CLINICAL MICROSCOPY Sir Carl Leoneill A.

Baroma, RMT, DTA, MPH

CSF & SEMEN

CSF

I Formation and Physiology

II Specimen Consideration
III Gross Examination
IV Microscopic Parameters
V Chemistry Tests
VI Other Tests

I. CSF FORMATION AND PHYSIOLOGY

Cerebrospinal fluid
• Imperfect ultrafiltrate of plasma produced in the
choroid plexuses within the ventricles of the brain at
a rate approximately 500 mL per day maintaining
total volume 90-150 mL/day for adults and 10-60 mL
B. Layers of the meninges in the spinal cord
for neonates
• 3rd major body fluid
FORMATION
 CSF has particles not seen in urine; not thoroughly
selective • CSF is produced in the choroid plexuses of the
FUNCTIONS: two lumbar ventricles and the third and fourth
• Supply the nutrients to the nervous system ventricles.
• Remove metabolic waste • Blood–brain barrier
• Produce a mechanical barrier to cushion the brain o is the tight-fitting structure of the endothelial cells
and spinal cord against trauma (Protection) in the choroid plexuses that prevent the passage
Meninges of many molecules
• Lines the brain and spinal cord  Blood-brain barrier prevents the passage of excessive
• Composed of three layers molecules; since an increase of certain molecules or
o Dura mater (outer layer) particles would indicate an abnormality or malignancy
 Counterpart of the glomerulus in the CSF
▪ lines the skull and vertebral canal
• Rate: 500 mL/day or 20 mL/hr
o Arachnoid mater (Spiderweb-like)
• TCV (Total Circulating Volume)
▪ filamentous inner membrane
o Neonates: 10-60 mL/day;
 Subarachnoid space – where CSF flows o Adults: 90-150 mL/day
o Pia mater (innermost layer)
▪ lines the surface of the brain and spinal cord II. SPECIMEN CONSIDERATIONS

PHYSIOLOGY SPECIMEN COLLECTION

A. Methods of collection:
• Lumbar Tap
• Cisternal puncture, lateral cervical puncture or
ventricular cannulas
B. Volume collected

A. Layers of the meninges in the brain

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SPECIMEN COLLECTION 4. BLOODY

CSF ORDER OF DRAW • Cause: RBC (↑ >6,000/uL)
• Major Significance:
Purpose Preservation
• Traumatic Tap (Puncture of blood vessel)
TUBE 1 chemical and serologic tests Frozen • Intracranial Hemorrhage (Bleeding within the
braincase)
TUBE 2 microbiology Room Temperature
TRAUMATIC TAP VS. INTRACRANIAL HEMORRHAGE
TUBE 3 Hematology Refrigerated
Distribution of Blood Uneven Even
TUBE 4 Microbiology/ Serology Clot Formation + -
Supernatant Clear Xanthocromic
If only one CSF tube only: Microbiology → HEMATOLOGY →
Erythrophages - +
CHEMICAL AND SEROLOGIC TESTS
D-dimer Negative Positive

5. CLOTTED
• Cause: Due to protein and clotting factors
• Major Significance:

6. XANTHOCHROMIC (YELLOW/PINK/ ORANGE)

• Indicates the presence of RBC degeneration products
or other pigments

SUPERNATANT ASSOCIATED
COLOR DISEASE/DISORDER
• RBC lysis
• Hemoglobin breakdown products
(oxyhemoglobin)

• RBC lysis
• Hemoglobin breakdown products
III. GROSS EXAMINATION (bilirubin)
• Hyperbilirubinemia
• CSF protein >150 mg/dL (1.5 g/L)
A. APPEARANCE
1. NORMAL • RBC lysis (Heavy hemolysis
2. HAZY/TURBID/MILKY/CLOUDY • Hemoglobin breakdown products
3. OILY • Hypervitaminosis
4. BLOODY
5. CLOTTED • Hyperbilirubinemia (biliverdin)
6. XANTHOCHROMIC • Meningeal melanoma
7. PELLICLE

