Title: OSMOTIC FRAGILITY Introduction

The osmotic fragility test is a measure of the resistance of erythrocytes to hemolysis by osmotic stress. The test consists of exposing red blood cells to decreasing concentration of NaCl solution and measuring the haemolytic effect on the cells at room temperature. The percentage of hemolysis is plotted on the vertical axis against decreasing NaCl concentration on the horizontal axis. A symmetrical curve, sigmoidal in shape, is obtained in most subjects.

Objective
To determine the fragility of the erythrocyte membrane against the haemolytic effect of NaCl solutions of varying concentrations.

Materials
Test tubes NaCl solution of different concentrations; 0.85%, 0.75%, 0.65%, 0.55%, 0.50%, 0.40%, 0.35%, 0.20%, 0.10%. Distilled water Blood Pipette Cuvettes Spectrophotometer

Procedure
1. Nine test tubes were prepared and labelled as followed: 0.85%, 0.75%, 0.65%, 0.55%, 0.50%, 0.40%, 0.35%, 0.20%, 0.10% and 0.00% NaCl. (See figure below) 2. 5ml of NaCl was pipetted into each test tube according to the label. 3. 10L blood which was already added with an anti-coagulant; heparin or sitrat was pipetted into each test tube. 4. The test tubes were then mixed carefully and were incubated for 20 minutes at room temperature. 5. The test tubes were then again shake carefully and were centrifuged for 9 minutes at a velocity of 2000rpm. 6. Supernatant were removed and transferred to labelled cuvettes. 7. OD of each supernatant was calculated using spectrophotometer with the wavelength 540nm. 8. Percentage of haemolysis was calculated using the formula:

(OD Sample/ OD of the Sample with 100% lysis) X 100

9. OD was recorded and the percentage of NaCl was converted to mole/L 10. A graph of haemolysis against concentration of NaCl was then plotted. The graph drawn is known as osmotic fragility curve.

Results
Percentage of NaCl (%) 0.00 Concentration of NaCl (mole/L) Percentage of haemolysis (%)

OD Sample

0.10

0.20

0.35

0.40

0.50

0.55

0.65

0.75

0.85

Discussion
1. The osmotic fragility test is a measure of the resistance of erythrocytes to hemolysis by osmotic stress. 2. The red blood cells are placed in hypotonic solutions of varying concentrations from 0.1% until 0.85% to which a small amount of blood is added. 3. The red blood cell expands causing the membrane to be stretched and leaks allowing haemoglobin to exit the cell. 4. Equal volumes of blood are put into a series of hypotonic solution, allowed to reach equilibrium, centrifuged and the optical density is determined. 5. The lower the surface to volume ratio, the more likely the cell is to lyse.

Conclusion
Based on the experiment done, it can be concluded that the osmotic fragility of red blood cell varies when it is put into NaCl solutions of different concentrations.