WHO/BS/2016.2281

ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 17 to 21 October 2016

COLLABORATIVE STUDY ON THE 2ND

INTERNATIONAL STANDARD FOR

BLOOD COAGULATION FACTOR XI, PLASMA (15/180): ASSIGNMENT OF

FUNCTIONAL AND ANTIGEN VALUES

Helen Wilmot1, Peter Rigsby

2, Jason Hockley

2 and Elaine Gray

1

1Haemostasis Section and

2Biostatistics Group,

National Institute for Biological Standards and Control,

Potters Bar, Hertfordshire, EN6 3QG, UK.

helen.wilmot@nibsc.org

NOTE:

This document has been prepared for the purpose of inviting comments and suggestions on the

proposals contained therein, which will then be considered by the Expert Committee on

Biological Standardization (ECBS). Comments MUST be received by 16 September 2016 and

should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:

Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to

the Responsible Officer: Dr C M Nübling at email: nueblingc@who.int

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WHO/BS/2016.2281

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Summary

The aims of this study were to assign Factor XI functional activity to the 2nd

International

Standard (IS) for Blood Coagulation Factor XI, Plasma, and to additionally assign a new analyte,

FXI antigen, to the same International Standard. Assignment of the functional activity value in

International Units (IU) was performed by one-stage clotting assay by 29 laboratories, relative to

the 1st IS. The majority of intra-laboratory GCVs were less than 5% and the inter-laboratory

variability was extremely low at 1.8%, with an overall geometric mean of 0.71 IU/ampoule. The

antigen value assignment was performed by 11 laboratories and was calculated relative to normal

plasma pools. As is customary with new coagulation factor analytes, the amount of analyte

present in 1 ml of normal plasma was taken to be 1 unit, therefore the assignment of antigen is in

IU/ampoule relative to normal plasma pools. The inter-laboratory variation of the antigen assays

was acceptable at 10%, with an overall geometric mean of 0.78 IU/ampoule.

Proposals for establishment:

To establish candidate 15/180 as the 2nd International Standard for FXI, Plasma, with

assigned values of:

o FXI functional activity (FXI:C) of 0.71 IU/ampoule

o FXI antigen value (FXI:Ag) of 0.78 IU/ampoule

Introduction

Factor XI deficiency is generally mild and bleeding is most often associated with surgery or

trauma, though bleeding phenotype can vary and is not always correlated to FXI clotting activity.

Deficiency is most common in Ashkenazi Jews (around 1 in 190 are homozygous for mutation in

the F11 gene and around 1 in 8 are heterozygous), but has now been identified in a wide variety

of populations. There are a number of inherited mutations that cause FXI deficiency, most of

which lead to a decrease in antigen and functional activity, though some patients (around 4%)

experience a decrease in functional activity only.1

The 1st International Standard (IS) for Blood Coagulation Factor XI, Plasma (04/102), was

established by the Expert Committee on Biological Standardisation (ECBS) of the World Health

Organisation (WHO) in October 20052. The standard is used to aid diagnosis of factor XI (FXI)

deficiency and to assign potency to licensed FXI concentrates as well as control of virus

inactivated plasma products, both of which are used for treatment of patients. As with most

coagulation factors and inhibitors where the International Unit (IU) is defined as the amount of

activity in 1 ml of pooled normal plasma, this first IS was assigned with FXI functional activity

(FXI:C) relative to local normal plasma pools only. The stock levels of the 1st IS are now near

depletion and a replacement standard is required. For continuity of the IU, the replacement IS

for FXI functional activity (FXI:C) was value assigned relative to the 1st IS in this collaborative

study. The relationship of the IS to normal plasma was also examined. In addition, this study

also aimed to establish an antigen value for FXI (FXI:Ag) for the same candidate relative to

WHO/BS/2016.2281

Page 3

laboratories’ local normal pooled plasma. This will enable differentiation between patients who

have low functional activity but normal antigen FXI levels and patients who have both low

functional and antigen FXI levels.

The replacement of the 1st IS for FXI, Plasma (FXI:C) and the FXI:Ag value assignment projects

were endorsed by the WHO ECBS in October 2014.

Candidate WHO 2nd

International Standard for Factor XI, Plasma (15/180)

Bulk material was purchased from the United Kingdom Blood Service in the form of plasma

which had been prepared by centrifugation of whole blood collected into CPD adenine

anticoagulant. A second centrifugation step was performed to remove all cellular material and

the plasma rapidly frozen at -70°C. Individual donations were tested at source and found to be

negative for HBsAg, anti-HIV-1 and HIV-2 antibodies, and anti-HCV. The material was

prepared for filling by thawing immediately prior to use in a 37°C waterbath and then pooled.

Glycine and HEPES were added at a final concentration of 1% w/v and 40 mM, respectively. To

avoid activation of FXI by cold activation or contact with glass, plastic vessels were used and the

plasma was maintained at room temperature after thawing and throughout the duration of the fill.

The product was filled into siliconized glass ampoules and freeze dried over a 5 day cycle. The

finished product summary is as follows:

Code number 15/180

Presentation Sealed, siliconized glass ampoules

Number of ampoules available 6000

Date filled 01 October 2015

Mean fill mass (n=410) 1.0094 g

Precision of fill (CV of fill mass) (n=410) 0.300%

Residual moisture (n=12) 0.605%

Mean dry weight (n=3) 0.0928 g

Mean oxygen head space (n=12) 0.23 %

Storage conditions -20°C

Address of processing facility NIBSC, Potters Bar, EN6 3QG, UK

Address of custodian NIBSC, Potters Bar, EN6 3QG, UK

Activation status of the proposed 2nd

IS

Non-activated partial thromboplastin time (NAPTT) is sensitive to activated clotting factors and

was used to profile the activation status of the finished product. The clotting times of the

propsed 2nd

IS (15/180) and the 1st IS (04/102) are shown below. A clotting time of greater than

250 s indicates that the plasma is relatively unactivated.

Mean clotting time (s) n=9 SD

15/180 300 2.26

04/102 310 6.80

WHO/BS/2016.2281

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The clotting time of the proposed 2nd

IS was similar to that of the 1st IS and therefore is deemed

to be suitable. A chromogenic assay for FXIa was also performed, however the assay kit is not

designed for use with plasma and no dose response was observed. Taking single point

estimations did give an estimation of FXIa activity of around 2.3 mIU/ml for the proposed 2nd

IS.

Spiking this amount of FXIa into the 1st IS and assaying by FXI clotting assay did not alter the

potency estimate of the 1st IS, so it is highly unlikely that any low level FXIa which may be

present in the proposed 2nd

IS would affect the overall potency estimates.

Stability studies

On-bench and accelerated degradation studies have been carried out. The results of the FXI:C

on-bench stability are shown below, with potencies representing 2 assays at each time-point and

determined relative to a fresh ampoule of 15/180 at each time. Potency is expressed as

percentage relative to the fresh ampoule. The potency after 4 hours storage at room temperature

in a capped plastic tube overlaps well with that at 0 h, indicating the material is stable for at least

4 hours when stored at room temperature.

FXI functional activity (FXI:C)

Time % potency – combined

(95% confidence intervals)

0 h 97.70

(93.58-102.00)

1 h 96.83

(94.15-99.58)

2 h 96.13

(91.38-101.13)

3 h 96.99

(92.51-101.69)

4 h 98.02

(93.33-102.95)

On-bench stability studies of FXI antigen were carried out, with potencies shown below

representing one assay performed with duplicate dilutions. Potency is expressed in percent,

relative to time 0 (assigned to be 100%). The 95% confidence limits at 4 h overlap 100%,

indicating the product is stable for at least 4 hours when stored at room temperature.

FXI antigen (FXI:Ag)

Time % potency – combined

(95% confidence intervals)

1 h 116.09

(105.99-127.24)

2 h 106.48

(97.22-116.65)

3 h 103.16

(94.19-113.00)

4 h 106.57

(97.31-116.76)

WHO/BS/2016.2281

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Accelerated degradation studies have been performed after 1.5 and 4 months storage at low and

high temperatures. The predicted loss per year for FXI:C at each temperature is shown below,

based on cumulative results from both time-points. The predicted percentage loss for FXI

functional activity at the normal storage temperature of -20 °C is 0.002%, showing that the

material is very stable at this temperature. Degradation studies are on-going.

FXI functional activity (FXI:C)

Storage

temperature

(°C)

Predicted loss per

year (%) (relative to

-150)

95% upper confidence

limit (% loss)

-150 <0.001 <0.001

-70 <0.001 <0.001

-20 0.002 0.502

4 0.020 5.687

20 0.068 19.054

37 0.222 44.322

The predicted stability of the antigen is as shown below, though it should be noted that the data

is based on one time-point only. The 95% upper confidence limits for the predicted loss for

antigen are relatively high, indicating a degree of assay variability, which in turn is reflected in

the predicted loss. More data is required to obtain an accurate measure of stability and this will

be achieved by both accelerated and real-time monitoring.

