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Detection of hemoglobin variants in erythrocytes by flow cytometry

1999; Wiley; Volume: 35; Issue: 3 Linguagem: Inglês

10.1002/(sici)1097-0320(19990301)35

1097-0320

Thomas A. Campbell, Russell E. Ware, Marsha L. Mason,

Blood groups and transfusion

Background: 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. Methods: 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. Results: 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. Conclusions: 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. Cytometry 35:242–248, 1999. © 1999 Wiley-Liss, Inc.