B. VISCOSITY 7. PELLICLE FORMATION

1. NORMAL • Causes:
2. CLINICAL SIGNIFICANCE OF VISCOUS CSF
3. METASTATIC MUCIN PRODUCING • Major Significance
ADENOCARCINOMAS
4. CRYPTOCOCCAL MENINGITIS B. VISCOSITY
5. LIQUID NUCLEUS PULPOSUS
• Normal
A. APPEARANCE • Clinical Significance of Viscous CSF
o Metastatic mucin producing adenocarcinomas
1. NORMAL CSF APPERANCE o Cryptococcal meningitis
• Colorless, crystal clear o Liquid nucleus pulposus

2. HAZY/TURBID/MILKY/CLOUDY IV. MICROSCOPIC PARAMETERS

Cause:
• ↑ WBC (>200/ uL)
CSF CELL COUNT
• ↑ RBC (>400/uL) 1. Total Cell Count
• ↑ LIPIDS AND PROTEIN 2. WBC Count
• ↑MICROORGANISMS 3. RBC Count
4. Differential Count
3. OILY
• Major Significance: Radiographic Contrast Media

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1. TOTAL CELL COUNT o -8 mg/dL Total protein concentration for every

• Dilution (using NSS), counting and calculation of the 10,000 RBCs/uL (Henry)
number of uL are done using the same procedure as o -1 mg/dL protein concentration for every 1,200
WBC count RBCs/uL (stras)
• Fuchs-Rosenthal-type chamber may be used • To correct WBC Error:
• An improved Neubauer counting chamber is
routinely used for performing CSF cell counts.
• Cells are counted in the four corner squares and the
center square on both sides of the hemocytometer
Neubauer Counting Chamber
4. DIFFERENTIAL COUNT
a. Concentration technique
• Filtration
o for large volumes of specimens
• Cytocentrifugation
o associated with decreased cellular distortion and
requires addition of _____ to not increase yield
and minimize distortion
b. Smear preparation
c. Examination
• WBC differential count

PREDOMINANT CELLS IN CSF

1. Lymphocytes and Monocytes
2. Neutrophils
3. Erythrophages
2. WBC COUNT 4. RARE: Blast forms, Macrophage, Plasma cells
Procedure:
1. Dilute (3% Acetic acid, NSS)
2. Charge Both side of Hemocytometer
LYMPHOCYTE AND MONOCYTES
• NORMAL VALUES
3. Count in 5 large squares per side
• CLINICAL SIGNIFICANCE:
4. Calculation
Predominant Cells Seen in CSF
Major Clinical Microscopic
Type of Cell
Significance Findings
• Normal • All stages of
Lymphocytes • Viral, tubercular, and development
fungal meningitis may be found
• Multiple sclerosis
• Normal Values:
• Bacterial meningitis • Granules may
• Adult:
• Early cases of viral, be less
• Neonates: prominent
tubercular, and
Neutrophils • fungal meningitis
than in blood
CSF DILUTION • Cerebral hemorrhage • Cells
APPEARANCE DILUTION
AMOUNT OF AMOUNT OF disintegrate
SAMPLE DILUENT rapidly
Clear UNDILUTED
• Normal • Found mixed
Slightly Hazy 1:10 270 uL • Viral, tubercular, and with
Monocytes
fungal meningitis lymphocytes
Hazy 1:20 250 uL
30 uL • Multiple sclerosis
Slightly cloudy 1:100 2,970 uL • RBCs in spinal fluid • May contain
Slightly bloody 1:200 5,970 uL phagocytized
Cloudy, RBCs
1: 10,000 1 mL 9 mL appearing as
Bloody, Turbid
empty
Macrophages • vacuoles or
3. RBC COUNT ghost cells,
• Done in cases of Traumatic Tap hemosiderin
• TCC – WBC ct granules, and
• To correct WBC error: • hematoidin
o -1 WBC for every 700 RBC seen crystals

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• Acute leukemia • Lymphoblasts

Blast forms , myeloblasts,
or monoblasts
• Disseminated • Resemble
Lymphoma lymphomas lymphocytes
cells with cleft
nuclei
• Multiple sclerosis • Traditional
• Lymphocyte and classic
Plasma cells reactions forms seen
• Reactive
lymphs
• Diagnostic • Seen in
Ependymal, procedures clusters with
choroidal, distinct nuclei
and spindle- and distinct
shaped cells cell
• walls
• Metastatic • Seen in
carcinomas clusters with
Malignant • Primary central fusing of cell
cells nervous system borders and
carcinoma nuclei