FXI antigen (FXI:Ag)

Storage

temperature

(°C)

Predicted loss per

year (%) (relative to

-70)

95% upper confidence

limit (% loss)

-150 <0.001 <0.001

-70 <0.001 <0.001

-20 0.194 3.100

4 1.775 15.786

20 6.229 31.151

37 19.462 41.168

Participants

Twenty nine laboratories took part in the study for the FXI:C assignment, and eleven for the

FXI:Ag assignment. For the functional assays, the participating laboratories were from Austria

(3), Canada (1), Croatia (1), Denmark (1), France (4), Germany (4), Italy (1) The Netherlands

(1), Spain (2), United Kingdom (9) and USA (2). These comprised of 8 clinical laboratories, 9

therapeutics producers, 6 diagnostics manufacturers, 5 regulatory laboratories and 1 research

laboratory. All participants returned data in time for analysis, however one laboratory (lab 32)

performed single point estimates only, hence was excluded from the overall analysis (these

WHO/BS/2016.2281

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results are shown in Appendix III, Table 3; reported potencies were approximately 6% lower

than the overall geometric mean for the candidate). For the FXI antigen study, the participating

laboratories were from Austria (1), Canada (1), Denmark (1), France (2), Germany (1), Spain (1)

United Kingdom (2) and USA (2), comprising 2 clinical laboratories, 1 diagnostics

manufacturer, 6 plasma therapeutics producers and 2 regulatory laboratories. One other

laboratory withdrew due to problems with supply of the kits from the manufacturers, as the

assays could not have been completed in time for analysis. Each participating laboratory is

referred to in the report by an arbitrarily assigned number and is listed in Appendix I, the order

of which does not represent the assigned laboratory number.

Collaborative study design

The samples sent out for study were as follows:

S (04/102): 1st IS for Factor XI, Plasma, FXI:C potency 0.86 IU/ampoule (used for functional

activity assays only)

A: (15/180): Candidate for 2nd

IS for Factor XI, Plasma

B: (15/180): Coded duplicate

P: Local plasma pool

Although the 2nd

IS factor XI potency will be established relative to the 1st IS, the relationship

between the plasma pool unit was also examined. Since the FXI antigen assignment is a new

value, the unitage of the standard will be assigned relative to normal plasma pools. For these

reasons, participants were asked to collect fresh local plasma pools for use both fresh and

subsequently frozen during the study. Laboratories were asked to follow a supplied protocol for

collection of plasma pools (see collaborative study protocol in appendix II). NIBSC in-house

studies on both FXI functional activity and FXI antigen assays have shown that there is no

significant difference in results when using fresh plasma pools compared to the same pool of

plasma used after freezing, therefore if participants were unable to collect fresh plasma pools,

then frozen plasma could be used as an alternative. A total of 5 laboratories used fresh plasma

pools for the FXI functional assays (18 assays in total), and 2 laboratories used fresh plasma

pools in the FXI antigen assays (4 assays in total). Across the pools used in the study (fresh and

frozen) for either functional activity or antigen value, plasma from more than 20,000 donors was

used.

The issue of commutability was considered, and sourcing of appropriate samples was

investigated. The samples would need to be plasma from Factor XI deficient patients with FXI

levels of around 1-5% of normal. Due to the fact that FXI deficieny is relatively rare and that a

large volume of plasma would be required for the study, it was not possible to source samples for

this purpose. Commerical sources of deficient plasma were either completely deficient

(immune-depeleted) and therefore not appropriate, or were pooled plasmas, which would not

address commutability.

WHO/BS/2016.2281

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Assay methods

Participants were asked to perform their in-house method for factor XI functional activity and

antigen. Four assays for each were requested, using freshly reconstituted ampoules of samples

for each assay. A balanced order of testing was suggested (for details of assay design refer to

collaborative study protocols in appendix II). All participants assaying for factor XI functional

activity employed the one-stage clotting method using an activated partial thromboplastin time

(APTT) reagent. In total, 13 different APTT reagents were used across a variety of instruments

and sources of deficient plasma. For antigen assays, 5 different kits or paired antibody sets were

used. A list of reagents and details of local plasma pools used is given in Tables 1 and 2.

Statistical Analysis

An independent statistical analysis of raw data was performed at NIBSC. Relative potency

estimates were calculated by fitting a parallel-line model3 based on a linear section of the

response range using a minimum of three dilutions for all samples. Non-linearity and non-

parallelism were considered in the assessment of assay validity. All data were plotted and a

visual assessment was used to determine linearity. Non-parallelism was assessed by calculation

of the ratio of fitted slopes for the test and reference samples under consideration. A log10

transformation of the assay response was used for the analysis of the factor XI antigen assays.

No transformation was necessary for the factor XI functional assays.

For the factor XI functional assays, candidates A and B (coded duplicates) were analysed and

potency was assigned relative to the current IS (coded S). Functional activity of these coded

duplicates were also analysed relative to local plasma pools (assuming potency value of 1 u/ml)

and compared with potencies obtained against the current IS. For the factor XI antigen assays,

coded samples A and B were analysed relative to the plasma pool sample (assuming potency

value of 1 u/ml) in each assay to provide a relative potency estimate.

All mean potencies given in this report are unweighted geometric mean (GM) potencies.

Variability between assays and laboratories has been expressed using geometric coefficients of

variation (GCV = {10s-1}×100% where s is the standard deviation of the log10 transformed

potency estimates).

Grubbs’ Test4 was applied to the log transformed laboratory mean estimates in order to detect

any significant outliers. Comparisons between methods were made by appropriate t-tests of log

transformed laboratory mean estimates.

Parallelism of dose-response slopes was evaluated by setting an acceptable range of values for

the ratio of fitted slopes for the test and reference samples. To determine this range for the factor

XI functional assays a parametric tolerance interval (95% confidence, 95% coverage) was

calculated using the ratio of the fitted slopes of coded duplicate samples A and B, and the more

extreme of the upper and lower bounds used with its reciprocal value to set symmetrical limits on

the acceptable ratio of slopes around 1. This result was an acceptable ratio of 0.91 to 1.10.

WHO/BS/2016.2281

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The same calculation method was used for the factor XI antigen assays, with a result of 0.79 to

1.27, and it was decided to use limits of 0.80 to 1.25 to demonstrate acceptable parallelism in

these assays. This narrower range was selected because it is generally considered to be the

widest range acceptable for these types of assays.

Assay validity

FXI functional assays

All laboratories performed 4 assays using one-stage clotting assay, with a total of thirteen

different APTT reagents used across 140 assays. Where a laboratory had performed more than

one set of assays using a different APTT reagent or coagulometer, these are designated as a, b

etc. In the case of labs 26 and 31, a and b refer to the two different sources of plasma pools used

within the same assays therefore these labs have one set of results relative to S, but two results

relative to P (one set relative to each plasma pool). Assay 3 from Lab 17 was excluded from the

analysis due to poor replicates and, for the analysis relative to S, all of the assays from Lab 17

were excluded because the results were found to be outliers. In addition, for the whole study, a

total of 8 assays for A against S, 6 for B against S, 19 for A against P and 13 for B against P

were excluded from the analysis for non-parallelism.

FXI antigen assays

With the exception of Lab 26, which performed 6 assays, all laboratories performed 4 antigen

assays for FXI antigen. In the case of lab 26, a and b refer to the two different sources of plasma

pools used within the same assays, therefore lab 26 has two set of results for FXI antigen against

P. Of the 52 assays performed, 4 assays of B against P were excluded for non-parallelism. No

assays for A against P were excluded for non-parallelism. One assay from Lab 24 was

withdrawn by the laboratory.

RESULTS AND DISCUSSION

FXI functional assays

Analysis relative to S, the 1st IS for FXI, Plasma

Results for samples A and B were analysed relative to sample S, the 1st IS for FXI, and the

individual assay results together with the laboratory geometric mean and GCV are shown in

Tables 3 and 4. For comparison, the laboratories’ own calculated results relative to S are shown,

alongside the centrally calculated results, in Appendix III, Table 1. On the whole, the

laboratories’ own calculated data were similar to NIBSC’s calculated results, indicating that the

laboratories were able to analyse the data appropriately. The laboratories were also able to

perform precise assays, with the majority of intra-laboratory GCVs being less than 5% for A or

B. The overall potency of A compared to S was 0.71 IU/ampoule, with a GCV of 1.60%. For B,

it was also 0.71 IU/ampoule, with a GCV of 2.40%. The good agreement of potencies for

sample A and B, the coded duplicates, showed that there was no assay design bias and that the

WHO/BS/2016.2281

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laboratories were able to measure FXI:C accurately. As samples A and B were coded duplicates,

the results for both A and B vs S were combined to give an overall estimate (AB vs S). Each

laboratory’s geometric mean and GCV are shown in Table 5. The overall result of AB vs S was

0.71 IU/ampoule, the same as for A or B individually against S, and the inter-laboratory GCV

was 1.80%. Results from Lab 17 were detected as statistical outliers and were excluded from the

overall calculation of potency and inter-laboratory GCV. The excellent agreement between the

laboratories as shown by the close distribution of laboratory geometric means is demonstrated in

Figure 1.

