Clinical Microscopy

(Urinalysis and other Body Fluids)

CEREBROSPINAL FLUID
I. Introduction
– a clear and colorless fluid produced by the
highly vascular choroid plexuses in the
ventricles of the brain
– does not resemble an ultrafiltrate of
plasma

*Blood-Brain Barrier –
represents the control & filtration of
blood compo nents to the CSF and then
to the brain
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Functions: (PERMS)

a. protect against mechanical injury

b. excretory channel for the metabolic
products
c. regulates intracranial pressure
d. medium for exchange and transfer of
substances between blood stream &
tissue of the brain and spinal cord
e. supplies nutrients to nervous tissues
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Collection:

Sterile:

tube 1 - chem & sero frozen

tube 2 - microbiology room temp.
tube 3 - hematology refrigerated
tube 4 – additional test like micro

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Methods of Collection:

1. lumbar/spinal tap

*between 3rd & 4th lumbar vertebrae

– for adults

*between 4th & 5thlumbar vertebrae –

for young/neonates

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2. ventricular puncture – collected directly
from ventricles of the brain

3. cisternal puncture – for patients with

paralysis and meningitis

4. lateral – cervical puncture

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Utilities of Analysis

1. To detect and differentiate meningitis

2. To detect CNS disorders

3. To detect presence of subarachnoid

block

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II. Physical/Macroscopic

A. Volume
Normal: 120-150 ml/day or
20 ml/hr by the choroid
plexuses

Total Volume: 140-170 ml (adults)

10-60 ml (neonate)

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B. Color

Normal: Colorless
Abnormal:

• Hazy,cloudy,turbid,milky
- due to wbc, rbc, microorganisms,
increase protein and lipid

• Oily -radiographic contrast media

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• Bloody- traumatic spinal tap
(non-pathogenic); Subarachnoid
hemorrhage (pathogenic)

• Clotted, pellicle - protein, clotting

factors

• Xanthochromic - Hb, oxyHb,

bilirubin, methemoglobin,
merthiolate, carotene, protein,
melanin

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Xanthochromia

• yellowish discoloration of CSF

• presence of RBC degradation products (usually
signifies bleeding)
• dependent on the amount of blood and length
of time it has been present, maybe due to:
a. Pink - very slight amount of oxyhemoglobin
b. Orange - heavy hemolysis
c. Yellow - conversion of oxyHb to conjugated
bilirubin

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Differentiation:
Traumatic Tap Subarachnoid
Hemorrhage

a. distribution uneven even

b. supernatant clear and xanthochromic

colorless

c. clot formation (+) (-)

*erythrophagocytosis
-macrophage
containing rbc or
hemosiderin
granules

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Additional test of Differentiation:
▪ D-dimer test – detection of fibrin
degradation product (D-dimer) by latex
agglutination immunoassay

(+) Clot formation (because of increase

filtration of CHON and coagulation factors)
but not bloody

– suppurative meningitis
– tuberculous meningitis
– Froin’s Syndrome
– Blockage in CSF circulation
– Neurosyphilis

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C. Transparency

Normal: Crystal clear

Variations/Turbidity – usually caused by
the following:

1. Cellular Elements:
a. 200-500 wbc/mm3 – hazy/sl. Turbid
b. >400 cells/uL rbc – sl. Turbid
c. >500 wbc/mm3 – distinct turbidity

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 d. <200 wbc & 400 rbc/ul – may appear
clear thus there is a need to examine
microscopically

2. microorganisms like bacteria, fungi

and amoeba

3. contrast media

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D. Specific Gravity: 1.006 – 1.008
E. pH: 7.3 – 7.45
F. Pressure: 50 – 200 mm H20

• Queckenstedt
-method for subarachnoid block
-compress jugular vein

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G. Clot and Pellicle formation

• pellicles are macroscopically small fine clots

which may be seen on the surface of CSF
after 12-24 hrs

• microscopically, it consists of white cells

against a fibrinous background and must be
examined for bacteria by culture, gram stain
and AFB

Normal: no clot due to absence of fibrinogen

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Conditions associated with Clot and
Pellicle Formation

• small clots – paresis

• large clot - purulent meningitis
• web-like clots – TB meningitis
• clotting en masse – blockage of CSF
circulation
• pellicle formation – suppurative meningit

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 III. Chemical Exam
• Increased in:
A. Protein: 15-45 1. damage to “blood-brain
barrier”
mg/dL
2. production of
immunoglobulins within the
*most frequently CNS
performed chem. 3. decreased clearance of
test on CSF normal protein from the fluid
4. meningitis and hemorrhage –
most common cause of
elevated protein CSF
• Decreased in: CSF
5. degeneration of neural tissue
leakage
6. multiple sclerosis
7. 7. endocrine/metabolic
disorder

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• Qualitative Tests

Test Reagents (+) Result

1. Nonne-Apelt/
Rose Jones ammonium sulfate white ring

3. Pandy’s phenol bluish white

cloud

4. Nogochi 10% butyric acid ppt’n

5. Colloidal red colloidal colorless (5+)

Gold test gold sol’n *pale blue (4+) (by
Lange) deep blue(3+)
purple(2+), blue (1+)

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• Quantitative Tests

1. Turbidimetric
-TCA - precipitates both albumin and globulin)
-SSA - precipitates only albumin, so add
Sodium Sulfate to ppt globulin

2. Nephelometric Method
-uses Benzalkonium chloride

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3. Dye-Binding Technique
Advantage – smaller sample size and less
interference
a. Coomasie Brilliant Blue G250 – uses
principle of “protein error of indicators”
*red to blue intensity
b. Ponceau S

4. Biuret Method
- uses Copper Sulfate plus NaOH or KOH
- (+) violet/purple
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5. Ultraviolet (Spectrophotometry)
6. Lowry Method using Folin-Ciocalteau
7. Immunologic Methods
a. Immunodiffusion
b. RID
c. ultracentrifugation
d. electrophoresis
- method of choice when it is necessary to
determine if fluid is actually CSF (‘tau”)
- primary purpose of use is for detection of
oligoclonal bands representing inflammation
within the CNS

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Notes:
1. Myelin Basic Protein

- protein not normally found in CSF

detected by RID

- presence is indicative of
demyelination

- can be used to monitor the course of

Multiple Sclerosis

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2. Proteins found in CSF (G-PATTCH)
a. albumin – major CSF protein
b. prealbumin – 2nd most prevalent fraction
c. haptoglobin – alpha-globulin
d. ceruloplasmin – alpha-globulin
e. transferrin – major beta-globulin
f. tau – aka B2 transferrin / transthyretin
-carbohydrate-deficient transferrin fraction
seen only in CSF and not in serum
g. gamma globulins – primarily IgG and small
amounts of IgA

EXCEPT: IgM, fibrinogen and beta-lipoprotein (BlIF)

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3. Multiple Sclerosis
- characterized by oligoclonal banding* which
remains positive during remission of MS but
disappears in other disorders
- best accomplished by measuring amount of
myelin basic protein
- has increased IgG

*Other disorders with oligoclonal banding

– encephalitis, neurosyphilis,
Guillain-Barr syndrome and neoplastic
disorders
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B. Glucose

- 60-70% of plasma glucose (i.e. 50-80

mg/dl or 2.75-4.4 mmol/L)

- blood glucose should be drawn 2 hrs

prior to spinal tap to allow time for
equilibrium between blood and fluid

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Clinical Significance:
1. decreased glucose value – considerable diagnostic
value in determining the causative agents of
meningitis

a. bacterial meningitis – markedly decreased CSF

glucose accompanied by increased WBC with large
percentage of neutrophils

b. tubercular meningitis – if lymphocytes have

greater percentage instead of neutrophils

c. viral meningitis – normal CSF glucose with

increased lymphocytes

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 Bacterial Tubercular Viral

Cells PMNs Lymph Lymph

Glucose Mdec dec N

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others:
hypoglycemia, pyogenic meningitis, fungal
meningitis, subarachnoid block,
toxoplasmosis, primary tumor of brain,
disorders which affect blood brain barrier

2. Increased glucose in
a. diabetes
b. encephalitis
c. conditions associated with intracranial
pressure

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C. Lactate: 10-22 mg/dL

- sensitive method for evaluating effectiveness of

antibiotic therapy
- used to monitor severe head injuries

Clinical Significance:
<25 mg/dL – viral meningitis
>25 mg/dL – bacterial, tubercular, fungal meningitis
>35 mg/dL – bacterial

others (increased) – idiopathic seizures, head injury, brain

tissue damage, hypoxia, respiratory alkalosis,
hydrocephalus, brain abscess, cerebral ischemia due to
arteriosclerosis, low bp, low arterial PO2

