Osmotic fragility test

Osmotic Fragility Test and NESTROFT

1. Osmotic fragility test Presenters : Dr. Sivaranjini N Dr. Krina Patel

2. Reagents Prepare a stock solution of buffered sodium chloride, osmotically equivalent to 100 g/l In preparing hypotonic solutions for use, it is convenient to make first a 10 g/l solution from the 100 g/l NaCl stock 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 and 1.0 g/l are then made.

3. Preparation of serial dilutions Prepare a stock solution dissolving 10g NaCl in 1litre of distilled water; i.e. 10g in 1000mL or 10x1000 mg in 1000 mL 1mL of such a solution will contain 10mg NaCl From this stock solution prepare further dilutions.

4. Concentration of solution Volume of stock solution (mL) Volume of distilled water (mL) 9.0 9.0  4.5 1.0  0.5 7.5 7.5 3.75 2.5  1.25 6.5 6.5 3.25 3.5  1.75 6.0 6.0  3.0 4.0  2.0 5.5 5.5  2.75 4.5  2.25 5.0 5.0  2.5 5.0  2.5 4.0 4.0  2.0 6.0  3.0 3.5 3.5  1.75 6.5  3.25 3.0 3.0  1.5 7.0  3.5 2.0 2.0  1.0 3.0  1.5 1.0 1.0  0.5 9.0  4.5

5. Procedure Deliver 5.0 ml of each of the 11 saline solutions into 12 test tubes. Add 5.0 ml of water to the 12 th tube. Add to each tube 50 µl of well-mixed blood and mix immediately by inverting the tubes several times, avoiding foam. Leave the suspensions for 30 min at room temperature. Mix again and then centrifuge for 5 min at 1200 rpm .

6. The sigmoid shape of the normal osmotic fragility curve indicates that normal red cells vary in their resistance to hypotonic solutions.

7. Osmotic Fragility after incubating the Blood at 37 C for 24 Hours Defibrinated blood should be used, care being taken to ensure that sterility is maintained. Incubate 1 ml or 2 ml volumes of blood in sterile 5 ml bottles. After 24 h, if no infection is evident, after thoroughly mixing the sedimented red cells in the overlying serum, estimate the fragility as previously described.

8. Because the fragility may be markedly increased set up additional hypotonic solutions containing 7.0 g/l and 8.0 g/l NaCl. In addition, use a solution equivalent to 12.0 g/l NaCl because sometimes, as in HS, lysis may take place in 9.0 g/l NaCl. In this case, use the supernatant of the tube containing 12.0 g/l NaCl as the blank in the colorimetric estimation.

9. Principle

10. The osmotic fragility of freshly taken red cells reflects their ability to take up a certain amount of water before lysing. This is determined by their volume-to-surface area ratio. 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 this limit is reached lysis occurs.

11. Interpretation of results

12. Spherocytes have an increased volume-to-surface area ratio. Their ability to take in water before stretching the surface membrane is thus more limited than normal and they are therefore particularly susceptible to osmotic lysis. The increase in osmotic fragility is a property of the spheroidal shape of the cell and is independent of the cause of the spherocytosis.

13. Decreased osmotic fragility indicates the presence of unusually flattened red cells (leptocytes) in which the volume-to-surface area ratio is decreased. Such a change occurs in iron deficiency anaemia and thalassaemia in which the red cells with a low mean cell haemoglobin (MCH) and mean cell volume (MCV) are unusually resistant to osmotic lysis

14. Reticulocytes and red cells from patients who have been splenectomized also tend to have a greater amount of membrane compared with normal cells and are osmotically resistant. In liver disease, target cells may be produced by passive accumulation of lipid and these cells, too, are resistant to osmotic lysis.

15. The osmotic fragility of red cells that have an abnormal membrane or have enzyme defects, increases abnormally after incubation. In thalassaemia major and minor, osmotic fragility is frequently markedly reduced after incubation, again owing to a marked loss of potassium. A similar, although usually less marked, change is seen in iron deficiency anaemia.