Several of the laboratories used Actin FS, SynthASil or APTT-SP as the APTT reagent in their

assays. To determine if these reagents produced any method bias, the AB vs S geometric means

of these laboratories were combined and compared to the overall geometric mean. Table 6

shows that the results for Actin FS (0.70 IU/ampoule; 5 laboratories), SynthASil (0.71

IU/ampoule; 7 laboratories) and APTT-SP (0.72 IU/ampoule; 5 laboratories) compared well with

the overall geometric mean of 0.71 IU/ampoule for AB, indicating no method bias. The overall

geometric mean of the other APTT reagents (excluding Actin FS, SynthASil and APTT-SP) was

0.71 IU/ampoule, with 95% confidence limits of 0.701-0.716 IU/ampoule.

Analysis relative to plasma pools (P)

Participants were asked to collect two plasma pools (P1 and P2) for use both as fresh and frozen

pools in the assays. Where participants were unable to collect fresh plasma pools, it was

requested that two different batches of local frozen plasma pools were used. In order to assess

the relationship with the plasma unit (where the amount of FXI in 1 ml of plasma is taken to be 1

u/ml), the data were analysed relative to P. The assay results for A and B vs P for each

laboratory are shown in Tables 7 and 8, together with the laboratory geometric means and intra-

laboratory variation (GCV). The intra-laboratory variations were higher than that of the data

compared to S, most likely due to the differences between the batches of plasma pools used

within each laboratory. However, the majority of the results still had a GCV of less than 10%,

showing that the laboratories were performing the assays with good precision. The overall

geometric mean for sample A or B relative to P was 0.72 u/ampoule, agreeing well with the

potency of 0.71 u/ampoule relative to S. As before, the results for A and B relative to P were

combined, and the overall results shown in Table 9 and Figure 2. The inter-laboratory variation

was higher than for AB vs S, at 7.20% overall, but still below 10%. The overall geometric mean

for AB vs P was 0.72 u/ampoule, compared to 0.71 IU/ampoule relative to S. The results for AB

vs S and AB vs P were compared using a paired two-tailed t-test and the result showed no

significant difference between the two values (p=0.215), indicating that a good relationship

between the IU and the plasma unit had been maintained. A comparison of assays performed

using fresh plasma pools to assays where frozen plasma pools were used showed there was no

difference in the results between fresh and frozen pools (p=0.704).

WHO/BS/2016.2281

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FXI antigen assays

Participants were asked to collect two plasma pools (P1 and P2) for use both as fresh and frozen

pools in the assays. Where participants were unable to collect fresh plasma pools, it was

requested that two different batches of local frozen plasma pools were used. As there is no

current International Standard for FXI antigen, the units of FXI antigen in 1 ml of plasma pool

was taken to be 1, and the potencies of A and B calculated relative to the laboratories’ local

plasma pool. The individual assay results for samples A and B, together with the laboratory

geometric mean and GCV are shown in Tables 10 and 11. The laboratories own calculated

results are shown in Appendix III, Table 2, alongside the centrally calculated results. As with

the FXI:C results, in general, the locally calculated data were similar to those obtained by

NIBSC. The overall geometric mean for A against P was 0.78 u/ampoule, compared to 0.79

u/ampoule for B against P. The inter-laboratory variation was 10% for A and B, showing

acceptable agreement between laboratories. Overall, the intra-laboratory variations ranged

between 0.5 and 18%, with most laboratories having a GCV of less than 10%. The variation of

these assays would be expected to be higher than that of the FXI functional assays, by virtue of

the fact that the calculations are relative to the local plasma pools, which themselves have

inherent variability. Due to the low number of assays performed using fresh plasma pools (5) it

was not possible to compare fresh and frozen plasma pools.

Since A and B were coded duplicates, the results were combined to give an overall result for AB

against P. The results are shown in Table 12. The overall geometric mean was 0.78 u/ampoule,

in good agreement with the results for A or B separately against P. Again, the variability of the

assays was acceptable, at 10% overall. No statistical outliers were detected. The individual

assay results from each lab are shown graphically in Figure 3, and the laboratory geometric

means are shown plotted in Figure 4. These graphs show a fairly even distribution of results

around the overall geometric mean.

Of the 12 laboratories that took part, 5 used an antigen kit from Affinity Biologicals. The

geometric means for AB against P of these laboratories were combined and resulted in a

geometric mean of 0.72 u/ampoule (with 95% confidence limits of 0.695-0.749 u/ampoule).

This is slightly lower than the overall result for AB against P (0.78 u/ampoule). The result for

the other laboratories not using the Affinity Biologicals kit was 0.82 u/ampoule, with 95%

confidence limits of 0.754-0.899 u/ampoule, which overlap the overall geometric mean of AB vs

P, but not that of the laboratories using the Affinity Biologicals kit. A comparison of the two

groupings was made using the unpaired two-tailed t-test and the difference was found to be

significant (p<0.05). The difference suggests that the Affinity Biologicals kit may yield lower

results than other kits, however this could also be a reflection of the different plasma pools used

within each laboratory. Due to the small number of laboratories taking part in the study and the

fact that the two groupings had 95% confidence limits that were extremely close to being

overlapping, the consensus geometric mean was taken as the antigen value for the candidate

preparation. Any effect of minor method bias on the results is likely to be small.

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RECOMMENDATIONS

The recommendations to the Expert Committee for Biological Standardization (ECBS) of the

World Health Organization (WHO) are:

To establish candidate 15/180 as the 2nd International Standard for FXI, Plasma, with

assigned values of:

o FXI functional activity (FXI:C) of 0.71 IU/ampoule

o FXI antigen value (FXI:Ag) of 0.78 IU/ampoule

A draft Instruction For Use (IFU) is shown in Appendix IV.

The collaborative study has been reviewed by the Participants and the Experts nominated by the

Scientific and Standardisation Committee (SSC) of the International Society on Thrombosis and

Haemostasis and their comments and the NIBSC corresponding responses are shown below. All

participants agreed with the recommendation and the SSC has also endorsed the proposal at the

Business Meeting of the 62nd

Annual SSC meeting, June 2016 to go forward for establishment by

the ECBS.

Participants’ Review

All participants were sent a copy of the report along with a questionnaire asking for their

agreement or otherwise with the recommendations to establish candidate 15/180 as the 2nd

IS for

Factor XI, Plasma, with a FXI:C value of 0.71 IU/ampoule and a FXI:Ag value of 0.78

IU/ampoule. The participants were requested to respond within two weeks if they had any

comments or objections to the recommendations, otherwise a null response would be taken as

agreement with no comments. Twenty-two of the participants chose to respond, all giving their

agreement to the recommendations. Any comments were related to the names or affiliations of

the participants and one requested a minor change to Figure 2, all of which have been completed.

SSC Experts’ Review

The proposals and recommendations presented in this report were approved at the SSC Board

Meeting in Montpellier in May 2016. Nine responses from the expert review were received and

all endorsed the project. Some minor typographical errors were highlighted; all of which have

been corrected. Other comments and NIBSC responses are shown below.

Reviewer A:

“The number of data is impressive and the study design is the best as possible.

I have only some minor remarks

Table 1:

In column 4 the company/source of the deficient plasma is mentioned.

Is this also the company of the APTT reagent? This information is missing in the table.

WHO/BS/2016.2281

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For the deficient plasma much more important information is the properties of the deficient

plasma used; is it immune depleted plasma or congenital deficient plasma. It is interesting if

there is a difference in result. If not this could also be mentioned as a remark.

Appendix III Table 1:

The calculated result by the own laboratory or by the NIBSC calculation is not always the same.

Therefore in the development of an international standard not only the comparison of the rough

measured data is important, but also to teach the laboratories the method to extract the final

result.”

NIBSC response:

The source of APTT reagent has been added to Table 1.

For the deficient plasma, 6 laboratories used plasma from deficient donors (congenital) and there

was no difference in potency from these labs (average 0.70 IU/ampoule with 95% confidence

limits of 0.68-0.73 IU/ampoule) compared to the overall assigned potency.

On occasion there are some discrepancies between the laboratory reported results and the NIBSC

calculated results. In these instances the participating laboratory is contacted and possible

reasons for the discrepancies are discussed. Usually the discrepancies are identified as being

caused by a reporting error in the raw data. The laboratories taking part in such studies are

usually expert laboratories that are adept at using appropriate data analysis methods.

Reviewer B:

There are two issues the authors need to address.

1) Please justify why the blood for this study was centrifuged at 4 degrees Celsius? This fact can

result in C1 inhibitor inactivation (Weiss R et al. Blood 68:239,1986) with increased in vitro

FXII contact activation on the tube surface resulting in prior activation and lowering of zymogen

FXI in the ampules. Perhaps that is the reason the level of FXI is only 0.71 U/ml in the 2nd

ampuole. It also leads to cold activation of FVII and FIX by FXIIa (Seligsohn U et al. Thromb

Res. 13:1049, 1978).

2) Should the results be expressed as “u/ml” or “U/ml”. What is the international standard.

NIBSC response:

1. From the pre-study questionnaires it was identified that only a few laboratories were able

to collect fresh plasma pools for the study (5 labs). The decision was taken to

recommend centrifugation at 4°C in order to maintain consistency with frozen plasma

pools which are routinely prepared by centrifugation in this manner. The potency of

FXI:C is assigned relative to the 1st IS and not the plasma pools, so the centrifugation of

local plasma pools at 4°C would not impact the overall assigned potency of FXI:C. The

bulk material itself was maintained at room temperature throughout the duration of the

fill in order to avoid cold activation and in-house studies using NAPTT and FXIa assays

indicated minimal activation of the product had occurred.

2. The results for FXI:C are expressed u/ampoule when referring to results calculated

relative to plasma pools because it is an accepted assumption that 1 ml of plasma contains

1 unit, rather than being an official unitage. The FXI:Ag results are expressed u/ampoule

throughout the report as there is no International Standard for FXI:Ag. The

WHO/BS/2016.2281

Page 13

recommendations refer to the assignment of units in IU/ampoule because this is the

terminology that would be used on establishment of the standard.

Reviewer C:

“A well designed study from an extensive number of laboratories yielding a conclusive

agreement on the potency of the FXI standard in terms of both activity and antigen. My only

slight concern is the large intra-laboratory variation of a couple of the reported laboratories

particularly number 27. However, the data from the remaining laboratories is sufficiently tight to

support the proposal.”

NIBSC response:

A small number of laboratories do have intra-laboratory variations that are higher than others;

however, it is the geometric mean from each laboratory that is used to calculate the overall

potency. The inter-laboratory agreement for FXI:C is excellent with a GCV of 1.8% and for

FXI:Ag is good at 10%. In this case, intra-laboratory variation has minimal impact on the

overall potency estimate and, in some cases, could be a reflection of the differences in the two

plasma pools used by each laboratory and/or an indication of the performance of the laboratory.

