1. CEREBROSPINAL FLUID
CHAPTER 9

2. Learning Objectives
Upon completing this chapter, the reader will be able to
State the three major functions of cerebrospinal fluid (CSF).
Distribute CSF specimen tubes numbered 1, 2, 3, and possibly 4 to their appropriate laboratory sections and correctly preserve them.  
Describe the appearance of normal CSF and the causes of abnormally appearing CSF.
Define xanthochromia and state its significance.
Differentiate between CSF specimens caused by intracranial hemorrhage and a traumatic tap.

3. Learning Objectives (cont’d)
Calculate CSF total, white blood cell (WBC), and red blood cell (RBC) counts when given the number of cells seen, amount of specimen dilution, and the squares counted in the Neubauer chamber.
Describe the leukocyte content of the CSF in bacterial, viral, tubercular, and fungal meningitis.
Describe and state the significance of macrophages in the CSF.
Differentiate between the appearance of normal choroidal cells and malignant cells.

4. Learning Objectives (cont’d)
State the reference values for CSF total protein and name three pathologic conditions that produce an elevated CSF protein.
Determine whether increased CSF albumin or immunoglobulin is the result of damage to the blood-brain barrier or central nervous system production.
Discuss the significance of CSF electrophoresis, immunophoresis, and isoelectric focusing findings in multiple sclerosis and the identification of CSF.
State the reference values for CSF glucose and name the possible pathologic significance of a decreased CSF glucose.

5. Learning Objectives (cont’d)
Discuss the diagnostic value of CSF lactate and glutamine determinations.
Name the microorganism associated with a positive India ink preparation.
Discuss the diagnostic value of the bacterial and cryptococcal antigen tests.
Determine whether a suspected case of meningitis is of bacterial, viral, fungal, or tubercular origin, when presented with pertinent laboratory data.
Describe the role of the Venereal Disease Research Laboratories test and fluorescent treponemal antibody-absorption test for syphilis in CSF testing.

6. Formation and Physiology
Brain and spinal cord lined by meninges
Three layers of meninges
Dura mater: outer
Arachnoid: middle
Pia mater: surfaces of brain and spinal cord
Cerebrospinal fluid (CSF) produced in choroid plexuses of the four ventricles
20 mL/min produced in adults
Volume adults 90 to 150 mL, neonates 10 to 60 mL

7. The Meninges

8. Formation and Physiology
CSF flows through subarachnoid space between arachnoid and pia mater
Reabsorbed into blood in arachnoid granulations/villae (one-way valves)
Formation by selective filtration
Hydrostatic pressure and active transport
Not an ultrafiltrate
Very tight-fitting endothelial cells, prevent filtration of large molecules—called the blood-brain barrier

9. Flow of Spinal Fluid Through the Brain

10. Formation and Physiology
Blood-brain barrier
Essential to protect brain
Chemicals and harmful substances do not pass
Antibodies and medications are excluded
CSF composition differs from plasma
Meningitis, multiple sclerosis disrupt membrane
Test for substances that pass through: cells, protein, bacteria, immunoglobulins

11. Specimen Collection & Handling
CSF collected between third to fifth lumbar vertebrae
Three sterile tubes in this order
Chemistry/serology
Microbiology (avoid skin contamination)
Hematology (avoid cells from tap)
Save leftover fluid/fourth tube for additional tests
Volume removed based on patient volume and opening pressure

12. CSF Specimen Collection Tubes

13. Specimen Collection & Handling
Usually STAT requests
Handle carefully to avoid repeat taps
Preservation
Hematology
Refrigerate
Microbiology
Room temperature
Chemistry/serology
Frozen

14. Appearance
Crystal clear, cloudy/turbid, milky, xanthochromic, hemolyzed/bloody
Cloudy = infection; milky = lipid or protein
Xanthochromic
Pink, orange, yellow
RBC degradation products
Also jaundice, ↑ ↑ protein, carotene
Pathologic = cerebral hemorrhage

