Erythrocyte osmotic fragility test

Erythrocyte osmotic fragility (EOF) was determined by subjecting erythrocytes to varying concentrations of buffered saline solutions with slight modification [38, 47]. Briefly, 5 mL of different buffered saline concentrations of pH 7.4 ranging from 0.1 to 0.9% were pipetted into 5 test tubes arranged serially in a test tube rack. A set of 5 test tubes was used to analyse each sample. Whole blood (20 μL) was pipetted into each test tube and mixed thoroughly by inverting the test tubes several times to mix the content. The mixed blood was incubated at room temperature for 20 min. The content was mixed again before spinning at 1500 x g for 15 min. The supernatant was transferred into cuvette and read at an absorbance of 540 nm using a spectrophotometer (Tecan Biochem, USA). The percentage haemolysis was calculated with the following formula:

The EOF curve was obtained by plotting the haemolysis percentage against the different saline concentration. The result of the EOF is expressed as the saline concentration that caused 50% haemolysis, which is the mean cell fragility (MCF).