Value Assignment of the Proposed 6 th International

WHO/BS/09.2116

ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 19 to 23 October 2009

Value Assignment of the Proposed 6th

International Standard for

Blood Coagulation Factor VIII and von Willebrand Factor in

Plasma, Human (07/316)

Anthony R. Hubbard

1,3, Michelle Hamill

2, Malinee Beeharry

1, Sally Bevan

1 &

Alan B. Heath

1 Haemostasis Section, Biotherapeutics Group, and

2 Biostatistics Section,

National Institute for Biological Standards and Control,

Blanche Lane, South Mimms,

Potters Bar, Herts EN6 3QG, UK

3Principal Investigator

© World Health Organization 2009

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WHO/BS/09.2116

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Summary

Background and aim The current WHO 5

th International Standard (5

th IS) Factor VIII/von Willebrand factor, plasma

(02/150) is used for the potency estimation of 5 analytes in plasma (Factor VIII:coagulant

activity; Factor VIII:antigen, VWF:antigen, VWF:ristocetin cofactor, VWF:collagen binding).

The 5th

IS provides a common traceable source of calibration to promote harmonisation between

secondary working plasma standards and is primarily used for the estimation of these analytes in

relation to the diagnosis of disease (eg. haemophilia A and von Willebrand disease). Stocks of

the WHO 5th

IS are extremely low and a replacement preparation is required. This report

describes the value assignment of the proposed WHO 6th

IS by assay relative to the WHO 5th

IS

and locally collected normal plasma pools.

Methods and Results Value assignment of the replacement WHO IS has been achieved through an international multi-

centre study involving 44 laboratories from 14 countries.

Estimates for FVIII:C, relative to the 5th

IS, by all three methods (one-stage clotting, two-stage

clotting, chromogenic), showed good inter-laboratory agreement with overall geometric

coefficient of variation (GCV) of 4.06% and a combined mean value of 0.68 IU per ml (n=52).

A significant difference was found between estimates relative to the fresh (mean 0.57 IU/ml) and

the frozen normal pools (mean 0.72 IU/ml). Estimates relative to the fresh normal plasma pools

were significantly lower (p<0.0001) and more variable (GCV 13.9%) than estimates relative to

the 5th

IS (GCV 4.06 %). In consideration of the known stability of the 5th

IS, the variability of

estimates relative to the fresh normal pools, and in order to maintain continuity between the 5th

and 6th

IS it is proposed that the 6th

IS be assigned the mean value obtained relative to the 5th

IS

of 0.68 IU per ampoule.

Estimates for FVIII:antigen, relative to the 5th

IS, showed good agreement between laboratories

with GCV of 4.24% and an overall mean of 1.04 IU per ml (n=10). These estimates were

significantly different from the estimates obtained relative to the normal plasma pools (mean

0.93 IU per ml) which were also more variable (inter-laboratory GCV 10.5 %). In the interest of

continuity it is proposed that the 6th


IS

of 1.04 IU per ampoule.

Estimates for VWF:antigen, relative to the 5th

IS, showed excellent agreement between the

ELISA and immuno-turbidimetric methods and produced an overall mean of 1.00 IU/ml (n=31)

with low inter-laboratory variability (GCV 4.29%). Estimates relative to the normal pools were

significantly lower (mean 0.89 IU/ml) and more variable (GCV 12.92%). In consideration of the

variability of the estimates relative to the normal pools and in order to maintain continuity of the

IU it is proposed that the 6th


IS of 1.00

IU per ampoule.

Estimates for VWF:RCo relative to the 5th

IS by the aggregometric methods were less variable

(GCV 6.52%) but not significantly different to estimates by visual agglutination (GCV 12.0%).

Combination of all estimates relative to the 5th

IS produced a mean of 0.87 IU/ml (n=28) with

acceptable inter-laboratory variability (GCV) of 8.13 %. In contrast the estimates relative to the

normal pools were much more variable between laboratories with GCVs from 10 – 28% and this

hindered meaningful statistical analysis comparing fresh and frozen pools. There was no

significant difference between the overall combined estimates relative to the 5th

IS (0.87 IU/ml,

WHO/BS/09.2116

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n=28) and the normal pools (0.90 IU/ml, n=28). It is proposed that the 6th

IS be assigned the

mean value obtained relative to the 5th

IS of 0.87 IU per ampoule.

Estimates for VWF:collagen binding, relative to the 5th

IS were associated with very good

agreement between estimates obtained using type III and type I/III mix collagen reagents and

produced an overall mean of 1.03 IU/ml (n=21) with inter-laboratory variability (GCV) of 5.80

%. Estimates relative to the normal plasma pools were associated with greater inter-laboratory

variability (GCV 12.24%, mean 0.99 IU/ml, n=20) but were not significantly different to

estimates vs the 5th

IS. It is proposed that the 6th

IS be assigned the mean value obtained relative

to the 5th

IS of 1.03 IU per ampoule.

Estimates for VWF:collagen binding in the 1st IS VWF Concentrate relative to the 5

th IS

indicated no significant difference between the use of type III and type I/III mix collagen

reagents. The overall combination of estimates produced a mean value of 10.74 IU/ml with

inter-laboratory variability (GCV) of 19.5%. These results indicate improved harmonisation

between laboratories compared to the original attempt to assign a value to the 1st IS VWF

Concentrate in 2000 and may be an indication that it will be possible to assign a value for

VWF:CB to the proposed 2nd

IS VWF Concentrate in 2010.

Stability of the proposed 6th

IS has been assessed through an accelerated degradation study after

storage of ampoules at elevated temperatures (+4, +20, +37, +45 °C) for up to 9 months. The

results from the initial testing are very encouraging and indicate that all analytes in the proposed

6th

IS are extremely stable when stored at the bulk storage temperature of -20 oC with the mean

predicted losses per year of 0.074% for FVIII:C 1-stage, 0.146% for FVIII:C chromogenic,

0.049% for FVIII:antigen, 0.00% for VWF:antigen, 0.022% for VWF:ristocetin cofactor and

0.002% for VWF:collagen binding.

Proposal It is proposed that the preparation coded 07/316 be accepted as the WHO 6

th International

Standard Factor VIII/von Willebrand Factor, Plasma with the following assigned values

estimated relative to the 5th

IS FVIII/VWF Plasma (02/150):

Factor VIII:C 0.68 IU per ampoule

Factor VIII:Antigen 1.04 IU per ampoule

VWF:Antigen 1.00 IU per ampoule

VWF:Ristocetin cofactor 0.87 IU per ampoule

VWF:Collagen binding 1.03 IU per ampoule

Responses from study participants and SSC/ISTH sub-committees

on Factor VIII/IX and von Willebrand factor Responses have been obtained from all of the 44 participants and all have approved of the

proposed value assignments for the 5 analytes. Additional comments were received relating to

errors or omissions in the description of methodologies (Appendix 3) or the names of

participants (Appendix 2) and these have been corrected. Several participants commented on the

limited value of including local normal pools and questioned if this comparison could be

excluded from future studies. One participant commented on the proposed value for FVIII:C

(0.68 IU per ampoule) and indicated they would have preferred a value around 1.00 IU per

ampoule. The participant was informed that the processing of the lyophilised standard together

with the use of citrate-based anticoagulant were the main reasons for the lower FVIII:C value;

similar values have been found for previous standards and have not affected their validity for

WHO/BS/09.2116

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FVIII:C estimation. One participant emphasised the need to exclude estimates for VWF:RCo

obtained using an immuno-turbidimetric method since this method is based on antigen

determination rather than platelet agglutination; results from this method have not been included

in the combined mean values for value assignment.

Responses have also been sought from experts associated with the Factor VIII/Factor IX and von

Willebrand factor sub-committees of SSC/ISTH. All responses were in favour of the proposal

and the following comments were also noted:

1) One comment suggested the number of laboratories using the immuno-turbidimetric method

for VWF:RCo should be increased in future studies, however, this is not in agreement with

current opinion (or the participant comment above) which does not support the inclusion of

surrogate measurements of VWF function based on antibodies directed against the Glycoprotein

1b binding site on VWF.

2) A comment expressed concern over the comparison of lyophilised and fresh plasma, in the

estimation of VWF:RCo, due to the increased opacity of lyophilised plasma which made it

difficult to “set up” aggregometers. The response to this comment attempted to provide

reassurance that the design of the study, which required the testing of 3 dilutions of all samples,

would allow the detection of such an effect by non-parallelism of the dose-response relationships.

Since no significant non-parallelism was detected between lyophilised plasma and fresh plasma

samples it was concluded that the comparisons were valid. Any such effect would have no

impact on comparisons of the 5th

IS and the proposed 6th

IS which are both lyophilised.

3) One comment recommended that normal plasma pools should be retained in future

collaborative studies as a “reality check” to provide contact with the “real world”.

The proposal to accept the preparation coded 07/316 as the 6th

IS Factor VIII/VWF Plasma with

values assigned relative to the 5th

IS was discussed at the WHO-ISTH Liaison Group Meeting

on 12 July 2009 and subsequently endorsed at the SSC Business Meeting, held in Boston MA,

on 15 July 2009.

Introduction and objectives of the study Stocks of the current WHO 5

th IS Factor VIII/VWF Plasma (02/150) will be exhausted by the

end of 2009 and the primary objective of this study is the assignment of values to the

replacement preparation, the proposed WHO 6th

IS Factor VIII/VWF Plasma (07/316). Values

will be assigned for the same five analytes attached to the 5th

IS, namely, factor VIII coagulant

activity (FVIII:C), factor VIII antigen (FVIII:Ag), von Willebrand factor antigen (VWF:Ag),

von Willebrand factor Ristocetin Cofactor activity (VWF:RCo) and von Willebrand factor

collagen binding (VWF:CB). Value assignment is achieved by the assay of the proposed WHO

6th

IS relative to the current WHO 5th

IS and locally collected normal plasma pools. The former

comparison provides continuity of the value of the International Unit (IU) between successive

standards and the latter comparison provides a check on the value of the IU relative to the

original definition of the “plasma unit” as the amount of analyte in 1 ml of pooled normal plasma.

In accordance with WHO principles the objective is to assign a single value for each analyte

which is valid for use with all current methodologies. This standard is intended to be used in the

in vitro diagnostics field and relates to category 4 of the principles set out in ISO 17511.

