Detection of hemoglobin variants in erythrocytes by flow cytometry

✍ Scribed by Thomas A. Campbell; Russell E. Ware; Marsha Mason

• Publisher: John Wiley and Sons
• Year: 1999
• Tongue: English
• Weight: 182 KB
• Volume: 35
• Category: Article
• ISSN: 0196-4763
• DOI: 10.1002/(sici)1097-0320(19990301)35:3<242::aid-cyto7>3.0.co;2-y

✦ Synopsis

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 chromatogra-phy (HPLC). In addition, a number of samples were fixed and permeabilized using this method as well as a previouslydescribed method that uses dimethyl 3,3Ј-dithiobispropionimadate (DTBP) as a fixative as well as a different anti-Hb F monoclonal. Good correlation (r ϭ 0.96, r 2 ϭ 0.93, P Ͻ 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,