Chrissa Mae T.

Catindoy BS Medical Technology 3A

FINALS [LECTURE III]: AUBF: Cerebrospinal fluid

Formation and Physiology:

- Brain and spinal cord lined by meninges.

- 3 layers of meninges:
o Dura mater: outer
o Arachnoid: middle
o Pia mater: surfaces of brain and spinal cord
- Cerebrospinal fluid (CSF) produced in choroid plexuses of the 4 ventricles from plasma.
- 20 mL/min produced in adults.
- Volume:
o Adults: 90-150 mL
o Neonates: 10-60 mL

- CSF flows through subarachnoid space between arachnoid and pia mater.
- Reabsorbed into blood in arachnoid granulations/villae (one-way valves).
- Formation:
o Hydrostatic pressure and active transport.
o Not an ultrafiltrate.
o Very tight-fitting endothelial cells, prevent filtration of large molecules – blood-brain
barrier.
- Blood-brain barrier
o Essential to protect brain.
o Chemicals and harmful substances do not pass.
o Ab and medications are excluded.

**NOTE: CSF composition differs from plasma.

o Meningitis, multiple sclerosis disrupt membrane.

o Test for substances that pass through:
▪ Cells ▪ Bacteria
▪ Protein ▪ Immunoglobulins

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Specimen Collection and Handling:

- CSF collected between 3rd, 4th, 5th ventricles.

- Referred to as a spinal fluid tap.
- 3 sterile tubes in this order:
o 1: Chemistry/serology.
o 2: Microbiology (avoid skin contamination).
o 3: Hematology (avoid cells from tap).
- Save leftover fluid for additional tests.
- Volume removed based on patient volume and opening pressure.
- Often a small volume is collected.

- Usually STAT requests.

- Handle carefully/avoid repeat taps.
- Preservation:
o Hematology Refrigerate
o Microbiology Room temperature
o Chemistry/serology Frozen
- Extra fluid is frozen.
- Always wear gloves; meningitis can be very contagious; centrifuge capped tubes.

Appearance

- Terminology:
o Crystal clear o Xanthochromic
o Cloudy/turbid = infection o Hemolyzed/bloody
o Milky = lipid or protein
- Xanthochromic
o Pink, orange, yellow
o RBC degradation products.
o Jaundice, ↑ protein, carotene.
o Pathologic = cerebral hemorrhage

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Chrissa Mae T. Catindoy BS Medical Technology 3A
Description Possible Cause/s
Slightly Hazy 200-500 WBC/µL.
Cloudy Above 500 WBC/µL.
WBC, RBC, proteins, increased lipid concentration, microorganisms,
Turbid/Milky
aspirated epidural fats, radiographic contrast media.
Clotted Froin’s disease (increased proteins and clotting factors).
Non-pathologic: traumatic tap.
Bloody
Pathologic: intracranial hemorrhage or subarachnoid hemorrhage.
Cryptococcal meningitis.
Viscous
Metastasizing mucin producing adenocarcinoma.
Oily Radiographic contrast media.
Pellicle formation Tubercular meningitis.
Presence of RBC degradation product.
• Pink - very slight amount of oxyhemoglobin.
• Orange - heavy hemolysis.
• Yellow - conversion of oxyhemoglobin to unconjugated bilirubin.
Other Causes:
Xanthocromia
• Increased serum bilirubin
• Presence of carotene
• Increased proteins (>250mg/dL)
• Melanoma pigment (meningeal melanosarcoma)
• Immature liver function (seen in infants)
Brown Methemoglobin, hematoma, melanin.

Traumatic Tap

- Blood vessel punctured during tap.

- Differentiate from cerebral hemorrhage.
- Uneven blood distribution in tubes with traumatic tap.
o D-dimer test for hemorrhage.
o Erythrophagocytosis, hemosiderin granules.
o Hemorrhage = even distribution in all tubes.
o Traumatic tap = decreasing tubes 1 through 3.
- Xanthochromia
o Not present in a recent traumatic tap.
o Indicates older hemorrhage.
- Clot formation
o Clots presents = traumatic tap (plasma)
o Hemorrhage does not have enough fibrinogen.
o Other causes:
▪ Non-bloody CSF = damage to blood-brain barrier.
▪ TB meningitis – Web-like pellicle after refrigeration.

