APPARATUS (Specimen and Reagents)
15 small test tubes, disposable syringe, rack for holding tubes, long nozzle dropper, 0.5% NaCl solution, fresh heparinized blood and distilled water.
PRINCIPLE:
It is designed as the ease with which the cells are broken down in hypotonic solutions. It is expressed in terms of concentration of saline solutions in which cells are haemolysed. RBCs in the anticoagulant blood are exposed to the various concentrations of sodium chloride. Isotonic saline has no effect on RBC fragility because no fluid moves out of or into the RBC. When these RBCs are exposed to the hypotonic saline, fluid moves into the RBCs in accordance with the law of osmosis. The RBCs swell up and rupture when the hypotonicity reaches upto 37%. Haemolysis begins at 0.42% saline solution and completes at 0.35% saline solution. Spherocytes are haemolysed earlier because they are already of rounded shape as compare to the normal RBCs which are of biconcave shape. RBCs in iron deficiency anaemia, thalassaemia (target cells) and sickle cell anaemia are haemolysed at higher dilution. Indicator of haemolysis is appearance of red color in mixture and when colour becomes saturated shows completion of haemolysis.
PROCEDURE:
- 2 Mark the test tubes W, 12, 13, 14.. .23, 24 and S from 1St test tube to 15th.
- Take 15 small test tubes and keep them in rack.
- Now add number of drops of 0.5% NaCl solution and distilled water.
- Add a drop of anticoagulant mixed blood with the help of a dropper in each test tube. 5. Shake each test tube gently to mix the blood and keep them for one hour.
- After one hour check for haemolysis by obserVing the color of mixture.
- Note down the concentration of saline in which red colour just appears and concentration of saline in which intensity of color becomes maximum (saturation of color).
- Just appearance of color indicates beginning of haemolysis and saturation of color indicates completion of heamolysis. 9. Residue at the bottom of tube can be used to see haemolysed cell under microscope.
Observation:
Haemolysis starts at………. % saline solution.
Haemolysis complete at ………..%saline solution.
RESULT :
Osmotic fragility of RBCs ranges from …… % saline to……..% saline solution.
PRECAUTIONS:
- Perform the test as soon as possible.
- Take blood sample with minimum trauma and stasis.
- The temperature of the environment should be kept constant during the test.
- Don’t shake the tube vigorously for mixing blood drop in saline.
- Saline solutions should be pure chemically and their dilutions should be accurate.
- Use the same dropper for adding 0.5% saline, distilled water and blood in all the test tubes.
- Take the reading exactly after one hour.
Clinical Significance:
Test detects increased red cell fragility in patient with intrinsic or acquired red cell abnormality.
very helpful