A secondary objective is to obtain further data on the inter-laboratory variability of VWF:CB

estimates for the WHO 1st IS VWF Concentrate (00/514) relative to the WHO 5

th IS plasma. A

previous attempt to assign a value for VWF:CB to the WHO 1st Concentrate, in 2000, was

unsuccessful due to extremely large variability between laboratories and methods. The

information obtained in the present study will indicate the current status of VWF:CB testing and

WHO/BS/09.2116

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will help in formulating the strategy for replacement of the WHO 1st IS VWF Concentrate which

is scheduled for 2010.

Samples included in the collaborative study

Sample A: WHO 5th

IS Factor VIII/VWF Plasma (02/150) The WHO 5

th IS was established in 2003 with assigned values for FVIII:C (0.68 IU/ampoule),

FVIII:Ag (0.94 IU/ampoule), VWF:Ag (0.91 IU/ampoule), VWF:RCo (0.78 IU/ampoule) and

VWF:CB (0.94 IU/ampoule) (Hubbard and Heath, 2004).

Sample B: Proposed WHO 6th

IS Factor VIII/VWF Plasma (07/316) The proposed WHO 6

th IS was prepared from a pool of 80 donations from normal healthy donors

(UK National Blood Service) collected by conventional venepuncture into citrate-phosphate-

dextrose-adenine anticoagulant. Each donation was checked and found negative for HBsAg,

anti-HIV-1 and -2, anti-HCV, HCV-RNA (mini-pool testing) and syphilis. Each donation

underwent leuco-filtration before being centrifuged twice to remove all cellular components.

The units were then frozen and stored at -70oC. Plasma was thawed on the day of filling, pooled

and then buffered by the addition of HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-

ethanesulfonic acid]) to a final concentration of 40 mmol/l. One ml of the pooled plasma was

dispensed into each of approximately 20,000 ampoules. Freeze-drying was performed in

accordance with the conditions required for International standards (Campbell 1974). Details of

the filling procedure and the characteristics of the lyophilised preparation are given in the table

below. A comparison of the multimeric profile of von Willebrand factor in the proposed 6th

IS,

the current 5th

IS and frozen normal plasma has revealed a similar distribution for all three

preparations (Figure 1).

WHO/BS/09.2116

Page 6

DETAILS OF CANDIDATE FILL (07/316)

Presentation

sealed glass DIN ampoules

Excipients/additives

Recovered normal human plasma with additive HEPES (N-[2-

Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid]) to final

concentration of 0.04 mol/l

Liquid filling weight (g)

Mean 1.1056 g

(range 1.1010 g – 1.1095 g)

Coefficient of variation of the

liquid fill (%)

0.118 % based on 786 check-weight ampoules

Residual moisture after

lyophilisation (%)

Mean 0.30%, CV 11.8% (n = 12)

Dry weight (mg)

Mean 93.4 mg, CV 0.39% (n = 6)

Headspace oxygen (%)

Mean 0.13%, CV 57.4% (n = 12)

Reconstitution volume and fluid

1.0 ml distilled water

Number of ampoules in stock

19,300

Manufacturing site

NIBSC, Potters Bar, UK

Custodian

NIBSC, Potters Bar, UK

Storage temperature

-20 °C

Sample C: WHO 1st IS von Willebrand Factor Concentrate (00/514)

This standard was established in 2001 and was included only in the VWF:CB assays. This

standard has no assigned value for VWF:CB (Hubbard et al, 2002).

Sample N: Locally collected normal plasma pools Participants were requested to prepare two fresh normal plasma pools N1 and N2 using at least 16

different donors in total according to the instructions given in the study protocol (Appendix 1).

Fresh normal pools were preferred for the estimation of FVIII:C and VWF:RCo whereas frozen

pools were acceptable for the testing of other analytes. For FVIII:C estimates 11 laboratories

prepared fresh pools (total 227 donors), 16 laboratories prepared frozen pools (total exceeding

4,700 donors), 2 laboratories used a mixture of fresh and frozen pools (analysed as frozen pools)

(total 28 donors) and one laboratory used a commercial lyophilised normal plasma pool. For the

VWF:RCo estimates 9 laboratories prepared fresh pools (total 146 donors), 21 laboratories

prepared frozen pools (total exceeding 4,400 donors), 2 laboratories used a mixture of fresh and

WHO/BS/09.2116

Page 7

frozen pools (analysed as frozen pools) (total 26 donors) and one laboratory used a commercial

lyophilised normal plasma pool.

Participants Samples were despatched in September 2008 to 44 laboratories in 14 different countries. The

participants comprised 20 clinical laboratories, 21 manufacturers’ laboratories and 3 regulators

(Appendix 2). Results were received from all laboratories. Participating laboratories have been

assigned code numbers to retain confidentiality in the report.

Assay methods and study design Participants were asked to follow their routine assay methodology as far as possible within the

specified assay design. Most laboratories assayed more then one analyte in the study. A

summary of the assay methods used in the study and the number of estimates is given in the

following table: Analyte Total

data sets

Methods

VIII:C

53

1-stage clotting (31), 2-stage clotting (1), chromogenic (21)

VIII:Ag

11

ELISA

VWF:Ag

31

ELISA (21), immuno-turbidimetric (10)

VWF:RCo

29

aggregometry (21), visual agglutination (7), immuno-turbidimetric (1)

VWF:CB

21

ELISA-type with detection using anti-VWF conjugate

Details of the methods used by the individual laboratories are given in Appendix 3.

Participants were requested to carry out four assays for each analyte using fresh ampoules of A

and B for each assay (and fresh ampoules of sample C for each VWF:CB assay) (Appendix 1).

The four assays were split into two sessions of two assays each. It was requested that a different

normal plasma pool (N1, N2) was included in each session if possible. Participants were

requested to follow a balanced assay design in which three different dilutions of each assay

material were tested in replicate wherever possible. Since most laboratories performed more

than one assay method on the same ampoules it was specified that assays of FVIII:C and

VWF:RCo must be carried out on freshly reconstituted ampoules whereas assays for FVIII:Ag,

VWF:Ag and VWF:CB could be carried out on frozen aliquots if this was unavoidable.

Statistical analysis All assays were analysed as multiple parallel line bioassays comparing response to log

concentration (Finney 1978). Linear and parallel response lines are required for this type of

analysis. If necessary, the responses were log transformed to achieve this. The parallelism of

the assays was assessed by comparing the slopes of the dose-responses across the assays.

For each assay the following potency estimates were calculated:

• proposed 6th

IS FVIII/VWF Plasma (B) vs 5th

IS FVIII/VWF Plasma (A) using the

assigned values for the 5th

IS FVIII/VWF Plasma

• proposed 6th

IS FVIII/VWF Plasma (B) vs local normal plasma pools (N) using an

arbitrary value of 1.0 unit per ml for the local normal plasma pools.

WHO/BS/09.2116

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• 1st IS VWF Concentrate (C) vs 5

th IS FVIII/VWF Plasma (A) and vs local normal plasma

pools (N) (VWF:CB assays only).

Estimates of FVIII:C and VWF:RCo were also sub-divided into groups using fresh or frozen

normal plasma pools. This is particularly important for FVIII:C estimates where levels can be

affected by freeze-thawing. No sub-division was made for FVIII:Ag, VWF:Ag and VWF:CB.

Estimates for any analyte, where the normal pool was a lyophilised preparation (Laboratory 10)

were not included in the calculation of means.

Combined potency estimates for each laboratory were obtained by taking unweighted geometric

means of results from all assays. Overall combined estimates were obtained by taking

unweighted geometric means of the mean results from the different laboratories. Where a

laboratory performed more than one assay method, the results for each method were analysed as

if from separate laboratories. Differences in potency estimates between laboratories (outlier

detection) were assessed using a Duncan's multiple range test (Duncan 1975). Intra- and inter-

laboratory variability is expressed as the geometric coefficient of variation (GCV%) (Kirkwood

1979).

Some estimates of VWF:RCo by visual agglutination methods (titres) were not suitable for

parallel line analysis. The results returned by the participants have been included in the tables

with an indication that raw data analysis was not carried out by NIBSC.

Results and conclusions

General comments on statistical analysis The parallelism of the proposed 6

th IS FVIII/VWF Plasma and the 1

st IS VWF Concentrate

against the 5th

IS and the normal plasma pools was assessed by calculating the ratio of the slopes

of the dose-response relationships and by visual inspection of all individual assays. The overall

mean ratios of slopes for all assay methods ranged from 96.5% - 100% for estimates against the

5th

IS and from 97.3% - 105.7% for estimates against the normal plasma pools. All assays were

considered valid for inclusion in the analysis except for the exclusions detailed below. In some

cases it was necessary to exclude single data points at the extreme ends of the dose-response

relationships in order to achieve linearity. The following sets of data were excluded from the

study:

- VWF:CB estimates of the 1st IS VWF Concentrate: exclusion of 4 assays by laboratories 3, 20

and 32 due to poor dose-response and non-parallelism of the 1st IS VWF Concentrate with the 5

th

IS; exclusion of single assays by laboratories 15, 30, 44 due to possible dilution errors causing

either non-parallelism and/or non-linearity.

- VWF:Ristocetin Cofactor: exclusion of two assays for the proposed 6th

IS vs the normal pools

by laboratory 28c due to lack of dose-response of the normal pools.

- VWF:Antigen: exclusion of two assays for the proposed 6th

IS vs the normal pools by

laboratory 38 due to lack of dose-response of the normal pools.

The following estimates were identified as “outlying" results and excluded from the calculation

of the overall mean values:

- FVIII chromogenic Lab 22 for the proposed 6th

IS vs the 5th

IS

- FVIII antigen Lab 27 for the proposed 6th

IS vs the 5th

IS

- VWF collagen binding Lab 24 for the proposed 6th

IS vs the normal pools

WHO/BS/09.2116

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Intra- and Inter-laboratory variability of estimates A summary of intra-laboratory variability for estimates of the proposed 6

th IS by all methods is

given in Table 1. For all methods there was a higher percentage of laboratories with GCVs

below 5% for estimates relative to the 5th

IS than for estimates relative to the normal pools.