V. CHEMISTRY TESTS
1. TOTAL PROTEIN
• Normal values:
• ↑ in Cases of:
• ↓ in cases of:
• Major CSF protein: Albumin
• 2nd most abundant
• Alpha globulins: Haptoglobin, ceruloplasmin
• Beta-globulins: Beta2-transferrin (TAU) protein
• Gamma globulins- IgG and some IgA
• Not found in CSF: IgM, Fibrinogen, Lipids

𝑚𝑔
𝐶𝑆𝐹 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 ( )
A𝑙𝑏𝑢𝑚𝑖𝑛 𝑖𝑛𝑑𝑒𝑥 = 𝑑𝐿
𝑔
𝑆𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 ( )
𝑑𝐿
CSF ELECTROPHORESIS
• Done in conjunction with serum electrophoresis
𝑚𝑔
𝐶𝑆𝐹 𝐼𝑔𝐺 ( ) • For the detection of oligoclonal
𝑑𝐿
𝑔 bands in the gamma region
𝑆𝑒𝑟𝑢𝑚 𝐼𝑔𝐺 ( )
I𝑔𝐺 𝑖𝑛𝑑𝑒𝑥 = 𝑑𝐿 • The presence of 2 or more
𝑚𝑔
𝐶𝑆𝐹 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 ( ) oligoclonal bands in CSF but not
𝑑𝐿
𝑔 in serum is valuable diagnosis of
𝑆𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 ( )
𝑑𝐿 MS
• Other conditions with oligoclonal
banding in CSF but not in serum
are: Neurosyphilis, encephalitis,
neoplastic disorders, Guillain
barre syndrome

MYELIN BASIC PROTEIN

• Protein component of the lipid-protein complex that
insulate the
• nerve fibers
• Presence of MBP in CSF indicate destruction of
myelin sheath
• Used to monitor the course of MS

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2. GLUCOSE 5. CSF ENZYMES

• Normal values: • Lactate dehydrogenase
• ↑ in Cases of: • CK-BB – increases about 6 hours following ischemia
• ↓ in cases of: and anoxia
• Normal in cases of: • Adenosine deaminase – increased in tubular
meningitis
3. LACTATE • Lysozyme – increased in patients with both bacterial
• Inversely proportional to glucose and tubercular meninges
• Normal values:
• ↑ in Cases of: LACTATE DEHYDROGENASE
• Normal in cases of: • Serum LDH
o Normal: LD 2 > 1 > 3 > 4 > 5
4. GLUTAMINE o Abnormal: LD 1 > 2 > 3 > 4 > 5
• Product of ammonia and alpha-ketoglutarate
• Indirect test for the presence of excess ammonia in • CSF LDH
CSF o Normal: LD 1 > 2 > 3 > 4 > 5
• Normal values: o Neurological M: LD 2 > 1
• ↑ in Cases of: o Bacterial M: LD 5 > 4 > 3 > 2 > 1
SPECIMEN CONSIDERATIONS
• GLUCOSE
• LACTATE

MAJOR LABORATORY REULTS FOR THE DIFFERENTIAL DIAGNOSIS OF MENINGITIS

Bacterial Viral Tubercular Fungal

Predominant Lymphoctes and Lymphocytes and

Neutrophil Lymphocytes
WBC Monocytes Monocytes

Moderate to markedly Moderate to markedly

Protein Markedly elevated Moderately elevated
elevated elevated
Glucose Marked decrease (<40
Normal Decreased (<45 mg/dL) decreased
mg/dL)
Lactate > 35 mg/dL Normal >25 mg/dL > 25 mg/dL

Agents: Enteroviruses Agent: M. tuberculosis Agent: C. neoformans

+ Gram stain ✓ Poliovirus + AFB stain + gram stain = classic
+ culture + Pellicle weblike clot starburst pattern
Other tests: ✓ Echovirus
+ Limulus lysate formation after 12-24 hr + india ink = capsule
✓ Coxsackievirus refrigeration (unstained); background
(black)

