AUBF: ANALYSIS OF URINE AND OTHER BODY FLUIDS

LECTURE 5: CEREBROSPINAL FLUID

1st SEMESTER | S.Y. 2024-2025

Why examines CSF?

 Indicated in suspicions of:
- Encephalitis
- meningitis
- multiple sclerosis
- neurosyphilis
- subarachnoid hemorrhage

Functions of CSF
1. Produces a mechanical barrier to cushion the brain and spinal cord against trauma.
2. Supplies nutrients to the nervous tissues
3. Removes metabolic wastes

CSF Production
 90% of the CSF
- produced by the choroid plexus epithelium found in 4 ventricles (2 lateral ventricles, third ventricle, fourth
ventricle).
- 35% is produced within the third ventricle
- 23% via the fourth ventricle
- 42% from general ependymal cell filtration
 10% - from brain interstitial fluid.

Composition
 CSF- very low protein constituent and only albumin is present with a very low level of cellularity.
 Biochemistry of CSF
1. sodium and chloride - high concentration
2. magnesium - very high concentration
3. potassium, calcium and glucose - Low concentration
 CSF flows through the subarachnoid space around the brain and the spinal cord
 Reabsorbed by the Arachnoid villi/ granulations into the superior sagittal sinus

The meninges
- system of membranes which envelopes the CNS (brain and spinal cord).
- Primary function of the meninges and CSF is to protect the CNS.
Three layers
1. DURA MATER (Latin for hard mother)
- outermost layer
- Lines the skull and vertebral canal
2. ARACHNOID MATER (spiderweb-like)
- Middle layer
- Filamentous inner membrane
3. PIA MATER (Latin for gentle mother)
- Innermost layer
- Adheres to the cerebrum (brain tissue)
Volume:
- 20 mL produced every hour
- Adult:
 140-170 mL (Strasinger 4th ed)
 90-150 mL (5th & 6th ed)
- Neonates: 10 – 60 mL (4th & 5th ed)

SPECIMEN COLLECTION
Lumbar puncture: Patient is in FETAL position or LATERAL DECUBITUS position
 Site of lumbar tap: between 3rd and 4th or between 4th and 5th lumbar vertebrae
- Adults - between 3rd & 4th LV
- Children – between 4th & 5th LV
- Up to 20mL can be collected

CABAY, J.B. | BMLS3 | PLT COLLEGE, INC.

Alternative Sites of Specimen Collection in Case of
Infection or Back Deformity
1. Cisternal Puncture
- needle is placed below the occipital bone (back of the skull) or suboccipital region.
- dangerous - so close to the brain stem.
- always done with fluoroscopy
- Fluoroscopy is a type of medical imaging that shows a continuous X-ray image on a monitor, much like an X-
ray movie. During a fluoroscopy procedure, an X-ray beam is passed through the body. -
2. Shunt or Ventricular Drain: CSF may be collected from a tube or shunt.
3. Ventricular Puncture
- rarely done
- used in infants with open fontanels
- recommended in people with possible brain herniation.
- done in the OR
- A hole is drilled in the skull, and a needle is inserted directly into one of brain's ventricles.

BLOOD BRAIN BARRIER

- Used to represent the control and filtration of blood components to the CSF and then to the brain.
- Composed of capillary endothelium held together by tight junctions and choroid plexuses (specialized ependymal
cells, their connective tissues and fenestrated capillaries)
- Allows essential metabolites (such as oxygen and glucose) to pass from the blood to the CNS
- Blocks most molecules >500 Daltons
- Damage to BBB allows entrance of increased amount of proteins and coagulation factors

Specimen Collection and Handling

 Handle CSF specimen with utmost care.
 Consider it highly contagious. Always wear protective gadgets.
 CSF for centrifugation should be capped.
 Tests are performed on STAT basis (within 30 min – Alba’s) - Excess CSF SHOULD NOT BE DISCARDED until
no further use of it.
Tube Examination Storage Temperature
Tube 1 Chemical and Serologic Frozen
Tube 2 Microbiologic Room Temperature
 Hematologic: (least cellular content
Tube 3 Refrigerate
introduced by the Lumbar tap procedure).
Reserved/ Cytology: to eliminate skin
Tube 4
contamination

PHYSICAL EXAMINATION/ APPEARANCE OF CSF

 Examine fluid at bedside immediately after collection
 Record
 Normal Values
- Color: Colorless
- Viscosity: Same as Water
- Clarity: Crystal Clear
- Specific Gravity: 1.006 to 1.008
- pH: 7.30 to 7.45
- Pressure: 50 to 200 mmHg

