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Multiplex real‐time quantitative polymerase chain reaction assay for rapid and sensitive detection of hematopoietic chimerism

The increase of mixed chimerism (MC) after allogeneic hematopoietic stem cell transplantation has been associated with a high risk of relapse. A variety of techniques that use polymorphic markers have been established to survey hematopoietic chimerism status. The highest sensitivity is achieved using real‐time quantitative polymerase chain reaction (RQ‐PCR) analysis of insertion/deletion polymorphism, which allows the detection of disease recurrence and subsequently the earlier initiation of therapeutic intervention. The purpose of this study is the evaluation of multiplex RQ‐PCR for MC assessment (six biallelic genetic systems and Y‐specific locus), allowing the amplification and detection of target gene of interest and glyceraldehyde‐3‐phosphate dehydrogenase reference housekeeping gene in a single microtube. With optimized amounts of primers and probe, the quantification of target DNA was shown to be linear throughout the tested range (100%‐0.05%). The efficiencies of multiplex RQ‐PCR were in a range of 0.89 to 1.07. The sensitivity of individual systems ranged 0.02% to 0.04% with an average of 0.034%. A high degree of linear correlation between the chimerism results obtained by multiplex RQ‐PCR vs singleplex RQ‐PCR was observed (P < 0.0001, Spearman's coefficient = 0.9927), while correlation between multiplex RQ‐PCR vs short tandem repeat analysis was also statistically significant (P < 0.0001, Spearman's coefficient = 0.9769). This new multiplex RQ‐PCR assay is a quick, sensitive, reproducible, and cost‐effective method for accurate MC assessment.