Similarly there were a higher percentage of laboratories with GCVs greater than 10% for

estimates relative to the normal pools. The larger intra-laboratory variability for estimates

relative to the normal pools is not surprising when we consider that most of the laboratories used

two different normal pools for their assays. Considering the intra-laboratory variability for

estimates relative to the 5th

IS it is apparent that the lowest variability (eg. most labs with GCVs

<5%) is associated with the estimates of FVIII:C, VWF:Ag and FVIII:Ag whereas the highest

variability was associated with estimates of VWF:CB. The GCVs for most of the VWF:CB

estimates (53%) fell in the range of 5 – 10%. The VWF:RCo method is generally regarded as

one of the most variable and this is supported by the finding that the GCV exceeded 10% in 29%

of laboratories.

A summary of inter-laboratory variability for estimates of the proposed 6th

IS by all methods is

given in Table 2. It is not surprising that the variability relative to the normal pools is greater

than the variability relative to the 5th

IS, for all methods, considering that each laboratory used a

different normal pool. Estimates of inter-laboratory variability based on assays relative to the 5th

IS indicated very good agreement with GCVs around 6% and below for sub-set methods and

overall combinations for FVIII:C, FVIII:antigen, VWF:antigen and VWF:CB. VWF:RCo

estimates by aggregometry also displayed low inter-laboratory variability (GCV 6.52%) whereas

the semi-quantitative estimates by visual agglutination were associated with the largest

variability (GCV 12.00%) and this has influenced the estimation of overall variability for

VWF:RCo of 8.13%.

Factor VIII:C in the proposed 6th

IS FVIII/VWF Plasma (Sample B) The mean results from the individual laboratories together with the means from each method are

given in Table 3 and Figures 2a and 2b.

a) estimates vs the 5th

IS FVIII/VWF Plasma (A) One estimate was identified as an outlier (chromogenic estimate from Lab 22) and this was

excluded from the calculation of overall mean values. There was very good inter-laboratory

agreement within the 1-stage and chromogenic methods with GCVs of 2.80% (n=31) and 3.57%

(n=20) respectively. The mean values from each method were also very similar at 0.67 IU/ml

(1-stage), 0.72 IU/ampoule (2-stage) and 0.70 IU/ampoule (chromogenic), however, there was a

significant difference between the estimates for the 1-stage and chromogenic methods

(p<0.0001). Combination of potencies from all three methods (52 estimates) gave an overall

mean of 0.68 IU per ml with low inter-laboratory variability (GCV 4.06%).

b) estimates vs the normal plasma pools (N) Inter-laboratory variability (GCV) exceeded 10% within the 1-stage and chromogenic methods

for estimates relative to both fresh and frozen plasma pools. There was good agreement between

the mean values calculated relative to the fresh normal plasma pools for the 1-stage (0.56 IU/ml,

n=11) and chromogenic methods (0.61 IU/ml, n=5). Estimates calculated relative to the frozen

pools were greater for the 1-stage (0.70 IU/ml, n=19) and chromogenic methods (0.76 IU/m,

n=12) and there was a highly significant difference (p<0.0001) between all estimates calculated

relative to the fresh (mean 0.57 IU/m, n=16) and frozen pools (mean 0.72 IU/ml, n=31).

WHO/BS/09.2116

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c) comparison of estimates vs 5th

IS and fresh normal pools Comparing estimates from all laboratories and methods, relative to the 5

th IS (mean 0.68 IU/ml,

n=52), with estimates from all methods, relative to the fresh normal pools (mean 0.57 IU/ml,

n=16) indicated a highly significant difference (p<0.0001). Comparison of estimates from only

those laboratories which used both fresh normal plasma pools and the 5th

IS in the same assays

(paired t test, n=16) also indicated a highly significant difference (p<0.0001).

FVIII:C value assignment: Estimates for FVIII:C relative to the 5th

IS showed good

overall agreement both within and between the three different methods producing an overall

combined mean of 0.68 IU per ml with inter-laboratory variability (GCV) of 4.06%. Although

the mean estimates by the one-stage and chromogenic methods were similar (0.67 and 0.70

IU/ml respectively) there was a significant difference between these estimates and this reflects

the extremely good agreement between laboratories within each method (GCVs 2.80% and

3.57%). In practice the small difference between the mean values supports the calculation of a

valid overall mean value from all estimates from the 3 methods.

Estimates relative to the normal plasma pools were associated with greater inter-laboratory

variability (GCV >10%) as might be expected considering that all laboratories used different

normal pools. Moreover there was a clear and highly significant difference (p<0.0001) between

all estimates calculated relative to the fresh pools (0.57 IU/ml) and the frozen pools (0.72 IU/ml).

This was also observed in the calibration of the 5th

IS and probably reflects the lability of

FVIII:C and loss of activity following freezing and thawing. This finding invalidates the

combination of estimates calculated relative to fresh and frozen pools and limits the calculation

of the overall mean to 16 laboratories which used fresh normal pools and gives a mean value of

0.57 IU per ml (GCV 13.90 %). Comparison of estimates from those laboratories (n=16) which

included both fresh normal pools and the 5th IS in the same assays indicated a highly significant

difference (p<0.0001) with means of 0.68 and 0.57 IU per ml relative to the 5th

IS and the fresh

normal pools respectively.

Assignment of a value for FVIII:C has to reconcile the need for continuity of the unitage

between the 5th

IS and the 6th

IS and also to recognise possible drift of the IU away from the

original "plasma unit". Previously, in the calibration of the 4th

IS there was a 15% discrepancy

between the estimates relative to the 3rd IS (0.60 IU per ml) and the fresh normal pools (0.52 IU

per ml) and an attempt was made to partially rectify the difference by assigning a mean value of

0.57 IU per ampoule to the 4th IS (Hubbard et al, 2001). This correction resulted in a change in

value of the IU for FVIII:C of around 7 – 8% and was a cause of confusion for several years

until it had been assimilated by manufacturers of secondary standards. Despite this correction

the subsequent calibration of the 5th

IS revealed a discrepancy of 11% between estimates relative

to the 4th

IS and the fresh normal pools. In consideration of the extremely good stability for

FVIII:C in the 4th

IS it was accepted that the varying discrepancy was caused by “oscillation” of

the mean FVIII:C in the normal pools between studies rather than by instability of FVIII:C in the

freeze-dried 4th

IS. It was therefore agreed that the 5th

IS should be assigned the consensus mean

value from estimates against the 4th

IS (Hubbard and Heath, 2004). The results in the present

study have presented a similar scenario with the mean value for the proposed 6th

IS relative to the

fresh normal pools (0.57 IU/ml) approximately 16% lower than the mean relative to the 5th

IS

(0.68 IU/ml). This is unlikely to have been caused by instability of the 5th

IS since stability

studies have indicated that no detectable loss of FVIII:C has occurred during its lifetime

(Appendix 4). Based on previous experience with the correction applied to the 4th

IS it is

unlikely that a further re-valuation of the IU would provide a permanent “fix” to reconcile the IU

and the “fresh plasma unit”. It is therefore proposed that continuity of the IU should take

WHO/BS/09.2116

Page 11

precedence and that the 6th

IS should be assigned the mean value calculated relative to the 5th

IS

of 0.68 IU/ml.

Factor VIII: antigen in the proposed 6th

IS FVIII/VWF Plasma (Sample B) All 11 laboratories used ELISA methods to estimate FVIII:Ag (Table 4 and Figures 3a and 3b).

Estimates calculated relative to the 5th

IS showed good inter-laboratory agreement when an

outlying result (Lab 27) was excluded to give a GCV of 4.24% and an overall mean value of

1.04 IU/ml (n=10). Estimates calculated relative to the normal pools were more variable with

inter-laboratory variability (GCV) of 10.53% and an overall mean value of 0.93 IU/ml (n=11).

Estimates calculated relative to the 5th

IS were significantly different to estimates relative to the

normal pools when analysed by unpaired “t” test (p<0.01, n=10 vs n=11) and paired “t” test

(p<0.05, n=10 vs n=10).

FVIII:antigen value assignment: There was very good agreement between laboratories

for estimates relative to the 5th

IS (GCV 4.24%) and a mean value of 1.04 IU/ml was obtained.

Estimates relative to the normal plasma pools were more variable (GCV 10.5%) as expected

from the use of different pools in each laboratory and a mean of 0.93 IU/ml was obtained. The

discrepancy between estimates relative to the 5th

IS and the normal pools in the present study

(11%) is slightly larger than that found in the previous calibration of the 5th

IS (8%) and

probably reflects variability in the normal plasma pools between the two studies. In the interests

of continuity it is proposed that the 6th

IS is assigned the overall mean estimate relative to the 5th

IS of 1.04 IU/ml.

von Willebrand Factor: antigen in the proposed 6th

IS FVIII/VWF Plasma

(Sample B) Twenty-one estimates were obtained using ELISA methods and 10 using immuno-turbidimetric

methods to estimate VWF:Ag (Table 5 and Figures 4a and 4b). There were no significant

differences between the two methods for estimates calculated relative to either the 5th

IS or the

normal plasma pools. Estimates calculated relative to the 5th

IS showed excellent inter-

laboratory agreement within both methods with inter-laboratory variability (GCV) of 5.17% and

1.46 % for the ELISA and immuno-turbidimetric methods respectively. Combining all estimates

relative to the 5th

IS gave an overall mean of 1.00 IU/ml (n=31) with inter-laboratory variability

of 4.29%. Estimates relative to the normal pools showed increased inter-laboratory variability

(overall GCV 12.92%) with an overall mean of 0.89 IU/ml (n=30). There was a highly

significant difference (p<0.0001) between the estimates calculated relative to the 5th

IS and the

normal plasma pools.

VWF:Antigen value assignment: Good agreement was found between the two methods

for estimates of the proposed 6th

IS vs the 5th

IS and for the proposed 6th

IS vs the normal pools.

However, there was a significant difference between estimates of the proposed 6th

IS vs the 5th

IS

(mean 1.00 IU/ml) and the normal pools (mean 0.89 IU/ml). This represents an 11%

discrepancy between the IU and the “plasma unit” and is much larger than that found in the

calibration of the previous 5th

IS where there was only a 2% difference, however in the earlier

calibration of the 4th

IS there was a 12% difference (Hubbard, 2006). Considering the good

stability of VWF:antigen in the lyophilised standards it is once again most likely that the

discrepancies are caused by variability in the normal pools between studies and decisions to

adjust the value of the IU should be undertaken with caution. It is proposed that continuity of the

IU should once again take precedence and it is recommended that the 6th

IS is assigned the mean

value relative to the 5th

IS of 1.00 IU per ml.

