WHO International Standard
1st WHO International Genetic Reference Panel for quantitation of
BCR-ABL translocation by RQ-PCR
NIBSC code: 09/138
Instructions for use
(Version 4.0, Dated 13/12/2012)
1. INTENDED USE
The panel comprises four individually coded ampoules, each containing
freeze-dried cells. Each ampoule has a different defined value for %BCRABL/control gene and they are intended to be used as a primary standard
for calibrating secondary standards by RQ-PCR (see pages 3&4). This
panel was established in 2009 by the Expert Committee on Biological
Standardization (ECBS) of the World Health Organization (WHO) as the
1st WHO International Genetic Reference Panel for the Quantitation of
BCR-ABL Translocation, NIBSC code 09/138 [1].
These materials should not be put to any other use. Please note that the
materials have been validated only for BCR-ABL detection in the range
0.01% to 10% on the International Scale. They have not been validated
for other applications, e.g. International Scale measurements >10% or for
determining assay sensitivity.
2. CAUTION
This preparation is not for administration to humans.
The preparations contain material of human origin. They have been tested
for HIV, HBV, HCV, CMV, EBV, HTLV-I/II, HHV-8 and mycoplasma by
PCR and none were detected.
Routine microbiology testing of the free-dried materials showed
contamination with Staphylococcus haemolyticus, which is classified in
Hazard Group 2. Experiments carried out at NIBSC showed that the
organism was completely killed after exposure to Trizol (30-60% Phenol)
which is the first step of the RNA extraction process. As with all
materials of biological origin, this preparation should be regarded as
potentially hazardous to health. It should be used and discarded
according to your own laboratory's safety procedures. Such safety
procedures should include the wearing of protective gloves and
avoiding the generation of aerosols. Care should be exercised in
opening ampoules or vials, to avoid cuts.
3. UNITAGE
The panel was tested in an international collaborative study involving 10
laboratories and the following mean %BCR-ABL / control gene values
were obtained following conversion to the international scale (IS);
Ampoule
Code no.
08/192
08/194
08/196
08/198
%BCR-ABL
/ ABL
0.0118
0.1112
1.1672
10.7469
%BCR-ABL
/ BCR
0.0195
0.1753
1.6627
16.3129
%BCR-ABL
/ GUSB
0.0071
0.0749
0.8295
10.1235
4. CONTENTS
Country of origin of biological material: Germany & United Kingdom.
The ampoules contain freeze-dried K562 cells (expressing the BCR-ABL
translocation b3a2) and HL60 cells (BCR-ABL negative) in varying
proportions. The total number of cells per ampoule is 1.5 x 10E6. The
cells were suspended in 2x PBS before freeze-drying.
5. STORAGE
Store all unopened ampoules at -20°C or below.
Please note: because of the inherent stability of lyophilized material,
NIBSC may ship these materials at ambient temperature.
6. DIRECTIONS FOR OPENING
DIN ampoules have an „easy-open‟ coloured stress point, where the
narrow ampoule stem joins the wider ampoule body.
Tap the ampoule gently to collect the material at the bottom (labeled)
end. Ensure that the disposable ampoule safety breaker provided is
pushed down on the stem of the ampoule and against the shoulder of the
ampoule body. Hold the body of the ampoule in one hand and the
disposable ampoule breaker covering the ampoule stem between the
thumb and first finger of the other hand. Apply a bending force to open
the ampoule at the coloured stress point, primarily using the hand holding
the plastic collar.
Care should be taken to avoid cuts and projectile glass fragments that
might enter the eyes, for example, by the use of suitable gloves and an
eye shield. Take care that no material is lost from the ampoule and no
glass falls into the ampoule. Within the ampoule is dry nitrogen gas at
slightly less than atmospheric pressure. A new disposable ampoule
breaker is provided with each DIN ampoule.
7. USE OF MATERIAL
No attempt should be made to weigh out any portion of the freeze-dried
material prior to reconstitution.
a. Open ampoules as described in section 6. above.
b. Reconstitute freeze-dried material at room temperature with
1mL Trizol or 600 µL RLT buffer.
c. Ensure all cells are lysed by repeated aspiration with a pipette
tip or a needle
d. Transfer the entire contents to nuclease-free tubes.
e. Using your local method, extract RNA from the 4 ampoules of freeze
dried material and the secondary standards to be calibrated at the same
time.
f. Primary and secondary material should be analysed in the same assay
to assign value to the secondary standard. The dose response curves of
the WHO panel and secondary standard should be parallel to each other
when log %BCR-ABL is plotted against log dilution.
If you require further information on how to use these materials please
contact NIBSC.
