Cell sorter immunofluorescence detection of human erythrocytes labelled in suspension with antibodies specific for hemoglobin S and C

Abstract

We have developed an immunochemical method for labeling human red blood cells in suspension with hemoglobin-specific antibodies. A membrane permeable cross-linking reagent, dimethyl suberimidate, is used to covalently bind, in situ, a fraction of the intracellular hemoglobin to integral membrane proteins. Hypotonic lysis and washing of the cells removes the unbound hemoglobin resulting in red blood cell ghosts which are permeable to macromolecules. Fluorescein-labeled antibodies for the hemoglobin variants S and C bind specifically to hemoglobin AS and AC ghosts, respectively, and not to normal hemoglobin AA ghosts. This technique can be used to prepare ghost suspensions for cell sorter analysis in which large numbers (10 9–10 10) of normal ghosts can be rapidly screened for the presence of rare anti-hemoglobin S and anti-hemoglobin C binding ghosts. In reconstruction experiments using mixtures of AS and AA cells and anti-hemoglobin S, AS ghosts as rare as 3 × 10 −5 were quantitatively recovered. Fluorescence artifacts prevented direct enumeration of AS ghosts at lower frequencies, but a two-step flow sorting-fluorescence microscope visual scanning procedure allows semiquantitative detection of anti-hemoglobin S-labeled ghosts as low as 10 −7. This method can be used for rapidly screening blood samples from individuals of normal hemoglobin A genotype for the presence of rare anti-hemoglobin S and anti-hemoglobin C binding ghosts.