Protocol:Osmotic fragility test

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Osmotic fragility test (OFT) was the first method used for screening of thalassaemia and was introduced as simple approach to detect thalassaemia carriers by Silvestroni and Bianco in the 1940s. This fast and simple method has been applied as a screening test in large populations. The availability of electronic counters for the measurement of MCV and MCH has decreased the use of OFT. It is still used mainly in India and some Middle East Countries. Several variants of the basic method have been proposed. The most used test at present is NESTROFT, the acronym for Naked Eye Single Tube Redcell Osmotic Fragility Test. (5-7).



Microcytic red blood cells are resistant to lysis when exposed to hypotonic solutions.


Blood in EDTA.


  1. 0.36% buffered saline:
    Dilute a 10% stock solution of sodium chloride (90 g), disodium hydrogen phosphate (13.65 g) and sodium dihydrogen phosphate (2.43 g) in 1000 ml of distilled water (pH 7.4).
    The original method (kindly given by Prof. Ida Bianco) uses the Tyrode’s solution, diluted 4:10 with distilled water.
  2. Tyrode’s solution (1Litre):
    NaCl 8.2 g
    KCl 0.2 g
    MgCl 2.6 H2O 0.2 g
    CaCl 2.2 H2O 0.2 g
    NaH2PO4.H2O 0.1 g
    NaHCO3 0.05 g
    Tyrode’s solution should be stored at +4°C and the work solution prepared few hours before use.


  1. Twenty μl of whole blood collected in EDTA is pipetted into a glass test tube (100 x 10 mm) containing 4 ml of 0.36% buffered saline solution.
  2. Shake the tube and leave at room temperature for 30 minutes.
  3. Shake again the tube and immediately hold the tube in front of a piece of paper with text.


If the words on the paper are clearly visible through the tube, the test is negative; whereas if the words are not clearly visible the test is positive (thalassaemia trait) due to the turbidity of the solution.


The method is easy to perform, fast, cheap and does not require sophisticated equipment. However, it needs careful standardization. It is particularly useful in places where the electronic cell counters are not available. The test is positive both in beta and in α-thalassaemia carriers, in sickle cell trait and iron deficiency anaemia. False positive results are obtained in patients with iron deficiency and therefore subjects positive with NESTROFT need further investigation to define the diagnosis. False negative results have also been reported (8).


  1. Rosatelli, C., Leoni, G.B., Tuveri, T., et al (1989) Heterozygous β-thalassemia: relationship between the hematological phenotype and the type of β-thalassemia mutation. American Journal of Hematology, 39, 1.
  2. Rund, D., Filon, D., Strauss, Rachmilewitz, E.A., Oppenheim, A. (1992) Mean corpuscolar of heterozygotes for beta-thalssemia correlates with the severity of mutations. Blood, 79(1), 238-243.
  3. Melis, M.A., Pirastu, M., Galanello, R., Furbetta, M., Tuberi, T., & Cao, A. (1983) Phenotypic effect of heterozygous α and β°-thalassemia interaction. Blood, 62, 226.
  4. Galanello, R., Paglietti, E., Giagu, L., Melis, M.A., Scalas, M.T., Cao, A. (1985) Hematological phenotype of carriers of deletion α-thalassemia according to the α-globin genotype. Haematologica, 70, 191-198.
  5. Thomas S, Srivastava A, Jeyaseelan L, Dennison D, Chandy M. NESTROFT as a screening test for the detection of thalassemia & common haemoglobinopathies—an evaluation against a high performance liquid chromatographic method. Indian J Med Res. 1996; 104:194-7.
  6. Bobhate SK, Gaikwad ST, Bhaledrao T. NESTROFT as a screening test for detection of Beta-thalassemia trait. Indian J Pathol Microbiol. 2002; 45:265-7.
  7. Thool AA, Walde MS, Shrikhande AV, Talib VH. A simple screening test for the detection of heterozygous beta thalassemia. Indian J Pathol Microbiol. 1998; 41:423-6.
  8. Galanello R, Angius A, Melis MA, Tuveri T, Cao A. Valutazione Clinica Dello Studio Delle Resistenze Globulari Osmotiche Come Metodo Di Screening Del Tratto β-Talassemico.Rivista Italiana di Pediatria, 2: 241-3, 1976.
  9. Carrell, R.W., Kay, R. (1972) A simple method for the detection of unstable heamoglobins. British Journal of Haematology, 23(5), 615-619.
  10. Galanello, R., Barella, S., Gasperini, C., Perseu, L., Paglietti, E., Sollaino, C., Paderi, L., Pirroni, M.G., Maccioni, L., Mosca, A. (1995) Evalution of a new automatic HPLC analyser for thalassemia and hemoglobin variants screening. Journal of Automatic Chemistry, 17, 73-76.
  11. Clarke, G.M., Higgins, T.N. (2000) Laboratory investigation of hemoglobinopathies and thalassemias: review and update. Clinical Chemistry, 46, 1284-1290.
  12. Pembrey, M.E., McWade, P., & Weatherall, D.J. (1972) Reliable routine estimation of small amounts of foetal haemoglobin by alkali denaturation. Journal Clinical Pathology, 25, 738.
  13. Kleihauer, E., Braun, H. & Betke, K. (1957) Demonstration von fetealem haemoglobin in den Erythrocyten eines Blutausstriche. Klein. Wochenschr., 35, 635.
  14. Kleihauer, E. (1974) Determination of fetal hemoglobin: elution technique in: The detection of hemoglobinopathies CRC press, with contribution: Schmidt, R.M., Huisman, T.H.J., Lehman, H. 20-22.
  15. Stamatoyannopoulos, G., Farquhar, M., Brice, M., Pappayannopoulou, Th., Nute, P.E. (1983) Monoclonal antibodies specific for globin chains. Blood, 61, 530-539.
  16. Thein, S.L., Reittie, J.E. (1998) F cells immunofluorescent staining of erythrocyte smears. Hemoglobin, 22, 415-417.
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  18. Paterakis, G.S., Thein, S.L., Fibach, E., Cappellini, M.D. (1998) Cross evalution of three flow cytometric F cell counting methods performed by different laboratories. Hemoglobin, 22, 427-444.
  19. Schimidt, R.M., Brosius, E.M. (1975) Basic laboratory methods of hemoglobinopathy detection. Center for disease control bureau of laboratories haematology division. 7th edition.
  20. Frischer H., Bowman J. (1975) Hemoglobin E, an oxidatively unstable mutation. Journal of Laboratory and Clinical Medicine, 85, 531-538
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