Aim To develop of reference reagents for BCR-ABL RQ- Why?

• 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
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??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
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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