Development of a Microscopic Method to Identify Hemoglobin C Conditions for Use in Developing Countries

Abstract

Hemoglobin C (HbC) is one of the most prevalent hemoglobinopathies worldwide along with HbS (sickle cell) and HbE. HbC disease (HbCC) produces mild symptoms but is a life-threatening disorder if inherited with HbS (HbSC). HbS and HbC are most prevalent in sub-Saharan countries underequipped to diagnose its presence through gold standard methods like electrophoresis, HPLC or isoelectric focusing. This study aims to create a simple and inexpensive method to identify the presence of HbC in human blood using limited resources with the potential to determine zygosity. HbC crystals that form when blood is incubated in a hypertonic salt solution become visible microscopically when stained with New Methylene Blue. The method was optimized by modifying the salt type, salt concentration, incubation time, and incubation temperature. The optimized method incubates RBCs in a 5x Dulbecco's phosphate buffered saline (DPBS) at 37°C for 4 hours. Blood samples with the HbSC genotype yielded between 600-750 HbC crystals for every 1000 RBCs counted while negative control samples (AA) do not produce HbC crystals. Preliminary results indicate that AS samples produce between 80-150 HbC crystals/1,000 RBCs. Future studies will include testing of blood samples of AC, CC, SC, and AA genotypes to determine if the number of crystals that form using this method will differentiate genotype. If so, this inexpensive, simple, and relatively rapid method can be used to identify patients with HbC and determine genotype in underdeveloped countries where HbC is prevalent.