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D. Glutamine: 8-18 mg/dL

- chem. test frequently performed in CSF and

not in blood

- produced in CNS by brain cells from ammonia

and a-ketoglutarate

- increased in liver disorders that result in

increased blood and CSF ammonia, Reye’s
Syndrome, coma of unknown origin (>35
mg/dL also in hepatic encephalopathy and
disturbance of consciousness)
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E. Enzymes
LD isoenzymes: 40 U/L (normal adults),
70 U/L (neonates)
Significance: increased during infection
(another useful method in detecting
different kinds of meningitis

LD 1&2 – brain tissue

LD 2&3 – lymphocytes
LD 4&5 – neutrophils

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2. CKBB: 17 mg/dL or <5 U/L
increased in seizure, stroke, head injury

3. chloride: 113-130 mEq/L

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IV. Microscopic
-usually WBC count; RBC is done only when
traumatic tap has occurred and a
correction for WBC or protein is needed

A. Cell Count
Normal: 0-5 wbc/uL (adult);
higher in children
0-30 mononuclear/uL (newborn)

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1. Fuchs-Rosenthal Counting Chamber

-undiluted and phase microscopy

-18 large squares, 0.2 mm depth

-formula: # of cells/3

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2. Neubauer Counting Chamber
- for both diluted and undiluted

a. cells/uL= # of cell counted x dilution

# of squares counted x vol. of 1 square

b. fixed formula (4 large squares + 1 large center

square) cells/uL=

# of cells counted x dilution x 1 uL

1 uL (0.1 x 10)
[volume counted]
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• Dilution
-not needed for clear Diluting Fluids
specimen
• NSS – total cell counts
Clarity Dilution • 3% HAc – WBC count
Sl. Hazy 1:10 • toluidine blue O - WBC
Hazy 1:20 count
Sl. Cloudy 1:100 • saponin sol’n - WBC
Sl. Bloody 1:200 count
Cloudy/bloody/
Turbid 1:10,000 others – glacial HAc
methylene blue
crystal violet
gentian violet
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• Total Cell Count and WBC Count –
count on 4 large squares + center
large square x dilution

• RBC count/uL = total cell count – wbc

count

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B. Differential Count
-performed in a stained smear and not on
counting chamber (allowed when there is
insufficient fluid to perform both cell count
and differential)

Concentration Methods for CSF before Differential

Count:
*1. filtration
*2. sedimentation
*3. cytocentrifugation – add 30%albumin to prevent
cell lysis
4. routine centrifugation (Wright’s after)

*Better yields and less cellular distortion

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• Romanowsky Stain ( e.g. Wright’s Stain)
More PMNs – bacterial
More lymph – viral

1. Lymphocytes and Monocytes – predominant

cells found in CSF
* Lymphocytes - predominant in adults
(70:30 ratio)
* Monocytes - prevalent in children

2. Neutrophils
-increased #s are seen in early stages of viral, fungal,
tubercular and parasitic meningitis
-also in bacterial meningitis and CNS hemorrhage
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*Pleocytosis – presence of increased numbers of
normal cells and is considered abnormal

-perform differential count when increased cells are

present

▪ increased CSF WBC count; increased neutrophils –

bacterial meningitis

▪ moderate increase CSF WBC with increased

lymphocytes and monocytes – viral, tuberculous,
fungal or parasitic origin

▪ decreased cell count (below 25 cells/uL) with

increased lymphocyte – multiple sclerosis
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3. macrophages 4. Ependymal Cells and
Choroid Plexus Cells
-appear within 2-4 hrs after
introduction of RBCs into -normally seen and not
the fluid clinically significant

-indicative of previous -frequently seen in:

hemorrhage (macrophage a. PneumoEncephalography
with rbc inside); b. fluid from ventricular taps
intracranial hemorrhage c. during NeuroSurgery

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5. Eosinophils – increased in:
a. parasitic infections
b fungal infections primarily C. immitis.
c. reactions to foreign protein in CSF
d. intracranial shunt malfunctions

6. Blast Forms and immature WBC – seen

in CNS involvement:
a. Leukemias
b. lymphomas

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7. nRBC – result of BM contamination during
spinal tap

8. Reactive Lymphocytes, containing increased

cytoplasm and clumped chromatin and
plasmacytoid, lymph – viral infections and
multiple sclerosis

9. mixed reaction containing

neutro,lymph,mono,pia arachnoid
mesothelial cells – viral, fungal, and tubercular
meningitis and later stages of bacterial
meningitis
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V. Microbiology Test

• G/S –70-80% sensitive

*failure to isolate organisms on smear should be not
interpreted to mean that organisms are absent,
because there must be 105/ml in order for them to
be demonstrated by this technique

• Acid-Fast or Fluorescent Antibody Stain

-for MTB

• India ink – Cryptococcus neoformans 50%

sensitive

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• Reverse Latex Agglutination –
Cryptococcus neoformans 90% sensitive

• Limulus Lysate Test

-Rgt: from blood cells (“amebocytes”) of
horsecrab (Limulus polyphemus)
-sensitive to minute amounts of endotoxin
and will detect all g(-) bacteria

VI. Serology (for Neurosyphyllis)

– VDRL – recommended by CDC
– FTA-ABS
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SEMINAL FLUID ANALYSIS
Utilities of Analysis
• investigate cases of infertility
• for medico-legal cases
• evaluate the effectiveness of vasectomy
• support or disapprove a denial of
paternity
• screen donor for artificial insemination
program
• to evaluate semen quality for semen and
sperm banking
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 I. Seminal Fluid / Ejaculate

A. Origin

a. Testes (5%) – produces spermatozoa

b. Seminal Vesicle (60%)

-produces slightly alkaline fluid; where fructose is being
produced
-produces flavin (yellow color of fluid responsible for the
fluorescence under UV)

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c. 10-15% from:

Epididymis - where spermatozoa mature and

are stored

Vas Deferens

Bulbo-Urethral - contribute to the thick and

alkaline mucus

Urethral Glands

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 d. Prostate Gland (20%)
-milky to slightly acidic fluid pH 6.5
-zinc, ALP, citric acid, proteolytic enzymes

A. Composition

1. sperm cells
2. Secretions
a. seminal plasma
-provides the nutritive medium for proper
osmolality and volume
-activates sperm cell motility for fertilization

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b. choline and spermine
-makes semen sterile by inhibiting the growth
of bacteria
-secreted by prostate glands

c. fructose
-main sugar of ejaculate

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d. proteolytic enzymes (fibrinolysin)
-responsible for complete liquefaction and
coagulation of seminal fluid

e. prostatic secretions
-citrates and carbonates

f. ACP and LDH

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Collection

– masturbation – best and most common

– coitus interruptus – for observing sperm-cervical

mucus studies

– vaginal vault aspiration – after coitus; in

medico-legal cases e.g. rape

– condom method – least common due to

spermicides present in condoms ; used in post
vasectomy cases

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Requirements

• Abstinence period is set empirically at 3-5 days

-Shorter – “reserve depletion”

-Longer – senescence and decreased motility

• For fertility testing, 2-3 samples must be

examined at 2 weeks intervals with two
abnormal samples considered significant

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• must empty bladder before ejaculation because
urine is toxic to sperm

• During transport – maintained at body

temperature

• Time of collection – preferably in the morning,

brought to lab within 30 mins and examined
within an hour

• Specimen is collected in a wide-mouthed jar to

prevent spillage

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II. Routine Examination
A. Color
Normal: “pearly white”, colorless to
creamy white
Variations:
• Rusty/red to brown – bleeding
• Yellowish – urine contamination,
antibiotics, prolonged abstinence due to
increased flavin, pyospermia
• Turbid – infection due to increased WBC
• Clear - infertility
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B. Volume: 2-5 ml/ejaculation
– Increased – prolonged abstinence
– Decreased – infertility ; improper functioning
of one of the semen-producing organs

C. Odor: fishy, “chlorox-like”, musty

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D. Viscosity: highly viscous, pours in droplets
Liquefaction time: within 30 mins
*increased viscosity and incomplete
liquefaction will impede sperm motility

E. pH: 7.3-8.3
– if pH is acidic – possible seminal vesicle
obstruction, absence of seminal vesicle or
increase prostatic fluid
– if pH is basic – possible infection within
reproductive tract