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VI. OTHER TESTS

Gram stain
✓ S. pneumonia
✓ H. influenzae
✓ E. coli
✓ N. meningitidis

Lymulus Lysate
• Detects Gram – bacterial endotoxin in body fluids and
surgical instruments
• Regent: Blood of Horseshoe crab REASONS FOR SEMINAL FLUID ANALYSIS
• Principle: in the presence of endotoxin, the • Fertility testing
amoebocytes (WBCs) will release lysate (protein), + • Postvasectomy semen analysis
clumping/ Clot formation • Forensic analysis (alleged rape)
2. COMPOSITION OF SEMEN
SEROLOGY
• ELISA COMPOSITION OF SEMEN
• VDRL (Venereal disease research laboratory test)
• 1. Seminiferous tubules (testes)
• FTA- ABS (fluorescent treponemal antibody o Spermatogenesis site
absorption) 5% Spermatozoa o Sertoli cells
• CAT • 2. Epididymis
• Bacterial antigen test • Seminal vesicles
60-70% Seminal o Provide nutrients for sperm & fluid
SEMEN Fluids o Rich in FRUCROSE
o Flavin
• Acidic fluid
20-30% Prostate • Contains ACP, zinc, citric acid & other
I Physiology fluid enzymes
II Composition of Semen • For Coagulation & Liquefaction
III Specimen Collection
5% Bulbourethral • Secretes thick alkaline mucus that
IV Semen Analysis
neutralizes acidity from the prostatic
A. Macroscopic Examination gland
secretions & vagina.
B. Sperm Concentration
C. Sperm Counting
D. Sperm Motility 3. SPECIMEN COLLECTION
E. Sperm Morphology
F. Sperm Viability 1. Abstinence of 2-3 days but not >7 days
G. Seminal fluid fructose 2. Collect the entire ejaculate
H. Antisperm Antibodies Methods of collection:
I. Chemical Testing
J. Microbial Testing o Masturbation
K. Additional Tests o Coitus interruptus
o Condom method
3. Specimen should be delivered to the laboratory within
1. PHYSIOLOGY 1 hour of collection
4. Take note of the time of specimen collection, specimen
receipt and liquefaction.
5. Analysis should be done after liquefaction (usually 30-
60 minutes)
6. Specimen awaiting analysis should be kept at 37° C.
4. SEMEN ANALYSIS

A. Macroscopic Examination
B. Sperm Concentration
C. Sperm Counting
D. Sperm Motility
E. Sperm Morphology
F. Sperm Viability
G. Seminal fluid fructose
H. Antisperm Antibodies
I. Chemical Testing
J. Microbial Testing
K. Additional Tests

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A. MACROSCOPIC EXAMINATION Sperm Count

• Normal Value = ≥ 40 M/ ejaculate
MACROSCOPIC EXAMINATION • Formula: Sperm concentration x specimen volume
Gray-white, • Normal
translucent = ___________
(with musty or • Reporting:
bleach odor) • 0 =
Appearance Increased white Viscosity _________
turbidity = • 4 =
Red or brown _________
coloration =
Yellow coloration
• Normal =
_________
Normal
• ↑pH =
Volume Increased in pH
_________
Decreased in
• ↓pH =
_________

A. Macroscopic Examination

• Liquefaction
o Normal: Clotted semen must liquefy within 30-60
mins after collection
o >60 mins – deficiency on prostatic enzyme
o > 2 hours – Add equal volume of physiologic
Dulbelcco’s phosphate-buffered saline or alpha
chymotrypsin or bromelain (proteolytic enzymes)
D. SPERM MOTILITY
B. SPERM CONCENTRATION
• Normal Values:
• Factors affecting sperm concentration: o Within 1 hour = 50% Motile
o Days of sexual abstinence before collection o Quality = ≥ 2.0
o Infection
o Stress • Computer – Assisted Semen Analysis (CASA)
o Provides objective determination of both sperm
Normal value = 20-160 million/mL o Velocity & trajectory (direction of motion)
1 000 000 000 leukocytes/mL = inflammation or infection o Sperm concentration & morphology
of the reproductive organs
>1 000 000 000 spermatids/mL = disruption of
spermatogenesis

