Abstract
Serial quantification of BCR–ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR–ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 106, 1.08±0.11 × 105, 1.03±0.10 × 104, 1.02±0.09 × 103, 1.04±0.10 × 102 and 10.0±1.5 copies/μl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR–ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR–ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).
Highlights
The BCR–ABL1 fusion gene is the primary pathogenic driver of chronic myeloid leukaemia (CML) and characterizes a subset of patients with acute lymphoblastic leukaemia
For samples that test negative for BCR–ABL1, the number of control gene (CG) transcripts gives an indication of the sensitivity with which residual disease can be excluded for that particular specimen.[1]
Several different in-house and commercial plasmid calibrators for BCR–ABL1 and CG measurement by reverse-transcription quantitative PCR (RT-Quantitative real-time PCR (qPCR)) have been developed. These calibrators have independently assigned copy number concentrations based on their molecular weight and DNA concentration, but in the absence of an internationally accepted certified reference material (CRM) it is inevitable that variation between calibrators has become established
Summary
The BCR–ABL1 fusion gene is the primary pathogenic driver of chronic myeloid leukaemia (CML) and characterizes a subset of patients with acute lymphoblastic leukaemia. Results for specimens that test positive for BCR–ABL1 are expressed as the ratio of BCR–ABL1/CG transcript numbers in the same volume of cDNA, subject to previously described performance criteria.[5,6] For samples that test negative for BCR–ABL1, the number of CG transcripts gives an indication of the sensitivity with which residual disease can be excluded for that particular specimen.[1] despite the established clinical utility of RT-qPCR for monitoring of CML patients, the comparability of results between testing laboratories may vary widely.[7] A major contributor to this variability is the use of different CGs to normalise results.
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