Reviewer D:

This is a carefully performed study and the recommendations of the Expert Committee for

Biological Standardization of the World Health Organization are sound.

Reviewer E:

Study & analyses appear to have been performed in a careful manner and the conclusions are

well supported by the study results.

WHO/BS/2016.2281

Page 14

Table 1: List of APTT reagent, incubation time, FXI deficient plasma, coagulometer, and

plasma pool used by each participant for FXI functional assays

Lab APTT reagent

(manufacturer)

Incubation

time

Deficient plasma

source

Coagulometer Plasma pool (no.

donors)

1 Actin FS

(Siemens)

180 s Siemens BCS-XP Local frozen (450)

2 SynthASil

(IL)

180 s IL ACL TOP 700 Local fresh (P1-13,

P2-14)

3 Actin FS

(Siemens)

180 s Dade Sysmex CS5100 Local frozen (6, 20,

10, 20)

4 Cephen

(Hyphen

BioMed)

240 s Hyphen BioMed STAR Commercial frozen

(P1-20, P2-20)

5a Pathromtin SL

(Siemens)

120 s Siemens BCS XP Local fresh (P1-10,

P2-10)

5b Actin FSL

(Siemens)

180 s Siemens BCS XP Local fresh (P1-10,

P2-10)

5c Pathromtin SL

(Siemens)

180 s Siemens CA-1500 Local fresh (P1-10,

P2-10)

5d Actin FS

(Siemens)

180 s Siemens CA-1500 Local fresh (P1-10,

P2-10)

8 APTT-SP

(Werfen)

165 s Helena Biosciences ACL 200/3000

Plus

Local frozen (P1-

1000, P2-1000)

9 Cephascreen

(Stago)

300 s Stago BCS-XP Commercial frozen

(P1-20, P2-10)

10 SynthASil

(IL)

180 s IL ACL TOP 500 Local fresh (P1-8,

P2-9)

11 SynthASil

(IL)

200 s American

Diagnostica

ACL TOP 500 Commercial frozen

(P1-20, P2-20)

12 Actin FSL

(Siemens)

180 s Siemens BCS-XP Commercial frozen

(20)

13 DG-APTT

Synth

(Diganostic

Grifols)

300 s Diagnostic Grifols Q Hemostasis

Analyzer

Local frozen (P1-

21, P2-21)

14 APTT-SP

(IL)

360 s Precision Biologic

(frozen)

STA-R

Evolution

Local frozen (1800)

16 APTT-SP

(IL)

300 s Diagnostic Grifols ACL Elite Pro Local frozen (P1-

4500, P2-4500, P3-

4500, P4-4500)

17 CK Prest

(Stago)

240 s Affinity

Biologicals

(frozen)

STA-R

Evolution

Local frozen (P1-

20, P2-20)

18 Pathromtin SL

(Siemens)

120 s Siemens BCS-XP Commercial

(P1-not specified,

P2-20, frozen)

19 CK Prest

(Stago)

240 s Diagnostica Stago STAR Commercial

lyophilised (1)

WHO/BS/2016.2281

Page 15

20 SynthASil

(IL)

180 s Technoclone ACL TOP 700 Commercial frozen

(P1-20, P2-20)

21 Actin FSL

(Siemens)

180 s Siemens Sysmex

CA7000

Local frozen (35)

22 SynthASil

(IL)

180 s IL ACL TOP 500 Fresh (P1-6, P2-6)

23 SynthASil

(IL)

200 s IL ACL TOP 700 Fresh (p1-8, P2-8)

24 APTT-SP

(IL)

300 s Stago ACL TOP 500 Fresh (P1-10, P2-

10)

25a Dapttin

(Technoclone)

240 s Technoclone Ceveron Alpha Frozen (P1-12, P2-

12)

25b Siron LS

(Technoclone)

240 s Technoclone Ceveron Alpha Frozen (P1-12, P2-

12)

26a SynthAFax

(IL)

300 s Affinity

Biologicals

Elite Pro Commercial frozen

(20)

26b SynthAFax

(IL)

300 s Affinity

Biologicals

Elite Pro Commercial frozen

(30)

27a CK Prest

(Stago)

240 s Stago STA-R Local frozen (P1-

20, P2-55)

27b Cephascreen

(Stago)

240 s Stago STA-R Local frozen (P1-

20, P2-55)

27c PTT A

(Stago)

240 s Stago STA-R Local frozen (P1-

20, P2-55)

27d CK Prest

(Stago)

240 s Stago

(ImmunoDeficient)

STA-R Local frozen (P1-

20, P2-55)

28 APTT-SP

(IL)

300 s IL ACL 9000 Fresh (P1-8, P2-8)

29 Actin FS

(Siemens)

180 s Diagnostica Stago BCS-XP Fresh (P1-8, P2-8)

30 Actin FS

(Siemens)

180 s Precision Biologic

(frozen)

Sysmex CS5100 Commercial frozen

(P1-20) and local

frozen (P2-not

specified)

31a SynthASil

(IL)

200 s Haematologic

Technologies

(frozen)

ACL TOP 500 Local frozen (1134)

31b SynthASil

(IL)

200 s Haematologic

Technologies

(frozen)

ACL TOP 500 Local frozen (388)

WHO/BS/2016.2281

Page 16

Table 2: List of FXI antigen kit/paired antibodies and local plasma pool used by each laboratory

Lab Antibody/kit source Plasma pool (no. donors)

Lab 6 Cedarlane paired

antibodies

Commerical frozen (P1-20, P2-20)

Lab 7 Cedarlane Lyophilised (donors not specified)

Lab 14 Coachrom paired

antibodies

Fresh (P1-1800, P2-1800)

Lab 15 Molecular Innovation kit Commercial frozen (p1-10, P2-10, P3-

10, P4-10)

Lab 16 Dunn Labortechnick kit Local frozen (P1-4500, P2-4500, P3-

4500, P4-4500)

Lab 17 VisuLize ELISA Kit,

Affinity Biologicals

Local frozen (P1-20, P2-20)

Lab 18 CoaChrom paired

antibodies

Commercial frozen (P1-not specified,

P2-20)

Lab 24 VisuLize ELISA Kit,

Affinity Biologicals

Fresh (P1-10, P2-10)

Lab 26a VisuLize ELISA Kit,

Affinity Biologicals

Commercial frozen (single batch-20)

Lab 26b VisuLize ELISA Kit,

Affinity Biologicals

Commercial frozen (single batch-30)

Lab 28 VisuLize ELISA Kit,

Affinity Biologicals

Local frozen (P1-8, P2-8)

WHO/BS/2016.2281

Page 17

Table 3: Individual assay results for FXI functional activity in IU/ampoule for Sample A against

Sample S, the 1st IS for FXI, Plasma. Each laboratory’s geometric mean is reported and intra-

laboratory variation is shown as GCV (%). Lab 17 (shaded) was excluded from the overall

analysis as an outlier. The overall geometric mean and inter-laboratory GCV is also shown.

Figures in brackets indicate the 95% confidence limits. NP: non-parallel.

Sample A vs Sample S

Lab Assay

1

Assay

2

Assay

3

Assay

4

Geometric

mean

(IU/amp)

GCV

1 0.70 0.66 0.70 0.68 0.68 3.10%

2 0.74 0.75 0.73 0.72 0.74 2.10%

3 0.70 0.66 0.73 0.71 0.70 4.80%

4 0.70 0.72 0.74 0.70 0.71 2.70%

5a 0.71 0.70 0.73 0.74 0.72 2.70%

5b 0.70 0.71 0.73 0.74 0.72 2.50%

5c 0.71 0.70 0.70 0.72 0.71 1.50%

5d 0.73 0.72 0.70 0.70 0.71 2.00%

8 0.70 0.73 0.71 0.73 0.72 2.10%

9 0.69 0.70 0.74 0.69 0.71 3.30%

10 0.71 0.68 0.71 0.75 0.71 3.90%

11 0.69 0.70 0.71 0.70 0.70 1.10%

12 0.74 0.71 0.70 0.73 0.72 2.50%

13 0.72 0.70 0.69 0.72 0.71 2.00%

14 0.75 0.73 0.69 0.72 0.72 3.80%

16 0.70 NP 0.70 0.71 0.70 0.55%

17 0.74 0.80 - 0.81 0.78 4.80%

18 0.73 0.73 0.72 0.71 0.72 1.10%

19 0.70 0.72 0.70 0.72 0.71 1.40%

20 0.70 0.75 0.67 0.70 0.70 4.70%

21 0.71 0.73 0.70 0.69 0.71 2.40%

22 0.73 0.69 0.70 0.74 0.72 3.50%

23 0.78 0.71 0.71 0.71 0.73 5.10%

24 0.69 0.75 0.73 0.70 0.72 3.70%

25a 0.66 NP NP 0.77 0.71 -

25b 0.73 0.70 0.73 NP 0.72 2.50%

26 NP 0.74 0.73 NP 0.73 -

27a NP 0.70 0.70 NP 0.70 -

27b 0.68 0.69 0.74 0.87 0.74 12.00%

27c 0.70 0.65 0.69 0.79 0.70 8.10%

27d 0.74 0.68 0.72 0.71 0.71 3.20%

28 0.68 0.73 0.70 0.71 0.71 3.10%

29 0.71 0.70 0.70 0.71 0.70 1.20%

30 0.70 0.71 0.72 0.73 0.71 1.60%

31 0.70 0.72 0.69 0.70 0.70 2.10%

Overall result for A vs S

0.71

(0.708-0.719)

2.30%

Overall result for A vs S, excluding outliers 0.71

(0.708-0.716)

1.60%

WHO/BS/2016.2281

Page 18

Table 4: Individual assay results for FXI functional activity in IU/ampoule for Sample B against

Sample S, the 1st IS for FXI, Plasma. Each laboratory’s geometric mean is reported and intra-

laboratory variation is shown as GCV (%). Lab 17 (shaded) was excluded from the overall

analysis as an outlier. The overall geometric mean and inter-laboratory GCV is also shown.

