Erythrocyte Antibody EA rosette

The identification of hematopoietic cells by their formation of rosettes with erythrocytes coated with antibody molecules is complex. Careful attention to the type of erythrocyte and of the antibody coating on that cell is required. Seven types of hematopoietic cells have receptors for immunoglobulin (Ig) on their cell membrane. An additional layer of complexity is that there are receptors for IgM, IgA, IgE and IgG and its subclasses. The difference between these receptors and their affinities is covered in the entry on Fc receptors. The most prominent Fc receptor-bearing cells are polymorphonuclear leukocytes and monocytes. In most immunologic studies of lymphocyte function the neutrophils are removed by density gradient centrifugation. They are readily identified by their characteristic morphologic appearance in smears or cytocentrifuge preparations. Monocytes are similarly identified as EA rosette-forming cells in such preparations. Monocytes are removed by glass or plastic adherence or passage through columns of Sephadex G-10. Platelets have Fc receptors and care must be taken to remove these cells as well from preparations. Eosinophils, basophils and mast cells are removed along with the neutrophils. B Lymphocytes may be identified and isolated once contaminating cells are removed by their rosette formation with ox erythrocytes coated with anti-ox cell antibody. This method is inferior to their identification by anti-surface membrane immunoglobulin antibody techniques, but may be performed when fluorescence microscopy is not available. The results obtained should be correlated with the results of complement receptor assays (see below).

The T cell population can be subdivided into two populations by rosette formation with erythrocytes coated with IgM or erythrocytes coated with IgG. It is important in this assay to incubate freshly isolated T cells overnight in immunoglobulin free media to allow the iti vivo passively absorbed IgM and IgG to be shed. Then rosette assays with appropriately coated erythrocytes can be performed. T cells with receptors for IgM were originally described as helper T cells, whereas cytotoxic T cells were said to have receptors for IgG. Subsequently it has been shown that these two classes of T cells cannot be distinguished on this basis. However, multiple cell types

Table 1 Rosette markers of human peripheral blood mononuclear cells
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