Flow cytometry analysis of dual red blood cell populations after bone marrow transplantation

Abstract

Flow cytometry represents an alternative method to agglutination assays for the accurate quantification of mixed field populations of erythrocytes observed after bone marrow transplantation. Murine monoclonal antibodies directed against the blood group ABH antigens were selected and processed in order to prepare ready-to-use fluorescent reagents. Anti-A (NaM8 7-1F6; IgG3), anti-B (NaM9-2E11: IgG3) and anti-H (NaM19-7Ell; 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 Anti-A1 (NaM1-1C9; IgG3) was no longer active after FITC-labelling and then was used in an indirect assay. The agglutination was prevented by formaldehyde pretreatment of erythrocytes.Using artificially-made double populations of erythrocytes, measured values with mixtures of 1-100% of cells were very closely related to expected values, showing both the sensitivity and the accuracy of the method. From careful investigation of a series of bone-marrow transplanted patients, we conclude that engraftments could be demonstrated earlier by flow cytornetry 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 graft failure in one case. The proposed method provides appreciable help to follow engraftment in patients and may have more general applications for the study of other haemopoietic chimaeras.

Clinical Utility of Flow Cytometry in the Study of Erythropoiesis and Nonclonal Red Cell Disorders

“…Continuing quantitation of the differences can give early warning of loss of the graft or confirmation of a stable chimera (Arnold et al, 1986;Blanchard et al, 1995;Lazarus et al, 1992). Continuing quantitation of the differences can give early warning of loss of the graft or confirmation of a stable chimera (Arnold et al, 1986;Blanchard et al, 1995;Lazarus et al, 1992).…”

Comparison of multiple red cell volume methods performed concurrently in premature infants following allogeneic transfusion

“…This method relies on its ability to discriminate transfused from endogenously produced RBC populations. Flow cytometry has been used to detect fetomaternal hemorrhage (4, 5), determine RBC phenotype following bone marrow transplant (6), measure RCV (7-9), detect illicit blood transfusions in athletes (10), and determine RBC survival (11, 12). Recently, our group has for the first time demonstrated that RCV can be accurately determined in adult humans and sheep using multiple distinct populations of biotin–labeled RBCs (BioRBCs) enumerated by flow cytometry (8, 13).…”

Blood doping in athletes—Detection of allogeneic blood transfusions by flow cytofluorometry

“…Use nonagglutinating (IgG) antibodies Chemically convert agglutinating IgM pentamers into nonagglutinating IgM monomers, e.g., using dithiothreitol [48,57] or 2-mercaptoethanol Purify IgG fractions of sera on Protein G [49] Dilute antisera past point of agglutination Avoid use of potentiators Chemically fix the RBCs prior to incubation with anti-A, -B, -M, or -N, e.g., with dimethylsuberimidate [74], formaldehyde [47,75], or glutaraldehyde [13,21,44,49] To avoid agglutination caused by secondary antibody:…”

“…The analysis of mixed RBC populations occurring as a result of hematopoietic chimerism in twins [42][43][44][45][46] and in bone marrow transplant recipients [47][48][49][50][51][52] is easily performed by flow cytometry. This technique has also been used in studies of individuals with blood group mosaicism [26,[53][54][55].…”

“…Fixation prevents conformational changes of the red cell membrane required for RBCs to be crosslinked by multivalent antibodies [76]. Formaldehyde [47,75] is a satisfactory fixative for the A, B, M, and N antigens though rather tedious (a two-day, three-step process) and causes some hemolysis. Fixation with glutaraldehyde [13,21,44,49] is rapid and nonhemolytic, and although the A, B, and M blood groups are not destroyed, the N antigen is sensitive (A.…”