8. STABILITY
Reference materials are held at NIBSC within assured, temperature
controlled storage facilities and they should be stored on receipt as
indicated on the label. It is the policy of WHO not to assign an expiry date
to their international reference materials. Accelerated degradation studies
have indicated that this material is suitably stable, when stored at -20ºC
or below, for the assigned values to remain valid until the material is
withdrawn or replaced. These studies have also shown that the material
is suitably stable for shipment at ambient temperature without any effect
on the assigned values. Users who have data supporting any
deterioration in the characteristics of any reference preparation are
encouraged to contact NIBSC.
9. REFERENCES
1. WHO document WHO/BS/09.2106
2. White H.E. et al, Establishment of the 1st World Health Organization
International Genetic Reference Panel for quantitation of BCR-ABL mRNA.
Blood, 2010.
10. ACKNOWLEDGEMENTS
We would like to thank the German Collection of Microorganisms and Cell
Cultures (DSMZ), the Hammersmith Hospital, London and the UK National
Genetics Reference Laboratory (Wessex) for supplying materials and
assistance with the collaborative study.
11. FURTHER INFORMATION
Further information can be obtained as follows;
This material: [email protected]
National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, EN6 3QG
WHO International Laboratory for Biological Standards, UK Official Medicines Control Laboratory
T +44 (0)1707 641000
nibsc.org
Page 1 of 4
WHO Biological Standards:
http://www.who.int/biologicals/en/
JCTLM Higher order reference materials:
http://www.bipm.org/en/committees/jc/jctlm/
Derivation of International Units:
http://www.nibsc.org/products/biological_reference_materials/frequentl
y_asked_questions/how_are_international_units.aspx
Ordering standards from NIBSC:
http://www.nibsc.org/products/ordering_information/frequently_asked_
questions.aspx
NIBSC Terms & Conditions:
http://www.nibsc.org/terms_and_conditions.aspx
12. CUSTOMER FEEDBACK
Customers are encouraged to provide feedback on the suitability or
use of the material provided or other aspects of our service. Please
send any comments to [email protected]
13. CITATION
In all publications, including data sheets, in which this material is
referenced, it is important that the preparation's title, its status, the
NIBSC code number, and the name and address of NIBSC are cited
and cited correctly.
14.
MATERIAL SAFETY SHEET
Physical and Chemical properties
Physical appearance:
Corrosive:
No
Freeze-dried solid
Stable:
Yes
Oxidising:
No
Hygroscopic:
Yes
Irritant:
No
Flammable:
No
Handling:See caution, Section 2
Other (specify):
Contains material of human origin
Toxicological properties
Effects of inhalation:
Effects of ingestion:
Effects of skin absorption:
Not established, avoid inhalation
Not established, avoid ingestion
Not established, avoid contact with skin
Suggested First Aid
Inhalation:
Ingestion:
Seek medical advice
Seek medical advice
Contact with eyes:
Contact with skin:
Wash with copious amounts of water. Seek
medical advice
Wash thoroughly with water.
Action on Spillage and Method of Disposal
Spillage of contents should be taken up with absorbent material
wetted with an appropriate disinfectant. Rinse area with an appropriate
disinfectant followed by water.
Absorbent materials used to treat spillage should be treated as
biological waste.
15. LIABILITY AND LOSS
In the event that this document is translated into another language, the
English language version shall prevail in the event of any inconsistencies
between the documents.
Unless expressly stated otherwise by NIBSC, NIBSC‟s Standard Terms
and Conditions for the Supply of Materials (available at
http://www.nibsc.org/About_Us/Terms_and_Conditions.aspx or upon
request by the Recipient) (“Conditions”) apply to the exclusion of all other
terms and are hereby incorporated into this document by reference. The
Recipient's attention is drawn in particular to the provisions of clause 11
of the Conditions.
16. INFORMATION FOR CUSTOMS USE ONLY
Country of origin for customs purposes*: United Kingdom
* Defined as the country where the goods have been produced and/or
sufficiently processed to be classed as originating from the country of
supply, for example a change of state such as freeze-drying.
Net weight: 0.0114g per ampoule
Toxicity Statement: Non-toxic
Veterinary certificate or other statement if applicable.
Attached: No
17. CERTIFICATE OF ANALYSIS
NIBSC does not provide a Certificate of Analysis for WHO Biological
Reference Materials because they are internationally recognised primary
reference materials fully described in the instructions for use. The
reference materials are established according to the WHO
Recommendations
for
the
preparation,
characterization
and
establishment of international and other biological reference standards
http://www.who.int/bloodproducts/publications/TRS932Annex2_Inter_biol
efstandardsrev2004.pdf (revised 2004). They are officially endorsed by
the WHO Expert Committee on Biological Standardization (ECBS) based
on the report of the international collaborative study which established
their suitability for the intended use.
