IMPLEMENTING A CERTIFIED PLASMID REFERENCE MATERIAL FOR COPY NUMBER CALIBRATION DEMONSTRATES GOOD REPORTING CORRELATION BETWEEN %BCR-ABL/ABL (IS) AND %BCR-ABL/ABL (COPY NUMBER) IN XPERT® BCR-ABL ULTRA
(Abstract release date: 05/19/16)
EHA Library. Day G. 06/09/16; 132644; E1095
Dr. Gwo-Jen Day
Contributions
Contributions
Abstract
Abstract: E1095
Type: Eposter Presentation
Background
Development of a quantitative BCR-ABL monitoring assay with sensitivity down to 4.5 logs below baseline (MR4.5) is important for patients being considered for tyrosine kinase inhibitor (TKI) discontinuation, and it is essential to ensure that variability in testing methodologies across laboratories is tightly controlled. Through analytical and clinical evaluations, Xpert BCR-ABL Ultra, an automated cartridge-based real-time qPCR BCR-ABL monitoring assay, has demonstrated sensitivity reaching MR4.5, and every production lot of cartridges is assigned a unique conversion factor assuring substantial alignment to the WHO International Scale (IS).
Aims
Here we describe the implementation of a certified plasmid reference material (IRMM_ERM-AD623 BCR-ABL Calibrator) for the standardization of BCR-ABL mRNA quantification through copy number calibration to ensure sufficient BCR-ABL and ABL copies are present to support the MR4.5 sensitivity claim for Xpert BCR-ABL Ultra.
Methods
To establish the correlation between Cycle threshold (Ct) reporting and copy number for BCR-ABL and ABL, and to understand the assay sensitivity limit in terms of relative copy number, the IRMM plasmid was used in a titration study involving three independent BCR-ABL Ultra assay lots. Based on the titration study, a copy number calibration curve was derived and used to estimate the copy number for BCR-ABL and ABL in analytical studies previously performed for CE registration, including the Linearity/Dynamic range, Analytical LoD and Clinical LoD studies.
Results
The results demonstrate that a linear detection range between 1x106 copies down to 3 copies was achieved with 5 copies being detected in 24 out of 24 replicates and determined as the Limit of Detection (LoD) for both BCR-ABL and ABL. The overall ABL copy number present in clinical samples in each study was at least 5-10 times the required minimal ABL copy number of ≥32,000 to support a claim of MR4.5 and ≥100,000 for MR5.0. This is explained by the fact that 4ml whole blood is used to prepare the sample lysate and an aliquot of which equivalent to 0.6ml whole blood is then processed completely in a fully automated and integrated GeneXpert system for nucleic acid extraction, target amplification, and quantification, resulting in improved target recovery. The %ratio of the derived BCR-ABL and ABL copy numbers was further calculated and compared to the original assay lot-specific %reporting (IS) per WHO calibration, revealing an un-biased correlation between %BCR-ABL/ABL (Copy number) and %BCR-ABL/ABL (IS) across the range 100% to 0.001% (IS). To validate the assay performance, Xpert BCR-ABL Ultra was further evaluated in field studies compared to three independent laboratory developed tests (LDTs) previously calibrated with the IRMM plasmid using either ABL or GUSB as control genes, demonstrating a correlation of >0.91, covering samples in the range 100% - 0.001% (IS).
Conclusion
These studies, using two different calibration systems (WHO/IS and IRMM/Copy number) and two different control genes (ABL and GUSB), carried out in four independent research labs, demonstrate, for the first time, good reporting correlation between %BCR-ABL/ABL (IS) and %BCR-ABL and ABL (Copy number) in Xpert BCR-ABL Ultra. Furthermore, these data demonstrate that from a typical clinical specimen, the BCR-ABL Ultra assay captures more than enough ABL copies to support a sensitivity claim of MR4.5.
Session topic: E-poster
Keyword(s): BCR-ABL, Monitor, Quantitative RT-PCR, Standardization
Type: Eposter Presentation
Background
Development of a quantitative BCR-ABL monitoring assay with sensitivity down to 4.5 logs below baseline (MR4.5) is important for patients being considered for tyrosine kinase inhibitor (TKI) discontinuation, and it is essential to ensure that variability in testing methodologies across laboratories is tightly controlled. Through analytical and clinical evaluations, Xpert BCR-ABL Ultra, an automated cartridge-based real-time qPCR BCR-ABL monitoring assay, has demonstrated sensitivity reaching MR4.5, and every production lot of cartridges is assigned a unique conversion factor assuring substantial alignment to the WHO International Scale (IS).
