[A novel single nucleotide polymorphism-based method for quantitative assessment of chimerism after allogeneic stem cell transplantation.].

ABSTRACT

OBJECTIVE: To develop a novel single nucleotide polymorphism (SNP)-PCR based method for quantitative detection of chimerism after allogeneic haemopoietic stem cell transplantation (allo-HSCT), and to explore its feasibility, accuracy and superiority. METHODS: 18 SNP loci were sereened to identify informative markers for detecting chimerism in each donor/recipient pair before transplantation. Then the chimerism rate of each informative marker was analyzed by real-time quantitative PCR (RQ-PCR). The accuracy and sensitivity were verified by multiple proportion dilution and analogy chimerism compared with quantitative detection of short tandem repeat (STR)-PCR, fluorescence in situ hybridization (FISH) and fusion gene. RESULTS: (1) The average slope of the 17 time amplications of the internal control plasmid was -3.39, the average intercept was 39.97, correlation coefficients were more than 0.995, which was close to the theoretical level. The intra- and interassay variability was 0.50% and 1.1%, respectively, which were both in the allowed ranges. A linear correlation with artificial mixed chimerism is above 0.99 and a sensitivity of 0.01% proved reproducible. (2) At least one informative marker could be found in over 95% of 40 donor/recipient pairs. The results of the chimerisms derived from SNP-PCR were consistent with that from STR-PCR (96.7%), FISH and fusion gene analasis (P > 0.05); the quantitative results of special fusion gene transcripts were negtive in complete chimerism samples, and positive in mixed chimerism samples. CONCLUSIONS: This new assay which overcome the PCR competition and plateau biases of STR-PCR provides an accurate, reliable and rapid quantitative assessment of mixed chimerism after allo-transplantation. It is highly promising for of clinical application and may take the place of STR-PCR in the conventional chimerisim assessment.