15. Tubes of CSF

16. Traumatic Tap Blood vessel punctured during tap
Differentiate from cerebral hemorrhage
Uneven blood distribution in tubes with traumatic tap
Erythrophagocytosis, hemosiderin granules
Hemorrhage = even distribution in all tubes
Traumatic tap = decreasing tubes 1 through 3

17. Traumatic Tap (cont’d)
Clot formation
Clots present = traumatic tap (plasma)
Hemorrhage does not have enough fibrinogen
Other causes of clot formation
Nonbloody CSF = damage to blood-brain barrier
TB meningitis: web-like pellicle after refrigeration
Xanthochromia
Not present in a recent traumatic tap
Indicates older hemorrhage
D-dimer test for hemorrhage

18. Cell Count White blood cell (WBC) and total cell count
Red blood cell (RBC) count seldom done
Granulocytes lyse within 1 hour; STAT
Normal adult 0 to 5 WBCs/µL
Neonates up to 30 mononuclear cells/µL
Neubauer counting chamber
Automated cell counters can be used
Body fluid specific automation is available

19. Neubauer Counting Chamber

20. Calculating CSF Cell Count
Standard Neubauer calculation formula (cells/µL)
Number of cells counted × dilution
Number of cells counted × volume of 1 square
= cells/µL
Can be used for diluted and undiluted samples

21. Calculating CSF Cell Count (cont’d)
Example
# cells counted × dilution × μL = cells/µL
1 μL (0.1 × 10)
(volume counted) </exeq)

22. Calculating CSF Cell Count (cont’d)
Total cell count
Clear specimens count undiluted unless overlapping cells are seen
Load with transfer pipette
Dilute with normal saline if necessary
WBC count
Dilute with 3% acetic acid; methylene blue helps to see cells; undiluted rinse transfer pipette with acetic acid, gently rotate pipette

23. Quality Control Commercial cell controls are available
Check diluents for contamination biweekly
Monthly check on cytocentrifuge speed and timing
Soak nondisposable chambers in bactericidal solution for 15 minutes; rinse; clean with isopropyl alcohol

24. Differential Count Valuable diagnostic aid Stained smear only
Must concentrate specimen
Sedimentation, filtration, centrifugation, and cytocentrifugation
100 cells should be counted, classified, and reported in terms of percentage

25. Cytocentrifugation Cytocentrifuge
Forces cells onto a slide in a monolayer
Filter paper absorbs moisture
0.1 mL CSF to 1 drop 30% albumin
Albumin increases the cell yield and decreases the cellular distortion
Positively charged slides to attract cells
Daily control of 0.2 mL saline and two drops of albumin stained for bacterial contamination

26. Cellular Constituents
Normal lymphocytes and monocytes
Adults: normal lymphocytes:monocytes = 70:30
Children’s ratio is reversed
Occasional neutrophils are normal
Pleocytosis: increased amounts of normal cells
Pleocytosis of normal cells is valuable in determining the cause of meningitis
Neutrophils = bacterial
Lymphocytes = viral, tubercular, fungal, parasitic

27. Prominent Cells Seen in CSF
Type of Cell
Major Clinical Significance
Microscopic Findings
Lymphocytes
Normal
All stages of development may be found
Viral, tubercular, and fungal meningitis
Multiple sclerosis
Neutrophils
Bacterial meningitis
Granules may be less prominent than in blood
Early cases of viral, tubercular, and fungal meningitis
Cells disintegrate rapidly
Cerebral hemorrhage
Monocytes
Found mixed with lymphocytes
Table 9-3

