Apparent loss of 2H4+ T cells in peripheral blood lymphocytes of normal donors: a 2H4-specific artefact unique to T cells.

The antigenic cluster designated CD45R is recognized by a family of monoclonal antibodies. However, one of those most frequently used because of its commercial availability is 2H4. In a series of experiments we show that 2H4 immunofluorescence is almost completely lost from CD4+ or CD8+ T cells of human origin if they are fixed after staining with 2H4. Loss of fluorescence intensity of the total population of 2H4+ lymphocytes and nearly complete quantitative loss of CD45R+ T cells is observed after fixation. This apparent loss of CD45R as detected by 2H4 is not seen if other CD45R-specific monoclonal antibodies are used. Fixation of B cells previously stained with 2H4 gives rise to slightly diminished fluorescence intensity, but no reduction in number of 2H4+ B cells. This appears to be due to a lower antigen density on T cells as compared to B cells. The effect is unique to 2H4, as other monoclonal antibodies recognizing CD45R, while exhibiting a decrease in fluorescence intensity after fixation, still unequivocally detect CD45R+ CD4+ or CD8+ T cells. The effect is restricted to human T cells, as primate lymphocytes, fixed after staining with 2H4, show no loss in fluorescence intensity. We conclude that if it is necessary to fix human T cells after fluorescent staining, the use of CD45R-specific antibodies other than 2H4 is mandatory.