom nguy
co cao hay khong ?
De xac dinh than nhan ngtfdi cho mau, neu co the dtfdc ta nen tham khao
them giay td tuy than cua ho de khang dinh dtfdc tinh chinh xac ve cac thong tin
co dtfdc. Vdi ngtfdi cho mau ho den de trd thanh ngtfdi cho mau chuyen nghiep thi
viec dieu tra nay vdi lan den dau tien la rat can thiet.
2.2. Dien bien sue khoe qua moi lan cho mau
De cd the dat dtfdc yeu cau nay, chung ta can cd va ltfu gitf cac thong tin ve
kham lam sang, ket qua xet nghiem, ngay kham va cho mau, ltfOng mau dtfdc ltfy
sau mdi lan cho mau.
2.2.1. Cac ket qua kham lam sang
- Trtfdc mdi lan cho mau, dac biet vdi lan cho mau dau tien, ngtfdi cho mau
can dtfdc trao doi can than vdi thay thuoc cua cd sd lay mau. Qua dd co the th&y
dtfdc hoac khdng d ngtfdi den cho cac hanh vi nguy cd cao trtfdc khi den cho mau
hoac gitfa hai lan cho mau), cac chong chi dinh tam thdi hoac vinh vien. Dong thdi
giao due cho ho ve stf can thiet cua mau trong dieu tri, song song vdi no la stf an
toan cua mau dtfdc cho y nghia cua giai doan ciia so’ cua cac benh lay truyen qua
dtfdng truyen mau. Chung ta can cd cac noi dung dieu tra cu the dtfdc chuan bi
trtfdc mdt cach ky ltfdng, cd the vtfa hdi trao doi vtfa ghi nhan hoac ngtfdi cho mau
se ttf ghi sau khi dtfcfc htfdng dan can than. De thtfc hien dtfdc mot cach hieu qua
cong viec nay, chung ta phai dam bao dtfdc nguyen tac bi mat rieng ttf cua ngtfdi
cho mau va phai lam sao de ho tin cay va tra ldi mot cach trung thtfc. Dieu nay
rat khd thtfc hien dtfdc vdi ngtfdi cho mau chuyen nghiep, chi cd the lam tot khi
mot ngtfdi den vdi chung ta de cho mau mot cach hoan toan ttf nguyen, nhan dao
va khdng lay tien.
- Tinh trang chung ve stfc khoe:

+ Chieu cao
+ Can nang
+ Da, niem mac
+ Cac bieu hien cd nang sau mdi lan cho mau
+ Tinh trang he thong tim mach trtfdc mdi lan cho
- Khoang thdi gian gitfa hai lan cho mau
2.2.2. M ot so cac xet nghiem ve sire khoe ngudi cho m au
- Huyet sac to'
- Dtfdng nieu. protein nieu
- X quang tim phoi
240
2.2.3. Cac x6t nghi§m sang loc cac benh lay truyen qua dudng truyen mau
- Cac ben h do virus:
+ Viem gan B
+ Viem gan C
+ H IV
- Cac benh do virus va ky sinh trung:
+ Sot ret
+ Giang mai
Tuy theo cac dieu kien co the ve nhan luc, ky thuat va kha nang tai chinh, tting
cO scf truyen mau co the tien hanh them mot so xet nghiem cf mtic do chinh xac cao
hdn, khi da co thi cung can ltfu gitf cac ket qua nay trong ho sd ngtfdi cho mau.
Cung tuy theo nhu cau, kha nang ky thuat, nhan ltfc va tai chinh, de ltfu gitf
cac thong tin noi tren ve ngtfdi cho mau ca ve ly lich ngtfdi cho, cac theo ddi ve lam
sang va xet nghiem, chung ta cd the phoi hop hoac sti dung mot trong cac hinh
thtfc sau:
- B in g ho so
- Bang phieu lo
- Hoac tin hoc
Can ltfu y cac thong tin ve ngtfdi cho mau phai dtfdc cap nhat kip thdi, chinh
xac de cd the theo ddi dien bien stfc khoe ngtfdi cho qua mdi lan cho mau va ttf van
cho ho mot cach kip thdi. M at khac theo doi dtfcfc chat ltfdng va stf an toan cua
ddn vi mau dtfdc lay de cd bien phap xti ly phu hcfp.
Viec quan ly ho so ngtfdi cho mau phai dam bao nguyen tac bi mat cho ngtfdi
cho mau. Cac thong tin thu dtfdc chi nh&m muc dich chuyen mon, ttf van ngtfdi cho
mau, tranh tuyet doi gay ra stf hieu lam va nhtfng phien phtfc doi vdi ngtfdi cho.
3. C ac tie u c h u a n c h o m au
Cac tieu chuan sau day chu yeu v&n dtfa theo quy che truyen mau do Bo Y
te ban hanh nam 1992
3.1. Tuoi: Doi vdi nam: 18-60 tuoi
Doi vdi ntf: 18-55 tuoi
3.2. Can nang: Doi vdi nam: > 45 kg
Doi vdi ntf: > 42 kg
3.3. Khoang cach giua hai lan cho mau: la 2,5 thang. Tot nhat nen ap dung them
mot yeu cau la khdng qua 5 lan/ nam doi vdi nam, va khdng qua 3 lln 1 nam doi
vdi ntf.
3.4. The tich mau dupe lay: Khdng vtfOt qua 7 ml/ 1 kg the trong; hoac khdng qua
13% the tich mau tfdc tinh.
241
3.5. Ceic tieu chuan ve lam sang
- Ngtfdi cho mau phai hoan toan tinh nguyen, khoe manh.
-La nhtfng ngtfdi khong co bieu hien ve cac hanh vi nguy cd cao (khong thuoc
nhom ngtfdi nguy cO cao).
- Khong co cac chong chi dinh cho mau tam thdi hoac vinh vien
- He thong tim mach:
+ Mach: 60 nhip < Mach < 100 nhip/ phut
+ Huyet ap: 90 mmHg < Huyet ap toi da < 140 mmHg
3.6. C4c tieu chuan ve xet nghiem

3.6.1. Cac xet nghiem huyet hoc
Huyet s ic to > 1 10g/l doi vdi ca nam va ntf, tot nhat la dtfdc >125g/l doi vdi ca
nam va ntf
3.6.2. Cac xet nghiem sang loc cac benh virus

- Viem gan B: am tinh
- Viem gan C: am tinh
- H IV : am tinh
3.6.3. Cac xet nghiem sang loc cac benh vi trung va ky sinh trung
- Sot ret: am tinh
- Giang mai: am tinh
3.6.4. Cac xet nghiem khac

- Dtfdng nieu, protein nieu: am tinh
- X quang tim pho’i: binh thtfdng
4. Tieu chuan ngirdi cho huyet ti/dng va te bao

Trtf cac trtfdng hdp dac biet thi ngtfdi cho huyet ttfOng hoac cho te bao cung
phai cd cac tieu chu&n theo thong le nhtf ngtfdi cho mau toan phan binh thtfdng ve
tuyen tron, quan ly ngtfdi cho.
4.1. Cho te bao: ngtfdi cho mau cung phai dtfOc kiem tra
- Kiem tra lam sang
- Mach, huyet ap
- Dtfdng, protein nieu
- Cac xet nghiem sang loc cac benh lay truyen
- Cac xet nghiem huyet hoc, sinh hoa:
242
+ D£m so ltfdng hong clu, bach clu, tieu cau
+ Protein huyet thanh, protein toan phan
+ Dien giai do
+ Dinh ltfdng mot so'protein huyet thanhvi du: albumin, IgG..)
Cac x6t nghiem tren phai co ket qua binh thtfdng. Ve so' lan cho te bao
khdng qua 2 lln/ nam. Trtf cac trtfdng hdp het stfc dac biet c ln thiet vi du trong
cac trtfdng hdp m iln dich dac biet hoac ngtfdi cho mau cd quan he ruot thit vdi
ngtfdi nh&n, thi so' lan cd the tang len to'i da la 4 lan/ nam.
C ln chu y rang viec cho te bao chi thtfc hien vdi ngtfdi cho dtfdi 50 tuoi.
4.2. Cho huyet ttf&ng: ngtfdi cho c ln phai dtfdc kiem tra
- Kiem tra lam sang

- Mach, huyet ap
- Huyet sac to' hoac hematocrit
- Cac xet nghiem sang loc cac benh lay truyen
- Cac xet nghiem sinh hoa: phan tich protein huyet thanh (protein toan
phan hoac dinh ltfdng mot so' protein nhtf albumin va IgG).
- Dem so' ltfdng tieu cau > 150 G/1
Cac ket qua tren phai nam trong gidi han binh thtfdng.
Khi cho huyet ttfdng ngtfdi cho cd the cho toi da:
+ 10 lit trong 1 nam
+ 2 lit trong 1 thang
+ Khdng qua 500ml trong 1 lan
Thdi gian toi thieu de cd the cho mau toan phan sau khi cho huyet ttfdng la
48 gid.
KY THUAT THU GOM MAU
I. K Y T H U A T L A Y M A U BAN G T U I C H A T DEO
1. Md dau
Viec sii dung tui deo trong truyen mau la mot phtfdng tien phu hdp rat tot
cho hien tai va lau dai. Tui nhtfa vdi nhieu tfu diem cua no tao dieu kien thuan ldi
cho viec lay mau, san xuat cac che pham mau, cung nhtf trong viec bao quan va
phan phoi.
243
Tui lay mau cung cac dung cu kem theo sU dung mot lln tao stf de chiu va
an toan cho ngtfdi cho mau, khong gay cac tac hai do qua trinh cho mau.
2. Nguyen tac
- Lay mau bang tui la lay mau theo mot quy trinh kin do vay khong ddi hoi
phai co buong vo trung, co the thtfc hien dtfOc 6 nhieu noi (vi du nhtf trong la'y
mau ltfu dong).
- De dam bao dtfOc tinh nang va cac tfu diem cua tui lay mau doi hoi ky
thuat phai dtfOc thtfc hien theo mot trinh ttf chat che, chinh xac.
3. Vj tri to churc lay mau

Vdi viec stf dung tui deo trong lay mau, chung ta f e khdng can den buong vo
trung nhtf doi vdi lay mau bang chai nhtf trtfdc day. Tai cac CO sd lay mau co' dinh
chi can cd mot buong to phu hop vdi nhu cau cua minh. Vdi lay mau ltfu dong ta cd
the tien hanh tai nhieu noi nhtf hoi trtfdng, ldp hoc...tham chi cd the thtfc hien
dtfOc ca d vtfdn hoa, trong cong vien..v.v
Nhtfng cung c ln chu y vi tri to chtfc phai dat tai mot ndi rong rai, thoang
mat, sach se, khdng khi trong lanh, day du anh sang, de cd the thtfc hien dtfpc ky
thuat mot cach chinh xac, tranh cac lay nhiem va tao dtfOc stf thoai mai de chiu
doi vdi ngtfdi cho mau trtfdc, trong va sau khi cho mau.
4. Phu’dng tien, dung cu: d day chung ta chi de cap den nhtfng phtfdng tien va
dung cu phuc vu trtfc tiep cho ky thuat lay mau.
1. Gitfdng lay mau: DtfOc san xuat chuyen dung cho lay mau, vdi cac ndi
khdng cd dieu kien cd the sti dung gitfdng phang ngang thay the. Di lafy mau ltfu
dong neu khdng stf dung o to lay mau chuyen dung thi nen dung gitfdng gap de de
van chuyen va lap dat.
2. M ay lac mau: Vtfa cd tac dung tron lan chat chong ddng vdi mau vtfa cd
tac dung xac dinh the tich mau dtfOc lay.
3. Dung cu xac dinh the tich mau dinh lay: Neu khdng cd may lac mau, phai
thtfc hien dong tac nay bang tay thi can cd cac dung cu xac dinh the tich mau
dinh lay nhtf: can bang, can treo hoac hop long.
4. Tui lay mau: don. ddi, hoac tui ba tuy theo nhu cau cO sd.
5. M ay han day (neu cd the) cd hai loai de co' dinh va di dong.
6. Kim vuot day lay mau
7. Khoa nhom
8. Kep hoac panh nhtfa
9. Bong gac vd trung va hop dtfng

10. Bang dinh
11. Dung cu sat trung
244
- Con Iod ho$c con 70° sat trung cho l&'y ven.
- Ntfdc Javen: lau va ta’y ue nhtfng vet mau day, dinh ra khu vtfc lay mau.
12. Panh, k6o kim loai

13. Day garo
14. Qua n&m cho ngtfdi cho n lm khi lay mau
15. But da
16. 6 n g nghiem va gia de o'ng nghiem

17. Ban lay mau
18. Dung cu dtfng rac thai: hop, thung, tui nilon
5. Xac djnh the tich mau djnh lay

5.1. Sir dung may lac mau
Thtfdng thi cac may l&c mau cd kem tinh nang xac dinh the tich mau dinh
lay (theo nguyen ly the tich hoac trong ltfOng). M ay se ttf dong khoa day, bao hieu
ngtfng lay khi ltfOng mau dinh lay da dtfdc lay du. C ln dieu chinh may theo ltfdng
mau dinh lay trtfdc khi tien hanh lay mau.
5.2. Hop long

Dung cu nay co cau tao ddn gian, re tien, de bao quan. Cung phai chu y kiem
tra va dieu chinh the tich hop long bang cac mieng nhtfa (adapter) tuy theo the
tich mau dinh lay trtfdc khi tien hanh ky thuat. Khi tui mau nam chat trong hop
long, ttfc la ltfOng mau dinh lay da du thi mau cung khdng vao them dtfdc ntfa.
5.3. Can
Cd the sti dung can ban hoac can treo, chu y phai sti dung loai can dtfng de
can cac vat cd trong ltfdng nhd (nen ttf 500-1000g) de dam bao dtfdc do chinh xac
cao cua can. Vdi dung cu nay xac dinh the tich mau dinh lay bang trong ltfdng cua
no, dtfa theo cach tinh toan sau:
- Ty trong mau = l,01g /ml.
- Trong ltfOng ttfi = Trong ltfdng bao bi + trong ltfdng dung dich chong ddng
- Trong ltfdng mau = So' ml mau dinh lay x l,01g.
Vay:
Trong ltfdng cud'i cung = Trong ltfdng tui + Trong ltfdng the tich mau dinh lay
V i du: Gia stf tui lay mau 250ml cd trong ltfdng 75g
The tich mau dinh lay la 250ml vay trong ltfOng se la: 250ml x l,01g = 252,5g
Do dd tong cong se la 7,5g + 252,5g = 327,5g
Khi trong ltfdng tui mau dat hoac treo tren can dat tdi 327,5g ttic la da lay
du so' ltfdng mau dtf dinh la 250ml.
245
6. Chuan bj trirdc khi lay mau: (Khong de cap den viec chuan bi khu vtfc
lay mau)
- Kiem tra dung cu lay mau: da d ly du ve chung loai, so' ltfdng va ch&'t
ltfdng. Sap xep dung cu da ngan nap va thuan tien.
- Kiem tra tui lay mau: kiem tra ve chung loai, so' ltfdng ltfu y chi md cac
hop dung tui lay mau sat vdi so ltfOng don vi mau dinh lay) do toan ven cua cac
tui lay mau. Kiem tra thanh p h ln dung dich chong ddng trong tui de xac dinh
dtfdc han luu gitf bao quan mau. Xac dinh trong ltfOng cua loai tui dtfdc dung.
- Cho ngtfdi cho mau n im len tren gitfdng trtfdc khi lay mau khoang v&i phut.
- BSng viec hdi trtfc tiep ngtfdi cho mau de kiem tra stf phu hop ve cac
thong tin ngtfdi cho mau so sanh vdi the hoac phie'u cho mau cua ho (ho ten, tuoi,
dia chi, nhom mau..). Neu cd bat ky mot stf khdng phu hop nao c ln ngtfng ngay
lai de tien hanh kiem tra xac dinh nguyen nhan va cd bien phap xti ly phu hop.
- Xac din h ltfdng m a u din h lay, y eu cau la y m a u n h l m muc dich gi khac
thong le khdng, de chu&n bi tui la y m a u ttfOng tfng.
V i du: + Lay mau bang tui ddi hay ba?

+ The tich mau dinh lay 250ml, 350ml hay 450ml?
+ Neu ltfdng mau dinh lay it hdn, can dieu chinh ltfdng dung dich
chong ddng, thi tinh toan de loai bd bdt mot p h ln ttfdng tfng dung
dich chong ddng cd s in trong tui.
- Dien cac thong tin dtfdc yeu cau len nhan cua tui nhtfa:
+ Ho ten va/ hoac so' ngtfdi cho mau.
+ Nhom mau: neu cd
+ Ngay lay mau va / hoac han sti dung
+ Ten ky thuat vien hoac y ta thtfc hien ky thuat lay mau
+ K et qua xet nghiem neu cd.
Neu la cac trtfdng hop lay mau cd tinh chat dac biet can ghi cu the muc dich,
yeu cau cu the len nhan tui va cd danh da'u ro rang.
- Xac dinh trong ltfdng toan bo tui mau khi du mau nhtf da htfdng d in d tren.
- Dien thong tin theo quy dinh len o'ng mau m iu (pilot) hoac o'ng mau dung
cho xet nghiem.
7. Tien hanh lay mau

7 .1. Ngay trirdc khi lay mau
- Neu thay can thiet cd the kiem tra lai cac thong tin ngtfdi cho mau da neu
d tren.
- Hdi xem ngtfdi cho mau cd thoai mai, de chiu va da sin sang cho mau chtfa.
- Kiem tra lai ttf the n lm cua ngtfdi cho da thoai mai hop ly chtfa.
246
- Boc lo that cao canh tay ngtfdi cho d ben dtf dinh la'y mau.
- Dat day garo: do chat vtfa phai, phia tren nep gap khuyu tay khoang 5-7cm.
- Chon vi tri choc ven: chon ven to, cang, ft di dong, thtfdng nam d gitfa nep
lan khuyu tay. Ltfu y viec ltfa chon vi tri choc ven c ln ra't cj£n than de tranh gay
cac true trac trong giai doan sau va gay dau cho ngtfdi cho mau. Neu chtfa that
thoai mai vdi ven da chon thi co the so sanh vdi ven d tay ben kia de chon dtfdc
ven tot nhat.
- Mot lan ntfa kiem tra lai do toan ven cua tui mau va kim, dung dich
chong dong, cac noi dung da ghi.
- Lam mot nut that long tren day lay mau cach kim la'y mau khoang 15-20cm
sau dd dung panh kep chat day lay mau lai. Cd the khdng cln lam nut that nay neu
ta sti dung ky thuat hai panh kep khi hoan thien qua trinh lay mau, nhtfng ky
thuat nay hoi rtfdm ra va cd mot so' nhtfdc diem nen chung tdi khdng de cap d day.
7.2. Ti§'n hanh lay mau

- Trong nhtfng trtfdng hdp la'y mau ltfu dong, so' ltfdng ngtfdi cho nhieu, cd
the gay ma't trat ttf, ngtfdi ky thuat vien hoac y ta phu trach la'y mau nhieu
gitfdng nen kiem tra stf phu hdp ve cac thong tin ngtfdi cho vdi cac noi dung dtfdc
ghi tren the va tui la'y mau. Mot lan ntia hdi ngtfdi cho mau da s in sang chtfa..
- Tien hanh choc ven, dung con Iod hoac con 70° sat trung vi tri dinh tien
hanh choc ven. Dong tac choc ven can tien hanh chinh xac, nhanh chong va dtit
khoat. Di qua da d vi tri canh ven (tranh di thang vao vi tri cd ven do de gay loang
mau ra xung quanh vi tri choc, rat xau ve mat ky thuat) sau dd dtfa kim vao long
ven, chu y can dtfa kim sau vao trong long ven de kim dtfdc co' dinh tot tranh gay
di lech ve sau.
Khi da cam giac dtfOc kim n a m trong ven roi, ta md p a n h kep d a y la'y m a u se
thay m a u ch ay n h a n h ttf d a y la'y m a u v a o tui.
- Co' dinh tot kim bang bang dinh sau khi theo ddi thay mau chay tot.
- Chu y neu dong tac choc ven khdng thanh cong, cd the dd tim tinh mach
(ven) can phai co gang het stfc, tranh gay dau ddn cho ngtfdi cho. Sau mot hoac
hai lan dd khdng dtfOc, thi khdng nen co' qua ma nen doi cho ngtfdi ky thuat vien
hoac y ta khac lam giup. Neu cam giac khdng tin ttfdng thanh cong thi nen doi
sang tay khac wa cho ngtfdi cho mau nghi mot lat. Can ltfu y trtfdc khi rut kim ra
khdi tay ngtfdi cho mau can kep lai day lay mau (thao garo, dat bang vo trung vao
cho choc). Khi rut kim kiem tra xem cd mau ddng d d lu kim hay khdng, neu
khdng cd can day ngay mui kim lai. Neu cd cuc ddng d dau kim thi doc ngtfdc tui
mau, md nhe panh kep day dung mot ltfdng rat nhd dung dich chong ddng trong
tui de day cuc ddng ra khdi mui kim, sau dd kep panh day lay mau va day mui
kim lai. Thtfc hien lai tti dau vdi lan choc thti hai vdi stf can than cao hon.
7.3. Theo ddi qua trinh lay mau

- Theo ddi tinh trang ngtfdi cho mau trong qua trinh lay mau: vdi cac bieu
hien ve net mat, sac da.. Thinh thoang cd the hdi ngtfdi cho mau mot so' cau xa
giao. Neu cd bieu hien khong binh thtfdng thi can ngtfng ngay qua trinh cho mau,
goi bac si phu trach de cd htfdng xti ly, phu hdp, kip thdi.
247
- Tron chat chong dong va mau: dong tac n&y khong phai 16m neu lav bang
may lac mau. Neu sti dung bang tay thi co the lam dong tac xoa bop, nhoi 6p hoac
dao chieu tui mau mot cach nhe nhang. Dong tac nay can dtfOc lam lien tuc trong
phut dau tien, sau do co the chi can lam khoang 50ml/l lan (1-2 phut/1 l4n).
- Nen yeu cau ngtfdi cho mau nam chat ban tay, hoac co bop ban tay (co the
dung qua nam) de mau chay dtfOc tot hon. Neu thay dong chay kem. can kiem tra
lai garo hoac long hoac chat qua, kiem tra lai vi tri kim dtfdc co dinh co tot khong.
- Theo doi the tich mau dtfdc lay: nhtf da trinh bay 0 tren.
+ Neu dung may lie mau: se ttf dong khoa day lay mau va bao dong khi du
ltfOng mau dinh lay.
+ Vdi lay mau bang can: tong trong ltfdng tui mau da den trong ltfdng yeu
cau ttfc la da lay du mau theo dtf tinh.
+ Vdi lay mau bang hop long: khi la'y du mau ta se thay tui mau nam chat
trong hop long, lac tui mau se co cam giac chat.
7.4. Hoan thien qua trinh lay mau: khi da lay du luong mau dtf dinh.
- T h a t chat n u t that lo ng dtfOc la m luc trtfdc den k hi tha'y nu t that t r a n g ra
k h o n g co m a u n a m d n u t la d a t y eu cau.
- Kep day lay mau sat vdi nut that ve phia kim.
- Keo panh kep ve phia kim, cach nut that khoang 2-3cm. Khi keo panh ra ta
se thay phan day gitia nut that va panh kep lai va khong co mau d doan do la dtfdc.
- Dung keo cat day la'y mau 0 doan gitfa nut that va tay kep tach rdi tui lay
mau khoi ngtfdi cho.
- Lay mau vao o'ng nghiem: md nhe panh kep la'y mot ltfdng mau vtia du,
sau do lai kep lai, thao day garo, dat bong vo trung vao vi tri choc ven, rut kim ra
tti tti (tranh rtit kim nhanh gay phan ting cho ngtfdi cho mau), phan mau trong
doan day con lai cho not vao o'ng nghiem thti lai. Day nut kim va bo doan day va
kim vao hop chtia do thai.
- Co' dinh bong cam mau bang bang dinh, nhtfng nen nhac ngtfdi cho duoi
tay, lay tay kia an bong cam mau khoang vai phut. Khong nen gap tay de gay
chay mau do bong khong ep chat vao vet choc kim.
- Hoi ngtfdi cho mau co de chiu sau khi cho mau khong. Nen de ngtfdi cho
mau n im them mot vai phut sau khi cho, tra cho ho the hoac phieu cho mau sau
khi dien ltfdng mau da cho. Htfdng dan ngtfdi cho mau ra noi an diem tam nhe sau
khi cho mau. Can cam On ngtfdi cho mau.
- Dting kim vuo't vuo't phan mau trong day ve tui mau lam tti mot den hai
lan de mau trong day dtfdc tron lan vdi chat chong dong.
- Gia co' them nut that tren day la'y mau bang may han hoac khoa nhom (d
vi tri sat vdi nut that da dtfdc lam) .
- Ghi so' ltfdng mau dtfdc lay len nhan tui, hoan chinh not cac thong tin yeu diu
neu con thieu. Chuyen cac tui mau va o'ng nghiem sang bo phan tiep theo.
II. K Y T H U A T T H U G O M M A U , C A C T H A N H P H A N M A U B A N G M A Y T U D O N G
(xem chtfOng 6)
248
KY THUAT SANG LOC CAC BENH NHIEM TRUNG
TRUYEN QUA DlfOfNG TRUYEN MAU
1. Sang loc HIV, HBV, HCV

• Hien nay co ba loai th ii nghiem hay diidc sii dung nhat de sang loc H IV ,
HBV, H C V cho ngiicfi cho mau la:
- Thti nghiem mien dich gan men (ELISA; Enzyme-Linked Immuno-Sorbent-Assay).
- Thti nghiem ngtfng ket hat.
- Thti nghiem nhanh.
• K h i lUa chon sii dung mot trong ba th ii nghiem tren, co mot so diem cdn
chu y n h ii sau:
- Nguyen ly ky thuat
- Su phtic tap cua thti nghiem
- Thdi gian phan ting
- Do nhay
- D o dac h ieu
- Kha nang thich hop cua tting phong xet nghiem

- Su tien loi
- Gia ca
• Nguyen ly cua th ii nghiem: Ca ba thti nghiem tren deu dtfa tren cung mot
nguyen ly sinh hoc, bao gom hai budc cd ban:
- Sii co mat cua khang nguyen hay khang the dac hieu trong mau mau can
x6t nghiem the hien b in g phan ting mien dich vdi khang the hay khang nguyen
tUdng ting da dtfdc g in tren pha r&n de tao thanh phtic hdp mien dich khang
nguyen- khang the.
- N h a n biet phtic hdp m i l n dich k h a n g n g u y e n - k h a n g the nhd he th o n g chi
thi mau.
• M au xet nghiem :
Hien nay, hau het cac thti nghiem co the sti dung huyet thanh hoac huyet
ttfOng, tuy nhien phai tuan thu theo stf htfdng dan cua nha san xuat la dting
huyet thanh hay huyet ttfOng.
• D u ng cu vd thuoc thii:
- Dung cu:
+ M ay ly tam
249
+ Tu lanh
+ He thong may E L IS A ttf dong hoac ban ttf dong
+ Pipetm an
+ Tu say
+ Binh cach thuy
+ D lu con cac loai, gia de dau con
+ Ong nghiem tan mau
+ Gia de g in gieng nhtfa
+ Buong to’i de u trong giai doan hien mau
- Thuoc thti:
+ Cac gieng nhtfa da dtfdc g in s in khang nguyen H I V l va H IV2, khang
nguyen loai IgG va IgM .
+ Dung dich hoa loang mau
+ Cong hop (Khang the khang y-globulin ngtfdi g in enzym hoac khang the
dac hieu g in enzym hoac khang nguyen da biet gan enzym).
+ Dung dich cd chat
+ Dung dich acid de ngting phan ting
+ Dung dich rtia
+ Chting am
+ Chting dUOng
+ Chting ngtfdng
+ Nude cat
1.1. Thir nghiem ELISA