28. Prominent Cells Seen in CSF (cont’d)
Type of Cell
Major Clinical Significance
Microscopic Findings
Macrophages
RBCs in spinal fluid from hemorrhage
Contrast media
May contain phagocytized RBCs appearing as empty vacuoles or ghost cells, hemosiderin granules, and hematoidin crystals
Blast forms
Acute leukemia
Lymphoblasts, myeloblasts, or monoblasts
Lymphoma cells
Disseminated lymphomas
Resemble lymphocytes with cleft nuclei
Plasma cells
Multiple sclerosis
Traditional and classic forms seen
Lymphocyte reactions
Reactive lymphs
Ependymal, choroidal, and spindle-shaped cells
Diagnostic procedures
Seen in clusters with distinct nuclei and distinct cell walls
Malignant cells
Metastatic carcinomas Primary central nervous system carcinoma
Seen in clusters with fusing of cell borders and nuclei
Table 9-3

29. Neutrophils Primarily in bacterial meningitis
Often contain phagocytized bacteria
Increased early viral, fungal, tubercular, parasitic
Vacuoles may be present

30. Cellular Constituents

31. Nucleated RBCs (NRBC)
Seen with bone marrow contamination from tap in 1% of specimens
Neutrophils with pyknotic nucleii may resemble NRBCs
Capillary structures and epithelial cells from traumatic taps

32. Cellular Constituents

33. Lymphocytes and Monocytes
Lymphs and monos in viral, fungal, tubercular
Reactive lymphocytes with viral
Multiple sclerosis has 50 or fewer lymphocytes/μL, both normal and reactive
Seen in HIV and AIDS

34. Eosinophils Parasitic and fungal infections
(primarily Coccidioides immitis)
Medications and shunts into the central nervous system

35. Macrophages Purpose is to remove cellular and other debris
May be seen after repeated taps
Hemorrhage: enter CSF within 2 hours to phagocytize RBCs
RBCs degraded to hematoidin crystals representing unconjugated bilirubin

36. Macrophages (cont’d)

37. Nonclinically Significant Cells
Seen after diagnostic procedures
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 seen in clumps

38. Malignant Cells of Hematologic Origin
Leukemias
Lymphoblasts, monoblasts, and myeloblasts
Nucleoli may be more prominent than in blood
Lymphomas
Dissemination from lymph organs
Cleaved nucleii and prominent nucleoli

39. Malignant Cells of Hematologic Origin (cont'd)

40. Malignant Cells of Hematologic Origin (cont'd)

41. Malignant Cells of Nonhematologic Origin
Metastatic carcinoma cells
Lung, breast, renal, gastrointestinal, and melanoma
Fused cell walls, nuclear irregularities, and hyperchromatic nucleoli
Primary tumors
Astrocytomas, retinoblastomas, medulloblastomas

42. Chemistry Tests CSF formed by plasma filtration
Normal values differ from plasma because of selectivity of blood-brain barrier
Abnormal values result
Alterations in the permeability of the blood-brain barrier
Increased production or metabolism by the neural cells in response to a pathologic condition

43. Cerebrospinal Protein
Total protein is the most common test
Normal 15 to 45 mg/dL (mg, not grams)
Method dependent
Increased in infants and persons >40
Albumin is predominant, prealbumin is second
Alpha globulins-haptoglobin and ceruloplasmin
Transferrin is major beta globulin
TAU, carbohydrate-deficient transferrin seen in CSF, not in blood; used to identify CSF
IgG major gamma globulin

44. 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

45. Clinical Significance of Elevated Protein Values
Elevated Results
• Meningitis
• Hemorrhage
• Primary CNS tumors
• Multiple sclerosis
• Guillain-Barré syndrome
• Neurosyphilis
• Polyneuritis
• Myxedema
• Cushing disease
• Connective tissue disease
• Diabetes
• Uremia
Decreased results
• CSF leakage/trauma
• Recent puncture
• Rapid CSF production
• Water intoxication
* Reference values for protein are usually 15 to 45 mg/dL, but are method dependent, and higher values are found in infants and people older than 40 years.
Table 9-4

