who/bs/2015.2261 english only expert committee on

WHO/BS/2015.2261

ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION

Geneva, 12 to 16 October 2015

COLLABORATIVE STUDY REPORT ON BLOOD COAGULATION FACTOR IX A. Value assignments:

1. Addition of Factor IX antigen value to The 4th

International Standard for Blood

Coagulation Factors II, VII, IX, X, Plasma, 09/172

2. The WHO 5th International Standard for Blood Coagulation Factor IX, Concentrate and

The Ph Eur BRP for Human Coagulation Factor IX, Concentrate, Batch 3

B. Investigation of the suitability of the 4th

International Standard for Blood Coagulation Factor

IX, Concentrate as a potency standard for purified full length recombinant FIX

1Elaine Gray, John Hogwood, Thomas Dougall* and Peter Rigsby*

Haemostasis Section and *Biostatistics Section

National Institute for Biological Standards and Control

Potters Bar, Hertfordshire, UK.

Eriko Terao

Department of Biological Standardisation, OMCL network and HealthCare (DBO)

European Directorate for the Quality of Medicines and HealthCare (EDQM)

Council of Europe, Strasbourg, France 1Principal Investigator

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 14 September 2015 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 M Nübling at email: [email protected]

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WHO/BS/2015.2261

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Summary

There are three main aims for this collaborative study:

1. to add a FIX antigen value to the 4th

International Standard for Blood Coagulation Factors II,

VII, IX and X, Plasma, Human (09/172);

2. to value assign a World Health Organisation (WHO) replacement International Standard (IS)

for Blood Coagulation Factor IX, Concentrate, Human and a replacement Pharmacopoeial (Ph

Eur) Biological Reference Preparation (BRP) for Blood Coagulation FIX, and

3.To investigate the suitability of the 4th

International Standard for Blood Coagulation Factor IX,

Concentrate as a potency standard for purified full length recombinant FIX.

Assignment of antigen value to the 4th International Standard for Blood Coagulation Factors II,

VII, IX and X, Plasma, Human (09/172) relative to local normal plasma pool was carried out by

15 laboratories returning 17 sets of data in total for analysis. Only 5 sets of data gave intra-

laboratory GCVs greater than 10%. There was good inter-laboratory agreement, GCV was 7.9%.

It was clear that local pools or the 4th

IS for FIX Plasma could not be used as an antigen

measurement standard for recombinant products since some reagent kits gave much lower results

than other kits and this is reflected in the high inter-laboratory variation (GCVs A: 26.6%; D;

31.5%). It is recommended that a FIX antigen value of 0.90 IU/ampoule be added to the label of

the 4th

IS for International Standard for Blood coagulation Factors II, VII, IX, X, Plasma.

Fifty laboratories from 18 countries took part for the value assignment of the plasma derived 5th

IS for FIX, Concentrate and Ph Eur BRP for FIX Concentrate, batch 3 relative to the 4th

IS for

FIX Concentrate, with 55 sets of clotting assay and 15 sets of chromogenic assay results returned

for analysis. The intra-laboratory variability was reasonably low, with the majority of geometric

coefficient of variation (GCV) below 10%. Lower inter-laboratory agreement was obtained for

sample B, 14/148 than for sample C, 14/162. Although there were no assay discrepancies

between clotting and chromogenic assays for either samples, a significantly lower (~6%) potency

was obtained for sample C with clotting assays when buffer rather than FIX deficient plasma was

used as pre-diluent. It is recommended that sample B, 14/148 be established as the 5th

International Standard for Blood Coagulation Factor IX, Concentrate and the Ph Eur BRP for

Blood coagulation Factor IX Batch 3, with an assigned value of 10.5 IU/ampoule.

The study also investigated the comparability of the plasma derived concentrate standard with the

full length recombinant FIX products and considered the feasibility of establishing an

International Standard for Recombinant FIX. There are currently 3 full length recombinant FIX

WHO/BS/2015.2261

products licenced and these were all represented in this collaborative study. Data from 49

laboratories (55 sets of clotting, 15 sets of chromogenic results) for two candidate recombinant

samples, A and D were received with additional results for another full length recombinant test

sample (Purple) returned by 6 laboratories. The intra-laboratory variability when samples A, D

and Purple were assayed against the 4th

IS for FIX Concentrate was acceptably low, majority

being less than 10%. In terms of inter-laboratory agreement, the clotting assay variability was

markedly high. For recombinant sample A, the potencies ranged 7.7 - 12.4 IU/ampoule (up to

62% discrepancy), with overall inter-laboratory GCV of 11.6% and for sample D, the potencies

ranged 6.6 – 12.4 IU/ampoule (up to 88% discrepancy), with overall inter-laboratory GCV of

13.4%. There were no major differences in the inter-laboratory GCVs for the chromogenic

assays. The inter-laboratory GCVs for overall potency estimates (including both clotting and

chromogenic assays) relative to the 4th

IS were high for both recombinant samples A (15.5%) and

D (16.8%); however, the GCV was reduced to 5.7% for Sample A when it was reanalysed using

sample D as the putative standard. In addition, reanalysis of clotting and chromogenic data for

sample A relative to sample D also markedly reduced the clotting and chromogenic discrepancy

observed when sample A was assayed against the 4th

IS for FIX Concentrate

(clotting/chromogenic ratios: vs 4th

IS – 1.25 (p <0.001) vs D – 0.98 (p=0.282). Although the full

length recombinant FIX could be assayed against the plasma derived 4th

IS Concentrate and

provided statistically valid results, there was high assay discrepancies amongst the clotting

assays using different APTT reagents and there was also significant clotting and chromogenic

assay discrepancies. The data from the present study indicated that a recombinant standard for

recombinant products will minimise assay discrepancies and improve inter-laboratory agreement.

Except for one laboratory who disagreed with a proposal of establishing an International Standard

for Recombinant FIX on the grounds that this may cause a shift in the amount of proteins in the

final products, all other participants agreed with the proposal. The data have also been evaluated

by 8 experts nominated by the Scientific and Standardisation Committee (SSC) of the

International Society for Thrombosis and Haemsotasis (ISTH). Seven SSC experts agreed with

the proposal, with 2 experts strongly support the establishment of an International Standard for

Recombinant FIX based on the reduction in assay discrepancies. One SSC Expert strongly

opposed and indicated that the use of multiple International Standards for the same coagulation

factor should be avoided whenever possible. Because of these 2 objections and after discussion

with the ISTH/WHO Liaison Group, it is unlikely that SSC will endorse such a proposal and

therefore the ECBS will not be requested to consider the establishment of an International

Standard for Recombinant FIX. The results and analysis for this investigation presented in this

report is for information only.

Proposals for establishment:

Sample P, NIBSC code 09/172: FIX antigen value: 0.90 IU/ampoule

WHO 4th

International Standard for Blood Coagulation Factors II, VII, IX, X, Plasma

Sample B, NIBSC code 14/148: Functional activity - 10.5 IU/ampoule

WHO 5th

International Standard for Blood Coagulation Factor IX, Concentrate

WHO/BS/2015.2261

Introduction

The 4

th International Standard (IS) for Blood Coagulation Factor IX, Concentrate, Human

(07/182) was established by the Expert Committee on Biological Standardisation (ECBS) of the

World Health Organisation (WHO) in October 20081,2

. Part of this batch of material was also

established as the European Pharmacopoeia Biological Reference Preparation (BRP) Batch 2 and

the US FDA reference standard for Blood Coagulation Factor IX Concentrate. The stock level of

the WHO 4th

IS and the Ph Eur BRP reference standards are now near depletion and replacement

standards are required. The aim of the study is to assay factor IX concentrate candidate

preparations against the 4th

International Standard, 07/182, with a view to establish a new

material as the 5th

International Standard for Blood Coagulation Factor IX, Concentrate, and EP

Human Coagulation Factor IX Concentrate Batch 3. In addition, as there are now three

recombinant FIX products licensed, this study also included these 3 recombinant preparations (2

of which could be candidate International Standard for Recombinant FIX) with a view to assess

the need and possible establishment of an International Standard for recombinant FIX.

This study also serves to value assign a factor IX antigen value to the 4th International Standard

for Blood Coagulation Factors II, VII, IX and X, Human, Plasma, 09/172 and provides an

opportunity to assess the factor IX unitage as defined by the concentrate and plasma standards.

The replacement of 4th

IS for FIX, Concentrate project and the antigen value assignment to the 4th

IS for FII, VII, IX and X, plasma were endorsed by the WHO Expert Committee on Biological

Standardisation in October 2012 and October 2014 respectively.

Participants

Fifty laboratories agreed to participate, with 49 laboratories (6 Austria, 1 Australia, 4 Canada, 1

China, 1 Croatia, 2 Denmark, 5 France, 6 Germany, 1 Italy, 1 Japan, 1 Korea, 3 Netherlands, 1

Portugal, 2 Spain, 1 Sweden, 1 Switzerland, 6 UK, 6 USA) returning data for the study. The

participants included 7 diagnostic reagents manufacturers, 19 therapeutic manufacturers, 14

regulatory authorities and 9 clinical laboratories. A list of participants is given in Appendix I at

the end of this report. Each laboratory is referred to in this report by an arbitrarily assigned

number, not necessarily representing the order of listing in the Appendix.

Samples

Coded samples included in the study were:

S – the 4th

I.S. Factor IX, Concentrate, 07/182, potency 7.9 IU/ampoule

WHO/BS/2015.2261

P – the 4th

I.S. Blood Coagulation Factor II, VII, IX, X, Plasma, 09/172, FIX potency 0.86

IU/ampoule

A – candidate sample, 07/142, recombinant FIX, nominal potency 8 – 10 IU/ampoule

B – candidate sample, 14/148, plasma derived FIX, nominal potency 10 – 12 IU/ampoule

C – candidate sample, 14/162, plasma derived FIX, nominal potency 8 – 10 IU/ampoule

D – candidate sample, 14/180, recombinant FIX, nominal potency 8 – 10 IU/ampoule

An additional frozen FIX concentrate sample, coded Pr (Purple) was also sent and assayed by 6

consenting participants.

The plasma pools for both the plasma derived candidates, the final products, the excipient human

albumin and the two International Standards have been tested negative for HBsAg, anti-HCV,

anti-HIV 1/2, and HCV RNA by PCR. All the candidates were prepared and freeze-dried

according to recommendations for the preparation, characterization and establishment of

international and other biological reference materials3. The product characteristics of each of the

candidates are summarised in Table 1. In addition, clotting times from activated coagulation

factors test (Non-activated partial Thromboplastin times, NAPTT), carried out in accordance with

Ph Eur method (2.6.22) and levels of activated factor IXa in the candidates are presented in

Tables 13 and 14 respectively.

With the exception of the local normal plasma pools, all samples were provided by NIBSC and

the participants were requested to reconstitute the samples according to protocol provided

(Appendix II).

Study design and assay methods

Details of the assay design were as stated in the protocol which is attached as Appendix II.

Briefly, each participant was requested to carry out 4 independent assays on 4 sets of samples and

to follow one of the suggested balanced assay designs as described in the study protocol.

Each participant was requested to perform their routine in-house method for FIX activity.

Multiple result sets returned by a participating laboratory were treated as results from an

independent laboratory and were given a separate lab code, e.g. Lab 2a, Lab 2b.

Statistical Analysis

Relative potencies of all samples in all assays were calculated by parallel line analysis with a log

transformation of assay response, using a minimum of three dilutions on a linear section of the

dose-response curve. Calculations were performed using the EDQM software CombiStats

Version 5.04. Non-linearity and non-parallelism were considered in the assessment of assay

validity. All dose-response lines showing no significant non-linearity (p>0.01) were accepted for

further analysis. All instances of significant non-linearity (p<0.01) were assessed visually and

those showing clear departures from linearity were excluded from further analysis. Non-

parallelism was assessed by calculation of the ratio of fitted slopes for the test and reference

WHO/BS/2015.2261

samples under consideration. The samples were concluded to be non-parallel when the slope ratio

was outside of the range 0.80 – 1.25 and no estimates are reported.

Relative potency estimates from all valid assays were combined to generate an unweighted

geometric mean (GM) for each laboratory and these laboratory means were used to calculate

overall unweighted geometric means for each sample. Variability between assays within

laboratories and between laboratories has been expressed using geometric coefficients of

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

estimates). Comparisons between assay methods were made by two-tailed t-test of log

transformed laboratory mean estimates (paired or unpaired as appropriate for the comparison

being made).

The relative contents of the accelerated thermal degradation samples were used to fit an

Arrhenius equation relating degradation rate to absolute temperature assuming first-order decay5

and hence predict the degradation rates when stored at -20°C.

Results and Discussions

FIX Antigen value assignment to 4

th IS for FII, VII, IX, X, Plasma relative to

local normal plasma pools

The Candidate, 4th

IS for FII, VII, IX, X, Plasma, 09/172 The candidate 09/172, coded sample P in the study was established by the ECBS/WHO in 2010

as the 4th

IS for FII, VII, IX, X, Plasma, with functional potencies assigned for these 4 factors. It

was produced from a pool of 85 donations of platelet poor normal plasma (The Welsh Blood

Service), each collected in CPD-adenine and buffered with 0.05 M HEPES. The final product

was filled and freeze-dried according to guidelines for production of international biological

standards (WHO TRS, 20063). Each individual plasma donation has been tested and found

negative for anti-HIV 1/2, HBsAg and anti-hepatitis C. The product characteristics are shown in

the following table:

NIBSC Code 09/172

Presentation Sealed, glass 5 ml DIN ampoules

Number of Ampoules available 16,000

Liquid filling weight (g) Mean=1.1078; Range=1.1000 – 1.1140

CV of fill mass (%) 0.20 (n=849)

Mean dry weight (g, n = 5) 0.1034

Mean head space oxygen (%) 0.14 (n =12)

Residual moisture (%) 0.225 (n = 12)

Storage temperature -20°C

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

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

WHO/BS/2015.2261

Assay methods

The FIX antigen value assignment was relative to the local normal pooled plasmas. Participants

were requested to collect fresh plasma on two separate days to prepare two normal plasma pools.

It was requested that each fresh pool was tested in the study on the day of collection and that a

sample of the same pool should be frozen for use on a second day. The normal pool was to be

used as the standard in the antigen assays. With the exception of Lab 28 and Lab 40, the

laboratories were not able to prepare fresh plasma pools and used in-house frozen pools or

commercial normal plasma pools; the number of donors across all pools was > 1800.

