Presence of HL.A Antigens on Human Reticulocytes as demonstrated by Electron Microscopy

Abstract

THE presence of human histocompatibility antigens (HL.A) has been demonstrated on all the human nucleated cells so far studied, but unlike mouse H2 antigens they have not been found on erythrocytes. Harris et al.¹, using serological methods, have suggested that they might be present on human reticulocytes. By electron microscopy and an indirect Ferritin labelled antibody technique², we have studied these antigens at the membrane of bone marrow erythroblasts and erythrocytes. We have observed them on erythroblasts and reticulocytes and their disappearance on mature erythrocytes. Heparinized bone marrow was obtained by sternal aspiration from three subjects, two normal (A Rh⁺ and O Rh⁺) and one with mild aplastic anaemia (A Rh⁺). The supernatant rich in leucocytes was collected after partial sedimentation of erythrocytes. Cells were washed three times in phosphate buffered saline pH 7.2 (PBS) containing 20 per cent foetal calf serum. Aliquots of 5 × 10⁶ cells were incubated for 30 min at 37° C in a 1/3 dilution of an anti-HL.A antiserum, washed three times in PBS, and incubated for a further 30 min at 37° C in ferritin labelled sheep globulin antihuman IgG, prepared as previously described³. After three more washings, cells were fixed with glutaraldehyde postfixed in osmium, dehydrated in alcohol and embedded in Epon. Sections were stained with uranyl acetate and lead acetate⁴ and examined using a Siemens E IA electron microscope with a magnification of 8,000 to 40,000 times. The anti-HL.A antiserum (provided by Professor J. Dausset) was a highly potent polyspecific anti-HL.A antiserum from a polytransfused patient. It reacted with the lymphocytes of 98 per cent of the French donors in cytotoxic tests up to a dilution of 1/500 to 1/1,000. The donor's blood, being group A, contained natural anti-B antibodies and no other detectable anti-erythrocyte agglutinins. In this respect it was tested against 12 isogroup donors using the immunoferritin technique and never reacted with erythrocytes.