The evaluation of erythrocyte and platelet engraftment in peripheral blood following bone marrow transplants in mice

Hematopoietic stem cell and progenitor populations (HSPG) within the bone marrow compartment consist of multiple groups of cell that vary in function when transplanted. The subsets of HSPG are beginning to be separated to define their engraftment characteristics, with the aim to improve current transplant management for patients with malignant disease. In this study, methods were developed to identify hemoglobin (Hbb) and glucose phosphate isomerase (Gpi) allelic variants in lysates of mouse peripheral blood cells, in order to facilitate evaluation of erythrocyte and platelet engraftment after bone marrow transplantation. The long-term repopulation assay (LTRA) was then used to demonstrate the efficacy of these techniques to distinguish donor-derived engraftment of bone marrow transplanted into lethally irradiated mice. Donor-derived Hbb[d] isoforms in recipient hemolysates, when pretreated with a cystamine lysing agent, could be separated by agarose gel electrophoresis, then stained and quantitated by densitometric analysis. A detection limit of 5% was established when mixtures to known hemolysates were assayed. As a second approach, transplant hemolysates were evaluated by HPLC. This cost-effective assay was demonstrated to have a direct correlation with the electrophoresis assay (r=0.998). Agarose gel electrophoresis and a specific stain for Gpi distinguished the platelet variants, Gpi-1a and Gpi-1b, with a limit of detection corresponding to 100,000 total platelets. Two contamination assays were performed to evaluate the contribution of residual leukocytes and erythrocytes in platelet preparations to the platelet Gpi signals. The results indicated that the separation protocol utilized in these studies was sufficient to exclude contaminating leukocytes and erythrocytes from platelet preparations. Platelets were also stained with monoclonal antibodies detecting allelic variants of Ly5 and analyzed by flow cytometry to detect post-transplant chimerism. The absence of fluorescence indicated a down-regulation of Ly5 during platelet maturation precluding the use of this method. The techniques developed in this project successfully measured erythrocyte and platelet engraftment kinetics using the LTRA. One million transplanted morrow cells repopulated lethally irradiated mice with multi-lineage progeny and sustained donor-derived hematopoiesis was observed one year post-transplant. In conclusion, the assays developed in this study can successfully evaluate erythrocyte and platelet repopulation of transplanted HSPG in mouse models. Defining HSPG will establish a better understanding of bone marrow transplantation in humans.