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Chrissa Mae T. Catindoy BS Medical Technology 3A
Traumatic Collection Differentiation

Differences Intracranial hemorrhage Traumatic tap

Even Uneven
Distribution of blood
all 3 tubes Tube 1 has greatest concentration of blood
Clot formation Absent Present
Xanthochromic supernatant Common Common
Erythrophagocytosis
Present Absent
(hemosiderin-laded macrophages)
D-dimer test + -

Cell Count

- WBC and total cell count; RBC count seldom done.

- Granulocytes lyse within 2 hours; STAT.
- Normal adult 0-5 WBCs/µL.
- Neonates up to 30 mononuclear cells.
- Standard Neubauer formula used for counts.

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛

= 𝑐𝑒𝑙𝑙𝑠/µ𝐿
𝑛𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑠𝑞𝑢𝑎𝑟𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑 𝑥 𝑣𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 1 𝑠𝑞𝑢𝑎𝑟𝑒 (0.1)

- Modification:
o Count 5 large squares on both sides of the chamber = 10 squares

10 squares x 0.1 = 1µL (volume counted)

Number of cells counted x dilution x 1µL = cells/µL in CSF

- Total cell count

o Clear specimens count undiluted unless overlapping cells are seen.
o Load with transfer pipette.
o Dilute with normal saline if necessary.
- WBC count
o Dilute with 3% acetic acid.
o Methylene blue helps to see cells.
o Undiluted rinse transfer pipette with acetic acid, gently rotate pipette.

Quality Control

- Commercial cell controls are available.

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- Check diluents for contamination biweekly.
- Soak non-disposable chambers in bactericidal solution for 15 minutes.
- Rinse.
- Clean with isopropyl alcohol.
- Monthly check on cytocentrifuge speed and timing.

Differential Count

- Very important.
- Do not use counting chamber.
- Must centrifuge specimen.
- Cytocentrifuge
o Forces cell onto a slide in a monolayer.
o Filter paper absorbs moisture.
o Positively charged slides to attract cells.
o 0.1 mL CSF and 1 drop 30% albumin.
- Albumin increases cell yield.
- Daily control of 0.2 mL saline and 2 drops of albumin stained for bacterial contamination.
- Cytocentrifuge produces some cellular distortion.
- Count and classify 100 cells if possible.

Cellular Constituents

- Normal lymphocytes and monocytes.

o Adults: 70% lymphocytes, 30% monocytes.
o Children ration is reversed.
- Pleocytosis: increased amounts of normal cells.

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- Pleocytosis of normal cells in valuable in determining the cause of meningitis.
o Neutrophils = bacterial
o Lymphocytes and monocytes = viral, tubercular, fungal, parasitic.

Neutrophils

- Primary in bacterial meningitis.

- Often contain phagocytized bacteria.
- Increased early viral, fungal, tubercular, parasitic.
- Vacuoles may be present.

Lymphocytes and monocytes

- Viral, tubercular, fungal, parasitic.

- Reactive lymphocytes with viral.
- Multiple sclerosis has 50 or fewer lymphocytes/µL, both
normal and reactive.
- Seen in HIV and AIDS.

Nucleated RBCs (NRBC)

- Seen with bone marrow contamination from tap in 1% of

specimens.
- Neutrophils with pyknotic nuclei may resemble NRBCs.
- Capillary structures and epithelial cells from trauma taps.

Eosinophils

- Parasitic and fungal infections.

- Medications and shunts into the CNS.

Macrophages

- Purpose is to remove cellular and other debris.

- May be seen after repeated taps.
- Hemorrhage: enter CSG within 2 hours to phagocytize RBCs.
- RBCs degraded to hemosiderin granules and further degraded
to hematoidin crystals representing unconjugated bilirubin.

