Detection by flow cytometry of antibodies against surface and intracellular granulocyte antigens

A flow cytometric technique was used to detect granulocyte antibodies, with attention to the distinction between antibodies directed against surface and intracellularly expressed antigens. Ten serum samples with positive results and 10 with negative results detected by the granulocyte immunofluorescence test (GIFT ), together with 10 positive serum samples detected by an indirect immunofluorescence test (IIF) were analyzed against leukocytes from healthy blood donors tested by flow cytometry (FC). Unpermeabilized and permeabilized cells were used to allow identification of surface and intracellular binding, respectively. The results of testing by GIFT corresponded with those by FC, with the exception of the results for four sera: one serum sample was FC negative, GIFT positive, and three samples were FC positive, GIFT negative. The IIF-positive sera were all FC positive, analyzed against permeabilized granulocytes, and one serum was also positive against non-permeabilized granulocytes. Analysis by FC is a readily performed technique, which can be used for the routine detection of antibodies against leukocyte antigens. Screening for granulocyte-specific antibodies can be carried out with pools of granulocytes from three donors. Analysis by FC allows detection of both HLA antibodies and granulocyte-specific antibodies and by using both unpermeabilized and permeabilized cells, antibodies against surface and intracellular antigens, respectively, can be identified.