- Day la loai thti nghiem phtic tap nhat trong ba loai thti nghiem, tuy nhien
neu da nam dtfdc nguyen ly cua thti nghiem E L IS A thi se nhanh chong hieu dtfdc
nguyen ly cua thti nghiem ngtfng ket va thti nghiem nhanh.
- Hien nay co nhieu loai thti nghiem E L IS A de phat hien khang the, hau
het cac thti nghiem de dtfa tren nguyen tac dung khang nguyen cua virus da dtfdc
co dinh tren pha ran de phat hien khang the co trong mau xet nghiem hoac g in
K T de phat hien KN.
- Cac thti nghiem E L IS A co the lam bang tay, tren cac he thong may
E L IS A ttf dong hoac ban ttf dong.
1.1.1. ThCf nghiem ELISA loai khang y-globulin
a. Nguyen ly cua phan dng
- Stf co mat cua khang the dac hieu trong m iu mau c in xet nghiem the ket
hdp vdi khang nguyen da biet gan tren pha ran de tao thanh phtfc hdp mien dich
khang nguyen- khang the.
250
- Nhan biet phtic hdp mien dich (khang nguyen-khang the) nhd he thong
chi thi mau.
b. Cac biidc tien hanh th ii nghiem
Budc 1: Kiem tra may rtia, u va may doc, dua sinh pham va thuoc thti ve
nhiet do phong thi nghiem (22°C), chua’n bi dung dich rtia.
Btfdc 2: Lap sd do xet nghiem (Vi tri cac gieng nho chting, nho m iu xet nghiem).
Btfdc 3: L&y cac gieng nhtfa da dtfdc gan ban khang nguyen H IV ra khoi tui
giay bac va g in vao phien nhtfa (Hinh 6.1).
A Khang nguyen
Hinh 6.1: Khang nguyen HIV dupe gan tren phien nhi/a
Budc 4: Nho dung dich hoa loang mau vao tat ca cac g ie n g
Budc 5: Nho chting va huyet thanh can xet nghiem vao cac gieng theo dung
sO do da lap va u trong thdi gian nhat dinh d nhiet do xac dinh. Trong thdi gian
nay, cac khang the dac hieu co trong huyet thanh can xet nghiem se phan ting vdi
khang nguyen da biet (H IV , H CV) da dtfpc gan tren be mat gieng nhtfa (Hinh 6.2).
A Khang nguyen
A Khang the HIV
Hinh 6.2: Khang the HIV dtfdc gan vdi khang nguyen HIV
Btfdc 6: Loai bo K T thtia vao cuoi giai doan u, rtia gieng de loai bo K T thtia
va huyet thanh, chuan bi cho btfdc tiep theo (Hinh 6.3). Co the tien hanh rtia tren
may rtia ttf dong hoac ban ttf dong, lan rtia cuoi cting can lam kho day gieng.
A Khang nguyen
A Khang the HIV

mau thti
Hinh 6.3: Loai bo huyet thanh va khang the thtia
251
Budc 7: Them dung dich cong hop vao tat ca cac gieng roi u 0 nhiet do nha't
dinh trong thdi gian xac dinh (Hinh 6.4). Trong giai doan nay cong hdp co ban
chat la mot khang khang the gan enzym, cong hop se la c lu no'i giiia phtfc hdp
khang nguyen -kh an g the dac hieu va chat hien mau. Cong hdp chi gan vdi khang
the ma da dtfdc g in vdi khang nguyen g in ban (Hinh 6.4).
A Kh3ng nguy6n
m x A Khang the’ HIV
x x x
^ CGnghop
Hinh 6.4: Cong hdp dtfdc gan vdi phtfc hop khang nguyen-khang the
Btfdc 8: Cuoi giai doan u, rtfa gieng loai bo thanh phan thtfa, cong hop khong
g in va chuan bi btfdc tiep theo. Qua trinh rtfa cung giong nhtf mo ta d btfdc 6
(Hinh 6.5).
A Khang nguyen
A Khang the HIV
^ COnghpp
Hinh 6.5: Loai bo cong hop thi/a
Btfdc 9: Them ngay dung dich cd chat vao tat ca cac gieng va u d nhiet do xac
dinh, thtfdng la ttf 18 - 25°C, trong thdi gian nha't dinh. Dung dich cd chat chtfa
mot chat hoa hoc la chat hieu mau. Khi them dung dich cO chat vao gieng co chtia
cac thanh phan gan cong hop, enzym se hoat hoa cd chat va do vay hinh thanh
chat mau trong gieng. Cac gieng khong chtfa thanh phan g in cong hdp se khong
thay doi mau khi them cd chat vao gieng (H inh 6.6). Cac gieng co phan tfng (gieng
ban dau chtfa huyet thanh H IV dtfOng tinh) thi co mau, trong khi gieng khong
phan tfng (gieng ban dau chtfa huyet thanh am tinh) thi khong mau. Cac gieng
nho chtfng am, chtfng dtfOng thi co mau khac nhau.
A Khang nguyen
A Khang the HIV
5^ Cong hep
Hinh 6.6: Enzym hoat hoa co chat
252
Btfdc 10: Cuoi giai doan u, them dung dich acid hoa loang vao tat ca cac
gieng de dtfng phan ting. Acid co tac dung bat hoat enzym va co dinh mau.
Btfdc 11 : Doc mat do quang (gia tri OD) cua dung dich trong gieng va danh
gia ket qua thti nghiem.
1.1.2. ThCf nghiem ELISA canh tranh
a. Nguyen ly phan ting

Thti nghiem E L IS A canh tranh cung giong nhu kieu E L IS A khang globulin.
Khang the g in vdi khang nguyen co' dinh va sau do stf co mat cua khang the dtfpc
phat hien, ket qua ngUpc lai v6i ky thuat tren phan ting (+) khong co K T dac hieu,
phan ting (-) co K T dac hieu, ket luan: ket qua (+).
b. Cac btidc tien hanh
Btfdc 1: Chuan bi : Kiem tra may rtia, u va may doc, dtfa sinh pham va thuoc
thti ve nhiet do phong thi nghiem (22°C), chuan bi dung dich rtia.
Btfdc 2: Lap sO do xet nghiem (Vi tri cac gieng nho chtfng, nho mau xet nghiem)
Btfdc 3: Lay cac gieng nhtfa da dtfpc gan ban khang nguyen dac hieu ra khdi
tui giay bac va gan vao phien nhtfa (Hinh 6.1).
Btfdc 4: Nhd chtfng va huyet thanh can xet nghiem vao cac gieng theo dung
sO do da lap va u trong thdi gian nhat dinh d nhiet do xac dinh. Trong thdi gian
nay, cac khang the dac hieu co trong huyet thanh c&n xet nghiem se gan vdi
khang nguyen da biet gan tren be mat gieng nhtfa.
Btfdc 5: Them dung dich cong hop K T dac hieu vdi K N da gan men vao tat ca
cac gieng roi u d nhiet do nhat dinh trong thdi gian xac dinh.
Btfdc 6: Cac gieng dem u d nhiet do xac dinh trong thdi gian nhat dinh.
Trong thdi gian nay, khang the dac hieu nao cd mat trong huyet thanh c in xet
nghiem se canh tranh vdi khang the dac hieu gan men cong hop ve vi tri gan vdi
khang nguyen virus (H inh 6.7)
A Khang nguyen
A Khang the
X c<?n9 hcJP
Hinh 6.7: Canh tranh gii/a cong hop va khang the HIV
Btfdc 8: Cuoi giai doan u, rtia gieng loai bd huyet thanh, cong hpp khdng g in
va chuan bi btfdc tiep theo.
Btfdc 9: Them ngay dung dich cO chat vao tat ca cac gieng va u d nhiet do xac
dinh, thtfdng la ttf 18 - 25°C, trong thdi gian nhat dinh.
253
Btfdc 10: Cuoi giai doan u, them dung dich acid v&o t&'t ca cac gieng de dtfng
phan tfng. Acid co tac dung bat hoat enzym va co dinh mau.
Btfdc 11 : Doc mat do quang (gia tri OD) cua dung dich trong gieng vh danh
gia ket qua thti nghiem.
•) — ?
Neu khdng cd khang the dac hieu trong mau xet nghiem, thi khang the cong
hop se g in vdi khang nguyen trong gieng va stf co mat cua enzym se hoat hoa co
chat thanh san pham cd mau. 0 gieng cd khang the dac hieu trong m iu x6t
nghiem, stf cd mat cua khang the se ngan can khang the cong hpp gan vdi khang
nguyen va vi vay cd it hoac khdng cd mat enzym trong gieng va dan den co it hoSc
khong hoat hoa co chat va khdng co mau (H inh 6.8).
A Khang nguyen
A Khang th i
^ Cdnghop
+
Khong mau
Hinh 6.8: Loai bo huyet thanh va cong hpp thtia
1.1.3. ThCf nghiem ELISA kieu banh kep (sang loc khang the nhtf H IV hoac khang
nguyen nhtf HBsAg).
Hien nay phan ldn cac thti nghiem phat hien anti-H IV va H B sAg cua cac
hang san xuat quoc te la loai nay.
a. Phat hien khang the (khdng the H IV )
• Nguyen ly phan dng: Nguyen ly co ban cua phtfOng phap E L IS A kep gitia
giong vdi E L IS A khang globulin: khang the H IV gan vdi khang nguyen co'dinh va.
stf cd mat cua khang the dtfpc phat hien. Thti nghiem nay khac ve cach phat hien
khang the H IV. Khang nguyen H IV (thtfdng la cac peptid tong hpp) dtfpc gan tren
be mat gieng. Them huyet thanh xet nghiem vao gieng va u. Cuoi giai doan u. rtia
loai bd huyet thanh, them cong hpp roi u tiep tuc. Cong hpp la khang nguyen tong
hpp gan enzym, chti khdng phai la khang the khang IgG ngtfdi gan enzym (nhu
trong E L IS A khang globulin). Trong thdi gian u, khang nguyen cong hpp gan vdi
phtfc hpp khang the khang H IV da gan vdi khang nguyen co" dinh trong gieng.
Mot phtfc hpp “kep” : khang nguyen - khang the - khang nguyen dtfpc tao thanh.
Rtia bd cong hpp thtfa va them chat hien mau vao giong nhtf trong thtf nghiem
khang globulin. Tien bo cua loai thti nghiem nay la tinh dac hieu cua no va ty le
dtfOng tinh gia giam di.
• Cac btidc tien hanh:

Btfdc 1: Kiem tra may rtia, u va may doc, dtfa sinh pham va thuoc thtf ve
nhiet do phong thi nghiem (22°C), chu&n bi dung dich rtfa.
254
Budc 2: Lap sd do xet nghiem (Vi tri cac gieng nho chting, nho m iu xet nghiem)
Btfdc 3: La'y cac gieng nhiia da dtfpc g in ban khang nguyen H IV ra khdi tui
giay bac va g in vao phien nhiia (Hinh 6.9).
A Khang nguyen
Hinh 6.9: Khang nguyen d iid c gan tren phien nhiia
Biidc 4: Nhd dung dich hoa loang mau vao tat ca cac gieng
Biidc 5 : Nhd chiing va huyet thanh can xet nghiem vao cac g i e n g theo dung
sO do da lap va u trong thdi gian nha't dinh 0 nhiet do xac dinh (Theo quy dinh cua
ttfng hang san xuat). Trong thdi gian nay, cac khang the dac hieu cd trong huyet
thanh can xet nghiem se gan vdi khang nguyen dac hieu da dtfpc gan tren be mat
gieng nhtfa (Hinh 6.10).
A Kh^ng nguyen
A Khang the’ HIV
Hinh 6.10: Khang the dac hieu d iid c gan vdi khang nguyen (+),
khong co KT dac hieu (-)
Btfdc 6: Vao cuoi giai doan u, rtia gieng de loai bd huyet thanh va chuan bi
cho btfdc tiep theo (H inh 6.11). Cd the tien hanh rtia tren may rtia ttf dong hoac
ban ttf dong, lan rtia cuo'i cung can lam khd day gieng.
A Khang nguyen
A Khang the’ HIV

(Miu thCr)
Hinh 6.11: Loai bo huyet thanh va khang the thtia
Btfdc 7: Them dung dich cong hpp vao tat ca cac gieng roi u d nhiet do nhat
dinh trong thdi gian xac dinh (Theo quy dinh cua ttfng hang san xua't). Trong giai
255
doan nay cong hpp co ban chat la mot khang nguyen g in enzym, cong hpp se 16
cau noi gitia phtic hpp khang nguyen - khang the dac hieu va chat hien mau. Cong
hpp chi gan vdi khang the da dtfpc gan vdi khang nguyen g in ban (H inh 6.12).
A Khang nguyen
A Khang the’ HIV (Mau thir)
^ Khang nguyen HIV gin enzym

(Cong hpp)
Hinh 6.12: Cong hpp dtfpc gan v 6 i phtic hpp khang nguyen - khang the
Btfdc 8: Cuo'i giai doan u, rtia gieng loai bo thanh phan thtia, cong hpp khong
g in va chuan bi btfdc tiep theo. Qua trinh rtia cung giong nhtf mo ta 6 btfpc 6 (xem
hinh 6.13).
A Khang nguyen
A Khang the
□
^7 Khang nguyen gan enzym
(Cong hap)
Hinh 6.13: Loai bo cong hpp thtia
Btfdc 9: Them ngay dung dich cO chat vao tat ca cac gieng va u d nhiet do xac
dinh, thtfdng la tti 18 - 25°C, trong thoi gian nhat dinh (Theo quy dinh cua tting
hang san xuat). Dung dich cO chat chtia mot chat hoa hoc la chat hieu mau. Khi
them dung dich co chat vao gieng co chtia cac thanh phan gan cong hpp, enzym se
hoat hoa cO chat va do vay hinh thanh chat mau trong gieng. Cac gieng khong
chtia thanh phan g in cong hpp se khong thay doi mau khi them cd chat vao gieng
(Hinh 6.13). Cac gieng co phan ting (gieng ban dau chtia huyet thanh H IV dtfOng
tinh), thi co mau trong khi gieng khong phan tfng (gieng ban dau chtfa huyet
thanh am tinh) thi khong mau. Cac gieng nho chtfng am, chtfng dtfOng thi co mau
khac nhau (Hinh 6.14).
A Khang nguyen
A Khang the
□
^7 Khang nguyen gan enzym
(Cong hop)
+
Co mau Khong hien mau
Hinh 6.14: Enzym hoat hoa cd chat
256
Bade 10: Cuo'i giai doan u, them dung dich acid hoa loang vao tat ca cac
gieng de dtfng phan ting. Acid co tac dung bat hoat men va co dinh mau.
Bade 11 : Doc mat do quang (gia tri OD) cua dung dich trong gieng va danh
gia ket qua thti nghiem. Trong cac gieng chtia m iu co khang the, cong hop dtfO c
gin vdi khang the va men hoat hoa cO chat de tao thanh san pham mau.
b. Ky thuat phat hien khdng nguyen (H BsAg) (banh kep)
• Nguyen ly cua phan ting : N g u y e n l y cO b a n c u a t h t i n g h i e m E L IS A p h a t
E L IS A p h a t h i e n k h a n g t h e n h t f n g
h i e n k h a n g n g u y e n tu fO n g t u n h u t h t i n g h i e m
6 d a y , v a i t r o c u a k h a n g n g u y e n v a k h a n g t h e n gtfO c n h a u , k h a n g n g u y e n d a c
h i e u g i n v d i k h a n g t h e co" d i n h v a p h a t h i e n s t f c o m a t c u a k h a n g n g u y e n .
• Cac bitdc tien hanh:

Btfdc 1: Kiem tra may rtia, u va may doc, dtfa sinh pham va thuoc thti ve
nhiet do phong thi nghiem (22°C), chuan bi dung dich rtia.
Btfdc 2: Lap so do xet nghiem (Vi tri cac gieng nho chtfng, nho mau xet nghiem)
Btfdc 3: Lay cac gieng nhtfa da dtfOc gan ban khang the HBs ra khoi tui giay
bac va gan vao phien nhtfa (Hinh 6.15)
Y Khang the Hbs
Hinh 6.15: Khang the HBs dUdc gan tren phien nhtfa
Btfdc 4: Mau nghiem de nguyen hoac hoa loang dtfdc cho vao cac gieng theo
dung so do da lap va dtfdc u dung nhiet do va dung thdi gian (Theo quy dinh cua
ttfng hang san xuat). Trong thdi gian nay, khang nguyen dac hieu (neu cd) se g in
vdi khang the (Hinh 6.16).
Y Khang the Hbs

♦ Khang nguyen
(M iu thti)
Hinh 6.16: Khang nguyen se dtidc gan vdi khang the HBs
Btfdc 5: Sau khi u, cac gieng dtfdc rtia de loai bo khang nguyen thtia.
Btfdc 6: Dung dich cong hop dtfdc them vao moi gieng. Sau dd cac gieng dtfdc
u dung nhiet do va du thdi gian (Theo quy dinh cua ttfng hang san xuat). Trong
thdi gian nay, cong hdp ket hop vdi khang nguyen dac hieu da gan tren gieng.
(Hinh 6.17).
257
Y Khang th6 Hbs
K « Kh^ng nguyen (miu thir)
K >-
Khang the Hbs gan men
X (cdng hop)
Hinh 6.17: Cong hop ket hop vdi phtic hop khang nguyen-khang the
Btfdc 7: Cuoi giai doan u, cac gieng dtfOc dem rtia de loai bo cong hop khong g&n.
Btfdc 8: Dung dich co chat nen dtfOc them vao moi gieng va lai dtfOc u 6 nhiet
do xac dinh. Khoang 18 - 25°C va dung thdi gian.
Btfdc 9: Cuoi giai doan u, dung dich acid dtfOc them vao de lam ngtfng phan tfng.
Btfdc 10: Doc mat do quang (OD) cua dung dich trong gieng va danh gia ket
qua thti nghiem.
Btfdc 11: Trong cac gieng co khang nguyen dac hieu thi cong hop dtfOc gin
vdi khang nguyen va men hoat hoa cd chat de tao mau (Hinh 6.18).
Y Khang the HBs
+ Khang nguyen HBsA (mau thii)
^ Khang the’ HBs gin men

(cong hop)
Co mau Khong mau
Hinh 6.18: Enzym hoat hoa co chat
1.1.4. Danh gia ket qua thCr nghiem ELISA
• Gia tri OD va gia tri ngtfdng

M at do quang (gia tri OD) cua moi gieng dtfcfc xac dinh khi chum anh sang d
btfdc song nao do chieu qua day gieng va do mtfc do hap thu anh sang cua dung
dich. De lam viec nay ngtfdi ta dung phtfOng tien dac biet goi la may doc phien,
thtfc chat la mot quang pho ke phtfc hop nhieu btfdc song. M ay doc phien doc cac
gia tri OD theo ttfng hang 8 gieng va doc dong thdi 8 gieng mot luc. Ket qua nay
in ra trtfc tiep hoac ndi sang mot may vi tinh de xti ly so lieu.
K et qua cuoi cung cua thtf nghiem E L IS A la tap hop cac gia tri OD hoac
danh gia luon la am tinh va dtfOng tinh. Bang 6.1 cho thay ket qua thu dtfOc tti
thti nghiem tim khang the H IV d cac m iu huyet thanh ngtfdi cho mau.
De sti dung dtfOc cac gia tri OD, ngtfdi ta phai so sanh chung vdi ket qua
chuan da biet. Ket qua nay gom tap hop cac chtfng va mau xet nghiem. Cd nhieu
258
cach tinh ket qua khac nhau, toi thieu c&n xac dinh 3 diem cho moi thii nghiem
ELISA. Trong do 2 diem la gia tri trung binh cua chting am va chting dtfOng - de
xac dinh xet nghiem la co y nghia va chung phai nSm trong khoang quy dinh cua
nha san xuat. Cuoi cung, de danh gia ket qua phan tfng hay khong phan tfng,
phai tmh toan gia tri ngtfdng.
Trong kieu E L IS A l (khang globulin va banh kep gia tri OD cao se co mau,
va cho ket qua “phan tfng” , con gia tri OD thap hay khong mau la ket qua “khong
phan tfng” . Vi v&y, cac gia tri tren ngtfdng la phan tfng v& gia tri dtfdi ngtfdng la
khong phan tfng.
Trong kieu ELISA2 (canh tranh) gia tri OD cao hay co mau la ket qua
“khong phan tfng” , gia tri OD th&'p hay khong mau la ket qua “phan ting” . Vi v&y
cac gia tri dtfdi ngtfdng la cac ket qua “co phan tfng” con cac gia tri tren ngtfdng la
cac ket qua “khong phan tfng” .
Chon gia tri ngtfdng dung rat quan trong doi vdi thti nghiem ELISA. Tinh
toan gia tri ngtfdng thtfdng can ctf gia tri OD chtfng am. Gia tri trung binh dtfdc
tinh ttf moi chtfng am - trung binh chtfng am (Ncm ) va don gian hoa b ln g cach
tinh gia tri ngtfdng thtfc te cho phien phan ting theo cong thtic sau:
Gia tri ngtfdng = Ncm + 0,2
Trong thi du dtfdi day (Bang 6.1), gia tri trung binh cua chting am la 0,16.
Gia tri ngtfdng vi vay se la 0,36 (0,16 + 0,2). Cac gia tri OD nao tren 0,36 thi dtfOc
coi la co phan tfng va cac gia tri OD nao dtfdi 0,36 d tfO c coi la khong phan ting.
B a ng 6.1: Vi du ket qua thu di/oc tti kieu ELISA khang globulin/ kep.
1 0,141
2 0,158
3 0,903
4 0,161
5 0,148
Chting am 0,156
Chting am 0,167
Chting am 0,157
Chting duong 1,352
Chting duong 1,283
A. Gia trj OD cua mau va chting.
B. Ket qua tuong ting duong tinh va am tinh (bay gid chUa can ghi).
C. Ty so da'u hieu/ngudng (bay gid chUa can ghi).
259
M at khac, gia tri ngtfdng co the bao gom cach ti'nh ttf gia tri OD cua chtfng
dtfOng va chtfng am hoac tham chi gia tri ngtfdng co dinh cua nhk san xuat. Tuy
nhien, cong thtfc de xac dinh gia tri ngtfdng dtfdc ap dung ttfdng ttf nhtf nhau.
1.2. Thur nghiem ngung ket hat

1.2.1. Nguyen ly cua phan Cmg
Thii nghiem ngtfng ket hat phat hien stf co mat cua khang the H IV nhd sU
ngtfng ket cac hat gelatin hoac latex da g in khang nguyen HIV. Thtf nghiem nay
co the dtfdc sii dung de phat hien HBsAg. Thtf nghiem thtfc hien trong cac gieng,
nhtfng giong nhtf thii nghiem gan tren bi, cac gieng chi don gian la ndi thtfc hien
phan tfng chtf khong gan gi ca.
Mot tfu diem chung cua loai thii nghiem nay la khong can cac dung cu dat
tien. Thii nghiem nay chi qua it giai doan, khong can riia gieng phan tfng va c6
the doc b ln g m it. Hat ngtfng ket khang nguyen va khang the dtfdc san xuat cung
mot kieu.
a. Phat hien khang the: Nguyen tac cua xet nghiem la co dinh khang nguyen
H IV tren hat vi ltfOng, ttfOng ttf nhtf tren gieng hay bi dtfdc mo ta trtfdc day doi
vdi thii nghiem ELISA. Nguon va loai khang nguyen cung giong nhtf doi vdi
E L IS A mac du khong dung cac peptid tong hop (Hinh 6.19).
A Khang nguyen
Hinh 6.19: Khang nguyen dUdc g§n tren hat vi ltfdng
b. Phat hien khdng nguyen HBsAg: Co sd cua xet nghiem ngtfdc vdi xet
nghiem phat hien khang the: Khang the dac hieu dtfdc co dinh tren be mat hat.
Nguon va loai khang the c in thiet giong nhtf trong thii nghiem E LIS A (Hinh 6.20).
Y Khang the
Hinh 6.20: Khang the dUdc gan tren hat vi luong
1.2.2. Cac budc tien hanh (Hinh 6.22)
Btfdc 1: Mau xet nghiem va chtfng dtfdc hoa loang bang dung dich hoa loang
mau (Tat ca cac thii nghiem ngtfng ket hat deu yeu cau phai hoa loang m iu trtfdc
de han che den mtfc thap nhat phan tfng dtfOng tinh gia). Trong mot so thii
nghiem dung dich hoa loang rat phtfc tap va luon luon dtfOc cung cap trong hop
sinh pham.
260
Bade 2: Thac hien qua trinh hoa loang trong cac gieng. Chuyen ra khoi
phien phan ang the tich m iu hoa loang 0 gieng cuo’i cung.
Bade 3: Them cac hat vao m iu da hoa loang va u, thudng la d nhiet do
phong (18 - 25°C) trong thdi gian khoang 1 - 2 gid 30 phut. Trong thdi gian u, cac
hat ngUng ket lai tUOng tu nhu phan ting gitia hong cau va khang huyet thanh
dinh nhom mau do co khang the H IV trong mau (Hinh 6.21).
A Khang nguyen
Y Khang the
Hinh 6.21: Khang the ket hop vdi khang nguyen tao phan ting ngung ket
Trong mot so thti nghiem, mau duoc kiem tra kep b ln g cac hat co gan va
khong gan khang nguyen. Nhting hat khong gan khang nguyen dung de xac dinh
phan ting khong dac hieu.
Bade 4: Cuo'i giai doan u, doc ket qua xet nghiem. Thti nghiem ngang ket ndi
chung dUOc doc bang m lt thudng va ket qua ghi lai la dUOng tinh hay am tinh.
Doc b ln g mat thich hop cho nhting thti nghiem nay vi ngUng ket cua cac hat co
the nhin thay ro rang bang mat dUOc. Neu dung hong cau gan khang nguyen thi
sU ngUng ket hoan toan co the thay dUOc trong gieng.
Neu dung cac hat gelatin gan thi cung thay duoc nhd mau xanh nhat. Con
neu dung hat latex thi cung thay ro duoc cac hat mau trang khi dat tren nen den.
Ket qua cua phan ting ngUng ket duoc trinh bay trong hinh 6.22. Ket qua “co
phan ting” se xuat hien mot mat phang md, trai deu cac hat ngUng ket hoac tao thanh
bd nham nhd 0 day gieng . Ket qua “khong phan ting” se xuat hien hinh nut hoac hinh
vong nhan cua cac hat khong ngUng ket lin g xuong day gieng (Hinh 6.22).
261
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Hinh 6.22: Cac bi/dc tien hanh va nhan djnh ket qua trong phan ung ngung ket hat
262
1.3. C4c thur nghiem nhanh
Loai thii 3 cua thti nghiem dtfdc quan tam la thti nghiem nhanh, cac thti
nghiem nay chi dung mot lln va cho ket qua nhanh vdi do nhay cao. Thti nghiem
nay dtfOc sti dung de phat hien ca khang the (khang the H IV, HCV) va khang
nguyen (nhu HBsAg). Thti nghiem don gian co mot so kieu khac nhau. Co sd la
khang nguyen hay khang the (tuy thuoc vao thti nghiem), dtfO c bat hoat tren
mang tham hoac mang ban tham, hoac tren mot dai bang. Loai nay cung cap cac
dang don gian de de dang cho mau thti hoac cac chat thti khac them vao. Phan ldn
cac thti nghiem don gian d tfO c chtia trong mot bo kit gom tat ca cac chat thti can
thiet de tien hanh ky thuat, cac thti nghiem nhanh nay co the dung huyet thanh
hoac huyet tuong va trong mot so' trtfdng hop cd the dung ca mau toan phan. Cac
thti nghiem nay cd the chia thanh 3 nhom theo nguyen tac sau day. Nhin chung
cac nguyen tac nay bao gom stf trinh bay cua ky thuat.
- Mien dich sac ky
- Loc mien dich

- E L IS A thong thtfdng
Sau day la cac vi du ve nguyen tac chung, phtfOng phap va tfng dung cua ba
nhom khac nhau. Stf khac nhau cua ba nhom dtfOc phan anh trong thiet ke va xay
dUng bdi cac nha san xuat hon la stf khac nhau ve nguyen tac dtfOc chap nhan.
1.3.1. ThCr nghiem miSn djch s ic ky (De phat hien khang the HIV, HCV, HBsAg)
a. Nguyen ly cua phan ting: Thti nghiem mien dich sac ky dtfa tren nguyen
tac ddng mau thtf di theo mot chieu tham dac biet cd chtfa cac chat thti hoa tan,
vdi stf lang dong cac chat va quan sat dtfO c cac phtfc hop mien dich 0 cac vi tri doc
theo thanh thti. Loai thti nghiem nay phan ldn dang cd ban san hien nay.
b. Cac btidc tien hanh
Btfdc 1: Mau thti dtfO c them vao va hap thu qua mot tam dem 0 dau thanh
thti. Mau di chuyen doc theo thanh thti.
Btfdc 2: Thanh thti co chtia cong hop khd, cong hop khang nguyen virus ket
hop vdi muoi vang, hoac ltfu huynh hoa tan trong mau doc theo thanh thti. Muoi
vang hoac ltfu huynh (yeu to' khac cung cd the dtfOc stf dung) la stf chuan bi cua
vang hoac ltfu huynh bao gom cac hat xac dinh dang trong dung dich cd the bi hoa
chat ket hop thanh phan tti ldn hOn nhtf la khang nguyen va khang the. Trong
khi cac hat rieng cd the qua nhd de nhan biet b ln g m&t thtfdng, cac ngtfng tap va
ngtfng ket cua cac hat cd the xem de hOn.
Btfdc 3: Khang the dac hieu trong mau ket hcfp vdi cong hop va tiep tuc di
doc theo thanh thti nhtf la phtfc hop mien dich.
Btfdc 4: Khang nguyen virus dtfOc co dinh d mot vach xa hOn cac thanh thti.
M iu thti chav qua vach nay, khang the dac hieu cd mat ket hop vdi khang nguyen cd
dinh. Nhtf ph ln ldn cac khang the dac hieu san sang ket hop vdi no, mot vach xuat
hien ngang thanh thtf nhtf la muoi vang hoac ltfu huynh dtfOc tao ra d vi tri dd.
263
Budc 5: Nhan dinh ket qua (H in h 6.23).
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H inh 6.23: Nhan dinh ket qua cua th if nghiem sac ky mien dich
1.3.2. Cac thti nghiem loc miSn djch
a. Nguyen ly ky thuat: Thu nghiem loc mien dich dtfa tren nguyen ly mau
thu va hoa chat d tfO c loc qua mang loc tren do co co" dinh khang nguyen va khang
the trong mot dia phan ting. Khi co khang nguyen hoac khang the dac hieu trong
mau se gan vdi mang qua viec tao phtic hop mien dich. Khi them cong hop, se ket
hop vdi phtic hop mien dich. mau se xuat hien d dia phan ting.
b. Cac biidc tien hanh
Budc 1: Nho mau vao dia phan ting va khang the co trong mau thti se gan
vdi khang nguyen d tfO c co' dinh san khi tham qua mang.
Budc 2: Sau khi u mau, mang phan ting dtfOc rtia bang dung dich rtia dtfO c
cung cap san, khi qua mang se lay di cac khang the thtia trong mau. Mot vai loai
sinh pham khong can btfdc nay.
Btfdc 3: Cong hop d tfO c them vao sau do. chat nay d tfO c cung cap d dang kho.
Chat tong hop cua cong hop khac nhau tuy theo ky thuat. Nhtfng dung protein A
gan vdi muoi vang la cach pho bien. Protein A la mot protein d tfO c san xuat b d i v i
khuan Staphylococcus aureus. Protein A co phan gan vdi Fc (gan vdi bd the) cua
phan tti IgG. Khi cong hop d tfO c them vao. protein A se ket hop vdi khang the dac
hieu co mat trong mau thti. Stf ket hop nay se xuat hien mau n O i mang phan t f n g
khi co khang the dac hieu gan (Hinh 6.24).
264
Ket qua am tinh Ket qua difcfng tfnh
Hinh 6.24: Thir nghiem loc mien djch phat hien khang the HCV
1.3.3. Thti nghiem nhanh loai ELISA thong thudng
a. Nguyen ly: Thti nghiem nhanh ddn gian nay dua tren nguyen ly cua
phUdng phap E L IS A thong thudng bao gom mot khay phan ting tren do co m iu
thti va cac hoa chat khac. Thong thudng, chat long ngam cham qua mang roi
chuyen sang bo phan thai qua vat lieu tham hoac dUdc lay ra khoi dia phan ting
sau moi budc cua ky thuat. Su ket hdp khang the hoac khang nguyen trong m iu
thti vdi khang nguyen hoac khang the co dinh xuat hien, sau do la stf ket hop vdi
cong hop, co chat dtfOc them vao va mau sac se xuat hien neu co cac marker dac
hieu trong mau.
b. Cac biidc tien hanh:
Budc 1: Mau thti d tfO c nho vao gieng phan ting khang the se ket hop vdi
khang nguyen co" dinh san. Dung dich hoa loang mau co the d tfO c cung cap trong
mot lo rieng de hoa loang mau thti trUdc khi nho vao gieng phan ting.
Budc 2: Sau khi u, m iu thti thtia dtfOc loai bo b ln g cach rtia gieng phan ting.
Mot vai loai kit khong can budc nay.
Budc 3: Cong hop se dtfOc them vao, cac ky thuat khac nhau thi cong hop
khac nhau. Mot vai ky thuat sti dung cong hop khang globulin ngudi (IgG ) giong
nhu trong ky thuat ELISA.
Budc 4: Sau khi them cong hop, rtia lai dia phan ting va them chromogen,
roi doc ket qua b ln g mat thudng. Co chat hoat hoa roi lang dong tren mang tao
m iu thich hop.
1.3.4. M ot so diSm ve s ti dung th ti nghiem nhanh ddn gian
- Khi xuat hien vach hay diem dac hieu (ket qua duong tinh). P h ln ldn cac
thti nhiem nhanh hien nay deu co phan ttf kiem soat di kem. dung de phat hien
protein hay albumin trong mau. Dieu nay chting to r in g kit hoat dong tot.
- Ket qua la am tinh khi co vach hoac diem d o kiem chting ma khong co
vach hay diem d o thti nghiem. K et qua d U O n g tinh khi co mat ca hai vach hoac
diem d o thti nghiem va o kiem chting. Neu vach 0 o kiem chting khong co coi nhu
xet nghiem khong co gia tri (Hinh 6.23).
265
- Mac d u k h o n g c a n c h t f n g k i e m t r a b e n n g o a i , n h u n g c l n c h u & n bi c h t i n g
d t f O n g y e u c h o m o i m e t h t i , ly t t f O n g h o n c a n c o c h t i n g a m c h o ky t h u a t . Sti d u n g
m iu da b i e t k e t q u a l a c a n t h i e t t r o n g vai t r t f d n g h d p c l n d o i c h i e u k e t q u a . V i d u
n h t f mau k h o n g r d r a n g .
- Nhin chung, toan bo quy trinh xet nghiem ttf khi nho m iu thtf den khi c6
ket qua la 10 -15 phut. Tuy nhien, thdi gian cua nha san xuat phai dtfdc ch&'p
hanh, tranh u qua thdi gian co the d in den ket qua dtfdng tinh gia trong mot so
trtfdng hop. Chung ta vtfa xem xet cac nguyen ly cua ba loai thtf nghiem chinh
sang loc H IV, HCV, HBV. Mac du co the chi dung mot trong so cac thti nghiem,
nhtfng dieu quan trong la hieu nguyen ly cua tat ca ba loai do va can ttf danh gia
loai nao cho phu hop.
2. Sang loc giang mai