Clinical Significance of Variation in CSF Appearance

Appearance Cause Major Significance
Crystal clear NORMAL
Hazy WBCs (200-500/µL) Meningitis
cloudy Above 500 WBC/µL Traumatic tap
milky/ turbid WBC, RBC
Microorganisms Meningitis
Protein Disorders that affect BBB
Production of IgG w/in the CNS
Xanthochromic Carotene Increased serum levels
Hemoglobin Old Hemorrhage
lysed cells from traumatic tap
Protein Disorders affecting BBB
Bilirubin Elevated Serum Bilirubin Level
Melanin Meningeal melanosarcoma
Oily Radiographics Contrast Media
Bloody RBC’s Hemorrhage
Traumatic Tap
Clotted Protein Disorders that affect BBB
Clotting factors Intro by traumatic tap
Pellicle Protein Disorders that affect BBB
Clotting factors Tubercular meningitis

XANTHOCHROMIA: most commonly due to presence of RBC degradation product

 PINK – very slight amount of oxyhemoglobin
 ORANGE – heavy hemolysis
 YELLOW – conversion of oxyhemoglobin to unconjugated bilirubin
 BROWN – Methemoglobin, Melanin
 RBCs must remain in the CSF for 2 hours before noticeable hemolysis begins, a xanthochromic supernatant maybe
the result of blood present in the CSF prior to the traumatic tap.
- A recent hemorrhage may produce a clear supernatant
- Introduction of serum protein from a traumatic tap can cause the fluid to appear xanthochromic.
- maybe seen in premature infants due to immature liver function

-
 INTRACRANIAL HEMORRHAGE VERSUS TRAUMATIC TAP
INTRACRANIAL
DIFFERENCES TRAUMATIC TAP
HEMORRHAGE
Tube 1 Has Greatest Conc of Blood
Distribution Of Blood Even In All 3 Tubes
W/ Diminishing Conc.
Clot Formation Absent Present
Xanthochromic Supernatant Common Not Common
Erythrophagocytosis (Hemosiderin –
Present Absent
Laden Macrophages)
D-Dimer Test Positive Negative

Clot Formation
 Intracranial hemorrhage - does not contain enough fibrinogen to clot
 Traumatic tap - forms clot due to plasma fibrinogen introduced into the CSF specimen
 Note: Damage to BBB allows increase filtration of proteins and coagulation factors that causes clot formation but
doesn’t produce a bloody fluid.

Xanthochromic Supernatant: Centrifuge a bloody sample in a microhematocrit tube and examine the supernatant
against a white background.

MICROSCOPIC EXAMINATION: macrophages containing RBCs (erythrophagocytosis), or hemosiderin granules

indicates intracranial hemorrhage.

 D-dimer test: detection of D-dimer (fibrin degradation product) by Latex Agglutination Immuno Assay indicates
the formation of fibrin at a hemorrhage site.
 Cell count
 RBC count – done in bloody specimens in cases of traumatic tap to correct WBCs or protein.
Note: Any cell count should be done immediately since WBCs (granulocytes) and RBCs begin to lyse within
1 hour. Refrigerate
 WBC/Leukocyte count – is routinely performed on CSF specimen
Normal Value
- Adult: 0 – 5 WBCs/uL
- 30 mononuclear cells/uL considered normal in newborn
- Note: It is NECESSARY to examine all specimens microscopically since specimens with 200 WBCs or
400 RBCs/uL may appear clear.
- Methodology: Improved Neubauer Counting
Chamber

1 large corner square contains 0.1 uL so that the 4 large corner

squares is equivalent to 0.4 uL

 Total cell count

- Clear specimens – count undiluted
- Use saline solution in diluting CSF specimens
1. Lyse the RBCs with 3% acetic acid in diluted or undiluted specimen
2. Add methylene blue to stain the WBCs for better identification

Clarity Dilution Amount of sample Amount of diluent

Slightly hazy 1:10 30uL 270 uL
Hazy 1:20 30uL 570 uL
Slightly cloudy 1:100 30uL 2970 uL
Slightly bloody | Cloudy 1:200 30uL 5970 uL
 Bloody | Turbid 1:10,000 0.1 mL of a 1:100 9.9 mL
dilution

Undiluted specimen
1. Place 4 drops of mixed specimen in a tube
2. Rinse a Pasteur pipet with glacial acetic acid
3. Drain thoroughly
4. Draw 4 drops of CSF into the rinsed pipet
5. Allow to stand for 1 minute
6. Mix solution in the pipet
7. Discard first drop and load the hemocytometer
8. Count WBCs in 4 large corner squares on both sides of the hemocytometer.
9. Number is multiplied by the dilution factor to obtain the number of WBCs per microliter.

