Hemoglobinopathy Screening by Osmotic Fragility Test Based on Flow Cytometer or Naked Eye

Diagnosis of hemoglobin disorders is based mostly on abnormal red blood cell indices, elevated levels of HbA2, HbF or any other Hb on the Variant 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 NESTROFT and the osmotic fragility described as percentage 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 hemoglobins 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 normals from the group of hemoglobinopathy carriers as compared to NESTROFT which missed majority of Hb S carriers. However, in view of feasibility and cost effectiveness, NESTROFT could still be used for population screening of thalassemia. This article is protected by copyright. All rights reserved.