2.1. Tac nhan nhiem trung: Tac nhan gay benh giang mai la xoan khuan
Treponema pallidum la thanh vien cua nhom xoan kh u in da biet la Spirochaete.
Co bon nhom chinh co lien quan chat che dtfOc mo ta la dtfdi nhom cua T. Pallidium.
- Treponema.Pallidum - pallidum
- Treponema.Pallidum - perteneve
- Treponema.Pallidum - carateum
- Treponema.Pallidum - endemicum
6 day chung ta chi can quan tam den nhom gay benh do Treponema.
Pallidum - pallidum (goi tat la T. Pallidum) d tfO c coi la tac nhan gay benh quan
trong nhat.
2.2. y nghia trong thuc hanh truyen mau
Giang mai la tac nhan lay nhiem dau tien d tfO c phat hien la lay nhiem qua
dtfdng truyen mau (1910), trtfdc day co rat nhieu trtfdng hop bi lay nhiem giang
mai qua dtfdng nay. Hien nay, nhting trtfdng hop nhtf vay thinh thoang v ln xuat
hien d cac ntfdc co ty le nhiem giang mai cao. Tuy nhien, chtfa bao gid truyen mau
la nguyen nhan chinh cua viec lan rong benh giang mai. O cac ntfdc co it trtfdng
hop bi mac giang mai, phan ldn cac ca bi nhiem d tfO c xac dinh trong so ngtfdi cho
mau la do bi lay nhiem ttf trtfdc va da d tfO c dieu tri thanh cong va khong c o dau
hieu nguy cO lay nhilm qua dtfdng truyen mau. Tuy nhien vdi viec loai trtf ngtfdi
cho mau co nguy CO, sang loc giang mai va bao quan mau d 4°C trtfdc khi truyen
thi nguy cd nhiem giang mai sau truyen mau la rat thap 0 nhieu ntfdc.
2.3. Ngan ngira lay lan
Viec ngan ngtfa lay nhiem giang mai chu yeu bang giao due va viec xay dtfng
phat trien cac chtfdng trinh dieu tri va sang loc hieu qua. Benh lay nhiem qua
dtfdng tinh due la nguyen nhan chinh lay benh. Viec sang loc tat ca mau. che
pham mau, mo va cac cd quan la rat quan trong. truyen mau chi la mot con dtfdng
rat nhd lay nhiem giang mai.
266
2.4. Sdng Igc giang mai
2.4.1. Phuong phap soi true tiep
Cd t h e d u n g k i n h h i e n v i n e n d e n d e q u a n s a t t r t f c t i e p x o i n k h u a n t t f d i c h
lay d v e t xtfdc, d i e u n a y c h i c o t h e t i e n h a n h d g i a i d o a n n h i e m t r u n g c h i c c h a n .
Do v & y , h u y e t t h a n h h o c l a p h t f O n g p h a p c h i n d o a n c h i n h .
2.4.2. Phuong phap huyet thanh hoc
Cd hai loai: Xet nghiem khong dac hieu va xet nghiem dac hieu.
• Xet nhiem khong dac hieu (VDRL hoac RPR): Sti dung cardiolipin de phat
hien khang the chong Treponema pallidum. Cardiolipin la mot thanh phan binh
thtfdng cua mo, do vay co khoang 1% ngtfdi trtfdng thanh binh thtfdng san xua't
khang the khong dac hieu chong lai khang nguyen nay nen co the cho ket qua
phan tfng V D R L (+) gia. K y thuat nay hien nay dang dtfOc sti dung rong rai d ntfdc
ta de sang loc giang mai cho ngtfdi cho mau vi day la mot thti nghiem cho ket qua
nhanh, co the dung ca huyet thanh hoac huyet ttfOng, ky thuat don gian, ket qua
dtfcfc nhan dinh de dang bang mat thtfdng.
- Nguyen ly ky th u a t: Hat latex co gan khang nguyen Cardiolipin se bi
ngtfng ket neu trong huyet thanh, huyet ttfOng cua ngtfdi cho mau hoac benh
nhan co khang the giang mai.
- Dung cu va thuoc thti :
+ Pipette nhd giot
+ Tam Kline
+ M ay lac Kline
+ Kinh hien vi
+ Khang nguyen Cardiolipin d tfO c pha s in trong c a c chai nhd giot

+ Chtfng am
+ Chtfng d t f O n g
M iu h u y e t t h a n h v a h u y e t t t f O n g k h d n g d tfO c l l n te bao, k h d n g n h i e m
k h u an , k h d n g ta n m a u , k h d n g v a n due.
- Cac btfdc t i e n hanh:
Btfdc 1: Dtfa bo thuoc thti ve nhiet do phong thi nghiem 10 phut va lie deu
trtfdc khi sti dung.
Btfdc 2: Nhd len mot tam Kline 30^1 mau thti, chting am va chting dtfOng
Btfdc 3: Nhd tiep mot giot V D R L - latex

Btfdc 4: Tron deu huyet thanh can thti, chting dtfOng vdi hat khang nguyen
VD RL-Latex b ln g mot que nhtfa.
Btfdc 5: Lac 6 phut tren may lie Kline
267
Btfdc 6 : Doc va nhan dinh ket qua b ln g mat thtfdng v& tren kinh hien vi
+ Cac hat latex phan bo' dong nha't (Khong ngtfng ket) : Phan tfng am tinh
+ Cac hat latex ngtfng k e t : Phan tfng dtfOng tinh
• Xet nghiem dac h ie u (T P H A ): Dung xoan khuln Treponema pallidum 16m
khang nguyen de phat hien khang the dac hieu khang lai xo&n khuan giang mai
trong huyet thanh hoac huyet ttfOng.
Ky thuat E LIS A cung dtfOc ap dung de phat hien khang th i dac hieu nhtfng
chi phi so vdi thtf nghiem ngtfng ket hat thi dat hdn, do vay p h ln ldn cac ntfdc
dang phat trien van dung thti nghiem ngtfng ket hat de sang loc giang mai cho
ngtfdi cho mau.
3. Sang loc sot ret

Sot ret gay ra do nhiem trung mot trong cac loai dong vat nguyen sinh goi la
ky sinh trung. Hang nam co khoang 150 trieu ngtfdi bi nhiem sot ret va khoang 2
trieu ngtfdi chet vi sot ret. Nhiem trung truyen qua vet can cua muoi Anopheles
cai. Co tren 200 loai Anopheles va khoang 60 loai la yeu to' gay benh sot ret. Ban
than con ngtfdi la cac o nhiem trung, mot con muoi can mot ngtfdi bi sot ret co the
truyen sot ret sang cho ngtfdi khong bi nhiem khi dot ho.
3.1. Tac nhan nhiem trung

Ky sinh trung la mot loai dong vat nguyen sinh truyen benh theo nhieu
cach, nhung chung den ky sinh tren mot hoac nhieu dong vat khac. Dang chu y la
chung can hai vat chu khac nhau de hoan thanh chu ky sinh san : ngtfdi va muoi.
Co bon loai ky sinh trung la nguyen nhan gay sot ret
- Plasm odium vivax
- Plasm odium ovale
- Plasm odium m alariae
- Plasm odium falciparum
Nhiem do P .fa lciparum thtfdng gay ra tti vong d ngtfdi
3.2. Sang loc sot ret

Cach chan doan chac chan nha't la soi mau tim ky sinh trung sot ret trong
hong cau. Tuy vay khong thich hdp cho sang loc so' ltfdng ldn ngtfdi cho mau.
Nhuom lam mau va quan sat la pho’ bien, phtfOng phap nhuom khang the gan
huynh quang dtfdc dung 0 mot vai phong thi nghiem.
Nhtfng nam gan day, ky thuat phat hien khang the chong mot protein be
mat da dtfOc san xua't, phan ldn la ky thuat ngtfng ket hat, mac dau da co mot vai
ky thuat ELISA.
3.3. y nghia trong thuc hanh truyen mau

Nhieu ttf lieu noi ve truyen mau la nguyen nhan gay sot ret, chu yeu la
P .fa lcip a ru m . Mtfc do lav truyen do truyen mau tuy thuoc hoac noi cd ty le cao sot
268
r6t ltfu h^nh d so ngtfdi cho mau. 6 cac ntfdc co sot ret lan tran, phan ldn co ca the
bi nhiem b tuo’i tre. Viec truyen so't ret do truyen mau khong that stf co y nghia
bdi vi phln ldn benh nhan da bi nhilm trtfdc. C) cac ntfdc ty le sot ret thap, sang
loc trd nen quan trong hon.
That khong may, thti nghiem sang loc thich hdp khong dtfOc pho bien rong
rai. Gan day, cach sang loc hieu qua nhat la hoi ho co du lich den vung co sot ret
va cach de phong sot ret cua ho. Viec tri hoan ngtfdi cho mau da den vung sot ret
la bien phap hieu qua nha't de ngan truyen so't ret do dtfdng truyen mau. Ngtfdi
cho mau co the cho muon hon tuy theo chinh sach cua ttfng dia phtfOng hoac quoc
gia, tuy thuoc vao tien sti benh va cach phong cua ho, tot nhat la tam hoan khong
la'y mau d do'i ttfOng nay trong 46 thang. Trong vai trtfdng hop, cd the dung dieu
tri phong so't ret sau truyen mau.
KY THUAT DjNH NHOM MAU ABO VA PHAT MAU AN TOAN
1. Nguyen ly
Nhom mau he ABO dtfO c x a c dinh nhd stf co mat cua khang nguyen tren be
mat hong cau va khang the trong huyet thanh. Hai thanh phan nay khi gap nhau
se xay ra phan tfng ngtfng ket dac hieu (bang 6.2)
T t f O n g t t f n h t f v a y , k h a n g t h e c h o n g A va c h o n g B k h i g a p h o n g c l u m i u A,
B c u n g se x a y ra p h a n tin g n g tfn g k e t h o n g c a u d a c h ie u .
Bang 6.2 : S ir co mat cua khang nguyen va khang the nhom mau ABO trong mot ca the
Nhom mau Khang nguyen hong cau Khang the trong huyet thanh
A A Chong B
B B Chong A
AB AB Khdng cd khang the chong A va B
0 Khong cd khang nguyen A va B Cd khang the chong A va B
2. Dung cu va hoa chat

2.1. Dung cu
- M ay ly tam
- Kinh hien vi
- Pipette Pasteur
- Phien da 12x12cm
- Que thuy tinh
269
- Tu lanh de sinh ph im
- Binh cach thuy 37°C
- Keo
- Panh co mau, khong m&'u
/s'
- Ong nghiem tan mau
- Lam kinh
- Qua bop cao xa
- Coc mo thuy tinh
- Bong tham ntfdc
- Tu a'm 37°C
- Tu say kho
- Giay tha'm
- Ba coc thuy tinh 500ml
- But viet tren kinh
2.2. Thuoc thur

- Huyet thanh m iu : khang the chong A, chong B, chong A B (Dtfdc san
xua't theo phtfOng phap khang the ddn ddng).
- Hong cau mau A 10%, 5%
- Hong cau m iu B 10%, 5%
- Ntfdc muoi 9%o

- Ntfdc cat
2.3. Mau mau xet nghiem

- Mot o'ng nghiem lay 5ml mau tinh mach khdng cd chong dong, mot dng
khac lay 2ml cd chong ddng b ln g ACD theo ty le 1/5 the tich mau. Ong mau nay
dung lay hong cau.
- Ly tam tach huyet thanh o'ng mau khdng chong ddng
- Rtia hong cau benh nhan b ln g ntfdc muo'i 9%o 3 lan, pha thanh dung dich
hong cau 5% trong ntfdc muoi 9%o.
3. Q u y trinh ky t h u a t : Co 3 ky thuat
- Dinh nhom mau ABO tren phien (da, nhtfa, thuy tinh)
- Dinh nhom mau trong o'ng nghiem
- Dinh nhom mau trong gelcard
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3.1. Dinh nhdm mau tren phi§n: Tien hanh hai phtfdng phap: huyet thanh m iu
va hong clu miu.
- l/u diem: nhanh, ddn gian, d i doc ket qua.

- Nhtfdc diem: kho danh gia khi gap khang the yeu, hoac khang nguyen &n
phan tfng ngtfng ket khong ro rang.
3.1.1. Phuong phap huyet thanh m iu
- Tren phien da sach m3, kho nho 1 giot huyet thanh m iu (khang the
chong A, chong B, chong AB) vao 3 vi tri khac nhau tren phien : 1,2,3.
- Them 1 giot dich treo hong cau c ln dinh nhom 10% vao 3 vi tri 1,2,3
- Tron deu huyet thanh mau vdi hong cau can dinh nhom bang cac que
thuy tinh de co mot vong tron co dtfOng kinh 20-30cm lac nhe lien tuc trong vong
2 phut roi doc va ghi lai ket qua.
3.1.2. Phuong phap hdng cau mau
- Tren phien da nho mot giot hong cau m iu A va mot giot hong clu m iu B
10% vao vi tri 4,5.
- Them hai giot huyet thanh can dinh nhom vao vi tri 4,5.
- Tron deu huyet thanh can thti vdi hong cau mau lam thanh mot vong
tron co dtfdng kinh 20-30cm. Lfic nhe lien tuc trong vong 2 phut roi doc va ghi lai
ket qua.
3.2. Phuong phap djnh nhom tren ong nghiem
La mot phtfdng phap tot cho cac trtfdng hcfp dinh nhom tren phien da khong
ro rang va trong cac trtfdng hdp muon xac dinh nhanh nhom mau, day la phtfdng
phap xac dinh nhom mau he ABO hien nay hay dtfOc sti dung nhat.
3.2.1. Phuong phap huyet thanh m iu

- Nho mot giot huyet thanh mau khang the A, khang the B, khang the AB
vao 3 o'ng nghiem da dtfdc chuan bi d tren .
- Them vao moi ong nghiem mot giot hong cau can dinh nhom da dtfOc hoa
loang (5% trong ntfdc 9%o).
- Tron deu va ly tam 1000 vdng/phut/1 phut
- Nghieng nhe thanh ong, doc ngtfng ket va hien ttfOng tan mau b ln g m lt
thtfdng va tren kinh hien vi
- Ghi lai ket qua.
3.2.2. Phuong phap hdng cau m iu
- Nho mot giot hong cau mau A (5%), mot giot hong cau m iu B (5%) vao
hai o'ng nghiem
271
- Them vao moi ong nghiem mot giot huyet thanh b$nh nhan
- Tron deu, ly tam 1000 vdng/phut/1 phut.
- Nghieng nhe thanh o'ng, doc hien ttfdng tan mau vk ngtfng ket b ln g m lt
thtfdng va tren kinh hien vi.
- Ghi lai ket qua (bang 6.3).
3.2.3. Nhan djnh ket qua djnh nhom mau he ABO: Ket qua dtfdc xac nhan theo bang 6.3
Bang 6.3: Ket qua djnh nhom mau ABO
Phtfdng phap HTM Phtfdng phap HCM

Chtfa biet
Hong cau xet nghiem Huyet thanh c ln xet nghiem
Da biet Chong A Chong B Chong AB Hong c lu A Hong c lu B

Nhom A (+) (-) (+) (-) (+)
Nhom B (-) (+) (+) (+) (-)
Nhom O (-) (-) (-) (+) (+)
Nhom AB (+) (+) (+) (-) (-)
Chu thich: (+): Ngi/ng ket
(-): Khong ngifng ket
3.3. Phuong phap dinh nhom ABO trong cot gelcard
Day la mot ky thuat co do nhay va do dac hieu cao, cho ket qua ngtfng ket ro
rang, thdi gian u ngan, co quy trinh tien hanh ky thuat chuan, d i dang ltfu gitf
ket qua va sti dung, an toan cho ngtfdi lam xet nghiem.
3.3.1. Nguyen ly ky thuat: Trong mot cot gel co buong phan tfng, co s in khang
huyet thanh chu&n de dinh nhom, co cac vien bi thuy tinh lam mang ngan dam
ngtfng ket. Neu co phan tfng khang nguyen - khang the dac hieu xay ra se tao
thanh cac dam ngtfng ket va hong cau ttf do khong con de lot qua ke cac vien bi
thuy tinh di xuong day cot gel (phan tfng dtfOng tinh), neu khong co phan ting
khang nguyen - khang the dac hieu xay ra, cac hong cau se lot qua ke cac vien bi
thuy tinh de di xuong day cot gel (phan ting am tinh).
3.3.2. Dung du va thuoc thCf

- Dung cu
+ M ay ly tam chuyen dung
+ M ay ly tam thtfdng de tach huyet thanh
+ M ay u chuyen dung
+ Gia dtfng gelcard
+ Pipettm an
- Thuoc thtf
272
+ Gelcard, da co sin cac khang huyet thanh chuan (khang the A, B, AB)
+ Dung dich de pha hong cau
- Chuan bi: Pha hong cau thanh huyen dich 0,8% trong dung dich pha
hong clu.

- Ghi ho ten benh nhan c ln dinh nhom vao gelcard
- Nho 50 dich treo hong cau vao cac gieng co ghi khang the A, B, AB.
- Ly tam 900 vong/phut/10 phut
- Nhan dinh ket qua: Trinh bay trong hinh 6.25.
Phan ting dUOng tinh: hong cau ngtfng ket nam tren cot gel
Phan tfng am tinh: hong cau lang toan bo xuong (tay cot gel
Hinh 6.25: Ket qua djnh nhom mau ABO bang gelcard neu co phan ting ngting ket thi
cac hat ngi/ng ket se dupe g iti lai tren cot gel (+); neu khong co ngi/ng ket hong cau Icing
xuong day hoan toan (-). M tic do ngting ket dtfpc danh gia bang 4+, 3+, 2+, 1+, (±), (-)
dtia vao hat ngtfng ket g iti lai tren be mat cot gen (A va AB (4+); D (1 +, 2+).
4. Nhung nguyen nhan sai lam trong djnh nhom mau ABO
4.1. Do cac thu tuc hanh chinh: nham ten hoac trung ten benh nhan, lay nham
ong mau, ghi ten sai...
4.2. Nham lan huyet thanh hoc
- Ngtfng ket khong dac hieu: do hong cau m iu, hoac huyet thanh m iu da
cu, nhiem trung. bien chat.
- Mau mau xet nghiem co hien ttfdng ket dinh hong cau chuoi tien gap d
mot so” benh ttf mien, myeloma.
273
- Ty le huyet thanh m iu, hong clu m iu khong ttfdng tfng
- De qua lau mdi doc ket qua (mau phan tfng kho).
4.3. Cac nguyen nhan khac

Dung cu khong sach, tay nghe cua ngtfdi xet nghiem vien chtfa thanh thao,
hoac thieu c ln than.
5. Nhumg kho khan trong djnh nhom mau ABO, giai phap
5.1. Do khdng phu hpp ket qua gidra hai phuong phap: huyet thanh m iu, hdng
cau mau.
G idi phap: phai tim nguyen nhan b ln g cac btfdc sau:
- Rtfa hong clu benh nhan va hong cau nhom 0 (co d ly du khang nguyen
hong cau ngoai he ABO ) bang ntfdc muoi 9% ba lan, pha thanh dung dich hong
cau 5%.
- Tien hanh ba chtfng:
+ Chtfng ttf than: phan tfng gitfa huyet thanh benh nhan va hong c lu cua
benh nhan da rtfa 3 lan.
Nho vao ong nghiem tan mau 2 giot huyet thanh cua benh nhan va 1 giot
hong cau benh nhan da rtia pha thanh 5%. Tron deu, ly tam 1000 vdng/phut/1
phut, doc ket qua bang mat thtfdng va tren kinh hien vi
+ Chtfng AB: (Phan tfng gitfa huyet thanh A B va hong c lu benh nhan)
Nhd vao ong nghiem tan mau 2 giot huyet thanh AB va 1 giot hong cau benh
nhan da rtia pha thanh 5%.Tron deu ly tam 1000 vong/phut/ 1 phut, doc ket qua
b ln g m lt thtfdng va tren ki'nh hien vi. Neu phan tfng am tinh thi phtfdng phap
dinh nhom bang huyet thanh mau la cd gia tri.
+ Chtfng dong loai: (Phan tfng gitia huyet thanh benh nhan va hong cau 0 ):
Nhd vao ong nghiem tan mau 2 giot huyet thanh benh nhan va 1 giot hong
cau O da rtia pha thanh 5%. Tron deu, ly tam 1000 vong/phut/1 phut, doc ket qua
b ln g m lt thtfdng va tren kinh hien vi. Neu phan tfng am tinh thi phtfdng phap
dinh nhom bang hong cau mau dtfpc bao dam.
Dtfa vao ket qua 3 chtfng: ttf than, dong loai va A B 0 tren ta cd the chia cac
trtfdng hpp khd khan trong dinh nhom mau he ABO thanh hai nhom chinh de tim
nguyen nhan.
Khi ca 3 chtfng ndi tren deu am tinh: trtfdng hpp nay cd the do cac nguyen
nhan sau:
Q Cd tieu huyet to:

V i du:
Khang the khang A Khang the khang B Khang the khang AB Hong cau A Hong cau B C h iin g AJIo (-)
(-) (-) (-) (+) +++ Chufng AB (-)
Chting Auto (-)
274
N han xet:
- Benh nhan co the co nhom mau 0 nhtfng khong co stf phu hpp gitfa 2
phtfOng phap huyet thanh m iu va hong clu m iu vi khang the khang A cua benh
nhan nay rat yeu hoac am tinh.
- Benh nhan co the co tieu huyet to khang A
- Tieu huyet to' chi xay ra khi co mat bo the
Cach gid i quyet:

Khti bo the co trong huyet thanh cua benh nhan 0 56°C / 30 phut
Thti l a i p h t f O n g p h a p h o n g c a u m a u , n e u c o t i e u h u y e t t o t h i s a u k h i k h t i b o
th e p h a n tin g s e trd la i b in h th tfd n g n h tf sa u :
Khang the Khang the Khang the Hong cau A Hong cau B
khang A khang B khang AB
(-) (-) (-) ++++ +++
( 2 ) Co hai quan the hong cau

V i du:
Khang the Khang the Khang the Hong cau A Hong cau B Chting Allo(-)
± (-) ± (-) +++ Chting AB(-)
Chting Auto(-)
Nhan xet: Nhom mau cua benh nhan co the la nhom A, nhtfng hong cau cua
benh nhan ngtfng ket vdi huyet thanh mau khang A, khang AB khong hoan toan,
cdn nhieu hong cau ttf do.
- Cd the gap hien ttfpng hai quan the hong cau trong nhting trtfdng hpp sau:
+ Nhtfng ngtfdi dtfpc truyen mau, truyen tuy khac nhom he ABO.
+ Nhting ngtfdi cd nhom mau A yeu, B yeu

+ Benh nhan da u tuy x t f O n g , leukemia
+ The kham hoac ghep cac gen cua he nhom mau ABO
Cach g id i quyet:
+ Hdi trieu chtfng lam sang cua benh nhan, chan doan lam sang cua benh nhan
+ Hdi tien sti truyen mau, truyen tuy cua benh nhan
+ Tim chat ABH trong ntfdc bot
+ Xac dinh kieu hinh trong cac trtfdng hpp kham hoac ghep cua gen bang
nghien ctfu di truyen.
+ Xac dinh cac trtfdng hpp A yeu, B yeu

© Do khang the yeu (suy giam mien dich b^m sinh ho&c mac phai):
V i du:
Khang the Khang the Khang the Hong cau A Hong cau B Chting Allo(-)
+++ (-) +++ (-) (-) Chting AB(-)
Chting Auto(-)
N han xet:
- Benh nhan co the co nhom mau A, nhung co khang the khang B rat yeu
hoac am tinh.
- Nhiing trtfdng hpp nay co the gap 0 tre so sinh dtfdi 6 thang tuoi do khang
the khang A, khang B chtfa dtfpc hinh thanh mot cac day du.
- Thieu hut mien dich bam sinh
- Thieu hut mien dich mac phaiLeukemia cap. ngtfdi gia..)
Cach g id i quyet:
- Dien di mien dich de khang dinh
- Lam cac xet nghiem de ch in doan leukemia
5.2. Do khang the lanh

Vi du:
Khang the Khang the Khang the Hong cau A Hong cau B Chting Alio +++
+++ +++ +++ +++ +++ Chting AB +++
Chting Auto +++
Nhan xet:
- Benh nhan co the co khang the lanh khi nhiet do cua moi trtfdng giam
dtfdi 20°C.
- Khi de hong cau benh nhan vao binh cach thuy 37°C thi hien ttfpng
ngtfng ket mat d in
Cach g ia i quyet:
- Rtia hong cau benh nhan b ln g ntfdc muoi 0,9% da dtfpc de am 37°C.
- Dinh nhom tren phien kinh nong 37°C hoac trong ong nghiem d 37°C.
5.3. Do cd khang the tu mien
V i du:
Khang the Khang the Khang the Hong cau A Hong cau B Chting A lio +++
+++ +++ +++ +++ +++ Chting AB +++
Chting Auto +++
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Nhan xet: Benh nhan co the co khang the ttf mien. Cac khang the nay da
dtfpc co dinh tren be mat hong cau, thtfdng gap d nhtfng benh nhan thieu mau tan
mau mien dich,benh nhan bi benh he thong.
Cach gid i quyet:
- Xem lai chan doan lam sang cua benh nhan
- Rtia hong cau benh nhan nhieu l&n b ln g ntfdc muoi 9%o da dtfpc de d
37°C, roi dinh lai nhom mau cho benh nhan.
5.4. Hien twang hdng cau chuoi tien

V i du:
Khang the Khang the Khang the Hong cau A Hong cau B Chting Alio +++
+++ +++ +++ +++ +++ Chting AB +++
Chifhg Auto +++
Nhan xet:
- Benh nhan cd stf tang bat thtfdng cua protein trong huyet thanh thtfdng
gap d benh nhan da u tuy xtfOng, tang spi huyet.
- Cd stf ngtfng ket tang nhanh cua hong cau giong nhtf mot ngtfng ket va
no se dtfpc phan tan rat nhanh khi ta nhd vao hong c&u benh nhan 1 giot ntfdc
muoi 1,5%.
Cach g ia i quyet:
Rtia hong cau benh nhan b&ng ntfdc muoi 0,9%, roi dinh lai nhom mau
bang phtfOng phap huyet thanh mau hoac pha loang nhe nhang huyet thanh benh
nhan trong ntfdc muoi sinh ly 0,9% cho den khi nong do protein trong huyet thanh
khong du de ket dinh cac hong cau mau thanh hinh chuoi tien thi dinh lai vdi
phtfOng phap hong cau miu.
KY THUAT DINH NHOM MAU HE Rh

■ ■ ■
1. Nguyen ly
He nhom mau Rh cd tam quan trong trong thtfc hanh truyen mau chi sau he
nhom mau ABO. Sau khi he thong nhom mau he ABO dtfpc phat hien, viec xay ra
cac tai bien truyen mau dtfpc giam nhieu so vdi trtfdc day, tuy nhien ngtfdi ta van
gap nhting tai bien truyen mau mac du da lam xet nghiem hoa hpp nhom mau he
ABO. Nam 1940, Lanstainer va W iener da tien hanh mot thtfc nghiem tren khi
Macacus Rhesus, va da phat hien ra he nhom mau Rh. Stf phat hien ra nhom mau
he Rh da giai thich dtfpc nhtfng trtfdng hpp vang da tan mau d tre so sinh. Khac
v6i he nhom mau ABO, he Rh chi co K N ma khong co K T tu nhien. K T dac hieu
K N he Rh chi co mat do co stf m ln cam K N ttf ngoai vao, vi vay con goi 16 K T
m iln dich. Khang nguyen dau tien cua he Rh dtfpc phat hien la khang nguyen D,
ngtfdi co khang nguyen D tren be mat hong cau dtfpc goi la ngtfdi co nhom mau Rh
dtfOng va ngtfdi khong cd khang nguyen D tren be mat hong cau dtfpc goi la ngtfdi
cd nhom mau Rh am.
Cac khang nguyen chinh cua he Rh bao gom 6 khang nguyen chinh la D,d,
C,c,E,e. Cac khang nguyen nay dtfpc ton tai va di truyen theo cap Dd. Cc, Ee.
Tren thtfc te khang nguyen d chi la gia thuyet vi cho den nay ngtfdi ta van chtfa
ro cau true cua khang nguyen d.
2. Cac ky thuat xac djnh nhom mau he Rh

Hien nay cd nhieu ky thuat xac dinh nhom mau he Rh, tuy thuoc ttfng loai
sinh pham cua cac hang ma ta cd cac ky thuat xac dinh nhom mau he Rh khac
nhau. Tuy nhien cd bon ky thuat hien hay dtfpc sii dung nhat la:
2.1. Ky thuat xac djnh nhom mau he Rh tren phien kinh nong (40°C): La ky
thuat hay dtfpc sti dung nhat.
- Khang the cua he Rh thtfdng la khang the thieu do vay no rat thich hpp hoat
dong trong mdi trtfdng 37°C, mdi trtfdng dai phan tti, hoac mdi trtfdng albumin.
- Dung cu va thuoc thti:
-t- Thuoc thti: khang the chong D- Albumin
+ Phien kinh
+ Que tron
+ Hop sang de dinh nhom he Rh
- Tien hanh ky thuat:
Btfdc 1: Dat hai giot thuoc thti anti-D - albumin vao mot phien kinh sach, cd
nhan 0 3 vi tri.
Btfdc 2: Them vao ben canh giot thuoc thti anti-D - albumin mot giot dich
treo hong cau 40-50% cua benh nhan, cua ngtfdi Rh(+), cua ngtfdi Rh (-)Cac hong
cau nay cd the treo trong dung dich ntfdc muoi 0,9% hoac trong huyet ttfOng cua
benh nhan).
Btfdc 3: Tron anti-D - albumin vdi dich treo hong cau de cd dtfdng kinh
khoang ttf 20 mm tren phien kinh nong, lie phien kinh nong ttf trtfdc ra sau cho
den khi xuat hien ngtfng ket.
Btfdc 4: Quan sat hien ttfpng ngtfng ket va doc ket qua trong vdng 2 phut
Btfdc 5: Danh gia va ghi lai ket qua
- Danh gia ket qua:
+ Phan tfng ngtfng ket: Chi ra la ngtfdi dd cd nhom mau Rh dtfOng tinh
+ Phan tfng khong ngtfng ket: Chi ra la ngtfdi dd nhom mau Rh (-)
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2.2. Ky thuat xac djnh nhom mau he Rh tren dng nghiem trong moi truong albumin
- Khang the cua he Rh thifdng la khang the thieu do vay no rat thich hpp hoat
dong trong moi trUdng 37°C, moi trtfdng dai phan tii, mdi trtfdng albumin.
- Dung cu va thuoc thii:
+ Thuoc thii anti - D
+ Ong nghiem
+ May ly tam
+ Pipette Pasteur
+ Ntfdc muoi 0,9%
+ Albumin bd 20-30%
+ 2ml mau chong dong
- Tien hanh ky thuat:
+ Btfdc 1: Nhd mot giot thuoc thii anti-D vao mot ong nghiem sach, cd nhan
+ Btfdc 2: Them vao ong nghiem tren mot giot dich treo hong cau can dinh
nhom 2-4%.
(Hong cau nay cd the treo trong huyet thanh AB hoac trong huyet thanh
cua benh nhan).
+ Btfdc 3: Tron deu anti-D vdi dich treo hong cau, u d 37°C ttf 45-60 phut.
+ Btfdc 4: Them 1 giot albumin bd 20-30% vao ong nghiem tren
+ Btfdc 5: U them 15 phut d 37°C.