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III. Microscopic
A. Motility - performed undiluted microscopically
; approximately 20 hpf should be examined
NV: minimum motility of 50-60% with quality
of fair (2) within 3-hr time
% motility = total sperm – nonmotile
total sperm X
100
Grading:
4.0 – rapid forward movement
3.0 – slower speed movement, some lateral movement
2.0 – slow movement, noticeable lateral movement
1.0 – no forward progression
0 - immotile
Normal Grading: 2.0-4.0
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Factors Affecting Motility

• complete liquefaction of seminal fluid

• temperature
• period of abstinence
• manner of collection and preparation
• # of cells present
• type of movement

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B. Sperm Count

NV: 20-160 M/ml

2 Types of Counting Chamber

1. Makler –undiluted; sperms are
immobilized by heating part of
specimen prior to charging the
chamber
2. Neubauer Hemocytometer

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Diluting Fluids (to immobilize the
sperm cells)

• 5% NaHCO3
• 1% formalin
• cold distilled water
• 0.5% chlorezene
• 1% formalin in trisodium citrate
• 5% naHCO3 in 1% phenol in distilled water
• tap water
• 5% NaHCO3 in formalin

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Usual dilution= 1:20
a. count in 5 rbc squares x I M = /ml
b. count in 2 large wbc squares x 100,000 = /ml
c. CSF= # of sperms x dilution = /ul x 1,000 = /ml
squares counted X vol counted

Total Sperm Count

Sperm cells/ejaculate
= sperm count/ml x vol of specimen
Normal: >40M/ejaculate

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Clinical Significance

• Azospermia – complete or total absence

of spermatozoa seen in:

• underdeveloped testes
• obstruction from previous
operation/traumatic procedure
• infection with Gonorrhea

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• necrospermia – presence of sperm cells
whether completely dead or immobile

• oligospermia – deficiency in the number

of sperm cells or presence of few motile
cells seen in:
– hypotropic lesions
– hypothyroidism

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C. Morphology
-stained, examined under oil immersion, at least
200 spermatozoa should be examined
-Stains: Paps (best), hematoxylin, crystal violet,
Giemsa

-Normal: <30% abnormal forms (3rd ed)

<50% abnormal forms (routine criteria)
to <70% abnormal forms (strict
criteria)

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3 Distinct Parts
1. head – ovum penetration
2. neck/middle piece – contains
mitochondria that provides energy for
flagellar tail motion
3. tail – motility

*oval shaped head approx 5um long and

3um wide
*acrosomal cap approx ½ of head

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• WHO Strict Criteria

->70% should be normal in appearance

-head size of 4-5.5 um x 2.5-3.5 um
-oval form with smooth contour
-acrosome 40-70%
-tail 45 um
-no abnormalities in neck, midpiece or tail
-no big cytoplasmic droplet

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V. Medico-Legal Cases
• Florence Test (FC)
-to determine the presence of Choline
-based on the presence of brown rhombic
crystals
Rgt: potassium iodide and iodine crystals

• Barbieros (BS)
- to determine the presence of spermine
-Rgt: picric acid and TCA
-(+) yellow leaflike structure

• Fluorescence/UV test
• ACP test – for rape cases

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• Others:

1. Viability
-done when decreased motility with normal count
-specimen mixed with Eosin-Nigrosin stain, smear,
count # of dead cells in 100 sperm
-cells that stain red against purple background (live
cells don’t take up stain and appear bluish
white in color)
-normal viability requires 75% living cells and
should correspond to the previously evaluated
motility

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2. Fructose level

-decreased count maybe caused by lack of

support medium produced in the seminal
vesicle
which is indicated by low to absent fructose
level

-uses Resorcinol for the presence of fructose

-Normal level of fructose = > 13

ummol/ejaculate

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3. Antisperm Antibodies

-result to clumping and inactivation of sperm

-Female has normal analysis with continued

infertility – tested by mixing semen with
female serum or cervical mucosa then
observe for agglutination

-Male has decreased motility with clumping

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TESTS
a. Gelatin agglutination test:
semen with sperm + 10% gelatin + px serum
(+) white particles in clear surrounding

b. Mixed Antiglobulin Reaction:

-screening procedure used primarily to detect
the presence of IgG antibodies

-semen sample containing motile sperm is

incubated with IgG AHG and a suspension of
latex particles or treated RBCs coated with IgG

-Normal=<10% of the motile sperm attached to

the particles
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c. Immunobead Test:

-more specific that it can be used to detect the

presence of IgG, IgM and IgA antibodies and will
demonstrate what area of the sperm (head,
neck or tail) the autoantibodies are affecting

-sperm are mixed with polyacrylamide beads

known to be coated with either anti-IgG,
anti-IgM or anti-IgA.

-Reporting: IgG tail antibodies, IgM head

antibodies and so forth

-Normal = <20% of the sperm

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d. Sperm immobilization

e. Double-Fluorochrome
Sperm-Cytotoxicity Antibody Assay

f. ELISA

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4. Sim Huhner Test
-post coital test
-tests the ability of sperm cells to penetrate
the cervical mucosa
-6-8 hours after coitus,aspirate from vaginal
vault
NV: 10 motile sperm cells after 6-8 hours

5. Spinbarkeit Method
-test for tenacity of mucus
->10 cm – fertile (girls)
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Microbial Testing

*>1M leukocytes/ml indicates infection within

reproductive system frequently the prostate

Perform routine aerobic and anaerobic cultures

and test for Chlamydia trachomatis,
Mycoplasma hominis and Ureaplasma
urealyticum

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SYNOVIAL FLUID ANALYSIS
• Synovial fluid

-often referred as “joint fluid” is an

ultrafiltrate plasma with a characteristic
mucoidal substance “hyaluronic acid” that is
produced as a product of the secretory
activity of the synovial lining

-supplies nutrients to the cartilage and proper

lubrication of joints

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• Method of Collection: Arthrocentesis

1st tube -heparinized Microbiology

2nd tube-liquid EDTA Hematology

3rd tube -non anticoagulated Chem,etc

*powdered anticoagulants should not be

used!

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Synovial Diluent (no HAc bec it
will lyse wbc)
– NSS + methylene blue
– 1% saponin
– 0.1 N HCl

• Characteristics:
A. Volume: 3.5ml
-increased dependent on the severity of
joint involvement

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B. Transparency: clear
Variations:
– cloudy to purulent – septic joint disease
– clear or slightly turbid – cell count
elevated; inflammatory disease
– black particles – onchronosis
– metallic particles – after prosthetic knee
Arthroplasty
– Milky- presence of crystals

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C. Color: colorless to pale yellow
Variations:
– green tinge – bacterial infection
– deep yellow – presence of inflammation
– reddish – hemorrhagic arthritis / traumatic tap

D. Viscosity: High (due to polymerization of

hyaluronic acid)
Tests:
1. Falling Drop / String Test (normal: 4-6 cm)
*Falls like water
Low Viscosity seen in:
– Rheumatoid arthritis
– Septic arthritis
– gout

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 2. Mucin Clot Formation / Ropes
-by addition of 2-5% HAc
-tests the degree of hyaluronate
• Grading:
Good (solid clot)
Fair (soft clot)
Poor (friable clot)
Very poor (no clot)

E. pH: 7.1

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F. Total Cell Count

– <200 WBC/mm3; rbc usually present in very

low numbers but may be present because
of Trauma from aspiration

– 100,000 cells/ul seen in severe

inflammation

G. Mucin clot: firm

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Cells and Inclusions seen in Synovial Fluid
• neutrophils (Normal:<20%)– indicates
– bacterial sepsis
– crystal-induced inflammation

• lymphocyte (normal:<15%)– non-septic

inflammation

• macrophage (monocyte) – normal /

viral infections

• synovial lining cell – normal

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• LE cell – PMN with characteristic ingested
“round body” ; seen in SLE

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• Reiter cells – vacuolated macrophage with
ingested neutrophils; seen in
– Reiter’s Syndrome
– Non-specific inflammation

• RA cells (Ragocytes) – neutrophil with dark

cytoplasmic granules containing immune
complexes ; seen in Rheumatoid Arthritis and
immunologic inflammation

• Cartilage cells – large multinucleated cells ;

seen in osteoarthritis

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• Rice bodies
Macroscopically – resemble polished rice
Microscopically – show collagen and fibrin
Seen in: tuberculosis, septic and
rheumatoid arthritis