Figures in brackets indicate the 95% confidence limits. NP: non-parallel.

Sample B vs Sample S

Lab Assay

1

Assay

2

Assay

3

Assay

4

Geometric

mean

(IU/amp)

GCV

1 0.71 0.65 0.68 0.68 0.68 3.90%

2 0.70 0.72 0.73 0.71 0.72 1.50%

3 0.70 0.670 0.76 0.71 0.71 3.80%

4 0.70 0.72 0.73 0.71 0.71 1.60%

5a 0.69 0.71 0.74 0.75 0.72 3.80%

5b 0.69 0.71 0.70 0.73 0.71 2.50%

5c 0.72 0.70 0.67 0.72 0.70 3.30%

5d 0.72 0.72 0.69 0.70 0.71 2.40%

8 0.73 0.73 0.73 0.76 0.74 1.70%

9 0.72 0.68 0.71 0.71 0.70 2.50%

10 0.75 0.68 0.71 0.74 0.72 4.70%

11 0.70 0.70 0.70 0.70 0.70 0.15%

12 0.73 0.73 0.70 0.70 0.72 2.20%

13 0.74 0.71 0.69 0.72 0.72 2.90%

14 0.77 0.71 0.70 0.68 0.72 5.50%

16 0.73 0.73 0.72 0.71 0.72 1.70%

17 0.74 0.79 - 0.80 0.77 4.60%

18 0.72 0.71 0.68 0.72 0.71 2.90%

19 0.73 0.70 0.68 0.72 0.71 3.00%

20 0.67 0.74 0.68 0.68 0.69 4.60%

21 0.74 0.74 0.69 0.71 0.72 3.70%

22 0.75 0.73 0.70 0.72 0.73 3.00%

23 0.70 0.71 0.72 0.69 0.70 1.50%

24 0.72 0.74 0.71 0.73 0.72 1.60%

25a 0.73 0.65 0.58 0.80 0.69 15.00%

25b NP 0.72 0.75 0.72 0.73 2.10%

26 NP 0.71 0.76 NP 0.74 -

27a NP 0.70 0.71 NP 0.70 -

27b 0.65 0.69 0.63 NP 0.66 4.20%

27c 0.67 0.62 0.61 0.85 0.68 16.00%

27d 0.73 0.67 0.70 0.71 0.70 3.60%

28 0.69 0.72 0.72 0.70 0.71 2.50%

29 0.70 0.69 0.68 0.70 0.69 0.86%

30 0.71 0.71 0.70 0.72 0.71 0.99%

31 0.71 0.72 0.68 0.71 0.71 2.80%

Overall result for B vs S

0.71

(0.703-0.717)

2.80%

Overall result for B vs S, excluding outliers

0.71

(0.702-0.714)

2.40%

WHO/BS/2016.2281

Page 19

Table 5: The geometric mean for each laboratory (IU/ampoule) for samples A and B (coded

duplicates) combined and analysed relative to sample S, the 1st IS for FXI, Plasma. Intra-

laboratory variation is shown as GCV (%). Lab 17 (shaded) was excluded from the overall

analysis as an outlier. The overall geometric mean and inter-laboratory GCV is also shown.

Figures in brackets indicate the 95% confidence limits.

Samples AB vs Sample S

Lab Geometric mean

(IU/amp)

GCV

1 0.68 3.40%

2 0.73 1.40%

3 0.71 4.00%

4 0.71 2.10%

5a 0.72 3.20%

5b 0.71 2.30%

5c 0.71 2.40%

5d 0.71 2.20%

8 0.73 1.70%

9 0.70 2.10%

10 0.71 4.00%

11 0.70 0.54%

12 0.72 1.90%

13 0.71 2.30%

14 0.72 4.40%

16 0.71 1.60%

17 0.78 4.70%

18 0.72 1.60%

19 0.71 1.80%

20 0.70 4.30%

21 0.71 2.90%

22 0.72 2.70%

23 0.72 2.30%

24 0.72 2.30%

25a 0.67 13.00%

25b 0.72 1.60%

26 0.74 -

27a 0.70 -

27b 0.72 13.00%

27c 0.69 12.00%

27d 0.71 3.30%

28 0.71 2.60%

29 0.70 0.96%

30 0.71 1.10%

31 0.70 2.30%

Overall results for AB vs S

0.71

(0.706-0.718)

2.30%

Overall results for AB vs S,

excluding outliers

0.71

(0.706-0.715)

1.80%

WHO/BS/2016.2281

Page 20

Figure 1: Histogram showing each laboratory’s geometric mean for FXI:C of samples A and B

combined, relative to sample S, the 1st IS for FXI, Plasma. Outliers are shown in red. The

overall geometric mean was 0.71 IU/ampoule with a GCV of 1.8%.

Table 6: Laboratory geometric means for AB vs S for APTT reagents Actin FS, SynthASil and

APTT-SP, their overall geometric mean and comparison to overall results for AB vs S

Lab number

(geometric mean of AB vs S, IU/amp)

Overall geometric

mean, IU/amp

(95% CL)

Overall geometric

mean of AB vs S (all

APTT), IU/amp (95%

CL)

Actin FS

(n=5)

1

(0.68)

3

(0.71)

5d

(0.71)

29

(0.70)

30

(0.71) -

- 0.70

(0.685-0.718)

0.71

(0.706-0.715) SynthASil

(n=7)

2

(0.73)

10

(0.71)

11

(0.70)

20

(0.70)

22

(0.72)

23

(0.72)

31

(0.70)

0.71

(0.700-0.722)

APTT-SP

(n=5)

8

(0.73)

14

(0.72)

16

(0.71)

24

(0.72)

28

(0.71) -

- 0.72

(0.707-0.728)

CL: Confidence limits

Num

ber

of Labora

tories

0

2

4

6

8

10

12

14

16

18

20

Potency (IU/ampoule)

0.5 0.6 0.7 0.8 0.9 1.0

1

25a

3

9

11

13

19

20

28

29

30

31

27a

27c

27d

5c

5d

2

4

8

10

12

14

16

18

21

22

23

24

26

25b

27b

5a

5b

17

WHO/BS/2016.2281

Page 21

Table 7: Individual assay results for FXI functional activity for Sample A against Sample P,

each laboratory’s local plasma pool. Each laboratory’s geometric mean is reported and intra-

laboratory variation is shown as GCV (%). The overall geometric mean and inter-laboratory

GCV for A against P is also shown. Figures in brackets indicate the 95% confidence limits. NP:

Non-parallel

Sample A vs Sample P

Lab Assay

1

Assay

2

Assay

3

Assay

4

Geometric

mean

(u/amp)

GCV

1 0.75 0.62 0.71 0.65 0.68 8.90%

2 0.64 NP 0.72 NP 0.68 -

3 0.71 0.70 0.65 0.70 0.69 4.10%

4 0.85 0.83 0.75 NP 0.81 6.90%

5a 0.67 NP 0.67 0.68 0.67 1.20%

5b 0.71 0.71 0.73 0.74 0.72 2.50%

5c 0.66 0.67 0.66 0.68 0.67 1.70%

5d 0.64 0.67 0.67 0.64 0.65 2.20%

8 0.75 0.75 NP 0.71 0.74 3.70%

9 0.70 NP 0.84 0.80 0.78 10.00%

10 0.64 0.63 0.69 0.68 0.66 4.60%

11 0.81 0.81 0.76 0.74 0.78 4.50%

12 0.76 0.74 0.73 0.75 0.74 1.80%

13 0.64 0.70 0.62 0.69 0.66 6.10%

14 0.78 0.79 0.81 0.82 0.80 2.50%

16 0.74 0.79 NP 0.82 0.78 5.20%

17 0.72 0.70 - 0.72 0.71 1.40%

18 0.81 0.87 0.81 NP 0.83 4.00%

19 0.73 0.72 0.76 NP 0.74 3.20%

20 0.74 0.77 0.73 0.81 0.76 5.10%

21 0.69 0.76 0.71 0.68 0.71 4.90%

22 0.69 0.68 0.70 0.72 0.70 2.50%

23 0.77 0.67 0.70 0.70 0.71 5.80%

24 0.64 0.68 0.69 0.69 0.68 3.70%

25a NP 0.80 0.78 NP 0.79 -

25b NP 0.78 0.81 NP 0.80 -

26a NP NP 0.68 NP 0.68 -

26b 0.70 0.78 0.70 0.71 0.72 5.60%

27a NP 0.72 0.71 0.71 0.71 1.20%

27b 0.76 NP 0.76 NP 0.76 -

27c 0.75 NP 0.67 0.76 0.73 6.90%

27d 0.73 0.72 0.69 0.70 0.71 3.00%

28 0.64 0.67 0.68 0.64 0.66 3.20%

29 0.70 0.67 0.75 0.73 0.71 4.90%

30 0.69 0.72 0.65 0.66 0.68 4.70%

31a 0.75 0.78 0.77 0.76 0.77 1.90%

31b 0.72 0.76 0.74 0.72 0.73 2.50%

Overall result for A vs P 0.72

(0.706-0.738)