46. Methodology
Turbidity
Automated instrumentation available
Nephelometry

47. Protein Fractions
Comparisons between serum and CSF levels of albumin and IgG
CSF/serum albumin index
Blood-brain barrier integrity
CSF IgG index
Comparison of the CSF/serum albumin index with the CSF/serum IgG index
Values for CSF albumin and globulin adapted for automated instruments

48. Protein Fractions (cont’d)
IgG index =
CSF IgG (mg/dL)/serum IgG (g/dL)
CSF albumin (mg/dL)/serum albumin (g/dL)
Values >0.70 indicate IgG production within the CNS

49. Electrophoresis and Immunophoretic Techniques
Detection of oligoclonal bands
Represent inflammation within the CNS
Located in the gamma region of the protein electrophoresis
Simultaneous serum electrophoresis must be performed

50. Electrophoresis and Immunophoretic Techniques (cont'd)
Multiple sclerosis (MS) = no bands in serum, bands in CSF
Leukemia, lymphoma, viral, HIV: bands in both
Primary purpose for MS, compare also to IgG index
Encephalitis, neurosyphilis, Guillain-Barré, and neoplasms may give same pattern
Consider symptoms

51. Myelin Basic Protein
Presence in CSF indicates demyelination of myelin sheath around axons of neurons
Monitors the course of multiple sclerosis
Effectiveness of treatment
Immunoassay procedures available

52. CSF Glucose Selective transport across blood-brain barrier
Approximately 60% to 70% plasma glucose
Plasma = 100 mg/dL; CSF = 65 mg/dL
Draw blood 2 hours before spinal tap
Significance
Values that are decreased relative to plasma values
Elevated CSF glucose values are always a result of plasma elevations

53. CSF Glucose Clinical Significance
Markedly decreased with increased neutrophils in bacterial meningitis
Tubercular meningitis decreased with increased lymphocytes
Viral/fungal meningitis, normal glucose and increased lymphocytes

54. CSF Lactate Diagnosis and management of meningitis
Bacterial, TB and fungal levels >25 mg/dL
Viral <25 mg/dL
More reliable than CSF glucose
Levels remain elevated until treatment becomes effective, then fall rapidly
Can result from any condition that decreases oxygen flow to the tissues
Monitor severe head injuries

55. CSF Glutamine
Produced by brain cells from ammonia and α-ketoglutarate to remove toxic ammonia
Indirect test for the presence of excess ammonia in the CSF
Normal: 8 to 18 mg/dL
Elevated in liver disease
Elevated in children with Reye syndrome
Disturbance of consciousness when glutamine levels are more than 35 mg/dL
More reliable than direct CSF ammonia

56. Microbiology Tests
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: S. pneumocystis<<AU: Should this be Streptococcus pneumocystis or pneumoniae?>>, Haemophilus influenza, Escherichia coli, Neisseria meningitidis, Listeria monocytogenes, S. agalactiae

57. Microbiology Tests (cont’d)
Cultures also plated on chocolate agar
TB smears very important because of growth time delay
Latex agglutination tests are available for
Group B streptococcus, Haemophilus influenzae, S. pneumocystis<<AU: Should this be Streptococcus pneumocystis or pneumoniae?>>, Neisseria meningitidis, and Escherichia coli
Gram stain is the best for detection
Compare with hematology and chemistry results

58. Parasites Naegleria fowleri Found in ponds, lakes, and some pools
Enters nasal passages and migrates to the brain
Motile amoeba seen in wet preps, nonmotile in cytospin preps
Elongated with tapered posterior

59. Serologic Testing Primary test is for neurosyphilis, third stage
Performed less now that people have been treated early with penicillin
Detect active cases within the CNS
The Venereal Disease Research Laboratories (VDRL) produces the recommended test for specificity
Should be accompanied by a positive serum Fluorescent Treponemal Antibody Absorption (FTA-ABS)
<<AU: defined FTA-ABS: ok?>>