CHAPTER 9: CEREBROSPINAL FLUID - Supernatant fluid:

leftover fluid that can be used for additional chemical or

Functions: serological tests
1. provides a physiologic system to supply nutrients to the - CSF test: STAT basis
nervous tissue - Preservation:
2. remove metabolic wastes 1. Hematology Refrigerate
3. produce a mechanical barrier to cushion the brain and 2. Microbiology Room temperature
spinal cord against trauma. 3. Chemistry/Serology Frozen

Formation and Physiology Appearance

- Meninges – line the Brain and spinal cord Crystal clear WBCs Normal
- Three layers of meninges Meningitis
1. Dura mater:
o outer layer Hazy, turbid, Microorganisms Meningitis
o lines the skull and vertebral canal milky, cloudy Protein Disorders affecting blood–brain
o name means: hard mater barrier
2. Arachnoid: Production of IgG within the CNS
o middle layer Oily Radiographic
o filamentous inner membrane contrast media
o name means: spiderweb-like Bloody RBCs Hemorrhage
3. Pia mater: Traumatic tap
o surfaces of brain and spinal cord Xanthochromic Hemoglobin Old hemorrhage
o name means: gentle mater Lysed cells from traumatic tap
Bilirubin RBC degradation
Cerebrospinal fluid (CSF) Elevated serum bilirubin level
- a major fluid of the body Carotene Increased serum levels
- Choroid plexuses Protein Disorders affecting blood–brain
are capillary networks that produce CSF Fluid In the two barrier
lumbar ventricles and the 3rd and 4th ventricles Melanin Meningeal melanosarcoma
o Adult CSF production: 20 mL/hr Clotted Protein Disorders affecting blood–brain
forms the CSF from plasma by: mechanisms of selective barrier
filtration under hydrostatic pressure and active transport Clotting factors Introduced by traumatic tap
secretion Pellicle Protein Disorders affecting blood–brain
(CSF is not an ultrafiltrate) barrier
- Subarachnoid space Clotting factors Tubercular
located between the arachnoid and pia mater meningitis
where the fluid flows
- Arachnoid granulations/villae (one-way valves) - Cloudy = infection (WBC)
Needed to maintain the volume: - Cloudy, Turbid or Milky = inc. lipid or protein
Adults: 90-150 mL, Neonates: 10-60 mL concentration
Fluid is reabsorbed into blood - Xanthochromia
Cells respond to pressure within the central nervous describe CSF supernatant that is colored:
system (CNS) and prevent reflux of the fluid 1. pink (very slight amount of oxyhemoglobin)
2. orange (heavy hemolysis)
Blood-brain barrier 3. yellow (conversion of oxyhemoglobin to
- the tight-fitting structure of the endothelial cells in the unconjugated bilirubin)
choroid plexuses - Caused by RBC degradation products
- essential to protect the brain from chemicals and other - Caused by elevated bilirubin due to immature liver
substances circulating in the blood function (premature infant)
- disruption cause diseases such as meningitis and multiple - Other causes:
sclerosis 1. presence of the pigment carotene
2. increased protein concentrations
Specimen Collection and Handling 3. melanoma pigment
- CSF collected by lumbar puncture
(3rd, 4th, 5th ventricles) Traumatic Collection (Tap)
- Referred to as a spinal fluid tap - Grossly bloody CSF – indicate intracranial hemorrhage or
- Three sterile tubes in this order: due to the puncture of a blood vessel
1: Chemistry/serology - Can determine if the blood is the result of hemorrhage or
(least affected by blood or bacteria) a traumatic tap.
2: Microbiology 1. Uneven blood distribution
3: Hematology (cell count) - Cerebral hemorrhage (blood evenly distributed)
(least likely to contain cells introduced by the procedure) - Traumatic tap: (blood decreases)
4th tube (optional): Microbiology (exclude skin Test tube 1: leave the heaviest concentration of blood
contamination) and Serology (Addional test) Test tube 2 and 3: diminished amounts
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 2. Clot formation - Dilutions required:
- Hemorrhage does not have enough fibrinogen so it does o use calibrated automatic pipettes
not clot o made with normal saline, mixed by inversion, and
- Traumatic tap fluid forms clots = caused by plasma loaded into the hemocytometer with a Pasteur
fibrinogen pipette.
- Other causes of clot formation:
 Nonbloody CSF = damage to blood-brain barrier 4. WBC count
Includes meningitis, Froin syndrome, and blocked CSF - Dilute with 3% glacial acetic acid to lyse RBCs
circulation through the subarachnoid space - Add methylene blue to stain WBCs
 TB meningitis – Web-like pellicle after overnight
refrigeration 5. Quality Control
- Commercial cell controls are available
3. Xanthochromic Supernatant - Check diluents for contamination biweekly by examining
- a result of blood that has been present longer than that them in a counting chamber under 400× magnification.
introduced by the traumatic tap. - Check the speed of the cytocentrifuge monthly with a
- RBCs: remain in the CSF for 2 hours before hemolysis tachometer, and the timing with a stopwatch.
- Clear supernatant: a very recent hemorrhage - Soak nondisposable chambers in bactericidal solution for
- Xanthochromic supernatang: introduction of serum 15 minutes; rinse; clean with isopropyl alcohol
protein from a traumatic tap
- Fluid is centrifuged in a microhematocrit tube and Differential Count
examined against a white background. - 100 cells should be counted
- Macrophages containing ingested RBCs - Do not use counting chamber; only stained smear
(erythrophagocytosis) or hemosiderin granules - specimen should be concentrated:
o indicates intracranial hemorrhage. sedimentation, filtration, centrifugation, and
- Additional test: D-dimer test. cytocentrifugation.
o Detect fibrin degradation product D-dimer by latex
agglutination immunoassay - indicate fibrin formation 1. Cytocentrifuge
at a hemorrhage site. - centrifuged for 5 to 10 minutes
- supernatant fluid is removed
Cell Count - slides made from the suspended sediment are air dry and
- Leukocyte/WBC count: most routinely performed on CSF are stained with Wright’s stain.
specimens - cells present in the fluid are forced into a monolayer
- RBC counts are seldom only used when: within a 6-mm diameter circle on the slide.
o a traumatic tap has occurred and a correction for - Forces cells onto a slide in a monolayer
leukocytes or protein is desired. - Filter paper absorbs the fluid
- RBC count calculation: - 0.1 mL CSF and 1 drop 30% albumin: produces an
o subtract the WBC count from the total count. adequate cell yield
- WBCs (granulocytes) and RBCs: - Albumin increases cell yield and decreases the
o begin to lyse within 1 hour cellular distortion
o 40% of the leukocytes disintegrate after 2 hours. - Positively charged coated slides to attract cells
- Should be refrigerated if not STAT - Cellular distortion: cytoplasmic vacuoles, nuclear clefting,
prominent nucleoli,indistinct nuclear and cytoplasmic
1. Methodology borders, and cellular clumping that resembles malignancy.
- Normal adult: 0-5 WBCs/uL - Daily control slide for bacterial contamination:
- Neonates: < 30 mononuclear cells/Ul 0.2 mL saline and 2 drops of 30% albumin
- Appear clear: 200 WBCs or 400 RBCs/uL
2. Cellular Constituents
2. Calculating CSF Cell couts - Primary: lymphocytes and monocytes
- Standard Neubauer counting chamber - Adults: 70% lymphocytes, 30% monocytes
(routinely used for performing CSF cell counts) - Children: 30% lymphocytes, 70% monocytes
- Formula: - Pleocytosis: increased amounts of normal cells
No. of cells counted × dilution = cells/µL determining the cause of meningitis
No. of squares counted × vol. of 1 sq.
- can be used for both diluted and undiluted specimens Lymphocytes Normal
Viral, tubercular, and All stages of development
fungal meningitis may be found
Multiple sclerosis
Neutrophils Bacterial meningitis
Granules may be less
Early cases of viral, prominent than in blood