Aims
Here we describe the implementation of a certified plasmid reference material (IRMM_ERM-AD623 BCR-ABL Calibrator) for the standardization of BCR-ABL mRNA quantification through copy number calibration to ensure sufficient BCR-ABL and ABL copies are present to support the MR4.5 sensitivity claim for Xpert BCR-ABL Ultra.
Methods
To establish the correlation between Cycle threshold (Ct) reporting and copy number for BCR-ABL and ABL, and to understand the assay sensitivity limit in terms of relative copy number, the IRMM plasmid was used in a titration study involving three independent BCR-ABL Ultra assay lots. Based on the titration study, a copy number calibration curve was derived and used to estimate the copy number for BCR-ABL and ABL in analytical studies previously performed for CE registration, including the Linearity/Dynamic range, Analytical LoD and Clinical LoD studies.
Results
The results demonstrate that a linear detection range between 1x106 copies down to 3 copies was achieved with 5 copies being detected in 24 out of 24 replicates and determined as the Limit of Detection (LoD) for both BCR-ABL and ABL. The overall ABL copy number present in clinical samples in each study was at least 5-10 times the required minimal ABL copy number of ≥32,000 to support a claim of MR4.5 and ≥100,000 for MR5.0. This is explained by the fact that 4ml whole blood is used to prepare the sample lysate and an aliquot of which equivalent to 0.6ml whole blood is then processed completely in a fully automated and integrated GeneXpert system for nucleic acid extraction, target amplification, and quantification, resulting in improved target recovery. The %ratio of the derived BCR-ABL and ABL copy numbers was further calculated and compared to the original assay lot-specific %reporting (IS) per WHO calibration, revealing an un-biased correlation between %BCR-ABL/ABL (Copy number) and %BCR-ABL/ABL (IS) across the range 100% to 0.001% (IS). To validate the assay performance, Xpert BCR-ABL Ultra was further evaluated in field studies compared to three independent laboratory developed tests (LDTs) previously calibrated with the IRMM plasmid using either ABL or GUSB as control genes, demonstrating a correlation of >0.91, covering samples in the range 100% - 0.001% (IS).
Conclusion
These studies, using two different calibration systems (WHO/IS and IRMM/Copy number) and two different control genes (ABL and GUSB), carried out in four independent research labs, demonstrate, for the first time, good reporting correlation between %BCR-ABL/ABL (IS) and %BCR-ABL and ABL (Copy number) in Xpert BCR-ABL Ultra. Furthermore, these data demonstrate that from a typical clinical specimen, the BCR-ABL Ultra assay captures more than enough ABL copies to support a sensitivity claim of MR4.5.
Session topic: E-poster
Keyword(s): BCR-ABL, Monitor, Quantitative RT-PCR, Standardization
Abstract: E1095
Type: Eposter Presentation
Background
Development of a quantitative BCR-ABL monitoring assay with sensitivity down to 4.5 logs below baseline (MR4.5) is important for patients being considered for tyrosine kinase inhibitor (TKI) discontinuation, and it is essential to ensure that variability in testing methodologies across laboratories is tightly controlled. Through analytical and clinical evaluations, Xpert BCR-ABL Ultra, an automated cartridge-based real-time qPCR BCR-ABL monitoring assay, has demonstrated sensitivity reaching MR4.5, and every production lot of cartridges is assigned a unique conversion factor assuring substantial alignment to the WHO International Scale (IS).
Aims
Here we describe the implementation of a certified plasmid reference material (IRMM_ERM-AD623 BCR-ABL Calibrator) for the standardization of BCR-ABL mRNA quantification through copy number calibration to ensure sufficient BCR-ABL and ABL copies are present to support the MR4.5 sensitivity claim for Xpert BCR-ABL Ultra.
Methods
To establish the correlation between Cycle threshold (Ct) reporting and copy number for BCR-ABL and ABL, and to understand the assay sensitivity limit in terms of relative copy number, the IRMM plasmid was used in a titration study involving three independent BCR-ABL Ultra assay lots. Based on the titration study, a copy number calibration curve was derived and used to estimate the copy number for BCR-ABL and ABL in analytical studies previously performed for CE registration, including the Linearity/Dynamic range, Analytical LoD and Clinical LoD studies.
