1. CYTOPATHOLOGY-14 DR. MAHA AL-SEDIK

2. CEREBROSPINAL FLUID (CSF) A. Composition and formation: 1. CSF is one of the major fluid of the body a. Adult total volume 90 - 150 ml b. Neonate total volume 10-60 ml 2. Cerebrospinal fluid formed by the choroid plexus cells and ependymal cells occupies this space and Absorbed by the arachnoid villus.

3. Blood Brain Barrier : It restricts entry of molecules to the brain such as blood cells, large proteins, lipids, H, K, Ca and bicarbonate. Therefore, the composition of CSF does not resemble plasma.

5. B. Functions: 1. To supply nutrients to the nervous tissue. 2. To remove metabolic wastes. 3. Serves as a mechanical barrier to cushion the brain and spinal cord against trauma. C. Indications: CSF analysis is performed to diagnose meningitis, intracranial hemorrhage, leukemia, malignancies, and central nervous system disorders.

6. Site of taking sample:  Between the third and fourth lumbar vertebrae.  Please do not forget to take glucose sample 2 hours before CSF sample. VIP

8. D. Specimen Collection 1. Routinely via lumbar puncture under sterile conditions 2. Three sterile tubes collected a. Tube 1: Chemistry and serology. b. Tube 2: Microbiology. c. Tube 3: Cytology and differential. 3. All CSF should be treated with extreme caution as they can be highly infectious.

12. VIP  If only tube 1 is hemorrhagic : traumatic puncture, It can not be used for protein analysis, We try to use tube 3 in almost all tests.  Tube 1 in this case is never to be used for bacterial culture.

14. COLLECTION AND PREPARATION OF SPECIMENS:  CSF is usually collected by aseptic lumbar puncture.  As cells in the CSF tend to degenerate quickly, the fluid samples must be processed within 30 min of sampling.  Usually, a 3-tube technique is used.  Tube # 1 is used for serology and biochemical tests.  tube # 2 is used for microbiological cultures.  tube # 3 is used for cytologic evaluation.

16.  Refrigeration at 4 0 C may preserve cell details up to 48 hr. If a delay in processing CSF is anticipated, the addition of an equal amount of 50% ethanol is recommended.  Centrifuge at a relatively slow speed: 500 rpm for 5 min.  The smears obtained are air-dried or fixed in 95% ethanol for staining with the Wright method or the Papanicolaou technique.

17.  A repeat lumbar puncture should be made if the first CSF sample is negative and there is strong clinical suspicion of disease.

18. Very important note: The spinal cord extends down to the space between the first and second lumbar vertebrae.

20. Recommended tests for CSF sample: Routine:  Opening CSF pressure.  Total cell counts.  Differential cell count ( after staining ).  Glucose ( CSF / Plasma ratio ).  Total protein.

21. Tests useful under certain conditions:  Culture.  Gram stain, Acid fast stain  Enzymes e.g. LDH  PCR for TB or viruses  Cytology  Electrophoresis

22. CSF Physical Characteristics /Appearance, and Gross Examination: It may be : 1.Turbidity or cloudiness: Normally crystal clear and colorless. It start to appear when leucocytes count over 200 cells / microliter. RBCs more than 200 cells / microliter. Also micro organisms may cause turbidity.

23. 2. Clot formation: May be present in patients with traumatic taps or suppurative or tuberculous meningitis. 3. Pink – red CSF : It means presence of RBCs. It may be due to : Subarachnoid hemorrhage. Intracerebral hemorrhage. Traumatic tap.

24. 4. Xanthochromic: Xanthochromia: term used only for CSF to describe a pale pink to yellow coloration of the supernatant of centrifuged. To detect it: You have to centrifuge the CSF then compare the supernatant with distilled water.

25. 2) Causes of xanthochromia: a)Pink : RBCs lysis and Hemoglobin presence. b)Yellow: Hemoglobin has been converted to bilirubin. Or serum hyperbilirubinemia. c)Orange: May be due to RBCS or hyper vitaminosis A. d)Yellow green: Elevated serum ( biliverdin ). e)Brown: Meningeal melanoma.

26. xanthochromia

27. How to differentiate between traumatic tap and subarachnoid hemorrhage?  In traumatic tab: The hemorrhagic fluid usually clears between the first and third tube.  In subarachnoid hemorrhage: It remains unchanged in the all 3 tubes.

29. TOTAL CELL COUNT The normal leukocyte cell count in adults is 0 - 5 cells/ul. Increased neutrophils :  Bacterial meningitis.  Cerebral abscess.  CNS hemorrhage.  Traumatic tap.

30. Increase Lymphocytes: Viral meningitis. TB meningitis. Multiple sclerosis. Fungal meningitis. Increased eosinophil: Very rare. Idiopathic eosinophilic meningitis. Parasitic infection.

31. RBCs:  Normally there are no RBCs in the CSF.  IF present it may be traumatic tap, intra cranial hemorrhage or malignancy.  Although red cell counts have limited diagnostic value, they may give a useful approximation of the true CSF WBCs or total protein.  By making correction for the leucocytes or proteins.  To be sure, you have to make protein, WBCs and RBCs from the same tube.

32. WBCs corrected = WBCs obs --- WBCs added WBCs added = WBCs bld X RBCs csf / RBCs bld WBCs obs : CSF leucocytic count. WBC added : Leucocytes added to CSF by the traumatic tap. WBCs bld : peripheral blood leucocytic count. RBCs csf : CSF erythrocyte count. RBCs bld : peripheral blood erythrocyte count.

33. GLUCOSE The normal Glucose is about 60% compared to serum level before 2 hours. Normal fasting CSF glucose level: 50 - 80mg/dl. Elevated Glucose: Diabetes mellitus or traumatic tap. Decreased Glucose: Bacterial Meningitis, TB or fungal meningitis.

34. TOTAL PROTEIN Over 80% of CSF protein content is derived from the plasma. In concentration less than 1 % of the plasma level. An increased CSF protein serves as a useful but nonspecific indicator of disease. Normal level 15 - 45mg/dl.

35. ELEVATED CSF PROTEIN:  Increased permeability of the blood-brain barrier.  Decreased resorption at the arachnoid villi.  Mechanical obstruction of CSF flow.  Traumatic tap. DECREASED CSF PROTEIN:  Normally in young children 6 months – 2 years.  In condition associated with increase CSF turnover : A) removal of large volume of CSF. B) CSF leak by any trauma.

36. SERUMCSFParameter 65-85 g/l0.2-0.4 (0.6) g/lTotal protein 35-53 g/l120-300 mg/lAlbumin 97-108 mmol/l113-131 mmol/lCl- 3.9-5.6 mmol/l2.2-4.2 mmol/lGlucose 3.8-5.0 mmol/l2.4-3.4 mmol/lK+ 137-146 mmol/l145-165 mmol/lNa+