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1. Cerebrospinal Fluid (CSF) Examination 1 By: Kibruyesfa B.

2. Introduction CSF : Clear colorless fluid placed in intraventicular & subarachnoidal spaces. It is formed as an ultra filterate of the plasma by the choroids plexuses of the brain. Circulates round brain and spinal cord. 2 By: Kibruyesfa B.

3. The two Chief Functions of CSF 1. To protect and cushion the brain and the spinal cord against possible injury . 2. To deliver nutrients from the blood to the CNS and removes wastes . • transport of biomolecules to the brain • clearance of catabolites (CO2, lactate) 3 By: Kibruyesfa B.

4. Indications to CSF Diagnostics/Examination The studies on CSF were majorly carried out on the experimental animals. But now-a-days CSF is frequently examined for diagnostic purpose & for understanding the severity and nature of the disease process involving the CNS. The composition of CSF reflects many biochemical and cell-shedding alterations in CNS diseases for example, it can reveal presence of infection, tumors, neurological disease or leukemia. 4 By: Kibruyesfa B.

5. Collection of CSF For collection of CSF, the main materials required are : Spinal needle : 14-22 G; 1.5 to 4 inch Syringes (2 ml...) Sterile plain collection tubes Sharp razor/scalpel blade Cotton swab. 5 By: Kibruyesfa B.

6. Methods of collection The CSF is collected from two sites : 1. Cisterna magna or atlanto-occipital puncture :  (horse, cat and dog) 2 . Sub lumbar or lumbosacral puncture :  (cow, sheep and goat) 6 By: Kibruyesfa B.

7. 1. Atlanto -occipital puncture (horse, cat and dog) The collection can be done either in the standing position or in the lateral recumbency with following steps: 1. Restrain the animal, casting with rope. 2. Sedation of the animal by giving local anesthesia such as 2% xylocain, lignocain and tranquilization with siquil or largactil. 3. Flex the head by bending & stretching the neck, so that it forms a 90 degree with the longitudinal axis of the neck & hold it in this position. 4. Clip, clean and sterilize the selected area. 7 By: Kibruyesfa B.

8. 5. Use a 3-4” long and 16 G spinal needle with a stylet and insert it slowly at the cranial edge of the wings of atlas . Direction of the needle should be parallel to the long axis of the head . When the needle enters subarachnoidal space resistance is not felt. 6. As the needle enters into atlanto-occipital joint, remove the stylet so that the CSF flows out and collect approximately 1 to 2 ml of CSF. 8 By: Kibruyesfa B.

9. 9 By: Kibruyesfa B.

10. 2. Sub lumbar puncture (cow, sheep and goat) The collection is done in the standing position . 1. Animal must be tied firmly to avoid damage to the spinal cord. 2. Presurgical and aseptic precautions are taken and The depression between the dorsal process of last lumbar vertebra and cephalic end of median crest of sacrum is palpated. . 10 By: Kibruyesfa B.

11. 3 . Needle is passed & punctured at this site . Insert the needle vertically, then slightly oblique . 4. Collection is done by removing the stylet , apply a syringe & suck the fluid. 11 By: Kibruyesfa B.

12. 12 Fluid is collected aseptically from the subarachnoid space and placed in 3 sterile tubes numbered in the order of collection (Tubes 1, 2, 3). TUBE #1 Contains debris from the puncture. Since it is the most likely to be contaminated with microbes, tissue fluid and blood cells which could yield misleading results it should not be used for micro or hematology studies. It is best used for chemistry and immunological determinations. By: Kibruyesfa B.

13. 13 TUBE #2 May contain some blood cell contaminants but is suitable for microbiological studies. TUBE #3 Has the least cellular or debris contamination and therefore is used for cell counts, white cell differentials and the examination of abnormal cells e.g. tumor cells. By: Kibruyesfa B.

14. Components of a routine CSF analysis - Physical characteristics of CSF (e.g., color, turbidity…) - Biochemical analysis (e.g., protein concentration , glucose, creatine kinase, lactate dehydrogenase… ) - Cytologic evaluation of sediment smears Total nucleated cell count (TNCC) Erythrocyte count - Bacteriological examination . 14 By: Kibruyesfa B.

15. 15 Physical Examination of CSF & Interpretation All fluids are measured and the volume is recorded. The color and clarity before and after centrifugation is noted. The presence of a clot should also be reported.  Normally, CSF is perfectly clear, colorless, and transparent.  Pathologically, it may be turbid/cloudy, bloody, or xanthochromic. By: Kibruyesfa B.

16. 16 Turbidity or cloudiness may result from the accumulation of protein, microorganisms or cells and generally suggests infection. Bloody fluids may result from a "traumatic tap" in which blood from vascular damage during the performance of the lumbar puncture has occurred or from a hemorrhage in the CNS. It is imperative that these two situations are accurately differentiated. Centrifuge and examine supernatant. Xanthochromia = yellow color - in the supernatant denotes pathological bleeding and occurs as a result of hemoglobin degradation and bilirubin formation in the subarachnoid space. By: Kibruyesfa B.