+ Btfdc 6: Doc va ghi lai ket qua
- Danh gia ket qua:
+ Phan tfng ngtfng ket: Chi ra la ngtfdi dd cd nhom mau Rh dUdng tinh
+ Phan tfng khong ngtfng ket: Chi ra la ngtfdi dd nhom mau Rh (-)
2.3. Ky thuat djnh nhom D yeu

Khi can xac dinh nhtfng trtfdng hpp D yeu, b ln g cac bien phap thong thtfdng
khd xac dinh. Trong nhtfng trtfdng hdp dd cd the xac dinh khang nguyen D yeu
b&ng nghiem phap Coombs gian tiep, ky thuat xii ly enzym.
- Dung cu va thuoc thii:
+ M ay ly tam
+ Binh cach thuy 37°C

+ Pipette Pasteur
+ Ntfdc muoi 0,9%

+ Huyet thanh Coombs
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- Cac btfdc tien hanh
+ Btfdc 1: Trong o'ng nghiem tan mau, nho 1 giot dich treo hong c&u 2-4% vh
them vao o'ng nghiem dd 2 giot anti-D.
+ Btfdc 2: U 37°C trong 30 phut

+ Btfdc 3: Quan sat hien ttfpng ngtfng ket
+ Btfdc 4: Neu dtfOng tinh ghi lai ket qua, neu am tinh thi rtfa hong c6u 3
lan bang ntfdc muo'i 0,9%.
+ Btfdc 5: Them 2 giot huyet thanh Coombs
+ Btfdc 6: Ly tam 1000 vong/ phut/ 1 phut
+ Btfdc 6: Doc ket qua phan tfng tren kinh hien vi
2.4. Ky thuat xac dinh nhom mau he Rh trong ong nghiem b£ng khang t h i ddc
hieu khang nguyen D dupe san xuat theo phuvng phap dom ddng
- Hien nay hau het cac anti-D deu dtfpc san xua't theo phtfdng phap ddn
ddng do vay rat thuan tien cho viec xac dinh nhom mau he Rh vi no cd do nhay va
do dac hieu rat cao va cd the dtfpc san xua't vdi so' ltfdng ldn.
- Dung cu va thuoc thtf:
+ Anti-D dtfpc san xua't theo phtfOng phap don ddng
+ Ntfdc muo'i 0,9%

+ M ay ly tam
+ Kinh hien vi
+ Pipette Pasteur
+ Que thuy tinh

+ Mau mau : 2 ml mau cd chong ddng
- Tien hanh ky thuat :
+ Btfdc 1: Nhd mot giot anti-D vao mot o'ng nghiem sach da dtfpc ghi nhan.
+ Btfdc 2: Them vao o'ng nghiem tren mot giot hong cau (Dich treo hong
cau dtfpc quy dinh theo ttfng loai sinh ph&m va dtfpc treo trong huyet ttfdng cua
benh nhan).
+ Btfdc 3: Tron deu anti-D va hong cau can dinh nhom he Rh(D).
+ Btfdc 4: L y tam 1000 vong/phut/1 phut
+ Btfdc 5: Quan sat hien ttfpng ngtfng ket va ghi lai ket qua
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KY THUAT DjNH NHOM MAU CAC HE KHAC
Trong mot vai trtfdng hpp ngoai viec dinh nhom mau ABO va Rh, ngtfdi ta
phai xac dinh them khang nguyen cua cac he nhom mau khac cua hong cau.
1. Cac trifdng hdp can djnh nhom mau khac ngoai he ABO
- Benh nhan se phai truyen mau nhieu lan:
Ngtfdi ta khuyen can xac dinh nhom mau cua nhtfng he de gay mien dich
nhat nhtf he Rhesus, he Kell, he Duffy, he Kidd
- Nghien ctfu di truyen:
+ Xac dinh sinh ddi cung trtfng hoac khac trtfng
+ Nhtfng trtfdng hpp bat thtfdng cua nhiem sac the
+ Xac dinh hong cau cua nhtfng ngtfdi cho mau thtfdng xuyen (Rh,Kell,
Duffy, Kidd, Lew is) de xay dtfng panel hong c&u.
+ Nghien ctfu quan the
+ Nghien ctfu pha he
2. Nhung thuoc thur thudng dung de dinh nhom mau khac cua he hong cau
- Cac thuoc thtf cd nguon goc ttf ngtfdi: Phan ldn la cac khang the m iln dich
+ He Rh: anti D, anti C, anti c, anti E, anti e
+ He Kell: anti K
+ He Duffy: anti Fya, anti Fyb
+ He Kidd: anti Jka, anti Jkb
+ He S,s: anti S, anti s
+ He Lutheran: anti Lua, anti Lub
- Nhtfng khang the ttf nhien (hiem gap hon)
+ He Lewis: anti Lea, anti Leb, anti Lex
+ He P: anti P i
- Nhtfng khang the cd nguon goc dong vat:
+ He M,N: Anti M, anti N cd nguon goc ttf thd
+ He P: anti P i cd nguon goc ttf lpn va ngtfa
+ He Lew is: anti Lea, anti Leb cd nguon goc ttf de
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Khong co mot nguyen tac chung nao cho viec sti dung nhiing thuoc thtf n&y.
Moi thuoc thu deu co stf chi dan sti dung rieng cua tting hang san xuat. Cac ky
thuat khac nhau co the dupe sti dung nhtf: dinh nh6m tren phien kinh ho&c trong
ong nghiem, dinh nhom trong moi trtfdng ntfdc muoi hoac dinh nhom trong moi
trtfdng enzym, hoac sti dung nghiem phap Coombs. Tuy nhien cd mot diem r&'t
quan trong la phai luon thtfc hien phan tfng kem theo mot chtfng am va chtfng
dtfOng cho mdi mot he thong nhom mau.
Viec dinh nhom cd the cung dtfpc thtfc hien tren may dinh nhom mau ttf
dong hoac bang tay.
PHAN UMG CHEO (PHAN |}NG HOA HOP)
1. Nguyen tac
De kiem tra stf hoa hpp nhom mau gitfa ngtfdi cho va ngtfdi nhan, ngtfdi ta
tron huyet thanh ngudi nhan vdi hong cau ngtfdi cho hoac hong cau cua ngtfdi
nhan vdi huyet tUOng cua ngtfdi cho roi xem xet hien ttfpng ngtfng ket.
2. Dung cu, phudng tien,hoa chat

- Lam kinh
- Pipette Pasteur
- Kinh hien vi
- M ay ly tam
- Ntfdc muoi 0,9%, ntfdc cat
- Huyet thanh mau, hong cau mau
3. Mau mau xet nghiem: gom hai ong

- Lay 5ml mau tinh mach vao mot o'ng nghiem sach khdng chong dong de
lay huyet thanh lam xet nghiem.
- Lay 2ml mau tinh mach vao mot o'ng nghiem nhtfa sach da cd san chat
chong ddng de lay hong cau lam xet nghiem.

- Mau ngtfdi cho va ngtfdi nhan tach thanh hai phan rieng biet: huyet
thanh va hong cau. Pha hong cau ngtfdi cho va hong cau ngtfdi nhan 5% b&ng
ntfdc muoi 0,9%(1 giot hong cau khoi + 19 giot ntfdc muo'i).
- Dinh lai nhom mau ngtfdi cho va ngtfdi nhan bang hai phtfOng phap huyet
thanh mau va hdng clu mau xem cd phu hdp khdng.
- Danh so ong nghiem 1, ong nghiem 2 va ghi ten ngtfdi nhan, khoa phong
len 2 ong nghiem dd. Ong nghiem 1 cho: 2 gipt huyet thanh ngtfdi nhan va 1 giot
hong clu ngtfdi cho 5%. Ong 2 cho: 2 giot huyet thanh ngtfdi cho va 1 giot hong cau
ngtfdi nhan 5%. L ie deu 2 o'ng, ly tam 1000 vong/phut/1 phut. Lay ra doc ket qua
b&ng mat thtfdng va tren kinh hien vi.
5. Danh gia ket qua

- Neu d ca ong 1 va 2 khdng cd hien ttfpng ngtfng ket thi mau ngtfdi cho va
ngtfdi nhan phu hpp.
- Trtfdng hpp ca ong 1 va 2 deu ngtfng ket hoac ong 1 ngtfng ket (khi truyen
khoi hong c lu ), ong 2 ngtfng ket khi truyen huyet ttfOng deu khdng truyen dtfpc.
Chu y:
- Trtfdng hpp truyen mau toan phan phai lam day du ca 2 ong cheo nhtf tren.
- Neu truyen khoi hdng cau chi can lam cheo ong 1, neu truyen huyet
ttfOng va khoi tieu cau chi can lam cheo o'ng 2.
- Hdng clu ngtfdi cho va benh nhan dtfpc pha dung ty le

- Mau mau bi nhiem trung
6. Cac yeu to anh hUdng

- Lam kinh, o'ng nghiem ban cd the gay ngtfng ket gia
- Ly tam vdi to'c do qua cao.
- Ngtfng ket lanh chu y dam bao nhiet do phong thi nghiem)
- Mau mau lay khdng dung quy cach hoac bi ddng day
Chu y: phai ltfu o'ng mau cheo gitfa ngtfdi cho va ngtfdi nhan 0 4°C, sau 24
gid mdi dtfpc huy.
PHUONG PHAP HIEU GIA KHANG THE M liN DjCH
1. Nguyen tac: K h a n g the m ien dich thtfdng k et hpp v6i k h an g the ttf
nhien.V i v a y m uon hieu gia k h a n g the m ien dich ph ai tien hanh qua hai
giai doan:
- Trung hoa khang the ttf nhien bang chat W itebsky hoac khtf khang the ttf
nhien bang cach dun nong d 70°C trong 10 phut.
- H ie u gia k h a n g the m ien dich cd the thtfc h ie n b a n g mot so ky t h u a t sau:
+ PhtfOng p h a p hieu gia d mdi trU dng a lbu m in .
+ Nghiem phap Coombs gian tiep.
+ K y thuat xU ly hong cau bang emzym papain, trypsin, bromelin.
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2. Chuan bj
- Dung cu: Ong nghiem nho, lam kinh, pipette.
- Huyet thanh xet nghiem 5 ml.
- H o n g cau nhom A v a B mdi dtfOc lay vao ong n g h iem sach d a c6 s i n d u ng
dich chong dong
- Huyet thanh nhom mau AB.

- Chat Witebsky.
- Huyet thanh khang globulin ngtfdi loai y globulin
- Huyet thanh chtfng lay 6 ngtfdi binh thtfdng khong co khang the mien dich.
3. Phudng phap tien hanh

3.1. Ky thuat trung hoa khang the ti/nhien
- Loai trtf khang the ttf nhien bang nhiet: trong ong nghiem da dtfdc ghi
nhan (so' 1) cho:
+ 0,5ml huyet thanh xet nghiem.
+ 0,5ml dung dich ntfdc muoi 0,9%.
Trong o'ng nghiem thtf 2 cho:
+ 0,5ml huyet thanh ngtfdi binh thtfdng (lam chtfng).
+ 0,5ml dung dich ntfdc muoi 0,9%
L&c deu hai ong nghiem, dat vao binh cach thuy cd nhiet do 70°C trong 10 phut.
Kiem tra xem d a khti het khang the ttf nhien chtfa, bSng cach nho huyet
thanh tren vdi hong cau A va B (giong nhtf hieu gia khang the ttf nhien) O. Neu
ong 2 khong thay ngtfng ket, chtfng to da loai trtf dtfOc khang the ttf nhien.
- Hut khang the ttf nhien bang chat Witebsky: trong ong nghiem thtf 3 cho:
+ 0,5ml huyet thanh xet nghiem.
+ 0,5ml chat Witebsky.
Trong o'ng so' 4 cho:
0,5ml huyet thanh ngtfdi binh thtfdng (lam chtfng).
0,5ml chat Witebsky.
Lac deu 2 o'ng tren, de tu lanh 4°C trong mot dem, tuy theo quy dinh cua
hang san xua't chat Witebsky, kiem tra xem da hut het khang the ttf nhien chtfa
(giong nhtf tren). Sau do hieu gia huyet thanh da khti khang the ttf nhien ma da
dtfdc hut bdi chat Witebsky.
3.2. Ky thuat hieu gia khang the mien djch khang A va khang B
Tien hanh theo ba phtfdng phap sau day:
3.2.1. Phuong phap trong ong nghiem
- Chuan bi: Huyet thanh AB.
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- Tien hanh: Rtia hong clu A va B 3 lln b ln g dung dich ntfdc muoi 0.9%.
Sau lln rtia cuo'i cung thi hut het ntfdc 6 tren ra va pha thanh dung dich hong clu
5% vdi huyet thanh AB.
Dung 20 o'ng nghiem, chia lam hai day, pha loang huyet thanh AB de co do
pha loang 1/1, 1/2, 1/4, 1/8... 1/512, moi o'ng co 4 giot huyet thanh AB da dtfOc pha
loang. Sau do cho hai giot dung dich hong c lu 5% vao moi o'ng. Do'i vdi day A nho
hong clu nhom A. Do'i vdi day B nhd hong c lu nhom B.
L ie deu. De tat ca hai day o'ng nghiem vao trong binh cach thuy 37°C/lgid.
Quay ly tam 2000vong/phut/lphut trong dieu kien nhiet do cua phong thi nghiem.
Sau khi ly tam xong, phai de nhting o'ng nghiem dd d nhiet do 37°C trong 3 phut.
- Doc ket qua: quan sat hien ttfOng tan mau va ngtfng ket bang mat thtfdng.
3.2.2. Nghiem phap Coom bs gian tiep: Huyet thanh xet nghiem da dtfoc tfc che
khang the ttf nhien he ABO b ln g chat W itebsky hoac bang nhiet. PhtfOng phap
tien hanh nhtf sau:
- Trong 10 o'ng nghiem pha loang huyet thanh cln xet nghiem d nong do
1/1,1/2,1/4...1/512 bang dung dich nUdc muo'i 0,9% (mdi o'ng cd 4 giot huyet thanh
da dtfdc pha loang bang ntfdc muo'i 0,9%).
- Cho them vao moi o'ng 2 giot dung dich hdng clu 5% (hong cau nhom A va
B da dtfOc rtia 3 lln va dtfOc pha bang dung dich ntfdc muo'i 0,9%).
- Quay ly tam lln thti nhat 10 o'ng xem cdn khang the ttf nhien khdng (thtfdng
khdng qua 1/4). Lay nhtfng o'ng khong ngtfng ket de vao thung cach thuy 37°C/1 gid,
lay ra quay ly tam 2000 vong/phut/lphut. Quan sat hien ttfOng ngtfng ket bang mat
thifdng. Neu thay ngtfng ket, chtfng to cd khang the mien dich loai du.
- Lai lay nhtfng o'ng khdng ngtfng ket dem ly tam d to'c do 2000 vdng/
phut/3 phut. Hut het dich noi d tren, roi rtia hong clu 3 lln b ln g dung dich ntfdc
muo'i 0,9%. Sau lln rtfa cuo'i cung thi hut het dich noi, roi nhd vao moi o'ng 2 giot
huyet thanh Coombs, lac deu ly tam 1000 vong/phut/lphut. Roi quan sat hien
ttfdng ngtfng ket. Hien ttfdng ngtfng ket d nghiem phap Coombs chting to stf cd
mat cua khang the mien dich loai thieu.
3.2.3. Phuong phap dung enzym: de xac dinh khang the mien dich chong A va B.
Hong clu da dtfdc xti ly bang enzym cho tiep xuc vdi khang the mien dich loai
thieu (khang the A va khang the B). Neu xua't hien hien ttfdng ngtfng ket vdi hong
clu ttfdng tfng cd nghia trong huyet thanh cua benh nhan cd khang the mien dich
chong A hoac B.
- PhtfOng phap tien hanh: chuan bi ngay dung dich papain va khoi hdng
cau rtia (A va B). Trong mot so' o'ng nghiem nhd: khoi hong clu 0,5 ml: dung dich
papain 0.5 ml.
Lac deu roi de vao binh cach thuy 37°C, thdi gian tuy thuoc ttfng loai papain
ma cac hang san xuat quy dinh.
Rtia hong cau 3 lan bang ntfdc muo'i 0.9% de loai trti het papain (Roi kiem
tra hdng cau sau khi xti ly enzym xem cd bi ngung ket khdng. neu cd ngtfng ket
phai xem lai cac khau tren).
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Pha dung dich hong cau da xti ly bang papain 3% trong dung dich ntfdc
muoi 0,9%.
Huyet thanh xet nghiem da dtfdc khti khang the ttf nhien dem pha loang
1/1,1/2,1/4...1/512. Sau do cho vao moi ong 2 giot dung dich hong c iu da xti ly
bang papain 3%.
L ie deu, de 0 binh cach thuy 37°C/ lgid.
Quan sat hien ttfdng ngtfng ket ngay d dieu kien a'm, khong quay ly tam.
- K et qua: Hien ttfOng ngtfng ket hong cau xti tri bang papain chting to c6
mat cua khang the mien dich loai thieu.
Xac dinh khang the mien dich he ABO bang nghiem phap Coombs gian tiep
va bang hong cau da dtfdc xti ly bang men, thtfdng dtfdc tien hanh cung mot luc de
doi chieu vdi nhau cho them phan chinh xac, ket qua thtfdng giong nhau.
PHIT0NG PHAP TACH KHANG NGUYEN
1. Nguyen ly
K h i ng h iep p h a p C o om bs trtfc tiep dtfOng tinh, ta p h a i tien h a n h ky thuat
tach k h a n g n g u y e n de xac din h b a n chat cu a k h a n g the. T a c h k h a n g n g u y e n la
phtfOng p h a p tach cac k h a n g the da dtfdc ca m n h iem r a khdi k h a n g n g u y e n ttfdng
tfng tren be m a t h o n g c l u . K y t h u a t tach k h a n g n g u y e n cd h ie u q u a cd n g h ia la
lam cho ltfc lien ket gitia k h a n g n g u y e n v a k h a n g the dac h ie u dd b i p h a vd hoac
dao ngtfdc. Cd n h ieu phtfOng p h a p tach k h a n g n g u y e n nhtf phtfOng p h a p tach:
b a n g nhiet, ch loroqu in disp h o sp h a t, acid citric, son g sieu am , la m t a n ddng, ether.
Cd h a i phtfOng p h a p h a y dtfcfc sti d u n g la tach k h a n g n g u y e n b a n g nh iet v a ether.
2. Dung cu va thuoc thur

2.1. Dung cu
- M ay ly tam
- Kinh hien vi
- Pipette Pasteur
- Binh cach thuy
- Lam kinh
2.2. Thuoc thu
- Ether
- Huyet thanh Coombs
286
- Niidc muoi 9%o
- Huyet thanh AB

3.1. Phuong phap tich khdng nguyen bang nhiet
- Trong mot ong nghiem tan mau. Nho mot the tich khoi hong cau da rifa 6
l£n bang ntfdc muoi 0,9%. Giut lai dich noi cua lan rifa cuoi cung de tien hanh thu
nghiem song song vdi nUdc tach.
- Them vao ong nghiem 1 the tich niidc muoi 0,9% hoac huyet thanh AB
- De 15 phut d 56°C
- Ly tam ong nghiem trong dieu kien nong d 56°C
- Thu hoi niidc tach cd chtia khang the
3.2. Tach khang nguyen biing ether

- Trong mot o'ng nghiem nhd mot the tich khoi hong cau da rtia 6 lan bang
niidc muoi 9%o. Giti lai dich noi cua lan rifa cuoi cung de tien hanh thti nghiem
song song vdi niidc tach.
- Them vao ong nghiem 1 the tich nUdc muoi 0,9% hoac huyet thanh AB
- Them 2 the tich ether vao o'ng nghiem tren
- Day nut o'ng nghiem va lac manh tti 3 den 4 phut
- Md nut va dat o'ng nghiem 0 37°C/15 phut.
- Ly tam to'c do 1000 vong/phut/5 phut
- Dung pipette Pasteur chuyen phan niidc tach sang mot o'ng nghiem sach
- De 0 37°C va dung pipette Pasteur hut bd phan ether thifa noi bot tren
phan ntfdc tach.
- Khi het mui ether, quay ly tam 1000 vong/ 5 phut thu lay phan dich ndi 0 tren.
3.3. Ky thuat tim khang the dac hieu cua nude tach
- Mot the tich ntfdc tach
- Mot the tich hong cau ma ta biet trtfdc cac nhom he hong cau panel
- U d 37°C trong 1 gid
- Ly tam, doc tren kinh hien vi va ghi lai ket qua
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Chifdng 7
SAN XUAT, BAO QUAN VA PHAN PHOI MAU,

CAC SAN PHAM MAU
CACH SAN XUAT HONG CAU MAU VA BAO QUAN
1. Hong cau mau

La nhiing hong cau mang khang nguyen co do dac hieu, do nhay cao da dtfdc
xac dinh trUdc. V i du: Hong cau mau A la hong cau mang khang nguyen A, hong
cau m iu B la hong cau mang khang nguyen B da dtfdc chon loc tii nhieu ca the de
cd hong cau cd do nhay, do dac hieu cao nhat.
Dung cac hong cau mang khang nguyen da biet trUdc (khang nguyen A,
khang nguyen B) dtfdc chuan bi s in de xac dinh huyet thanh chtfa biet.
2. Nguyen tac: Cac hong cau m au tro n g m oi trtfdn g nuoi dtfdng dac biet
van gitf n gu yen dac tinh k h a n g nguyen.
3. Dung cu, phuong tien, hoa chat

- Ong nghiem to nhon day Complexon III
- Pipette Pasteur Na H P 0 .2 H 0
- May ly tam Cloramphenicol
- Ntfdc muoi 0,9% Albumin huyet thanh bd (BSA) 30%
- Hong cau A va hdng cau B Ntfdc cat
- Huyet thanh AB NaO H IN .
- Glucose

4.1. Pha dung djch bao quan
Can chinh xac
- 7.44g complexon III - 33,3ml BSA 30%
- 3,56g Na H P 0 .2 H 0 - 200 ml huyet thanh AB
- Ig cloramphenicol
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- Niidc ck't vita du 1000ml
- 17,5g glucose
Sau do dieu chinh b ln g NaOH IN den pH=7.
4.2. Chon hdng cau mau

Tuyen chon ngudi cho mau: khoe manh, tuoi 18-40, khong co benh man
tinh, HIV, HCV, HBV(-)
- L a y m a u tti 10 ngudi ho n g cau nhom A v a 10 ngudi h o n g cau nhom B vao
cac o'ng nghiem nhon d a y co chong dong ACD hoac CPD. L y tam hut bo huyet
tUOng. Rtfa hong cau 3 lan b a n g nUdc muoi 0 ,9 % .S a u lan rtia cuo'i cu n g hu t kiet
nUdc muo'i de co dUOc kho'i ho n g cau.
- Tiep theo la tim hong cau co do nhay cao bang khang huyet thanh khang
A, khang B pha loang dan theo ty le 1/2, 1/4.... 1/512. Chon cac hong cau co do
nhay tti 1/128 trd len lam hong cau mau.
4.3. Bao quan
- Tron cac hong c&u A da dUOc chon loc vdi nhau, cac hong clu B da dUOc
chon loc vdi nhau (pool) roi phan pho'i vao cac ong nghiem say vo trung da danh
da'u nhom san. Them dung dich bao quan vao de duoc dung dich hong cau 5% (ty
le: 1 giot kho'i hong cau them 19 giot dung dich bao quan). Dung dich hong cau
nay co the giti duoc 4 tu&n d 4°C. Khi lay ra sti dung: ly tam, hut kiet dung dich
bao quan va them nUdc muo'i de co dung dich hong c&u 10-20%.
5. Cac yeu to anh hUdng

- De tranh nhiem trung hong cau mau
+ Dung cu (o'ng ly tam, o'ng nghiem dung hong cau mau, pipette...) phai say
tiet trung.
+ Hap t i e t t r u n g n U d c c a t t r U d c khi pha d u n g dich b a o q u a n
+ Thao tac ky thuat phai nhanh, chinh xac, tot nha't la tien hanh trong
buong vo trung.
- Hong cau mau muon phat hien dUOc cac khang the trong huyet thanh can
xac dinh phai duoc chuan bi tti nhieu ngudi (tot nha't la 10 ngiidi) de co mat du cac
khang nguyen can thiet.
KY THUAT DIEU CHE KHOI HONG CAU
1. Nguyen tac
Khoi hong cau thu duoc sau khi tach huyet tUOng tti mau toan phan da ly
tam hoac de lang.
289
2. May moc, dung cu, vat lieu
- Mau toan phan dtfOc lay vo trung vao bo tui deo c6 g in them mot hoSc
nhieu tui chuyen.
- B ^ n e p h u y e t tU O ng
- Kim vuo't day

- Keo
- Kep hoac khoa nhtfa
- May han day hoac vong nhom han day
- May ly tam lanh
- Can dia thang b ln g
- Can ban hoac can lo xo
3. Quy trinh thuc hien

- Xep cac tui mau vao o'ng ly tam. Can b ln g tting cap ong ly tam va dat vao
may ly tam d cac vi tri doi xting. Ly tam manh 6 4°C. Neu tui mau toan phln da
de l&ng vai ngay thi khong c ln phai ly tam.
- Dat tui mau toan phln da ly tam hoac de lang ro thanh hai ldp hong clu
va huyet ttfOng len ban ep huyet tUOng. Chu y de mat tui khong dan nhan quay ra
ngoai. Thao can ham 16 xo nen tam ep vao be mat tui mau.
- Md t h o n g d a y n o i t u i m a u t o a n p h a n v d i t u i c h u y e n b l n g c a c h b e k h o a d
d a u d a y n o ’i v d i t u i m a u t o a n p h l n d e h u y e t t t f O n g c h a y s a n g t u i c h u y e n .
- K h o a d a y noi tui m a u chtia khoi ho n g cau v a tui chtia h u y et ttfOng b l n g

kep hoac k ho a nhtfa k hi tach du ltfdng hu y et ttfdng yeu cau.
- Ngting ep bang cach ha can ham 16 xo va thao tui khoi hong cau khoi ban ep.
- Bo sung tti tui chuyen chtia dung dich bao quan hong cau (neu co) vao tui
kho'i hong cau.
- H a n day no'i gitia tui kho'i h o n g cau v a tui h u y et ttfOng d h a i vi tri cach
n h a u 0,5 cm.
- Dien d l y du thong tin d nhan tui kho'i hong cau va tui huyet ttfOng
- Cat rdi day no'i 0 gitia hai vi tri han
4. Bao quan va cach dung

