Osmotic fragility test of red blood cells: principle, procedure, precautions, and its clinical significance

Principle

Principle: when the RBCs are suspended in a hypotonic solution they will swell and critical volume reached then hemolysis occurs. Red cell fragility can be helpful in establishing the functional state of RBC. The diagram below shows the reaction of RBCs in different types of solution. As it gets shrink in the hypertonic solution and swells in the hypotonic solution but no change in shape in the isotonic solution.

Equipment

• Clean test tubes
• Test tube rack
• Distilled water
• 1% NaCl solution
• dropper

Procedure

1. Arrange the test tubes in the rack and number them serially from 1 to 12.
2. Prepare solutions of increasing hypotonicity by mixing the required number of drops of 1 percent sodium chloride solution and distilled water in the test tubes serially from 1 to 12, as given in the figure.
3.  Use one dropper for all saline solutions and another for distilled water.
4. Note that the first tube contains saline, which is nearly isotonic and the last tube is filled with only distilled water (tonicity nil).
5. Shake the tubes thoroughly and add a drop of blood to each tube.
6. Invert each tube gently once to mix the blood with saline, and then place them in a rack.
7. After 30 minutes observe the tubes against a white background without disturbing the tubes.

Note the number of the first tube that shows partial hemolysis and the number of the tube in which hemolysis is complete. A tube with partial hemolysis shows an upward supernatant fluid with pink color proportionate to the degree of hemolysis and a lower layer of sedimented red cells at the bottom of the tube. A tube with complete hemolysis shows a clear, uniformly pink solution in the absence of red cells at the bottom. A tube with no hemolysis shows a clear, straw-colored supernatant fluid with few red cells settled at the bottom.

Observation from the above diagram

Beginning of hemolysis in 0.48 % NaCl

Completion of hemolysis in 0.32 % of NaCl

Normal value: 

• Osmotic fragility begins at 0.45- 0.50 and completes at 0.30 to 0.33 percent saline

Precautions:

• Separate droppers should be used for pouring distilled water and saline
• A minimum of 30 minutes should be allowed for hemolysis to occur.
• Hemolysis should be checked against the white background.

Clinical significance

Diminished red cells fragility

• Iron deficiency anemia
• Thalassemia
• Sickle cell anemia
• After splenectomy

Increased red cells fragility

• Hereditary spherocytosis
• Congenital hemolytic anemia

Source: Textbook of Practical Physiology.GK pal 3rd edition