• fat droplets – stains with Sudan dyes ; indicates

traumatic injury

• hemosiderin – inclusions with clusters of

synovial cells ; seen in pigmented Villonodular
cells

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Crystals
• Causes of formation
-metabolic disorder
-decreased renal excretion that produce
elevated blood levels of crystallizing
chemicals
-degeneration of cartilage and bone
-injection of medication e.g. corticosteroids

1. monosodium urates (uric acid)

-seen in “gouty arthritis”
-increase in refrigeration
-appear as needlelike
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2. calcium pyrophosphate dehydrate
-seen in pseudogout
-rhombic shape, rectangles, rods, needles

3. Cholesterol – notched plates

4. apatite
-major mineral found in cartilage
-short needle crystals; petite crystal

5. corticosteroid- results from drug

injections
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Chemistry
• Glucose – most frequently requested test
; <10 mg/dl lower than blood volume
• Lactate - <7.5 mmol/L exclusion for
septic arthritis
• tCHON - <3 g/dL

Microbiology
• G/S and culture – 2 most important tests
• Organisms- Staph, Strep, Haemophilus,
Neisseria

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 SEROUS FLUIDS

-fluids between the membranes, which provides

lubrication as the surfaces move against each
other

- formed as ultrafiltrates of plasma, with no

additional material contributed by the
membrane cells
Utilities of Analysis
• determine the cause of effusion
• differentiating transudate from exudative effusions
• identify malignancy or infection in exudative
effusion
• establish specific diagnosis and alleviate symptoms

Causes of Effusion
• increase hydrostatic pressure
• decrease oncotic pressure
• increase capillary permeability
• lymphatic obstruction

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Collection: needle aspiration
• pleural fluid – thoracentesis
• pericardial fluid – pericardiocentesis
• peritoneal fluid – paracentesis

Tubes

• EDTA 5-7ml aspiration for gross appearance, cell

counts, morphology and differential count

• heparin 7-10 ml for chemical, serological and

cytologic tests

• sterile heparinized for culture, G/S, AFS, cytocentri

smear

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Transudates – effusions that form because
of a systematic disorder that disrupts the
balance in the regulation of fluid
filtration and reabsorption; results from
“mechanical process”

Exudates – produced by conditions that

directly involve the membranes of the
particular cavity including infection and
malignancies; result of an “inflammatory
process”

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Laboratory Differentiation:
Transudates Exudates

1. Appearance clear cloudy

2. Color colorless depends on condition
3. Odor odorless depends
4. pH alkaline acidic
5. Specific gravity <1.015 >1.015
6. total CHON <3 g/dL >3 g/dL
7. LDH <200 IU >200 IU
8. cell count <1000/ul wbc >1000/ul wbc
<100,000/ul rbc
9. spontaneous no possible due to
clotting fibrinogen
10. pleural fluid <60 mg/dl >60 mg/dl
cholesterol
11. origin non-inflammatory Inflammatory
12. condition CHF, liver cirrhosis infection, SLE,
nephrotic syndrome malignancy

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13. glucose as in plasma <plasma level
14. chloride 98-106 mEq/l <plasma level

15. differential count few lymphocytes, lymphocytes,

rbcs, mesothelial mesothelial cells,
cells eosinophils

16. crystals none (presence: stasis) plenty: cholesterol

(fatty degradation),
hematoidin
(hemorrhagic cases)

17. fluid:serum CHON <0.5 >0.5

18. fluid:serum LD <0.6 >0.6
19. pleural fluid:serum <0.3 >0.3
Cholesterol ratio
20. pleural fluid: <0.6 >0.6
serum bilirubin
21. serum ascite >1.1 <1.1
albumin gradient

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I. PLEURAL FLUID

Normal Volume: <10ml

Cases Causing Pleural Effusion
• systemic disorder (transudate)
– congestive heart failure
– hypoalbuminemia
• localized damage (exudates)
– pneumonia
– carcinoma

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• useful tests for differentiating
pleural fluid transudate and
exudates:

1. Pleural fluid cholesterol and

fluid-to-serum cholesterol ratio

2. Pleural fluid-to-serum total

bilirubin ratio

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• Organ involved – lungs
• Normal Appearance – clear, pale yellow

• Turbidity – WBC and microorganisms; indicates bacterial

infection, TB or Immunologic diseases e.g. RA

• Blood – traumatic injury, malignancy, traumatic tap

*Hemothorax – Hct is same with whole blood; occurs from
inpouring of blood from injury
*Hemorrhagic Exudate – lower Hct than whole blood

• Milky – chylous (from thoracic duct leakage) or

pseudochylous material (produced in chronic inflammatory
conditions)
*to distinguish the two, fluid is mixed with ether, chylous
material is extracted in ether leaving the clear layer

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• Sudan III – stains chylous material,does not stain pseudochylous
material
*chylothorax fluid – increase TAG, no cholesterol crystal

• Neutropils – bacterial infection e.g. pneumonia, increase in

disseminated LE, pancreatitis, pulmonary infarction

• Lymphocytes – Tuberculosis (with plasma cells); malignancy, viral

infections and autoimmune disorders e.g. LE

• Eosinophils (>10%) – trauma, allergic reactions and parasitic

infections

• Mesothelial cells – increased in pneumonia and malignancy;

decreased in TB

• Plasma cells – TB

• Malignant cells – primary adenocarcinoma and small-cell

carcinoma; metastatic carcinoma

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• Normal Glucose – parallels serum glucose
• Low Glucose – TB, rheumatoid inflammation,
malignancy, purulent infections

• Lactate – increased in bacterial infections

• Low pH – TB, esophageal rupture (pH as low as 6.0)

*pH lower than 7.3 indicates the need for chest tube
drainage and antibiotics in cases in pneumonia
*greater than 7.4 – malignancies

• Elevated Amylase - pancreatitis

• CEA – malignancy

• Microbiology tests – ANA and RF

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II. PERICARDIAL FLUID
*Tests differentiating transudate and exudate:
Fluid to serum CHON and LD ratios

• Amount – 10-50 ml

• Normal appearance – clear, pale yellow

• Turbidity – infection, malignancy

• Blood – TB, tumor, cardiac puncture (grossly bloody),

misuse of anticoagulant drugs

• Milky – lymphatic drainage

• Malignant cells – metastatic carcinoma

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• Neutrophils – bacterial endocarditis
*WBC count >1000 cells/ul are indicative of infection
Low Glucose – bacterial infection, malignancy

• CEA – malignancy; metastatic carcinoma

• G/S and culture – bacterial endocarditis

• AFS – tubercular effusion

• Adenosine deaminase – tubercular effusion

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III. PERITONEAL FLUID
Normal volume: <100ml
-commonly referred to as “ascitic fluid”

*Ascites – accumulation of fluid in the peritoneal cavity

*Normal Saline – sometimes introduced into the
peritoneal cavity to act as Lavage for the detection of
abdominal injuries that have not yet resulted in the
accumulation of fluid

*The Peritoneal Lavage fluid is particularly sensitive test

for the detection of intra-abdominal bleeding in “blunt
trauma cases” ; RBC and WBC count are used to aid in
determining the need for surgery.

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*serum-ascites albumin gradient

- recommended over the fluid to serum total

CHON and LD ratios for the detection of
transudates and exudates of hepatic origin

- fluid and serum albumin levels are measured

concurrently and the fluid albumin level is
then subtracted from the serum albumin
level

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• Normal Appearance – clear, pale yellow

• Turbidity – bacterial or fungal infection

• Blood – trauma
*RBC count <100,000 cells/ul is considered normal
*WBC count normal - <500 cells/ul ; increase in
bacterial peritonitis and cirrhosis
*Absolute granulocye count >250 to 500 cells/ul or
>50% of the total WBC count is indicative of
infection

• Milky, chylous and pseudochylous material

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• Green – bile – (tested using standard chem.
screening for bilirubin)
• Neutrophil – peritonitis

• Psammoma bodies – has concentric striations

of collagen-like material can be in benign
conditions and also associated with ovarian and
thyroid malignancies

• Low Glucose – tubercular peritonitis,

malignancy
• Elevated amylase – pancreatitis,
gastro-intestinal perforation
• Elevated ALP – highly diagnostic of intestinal
perforation
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• Elevated urea or creatinine – ruptured bladder or
accidental puncture of bladder during paracentesis

• CEA and CA125 – for identifying primary source of

tumors producing the exudates
*presence of CA 125 antigen with negative CEA –
suggests that the source is from the ovaries,
fallopian tubes or endometrium

