YAZHEN QIN, MS

China Associate Professor, Peking University People’s

Hospital, Peking University Institute of Hematology

Dr. Qin specializes in the molecular diagnostics in

hematologic malignancies. Her field of research

focuses on mechanism of leukemogenesis.

“China Standardization Project of BCR-ABL

detection in CML”

——an efficient way for multi-centers to

simultaneously derive and validate CFs

Ya-Zhen Qin

Peking University People's Hospital,

Peking University Institute of Hematology,

Beijing, China

Standardization of BCR-ABL detection in CML

BCR-ABL transcript levels detected by real-time PCR

(RQ-PCR)reflect molecular response to TKI for CML

patients

Variation exists among the individual laboratory’s

report, which limits its clinical application

BCR-ABLIS

Proposed in October 2005

Harmonizing the differing methodologies for measuring

BCR-ABL transcripts

using an individual laboratory’s conversion factor (CF)

to express BCR-ABL transcript levels on an

internationally agreed scale

MMR: BCR-ABLIS=0.1%

Blood 2006; 108: 28-37

Acquisition of CF

——sample exchange with reference lab

Derivation of CF

Validation of CF

Great variation existed in the BCR-ABL

results from different laboratories in China

Four samples (same BCR-ABL levels prepared in TRIzolby one lab)

Detected in 10 labs in China, May 2010

Variation caused by:

different protocols

problems in protocol

0.01

0.1

1

10

100

1000

0

sample 1BC

R/A

BL

tra

nscri

pt

levels

(%

)

0.01

0.1

1

10

100

sample 2BC

R/A

BL

tra

nscri

pt

levels

(%

)

0 0.001

0.01

0.1

1

10

sample 3BC

R/A

BL

tra

nscri

pt

levels

(%

)

0 0.00001

0.0001

0.001

0.01

0.1

1

0

sample 4BC

R/A

BL

tra

nscri

pt

levels

(%

)

“China Standardization Project of RQ-

PCR detected BCR-ABL in CML”

Initiated in 2010

Aim

To test the accuracy and stability of BCR-ABL

detection of the participated laboratories and

help to improve the performance of those

laboratories not well done

To derive and validate laboratory specific CFs

of the participants to make the results from

different laboratories comparable

Participants of Project

A regional reference laboratory:

Peking University People’s Hospital, Peking

University Institute of Hematology (PUIH)

3 batches of laboratories :

36 laboratories from hospitals and

commercial organizations

Summary of the five comparisons of the 1st batch

laboratories

0.000001

0.00001

0.0001

0.001

0.01

0.1

1

10

1:1000 1:100 1:10 1

样本稀释度

BC

R-A

BL

/AB

L

0.00001

0.0001

0.001

0.01

0.1

1

1:3000 1:300 1:30 1:3

样本稀释度B

CR

-AB

L/A

BL

0.00001

0.0001

0.001

0.01

0.1

1

1:400 1:40 1:4

样本稀释度

BC

R-A

BL

/AB

L

0.0001

0.001

0.01

0.1

1

1:1000 1:100 1:10 1

样本稀释度

BC

R-A

BL

/AB

L

0.0001

0.001

0.01

0.1

1

1:1000 1:100 1:10 1

样本细胞稀释度

BC

R-A

BL

/AB

LSamples May 2010 Samples August 2010 Samples April 2011

Samples July 2011 Samples September 2012

Accuracy of

BCR-ABL

detection by RQ-

PCR is greatly

improved

Acquisition of

CF

Qin, YZ. et al. Chinese J of Hematology 34: 104-108 (2013)

A sample exchange between Adelaide international

reference laboratory (IMVS) and PKUPH in 2012

PUIH: regional reference lab

Distributing samples for calculating CF

Preparing and distributing samples by PUIH

Performing BCR-ABL detection according to the laboratory’s

own protocols

Analyzing results and calculating CF by PUIH

Usual method of validation in the world

Participating laboratory sends 20-30 samples to reference laboratory, they simultaneously test these samples and CF is validated

Validation is a heavy work for a reference laboratory

we used a simultaneously distributing

same prepared samples method to

perform validation

Validating CF in China

Preparing validation samples by PUIH

20~30 times of dilution of fresh BCR-ABL (+) nucleated

cells by BCR-ABL (-) cells.

The dilutions covered 3-log BCR-ABL levels range, with

the highest level lower than 10%IS

Each set contained 21 samples with different BCR-ABL

levels

The samples were distributed and were

simultaneously tested by PUIH and the other

participating laboratories over 2-3 months

Criterion of successful validation in China

Bias≤±1.4 fold

95% limits of agreement≤±6 fold

Difference and agreement analysis

Bias(fold)=1/10-0.0032

Lower limit(fold)= 1+(-1/10-0.0032)-10((0.42+0.41)/2)

Higher limit(fold)=1+(-1/10-0.0032)+10((0.42+0.41)/2)

Bias -0.00322306

SD of bias 0.211994

95% Limits of Agreement

From -0.418731

To 0.412285

0.4

0.2

0.0

-0.2

-0.4

Difference

Average

QiLu13.12

-3 -2 -1 0 1 2

Log(QL)IS

-3

-2

-1

0

1

2

-3 -2 -1 0 1 2

PUIH vs QiLuLog(PKUPH)IS

30 laboratories acquired their validated CFs

Beijing

Reference lab

1st batch

2nd batch

3rd batch

Next step

There are still some more laboratories which want to acquire a validated CF for conversion

of BCR-ABLIS

The stability of BCR-ABL detection of the

laboratories that have validated their CFs

needs to be tested regularly——revalidation

Summary

The approach we derive and validate CF is practical and efficient when a number of centers need to be performed simultaneously

Our simultaneously distributing same prepared samples method is also the way to regularly test the stability of BCR-ABL detection of multi-centers

Acknowledgements

Hospitals involved in the Project

Adelaide reference laboratory

Susan Branford

Novartis China