Cerebrospinal fluid (CSF) plays a crucial role in protecting the brain and spinal cord, with normal volumes ranging from 140-170 ml in adults. It is produced at a rate of 500 ml/day and is examined for diagnosing various conditions such as meningitis and malignancies, using methods like lumbar puncture. Clinical significance includes assessments of protein levels, glucose concentration, and cell counts, which aid in identifying pathological conditions affecting the central nervous system.

1. Cerebrospinal Fluid Analysis BY BIJO AUGUSTINE

2. Anatomy and Physiology Cerebrospinal fluid (CSF) is present within the subarachnoid space surrounding the brain in the skull and the spinal cord in the spinal column. Total volumes: Adults: 140 - 170 mL Children: 10 - 60 mL

3. Functions of CSF It’s main function is to protect the brain and the spinal cord from injury by acting as a fluid cushion. It is the medium through which nutrients and the waste products are transported between brain/spinal cord and the blood.

4. Formation and composition of CSF CSF is derived by ultra filtration of plasma and by secretion through the choroid plexus located in the ventricles of the brain. Reabsorbtion of CSF occurs at the arachnoid villi which projects in the venous sinuses in the duramater. CSF is produce at the rate of 500 mL/day.

5. Formation andComposition of CSF Blood brain barrier maintains the relative homeostasis of CNS environment by tightly regulating the concentration of substances by specific transport systems for H+, K+, Ca2+, Mg2+, HCO3-. Glucose, urea and creatinine diffuse freely between blood and the CSF.

6. Proteins cross freely by passive diffusion along the concentration gradient and is also influenced by molecular weight. Formation and Composition of CSF

8. Composition of Normal CSF Protein - 15 - 45 mg/dL Glucose - 50 - 80 mg/dL Urea - 6.0 - 16 mg/dL Uric acid - 0.5 - 3.0 mg/dL Creatinine - 0.6 - 1.2 mg/dL Cholesterol - 0.2 - 0.6 mg/dL Ammonia - 10 – 35 μ g/dL

9. Composition of Normal CSF Sodium - 135 – 150 mEq/L Potassium - 2.6 – 3.0 mEq/L Chloride - 115 – 130 mEq/L Magnesium - 2.4 – 3.0 mEq/L Cells - 0 – 5 Lymph/ μ L

10. Clinical Application of CSF Examination In the diagnosis of Bacterial, viral or fungal meningitis. Encephalitis. Malignant infiltrates like in acute leukemia, lymphoma. Subarachnoid hemorrhage. Spinal canal blockage leading to elevated intracranial tension. Sub acute sclerosing pan encephalitis (SSPE)

11. Characteristics of normal CSF Color - Colorless PH - 7.28 – 7.32 Appearance - Clear Sp. Gravity - 1.003 – 1.004 No clot formation on standing Total solids - 0.85 – 1.70 g/dL PO2 - 40 – 44 mmHg

12. Collection and Processing Lumbar puncture, cisternal puncture, lateral cervical puncture, shunts & cannulas Opening pressure = 90 - 180 mm H 2 O (+/-) Approximately 15 - 20 cc fluid collected Process within 1 hour without refrigeration - STAT Three tube set-up: Tube 1: Chemistry and Immunology (Frozen) Tube 2: Microbiology (Room temperature) Tube 3: Cell count, differential, cytology (Refrigerated)

15. Diagnosis by CSF High sensitivity, high specificity Bacterial, TB, and fungal meningitis High sensitivity, moderate specificity Viral meningitis, SAH, CNS syphilis, abscess Moderate sensitivity, high specificity Meningeal malignancy Moderate sensitivity, moderate specificity Intracranial hemorrhage, viral encephalitis, subdural hematoma

16. Routine Lab Tests Required Opening CSF pressure Macroscopic Examination Total cell count and differential (stained) Glucose (CSF/plasma ratio) Protein Optional Cultures, gram stain, antigens, cytology Protein electrophoresis, VDRL, D-dimers

17. Gross Examination Normal CSF is clear, colorless Viscosity equal to water Clot seen in traumatic tap, not SAH Viscous CSF with increased protein exudate Turbidity: WBC > 200 cells/  L RBC > 400 cells/  L Microorganisms, increased protein

18. Clot/Coagulation formation Allow the specimen of CSF to stand over over night and examine the sample for fibrin clot, which is formed if the sample contains fibrinogen. Also note the nature of the clot. - Delicate clot, which resembles a cobweb, is characteristically seen in tubercular meningitis due to marked increased in CSF proteins. The clot may have entrapped tubercle bacilli, which could be demonstrated microscopically by staining for acid-fast bacilli.

