The GLAM test: a flow cytometric assay for the detection of leukocyte antibodies in autoimmune neutropenia

The GLAM assay, a combined flow cytometric immunofluorescence test that simultaneously detects antibodies to granulocytes, lymphocytes, and monocytes, was used in the investigation of autoimmune neutropenia. This method avoids the need for a succession of immunofluorescence tests, first against granulocytes and then against lymphocytes, in order to distinguish granulocyte-specific from granulocyte/lymphocyte-reactive antibodies, such as noncomplement-fixing anti-HLA sera. Samples from 18 patients with a suspected diagnosis of autoimmune neutropenia were referred for investigation. Leukocytes were harvested in sufficient quantities from 10 of the patients such that neutrophil and lymphocyte direct antiglobulin tests (DATs) and antibody screening and identification could be undertaken (in one case the results were inconclusive). Only three of these 10 patients had DAT-positive granulocytes, and one of these three also had DAT-positive lymphocytes. One further patient demonstrated DAT-positive lymphocytes in the absence of granulocyte-bound IgG, despite a presumed diagnosis of autoimmune neutropenia, rather than pancytopenia. This was the only patient in this cohort who had demonstrable leukocyte antibodies reacting with lymphocytes but not granulocytes. Of the remaining eight patients (not evaluable for granulocyte or lymphocyte DATs), five had free leukocyte antibodies in the serum; three of these five had both granulocyte- and lymphocyte-reactive antibodies free in the serum and two only had granulocyte-specific antibodies.