Antibody Identification - II

Blood Group Antibody Identification
Jim Perkins, M.D.

NorthShore University Health System Blood Banks
2006, James T. Perkins, all rights reserved
Results which prompt ABID Initial identification tests Analyzing a panel (forming a hypothesis) Confirmatory tests:
Rule out cells Rule in cells Antigen typing Elution/adsorption Neutralization tests
06, James T. Perkins
Standard test results: Normal Person

Forward and Reverse ABO types match
Antibody screen negative

Crossmatch is negative with ABO
compatible RBCs
(Direct Antiglobulin Test negative)
Identify cause of abnormality when:

Antibody screen is positive
Forward and reverse ABO typing dont match Crossmatch positive; negative antibody screen
(Positive DAT)
Typical problem
<A <B A cells B cells >D Interpretation
+
37o, 30
O Pos
IS OI OII
AHG
CC
Interpretation
0 0
0 0
0 3+
2+ Positive NT
Initial ABID Tests

ABID panel
Same IAT technique as antibody screen
DAT More specimen Patient history

What is the urgency of transfusion? Has the patient been pregnant or transfused? Is there an autoimmune disease?
Antibody Identification panel D 1 + 2 + 3 + C + + 0 c 0 0 + E 0 0 + e + + 0 K + 0 + k + + + S 0 0 + s + + 0 AHG 0 0 3+
4 +
5 0 6 0 7 0 8 0
0
0 0 0 0
+
+ + + +
+
0 0 0 +
0
+ + + +
0
0 + 0 0
+
+ + + +
+
0 0 + 0
+
+ + + +
3+
0 0 0 2+
Analyzing a panel
Crossout all antigens NON-REACTIVE in double dose
Corresponding ANTIBODIES are ruled out

C+c- (C/C) Double dose C
C+c+ (C/c) Single dose C

D
1 +
C
+
c
0
E
0
e
+
K
+
k
+
S
0
s
+
AHG
0
2
3 4 5 6 7 8
+
+ + 0 0 0 0
+
0 0 0 0 0 0
0
+ + + + + +
0
+ + 0 0 0 +
+
0 0 + + + +
0
+ 0 0 + 0 0
+
+ + + + + +
0
+ + 0 0 + 0
+
0 + + + + +
0
3+ 3+ 0 0 0 2+
D
1 +
C
+
c
0
E
0
e
+
K
+
k
+
S
0
s
+
AHG
0
2
3 4 5 6 7 8
+
+ + 0 0 0 0
+
0 0 0 0 0 0
0
+ + + + + +
0
+ + 0 0 0 +
+
0 0 + + + +
0
+ 0 0 + 0 0
+
+ + + + + +
0
+ + 0 0 + 0
+
0 + + + + +
0
3+ 3+ 0 0 0 2+
D
1 +
C
+
c
0
E
0
e
+
K
+
k
+
S
0
s
+
AHG
0
2
3 4 5 6 7 8
+
+ + 0 0 0 0
+
0 0 0 0 0 0
0
+ + + + + +
0
+ + 0 0 0 +
+
0 0 + + + +
0
+ 0 0 + 0 0
+
+ + + + + +
0
+ + 0 0 + 0
+
0 + + + + +
0
3+ 3+ 0 0 0 2+
D
1 +
C
+
c
0
E
0
e
+
K
+
k
+
S
0
s
+
AHG
0
2
3 4 5 6 7 8
+
+ + 0 0 0 0
+
0 0 0 0 0 0
0
+ + + + + +
0
+ + 0 0 0 +
+
0 0 + + + +
0
+ 0 0 + 0 0
+
+ + + + + +
0
+ + 0 0 + 0
+
0 + + + + +
0
3+ 3+ 0 0 0 2+
D
1 +
C
+
c
0
E
0
e
+
K
+
k
+
S
0
s
+
AHG
0
2
3 4 5 6 7 8
+
+ + 0 0 0 0
+
0 0 0 0 0 0
0
+ + + + + +
0
+ + 0 0 0 +
+
0 0 + + + +
0
+ 0 0 + 0 0
+
+ + + + + +
0
+ + 0 0 + 0
+
0 + + + + +
0
3+ 3+ 0 0 0 2+
D
1 +
C
+
c
0
E
0
e
+
K
+
k
+
S
0
s
+
AHG
0
2
3 4 5 6 7 8
+
+ + 0 0 0 0
+
0 0 0 0 0 0
0
+ + + + + +
0
+ + 0 0 0 +
+
0 0 + + + +
0
+ 0 0 + 0 0
+
+ + + + + +
0
+ + 0 0 + 0
+
0 + + + + +
0
3+ 3+ 0 0 0 2+
D
1 +
C
+
c
0
E
0
e
+
K
+
k
+
S
0
s
+
AHG
0
2
3 4 5 6 7 8
+
+ + 0 0 0 0
+
0 0 0 0 0 0
0
+ + + + + +
0
+ + 0 0 0 +
+
0 0 + + + +
0
+ 0 0 + 0 0
+
+ + + + + +
0
+ + 0 0 + 0
+
0 + + + + +
0
3+ 3+ 0 0 0 2+
D
1 +
C
+
c
0
E
0
e
+
K
+
k
+
S
0
s
+
AHG
0
2
3 4 5 6 7 8
+
+ + 0 0 0 0
+
0 0 0 0 0 0
0
+ + + + + +
0
+ + 0 0 0 +
+
0 0 + + + +
0
+ 0 0 + 0 0
+
+ + + + + +
0
+ + 0 0 + 0
+
0 + + + + +
0
3+ 3+ 0 0 0 2+
D
1 +
C
+
c
0
E
0
e
+
K
+
k
+
S
0
s
+
AHG
0
2
3 4 5 6 7 8
+
+ + 0 0 0 0
+
0 0 0 0 0 0
0
+ + + + + +
0
+ + 0 0 0 +
+
0 0 + + + +
0
+ 0 0 + 0 0
+
+ + + + + +
0
+ + 0 0 + 0
+
0 + + + + +
0
3+ 3+ 0 0 0 2+
Hypothesis
The positive antibody screen was due to anti-E
Anti-E explains all of the reactions. (Note dosage effect)
When is the hypothesis regarding the antibody specificityProved?

