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

Lavrinenko V., Savitskaya T.V., Mareika Y.

V. Lavrinenko, T. Savitskaya, Y. Mareika

Belarusian Research Сenter for Pediatric Oncology, Hematology and Immunology, Minsk, Belarus.

viallav@mail.ru

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. ResultsRecipient-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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