Detection of hemoglobin variants in erythrocytes by flow cytometry

With the emergence of fetal hemoglobin (Hb F)stimulating agents as potential treatments for sickle-cell disease and thalassemias, procedures to monitor the effect of these agents on Hb F levels in individuals will be needed. We developed a rapid procedure that detects fetal hemoglobin in erythrocytes (F cells) using a fluorescein isothiocyanate (FITC) conjugated monoclonal antibody against Hb F.

Ten microliters of washed blood was fixed in formaldehyde and glutaraldehyde, then permeabilized in a Triton X-100/PBS solution containing a FITC-labeled monoclonal antibody to Hb F. The blood was analyzed by flow cytometry to determine the percentage of F cells.

Nearly 200 Hb F-containing samples were analyzed by this protocol and demonstrated good correlation to percent Hb F results determined by high pressure liquid chromatography (HPLC). In addition, a number of samples were fixed and permeabilized using this method as well as a previously-described method that uses dimethyl 3,3'dithiobispropionimadate (DTBP) as a fixative as well as a different anti-Hb F monoclonal. Good correlation (r = 0.96, r2 = 0.93, P<0.001) was observed between the two protocols.

This procedure is easy, reproducible, and gives accurate F cell results. It can be used to measure a wide range of F cell percentages and may also be used to dual-stain Hb F along with other hemoglobin variants and erythrocyte surface antigens.