1. 3 antigen positive cells are reactive 2. 3 antigen negative cells are non-reactive 3. Clinically significant and common antibodies are ruled out
Rh (anti-D, -C, -c, -E, -e) Duffy (anti-Fya, -Fyb) MNSs (anti-M, -N, -S, -s) P system (anti-P1) Kell (anti-K, -k) Kidd (anti-Jka, -Jkb) Lewis (anti-Lea, -Leb)
4. The patient LACKS the corresponding antigen

D
1 +
C
+
c
0
E
0
e
+
K
+
k
+
S
0
s
+
AHG
0
2
3 4 5 6 7 8
+
+ + 0 0 0 0
+
0 0 0 0 0 0
0
+ + + + + +
0
+ + 0 0 0 +
+
0 0 + + + +
0
+ 0 0 + 0 0
+
+ + + + + +
0
+ + 0 0 + 0
+
0 + + + + +
0
3+ 3+ 0 0 0 2+
Need to rule out anti-S Rule-out cells are NON-reactive cells
D
+
C
+
c
0
E
0
e
+
K
+
k
+
S
+
s
0
AHG
0
Antigen Typing
Hypothesis: ALLO-anti-E
Patient
CC
Pos Neg control control

4+ 0
CC
Anti-E
2+
2+
Immunohematologic diagnosis: alloanti-E

1. 2. 3. 3 E positive cells are reactive 3 E negative cells are non-reactive The following antibodies are ruled out
Rh (anti-D, -C, -c, -e) Duffy (anti-Fya, -Fyb) MNSs (anti-M, -N, -S, -s) P system (anti-P1) Kell (anti-K, -k) Kidd (anti-Jka, -Jkb) Lewis (anti-Lea, -Leb)
4.
The patient LACKS the E antigen

D
1 +
C
+
c
0
E
0
e
+
K
+
k
+
S
0
s
+
AHG
4+
2
3 4 5 6 7 8
+
+ + 0 0 0 0
+
0 0 0 0 0 0
0
+ + + + + +
0
+ + 0 0 0 +
+
0 0 + + + +
0
0 + 0 + 0 0
+
+ + + + + +
0
+ + 0 0 + 0
+
0 + + + + +
4+
0 3+ 0 3+ 0 0
Hypothesis: anti-C plus anti-K