In total, 17 sets of antigen results (66 assays in total) from 15 labs were returned. Five

commercial ELISA kits were used (Table 2C). Laboratory 2 returned three sets of assays coded

2a, 2b and 2c. All laboratories performed four independent assays except Lab 48 which was only

able to perform 2 assays.

This study also offered the opportunity to assess whether a plasma antigen standard would be an

appropriate comparator for therapeutic concentrates as purified plasma derived and recombinant

FIX concentrates (samples A, B, C and D) were included in this study.

Assay validity

The majority of antigen assays gave valid estimates of relative potency. A slightly higher

proportion of non-linear and non-parallel exclusions (both 2-4%) was observed, but this partially

reflects the use of a log transformation of assay response which was consistently applied in all

laboratories and this may not reflect the best transformation choice in a small number of cases.

Individual cases of exclusions due to non-linearity or non-parallelism are indicated in Appendix

III, Table S3.

Results

The antigen value was assessed relative to the local normal plasma pools (L). Individual assay

results are shown in Appendix III, Table S3. Individual laboratory mean and overall potency

estimates for antigen are presented in Tables 8 and 9A.

Antigen relative to local normal plasma pools

o The intra-laboratory variation (%GCV) ranged 1.8 – 27.1%, with 5 laboratories

having %GCV greater than 10% (Table 8, 9A).

o The inter-laboratory variability was 7.9% (Table 8, 9A) and there was no obvious

assay kit bias or any detectable outliers.

o The overall FIX antigen value for the 4th

IS for FII, VII, IX, X, plasma against

local pool was 0.90 u/ml (Table 8, 9A).

Antigen values for samples S, A, B, C, D relative to local normal pooled plasma, L and

sample P, assuming antigen value of 0.90 u/ml

o When samples S, B and C, the plasma derived samples were assayed against L, the

local normal pooled plasma, there was good laboratory agreement of antigen

values for sample S, B and C; the inter-laboratory %GCVs were 9.2%, 9.8% and

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8.6% respectively (Table 9A) and these were slightly reduced when these samples

were assayed against P, the plasma IS, with an assumed value of 0.90 u/ml.

o The inter-laboratory %GCVs for A and D, the recombinant samples, were

extremely high (28.9% and 30.7% for samples A and D) and they were not

reduced when the samples were reanalysed against sample P (25.2% for A and

30.2% for D). The ranges of potencies against P for samples A and D were found

to be 3.8 – 9.4 u/ml and 3.1 – 8.1 u/ml respectively.

Discussion

Antigen measurements are carried out to assess phenotypes of haemophilia B, expression of

antigen in gene therapy and characterisation of therapeutic products. Since this is the first time a

FIX antigen value is assigned to an International Standard, the calibration is relative to local

normal plasma pool. Ideally, fresh normal plasma pool with large numbers of donors is used for

the comparison. However, for logistical reasons, it is difficult for laboratories to collect and

prepare fresh plasma to be used for assays on the same day. NIBSC have carried out in-house

studies to show that there was no significant difference in the antigen values of fresh plasmas and

their corresponding frozen/thawed samples (data not shown), the study therefore allowed the use

of fresh frozen plasmas as calibrators. With the exception of 2 laboratories (labs 28 and 40), all

laboratories used frozen pools (Table 2C) and although the total number of donors was not

known, it was >1800. Five different commercial reagents/kits were used: Asserachrom (8),

Visulize (5) and Zymutest (2), AssayPro (1) and Cedarlane (1) by 17 laboratories. The following

summarises the essential points for consideration:

Antigen value of sample P relative to local pooled plasma, L

o Intra-laboratory variability: 5 laboratories gave GCVs greater than 10% and only one

laboratory obtained GCV of 27% (Tables 8A, 9A). This indicates the kits and the

laboratories were able to measure antigen in plasma with reasonable precision.

o Inter-laboratory agreement: There was no significant difference in values by different

assay kits and outliers were not detected. Considering that the calibration was against

local pools, good agreement of antigen values was obtained; GCV was 7.9% (Tables

8, 9A). This may be partly aided by the sufficiently large number of donors in each

pool to offset the differences in individual donors. The overall value for sample P was

0.90 u/ampoule.

Antigen values of samples S, A, B, C and D relative to local normal pool plasma, L or

sample P, assuming assigned value of 0.90 u/ampoule

Low intra-laboratory variability was observed with the majority of GCVs less than

10% for all 4 samples (Table 9A) when assayed against L. The GCVs were

slightly reduced when these samples were assayed against P. (Table 9B)

The inter-laboratory GCVs for samples B and C, the plasma derived preparations

was reasonable when assayed against the local pool and were only marginally

reduced when assayed against P (Tables 9A, 9B).

The inter-laboratory GCVs for A and D, the recombinant samples, were extremely

high, being close to 30% when assayed against L (Table 9A) and were not reduced

when the samples were reanalysed against sample P (A: 26.6%; D 31.5%). The

WHO/BS/2015.2261

ranges of potencies against P for samples A and D were found to be 3.8 – 9.4 U/ml

and 3.1 – 8.1 U/ml respectively. This indicates that antigen measurement of

recombinant products, including FIX expressed by gene therapy vectors, against

normal plasma, using different antigen kits or antibody pairs may give

substantially different results and product specific standards may be required for

accurate measurement of antigen in recombinant FIX products.

Long term and on-bench stability of the 4th

IS for FII, VII, IX and X Plasma

The 4th

IS for FII, VII, IX, X, Plasma, 09/172 was established in 2010. Accelerated degradation

study for FIX antigen at 6 month time point showed no loss of activity. Stability data on other

analytes (functional activities of FII, VII, IX and X) showed that this reference standard is highly

stable. As with all International Standards, the proposed International Standards will also be

under real time stability monitoring throughout the life time of the standard.

Assessment of on-bench stability was carried out at NIBSC by storage of the reconstituted sample on

melting ice. Samples were reconstituted at 0 h, 2 h, and 3 h and stored on melting ice. After storage

of the first sample for 4 hours on melting ice, all samples were assayed relative to freshly

reconstituted ampoules of 09/172. The results indicated that FIX antigen would be stable for up to 4

hours after reconstitution when stored on melting ice.

Time point Potency % fresh ampoule

(95% confidence intervals)

2 h 96.1 (93.3 -100.0)

3 h 101.0 (98.1 – 103.8)

4 h 98.1 (95.2 -101.0)

Conclusions Intra- and inter-laboratory variability were low when P, the 4

th IS for FIX Plasma was

assayed against local normal pooled plasma (L).

Sample P improved antigen estimates agreement for samples S, B and C, the plasma

derived concentrates when used as a putative standard.

Both local pooled plasma (L) and the 4th

IS for plasma FIX (P) gave highly variable

results for samples A and D, the recombinant products.

It is recommended that an antigen value of 0.90 IU/ampoule be included in the labelling of the

4th

International Standard for FII, VII, IX and X, Plasma.

However, it should be noted that the use of this International Standard for measurement of

antigen in recombinant/modified products, post-infusion plasma from patients who have been

treated with recombinant/modified products or gene therapy products is not recommended. If this

standard is used for these purposes, it needs to be qualified and validated by the end-user with

their own reagents and methods. The draft Instruction for Use for this proposed IS is illustrated in

Appendix IV.

WHO/BS/2015.2261

Value assignment of the 5th

IS for IX, Concentrate and Ph Eur BRP for FIX, Batch

3 relative to the 4th

IS for FIX, Concentrate, 07/182 and comparability of full

length recombinant FIX with plasma derived concentrate IS

The Candidates, Samples B (14/148) and C (14/162) The characteristics of the candidates are as described in the Sample Section.

Recombinant FIX, Samples A (07/142), D (14/180) and Purple (Pr) The characteristics of the candidates are as described in the Sample Section.

Assay methods

Each participant was requested to perform their routine in-house method for FIX activity.

Multiple result sets returned by a participating laboratory were treated as results from an

independent laboratory and were given a separate lab code, e.g. Lab 2a, Lab 2b.

FIX functional activity assays

Clotting: The details of the instruments and reagents used by the participants are listed in Table

2A. With the exception of 3 laboratories that returned more than one set of results (Lab 2 – 5 sets;

Lab 8 – 4 sets; Lab 28 – 3 sets with 1 assay for two sets and 2 assays for the third set), all other

laboratories returned one set of results. In total 55 sets of clotting assays were analysed. Of the

clotting assays, 35 sets of results were obtained using FIX deficient plasma as pre-diluent for the

concentrate preparations (samples A, B, C and D), as stated in the European Pharmacopoeia

monograph for assay of human coagulation factor IX (01/2008:20711). Twenty sets of results

were from assays using buffer for pre-dilution (Table 2A). Sample P, the plasma IS did not

require pre-dilution as its activity is less than 1 IU/ml.

Chromogenic: In total, 15 sets of chromogenic assay results from 13 labs were returned. Two

commercial kits, Biophen Factor IX (Hyphen Biomed) and Rox Factor IX (Rossix AB) were

employed (Table 2B). The participants carried out the assays as described by the instruction given

by the manufacturers of the kit and pre-dilutions were carried out using kit buffers recommended.

Purple sample

In total, 10 sets of clotting results were returned from 5 labs (1 lab returned 5 sets), 4 sets of

chromogenic results from 3 labs (1 lab returned 2 sets) and 2 sets of antigen assays from 1 lab.

Assay data returned Clotting assays

Fifty-five sets of results from clotting assays (213 assays in total) were returned. Laboratory 2

performed five sets of assays which are coded 2a, 2b, 2c, 2d and 2e. Laboratory 8 performed four

sets of assay assigned codes 8a, 8b, 8c and 8d. Laboratory 31 performed 2 sets of assays, coded

31a and 31b. All laboratories performed four independent assays, apart from laboratory 33 which

performed 5 assays (extra assay performed using samples supplied for antigen testing).

WHO/BS/2015.2261

Laboratory 28 performed four assays, but with three different APTT agents and results were 28a,

28b and 28c. Sample P was not tested by laboratory 42.

Chromogenic assays

Thirteen laboratories performed chromogenic assays (59 assays in total). Laboratory 2 and 12

both performed two sets of assays, coded 2a & 2b and 12a & 12b respectively. All laboratories

performed four independent assays, except laboratory 48 which was only able to perform 3

assays.

Assay validity

The majority of clotting and chromogenic assays gave valid estimates of relative potency when

assessed as described in the analysis section above. Samples omitted for showing a non-linear

dose-response accounted for around 1% of cases. Reference-test pairs concluded to be non-

parallel accounted for only 1-2% of cases. A high number of slope ratios were in the range 0.90 –

1.11 indicating that more stringent criteria for parallelism could be applied to these assays.

Individual cases of exclusions due to non-linearity or non-parallelism are indicated in Appendix

III. In general, relative to the plasma derived 4th

International Standard for FIX Concentrate,

statistically valid assays by all method types were obtained for all the test samples, including

recombinant preparations.

Results

Samples A, B, C and D relative to the 4th


The potencies of samples A, B, C and D have been calculated relative to sample S, the 4th

IS for

FIX Concentrate. Individual assay results are shown in Appendix III. Laboratory mean and

overall potency estimates for clotting and chromogenic assays are presented in Tables 3A and 3B

respectively and are also illustrated as histograms in Figures 1 – 4.

Clotting assays

o Of the 55 laboratories, <10 laboratories had an intra-laboratory GCV >10% and over

60% of laboratories had intra-laboratory GCV <5% for all 4 samples.

o Inter-laboratory variability for the clotting assays was 4.8% and 8.4% for the plasma

derived samples, B and C and higher at 11.6% and 13.4% for the recombinant

samples, A and D. Figure 5 shows that there was no obvious effect with regards to

APTT reagents on the potencies obtained, but wider ranges of estimates were

observed for the recombinant samples A and D as confirmed by the higher inter-

laboratory variation for these two samples.

o With the exception of sample C, there was no effect of the diluents used for pre-

dilution of the samples on the overall potency estimates by clotting assay (Table 4).

For sample C, there was a 6% (including all results) - 8% (excluding Lab 15) decrease

in potency when the samples were diluted in buffer rather than in FIX deficient

plasma.

WHO/BS/2015.2261

o Tables 5A and 5B show the overall potency estimates by clotting methods were 9.8,

10.5, 8.7 and 9.4 IU/ml for samples A, B, C and D. Results from Lab 15 were

identified as outliers for samples B, C and D. Mean potency estimates excluding

results from Lab 15 were marginally increased for samples A (9.9 IU/ml) and C (8.8

IU/ml), with a reduction in %GCV for samples B, C and D.

Chromogenic assays

o Intra-lab variability for chromogenic assays was higher than for clotting assays, but

GCVs in the majority of cases were still <10%.

o Inter-laboratory variability for the chromogenic assays was 9.3% and 8.6% for the

plasma derived samples, B and C, and only slightly higher at 10.2% and 10.4% for the

recombinant samples, A and D.

o The overall potencies by chromogenic methods were found to be 7.8, 10.6, 9.1 and 7.3

IU/ml for samples A, B, C and D respectively. Results from Lab 21 were identified as

outliers for samples B and C. Potency estimates excluding results from Lab 21 were

marginally reduced for samples A, B and C, with a marked reduction in %GCV also

for samples B and C (Tables 5A and 5B).

o The results from the 2 chromogenic assay kits used in the study showed no significant

differences in the potencies obtained for plasma derived samples B and C, although

the Hyphen kit gave approximately 10% lower potencies for the recombinant samples

A and D.

Comparison of clotting and chromogenic assays (Table 5A and 5B)

o It is clear that there were no clotting and chromogenic assay discrepancies for the

plasma derived samples B and C, including or excluding the outliers.

o For recombinant samples A and D, significantly higher potencies (approximately

30%) were obtained by clotting assays.

Overall potency estimates by clotting and chromogenic assays

o Table 7 presents the overall geometric mean potencies obtained by both clotting and

chromogenic assays for samples A, B, C and D, including and excluding Lab 15 for

clotting assay and Lab 21 for chromogenic assay. The GM estimates were 9.3, 10.5,

8.8, 8.9 IU/ml for samples A, B, C and D when all the assays were included. With the

exception of sample A, which gave a potency estimate of 9.4 IU/ml, the GM potency

did not change when labs 15 (clotting) and 21 (chromogenic) were excluded. The

inter-laboratory variation was markedly lower for samples B and C (5.9% and 8.6%

respectively) than for samples A (15.4%) and D (17.1%). The %GCV for samples A

and D were not changed by the exclusion of labs 15 and 21, but they were reduced by

approximately 2% for samples B and C.

