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

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 facilitate...

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Autores principales: Stahl, Tanja, Rothe, Caroline, Böhme, Manja U., Kohl, Aloisa, Kröger, Nicolaus, Fehse, Boris
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2016
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5037792/
https://www.ncbi.nlm.nih.gov/pubmed/27618030
http://dx.doi.org/10.3390/ijms17091515
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author Stahl, Tanja
Rothe, Caroline
Böhme, Manja U.
Kohl, Aloisa
Kröger, Nicolaus
Fehse, Boris
author_facet Stahl, Tanja
Rothe, Caroline
Böhme, Manja U.
Kohl, Aloisa
Kröger, Nicolaus
Fehse, Boris
author_sort Stahl, Tanja
collection PubMed
description 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.
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spelling pubmed-50377922016-09-29 Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation Stahl, Tanja Rothe, Caroline Böhme, Manja U. Kohl, Aloisa Kröger, Nicolaus Fehse, Boris Int J Mol Sci Article 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. MDPI 2016-09-09 /pmc/articles/PMC5037792/ /pubmed/27618030 http://dx.doi.org/10.3390/ijms17091515 Text en © 2016 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC-BY) license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Article
Stahl, Tanja
Rothe, Caroline
Böhme, Manja U.
Kohl, Aloisa
Kröger, Nicolaus
Fehse, Boris
Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation
title Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation
title_full Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation
title_fullStr Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation
title_full_unstemmed Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation
title_short Digital PCR Panel for Sensitive Hematopoietic Chimerism Quantification after Allogeneic Stem Cell Transplantation
title_sort digital pcr panel for sensitive hematopoietic chimerism quantification after allogeneic stem cell transplantation
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5037792/
https://www.ncbi.nlm.nih.gov/pubmed/27618030
http://dx.doi.org/10.3390/ijms17091515
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