Examination of body fluids

EXAMINATION OF BODY FLUIDS

  • The body fluids include blood, urine, cerebrospinal fluid, synovial fluid, serum and also effusions formed in cavities during disease process.

Examination of CSF

  • Examination of CSF is advised in the following conditions:
  • Whenever there is clinical evidence suggesting CNS disease
  • As a prognostic method for evaluation of disease
  • To assess the response to treatment
  • In X- ray procedure to visualize the CNS

Methods of collection of CSF

The methods advised for collection of CSF are suboccipital puncture (horse, cattle, sheep, dogs and cat) and lumbar puncture (cattle, sheep & goats).

  • Suboccipital puncture
    • The site is immediately anterior to the wing of atlas in the midline. Cast the animal on the right side and flex the head to left so that its longitudinal axis forms a right angle with the longitudinal axis of the neck. Shave, clean and sterilise the area. Take a 3-4 inch long 16 gauge, sterile needle with a stylet and insert it at the cervical midline at the level of cranial edge of the wings of atlas. The direction of the needle should be parallel to the long axis of the head. When the needle enters the subarachnoid space you suddenly feel no resistance. Hold the head firmly to avoid any sudden jerk. Remove the stylet, CSF will flow out. If not, withdraw 5ml of fluid by means of a sterile syringe. If the animal is furious and violent it is better to anaesthetize it.
  • Lumbar puncture
    • In this procedure CSF can be collected while the animal is in standing position. The site is a depression in the midline between the dorsal spinous processes of the last lumbar vertebrae and the cranial edge of the median sacral crest. Shave, clean and sterilise the area and insert a 5 inch, 14 gauge needle with stylet in the midline. On entering into the subarachnoid space CSF will flow out when the stylet is withdrawn. If not, you can use a sterile syringe.

Analysis

  • It involves various physical, chemical and cytological examinations. Invariably, physical examination, total cell count and protein determination should be conducted on all the samples. If these tests reveal abnormalities, then additional test such as microbiological examination, differential cell count, quantitative protein determination, electrolytes, glucose and other tests may be carried out.

Physical examination

  • Colour
    • Normally, CSF is clear and colourless like distilled water. It will be dull red or brown in case of haemorrhages, yellow (xanthochromic) in case of icterus and grey or green in case of suppurative conditions.
  • Turbidity
    • Normally CSF is perfectly transparent without any turbidity. Turbidity does not become grossly evident until there are 500 or more number of cells/ m L.
  • Coagulation
    • Normal CSF does not coagulate. If the fluid contains abnormal amounts of proteins, especially fibrinogen, it may coagulate. Internal haemorrhage or contamination during collection may also cause its coagulation. Heavy coagulation in acute suppurative meningitis.
  • Specific gravity
    • Though its results are not considered very precisely useful in the investigation of neurological diseases, there is a significant correlation between CSF protein concentration and specific gravity.

Chemical examination

    • The normal protein content of the CSF is very less (12-40mg/dL) and constitutes entirely of albumin. Globulins are the fractions, which are usually increased in pathological conditions, total protein concentration is increased in inflammatory and non inflammatory conditions.
    • A crude test to find out the excess of protein is foam test. In this test, the foams formed after shaking the CSF will disappear within a few minutes if it is normal. Persistence of foam even after 5 minutes indicates the presence of excess proteins.
      • Test for globulin
      • Pandy’s test
  • Pandy’s reagent
    • 10 g of carbolic acid (phenol) in 100ml of distilled water.
    • To 1 mL of saturated aqueous solution of Pandy’s reagent add a few drops of CSF and shake the mixture. If turbidity develops, then it is an indication of the presence of globulins. Development of very faint turbidity is taken as negative. Result should be graded from + to +++ depending on turbidity.
    • Nonne-Apelt (Ross-Jones) test:
    • Take 1ml of filtered saturated aqueous solution of ammonium sulphate. On this, layer carefully add 1 mL of the CSF. Keep for few minutes. Development of a white ring at the junction of two liquids indicates the presence of globulin.
    • Increased globulin in CSF indicates the presence of inflammation in CNS, increase in the capillary permeability, haemorrhages or tissue destruction occasionally resulting from congestion, haemorrhages or edema.
  • Glucose
    • Method of estimation is similar to the estimation of blood glucose and must be done in fasting animals. Dipstick method for glucose estimation in urine can be used to screen CSF samples also.
    • The concentration of glucose in CSF is approximately 60-70% of the blood glucose level. In normal animals CSF glucose values range from 40-80 mg/dL.
    • Increased glucose levels (hyperglycorrhachia) occurs in hyperglycemia, encephalitis, compression of spinal cord, brain tumours and abscess of brain, whereas decreased glucose level (hypoglycorrhachia) occurs in hypoglycemia and acute pyogenic infections.

Cytological  examination

The total cell count in the CSF must be estimated within 20 minutes of collection, since cells degenerate rapidly. Normal fluid contains small number of lymphocytes and rarely, histiocytes and endothelial cells.

  • WBC count:
  • Diluting fluid:
  • Crystal violet 0.1. mL
  • Glacial acetic acid 10.0 mL
  • Distilled water 90.0 mL

Procedure

  • Use white cell pipette of haemocytometer.
  • Draw the diluting fluid up to 1 mark.
  • Draw the CSF upto 11 mark.
  • Mix thoroughly.
  • Discard the first 2 or 3 drops.
  • Charge the chambers on both sides of the standard haemocytometer.
  • Wait for 2 minutes for the cells to settle.
  • Count the cells on all squares on the 2 sides (that is on 18 sq. mm of the area)
  • Multiply the total number obtained by 0.6 to give the number of cells in a m l of the CSF

Normal count

  • Cattle, sheep and pig – 0 to 15 cells/ m L
  • Dog – Up to 25 cells/ m L
  • Horse – Up to 23 cells/ m L
  • Increased number of white blood cells (pheocytosis) occurs in inflammatory lesions or irritation of brain, spinal cord or meninges.

Differential count

  • If the total count is less than 500 cells/ mL, centrifuge the fluid and make a smear of the sediment. Dry in air, stain with Leishman’s stain and examine. Normally only lymphocytes or occasionally a few mononuclear cells or histocytes may be found. Presence of neutrophils indicates suppurative conditions.

Bacteriological examination

  • Increase in lymphocytes than the normal value indicates viral infection, fungal infection and toxic conditions. If the cell count and the protein content are high, the aseptically collected CSF must be subjected to various microbiological studies to ascertain the aetiologic agent.
Last modified: Saturday, 24 March 2012, 7:27 AM