EXAMINATION OF CEREBROSPINAL FLUID (CSF)
Cerebrospinal fluid (CSF) is contained in the 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. Choroid plexuses present in ventricles of the brain secrete it continuously at a rate of 500 ml/day. From here it circulates the sub-arachnoid space of both brain and spinal cord and is absorbed into the blood of dural venous sinuses by arachnoid villi. CSF is composed of substances present in plasma but its composition differs, as it is not formed by simple filtration. The entry of many substances into CSF is controlled by the so-called Blood Brain Barrier, which allows free entry of some substances into CSF but inhibits the entry of others. This barrier is however, deranged in inflammation. Therefore, changes in composition of CSF may occur not only in diseases of the brain and spinal cord but also in metabolic diseases like diabetes, etc. The main function of CSF is protective. It provides a fluid cushion for the brain to protect it from injuries that may otherwise occur due to sudden movements inside the bony cavity. It also maintains the volume of the brain inside the cranial cavity and provides some nutrition. It absorbs the waste of neuronal tissue. The normal volume of the CSF is 100-150 ml.
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. The chemical composition is as follows:
Proteins: 0.2-0.45 g/L (20-45 mg/dl). Higher the level of collection of CSF, lower the protein. Therefore, in ventricular fluid these are only 50-150 mg/L. In neonates, protein concentration may be as high as 1.7 g/L.
Glucose: It is 2.5-4.5 mmol/L (45-80 mg/dl) and the value is usually 2/3 of the blood glucose level at any time. In diabetes or continuous intravenous glucose infusion, the value may be high. It is better that a sample for blood glucose also be collected simultaneously to make the interpretation easier.
Chlorides: 118-127 mmol/L. The estimation of chlorides is of some value in tuberculous, meningitis and heat stroke. In addition, CSF contains other plasma crystalloids too but these are not determined in routine examination.
CSF sample collection
SAMPLE COLLECTION AND STORAGE
CSF is normally collected from the sub-arachnoid space of the spinal cord at lumbar level by puncture with a long needle. A physician in the ward, under strict aseptic conditions, performs the procedure. The specimen is to be collected in 2-4 ml quantities in 3-4 sterile screw -capped bottles that are serially numbered and must be sent to the laboratory immediately. In case the CSF is to be cultured for M. tuberculosis, then at least a 5 ml sample is needed. CSF should be tested as soon as it arrives in the laboratory. The CSF in the first bottle is sometimes contaminated with blood and should be kept aside. Fluid from the second bottle is used for routine tests while the fluid from the third bottle is used for bacterial culture etc. If tuberculous meningitis is suspected, the 4th bottle is kept undisturbed in a refrigerator to see whether a pellicle or coagulum forms. Otherwise CSF must never be refrigerated (if for bacterial culture as it kills H.influenzae) and should be kept at 37°C.
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 the first bottle and then disappears. The amount of blood should also be noted. If there is gross contamination of CSF with blood in all of the bottles then the chemical values will not be true. If no blood is seen, then note the colour. A yellowish colour (Xanthochromia) is commonly seen in sub-arachnoid haemorrhage persisting for several weeks, in the neonatal period, brain tissue destruction and sometimes in long-standing jaundice. Pseudomonal meningitis may be associated with bright green CSF. Note the translucency or turbidity. If the number of WBCs is high in the CSF, then the fluid becomes turbid. In such cases a cell count can be omitted with the main emphasis on gram stain and culture. Finally, check if there is clot or pellicle formation in the CSF. It indicates increased fibrinogen in the CSF, which is a sign of inflammation.
The CSF may contain WBCs in varying quantities in certain diseases. The cell count should be carried out as soon as possible after collection of the specimen, since the cells are rapidly lysed. Table 1 depicts the WBC counts in different CSF samples.
WBC count in various conditions
WBC count /mm3
Predominant cell type
Normal adult CSF
Normal neonatal CSF
If CSF is clear then the cells can be counted by charging a Neubauer counting chamber with well-mixed, uncentrifuged, undiluted fluid. Cells in all of the nine WBC squares should be counted. The number of cells counted is approximately the number of cells per mm3 of CSF. 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. The method for counting and calculation is the same as for counting WBCs in peripheral blood. In the case of gross contamination of CSF with blood, blood-derived leucocytes will be present in the CSF, therefore, the count is to be corrected. For this purpose perform a RBC and WBC count in both the CSF and the peripheral blood.