Differential count on CSF

 Concentrate specimen prior to smear
 Performed on stained smear to identify type of cell present in the specimen. Don’t do differential count in
Neubauer Counting chamber.
Methods of concentration
1. Sedimentation
2. Filtration
3. Centrifugation
4. Cytocentrifugation -read
 Procedure:
a. Centrifuge for 5-10 min
b. Remove supernatant and saved for additional tests
c. Smear suspended sediment onto slide
d. Air dry
e. Stain with Wright’s stain
f. Count and classify 100 cells and report in percentage

CSF cellular constituents

 Lymphocytes and Monocytes
- Primary cells in CSF
- Adults (70:30)
- Children (monocyte > lymphocytes)
- Occasional Neutrophils- normal
 Abnormal cells in CSF (pleocytosis)
- Eosinophils
- Plasma cells
- Macrophages
- Immature WBCs (rare)
 Significance: Pleocytosis of Neutrophils, Monocytes, lymphocytes provide diagnostic information on the
microorganism causing infection of the meninges (meningitis).
Type of cell Major Clinical Significance
Lymphocytes | Monocytes Normal
Viral, tubercular, fungal meningitis
Multiple sclerosis
Neutrophils Bacterial meningitis
Early cases of viral, tubercular, and fungal meningitis, parasitic
Cerebral hemorrhage, repeated lumbar puncture, injection of medication or
radiographic dye
Macrophages RBCs in spinal fluid
Contrast media
Lymphoma Cells Disseminated Lymphoma
Blast forms Acute Leukemia
Plasma cells Multiple sclerosis
Lymphocyte reactions
Ependymal, choroidal, and spindle Diagnostic procedures
shaped cells
Malignant cells Metastatic carcinomas Primary CNS carcinoma

Parameters Bacterial Meningitis Viral Tubercular Fungal

Predominant Neutrophils Lymphocytes Lymphocytes Lymphocytes
Cells Monocytes Monocytes
Protein Increased Increased Increased Increased
 Glucose Decreased Normal Decreased Decreased
Lactate Increased Normal Increased Increased
Other Information (+) Gram Stain Enterovirus Mycobacterium Cryptococcus
(+) Culture  Poliovirus tuberculosis neoformans
(+) Litmulus Lysate Test  Echovirus (+) AFB (+) gram stain=
 Coxsackievirus (+) Pellicle starburst pattern
formation: weblike (+) india ink
pellicle after (+) immunologic test
overnight
refrigeration of
sample.

MICROSCOPIC EXAMINATION

Cryptococcus Erythrophagocytosis, Subarachnoid Metastatic small cell

which may mimic haemorrhage carcinoma from the lung.
Cryptococcus Note nuclear moulding

Unsatisfactory. Viral meningitis with

Degenerate cells with Bacterial meningitis with Chronic asceptic
neutrophils and bacterial lymphocytes and meningitis with
smudged nuclear detail. monocytes
cocci lymphocytes and
vacuolated monocytes

Metastatic squamous cell Metastatic small cell Metastatic melanoma

carcinoma, elicits carcinoma from the lung.
prominent acute Note nuclear molding
inflammation
Normal brain smears smoothly as a monolayer. There may be small clumps in lymphoma and carcinoma. More difficult
to smear in meningioma and schwannoma with larger aggregates.

CHEMICAL EXAMINATION
CSF PROTEIN
 The most commonly performed chemical test.
 Normal Value: 15-45 mg/dL
 Higher values in infants and older persons
 Proteins normally found in CSF
- Albumin – major CSF protein
- Prealbumin – 2nd most prevalent
- Ceruloplasmin & Haptoglobin – alpha globulins
- Transferrin – major beta globulin
- “TAU” protein – separate carbohydrate deficient transferrin fraction seen ONLY in CSF
- IgG & small amount of IgA – gamma globulins
 Proteins NOT normally found in CSF
- IgM
- Fibrinogen
- Beta lipoproteins
 Clinical Significance
- Decreased in: Leakage of fluid from the CNS
 ELEVATED IN:
- Meningitis and hemorrhage – most common causes
- Damage to BBB
- Production of Ig within the CNS
- Decreased clearance of normal protein from the fluid
- Degeneration of neural tissue
- Multiple sclerosis
- Endocrine / metabolic disorders