3. Total cell count tubercular, and fungal Cells disintegrate rapidly
- Clear specimens: meningitis
o Counted undiluted unless overlapping cells are seen
Cerebral hemorrhage
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 Monocytes Normal Malignant Cells of Hematologic Origin
Viral, tubercular, and Found mixed with
fungal meningitis lymphocytes 1. Leukemias
Multiple sclerosis - Lymphoblasts, monoblasts, and myeloblasts
Macrophages RBCs in spinal fluid May contain phagocytized - Nucleoli may be more prominent than in blood
RBCs appearing as empty
vacuoles or ghost cells, 2. Lymphomas
hemosiderin granules, and - Dissemination from lymph organs
hematoidin crystals - Cleaved nucleii and prominent nucleoli
Blast forms Acute leukemia Lymphoblasts,
myeloblasts, monoblasts Metastatic and Primary Carcinoma Cells
Lymphoma Disseminated Resemble lymphocytes
cells lymphomas with cleft nuclei 1. Metastatic carcinoma cells
Plasma cells Multiple sclerosis Traditional and classic - Lung, breast, renal, gastrointestinal, and melanoma
forms seen - Fused cell walls, nuclear irregularities, and hyperchromatic
Lymphocyte reactions Reactive lymphs nucleoli
Ependymal,ch Diagnostic procedures Seen in clusters with
oroidal, and distinct nuclei and distinct 2. Primary tumors
spindleshape cell walls - Astrocytomas, retinoblastomas, medulloblastomas
Malignant Metastatic carcinomas Seen in clusters with - Occur in children
cells Primary central fusing of cell borders and
nervous system nuclei Chemistry Tests
carcinoma
Chemical Reference Significance Significance
Neutrophils Substance Concentration of Increased of Decreased
- Primary in bacterial meningitis Value, Normal Concentration Concentration
- Often contain phagocytized bacteria CSF
- Increased early viral, fungal, tubercular, parasitic Protein 15 to 45 mg/dL Meningitis CSF leakage
- Vacuoles may be present Hemorrhage
Multiple
Lymphs and monos in viral, fungal, tubercular sclerosis
- Reactive lymphocytes with viral Glucose 60% to 70% of None Bacterial,
- Multiple sclerosis has 50 or fewer lymphocytes/μL, both plasma tubercular,
normal and reactive concentration and fungal
- Seen in HIV and AIDS meningitis
Lactate 10 to 24 mg/dL >35 mg/dL: None
Nucleated RBCs (NRBC) Bacterial
- Seen with bone marrow contamination from tap in 1% of meningitis
specimens Glutamine 8 to 18 mg/dL >35 mg/dL: None
- Neutrophils with pyknotic nucleii may resemble NRBCs Some
- Capillary structures and epithelial cells from traumatic taps disturbance
of
Eosinophils
- Parasitic and fungal infections - Normal values differ from blood because of blood-brain
- Medications and shunts into the central nervous system barrier
- Total protein is common test
Macrophages  Normal 15-45 mg/dL (mg, not grams)
- Purpose is to remove cellular and other debris  Fractions differ, prealbumin is second
- May be seen after repeated taps  Transferrin is major beta globulin
- Hemorrhage: enter CSF within 2 hours to phagocytize RBCs  TAU, carbohydrate-deficient transferrin seen in CSF, not
- RBCs degraded to hemosiderin granules and further in blood; used to identify CSF
degraded to hematoidin crystals representing  IgG major gamma globulin
unconjugated bilirubin
Clinical Significance
Nonclinically Significant Cells - Decreased protein levels = fluid leakage
1. Choroidal cells - Elevated levels = damage to blood-brain barrier, Ig
- Epithelial lining of choroid plexus, singular and in clumps, production within CNS, decreased clearance, degeneration
uniform cells of neural tissue
- Ependymal cells lining ventricles and neural canal; less - Meningitis/hemorrhage most common causes of increased
defined cell membranes in clumps damage to blood-brain barrier
- Spindle cells lining arachnoid - Find abnormal results on clear fluid with low cell counts
- All seen after encephalography procedures; no significance from neurologic disorders