• G/S and culture – bacterial peritonitis

• AFS – tubercular peritonitis

• Adenosine deaminase – tubercular peritonitis

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AMNIOTIC FLUID
• formed from the metabolism of fetal
cells, transfer of water across placental
membrane and later stages of
development of fetal urine

• by the time fetal urine production

occurs, the fetus begins swallowing the
amniotic fluid in an amount
approximately equal to the urine output

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• build up of amniotic fluid to a volume of
500 to 2500 ml is produced primarily by
increased cell metabolism and placental
water exchange (other books: 500-1500
ml)

• Volume
-maintained by fetal swallowing of fluid
-200-500 ml/day of amniotic fluid

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• Purposes of amniotic fluid

1. to protect the fetus

2. allow fetal movement and growth
3. maintain an even temperature
4. to participate in fetal
biochemical homeostasis

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• 2 Procedures of
Collection

1. transabdominal –
preferred method

2. vaginal – greater
risk of infection

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• Amniocentesis

-needle aspiration of amniotic fluid by

a doctor
-max of 30 ml is collected in sterile
syringes
-performed at 14th week of gestation

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• Utilities of Analysis

1. Predict severity of HDN in Rh Erythroblastosis

Fetalis

2. Assess IntraUterine Fetal Maturity before

Ceasarian Section to assure delivery of an
infant with good chance of survival

3. Detect fetal sex in pregnant women

heterozygous for x-linked recessive disorders
such as Hemophilia and Muscular Dystrophy

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4. Discover Genetic Fetal Disorders in Genetic
High Risk patients e.g. Down’s Syndrome,
Pompe’s and Tay-Sachs, Lesch-Nyhan
Syndrome (def. in HGPRT)

5. To assess Pulmonary Maturity

6. Predict spontaneous onset of labor

7. Determine fetal jeopardy e.g. Rh

sensitization, Diabetes Mellitus,
Pre-Eclampsia and Eclampsia
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Terminologies

• Hydramnios – increase amniotic fluid or

polyhydramnios; an indication of fetal
distress and often associated with neural
tube disorders

• Acute hydramnios – increase amniotic

fluid associated with rapidly developing
fetal edema and in Hydrops Fetalis and
Fetal Heart Failure
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• Chronic hydramnios – associated with
fetal disorders by poor fetal swallowing,
maternal diabetes and toxemia of
pregnancy

• Oligohydramnios - <300 ml of amniotic

fluid volume
-would cause fetal distress and indicate the
need for measurement of fetal well being
-causes increased fetal swallowing, urinary
tract deformities and membrane leakage

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• Color: colorless to slight to moderate
turbidity

a. yellow orange – bilirubin

b. red – traumatic tap, abdominal trauma or

intra-amniotic hemorrhage

c. brown – severe hemolysis ; darkness may

indicate fetal death

d. green – meconium

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• Fetal Lung Maturity
1. L/S Ratio - reference method
– Lecithin
• primary component of the surfactant
(phospholipids, neutral lipids and protein) that
make-up the majority of alveolar lining

• account for alveolar stability

• relatively low and constant rate until the 35th

week of gestation, at which time noticeable
increase in its production is observed, resulting
in stabilization of the fetal lung alveoli

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– SphigoMyelin

• is a lipid that is produced at a constant

rate after about 26 weeks gestation

• serves as a Control on which to base the

rise of Lecithin

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• Pattern of Phospholipids during
Gestation:

-up to 26th week of gestation - the amount of

Lecithin is < the amount of Sphingomyelin

-36th week - amount of both phospholipids is

about equal

-after 36th week – amount of Lecithin

increases markedly; sphingomyelin remains
relatively constant or may decrease

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• L/S ratio >2 – fetal lung maturity

• L/S ratio about 1.5

– infant develops some degree of RDS
(respiratory distress syndrome) but will
survive with adequate treatment
-Lecithin is deficient

*falsely elevated results occur in fluid

contaminated by blood or meconium
because both have L/S ratio of at least
2.0
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2. Phosphatidylglycerol/
Phosphatidylinositol
(performed by TLC together with LS)

- maybe used in place of L/S ratio when

fluid is contaminated because these are
not found in blood or meconium

-normally parallels that of lecithin but

delayed in diabetic mother

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3. Aminostat-FLM

– uses antisera specific for phosphatidyl

glycerol and not affected by specimen
contamination with blood and
meconium

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4. Foam/Shake Test (Mechanical Method)

– done by shaking amniotic fluid with 95%

ethyl alcohol for 15 secs and observe
continuous bubble formation that should
persist for 15 mins.

– Principle*

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5. Foam Stability Index

– amniotic fluid with varying amount of 95%

ethanol (gradient of ethanol/fluid ratios
ranging from 0.42 ml to 0.55 ml in 0/01 ml
increments)

– value of >47 – mature

– value of <47 - immature

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6. Microviscosity

– presence of phospholipids decreases the

microsviscosity of amniotic fluid, this
change in microviscosity can be measured
using the principle of fluorescence
polarization by Abbott TDx analyzer

– measure polarization of fluorescent

indicator that partitions between
surfactant lipids (low polarization) and
albumin (high polarization)
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– albumin – used as internal standard
- same manner as
Sphingomyelin (constant throughout
gestation)

– >70 mg/g – FLM

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7. Lamellar Bodies and Optical Density

– the surfactants responsible for FLM are

produced and secreted by the type II
pneumocytes of the fetal lung in the form of
structure termed “Lamellar Bodies”

– lamellar bodies enter the alveolar spaces to

provide surfactant and also enter the
amniotic fluid

– therefore, the # of LB present in AF

correlates with amount of phospholipids
present in fetal lungs

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– presence of LB increases OD of AF

– examined at 650 nm (wavelength that rules

out interference from Hb but not meconium)

– OD 0.150 – correlate with a L/S ratio of >2.0

and presence of phosphatidylglycerol

– a count of 32,000 or more particles/ml

represents adequate FLM (when LB are
counted using resistance-pulse counting such
as that employed by Coulter Cell Counting
Instruments)
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• Fetal Distress

1. Bilirubin Analysis

– performed by spectrophotometric analysis

and plotted on Liley curve

– evaluates fetal distress/fetal anemia

produced by HDN

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– to prevent Hb and meconium interference,
add chloroform

– in the course of normal pregnancy, bilirubin

pigment in amniotic fluid decreases, but if
maternal antibodies that cross the placenta
destroy fetal red cells, bilirubin fails to
decline and may even further increase

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 0.6 Oxyhemoglobin
peak at 410 Bilirubin
peak at 450
A
0.5
B
S
O 0.4
R
B Ba
0.3 se
A
li
N ne
C 0.2
E

0.1

365 410 450 500 550

W A V E L E N G T H (nm)
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2. Infection of mother and fetus

-perform fluid WBC count or Leukocyte

Esterase Reagent Strip

*WBC >50/uL is indicative of infection

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3. Alpha Fetoprotein (<2.0 MoM –
multiple of the median)

-detects neural tube disorder e.g.

anencephaly, spina bifida, strangulation

-major CHON produced by fetal liver during

early gestation (prior to 18 weeks) and
found in maternal serum due to the
combined circulation and in amniotic fluid
by excretion in fetal urine

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4. Acetylcholinesterase Level

-elevated in amniotic fluid in neural tube

disorder because cholinesterase is a
component of nerve tissue

-confirmatory test after elevated AFP bec it

is more specific

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• Fetal Age
1. Creatinine Concentration

-rises as patient near term

-measured by serum methods (Jaffe’s

Reaction)

*Jaffe Reaction – quantitative, reddish color

formed when an alkaline solution is added
to a creatinine solution

-2.0 mg/dl – pregnancy over 36 weeks

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• Differentiation of Amniotic Fluid from
Maternal Urine

1. creatinine and urea are much lower in

amniotic fluid than in urine

2. creatinine does not exceed 3.5 mg/dl

and urea 30 mg/dl in amniotic fluid

3. creatinine of 10 mg/dl and 300 mg/dl

urea in urine

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 EXAMINATION OF SWEAT

• Cystic Fibrosis
-patients characteristically sweat with
much higher sodium and chloride
content

❑ Normal Sweat Chloride:

5-45 mmol/L (children)
10 mmol/L higher (adults)

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BRONCHOALVEOLAR LAVAGE
• Pneumocystis carinii (rats) – now
Pneumocystis jiroveci (human)

