DEVELOPMENT AND CLINICAL EVALUATION OF DIGITAL PCR ASSAYS FOR SENSITIVE CHIMERISM ANALYSIS BASED ON DELETION/INSERTION POLYMORPHISMS
(Abstract release date: 05/19/16)
EHA Library. Mersmann S. 06/09/16; 133076; E1527
Dr. Sophia Mersmann
Contributions
Contributions
Abstract
Abstract: E1527
Type: Eposter Presentation
Background
Examination of chimerism has become a crucial means to monitor engraftment and, in the absence of specific disease markers, detect recurrence of leukemic cells after allogeneic stem cell transplantation. Various techniques have been established for chimerism analysis; the most often used STR amplification has high power of discrimination, but its detection limit is restrained to 1%>5%. Consequently, more sensitive approaches are required to ensure earlier detection of engraftment failure or relapses.
Aims
We have previously shown that digital PCR assays detecting deletion/insertion polymorphisms (DIPs) combine excellent sensitivity (routinely ≤0.1%) with accurate quantification over a large dynamic measurement range. Moreover, built on the dPCR principle, there is no need for standard curves and replicate measurements. Here we aimed at introducing a whole panel of digital-PCR based assays for routine chimerism measurement.
Methods
We selected a set of 53 DIPs and tested their suitability for duplex analysis in combination with a reference gene and a Y-chromosome specific locus. We identified 29 DIPs with high power of discrimination and good performance in duplex PCR.
Results
Assays were optimized and technically validated; applicability for diagnostics was confirmed on clinical samples in direct comparison with STR and qPCR. Finally, we established a screening plate for initial genotyping with DIP-specific digital duplex assays for convenient application of the system in every-day diagnostics.
Conclusion
In conclusion, we have developed and validated assays for regular chimerism determination including a screening plate for initial DIP assessment. The new tool ensures precise, robust and fast analysis of hematopoietic chimerism with a regular sensitivity of ≤0.1% (for ≥20 ng of genomic DNA). We propose that the new method will be highly useful for clinical routine diagnostics.
Session topic: E-poster
Keyword(s): Chimerism quantification, Clinical data, Diagnosis
Type: Eposter Presentation
Background
Examination of chimerism has become a crucial means to monitor engraftment and, in the absence of specific disease markers, detect recurrence of leukemic cells after allogeneic stem cell transplantation. Various techniques have been established for chimerism analysis; the most often used STR amplification has high power of discrimination, but its detection limit is restrained to 1%>5%. Consequently, more sensitive approaches are required to ensure earlier detection of engraftment failure or relapses.
Aims
We have previously shown that digital PCR assays detecting deletion/insertion polymorphisms (DIPs) combine excellent sensitivity (routinely ≤0.1%) with accurate quantification over a large dynamic measurement range. Moreover, built on the dPCR principle, there is no need for standard curves and replicate measurements. Here we aimed at introducing a whole panel of digital-PCR based assays for routine chimerism measurement.
Methods
We selected a set of 53 DIPs and tested their suitability for duplex analysis in combination with a reference gene and a Y-chromosome specific locus. We identified 29 DIPs with high power of discrimination and good performance in duplex PCR.
Results
Assays were optimized and technically validated; applicability for diagnostics was confirmed on clinical samples in direct comparison with STR and qPCR. Finally, we established a screening plate for initial genotyping with DIP-specific digital duplex assays for convenient application of the system in every-day diagnostics.
Conclusion
In conclusion, we have developed and validated assays for regular chimerism determination including a screening plate for initial DIP assessment. The new tool ensures precise, robust and fast analysis of hematopoietic chimerism with a regular sensitivity of ≤0.1% (for ≥20 ng of genomic DNA). We propose that the new method will be highly useful for clinical routine diagnostics.
Session topic: E-poster
Keyword(s): Chimerism quantification, Clinical data, Diagnosis
Abstract: E1527
Type: Eposter Presentation
Background
Examination of chimerism has become a crucial means to monitor engraftment and, in the absence of specific disease markers, detect recurrence of leukemic cells after allogeneic stem cell transplantation. Various techniques have been established for chimerism analysis; the most often used STR amplification has high power of discrimination, but its detection limit is restrained to 1%>5%. Consequently, more sensitive approaches are required to ensure earlier detection of engraftment failure or relapses.
Aims
We have previously shown that digital PCR assays detecting deletion/insertion polymorphisms (DIPs) combine excellent sensitivity (routinely ≤0.1%) with accurate quantification over a large dynamic measurement range. Moreover, built on the dPCR principle, there is no need for standard curves and replicate measurements. Here we aimed at introducing a whole panel of digital-PCR based assays for routine chimerism measurement.
Methods
We selected a set of 53 DIPs and tested their suitability for duplex analysis in combination with a reference gene and a Y-chromosome specific locus. We identified 29 DIPs with high power of discrimination and good performance in duplex PCR.
Results
Assays were optimized and technically validated; applicability for diagnostics was confirmed on clinical samples in direct comparison with STR and qPCR. Finally, we established a screening plate for initial genotyping with DIP-specific digital duplex assays for convenient application of the system in every-day diagnostics.
Conclusion
In conclusion, we have developed and validated assays for regular chimerism determination including a screening plate for initial DIP assessment. The new tool ensures precise, robust and fast analysis of hematopoietic chimerism with a regular sensitivity of ≤0.1% (for ≥20 ng of genomic DNA). We propose that the new method will be highly useful for clinical routine diagnostics.
Session topic: E-poster
Keyword(s): Chimerism quantification, Clinical data, Diagnosis
Type: Eposter Presentation
Background
Examination of chimerism has become a crucial means to monitor engraftment and, in the absence of specific disease markers, detect recurrence of leukemic cells after allogeneic stem cell transplantation. Various techniques have been established for chimerism analysis; the most often used STR amplification has high power of discrimination, but its detection limit is restrained to 1%>5%. Consequently, more sensitive approaches are required to ensure earlier detection of engraftment failure or relapses.
Aims
We have previously shown that digital PCR assays detecting deletion/insertion polymorphisms (DIPs) combine excellent sensitivity (routinely ≤0.1%) with accurate quantification over a large dynamic measurement range. Moreover, built on the dPCR principle, there is no need for standard curves and replicate measurements. Here we aimed at introducing a whole panel of digital-PCR based assays for routine chimerism measurement.
Methods
We selected a set of 53 DIPs and tested their suitability for duplex analysis in combination with a reference gene and a Y-chromosome specific locus. We identified 29 DIPs with high power of discrimination and good performance in duplex PCR.
Results
Assays were optimized and technically validated; applicability for diagnostics was confirmed on clinical samples in direct comparison with STR and qPCR. Finally, we established a screening plate for initial genotyping with DIP-specific digital duplex assays for convenient application of the system in every-day diagnostics.
Conclusion
In conclusion, we have developed and validated assays for regular chimerism determination including a screening plate for initial DIP assessment. The new tool ensures precise, robust and fast analysis of hematopoietic chimerism with a regular sensitivity of ≤0.1% (for ≥20 ng of genomic DNA). We propose that the new method will be highly useful for clinical routine diagnostics.
Session topic: E-poster
Keyword(s): Chimerism quantification, Clinical data, Diagnosis
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