A certified plasmid reference material for the standardisation of BCR-ABL1 mRNA quantification by real-time quantitative PCR

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 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) 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).

Figure 1

Schematic map of the multitarget plasmid pIRMM0099. The arrows represent the inserts from the transcript fragments of BCR, GUSB and BCR–ABL1. The rectangles show the location of the PCR targets BCR–ABL1 and ABL1 (the ABL1 CG is within the BCR–ABL1 fragment) used quantify the copy number concentration of the plasmid. The single restriction site for EcoRV is also shown.

Figure 2

Comparison of measured % BCR-ABL1/ABL1 ratio between laboratory calibrators (Lab) and ERM-AD623 for the cell line dilutions. Data from all centres in the second suitability study that used ABL1 as a control gene are included; median values are indicated.

Figure 3

Comparison of measured % BCR-ABL1/ABL1 ratio between laboratory calibrators (Lab) and ERM-AD623 for the aRNA mixtures. Data from all centres in the second suitability study that used ABL1 as a control gene and returned aRNA data are included; median values are indicated.

Figure 4

Reported numbers of BCR–ABL1 and ABL1 transcripts using laboratory-specific methods (which vary with regard to the amount of material analysed), laboratory calibrators (Lab) and ERM-AD623 for cell line dilutions. Data from all centres in the second suitability study that used ABL1 as a control gene are included; median values are indicated. Values for ABL1 are for all three dilutions combined; values for BCR–ABL1 differ between the three levels and are shown separately.

Figure 5

Estimates of copy numbers of BCR–ABL1 and ABL1 transcripts using laboratory calibrators and ERM-AD623 for the aRNA dilutions in the second suitability study. Individual laboratory protocols have been taken into account to derive estimate per μl of aRNA. Data from all centres in the second suitability study that used ABL1 as a control gene and returned aRNA data are included; median values are indicated. Values for ABL1 are for both dilutions combined; values for BCR–ABL1 differ between the two levels and are shown separately. Expected values for ABL1 if the lysis, reverse transcription and qPCR were all perfect is 305 500; expected BCR–ABL1 values for levels 1 and 2 are 300 and 30, respectively.