WHO/BS/09.2116

Page 12

von Willebrand Factor: Ristocetin Cofactor in the proposed 6th

IS FVIII/VWF

Plasma (Sample B) Twenty-one estimates were obtained using aggregometric methods and 7 estimates were

obtained using visual agglutination methods (Table 6 and Figures 5a and 5b). One laboratory

used an immuno-turbidimetric method for VWF activity and this result was not included in the

calculation of overall means. Estimates calculated relative to the 5th

IS using the aggregometric

methods gave a mean value of 0.86 IU/ml (n=21) and good agreement between laboratories

(GCV 6.52%); these estimates were not significantly different to the estimates obtained relative

to the 5th

IS using visual agglutination methods (mean 0.90 IU/ml, n=7). Estimates calculated

relative to the normal pools were much more variable with inter-laboratory GCVs ranging from

10 to 28%. There was no significant difference between the use of fresh and frozen pools for

estimates obtained using aggregometric methods, however, there was a significant difference

(p<0.05) between fresh (mean 0.80 IU/ml, n=8) and frozen pools (mean 0.94 IU/ml, n=20) when

estimates from all methods where included (excluding the immuno-turbidimetric method).

There was no significant difference between all estimates relative to the 5th

IS (mean 0.87 IU/ml,

n=28) and all estimates relative to the normal pools (mean 0.90 IU/ml, n=28).

VWF:Ristocetin cofactor value assignment: Estimates calculated relative to the 5th

IS

using aggregometric methods showed low inter-laboratory variability (GCV 6.52%) whereas

there was larger variability for the estimates based on visual agglutination (GCV 12.0%). This is

not surprising since the visual agglutination method is a semi-quantitative procedure with a

subjective aspect to end-point detection. However, combination of all estimates relative to the

5th

IS provided a valid mean of 0.87 IU/ml with acceptable overall inter-laboratory variability

(GCV) of 8.13 %. In contrast the estimates relative to the normal pools were much more

variable between laboratories (GCVs 10 – 28%) and this has compromised the conclusions from

statistical analysis. The variability of the method coupled with the variability between the

different normal pools has obscured any meaningful conclusions regarding the use of fresh or

frozen pools or agreement between methods relative to the normal pools. It is therefore not

possible to consider the estimates relative to the normal pools in the proposed assigned value for

the 6th

IS, except to note that the combined estimates relative to the normal pools (mean 0.90

IU/ml) are not significantly different to the combined estimates relative to the 5th

IS (mean 0.87

IU/ml). It is proposed that the 6th

IS is assigned the mean value relative to the 5th

IS of 0.87

IU/ml.

von Willebrand Factor: collagen binding in the proposed 6th

IS FVIII/VWF

Plasma (Sample B) Twenty one estimates were obtained using a range of different collagen reagents (Table 7 and

Figures 6a and 6b). There were no significant differences between estimates obtained with the

type III collagen reagents and those using type I and type I/III mix reagents when calculated

relative to the 5th

IS or the normal plasma pools. Estimates calculated relative to the 5th

IS, were

associated with very good inter-laboratory agreement (GCV 5.8%) with an overall mean of 1.03

IU per ml (n=21). Analysis of estimates calculated relative to the normal plasma pools revealed

one outlier (Lab 24) which was not included in the calculation of the overall means. Estimates

relative to the normal pools were more variable (GCV 12.24%, n=20) with an overall mean of

0.99 IU per ml, however, these estimates were not significantly different to the estimates

calculated relative to the 5th

IS.

VWF:collagen binding value assignment: There was good agreement between the use

of different collagen reagents and between the estimates relative to both the 5th

IS (mean 1.03

WHO/BS/09.2116

Page 13

IU/ml, GCV 5.80 %) and the normal pools (mean 0.99 IU/ml, GCV 12.24%). These results

provide a robust basis for the proposal that the 6th

IS is assigned the mean value relative to the 5th

IS of 1.03 IU/ml.

von Willebrand Factor: collagen binding in the 1st IS VWF Concentrate (C)

Eighteen estimates were obtained using a range of different collagen reagents (Table 8 and

Figures 7a and 7b). There were no significant differences between estimates obtained with the

type III collagen reagents and those using type I and type I/III mix reagents when calculated

relative to the 5th IS or the normal plasma pools. Estimates calculated relative to the 5th

IS, were

slightly less variable between laboratories (GCV 19.51%, mean 10.74 IU/ml, n=18) and not

significantly different to estimates relative to the normal pools (GCV 25.39%, mean 10.75 IU/ml,

n=18).

Comments on VWF:collagen binding estimates for the 1st IS VWF Concentrate:

The 1st IS VWF Concentrate was included in the present study to provide further information on

the inter-laboratory variability associated with the comparison of concentrate vs plasma samples

prior to the collaborative study to calibrate the proposed 2nd

IS VWF Concentrate which is

scheduled for Autumn 2009. As expected the inter-laboratory variability for estimates of the 1st

IS Conc vs the 5th

IS Plasma (GCV 19.5%) was greater than estimates of the proposed 6th

IS

Plasma vs the 5th

IS Plasma (GCV 5.8%). These data support the “like vs like” principle in that

a plasma standard is not ideal for the estimation of VWF:CB in concentrates. The previous

attempt to assign a value to the 1st IS Conc by assay relative to the 4

th IS Plasma in 2000 was

unsuccessful due to the extremely large variability between laboratories (GCV 40%) and a

marked difference between estimates obtained using type III and type I/III mix collagen reagents

(Hubbard et al, 2002). In contrast the results from the present study indicated much better

agreement between the use of different collagen types and improved overall inter-laboratory

variability relative to the 5th

IS (GCV 19.5%). These results are very encouraging and, if

reproducible, they show promise for the value assignment of the proposed 2nd

IS VWF

Concentrate for VWF:CB in 2010.

Stability of the proposed WHO 6th

IS FVIII/VWF Plasma

Accelerated degradation study Stability of the proposed 6

th IS has been assessed in an accelerated degradation study which

allows the estimation of predicted loss per year based on the observed loss occurring in ampoules

stored at elevated temperatures (Kirkwood and Tydeman 1984). The study involved 8

laboratories and the testing of 6 analytes (FVIII:C 1-stage, FVIII:C chromogenic, FVIII:antigen,

VWF:antigen, VWF:RCo, VWF:CB). Results were obtained from 2 different laboratories for

each analyte. The residual potencies of ampoules stored at 4, 20, 37 and 45 oC after storage for 7

or 9 months are given in Table 9 expressed as a % relative to ampoules stored at -20oC. Some

laboratories reported problems in the reconstitution and testing of the ampoules stored at +45 °C.

The results from this initial testing are very encouraging and indicate that all analytes in the

proposed 6th

IS are extremely stable when stored at the bulk storage temperature of -20oC with

the mean predicted losses per year of 0.074% for FVIII:C 1-stage, 0.146% for FVIII:C

chromogenic, 0.049% for FVIII:antigen, 0.00% for VWF:antigen, 0.022% for VWF:ristocetin

cofactor and 0.002% for VWF:collagen binding.

Stability after reconstitution Although the Instructions for Use will recommend that assays are performed as soon as possible

after reconstitution it is useful to indicate a suitable period of use. In common with previous

WHO/BS/09.2116

Page 14

WHO Plasma Standards for blood coagulation factors it is recommended that the standard is

transferred, after reconstitution, to a plastic tube in order to prevent activation by the glass

surface of the ampoule. Recommendations for the storage after reconstitution have been limited

to the period of storage on melting ice since local ambient temperature can vary considerably.

Results from two separate tests on the most labile analyte (FVIII:C), performed at NIBSC,

indicated that 99% and 101% respectively, of the starting concentration of FVIII:C was retained

after 3 hours for the freshly reconstituted standard when stored in melting ice in plastic tubes.

This period is sufficient for numerous assays to be performed. The use of frozen aliquots of the

proposed 6th

IS for the estimation of FVIII:C, VWF:RCo and VWF:CB is not recommended.

The use of frozen aliquots for the estimation of FVIII:antigen and VWF:antigen requires local

validation.

References Campbell PJ (1974) International biological standards and reference preparations. 1. Preparation

and presentation of materials to serve as standards and reference preparations. J Biol

Standardisation 2; 249-67

Duncan DB (1975) T-tests and intervals for comparisons suggested by the data. Biometrics 31;

339-359

Finney DJ (1978) Statistical methods in biological assay. 3rd

edition Charles Griffin. London.

Hubbard AR, Rigsby P & Barrowcliffe TW (2001) Standardisation of Factor VIII and von

Willebrand Factor in plasma: calibration of the 4th International Standard. Thrombosis and

Haemostasis 85; 634-638

Hubbard AR, Sands D, Chang AC & Mazurier C (2002) Standardisation of von Willebrand

Factor in therapeutic concentrates: calibration of the 1st International Standard for von

Willebrand Factor Concentrate (00/514). Thrombosis and Haemostasis 88; 380-386

Hubbard AR and Heath AB (2004) Standardization of factor VIII and von Willebrand factor in

plasma: calibration of the WHO 5th International Standard (02/150). Journal of Thrombosis and

Haemostasis 2, 1380-4

Hubbard AR (2006) von Willebrand Factor standards for plasma and concentrate testing. Semin

Thromb Hemost 32; 522-528

Kirkwood TBL (1979) Geometric means and measures of dispersion. Biometrics 35; 908-9

Kirkwood TBL & Tydeman MS (1984) Design and analysis of accelerated degradation tests for

the stability of biological standards II. A flexible computer program for data analysis. J Biol

Standardisation 12; 207-14

Acknowledgements We are very grateful to the participants in this calibration exercise for their considerable efforts

in performing the assays; to Prof Dr Ulrich Budde for the VWF multimer gel electrophoresis; to

the Standards Processing Division (NIBSC) for preparing the ampouled materials and despatch

of samples, and finally to the chairs and members of the ISTH/SSC sub-committees for

FVIII/FIX and von Willebrand Factor for their support.

WHO/BS/09.2116

Page 15

Instructions for Use The draft Instructions for Use for the proposed WHO 6

th IS Factor VIII / von Willebrand factor,

plasma are found in Appendix 5.