Blood RBC count = RBC(B)
CSF RBC count = RBC(F)
Blood WBC count = WBC(B)
CSF WBC count = WBC(F)
The finding of >1 WBC/1000 RBCs will suggest the presence of meningitis.
If the CSF does not contain numerous cells (<200×109/L), centrifuge 2-4 ml CSF in a conical test tube, preferably, at a slow speed for 5-10 min. Save most of the supernatant in a clean test tube for chemical analysis. Re-suspend the sediment in a drop of remaining CSF. Prepare at least three smears on glass slides and dry these in the air.
Stain one smear with Leishman Stain (for the type of WBC), one with Gram Method (for presence and type of bacteria) and the third with the Ziehl-Neelsen Method of staining (for acid-fast bacilli).
Special preparations can be made, if required, e.g., India ink preparation or Nigrosine staining if Cryptococcus is suspected or direct wet preparation for trypanosomes and Neglaria spp.
ESTIMATION OF PROTEINS
An increase in protein is the commonest abnormality of CSF. Protein should always be estimated quantitatively. Various methods are available for this purpose. The easiest is the turbidimetric method using a proteinometer. A Proteinometer is a set of standard tubes showing the turbidity of known amounts of proteins in CSF.
Mestrezat’s Diaphenometric Procedure
Place 2 ml CSF in a small test tube (sugar tube) and add 0.3 ml 30% trichloracetic acid to it. Shake well and place in a water bath at 100°C for 2 min. Set aside for 20 minutes or longer. Then, compare the turbidity with standard tubes.
Sulfosalicylic Acid Test
Take 3 ml of 3% sulfosalicylic acid in a tube and add 1 ml of supernatant clear CSF in it. The cloudiness of the test is compared with that of a standard tube.
Principle: CSF proteins can be estimated calorimetrically by using the Biuret or Kingsbury methods.
Reagents: Trichloracetic acid 10%, Sodium hydroxide 15%, Copper sulphate 5%
Procedure: To 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”. 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, marked tubes. Read the absorbance of the “test” against “blank” in a colorimeter at 550 nm. Read the quantity of proteins from the calibration curve.
Preparation of a Standard Curve: Take pooled serum and determine its protein content by the standard method for serum. Dilute with normal saline so as to obtain a concentration of 2 g/L. Set up a series of tubes as shown in Table Treat each tube as “test” making only one blank and add 2 ml of 10% trichloracetic acid. Shake well and let stand for 5 minutes. Note the absorbance. Plot these on linear graph paper against concentration for detail.
There are certain dyes that bind with protein to give colour complexes. These have been used for measuring small amounts of protein in body fluid such as CSF. Initially Coomassie Brilliant Blue (CBB) was used for this purpose. Although it was very sensitive and specific, it had the disadvantage of staining all of the glassware. The method has now been replaced with other dyes.
Pyrogallol Red Method:
Doubt is often expressed about the values of protein estimation when CSF contains red cells and therefore added plasma protein. Calculation shows that 1400 red cells per ml (mm3) of CSF fluid correspond approximately to 1 mg of added proteins per 100 ml of CSF. The adjustment can be made as follows:
RBC = 30000 per 1 mm2
Protein = 220 mg per 100 ml
The Estimation of Globulins
This test is quite useful and, in the 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. The 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 varies in intensity. Only a slight opalescence is significant and indicates increased globulins. A coat of white precipitate forms around a drop of CSF when it travels through the reagent.
This test will also determine the globulin in the CSF. In this test, 1 ml CSF is mixed with 1 ml saturated ammonium sulphate solution and shaken well. Keep the mixture aside for 3-4 min. Normal CSF will remain clear, whereas, increased globulins produce opalescence, turbidity or precipitate. (Normal CSF may be slightly opalescent).
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 be further reduced due to disease, the volume of CSF used in the test should be twice that of blood used in the same procedure.