- Bao quan kho'i hong cau 0 tu lanh nhiet do 2 - 6°C
- Thdi gian bao quan:
+ Neu khong bo’ sung dung dich bao quan hong cau, dieu che vo trung,
hematocrit < 75%: thdi gian bao quan nhtf mau toan phan.
+ Neu co bd sung dung dich bao quan hong cau trong he thong kin: thdi
gian bao quan theo quy dinh cua nha san xuat.
+ Neu co bd sung dung dich NaCl 0,9% va/hoac ky thuat dieu che khong bao
dam vo trung: thdi gian bao quan to'i da 24 gid sau khi dieu che.
- C6 the bd sung 50 - 100 ml dung dich natri chlorua 0,9% vao khdi hong cau
ngay trUdc khi truyen mau lam sang (khong tiep tuc bao quan them). Khong dtfdc sii
dung ba't cti dung dich nao khac de pha tron vdi khoi hong cau.
- Sii d u n g b o d a y c o b a u l o c vdi d t f d n g k i n h l o l o c 170 - 200 ( i m d e t r u y e n .
Co t h e sii d u n g b o l o c vi n g t f n g k e t vdi d t f O n g k i'n h l o l o c 20 - 40 jim, h o a c b o d a y
tr u y e n k e m b o lo c b a c h c a u .
5. Chi djnh lam sang

Cac trtfdng hdp thieu hong cau cap va man tinh do cac loai nguyen nhan.
6. Mot so thong so tham khao

Mot don vi kho'i hong cau dieu che ttf don vi mau toan phan 250 ml bd sung
dung dich bao quan hong cau hoac NaCl 0,9% co mot so' dac tinh sau:
- The tich khdi hong ciu: 190 ± 30 ml
- Hematocrit: 0,60 ± 0,10
7. Lilli y
- TtfOng quan gitfa ltfc ly tam va to'c do ly tam:
Ltfc ly tam (g) = 1,118 x 10 5 x N 2. r
g: Don vi do ltfc ly tam
N: To'c do ly tam (vong/phut)
r: Ban ki'nh roto may ly tam (cm)
- Ly tam manh: Ly tam vcii to'c do cao va/hoac thoi gian dai nham lam lang
moi loai te bao. De dieu che khdi hdng cau, thong so' tham khao co the la:
+ 5000 x g trong 5 phut.
+ 3000 x g trong 8 phut 30 giay
KY THUAT DIEU CHE KHOI HONG CAU NGHEO BACH CAU
1. N g u y e n ta c
T u y phtfOng thtfc ly tam, bach cau se n am 6 ldp tren cun g vdi huyet ttfOng hoac
d mat ph an cach huyet ttfdng - hdng cau. Khdi hdng cau thu dtfdc sau khi tach bo
bach cau cung vdi huyet ttfOng ttf m a u toan p h a n hoac b a n g thao tac bd sung.
2. M a y m o c , d u n g cu, v a t lieu
- M a u toan p h a n dtfdc lay mot cach vo t r u n g v ao bo tui deo co g a n them

mot hoac n h ieu tui chuyen.
291
- D u n g dich bao q u a n ho n g cau co s i n trong bo tui lay m a u ho&c d u n g dich
muo'i d i n g trUOng n a tri cloru a 0,9%.
- Ban ep huyet ttfOng

- Kim vuo't day
- Keo
- Kep ngoai khoa canh dai 20 - 30 cm
- May ly tam lanh
3. Quy trinh thi/c hien: Theo hai phtfOng phap

3.1. Tach bach cau bang cach tach huyet tuong giau bach cau, tieu cau
- Lay mau ngtfdi cho vao he thong tui doi (tui lay mau va 1 tui chuyen),
hoac tui ba (tui lay mau va 2 tui chuyen).
- Xep cac tui mau vao o'ng ly tam. Can bang ttfng cap o'ng ly tam va dat vao
may ly tam 0 cac vi tri do'i xtfng. Ly tam nhe 6 22°C. Neu tui mau toan ph in da de
lang 2 - 12 gid thi khong can phai ly tam.
- Dat tui mau toan phan da ly tam hoac de lang ro thanh hai ldp hong c i u
va huyet ttfOng len ban ep huyet ttfOng. Chu y de mat tui khong dan nhan quay ra
ngoai. Thao can ham 16 xo nen tam ep vao be mat tui mau.
- M6 thong d a y no'i tui m a u toan p h a n vdi tui ch u yen b l n g cach be k ho a 6
d a u day no'i v6i tui m au toan p h a n de h u y et ttfOng g ia u b ac h cau, tieu c l u chay
s a n g tui chuyen.
- K h o a day noi tui m a u chtfa kho'i h o n g cau v a tui chtfa hu y et ttfOng b a n g

kep hoac k ho a nhtfa khi m at p h a n cach h o n g cku - hu y et ttfOng d a n g len sat
th a n h tren tui m au.
- Ngtfng ep b in g cach ha can ham lo xo va thao tui kho'i hong cau khoi
ban ep.
- Bo sung ttf tui chuyen chtfa dung dich bao quan hong cau (neu co) hoac ttf
binh chtfa natri clorua 0,9% vao tui khoi hong cau. LtfOng dich bo sung 50 - 75 ml
cho 1 don vi khoi hong cau ttf 250 ml mau toan phan.
- Han day noi gitfa tui kho'i hong cau va tui huyet ttfOng 6 hai vi tri cach
nhau 0,5 cm.
- Dien day du thong tin 6 nhan tui kho'i hong cau va tui huve't tuong.
- Cat rdi day no'i 6 gitfa hai vi tri han
292
3.2. Tdch bach cau biing cach loai bd buffy coat
- La'y mau ngtfdi cho vao he thong tui ba (1 tui lay mau va 2 tui chuyen),
hoac tui bon (tui lay mau va 3 tui chuyen). Trong he thong tui can co 1 tui chuyen
chtia dung dich bao quan hong clu.
- Xep cac tui mau vao o'ng ly tam. Can bang tting cap o'ng ly tam va dat vao
may ly tam cl cac vi tri do'i xting. Ly tam manh 0 22°C.
- Dat tui mau toan phan da ly tam thanh hai ldp hong clu va huyet ttfdng
len ban ep huyet tUOng. Chu y de mat tui khong dan nhan quay ra ngoai. Thao
can ham lo xo nen tam ep vao be mat tui mau.
- Met thong day no'i tui m au toan phan vdi tui chuyen b a n g cach be khoa 0 dau
day no'i v6i tui m au toan ph an de huyet ttfOng ngheo te bao chay san g tui chuyen.
- K h o a d a y no'i tui m au chtia kho'i ho n g cau v a tui chtia huyet ttfOng b a n g

kep hoac k ho a nhtfa khi m at p h a n cach ho n g cau - huyet ttfOng d a n g len sat
thanh tren tui m au.
- Dung kep ngoai khoa canh dai luon dtfdi mat phan cach hong clu - huyet
ttfOng khoang 1 cm.
- Siet kep dong thoi vdi viec:
+ Ha can ham lo xo,
+ Nhac tui mau khoi ban ep,
+ Quay kep mot goc 90°,
+ Ndi khoa nhtfa md thong day no'i tui mau va tui chuyen thti 2.
+ D e ldp b u ffy coat (gom p h a n ldn b ach cau, tieu cau, mot p h a n nho ho n g
cau va hu y et ttfOng) ch ay s a n g tui chuyen ttfOng tfng.
- Dung kep nhtfa khoa day no'i tui mau va tui chuyen chtfa buffy coat.
- Bo sung tti tui chuyen chtia dung dich bao quan hong cau (neu co) hoac tti
binh chtia natri chlorua 0,9% vao tui kho'i hong cau. LtfOng dich bo’ sung 5 0 - 7 5
ml cho 1 don vi kho'i hong cau ttf 250 ml mau toan phan.
- H a n d a y no'i gitia tui kho'i h o n g c l u v a tui h u y et ttfOng d h a i vi tri cach
n h au 0,5 cm.
- Dien day du thong tin d nhan tui kho'i hong cau va tui huyet ttfdng.
- Cat rdi d a y no'i 0 gitia h a i vi tri han

- Bao quan kho'i hong cau d tu lanh nhiet do 2 - 6°C
- Thdi gian bao quan:
+ Neu co bo sung dung dich bao quan hong cau trong he thong kin: thdi
gian bao quan theo quy dinh cua nha san xuat.
293
+ Neu bd sung dung dich natri chlorua 0,9% hoac bd sung dung dich bao
quan hong cau trong he thong hd: thdi gian bao quan la 24 gid ke tti luc dieu ch£.
- Sti d u n g b o d a y c o b a u l o c vdi d t f d n g k i n h lo l o c 170 - 200 nm de t r u y e n .
Co t h e vi n g U n g t a p vdi d t f d n g k i n h l o l o c 20 - 40 n m . h o a c b o d a y
s ti d u n g b o lo c
t r u y e n k e m b o lo c b a c h c a u .

Benh nhan thieu hong cau cap va man tinh do nhieu nguyen nhan cln
truyen mau nhieu lan va / hoac co nguy cO gap cac phan ting so't, ngtia. noi man,
me day do bach cau.

Mot don vi kho'i hong cau ngheo bach cau dieu che tti dOn vi mau toan phln
250 ml co mot so' dac tinh sau:
- The tich kho'i hong cau: 190 ± 30 ml
- Hematocrit: 0,60 ±0,10
- So' lt f O n g bach cau: < 0,67 x 109.
- So' lt f O n g tieu cau: < 10 x 109.
7. Liru y
- Ly tam manh: Ly tam vdi to'c do cao va/hoac thdi gian dai nham lam lang
moi loai te bao. Dieu che kho'i hong cau tti d O n vi mau the tich 250 ml, thong sd
tham khao co the la:
- Ly tam nhe: Ly tam vdi to'c do thap va/hoac thdi gian ngan nham lam
lang mot loai te bao trong khi mot hoac nhieu te bao khac chtfa bi lang. Dieu che
khoi hong cau ttf ddn vi mau the tich 250 ml, thong so' tham khao co the la:
KY THUAT DIEU CHE KHOI HONG CAU RITA
1. N g u y e n ta c
Khoi h o ng cau thu dtfOc s a u khi loai bo h a u het protein hu y et ttfdng b a n g

cach rtia ho n g cau n h ieu la n vdi d u n g dich m uoi d a n g trtfOng n a tri clo ru a 0.9%.
294
- Mau toan phln hoac khoi hong clu.
- B ^ n e p h u y e t ttfO n g
- Kim vuo't day

- Keo
- May ly tam lanh
- Can ban hoac can 16 xo
- Dung dich natri clorua 0,9%

- Dieu che kho'i hong clu: xem ky thuat dieu che kho'i hong clu.
- No'i day vo trung va bo’ sung 150 - 200 ml dung dich natri clorua 0,9% vao
tui chtfa kho'i hong clu, lac deu.
may ly tam 6 cac vi tri do'i xtfng. Ly tam manh 6 4°C.
- Lay tui kho'i hong clu da ly tam khoi may ly tam. Thtfc hien no'i vo trung no'i
tui mau va binh chtfa chat thai. Khoa day no'i tam thoi bang khoa nhtfa.
- Dat tui kho'i hong clu len ban ep huyet tUOng. Chu y de mat tui khong dan
nhan quay ra ngoai. Thao cln ham lo xo nen tam ep vao be mat tui mau.
- M6 thong day no'i tui mau vdi binh chtfa chat thai b ln g cach md khoa
nhtfa de ntfdc trong tren chay sang binh chtfa chat thai.
- Khoa day no'i khi mat phan cach hong clu dang sat thanh tren tui mau
blng khoa nhtfa.
- Ngtfng ep bang cach ha c ln ham lo xo va thao tui kho'i hong c lu khoi
ban ep.
- Lap lai cac btfdc them 2 - 5 l ln ntfa.
- No'i day vo trung va bo’ sung l l n cuo'i 50 - 75 ml dung dich natri clorua
0,9% vao tui chtfa kho'i hong clu.
- Dien d l y du thong tin d nhan tui kho'i hong c lu rifa.
- Han day va c i t rdi day no'i d gitfa hai vi tri han

- Bao quan kho'i hong clu rifa d tu lanh nhiet do 2 - 6°C
- Thdi gian bao quan: C ln sti dung sdm, bao quan to'i da 24 gid ke tti luc
dieu che.
295
- Sti dung bo day co b lu loc vdi dtfdng kinh lo loc 170 - 200 nm de truy§n.
Co the sti dung bo loc vi ngtfng tap vdi dtfdng kinh lo loc 20 - 40 |j.m, hoac bo day
truyen kem bo loc bach clu.

- Cac trtfdng hop thieu hong cau cap va man tinh do cac loai nguyen nhan d
benh nhan co m ln cam vdi cac protein huyet ttfOng.
- Thieu mau tan mau phu thuoc bo the (thi du: benh dai huyet s ic to kich
phat ban dem).
- Chi dinh tuyet doi d benh nhan man cam vdi Ig A (co nguy CO gay soc phan
ve) d ngtfdi thieu hut IgA bam sinh.

Mot dOn vi khdi hong cau dieu che tti dOn vi mau toan phan 250 ml co mot so
tmh cha't:
- The tich khoi hong clu: 210 ± 30 ml
- Hematocrit: 0,60 ±0,10
- Protein ngoai hong clu: < 0,5 g / dOn vi khoi hong clu.
7. Luu y
- Ly tam manh: Ly tam vdi toe do cao va/hoac thdi gian dai nham lam lang
moi loai te bao. Dieu che khoi hong clu rtia tti dOn vi mau 250 ml, thong so' tham
khao co the la:
+ 5000 x g trong 5 - 6 phut.
+ 3000 x g trong 8 phut 30 giay - 9 phut.
- Do nguy co nhiem khuan cao nen che pham c ln dtfOc sti dung cang sdm
cang tot.
- Khong dtfOc bo sung bat cti dung dich nao khac vao khoi hong c lu (trti
dung dich bao quan hong clu da dtfoc chuan hoa).
KY THUAT DIEU CHE KHOI TIEU CAU
1. Nguyen tac
K h o ’i tieu c l u dieu che tti hu y et ttfOng g ia u tieu c l u hoac ttf b u ff y coat sau
k hi loai bo p h l n ldn h o n g c l u , b a c h c l u v a hu y et ttfOng.

- Mau toan phln ttfoi (thdi gian trong vong 8 gid ke tti luc lay mau) bao
quan d nhiet do phong (22 - 24°C) trtfdc khi tach tieu clu. Mau toan ph ln duoc lay
trong he thong 3 hoac 4 tui.
296
- B ^ n 6p huyet ttfdng
- Kim vuot day

- K6o
- Kep ruot ngoai khoa canh dai 15 - 20 cm
- May ly tam lanh
- Can dia thang bang
3. Quy trinh thirc hien: Theo hai phtfdng phap

3.1. Dieu che khoi tieu cau tCr huyet tuong giau bach cau, tieu cau
- Lay mau ngtfdi cho vao he thong tui 3 (tui la'y mau va 2 tui chuyen), hoac
tui 4 (tui lay mau va 3 tui chuyen).
may ly tam 6 cac vi tri do'i xting. Ly tam nhe 6 22°C.
- Dat tui mau toan phan da ly tam len ban ep huyet ttfdng. Chu y de mat tui
khong dan nhan quay ra ngoai. Thao can ham lo xo nen tam ep vao be mat tui mau.
- Mcl t h o n g day no'i tui m a u toan p h a n vdi tui chuyen thtf n h a t b a n g cach
be khoa 6 d a u d a y no'i cua tui m a u toan p h l n de hu y et ttfOng g ia u b ach cau, tieu
cau chay s a n g tui chuyen.
- K h o a d a y no'i tui m a u chtfa kho'i ho n g c l u v a tui chtfa huyet ttfOng giau

bach c l u , tieu c l u b a n g kep hoac k ho a nhtfa khi m at p h a n cach ho n g c l u - huyet
ttfOng d a n g len s i t th a n h tren tui mau.
- Ngtfng ep bang cach ha c ln ham Id xo va thao tui kho'i hong c lu khoi

ban ep.
- Xti ly tui chtfa kho'i hong clu nhtf ky thuat dieu che kho'i hong clu.
- Han day no'i gitia tui kho'i hong c lu va tui huyet ttfdng giau tieu clu. bach
clu d hai vi tri cach nhau 0,5 cm.
- D ie n d l y du thong tin 6 n h a n tui kho'i h o n g c l u v a tui hu y et ttfOng gia u
tieu c l u , b ac h c l u .
- Cat rdi day no'i 0 gitia hai vi tri han

- Xep cac tui chtia huyet ttfdng giau tieu clu vao o'ng ly tam. Can bang tting
cap o'ng ly tam va dat vao may ly tam 0 cac vi tri doi xting. Ly tam manh 6 22°C.
- L a y nhe n h a n g tui hu y et ttfOng g ia u tieu c l u , b ac h c l u khoi m a y ly tam
va dat len b a n ep hu y et ttfOng.
- Thao c ln ham Id xo nen tam ep vao be mat tui mau.
297
- Md khoa cho phan huyet ttfdng tren nghdo t£ bao chay sang tui chuyen
thtf hai.
-Khi ltfdng huyet ttfdng trong tui con 30 - 50 ml, ngtfng 6p bang cach ha
can ham lo xo va thao tui khoi ban ep.
- Dien day du thong tin 0 nhan tui khoi tieu cau.
- Han day noi gitfa hai tui va c it rdi day noi gitfa hai vi tri han.
- D u n g cac ngon tay hoac vat mem cha sat can de tai huyen dich tieu c l u v&
de tui khoi tieu cau 1 gid khong lac trUdc khi truyen cho b en h nh an hoac bao quan.
3.2. Dieu che khoi tieu cau tir buffy coat

- Lay mau ngudi cho vao he thong tui 4 (tui lay mau va 3 tui chuyen). Trong
he thong tui can co 1 tui chuyen chtfa dung dich bao quan hong clu.
- Xep cac tui mau vao ong ly tam. Can bang ttfng cap o'ng ly tam va dat vao
may ly tam d cac vi tri doi xtfng. Ly tam manh d 22°C.
- D a t tui m a u toan p h l n da ly tam th a n h h a i ldp h o n g c l u v a h u y e t ttfOng
len b a n ep huyet ttfOng. C h u y de m at tui k h o n g d a n n h a n q u a y ra ngoai. T h a o
c l n h a m lo xo nen tam ep vao be m at tui mau.
- Md thong day no’i tui mau toan phln vdi tui chuyen bang cach be khoa d
d lu day noi vdi tui mau toan phln de huyet tuong ngheo te bao chay sang tui
chuyen thtf nhat.
- K h i m at p h a n cach h o n g c l u - huyet ttfOng d a n g len cach t h a n h tren tui
m a u k h o a n g 2,5 - 3 cm, k ho a day no’i tui m a u chtfa khoi h o n g c l u v a tui chtfa
huyet ttfOng b a n g kep hoac k ho a nhtfa.
- D u n g kep ngoai k ho a canh dai luon dtfdi m a t p h a n cach h o n g c l u - huyet

ttfOng k h o a n g 0,5 - 1 cm.
- Siet kep dong thdi vdi viec:

+ Ha c ln ham lo xo
+ Nhac tui mau khdi ban ep
+ Quay kep mot goc 90°
+ Ndi khoa nhtfa md thong day noi tui mau va tui chuyen thtf 2
+ De ldp buffy coat (gom phln ldn bach clu, tieu clu, mot p h ln nhd hong
clu va huyet ttfOng) chay sang tui chuyen.
- Dung kep nhtfa khoa day no’i tui mau va tui chuyen chtfa buffy coat.
- Bo" s u n g ttf tui ch u yen chtfa d u n g dich b a o q u a n h o n g c l u (n e u co) hoac ttf

b in h chtfa n a tri cloru a 0.9% v ao tui kho’i h o n g c l u nhtf k y t h u a t d ieu che kho’i
ho n g c l u .
- Han va cat day ndi vdi tui khoi hong c lu v a tui huyet ttfOng.
- D ie n d l y du thong tin d n h a n tui khdi h o n g c l u , tui h u y e t ttfOng v a tui

chtfa b u ffy coat.
298
- Cat rdi cac day no'i b gitfa hai vi tri han trong khi v l n de tui chtfa buffy
coat con no'i vdi tui chuyen thii 3.
- Xep cac tui chtia buffy coat vao o'ng ly tam. Can bang ttfng cap o'ng ly tam
va dat vao may ly tam b cac vi tri do'i xtfng. Ly tam nhe d 22°C.
- L a y nhe nhan g tui buffy coat khoi may ly tam va dat len ban ep huyet ttfOng.
- Thao c ln ham 16 xo nen tam ep vao be mat tui buffy coat.

- Md khoa cho phan kho'i tieu cau chay sang tui chuyen thtf 3.
- Khi can hong cau dang sat mieng tui, ngtfng ep bang cach ha can ham lo
xo va thao tui khoi ban ep.
- Dien day du thong tin 6 nhan tui kho'i tieu cau.
- Han day no'i gitfa hai tui va cat rdi day no'i gitfa hai vi tri han.

- Kho'i tieu cau dieu che trong he thong tui kin bao quan 6 22°C co lac trong
3 den 7 ngay ke ttf ngay lay mau (thoi gian tuy theo loai tui deo va loai may lac
bao quan tieu cau).
- Kho'i tieu cau dieu che trong he thong ho bao quan 6 22°C trong 24 gid ke
ttf luc dieu che.
- Trtfdc khi truyen cho benh nhan: cac dOn vi kho'i tieu cau can dtfdc pool
lai du lieu theo yeu cau cua dieu tri lam sang. So' ltfdng ddn vi tieu cau cho 1 pool
thay doi tuy thuoc nhu cau dieu tri ttfng benh nhan cu the.
- Sii dung bo day co bau loc vdi dtfOng kinh lo loc 170 - 200 jim de truyen.
Co the truyen bang day truyen kem bo loc bach cau.

- Cac trtfdng hop giam tieu cau gay xua't huyet nguy hiem gap trong cac
benh leukemia cap, suy tuy, Dengue xua't huyet...
- Cac ro'i loan dong mau co giam tieu cau nang.

Mot dOn vi mau toan phan 250 ml dieu che dtfdc 1 ddn vi kho'i tieu cau vdi
mot so' dac tinh sau:
- The tich: 45 ± 15 ml
- So ltfdng tieu cau: > 3,0 x 1010 / dv (neu Pool > 1,3x10” )
- So ltfdng bach cau: < 0,1 x 109 trong 75% m iu kiem tra (neu Pool < 0,4 x 109).
- So ltfdng hong cau: < 2,2 x 109trong 75% mau kiem tra (neu Pool < 0,01 x 1012).
- pH > 6.5
299
7. Liru y
- Ly tam manh: Ly tam v6i toe do cao va/hoac thdi gian dai nham lam lang
moi loai te bao. Dieu che buffy coat ttf ddn vi mau the tich 250 ml, thong so tham
khao co the la:
- Ly tam nhe: Ly tam vdi toe do thap va/hoac thdi gian ngan nham lam
lang mot loai te bao trong khi mot hoac nhieu te bao khac chtfa bi lang. Dieu che
huyet ttfOng giau tieu cau ttf ddn vi mau the tich 250 ml, thong so tham khao c6
the la:
KY THUAT DIEU CHE HUYET TlfflNG
1. Nguyen tac
Huyet ttfOng dtfdc tach khoi cac thanh pham te bao de bao quan lanh dai ngay.

- Mau toan phan dtfdc lay mot cach vo trung vao bo tui deo co g in them
mot hoac nhieu tui chuyen.
- B a n e p h u y e t ttfO n g
- Kim vuo't day

- Keo

- May ly tam lanh
- Can ban hoac can 16 xo

3.1. Dieu c h i huyet tuung khi dieu che khdi hdng cau thong thudng
Xem ky thuat dieu che khoi hong cau
3.2. Dieu che huyet tuong khi tach buffy coat
Xem ky thuat dieu che khoi tieu cau (phan dieu che khoi tieu cau ttf buffy coat).
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- Ngay sau khi dieu che, huyet ttfdng dtfdc dong lanh va bao quan d nhiet
do th&'p hdn - 18°C hoac tot nha't la dtfdi -35°C. Thdi gian bao quan la 24 thang ke
ttf luc dieu che.
- Huyet ttfdng ttfdi: la huyet ttfdng dtfdc dieu che trong vong 8 gid ke ttf luc
l&y mau.
- Huyet ttfdng ttfdi dong lanh: la huyet ttfdng ttfdi dtfdc dieu che va dong lanh
trong vdng 8 gid ke ttf luc lay mau va bao quan ddng lanh trong vdng 12 thang.
- S a u khi p h a ddng, huyet ttfOng can dtfdc tru y en tron g v dn g 6 - 8 gid.
- Sti dung bo day co bau loc vdi dtfdng kinh lo loc 170 - 200 nm de truyen.

- Dieu tri thay the trong cac hoi chtfng roi loan ddng mau bam sinh hoac
mac phai cd suy giam cac yeu to ddng mau. Huyet ttfOng ttfOi va huyet ttfdng ttfdi
ddng lanh co the dung dieu tri hemophilia A.
- Bong nang
- Truyen nhieu khoi hong cau trong mat mau cap.
- Dieu tri thay mau, thay huyet ttfOng.

- The tich: 120 ± 15 ml
- Protein huyet ttfdng: > 50 g/1.
7. Luu y
- Pha ddng huyet ttfdng ddng lanh d nhiet do 37°C trong binh cach thuy.
- Tranh ddng lanh va pha ddng huyet ttfdng nhieu lan.
KY THUAT DIEU CHE TUA LANH GIAU YEU TO VIII
1. Nguyen tac
Yeu to' V I I I (yeu to' chong hemophilia A) cd the dtfdc dieu che ttf huyet ttfdng
ttfdi bang cach ddng lanh nhanh va pha ddng cham d nhiet do thap.

- H u y e t ttfOng ttfOi ddng la n h (xem p h a n k y t h u a t dieu che h u y e t ttfdng).
- Ban ep huyet ttfdng
- Kim vuo't day
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- Keo
- May ly tam lanh

1. Pha dong huyet ttfOng d 1 - 6°C trong binh cach thuy 45 - 90 phut ho&c
trong tu lanh 1 5 -1 8 gid.
2. Khi huyet ttfOng tan dong gan hoan toan va con mot so' cuc da nho, tua
lanh hinh thanh dtfdi dang cac van nhd nhtf khdi thuoc. Tach tua cd the thtfc hien
tiep tuc b in g 1 trong 2 cach sau.
3. Tach tua lanh:
• Cach 1:
- Ly tam m a n h tui huyet ttfOng ttfOi p h a ddn g d nhiet do 5°C.
- Lay tui huyet ttfOng da t len b a n ep.
- N o i vd t r u n g tui hu y et ttfOng vdi 1 tui chuyen.
- Thao can ham lo xo nen tam ep vao be mat tui huyet ttfOng. Phan huyet
ttfOng tan ddng noi phia tren dtfOc chay sang tui chuyen, de lai tua lanh d day tui.
- Gitf lai 10 - 15 ml huyet ttfOng de hoa tan tu a lanh.
• Cach 2:
- Treo tui hu y et ttfOng tren b a n ep
- Noi vd t r u n g tui hu y et ttfOng vdi 1 tui chuyen.
- Md thong day noi de huyet ttfOng tan ddng chay sang tui chuyen. Cac
mau da chtfa tan noi tren be mat cd tac dung nhtf t§fm loc ngan can tua lanh troi
cung huyet ttfOng.
- Khoa day noi khi phan ldn huyet ttfdng chay sang tui chuyen. Ltfdng
huyet ttfdng cdn lai trong tui khoang 1 0 -1 5 ml.
4. Hoa tan tua bln g cach de tui tua trong binh cach thuy 37°C trong 1-5 phut
5. Pool tua lanh: Tuy theo yeu cau dieu tri, cd the pool ttf 4 - 16 tui tua
thanh mot pool
6. Han day noi cua tui chtfa pool tua lanh.