• BAL – can be as effective in

diagnosis of PCP (Pneumocystis
carinii Pneumonitis) as open lung
biopsy but sensitivity of test is
dependent on methods used to
collect, process and stain the
samples
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GASTRIC FLUID ANALYSIS
I. Physiology

• presence of food and fluid in the

stomach promotes the G cells to
produce Gastrin

• Gastrin (hormone) stimulates Parietal

Cells to produce acid

• HCl converts Pepsinogen (by Zymogen

chief cells) to Pepsin, which catalyzes
Protein
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• Gastric acidity results from secretion of
HCl by the Parietal Cells in the stomach

• Parietal cells are responsible for the

production of Intrinsic Factor necessary
for intestinal absorption of Vit. B12

• aside form HCl and Pepsin, gastric

secretions may also contain saliva,
mucus, acid-neutralizing chemicals and
pancreatic secretions.
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• Utilities of Analysis

1. determine whether the patient can

secrete gastric acid

2. useful in patient with PA and ulcer

3. measure amount of acid produced by a

patient with symptoms of Peptic Ulcer,
suspected Duodenal Ulcer or
Postoperative Marginal Ulcer who has a
demonstrable lesion by Roentgenography

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4. to support a hypersecretory state
characteristic of Zollinger-Ellison
Syndrome (fulminate peptic ulcer or
non-B cell tumor of pancreas)

5. determine “completeness of
vagotomy”

6. aid in differential diagnosis of gastric

from duodenal ulcer

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II. Composition

A. Hydrochloric Acid

-secreted by the parietal cells

-its major role in digestion is to provide the high

acidity necessary for the activation of pepsin from
pepsinogen

-also hydrolyzes directly to a limited extent,

polypeptides and disaccharides

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B. Electrolytes

-gastric secretion contains all the

electrolytes found in other body fluids
in a combined osmolar concentration
equal to or slightly greater than the
plasma

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C. Mucus

-complex mixture of mucoproteins and

mucopolysaccharides

-secreted by specialized cells of the :

1. gland necks in the fundus and body of stomach
2. cells of the surface epithelium
3. acinar cells of the cardia, antrum and pylorus

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D. Digestive and Non-Digestive Enzymes

1. Pepsin

-major digestive enzyme of the gastric secretion

-secreted as Zymogen and Pepsinogen (activated

by gastric acid at an optimal pH of 1.6 to 2.4)

-catalyzes the degradation of proteins

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2. gastricsin – proteolytic activity at pH 3.2,
higher than pepsin

3. rennin- weak proteolytic activity ;

coagulates caseinogen in milk

4. Gastric lipase
-important in the digestion of dietary fat
especially when pancreatic function is
not well developed (in neonates) or is
compromised (cystic fibrosis)

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5. non-digestive enzymes – LDH, AST and ALT
and ribonuclease

6. miscellaneous substances

-small amounts of serum albumin and

gamma-globulin are normally present

-intrinsic factor which promotes B12 absorption

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III. Collection

• done by physician or well trained

personnel

• Preparation of the Patient:

1. should fast for 12 hrs with no medications
during the last 24 hrs.
2. should not swallow excessive amount of
saliva during collection (saliva neutralizes
gastric acidity)
3. should be resting and relaxed

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• Methods of Collection

1. Tube or Intubation
Method
- gastric fluid is
obtained by
inserting a gastric
tube into the
stomach through
the buccal cavity
or the nasal cavity.

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• proper position:

a. sitting – for best recovery

b. lie on left side with head elevated

approximately 45 degrees

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• Kinds of Evacuation / Gastric Tubes:

a) Ewald’s or Boa’s tube

- for washing and emptying stomach in case
of poisoning
-with large diameter

b) Rehfuss tube
– for gastric and duodenal contents collection
- has metal tip, swallowed by gravity

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c) Sawyer’s tube –gastric collection; longest tube

d) Levine tube – gastric collection; rubber; has

the smallest diameter; inserted through the
nose

e) Kaslow tube – gastric collection

f) Miller Abbott – has mercurial tip

g) Lyn
h) Einbor
i) Jutte
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• Contraindications of Evacuation Tubes:

a) pregnant and severely ill patients

b) liver cirrhosis
c) stenosis or malignant tumor of the
esophagus
d) severe gastric hemorrhage
e) cardiac decompensation
f) marked arteriosclerosis
g) esophageal varices or diverticilium

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2. Tubeless Method / Diagnex Blue tests

Principle: An ion-exchange resin (amberlite

cation) that is coupled with a blue dye. Azure
A is given by mouth. In the presence of free
gastric acid (HCl), the Azure Blue is released
from its resin binding in exchange for H+.
Azure Blue is rapidly reabsorbed from the
intestines and travels into the blood, to the
kidneys, and excreted in the urine.
Appearance of azure blue is indicative that
free HCl is present in the stomach.

Note*

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• Test Meals:

- These are definite quantity and quality of

food or drug taken to stimulate gastric flow.

- They are poor gastric stimulants and have

become obsolete

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• Examples:

1. Ewald’s Test Meal

-a.k.a. Breakfast Test Meal; composed of

toasted bread, water or tea without sugar

-routinely used test meal for gastric analysis

-not advisable if lactic acid is detected

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2. Boa’s

-composed of oatmeal and small amount of salt

-Boa’s Oppler’s Bacillus- increase lactic acid
-recommended for lactic acid

• other tests for lactic acid

1. Uffelman phenol (+) canary yellow

2. Kelling 10% ferric chloride (+) deep yellow
3. Strauss ether + 10% FeCl(+) green

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3. Reigel Test Meal

-recommended for detection of achylia

and hypoacidity

-composed of broiled beef steak,

mashed potato and Bouillon soup

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4. Alcohol Test Meal

-helps in detecting regurgitation of

alkaline material from duodenum in
stomach by the change of blue to
greenish hue

-composed of ethyl alcohol and

methylene blue

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5. Histamine
– exerts unpleasant systemic effects
on blood vessels and smooth muscles

6. Histalog/ Betazole
– histamine isomer with preferential effect
on gastric secretion
- less severe systemic effect

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7. Insulin

8. Pentagastrin
- stimulant of choice resembling gastrin
- causes least troublesome and shortest-lived
side effects
- occurs with histamine administration
- has more rapid response than Histalog

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9. Others:

– Heckman’s – egg albumin, dH20 & methylene blue

– Dock’s – shredded wheat bread

– Fisher – gives higher acidity than Ewald’s

– Moter- spinach or raisin + water

– Salzer Motility Test Meal – roast beef or broiled

lamb chop, milk, rice, 1 soft broiled egg

– Stasis Meal – half cooked rice and 12 chewed raisins

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IV. Physical Examination of Gastric Juice

A. Color
• Normal – colorless or pale gray and
translucent and contains mucus

• Variations:
1. green – old bile
2. yellow – fresh bile
3. red – blood
4. coffee ground – old blood
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B. Volume
– Fasting State – 20-50 ml
– After a test meal – 20-80 ml
– Chemical stimulant – 45-150 ml

• Increased in:
– hypomotility
– pyloric obstruction
– Zollinger-Ellison Syndrome

• Decreased in:
– gastric hypermotility

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C. Odor
• Normal: usually odorless or slightly sour

• Variations:
1. foul odor – necrotic lesions
2. fecaloid – intestinal obstruction and regurgitation
3. rancid – increased lactic acid ; benign stenosis
4. ammoniacal – uremia
5. putrid – malignant stenosis

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D. Viscosity and Character

• Normal: it separates into 3 layers on

standing, namely:
– top layer – mucus (responsible for
viscosity)
– center layer – opalescent fluid
– bottom layer – sediments

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E. pH: 1.6-1.9

F. Food Particles – any recognizable

food particles from previous meal
indicates obstruction, may give
fecaloid odor

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G. Bile
– Normal: minute particles due to strain
while tube is in the stomach
– Abnormal: large amounts – hyperintestinal
obstruction

H. Blood
– trauma and hemorrhage
– maybe caused by lesions in the stomach

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V. Chemical Examination
A. Total Acidity – HCl + combined acids
NV: 40-70 mEq/L

1. phenolphthalein test
• Rgt: alcoholic phenolphthalein
• End color: deep pink

2. Topfer’s test
• Titrant: 0.1 N NaOH
• Indicator: phenolphthalein
• End color: salmon pink
• NV: 50-75 degrees
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b. Free HCl
NV: 20-40 mEq/L