Table 1: Intra-laboratory variability for estimates of the proposed 6th

IS by all methods

assays vs 5th

IS

assays vs normal pools Analyte

GCV < 5% GCV > 10%

GCV < 5% GCV > 10%

Factor VIII: C

41/53 (77%) 3/53 (6%) 21/49 (43%) 10/49 (20%)

Factor VIII: Ag

7/11 (64%) 2/11 (18%) 2/11 (18%) 2/11 (18%)

VWF: Ag

21/31 (68%) 4/31 (13%) 11/31 (35%) 9/31 (29%)

VWF: RCo

13/28 (46%) 8/28 (29%) 10/28 (36%) 9/28 (32%)

VWF: CB

6/21 (29%) 3/21 (14%) 3/21 (14%) 9/21 (43%)

Figures indicate numbers of laboratories with GCV <5% or >10%

Table 2: Summary of inter-laboratory variability (GCV%) for estimates of the proposed 6

th IS by

all methods

Analyte and method

assays vs 5th IS assays vs normal pools

Factor VIII:C

1-stage clotting

Chromogenic

Overall

2.80%

3.57 % (ex lab 22)

4.06% (ex lab 22)

fresh 10.29%; frozen 14.16%



Factor VIII: antigen

4.24% (ex lab 27) 10.53%

VWF: antigen

ELISA

Immuno-turbidimetric

Overall

5.17%

1.46%

4.29%

13.14%

12.51%

12.92%

VWF: ristocetin cofactor

Aggregometry

Visual agglutination

Overall

6.52%

12.00%

8.13%


fresh* ----; frozen 24.16%


VWF: collagen binding

Type I/III mix collagen

Type III collagen

Overall

5.09%

6.37%

5.80%

15.58% (ex lab 24)

11.77%

12.24% (ex lab 24)

* - estimates from only 1 laboratory

WHO/BS/09.2116

Page 16

Table 3: Estimates for FVIII:C (IU per ml) in the Proposed 6th IS FVIII/VWF Plasma (07/316)

relative to the 5th IS FVIII/VWF Plasma (02/150) and the normal plasma pools Method Laboratory vs 5th IS (A)

n mean GCV%

vs normal pools (N)

n mean GCV%

2

3

4

5

6

7

10

11 12

13

16 17

18

19 20

21 23

24

25 27

29

30

32

33

34

35

36

37 38

43

44

4 0.68 3.28

4 0.65 3.47

4 0.66 3.41

4 0.68 2.09

4 0.67 2.06

4 0.67 2.24

4 0.64 7.98

4 0.65 4.69 4 0.67 2.00

4 0.67 5.62

4 0.67 7.83 4 0.70 3.50

4 0.68 4.11

4 0.64 11.91 4 0.66 0.26

4 0.69 3.46 4 0.65 5.79

4 0.66 0.62

4 0.64 4.22 4 0.68 4.33

4 0.66 2.01

4 0.63 1.77

4 0.69 3.35

4 0.67 2.19

4 0.66 1.09

4 0.67 2.19

5 0.69 4.20

4 0.69 3.12 4 0.67 6.29

4 0.70 3.35

4 0.69 3.07

4 0.70 12.09*

4 0.88 8.45†

4 0.86 2.97†

4 0.59 4.91*

4 0.65 6.66†

4 0.62 7.07†

4 0.79 1.81‡

4 0.64 6.23† 4 0.63 2.88†

3 0.74 12.04†

4 0.64 4.35† 4 0.72 3.63†

4 0.71 13.14†

4 0.50 20.31* 4 0.64 4.34†

4 0.55 9.14* 4 0.90 12.06†

4 0.67 1.50†

4 0.65 5.19† 4 0.68 22.61†

4 0.57 5.59*

4 0.55 5.54†

4 0.65 8.11†

4 0.49 5.38*

4 0.57 4.46*

4 0.60 4.93*

5 0.52 42.71*

4 0.54 7.43* 4 0.80 12.32†

4 0.75 3.48†

4 0.57 6.22*

1-stage clotting

Mean 31 0.67 2.80 Fresh 11 0.56 10.29

Frozen 19 0.70 14.16

26

4 0.72 2.86

no pool

2-stage clotting

Mean 1 0.72 ------ -- ----- -----

3

5

8

10

13

14

16 18

21

22 23

24

28 29

34

39

41

42

43a

43b

44

4 0.73 3.81

4 0.70 2.18

4 0.71 6.51

4 0.70 2.45

4 0.71 0.89

4 0.69 7.39

4 0.72 5.83 4 0.74 4.63

4 0.68 2.70

4 1.14 12.46 4 0.66 10.10

4 0.71 1.33

4 0.71 2.26 4 0.77 2.20

4 0.68 1.38

4 0.71 3.04

4 0.72 0.66

4 0.69 3.08

4 0.67 2.40

4 0.70 4.56

4 0.69 8.32

4 0.89 8.36†

4 0.57 5.94*

4 0.74 2.14†

4 0.76 1.00‡

4 0.76 3.86†

no pool

4 0.67 3.84† 4 0.76 21.31†

4 0.59 9.11*

4 0.73 17.48† 4 0.96 6.79†

4 0.70 1.80†

4 0.77 10.73† 4 0.82 7.33*

4 0.59 3.18*

no pool

no pool

4 0.67 2.76†

4 0.74 2.10†

4 0.73 3.83†

4 0.50 3.28*

Chromogenic

Mean

Exc Lab 22

21 0.72 11.75

20 0.70 3.57

Fresh 5 0.61 20.28

Frozen 12 0.76 11.04

Overall mean estimate

Exc Lab 22

52 0.68 4.06

Fresh 16 0.57 13.90

Frozen 31 0.72 13.54

* - fresh normal pools, † - frozen normal pools, ‡ - lyophilised pools

NOTES: GCV – geometric coefficient of variation for intra-laboratory variability (individual laboratories) and inter-laboratory

variability (combined mean values). Labs 17 and 38 used a mixture of fresh and frozen pools – the laboratory mean values have

been included as frozen pools in the calculation of overall mean values. Lab 10 used a lyophilised pool – this result has not been

included in the combined results.

WHO/BS/09.2116

Page 17

Table 4: Estimates for FVIII:antigen (IU per ml) in the Proposed 6

th IS FVIII/VWF Plasma

(07/316) relative to the 5th

IS FVIII/VWF Plasma (02/150) and the normal plasma pools Method Laboratory vs 5

th IS

n mean GCV%

vs normal pools

n mean GCV%

ELISA

3

9

11

15

17

22

24

27

28

29

40

4 1.01 1.98

4 1.06 4.33

4 1.02 1.45

4 1.10 4.05

4 0.99 7.67

4 1.11 13.57

4 1.01 2.39

4 1.26 21.46

3 1.05 4.64

4 0.99 3.46

4 1.06 12.82

4 0.93 8.41

4 0.94 7.81

4 0.80 5.52

4 0.79 33.36

4 1.01 5.63

4 1.03 6.60

4 0.85 1.83

4 0.99 24.99

3 0.90 4.26

4 1.06 5.24

4 0.99 9.28


Excluding Lab 27

11 1.06 7.35

10 1.04 4.24

11 0.93 10.53

GCV – geometric coefficient of variation for intra-laboratory variability (individual laboratories) and inter-laboratory variability

(combined mean values).

WHO/BS/09.2116

Page 18

Table 5: Estimates for VWF:antigen (IU per ml) in the Proposed 6th

IS FVIII/VWF Plasma

(07/316) relative to the 5th

IS FVIII/VWF Plasma (02/150) and the normal plasma pools Method Laboratory vs 5

th IS

n mean GCV%

vs normal pools

n mean GCV%

3

8

9

11

12

13

16

18

19

22

23

26

27

28

30

31

35

36b

37

42

43

4 0.94 4.08

4 0.94 1.09

4 0.99 2.51

4 0.99 3.52

4 0.93 23.07

4 0.98 4.86

8 1.01 5.26

4 1.06 15.58

4 0.93 8.58

4 1.02 7.72

4 1.13 3.87

4 1.02 5.54

4 1.05 3.14

4 1.01 4.71

4 0.92 16.32

4 1.06 12.78

4 1.00 1.67

5 1.01 5.78

4 1.01 1.59

4 1.02 3.17

4 1.00 5.25

4 0.95 7.05

4 0.89 1.62

4 1.01 3.82

4 0.90 7.63

4 0.99 31.86

4 0.76 2.16

8 0.83 21.48

4 0.79 14.90

4 0.89 20.25

4 1.10 3.75

4 0.95 8.49

2 0.70 6.06

4 0.98 24.73

4 0.88 21.89

4 0.83 7.31

4 1.11 8.79

4 0.92 9.89

5 0.79 37.27

4 0.78 3.19

4 0.81 3.23

4 0.76 3.01

ELISA

Mean 21 1.00 5.17 21 0.88 13.14

2

4

6

7

10

33

34

36a

38

44

4 1.00 1.37

4 1.00 3.10

4 1.00 2.60

4 0.99 1.04

4 0.98 1.78

4 0.99 2.88

2 0.98 1.07

5 1.01 2.67

4 1.03 2.96

4 1.00 1.37

4 0.90 5.36

4 1.09 5.42

4 0.85 9.02

4 1.04 2.85

4 0.89 1.93

4 0.90 3.33

2 0.81 12.41

5 0.80 34.69

2 1.06 0.51

4 0.90 5.36

Immuno-

turbidimetric

Mean 10 1.00 1.46 9 0.92 12.51


31 1.00 4.29

30 0.89 12.92


(combined mean values). Lab 10 used a lyophilised pool – this result has not been included in the combined result for estimates vs the normal pools.