Results
The results demonstrate that a linear detection range between 1x106 copies down to 3 copies was achieved with 5 copies being detected in 24 out of 24 replicates and determined as the Limit of Detection (LoD) for both BCR-ABL and ABL. The overall ABL copy number present in clinical samples in each study was at least 5-10 times the required minimal ABL copy number of ≥32,000 to support a claim of MR4.5 and ≥100,000 for MR5.0. This is explained by the fact that 4ml whole blood is used to prepare the sample lysate and an aliquot of which equivalent to 0.6ml whole blood is then processed completely in a fully automated and integrated GeneXpert system for nucleic acid extraction, target amplification, and quantification, resulting in improved target recovery. The %ratio of the derived BCR-ABL and ABL copy numbers was further calculated and compared to the original assay lot-specific %reporting (IS) per WHO calibration, revealing an un-biased correlation between %BCR-ABL/ABL (Copy number) and %BCR-ABL/ABL (IS) across the range 100% to 0.001% (IS). To validate the assay performance, Xpert BCR-ABL Ultra was further evaluated in field studies compared to three independent laboratory developed tests (LDTs) previously calibrated with the IRMM plasmid using either ABL or GUSB as control genes, demonstrating a correlation of >0.91, covering samples in the range 100% - 0.001% (IS).
Conclusion
These studies, using two different calibration systems (WHO/IS and IRMM/Copy number) and two different control genes (ABL and GUSB), carried out in four independent research labs, demonstrate, for the first time, good reporting correlation between %BCR-ABL/ABL (IS) and %BCR-ABL and ABL (Copy number) in Xpert BCR-ABL Ultra. Furthermore, these data demonstrate that from a typical clinical specimen, the BCR-ABL Ultra assay captures more than enough ABL copies to support a sensitivity claim of MR4.5.
Session topic: E-poster
Keyword(s): BCR-ABL, Monitor, Quantitative RT-PCR, Standardization
Type: Eposter Presentation
Background
Development of a quantitative BCR-ABL monitoring assay with sensitivity down to 4.5 logs below baseline (MR4.5) is important for patients being considered for tyrosine kinase inhibitor (TKI) discontinuation, and it is essential to ensure that variability in testing methodologies across laboratories is tightly controlled. Through analytical and clinical evaluations, Xpert BCR-ABL Ultra, an automated cartridge-based real-time qPCR BCR-ABL monitoring assay, has demonstrated sensitivity reaching MR4.5, and every production lot of cartridges is assigned a unique conversion factor assuring substantial alignment to the WHO International Scale (IS).
Aims
Here we describe the implementation of a certified plasmid reference material (IRMM_ERM-AD623 BCR-ABL Calibrator) for the standardization of BCR-ABL mRNA quantification through copy number calibration to ensure sufficient BCR-ABL and ABL copies are present to support the MR4.5 sensitivity claim for Xpert BCR-ABL Ultra.
Methods
To establish the correlation between Cycle threshold (Ct) reporting and copy number for BCR-ABL and ABL, and to understand the assay sensitivity limit in terms of relative copy number, the IRMM plasmid was used in a titration study involving three independent BCR-ABL Ultra assay lots. Based on the titration study, a copy number calibration curve was derived and used to estimate the copy number for BCR-ABL and ABL in analytical studies previously performed for CE registration, including the Linearity/Dynamic range, Analytical LoD and Clinical LoD studies.
Results
The results demonstrate that a linear detection range between 1x106 copies down to 3 copies was achieved with 5 copies being detected in 24 out of 24 replicates and determined as the Limit of Detection (LoD) for both BCR-ABL and ABL. The overall ABL copy number present in clinical samples in each study was at least 5-10 times the required minimal ABL copy number of ≥32,000 to support a claim of MR4.5 and ≥100,000 for MR5.0. This is explained by the fact that 4ml whole blood is used to prepare the sample lysate and an aliquot of which equivalent to 0.6ml whole blood is then processed completely in a fully automated and integrated GeneXpert system for nucleic acid extraction, target amplification, and quantification, resulting in improved target recovery. The %ratio of the derived BCR-ABL and ABL copy numbers was further calculated and compared to the original assay lot-specific %reporting (IS) per WHO calibration, revealing an un-biased correlation between %BCR-ABL/ABL (Copy number) and %BCR-ABL/ABL (IS) across the range 100% to 0.001% (IS). To validate the assay performance, Xpert BCR-ABL Ultra was further evaluated in field studies compared to three independent laboratory developed tests (LDTs) previously calibrated with the IRMM plasmid using either ABL or GUSB as control genes, demonstrating a correlation of >0.91, covering samples in the range 100% - 0.001% (IS).
Conclusion
These studies, using two different calibration systems (WHO/IS and IRMM/Copy number) and two different control genes (ABL and GUSB), carried out in four independent research labs, demonstrate, for the first time, good reporting correlation between %BCR-ABL/ABL (IS) and %BCR-ABL and ABL (Copy number) in Xpert BCR-ABL Ultra. Furthermore, these data demonstrate that from a typical clinical specimen, the BCR-ABL Ultra assay captures more than enough ABL copies to support a sensitivity claim of MR4.5.
Session topic: E-poster
Keyword(s): BCR-ABL, Monitor, Quantitative RT-PCR, Standardization
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