Flow cytofluorometric analysis in red blood cell immunology

Flow cytofluorometry (FC) is primarily used in the clinical laboratory for the analysis of white blood cells but also has applications in the study of red blood cell (RBC) immunology. When studying RBCs by FC it is important to avoid agglutination of the RBCs, which ironically is the endpoint for most immunohematologic assays. When studying low levels of RBC-bound antibody or RBC antigens with low copy number, background noise due to autofluorescence and nonspecific binding need to be taken into account. It is also important to ensure that reagents ( e. g. antisera) used by FC have been standardized for use by FC. Applications of FC in RBC immunology include i) detection and quantitation of globulin (RBC-bound or free in serum), ii) detection and quantitation of RBC antigens, and iii) detection and quantitation of mixed cell populations. FC can be used to differentiate the amount of IgG on strongly IgG-coated RBCs, as well as to detect small amounts of RBC-bound IgG. FC has been used to quantitate antibody that is free in serum, e. g. anti-D in a pregnant woman or in immune globulin preparations. Many RBC antigens have been studied by FC to determine the number of antigen sites, the density distribution, and phenotype-related differences ( e. g. zygosity). FC has been found to be very useful for studying mixed cell populations; applications include fetomaternal hemorrhage quantitation, determination of survival/clearance of transfused RBCs, phenotyping of transfused patients' RBCs, evaluation of bone marrow transplant engraftment, and study of RBC chimerism and mosaicism.