1. Topfer’s
• Titrant: 0.1 N NaOH
• (+) canary yellow

2. Boa’s
• Titrant: resublimed resorcinol, canesugar
alcohol
• (+) rose red

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 3. Gunzberg’s
• Rgt: phloroglucin, vanillin, 95% ethyl
alcohol
• (+) purple red

C. Free Acidity – free HCl, organic

acids and acid salts
– Rgt: sodium alizarin
– (+) violet color with bluish tinge

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VI. Microscopic

• pus cells or WBC – stomach abscess, chronic gastritis,

gastric ulcer

• rbc – ulcer, tauma

• yeast cells – in cases of fermentation because there

is a large amount of food left

• bacteria – g+ mostly

• food residue – pyloric obstruction

• parasites

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• Clinical Significance

– Hyperchlorhydria – increased free HCl (above

60 mEq/L); seen in peptic ulcer

– Hypochlorhydria – decreased free HCl; seen

in chronic gastritis, gastric ulcer and
carcinoma of the stomach

– Achlorhydria – absence of free HCl; seen in

Pernicious Anemia, Advanced Gastric Ulcer
and Pellagra-Niacin Deficiency

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• Notes:

1. Lactic acid
-normally absent however maybe seen in small
amounts from the fermentation of CHO or
from foods like sour milk

-present in gastric cancer

-associated with achlorhydria and presence of

Boas-Oppler bacilli

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2. Pernicious Anemia and some cases of
gastric CA

- can’t produce gastric acidity

-pH doesn’t fall below 6.0 (anacidity)

-no response to stimulation

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FECALYSIS
• Utilities of Analysis

1. to detect the presence of specific disease

agents such a intestinal parasites

2. detect malfunction in GIT, Liver and pancreas

3. investigate GI bleeding

4. evaluate steatorrhea

5. clue for medical and surgical diagnosis

6. screen colorectal cancer

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• Composition

– ¾ water

– ¼ solid: 50% bacteria; 10-20% inorganic

matter and fats; 2-3% protein; 30%
undigested roughage (course food) and
sloughed epithelial cells

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• Preservation

1. physical – ref

2. chemical:
formalin
95% ethanol
glycerol in NSS
Merthiolate Iodine Formaldehyde
Polyvinyl Alcohol

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• Collection
– Cammidge – scrape stool from diapers
– Jelliffe – insert thick walled glass in rectum
of pediatric patients

I. Macroscopic/Physical Exam
A. Quantity: 100-250 gms/day
– Increase CHO – increase output
– Increase meat – decrease output

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B. Color
• Normal: Light to dark brown
• Causes of color:
– stercobilin
– urobilin
– Hydrobilin

• Variations:
– yellow – milk diet, corn meal, santonin, rhubarb and
fats

– green – spinach, calomel, cooked green chlorophyll

from vegetables, unchanged biliverdin
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– clay/putty – absence of urobilin in case of liver
obstruction, excess fats in pancreatic disease,
barium, and fibrocystic disease of pancreas

– bright red – bleeding in lower GIT (pathologic),

pyridium compounds, rifampin and undigested beets
and tomatoes

– dark red/chocolate brown – excess urobilin, coffee,

cocoa, chocolate, blackberries and cherries and
Hemolytic Anemia

– black/tarry – after taking iron, bismuth or charcoal

or digested blood, suggest upper GIT bleeding

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C. Odor
• Normal: Foul to offensive due to the following:
• skatole
• indole
• butyric acid

• Variations:
– putrid – found in ulcerated & malignant tumors of
lower bowel

– extremely foul – indicates putrefaction due to

undigested CHONs; usually associated with alkaline
reaction of feces and bacterial contamination

– sour/rancid – gas formation and fermentation of

CHOs; due to unabsorbed fatty acids

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D. pH Reaction

• Normal pH: neutral / slightly alkaline

/ slightly acidic

– CHO fermentation – acidic feces

– CHON fermentation – alkaline feces

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E. Form and Consistency
• Normal: soft to well-formed

• Variations:
– watery –diarrhea, intestinal tract irritation

• “pea-soup” – typhoid fever

• “rice-water” –cholera

– goat droppings/scybalous – constipation,

decrease fluid intake
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– flattened/ribbon-like – spastic colitis, obstruction in
lower colon, syphilis, stricture

– gaseous – excessive CHO fermentation

– unformed – lack of proper absorption by the large

intestine

– bulky/frothy – steatorrhea

– small caliber – cancer, ulcer, tumor

– large caliber – Hirschprung’s disease (massive

enlargement of intestine)

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F. Presence of Mucus
• Normal: trace amounts (large intestine)
Abundant (small intestine)

• Clinical Significance:
1. excessive irritation/inflammation of
intestinal wall
2. dysentery

• *Hecht Test (staining method)

– Reagent: 2% brilliant green & neutral red
– Result: light red color against green
background; nuclei and cell membranes
appear reddish violet
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G. Presence of Blood
– *bleeding in excess of 2ml/150 grams stool -
pathologically significant

H. Presence of Pus cells

– Normal: insignificant numbers are present
– 3/hpf – indicative of invasive condition

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II. Chemical Method
A. Fecal Fats
• Normal: 5 grams/day (fatty diet)
1-4 grams/day (fat-free diet)

• Main Source: Dietary

• Other sources: bacterial metabolism,

GI excretions; cellular desquamations

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• Neutral fats (TAG) – stains with Sudan III
& appear as large orange-red droplet

• Fatty acid salts (soaps) and fatty acids –

don’t stain with Sudan III; mixed with HAc
and heated
– Normal: 100 small droplets, <4u/hpf
– sl. Increased: 1-8 u
– increased: 6-75 u

• cholesterol – stained by Sudan III after

heating and forms crystal when it cools
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• *Steatorrhea
– increased of fats in feces indicating
pathological condition
- >60 droplets/hpf of large orange red (neutral
fats)

• Indicates:
– deficiency of lipase in Fibrocystic Disease of
Pancreas
– deficiency of bile salts in Obstructive
Jaundice
– Lymphatic Obstruction (in abdominal TB)
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-examined using
a. Sudan III or IV (deep red or orange) or
Oil Red O stain

b. Van de Kamer Method – measure total

Fat
Specimen: 3 days output
NV: 4-6% ingested fats

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B. Occult Blood – screening procedure for
early detection of colorectal CA
– Principle: pseudoperoxidase activity of
hemoglobin

Hb + H2O2 + Benzidine Blue color

(pseudo Orthotolidine
peroxidase Guaiac
activity)

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• False (+) – turnips, raw broccoli,
cauliflower, radish, bananas, apples,
sardines, salmons, fish in general,
aspirin, aspilet (promote GI bleeding),
red meat, horseradish, melons

• False (-) – vit. C, iron therapy, low-bulk

diet

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• Tests:
– Benzidine – too sensitive and
carcinogenic
– O-Toluidine – too sensitive
– Guaiac’s test – preferred for routine
testing
– Hemoccult II kit – for Hb
– Hemoquant – converts Hb to porphyrin
– Immunologic test – specific for human
Hb

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C. Bile Pigments
– oxidation test
– Gmelin’s test
– Fouchet’s

D. Fecal Urobilinogen and Urobilin

– Schwartz-Watson
• (+) urobilinogen – pink color
• (+) urobilin – greenish fluorescence

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E. APT test
-distinguish between presence of fetal blood
or maternal blood in infant’s stool or
vomitus

Reagent: NaOH (fetal Hb is resistant to both

alkali and acid)
– Retain reddish to pink- fetal
– Yellow to brown - maternal

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F. Trypsin (protein digesting enzyme)
-uses xray film with gelatin
– (+) clear
– (-) opaque
-detects only severe cases of pancreatic
insufficiency

• False (-) – intestinal degradation of trypsin,

possible presence of trypsin inhibitors

• False (+) – proteolytic activity of bacteria

enzymes

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G. Fecal Chymotrypsin

– more resistant to intestinal degradation

and is more sensitive indicator of less
severe cases of pancreatic insufficiency

– remains stable in specimens for up to 10

days at room temperature

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H. Elastase I

– isoenzyme of the enzyme elastase and is

the enzyme form that the pancreas
produces

– present in high concentrations in

pancreatic secretions and is strongly
resistant to degradation

– measured by immunoassay and provides a

very sensitive indicator of pancreatic
insufficiency
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I. CHO Metab