National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, EN6 3QG
WHO International Laboratory for Biological Standards, UK Official Medicines Control Laboratory
T +44 (0)1707 641000
nibsc.org
Page 2 of 4
Suggested method for calibration of secondary standards
This method is based on the publication Branford et al [Blood 2008;112(8):3330-8] but uses Grubbs‟ test for the
identification of outliers and regression analysis to determine if there is a trend in bias.
The method will allow you to assign International Scale (IS) values to your materials. Either of the control genes BCR,
ABL or GUSB can be used for this calculation and the corresponding assigned IS values for each of the four panel
members are shown below;
% BCR-ABL / ABL
% BCR-ABL / BCR
% BCR-ABL / GUSB
BCR-ABL 1 08/192
0.0118
0.0195
0.0071
BCR-ABL 2 08/194
0.1112
0.1753
0.0749
BCR-ABL 3 08/196
1.1672
1.6627
0.8295
BCR-ABL 4 08/198
10.7469
16.3129
10.1235
1. Order 5 panels of materials (i.e. 20 ampoules).
2. On 5 different days, take one complete panel of 4 materials, reconstitute them according to the Instructions For
Use supplied and extract the RNA.
3. Using these samples and your own secondary reagents, make two batches of cDNA on different days and test
these in duplicate/triplicate by RQ-PCR on separate days i.e. testing is over 10 days.
4. You should now have 40 data points from the primary standard. Exclude all failed reactions, but if you have less
than 6 data points for each level of BCR-ABL then start again from step 3.
5. Determine the conversion factor using the following procedure and using the example on the following page as
a guide;
a) Convert IS and testing lab % BCR-ABL/control gene values to log10
b) For each sample tested, calculate the mean of the assigned IS value and the testing lab result:
m = ½ (log10 assigned IS value + log10 testing lab result).
c) For each sample tested, calculate the difference (d) between the assigned log 10 IS value and the testing
lab log 10 value:
d = log10 assigned IS value - log10 testing lab result.
d) Calculate the mean (Md) and the standard deviation (Sd) of the differences (d).
e) For each sample tested, calculate the outlier test statistic:
f)
z = | Md – d | / Sd.
If the maximum value of z exceeds the relevant value in the table of critical values for Grubbs‟ outlier test
(overleaf), delete all data for the corresponding sample and repeat steps d) and e).
g) Perform regression analysis of differences (d) against mean values (m). Assignment of IS values to your
materials will be valid ONLY if no significant trend is observed (p>0.05).
h) The correction factor (analogous to a laboratory-specific conversion factor) is defined as the anti-log of the
mean difference i.e. antilog (Md).
6. Multiply results obtained with your secondary standard by the correction factor to obtain results on the
International Scale.
National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, EN6 3QG
WHO International Laboratory for Biological Standards, UK Official Medicines Control Laboratory
T +44 (0)1707 641000
nibsc.org
Page 3 of 4
IS
Ampoule
1
BCR-ABL/ABL %
IS scale
Testing lab
0.0118
0.0261
0.0118
0.0177
0.0118
0.0261
0.0118
0.0290
0.0118
0.0119
0.0118
0.0188
0.0118
0.0160
0.0118
0.0135
0.0118
0.0161
0.0118
0.0185
0.1112
0.2311
0.1112
0.1973
0.1112
0.1892
0.1112