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Methodology CSF Lactate
- Diagnosis and management of meningitis
1. Turbidity - TB and fungal levels >25 mg/dL
- Trichloroacetic acid precipitates both albumin and globulin - Bacterial >35 mg/dL
- Viral <25 mg/dL
2. Dye – binding - More reliable than glucose
- Coomassie blue dye uses protein error of indicators - Levels remain elevated until treatment becomes effective,
principle then fall rapidly
- Red to blue: measure intensity of blue color - No need to compare with plasma

3. Automated instrumentation available CSF Glutamine

- Pyrogallol red-molybdate - Produced by brain cells from ammonia and α-
ketoglutarate to remove toxic ammonia
Protein Fractions - Elevated in liver disease
- CSF/serum albumin index - Determines ↑ ammonia in CSF; after α- ketoglutarate is
 Blood-brain barrier integrity depleted = coma
- CSF IgG index - >35 mg/dL = disturbance of consciousness
 Measure IgG synthesis in CNS - More reliable than blood ammonia
 Multiple sclerosis - Seen in children with Reye syndrome
 Stimulates immunocompetent cells in CNS
Microbiology Tests
Values for CSF albumin and globulin available on automated - Gram stain and cultures must be performed on sediment
instruments from centrifuged CSF; cytocentrifuge helps Gram stains
- CSF to Albumin Index - Blood cultures also must be drawn
CSF albumin (mg/dL) = Index - Difficult to interpret Gram stains, few organisms and often
Serum albumin (g/dL) debris
- Normal value less than 9 = no damage - Organisms: S. pneumocystis, H. influenza, E. coli, N
- IgG Index meningitidis, Listeria monocytogenes, S. agalactiae
CSF IgG (mg/dL) / serum IgG (g/dL) = Index - Cultures also plated on chocolate agar
CSF albumin(mg/dL) / serum albumin (g/dL) - TB smears very important because of growth time delay
- Normal: > 0.77 indicates IgG production - Latex agglutination tests are available for
 Group B streptococcus, H. influenza, S. pneumocystis, N.
Protein Electrophoresis meningitidis, and E. coli
- Detection of oligoclonal bands - Gram stain is the best for detection
- Indicate inflammation within the CNS - Compare with hematology and chemistry results
- Must run serum electrophoresis
- Multiple sclerosis (MS) = no bands in serum, bands in CSF Cryptococcus neoformans
- Leukemia, lymphoma, viral, HIV: bands in both - Frequent complication of AIDS
- Primary purpose for MS, compare to IgG index also - Gram stain produces a starburst pattern
- Encephalitis, neurosyphilis, Guillain-Barre, and neoplasms - India ink stain on CSF to detect the capsule and budding
may give same pattern yeast; beware of air bubbles
- Consider symptoms - Latex agglutination tests and immunoassay procedures are
the most sensitive detection method
Myelin Basic Protein - Latex agglutination primary interference is from
- Presence in CSF indicates demyelination of myelin sheathe rheumatoid factor
around axons of neurons - Always confirm immunologic results with culture
- Monitors the course of multiple sclerosis
- Effectiveness of treatment Serologic Testing
- Immunoassay procedures available - Primary test is for neurosyphilis, third stage
- Less performed now that people have been treated early
CSF Glucose with penicillin
- Selective transport across blood-brain barrier - Detect active cases within the CNS
- Approximately 60%-70% plasma glucose - The Venereal Disease Research Laboratories (VDRL)
 Plasma = 100 mg/dL; CSF = 65 mg/dL produces the recommended test for specificity
 Draw blood 2 hours before tap - Should be accompanied by a positive serum FTA-ABS
- Significance:
 Very low levels in bacterial meningitis, altered transport
and use by neural cells, not bacteria
 TB meningitis also lowers glucose values and lymphocytes
 Viral/fungal meningitis, normal glucose and lymphocytes

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