WHO/BS/09.2116

Page 19

Table 6: Estimates for VWF:ristocetin cofactor (IU per ml) in the Proposed 6th

IS FVIII/VWF

Plasma (07/316) relative to the 5th

IS FVIII/VWF Plasma (02/150) and the normal plasma pools

Method Laboratory vs 5th IS

n mean GCV%

vs normal pools

n mean GCV%

1

2

3

4

5

12

18

19

20

21

25

28b

28c

29

30

31

33

34

37

38

42

4 0.90 5.69

4 0.80 2.22

4 0.87 1.50

4 0.83 15.01

4 0.82 12.10

4 0.81 1.34

4 0.92 1.60

4 0.85 5.68

4 0.81 27.66

4 0.90 4.90

4 0.85 4.62

4 0.88 1.24

4 0.90 6.55

4 0.88 1.32

4 0.90 13.15

4 0.99 5.62

4 0.74 23.55

2 0.92 4.56

4 0.88 2.49

4 0.89 8.50

4 0.83 12.21

4 1.05 4.34†

4 0.88 11.05*

4 1.13 6.09†

4 1.03 4.95†

4 0.55 25.76†

4 0.81 5.39†

4 0.91 7.43†

4 0.82 13.86*

4 0.86 20.92†

4 0.84 16.84*

4 1.13 4.57†

4 1.30 0.83†

2 1.36 3.51†

4 0.90 9.29*

4 0.59 16.41†

4 0.80 11.35†

4 0.69 26.17*

2 0.79 2.09*

4 0.72 1.56*

4 1.00 5.62†

4 0.77 12.70†

aggregometry

Mean 21 0.86 6.52 Fresh 7 0.80 10.42

Frozen 14 0.92 30.60

All 21 0.88 26.08

7+

8+

22

26

28a+

43+

44+

4 0.78 2.70

4 0.78 0.00

4 0.87 8.51

4 0.92 12.80

4 0.92 4.55

4 1.03 7.28

4 1.02 42.57

4 0.84 1.38†

4 0.99 0.00†

4 0.95 5.69†

4 0.72 7.93†

4 1.15 5.71†

4 1.32 7.63†

4 0.80 3.84*

visual

agglutination

Mean 7 0.90 12.00 Fresh 1 0.80 -----

Frozen 6 0.98 24.16

All 7 0.95 23.53

immuno-

turbidimetric

10 4 0.85 5.14 4 0.99 2.69‡


(excluding Lab 10)

28 0.87 8.13

Fresh 8 0.80 9.62

Frozen 20 0.94 28.26

All 28 0.90 25.29

* - fresh normal pools, † - frozen normal pools, ‡ - lyophilised pools + - laboratory's own analysis

NOTES: GCV – geometric coefficient of variation for intra-laboratory variability (individual laboratories) and inter-laboratory

variability (combined mean values). Lab 38 used a mixture of fresh and frozen pools – the laboratory mean value has been

included as a frozen pool in the calculation of overall mean values. Lab 10 used an immuno-turbidimetric method and a

lyophilised pool – these results have not been included in the combined results.

WHO/BS/09.2116

Page 20

Table 7: Estimates for VWF:collagen binding (IU per ml) in the Proposed 6th

IS FVIII/VWF

Plasma (07/316) relative to the 5th


Collagen Reagent Laboratory vs 5th IS (A)

n mean GCV%

vs normal pools (N)

n mean GCV%

5

15

18

24

28a

31

44

4 0.98 5.70

4 1.11 6.89

4 1.03 9.65

4 1.00 11.28

4 1.06 5.33

4 0.96 10.11

4 1.00 3.63

4 1.01 4.37

4 0.91 40.10

4 0.87 17.79

4 1.70 60.64

4 1.28 6.77

4 1.02 9.14

4 0.88 14.16

Type I and Type

I/III mix

Mean 7 1.02 5.09 7 1.07 27.72

6* 0.99 15.58 *exc Lab 24

1

3

11

20

23

25

28b

30

32

34

37

40

43

4 1.09 5.21

4 1.16 6.48

4 1.03 4.59

4 1.06 2.02

4 1.10 1.60

4 1.02 9.14

4 1.01 6.62

4 0.91 9.68

4 1.06 4.45

4 1.12 2.16

4 1.01 3.40

4 1.01 13.27

4 1.00 6.74

4 1.04 7.33

4 1.10 30.33

4 0.94 16.28

4 1.02 9.95

4 0.96 5.96

4 1.15 5.05

4 1.12 5.47

4 0.90 20.08

4 0.88 3.01

4 0.89 9.95

4 0.79 1.06

4 1.01 17.32

4 1.08 6.62

Type III

Mean 13 1.04 6.37 13 0.99 11.77

Not specified 27 4 1.01 6.62 4 0.99 14.78


21 1.03 5.80

21 1.01 17.79

20* 0.99 12.24 *exc Lab 24



WHO/BS/09.2116

Page 21

Table 8: Estimates for VWF:collagen binding (IU per ml) in the 1st IS VWF Concentrate (00/514)

relative to the 5th


Collagen Reagent Laboratory vs 5th IS (A)

n mean GCV%

vs normal pools (N)

n mean GCV%

5

15

18

24

28a

31

44

4 11.16 8.04

4 11.75 32.32

4 10.69 12.49

4 8.08 19.55

4 11.97 6.12

4 9.67 11.35

4 7.10 4.60

4 11.52 12.52

4 10.28 35.92

4 9.02 10.26

4 14.83 22.96

4 14.24 8.22

4 10.25 11.15

4 6.43 22.91

Type I and Type

I/III mix

Mean 7 9.90 22.04 7 10.58 32.88

1

11

23

25

28b

30

34

37

40

43

4 13.26 4.48

4 11.18 2.57

4 13.22 8.94

4 12.96 5.56

4 12.02 6.23

4 8.93 4.96

4 13.09 1.84

4 9.70 2.66

4 10.16 14.15

4 11.87 9.93

4 12.69 3.86

4 10.23 12.82

4 11.56 5.68

4 14.61 7.55

4 13.28 5.88

4 9.28 7.75

4 10.37 9.87

4 7.58 2.02

4 10.08 15.05

4 12.82 3.53

Type III

Mean 10 11.54 15.31 10 11.06 21.66

Not specified 27 4 9.28 5.04 4 9.05 20.40


18 10.74 19.51

18 10.75 25.39



Table 9: Results from the accelerated degradation study on the proposed WHO 6th

IS

Residual potencies after storage

(% vs -20 °C ampoules)

Analyte Lab ID

+4°C +20°C +37°C +45°C

Mean predicted % loss per

year at -20 °C (and 95 %

upper conf limit of loss)

FVIII:C

1-stage clotting

A†

B*

98

97

90

90

36

43

17

18

0.074% (0.179%)

FVIII:C

chromogenic

A†

B*

100

104

88

91

32

41

26

33

0.146% (0.546%)

FVIII:Antigen

A*

B*

94

96

89

93

33

49

----

----

0.049% (0.403%)

VWF:Antigen

A†

B*

97

103

97

101

42

38

2

----

0.000% (0.000%)

VWF:RCo

A*

B*

101

96

102

76

30

----

----

----

0.022% (0.516%)

VWF:CB

A†

B*

102

98

105

97

69

47

76

----

0.002% (0.021%)

† - tested after storage for 7 months; * - tested after storage for 9 months;

Results are the mean values from 3 independent assays

WHO/BS/09.2116

Page 22

Figure 1: Von Willebrand factor multimer profile for the proposed WHO 6th

IS, the

current WHO 5th

IS and frozen normal plasma (agarose gel medium resolution)

1 Frozen normal plasma

2 Proposed WHO 6th

IS (07/316)

3 WHO 5th

IS (02/150)

WHO/BS/09.2116

Page 23

Figure 2a: Factor VIII:C in the proposed 6th

IS relative to the 5th

IS

0

2

4

6

8

10

12

14

16

18

20

22

24

% of Overall Mean

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

03

04

10

11

19

20

23

23

24

25

29

30

34

02

05

06

07

12

13

14

16

18

21

21

27

32

33

34

35

36

37

38

42

43.1

44

44

03

05

08

10

13

16

17

24

26

28

39

41

43

43.2

18 29

Figure 2b: Factor VIII:C in the proposed 6th

IS relative to the normal pools

0

1

2

3

4

5

6

7

8

9

10

% of Overall Mean

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

19*

33*

36*

44*

21*

37*

05*

29*

30

34*

44*

05*

07

21*

34*

35*

06

11

12

16

20

25

32

16

24

27

42

02*

18

24

08

13

17

22

43

43.1

43.2

10‡

13

18

28

10‡

38

29* 03

04

03

23

23

* - fresh pools ‡ - lyophilised pool

*

WHO/BS/09.2116

Page 24

Figure 3a: Factor VIII:antigen in the proposed 6th


IS

0

1

2

3

4

5

6

7

8

9

10

% of Overall Mean

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155

03

17

24

29

09

11

28

40

15

22

27

Figure 3b: Factor VIII antigen in the proposed 6th


0

1

2

3

4

5

6

7

8

9

10

% of Overall Mean

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

11

15

24 28 03

09

27

40

17

22

29

WHO/BS/09.2116

Page 25

Figure 4a: VWF:antigen in the proposed 6th


IS

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

% of Overall Mean

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

30 03

08

12

19

02

04

06

07

09

10

11

13

16

22

26

28

33

34

35

36a

36b

37

42

43

44

18

27

31

38

23

Figure 4b: VWF:antigen in the proposed 6th


0

1

2

3

4

5

6

7

8

9

10

% of Overall Mean

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

26 13

43

18

34

36a

36b

37

42

06

16

30

02

08

10‡

11

19

28

33

44

03

23

35

12

27

07

09

38 04

22

31

WHO/BS/09.2116

Page 26

Figure 5a: VWF:Ristocetin Cofactor in the proposed 6th


IS

0

1

2

3

4

5

6

7

8

9

10

% of Overall Mean

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

33 02

07

08

04

05

12

20

42

03

10

19

22

25

28b

29

37

38

01

18

21

26

28a

28c

30

34

31

44

43

Figure 5b: VWF:Ristocetin Cofactor in the proposed 6th


0

1

2

3

4

5

6

7

8

9

10

% of Overall Mean

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150 155

05* 30 26

33*

37*

42 12

19*

31

34*

44*

07

20

21*

02*

29*

18

22

08

10‡

04

38

01 03

25

28a 28b 28c

43

WHO/BS/09.2116

Page 27

Figure 6a: VWF:Collagen Binding in the proposed 6th


IS

0

1

2

3

4

5

6

7

8

9

10

% of Overall Mean

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

30

32

05

24

31

43

44

11

18

25

27

28B

37

40

01

20

23

28A

15

34

03

Figure 6b: VWF:Collagen Binding in the proposed 6th


0

1

2

3

4

5

6

7

8

9

10

% of Overall Mean

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

37 15

18

30

32

34

44

11

23

05

27

40

01

20

31

03

43

25

28B

28A

WHO/BS/09.2116

Page 28

Figure 7a: VWF:Collagen Binding in the 1st IS VWF Concentrate relative to the 5

th IS

0

1

2

3

4

5

6

7

8

9

10

% of Overall Mean

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

44 24 27

30

31

37

40 18 05

11

15

28A

28B

43

25

34

01

23

Figure 7b: VWF:Collagen Binding in the 1st IS VWF Concentrate relative to the normal

pools

0

1

2

3

4

5

6

7

8

9

10

% of Overall Mean

50 55 60 65 70 75 80 85 90 95 100 105 110 115 120 125 130 135 140 145 150

44 37 18

27

30

11

15

31

34

40

05 23 01

43

28B 28A 25 24

WHO/BS/09.2116

Page 29

Appendix 1: Study protocol provided to participants

1 SAMPLES INCLUDED IN THE ASSAYS

A - 5

th International Standard Factor VIII/VWF Plasma (02/150)