Qualitative tests
Tests Reagent Positive Result
Nonne-Apelt Ammonium Sulfate Cloudy Precipitate
Rose Jones Ammonium Sulfate White Ring
Pandy’s Phenol Bluish White Cloud
Nogochi 10% Butyric Acid Precipitate
Colloidal Gold Test Colloidal Gold Solution Blue = 1+
PURPLE = 2+
DEEP BLUE = 3+
PALE BLUE = 4+
COLORLESS = 5+

QUANTITATIVE TESTS
1. TURBIDIMETRIC
 Precipitation of protein using
 TRICHLOROACETIC ACID (TCA) – reagent of choice; precipitates both albumin and globulins
 SULFOSALICYLIC ACID (SSA) – precipitates albumin only unless combined with sodium sulfate
2. DYE- BINDING TECHNIQUES
 Protein error of indicators”
o Advantages:
- Smaller sample size
- Less interference from external sources
 COOMASSIE BRILIANT BLUE (CBB) G250
- binds to variety of proteins
- color change from RED to BLUE
- uses BEER’S LAW
 PONCEAU’s
3. NEPHELOMETRY: uses Benzalkonium Chloride
4. ELECTROPHORESIS
 AGAROSE GEL electrophoresis followed by CBB staining- most frequently performed
 method of choice when it is necessary to determine if the fluid is CSF (“tau” method)
 Detection of OLIGOCLONAL BANDS
 MULTIPLE SCLEROSIS
- PRESENCE of 2 or more oligoclonal bands in CSF but not present in serum part when accompanied by
increased IgG index.
- oligoclonal bands remain positive during remission.
 Other disorders with oligoclonal bands but not in serum
- Encephalitis, neurosyphilis, Guillain-Barre Syndrome and neoplastic disorders
5. PROTEIN FRACTIONS
 To accurately determine whether IgG is increased because it is being produced within the CNS or because of a
defect in BBB, comparisons between serum & CSF levels of albumin & IgG must be made
 CSF/SERUM ALBUMIN Index
- evaluates BBB integrity
- Index value of <9= INTACT BBB

 CSF IgG index

- measures IgG synthesis within the CNS
- index value of >0.77=indicates IgG production within CNS

6. MYELIN BASIC PROTEIN

 Presence indicates recent destruction of myelin sheath that protects the axons of neurons (demyelination)
 Used to monitor course of multiple sclerosis
 Measure of the effectiveness of current and future treatments.
 Detection of radio immunodiffusion
7. Automated chemistry analyzer for CSF protein

GLUCOSE DETERMINATION
- NORMAL VALUE: 60 – 70% of plasma glucose
- blood glucose should be drawn 2 HOURS prior to spinal tap – equilibration of two fluids prior to tap for comparison
- specimen should be tested immediately because glycolysis occurs rapidly in CSF
- Same procedure as blood
- CLINICAL SIGNIFICANCE
 Alteration in the mechanisms of glucose transport across BBB
 Increased glucose uses by brain cells
 increased in DM, encephalitis and conditions associated with intracranial pressure
- always a result of plasma glucose elevation
 aids in determining the causative agent of meningitis

LACTATE DETERMINATION
 Aids in the diagnosis and management of meningitis cases
 Sensitive method for evaluating effectiveness of antibiotic therapy
 Used to monitor severe head injuries (usually increased levels)
 RBCs have increased concentration of lactate
 Clinical significance
- Viral meningitis - <25mg/dL
- Tubercular & Fungal meningitis - >25 mg/dL
 - Bacterial meningitis - > 35 mg/dL
- Note:
1. Destruction of tissue due to O2 deprivation – increased CSF lactate
2. Falsely elevated in bloody/ xanthochromic/hemolyzed specimen

CSF GLUTAMINE
 Glutamine: produced by brain cells in the CNS from ammonia and alpha ketoglutarate.
 As the concentration of ammonia in the CSF increases, the supply of alpha ketoglutarate becomes depleted,
glutamine no longer be produced to remove the toxic ammonia and coma ensues.
 Chemical test frequently performed in CSF but not in blood
 Produced by brain cells from ammonia and alpha-ketoglutarate
 Increased synthesis of glutamine is caused by excess ammonia in the CNS. Indirect test for the presence of
ammonia in CSF
 As CSF ammonia increases- alpha- ketoglutarate decreases - COMA
Elevated in:
 Liver disorder that results in increased blood ammonia & CSF ammonia
 Coma of unknown origin
 Reye’s syndrome (75% of children with this disease have increased glutamine)
 > 35mg/dL – disturbance of consciousness
Test for glutamine
 Glutamine Assay instead of test for Ammonia (volatile) since glutamine concentration is more stable in collected
specimen.
 Requested in coma patients of unknown origin