Sample A relative to Sample S, the 4th

IS for FIX Concentrate and sample D, the putative

recombinant standard

Results for sample A were reanalysed relative to sample D with an assumed assigned value of 9.4

IU/ml based on clotting assays (Dcl) only or 8.9 IU/ml based on overall potency estimates

(Dcl+ch).

WHO/BS/2015.2261

Table 10A shows comparison of potencies for sample A relative to sample S, the 4th

IS

for FIX Concentrate and sample D, the putative recombinant standard. For clotting

assays, similar ranges of intra-laboratory variability as expressed by %GCVs were

obtained when A was assayed against S or D, with 5 labs giving >10% GCV. For

chromogenic assays, the majority of the labs gave intra-laboratory GCVs of <10% (Table

10B).

There was no significant difference between the potency estimates by clotting assays

obtained against sample S and sample Dcl (Table 10A; p = 0.985), but there was

significant difference when compared with results against sample Dcl+ch (Table 10A;

p=0.003). The inter-laboratory variability as expressed by GCVs was markedly reduced

for both cases from 11.6% against sample S to 6.1% against samples Dcl or Dcl+ch.

There was a significant difference between the potency estimates by chromogenic assays

obtained against sample S, sample Dcl or sample Dcl+ch (Table 10B, p<0.001); however,

the inter-laboratory variation was reduced from 10.2% against sample S to 3.6% against

both samples D.

Clotting and chromogenic assays discrepancy (clotting and chromogenic ratio= 1.25, p

<0.001) was apparent when sample A was assayed against sample S. There was no

clotting and chromogenic discrepancy when sample A was assayed against either sample

Dcl or Dcl+ch (clotting and chromogenic ratio of 0.98; p = 0.282).

Sample Purple relative to Sample S, the 4th

IS for FIX Concentrate and using sample A and

sample D as putative recombinant standards

In total, 14 sets of results, 10 clotting and 4 chromogenic assays, were returned for analysis.

Results for sample Pr were analysed relative to sample S and then recalculated against samples A

(Acl) and D (Dcl) using assigned values (based on clotting assays only, 9.8 IU/ml for Acl and 9.4

IU/ml for Dcl)) and against samples A (Acl+ch) and D (Dcl+ch) using assigned values (based on

overall potency estimates, 9.4 IU/ml for Acl and 8.9 IU/ml for Dcl))

Similar ranges of intra-laboratory GCVs were obtained for both clotting and chromogenic

assays when sample Pr was assayed against samples S, A or D (Tables 11A, 11B). Only 1

lab gave GCV >10% for chromogenic assay when Purple was assayed against A; for

clotting assays, 2 labs gave GCVs just over 10% when assayed against sample A and 3

labs gave GCVs over 10% when assayed against sample D (Tables 11A, 11B).

Some significant clotting and chromogenic assay discrepancies were observed (Table

11C: vs S, p < 0.001; vs Acl, p = 0.038; vs Dcl, p = 0.02). However, the clotting and

chromogenic ratio was reduced from 1.26 when sample S was used as the standard to 0.90

and 0.88 when against samples A and D.

The overall potency was around 4.8% higher against Acl and was higher, at 6.0%, when

assayed against Dcl (Table 11A). Good agreement of overall estimates was obtained

when Pr was assayed against samples Dcl+ch and Acl+ch and it was only 1.2% higher

than against sample S (Table 11B). The inter-laboratory variability as expressed by

GCVs were reduced from 14.6% against sample S to 9.5% against A and 10.7% against

sample D.

WHO/BS/2015.2261

Sample P, the 4th

IS for FII, VII, IX and X Plasma relative to the 4th


The FIX potency of sample P was calculated relative to sample S, the 4th

IS for FIX concentrate.

Individual assay results are shown in Appendix III. Individual laboratory mean and overall

potency estimates for clotting and chromogenic assays are presented in Table 8 and are also

shown in Figure 6.

Clotting and chromogenic assays against 4th


o The intra-laboratory variation (%GCV) ranges were 0.7 – 14.9% for clotting

assays and narrower, 1.6 – 7.9%, for the chromogenic assays. Only 4 out of 54

laboratories had %GCV >10% and 25 laboratories had %GCVs <5% for the

clotting assays (Table 8).

o The inter-laboratory variability was high, at 16.3% for the clotting assays and

lower, at 8.6% for the chromogenic assays (Tables 8, 5A, 5B).

o Overall potency estimates for clotting and chromogenic assays were 0.91 and 0.81

IU/ml and remained the same when Lab 15 for clotting and Lab 21 for

chromogenic were excluded. (Tables 5A, 5B).

o A significant assay discrepancy was observed with estimates by clotting methods

(12% higher than chromogenic assay results) yielding a clotting to chromogenic

ratio of 1.11 (Table 5B).

o The overall potency by both clotting and chromogenic assay was 0.88 IU/ml and

this equates to 2.3% difference to the labelled potency of 0.86 IU/ml. However

this difference is not statistically significant (unpaired t-test p=0.195). The inter-

laboratory GCV, including both clotting and chromogenic assay results was 15.4%

(Table 7)

Long term stability of the candidates and samples

Accelerated degradation studies have been initiated for all four concentrate preparations. Table 12

shows the predicted loss of clotting activity for samples A, B, C and D after being stored at

various elevated temperatures (-70, –20, +4, +20, +37 and +45°C). All samples showed low

predicted loss of activity at storage temperature of -20 °C. However, it should be noted that these

data are preliminary and further results are required to fully evaluate the long term stability of

these samples.

Discussion The most important prerequisite for a potency reference standard is improvement of laboratory

agreement and this is evidenced by its ability to improve agreement of potency estimates within a

method and between method types. This study generated data from 55 laboratories, using

different methods to support the choice of candidates and value assignment of these candidates as

potency standards for FIX. There were two main aims to this part of the study:

a. to assign the blood coagulation factor IX functional activity values to the replacement

WHO International Standard for Blood Coagulation Factor IX, Concentrate and Ph

WHO/BS/2015.2261

Eur. BRP for Human Blood Coagulation Factor IX, relative the 4th

IS for FIX,

Concentrate

b. To assess the comparability of the plasma derived IS for FIX concentrate with

recombinant FIX preparations and calibrate an IS for recombinant FIX relative to the

4th

IS for FIX, Concentrate

Overall, the majority of the assays were statistically valid even when the recombinant samples (A,

D, Purple) were compared with the plasma derived International Standard for FIX concentrate,

thus supporting potency labelling of recombinant FIX products in IU. The majority of the intra-

laboratory GCVs were under 10% and a high proportion were less than 5% for all assay method

types, thus indicating the laboratories were able to perform the assays with good reproducibility.

Results from only 2 laboratories were identified as outliers, Lab 15 for clotting assays and Lab 21

for chromogenic assays, and these were excluded in the value assignment of the proposed

International Standards and Ph Eur BRP.

Value assignment to the 5th

IS for FIX, Concentrate and the Ph Eur BRP for FIX, Batch 3

The current (4th

IS) and the previous International Standards were value assigned using 1-stage

clotting assays only. In the 2008 collaborative study that value assigned the 4th

IS, the Ph Eur

BRP Batch 2 and the FDA/CBER standard, two chromogenic kits were used, however, since

there was only one set of data for each assay kit returned for analysis, it was decided that

assignment of the three standards should exclude results from chromogenic assays. Since the

2008 study, the 2 commercial chromogenic assay kits have become more widely used, especially

in the development of new generation products and in the estimation of low levels of FIX in

haemophilic patients. In the present study, 15 laboratories returned data for chromogenic assays

and since discussions with manufacturers and regulators indicate that chromogenic assays are

used as part of the characterisation package for FIX products, value assignment for the 5th

IS for

FIX, Concentrate also considered results from chromogenic assays as well as those obtained

using 1-stage clotting assays. For the Ph Eur BRP, it is clear that the calibration should consider

results only from the Monograph method and this is the 1-stage clotting assay, using FIX

deficient plasma as diluent. There are 2 plasma derived candidates, samples B and C, both could

be considered as the replacement IS and Ph Eur BRP. The following summarises the essential

points for consideration:

o Intra-laboratory variability: Both samples gave similar ranges of GCVs for all assay

methods when assayed against sample S, the 4th

IS for FIX, Concentrate (Tables 3A, 3B)

o Effect of pre-diluent: For sample B, the potency estimates were 10.5 IU/ml, using either

FIX deficient plasma or buffer as pre-diluent. However, for sample C, the estimates were

9.0 IU/ml when FIX deficient plasma was used as pre-diluent and 8.5 IU/ml, 6% lower,

when buffer was used (Table 4). The inter-laboratory agreement was better for sample B

(GCVs – FIX deficient plasma: 3.4%, buffer: 4.6%) than sample C (GCVs –FIX deficient

plasma: 4.8%, buffer: 7.8%) regardless of diluent, and the agreement was improved when

FIX deficient plasma was used, variation as expressed by GCVs was lower. Both samples

were prepared from the same FIX therapeutic product and the major difference between

WHO/BS/2015.2261

samples B and C is the formulation. It is possible that sample B which has a similar

formulation as the current IS, and different formulation of sample C caused this discrepant

result. This interesting effect will be further investigated by NIBSC.

o Effect of APTT reagents: There was no obvious bias with the 15 APTT reagents

employed in this study on the potency estimates obtained using different APTT reagents

for either samples B and C (Figure 5).

o Effect of Chromogenic assay kits: For samples B and C, when assayed against the 4th

IS,

Concentrate , there was no significant difference in potency estimates obtained using

either kits; the Hyphen to Rossix potency ratios were 0.98 and 0.99 for samples B and C

respectively. The Inter-laboratory GCVs were less than 8% for both samples and both

kits (Table 6).

o Stability: Preliminary accelerated degradation studies on both samples B and C predicted

no loss of activity when stored at -20°C.

o Number of ampoules available: For harmonisation purpose, it would be ideal for the IS

and BRP to be the same batch of material, providing there is sufficient stock to support

this. There are approximately 20,000 ampoules of sample B and 24000 ampoules of

sample C. Although more ampoules of sample C are available, there should be sufficient

ampoules of sample B to cover both reference standards.

In summary, given that sample B gave agreement of potencies by all assay method types and that

sample C gave discrepant results in clotting assays using FIX deficient plasma or buffer as

diluents, it is proposed that sample B be the 5th

IS for FIX, Concentrate and the Ph Eur BRP

Batch 3. The assignment of the 5th

IS is based on results from 1-stage clotting with both FIX

deficient plasma and buffer as diluents and chromogenic assays, while the value for the BRP is

assigned with value from 1-stage clotting assays using only FIX deficient plasma as diluent. For

both standards, the unitage proposed is 10.5 IU/ampoule.

Assessment of on-bench stability of sample B was carried out at NIBSC by storage of the

reconstituted sample on melting ice. The potency at 1 h, 2 h, 3 h and 4 h were estimated relative to

freshly reconstituted 14/148 at each time-point. Two assays were carried. No significant difference

was observed between the potency values for the time points. This indicates that the material would

be stable for up to 4 hours storage on melting ice.

Time point Potency % fresh ampoule

(95% confidence intervals)

1 h 100.0 (96.5 – 104.4)

2 h 101.7 (98.2 - 105.3)

3 h 100.0 (94.7 - 105.3)

4 h 98.2 (93.8 – 103.5)

Rationale for route of value assignment to an International Standard for Recombinant FIX

Results from a previous international collaborative study (NIBSC Phase II study 2013) indicated

that although the 3 recombinant FIX products could be assayed validly against the 4th

IS for FIX

Concentrate, there were substantial potency discrepancies within 1-stage clotting assays and

between clotting and chromogenic methods. Agreement of potencies was obtained when the 3

WHO/BS/2015.2261

recombinant products were assayed against a recombinant reference preparation. The current

study aimed to investigate these discrepancies further and confirm the need for a recombinant

reference standard to harmonise assay methods and improve agreement of potency estimates

within and between laboratories. The present study included 2 recombinant products, samples A

and D. Additionally, a third recombinant product, coded Purple, was also sent to 6 laboratories so

that the 3 recombinant products could be compared.

The current practice for potency labelling of recombinant FIX products is by one-stage clotting

assays against the 4th

IS for FIX Concentrate or a reference standard that is traceable to the 4th

IS.

The impact of value assignment using a recombinant putative standard labelled with overall

potency by both clotting and chromogenic assay results or labelled with estimate by clotting

assays only was evaluated. For sample A, in addition to analysis of data exploring the effect of

using sample D as a putative standard with value assigned from all assays (Dcl+ch), with the

overall potency estimate, 8.9 IU/ml against sample S, the results were also reanalysed using the

clotting assay GM for sample D (Dcl) at 9.4 IU/ml.

Potency estimates for sample A relative to sample S, the 4th

IS for FIX Concentrate and

sample D:

o The intra-laboratory variation was similar when sample A was assayed against

sample S or sample Dcl or sample Dcl+ch (Table 10A, 10B)

o The inter-laboratory variability for both clotting and chromogenic assays were

reduced by approximately two-fold (Table 10C).

o There was significant clotting and chromogenic assay discrepancy when sample A

was assayed against sample S, with chromogenic assays giving 25% lower

potency than the clotting assays (p<0.001) This discrepancy was eliminated when

sample A was reanalysed against either sample Dcl or Dcl+ch (clotting and

chromogenic ratio 0.98, p = 0.282).

o Potency estimates

The clotting potency estimates for sample A against samples S and Dcl

were the same, giving 9.8 IU/ml, but approximately 5% lower, at 9.3 IU/ml

when assayed against Dcl+ch (Table 10C).

The estimates by chromogenic assays against S were over 20% lower than

those obtained against Dcl or Dcl+ch.

It is clear that there is improvement in agreement of potency by both

clotting and chromogenic assays when sample A was assayed against

sample D (compare Figures 1 and 7). While the overall potency estimate

generated against Dcl+ch was not significantly different to values from

assays against S (Table 10C, p =0.956), potency obtained against Dcl was

significantly different (Table 10C, p = 0.003).

