An Immunoelection Microscopic Study on the Expression of Blood Group B Antigens in Human Bone Marrow Cells

, Nam-Jin Yoo; , Choo-Soung Kim

Original Article

Korean Journal of Legal Medicine 1985;9(Suppl DB Error: syntax error):11-16.

Department of Pathology, Catholic Medical College, Seoul, Korea

Abstract

Membrane antigens are extensively studied on different mammalian cell lines. Antigens of the erythrocyte series are often chemically well defined heterosaccharide determinants. Since Yunis and Yunis (1963) gave strong evidence of the presence of A, B and H alloantigens on human normoblasts by indirect agglutination methods, several workers have detected A antigens by immunoelectron microscopy using peroxidase labelled antibodies(Reyes et al., 1974), staphylococcal protein A technique(Karhi et al., 1981) and fluorescence activated cell sorter(Sieff et al., 1981, 1982). Here we report immunoelectron microscopy observation of blood group B antigen on human normoblasts using peroxidase-labelled antibodies which allow detection of surface antigens on separated cells. The bone marrow specimens were selected from the ones which had been depleted of erythrocytes by a Ficoll-Hypaque technique and stored in liquid nitrogen (-1960 °C) as described (Seung & Kim, 1985). The selected specimens were from eight blood type B individuals with no disturbances in erythropoiesis. These experiments were carried out with cytocentrifuge smears and cell suspensions for light microscopic and electron microscopic observations respectively. The cell suspensions were fixed prior to incubation with reagents. Fixation was achieved by resuspending the cell pellet in phospate-buffered saline solution of 1.25% glutaraldehyde for 30 min: followed by washes with a large quantity of the same buffer. They were resuspended in methanol containing 0.3% H202 for 30 min. to block endogenous peroxidase and washed. And they were labelled by an indirect method using human Anti-B (IgM) and rabbit antihuman IgM antibodies labelled with horseradish peroxidase. The cytochemical detection of horseradish peroxidase was carried out by resuspending the cells for 30 min. in the dark in the usual medium containing diamino- benzidine and H2O2. Subsequently specimens were washed, postfixed with 10% 0s04 for 30 min. and processed for dehydration in alcohol and acetone. For transmission electron microscopy they were embedded in Epon, sectioned and stained with uranyl acetate and lead citrate. The results were as cirtate 1. Under a light microscope the smears showed that brown positive cells were mixed with negative cells, but the precise identification of the cell types was difficult. 2. Under anelectron microscope a continuous or incontinuous labelling of blood group B antigens was seen in contact with the membrane of all normoblasts. 3. The myeloid precursor cells are not labelled, but occasionally limited labelling of the membrane of granulocytic and monocytic cells could be seen.