Digital PCR to assess hematopoietic chimerism after allogeneic stem cell transplantation
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Digital PCR to assess hematopoietic chimerism after allogeneic stem cell transplantation. / Stahl, Tanja; Böhme, Manja; Kröger, Nicolaus; Fehse, Boris.
in: EXP HEMATOL, Jahrgang 43, Nr. 6, 18.03.2015, S. 462-468.Publikationen: SCORING: Beitrag in Fachzeitschrift/Zeitung › SCORING: Zeitschriftenaufsatz › Forschung › Begutachtung
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TY - JOUR
T1 - Digital PCR to assess hematopoietic chimerism after allogeneic stem cell transplantation
AU - Stahl, Tanja
AU - Böhme, Manja
AU - Kröger, Nicolaus
AU - Fehse, Boris
N1 - Copyright © 2015 ISEH - International Society for Experimental Hematology. Published by Elsevier Inc. All rights reserved.
PY - 2015/3/18
Y1 - 2015/3/18
N2 - Analysis of hematopoietic chimerism after allogeneic stem cell transplantation represents a crucial method to evaluate donor-cell engraftment. Whereas sensitivity of classical approaches for chimerism monitoring is limited to ≥1%, quantitative polymerase chain reaction (qPCR)-based techniques readily detect one patient cell in >1,000 donor cells, thus facilitating application of chimerism assessment as a surrogate for minimal residual disease. However, due to methodologic specificities, qPCR combines its high sensitivity with limited resolution power in the state of mixed chimerism (e.g., >10% patient cells). Our aim was to overcome this limitation by employing a further development of qPCR, namely digital PCR (dPCR), for chimerism analysis. For proof-of-principle, we established more than 10 dPCR assays detecting Indel polymorphisms or Y-chromosome sequences and tested them on artificial cell mixtures and patient samples. Employing artificial cell mixtures, we found that dPCR allows exact quantification of chimerism over several orders of magnitude. Digital PCR results proved to be highly reproducible (deviation < 5%), particularly in the "difficult" range of mixed chimerism. Excellent performance of the new assays was confirmed by analysis of multiple retrospective blood samples from patients after allogeneic stem cell transplantation, in comparison with established qPCR (14 patients) and short-tandem repeat PCR (4 patients) techniques. Finally, dPCR is easy to perform, needs only small amounts of DNA for chimerism assessment (65 ng corresponds to a sensitivity of approximately 0.03%), and does not require the use of standard curves and replicate analysis. In conclusion, dPCR represents a very promising method for routine chimerism monitoring.
AB - Analysis of hematopoietic chimerism after allogeneic stem cell transplantation represents a crucial method to evaluate donor-cell engraftment. Whereas sensitivity of classical approaches for chimerism monitoring is limited to ≥1%, quantitative polymerase chain reaction (qPCR)-based techniques readily detect one patient cell in >1,000 donor cells, thus facilitating application of chimerism assessment as a surrogate for minimal residual disease. However, due to methodologic specificities, qPCR combines its high sensitivity with limited resolution power in the state of mixed chimerism (e.g., >10% patient cells). Our aim was to overcome this limitation by employing a further development of qPCR, namely digital PCR (dPCR), for chimerism analysis. For proof-of-principle, we established more than 10 dPCR assays detecting Indel polymorphisms or Y-chromosome sequences and tested them on artificial cell mixtures and patient samples. Employing artificial cell mixtures, we found that dPCR allows exact quantification of chimerism over several orders of magnitude. Digital PCR results proved to be highly reproducible (deviation < 5%), particularly in the "difficult" range of mixed chimerism. Excellent performance of the new assays was confirmed by analysis of multiple retrospective blood samples from patients after allogeneic stem cell transplantation, in comparison with established qPCR (14 patients) and short-tandem repeat PCR (4 patients) techniques. Finally, dPCR is easy to perform, needs only small amounts of DNA for chimerism assessment (65 ng corresponds to a sensitivity of approximately 0.03%), and does not require the use of standard curves and replicate analysis. In conclusion, dPCR represents a very promising method for routine chimerism monitoring.
U2 - 10.1016/j.exphem.2015.02.006
DO - 10.1016/j.exphem.2015.02.006
M3 - SCORING: Journal article
C2 - 25795523
VL - 43
SP - 462
EP - 468
JO - EXP HEMATOL
JF - EXP HEMATOL
SN - 0301-472X
IS - 6
ER -