WHO/BS/2015.2261

o This indicates that if sample D is a standard for sample A, it will reduce both inter-

laboratory variability and improve agreement of potencies by both clotting and

chromogenic assays.

o Taking into account that the current recombinant products are labelled against the

4th

IS for FIX concentrate using clotting assays, the clotting potency of sample A

relative to sample S, the 4th

IS for FIX concentrate was compared with the overall

potency estimates obtained against Dcl and Dcl+ch. Table 10C shows that there

was good agreement of potency against samples S and Dcl (A vs S clotting: 9.8

IU/ml; A vs Dcl overall: 9.8 IU/ml; p =0.779). However there was significant

differences in potencies when compared with the overall value obtained with

Dcl+ch (A vs S clotting: 9.8 IU/ml; A vs Dcl+ch overall: 9.3 IU/ml; p =0.003).

Therefore if sample D is to be the standard for recombinant products, value

assigned using clotting assays results only, this will ensure the continuity of the IU

and there should be minimal shift in potency labelling of recombinant products.

The recombinant standard will also help to reduce inter-laboratory variability and

harmonise clotting and chromogenic assay results.

Potency estimates for sample Purple relative to sample S, the 4th

IS for FIX Concentrate,

sample A and sample D:

o The intra-laboratory variation was similarly low when sample Purple was assayed

against samples S, A, or D (Table 11A, 11B), indicating that the laboratories were

able to assay the recombinant products with precision and reproducibility.

o While the inter-laboratory GCVs were slightly reduced for the clotting assay when

Purple was assayed against A or D than against S (Tables 11C, 11D; GCVs vs S:

8.3%; vs A: 7.1%; vs D: 8.0%;), the GCVs for the chromogenic assays were not

reduced (Tables 11C, 11D; GCVs vs S: 10.1%; vs A: 10.6%; vs D: 10.8%;).

o Although some clotting and chromogenic discrepancies were found to be

statistically significant, the clotting to chromogenic ratio was reduced from 1.26

when assayed against S to 0.90 against A and 0.88 against D, (Tables 11C, 11D).

o Although there was no significant difference to the overall potency estimates

against the different standards, using results from all methods (Tables 11A. 11B),

the overall potency agreement was improved when Purple was assayed against

samples A or D as exemplified by the approximately 5% reduction in the inter-

laboratory variability as expressed by GCVs. (Tables 11A, 11B, 11C, 11D).

o These results indicate that if sample A or D was used as a standard for sample

Purple, it will minimise clotting and chromogenic discrepancies and improve

potency agreement by reduction of overall inter-laboratory variability

The reanalysis of data for samples A, D and Purple confirms finding from the previous study that

assaying recombinant product against a recombinant standard helps to reduce assay discrepancies

and increase potency agreement between laboratories.

WHO/BS/2015.2261

Conclusions

The 5th

IS for Blood Coagulation Factor IX, Concentrate and Ph Eur BRP batch 3

The high inter-laboratory %GCV (Table 7, 15.4%) obtained when the 4th

IS for

FIX Plasma was assayed against the 4th

IS for FIX Concentrate support the

rationale of a concentrate potency reference standard for plasma derived FIX

products.

Both candidates B, C gave similarly low within laboratory variability.

There was no clotting and chromogenic discrepancy for either candidate.

Preliminary accelerated degradation studies showed that both candidates are

predicted to be equally stable.

Candidate B gave slightly lower inter-laboratory variability

Different pre-diluent gave discrepant clotting assay results for candidate C.

Since candidate B gave agreement of potencies by different methods and pre-diluents, it is

recommended that candidate B, 14/148 be the 5th

International Standard for Blood Coagulation

Factor IX, Concentrate and EP BRP for Human Coagulation Factor IX Concentrate, Batch 3.

Both reference standards are value assigned relative to the 4th


Coagulation Factor IX, Concentrate, with the 5th


FIX, Concentrate potency labelled based on all functional activity assays and the Ph Eur BRP

labelled based on one-stage clotting assays using FIX deficient plasma as pre-diluent. In both

cases, the value is 10.5 IU/ampoule. The draft Instruction for Use for this proposed IS is

illustrated in Appendix IV.

Should an International Standard for Recombinant FIX be established?

It is clear that statistically valid assays can be obtained when the current recombinant FIX

products are assayed against the plasma derived IS for FIX Concentrate and that one of these

products has been potency labelled against the IS for the last decade. Unlike FVIII, there is global

agreement that 1-stage clotting assays is the assay of choice for potency labelling and therefore

clotting and chromogenic assay discrepancies have not so far been an issue. Nonetheless,

chromogenic assay kits for FIX are now available and these kits will suit the clinical need for

assays with higher sensitivity for low level of FIX. The results from this study showed that there

are significant chromogenic and clotting assay discrepancies when the recombinant samples were

assayed against the current IS. In addition, the availability of new recombinant and modified

products has brought attention to assay discrepancies within the 1-stage clotting assays. The

package insert from one of the most recently licensed recombinant full-length FIX indicates that

the FIX potency results for this product can be affected by the type of APTT reagent and

reference standard used in the assay and that differences of up to 40% have been observed. The

current study, using a range of APTT reagents routinely used by manufacturers, regulators and

clinical laboratories indicated that up to >60 % and >80% differences in potencies could be seen

when recombinant samples A and D were assayed by clotting assays against the plasma derived

IS. The APTT reagent discrepancy for sample A was reduced to ~20% and was reflected by the

decrease of inter-laboratory GCV from 11.6 to 6.4% when sample D was used as the reference

WHO/BS/2015.2261

standard. In addition when sample D was used as the putative standard for samples A or Purple,

apart from lowering the overall inter-laboratory variability, the clotting and chromogenic

discrepancies was eliminated or reduced to an acceptable level.

Although having a standard for recombinant products in addition to the plasma derived

concentrate IS may cause some initial confusion, precedent has been set for a number of

Biotherapeutics (Prolactin: WHO 3rd

International Standard for Prolactin, Human, NIBSC code:

84/500 and WHO 1st International Reference Reagent for Prolactin, Human, recombinant. NIBSC

code: 97/714; Follicle Stimulating Hormone (FSH): WHO 2nd

International Standard for Follicle-

Stimulating Hormone, human, recombinant, for bioassay, NIBSC code: 08/282 and WHO 5th

International Standard for Follicle Stimulating Hormone, Luteinizing Hormone human, urinary

for bioassay, NIBSC code:10/286) which have co-existing International Standards for

plasma/human derived and recombinant proteins and both of these International Standards serve

well to support potency labelling and clinical measurement in patient samples of the different

forms of the same therapeutic product types. The results from the study also support the

continuity of the FIX IU when transferring to the recombinant standard. This is illustrated by the

same clotting potency of A, 9.8 IU/ml against S and sample D (when sample D is assigned using

the clotting potency value; Tables 10C). The replacement strategy for such a standard should also

be considered carefully and it is proposed that successive replacement recombinant standards

should follow the same route as other International Standards i.e. value assignment relative to the

previous recombinant standard but study should include current plasma derived concentrate

standard so that any drift of IUs would be monitored.

While the establishment of an IS for recombinant full length FIX based on the data from the

current study will serve well against the current licensed products, there is a possibility that more

recombinant FIX products will be on market and that the future recombinant FIX products may

not compare well with this recombinant IS. The NIBSC Phase II study has already identified that

the modified recombinant products (e.g. pegylated-FIX) do not compare well against full length

recombinant products and recombinant full length FIX should not be used as potency standard for

these modified products. This study showed that the 3 recombinant full length products compared

better against each other than against the plasma derived IS and the recombinant IS will promote

better comparability of potencies amongst these products. The likelihood that another

recombinant full length FIX will behave substantially different to these 3 products is low, but not

impossible and should be investigated should another recombinant full length FIX becomes

available. There is also another advantage of the availability of an IS for recombinant FIX.

There have been numerous discussions amongst regulators, manufacturers and clinical

laboratories on assay discrepancies related to clinical monitoring. The sensitivity and the

reproducibility of chromogenic assays are being considered alongside the conventional one-stage

clotting assay and is supported by the US National Hemophilia Foundation who urged the US

FDA to accelerate the approval of chromogenic assay kit and clinical laboratories to establish the

chromogenic assays. The clinical laboratories will require support to improve intra- and inter-

laboratory agreement and minimise assay discrepancies whenever they change reagents,

instruments and operators. Since product specific standards are not yet feasible, the availability

WHO/BS/2015.2261

of a global recombinant International Standard will go a long way to aid the development and

continual validation of assay methods for measurement of FIX activity following replacement

therapy with recombinant full length FIX. The following table summarises the pros and cons of

establishing an International Standard for Recombinant FIX and on balance, having a global

standard for recombinant FIX would be useful to the community as the disadvantages could be

minimised by careful and considered risk management:

Pros Cons

Improve intra-laboratory

agreement, less reliant on

calibration against the plasma

derived IS and stability of

manufacturers’ own in-house

standards

Minimize the substantial assay

discrepancies within and between

clotting and chromogenic assays

Improve inter-laboratory

agreement, giving more

confidence in label potencies and

interchangeability of products if

and when required

Useful independent resource for

clinical labs to develop assays for

measurement of recombinant FIX

in patient samples

Too many standards, can be

confusing to end-users

Future recombinant full length

FIX may not assay so well against

recombinant standard

Complex considerations for route

of value assignment

Replacement strategy require

careful consideration

Possible discontinuity of for

labelling of licensed recombinant

products

In consideration of which candidate should go forward as the 1st International Standard for

Recombinant FIX, both candidates have similar physical characteristics in terms of coefficient of

variation, residual moisture and oxygen headspace and passed all the recommended specifications

for a WHO international biological reference standards. They gave similar intra- and inter-

laboratory variability. Candidate A, 07/142, was ampouled in 2007 with fewer ampoules

available than candidate D, 14/180. In addition, although all the candidates including samples A

and D passed the activated coagulation factors test (all test clotting times >150s; Table 13), the

amount of FIXa estimated in sample A was 5 times higher than that obtained for sample D (Table

14). High levels of FIXa in a FIX reference standard may have some influence on the potency

estimates of test preparations. So it is recommended that candidate D be the 1st International

Standard for Recombinant FIX. In terms of value assignment, since the Ph Eur monograph

method for labelling is the one-stage clotting assay and globally the current licensed products are

labelled by one-stage clotting assay, to ensure good continuity of the IU, it is recommended that

the 1st IS for recombinant FIX be value assigned with overall clotting potency of 9.4 IU/ampoule,

against the 4th

IS for FIX concentrate. The established standard can be used as a reference

WHO/BS/2015.2261

standard for measurement of recombinant FIX activity by either 1-stage clotting or chromogenic

assays.

Based on the above rationale, it was proposed to the participants that Sample D, 14/180 be

established as the WHO 1st International Standard for Recombinant Human Blood Coagulation

Factor IX, with the assigned value for functional activity of 9.4 IU/ampoule.

Participants Comments and Responses Thirty out of the 49 participating laboratories returned comments on the study. Most of the

comments were associated with typos and queries related to analysis of results for individual

assays. Following investigation of the revised data, the statisticians concluded that there will not

be any major impact on the overall potency estimates or the recommendations for establishment

of the standards and the tables have been updated for the ECBS report. All responded participants

agreed with the proposal of the value assignment of the 5th


Coagulation Factor IX, Concentrate and the addition of the FIX antigen value to the 4th

International standard for Factors II, VII, IX and X, Plasma.

For the proposal of an International Standard for Recombinant FIX, with the exception of one lab

(see comment A), who did not agree with the proposal, all others agree with the proposed

establishment. There were 2 other significant comments (comments B and C).

Comment A

“At this time, we would recommend not supporting the proposal to establish an

International Standard (IS) for recombinant Factor IX (FIX) because this will complicate

the meaning of the International Unit (IU) as it has been understood for decades by users

of FIX products. Although we know that the IU is dependent on many factors, having one

IU for all FIX products provides a reference with which one can gauge the effect of the

variables on the resultant potency values. If we agree to having an IS for recombinant

FIX concentrates, manufacturers of currently licensed recombinant FIX products would,

in practice, need to recalibrate their in-house recombinant product-specific standards,

which may result in adjustment of FIX protein content in the final container. As a result,

we would have to re-assign the potency of all currently licensed recombinant FIX

products (both traditional and long-acting analogues), which would most likely

necessitate changes in dosing regimens. In addition, we can expect that these two FIX IS

will be replaced at different times and experience different drifts, which will further

exacerbate the differences between the two standards.

On the other hand, we know that plasma-derived and recombinant FIX preparations do

behave differently as we have seen in laboratory studies comparing different APTT

reagents. It may be worthwhile to investigate the possibility of harmonizing potency

assignment and product description in the labelling of different recombinant and long-

acting FIX products. To this end, the availability of a recombinant FIX reference reagent

would be helpful in such studies. Thus, we can support establishing the proposed

candidate as a WHO Reference Reagent for recombinant FIX concentrates.”

WHO/BS/2015.2261

Response from NIBSC We like to make clear that a recombinant FIX International Standard or

International Reference Reagent should not be used for assays of long-acting

products or any other modified products and this will be explicit in the Instruction

for Use to the end-users.

There is a possibility that a recombinant FIX International Standard may cause a

shift in labelling of the current licensed (or product in development) recombinant

FIX products. The current study data (Table 10A) showed that the overall potency

estimate for sample A (a recombinant product) against the 4th

International

Standard (sample S) was the same as the value against sample D (another

recombinant product). However, due to the variability of the APTT reagents, there

will be discrepancies within individual laboratories. So results on recombinant

products against the proposed International Standard from manufacturers will be

needed to assess the impact of the recombinant standard on the potency labelling

of their products. Even if there is a slight shift of the unit, this one-time transfer

will have the long-lasting advantage that a recombinant standard will diminish the

APTT reagents and the clotting and chromogenic discrepancies.

Comment B “1. The mere fact that only “the OS method” is prescribed by regulatory authorities

as the method to be used for release testing, and the results of which are hence proposed

for Sample D assignment, should not be taken as an argument to prevent introduction of

CS methods for release testing. We also want to state that there is no support for any

tentative view that the OS results for Sample D are more true than the CS results. The

ratio of close to 1.3 for OS/CS (close to 1.2 if only including the seven Rox Factor IX

results) when A and D were value assigned vs the 4th IS may encourage follow-up work to

investigate why the difference appears.

