Cerebrospinal Fluid Examination PDF

Cerebrospinal Fluid Examination CSF PREP BY AFNAN  EXAMINATION OF CEREBROSPINAL FLUID (CSF)  Cerebrospinal fluid (CSF) is contained in a cavity that surrounds the brain in the skull and the spinal column.  It nourishes the tissues of the central nervous system and helps to...

Cerebrospinal Fluid Examination CSF PREP BY AFNAN  EXAMINATION OF CEREBROSPINAL FLUID (CSF)  Cerebrospinal fluid (CSF) is contained in a cavity that surrounds the brain in the skull and the spinal column.  It nourishes the tissues of the central nervous system and helps to protect the brain and spinal cord from injury.  CSF is composed of substances present in plasma but its composition differs as it is not formed by  simple filtration. Entry of many substances into CSF is controlled by so called Blood Brain Barrier, which allows free entry of some substances into CSF but inhibits the entry of others. Topic: CSF | By: M.Danish | Date:  NORMAL CSF Normal CSF is a colourless, clear, watery fluid and no coagulum or pellicle is formed when it is allowed to stand undisturbed in a refrigerator. It contains only 1-5 cells/mm3 and these are lymphocytes. Chemical composition is as follows: Proteins: 0.2-0.45 g/L (20-45 mg/dl). Glucose: It is 2.5-4.5 mmol/L (45-80 mg/dl) Topic: CSF EXAMINATION | By: M.Danish | Date: the value is usually 2/3 of the blood glucose level at any time. Chlorides: 118-127 mmol/L. The estimation of chlorides is of some value in tuberculous meningitis and heat stroke.  SAMPLE COLLECTION AND STORAGE  CSF is normally collected from sub-arachnoid space of spinal cord at lumber level by puncture with a long needle  Specimen shall be collected in 2-4 ml quantities in 3-4 sterile screw capped bottles.  In case CSF is to be cultured for M. tuberculosis then at least 5 ml sample is needed  CSF shall be tested as soon as it arrives in the laboratory.  CSF in the first  bottle is sometimes contaminated with blood and should be kept aside.  Sample Collection Fluid from second bottle is used for routine tests. fluid from third bottle is used for bacterial culture. If tuberculous meningitis is suspected, 4th bottle is kept in refrigerator undisturbed to see whether a pellicle or coagulum forms. Otherwise CSF must never be refrigerate  (if for bacterial culture as it kills H.influenzae) and should be kept at 37°C.  ROUTINE EXAMINATION Appearance:First of all note the colour of CSF in all three bottles. If blood is visible it should be noted whether it is present in all bottles equally or it is present in first bottle and then disappears. If no blood is seen, then note the colour. A yellowish colour (Xanthochromia) is commonly seen in subarachnoid haemorrhage persisting for several weeks, in neonatal period, brain tissue destruction.  and sometimes in long standing jaundice.  Continue If the number of WBCs is high in the CSF, then the fluid becomes turbid. In such cases cell count can be omitted with main emphasis on Gram stain and culture. Finally check if there is clot or pellicle formation in the CSF. It indicates increased fibrinogen in CSF that is a sign of inflammation.  Cell Count The CSF may contain WBC in varying quantity in certain diseases. The cell count should be carried out as soon as possible after collection of specimen, since the cells are rapidly lysed.  Continue If CSF is clear then the cells can be counted by charging a Neubauer counting chamber with well-mixed, uncentrifuged, undiluted fluid. If the count is expected to be high then CSF has to be diluted for cell counting. Diluting fluid for CSF is prepared by dissolving 200 mg crystal violet in 100 ml of 10% acetic acid.  continue  In case there is gross contamination of CSF with blood,  blood derived leucocytes would be present in CSF, therefore, the count is to be corrected. For this purpose perform RBC and WBC count in both CSF and peripheral blood.  Blood RBC count = RBC(B)  CSF RBC count = RBC(F)  Blood WBC count = WBC(B)  CSF WBC count = WBC(F)  NOTE:The finding of >1 WBC/1000 RBCs will suggest the presence of meningitis.  Microscopic examination  If the CSF does not contain numerous cells ,  centrifuge 2-4 ml CSF in a conical test tube.  Re-suspend the sediment in a drop of remaining CSF. Prepare at least three smears on glass slides and dry these in air.  