17. 17 By: Kibruyesfa B.

18. Chemical Examinations & Interpretations Proteins : Normal protein content of CSF is 12-40 mg/100 ml and most of it is albumin. The protein conc. of CSF is too low to be accurately determined by a refractometer or by the methods used for serum proteins. Most clinics/labs do not have the machines to perform quantitative microprotein levels so a semiquantitative CSF protein measurement can be determined using a urine dipstick & other tests . 18 By: Kibruyesfa B.

19. 19 By: Kibruyesfa B.

20. Glucose : estimation of CSF glucose is done by Folin -Wu technique. The concentration of the glucose in CSF is approximately 60 -70 % of blood glucose level & ranges from 40-80 mg/100 ml in normal CSF. An increased glucose level in the CSF is called “ hyperglycorrhacia ” Associated with any disease having a hyperglycemia ( Diabetes mellitus ), encephalitis , spinal cord compression, brain tumors or brain abscess. 20 By: Kibruyesfa B.

21. A decreased glucose level in the CSF is termed as ‘ hypoglycorrhacia ’. Associated with systemic hypoglycemia or acute pyogenic infection. 21 By: Kibruyesfa B.

22. Chlorides : Normal CSF values in domestic animals ranges between 650-850 mg/100ml. Lower values are seen in pyogenic meningitis , protracted vomiting , advanced pneumonia & etc. Cholesterol : Usually normal cholesterol level is very low & values recorded in Horse: 0.36-0.55 mg/dl and Goat: 0.51 mg/dl. Hemorrhages in the CNS, tumors, meningitis and brain abscess lead to an increase in cholesterol content. 22 By: Kibruyesfa B.

23. Enzymes : Increased levels of CSF ALT (9-46 units) and AST (2-32 units) have been observed in dogs with distemper (CNS), purulent meningitis and cerebral infarction . Lactic dehydrogenase enzyme level of CSF are also increased in bacterial meningitis , metastatic carcinoma and cerebral infarction & etc . 23 By: Kibruyesfa B.

24. Cytological Examination The total cell counts of the CSF must be estimated within 20 minutes of collection, since the cells degenerate rapidly . Because normal CSF is of very low cellularity , some type of concentrated smear must be made using a cytocentrifuge. The estimation of the number of cells is done as for the determination of WBC’s of the blood . 24 By: Kibruyesfa B.

25. Normal Counts Cattle, sheep and pig................upto 15 cells/cu mm Dog..........................................upto 25 cells/cu mm Horse........................................upto 23 cells/cu mm 25 By: Kibruyesfa B.

26. Interpretation of Cytology smear made from CSF 1. Cytoalbuminemic dissociation An increased CSF protein con. often is associated with an elevated total nucleated cell count (TNCC) . The term cytoalbuminemic dissociation is used when the CSF protein concentration is increased but the TNCC is within the reference range. 26 By: Kibruyesfa B.

27. 27 Some Causes of Cytoalbuminemic Dissociation : A. Blood contamination B. Intervertebral disk disease C. Trauma D. Degenerative spinal cord disease E. Neoplasia (parenchyma of brain) 2. Presence of Erythrocytes (RBCs) The presence of RBCs in CSF indicates either blood contamination during sample collection (traumatic tap) or true intracavity hemorrhage . By: Kibruyesfa B.

28. 3. Pleocytosis An elevated total nucleated cell count in CSF is termed as pleocytosis . It is subclassified as neutrophilic, mononuclear, or mixed . Neutrophilia indicates abscesses in brain, bacterial meningitis, encephalitis and hemorrhage. Lymphocytosis is observed in uremia, toxemia, chronic viral and fungal infections. 28 By: Kibruyesfa B.

29. Neutrophilic Pleocytosis Steroid-responsive meningitis Necrotizing vasculitis Feline infectious peritonitis Infection: 1. Bacterial 2. Rickettsial (e.g., Ehrlichia ewingii ) 3. Mycotic (e.g., cryptococcosis) 29 By: Kibruyesfa B.

30. Mononuclear Pleocytosis Canine distemper virus Granulomatous meningoencephalitis Necrotizing meningoencephalitis Toxoplasmosis Neosporosis Bacterial meningitis 30 By: Kibruyesfa B.

31. Mixed Pleocytosis Include granulomatous meningoencephalitis and many of other diseases that cause a neutrophilic & other cells pleocytosis . 31 By: Kibruyesfa B.

32. Bacteriological Examination It is carried out when the CSF cell count and protein contents are high. The organisms are isolated in CSF and identified by cultural methods . 32 By: Kibruyesfa B.

33. 33 By: Kibruyesfa B.