B - Proposed 6th

International Standard Factor VIII/VWF Plasma (07/316)

C - 1st International Standard VWF Concentrate (00/514) (VWF:CB assays only)

N1, N2 - fresh normal plasma pools prepared locally by participants (see section 5)

Samples A and B are pooled normal plasmas. Sample C is a purified concentrate for inclusion

only in VWF:CB assays – it has a nominal VWF:CB content of 8 – 10 IU/ampoule. All plasma

donations used to prepare samples A, B and C have been tested and found negative for HBsAg,

antibodies to HIV-1 and -2, antibodies to HCV and for the presence of HCV RNA (mini-pools).

2 STORAGE AND RECONSTITUTION OF SAMPLES A, B AND C

Store the unopened ampoules of A, B and C at -20

oC or below. Allow the ampoules to warm to

room temperature before reconstitution. Tap ampoules gently to ensure that all of the contents

are in the lower part of the ampoules. Reconstitute by adding 1.0 ml of distilled water at room

temperature. Dissolve the contents with gentle agitation at room temperature. When

reconstitution is complete transfer the entire contents to stoppered plastic tubes and store at 4oC

during the assay period.

3 GENERAL PLAN OF THE STUDY

You are requested to carry out 4 assays by each method using fresh ampoules for each assay. If

you are measuring 1 or 2 different analytes only 4 ampoules of A, B (and C where applicable)

are provided (plus 1 spare ampoule of each) and it will therefore be necessary to carry out more

than one assay method on each ampoule (where applicable). If you are measuring 3 or more

analytes then extra ampoules of A, B and C will be supplied. Please ensure that sufficient

volume is available for all of the assay methods you are using. Please let me know if you require

more ampoules.

Assays for FVIII:C and VWF:RCo must be carried out on freshly reconstituted ampoules of A

and B and fresh normal pools (N1, N2). If both of these methods are being carried out they

should ideally be performed at the same time. If this is not possible please carry out the assays

of FVIII:C first followed by assays for VWF:RCo.

Assays for FVIII:Ag and VWF:Ag should preferably be carried out on fresh samples but may

be carried out on frozen aliquots where unavoidable.

Assays for VWF:CB should preferably be carried out on fresh samples but may be carried out

on frozen aliquots where unavoidable. Sample C should be included only in VWF:CB assays.

The 4 assays should be spread over 2 separate days/sessions as follows:

Assay session

Normal pool Ampoules of A and B

(and C where applic)

Assay number

1 1 Day 1 N1

2 2

3 3 Day 2 N2

4 4

WHO/BS/09.2116

Page 30

If you are unable to prepare the two fresh local pools (N1, N2) you may alternatively include

the same local frozen plasma pool in all four assays.

4 ASSAY DESIGN

All three preparations (A, B, N) are included in each of the 4 assays. (Sample C is also included

in the VWF:CB assays). A minimum of 3 dilutions of each preparation should be tested, in

replicate, within each assay. Please follow a balanced assay design such as the optimal 18-place

assay described in Design 1 below. If an 18-place assay is not possible please use an alternative

design such as Design 2 (15-place assay). Please contact me if you need help with the assay

design. A separate design is given below for the VWF:CB assays.

In the following designs, each letter refers to a separate set of three or more dilutions and A, A'

and B, B' etc. refer to fresh sets of dilutions (replicates) made from the same ampoule.

Design 1 (18-place assay)

Assay 1 A B N1 N1' B' A'

Assay 2 B N1 A A' N1' B'

Assay 3 B A N2 N2' A' B'

Assay 4 N2 B A A' B' N2'

Design 2 (15-place assay)

Assay 1 A B N1 N1' B'

Assay 2 N1 A B B' A'

Assay 3 B N2 A A' N2'

Assay 4 N2 B A A' B'

Design for VWF:CB assays (24-place assay)

Assay 1 A B C N1 N1' C’ B' A'

Assay 2 B C N1 A A' N1' C’ B'

Assay 3 C N2 A B B’ A’ N2' C'

Assay 4 N2 A B C C’ B’ A' N2'

5 COLLECTION OF FRESH NORMAL PLASMA

Collect fresh normal plasma on two separate days to prepare pools N1 and N2. The method of

collection for the fresh normal plasma is an important part of the study and should be

standardised as far as possible according to the following protocol.

Donors Normal healthy volunteers, excluding women taking oral contraceptives or pregnant.

Take blood from as many different individuals as possible, on two separate days. If possible, use

a minimum of 8 different donors for each pool; if this is not possible, some of the same

individuals can be used again, but the aim is to have as many different donors as possible from

each laboratory.

WHO/BS/09.2116

Page 31

Anticoagulant 0.109 mol/L tri-sodium citrate or a mixture of tri-sodium citrate and citric acid

with a total citrate concentration of 0.109 mol/L. Add 9 volumes of blood to 1 volume of

anticoagulant.

Centrifugation Blood should be centrifuged at 4oC as soon as possible after collection either at

50,000 g for 5 minutes or at 2,000 g for 20 minutes.

Storage Keep plasma pool in a plastic stoppered tube at 4oC during the assay session. Freeze

aliquots of each pool (N1, N2) for subsequent assays where applicable.

6 RESULTS

Please return the raw data from your assays and the methodology questionnaire, by e-mail to

[email protected], using the Excel results sheets by 28 November 2008. Please ensure

that your results are presented as true raw data (eg. clotting time, optical density) rather than as

% or units relative to an in house standard. You are also invited to calculate your own estimates

for B relative to A and for B relative to to N1 and N2 if you wish (see Excel results sheets). If

you are unable to use the Excel sheets please use the results sheets which are attached to this

document. Results returned by post should be addressed to:

Dr A R Hubbard, Haemostasis Section, NIBSC, Blanche Lane, South Mimms, Potters Bar,

Hertfordshire. EN6 3QG United Kingdom

Fax: International +44 1707 641050 UK 01707 641050 E-mail: [email protected]

WHO/BS/09.2116

Page 32

Appendix 2: List of participants

Dr M Shima

Nara Medical University

Kashihara City, Japan

Mr K Sukhu/Dr P Giangrande

Oxford Haemophilia & Thrombosis Centre

Churchill Hospital

Oxford, United Kingdom

Dr R Bader

Centro Emofilia e Trombosi "A Bianchi

Bonomi"

Milan, Italy

Ms E Duncan

Division of Haematology

Institute of Medical & Veterinary Science

Adelaide, Australia

Dr K Friedman / Ms J Endres

Blood Center of SE Wisconsin

Milwaukee, WI

United States of America

Dr C Eby

Washington University School of Medicine

Saint Louis, MO


Dr B Woodhams

Diagnostica Stago R&D

Gennevilliers

France

Dr A Lichte/Dr J Patzke/Mr M Timme

Siemens Healthcare Diagnostics

Products GmbH, Marburg

Germany

Dr C Andreini

Instrumentation Laboratory

Milan

Italy

Dr S Rosén

Rossix

Molndal

Sweden

Dr S Duff/Dr M Boylan

Precision Biologic Inc

Dartmouth, Nova Scotia

Canada

Dr S Cinotti

Centro Emofilia

Azienda Ospedaliera Careggi

Florence, Italy

Mr C Watson

Haemostasis Lab, Royal Infirmary

Leicester, United Kingdom

Dr B Kerbl

Technoclone GmbH

Vienna, Austria

Dr M G Riera

Instituto Grifols S.A.

Barcelona, Spain

Dr F Rodeghiero/Dr A Tosetto

San Bortolo Hospital

Vicenza, Italy

Dr M Chitlur / Dr J M Lusher

Children's Hospital of Michigan

Detroit, MI


Dr M Wolf

Hôpital Antoine-Béclère

Clamart, France

Dr P Turecek / Dr H Gritsch

Baxter Innovations GmbH

Vienna, Austria

Dr E J Favaloro

Institute of Clinical Pathology & Medical

Research

Westmead Hospital

Westmead, Australia

WHO/BS/09.2116

Page 33

Dr A Hunfeld/Ms J Fötisch

Ms A Schroda/Ms R Nawroth

Dept Haematology &Transfusion Medicine

Paul-Ehrlich-Institut, Langen, Germany

Dr K Trumbull

Instrumentation Laboratory

Lexington, MA


Dr B Samor

LFB Biotechnologies

Lille, France

Ms S Bevan/ Ms M Beeharry

Haemostasis Section

NIBSC

Potters Bar, United Kingdom

Prof C A Ludlam/Ms L Germain

Dept of Haematology

Royal Infirmary

Edinburgh, United Kingdom

Dr S Kitchen / Ms K Goodfellow

Dept of Coagulation

Royal Hallamshire Hospital

Sheffield, United Kingdom

Ms A Riddell

Haemophilia Centre & Thrombosis Unit

Royal Free Hospital

London, United Kingdom

Dr S Berrier

Diagnostica Stago QC

Franconville, France

Mme C Caron

Laboratoire d'Hématologie

Centre de Biologique Pathologie

Lille, France

Dr C M Eckmann

Sanquin Plasma Supply Foundation

Amsterdam, The Netherlands

Dr P Gärtner

Baxter AG

Vienna, Austria

Dr D Elmlinger

Octapharma SA QC

Lingolsheim, France

M Blum

Baxter AG

Orth/Donau, Austria

Dr J Amiral

Hyphen Biomed SAS

Neuville-sur-Oise

France

Dr M Bono

Haemostasis Dept.