CK-BB ISOENZYMES: WHEN levels are <17mg/dL, it predicts recovery from cardiac arrest after resuscitation
LD ISOENZYMES: HELP DIAGNOSE meningitis by confirming the presence of PMN and lymphocytes
Increased LD Condition
isoenzymes
LD1 and LD2 Brain tissue
destruction
LD2 and LD3 Viral meningitis
LD4 and LD5 Bacterial meningitis

MICROBIOLOGIC EXAMINATION
Preliminary tests:
1. Gram stain
2. Acid-fast stain
3. India ink preparation
4. Limulus lysate test
5. Latex agglutination test

Culture media – confirmatory test

- 24 hrs incubation – bacterial meningitis
- 6 wks incubation– tubercular meningitis

Gram’s Stain
- Routinely performed in all suspected meningitis cases
- RECOMMENDED METHOD for detection of microorganism
- Performed on concentrated specimens
- Centrifuge first at 1500 RPM for 15 minutes to obtain maximum concentration
- Presence of 10 /mL of organism is needed for demonstration by grams stain
- At least 10% chance exists that GS and culture will be negative

Organisms most frequently encountered

 Gram (+)
1. S. pneumoniae – adults
2. S. agalactiae, L. monocytogenes – newborns
 Gram (-)
1. Hemophilus influenzae
2. E. coli
3. N. meningitidis
 India ink preparation in suspected C. neoformans
- produces STARBURST PATTERN in Gram stain
- Acid-fast stain/ Fluorescent antibody test - not routinely done, only in suspected tubercular meningitis
 Latex Agglutination Test and ELISA: rapid means of identifying microorganisms (H. influenza type B,
Streptococcus group B, S. pneumoniae, N. meningitidis A, B, C, Y, W135 and E. coli K1 antigens)
 Bacterial antigen test (BAT)
- not sensitive for N. meningitidis
 - should be used in combination with results from the hematology and clinical chemistry laboratories for
diagnosing meningitis.
Reverse Latex Agglutination
- more sensitive than India ink for C. neoformans.
- false positive (+) occurs because of interference from Rheumatoid factor (most common)
- inhibited by incubation with dithiothreitol or pronase and boiling with EDTA
Limulus Lysate assay
- Limulus Lysate assay – for diagnosis of meningitis caused by gram neg bacteria by detecting ENDOTOXINS
found in their walls.
- Uses blood cells of the HORSESHOE CRAB (lymulus polyphemus) termed “amebocytes” which contains COPPER
COMPLEX responsible for the BLUE COLOR
- Coagulates the amebocyte lysate within 1 hour at 37⁰C
- Sensitive to minute amounts of endotoxins and will detect all gram neg bacteria

Serologic test: Test for tertiary syphilis or neurosyphilis

 VDRL – recommended for CSF to diagnose syphilis due to its ability to detect active cases of syphilis
 Fluorescent treponemal antibody- absorption test (FTA-ABS): used with care to avoid contamination with blood
because it remains positive in the serum of treated cases of syphilis
 RPR – NOT RECOMMENDED: less sensitive and specific for CSF

AUBF: ANALYSIS OF URINE AND OTHER BODY FLUIDS

LABORATORY 5: CEREBROSPINAL FLUID EXAMINATION

MRS. CHARI K. WACHAYNA, RMT

1st SEMESTER | S.Y. 2024-2025

I. AIM: To study the important characteristics of CSF and the various examinations associated with this
fluid.

II. MATERIALS:
Counting chamber Rgt. Kit for Chloride
Test tube Rgt. Kit for Glucose
5mL pipet Saturated Ammonium Sulfate
1mL pipet 0.45 NaCl Saturated Phenol Crystals Colloidal Gold Reagent
1% NaCl Litmus paper
Urinometer set
WBC diluting pipet
RBC diluting fluid

