A Method of HbF Determination for Potential Use in Underdeveloped Countries

Abstract

The objective of this study was to develop a simple, cost-effective method of HbF determination potentially useable in underdeveloped countries to determine sickle cell patient response to hydroxyurea treatment. Normal adult blood (HbA), cord blood (HbF), and a 50:50 mixture (HbA+F) were the three sample types used in procedure development. Normal blood samples were collected from the research team, and de-identified cord blood samples were provided by Cardinal Glennon Pediatric Research Institute, St. Louis, MO. The hematocrit of all blood samples was standardized to 35%. The method, based on the Kleihauer-Betke (K-B) test principle, used a citrate solution to selectively elute HbA from RBCs while HbF remained intracellular, and spectrophotometric absorbance of the eluate was the primary outcome measure. A procedure was developed and optimized utilizing a 395 nm wavelength, 30 sec centrifugation time, 6 min incubation time, 20 μL blood volume, and 0.07 M sodium citrate in a 0.06 M sodium phosphate buffer solution. Reproducibility was demonstrated (N = 39) with a mean HbA absorbance of 1.285 (SD 0.069), mean HbA+F absorbance of 0.690 (SD 0.050), and mean HbF absorbance of 0.035 (SD 0.005), also exhibiting linearity (r² = 0.99). This simple, cost-effective method of HbF determination shows potential as a basis for determining sickle cell patient response to hydroxyurea treatment in underdeveloped countries.

ABBREVIATIONS

HbS-sickle hemoglobin, HbA-normal hemoglobin, SCD-sickle cell disease, SCT-sickle cell trait, RBCs-red blood cells, HPLC-high performance liquid chromategraphy, HbF-fetal hemoglobin, K-B-Kleihauer-Betke.