- Ngay sau khi dieu che, tua lanh dtfOc ddng lanh va bao quan d nhiet do
thap hon - 18°C hoac tot nhat la dtfdi -35°C. Thdi gian bao quan la 12 thang ke ttf
luc dieu che.
- Sti dung bo day cd bau loc vdi dtfdng kinh lo loc 170 - 200 |im de truyen.
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- Dieu tri thay the trong benh hemophilia A, von Willebrand
- Dieu tri bo sung trong mot so' ro'i loan dong mau nhtf dong mau noi mach
rai rac, tieu sdi huyet c&'p.
6. Mot so thong so tham khao: T i i 1 ddn v i h uyet ttfdng 120 ml

- The tich: 10 ± 5 ml.
- Nong do yeu to' V III: > 2,5 dv VIII/ ml tua
7. Luu y
- Ly tam manh: Ly tam vdi toe do cao va/hoac thdi gian dai nh&m lam l&ng
moi loai te bao va/hoac cac tua protein. Dieu che tua lanh, thong so' tham khao cd
the la:
+ 3000 x g trong 9 - 10 phut.
- T u a lanh hinh thanh trong qua trinh pha ddng cham rat de hoa tan trd lai,
do vay can thao tac nhe n h an g vdi tui huyet ttfOng pha ddng trtfdc khi tach tua.
KY THUAT LUU TRUf, BAO QUAN VA PHAN PHOI MAU
1. Md dau
Ltfu trtf mau la cong doan cuo'i cung, no quyet dinh toan bo qua trinh, cong
stfc cua mot cO sd truyen mau. Cd ltfu trtf mau va cac che pham mau tot neu cd
the dam bao dtfoc chat ltfdng, hieu qua sti dung cua mau va cac che pham ttf mau.
2. Nguyen tac
- Mau toan phan hoac che pham mau phai dtfdc bao quan trong nhting dieu
kien quy dinh cho mdi loai de dam bao dtfdc chat ltfdng cua chung qua qua trinh
bao quan.
- Phai cd phtfOng phap, he thong theo ddi, quan ly cac dOn vi mau hoac che
pham mau mot cach quy cu, ro rang, chinh xac de cd the tranh dtfdc nh&m lan, sai
sot, luan chuyen mot cach hdp ly nha't.
3. Kho mau
- Mot gian phong dtfdc sti dung lam kho mau ddi hdi mot so yeu cau sau:
+ Rdng rai, sach se, thoang mat, khdng khi ltfu thong
+ Phai day du anh sang, nguon dien phai thtfdng xuyen on dinh
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+ Hau het cac phtfdng tien bao quan mau va che pham la cac may m6c lam
lanh viec trao doi nhiet la rat ldn. Vi vay nen trang bi cac may dieu hoa nhiet do,
de tranh tinh trang hoat dong qua tai cua cac may moc dot xua't gay mat on dinh
nhiet do ben trong ma hau qua la gay htf hong cac don vi mau hoac che pham
dang dtfdc ltfu gitf.
+ La khu vtfc danh rieng cho nhan vien phu trach cong viec ctfa ngo kin
dao, han che to'i da stf ra vao cua nhtfng ngtfdi khong co trach nhiem.
- Cac may moc trang thiet bi trong kho mau can dtfOc gitf d ttf the on dinh,
vtfng chtfc. Nen ke cach nhau va cach ttfdng khoang 40-50cm. Cd nhan ghi muc
dich sii dung cua ttfng cai, cd so sach theo ddi hoat dong cua may mot cach day du.
4. Dung cu va trang thiet bj

- Tu lanh chuyen dung, ltfu trtf mau d nhiet do 2-6°C
- Tu lanh -35°C
- May lac tieu cau +22°C
- Neu can thiet tuy ttfng cO sd cd the cd them:
+ Tu am +20°C
+ Dung cu lanh -150°C hoac —196°C
- Ho sd sd sach
5. Nhiet do va thdi gian bao quan mau toan phan va che pham mau
C) day chung tdi chi xin trinh bay mot so' nguyen tac chu yeu va vdi mot so
che pham chinh:
Ten Nhiet do bao quan Thdi gian bao quan

1. Mau toan phan
- Bao quan bang ACD 2°C den 6 °C 21 ngay
- Bao quan bang CPD 2°C den 6 °C 21 ngay
- Bao quan bang CPDA, 2°C den 6 °C 35 ngay
2. Cac loai khdi tieu cau (trong may 20°C den 24°C 3-5 ngay
lac lien tuc)
3. Huyet ti/ong ttfdi dong lanh <-30°C 12-24 thang
ttf -25 den -30°C <12 thang
t t f -18 den -25°C < 6 thang
Huyet ttfdng ttfdi 2°C den 6 °C < 24 gid
4 .Huyet ttfdng dong lanh da tan dong 2°C den 6 °C < 5 ngay
5. Huyet ttfOng tach ttf mau toan phan 2°C den 6 °C < 1 thang
6 . Cryo < -30°C 12 den 24 thang
-25 den -30°C < 12 thang
-18 den -25°C < 6 thang
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L:tu y:
- Vdi cac kho'i hong cau dtfoc san xua't theo he thong kin vao co d u n g dich
bao quan thi nhiet do va thdi gia n bao q u a n ttfOng tu n h u m a u toan phan.
- Vdi khoi hong cau san xua't theo he thong hd thi nen stf dung ngay sau
khi san xua't.
6. Quy trinh luu trdr

Khi mot ddn vi mau rieng le hoac mot lo tui mau dtfOc dUa den ngudi ky
thuat vien chiu trach nhiem d kho mau se thtfc hien qua cac budc sau:
6.1. Nhap kho

Ngudi chiu trach nhiem can phan loai ngay dtfdc cac tui mau dtfdc dtfa den.
6.1.1. Mau vtfa mdi lay ve chtfa lam xet nghiem
- Kiem tra so ltfdng ddn vi mau la bao nhieu tui

- Kiem tra cac noi dung ghi tren nhan tui co day du khong
+ Ho ten hoac so' ngtfdi cho
+ So' ltfOng ml m au trong tui
+ Ho ten ngtfdi lay m au
- Vi cac don vi mau nay con lien quan den nhieu van de nhtf xet nghiem
nhom mau, xet nghiem sang loc, mau huy, sii dung trong san xua't che pham
thanh pham. Vi vay nen vao mot so rieng, ghi lai cac noi dung ghi tren nhan tui.
Dong thdi can them mot sd noi dung:
+ Ngay gid lay mau
+ Ngay gid nhan mau
+ Noi lay mau
+ Ky nhan ban giao gitfa ngtfdi mang den va kho mau
- Cung vi cac ly do nen tren ma cac tui mau nay phai dtfdc xep vao mot tu
lanh bao quan rieng (tot nhat la co khoa) co ghi ro chtfc nang dac biet cua tu lanh
nay, vi du: “Mau chtfa xet nghiem - khong dtfcfc stf dung” .
Ngtfdi phu trach can co trach nhiem nhac nhd cac bo phan lien quan thtfc
hien sdm nha't nhiem vu cua ho vdi cac ddn vi mau nay. Khi can phai ban giao cho
ho, phai co giao nhan r5 rang.
6.1.2. Mau huy

Can nhap so' lieu ve cac dOn vi mau nay vao mot so theo ddi rieng cung vdi
cac noi dung neu d tren va nen bd sung them:
- Ly do huy
- Ket qua xet nghiem
305
- Phai ghi ro rang tren ttfng tui mau vdi but mau de de nhan biet vdi noi
dung “Mau huy, khong stf dung, ly do huy” .
- Cac tui mau nay nhat thiet phai de vao tu lanh rieng biet. co ghi ro noi
dung “Mau huy. khong stf dung” , can co khoa de dam bao an toan.
- Vdi cac tui mau dac biet co the stf dung cho muc dich khac (vi du huyet
thanh viem gan B dung cho san xuat...) can tap trung thanh ttfng nhom tuy theo
htfdng stf dung trong tu nay (neu co dieu kien thi co the xep trong mot tu khac).
- Ltfu y: du rat hiern gap song co the co trtfdng hop mau het han stf dung,
cung xtf ly theo cac btfdc tren.
- Tong ket bao cao ket qua vdi ngtfdi phu trach cO sd truyen mau hoac nhtfng
ngtfdi khac co lien quan de lam sdm xtf ly cac tui mau theo quy dinh.
6.1.3. Mau du tieu chuan, duoc luu trur cho sCf dung
Day la phan chi'nh trong cong viec. doi hdi ngtfdi chiu trach nhiem dau ttf
nhieu thdi gian va chap hanh dung cac quy dinh.
- Phan loai
+ P h a n loai theo noi d u n g ben trong tui m au: cac dOn vi m a u dtfOc dtfa den
kho m au vdi nhieu noi d u n g khac nhau, ngtfdi chiu trach nh iem (th u k ho ) can sdm
ph an loai theo ttfng loai nhtf m au toan phan. kho'i ho n g cau. huyet ttfOng. huyet
ttfOng ttfOi ddng la n h th a n h ttfng lo r ie n g biet, de tach biet n h au .
+ Phan loai theo nhom ABO: vdi mdi loai tren can tiep tuc phan thanh ttfng
nhom A, B, 0 hoac AB. Song song vdi viec phan loai can kiem tra ve sd ltfOng,
kiem tra cac noi dung ghi theo veu chu tren nhan tui cd day du hay khdng. kiem
tra stf toan ven cua tui. ltfu y cac thdi han sii dung hoac dac Ijiet cac yeu cau ltfu
trtf de cd stf chuan bi cho cac btfdc tiep theo. Neu khdng co van de gi khac thtfdng
se tien hanh ky nhan gitfa ngtfdi ban giao va kho mau. Viec ky nhan c&n tien
hanh nghiem chinh, cd sd sach rieng. theo ttfng loai mau hoac ttfng che pham vdi
cac nhom mau cua ttfng loai, ddng thdi ghi ro ngay thang giao va nhan.
- Nhap so sach: de thuan loi cho viec theo doi, luan chuyen. cap phat nen
theo cac yeu cau sau:
+ Mdi loai mau hoac che pham vao mot so nhap rieng
+ Mdi nhom (A.B.O.AB) cua ttfng loai theo mot so rieng

+ Noi dung can nhap:
Ho ten ngtfdi cho hoac so cua tui mau

Ten cua thanh phan trong tui (cd the can hoac khdng)
S d ltfOng tron g tui
Ten ngtfdi lay mau hoac san xuat
Ten ngtfdi dinh nhom mau
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Ndi ban giao
Ngay nhan
Han sii dung
Cac ket qua xet nghiem sang loc
Ngay phat ra
Noi nhan (ndi sii dung)
Ky nhan gitfa kho va ndi sii dung
Cdn li/u y:
- Giai doan nay can tien h a n h n g a y khi n h ap m au vao kho, thtfc hien mot
cach nhanh chong, k h a n trtfOng de co the d u a cac don vi m au hoac che ph am vao
ndi bao q u a n sdm nh at n h am dam bao chat ltfOng cua no.
- Vdi cac don vi mau nhan dtfdc ttf cac cO sd truyen mau khac dtfoc coi la
mau bo de sii dung cung thtfc hien theo quy trinh nay.
- Sap xep: can tien hanh ngay mot cach lien tuc vdi cac giai doan tren vi
mau hoac che pham chi dam bao dtfOc chat ltfOng khi dtfOc bao quan theo dung
dieu kien dtfdc yeu cau cua no.
+ Sap xep theo thtf ttf thdi gian, nhtfng tui mdi de trong, tui lau de ra ngoai
+ Cac tui mau toan phan can dat tren gia de tui dtfcfc d ttf the dtfng co
khoang cach gitfa cac tui de dam bao ltfu thong khong khi va nhiet do.
+ Ttfng loai mau hoac che pham vao ttfng tu lanh hoac ttfng ngan rieng biet
+ Ttfng loai mau hoac che phaim lai xep theo ttfng nhom (ABO) hay ttfng
ngan hoac ttfng day rieng biet. Vdi ttfng noi dung, ttfng nhom phai cd nhan in chtf
to, ro de de tim, hoac tim dtfdc nhanh nhat tranh md dung cu bao quan qua lau.
+ Sau mdi lan nhap hoac cap phat can luan chuyen thay doi vi tri cac tui
theo nguyen tac nhtf tren.
Lliu y:
- Phai thtfdng xuyen kiem tra hoat dong cua may moc trang thiet bi trong
kho mau.
- Dinh ky lam ve sinh trong va ngoai cac trang thiet bi ltfu trtf
- Tuyet ddi khdng de nhtfng thtf khdng phai la mau hoac cac che pham mau
vao trong cac thiet bi nay, ngay ca cac sinh pham.
6.2. Phat va phan phoi mau
- Moi qua trinh cap phat phai cd stf ddng y cua ngtfdi phu trach cd sd
truyen mau.
- Khi cd yeu cau linh mau trong va ngoai vien: ngtfdi phu trach kho mau
can kiem tra phieu yeu cau ve cac noi dung:
+ Ndi linh
+ Ho ten benh nhan
+ Nhom mau benh nhan
+ Noi dung xin linh: mau toan phan. khoi hong cau
+ Nhom mau cua thanh phan xin linh. so ltfOng
+ Ddn vi yeu cau: khoa, phong, hoac benh vien
+ Chtf ky cua ngtfdi phu trach ddn vi yeu cau stf dung
+ Chtf ky cua ngtfdi phu trach cd sd truyen mau
- Ltfa chon cac don vi m au hoac che ph&m phat ra p h u hop vdi yeu cau
th a n h phdn, nhom m au, so ltfOng v a thdi h a n sii d u n g (n e u co).
- Kiem tra chat ltfOng don vi mau dtfOc phat ra bang mat thtfdng. phat hien
nhtfng bien doi bat thtfdng xuat hien trong qua trinh bao quan. Chi phat ra
nhtfng don vi co the tin ttfdng ve chat ltfdng. Neu co bat ky stf e ngai nao tui mau
phai duoc gitf lai, bao vdi ngtfdi phu trach de co bien phap kiem tra xii ly kip thdi.
- Don vi phat mau ra phai dtfoc ghi vao so cua kho mau
+ Ngay gid phat mau

+ Noi nhan
+ Ky nhan cua ngtfdi linh
L itu y: Khi ndi nhan mau la cd sd truyen mau cua mot benh vien khac khi
don vi mau phat ra se khong qua bo phan phat mau an toan mien dich cua don vi
co kho mau thi can:
- Ngtfdi nhan mau phai la nhan vien y te phong truyen mau cua benh
vien do.
- P h a i co phtfOng tien b a o q u a n v a n c h u y e n theo q u y d in h d a m b a o du y
tri dtfoc n h ie t do t r o n g q u a t r in h b a o q u a n t r a n h n h tfn g tac d o n g g a y ton
thtfOng tui m a u hoac a n h htfdng ch at ltfOng c u a t h a n h p h a n t r o n g tui m a u .
- D in h lai nh om m a u b a n g mot phtfOng p h a p H T M de mot la n cuoi cun g

h a n che nhtfng sai sot du rat hiem khi x ay ra.
- Kho mau se gitf lai phieu linh mau cua benh vien nhan mau
N g o a i cac nh iem vu tren ngtfdi p h u trach kho m a u cdn p h ai co trach nhiem

bao cao theo quy dinh: so ltfOng m au. che p h a m n h a p v ao va p h at ra h a n g ngay;
h a n g thang: tinh h in h hoat d o n g cua cac t r a n g thiet bi tron g kho m au . T h o n g ke
so ltfOng don vi m a u h u y vdi moi n g u y e n n h a n (het han. bien doi chat ltfOng. ton
thtfdng tui m a u do b ao q u a n hoac tan d o n g ...), din h ky vdi ngtfdi p h u trach.
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Chitfcfng 8
MOT SO VAN BE CO BAN TRONG

■
XET NGHIEM
■ HUYET HOC
■ - TRUYEN MAU
PHI/ONG PHAP LAM SACH VA KHUT TRUNG DUNG CU LAM XET NGHIEM
Do chinh xac cua xet nghiem phu thuoc vao nhieu yeu to', trong do dung cu
de lam xet nghiem cung la mot yeu to can dtfOc quan tam, dac biet la cac dung cu
hang thuy tinh. bao gom nhieu loai nhtfng deu co dac diem chung:
- Trung tinh.
- Kho sach.
- Khong stft me hoac bien dang.
Do do rtfa. say. hap dung cu phai bao dam cac yeu c&u tren, nhtf vay mdi stf
dung lau dai va khong gay sai sot ve ket qua.
1. Rura lam kinh va la kinh

1.1. Vdi lam kinh va la kinh chua dung hoac da dung cho benh pham khong mau
- Rtfa sach bang nude thtfdng.

- Ngam vao dung dich sulfocromic trong 24 gid.
- Rtfa lai bang ntfdc- thtfdng that sach.
- Trang lai bang nude cat.
- Ngam vao bocan thuy tinh dtfng con 99° , day kin. khi dung mdi vdt ra.
- Lau kho bang gac hoac vai mem.
- Xep vao tu am 56"C cho kho hoac de kho ttf nhien. chu y d a y de tran h bui.
1.2. Vdi lam kinh da dung cd dinh dau hoac pham nhuom
- Ngam vao ntfdc xa phong 24 gid (ctf lOOg xa phong hoa trong 5lft ntfdc).
- Vdt ra lav ban eliai hoac choi duoi ngtfa co sach.
- Ngam vao dung dich sulfocromic hoac NaOH trong 24 gid.
- Rtfa bang ntfdc thtfdng 3,4 lan cho sach.
- Trang lai 1.2 lan bang ntfdc cat.
509
- Ngam vao bocan thuy tinh dtfng con 99°C, day kin. khi dung mdi vdt ra.
- Lau kho bang gac hoac vai mem. -
- Xep vao tu am 56°C cho kho hoac de kho tu nhien (chu y day tranh bui).
2. Rtfa cac loai ong nghiem

2.1. Ong nghiem chua dung
- Dung choi duoi ngtfa rtia sach bang ntfdc thtfdng vai lan.
- Trang lai bang ntfdc cat.
- Doc het ntfdc trong o'ng ra xep vao sot nhom co lo 0 day.
- De tu am 56°C cho kho hoac phoi kho tu nhien (chu y day tranh bui).
2.2. Ong nghiem da dung roi

- Dung choi duoi ngtfa rtia sach bang ntfdc thtfdng vai lan.
- Rtia lai bang xa phong.
- Rtia lai bang ntfdc thtfdng.
- Ngam vao dung dich sulfocromic hoac NaOH trong 24 gid.
- Rtia lai bang ntfdc thtfdng.
- Doc het ntfdc trong dng ra va xep vao sot nhom co lo d day.
- De tu am 56°C cho kho hoac de kho ttf nhien.
3. Rtfa cac ong hut (potain) hong cau, bach cau, ong mau lang, pipette
huyet sac to
- Thoi bd het hon dich mau trong o'ng hut.
- Rtia vai lan bang ntfdc thtfdng.
- Rtia bang con 99°.
- Rtia bang ether.
- De tu am 56° cho kho.
Mot tuan ngam dung cu mot lan vao dung dich sulfocromic trong 24 gid.
- Rtia lai vai lan bang ntf6c thtfdng.
- Rtfa bang con 99°.
- Rtia bang ether.
- De tu am 56° cho kho.

4. Cac loai buong dem
- Ngam vao dung dich sulfocromic 2-3 gid.
- Rtia bang ntfdc thtfdng vai lan.
- Lau sach.
- De trong hop co lot gac mem hoac giay loc.
PHA CAC HOA CHAT, DUNG DjCH SI/DUNG

TRONG XET NGHIEM HUYET HOC
1. Cach thur nirdc cat trung tinh de lam xet nghiem

1.1. Thubing dung djch do trung tmh
- Ntfdc cat 100ml.
- Dung dich dd trung tinh 1% vai giot.
Neu ntfdc acid se co mau vang chanh, nhd ttfng giet natri bicarbonat 1% vao
cho den khi ntfdc cd mau hdng la ntfdc trung tinh.
Neu ntfdc kiem se cd mau hong, nhd ttfng giot acid acetic 1% vao cho den khi
ntfdc chuyen thanh mau da cam la ntfdc trung tinh.
1.2. Thif bang giay do pH

Cho vao coc thuy tinh hoac o'ng nghiem ltfdng ntfdc dinh thti. Lay giay do pH
nhung vao ntfdc trong 5 phut. Lay giay ra dem so sanh vdi mau chuan quy dinh,
doc ket qua.
Neu mau giay thti khdng dung vdi mau chuan thi cho acid acetic 1% vao neu
ntfdc kiem hoac natri bicarbonat 1% vao neu ntfdc acid. Lay giay thti lai cho den
khi dat dtfOc mau quy dinh cua pH trung ti'nh.
1.3. Do bang pH met (pH ke)
- Cho mau ntfdc dinh thti vao cong, dat vao may pH ke.
- Bat nut dien quy dinh trong may, de on dinh trong 5 phut.
- Kim pH ke se chi cho biet ntfdc dinh thti la kiem, trung tinh hay acid.
- Cho tti tti acid acetic 1% vao (neu ntfdc kiem) hoac natri bicarbonat vao
(neu ntfdc acid), dieu chinh den khi may pH chi ntfdc dat tri so' trung tinh.
311
2. Cach bao quan va pha loang con
2 . 1. Cach bao quart con tuyet doi
Muon gitf dtfdc con tuyet do’i thi cti 1000ml con cho them vao 50g kali
carbonat hoac 50g dong sulfat khan, se giti dtfdc do con de lam xet nghiem.
2.2. Cach pha loang con
2.2.1. P hi/ong phap Lowi

, V *» . ^
- Cho vao coc ntfdc chia do so ltfOng mililit con dung de pha loang bang so
do con dinh pha loang.
- Cho them ntfdc cat vao cho den khi so mililit bang so do con dem pha loang.
T h i du: Dinh pha con 95° thanh con 60°:
- Cho vao coc chia do 60ml con 95°.
- Cho them ntfdc cat vao cho den khi dung dich co the tich = 95 ml thi dtfdc
con 60°.
2.2.2. Phi/ong phap G ay-Lussac
The tich ntfdc them vao 100 ml The tich ntfdc them vao
Con djnh pha
con 90°(ml) 100 ml con 95°(ml)
85° 6 13
O
o
00
14 21
75° 22 29
oo
31 39
6 5° 42 50
o
CO
O
54 65
55° 68 78
oo
cn
85 96
45° 105 117

o
O
131 144
cno
Ca)
163 178
30° 206 224
25° 2 66 287
20° 352 382
15° 500 540
10° 605 655
3. Dung dich chong dong

3.1. Dung djch natri citrat 3,8%
- Natri citrat 3.8g
- Ntfdc cat vtfa du 100ml
312
3.2. Chdng ddng heparin
- Heparin 5000 ddn vi
- Niidc cat 10ml
3.3. Dung djch complexon

- Muoi dipotassic cua acid ethylen diamin tetra acetic (EDTA): l,5g
- Nude cat vtia du : 100ml
3.4. Hon djch amoni oxalat va kali oxalat

De kiem, re tien, it lam thay doi hinh thai te bao mau vi amoni oxalat lam
nd te bao con kali oxalat lam co te bao.
- Amoni oxalat l,2g
- Kali oxalat 0,2g
- Nude cat 100ml
Dung dich (3), (4): Pha xong loc ky, dung dan. Co the lam thanh dang dong
kho de khoi lam anh hudng den the tich mau. Cach lam: cho 0,1 ml dung dich
tren vao ong nghiem tan mau, cho vao tu am 56° trong 6 gid. Dung dich kho di. Co
the chong dong cho lm l mau.
4. Cach pha Giemsa dam dac (Giemsa me)

- Giemsa bot 0,75g
- Glycerin 35ml
- Con methylic 65ml
Cho Giemsa bot vao col bang sti, cho tti tti glycerin vao va dung chay bang
sti nghien that min. Tiep tuc cho con vao hoa deu. Dung dung dich vao lo mau,
nut lo that kin. Boc lo bang giay den hoac de d cho to’i, ngay lac 3 lan, trong 4
ngay lien. Giemsa me de tren 6 thang mdi dem dung la tot nhat. Co the thay bot
Giemsa bang bot Wright theo cong thtic:
- Wright bot l,3g
- Glycerin 3ml
- Con methylic 97ml
5. Hon hop sulfo-cromic ngam dung cu va lam

- Potassium bicromat K 2Cr20 7 100g
- Nude 1000ml
Quay cho tan deu. Sau do tti tti them vao: acid sulfuric nguyen chat H_S(), 100ml
Dung dich phai day kin.
6. Dung djch nhuom hong cau ludi

6.1. Xanh cresyl trong con
- Xanh cresyl anh lg
- Con tuyet do'i 100ml
313
6.2. Xanh cresyl trong dem muoi citrat va clorua (OMS)
- Xanh cresyl anh lg
- Natri citrat 3% 20ml
- Natri clorua 9%o 80ml
LSc cho tan deu roi loc. Dung trong 1 thang
7. Dung djch dem so ltfdng hong cau

7.1. Dung djch Hayem
- Natri clorua lg
- Natri sulfat 5g
- Sublimat l,5g
- Nude cat 200ml
7.2. Dung dich Marcano

- Natri sulfat 50g
- Formol 40% 5ml
- Nude cat 1000ml
7.3. Dung dich nude muoi sinh ly

- NaCl 8,5 g
- Nude cat vtia du 1000ml
8. Dung djch dem so ltfdng bach cau

8.1. Dung djch Lazarus
- Acid acetic 50ml
- Xanh methylen 1% 20 giot
8.2. Dung djch Hayem

- Acid acetic 5ml
- Xanh methylen 0,25g
8.3. Dung djch xanh acetic
- Acid acetic 10ml
- Xanh Toludin 0.25% 10ml
- Nif6c cat vtia du 1000ml
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9. Dung djch dem so ltfdng tieu cau
9.1. Dung djch gi Ornguyen ca hdng cau va tieu cau
Dung dich Marcano:
- Natri sulfat 50g
- Formol 40% 5ml
- Nu6c cat vtia du 1000ml
9.2. Dung djch lam vd hong cau va gi Ornguyen tieu cau

9.2.1. Dung djch ure
- Ure nguyen chat 0,7g
- Nude muoi 9%0 7ml
- Nude cat 3ml
9.2.2. Dung dich cocain

- Cocain clohydrat 30g
- Natri clorua 2g
9.2.3. Dung dich hon hpp

- Dung dich A giti d 4°C Clohydrat procain 24,3g
Nude cat vtia du 100ml
- Dung dich B giti 0 4°C Clorua natri 200g
Nude cat vtia du 100ml
TrUdc khi dung thi pha nhu sau:
- Dung dich A lm l Khong de lau
- Dung dich B 9ml
9.2.4. Dung dich am onium oxalat 1% trong nude cat

Giti d nhiet do lanh, trUdc khi dung phai loc.
CACH SIT DUNG VA DAO QOAN KINH HIEN VI
Kinh hien vi la cong cu quan trong d moi phong xet nghiem huyet hoc. No co
tac dung phong dai anh cua vat can soi bdi mot he thong thau kinh.giup cho ta
quan sat vat duoc chi tiet hon.
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1. Cau tao (hinh 8.1)
H inh 8.1: Cac bo phan cua mot kinh hien vi phtic hpp dien hinh
1. Ong true chinh
2. Thau kinh (ISng kinh) dau kinh nghieng
3. Mam quay g in vat kinh
4. Vat kinh
5. Mam soi (mam kinh)
6 . Tu quang dudi mam kinh vdi man c h in sang
* 7. GUOng
8 . De (chan)
9. Thi kinh
10. Tay cam
11. Oc dai cap (dieu chinh tho)
12. Oc vi cap (dieu chinh tinh)
1.1. Ong true chi'nh: Cd hai loai

- Loai cd mot o’ng true chi'nh mang mot thi kinh(kmh mot mat)
- Loai cd hai o’ng true chi'nh mang hai thi kinh (kinh hai mat)
1.2. Dau ki'nh
Hinh tron hoac da giac. Trong dau ki'nh chtia cac thau ki'nh hinh lang tru
tam giac, cd tac dung hat anh cua vat tti vat kinh len thi ki'nh khdng bi dao ngUOc .
1.3. Ban xoay

Ban xoay cd the xoay tron 360° giup cho thay ddi vat kinh de dang
Ban xoay cd 4 lo mang 4 loai vat kinh khac nhau
316
1.4. Vat kinh
Co tac dung phong dai anh cua vat len nhieu lln. Co nhieu loai vat kinh vdi
he so phong dai khac nhau: x 4, x 6, x 8, x 10, x 17, x 40, x 100, nghia la cac vat
kinh do co the phong dai dtfpc 4 den 100 lln . Vat kinh co he so' phong dai 100 la
vat kinh dau.
1.4.1. Vat kinh cd he so phong dai nho thi kich thudc ngSn. Vat kinh cd he so phong
dai to: kich thitdc d d i
1.4.2. Khoang cach giCta vat kinh vdi m$t phSng (tieu ban)
- Vat kinh x 10 : khoang cach xa = 15,98 mm
- Vat kinh x 40 : khoang cach gan = 4,31 mm
- Vat kinh x 100 : khoang cach rat gan = 1,81 mm
1.4.3. CCfa so anh sang va kha nang phan ly cac loai vat kinh
- Vat kinh x 10: ctia so anh sang ldn, kha nang phan ly nho, co kha nang
nhin hai vat xa nhau
- Vat kinh x 40: kha nang phan ly ttfOng do'i ldn, co kha nang nhin hai vat
ttfOng do'i gan nhau
- Vat kinh x 100: ctia so anh sang rat nho, kha nang phan ly ldn, co kha
nang nhin dtfpc hai vat rat g&n nhau. Do kha nang phan ly ldn, anh sang khong
tap trung nen khi sti dung phai dung anh sang to'i da va dau soi de tang do chiet
quang, nhin vat ro net hOn
1.5. Mam ki'nh

Co tac dung nang do mau vat (tieu ban)
Tren mam kinh co:
- Mot lo de cho anh sang di ttf gtfOng qua tu quang len vat kinh
- Mot he thong kep giti tieu ban
- Mot bo phan di chuyen tieu ban goi la xa chuyen co tac dung dtfa tieu ban
len xuong, sang phai hoac sang trai
- Mot bo phan thtfdc do (duxich)
1.6. Tu quang
La he thong tha'u kinh co tac dung tap trung, hoi tu anh sang len vat dinh soi
- Neu 0 thap thi anh sang nho
- Neu d cao thi anh sang dtfpc tap trung cao
Tu quang co gan hai bo phan: la chan sang va loc sang
- La chan sang la nhting la nhtfa xep theo hinh dong tam. Muon anh sang
manh thi md chan sang. Muon anh sang yeu thi dong chan sang.
- Loc sang dat dtfdi tu quang, mau xanh, co tac dung lam diu anh sang khi
soi kinh.
317
1.7. Gwang
Gtfdng phang de lay anh sang gan

Gtfdng lom de lay anh sang xa hdn
Hien nay nhieu ki'nh sU dung nguon sang la den dat trong de kinh va co bo
phan dieu chinh ctfOng do anh sang.
1.8. De kinh
Giut cho kinh co dinh
1.9. Thi kinh

Cau tao bdi he thong thau ki'nh co tac dung phong dai. Thudng la thi kinh >'10
1.10. Than kinh - tay cam

Co tac dung nang do ong ki'nh va mam kinh. Tren than kinh co oc dai cap va
oc vi cap co tac dung dieu chinh khoang cach gitfa vat ki'nh va tieu ban.
Khi stf dung vat kinh x 10 dieu chinh bang oc dai cap
Khi stf dung vat kinh x 40, 100 dieu chinh oc vi cap
2. Cach sur dung