6.90%

WHO/BS/2016.2281

Page 22

Table 8: Individual assay results for FXI functional activity for Sample B against Sample P,

each laboratory’s local plasma pool. Each laboratory’s geometric mean is reported and intra-

laboratory variation is shown as GCV (%). The overall geometric mean and inter-laboratory

GCV for B against P is also shown. Figures in brackets indicate the 95% confidence limits. NP:

Non-parallel

Sample B vs Sample P

Lab Assay

1

Assay

2

Assay

3

Assay

4

Geometric

mean

(u/amp)

GCV

1 0.76 0.61 0.69 0.66 0.68 9.40%

2 0.62 0.64 0.71 NP 0.66 7.20%

3 0.71 0.74 0.66 0.70 0.70 4.80%

4 0.85 0.84 0.74 0.74 0.79 8.00%

5a 0.66 NP 0.68 0.69 0.68 2.60%

5b 0.70 0.70 0.70 0.74 0.71 2.80%

5c 0.67 0.67 0.63 0.68 0.66 3.50%

5d 0.64 0.67 0.66 0.64 0.65 2.00%

8 0.79 0.76 NP 0.73 0.76 3.90%

9 0.73 0.67 0.81 0.81 0.75 9.40%

10 0.67 0.63 0.69 0.68 0.67 4.40%

11 0.82 0.81 0.75 0.74 0.78 5.50%

12 0.75 0.76 0.73 0.73 0.74 2.40%

13 0.66 0.72 0.62 0.69 0.67 6.30%

14 0.80 0.77 0.82 0.78 0.79 3.10%

16 0.78 0.78 0.86 0.81 0.81 5.10%

17 0.72 0.69 - 0.71 0.71 2.20%

18 NP 0.85 0.77 0.89 0.84 7.50%

19 0.76 0.70 0.75 0.76 0.74 4.00%

20 0.70 NP 0.74 0.78 0.74 5.50%

21 0.71 0.77 0.70 0.70 0.72 4.70%

22 0.71 0.72 0.70 0.70 0.71 1.50%

23 0.68 0.66 0.71 0.69 0.69 2.90%

24 0.66 0.67 0.67 0.72 0.68 3.50%

25a NP 0.86 0.80 NP 0.83 -

25b 0.82 0.81 0.83 0.87 0.83 3.00%

26a NP NP 0.71 0.66 0.68 -

26b 0.72 0.74 0.74 0.70 0.72 3.00%

27a 0.69 0.72 0.71 0.73 0.71 2.40%

27b 0.72 NP NP NP 0.72 -

27c 0.72 NP 0.60 0.83 0.71 18.00%

27d 0.73 0.71 0.66 0.70 0.70 4.30%

28 0.65 0.66 0.70 0.63 0.66 4.80%

29 0.69 0.67 0.74 0.72 0.70 4.60%

30 0.70 0.73 0.64 0.65 0.68 6.00%

31a 0.76 0.78 0.76 0.78 0.77 1.40%

31b 0.73 0.75 0.72 0.74 0.74 1.70%

Overall result for B vs P 0.72

(0.705-0.739)

7.30%

WHO/BS/2016.2281

Page 23

Table 9: The geometric mean for each laboratory for samples A and B (coded duplicates)

combined and analysed relative to sample P, each laboratory’s local plasma pool. Intra-

laboratory variation is shown as GCV (%). The overall geometric mean and inter-laboratory

GCV for AB vs P is also shown. Figures in brackets indicate the 95% confidence limits.

Samples AB vs Sample P

Lab Geometric mean

(u/amp)

GCV

1 0.68 9.10%

2 0.66 6.70%

3 0.70 4.20%

4 0.79 7.60%

5a 0.67 1.80%

5b 0.72 2.50%

5c 0.66 2.50%

5d 0.65 2.10%

8 0.75 3.60%

9 0.75 10.00%

10 0.66 4.30%

11 0.78 5.00%

12 0.74 1.60%

13 0.67 6.10%

14 0.80 2.20%

16 0.80 5.70%

17 0.71 1.80%

18 0.84 5.40%

19 0.74 3.10%

20 0.76 4.70%

21 0.71 4.70%

22 0.70 0.91%

23 0.70 3.60%

24 0.68 3.20%

25a 0.81 -

25b 0.83 3.60%

26a 0.67 -

26b 0.72 3.80%

27a 0.71 2.10%

27b 0.75 -

27c 0.72 12.00%

27d 0.71 3.60%

28 0.66 3.90%

29 0.71 4.70%

30 0.68 5.30%

31a 0.77 1.40%

31b 0.74 1.90%

Overall results for AB vs P 0.72

(0.705-0.739) 7.20%

WHO/BS/2016.2281

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Figure 2: Histogram showing each laboratory’s geometric mean for FXI:C of samples A and B

combined, relative to sample P, the laboratory’s local plasma pool. The overall geometric mean

was 0.72 IU/ampoule with a GCV of 7.2%.

Num

ber

of Labora

tories

0

2

4

6

8

10

12

14

16

18

20

Potency (units/ampoule)

0.5 0.6 0.7 0.8 0.9 1.0

2

28

5d

1

10

13

24

30

26a

5a

5c

3

17

22

23

29

27a

27d

21

26b

27c

31b

5b

8

9

12

19

20

27b

11

31a

4

14

16

25a

18

25b

WHO/BS/2016.2281

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Table 10: Individual assay results for FXI antigen for Sample A against Sample P, each

laboratory’s local plasma pool. Each laboratory’s geometric mean is reported and intra-

laboratory variation is shown as GCV (%). The overall geometric mean and inter-laboratory

GCV for A against P is also shown. Figures in brackets indicate the 95% confidence limits.

Sample A vs P, u/ampoule

Lab Assay 1 Assay 2 Assay 3 Assay 4 Assay 5 Assay 6 Geometric

mean

(u/amp)

GCV

6 0.60 0.77 0.70 0.72 - - 0.70 11.00%

7 0.88 0.90 0.96 1.00 - - 0.93 6.10%

14 0.75 0.82 0.81 0.77 - - 0.79 4.10%

15 0.72 0.74 0.76 0.84 - - 0.76 6.80%

16 0.86 0.76 0.99 0.89 - - 0.87 11.00%

17 0.73 0.72 0.71 0.72 - - 0.72 1.00%

18 0.76 0.96 0.98 0.86 - - 0.89 13.00%

20 0.77 0.81 0.91 0.75 - - 0.81 8.60%

24 0.72 0.72 - 0.73 - - 0.72 0.47%

26a 0.72 0.68 0.72 0.70 0.84 0.73 0.73 7.70%

26b 0.71 0.60 0.78 0.77 0.82 0.78 0.74 12.00%

28 0.71 0.71 0.72 0.73 - - 0.72 1.20%

Overall result for A vs P

0.78

(0.731-0.827) 10.00%

Table 11: Individual assay results for FXI antigen for Sample B against Sample P, each

laboratory’s local plasma pool. Each laboratory’s geometric mean is reported and intra-

laboratory variation is shown as GCV (%). The overall geometric mean and inter-laboratory

GCV for B against P is also shown. Figures in brackets indicate the 95% confidence limits. NP:

non-parallel

Sample B vs P, u/ampoule

Lab Assay 1 Assay 2 Assay 3 Assay 4 Assay 5 Assay 6 Geometric

mean

(u/amp)

GCV

6 NP 0.73 0.71 0.77 - - 0.74 3.90%

7 0.91 0.89 0.99 1.00 - - 0.94 5.80%

14 0.89 0.74 0.91 0.80 - - 0.83 9.90%

15 0.85 0.75 0.74 0.98 - - 0.82 14.00%

16 NP 0.73 1.01 0.85 - - 0.86 18.00%

17 0.67 0.70 0.67 0.75 - - 0.70 5.30%

18 0.79 0.93 1.05 0.80 - - 0.89 15.00%

20 0.79 0.76 0.92 0.72 - - 0.79 11.00%

24 0.69 0.65 - 0.75 - - 0.69 7.30%

26a 0.68 0.76 NP 0.77 0.87 0.77 0.77 9.40%

26b 0.67 0.67 NP 0.84 0.85 0.82 0.77 13.00%

28 0.74 0.71 0.71 0.73 - - 0.72 1.90%

Overall result for B vs P

0.79

(0.743-0.840) 10.00%

WHO/BS/2016.2281

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Table 12: The geometric mean for each laboratory for samples A and B (coded duplicates)

combined and analysed relative to sample P, each laboratory’s local plasma pool. Intra-

laboratory variation is shown as GCV (%). The overall geometric mean and inter-laboratory

GCV for AB vs P is also shown. Figures in brackets indicate the 95% confidence limits.

Samples AB vs Sample P

Lab Geometric

mean (u/amp)

GCV

6 0.70 11.00%

7 0.94 5.80%

14 0.81 4.50%

15 0.79 9.70%

16 0.86 13.00%

17 0.71 2.80%

18 0.89 13.00%

20 0.80 9.40%

24 0.71 3.80%

26a 0.74 7.60%

26b 0.76 11.00%

28 0.72 1.10%

Overall result for AB vs P

0.78

(0.735-0.832) 10.00%

WHO/BS/2016.2281

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Figure 3: Histogram showing each laboratory’s individual assay results for FXI:Ag of samples

A and B combined, relative to sample P, the laboratory’s local plasma pool. The overall

geometric mean was 0.78 u/ampoule with a GCV of 10.0%.