19. Clot/Coagulation formation Corase clot is formed in pyogenic meningitis, traumatic tap and in case of complete spinal block. pH Determination. pH can be measured by using pH paper or using pH meter.

20. Xanthochromia Pink, orange, or yellow discoloration RBC lysis or hemoglobin breakdown May be seen within hours of LP Peak intensity at 24 - 36 hours RBC > 6000/ μ L (SAH, ICH, infarct, traumatic) Oxyhemoglobin, bilirubin, increased protein Carotinoids, melanin, rifampin therapy

21. Differential Dx of Bloody CSF Traumatic tap - blood clears between tubes Xanthochromia - pink tinge, RBCs SAH - blood does not clear or clot

22. Microscopic Exam of CSF Total WBC Count Normal CSF contains 0-8 lymph and no RBCs. Procedure Glass slides Counting chamber Cover slip of thickness with size of 22 ˟ 23 mm CSF diluting fluid – 1% Toludine blue or 1 % violet – stains the WBC without lysing the RBC, thus enabling to count both RBC and WBC in the

23. Same chamber. The stain is mixed with the CSF in the ratio 1:9 dil. Dilute acetic acid – 0.1 gm of crystal violet is added to 1 ml glacial acetic acid is made up to 50 ml by adding distilled water. Few drops of phenol is also added to this. As this fluid lyses the red cells it is useful in case of blood tinged CSF. In such case the RBC count estimated separately using undiluted CSF sample.

24. Procedure Dilution – if CSF is clear there is no need for dilution and both RBS and WBC can be counted simultaneously in the same chamber. If CSF is cloudy then make a dilution of 1:10 or 1:20.One can also pipette out 900 μ L of CSF diluting fluid in the tube and 100 μ L of CSF to it.

25. Counting of cells Charge the counting chamber properly without any air bubbles. Wait for 5 minutes before counting, to allow the cells in CSF to settle down. Count the cells in all 9 squares by using low power objective. Calculation WBC in CSF/cumm( μL)= No.of cellˣdep.ˣdilu. Area counted

26. Important points Cells in CSF should be counted immediately with out delay to prevent degeneration of cells which will give false low counts. Bloody/ traumatic tap adds approximately 1-2 WBC per 1000 RBCs. Hence in the estimation of total leucocytes count a deduction is made equivalent to 1 WBC for every 700 erythrocytes counted.

27. Differential leucocytes count Chamber differential. Differential cell count Leishman’s stained smear or Gimsa’s Stained smear.

28. Reference Intervals for CSF

29. Increased Neutrophils in CSF Meningitis (bacterial, early TB, fungal) Other infections Following seizures Following CNS hemorrhage Following CNS infarct Reaction to repeated LP Foreign materials Metastatic tumor

30. Increased Lymphocytes in CSF Meningitis (aseptic, viral, L monocytogenes) Parasitic infections Degenerative disorders Encephalopathy due to drugs, GBS Other inflammatory conditions Sarcoidosis, polyneuritis, periarteritis involving the CNS

31. Plasmacytosis in CSF TB meningitis Syphilitic meningitis Parasitic infection Sarcoidosis Acute viral infections

32. Immature cells in CSF

33. Eosinophilic pleocytosis in CSF Commonly associated with Parasitic infections Fungal infections Reaction to foreign material Infrequently associated with Bacterial or tuberculous meningitis Viral, rickettsial infection, lymphoma, sarcoidosis

34. Chemical Analysis Total protein non-specific marker of disease Turbidimetric methods based on TCA or SSA & sodium sulfate for precipitation Simple, rapid, no special instrumentation 300 different proteins have been isolated from CSF using two-dimensional electrophoresis and silver staining