2.1. Lay anh sang
Co the la anh sang thien nhien hoac anh sang nhan tao. Neu anh sang thien
nhien thi nen dat kinh 0 gan ctfa so hoac ctfa ra vao.
Muon sii dung anh sang thi sii dung:
- Dong hoac md chan sang
- Ha hoac nang tu quang
- Dieu chinh gtfdng phan chieu hoac den
2.2. S ti dung vat kinh
Luc dau xem khai quat thi dung vat kinh nho va dieu chinh bang oc dai cap.
Khi tim dtfdc khoang xem thich hdp thi chuyen sang vat kinh dau. Nho mot giot
dau soi len tieu ban. sau do vtfa nhin vao thi kinh vtfa dieu chinh oc vi cap cho
den khi thay ro chi tiet dinh soi.
2.3. S u d u n g oc dai cap va oc vi cap
Dung oc dai cap de dieu chinh tho. dung oc vi cap de dieu chinh tinh. Dung
oc dai cap de dieu chinh trtfdc roi mdi dung oc vi cap
2.4. S u'du ng la chan sang va tu quang
Khi soi bang vat kinh thtfdng thi dong la chan va ha tu quang. Khi soi bang
vat kinh dau de xem chi tiet thi md la chan va nang tu quang.
318
3. Cach bao quan
Phai lam hang ngay mot cach can than, ti mi
3.1. TrUdc k h i s o i
Dung choi long quet het bui. Dung vai mem min khong so long hoac giay
mem dac biet lau sach vat kinh, thi kinh, tu quang, gtfdng..Mat trong cua thi
ki'nh va vat ki'nh thi phai dung qua bop thoi khong khi' vao va dung choi long quet
het bui.
3.2. Sau k h i s o i
- Ha mam ki'nh
- Nhac tieu ban ra ngoai
- Lay vai mem lau sach dau d vat ki'nh roi tham l't xylen lau lai. Cuoi cung
lau lai bang vai mem khac
- Ha tu quang
- Dieu chinh cac oc ve so 0
- Dtfa ki'nh ve ttf the nghi
- P h u ki'nh b a n g chup vai sach
3.3. Nhung dieu can tranh
- L a u cac thau ki'nh b a n g con
- N h u n g vat kinh vao con, xylen se lam b o n g cac thau ki'nh
- D u n g g ia y thtfdng hay bo n g de lau ki'nh
- Sd tay vao m at soi cua vat ki'nh
- Lau de hoac thau ki'nh hien vi bang dung moi htfu cO (xylen, xang)
- Lau mat trong thau ki'nh(cua thi kinh, vat ki'nh)bang khan hoac giay se
lam mat ldp phan chieu
- Bo thi ki'nh ra khoi kinh hien vi ma khong co nap day
- Xep ki'nh hien vi vao hop go dong kin hoac phu kin bang tui chat deo se
tao thanh buong am lam cho nam moc de phat trien.
- Di chuyen kinh hien vi bang mot tay
3.4. C hong cac tac nhan ben ng oai
• Chong chan dong khi van chuyen:
Kinh hien vi cung nhtf cac may moc quang hoc khac khi van chuyen phai dtfng
trong hop go dtfOc bat vi't chat, chen ky bang vai hoac mieng dem. Ki'nh hien vi phai de
cho co dinh. khong nen di chuyen nhieu va tranh de d ndi co nhieu chan dong.
319
• Chong nong vd chong an mon:
Cac he thong thau kinh, cac doan tiep xuc thudng g in b ln g mot loai nhtfa
dac biet, neu de noi nhiet do cao thi nhtfa de chay ra gay di lech, giam do chinh
xac. Khong de gan cac chat acid hoac kiem manh. Khong de anh sang m§t trdi roi
thang vao kinh.
• Chong xiidc: Khi lau chui nen dung qua bop thoi khong khi cho het bui,
dung choi long quet va vai mem sach hoac giay dac biet de lau, khong dung vai
cutng hoac giay thudng de lau de lam xUdc va md he thong quang hoc.
• Chong dm: de kinh 0 phong kho rao, co dieu hoa nhiet do, co hut am, neu
khong co dieu kien phai bao quan trong chup bang thuy tinh co chat hut am thay
hang ngay.
• Chong moc:
Khong de kinh d nOi co do am cao vi de tao dieu kien cho nam moc phat
trien. May moc quang hoc chi chiu dtfOc do Im khong khi khoang 65% nen de
kinh hien vi trong phong co dieu hoa nhiet do va may hut am hoac de trong tu co
bong dien thap sang ca ngay dem vdi nhiet do cao hdn nhiet do phong khoang 5°C.
Neu khong co dien thi de kinh d cho ram, thoang, gan vi tri co anh sang, dat canh
tui dung silicagel co tac dung hut am. Silicagel khi cdn tac dung thi co mau xanh
lo, neu chuyen sang mau hong thi het kha nang hap thu ntfdc, luc do cho silicagel
vao tu am say khd roi dung lai
• Lau moc
Moc thudng phat trien tti ngoai vao gitia do cac bao tti nam rdi vao cac khe.
Muon biet cac he thong thau kinh cd bi moc hay khdng ta di chuyen (xoay) cac thi
kinh hay vat kinh, neu vet md xoay theo thi dung la moc.
- Cac phUOng tien lau moc:
+ Qua bop cao su
+ Choi long mem
+ Bong hut nUdc, v a i m em

+ Nude cat, xylen, con, ether
+ Tuoc no vit cac cd, k h o a md chot
- Lau chui: trtfdc khi lau ngtfdi lam phai rtfa tay sach, lau tay khd, trai mot
mieng vai sach de dtfng cac bo phan thao ra. Dung ntfdc cat lau qua mot lln , lay
vai mem lau khd, dung xylen hoac aceton lau lai, cuoi cung dung ether lau cho
that khd.
Chu y: Khi lau nen chon ngay khd rao, neu vao ngay am tfdt se gay moc
them. Di chuyen kinh phai dung hai tay, mot tay dd de kinh, mot tay cam tru
(than kinh) hien vi.
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Chtfcfng 9
DAM BAO CHAT LI/ONG XET NGHIEM • a
HUYET HOC VA TRUYEN MAU

■
Cung nhif cac hoat dong khac, hoat dong truyen mau va xet nghiem huyet
hoc cung co san pham.
S:in pham cua mot hoat dong la ket qua cuoi cung do hoat dong dem lai. Doi
vdi cac hoat dong san xuat thi san pham co chat lifdng co nghia la dap tfng mot
cach tot nhat muc dich sii dung san pham do. Ddi vdi cong tac xet nghiem huyet
hoc thi san pham la ket qua xet nghiem. Chat luong xet nghiem dtfdc dam bao cd
nghia la ket qua phan anh kip thdi. chinh xac va ro rang cac chi sd huyet hoc d
benh nhan ma lam sang yeu cau.
C o ng tac truyen mau dtfdc dam bao chat ltfdng cd nghia la m au va thanh
pham truyen cho benh nh an dap tfng tot nhat yeu cau dieu tri va han che den
mtfc thap nhat cac ket qua khong mong muon.
1. Mot so khai niem ve dam bao chat li/dng

1.1. Chinh sach ve cha t luo ng
Bat ky mot hoat dong gi muon cd cha’t ltfOng phai cd chinh sach phu hop, dd
la chinh sach q uan tam den chat ltfOng. C h in h sach cd tac d u n g chi dao va dinh
hudng tfu tien den chat ltfdng. chat ltfdng cua san ph am cdn cd gia tri bao ve
thtfdng hieu cua cd sd san xuat.
1.2. Quan ly cha t lu o n g hay he tho ng cha t lu o n g
Bao gom tat ca cac hoat dong nham cd san pham dat chat ltfdng tot. Nhtfng
hoat dong nay dude soan thao bang van ban gom ca nhtfng quy dinh ve quy trinh
va qua trinh hoat dong. Viec xem xet chat ltfdng san pham hay chat ltfdng ket qua
xet nghiem dtfoc coi la quan trong dtfa tren cac hoat dong ttf dau ttf, dao tao... den
1)6 tri nhan luc.
1.3. Dam bao cha t luong
Nhtfng hoat dong kiem tra. xem xet va danh gia cac quy trinh, qua trinh
nham ket qua cd chat lifting, ttfc la san pham dap tfng tot muc dich san xuat no.
bao gom viec de ra cac bien phap. cac khau de chat ltfdng cua san pham dtfdc coi
la tot nhat. hay phai co tieu chuan ro rang ve chat ltfdng cua mdi san pham.
321
1.4. Kiem tra chat luong
Gom cac kiem tra. danh gia dinh ky hay dot xuat viec thac hien quy trinh.
kiem tra chat ltfdng cua phudng tien san xuat. cua dung cu hay nguyen lieu cho
den san pham. Bao gom kiem tra chat ltfdng noi bo va danh gia chat ltfdng ttf ngoai.
1.4.1. Kiem tra chat luong noi bd
Kiem tra dinh ky co ke hoach. co bien phap va kiem tra viec thtfc hien kiem
tra. Kiem tra trinh do nhan vien. kiem tra thuc hien quv trinh. Kiem tra chat
luong san pham (kiem tra ket qua xet nghiem. chat ltfdng mau hoac phe pham)
theo tieu chuan da quy dinh.
1.4.2. Danh gia chat luong tu ngoai
Sii dung mot trung tam doc lap gui mau xet nghiem den nhieu phong xet
nghiem. sau do thu thap ket qua va xii ly bang thuat toan. de danh gia chat ltfdng
cua cac phong xet nghiem khac nhau. Hoac mot trung tam ben ngoai den cac cO sd
san xuat lay san pham ngau nhien va phan tich chat ltfOng.
1.5. Ghi chep va thong ke
Viec ghi chep ket qua co tinh khoa hoc. thong ke ket qua xet nghiem hang
ngay. hang tuan. hang thang cung giup phat hien nhtfng sai sot cd the xay ra.
1.6. Viec thanh tra, kiem tra
Cung la mot hinh thtfc de dam bao chat ludng xet nghiem hoac chat ltfOng
san pham mau qua danh gia thuc trang to chtfc. danh gia viec thtfc hien cac quy
trinh. quy dinh. kiem tra san pham tai cho.
1.7. T u d a n h gia lai
La viec tu xem xet lai qua trinh thtfc hien hoac xem xet lai ttfng khau da
dtfoc tien hanh co trao ddi de phat hien nhtfng khau cd the sai sot.
Noi tom lai. chat ltfOng cua thanh phan mau. hay thuc te hon la ket qua cua
hoat dong truyen mau (bao gom ttf tuyen chon, sang loc. thu gom mau. chiet tach.
bao quan va truyen mau lam sang) va ket qua cua xet nghiem cung nhu mot san
pham cua mot qua trinh san xuat phu thuoc vao nhieu yeu to: tu nang ltfc to chtfc
va chinh sach cua toan he thong den chat ludng cua nguyen lieu, loai quy trinh ky
thuat duoc ap dung, chat luong cua trang bi. nang luc cua nhan cong... Do dd hoat
dong de dam bao chat luong bao gom ttf nhtfng hoat dong co tinh vi mo den viec
thuc hien cac bien phap cu the cho quy trinh cu the.
2. Dam bao chat lifdng xet nghiem huyet hoc

Muc tieu cua xet nghiem la dap tfng dude yeu cau lam sang. Do vay cong tac
dam bao chat ltfdng xet nghiem huyet hoc la thuc hien nhtfng viec can thiet de
dam bao ket qua kip thdi. cd do tin cay cao. Trong cdng tac xet nghiem huyet hoc
cd bon hoat dong dam bao chat luong.
322
2.1. Kiem tra chat luong n o i bo (n oi kiem tra)
La cac hoat dong cua labo nham dam bao xet nghiem co do tin cay.
- Kiem tra do xac thuc: Vi du: sii dung mau chuan (vdi may dem te bao).
- Kiem tra do lap lai:

Vi du: + 1 mau duoc kiem tra 2-3 lan (kiem tra ti'nh on dinh)
+ Ngay sau xet nghiem lai cac mau cua ngay trtfdc
- Kiem tra phuong tien va sinh pham: vi du may do pH. may li tam, ki'nh
hien vi. may dem te bao tu ddng va sinh pham cua may...
- Kiem tra bang ky thuat khac.
Do tien hanh ndi kiem tra. ngtfdi chiu trach nhiem chat ltfOng xet nghiem
cua labo phai nghien ctfu ky quy trinh ky thuat, dat ra cac che do kiem tra thtfdng
quy co tinh djnh kv. ddng thdi de ra cac bien ph ap giai quyet cac tinh h u o n g khac
nhau. N e u stf d u n g cac phtfOng tien xet nghiem cd q u a n ly b a n g ph an mem, can
dat ra trong chtfOng trinh cac bao dong can thiet de nhac viec noi kiem tra.
Vdi cac labo thong thtfdng nhieu khi de bo q u a cong tac noi kiem tra nen can
dat ra mot nguyen tac. vi du ngtfdi chju trach nhiem chat ltfOng xet nghiem te bao
mau trtfdc khi ky tra ket qua xet nghiem phai ky vao to ket q u a xet nghiem lai
cac man ngay trtfdc. ddng thdi khi xay dtfng quy trinh cho mot n g ay lam viec can
co muc tieu kiem tra chat ltfdng noi bo.
2.2. Danh gia cha t lu o n g tu ngoai
Cd cd q u a n chat ltfOng du’Oc thtfc hien d u n g quy ch u an doc lap to chtfc kiem
tra dinh ky hoac dot xuat b a n g cach giii m au xet n ghiem den cac labo tron g he
thong va thu thap. xtf ly ket qua.
- Tac d u n g cua danh gia chat ltfOng ttf ngoai la thong nh at dtfOc he thong
hay noi cach khac him cho cac ph on g xet nghiem khac n h a u ctfng dtfa ra mot ket
qua ttfOng ttf ddi vdi mot mau nghiem.
- Vdi he thong chat ltfOng xet n gh iem chat che, ngtfdi ta yeu cau cac phong
xet nghiem phai tham gia v;\o he thong d a n h gia chat ltfOng.
Vi du:
323
Labo tham chieu dinh ky giii mau den cac labo 1. 2. 3. 4 . 5 va sau do thu
thap ket qua. xU ly. Labo nao co ket qua tach rdi xa cac labo khac thi can xem lai.
Cac labo 1. 2. 3. 4. 5 chi dtfdc phep hoat dong neu cd giay chung nhan chat
luong dinh ky cua labo tham chieu.
2.3. Giam sat quy trinh

Mot phong xet nghiem hoat dong phai co cac quy trinh:
- Quy trinh to chtfc. sap xep

- Quy trinh dao tao nhan ltfc
- Quv trinh thuc hien cong viec: ttf sang den chieu. ai l.am gi. ai chiu
trach nhiem
- Quy trinh tien hanh ky thuat
- Quy trinh luu. ghi chep. tra ket qua.
* Viec kiem tra thudng xuyen xem cd theo dung quy trinh nay la dieu can
thiet. can co nhan vien kiem tra thtfc hien quy trinh.
* Thudng xuyen phai xem xet lai va ban bac khau nao chua thtfc stf tot can
stfa chtia.
Quv trinh to chtfc labo tao mot day truyen (hay dudng di) cua mot xet
nghiem sao cho hop ly. tiet kiem nhan luc. tiet kiem thdi gian va tranh moi nham
lan. sai sot. quy trinh sap xep nhan luc de dam bao ket qua xet nghiem dtfOc thtfc
hien khach quan va duOc ngudi co trach nhiem kiem tra. danh gia.
Khi xay dung quy trinh to chuc labo can luu y tat ca cac khau ttf dja diem,
hinh thiic. dieu kien nhan benh pham den tra ket qua.
Quy trinh dao tao nhan luc: Dao tao nhan luc phai du'Oc luon chu y. vtfa de
cung cd kien thuc chuyen sau. vtfa cap nhat cac hieu biet Ung dung mdi. Thudng d
mot phong xet nghiem co nhieu loai cong viec tu don gian den phtfc tap. Can bo
khi ve nhan cong tac duoc bo tri cong viec va se duoc dao tao dan trong ca qua
trinh de thuc hien nhung cong viec ngay cang cao.
Quy trinh thuc hien cong viec va quy trinh tien hanh ky thuat cd mdi quan
he rat chat che. Tuy nhien. de thuc hien dung ky thuat mot khoi ltfOng xet
nghiem ldn lai phai dap ung ve mat thdi gian nen yeu cau bo tri cong viec khoa
hoc de moi lao dong deu duoc tan dung va co hieu qua.
Khi xay dung quv trinh ky thuat xet nghiem can luu y den cac dieu kien lien
quan nhu' cach lay benh pham. thdi gian giu benh pham tdi da. dieu kien van
chuyen. luu giu benh pham...
Sau khi cd ket qua xet nghiem. viec ghi chep so sach ltfu theo mau thong
nhat. khoa hoc de sao cho co the kiem tra danh gia dtfoc. Ngtfdi ta can ctf ket qua
ttfng thong sd sau do lap do thi. Nhieu khi dua vao dd thi cd the giup nhan ra quy
luat benh tat hay phat hien sai sot trong xet nghiem. Vi du may dem te bao trong
mdt thdi gian dai cho thay tat ca benh nhan deu co MCV (the tich trung binh
hong cau) tren 100 fl. luc do neu ve do thi cho thay do thi di len va duy tri mai d
do cao dd hoac cang ngay cang cao. K hi dd can xem ngay lai chat ltfOng cua xet
nghiem. cha't ltfOng cua may dem.
2.4. Tieu chuan va chuan hoa

La dat ra cac tieu chuan cua can bo. cua trang bi, cua ky thuat de tuan thu.
Tieu chuan va chuan hoa khong phai giong nhau cho tat ca cac labo ma tuy
cap. mtfc do phuc vu.
Tuy dieu kien trang bi ma dat ra tieu chuan cua labo can dat. Chuan hoa
dat ra yeu cau rat cao. nhieu thong sd dtfOc kiem tra chat che. Tuy nhien da la
labo huvet hoc du d mtfc do nao cung phai cd cac tieu chuan chat che de dam bao
ket qua xet nghiem dat do tin cay can thiet.
C h u a n hoa la d u n g cac phtfOng phap, quy trinh, vat lieu da biet, da dtfOc
danh gia la tot de ap d u n g trong cac labo.
Nh ie u thong so dat ra cho viec ch uan hoa cua labo xet nghiem :
- C h u a n tham chieu: mot chat, mot thiet bi, mot quy trinh dtfOc goi la
chuan khi no dap tfng dtfOc nhtfng yeu cau chat che ve chat ltfOng, ve do tin cay
khi tinh den moi yeu to.
- Vat lieu tham gia: vat lieu da dtfOc nhieu trung tam nghien ctfu danh gia
phu hop cho mot xet nghiem dac thu dung de lam chuain.
- P h u o n g ph ap tham chieu: ky thuat dUOc md ta chi'nh xac, ro r a n g cho mot
xet nghiem cu the dtfoc hoi ddng chuyen mon xem xet va xac nhan, p h u o n g ph ap
nay d u ng de danh gia ca phtfOng ph ap labo khac. PhtfOng p h ap ch u an quoc te la
phtfOng phap dtfoc thiet lap nhd hoi ddng khoa hoc quoc te.
- L u a chon phtfOng phap xet nghiem: dtfa vao p h u o n g p h ap chua!n tham

chieu, can ctf vao dieu kien kinh te. t r a n g bi, lao dong de ltfa chon phtfOng p h ap stf
dung h a n g n g ay thich hop vtfa dam bao mtfc do chi'nh xac can thiet vtfa ti'nh den
yeu to tiet kiem va kha thi. Vf du: p h u o n g p h ap ch u an de xet nghiem tham dd
benh hem ophilia la dinh luong yeu to' V I I I h a y yeu to' IX b a n g cac ky th u at nhay,
trtfc tiep. Mot labo ddng m au tuyen tinh cd the can ctf vao dieu kien thtfc te xet
thay xet nghiem A P T T la hop ly. vtfa kinh te, cd the thtfc hien dtfoc, lai khdng bd
sot benh nhan du khdng phai xet nghiem k h a n g dinh. Mot labo tuyen huyen cd
the phai chap nhan sii d u n g phtfOng p h a p xet nghiem thdi gia n H ow ell.
- N h ie u thong sd du'Oc ch u an hoa khac n h u kit chan doan, dieu kien

chuan... Tat ca phai dtfoc tm h den de lam can ctf cho cac thong so cu the cua labo
chon lua su dung.
3. Mot so bien phap dam bao chat li/dng trong truyen mau
3.1. Chu trinh truyen mau
- Ngtfdi cho mau: ngtfdi cho khoe manh, ttf nguyen
325
- T u y e n chon: k ham . ttf v a n ngtfdi cho
- Lay mau: ky thuat lay, dieu kien va dung cu lay mau

- Sang loc benh nhiem trung: cac trang bi, kit va ky thuat dtfOc ap dung
- Dieu che cac thanh phan mau
- LUu trtf va phan phoi
- Phat mau cho benh nhan va thtfc hanh truyen mau lam sang
3.2. Yeu cau cua an toan truyen mau
- Khdng lam lay benh

- Khdng gay phan tfng (mien dich va khdng mien dich)
- Khdng lam anh htfdng stfc khoe cua ngtfdi nhan mau. ngtfdi cho mau va
nhan vien truyen mau
- Cd hieu qua chtfa benh.
3.3. Mot so bien phap dam bao chat luong

3.3.1. Xay dung va dam bao quy trinh
Quy trinh ttf tuyen chon ngudi cho den kham va lay mau va phai dtfoc xay
dtfng va loai trtf cac yeu to nham cd ngtfdi cho hoan toan manh khoe, lay mau
an toan.
- Doi tuong cho mau: quan ly dtfoc ngtfdi cho mau, chu y lay d nhom ft nguy
cO lay nhiem benh truyen qua dudng mau nham an toan cho ngudi nhan. ngtfdi
cho, nhan vien.
- Quy trinh va thu tuc ghi chep: nham kiem tra dtfOc stfc khoe ngtfdi cho,
quan ly dtfOc ngtfdi cho, khdng nham lan mau ngudi nay vdi ngtfdi khac nhtfng
van dam bao thuan tien. tranh phien ha.
- Quy trinh nhan mau vao kho. thu tuc giao - nhan.
- Quy trinh sang loc: ttf to chtfc thtfc hien ky thuat den quy trinh dao tao
can bo, quy trinh chon kit, quy trinh xtf ly chat thai bd va tra. thong bao ket qua.
- Quy trinh san xuat de khdng bi lay nhiem ttf ngoai. khdng lan mau ngudi
nay vdi mau khac. thanh phan nay vdi thanh phan khac. Thanh phan mau phai
dude san xuat kip thdi. san pham cd cac thong tin can thiet.
- Quy trinh ltfu trtf. phat mau. quy trinh truyen mau lam sang: lam viec gi
trtfdc, viec gi sau, khi linh can chu y gi. khi phat can yeu cau gi, khi truyen mau
phai theo quy trinh da dtfoc xem xet va thong nhat.
3.3.2. Ap dung mot so bien phap tu kiem tra
- Ttf kiem tra phtfOng tien, dung cu: can. dung cu xet nghiem. dat ke hoach
kiem tra dinh ky cd sd theo doi.
326
- Tu kiem tra kft xet nghiem va may.
- Ta kiem tra dinh nhom bang ap dung dinh nhom hai phtfOng phap, hai
nhan vien doc lap. Kiem tra hang ngay cac sinh pham sU dung ve tmh dac hieu.
do nhay. co so. giay theo doi. co ngtfdi chiu trach nhiem ky ten.
- Kiem tra mau, san pham mau.
Khi cong tac truyen m au co sii d u n g he thong p h a n mem q u a n ly, can dat
cac che do tu kiem tra trong chUOng trinh de lam sao khi co bat ky mot sai sot nao
se co he thong bao dong. Vi du, ma so cua mot don vi m au tii mot ngudi cho lai co
hai nhom khac nhau 6 hai che ph am (vi du khoi hong c l u co nhom A, huyet tUOng
co nhom B) thi se co bao dong xuat hien.
3.3.3. Tham gia he thong chat li/png
- He thong kiem tra quy trinh

- He thong kiem tra san pham mau
- He thong kiem tra chat ltfdng sang loc.
De co mau va che pham mau dat chat ltfdng tot cln co mot trung tam hoat
dong doc lap. co nhiem vu to chtfc danh gia chat ltfdng cua cac trung tam truyen
mau trong toan he thong.
Can ctf vao quy trinh. phuong phap chuan da dtfOc xay dtfng, hang nam hay
hang quy theo dinh ky. trung tam nay den xem xet viec ap dung quy trinh,
phtfOng phap. Dong thdi hang quy hay hang thang trung tam nay giii cac mau xet
nghiem vi du dinh nhom kho, mau huyet thanh can sang loc da biet mat do quang
cu the (vdi ky thuat ELISA sang loc virus) tdi cac trung tam truyen mau.
Sau khi nhan dtfoc ket qua, trung tam chat ltfdng se thong bao lai va de
xuat hudng dieu chinh. Truong hop nhieu lan cd sai so hay sai so co he thong
khdng the dieu chinh thi trung tam chat ltfOng cd the yeu cau cac cd quan chtfc
nang dinh chi hoat dong cua trung tam truyen mau nay.
3.3.4. Ap dung cac tieu chuan chat lifdng: dat tieu chuan cu the (quy dinh cua Nha
nude) ddng thdi ttfng khu vtfc yeu cau them.
3.3.5. Ap dung ky thuat mdi trong sang loc, pho bien kien thtfc (dao tao) sii du n g mau.
Can cd ke hoach dao tao cu the, ttf don gian den phtfc tap, de cd doi ngu can
bo thanh thao vtfa cd tay nghe cao vtfa cd hieu biet can thiet de cd the xii ly d moi
tinh huong.
3.4. Tieu chuan GMP (Good Manifacturing Practice)
Hien nay trong linh vtfc truyen mau ngtfdi ta nhac nhieu den thtfc hien tieu
chuan GMP. Vay G M P la gi?
GMP (Good Manifacturing Practice) la moi hoat dong trong ca dich vu nham cd
san pham dat dtfoc muc dich san xuat ra no (cd the goi la qua trinh thtfc hanh tot).
327
Muc dich cua cong tac truyen mau la hieu qua chtfa benh 0 ngtfdi benh.
Muon ngudi benh dtfng mau. che pham mau dat hieu qua cao (nhan dung cai
minh can va an toan) thi phai co stf phoi hop cua tat ca cac hoat dong trong ca day
chuyen. cac cong tac ttf quan ly chat ltfOng. kiem tra chat ltfOng va tinh thanh
thao cua can bo. den viec to chtfc thtfc hien. to chtfc dao tao tuven ltfa nhan vien
cung nhtf cac can thiep co tinh kiem tra chat che. cua dtfdng loi chinh sach dau ttf
phoi hop.
Khong the kiem tra chat ltfOng het cac thong sd cho tat ca cac san pham do
vay danh gia mot co sd san xuat. mot ngan hang mau tot la dtfa vao viec co sd do
co dat GM P hay khdng? Cac trung tam truyen mau ldn tren the gidi hien nay
dang co gang dat GMP.
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Chifdng 10
CAC GIA TRI• SINH

• HOC VE HUYET
• HOC
VA M liN DjCH HUYET HOC
(G ia i doan 1995 - 2005)
1. Khang nguyen he hong cau

1.1. Khang nguyen nhom mau he ABO
Dan toe n A (%) B (%) o (%) AB (%)
Kinh 4025 21,14 28,34 45,08 5,44

Ede 540 29,2 31,9 23,6 15,3
Dao 1122 30,60 21,21 41,71 6,42
San Diu 619 26,60 28,10 37,31 7,90
Mong 683 33,70 13,6 48 4,7
Phi/ong phap Ngi/ng ket, khang the don dong cua Sanofi (Phap)
1.2. Khang nguyen he Rh (Khang nguyen D)

Khang nguyen (%)
Dan toe n
Khang nguyen D' Khang nguyen D'
Kinh 1483 99,93 0,07
San Diu 300 100 0
Mong 300 100 0
Tong 2083 99,977 0,023
1.3. Cac khang nguyen (gen) he Rh
Khang nguyen (%)

Dan tdc n
D d c c E e
Kinh 320 100 0 95,31 47,5 33,12 94,68

329
1.4. Ty le cap gen he Rh
f
Cap khang nguyen (%)
Dan toe n
cc cc CC EE ee Ee
Kinh 320 52,18 4,7 41,25 3.4 66,56 28,12
PhiTOng phap Ngtfng ket, khang the don dong cua Sanofi (Phap)
1.5. Khang nguyen he Kell, he Duffy, he Kidd, he Ss

Kell Ss
He Duffy (n=192) Kidd (n=204)
(n=181) (n=95)
K ha ng Jk Jk Jk Jk
Fy Fy Fy Fy
nguyen KK Kk kk SS Ss ss
(a+b-) (a+b+) (a-b+) (a-b-) (a+b-) (a+b+) (a-b+) (a-b-)
(%)
Kinh 86,46 1 1 .7 4 0 1.8 18 ,77 2.63 4 1.6 1 36,99 0 0 100 u 84 80,4
Phi/ong
N g im g ket. khang the den dong cua Sanofi (Pha p)
phap
2. Cac gia trj sinh hoc ve te bao mau ngoai vi

2.1. Cac thong so hong cau
Hematocrit- Hong cau
Hong cau Huyet MCV MCH MCHC
Nhom mang
Gidi n s ic to - Hct
tudi (T/l) (A) (pg) (g/i)
Hb (g/l) (%)
(I/I)
Nam 234 4 .8 8 *0 ,3 8 126+10 0.38 ±0 .0 2 79±5 25±2 332±18
2-6
Nur 218 4 ,8 5 ± 0 .4 4 127±10 0 .3 8 ± 0 .0 3 80± 5 26±2 333-36
Nam 399 4 ,78 + 0 ,4 4 128+10 0 ,38 8 ± 0 ,0 2 9 81 ±6 26±2 332±5

7 - 17
Nur 394 0 ,8 0 + 0 ,4 9 128±9 0 ,3 8 8 ± 0 ,0 2 9 81 ±6 2 6 ±3 329 *5
Nam 4 673 5 ,0 5 +0 ,3 8 1 51 ±6 0 ,4 4 + 0 ,0 3 8 8±4 30±2 339=17 1 ,2 ± 0 . 4

18-59
NO 3035 4,6 6 ± 0 ,3 6 , 1 35 ± 5 0 ,41 ± 0 ,0 3 8 7 ±4 29 +2 336r15 1 .7 ± 7
Nam 38 4 ,4 3 ± 0 ,3 6 1 4 1 ± 13 0 ,41 ± 0,0 3 91 ±5 31 ±1 3 4 7 :1 0 1 .0 ± 0 .5

