A SYBR Green - Based Real Time PCR Method for Detection of Haemopoietic Chimerism in Allogeneic Stem Cell Transplant Recipients.

Aim Detection of donor cells (chimerism) in patients post allogeneic stem cell transplantation is for monitoring engraftment, early detection of graft rejection and disease relapse. Current methods include cytogenetics, blood grouping and DNA microsatellite test. Our aim is to develop a reliable and rapid real-time quantitative PCR (Q-PCR) method using SYBR green for detecting chimerism in allogeneic haemopoietic stem cell transplant recipients.

Methods Twelve specific nucleotide polymorphisms (NPs) on 11 different chromosomes (including X, Y) were selected. Specific primers and fluorogenic probes were used for Q-PCR analysis. These NPs were screened in pre-transplantation donor and recipient pairs for informative markers. One informative marker was then used for Q-PCR to detect donor cell chimerism post transplantation using either SYBR green or TaqMan probe. Quantitation of donor cell percentage was calculated using a standard amplification curve generated from artificially mixed donor/recipient chimeric DNA samples made with 10 serial dilutions (0.01% ~ 100%), low, medium and high controls were included.

Results Thirty-seven donor/recipient pairs including 9 sex-mismatched allograft pre-transplantation DNA samples were examined. In all cases, it is possible to discriminate between recipient and donor genetic profiles. The detection limitation is 0.01%, which is more sensitive than the currently used Microsatellite and FISH methods. The results are highly reproducible with accurate quantitation. In 16 post transplant samples, donor-recipient chimerism were distinguishable using this method. Furthermore this new assay detected similar levels of chimerism compared with either FISH for sex-mismatch donor/recipient pairs or Microsatellite for sex match donor-recipient pairs. We also found that the SYBR green Q-PCR is less expensive and as accurate as the TaqMan probe Q-PCR.

Conclusion The new single platform Q-PCR method is capable of detecting haemopoietic chimerism after transplantation and has the potential to replace the three current methods (i.e. cytogenetics, blood grouping and microsatellite).