35. Conditions Associated with Increased CSF Total Protein Increased blood-CSF permeability Meningitis (bacterial, fungal, TB) Hemorrhage (SAH, ICH) Endocrine disorders Mechanical obstruction (tumor, disc, abcess) Neurosypilis, MS, CVD

36. Electrophoresis Identification of oligoclonal bands 2 or more discrete bands in the gamma region absent or of lesser intensity in concurrently run patient’s serum Silver stain more sensitive than paragon violet IFE better resolution and more specific Sensitivity = 83 - 94%

37. Glucose estimation in CSF CSF glucose is derived from blood glucose hence, ideally CSF glucose level should be compared with fasting plasma glucose level for adequate clinical interpretation. Clinical Significance. CSF glucose less than 40 mg/dL or CSF/plasma glucose less than 0.3 are considered abnormal (normal CSF/Plasma glucose ratio may very from 0.3 – 0.9)

38. Increased CSF glucose is of no clinical significance. Causes of decreased CSF glucose Meningitis-Bacterial, fungal tubercular and syphilitic meningitis. Tumors involving the meninges. Subarachnoid hemorrhage. Cerebral ameobiasis.

39. Bacterial Meningitis 0 - 1m: Group B strept & E. coli (GNR) 1m - 5y: H. influenzae 5 - 29y: N. meningitidis >29y: S. pneumoniae Listeria monocytogenes common in newborns, elderly, and other immunocompromised hosts

40. Bacterial Meningitis Gram’s stain sensitivity = 60 - 90% Depends on organism, experience, Culture sensitivity = 80 - 90% Latex agglutination becoming more widely used due to simplicity and accuracy

41. Test Appearance Pressure WBC/ μ L Protein mg/dL Glucose mg/dL Chloride Normal CSF Clear 90 – 180 mm 0-8 lymph. 15-45 50-80 115-130 mEq/L Acute bacterial meningitis Turbid Increased 1000 -10000 100 – 500 < 40 Decreased Viral meningitis Clear Normal to moderate increase 5-300, rarely >1000 Normal to mild increased Normal Normal Tubercular meningitis Slightly opaque cobweb formation Increased/ decreased, spinal block 100-600 mixed or lymph. 50-300 due to spinal block Decreased Decreased Fungal meningitis Clear Increased 40-400 mixed 50-300 Decreased Decreased Acute syphilitic Clear Increased About 500 lymph Increased but <100 Normal normal

42. Bacterial Meningitis

43. Neurosyphilis Darkfield microscopy for spirochetes CSF FTA-ABS 100% sensitive Negative test rules out diagnosis VDRL nearly 100% specific Positive test rules in neurosyphilis RPR unsuitable for CSF (higher FP than VDRL)

44. Neurosyphilis

45. Viral Meningitis Enteroviruses (echoviruses, coxsachie, polio viruses) account for 80% cases Diagnosis of exclusion, rarely use cultures Viral inclusions for CMV, HSV PCR for HSV available Usually requires brain biopsy

46. HIV Wide variety of abnormalities with or without neurological disease Lymphocytic pleocytosis, elevated IgG, and oligoclonal bands ID of opportunistic (fungal) infections main reason for examining CSF

47. Fungal Meningitis India ink for cryptococcal capsular halos 50% sensitivity LA and CF antibodies now available Sensitivity as high as 96%

50. Tuberculous Meningitis Early diagnosis extremely difficult Sensitivity for acid-fast stains 10% Large volumes of CSF recommended Higher levels of adenosine deaminase ELISA and PCR now available Sensitivity = 50 - 82% Specificity = 90 - 100%

51. Primary Amoebic Meningoencephalitis (PAM) Rare disease caused by free-living amoeba Naegleria fowleri or Acanthamoeba species Motile Naegleria trophozoites may be seen with light microscope Acridine orange stain can differentiate amoeba (brick red) from leukocytes (bright green)

53. CSF Cytology Cytological evaluation of CSF is an effective means for diagnosing many disorders involving the central nervous system. Preparatory methods for CSF examination are discussed and normal and reactive conditions involving lymphoma, leukemia, meningeal carcinomatosis and the subarachnoid spread of primary brain tumors are evaluated by primary cytological examination of CSF

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