60-80
N lt 36 4 , 3 8 ± 0 ,2 6 132±8 0 , 3 7 ± 0 ,0 2 8 8±4 30±1 3 43 ±6 1.3± 0 .4
NhuOm
P hu o n g phap M a y dem te bao tu don g Hycel ( P h a p ) , S y s m e x (Nha t)
xanh cresyl
MCV: Mean Cell Volume (fl: Feltolit)

MCH: Mean Cell Hemoglobin (pg: Picogam)
MCHC: Mean Cell Hemoglobin Concentration (g/1: gam/lit)
T/l: Tera/lit (x lO IJ te bao/h't)
G/1: Giga/b't (xlO* te bao/b't)
Het: H ematocrit
Hb: Hemoglobin (H ST)
330
2.2. Cac thong so bach cau
Bach c3u Bach cau

Nhom Bach cau Bach cau Bach c lu
7 Gidi n trung tinh
tuoi (G/l) acid (%) mono (%) lym pho
(%)
Nam 234 10,4±3,0 4 5 .0 ± 1 1,0 44,6+9,5
2-6
NCf 218 10,1±4,5 42,7+10,0 46,6±9,0
1 7 -1 7 Nam 399 9,7±2,4 45.9±8,9 43,8±7,9
NCr 394 9,2±2,1 47,1+9,4 45,9±8,0

Nam 4945 8,0±2,0 57.4±8.4 3,2±2,6 3,8±0,5 35±7,2
18-59
NCf 3326 8,1±2,0 57,4±8,4 2,8+2,1 3,8±0,5 35.6±6,4
Nam 38 6,1±0.8 64.7±7.0 3,9±3,0 1,7±1,7 30,5+7,0

60-80
Nil 36 6 ,1±1,2 6 1 ,7±7,0 2,8±2,0 1,09 ±1,09 32,3+8,0
May dem te bao ti/
Phi/ong phap dong Hycel (Phap), Kinh hien vi quang hoc
Sysmex (Nhat)
2.3. Cac thong so tieu cau

Do ngi/ng tap tieu c lu
Nhom So lifdng tieu cau
Gidi n C ollagen 1mg/ml
tuoi ' (G/1) ADP 10^mol
(MA %) (M A %)
Nam 234 344 ± 91

2-6
NCf 218 360 ± 98
Nam 399 339 ± 101

7 - 17
NCf 394 338 ± 103
Nam 4945 263 ± 61 66,69 ± 6.25 65,97 ± 7,1

18-59
NCf 3326 274 ± 63 67,26 ± 6,8 65,06 ± 6,3
Nam 38 233 ± 48
60-80 67,0 + 6,5 65,5 ± 6,7
NCr 36 267 + 63
May dem te bao ttr dong Hycel

Phi/ong phap Kinh hien vi quang hoc
(Phap), Sysmex (Nhat)
G/1 : Giga/lit (10u/h't)

ADP : Adenosine diphotphate
MA : Maximum Agregation
3. C ac gia trj s in h ho c v e te b a o tu y ngtfdi trtfd n g th a n h
3.1. So lupng te bao cd nhan va te bao goc sinh mau CD34+
Thong so n X+SD Phi/dng phap

(G/l)
Te b a o co n h a n tu y 79 5 7 ,3 6 ± 1 5 ,5 0 M ie n d ic h h u y n h q u a n g tr i/c tie p , kit
s im u lte s t c u a B e c to n - D ic k in s o n (M y )
T e b a o g o c C D 34< tu y 79 1 ,2 1 9 ± 0 , 7 9 8
331
3.2. Cac thong so ddng hong cau (n = 79)
- ' "
SD
Cac thong so
% So luong (G/l)
H C co n h a n 1 9 ,1 4 ± 3 ,3
N g u y e n tie n HC 0 ,0 9 ± 0,11
H C iia b a s e 2 ,4 ± 1,5
H C da s a c 6 ,4 ± 2 ,3
H C i/a a cid 1 0 ,7 ± 3 ,7
H C li/d i 1 ,2 5 ± 0,31 4 8 ,2 0 ± 1 3 ,6 7
H C tru o n g th a n h 4 ,8 0 ± 0 ,5 0 (T /l)
C h i so tru o n g th a n h d o n g HC 0 ,3 7 ± 0 , 1 5
•- M a y d e m te b a o to d o n g H y c e l (P h a p ), S y s m e x (N h a t).
P huong phap
- K in h h ie n vi q u a n g h o c .
3.3. Cac thong so bach cau (n=79)
A" + SD
Cac thong so
% So ludng (G/l)
B C h at 5 9 ,2 ± 7 ,5 3 0 ,9 9 ± 1 0 ,1 6
Lym pho 17,4 ± 7 ,6 9 ,9 7 ± 3,51
M ono 0 ,7 6 ± 0,71 0 ,4 4 x 0 ,4 4
- M a y d e m te b a o to d o n g H y c e l (P h a p ), S y s m e x (N h a t).
P h i/d n g p h a p
3.4. Cac thong so bach cau hat (n=79)
X + SD
Cac thong so
% So luong (G/l)
N g u y e n tu y b a o 0 .6 9 ± 0 ,6 9 0 ,7 9 ± 0 ,7 9
T ie n tu y b ao 0 ,8 2 ± 0 .8 2 0 ,7 6 ± 0 .7 5
T u y b a o tru n g tin h 5 ,6 ± 2 ,7 4 ,0 7 ± 3 ,4 9
H au tu y b a o tru n g tin h 8,1 ± 3 .6 5 ,2 9 ± 3 ,7 0
D u a tru n g tin h 8 .5 ± 3 ,7 5 ,5 3 ± 3 ,3 0
D o a n iia a cid 2 .2 ± 1.5 1,6 6 i 1 .4 7
D o a n tru n g tin h 3 3 ,2 - 8 .5 2 2 .0 3 t 14 85
C h i so truO ng th a n h 0 ,1 7 ± 0 ,1 3
- M a y d e m te b a o t i/ d o n g H y c e l (P h a p ), S y s m e x (N h a t).
P huong phap
3.5. Cac dudi nhom lym pho (n=79)
So lifdng
Cac thong so % Phi/dng phap
(Gfi) '
L y m p h o B (C D 1 9 ) 2 3 ,8 ± 8 ,9 7 2 ,13 1 ± Q ,697 M ien d ich h u yn h q u a n g tri/c tiep. kit

Te b a o N K (C D 1 6 1 1 ,8 6 ± 4 ,0 3 1 ,3 6 6 ± 0 ,5 2 8 s im u lte s t cu a B e c to n - D ic k in s o n
(M y), kinh hien vi h u yn h q uang.
+ C D 56)
L y m p h o T (T C D 3) 5 8 ,7 ± 1 1 , 3 3 6 ,6 1 9 ± 3 .5 9 0
t c d 4 2 3 ,8 2 ± 9 , 3 2 ,451 ± 1,261 M ien d ich h u yn h q u a n g trtfc tiep, kit

sim u lte s t cu a B e c to n -D ia c k in s o n
TCDe 3 1 ,1 8 ± 9 ,1 4 3 ,6 0 6 ± 1 .9 7 2 (M y), m a y F A C S count.
C D 4/C D 8 0 ,71 ± 0 ,2 2
3.6. Cac thong so ddng mau tieu cau (n=79)

Cac thong so % MTC Phi/dng phap
N g u ye n M T C 0 ,7 2 ± 0 ,6 9
M T C i/a b ase 9 ,2 6 ± 4 ,5 1
"," .
M TC co h at c h ira s in h tie u ca u 4 5 ,9 1 ± 7 ,7 3 K in h h ie n vi q u a n g h o c
M T C co h a t d a n g s in h tie u ca u 3 0 ,3 7 ± 9 ,4 5
M TC n h a n trd 1 3 ,6 2 ± 6 ,5 5
4. Te bao go c s inh m a u co dau an C D 34+ (H e m o p o ie tic S te m C ell)
Mau xet nghiem n Gia trj CD34+ (G/l) Phtfdng phap

M au n g o a i vi 63 0 ,2 2 ± 0 ,0 7
M ie n d ic h h u y n h q u a n g trtfc tie p , kit
M au d a y ron tre sd s in h 50 0 ,2 4 ± 0 ,1 3 s im u lte s t c u a B e c to n - D ic k in s o n ,
k in h h ie n vi h u y n h q u a n g
T u y x tfd n g 79 1 ,2 1 9 ± 0 ,7 9 8
5. Toe do m a u lang, thdi gian m a u c h a y , th d i gian m a u d o n g
5.1. Thong so tdc do mau lang nguoi truong thanh (n=520)
Pachenkow (mm/gid) Westergreen (mm/gid)

Gidi
Gid thtf nhat Gid thtf hai Gid thtf nhat Gid thtf hai
N am 4 ,7 ± 3 ,2 2 1 6 ,7 3 ± 5,31 3 ,0 5 ± 2 ,2 6 15,41 ± 9 ,3 3
NCr 7 ,3 5 ± 3 , 9 4 1 9 ,8 6 ± 1 5 ,0 0 6 ,1 2 ± 5 ,2 5 19,51 ± 1 4 ,6 7
5.2. Thong sd thdi gian mau chay, mau ddng (n=130)

I
Thong sd Gia trj Phtfdng phap
T h d i g ia n m a u c h a y 3 p h u t 30 g ia y D uke
. . . .
T h d i g ia n m a u d o n g 6 p h u t - 9 p h u t 3 0 g ia y L e e - W h ite
333
6. C ac th o n g so p ro te in h u y e t ttfdng
6.1. Protein, albumin, globulin va ty le A/G trong huyet thanh
Thong Don vi
Li/a tuoi Gidi n X * SD K y th u a t
so do
Ngudi trUdng Nam 225 73,10 ± 6.06 Phi/ong phap Biure sCl dung
Protein g/i thanh kit cua Boehrmger-
NCr 189 74,33 ± 4.82
Mannheim tren may 4010
■
Ngudi trUdng Nam 36 56,67 ± 5.28 Vert Bromocresol pH 4.2 tren
Albumin g/i thanh may Hitachi 704
NCr 38 53,72 ± 4,26
\ ...
2-5 Chung 92 44.21 ± 2.58
Albumin g/i 6-10 Chung 169 45.85 ± 3.49 Vert Bromocresol
11-15 Chung 112 47.27 ± 3.41
----------------
2-5 Chung 92 30.87 ± 3,38
Globulin g/i 6-10 Chung 169 31.64 ± 4.03
11-15 Chung 112 33.45 ± 3.55
---------------
2-5 Chung 92 1.45 ± 0,21
Ty le A/G 6-10 Chung 169 1,48 ± 0,30 Tinh ty le
11-15 Chung 112 1,43 ± 0,19
6.2. Dien di protein huyet thanh

...
T h an h phan Odn v) ,
Lira tu o i G idi n X + SD K y th u a t
p ro te in do
Albumin % % Ngudi truong thanh Chung 52 56,3 ± 3.52
Globulin u1% % Ngudi trUdng thanh Chung 52 2.5 ± 0,52
Globulin u2% % Ngudi trUdng thanh Chung 52 8,83 ± 1.34 Dien di tren
acetat
Globulin p% % Ngudi trUdng thanh Chung 52 11.6 + 1.22 cellulose
Globulin 7% % NgUdi trUdng thanh Chung 52 20.6 ± 2.82
A/G % NgUdi trUdng thanh Chung 52 1,31 ± 0,99
.
7. C ac th o n g so m ie n djch
7.1. Cytokin
T h o n g s d do D d n vi do n Ky th u a t
' .V + SD
ELISA (kit CLB club) (chuan
TNFu pg/ml 34 19.5 r 11.3
WHO: 11,7 pg/ml)
IL1|i pg/ml 34 27.4 ± 15.2 ELISA (kit CLB club)
IL6 pg/ml 34 36.0 ± 22,2 ELISA (kit CLB club)
TNFu ng/ml 30 0.13 ± 0.14 RIA, kit IMK-480 (CIAE)
MIF (yeu to Uc che George B va Clausen M. (1974)

Chi so di
30 63.2% i 6.9% thu1 vdi Con-A cua hang
di tan bach cau) tan (Ml)
Welcome - UK)
ELISA; kit Henning Berlin
NPT (neopterin) nmol/l 30 6,70 ± 2.50 GmBH
-------------------------
o.)4
7.2. Nong do globulin mien djch trong huyet thanh
7.2.1. IgG (m g % )
Nhom tuoi n X ±SD Cl 95% Ky thuat
Ngifdi trifdng Khuech tan trong thach: Mancini

223 1368 ± 93 1355,8 - 1380,2
thanh
Ngifdi trifdng 56 1780 ± 202 1745,9 - 1814,1 Khuech tan trong thach: Mancini
thanh 60 1707 ± 247 1668,9 - 1745,1 Do do due mien djch
5 - 1 0 tuoi 30 1549 ± 392 1432,4 - 1665,6 Khuech tan trong thach: Mancini
7.2.2. IgA (m g % )
Nhom tuoi n .V ± SD Cl 95% Ky thuat
Ngifdi trifdng
223 326 ± 11 324,6 - 327,4 Khuech tan trong thach: Mancini
thanh
Ngi/di trifdng 56 363 ± 30 357,9 - 368,1 Khuech tan trong thach: Mancini
thanh 60 263 ± 65 253,0 - 273,0 Do do due mien djch
5 - 1 0 tuoi 30 323 ± 49 308,4 - 337,6 Khuech tan trong thach: Mancini
7.2.3. IgM (m g % )
■
Nhom tuoi n A' ± SD Cl 95% Ky thuat

i
Ngifdi trifdng
223 93 ± 17 90,8 - 95,2 Khuech tan trdng thach: Mancini
thanh
Ngi/di trifdng 56 238 ± 48 229,9 - 246,1 Khuech tan trong thach: Mancini
thanh 60 277 ± 88 263,4 - 290,6 Do do due mien dich
5 - 1 0 tudi 30 172 ± 43 159,2 - 184,8 Khuech tan trong thach: Mancini
7.2.4. IgE (ng/ml)

"
Nhom tuoi n Cl 95% Ky thuat
X ± SD
NgiTdi trifdng thanh 62 435 ± 171 409,8 - 460,2 ELISA
.
7.3. Nong do thanh phan bo the C3, C4 va yeu to B

| -
Thong so do n Ky thuat
X ± SD
Hoat tinh b6 the toan phan 43 114,0 ± 10,0 Teresoi Aguado M CH50/1ml)
Hoat tinh C} 43 105,0 ± 19,0 Mancini (mg/dl)
I Hoat tinh yeu to B 38 629,0 ± 113,0 Teresoi Aguado M CH50/1ml
335
7.4. Cac te bao cd tham quyen mien djch
7.4.1. T C D , (tebao/fil)
Nhom tuoi Gidi n A' ± SD Ky thuat
18-55 Nam 47 1571,0 ± 350,0

NCr 46 1685,0 ± 261,0
19 - 25 Chung 40 1644,77 ± 306,25 FACS count
60 - 85 Nam 25 1282,0 ± 415,0

, .. . _
NCr 26 1381,0 ± 285,0

19 - 25 Chung 120 2007,35“ ± 662,97 Mien dich huynh quang tri/c tiep
19 - 25 Nam 34 1176 ± 479 M iln djch huynh quanh gian tiep

..
7.4.2. T C D 4 (te bao/jtl)
Nhom tuoi Gidi Ky thuat

, X ± SD
n
18-55 Nam 47 746,0 ± 171,0
! NCr 46 839,0 ± 158,0
19 - 25 Chung 40 781.87 i 176.45 FACS count
60 - 85 Nam 25 643,0 ± 199,0

NCr 26 774,0 ± 176,0
_
19 - 25 Chung 120 1023,08 ± 384,79 Mien djch huynh quang tri/c tiep
i 19-25 Chung 20 1247,30 ±434,76 Mien dich huynh quanh gian tiep
7.4.3. T C D 8 (te bao/ul)
Nhom tuoi Gidi n A' ± SD Ky thuat
18 - 55 Nam 47 636.0 + 173,0

Na 46 698,0 ± 173,0
;
19 - 25 Chung 40 681,9 ± 175,5 FACS count
60 - 85 Nam 25 601,0 r 220,0
NCr 26 | 516.0 z. 163,0
: .
l
19 - 25 Chung 120 862.27 ; 306,77 Mien dich huynh quang true tiep
19-25 Chung 20 1172.7 ± 357.5 Mien dich huynh quanh gian tiep
7.4.4. TC D /TC D 8
Nhom tuoi Gidi n X ±SD Ky thuat
18 - 55 Nam 47 1,12 ± 0,26

Ntf 46 1,25 ± 0,25
19 - 25 Chung 40 1,2 ± 0,34 FACS count
60 - 85 Nam 25 1,36 ± 0,57

Ntf 26 1,61 ± 0,52
19 - 25 Chung 120 1,25 ± 0,45 Mien dich huynh quang trtfc tiep
19 - 25 Chung 20 1,08 ± 0,24 Mien djch huynh quanh gian tiep
7.4.5. B C D 19 (te b a o /fJ )
Gidi n X ± SD Ky thuat
Chung 120 419,02 ± 206,99 Mien djch huynh quang trtfc tiep
337
PHIEU XET NGHIEM MS: 32/BV-99
So: ...............
TE BAO MAU NGOAI VI
n: Nam
Thtfdng □ Cap ctfu n So:

Ho ten ngtfdi benh: .............................................. Tuoi: ............ Nam/Ntf
Dia chi: ................................................................. So B H Y T ....................
So gitfdng: ........................B u on g:............ Khoa: ..................................
Chan doan: ..............................................................................................
C h i so S o lu o n g K e t qua C h i so K e t q ua
□ So luong HC - n 4,2-5.4 T /l □ So luo ng BC (5 -1 0 G /l)

- nur 4.0-4.9 T /l
n Huyet sac to - n 130-160 g/1 □ Thanh phan bach cau (ck )

- nu 125-142 g/l - Te bao mau bat thucrng
n H em atocril - n 0.40-0.47 1/1 + N guyen tuy bao

- nur 0.37-0.42 1/1 + Tien tu v bao
□ MCV 85-95 0 + T u y bao
n M CH 28-32 pg + Hau tu y bao
□ MCHC 320-360 - Bach cau dua

Hong cau co nhan (G /l) - Doan trung tinh
n Hong cau luoi 0.5-1.5% - Doan ua acid
□ So luong lieu cau 150-500 G /l - Doan ua base
n Mau lang - lh - M on ocvt

- 2h - L ym p h o cyt
- C hi so IK (< 15)
Nh an xet:
■gic/......... ngay ........ thang .........nam 200 .................... g id ..........ngay .........thang .........nam 200...
BAC Si DIEU T R I TRUONG KHOA XET NGHIEM
Ho ten: ............................................ Ho ten:
338
PHIEU XET NGHIEM M S: 33/BV-99
HUYET - TUY 0 0 S° :
n: Nam
So:
Ho ten ngtfdi benh: ................................... Tuoi: .............................Nam/Ntf
Dja c h i:..................................................... So B H Y T .................................
So gitfdng: .......................... Buong: ....... Khoa: ...................................
Tom t&t qua trinh benh ly, chtfng thtfc the (gan, lach, h a c h )..................
Chan doan lam sang: .................................................................................

Yeu cau xet nghiem: ..................................................................................
Lam xet nghiem:...,gid.... ngay... thang... nam 200... Ngay ...thang...nam 200..
Ket (j ua test xylocain 2^/c SI DIEU TR I
Bcic si doc test xylocain 2c/c
Ho t e n : ..............................
KET QUA
Chi so Ket qua Chi so Ket qua
So lu o n g h o n g cau ( T / l) So lu o n g bach cau ( G /l)
L ifa n g h u y e t sac to ( g /l) C h i so tru o n g th a n h BC hat
H e m a to c rit (l/D T y le h o n g c a u / bach cdu hat
MCV (I'D So lu o n g te bao tu y x u o n g ( G /l)
MCH (Pg) H o a h o c te bao:
MCHC ( g /l) + P e ro x y d a s e
H o n g cau co nhan ( G /l) + Sudan
H o n g cau lu o i m au (%) + E sterase k h o n g ure che
H o n g cau lu d i tu y (70 + E sterase ire che ban g N a F
C h i so tru o n g th an h H C + P .A .S
So lu o n g tie u cau ( G /l)
Nhan xet:
K et l u a n : ...............................................................................................
De nghj: ................................................................................................
N g a y ...... th a n g ....... nam 200...

TRUONG KHOA XET NGHIEM
Ho ten:
339
HUYET - TUY DO LA N THI/
-----------------------------
T u y b in h K e t qua
Te bao th u a n g
(% ) Tuv M au
T c bao bat th u d n g
M y e lo b la s t (N g u y e n tu y bao) 0-2
P ro m y e lo c y te (T id n tu v bao) 0-3
N e u tro p h il (T ru n g tin h ) 5-10
M y e lo c y te
E o s in o p h il ( U a a e id ) 0.5-3
... . -
( T u y bao)
B a s o p h il ( U a base)
N e u tr o p h il (T ru n g tm h ) 5-12
M e ta m y e lo c y te
E o s in o p h il ( U a a e id ) 0-1
(H a u tu y b ao )
N e u tro p h il (T ru n g tm h ) 5-12
Band
E o s in o p h il ( U a a e id ) 0-0,3
(B C d u a )
N e u tr o p h il (T r u n g tin h ) 20-40
S egm en ___
E o s in o p h il ( U a a c id ) 1-4
(D o a n )
B a s o p h il ( U a base) 0-1
L y m p h o b la s t (N g u y e n bao ly m p h o ) 0-0.1
P ro ly m p h o c y te (T ie n ly m p h o ) 0-0.1
L y m p h o c y te (L y m p h o ) 8-2.5
P la s m o b la s t (N g u y e n tu o n g b a o )
P ro p la s m o c y te ( T ie n tu o n g b ao )
P la s m o c y te (T u o n g b ao ) 0-1
M o n o b la s t (N g u y e n bao m o n o )
P ro m o n o c y te ( T iiin m o n o )
M o n o c y te (M o n o ) 0-2
h—
P ro e ry th ro b la s t (N g u y e n tie n h o n g c a u ) 0-0,3
r '
E r y th ro b la s t b a s o p h il (N g u y e n h o n g cau ua base) 1-4
E r y th ro b la s t p o ly c ro m a to p h il (N g u y e n h o n g cau da sac) 3-10
E ry th ro b la s t a c id o p h il ( N s u v e n hcSng cau ua a c id ) 7-15
M e e a k a rv o b la s t (N g u y e n m au tie u c a u ) 0-1
•-
M e g a k a ry o c y te b a s o p h il ( M a u tie u cau ua base) 5-14
M e g a k a ry o c y te g ra n u la r ( M T C hat ch u a sin h T C ) 38-53
M e g a k a ry o c y te m a tu re ( M T C hat d a n g s in h T C ) 20-40
Duvet mau 29-12-2000
340
PHIEU XET N G H IE M D O N G M AU So:
□ XN thifdng: Ho va ten: ......................................Tuoi................. Nam/NC(

□ XN cap- BHYT: ...............................So benh an: .....................
Khoa: .................................... Phong: .............. Gii/Ong......
Chan doan: .......................................................................
Ten x e t n g h ie m Ket qua Chi so b in h thtfdng

n Dong mau cd ban: - P T (%) 70-140 %
- Fibrinogen (g/1) 2-4 g/1
- A P T T : + T.gian (gia y) Chtfng: ..................
- Benh/chtfng B/ch: 0,85 - 1,2
- So ltfdng tieu cau (10!>/l) 150.109 - 400.10!71
□ Co cuc mau dong Cd hoan tdan
n Dau hieu day that A m tinh
n PT: - Thdi gian (giay) Chtfng: ..............
-% 70 - 140%
- IN R
n APTT: - Thdi gian (giay) Chtfng: ..............
n T T (T/gian Thrombin): - Thdi gian (gia y) Chtfng: ..............
□ Nghiem phap rtfdu (L'E thanol Test) A m tinh
n Thdi gian tieu Euglobulin (N.F.Von - Kaulla) >60 phut
n FDPs
□ D-Dimer
n Dinh ltfdng yeu to V I I I 30 - 180%
n Dinh ltfdng yeu to IX 30 - 150%
□ Dinh ltfdng yeu to khac:
n N gu ng tap tieu cau vdi cac chat khac
60 • 70%
(Collagen. A D P )
n A T III (A ntith rom bin I I I )
n Protein S
□ Protein C
n K h a ng dong ltfu hanh dtfdng ndi sinh
n K h a ng dong ltfu hanh dtfdng ngdai sinh
n Cac XX khac: ...........................
Nhan xet: ..................................................................................................
■gid....... ngay... thang...nam 200... N ga y...... thang...... nam 200...
BAC Si DIEU T R I TRUONG PHONG DONG MAU
Ho t e n : ........................................................ Ho ten:
341
TAI LIE U THAM KHAO
•
1. AABB (2006): Technical M anual

15th Edition, by AABB, 2006
2. Vu Trieu An va CT. (1982): Ky thuat cti ban v i mien dich, NXB Y hoc, Tap 1
3. Tran Van Be va CT. (2003): Thtfc hanh Huyet hoc - Truyen mau
NXB Y hoc, TPHCM, 2003
4. Van Dinh Hoa va CT. (2003): Cac gia tri sinh hoc ve mien dich
Trong: Cac gia tri sinh hoc ngtfdi Viet Nam binh thudng, thap ky 90/XX,
NXB Y hoc, Ha Noi, 2003
5. Nguyen The Khanh, Pham Ttf Dtfdng (1976): Ky thuat xet nghiem
6. Do Trung Phan (1979): Mien dich trung gian te bao
7. Do Trung Phan va CT (2003): Cac gia tri sinh hoc ve huyet hoc
Trong: Cac gia tri sinh hoc ngtfdi Viet Nam binh thtfdng thap ky 90/XX
8. Do Trung Phan (2000): An toan truyen mau

9. Hoffman R. (1995): Special tests and procedures in Hematology
In: Hematology, Basic principles and practice
Ed. by Hoffman, Pub. by churchill livingston, New York, 1995, p: 2201-2274
10. Michell lewis (1999): Labolatory practice
In: Posgraduate Hematology, Ed, by Hoffbrand V.,

Pub. by planta tree, Italy, 1999
11. Ivan Roitt et.al. 1998): Im m unological techniques.
Ed. by Roitt, Pub. by Mosby, London, 1998

12. Williams Hematology (1995): Labolatory techniques: Principles and
Interpretation.
Ed. Beutler E.. pub. by McGran-Hill, 1995
343
NHA XUAT BAN Y HQC
Ky thuat
XET NGHIEM HUYET HOC VA TRUYEN MAU
0NG DUNG TRONG liM SANG
■
Chju trdch nhi$m xuat ban
HOANG TRONG QUANG
Bien tap: BS. NGUYEN LAN

SCra ban in: NGUYEN LAN
Trinh bay bia: CHU HUNG
Kt vi tinh: TRAN THANH TU
In 1000 cuon, kho 19 x 27cm tai Xuong in Nha xuat ban Y hoc.
Giay phep xuat ban so: 25 - 2009/CXB/268- 168/YH

In xong va nop luu chieu quy II nam 2009.

Kỹ Thuật Xét Nghiệm Huyết Học Và Truyền Máu Ứng Dụng Trong Lâm Sàng

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VIEN HUYET HOC - TRUYEN MAU TRUNG JONG

Chu bien: GS.TSKH. D6 Trung Phan

KY THUAT XET NGHIEM

LfhG DUNG TROMG LAN ^ANG

NHA XUAT BAN Y HOC

HUYET HOC VA TRUYEN MAU

NHA X U A T BAN Y HOC

Tham gia bien soan

TS. BUI THI MAI AN

TS. TRUONG CONG DUAN

ThS. PHAM TUAN DUONG

ThS. NGUYEN TH| Y LANG

TS. NGUYEN TH( NLT

GS. TSKH. DO TRUNG PHAN

PGS.TS. THAI GUY

BSCKII. TRAN THI HONG THUY

BSCKI1. DO MANH TUAN

BSCKI. NGUYEN CHI TUY^N

PGS.TS. CUNG TH( TY

TS. NGUYEN TRlEU VAN

PGS.TS. PHAM QUANG VINH

Thu* ky bien soan

TS. NGUY§N TRlEU VAN

PGS.TS. PHAM QUANG VINH

Chifdng 1 : Te bao - To chtic hoc cd quan tao mau

Chifdng 2: Dong mau - Cam mau

Chifdng 3: Di truyen huyet hoc

Chifdng 4: Sinh hoa huyet hoc

Chifdng 5: M ien dich huyet hoc

Chifdng 6 : An toan truyen mau

Xin tran tron g cam On

THAY MAT TAP THf; BI^N SOAN

GS. TSKH. D d TRUNG PHAN

C h i/d n g 1: TE BAO - TO CHLfC HOC CO QUAN TAO MAU

Omh li/cng huyet s^ic to 21

Do the tich khoi hong cau 23

C hi/dng 3: Dl TR UYEN HUYET HOC

Sat huyet thanh 136

C hi/dng 5: M I§N D|CH HUYET HOC

Van dong hien mau nhan dao 235

Cach san xuat hong cau mau va bao quan 288

TE BAO - TO CHLfC HOC

CACH LAY VA BAO QUAN BENH PHAM XET NGHIEM TE BAO

1.2. Phircmg phap lay mau

PHUOTNG PHAP LAM TIEU BAN XET NGHIEM

1. Lam tieu ban mau ngoai vi

Hinh 1.1: Tieu ban mau dan

1.2. Tieu ban giot dac

1.3. Tieu ban soi tuoi

2. Lam tieu ban djch tu y xU dng

3. Lam tieu ban nUdc djch

4. Lam tieu ban mo

4.1.1. XCf ly manh sinh thiet

Co" dinh trong dung dich Helly: 5-6 gid

4.1.2. Nhuom H &E

- Tay nen bang xylen 3 lan: 5 phut 1 lan

4.2. Tieu ban mo mem ( Lach, hach,...)

DEM SO LlfflNG HONG CAU

2. Mau xet nghiem, hoa chat va dung cu

- Dung cu la y mau mao mach (dau ngon tay) va tinh mach.