16. The increased osmotic fragility of normal red cells, which occurs after incubation is mainly caused by swelling of the cells associated with an accumulation of sodium that exceeds loss of potassium. During incubation for 24 hr., the metabolism of the red cell becomes stressed and the pumping mechanisms tend to fail, one factor being a relative lack of glucose in the medium.

17. Increased osmotic fragility/ shift to right Hereditary spherocytosis Entire curve may be ‘shifted to the right’, or most of it may be within the normal range but with a ‘tail’ of fragile cells. After incubation for 24 h, abnormalities usually more marked. Hereditary elliptocytosis As in HS, but in general changes less marked. Hereditary stomatocytosis As in HS with large osmotically fragile cells with low MCHC Other inherited membrane abnormalities Results variable; with milder disorders curve more likely to be abnormal after incubation for 24 h Autoimmune hemolytic anaemia Tail of fragile cells roughly proportional to number of spherocytes; rest of curve normal (or even left-shifted on account of reticulocytosis)

18. Decreased osmotic fragility/ shift to left Thalassemia Osmotic fragility decreased in all forms of thalassaemia, usually the entire curve is left-shifted Enzyme abnormalities OF usually normal. After incubation for 24 h, there may be a tail of fragile cells. Hereditary xerocytosis Increased resistance to osmotic lysis and increased MCHC Iron deficiency Curve shifted to left, wholly or partly, depending on proportion of hypochromic red cells

19. Modified Osmotic Fragility Test/ NESTROFT Simple and inexpensive test for screening for ß thalassaemia trait It is useful when quantification of haemoglobin A2 is not possible and standardized automated analyzers are not available for accurate measurement of MCV and MCH.

20. Principle of NESTROFT

21. Normally, red cells put in saline solution begin to lyse at a saline concentration of 0.4-0.5% and lysis is complete at 0.32%. However, in beta thalssemia trait, due to alteration in osmotic resistance of the affected RBC’s due to volume/surface area ratio changes, lysis begins at a saline concentration between 0.4-0.35% and it may not be completed even at 0.1% solution.

22. Reagents NESTROFT is done at a saline concentration of 0.36%. 0.36% buffered saline (BS) prepared by diluting 36ml of 1% buffered saline with 64ml of distilled water (DW) to make 100ml

23. Procedure Two test tubes labelled as BS (2ml) and DW (2ml) are taken and a drop of blood is added to each of the tubes, which are then left undisturbed for half an hour at room temperature.

24. The line is clearly visible through DW tube, If it is the same in BS tube; it is considered negative, otherwise test result is interpreted as positive

25. The tubes are then left undisturbed for 3 hours. At the end of 3 hours, the DW tube is usually seen to be homogeneously pink with no sediments. In the BS tube the negative test shows similar findings as DW tube where as in a positive case, a clear supernatant and a sediment at bottom is observed.

26. Because the false-positive rate is around 10%, confirmation of a positive result requires referral of a sample to a laboratory able to quantitate haemoglobin A2. The test can also be used to screen for alpha thalassaemia trait, with positive samples being referred to a reference centre for DNA analysis.

27. About 50% of samples containing haemoglobin E also give a positive result. This is an advantage rather than a disadvantage because detection of haemoglobin E is important in predicting the possibility of thalassaemia major or intermedia in compound heterozygotes with ß thalassaemia.

28. Summary The osmotic fragility test gives an indication of the surface area/volume ratio of erythrocytes. Its greatest usefulness is in the diagnosis of hereditary spherocytosis. The test may also be used in screening for thalassaemia.

29. References Dacie and Lewis practical haematology. Piplani M. NESTROFT - A Valuable, Cost Effective Screening Test for Beta Thalassemia Trait in North Indian Punjabi Population. Journal of clinical and diagnostic research. 2013.