2. The data from the study clearly show a significantly lower inter-lab GCV for CS

methods when Sample A was value assigned vs Sample D, no matter whether Dcl or

Dcl+ch was used for D. Such results strongly indicate that CS methods may well improve

consistency in FIX potency assignments in manufacturers´QC labs. Again, it would we

quite unfortunate if regulatory authorities will not consider CS methods to be allowed for

potency assignments by referring to the Sample D value assignment.

3. The proposed value and use of Sample D (14/180) will not solve the problems with

OS methods on analysis of long-life rFIX concentrates. The data from CS methods,

although so far limited, clearly indicate that CS methods may be a better alternative.”

Response from NIBSC

The value assignment of the proposed standard based on clotting assays only is to

minimize the risk of a shift in potency labelling of current licensed products which were

licensed on potency labelling by the clotting assay. The recombinant standard should

reduce clotting and chromogenic assay discrepancy and allow the development of the

chromogenic assay not only as an alternative for potency labelling but also for clinical

monitoring.

Comment C

WHO/BS/2015.2261

“We understand that the European Pharmacopoeia focuses on the one-stage clotting

assay for blood coagulation factor IX. Nevertheless, we think that in light of the current

uncertainties around potency testing of coagulation factor products, alignment of assays

which are based on different assay principles should not be ignored. Thus, the proposed

value assignment may not only be substantiated by a majority driven decision.”

Response from NIBSC Pharmacopoeial standards are conventionally calibrated using only the pharmacopoeial

assay while WHO International Standards are value assigned by multiple methods. Assay

methods for value assignment of an International Standard for Recombinant FIX could be

further discussed.

SSC Experts Comments and Responses

Eight experts nominated by the SSC sent in responses to the SSC report. Two experts agreed with

all the proposals with no comments and 2 experts with minor comments related to the plasma

derived 5th

IS. There were 4 significant comments:

Comment A

“Is the evidence truly convincing that the definition of a factor IX unit is equivalent

between the plasma and recombinant standards?”

Response from NIBSC

The results from this study showed that the IU derived for the recombinant candidates was

directly traceable to the plasma derived 4th

IS for FIX, Concentrate. The potencies of

sample A, a recombinant preparation by clotting assays were similar against the 4th

IS or

against sample D (the other recombinant samples) used as the putative standard (Table

10A). This demonstrates that if one of the recombinant samples in the study is the

International Standard, the IU expressed by this standard is traceable and equivalent to the

IU expressed by the plasma derived IS.

Comment B

“I understand the argument for retaining just 1 concentrate standard, as for FVIII,

especially as potency is assigned currently using the same methodology (1-stage clotting

assay) in both Europe and US. However, the discrepancy between clotting and

chromogenic assay results with recombinant products when compared to a plasma-

derived concentrate standard, and especially the variability encountered within 1-stage

clotting assays with different reagents suggests that future use of plasma-derived

standards to assign potency of current and new recombinant products could lead to

problems. Therefore a recombinant concentrate standard with potency itself assigned

against a plasma-derived concentrate standard but able to reduce variability between

methods is a welcome step.”

WHO/BS/2015.2261

Response from NIBSC

The results from this study showed that a recombinant International Standard would

significantly reduce variability between methods whilst maintaining traceability to the

plasma derived concentrate IS.

Comment C

“1. Sample B- proposed plasma derived reference – Many of us expect that chromogenic

IX assays will be increasingly used in future when pegylated FIX and some other extended

half life products. Therefore it is useful that the chromogenic assay data is in agreement

with the one stage (as used for potency assignment. This predicts that labs can use

chromogenic assays for post infusion monitoring of plasma derived products whose

potency has been assigned against sample B in future.

2. Sample D as first recombinant standard. Probably sample A could have also been

suitable but the 3k extra stock of D and lower level of FIXa probably make it the better

candidate. It would be helpful if the failed candidates can be returned to the provider so

that they can take advantage of the collaborative data. This would assist future

standardization. Does this routinely occur?

3. There is an important difference between chromogenic and clotting assay results for

the recombinant materials (as in previous studies). Since potency assignment to

commercial batches of recombinant IX is by one stage I think its really important to have

this recombinant material and for potency assignment to be by the one stage assay (as

proposed). The data predict that post infusion monitoring of these (unmodified )

recombinants should be by one stage (not chromogenic) assays.

4. The GCV of recombinant materials (A and D) against plasma standards is

approximately three times as high as for recombinant against recombinant. This predicts

that potency assignments by manufactures will be less method dependent when assaying

recombinant against recombinant which in turn will help make post infusion monitoring

less dependent on the reagents in local use. Just another example of the like versus like

principle improving the agreement between methods. “

Response from NIBSC

1. Since there was no significant difference between clotting and chromogenic assay

methods for the plasma derived candidates relative to the 4th

IS for FIX Concentrate,

chromogenic assays could be used for potency estimation of purified concentrates.

There was significant clotting and chromogenic assay discrepancy (12% higher by

clotting, Tables 5A and 5B) when the plasma IS was assayed against the concentrate

IS. This indicates that if clinical labs use normal pooled plasma as a standard for

estimation of post infusion of plasma derived therapeutics, clotting and chromogenic

assay discrepancies may still be obtained. However, the high variability of assay

results from clinical labs may mask this assay discrepancy.

WHO/BS/2015.2261

2. The provider of donated materials always has an option to obtain part of the batch for

their in-house use should that particular material not be established as an IS.

3. The rationale for potency assignment of a proposed recombinant FIX IS using

clotting assay only was based on all 3 current commercial recombinant FIX are

potency labelled by one-clotting clotting assay. However, one of the participants

comment indicated that a combination of multiple methods i.e including chromogenic

assays should be considered. In terms of post infusion monitoring, if a recombinant IS

is used as the local calibrant for administered recombinant FIX, there should not be

assay discrepancy between clotting and chromogenic and between one-stage clotting

assays using different APTT reagents.

4. Using a recombinant FIX standard will reduce method dependent variability.

Comment D

“This is an important study that has been well performed. Unfortunately however, the

report is confusing because it contains multiple proposals. Some of these raise issues that

need to be clarified before I could recommend any SSC endorsement:

- Sample P (09/172) FIX antigen value: proposal supported

- Sample B (14/148) FIX concentrate: potency proposal supported. However, according

to the report “Sample B degradation data is not yet available” (see report page 12, see

also Table 12 on page 50). In absence of such date I cannot provide support for

recommending this material to WHO as the 5th

IS to the WHO.

The same holds for proposing this as the Ph Eur BRP, but I am not sure whether this

would need any formal SSC endorsement at all.

I would be in favor of proposing preparation B as 5th

IS for FIX provided that stability

data would be available to justify this.

- Sample D (14/180) recombinant FIX: I do not support this proposal because:

The use of multiple International Standards for the same coagulation factor should

be avoided whenever possible (see SSC recommendation by Hubbard et al, JTH

2013, 11(5); 988-9). The proposal for a separate recombinant FIX standard does

not fit into this policy.

This standard is not needed al all because plasma-derived and recombinant FIX

can well be assayed against the proposed 5th

IS.

Apart from these formal points, I fully support the comments from one of the

participants (see participants comment A) relating to the implications for

manufacturers of recombinant FIX products.

In view of these comments, I feel that the section on the establishment of an IS for recFIX

(Page 19-22(top)) is inappropriate and confusing.”

WHO/BS/2015.2261

Response from NIBSC

The stability data for sample B is now presented in Table 12 and it shows that this

candidate is highly stable and therefore suitable as a WHO IS.

With reference to Sample D, the recombinant FIX, the SSC publication quoted is a

recommendation and was written when limited data on the comparability of recombinant

and modified products with plasma derived standards were available. The publication

indeed mapped out routes for potency labelling and that if the product can be assayed

validly against the current IS using a particular method type, then it should be assayed

against the IS and labelled in IU. However, the recommendation did not give an

indication on how to deal with assay discrepancy within one method type, for example,

within the clotting method. The study results did show that both plasma derived and

recombinant FIX “can well be assayed against the proposed 5th

IS” i.e. gave statistically

valid assays, however, there is significant potency discrepancies (up to 63 and 88% for

samples A and D respectively) by clotting assays using different APTT reagents when the

recombinant FIX was assayed against the IS and this discrepancy was markedly lower

when the plasma derived FIX was assayed against the IS.

Proposal and Recommendation

In view of the objections raised by one participant and one SSC expert on the proposal to

establish an International Standard for Recombinant FIX, this proposal will not be put forward to

the ECBS and the data and discussion on this topic are for information only. The value

assignments for FIX antigen and the 5th

IS for FIX Concentrate have unanimous support and

therefore it is proposed to the ECBS that:

1. Sample P, the 4th

International Standard for Factors II, VII, IX, X, Plasma,

(09/172)

Addition of FIX antigen value to existing standard

The assigned value for FIX antigen: 0.9 IU/ampoule

2. Sample B, 14/148 be established as the:

WHO 5th

International Standard for Blood Coagulation Factor IX, Concentrate,

Human

The assigned value for functional activity: 10.5 IU/ampoule

WHO/BS/2015.2261

References

1. WHO technical report series no 964, Fifty-ninth report;

http://www.who.int/28iological/WHO_TRS_964_web.pdf?ua=1

2. WHO/BS/08.2097. http://whqlibdoc.who.int/hq/2008/WHO_BS_08.2097_eng.pdf

3. WHO Technical Report Series, No. 932, 2006. Annex 2 Recommendations for the

preparation,characterization and establishment of international and other biological

reference standards (revised 2004)

4. CombiStats v5.0, EDQM – Council of Europe, www.combistats.eu.

5. Kirkwood, TBL. Predicting the stability of biological standards and products. Biometrics.

1977; 33: 736-742.

Acknowledgements

We would like to acknowledge:

the participants of the study

Dr Paul Matejtschuk, Technology, Development and Infrastructure (TDI), NIBSC for the

formulation and trial fills of candidates

the staff of the Centre for Biological Reference Material (CBRM) for processing the

candidates

Wyeth BioPharma, USA; CSL Behring, USA; Baxter BioScience AG, Austria; Emergent

Biosolutions, Canada for the kind donation of candidate materials

.

http://www/

http://whqlibdoc.who.int/hq/2008/WHO_BS_08.2097_eng.pdf

http://www.combistats/

WHO/BS/2015.2261

Table 1: Product Summary

Code 07/142

Sample A

14/148

Sample B

14/162

Sample C

14/180

Sample D

Presentation Sealed, glass 3 ml DIN ampoules

Number of Ampoules

available 22,399 20,689 23,300 25,133

Date Filled 24 May 2007 19 Jun 2014 25 Sept 2014 02 Oct 2014

Excipients

50 mM Tris, pH 7.4

150 mM NaCl,

2 mg/ml Trehalose,

5 mg/ml human

albumin,

50 mM Tris, pH 7.4

150 mM NaCl,

2 mg/ml Trehalose,

10 mg/ml human

albumin

10 mM Tris, pH 7.4

1% sucrose,

4% mannitol,

5 mg/ml human

albumin,

10 mM Tris pH 7.4

1% sucrose,

4% mannitol,

5 mg/ml human

albumin,

CV of fill mass (%) 0.134 (n=653) 0.242 (n=869) 0.213 (n=839) 0.126 (n=825)

Mean dry weight (g, n = 6) 0.0242 0.0316 0.0560 (n=5) 0.0613

Mean head space oxygen

(%, n=12) 0.70 0.43 0.30 0.20

Residual moisture (%,

n=12) 0.139 0.220 0.842 0.65

Storage Conditions -20°C

Address of processing

facility NIBSC, Potters Bar, EN6 3QG, UK

Address of present

custodian NIBSC, Potters Bar, EN6 3QG, UK

WHO/BS/2015.2261

Table 2A: Methods and reagents used by the participants in the study – clotting assays

Lab

No Method

Pre-

dilution in

Plasma (P)

or Buffer

(B)

APTT

Reagent/Chromogenic

Kit source

Source of plasma Instrument

1 Clotting P Actin-FSL Siemens Sysmex CS2100i

2a Clotting P Actin-FS Technoclone ACL-TOP 500

2b Clotting P APTT-SP Technoclone ACL-TOP 500

2c Clotting P Cephascreen Technoclone ACL-TOP 500

2d Clotting P Dapttin Technoclone ACL-TOP 500

2e Clotting P Synthasil Technoclone ACL-TOP 500

3 Clotting P C.K. Prest Stago Coagulometer Start 4

4 Clotting P Dapttin Technoclone BCS-XP

5 Clotting P Pathromtin Siemens BCS-XP

6 Clotting P Actin FS Instrumentation Laboratory BCS-XP

7 Clotting B PTT-A George King STA-R Evolutic

8a Clotting P Actin-FSL Siemens BCS-XP

8b Clotting P Actin-FSL Siemens CA-1500

8c Clotting P Pathromtin Siemens BCS-XP

8d Clotting P Pathromtin Siemens CA-1500

9 Clotting P Actin-FS Siemens ACL-TOP 700

10 Clotting B Actin-FSL Precision Biologics BCS-XP

11 Clotting B CK-Prest Affinity Biologicals STA-R Evolution

13 Clotting B TriniClot aPTT HS Precision Biologics ACL-TOP

14 Clotting P Actin-FS Siemens BCS-XP

15 Clotting B PTT Stago Stago Compact

16 Clotting P Actin Siemens CL 8 (Behnk Electonik)

17 Clotting B TriniClot APTT Precision Biologics STA-R Evolution

18 Clotting B Actin-FS Stago ACL-9000

19 Clotting P Trinity George King ACL-TOP 500

20 Clotting P Actin-FSL HRF BCS-XP

22 Clotting B SynthASil Instrumentation Laboratory ACL-TOP 500

23 Clotting B APTT-SP Helena Biosciences ACL-9000

24 Clotting P PTT-A Precision Biologics STA-R Eveolution

25 Clotting P Pathromtin SL Siemens BCS

27 Clotting P APTT-SP Stago ACL Elite Pro

28 Clotting B Cephascreen Stago STAR

28 Clotting B CK-Prest Stago STAR

28 Clotting B PTTA Stago STAR

29 Clotting P Actin Siemens Sysmex CA-1500

30 Clotting P APTT Synth Grifols Grifols Amelug CS-190 Amax

31 Clotting B Cephascreen Stago STA-R Evolution

WHO/BS/2015.2261

Lab

No Method

Pre-

dilution in

Plasma (P)

or Buffer

(B)

