• Aim – To develop of reference reagents for BCR-ABL RQPCR • Why? – To provide a stable, independent definition of the International Scale – To enable more ready access to the International Scale • How? – By development of higher order, primary reagents that will be internationally accredited Primary reference standards WHO definition • Ideally be as close as possible to real samples. • Should cover all steps of the process, including RNA extraction. • Must be stable over several years (=freeze dried) and physically possible to prepare batches that last several years. • Must be applicable to all or most existing methods Primary reference standards • Prepared centrally and accredited – National Institute of Biological Standards and Controls (NIBSC) – Accreditation by the WHO – CE marked • Probably very limited in supply • Main use will be by reference labs or companies to calibrate secondary reference standards Formulation for primary reagents • [CML cells (primary or K562) diluted in normal leucocytes] • Cell line mixtures • K562 is fine for BCR-ABL • Non BCR-ABL: 30 lines tested: KG1 and HL60 had ratios of ABL:BCR:GUSB closest to that seen in normal leukocytes – – – – ??500 labs worldwide Median 3 runs/week = 78,000 runs pa Four dilutions, each containing 106 cells Would need 3x1011 cells (>600 litres culture volume) per annum to satisfy world wide demand for reference reagents to be included in each run Primary reference reagents: pilot trial • Pilot trial: – Cells grown and mixtures made in Salisbury – 4 dilutions of K562 in both KG1 and HL60; 10%-0.01%; 3x106 cells/vial – Freeze dried at NIBSC – Full performance evaluation involving 14 labs worldwide JuneSept 2007 Performance of Freeze Dried Cell Mixtures HL60/K562 Reference Reagents: full scale production • Both freeze dried cells combinations performed well in international field trials • Large scale grow ups of HL60 (40L) • Mixtures made with K562 to yield approx 10%, 1%, 0.1%, 0.01% BCR-ABLIS. • 3000 vials at each dilution freeze dried by NIBSC (Sept 2008) • Initial in house evaluations successful Primary Reference Reagents: evaluation and assignment of values • • • • Field trial Jan – March 2009 10 labs worldwide with validated CFs At least 3 labs per control gene (ABL, BCR, GUSB) Each lab received 3 vials at each of the four dilution levels • Each extracted and reverse transcribed twice on different days = 24 datapoints/lab • Amounts of RNA, absolute copy numbers of control gene, BCR-ABL/control gene before and after conversion • Values assigned to each dilution Primary Reference Reagents: evaluation and assignment of values Primary Reference Reagents: evaluation and assignment of values % BCR-ABL / ABL % BCR-ABL / BCR % BCR-ABL / GUS BCR/ABL 1 08/192 0.0118 0.0195 0.007 BCR/ABL 2 08/194 0.1112 0.1753 0.075 BCR/ABL 3 08/196 1.1672 1.6627 0.829 BCR/ABL 4 08/198 10.7469 16.3129 10.123 Primary reference materials: stability studies Fresh 1.00E+06 Freeze dried 2nd Oct -150 deg (6 month) 1.00E+05 45 deg (6 months) 56 deg (6 months) 1.00E+04 1.00E+03 1.00E+02 1.00E+01 1.00E+00 ABL Copy no. BCR Copy no. GUS Copy no. Primary reference reagents: next steps • Submission to WHO for accreditation by NIBSC July 2009 • Evaluation to determine if freeze dried materials can be used to derive CFs • Evaluation to determine if freeze dried materials can effectively calibrate secondary reference materials Other reference standards: High / low BCR-ABL standards – Adelaide model Make large batch of high and low level controls Trizol or RLT lysates stored at -80ºC Single use aliquots of RNA stored at -80 ºC Aliquot reverse transcribed and used in each RQ-PCR run %BCR-ABL / control gene values monitored over time for every run Result must fall within defined limits for run to be accepted Other reference standrads: High / low BCR-ABL standards – Adelaide model High level standard: approx 10% BCR-ABL / ABL HL60 (b3a2 –ve) plus 1:50 dilution of K562 (b3a2 +ve) 1.5 x 106 cells / 1ml Trizol or 1.5 x 106 cells / 600µl RLT Low level standard: approx 0.1% BCR-ABL / ABL 1:100 dilution of high level standard (in HL60) 1.5 x 106 cells / 1ml Trizol or 1.5x106 cells / 600µl RLT Other reference standrads: High / low BCR-ABL standards – Adelaide model Sue Branford Westgard (1981) Clin Chem, 27,493 Improved comparability using a common calibration plasmid Acknowledgements • Adelaide – Tim Hughes, Sue Branford • Mannheim – Andreas Hochhaus, Martin Müller • Salisbury – Helen White, Lin Feng • NIBSC • Asuragen • International BCR-ABL Standardization Group