Diagnostic Grifols

Barcelona, Spain

Dr J C J Eikenboom

Leiden University Medical Center

Dept Thrombosis and Hemostasis

Leiden, The Netherlands

Dr K Pock

Octapharma R & D

Vienna, Austria

Prof A Federici/Dr M T Canciani

Dept. Internal Medicine

A Bianchi Bonomi Hemophilia &

Thrombosis Centre

Milan, Italy

Dr J Conkie / Ms G Hickman

Dept Haematology

Royal Infirmary

Glasgow, United Kingdom

Dr M Telesca

Presbyterian Hospital

New York, NY


Mr G Longin

CAF-DCF Red Cross

Neder-Over-Heembeek (Brussels)

Belgium

Dr G Rautmann / Ms C Raphalen

EDQM, Council of Europe

Strasbourg, France

WHO/BS/09.2116

Page 34

Dr D Krause

QC Method Validation

Octapharma Prod. GmbH

Vienna, Austria

Dr A Blande

Bio Products Laboratory

Elstree, United Kingdom

WHO/BS/09.2116

Page 35

Appendix 3: Details of methodology

Factor VIII: Clotting activity

Method Laboratory APTT reagent Substrate plasma Instrument

1-stage clotting

2

3

4

5

6

7

10

11

12

13

16

17

18

19

20

21

23

24

25

27

29

30

32

33

34

35

36

37

43

44

Hemosil/IL APTT-SP

DAPTTIN (Baxter)

Stago CK Prest

Biomerieux Auto APTT

IL Synthasil

Biomerieux

Hemosil/IL APTT Synthasil

Stago CK Prest

IL Synthasil

Hyphen Cephen LR

DG-APTT

IL Synthasil

Platelin LS

Biomerieux Auto APTT

STA Cepascreen

Actin FS

DAPTTIN (Baxter)

Triniclot APTT

Silica

Hemosil/IL APTT-SP

Actin FSL

Hemosil APTT

Hemosil APTT

Stago STA-PTTA

Actin FSL

Hemosil/IL APTT Synthasil

Stago PTTA /CK Prest

Actin FS

Hemosil/IL APTT-SP

Hemosil/IL APTT-SP

immuno-depleted

immuno-depleted

Severe Haem Plasma

immuno-depleted

immuno-depleted

immuno-depleted

immuno-depleted

immuno-depleted

immuno-depleted

immuno-depleted

Immuno-depleted

Severe Haem Plasma

ng

immuno-depleted

immuno-depleted

immuno-depleted

Severe Haem Plasma

immuno-depleted

Immuno-depleted

immuno-depleted

immuno-depleted

Severe Haem Plasma

Severe Haem Plasma

immuno-depleted

immuno-depleted

immuno-depleted

immuno-depleted

Severe Haem Plasma

immuno-depleted

immuno-depleted

IL ACL 9000

KC4A

STA-R Evolution

STA-R Evolution

ACL TOP

STA-R

ACL TOP

STA-R

ACL TOP

STA-R

Amax mechanical

ACL TOP

Behring BCS

STA-R

STA-R Evolution

BCS-XP

KC10

Sysmex CA 1500

BCT Dade Behring

STA Compact

Sysmex CA 7000

ACL 3000

ACL 10000

STA-R

Sysmex CA 1500

ACL TOP

STA-R

BCS

STA-R

ACL TOP 500

Method Laboratory Reagents Samples adsorbed Instrument

2-stage clotting 26

Diagnostic Reagents

Yes

Sysmex CA 7000

Method Laboratory Kit Rate or end-point Instrument

Chromogenic

3

5

8

10

13

14

16

18

21

22

23

24

28

29

34

39

41

42

43

44

Baxter Immunochrom

Hyphen Biophen

Chromogenix Coamatic


Hyphen Biophen

Chromogenix Coatest SP4

DG-Chrom FVIII

Hyphen Biophen



Technochrom

Hyphen Biophen



Siemens FVIII Chromogenic






Rate

Rate

End-point

Rate

Rate

End-point

Rate

End-point

Rate

End point

Rate

Rate

End-point

End-point

Rate

End-point

End-point

Rate

End-point

Rate

Powerwave 340

STA-R Evolution

Versamax

ACL TOP

STA-R

T-Max microplate reader

Amax optical

Bio Tek plate reader

ACL Advance

Multiskan JX

Microplate reader

STA-R

Spectramax Plus

Biomek 2000

Sysmex CA-1500

iEMS

Spectramax

ACL 200/3000 plus

STA-R/MTP-Reader

ACL TOP 500

ng - details not given

WHO/BS/09.2116

Page 36

Factor VIII: Antigen

Method Laboratory Method/Kit Capture antibody

ELISA

3

9

11

15

22

24

27

28

29

40

Asserachrom FVIII:Ag



VisuLize FVIII:Ag (Affinity Biologicals)

In house


Haematologic Technologies


In-House


Mouse monoclonal

Mouse monoclonal

Mouse monoclonal

Sheep polyclonal

Human allo-antibodies

Mouse monoclonal

Sheep polyclonal

Mouse monoclonal

Mouse monoclonal

Mouse monoclonal

von Willebrand Factor: Antigen Method Laboratory Method/Kit Antibodies

ELISA

3

8

9

11

12

13

16

17

18

19

22

23

26

27

28

30

31

35

36b

37

42

43

In house

Asserachrom VWF:Ag

In house

Asserachrom VWF:Ag

In house

Hyphen Zymutest

DG-EIA VWF kit

Asserachrom VWF:Ag

In house

In house

In house

Technozym VWF:AG

In house

In house

Asserachrom VWF:Ag

In house

In house

In house

Asserachrom VWF:Ag

In house

In house

Asserachrom VWF:Ag

Dako Rabbit polyclonal

Rabbit polyclonal


Rabbit polyclonal


Rabbit polyclonal

Rabbit polyclonal

Rabbit polyclonal




Dako rabbit anti-human


PBI/HTI antibodies

Rabbit polyclonal


Rabbit polyclonal


Rabbit polyclonal



Rabbit polyclonal

Immuno-

turbidimetric

2

4

6

7

10

33

34

36a

38

44

Hemosil/IL VWF Antigen

Stago Liatest

Hemosil/IL VWF Antigen Stago

Liatest


Stago Liatest

Siemens VWF Ag

Stago Liatest

Stago Liatest


Rabbit polyclonal

Rabbit polyclonal

Rabbit polyclonal

Rabbit polyclonal

Rabbit polyclonal

Rabbit polyclonal

Rabbit polyclonal

Rabbit polyclonal

Rabbit polyclonal

Rabbit polyclonal

WHO/BS/09.2116

Page 37

von Willebrand Factor: Ristocetin cofactor

Method Laboratory Platelets Reagents Instrument

aggregometry

1

2

3

4

5

12

18

19

20

21

25

28b

28c

29

30

31

33

34

37

38

42

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Dade Behring/Siemens

Mascia Brunelli Ristocetin


ABP Ristocetin

Helena Ristocetin

American BP Ristocetin


Hart Biologicals

ABP Ristocetin



Dade Behring + Ristocetin A

(Mascia Brunelli S.P.A)

Dade Behring + Ristocetin A

(Mascia Brunelli S.P.A)



ng

Bio-Data

Siemens BC von Willebrand



Helena Biosciences

BCS

Chronolog

BCS

STA-R Evolution

Helena Pack-S 4

Bio Data PAP-4

BCS

Chronolog

Helena AggRam

BCS-XP

BCS

BCS-XP

ACL 10000

BCS

ACL 9000

Aggregometer

Bio-Data PAP-4D

BCS XP

BCS

BCS

Helena Pack-S 4

visual

agglutination

7

8

22

26

28

43

44

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed

Fixed




In house




--------------

--------------

--------------

--------------

--------------

--------------

--------------

immuno-

turbidimetric

10 Not applicable Hemosil/IL VWF activity ACL TOP

ng - details not given

von Willebrand Factor: Collagen binding

Method Laboratory Kit or in house collagen anti-VWF antibody

ELISA-type

1

3

5

11

15

18

20

23

24

25

27

28a

28b

30

31

32

34

37

40

43

44

In house

Technozym VWF:CBA

In house

Asserachrom VWF:CB

Technozym VWF:CBA

In house

Corgenix Kit

Technozym VWF:CBA

In house

In house

In- house

Technozym VWF:CBA

Technozym VWF:CBA

In-House

In-House

Technozym VWF:CBA

Technozym VWF:CBA

In-house

In house

Technozym VWF:CBA

In-House

Human type III

Human type III

Bovine type I/III mix

Human type III

Type I

Bovine type I/III mix

Equine type III

Human type III

Equine type I/III mix

Human type III

Proprietary

Human type I

Human type III

Human type III

Type I

Human type III

Human type III

Human type III

Human type III

Human type III

Equine type I/III mix

HRP conjugate

polyclonal HRP conjugate




















WHO/BS/09.2116

Page 38

Appendix 4: Real-time stability data for the 5th

IS FVIII/VWF

Plasma (02/150) Ampoules of the 5

th IS were stored for 6.67 years at -150, -70 and -20 °C. Factor VIII:C was

estimated by both 1-stage clotting and chromogenic methods and the potency of -70 and -20

°C ampoules has been expressed as a percentage of the vials stored at -150 °C.

Estimates of Factor VIII:C in ampoules stored for 6.67 years at -20oC and -

70°C relative to ampoules stored at -150oC (assigned 100%).

Chromogenic

One-stage clotting

Ampoule/Assay

number

-20 °C storage -70 °C storage -20 °C storage -70 °C storage

1

2

3

4

5

6

100.45

97.97

101.58

98.75

106.05

96.89

101.25

99.88

104.14

99.05

103.19

97.56

100.17

99.83

100.24

102.71

96.21

99.02

100.67

94.44

96.05

102.71

99.05

102.27

Mean

100.24 %

100.82 %

99.68 %

99.15 %

There was no significant difference between estimates for ampoules stored at -150, -70 and -20 °C.

WHO/BS/09.2116

Page 39

Appendix 5: Draft Instructions for Use for the proposed WHO 6th

International Standard Factor VIII and von Willebrand factor in

plasma (07/316)

WHO/BS/09.2116

Page 40

WHO/BS/09.2116

Page 41

Documents

Value Assignment of the Proposed 6 th International