VLD 421: Veterinary Clinical Biochemistry and Laboratory Diagnosis-II (0+1)

MATERIALS

• Triturated organ sample suspensions ( 20% tissue homogenate )
• Negative and positive control samples
• V-bottom micro well plate and lid
• Micropipette and tips to measure 25 µl
• 1 percent suspension of red blood cells
• Normal saline
• Alsever's solution
• Morter and pestle
• Cotton and spirit
• Forceps and/or small scissors
• Discard tray

Prepartion of 1% Chicken RBC

• Collect the chiken blood from wing vein aseptically in normal saline (O.85 g of sodium choride in 100 ml of distilled water.) or Alserver’s solution in the proportion of 1.1 ml of Alserver’ssolution for each ml of blood.
• Wash the erthrocyte thrice by gentle centrifugation in sterile normal saline at 1500 rpm for 5 minute. Prepare 1% suspension by adding 99 ml of sterile normal saline to 1ml of the packed erythrocyte and store at 4 °c.

Alserver’s solution

• Alserver’s solution is anticoagulant used during collection of blood for preparation of erythrocyte suspension.
  • Sodium chloride    : 4.020g
  • Sodium citrate       : 8.00g
  • Dextrose                  : 20.50g
  • Citric acid                : 0.55g
  • Distilled water        : 100ml
  • Sterilze at 10 lb pressure for 10 minutes and store at 4 c .

Red blood cell control in the haemagglutination test

• Every time while haemagglutination test is carried out, it is necessary to test the settling pattern of the suspension of red blood cells. This involves mixing normal saline with red blood cells and allowing the cells to settle.
  • Dispense normal saline.
  • Add red blood cells and mix by gently shaking.
  • Allow the red blood cells to settle and observe the pattern.
  • Observe the cells for normal settling pattern.
  • There should be clear distinct button formation in the bottom.
• There should be no signs of haemolysis in the red blood cell suspension.
• If there are signs of haemolysis, a fresh suspension must be prepared.
• There should not be any sign of auto-agglutination in the red blood cell control.
• If an agglutination pattern is observed, discard the suspension of red blood cells. Prepare a fresh suspension and test again.

Sample preparation

• Sample is liquid in nature use directly, or tissue in nature place a piece of suspected organ in mortar and grind with PBS then centrifuge the content in a centrifuge tube finally collect the supernatant for HA test.