OBJECTIVE: We aimed to create a molecular assay to monitor erythroid (red blood cell [RBC]) engraftment in any patient following allogeneic hematopoietic stem cell transplantation, independent of disease-specific mutations. MATERIALS AND METHODS: We identified 10 common single nucleotide polymorphisms (SNPs), expressed by genes encoding RBC antigens and structural proteins. These SNPs were polymerase chain reaction-amplified from total RNA extracted from peripheral blood, which contains nucleated erythroid progenitors. Mixing studies validated that each SNP can quantitatively measure donor/recipient DNA and RNA. RESULTS: We directly genotyped 23 patients who underwent hematopoietic stem cell transplantation and their human leukocyte antigen-matched donors and found a median of three informative SNPs (i.e., discordant between donor and recipient) per pair. By using the informative RBC SNPs to quantify donor-derived RBC transcripts, we compared rates of RBC engraftment in 13 patients with hemoglobinopathies vs donor mononuclear cell (white blood cell [WBC]) engraftment. Consistent with known ineffective erythropoiesis associated with hemoglobinopathies, we detected up to threefold greater RBC-specific compared to overall WBC engraftment in five of eight patients who were mixed chimeras by transplant day 30. The remaining three of eight who received ABH-incompatible grafts, demonstrated at least 0.5-fold lower RBC compared to WBC engraftment that was related to persistence of host-derived anti-isohemagglutinin antibodies. CONCLUSION: This RNA-based assay can be used to monitor RBC-specific engraftment regardless of a patient's specific hemoglobin mutation or even diagnosis. We propose that panels of expressed SNPs informative for other cell lineages can be created to comprehensively assess the impact of novel stem cell-based therapies on lineage-specific engraftment.
OBJECTIVE: We aimed to create a molecular assay to monitor erythroid (red blood cell [RBC]) engraftment in any patient following allogeneic hematopoietic stem cell transplantation, independent of disease-specific mutations. MATERIALS AND METHODS: We identified 10 common single nucleotide polymorphisms (SNPs), expressed by genes encoding RBC antigens and structural proteins. These SNPs were polymerase chain reaction-amplified from total RNA extracted from peripheral blood, which contains nucleated erythroid progenitors. Mixing studies validated that each SNP can quantitatively measure donor/recipient DNA and RNA. RESULTS: We directly genotyped 23 patients who underwent hematopoietic stem cell transplantation and their human leukocyte antigen-matched donors and found a median of three informative SNPs (i.e., discordant between donor and recipient) per pair. By using the informative RBC SNPs to quantify donor-derived RBC transcripts, we compared rates of RBC engraftment in 13 patients with hemoglobinopathies vs donor mononuclear cell (white blood cell [WBC]) engraftment. Consistent with known ineffective erythropoiesis associated with hemoglobinopathies, we detected up to threefold greater RBC-specific compared to overall WBC engraftment in five of eight patients who were mixed chimeras by transplant day 30. The remaining three of eight who received ABH-incompatible grafts, demonstrated at least 0.5-fold lower RBC compared to WBC engraftment that was related to persistence of host-derived anti-isohemagglutinin antibodies. CONCLUSION: This RNA-based assay can be used to monitor RBC-specific engraftment regardless of a patient's specific hemoglobin mutation or even diagnosis. We propose that panels of expressed SNPs informative for other cell lineages can be created to comprehensively assess the impact of novel stem cell-based therapies on lineage-specific engraftment.
Authors: Wandi Zhang; Jaewon Choi; Wanyong Zeng; Shelby A Rogers; Edwin P Alyea; James G Rheinwald; Christine M Canning; Vladimir Brusic; Tetsuro Sasada; Ellis L Reinherz; Jerome Ritz; Robert J Soiffer; Catherine J Wu Journal: Clin Cancer Res Date: 2010-05-11 Impact factor: 12.531