METHODS
1. Improved Neubauer Counting Chamber
o Dilution = 1:20
o Diluents: to immobilize sperm
o Formalin
o Sodium
o Saline
o Distilled water
o Tap water
2. Makler Counting Chamber
o For undiluted specimen – no dilution
o Uses heat to immobilize sperm cells

C. SPERM COUNTING

Sperm Concentration Computation

• 2 WBC squares = # of sperms counted x 100,000
• 5 RBC squares = # of sperms counted x 1,000,000

Sperm Concentration Method using Neubauer

Formula

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E. SPERM MORPHOLOGY H. ANTISPERM ANTIBODIES

1. Mixed agglutination reaction

o Detects the presence of IgG antibodies
o Semen sample + AHG + latex particles or treated
RBCs coated with IgG
o Normal = <10% motile sperm attached to the
particles.
2. Immunobead test
o Detects the presence of IgG, IgM & IgA
antibodies + location
o Demonstrate what area of the sperm (head, neck,
tail) the autoantibodies are affecting / attached.

I. CHEMICAL TESTING

Decreased
Analyte Normal value
Values Indicate
Fructose ≥ 13 umol / ejaculate ↓Seminal fluid

Neutral α- ≥ 20 mU / ejaculate Epididymis disorder

glucosidase
Zinc ≥ 2.4 umol / ejaculate
• Normal values: Citric acid ≥ 52 umol / ejaculate
o Rountine criteria ↓Prostatic fluid
Acid ≥ 200 units / ejaculate
o Kruger’s strict criteria phosphatase
• Stains for Sperm Morphology:
o Wright’s stain
o Giemsa stain J. MICROBICAL TESTING
o Papanicolau’s stain – Stain of choice
• Round cells
• Head
o WBCs and spermatids (immature cells)
o Normal
o Normal value = 1 million / µL
o Abnormal
▪ > 1 million WBCs/mL = Infection
• Midpiece
▪ > 1 million spermatids/mL = Distraption of
o Contains mitochondria
spermatogenesis
o Tail
• Test for Chlamydia Trachomatis, Mycoplasma
o Abnormal – poor motility
hominis & Ureaplasma urealycticum
F. SPERM VIABILITY/ VITALITY
K. OTHER TESTS
• Modified Bloom’s test
o Preparing a smear and counting the number of
MEDICO LEGAL TESTS
dead cells in 100 sperms Test for detection of semen:
1. Microscopic exam
o Reagents
2. Fluorescene under UV light
o Living sperms = unstained, bluish white
3. Acid phosphatase determination
o Dead sperms = red
o Normal Values: 50% more living cells 4. Glycoprotein p30 = more specific method
5. Florence test (not specific)
o Test for CHOLINE
G. SEMINAL FLUID FRUCTOSE o Reagents: Iodine crystals = KI
o (+) Dark brown rhombic crystals
• SEMINAL FLUID FRUCTOSE
6. Barbiero’s test (very specific)
• Tested within 2 hours or frozen to prevent fructolysis
o Test for SPERMINE
• Screening test
o Reagents: Saturated picric acid + TCA
• Resornicol test = (+) Orange-red color
o (+) Yellow leaf-like crystals
• a.k.a Seliwanoff’s Test
7. ABO blood Grouping
• Low fructose levels are caused by abnormalities of
8. DNA analysis
the seminal vesicles, bilateral congenital absence of
the vas deferens, obstruction of the ejaculatory duct, POSTVASECTOMY SEMEN ANALYSIS
partial retrograde ejaculation, and androgen
• Vasectonomy
deficiency
o Cutting of vas deferens so that the ejaculate will
not contain any sperm cell

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• The only concern is the presence or absence of

sperm
• Done 2 months after vasectomy & continued until 2
consecutive monthly specimens show NO SPERM

TERMINOLOGIES
Terminology Definition
Aspermia No ejaculate
Azoospermia Absence of sperm cells
Necrospermia Immotile/ dead sperm cells
Oligospermia Decreased sperm concentration

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