Non-clinically significant cells:

Choroidal cells

- Epithelial lining of choroid plexus, singular and in clumps, uniform cells.

Ependymal cells

- Lining ventricles and neural canal; less defined cell membranes in clumps.

Spindle cells

- Lining arachnoid.

**NOTE: All seen after encephalography procedures; no significance.

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Chrissa Mae T. Catindoy BS Medical Technology 3A
Malignant cells of hematologic origin:

Leukemias

- Lymphoblasts, monoblasts, and myeloblasts.

- Nucleoli may be more prominent than in blood.

Lymphoma

- Dissemination from lymph organs.

- Cleaved nucleii and prominent nucleoli.

Metastatic and primary carcinoma cells:

Metastatic carcinoma cells

- Lung, breast, renal, gastrointestinal, and melanoma.

- Fused cell walls, nuclear irregularities, and hyperchromatic
nucleoli.

Primary tumors

- Astrocytomas, retinoblastomas, medulloblastomas.

- Occur in children.

Chemistry Tests

- Normal values differ from blood because of blood-brain barrier.

- Total protein is common test.
o Normal 115-16 mg/dL (mg, not grams).
o Fractions differ, prealbumin is second.
o Transferrin is major β-globulin.
o TAU, carbohydrate-deficient transferrin seen in CSF, not in blood; used to identify
CSF.
o IgG major γ-globulin.

Clinical Significance:

- Decreased protein levels = fluid leakage

- Elevated levels = damage to blood-brain barrier, Ig production within CNS, decreased
clearance, degeneration of neural tissue.
- Meningitis/hemorrhage most common causes of increased damage to blood-brain barrier.
- Find abnormal results on clear fluid with low cell counts from neurologic disorders.

Methodology:

• Turbidity
o Trichloroacetic acid precipitates both albumin and globulin.
• Dye-binding
o Coomassie blue dye uses protein error of indicators principle.
o Red-blue: measure intensity of blue color.
• Automated instrumentation available
o Pyrogallol red-molybdate.

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Chrissa Mae T. Catindoy BS Medical Technology 3A
Protein Fractions:

• CSF/serum albumin index

o Blood-brain barrier integrity.

𝐶𝑆𝐹 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (mg/dL)

= 𝐼𝑛𝑑𝑒𝑥
𝑆𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (g/dL)

o Normal value <9 = no damage

• CSF IgG index
o Measure CSF IgG synthesis in CNS.
o Multiple sclerosis.
o Stimulates immunocompetent cells in CNS.

𝐶𝑆𝐹 𝐼𝑔𝐺(mg/dL) / 𝑠𝑒𝑟𝑢𝑚 𝐼𝑔𝐺 (g/dL)

= 𝐼𝑛𝑑𝑒𝑥
𝐶𝑆𝐹 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (mg/dL) / 𝑠𝑒𝑟𝑢𝑚 𝑎𝑙𝑏𝑢𝑚𝑖𝑛 (g/dL)

o Normal: > 0.77 indicates IgG production

**NOTE: Values for CSF albumin and globulin available on automated instruments.

Protein Electrophoresis

- Detection of oligoclonal bands.

o Indicate inflammation within the CNS.
o Must run serum electrophoresis.
o Multiple sclerosis (MS) = no bands in serum, bands in CSF.
o Leukemia, lymphoma, viral, HIV: bands in both.
o Primary purpose for MS, compare to IgG index also encephalitis, neurosyphilis,
Guilain-Barre, and neoplasms may give same pattern.
o Consider symptoms.

Qualitative Test for Protein

TEST 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
Blue = 1+
Purple = 2+
Colloidal Gold Test Colloidal gold solution Deep blue = 3+
Pale blue = 4+
Colorless = 5+

• Turbidimetric
o Precipitation of proteins using:
▪ Trichloroacetic acid (TCA) - reagent of choice; precipitates both albumin and
globulins.
▪ Sulfosalicylic acid (SSA) - precipitates albumin only unless combined with
Na2SO4.

