Detection of cytotoxic antibody to erythroblasts

A two-stage test of cytotoxicity for erythroblasts has been developed that is faster and more sensitive than a previous method. Release of 59Fe from erythroid precursors was used as an index of cytotoxic injury. Optimal release was obtained by pretreating the labeled marrow cells with 0.46M reduced glutathione (GSH) at pH 8.0 for 1 hour at 37 degrees C. The first-stage test plasmas or IgG globulins were incubated with the treated cells at 22 degrees C. for 1 hour and the second-stage source of complement was incubated with the cells for 1 hour at 37 degrees C. These changes permitted cytotoxic plasmas to be detected when they previously would not have been identified. Substitution of the GSH by trypsin or neuraminidase did not yield comparable results. Measurement of the cytotoxicity for GSH-treated cells by the trypan blue exclusion technique showed that the loss of exclusion of trypan blue by marrow erythroblasts increased with an increased release of 50Fe. No cytotoxicity was detected if complement was inactivated at 56 degrees C. The results observed with trypan blue and the occurence of cytotoxicity with the addition of IgG globulins and complement indicate that this system can be used to detect cytotoxic antibody to erythroblasts.