Num

ber

of A

ssays

0

2

4

6

8

10

12

14

16

18

20

Potency (units/ampoule)

0.5 0.6 0.7 0.8 0.9 1.0 1.1 1.2

6 26b 24 6

17

17

17

24

28

26a

26b

17

20

24

28

28

28

26a

26a

26a

6

6

15

15

16

26a

14

14

15

18

20

26b

20

26b

26b

14

18

26b

14

16

26a

16 7

7

15

20

18 7 7

16

18

WHO/BS/2016.2281

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Figure 4: Histogram showing each laboratory’s geometric mean for FXI:Ag samples A and B

combined, relative to the laboratory’s local plasma pool (sample P).

References

1. Gomez K and Bolton-Maggs P (2008). Factor XI deficiency. Haem 14: 1183-1189.

2. Assignment of potency to the WHO 1st International Standard for blood coagulation

factor XI in plasma, human (04/102) and calibration of blood coagulation factor XI in

SSC secondary plasma standard lot #3. WHO/BS/05.2017.

3. Finney DJ. Statistical Methods in Biological Assay. 3rd Edition. London: Charles Griffin

1978.

4. Grubbs F (1969). Procedures for Detecting Outlying Observations in Samples.

Technometrics: 11: 1-21.

Acknowledgements

The authors would like to thank all the participants of the study and the “Factor XI and the

contact system” sub-committee of the Scientific and Standardization Committee (SSC) of the

International Society on Thrombosis and Haemostasis (ISTH).

Num

ber

of Labora

tories

0

2

4

6

8

10

12

14

16

18

20

Potency (units/ampoule)

0.5 0.6 0.7 0.8 0.9 1.0

6

17

24

28 26a

26b

14

15

20

16

18

7

WHO/BS/2016.2281

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Appendix I – List of participants

Renata Zadro, University Hospital Center Zagreb, Zagreb, Croatia

Joergen Jespersen and Johannes Sidelmann, University of Southern Denmark, Esbjerg, Denmark

Caroline Lawrence and Grainne Hickman, Glasgow Royal Infirmary, Glasgow, United Kingdom

Sophie Desseauve and Jean Amiral, Hyphen Biomed, Neuville Sur Oise, France

Chris Hardway, Bio Products Laboratory, Elstree, United Kingdom

Chris Gardiner and Ian Mackie, University College London, London, United Kingdom

Regina Gebauer and Michael Timme, Siemens Healthcare Diagnostics Products GmbH,

Marburg, Germany

Anne Riddell, Royal Free London NHS Hospital Trust, London, United Kingdom

Frédéric Dhainaut, LFB Biotechnologies, Les Ulis, France

Aurelie Hazotte, LFB, Cedex, France

Amanda Blande, Bio Products Laboratory, Elstree, United Kingdom

Francois Hemery, LFB Biomedicaments, Lille, France

Roger Luddington, Addenbrooke’s Hospital, Cambridge, United Kingdom

Stephanie Eichmeir and Christoph Kefeder, AGES MEA, Vienna, Austria

Steffen Kistner, Jens Daufenbach and Karin Fuchs, Biotest AG, Dreieich, Germany

Mariona Bono and Begona Alonso, Diagnostic Grifols SA, Barcelona, Spain

Enrica Paladini, San Bortolo Hospital, Florence, Italy

Andrea Buchacher, Martina Schwartz and Pieter De Jong, Octapharma PPGmbH, Vienna,

Austria

Katherine Tull, Peter Vandeberg, Maria Cruz, Catherine Russ, Grifols Inc, North Carolina, USA

Berta Pons and Maite López, Instituto Grifols SA, Barcelona, Spain

Denise Foulon, Affinity Biologicals, Ontario, Canada

WHO/BS/2016.2281

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Colin Downey, Royal Liverpool Hospital, Liverpool, United Kingdom

Annette Feussner, CSL Behring GmbH, Marburg, Germany

Valérie LIEVRE, ANSM, Saint Denis, France

Carel Eckmann and Jeanette Rentenaar, Sanquin Blood Supply, Amsterdam, The Netherlands

Kathleen Trumbull, Instrumentation Laboratory, Massachusetts, USA

Annette Bowyer, Royal Hallamshire Hospital, Sheffield, United Kingdom

Nikolaus Binder, Technoclone GmbH, Vienna, Austria

Helen Wilmot and Stella Williams, NIBSC, Potters Bar, United Kingdom

Mikhail Ovanesov, Yideng Liang, Tseday Tegegn and Ravi Rasmi Jasti, FDA/CBER, Maryland,

USA

Claire Martin, Anne Lochu, Jerome Beltran, Marc Grimaux and Francois Nicham, Stago,

Gennevilliers, France

Andreas Hunfeld and Sylvia Rosenkranz, Paul Ehrlich Institute, Langen, Germany

WHO/BS/2016.2281

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Appendix II – Collaborative study protocols for FXI functional activity and FXI antigen

Appendix IIi – FXI functional activity protocol

CALIBRATION OF PROPOSED 2nd INTERNATIONAL STANDARD FOR

FACTOR XI, PLASMA (15/180).

CS546 STUDY PROTOCOL – FXI functional activity

1 SAMPLES FOR ASSAY

CODE PREPARATION

S 1st International Standard for Factor XI, Plasma (04/102), containing 0.86

IU FXI per ampoule

A Candidate A, containing approximately 0.7 units FXI per ampoule

B Candidate B, containing approximately 0.7 units FXI per ampoule

P1 – P2 Fresh normal plasma pools prepared locally according to the instructions

in Appendix one.

2 STORAGE AND RECONSTITUTION OF AMPOULES S, A AND B

Store all unopened ampoules at -20oC or below. For reconstitution, ampoules should first be

allowed to warm to room temperature and subsequently reconstituted by the addition of 1.0 ml of

distilled water. Allow the contents to solubilise for 10-15 minutes at room temperature with

gentle mixing, transfer contents to a plastic tube and store at ambient temperature (18-25oC).

Assays should be completed within 2 hours of reconstitution.

3 OUTLINE OF STUDY

If possible, please collect two sets of normal pooled plasma on days 1 and 3 of the study (see

appendix one) for use fresh and then subsequently frozen. A total of four assays should be

carried out over 4 separate days, using fresh ampoules of S, A and B in each assay. Please

perform Assay 1 on freshly prepared normal plasma pool (P1), Assay 2 on frozen plasma pool

P1, Assay 3 on a second freshly prepared normal plasma pool (P2) and Assay 4 on frozen plasma

pool P2. If freshly prepared normal pooled plasma cannot be collected, please use different

batches of freshly thawed normal pooled plasma as a substitute.

WHO/BS/2016.2281

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4 ASSAY DESIGN

A balanced order of testing should be followed, for example:

Assay 1 S A B P1 P1’ B’ A' S'

Assay 2* A B P1 S S’ P1’ B’ A’

Assay 3 B P2 S A A’ S’ P2’ B’

Assay 4* P2 S A B B’ A’ S’ P2’

*Using frozen plasma

where each letter refers to a set of different dilutions (please use four dilutions if possible, for

example 1/10, 1/30, 1/50, 1/100) and A, A' and S, S' etc. refer to separate sets of dilutions

(replicates) made independently from the same ampoule. The range of dilutions should be

chosen to lie on the most linear portion of the dose-response relationship. The same range of

dilutions should be used for all materials (S, A, B, P1/P2). The assays should be completed

within two hours of reconstitution. It is preferable for the whole study to be carried out over

four days with a fresh plasma pool prepared on two of the days, and then frozen for use in a

second assay on a different day.

If you are using two different APTT reagents for the study, 4 separate assays should be

performed for each reagent (total 8). You may, however, use the same set of reconstituted

samples for the second APTT reagent (therefore performing 2 assays on each day), but please

prepare fresh dilutions for each assay and complete the assays within 2 hours of reconstitution.

5 USE OF FROZEN PLASMA POOLS

Laboratories who are unable to prepare the fresh plasma pools (P1 – P2) may use frozen plasma

pools instead. Ideally different batches should be used. Please enter the details of the

preparations of frozen pool in the results sheets.

6 RESULTS

Raw data (e.g. clotting times) should be recorded on results sheets. You are also invited to

calculate the relative potencies of A, B and P vs S from your own assay results using the

assigned potencies of S given in section 1. Please return your raw data and calculated potency

estimates by 14th December 2015 to helen.wilmot@nibsc.org

APPENDIX ONE

WHO/BS/2016.2281

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PREPARATION OF FRESH NORMAL PLASMA POOLS

Collect fresh normal plasma as described below, on two separate days, giving pools P1 and P2.

The method of collection of 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 pregnant women or women taking oral contraceptives.

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

a minimum of eight 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 a total of at least 12 different donors for

each laboratory.

Anticoagulant

0.109 M tri-sodium citrate, i.e. 3.2% w/v of the dihydrate (or a mixture of tri-sodium citrate and

citric acid with a total citrate concentration of 0.109 M). Ratio of 9 volumes 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 2000 g for 20 minutes.

Pooling and Storage

Pool equal volumes of plasma from the different donors and mix gently. Snap-freeze aliquots of

each pool (P1, P2) for assays 2 and 4. Thaw frozen aliquots at 37oC before use.

Keep the plasma pool in a plastic stoppered tube at ambient temperature (18-25oC) during the

assay session.

Appendix IIii – FXI antigen protocol

CALIBRATION OF PROPOSED 2nd INTERNATIONAL STANDARD FOR

FACTOR XI, PLASMA (15/180).

STUDY PROTOCOL – FXI / FXII antigen

1 SAMPLES FOR ASSAY

CODE PREPARATION

A Candidate A, containing approximately 1.0 u FXI/FXII per ampoule

B Candidate B, containing approximately 1.0 u FXI/FXII per ampoule

WHO/BS/2016.2281

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P1 – P2 Fresh normal plasma pools prepared locally according to the instructions

in Appendix one.