APTT

Reagent/Chromogenic

Kit source

Source of plasma Instrument

31 Clotting B CK-Prest Stago STA-R Evolution

32 Clotting B CK Prest George King STA-R Evolution

33 Clotting P SynthaFax HRF ACL Elite Pro

34 Clotting P Pathromtin SL Siemens BCS-XP

35 Clotting B Actin Siemens CA-1500

36 Clotting B Dapttin Hyphen BCS-XP

37 Clotting B Cephen Hyphen BCS

39 Clotting P APTT-SP Helena Biosciences ACL 200 & ACL 3000Plus

40 Clotting P Actin-FS Siemens BCS-XP

41 Clotting B Actin-FSL Siemens BCS-XP

42 Clotting P SynthaFax Instrumentation Laboratory ACL Elite Pro

43 Clotting P APTT-SP Instrumentation Laboratory ACL-10000

44 Clotting P SynthaSil Instrumentation Laboratory ACL TOP 300

45 Clotting P Actin-FS Precision Biologic Sysmex CS5100i

46 Clotting P Actin-FSL Siemens Sysmex CA7000

47 Clotting P Actin-FS Technoclone Sysmex CS5100

48 Clotting B SynthaSil Instrumentation Laboratory ACL-TOP 700

49 Clotting P Cephen Hyphen STAR

WHO/BS/2015.2261

Table 2B: Methods and reagents used by the participants in the study – chromogenic assays

Lab

No

APTT

Reagent/Chromogenic

Kit source

Instrument

2a Hyphen ACL-TOP 500

2b Rossix ACL-TOP 500

4 Rossix Biotek Platereader

6 Rossix BCS-XP

12a Hyphen SpectraMax

12b Rossix SpectraMax

21 Hyphen BCS-XP

26 Rossix ThermoMax

33 Hyphen Biotek Platereader

36 Hyphen Elx808

40 Rossix BCS-XP

41 Rossix BCS-XP

43 Hyphen SpectraMax

*45 Hyphen* Sysmex CA7000

48 Hyphen ACL-TOP 700

49 Hyphen STAR

*Single point results, not included in analysis

WHO/BS/2015.2261

Table 2C: Methods and reagents used by the participants in the study – antigen assays

Lab

No ELISA Kit used Pool used

2a Asserchrom (Stago) Frozen pool, n = 10

2b Visulize (Affinity

Biologicals) Frozen pool, n = 10

2c Zymatest (Hyphen

Biomed) Frozen pool, n = 10

10 Visulize (Affinity

Biologicals) Local pool, n = 12


Biologicals) Two Local pools n = 20 (total)

21 Cedarane Commercial Pool, n > 240

23 AssayMax ELISA

(AssayPro) Frozen pool, n = 1134

24 Asserchrom (Stago) Two commercial pool, n = 21 & n = 26

28 Asserchrom (Stago) Two Local pools (2 days fresh and 2 days frozen), n =

20 (total)

31 Asserchrom (Stago) Two Local pools, n = 37 (total)


Biologicals) Two commercial pools, n > 50

34 Asserchrom (Stago) Commercial pool

38 Affinity Biologicals Commercial pool n > 30

40 Asserchrom (Stago) Two local pools (2 days fresh and 2 days frozen), n = 18

(total)

43 Asserchrom (Stago) Kit standard


Biologicals) Two commercial pools, n = 200 (total)

49 Zymutest (Hyphen) Two commercial pools, n > 40 (total)

WHO/BS/2015.2261

Table 3A: Clotting assays results for samples A, B, C and D calculated relative to S:

Laboratory mean (GM), geometric coefficient of variation (GCV) and overall GM and

GCV. Results using buffer as pre-diluent are denoted in red, all other laboratories used

haemophilia plasma as diluent.

Lab APTT reagent

A B C D

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

1 Actin-FSL 9.8 6.1% 4 10.8 7.2% 4 9.8 4.2% 4 9.5 4.3% 4

2a Actin-FS 12.3 3.1% 4 10.5 6.5% 4 9.0 5.0% 4 11.9 6.3% 4

2b APTT-SP 9.4 2.9% 4 10.5 5.3% 4 8.8 3.9% 4 8.9 5.4% 4

2c Cephascreen 12.4 4.2% 4 10.8 4.3% 4 8.8 3.7% 4 11.5 4.7% 4

2d Dapttin 11.2 6.4% 4 11.0 0.5% 3 9.2 2.9% 4 10.1

2

2e Synthasil 10.9 4.3% 4 10.4 7.0% 4 8.9 3.1% 4 10.6 5.6% 4

3 C.K. Prest 9.0 7.3% 4 10.9 6.6% 4 8.7 5.5% 4 8.3 7.0% 4

4 Dapttin 8.1 6.5% 4 10.5 4.3% 4 8.5 7.0% 4 7.6 3.0% 4

5 Pathromtin SL 9.5 1.5% 4 11.1 0.8% 4 8.8 2.2% 4 8.5 1.9% 4

6 ActinFS 12.4 7.1% 4 10.4 4.0% 4 9.2 6.7% 4 11.7 16.6% 4

7 PTT-A 10.4 3.4% 4 10.4 21.9% 4 9.4 7.0% 4 12.4 19.2% 4

8a ActinFSL-BSCXP 9.2 1.7% 4 10.3 4.1% 4 8.4 5.1% 4 9.2 4.3% 4

8b ActinFSL-CA1500 10.0 3.2% 4 10.3 1.9% 4 8.8 4.7% 4 10.2 2.1% 4

8c Pathromtin SL-

BSCXP 9.1 3.1% 4 10.4 4.3% 4 8.6 6.4% 4 8.7 5.3% 4

8d Pathromtin SL-

CA1500 8.7 5.1% 4 10.2 5.0% 4 8.7 8.0% 4 8.6 2.8% 4

9 ActinFS 9.2 5.8% 4 10.8 2.9% 4 8.9 4.2% 4 8.8 3.1% 4

10 ActinFSL 9.9 11.5% 4 9.3 17.7% 4 7.9 6.3% 4 9.7 9.1% 4

11 CK-Prest 10.1 3.8% 4 10.4 3.5% 4 8.7 4.3% 4 9.5 5.9% 4

13 TriniClot 10.3 2.9% 4 11.4 6.0% 4 8.2 3.8% 4 9.1

2

14 ActinFS 11.4 3.5% 4 10.5 1.4% 4 9.0 4.1% 4 11.2 3.7% 4

15 PTT-A 7.7

2 8.5 2.8% 3 6.0 4.6% 4 6.6

2

16 Actin 10.2 4.5% 4 10.4 4.7% 4 8.7 3.5% 4 9.5 2.8% 4

17 TriniClot 9.5 2.8% 4 10.5 5.3% 4 8.5 3.0% 4 9.1 1.6% 4

18 ActinFS 10.1 3.7% 4 10.4 5.5% 4 8.9 5.5% 4 9.8 3.8% 4

19 TriniClot 10.2 1.9% 4 10.7 4.1% 4 9.2 1.3% 4 9.3 2.6% 4

20 ActinFSL 11.1 4.4% 3 11.3 5.9% 4 9.2 3.9% 4 10.8 2.9% 4

22 SynthaSil 10.6 4.2% 4 10.9 10.4% 4 8.8 2.3% 4 10.9 13.0% 4

23 APPT-SP 11.0 8.4% 4 10.4 5.9% 4 8.5 10.3% 3 10.4 10.2% 4

24 PTT-A 10.2 13.1% 4 10.3 11.5% 4 8.5 10.4% 4 10.1 7.4% 4

25 Pathromtin SL 9.4 3.7% 4 10.4 1.4% 4 8.5 3.3% 4 8.8 3.8% 4

27 APTT-SP 10.6 2.7% 4 10.8 2.3% 4 8.9 3.9% 4 10.0 4.0% 4

28a Cephascreen 9.8

1 10.3

1 7.3

1 9.0

1

28b CK-Prest 10.0

2 10.7

2 7.8

2 8.6

2

28c PTT-A 10.8

1 11.2

1 9.4

1 11.0

1

29 Actin 8.8 8.5% 4 10.3 2.6% 4 8.6 3.7% 4 8.4 11.7% 4

30 APTT-synth Grifol 9.7 2.0% 4 10.6 1.7% 4 8.7 0.5% 4 9.2 0.5% 4

31a Cephascreen 9.4 16.1% 4 11.3 25.6% 3 8.6 9.6% 3 9.1 13.4% 3

31b CK-Prest 9.5 7.1% 4 9.9 6.4% 4 7.1 6.6% 3 8.0 13.9% 4

32 CK Prest 10.0 6.8% 4 10.8 5.1% 4 8.5 11.5% 4 9.3 5.9% 4

33 SynthaFax 7.6 9.3% 5 11.2 17.9% 5 9.5 16.5% 5 7.2 11.4% 5

34 Pathromtin SL 10.1 9.2% 4 10.9 8.4% 4 8.9 10.2% 3 9.0 3.7% 4

35 Actin 10.2 2.6% 4 10.4 1.5% 4 8.7 1.8% 4 10.1 2.4% 4

36 Dapttin 8.0 4.7% 4 10.6 4.7% 4 9.0 2.4% 4 7.6 4.1% 4

WHO/BS/2015.2261

Lab APTT reagent

A B C D

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

37 Cephen 9.9 1.5% 4 10.5 1.6% 4 9.0 1.7% 4 9.6 1.0% 4

39 APTT-SP 9.1 3.1% 4 10.4 2.3% 4 9.2 6.1% 4 9.2 4.5% 4

40 ActinFS 9.8 4.7% 4 9.7 7.2% 4 9.2 5.4% 4 9.5 9.9% 4

41 ActinFSL 10.1 4.3% 4 10.6 3.1% 4 8.2 1.9% 4 9.3 3.8% 3

42 SynthaFax 7.6 13.3% 4 10.3 19.5% 4 10.6 17.7% 4 7.5 23.6% 4

43 APTT-SP 9.7 2.3% 4 10.4 2.5% 4 9.0 2.4% 4 9.0 11.9% 4

44 Synthasil 10.5 2.9% 4 10.6 3.3% 4 9.1 2.5% 4 9.6 11.4% 4

45 ActinFS 9.0 8.2% 4 9.7 6.6% 4 8.6 6.9% 4 10.4 31.8% 4

46 ActinFSL 8.9 3.6% 4 10.4 2.3% 4 8.4 5.3% 3 8.4 0.6% 4

47 ActinFS 11.0 2.5% 4 10.5 2.6% 4 9.6 1.0% 4 10.5 1.6% 4

48 SynthaSil 9.8 4.8% 4 10.2 3.3% 4 8.6 5.5% 4 9.5 3.3% 4

49 Cephen 10.9 1.7% 4 10.5 1.2% 4 9.1 1.1% 3 10.2 1.0% 4

Overall GM / GCV/ n 9.8 11.6% 55 10.5 4.8% 55 8.7 8.4% 55 9.4 13.4% 55

Table 3B: Chromogenic assays results for samples A, B, C and D calculated relative to S:

Laboratory mean (GM), geometric coefficient of variation (GCV) and overall GM and

GCV.

Lab Chromogenic

Kit

A B C D

GM GCV n GM GCV n GM GCV n GM GCV n

12a Hyphen 7.2 4.3% 4 10.1 6.7% 4 8.5 5.5% 4 6.7 5.8% 4

12b Rossix 7.5 6.4% 4 10.3 2.0% 3 8.8 4.6% 4 7.5 7.9% 4

2a Hyphen 7.0 11.6% 4 9.5 32.5% 4 9.0 12.1% 4 6.5 11.9% 4

2b Rossix 7.9 5.9% 4 11.0 6.2% 4 8.6 8.0% 4 7.3 5.2% 4

21 Hyphen 9.3 8.8% 4 13.7 4.3% 4 11.4 7.4% 4 8.4 4.0% 3

26 Rossix 8.8 6.1% 4 10.4 2.8% 4 8.8 1.8% 4 8.6 4.0% 4

33 Hyphen 8.5 5.5% 4 11.8 3.5% 4 10.0 3.2% 4 8.3 4.4% 4

36 Hyphen 7.5 6.4% 4 10.5 6.4% 4 9.1 4.0% 4 7.1 3.9% 4

4 Rossix 7.9 7.9% 4 10.6 5.7% 4 8.9 6.1% 4 7.5 5.3% 4

40 Rossix 8.3 5.3% 3 10.4 4.8% 3 10.1 12.3% 3 7.8 10.3% 3

41 Rossix 7.9 7.9% 4 10.3 1.2% 4 8.7 4.6% 4 7.3 0.9% 4

43 Hyphen 7.5 12.8% 3 9.5 11.2% 4 8.4 7.3% 3 7.2 22.3% 3

48 Hyphen 6.7 6.3% 3 10.2 7.2% 3 8.8 7.0% 3 6.3 2.2% 3

49 Hyphen 6.7 4.7% 4 10.4 4.3% 4 8.7 1.8% 4 6.2 4.6% 4

6 Rossix 8.5 13.5% 4 10.7 12.6% 4 8.9 6.4% 4 7.5 8.3% 4

Overall GM / GCV/ n 7.8 10.2% 15 10.6 9.3% 15 9.1 8.6% 15 7.3 10.4% 15

WHO/BS/2015.2261

Table 4: Effect of pre-diluent on potency estimates by clotting assay for samples A, B, C

and D

Samples

FIX deficient plasma Buffer (without/with outlier

excluded)

P-value

(without/with

outlier

excluded) GM

IU/ml GCV n GM IU/ml GCV n

A 9.8 13.0% 35 9.8/9.9 9.0/6.9% 20/19 0.927/0.747

B 10.5 3.3% 35 10.4/10.5 6.7/4.6% 20/19 0.435/0.977

C 8.9 4.8% 35 8.3/8.5 11.0/7.8% 20/19 0.001/0.001

D 9.4 12.9% 35 9.3/9.5 14.6/11.9% 20/19 0.872/0.713

Table 5A: Overall potency estimates by clotting or chromogenic methods

Samples

Clotting Chromogenic Ratio

Clotting/Chromogenic P-value GM

IU/ml GCV n

GM

IU/ml GCV n

A 9.8 11.6% 55 7.8 10.2% 15 1.26 <0.001

B 10.5 4.8% 55 10.6 9.3% 15 0.99 0.546

C 8.7 8.4% 55 9.1 8.6% 15 0.96 0.087

D 9.4 13.4% 55 7.3 10.4% 15 1.29 <0.001

P 0.90 16.1% 54 0.81 8.6% 15 1.10 0.012

Table 5B: Overall potency estimates by clotting or chromogenic methods. Excluding

