Protocol:Osmotic fragility test

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).

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

Sample
Blood in EDTA.

Reagents

 * 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.
 * 1) 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.

Method

 * 1) Twenty &mu;l 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.

Interpretation
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.

Comments
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 &alpha;-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).