Useful Notes on the Procedure and Techniques of “CSF Analysis”

On gross examination, there is no sediment, reaction is alkaline and specific gravity is 1.006 to 1.008.

Cell Count

Leucocyte Count:

It is done within first half an hour after withdrawal, because, on longer standing, cells begin to disintegrate. Normally, 0-10 cells/mm3, all of which are lymphocytes, are present. In children, a normal count is slightly high, about 20 cells/mm3.

Polymorphs are found only in pathological conditions. Cell count should be done soon after collection of the fluid.

Differential Count:

Centrifuge a little CSF and make 3 smears from deposit:

1. First smear stain by Leishman stain, to observe leucocytes.

2. Second smear with Grams stain and look for meningococci.

3. Third smear with Ziehl-Neelsen stain and look for acid fast bacilli.

Total count— It is done as follows:

CSF Diluting Fluid:

Composition:

Crystal violet – 0.2 gms.

Glacial acetic acid – 10 ml.

Distilled water – 90 ml.

Filter if cloudy; add a trace of potassium oxalate to prevent clotting.

The Futchus-Rosenthal counting chamber is specially meant for CSF cell count, which has ruled area of 16 sq mm and depth 0. 2. mm. Count all cells in entire ruled area and divide the number of cells by 3 or multiply by 0.35 to get number of cells per mm3.

Tumor cells, when suspected, should be searched for after staining the smear of the centrifuged deposit with panpani- coloou stain.

1. Count is between 10 to 100 cells/mm3:

In syphilits disease of CNS, encephalitis, anterior poliomyelitis, in chronic degenerative diseases of CNS.

2. Count is between 100 to 500 cells/mm3:

In lymphocytic choriomeningitis, syphilitic meningitis, TB meningitis, aseptic meningitis, acute poliomyelitis, brain tumors.

3. Count is over 500 cells/mm3:

Count of 1000 or more with predominance of lymphocytes found in late TB meningitis, poliomyelitis, pyogenic meningitis, influenzal meningitis, brain abscess, etc.

The CSF serves as a medium for nutrient exchange in nervous system. The CSF is usually collected by a spinal puncture. The fluid should be collected in two sterile tubes.

Biochemical Examination of CSF:

Under routine biochemical examination of CSF, protein, sugar, chlorides and pH reactions should be done.

Protein:

Normal CSF contains 15 to 45 mgm of protein %. It includes albumin and globulin in the ratio 8:1.

A. Test for Total Protein:

Take 1 ml of CSF. Add 3 ml of 3% sulfosalicylic acid. Mix well and allow it to stand for 5 min. Now compare the turbidity with visual colorimeter against known standards.

Albumin level of CSF increases in cerebral tumor and encephalitis.

B. Test for globulin (Pandy’s test):

It involves use of Pandy’s reagent. The Pandy’s reagent is prepared by dissolving 10 gm of phenol in 150 ml of distilled water. It should be clear and colourless.

Pipette 2 ml of Pandy’s reagent in a small test tube. Add 2 to 3 drops of clear CSF specimen, do not mix. Observe for the formation of turbidity. A bluish white ring is immediately formed if excess of globulin is present.

Increase of globulin is found in complete cerebrospinal meningitis, card tumor, etc.

Sugar (Glucose):

Glucose concentration in CSF depends on blood glucose level at the time of withdrawal of fluid and the presence of cells and organisms in fluid.

The normal value is 45 to 70 mgm %. It is increased in diabetes mellitus, diabetic coma, viremia, and brain tumor. Decreased amounts are associated with acute meningitis, tubercles meningitis, insulin shock, viral meningitis, etc.

Tests for Sugar:

a. Qualitative Test:

In a test tube, take 1 ml of CSF and 1 ml. Benedict’s qualitative solution. Mix well and boil over spirit lamp. Presence of sugar deposits show yellow to brick red coloured precipitate in increasing amounts of sugar.

b. Quantitative Test:

Quantitative estimation of CSF sugar is done in the same way as in the case of blood sugar using 0.2 ml of CSF

Chlorides:

Normal value for adult 720 to 760 mgm %.

Normal value for children 625 to 670 mgm %.

Chloride levels are increased in conditions in which there is increase in plasma chloride levels. It is decreased in TB meningitis.

Test: Chloride can be estimated by titrometric method. Take 2 ml of CSF and 3 ml of distilled water in a small conical flask and add 2 drops of 5% potassium chromate in it. Titrate it with 0.5814% silver nitrate from a 10 ml graduated pipette till it gives red colour.

Calculation:

Amount of chloride = 1 ml. of silver nitrate used for titration ? 100

pH:

The normal pH of CSF is 7.4 to 7.8. It becomes acidic in acute meningitis, uremia, and tuberculous meningitis.

pH can be detected by pH meter or pH paper.

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