Monitoring of donor/recipient T-cell engraftment kinetics in myeloablative allogeneic stem cell transplantation using short tandem repeat amplification from cell lysates.

摘要

Molecular monitoring of donor/recipient T-cell kinetics early post-transplant can provide clues to the immunological events that govern host-versus-graft reaction (HVGR) and graft versus-host-disease (GVHD). We have previously used fluorescence in situ hybridization (FISH) with X and Y probes to monitor recipient T (R-T) cell clearance early after myeloablative allogeneic stem cell transplantation (ASCT). We demonstrated that impaired clearance of residual host-T-cells in the early days post-transplant was associated with graft rejection, while enhanced clearance could be an indicator of increased donor anti-host alloreactivity and predictive of acute GVHD. Although FISH is the most accurate quantitative molecular tool for the determination of the exact donor/recipient-T-cell numbers at any time points post-transplant, it has the disadvantage of being limited to sex mismatched donor/recipient pairs. Our goal was to develop a molecular approach that, irrespective of gender, would be comparable to FISH in accurately determining host residual T-cell clearance after myeloablative conditioning for ASCT. We have genotyped DNA from cell lysates using polymerase chain reaction (PCR) amplification of short tandem repeats (STR) with fluorescently labeled oligonucleotide primers, and used the Genescan 672 software for accurate quantitative analysis of the amplified alleles. Here, we show that this approach allowed us to achieve in T-cells accurate quantitative analyses of amplified donor/recipient alleles in sex matched patients on days +5, +8 and +12 post-transplant, despite severe leukopenia.