Detection of hemoglobin variants in erythrocytes by flow cytometry

Background: With the emergence of fetal hemoglobin (Hb F)stimulating agents as potential treatments for sickle-cell disease and thalassemias, procedur es 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) conjugate d monoclonal antibody against Hb F. Methods: Ten microliters of washed blood was fixed in formaldehyde and glut araldehyde, then permeabilized in a Triton X-100/PBS solution containing a FITC-labeled monoclonal antibody to Hb F. The blood was analyzed by flow cy tometry to determine the percentage off cells. Results: Nearly 200 Hb F-containing samples were analyzed by this protocol and demonstrated good correlation to percent Hb F results determined by hig h pressure liquid chromatography (HPLC). In addition, a number of samples w ere fixed and permeabilized using this method as well as a previously-descr ibed method that uses dimethyl 3,3-dithiobispropionimadate (DTBP) as a fixa tive as well as a different anti-Hb F monoclonal. Good correlation (r = 0.9 6, r(2) = 0.33, P < 0.001) was observed between the two protocols. Conclusions: This procedure is easy, reproducible, and gives accurate F cel l 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 a nd erythrocyte surface antigens. (C) 1999 Wiley-Liss,Inc.