Lab 15 (Clotting) and Lab 21 (Chromogenic)

Samples

Clotting Chromogenic Ratio

Clotting/Chromogenic

P-value

GM

IU/ml GCV n

GM

IU/ml GCV n

A 9.9 11.1% 54 7.7 9.0% 14 1.29 <0.001

B 10.5 3.8% 54 10.4 5.6% 14 1.01 0.330

C 8.8 6.6% 54 8.9 5.7% 14 0.99 0.337

D 9.4 12.5% 54 7.3 10.0% 14 1.29 <0.001

P 0.90 16.2% 53 0.81 8.1% 14 1.11 0.009

Table 6: Comparison of potency estimates obtained using Hyphen and Rossix

chromogenic kits (excluding Lab 21)

Samples

Hyphen Rossix Ratio

Hyphen/Rossix P-value GM

IU/ml GCV n

GM

IU/ml GCV n

A 7.3 8.5% 7 8.1 5.6% 7 0.90 0.014

B 10.3 7.7% 7 10.5 2.4% 7 0.98 0.415

C 8.9 6.1% 7 9.0 5.6% 7 0.99 0.810

D 6.9 10.7% 7 7.6 5.9% 7 0.91 0.032

P 0.78 9.4% 7 0.83 5.0% 7 0.94 0.133

WHO/BS/2015.2261

Table 7: Summary of overall potency estimates (GM) for functional activity (clotting

and chromogenic assays) and coefficient of variation expressed as %GCV

Samples All Excluding outliers

GM IU/ml GCV n GM IU/ml GCV n

A 9.4 15.4% 70 9.4 15.5% 68

B 10.5 5.9% 70 10.5 4.2% 68

C 8.8 8.6% 70 8.8 6.4% 68

D 8.9 17.1% 70 8.9 16.9% 68

P 0.88 15.4% 69 0.88 15.7% 68

WHO/BS/2015.2261

Table 8: Clotting, chromogenic and antigen results for sample P, the 4th

IS for FII, VII,

IX and X Plasma: Laboratory mean (GM), geometric coefficient of variation (GCV)

and overall GM and GCV. For clotting assays, results using buffer as pre-diluent are

denoted in red, all other laboratories used haemophilia plasma as diluent.

Clotting vs S Chromogenic vs S Antigen vs Local Pool

Lab APTT reagent GM

IU/ml GCV n Kit

GM

IU/m

l

GCV n Kit GM

u/ml GCV n

1 Actin-FSL 0.90 3.3% 4

2a Actin-FS 0.78 6.8% 4 Hyphen 0.77 7.8% 4 Asserachrom 0.94 3.8% 4

2b APTT-SP 0.90 2.6% 4 Rossix 0.81 4.4% 4 Visulize 1.02 10.5% 4

2c Cephascreen 0.82 5.5% 4 Zymutest 0.98 7.9% 4

2d Dapttin 0.85 3.3% 3

2e Synthasil 0.78 4.0% 4

3 C.K. Prest 0.68 4.5% 4

4 Clotting 0.81 6.9% 3 Rossix 0.84 4.6% 4

5 Pathromtin 1.15 1.2% 4

6 ActinFS 0.80 7.8% 4 Rossix 0.83 6.0% 3

7 PTT 1.10 14.9% 3

8a ActinFSL-BSCXP 0.81 4.4% 4

8b ActinFSL-

CA1500 0.77 2.3% 4

8c Pathromtin SL-

BSCXP 0.86 4.4% 4

8d Pathromtin SL-

CA1500 0.79 3.3% 4

9 ActinFS 0.88 3.3% 4

10 ActinFSL 1.09 9.0% 4 Visulize 0.87 2.6% 4

11 CK-Prest 1.02 3.0% 4 Visulize 0.85 8.3% 4

12a Hyphen 0.79 3.4% 4

12b Rossix 0.77 7.7% 4

13 TriniClot 1.22 1

14 ActinFS 0.87 2.9% 4

15 PTT 0.86 9.9% 4

16 Actin 0.94 5.7% 4

17 APTT 1.06 1.8% 4

18 ActinFS 0.92 5.2% 4

19 Trinity 0.80 0.7% 4

20 ActinFSL 0.79 8.0% 4

21 Hyphen 0.92 2.4% 4 Cedarlane 0.81 17.7% 4

22 SynthaSil 0.91 1.7% 4

23 APPT 0.94 14.4% 3 AssayPro 0.93 27.1% 4

WHO/BS/2015.2261

Clotting vs S Chromogenic vs S Antigen vs Local Pool

Lab APTT reagent GM

IU/ml GCV n Kit

GM

IU/m

l

GCV n Kit GM

u/ml GCV n

24 PTT-A 0.84 7.1% 4 Asserachrom 0.87 4.8% 4

25 Pathromtin SL 0.98 5.1% 4

26 Rossix 0.86 7.1% 4

27 APTT 0.84 2.9% 4

28a Cephascreen 1.07 1 Asserachrom 0.83 7.5% 4

28b CK-Prest 1.07 2

28c PTTA 1.17 1

29 Actin 0.82 4.5% 4

30 APTT 0.82 2.7% 4

31a Cephascreen 1.20 9.9% 4 Asserachrom 0.88 7.7% 4

31b CK-Prest 0.96 4.5% 4

32 CK Prest 1.09 5.6% 4

33 SynthaFax 1.00 12.5% 5 Hyphen 0.93 3.0% 4 Visulize 0.96 5.2% 4

34 Pathromtin SL 0.92 12.0% 4 Asserachrom 0.96 1.8% 4

35 Actin 1.10 3.7% 4

36 Dapttin 0.95 5.3% 4 Hyphen 0.80 3.3% 4

37 Cephen 0.84 3.0% 4

38 Affinity Bio’ 0.97 2.3% 4

39 APTT-SP 0.87 5.5% 3

40 ActinFS 0.81 7.8% 4 Rossix 0.83 7.9% 3 Asserachrom 0.96 1

41 ActinFSL 1.31 1 Rossix 0.90 1.6% 3

42 SynthaFax

43 APTT-SP 0.88 5.4% 3 Hyphen 0.73 2 Asserachrom 0.79 10.1% 4

44 SynthaSil 0.84 3.2% 3

45 ActinFS 0.70 5.2% 4

46 ActinFSL 0.71 3.2% 4

47 ActinFS 0.74 2.3% 4

48 SynthaSil 1.00 5.3% 4 Hyphen 0.71 5.9% 3 Visulize 0.83 2

49 Hyphen 0.80 4.1% 4 Hyphen 0.74 5.5% 4 Zymutest 0.88 11.1% 3

Overall GM / GCV / n 0.90 16.1% 54 0.81 8.6% 15 0.90 7.9% 17

Overall potency (clotting

& chromogenic) 0.88 IU/ml, GCV 15.4%, n = 69

WHO/BS/2015.2261

Table 9A: Antigen values for samples S, A, B, C, D, and P, assuming local plasma pool (L) value is 1.0 U/ml

S vs L A vs L B vs L C vs L D vs L P vs L

Lab Method GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

2 Asserachrom 10.7 10.2% 4 8.2 7.6% 4 12.4 16.4% 4 10.6 13.3% 4 7.2 6.6% 4 0.94 3.8% 4

2 Visulize 11.4 8.7% 4 7.0 4.6% 3 12.4 4.8% 4 10.4 7.1% 4 6.1 5.4% 3 1.02 10.5% 4

2 Zymutest 9.4 3.8% 4 9.5 4.2% 4 10.8 2.5% 4 9.2 3.3% 4 8.0 3.3% 4 0.98 7.9% 4

10 Visulize 9.7 4.0% 4 6.0 4.3% 4 10.7 1.9% 4 9.1 3.9% 4 5.5 3.3% 4 0.87 2.6% 4

11 Visulize 9.5 10.5% 4 6.1 8.1% 4 10.6 6.8% 4 9.0 8.5% 4 5.2 6.8% 4 0.85 8.3% 4

21 Cedarlane 11.3 17.6% 4 3.4 11.5% 4 10.5 23.7% 4 8.6 13.4% 3 2.9 15.1% 4 0.81 17.7% 4

23 AssayPro 9.8

2 6.9 14.8% 3 11.4 11.4% 4 10.4 15.3% 3 6.3

2 0.93 27.1% 4

24 Asserachrom 9.7 8.7% 4 7.9 8.4% 4 11.4 8.1% 4 9.6 4.9% 4 6.2 10.2% 4 0.87 4.8% 4

28 Asserachrom 9.3 10.6% 4 7.2 11.9% 4 10.3 9.6% 4 8.8 13.0% 4 6.0 13.2% 4 0.83 7.5% 4

31 Asserachrom 9.7 10.1% 4 7.6 9.3% 4 10.9 10.5% 4 9.3 10.8% 4 6.4 7.7% 4 0.88 7.7% 4

33 Visulize 9.4 1.3% 3 5.3 4.9% 4 9.3 12.9% 3 9.3 2.5% 3 4.6 2.6% 3 0.96 5.2% 4

34 Asserachrom 10.6 3.2% 4 8.0 3.0% 3 12.0 1.6% 4 10.5 4.4% 4 6.5 2.4% 4 0.96 1.8% 4

38 Affinity BIologicals 11.8 3.3% 4 6.8 3.2% 4 12.3 1.1% 4 10.4 2.9% 4 5.8 2.3% 4 0.97 2.3% 4

40 Asserachrom 9.5

1 9.9

1 12.3

2 11.2

2 7.7

2 0.96

1

43 Asserachrom 9.4 9.0% 4 6.4 7.8% 3 9.2

1 9.0 6.0% 4 5.0 28.0% 3 0.79 10.1% 4

48 Visulize 11.5

2 8.2

1 11.0

2 10.0

2 3.6

1 0.83

2

49 Zymutest 8.8 10.6% 3 9.0 9.4% 3 10.2 4.5% 3 8.7 9.2% 3 7.8 9.1% 3 0.88 11.1% 3

GM / GCV / n 10.0 9.2% 17 7.1 28.9% 17 11.0 9.8% 17 9.6 8.6% 17 5.7 30.7% 17 0.90 7.9% 17

WHO/BS/2015.2261

Table 9B: Antigen values for samples S, A, B, C and D relative to sample P, the 4th

IS for FII, VII, IX and X Plasma, assuming antigen

value of 0.9 U/ml

Lab Method

S vs P A vs P B vs P C vs P D vs P

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

2 Asserachrom 10.2 11.7% 4 7.8 9.1% 4 11.9 15.2% 4 10.2 17.0% 4 6.8 9.4% 4

2 Visulize 10.1 12.1% 4 6.1 10.6% 3 10.9 12.3% 4 9.2 10.1% 4 5.3 7.5% 3

2 Zymutest 8.6 11.3% 4 8.7 5.4% 4 9.8 6.5% 4 8.4 6.9% 4 7.3 5.9% 4

10 Visulize 10.0 4.4% 4 6.2 3.7% 4 11.1 3.4% 4 9.4 4.3% 4 5.7 4.2% 4

11 Visulize 10.0 3.2% 4 6.5 5.9% 4 11.2 6.2% 4 9.5 4.6% 4 5.6 5.2% 3

21 Cedarlane 12.4 4.7% 4 3.8 8.5% 4 11.6 9.4% 4 9.3 8.8% 4 3.1 15.5% 3

23 AssayPro 9.2 18.0% 4 6.4

2 9.9 14.2% 3 9.5 16.6% 4 5.7 21.0% 3

24 Asserachrom 9.8 10.8% 3 8.1 7.0% 4 11.7 8.7% 4 9.9 7.3% 4 6.5 9.5% 4

28 Asserachrom 10.0 5.1% 4 7.8 6.0% 4 11.1 3.2% 4 9.5 8.3% 4 6.5 7.1% 4

31 Asserachrom 9.9 2.6% 4 7.8 2.6% 4 11.2 2.8% 4 9.5 3.5% 4 6.6 0.6% 4

33 Visulize 8.9 5.4% 3 4.9 7.4% 4 8.7 11.4% 3 8.8 9.0% 3 4.4 8.6% 4

34 Asserachrom 9.9 2.6% 4 7.6 2.9% 3 11.3 1.7% 4 9.9 4.6% 4 6.1 3.4% 4

38 Affinity Biologicals 10.9 4.8% 4 6.3 1.5% 4 11.4 3.2% 4 9.6 5.1% 4 5.4 1.4% 4

40 Asserachrom 8.9

1

11.1

1 10.1

1 6.5

1

43 Asserachrom 10.8 6.9% 4 7.2 8.7% 3 11.4 9.7% 3 10.3 10.6% 4 5.6 19.9% 3

48 Visulize 10.7

2 7.4

1 10.3

2 9.3

2 3.1

1

49 Zymutest 9.2 7.6% 4 9.4 3.5% 4 10.4 5.6% 4 8.9 7.6% 4 8.1 3.8% 4

GM / GCV / n 9.9 9.1% 17 6.8 26.6% 16 10.9 8.2% 17 9.5 5.4% 17 5.6 31.5% 17

WHO/BS/2015.2261

Table 10A: Comparison of potency estimates by clotting assays for sample A, relative to

sample S, the 4th

IS for FIX concentrate and sample D with assigned potency of 9.4 IU/ml

based on clotting assays or value of 8.9 IU/ml based on overall potency estimate.