0.1839
0.1112
0.1842
0.1112
0.1894
0.1112
0.2204
0.1112
0.2336
0.1112
0.2389
0.1112
0.1611
1.1672
2.0203
1.1672
2.2358
1.1672
2.2934
1.1672
2.2099
1.1672
1.8396
1.1672
1.6579
1.1672
1.8037
1.1672
2.5416
1.1672
2.6970
1.1672
2.2330
10.7469
16.3350
10.7469
14.1758
10.7469
17.1538
10.7469
15.9942
10.7469
15.6497
1
2
3
4
5
2
1
2
3
4
5
3
1
2
3
4
5
4
1
2
3
10.7469
10.7469
10.7469
10.7469
10.7469
4
5
IS scale
-1.9281
-1.9281
-1.9281
-1.9281
-1.9281
-1.9281
-1.9281
-1.9281
-1.9281
-1.9281
-0.9539
-0.9539
-0.9539
-0.9539
-0.9539
-0.9539
-0.9539
-0.9539
-0.9539
-0.9539
0.0671
0.0671
0.0671
0.0671
0.0671
0.0671
0.0671
0.0671
0.0671
0.0671
1.0313
1.0313
1.0313
1.0313
1.0313
17.5578
18.2664
18.8170
17.8710
16.5343
Log10 transformed BCR-ABL/ABL %
Testing lab
Mean (m)
Difference (d) Outlier test
-1.5826
-1.7553
-0.3456
1.42
-1.7514
-1.8398
-0.1767
0.62
-1.5836
-1.7558
-0.3446
1.41
-1.5381
-1.7331
-0.3900
1.96
-1.9242
-1.9262
-0.0039
2.70
-1.7265
-1.8273
-0.2017
0.32
-1.7960
-1.8620
-0.1321
1.15
-1.8704
-1.8993
-0.0577
2.05
-1.7934
-1.8608
-0.1347
1.12
-1.7318
-1.8300
-0.1963
0.38
-0.6362
-0.7951
-0.3177
1.08
-0.7049
-0.8294
-0.2490
0.26
-0.7231
-0.8385
-0.2308
0.04
-0.7354
-0.8446
-0.2185
0.11
-0.7348
-0.8443
-0.2191
0.10
-0.7225
-0.8382
-0.2314
0.04
-0.6567
-0.8053
-0.2972
0.84
-0.6316
-0.7928
-0.3223
1.14
-0.6218
-0.7878
-0.3321
1.26
-0.7930
-0.8734
-0.1609
0.81
0.3054
0.1863
-0.2383
0.13
0.3494
0.2083
-0.2823
0.66
0.3605
0.2138
-0.2933
0.79
0.3444
0.2058
-0.2772
0.60
0.2647
0.1659
-0.1976
0.36
0.2196
0.1434
-0.1524
0.91
0.2562
0.1617
-0.1890
0.47
0.4051
0.2361
-0.3380
1.33
0.4309
0.2490
-0.3637
1.64
0.3489
0.2080
-0.2817
0.65
1.2131
1.1222
-0.1818
0.55
1.1515
1.0914
-0.1203
1.30
1.2344
1.1328
-0.2031
0.30
1.2040
1.1176
-0.1727
0.66
1.1945
1.1129
-0.1632
0.78
1.0313
1.0313
1.0313
1.0313
1.0313
1.2445
1.2617
1.2746
1.2521
1.2184
1.1379
1.1465
1.1529
1.1417
1.1248
-0.2132
-0.2304
-0.2433
-0.2209
-0.1871
0.18
0.03
0.19
0.08
0.49
N=40 so exclude sample if z> 3.04
Bias plot:
0.5
Mean difference (M d ):
-0.2278
Standard deviation (S d ):
0.0829
N:
0.4
40
0.3
Correction factor:
Dif f erence
0.2
0.5918
0.1
0.0
Critical vales for Grubbs‟ outlier test.
[Grubbs F: Technometrics 1969;11(1):1-21]
Critical value
N
N
Critical value
3
1.15
22
2.76
4
1.48
23
2.78
5
1.71
24
2.8
6
1.89
25
2.82
7
2.02
26
2.84
8
2.13
27
2.86
9
2.21
28
2.88
10
2.29
29
2.89
11
2.34
30
2.91
12
2.41
31
2.92
13
2.46
32
2.94
14
2.51
33
2.95
15
2.55
34
2.97
16
2.59
35
2.98
17
2.62
36
2.99
18
2.65
37
3
19
2.68
38
3.01
20
2.71
39
3.03
21
2.73
40
3.04
-0.1
-0.2
-0.3
-0.4
-0.5
-2.5
-2.0
-1.5
-1.0
-0.5
0.0
0.5
1.0
1.5
Mean
SUMMARY OUTPUT
Regression Statistics
Multiple R
0.0234
R Square
0.0005
Adjusted R Square
-0.0258
Standard Error
0.0839
Observations
40
ANOVA
df
Regression
SS
MS
F
1
0.0001
0.0001
Residual
38
0.2677
0.0070
Total
39
0.2679
Coefficients
Standard Error
t Stat
Significance F
0.0208
P-value
0.8860
Lower 95%
Upper 95%
Lower 95.0% Upper 95.0%
Intercept
-0.2284
0.0139
-16.4813
0.0000
-0.2564
-0.2003
-0.2564
-0.2003
X Variable 1
-0.0017
0.0120
-0.1444
0.8860
-0.0260
0.0225
-0.0260
0.0225
National Institute for Biological Standards and Control, Potters Bar, Hertfordshire, EN6 3QG
WHO International Laboratory for Biological Standards, UK Official Medicines Control Laboratory
T +44 (0)1707 641000
nibsc.org
Page 4 of 4