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The Osmotic Fragility Test (Part 1)

© Yasser Anathallee

THE OSMOTIC FRAGILITY TEST

The osmotic fragility test is a measure of the resistance of erythrocytes to hemolysis by osmotic stress. The test consists of exposing red cells to decreasing strengths of hypotonic saline solutions and measuring the degree of hemolysis colorimetrically at room temperature. (15 ° - 20 ° C). The percentage of hemolysis is plotted on the vertical axis against decreasing saline concentration on the horizontal axis. A symmetrical curve, sigmoidal in shape, is obtained in most subjects.

Reagents:

Stock Solution of buffered sodium chloride (AR) 100 g/L NaCl 90 g

Na2HPO4 13.65 g or Na2HPO4.H2O 17.115 g

NaH2P04.2H2O2.34g

Water to 1 L

From (the stock solution, prepare first a 10 g/L solution by dilution with water. Dilutions equivalent to 9.0, 7.5, 6.5, 6.0, 5.5, 5.0, 4.0, 3.5, 3.0, 2.0, 1.0 g/L are then prepared.

Method:

Heparinized venous blood is used. The test should be carried out within 2 hours of collection or within 6 hours if kept at 4° C.

1.Deliver 5.0 mL of the 11 saline solutions in testubes. Add 5.0 mL of water to tube 12.

2. Add to each tube 50 uL (microlitres) of well mixed blood and mix immediately by inverting the tubes for several times avoiding foam.

3. Incubate at room temperature for 30 min. Mix again and centrifuge.

4. Remove supernantant and estimate the amount of lysis in a colorimeter at 540 nm. Use as blank the supernatant in tube 1.

5. Assign a value of 100 % lysis to tube 12 (water).

Factors affecting osmotic fragility tests: Three variables capable of markedly affecting the results, apart from the accuracy of saline concentrations:

1. The relative volumes of blood and saline.

2. The final pH of the blood in saline suspension.

3. The temperature at which the tests are carried out.

A proportion of 1 volume of blood to 100 volumes of saline is chosen to render the effect of the plasma on the final tonicity of the suspension negligible. The fragility of the red cells is increased by a fall in pH. A rise in temperature decreases the fragility, a rise of 5° C being equivalent to an increase in saline concentration of about 0.1 g/L

Results recording:

Osmotic fragility can be described in terms of the saline concentration at which lysis begins (initial lysis or minimum resistance normally 4.5 - 5.0 g/L) and at which lysis appears to be complete ( complete lysis or maximum resistance, normally 3.0 - 3.3 g/L). It is essentially useful to record the concentration of saline causing 50 % lysis, i.e. the Median Corpuscular Fragility (MCF), normally 4,0 - 4.45 g/L. and to inspect the entire curve.

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