A comparison of muliplex short tandem repeat PCR and real-time PCR insertion/deletion polymorphisms methods for quantification of chimerism after hematopoietic stem cell transplantation
V. Lavrinenko, T. Savitskaya, Y. Mareika
Belarusian Research Сenter for Pediatric Oncology, Hematology and Immunology, Minsk, Belarus.
Introduction: Quantitative monitoring of chimerism after allogeneic hematopoietic stem cell transplantation (HSCT) by molecular methods has become an indispensable diagnostic tool in detection of engraftment/graft failure, predicting rejection and disease relapse. Despite the great utility of chimerism analysis there is no unique standard method for its quantification. Aim: The objective of the present investigation was to compare the sensitivity (detection limit) and the quantification accuracy of two perspective methods: multiplex short tandem repeat polymerase chain reaction (STR-PCR) and real-time PCR insertion/deletion polymorphisms (InDel-PCR) for the quantification of chimerism after stem cell transplantation. Methods: We performed a perspective study analyzing the chimerism status in 46 patients by STR-PCR using AmpFlSTR SGM Plus PCR Amplification Kit (ABI, UK) with capillary electrophoresis and by InDel-PCR with primers and probes to 20 allele-specific markers and reference gene albumin. Results: Recipient-donor discrimination was possible with STR-PCR in all patient-donor pairs (100%), whereas informative alleles for recipient were found in 94% pairs with InDel-PCR. The sensitivity (detection limit) of STR-PCR was 1–5% donor DNA with variation among STR markers. The sensitivity of InDel-PCR was more than 0.01% donor cells. The accuracy of quantification was higher for InDel-PCR than for InDel-PCR, when level of donor chimerism was <5% or >95% as it had higher sensitivity. The accuracy of quantification was higher for STR-PCR than for InDel-PCR, when level of chimerism was >10%, because there was 0.5 threshold cycle error for InDel-PCR and this corresponded to a variation in DNA of 50%. The results obtained by two methods showed a good agreement and correlated well (r=0.98; p<0.0001). Conclusions: These methods can be successfully used to determine chimerism after allogeneic HSCT. Considering the higher sensitivity and quantification accuracy of InDel-PCR it should be chosen if donor chimerism level is less than 5–10% or more 90–95% (i.e. after myeloablative conditioning) and in other cases STR-PCR should be chosen.
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