Need to RULE IN both antibodies
Rule-in cells are REACTIVE cells

D + + 0 C + + 0 c 0 0 + E 0 0 0 e + + + K 0 0 + k + + + S 0 0 0 s + + + AHG 4+ 4+ 3+
Antigen Typing
Hypothesis: ALLO-anti-C and -anti-K
Patient
CC
Pos Neg control control 4+ 0
CC
Anti-C
2+
2+
Anti-K
2+
4+
2+
D
1 +
C
+
c
0
E
0
e
+
K
+
k
+
S
0
s
+
AHG
4+
2
3 4 5 6 7 8
+
+ + 0 0 0 0
+
0 0 0 0 0 0
0
+ + + + + +
0
+ + 0 0 0 +
+
0 0 + + + +
0
+ 0 0 + 0 0
+
+ + + + + +
0
+ 0 0 0 + 0
+
0 + + + + +
4+
0 0 4+ 4+ 4+ 4+
Antigen Typing
Hypothesis: ALLO-anti-e Cant rule out: anti-C Patient Anti-C Anti-e 3+ 0 CC NT 2+ Pos Neg control control 4+ 4+ 0 0 CC 2+ 2+
Conclusion: anti-C ruled out

What if the DAT was positive?

Detects: In vivo sensitization by antibody or complement Autoimmune Hemolytic Anemia Hemolytic Transfusion Reaction Hemolytic Disease of Newborn Drug/RBC Antibodies Passive Immunization (esp. plts.)
Application:
Consider the following test results:

Group O, Rh positive
Antibody screen positive
Antibody identification; serum anti-e

Direct antiglobulin test positive
The patient HAS the e antigen (not

transfused)
DAT
Poly
Anti-IgG
Anti-C3 O
e+ e+
Eluate
e+
ee-
Conclusion: Auto-anti-e
Autoanti-e in serum and eluate.

D + C + c 0 E 0 e + K + k + S 0 s + Serum Eluate 2+ 3+
+ + +
0 0 0 0
+ 0 0
0 0 0 0
0 + +
+ + + +
0 + +
0 0 0 +
+ 0 0
+ + + +
0 + 0
0 + 0 0
+ + +
+ + + +
0 + +
0 0 + 0
+ 0 +
+ + + +
2+ 0 0
2+ 2+ 2+ 2+
3+ 1+ 1+
3+ 3+ 3+ 3+
Most Autoantibodies are Pan-agglutinins

D + C + c 0 E 0 e + K + k + S 0 s + Serum Eluate 2+ 3+
+ + +
0 0 0 0
+ 0 0
0 0 0 0
0 + +
+ + + +
0 + +
0 0 0 +
+ 0 0
+ + + +
0 + 0
0 + 0 0
+ + +
+ + + +
0 + +
0 0 + 0
+ 0 +
+ + + +
2+ 2+ 2+
2+ 2+ 2+ 2+
3+ 3+ 3+
3+ 3+ 3+ 3+
Auto-antibody can mask allo-antibody

D C c E e K k S s Autoab AntiK
+ +
+ + 0 0 0 0
+ +
0 0 0 0 0 0
0 0
+ + + + + +
0 0
+ + 0 0 0 +
+ +
0 0 + + + +
+ 0
+ 0 0 + 0 0
+ +
+ + + + + +
0 0
+ + 0 0 + 0
+ +
0 + + + + +
4+ 4+
4+ 4+ 4+ 4+ 4+ 4+
1+ 0
1+ 0 0 1+ 0 0
Auto-antibody + Allo-antibody
Separate serum and RBCs
Remove auto-antibody from the RBCs
ZZAP: DTT
Papain
PBS Chloroquine
Return serum to the RBCs
Incubate
Separate again
Adsorbed serum
Autoadsorbed serum may reveal an underlying ab

D C c E e K k S s Raw Ads Serum serum
+
+ + + 0 0 0 0
+
+ 0 0 0 0 0 0
0
0 + + + + + +
0
0 + + 0 0 0 +
+
+ 0 0 + + + +
+
0 + 0 0 + 0 0
+
+ + + + + + +
0
0 + + 0 0 + 0
+
+ 0 + + + + +
2+
2+ 2+ 2+ 2+ 2+ 2+ 2+
1+
0 1+ 0 0 1+ 0 0
Neutralizable Antibody
Neutralization Test
incubate
Neutralization may demonstrate allo-ab specificity

D C c E e K k Lea Leb Raw serum HPC neut.
+ +
+ + 0 0 0 0
+ +
0 0 0 0 0 0
0 0
+ + + + + +
0 0
+ + 0 0 0 +
+ +
0 0 + + + +
+ 0
+ 0 0 + 0 0
+ +
+ + + + + +
0 +
0 0 0 0 + 0
+ 0
+ + + 0 0 +
0 0
1+ 0 0 0 0 0
0 0
0 0 0 0 0 0
Neutralized serum may reveal an underlying ab

