FLOW-CYTOMETRY ANALYSIS OF DUAL RED-BLOOD-CELL POPULATIONS AFTER BONE-MARROW TRANSPLANTATION

Flow cytometry represents an alternative method to agglutination assay s for the accurate quantification of mixed field populations of erythr ocytes observed after bone marrow transplantation. Murine monoclonal a ntibodies directed against the blood group ABH antigens were selected and processed in order to prepare ready-to-use fluorescent reagents. A nti-A (NaM87-1F6; IgG3), anti-B (NaM9-2E11; IgG3) and anti-H (NaM19-7E 11; IgM) were purified, labelled with fluorescein isothiocyanate, and used in a direct flow cytometry assay. Anti-A1 (NaM1-1C9; IgG3) was no longer active after FITC-labelling and then was used in an indirect a ssay. The agglutination was prevented by formaldehyde pretreatment of erythrocytes. Using artificially-made double populations of erythrocyt es, measured values with mixtures of 1-100% of cells were very closely related to expected values, showing both the sensitivity and the accu racy of the method. From careful investigation of a series of bone-mar row transplanted patients, we conclude that engraftments could be demo nstrated earlier by now cytometry than by agglutination, because minor populations (1-10%) of cells could be determined accurately only with labelled reagents, In addition, the disappearance of the donor cells on a longterm follow-up of patients enabled an earlier detection of gr aft failure in one case. The proposed method provides appreciable help to follow engraftment in patients and may have more general applicati ons for the study of other haemopoietic chimaeras.