III. PROCEDURES:
N.B. The specimen is collected in three sterile clear bottles/ test tubes with caps and marked/ labeled 1, 2, 3.
Tube 1: Contains first few drops which should not be used unless necessary for Chemical and Serologic examination.
Tube 2: Contains about 7cc, for Microbiologic/ bacteriological examination.
Tube 3: Contains about 2c for cell count/ Hematologic examination and qualitative protein tests.
1. PHYSICAL EXAMINATION: Take note of the color, turbidity or transparency, reaction, coagulation or clot
formation, and specific gravity.
RESULTS AND INTERPRETATIONS:
 COLOR: Normally colorless, crystal clear
 TRANSPARENCY: A normal specimen is clear, fewer than 200 WBC/cu mm does not give rise to the cloudiness
of the fluid.
 REACTION: Normally alkaline
 COAGULATION/ CLOT FORMATION: Absent in normal specimen SPECIFIC GRAVITY: 1.003 - 1.008

2. CHEMICAL EXAMINATION
1. Qualitative test for Globulin (ROSS JONES OR NONNE APELT METHOD)
a. In a 13x100 test tube, overlay 2-3mL saturated NH4504 with 1cc of the supernatant of a centrifuged CSF
specimen. Read after 3minutes standing.
b. (+) Gray ring at the zone of contact
2. Qualitative test for Protein (PANDY'S METHOD)
a. To 1cc saturated aqueous solution of Phenol, add 1large drop of CSF. (+) Bluish white cloud forms as soon as
the drop of CSF mix with the reagent.
b. A normal CSF specimen shows a faint trace bluish-white color but should be reported as negative.
3. Test for Albumin Globulin ratio: (COLLOIDAL GOLD TEST)
a. Arrange twelve, 13 x 100 test tubes in a rack.
b. To the first tube, pipet 0.9cc of a freshly prepared 0.4% NaCl solution and 0.5 c in each of the next 10 tubes.
c. Make a serial dilution by pipetting 01. mL of the clear supernatant of a centrifuged CS specimen to the firs
tube and mix. Transfer 0.5 ml to the second tube and mix. Continue doing the same procedure discarding 0.5
ml from the tenth tube.
d. In tube 12, pipet 0.85 ml of %f 1 NaCI.

N.B. The 11th and 12th tubes are controls and contain no spinal fluid. The 1h tube serves as a control for the stability of
the
colloidal gold. (Colorless in 1hour time).

e. To each of the 12 tubes, add 2.5cc of the colloidal gold solution.

f. Let the preparation stand overnight and read the results the following morning from tubes 1- 10.

RESULTS AND INTERPRETATION:

0 No change
1 Bluish Red
2 Reddish Blue
3 Blue
4 Pale blue
5 Colorless

 The normal reaction of a normal specimen is 0000110000

 Paretic curve or Zone -1 marked change is seen in Multiple Myeloma patients- 5555543000.
 Luetic or Tabetic or Zone Il - greatest change in the middle tubes - seen in Syphilitic patients. 0003333000.
 Meningitic or Zone IIl -greatest change in the 5th - 9th tubes seen in TB meningitis patient - 0000244440.

4. Test for the Glucose Content:

- Determine the Glucose content of blood and CS of the patient. The procedure depends upon the available set
of reagents.
- Normally, the CSF value is 2/3 that of the concentration in blood.
5. Test for Chloride content:
 Determine the Chloride content of blood and CS of the patient. The procedure depends upon the available set
of reagents.
 Normally, the CS value is 25% higher than the concentration in the blood (serum chloride is 103mEq/L)

3. MICROSCOPIC EXAMINATION:

1. Cell Count
a. Method for low White Cell Count: Applied when the CSF appears clear.
a1. Fil in a WBC pipet with the undiluted CSF. Charge the counting chamber.
a2. Count the number of cells in all of the 9 squares.
a3. Compute
b. Method for moderate WBC: Employ when CSF appears hazy or slightly turbid
b1. Fil in a WBC pipet with the undiluted CS up to the 1 mark. Dilute the specimen using the WBC diluting fluid
up to the 11 marks.
 b2. Discard the first 3 to 4 drops and charge the counting chamber. Count WBC in the 4 large corner squares
and the large central square.
b3. Compute
c. Method for high WBC: Applied when CS is purulent or very turbid.
c1. Fill in a WBC pipet up to the 0.5 mark and dilute up to the 1 mark with WBC diluting fluid.
c2. Mix and discard the first 3- 4 drops and charge the counting chamber.
c3. Count the number of WBCs present on the 4 corner large squares.
c4. Compute
d. Method for counting mixture of WBCs and RBCs: Employed when the CSF is turbid with a reddish
tinge.
d1. Make WBC count on the patient's blood and CSF. d2. Make RBC count on the patient's blood and CSF.
d2. Compute. Normal Value: 0-5 cells per cu mm

2. Differential count:
a. When the total count is low, make a chamber differential count by identifying the cells in the counting
chamber using HPO.
b. When there are numerous cells, make a smear of the sediment after centrifugation in similar manner as in
making a blood smear.
 Normal value: 0 - 5 lymphocytes.

