Examination of CSF: Cerebro Spinal Fluid

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Examination of CSF: Cerebro Spinal Fluid 




Function of CSF:
  • Cerebro Spinal Fluid is formed by choroid plexus of ventricles of brain.
  • It protects the brain and spinal cord from external pressure injuries.
  • It also provides nutrients thus acting as a nutrient medium.
  • It is involved in the excreting waste product of metabolism of nervous tissues.
Features of CSF:
  • Color: CSF is clear, colorless fluid.
  • Volume: Total volume in adult is 100-150 ml.
  • Specific Gravity: 1.006-1.008.
  • Reaction: Alkaline in reaction.
Collection of specimen:
  • CSF is collected by puncturing the space between 3rd and 4th lumber vertebrae. It is called lumber puncture. Specimen collection may be done by expert doctor under sterilized conditions.
  • About 3-5 ml of CSF can be collected in 3 tubes (5 ml each).
  • It is sent immediately to the laboratory for tests.



Physical Examination :
  • Normally CSF is crystal clear.
  • Color of CSF is changed in the diseases. Presence of blood (reddish) in CSF mean bleeding (subarachnoid or intraventricular haemorrhage).It may be yellowish tinged. e.g. in jaundice.
  • A cloudy or tubid fluid may mean brain abscess, bacterial infection of covering of brain (Meningitis).
  • A fine pillicle of clot in fluid is found in tuberculosis of meninges.



Cytological Examination:
    Leukocyte (WBC) count: It should done immediately after collection of CSF as described here:
  • Draw Unna's polychrome methylene blue to mark 1 in a RBC pipette and fill pipette up to mark 101 with CSF.
  • The reagent colors- Red cell yellow and White cells blue.
  • Cover the counting chamber (Neurbauer) with coverslip and put a drop of fluid and allow the fluid to spread under the coverslip.
  • Wait for 1-5 minutes to settle cells.
  • Count 9 large squares in counting chamber for both RBCs and WBCs.
  • The total cell counted multiplied by 1.1 gives the number of cells per cu mm.


Differential count:
    Centrifuge the CSF and make smear from deposits and stain by Leishman's stain. Count about 100 or more cells and record the %  of cells.
    A large number of lymphocyte is observed in tuberculosis whereas large number of segmented leukocytes are seen in bacterial meningitis.


Gram's Stain:
    Staining the smear by Gram's method shows whether bacteria are gram- positive (violet color) or gram - negative (red color).

Ziehl- Neelsen (ZN) Stain:
    Staining the smear by Ziehl- Neelsen  method shows weather acid -fast bacilli (Tubercular bacteria) are present or absent.

Papanicolaou stain (PAP):
    Tumor's cells (cancer cells) may be seen by using PAP stain.

Chemical Examination :
- Estimation of Total Protein:
- Normal value is 15-45 mg%
  • Take 1 ml of the CSF in a tube.
  • Add 3 ml of 3% sulfosalicylic Acid.
  • Mix and allow the mixture to stand for 5 minutes.
  • Significant turbidity if present, Means present of Increased Protein.
  • Compare the turbidity in a colorimeter with standard.

Qualitative tests for increased in globulin:

Pandy's test:
  • Take 1 ml solution of carbonic acid (10gm + 150 ml water) in a Kahn's tube or Wassermann's tube.
  • Add to it 1 drop of CSF.
  • No change in Normal CSF.
  • If globulin is in excess, then bluish ring develops.
Nonne-Apelt Test:
  • Take 1 ml of saturated ammonium sulfate solution in a test tube.
  • 1 ml of CSF is  layred over it.
  • Allow it to stand for 3 minutes.
  • A white or gray ring at the point of contact indicates excess of gloublin.
Sugar Estimation:
  • Normal value is 45 to 70 mg %.
  • Increased values are observed in diabetes mellitus, uremia, brain tumor, etc.
  • Decreased values are seen in acute meningitis ( 30 mg%), tuberculosis meninges (6 to 40 mg%), etc.
  • Quantitative estimation of sugar may be done by O-toluidine method as in blood sugar estimation
  • Remember that tests for sugar must always be done on freshly collected CSF.
Chloride Estimation
  • Normal value for adults is 720 to 760 mg% and for children, it is 625 to 670 mg %.
  • Decreased levels are seen in tuberculosis of meninges.
  • Levels remain normal in tumors, brain abscess, etc.
  • Increased values are seen in hypertension.
        Silver Nitrate Titration Method:

Principle:
    Chloride Present in the CSF is directly titrated with silver nitrate.
    AgNO3 + NaCl gives AgCl + NaNO3
    When chloride ions are exhausted , the excess silver ions react with chromate ions to form red                 solution of silver chromate.
     2AgNO3 + K2CrO4 gives Ag2CrO4 / (red precipitates) + 2KNO3

Reagents:
  • Silver nitrate solution 25 mEq/L (4.25 g/L) check against sodium chloride 125 mEq/L (7.3125 g/L). 1 ml of this solution should require exactly 5 ml of silver nitrate.
  • Potassium chromate 10 %.
Procedure:
  • Take about 30 ml water ina small beaker or 28 mm x 150 mm test tube.
  • Add 1 ml CSF followed by 2-3 drops of potassium chromate.
  • Mixture is titrated with silver nitrate solution to a faint brick of red color.
  • Normal fluid requires about 5 ml of silver nitrate.

Calculation:
    CSF chloride (mEq/L) = ml of silver nitrate solution used x 25

Bacteriological Examination:

1. Culture in 10 % CO2
  • Cloudy fluid should be cultured on chocolate agar (bacterial), Sabouraud's agar (fungi), nutrient agar, blood broth and thioglycollate media. Incubation is done at 37 degree Celsius in candle jar to provide 10 % CO2.
  • Sediments of  centrifuged fluid should be cultured on Lowenstein-Jensen media to grow tubercle bacilli , if present.
  • Guinea pig injection of CSF in peritoneal cavity may be useful for the diagnosis of tuberculosis.
2. Serological test:
  • Wassermann test (complement fixation test (CFT)).
  • Kahn's test
  • VDRL test
  • Latex agglutination and CFT for fungal infection (Cryptococcal infection).
Reporting of CSF Examination:

1. Physical Examination:
  • Color
  • Transparency
  • Clot formation (Cobweb)
  • Pressure
2. Cytological examination:
  • Total Cell Count
  • Predominant cells
3. Staining: 
  • Leishman's stain
  • Gram's staining
  • Ziehl - Neelsen stain
  • Papanicolaou stain
4. Chemical Examination:

Proteins:
  • Total protein
  • Qualitative globulin
  • Sugar value
  • Chloride value
Microbiological Examination:
  • Culture of bacteria
  • Culture of tubercle bacilli
  • Culture for fungi
  • Animal Inoculation (Guinea pig and mice)
  • Serological tests




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