Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation

International Journal of Molecular Sciences

Manja U. Böhme¹  Caroline Rothe¹  Nicolaus Kröger²  Tanja Stahl²  Aloisa Kohl²  Boris Fehse² 

[1] Biotype Diagnostic GmbH, 01109 Dresden, Germany;Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany;

关键词: chimerism;    minimal residual disease;    digital PCR;    allogeneic stem cell transplantation;   

DOI  :  10.3390/ijms17091515

来源: DOAJ

【 摘 要 】

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.

【 授权许可】

Unknown