Stain one smear with Leishman stain (for type of WBC)  one with Gram method (for presence and type of bacteria)  third with Ziehl-Neelsen method of staining (for acid-fast bacilli).  Special preparation Special preparations can be made if required, India ink preparation or Nigrosine staining if Cryptococcus is suspected. or direct wet preparation for trypanosomes and Neglaria spp.  ESTIMATION OF PROTEINS Increase in protein is the commonest abnormality of CSF. Proteins should always be estimated quantitatively. Various methods are available for this purpose. Easiest is turbidimetric method using proteinometer. Proteinometer is a set of standard tubes showing turbidity of known amount of proteins in CSF  Sulfosalicylic acid test Take 3 ml of 3% sulfosalicylic acid in a tube And add 1 ml of supernatant clear CSF in it. Cloudiness of the test is compared with that of standard tube.  Biuret Method  Principle: CSF proteins can be estimated calorimetrically by using Biuret or Kingsbury method.  Reagents: Trichloracetic acid 10%, Sodium hydroxide 15%, Copper sulphate 5%.  Procedure:  2 ml CSF add 2 ml 10% trichloracetic acid,  mix well and allow to stand for 5 min.  Centrifuge at high speed and discard the supernatant.  Mark this tube containing precipitate as test.  Continue  Take another test tube  And mark it blank.  To both tubes add 1 ml 15% NaOH.  Shake the “test” tube to dissolve the precipitate.  Add 0.5 ml 5% Copper sulphate and 4 ml distilled water.  Mix thoroughly and centrifuge at high speed.  Transfer the supernatant to corresponding clean tubes marked.  Read absorbance of the “test” against “blank” in a colorimeter at 550 nm. Read the quantity of proteins from calibration curve.  Estimation of Globulins This test is quite useful and in absence of contamination by blood, a positive reaction is always pathological. Normal CSF contains traces of globulin (about 3 mg/100 ml), but not sufficient to react positively. The test is almost always positive, when total protein exceeds 100 mg/100 ml. The following test is performed  Pandy’s test  A qualitative Pandy’s Test is sufficient for routine purposes.  Pandy’s Reagent:  Dissolve 10g phenol in 150 ml distilled water.  Reagent should be clear and colourless.  Procedure:  Take 2 ml reagent in a test tube and add 2-3 drops of CSF.  Examine the solution after each drop  Opalescence will appear in the reagent that vary in intensity  Continue Only a slight opalescence is significant and indicates increased globulins.  A coat of white precipitate forms around drop of CSF when it travels through reagent.  ESTIMATION OF GLUCOSE Glucose in the CSF is rapidly destroyed once the fluid is collected, it is, therefore, important to carry out glucose estimation as soon as possible. If there is likely to be a delay, the CSF should be preserved in fluoride oxalate. Any method of blood glucose estimation can be used. Since the amount of glucose in CSF is less than that in blood and may further be reduced due to disease.  therefore volume of CSF used in the test should be twice that of blood used in the same procedure.  ESTIMATION OF CHLORIDES Readings above 760 mg/dl are most commonly encountered in renal inefficiency and below 700mg/dl in meningitis.  Although it is not usually performed for CSF but it may become useful in diagnosis of tuberculosis meningitis.  They are also valuable in cerebral abscess and in other complications of infections in ear and nose.  CSF CULTURE Findings of routine examination indicative of infection make the culture mandatory. Whether culture for Mycobacterium tuberculosis and procedures for viral diseases are required will depend upon findings of routine examination CSF in 3rd bottle is used for these. Flowchart for the procedure of CSF examination  Important points  If less CSF is available, most of CSF examination can be carried out with some limitations.  Microbiological analysis/procedures should be done first.  2. Usually contaminated with blood, cell count can be made even with some minor traumatic tap by making dilutions.  3. When brain abscess is present possibly due to anaerobic infection.  4. Uncentrifuged, undiluted sample.  5. Subject to availability of appropriate reagents.  6. If less CSF is available, i.e., no bottle 3, deposit can be used for culture purposes.  7. In suspected tuberculous meningitis.  8. In suspected Cryptococcus neoformans meningitis. THE End