D C c E e K k Lea Leb Raw Neut. serum serum
+ +
+ + 0 0 0 0
+ +
0 0 0 0 0 0
0 0
+ + + + + +
0 0
+ + 0 0 0 +
+ +
0 0 + + + +
+ 0
+ 0 0 + 0 0
+ +
+ + + + + +
0 +
0 0 0 0 + 0
+ 0
+ + + 0 0 +
2+ 0
3+ 3+ 2+ 0 0 3+
0 0
3+ 3+ 0 0 0 3+
ABO discrepancy
<A <B A cells B cells >D Interpretation
4+
1+
37o, 30
4+
3+
IS OI OI
AHG
CC
Interpretation
1+ 0
W+ 0
0 0
2+ 2+
Positive (? ab)
Antibody against a low-frequency antigen D 1 + 2 + 3 + C + + 0 c 0 0 + E 0 0 + e + + 0 K + 0 + k + + + S 0 0 + s + + 0 AHG 0 0 0
4 +
5 0
0
0
+
+
+
0
0
+
0
0
+
+
+
0
+
+
3+
0
6 0
7 0 8 0
0
0 0
+
+ +
0
0 +
+
+ +
+
0 0
+
+ +
0
+ 0
+
0 +
0
0 0
Antibody against a high-frequency antigen D 1 + 2 + 3 + C + + 0 c 0 0 + E 0 0 + e + + 0 K + 0 + k + + + S 0 0 + s + + 0 AHG 3+ 3+ 3+
4 +
5 0
0
0
+
+
+
0
0
+
0
0
+
+
+
0
+
+
3+
3+
6 0
7 0 AC
0
0
+
+
0
0
+
+
+
0
+
+
0
+
+
0
3+
3+ 0
(Patient cells + patient serum)
Key question: Does my answer explain all of the reactions?

Antibody Identification - II

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Antibody Identification - II

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Blood Group Antibody Identification

Jim Perkins, M.D.

Standard test results: Normal Person

Antibody screen negative

Identify cause of abnormality when:

Initial ABID Tests

DAT More specimen Patient history

Antibody Identification panel D 1 + 2 + 3 + C + + 0 c 0 0 + E 0 0 + e + + 0 K + 0 + k + + + S 0 0 + s + + 0 AHG 0 0 3+

Corresponding ANTIBODIES are ruled out

C+c- (C/C) Double dose C

C+c+ (C/c) Single dose C

06, James T. Perkins

06, James T. Perkins

06, James T. Perkins

06, James T. Perkins

06, James T. Perkins

06, James T. Perkins

06, James T. Perkins

06, James T. Perkins

06, James T. Perkins

When is the hypothesis regarding the antibody specificityProved?

4. The patient LACKS the corresponding antigen

06, James T. Perkins

Need to rule out anti-S Rule-out cells are NON-reactive cells

Pos Neg control control

06, James T. Perkins

Immunohematologic diagnosis: alloanti-E

The patient LACKS the E antigen

06, James T. Perkins

Hypothesis: anti-C plus anti-K

Rule-in cells are REACTIVE cells

Pos Neg control control 4+ 0

06, James T. Perkins

Conclusion: anti-C ruled out

What if the DAT was positive?

Consider the following test results:

Antibody identification; serum anti-e

The patient HAS the e antigen (not

Autoanti-e in serum and eluate.

Most Autoantibodies are Pan-agglutinins

Auto-antibody can mask allo-antibody

06, James T. Perkins

Separate serum and RBCs

06, James T. Perkins

Remove auto-antibody from the RBCs

Return serum to the RBCs

06, James T. Perkins

06, James T. Perkins

Autoadsorbed serum may reveal an underlying ab

06, James T. Perkins

06, James T. Perkins

Neutralization may demonstrate allo-ab specificity

Neutralized serum may reveal an underlying ab

Antibody against a low-frequency antigen D 1 + 2 + 3 + C + + 0 c 0 0 + E 0 0 + e + + 0 K + 0 + k + + + S 0 0 + s + + 0 AHG 0 0 0

Antibody against a high-frequency antigen D 1 + 2 + 3 + C + + 0 c 0 0 + E 0 0 + e + + 0 K + 0 + k + + + S 0 0 + s + + 0 AHG 3+ 3+ 3+

(Patient cells + patient serum)

Key question: Does my answer explain all of the reactions?

06, James T. Perkins

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