4. BACTERIOLOGIC EXAMINATION
1. Transfer a portion of the specimen from the appropriate tube into a sterile 13 x 100 test tube and cover it with
a cotton plug.
2. Centrifuge at 3000 rpm for 15 minutes and decant the supernatant.
3. Inoculate the thioglycolate broth and chocolate agar. Incubate and follow up.
4. Make a smear from the remaining sediment then stain using the Ziehl Neilsen method. Look for acid-fast
bacilli.
5. If tuberculous meningitis is suspected, allow a portion of the specimen to stand overnight and observe for the
formation of a cobweb-like or inverted pine tree-like clot.

IV. COMPUTATIONS, RESULTS, AND INTERPRETATIONS:

V. QUESTIONS:
1. What is the three-tube test?
 Tube 1 is used for chemical and serologic tests because these tests are least affected by blood or bacteria
introduced as a result of the tap procedure.
 Tube 2 is usually designated for the microbiology laboratory.
 Tube 3 is used for the cell count, because it is the least likely to contain cells introduced by the spinal tap
procedure.
 A fourth tube may be drawn for the microbiology laboratory to better exclude skin contamination or for
additional serologic tests.
2. Discuss the significance of Blood in CSF.
 Grossly bloody CSF can be an indication of intracranial hemorrhage, but it may also be due to the puncture of
a blood vessel during the spinal tap procedure. Three visual examinations of the collected specimens can
usually determine whether the blood is the result of hemorrhage or a traumatic tap.
3. In tabulation, differentiate CS from other body fluids (serum, urine, amniotic fluid, CSF) as to
serum urine amniotic fluid CSF
Total CHON content 6.0 – 8.3 g/dL <10mg/dL 0.16 – 10 g/L 15 to 45 mg/dL
Uric acid 4.0 – 8.5 mg/dL 500 -600 mg/L >2.0 mg/dL 0.2 – 0.72 mg/dL
Creatinine 0.7 – 1.3 mg/dL 10 mg/dL up to 3.5 mg/dL 20 -40 mg/dL
urea nitrogen 300 mg/dL 30 mg/dL 3.0 – 8.0 mmol/L
glucose 6-21 mg/dL 0-0.80 mmol/L 14 mg/dL 40 – 80 mg/dL
sodium 135-145 mEq/L 20 mEq/L 130 – 150 mEq/L 135 – 150 mEq/L
potassium 3.5 – 5.0 mEq/L 3.3g 4.8 – 6.5 mEq/L 2.0 – 3.0 mEq/L
urobilinogen <1 mg/dL <1 mg/dL <1 mg/dL >0.2 mg/dL
pH 7.35 – 7.45 Approximately 6.0 7.1 – 7.3 7.3 – 7.45
specific gravity 1.0621 – 1.0506 1.005 – 1.030 1.0078 1.006 – 1.008
total bilirubin 0.2 – 1.3 mg/dL 0.40 mg/dL 0.28 – 0.90 0-7 mAU
osmolality 275 – 295 mOsm/kg 500 – 850 mOsm/kg 80 – 140 mOsm/kg 281 mOsm/kg