2 STORAGE AND RECONSTITUTION OF AMPOULES A AND B

Store all unopened ampoules at -20oC or below. For reconstitution, ampoules should first be

allowed to warm to room temperature and subsequently reconstituted by the addition of 1.0 ml of

distilled water. Allow the contents to solubilise for 10-15 minutes at room temperature with

gentle mixing, transfer contents to a plastic tube and store at ambient temperature (18-25oC).

3 OUTLINE OF STUDY

If possible, please collect two sets of normal pooled plasma on days 1 and 3 of the study (see

appendix one) for use fresh and then subsequently frozen. A total of four assays should be

carried out over 4 separate days, using fresh ampoules of A and B in each assay. Please perform

Assay 1 on freshly prepared normal plasma pool (P1), Assay 2 on frozen plasma pool P1, Assay

3 on a second freshly prepared normal plasma pool (P2) and Assay 4 on frozen plasma pool P2.

If freshly prepared normal pooled plasma cannot be collected, please use different batches of

freshly thawed normal pooled plasma as a substitute.

4 ASSAY DESIGN

A balanced order of testing should be followed for each assay of FXI antigen and FXII antigen,

for example:

Assay 1:

1 2 3 4 5 6 7 8 9 10 11 12

A P1a P1b P1c P1d

B Aa Ab Ac Ad

C Ba Bb Bc Bd

D Ba’ Bb’ Bc’ Bd’

E Aa’ Ab’ Ac’ Ad’

F P1a’ P1b’ P1c’ P1d’

G X Y Z

H

X- plate blank (no liquids applied to well)

Y – Buffer blank (sample replaced by buffer; other reagents and substrate still applied)

WHO/BS/2016.2281

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Z – Substrate blank (please apply pooled plasma as the sample. Apply all other reagents as

normal but replace the substrate with buffer)

Assay 2:

1 2 3 4 5 6 7 8 9 10 11 12

A

B Aa Ab Ac Ad

C Ba Bb Bc Bd

D P1a P1b P1c P1d

E P1a’ P1b’ P1c’ P1d’

F Ba’ Bb’ Bc’ Bd’

G Aa’ Ab’ Ac’ Ad’

H X Y Z

*Using frozen plasma

Assay 3:

1 2 3 4 5 6 7 8 9 10 11 12

A Ba Bb Bc Bd

B P2a P2b P2c P2d

C Aa Ab Ac Ad

D Aa’ Ab’ Ac’ Ad’

E P2a’ P2b’ P2c’ P2d’

F Ba’ Bb’ Bc’ Bd’

G X Y Z

H

Assay 4:

1 2 3 4 5 6 7 8 9 10 11 12

A

B P2a P2b P2c P2d

C Aa Ab Ac Ad

D Ba Bb Bc Bd

E Ba’ Bb’ Bc’ Bd’

WHO/BS/2016.2281

Page 36

F Aa’ Ab’ Ac’ Ad’

G P2a’ P2b’ P2c’ P2d’

H X Y Z

*Using frozen plasma

where a,b,c and d refer to different dilutions (for example, 1/5, 1/10, 1/20, 1/40) and A, A' and B,

B' etc. refer to separate sets of dilutions (replicates) made from the same ampoule. The range of

dilutions should be chosen to lie on the most linear portion of the dose-response relationship.

The same range of working dilutions should be used for all three materials (A, B, P1, P2). It is

preferable for the whole study to be carried out over four days with a fresh plasma pool prepared

on two of the days, and then frozen for use in a second assay on a different day.

Please use fresh ampoules of A and B for each assay.

If you are performing both FXI and FXII antigen assays, please use the above testing protocol

for both.

5 USE OF FROZEN PLASMA POOLS

Laboratories who are unable to prepare the fresh plasma pools (P1 – P2) may use frozen plasma

pools instead. Ideally different batches should be used. Please enter the details of the

preparations of frozen pool in the results sheets.

6 RESULTS

Raw data (e.g. clotting times) should be recorded on results sheets. You are also invited to

calculate the relative antigen content of A and B vs P from your own assay results using a value

of 1 u/ml for P. Please return your raw data and calculated antigen estimates by 14th December

2015 to helen.wilmot@nibsc.org

WHO/BS/2016.2281

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APPENDIX ONE

PREPARATION OF FRESH NORMAL PLASMA POOLS

Collect fresh normal plasma as described below, on two separate days, giving pools P1 and P2.

The method of collection of 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 pregnant women or women taking oral contraceptives.

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

a minimum of eight 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 a total of at least 12 different donors for

each laboratory.

Anticoagulant

0.109 M tri-sodium citrate, i.e. 3.2% w/v of the dihydrate (or a mixture of tri-sodium citrate and

citric acid with a total citrate concentration of 0.109 M). Ratio of 9 volumes 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 2000 g for 20 minutes.

Pooling and Storage

Pool equal volumes of plasma from the different donors and mix gently. Snap-freeze aliquots of

each pool (P1, P2) for assays 2 and 4. Thaw frozen aliquots at 37oC before use.

Keep the plasma pool in a plastic stoppered tube at ambient temperature (18-25oC) during the

assay session.

WHO/BS/2016.2281

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Appendix III – Participants’ own calculated geometric mean values (where reported)

Table 1: Geometric means of all 4 FXI functional assays from each laboratory, as calculated by

the laboratory itself and centrally at NIBSC, relative to sample S (n=4 unless stated otherwise)

FXI functional activity relative to S, IU/ampoule

Laboratory’s own results NIBSC calculated results

Laboratory Sample A Sample B Sample A Sample B

Lab 1 0.68 0.68 0.68 0.68

Lab 2 0.73 0.72 0.74 0.72

Lab 3

(Used S as 1

IU/ml)

0.82 0.83

0.70 0.71

Lab 4 0.70 0.71 0.71 0.71

Lab 5a 0.73 0.73 0.72 0.72

Lab 5b 0.73 0.71 0.72 0.71

Lab 5c 0.72 0.71 0.71 0.70

Lab 5d 0.72 0.71 0.71 0.71

Lab 8 0.72 0.74 0.72 0.74

Lab 9 0.70 0.70 0.71 0.70

Lab 10 0.72 0.77 0.71 0.72

Lab 11 0.70 0.70 0.70 0.70

Lab 12* 0.84 0.84 0.72 0.72

Lab 13 0.70 0.72 0.71 0.72

Lab 14 0.71 0.70 0.72 0.72

Lab 16 0.71 0.72 0.70 (n=3) 0.72

Lab 17 0.81 0.76 0.78 (n=3) 0.77 (n=3)

Lab 18 0.72 0.71 0.72 0.71

Lab 19 0.72 0.72 0.71 0.71

Lab 20 0.67 0.68 0.70 0.69

Lab 21 0.71 0.72 0.71 0.72

Lab 22 Not reported Not reported 0.72 0.73

Lab 23 0.71 0.71 0.73 0.70

Lab 24 0.73 0.73 0.72 0.72

Lab 25a 0.64 0.67 0.71 (n=2) 0.69

Lab 25b 0.70 0.73 0.72 (n=3) 0.73 (n=3)

Lab 26 0.70 0.70 0.73 (n=2) 0.74 (n=2)

Lab 27a 0.71 0.73 0.70 (n=2) 0.70 (n=2)

Lab 27b 0.76 0.75 0.74 0.66 (n=3)

Lab 27c 0.71 0.69 0.70 0.68

Lab 27d 0.72 0.70 0.71 0.70

Lab 28 0.70 0.71 0.71 0.71

Lab 29 0.72 0.72 0.70 0.69

Lab 30 0.69 0.69 0.71 0.71

Lab 31 0.70 0.71 0.70 0.71

Lab 32** Not reported Not reported Not calculated Not calculated

* Laboratory used calibration plasma as the standard instead of sample S

** Laboratory reported single point estimates only

WHO/BS/2016.2281

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Table 2: Geometric means of all FXI antigen assays from each laboratory, as calculated by the

laboratory itself and centrally at NIBSC, relative to sample P (n=4 unless stated otherwise)

FXI antigen value activity relative to P, u/ampoule

Laboratory’s own results NIBSC calculated results

Laboratory Sample A Sample B Sample A Sample B

Lab 6 0.72 0.73 0.70 0.74 (n=3)

Lab 7 0.94 0.95 0.93 0.94

Lab 14 0.81 0.85 0.79 0.83

Lab 15 0.76 0.81 0.76 0.82

Lab 16 0.87 0.85 0.87 0.86 (n=3)

Lab 17 0.74 0.73 0.72 0.70

Lab 18* 0.72 0.70 0.89 0.89

Lab 20 Not reported Not reported 0.81 0.79

Lab 24 0.76 0.72 0.72 (n=3) 0.69 (n=3)

Lab 26a 0.75 (n=6) 0.78 (n=6) 0.73 (n=6) 0.77 (n=5)

Lab 26b 0.76 (n=6) 0.79 (n=6) 0.74 (n=6) 0.77 (n=5)

Lab 28 0.72 0.72 0.72 0.72

* Calculated relative to own internal standard instead of P

Table 3: Laboratory 32 reported results

FXI potency (IU/ml) Clotting time (sec.)

A B P A B P

Assay 1 0.67 0.66 1.09 56.8 56.9 50.8

Assay 2 0.68 0.66 1.10 56.5 56.8 50.7

Assay 3 0.67 0.64 1.08 56.8 57.4 50.8

Assay 4 0.68 0.65 1.11 56.6 57.2 50.7

WHO/BS/2016.2281

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Appendix IV: Instructions for use - draft

WHO/BS/2016.2281

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