1. 1 Urinalysis & Body Fluids CLS 431 2 ND Semester, 2015 Chapter 4 :  Cerebrospinal Fluid (CSF)

2. 2 Cerebrospinal Fluid (CSF)

3. CSF Cerebrospinal fluid (CSF) is 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

4. Functions of CSF  -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

5. CSF  CSF is produce at the rate of 500 mL/day. 5

6. Composition 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+, Ca 2+, Mg 2+, HCO 3 -.  Glucose, urea and creatinine diffuse freely between blood and the CSF.  Proteins cross freely by passive diffusion along the concentration gradient 6

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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 μg/dL 8

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/μL 9

10. 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 10

11. Processing 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 temprature) –Tube 3:Cell count, differential, cytology (Refrigerated) 11

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14. Diagnosis by CSF –Bacterial, TB, and fungal meningitis –Viral meningitis CNS syphilis, abscess –Meningeal malignancy 14

15. Routine Lab Tests Required Macroscopic Examination Total cell count and differential (stained) Glucose (CSF/plasma ratio) Protein Optional Cultures, gram stain, antigens, cytology Protein electrophoresis, VDRL, D-dimers 15

16. Gross Examination Normal CSF is clear, colorless Viscosity equal to water Viscous CSF with increased protein exudate Turbidity: –WBC > 200 cells/  L –RBC > 400 cells/  L –Microorganisms, increased protein 16

17. Clot/Coagulation formation Allow the specimen of CSF to stand 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 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. 17

18. Clot/Coagulation formation Corase clot is formed in pyogenic meningitis, in case of complete spinal block. pH Determination. pH can be measured by using pH paper or using pH meter. 18

19. Xanthochromia Pink, orange, or yellow discoloration RBC lysis or hemoglobin breakdown Oxyhemoglobin, bilirubin, increased protein Carotinoids, melanin 19

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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 X 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 Same chamber. 22

23. 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. 23

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 900μL of CSF diluting fluid in the tube and 100μL of CSF to it. 24

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 cell ˣ dep. ˣ dilu. Area counted 25

26. Important point Cells in CSF should be counted immediately with out delay to prevent degeneration of cells which will give false low counts. 26

27. Reference Intervals for CSF 27

28. Increased Neutrophils in CSF Meningitis (bacterial, early TB, fungal) Other infections Following CNS hemorrhage Following CNS infarct Foreign materials Metastatic tumor 28

29. Increased Lymphocytes in CSF Meningitis (aseptic, viral, Listeria monocytogenes ) Parasitic infections Degenerative disorders –Encephalopathy due to drugs Other inflammatory conditions 29

30. Plasmacytosis in CSF TB meningitis Syphilitic meningitis Parasitic infection Acute viral infections 30

31. 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 31

32. Chemical Analysis Total protein non-specific marker of disease 300 different proteins have been isolated from CSF using two-dimensional electrophoresis and silver staining 32

33. Conditions Associated with Increased CSF Total Protein Increased blood-CSF permeability –Meningitis (bacterial, fungal, TB) –Hemorrhage –Endocrine disorders –Mechanical obstruction (tumor, disc, abcess) –Neurosyphilis 33

34. 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) 34

35. Increased CSF glucose is of no clinical significance. Causes of decreased CSF glucose Meningitis-Bacterial, fungal tubercular and syphilitic meningitis. Tumors. Cerebral ameobiasis. 35

36. Bacterial Meningitis Listeria monocytogenes common in newborns, elderly, and other immunocompromised hosts 36

37. 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 37

38. TestAppearancePressureWBC/μLProtein mg/dL Glucose mg/dL Chloride Normal CSF Clear90 – 180 mm 0-8 lymph.15-4550-80115-130 mEq/L Acute bacterial meningitis TurbidIncreased1000 - 10000 100 – 500< 40Decreased Viral meningitis ClearNormal to moderate increase 5-300, rarely >1000 Normal to mild increased Normal Tubercular meningitis Slightly opaque cobweb formation Increased/ decreased, spinal block 100-600 mixed or lymph. 50-300 due to spinal block Decreased Fungal meningitis ClearIncreased40-400 mixed 50-300Decreased Acute syphilitic ClearIncreasedAbout 500 lymph Increased but <100 Normalnormal 38

39. Bacterial Meningitis 39

40. Neurosyphilis 40