Immunochemical estimation of haemoglobin types in red blood cells by FACS analysis

Abstract

A fixation and permeabilization procedure using formaldehyde and acetone has been developed which allows immunostaining of intracellular haemoglobin for fluorescence activated cell sorter (FACS) analysis of erythrocytes. The treatment preserves antigenicity and light-scattering properties. Validation of the method was given by the correlation of F cell number in adults determined by FACS analysis with that assessed by microscopic examination of cell smears, and by the direct relationship between beta chain synthesis and intensity of beta chain/Hb A immunofluorescence within fetal erythrocyte samples known to vary in their beta chain/Hb A content. The procedure is rapid, non-subjective and sensitive, and makes analysis of haemoglobin content, type and distribution amongst red cell populations possible.