Principle
The osmotic fragility test gives an indication of the surface area / volume ratio of erythrocytes. The ability of the normal red cell to withstand hypotonicity results from its biconcave shape, which allows the cell to increase its volume by about 70% before the surface membrane is stretched. Once the limit is reached, lysis occurs. Spherocytes have an increased volume-to-surface area ratio and are therefore particularly susceptible to osmotic lysis. Its greatest usefulness is in the diagnosis of hereditary spherocytosis.
Small volumes of blood are mixed with a large excess of buffered saline solutions of varying concentration. The fraction of red cells that lyse at each saline concentration is determined colorimetrically. The test is normally carried out at room temperature.
Reagents
a) Prepare a stock solution of buffered NaCL osmotically equivalent to 100 grams of NaCl as follows:
1) NaCl – 90 grams
2) Na2HPO4 – 13.65 grams
3) NaH2PO4, 2H2O – 2.34 grams
Adjust the final volume to 1 litre. This solution will keep for months at 4°C in a well-stopper bottle.
b) Dilutions equivalent to 9.0, 7.5, 6.5, 6.0, 5.5, 5.0, 4.0, 3.5, 3.0, 2.0 and 1.0 gram/litre are convenient concentrations of hypotonic solutions for use.
Primary sample
Freshly collected ( <2 hours) heparinized venous blood or defibrinated blood.
Method
1) Deliver 5 ml of each of the 11 saline solutions into 12 x 75 mm test tubes. Add 5 ml of water to the 12th tube
2) Add to each tube 50 µl of well-mixed blood and mix immediately by inverting the tubes several times, avoiding foam.
3) Leave the suspensions for 30 minutes at room temperature. Mix again, and then centrifuge for 5 minutes at 1200 grams.
4) Remove the supernatants and estimate the amount of lysis in each using a spectrometer at a wavelength setting of 540 nm.
5) Use as a blank the supernatant from tube 1 (osmotically equivalent to 9 grams/litre NaCl).
Assign a value of 100% lysis to the reading with the supernatant of tube 12.
Result
- Initial lysis at 5 grams/liter of NaCl.
- Complete lysis at grams/liter of NaCl.
Interpretation
Increased osmotic fragility occurs in:
- Hereditary spherocytosis
- Hereditary elliptocytosis
- Hereditary stomatocytosis
- Autoimmune hemolytic anemia
Decreased osmotic fragility occurs in
- Thalassemia
- Enzyme abnormalities
- Hereditary xerocytosis
- Iron deficiency anaemia
Factors affecting the test
- The relative volume of blood and saline.
- The final pH of the blood in saline suspension.
- The temperature at which the test is carried out.