4. State the principle of

a. Colloidal Gold Test
- This is one technique for finding antibodies. Protein A, which has the ability to bind to the antibody non-
specifically, is fixed on the conjugate pad after being tagged with colloidal gold via an indirect technique.
The fixed selection line of the Ne membrane is an antigen that can selectively attach to the antibody that
is being tested. The control of Ne membrane is tipped with an anti-protein antibody. It was shown that the
ideal concentration of the cloudy and capture antibody was around
 b.Dye binding method
- This is based on the premise that some strongly anionic dyes. Amido back, for instance, forms insoluble
complexes at low DH. under five charges, causing a dye protein complex to develop that is insoluble. This
method's technique for estimating protein levels includes adding too much dye to the sample in an acidic
environment, then filtering the mixture.
5. Give the disadvantages of using Biuret method
 We can't exactly quantify the number of proteins present in the sample.
 Only soluble proteins can be detected.
 Ammonium and magnesium ions, carbohydrates, fats, and turbidity can hinder the reaction.
 Amino acid histidine also gives a positive result
6. Enumerate the general methods of CHON determination in CSF. Give the principle of tests under
each.
 Turbidimetric: protein precipitation with TCAP SUA; SSA precipitates albumin alone, whereas ICA precipitates
albumin globulin.
 Dye-Binding Technique: It becomes blue when it attaches to particular proteins, changing its color from red.
 Nephelometry: Employs benzalkonium chloride as a protein detector.
 Electrophoresis: For oligoclonal band detection. The IgG index rises when two Oligoclonal bands are present
 Myelin Basic Protein: The presence of this indicates that the myelin sheath, which covers a neuron's axon, has
recently been destroyed.
 Protein Fractions: Since it is being generated, I determine the rise in IgG. comparing the levels of serum and
extracellular synthesized fuel (ESF) in the central nervous system.
 automated chemistry analyzer for CSF Protein: Immunologic, electrophoretic, colorimetric, and turbidimetric
tests.

NOTES:

1. What is the first and second most numerous body fluid?

a.
2. Formation and physiology of csf
a. Choroid Plexus: specific part of the brain that produces CSF (selective filtration) 20mL/hr is rate of production
or reabsorption
b. Arachnoid Villi: reabsorbs CSF
c. Blood Brain Barrier: protects the brain from chemical and other substances that circulating in the that can
harm the brain tissue. Disruption of BBB allows WBC’s protein and other chemicals to enter the CSF.
3. Why is it that albumin is the only protein present in csf?
 its elevation in the CSF is indicative of the dysfunction of the blood–brain barrier (BBB)
4. How CSF flows into the brain?

5. What are the epithelial cells present in the Central nervous system?
 Ependymal cells are glial cells with a ciliated simple columnar form that line the ventricles and central canal of
the spinal cord.
6. Liver to Xanthochromia, is it related to traumatic tap?
7. Causes of intracranial pressure
 Hydrocephalus, which is an abnormal buildup of cerebrospinal fluid. This is the fluid around your brain and
spinal cord.
 Bleeding into the brain
 Swelling in the brain
 Aneurysm
 Blood pooling in some part of the brain
 Brain or head injury
 Brain tumor
 Infections such as encephalitis or meningitis
 High blood pressure
 Stroke
8. Correction for contamination (WBC AND PROTEIN)
 -1 WBC for every 700 RBC’s seen
 -8 mg/dL Total Protein concentration for every 10,000 RBC’s/uL (Henry’s)
 -1mg/dL Total Protein concentration for every 1,200 RBC’s/uL (Strasinger)
9. Cloudy and Turbid dilution factor, amount of sample, amount of diluent

Clarity Dilution Amount of Sample Amount of Diluent

Cloudy 1:200 30 uL 5970 uL
Turbid 1:10,000 0.1 of a 1:100 dilution 9.9 mL
10. Will bacteria induce immune response?
 The beneficial gut microbes do this by ordering specialized immune cells to produce potent antiviral proteins
that ultimately eliminate viral infections. And the body of a person lacking these beneficial gut bacteria won't
have as strong an immune response to invading viruses
11. Virus that causes meningitis
 Enterovirus, Poliovirus, Echovirus, Coxackievirus
12. Lactate level in Viral meningitis-what is lactate for?
 below 2 mmol/L.
 help to distinguish post-neurosurgical bacterial meningitis from a 'chemical/aseptic meningitis' following
surgical intervention. CSF lactate can help to diagnose mitochondrial disorders and inherited metabolic
disorders.
13. Why does tubercular meningitis need to be refrigerated to have a weblike pellicle?
 This faintly visible "spider's web clot" is due to the very high level of protein in the CSF (1–8 g/L, or 1000–8000
mg/dL)
14. Stain for fungi C. neoformans
 India ink stain
15. Where are chondrocytes seen?
 in the cartilage where they synthesize type II collagen, proteoglycan, and other proteins such as enzymes that
degrade certain matrix components.
16. Protein Fraction/ Electrophoresis
 To look for the characteristic banding pattern that may be seen in multiple sclerosis (oligoclonal bands) To
evaluate a person with headaches or other neurologic symptoms and look for proteins suggestive of
inflammation or infection that would explain the symptoms.
17. Ammonia- glutamine-liver cirrhosis -coma