Copper Reduction Test

a. Clinitest Tablet + 1 part stool
emulsified in 2 parts water
Result: 0.5 g/dl – indicative

Confirmed by:
D-xylose
Lactose Tolerance Test

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III. Microscopic Exam
a. Parasites
b. Bacteria
c. Crystals: e.g. triple phosphate,
cholesterol, hematoidin, bilirubin,
CaOx,Charcot-Leyden

d. Cellular Elements
– Epithelial cells – few; increase during GIT
irritation

– WBC – few ; increased in bacillary

dysentery, ulcerative ulceration
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• *>3/hpf neutrophils – can be indicative of
invasive condition

• *Lactoferrin Latex Agglutination test – for fecal

leukocytes and remains sensitive in refrigerated
and frozen specimens; the presence of
lactoferrin (a component of granulocyte
secondary granules) is indicative of invasive
bacterial pathogen

• RBC – none; seen in hemorrhoids, parasitic

infections, lower GIT

• Macrophage – increase during ulcerative colitis,

bacillary dysentery, parasitic infection
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e. Miscellaneous Structures

– food remnants

– fungi and yeast cells

– starch granules

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– muscle fibers – presence of undigested
striated muscle is helpful in diagnosis and
monitoring of patient with Pancreatic
Insufficiency

• *increased amount (>10 significant undigested) –

biliary obstruction, gastrocolic fistula, pancreatic
insufficiency

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undigested

partially digested 10% alcoholic eosin (red)

well digested

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SPUTUM ANALYSIS
• Sputum
– secreted by the “Goblet Cells” of the
Bronchial lining and the mucus-secreting
glands of the bronchial tree

– exudates that is formed during tracheal,

bronchial, and pulmonary infection

– mixture of plasma, electroytes, mucin, and

water

– must be produced from the lower respiratory

tract
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• Characteristics of Sputum

1. visco-elastic
2. consistency depends on glycoprotein
molecular structure and degree of
hydration
3. viscosity depends on sialic acid
4. 95% water, 5% solid
5. solid particles are increased in
inflammation

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• Utilities of Analysis

– detect neoplastic diseases of the lungs and

other respiratory organs

– detect occurrence of infections e.g. TB,

upper respiratory tract infections

– detect other respiratory diseases e.g. allergic

reactions (asthma); inflammation (pulmonary
lesion)

– guidance in selection of appropriate therapy

for disease agent
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• Methods of Collection

– expectoration
• first morning – ideal because it represents
accumulation secreted overnight
• sputum induction

– bronchial lavage

– heated aerosol technique – especially for infants

and patients with pulmonary lesions

– throat swab – for babies

– tracheal aspirate – debilitated patients

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• Preservation
– refrigeration – preserve T. bacilli
– formalin – fix/kill bacteria

• To induce cough
– 10% NaCl (aerosol) – must be sterile
– 10% propyleneglycol in saline solvent

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• Contraindications
– uncooperative patient
– hemorrhagic diathesis
– cardiac arythmias
– severe arythmias

• Major Complications
– bleeding
– para-tracheal infections
– subcutaneous emphysema

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I. Macroscopic
A. Volume – dependent
on the number of
active secreting
mucus cells, type and
severity of the • *Increased in
disease 1. bronchiectasis
2. lung abscess
• Decreased in 3. edema
1. early pneumonia 4. gangrene
2. bronchial asthma 5. TB
3. acute bronchitis 6. lung cancer

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B. Color
• Normal: colorless and translucent or
whitish to faint yellow and orange to
purulent

• Variations:
1. white/yellow - when pus is present
a. pulmonary TB
b. chronic bronchitis
c. lobar pneumonia

2. gray – pus/ epithelial cells are present

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3. yellowish green
a. advance TB
b. chronic bronchitis

4. bright green (bile is present)

*due to breakdown of neutrophils releasing
the Verdoperoxidase
a. jaundice
b. caseous pneumonia
c. Pseudomonas infection
d. rupture of liver

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5. red/bright red
• due to fresh blood
• presence of blood streaks indicate Pulmonary TB

6. prune juice
a. pneumonia
b. chronic cancer of lungs

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7. anchovy/rusty (old blood)
a. lobar pneumonia
b. TB
c. Pulmonary gangrene
d. hemorrhage from lung due to pulmonary
infarction

8. brown – Congestive Heart Failure

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9. olive green/grass green – chronic
cancer

10. black
– due to dust, dirt, carbon or charcoal
– seen in heavy smokers
– Anthracosis

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C. Odor
• Normal: odorless

• Variations:
1. foul and putrid due to anaerobic infection
of lungs
• lung gangrene
• necrotizing tumors and lesions
• cavitary TB
• lung abscess

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2. sweetish
• bronchiectasis
• bronchomoniliasis
• TB with cavities

3. fecaloid
• necrosis
• rupture subphrenic
• liver abscess

4. cheesy – in necrosis or malignant tumors

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D. Consistency
• Normal: watery
– serous – generally frothy ; seen in
pulmonary edema

– mucoid
• acute bronchitis
• whooping cough
• asthma

– purulent – plenty of pus

• lung abscess
• lung gangrene

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 – seropurulent – transparent/thin

– mucopurulent – thicker/heavier

– sanguinous/bloody

– rusty & tenacious

• lobar pneumonia
• bronchomoniliasis

E. pH: 6.5-7.0

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F. Specific Gravity
– mucoid – 1.004-1.008
– purulent – 1.015-1.060
– serous – 1.037 or higher

G. Features
1. cheesy masses
-fragments of necrotic tissues
-seen in pulmonary gangrene, pulmonary TB and
lung abscess

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2. Dittrich’s Plugs

-yellowish or gray caseous matter about the

size of a pinhead to navy bean that gives a
foul odor when crushed
-composed of fatty acid, fat globules,
crystals, bacteria
-comes from the bronchi and bronchioles
-seen in Bronchiectasis, bronchitis,
bronchial asthma

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3. Curshman’s Spirals

-whitish to yellow waxy, spirally twisted

mucoid strands frequently coiled into little
balls
-consists of epithelial cells, wbc, chiefly
eosinophils and sometimes Charcot-Leyden
crystals
-seen in acute bronchitis and chronic
pulmonary TB

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4. Pneumoliths / Broncholiths / Lung Stones

- calcification of infected and necrotic pulmonary

tissue within bronchus cavity
-seen in TB, Histoplasmosis

5. Casts/ Bronchial Casts

-branching tree-like cast from bronchioles

-white or gray; mainly composed of fibrin
-seen in lobar pneumonia

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6. Parasites
• E. granulosus
• T. canis
• P. westermani
• A. lumbricoides

7. Mycetomas – rounded masses of fungal

debris (e.g. Aspergillus); opportunistic fungi

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II. Microscopic

1. Elastic Fibers

-from walls of alveoli and bronchioles that are

shed off during coughing out process

-denotes destructive disease of lungs

-highly refractile

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2. crystals

A. Charcot-Leyden Crystals

-most significant

-hexagonal in shape

-derived from eosinophil degradation

-stain black in hematoxylin and red with eosin

-pointed at both ends; needlelike; colorless

-seen in Bronchial Asthma (also Creola bodies)

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• Creola Bodies

-bronchial epithelial cells that

gather in larger clusters and have a
vacuolated cytoplasm with ciliated
borders

-seen in Bronchial Asthma

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B. Hematoidin

-rhombic shaped crystals

-seen in pulmonary infarction and lung abscess

C. Cholesterol

-broken edge stair; notched plate

-seen in lung abscess

D. Fatty acids

-colorless needlelike crystals in sputum

-seen in lung abscess
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3. pigmented cells – endothelial leukocytes
taking up pigmented granules

• Kinds:
a. Carbon-Laden or Dust Cells – contains black
granules ; anthracosis

b. Heart Failure Cells / Siderocytes

• large mononuclear cells which contain the
pigment hemosiderin
• due to poor compensated lung disease/
congestive heart failure

c. Asbestos
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4. Myelin Globules

5. yeast – budding forms seen in

antibiotic treatment

6. Fungi and molds

– C. albicans
– C. immitis
– C. neoformans
– actinomyces

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7. parasites

– E. histolytica
– T. hominis
– P. westermani
– Hookworm
– S. stercoralis

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• Stains

– Wright-Giemsa – wbc differential

– Gram Stain / AFS (Ziehl Nielsen)

– India ink

– Pap stain – for neoplastic/ malignant cells

– crystal violet – bronchial epithelium

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End of Other Body fluids …
perc_kmh_06