Hemoglobinopathy screening by osmotic fragility test based on flow cytometer or naked eye

Diagnosis of hemoglobin (Hb) disorders is based mostly on abnormal red blood cell (RBC) indices, elevated levels of HbA2, HbF, or any other Hb on the Variant high performance liquid chromatography (HPLC) system, and confirmation by molecular methods. However, large scale population screening is of prime importance and requires a simple, accurate, and cost effective technique. We have tried to compare the sensitivity of the widely used Naked Eye Single Tube Red Cell Osmotic Fragility Test (NESTROFT) and the osmotic fragility described as % residual RBCs through flow cytometry for population screening.

The count of residual red cells was measured sequentially in real-time using flow cytometry. NESTROFT was performed using a 0.36% buffered saline. HbA2 and HbF levels along with other abnormal Hbs were determined on the Variant HPLC System. Molecular studies were done to confirm the diagnosis.

The normal group showed a significantly lower percentage of residual RBCs (48.08 ± 11.87) as compared to cases (β thalassemia trait-82.97 ± 12.20, α thalassemia trait-72.58 ± 8.34, and HbS trait-85.00 ± 4.05). The sensitivity and specificity of NESTROFT was high for both β thalassemia traits (98.33 and 96.72%, respectively) and α thalassemia traits (100 and 96.72%, respectively) but very low sensitivity for HbS traits (54.84%).

Flow cytometric osmotic fragility was a more sensitive method to discriminate normal from the group of hemoglobinopathy carriers as compared to NESTROFT which missed majority of HbS carriers. However, in view of feasibility and cost effectiveness, NESTROFT could still be used for population screening of thalassemia. © 2014 International Clinical Cytometry Society.