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Int J Mol Sci. 2016 Sep 9;17(9). pii: E1515. doi: 10.3390/ijms17091515.

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

Author information

1
Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany. t.stahl@uke.de.
2
Biotype Diagnostic GmbH, 01109 Dresden, Germany. c.rothe@biotype.de.
3
Biotype Diagnostic GmbH, 01109 Dresden, Germany. m.boehme@biotype.de.
4
Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany. aloisakohl@hotmail.com.
5
Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany. n.kroeger@uke.de.
6
Department of Stem Cell Transplantation, University Medical Center Hamburg-Eppendorf, 20246 Hamburg, Germany. fehse@uke.de.

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.

KEYWORDS:

allogeneic stem cell transplantation; chimerism; digital PCR; minimal residual disease

PMID:
27618030
PMCID:
PMC5037792
DOI:
10.3390/ijms17091515
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

Conflicts of InterestCaroline Rothe and Manja U. Böhme are employees of Biotype Diagnostic GmbH, a company that aims at commercializing the presented dPCR-based chimerism assays. All other authors declare no conflict of interest. The founding body had no role in the design of the study, in the collection, analyses, or interpretation of data, in the writing of the manuscript, and in the decision to publish the results.

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