Osmotic fragility Objective: To determine the osmotic fragility of erythrocytes.

Principle: Tonicity is a measure of the osmotic pressure of two solutions separated by a semi permeable membrane. Like osmotic pressure, tonicity is influenced only by solutes which cannot cross the semi permeable membrane. Higher the concentration of solute higher will be the tonocity of solution. Red blood cells are bound by a membrane which allows water to pass through while generally restricting the solutes. This process, causes cells to shrink due to loss of water (called exosmosis) when placed in hypertonic solutions. On the other hand, red cells absorb water (called endosmosis) when in a hypotonic medium. This results in swelling and ultimately hemolysis as the cell bursts . The osmotic fragility test is a measure of the resistance of erythrocytes to hemolysis by osmotic stress. Osmotic stress is produced by exposing cell to NaCl solution of tonicity lower than that of plasma. The test consists of exposing red cells to decreasing strengths of hypotonic saline solutions and measuring the degree of hemolysis calorimetrically at room temperature.

Reagents: For the purpose of osmotic fragility, test stock solution of 9% NaCl concentration was prepared in phosphate buffer. To prepare stock solution of phosphate buffer – 9 g NaCl & 1.3654g of Na2HPO4 was dissolved in 40 ml distilled water and then pH was adjusted to 7.4 using NaH2PO4 solution. This is then finally raised to 100ml.

From this stock various aliquots are prepared as follow.

Concentration of NaCl(%) Volume of stock(ml) Volume of dd water(ml) 0.0 0.00 100.0 0.2 2.22 97.78 0.4 4.44 95.56 0.6 6.66 93.34 0.8 8.88 91.12

0.9 10.0 90.00

% H

emol

ysis

% Saline Solution

Method: Heparinized venous blood was used. The tests were carried out within 2 hours of collection or within 6 hours if kept at 4° C.

5.0 mL of respective saline was delivered in 13 test-tubes. 5.0 mL of water was added to tube.

50 uL (microliters) of well mixed blood was added to each tube and mixed immediately by inverting the tubes for several times avoiding foam.

Tubes were incubating at room temperature for 30 min, mixed again and centrifuged.

Supernatant was removed and the amount of lysis was estimated in a spectrophotometer at 540 nm.

Supernatant from tube 13 was taken as blank. Assign a value of 100 % hemolysis in to tube 12.

For each set %hemolysis was calculated using following formula. %Hemolysis= {(OD value of particular tube)/ (OD value of tube 1)}*100

While determining the osmotic fragility, each sample of blood was run in triplicates or in duplicates depending on availability of blood sample.

Using duplicated or triplicated values of %hemolysis mean hemolysis was calculated and finally standard deviation was also calculated.

Result

Importance of osmotic fragility test The osmotic fragility test ultimately tries to determine the concentration of solute

inside the cell subjecting it to the hemolytic effects of solutions of different concentrations.

In osmotic fragility test %hemolysis values are calculated and finally plotted against concentration to give and sigmoidal curve.

This sigmoidal shape of curve indicates that normal RBC varies in their resistance to hypotonic solution. This resistance varies gradually with the age of RBC age. Young cell are most resistant while old cells are most fragile. The reason is that old cells have high sodium content and low capacity to pump it out.

The osmotic fragility test is a very important diagnostic as well as research tool. It is used for diagnosis of many RBC membrane disorders like hereditary spherocystosis and screening of beta thalassemia. In research area it is being used by scientist for hematological research.

Factors modulating Osmotic Fragility

In carrying out osmotic fragility tests by any method, three variables capable of markedly affecting the results must be controlled, quite apart from the accuracy with which the saline solutions have been made up. These are:

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.