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Chrissa Mae T. Catindoy BS Medical Technology 3A
o Dye-binding techniques
▪ Coomassie brilliant blue G250
▪ Ponceaus

Myelin Basic Protein

- Presence in CSF indicates demyelination of myelin sheathe around axons of neurons.

- Monitors the course of multiple sclerosis.
- Effectiveness of treatment.
- Immunoassay procedures available.

CSF Glucose

- Selective transport across blood-brain barrier.

- Approximately 60%-70% plasma glucose.
o Plasma = 100mg/dL
o CSF = 65mg/dL.
o Draw blood 2 hours before tap.
- Significance:
o Very low levels in bacterial meningitis, altered transport and use by neural cells, not
bacteria.
o TB meningitis also lowers glucose values and lymphocytes.
o Viral/fungal meningitis, normal glucose and lymphocytes.

CSF Lactate

- Diagnosis and management of meningitis.

o TB and fungal levels > 25 mg/dL
o Bacterial >35 mg/dL
o Viral <25 mg/dL
- More reliable than glucose.
- Levels remain elevated until treatment becomes effective, then fall rapidly.
- No need to compare with plasma.

CSF Glutamine

- Produced by brain cells from ammonia and ɑ-ketoglutarate to remove toxic ammonia.
- Elevated in liver disease.
- Determines ↑ ammonia in CSF; after ɑ-ketoglutarate is depleted = coma.
- >35 mg/dL = disturbance of consciousness.
- More reliable than blood ammonia.
- Seen in children with Reye syndrome.

LD Isoenzymes

- Help diagnose meningitis by confirming the presence of PMN and lymphocytes.

Increased LD Isoenzymes Condition

LD1 and LD2 Brain tissue destruction
LD2 and LD3 Viral Meningitis
LD4 and LD5 Bacterial meningitis

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Chrissa Mae T. Catindoy BS Medical Technology 3A
- CK-BB isoenzyme
o When levels are <17 mg/dL, it predicts recovery from cardiac arrest after resuscitation.

Microbiology Test

- Gram stain and cultures must be performed on sediment from centrifuged CSF; cytocentrifuge
helps Gram stains.
- Blood cultures also must be drawn.
- Difficult to interpret Gram stains, few organisms and often debris.
- Organisms:
o S. pneumocystis o N. meningitidis
o H. influenza o L. monocytogenes
o E. coli o S. agalactiae
- Cultures also plated on chocolate agar.
- TB smears very important because of growth time delay.
- Latex agglutination tests are available for:
o Group B streptococcus o N. meningitidis
o H. influenza o E. coli
o S. pneumocystis
- Gram stain is the best for detection.
- Compare with hematology and chemistry results.

Organisms Most Frequently Encountered:

• G (+) – S. pneumonia (in adults), S. agalactiae (in newborns)

• G (-) – H. influenza, E. coli, N. meningitidis (in adults), L. monocytogenes (in newborns)

Other microbiologic tests:

Limulus Lysate Test

- Diagnosis of meningitis caused by Gram negative organisms by detecting endotoxins found in

their cell walls.
- Uses blood cells of the Horseshoe crab (Limulus polyphemus) termed “amebocyte” which
contains copper complex responsible for the blue color.
- Coagulates the amebocyte lysate within 1 hour at 37°C.
- Sensitive to minute amounts of endotoxin & will detect all gram-negative bacteria.

Cryptococcus neoformans

- Frequent complication of AIDS.

- Gram stain produces a starburst pattern.
- India ink stain on CSF to detect the capsule and budding yeast;
beware of air bubbles.
- Latex agglutination tests and immunoassay procedures are the
most sensitive detection method.
- Latex agglutination primary interference is from rheumatoid factor.

**NOTE: Always confirm immunologic results with culture.

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Serologic Testing

- Primary test is for neurosyphilis, 3rd stage.

- Less performed now that people have been treated early with penicillin.
- Detect active cases within the CNS.
- The Veneral Disease Research Laboratories (VDRL) produces the recommended test for
specificity.
- Should be accompanied by a positive serum FTA-ABS.

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