Abstract
Accurate and sensitive determination of hematopoietic chimerism is a crucial diagnostic measure after allogeneic stem cell transplantation to monitor engraftment and potentially residual disease. Short tandem repeat (STR) amplification, the current “gold standard” for chimerism assessment facilitates reliable accuracy, but is hampered by its limited sensitivity (≥1%). Digital PCR (dPCR) has been shown to combine exact quantification and high reproducibility over a very wide measurement range with excellent sensitivity (routinely ≤0.1%) and thus represents a promising alternative to STR analysis. We here aimed at developing a whole panel of digital-PCR based assays for routine diagnostic. To this end, we tested suitability of 52 deletion/insertion polymorphisms (DIPs) for duplex analysis in combination with either a reference gene or a Y-chromosome specific PCR. Twenty-nine DIPs with high power of discrimination and good performance were identified, optimized and technically validated. We tested the newly established assays on retrospective patient samples that were in parallel also measured by STR amplification and found excellent correlation. Finally, a screening plate for initial genotyping with DIP-specific duplex dPCR assays was designed for convenient assay selection. In conclusion, we have established a comprehensive dPCR system for precise and high-sensitivity measurement of hematopoietic chimerism, which should be highly useful for clinical routine diagnostics.
Highlights
Allogeneic hematopoietic stem cells transplantation (SCT) represents an established curative treatment for various malignant and non-malignant hematological disorders such as leukemia, aplastic anemia, multiple myeloma and lymphoma [1]
We and others have previously shown that this limitation can be overcome by a further advancement of quantitative PCR (qPCR), namely digital PCR, which combines the excellent sensitivity of qPCR with the high accuracy and reproducibility of short tandem repeats (STRs) [7,8]
The aim of this project was the establishment of a broad panel of Digital PCR (dPCR)-based assays for chimerism analysis
Summary
Allogeneic hematopoietic stem cells transplantation (SCT) represents an established curative treatment for various malignant and non-malignant hematological disorders such as leukemia, aplastic anemia, multiple myeloma and lymphoma [1]. The ratio of donor to recipient blood cells, the hematopoietic chimerism, needs to be closely observed, in the early engraftment phase. Different techniques are available for chimerism analysis, but PCR-based analysis of short tandem repeats (STRs) is most widely used [4] due to its very good accuracy and reproducibility. STR-PCR (“STR”) has a comparatively low detection threshold (approximately 1%–5%) limiting its usefulness in the early detection of impending cytological relapse. An inherent restraint of qPCR-based techniques is their comparatively low accuracy in the state of mixed chimerism (i.e.,
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