A vs D

Lab APTT reagent

A vs S

Assigned value of D

by clotting only: 9.4

IU/ml

Assigned value of D by

clotting and chromogenic:

8.9 IU/ml

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

1 Actin-FSL 9.8 6.1% 4 9.8 10.1% 4 9.3 10.1% 4

2a Actin-FS 12.3 3.1% 4 9.7 3.2% 4 9.2 3.2% 4

2b APTT-SP 9.4 2.9% 4 10.0 7.5% 4 9.4 7.5% 4

2c Cephascreen 12.4 4.2% 4 10.1 3.3% 4 9.6 3.3% 4

2d Dapttin 11.2 6.4% 4 10.2

2 9.7 2

2e Synthasil 10.9 4.3% 4 9.7 6.6% 4 9.2 6.6% 4

3 C.K. Prest 9.0 7.3% 4 10.2 3.3% 4 9.6 3.3% 4

4 Dapttin 8.1 6.5% 4 10.1 3.7% 4 9.5 3.7% 4

5 Pathromtin SL 9.5 1.5% 4 10.4 2.1% 4 9.9 2.1% 4

6 ActinFS 12.4 7.1% 4 9.9 10.0% 4 9.4 10.0% 4

7 PTT-A 10.4 3.4% 4 7.9 17.5% 4 7.4 17.5% 4

8a ActinFSL-BSCXP 9.2 1.7% 4 9.5 4.1% 4 9.0 4.1% 4

8b ActinFSL-CA1500 10.0 3.2% 4 9.2 2.3% 4 8.7 2.3% 4

8c Pathromtin SL-

BSCXP 9.1 3.1% 4 9.9 5.2% 4 9.3 5.2% 4

8d Pathromtin SL-

CA1500 8.7 5.1% 4 9.4 2.6% 4 8.9 2.6% 4

9 ActinFS 9.2 5.8% 4 9.9 7.4% 4 9.4 7.4% 4

10 ActinFSL 9.9 11.5% 4 9.6 7.1% 4 9.1 7.1% 4

11 CK-Prest 10.1 3.8% 4 10.0 6.1% 4 9.5 6.1% 4

13 TriniClot 10.3 2.9% 4 10.7 3.1% 4 10.1 3.1% 4

14 ActinFS 11.4 3.5% 4 9.64 1.5% 4 9.1 1.5% 4

15 PTT-A 7.7

2 10.6

1 10.1 1

16 Actin 10.2 4.5% 4 10.1 4.1% 4 9.6 4.1% 4

17 TriniClot 9.5 2.8% 4 9.81 3.3% 4 9.3 3.3% 4

18 ActinFS 10.1 3.7% 4 9.4

1 8.9 1

19 TriniClot 10.2 1.9% 4 10.3 2.9% 4 9.8 2.9% 4

20 ActinFSL 11.1 4.4% 3 8.8 3.4% 3 8.3 3.4% 3

22 SynthaSil 10.6 4.2% 4 9.1 11.0% 4 8.6 11.0% 4

23 APPT-SP 11.0 8.4% 4 9.9 7.2% 4 9.4 7.2% 4

24 PTT-A 10.2 13.1% 4 9.5 5.8% 4 9.0 5.8% 4

25 Pathromtin SL 9.4 3.7% 4 10.1 2.7% 4 9.6 2.7% 4

27 APTT-SP 10.6 2.7% 4 10.0 2.6% 4 9.5 2.6% 4

WHO/BS/2015.2261

A vs D

Lab APTT reagent

A vs S

Assigned value of D

by clotting only: 9.4

IU/ml



8.9 IU/ml

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

28a Cephascreen 9.8

1 10.2

1 9.7 1

28b CK-Prest 10.0

2 11.0 8.5% 2 10.4 8.5% 2

28c PTT-A 10.8

1 9.3

1 8.8 1

29 Actin 8.8 8.5% 4 9.8 2.9% 4 9.2 2.9% 4

30 APTT-synth Grifol 9.7 2.0% 4 9.9 2.2% 4 9.4 2.2% 4

31a Cephascreen 9.4 16.1% 4 9.9 9.2% 4 9.4 9.2% 4

31b CK-Prest 9.5 7.1% 4 11.2 7.7% 4 10.6 7.7% 4

32 CK Prest 10.0 6.8% 4 10.1 6.9% 4 9.6 6.9% 4

33 SynthaFax 7.6 9.3% 5 10.0 8.2% 5 9.5 8.2% 5

34 Pathromtin SL 10.1 9.2% 4 10.5 7.1% 4 9.9 7.1% 4

35 Actin 10.2 2.6% 4 9.5 1.0% 4 9.0 1.0% 4

36 Dapttin 8.0 4.7% 4 10.0 0.8% 4 9.4 0.8% 4

37 Cephen 9.9 1.5% 4 9.7 1.0% 4 9.2 1.0% 4

39 APTT-SP 9.1 3.1% 4 9.6

1 9.1 1

40 ActinFS 9.8 4.7% 4 9.7 7.3% 4 9.2 7.3% 4

41 ActinFSL 10.1 4.3% 4 10.4 6.8% 4 9.8 6.8% 4

42 SynthaFax 7.6 13.3% 4 9.5 9.3% 4 9.0 9.3% 4

43 APTT-SP 9.7 2.3% 4 10.1 14.2% 4 9.6 14.2% 4

44 Synthasil 10.5 2.9% 4 10.3 8.5% 4 9.7 8.5% 4

45 ActinFS 9.0 8.2% 4 8.2 33.7% 4 7.7 33.7% 4

46 ActinFSL 8.9 3.6% 4 10.0 3.0% 4 9.4 3.0% 4

47 ActinFS 11.0 2.5% 4 9.9 1.7% 4 9.3 1.7% 4

48 SynthaSil 9.8 4.8% 4 9.7 3.2% 4 9.1 3.2% 4

49 Cephen 10.9 1.7% 4 10.1 2.6% 4 9.6 2.6% 4

Overall GM / GCV / n 9.8 11.6% 55 9.8 6.1% 55 9.3 6.1% 55

Paired t-test p-value vs S 0.985 0.002

WHO/BS/2015.2261

Table 10B: Comparison of potency estimates by chromogenic assays for sample A, relative

to sample S, the 4th

IS for FIX concentrate and sample D with assigned potency of 9.4

IU/ml based on clotting assays or value of 8.9 IU/ml based on overall potency estimate.

A vs D

Lab Kit

A vs S Assigned value of D by

clotting only: 9.4 IU/ml



8.9 IU/ml

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

12 Hyphen 7.2 4.3% 4 10.1 4.8% 4 9.6 4.8% 4

12 Rossix 7.5 6.4% 4 10.1 3.4% 4 9.5 3.4% 4

2 Hyphen 7.0 11.6% 4 9.8 4.7% 4 9.3 4.7% 4

2 Rossix 7.9 5.9% 4 10.7 9.4% 4 10.1 9.4% 4

21 Hyphen 9.3 8.8% 4 10.1 7.4% 4 9.5 7.4% 4

26 Rossix 8.8 6.1% 4 9.4 11.8% 4 8.9 11.8% 4

33 Hyphen 8.5 5.5% 4 10.7 8.0% 3 10.2 8.0% 3

36 Hyphen 7.5 6.4% 4 9.7 8.4% 4 9.2 8.4% 4

4 Rossix 7.9 7.9% 4 9.6 1.1% 4 9.1 1.1% 4

40 Rossix 8.3 5.3% 3 9.9 4.6% 4 9.4 4.6% 4

41 Rossix 7.9 7.9% 4 9.9 11.3% 3 9.4 11.3% 3

43 Hyphen 7.5 12.8% 3 10.2 8.2% 4 9.6 8.2% 4

48 Hyphen 6.7 6.3% 3 9.8 8.8% 3 9.3 8.8% 3

49 Hyphen 6.7 4.7% 4 10.0 4.2% 3 9.4 4.2% 3

6 Rossix 8.5 13.5% 4 10.2 4.5% 4 9.7 4.5% 4

GM / GCV/n 7.8 10.2% 15 10.0 3.6% 15 9.5 3.6% 15

Paired t-test p-value vs S <0.001 <0.001

WHO/BS/2015.2261

Table 10C: Summary of sample A potencies against sample S, the 4th

IS for FIX

concentrate and sample D, assuming (assuming the assigned potency of 9.4 IU/ml based on

clotting assays).

Method

A v S A v D (9.4 IU/ml) A v D (8.9 IU/ml)

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

Clotting 9.8 11.6% 55 9.8 6.1% 55 9.3 6.1% 55

Chromogenic 7.8 10.2% 15 10.0 3.6% 15 9.5 3.6% 15

Clotting /Chromogenic ratio (unpaired

t test) 1.25 (p <0.001) 0.98 ( p = 0.282)

Overall potency (incl clotting and

chromogenic) 9.4 15.4% 70 9.9 5.7% 70 9.3 5.7% 70

paired t-test p value for A vs S against

A vsD 0.007 0.956

unpaired t-test p value for A vs S

Clotting only against overall potency

for A vsD

0.779 0.003

WHO/BS/2015.2261

Table 11A: Comparison of potency estimates for sample Purple against sample S, the 4th

IS for FIX concentrate, sample A (Acl), assuming the assigned potency of 9.8 IU/ml based

on clotting assays, and sample D (Dcl), assuming the assigned potency of 9.4 IU/ml based

on clotting assays.

Lab Type Method Purple v S Purple v Acl Purple v Dcl

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

2 Clotting Actin-FS 9.6 6.3% 4 7.7 3.3% 4 7.6 1.9% 4

2 Clotting APTT-SP 8.3 8.9% 4 8.6 10.5% 4 8.7 11.3% 4

2 Clotting Cephascreen 10.3 7.4% 4 8.1 5.1% 4 8.4 5.6% 4

2 Clotting Dapttin 9.3 7.4% 3 8.0 9.5% 3 9.2 2.9% 2

2 Clotting Synthasil 9.2 4.5% 4 8.3 8.1% 4 8.2 6.8% 4


37 Clotting Cephen 8.7 7.5% 4 9.0 4.9% 4 8.9 6.2% 4

44 Clotting SynthaSil 9.0 1.1% 4 8.5 2.3% 4 8.9 11.0% 4

45 Clotting ActinFS 8.5 7.3% 4 9.2 10.2% 4 7.7 26.3% 4


2 Chromo Hyphen 8.0 25.2% 4 11.1 24.6% 4 11.4 20.9% 4

2 Chromo Rossix 7.2 5.8% 4 9.0 3.7% 4 9.3 5.6% 4

36 Chromo Hyphen 7.0 4.2% 4 9.0 1.6% 4 9.3 4.8% 4

48 Chromo Hyphen 6.3 4.1% 3 9.2 2.8% 3 9.4 1.8% 3

GM / GCV / n 8.4 14.6% 14 8.8 9.4% 14 8.9 10.7% 14

paired t-test, S v A; S v D; p-

value 0.338 0.273

WHO/BS/2015.2261

Table 11B: Comparison of potency estimates for sample Purple against sample S, the 4th

IS for FIX concentrate, sample A (Acl+ch),assuming the assigned potency of 9.4 IU/ml

based on overall potency estimate, and sample D (Dcl+ch) assuming the assigned potency of

8.9 IU/ml based on overall potency estimate.

Lab Type Method

Purple v S Purple v Acl+ch Purple v Dcl+ch

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

2 Clotting Actin-FS 9.6 6.3% 4 7.3 3.3% 4 7.2 1.9% 4

2 Clotting APTT-SP 8.3 8.9% 4 8.2 10.5% 4 8.3 11.3% 4

2 Clotting Cephascreen 10.3 7.4% 4 7.8 5.1% 4 8.0 5.6% 4


2 Clotting Synthasil 9.2 4.5% 4 8.0 8.1% 4 7.8 6.8% 4


37 Clotting Cephen 8.7 7.5% 4 8.6 4.9% 4 8.6 6.2% 4


45 Clotting ActinFS 8.5 7.3% 4 8.8 10.2% 4 7.3 26.3% 4


2 Chromo Hyphen 8.0 25.2% 4 10.7 24.6% 4 10.8 20.9% 4

2 Chromo Rossix 7.2 5.8% 4 8.6 3.7% 4 8.8 5.6% 4

36 Chromo Hyphen 7.0 4.2% 4 8.7 1.6% 4 8.8 4.8% 4

48 Chromo Hyphen 6.3 4.1% 3 8.8 2.8% 3 8.9 1.8% 3

GM / GCV / n 8.4 14.6% 14 8.5 9.4% 14 8.5 10.7% 14

paired t-test, S v A; S v D; p-

value 0.832 0.847

WHO/BS/2015.2261

Table 11C: Summary of potency estimates for sample Purple against sample S, the 4th

IS

for FIX concentrate, sample Acl (assuming the assigned potency of 9.9 IU/ml based on

clotting assays), and sample Dcl (assuming the assigned potency of 9.4 IU/ml based on

clotting assays).

Table 11D: Summary of potency estimates for sample Purple against sample S, the 4th

IS

for FIX concentrate, sample A (Acl+ch),assuming the assigned potency of 9.4 IU/ml based

on overall potency estimate, and sample D (Dcl+ch) assuming the assigned potency of 8.9

IU/ml based on overall potency estimate.

Method Purple v S Purple v Acl Purple v Dcl

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

Clotting 9.0 8.3% 10 8.8 7.1% 10 9.1 8.0% 10

Chromogenic 7.1 10.1% 4 9.4 10.6% 4 10.4 10.8% 4

Overall (incl clotting and chromogenic) 8.4 14.6% 14 9.0 9.4% 14 9.5 10.7% 14

Clotting /Chromogenic ratio

(unpaired t-test p-value) 1.26 ( p < 0.001) 0.90 (p =0.038) 0.88 (p = 0.020)

Method

Purple v S Purple v Acl+ch Purple v Dcl+ch

GM

IU/ml GCV n

GM

IU/ml GCV n

GM

IU/ml GCV n

Clotting 9.0 8.3% 10 8.2 7.1% 10 8.1 8.0% 10

Chromogenic 7.1 10.1% 4 9.2 10.6% 4 9.3 10.8% 4

Overall (incl clotting and chromogenic) 8.4 14.6% 14 8.8 9.4% 14 8.5 10.7% 14

WHO/BS/2015.2261

Figure 1: Histogram showing estimated potency of sample A relative to sample S, the 4th

IS

for Blood Coagulation Factor IX, Concentrate, 07/182. Each box denotes overall geometric

mean potency (GM) from one laboratory

Figure 2: Histogram showing estimated potency of sample B relative to sample S, the 4

th IS



GM= 9.4 IU/ml; GCV= 15.5%, n = 68

Outliers: clotting, Lab15; Chromogenic , Lab 21

GM= 10.5 IU/ml; GCV= 4.2%, n = 68

Outliers: clotting, Lab15; Chromogenic , Lab 21

WHO/BS/2015.2261

Figure 3: Histogram showing estimated potency of sample C relative to sample S, the 4th

IS



Figure 4: Histogram showing estimated potency of sample D relative to sample S, the 4

th IS



GM= 8.8 IU/ml; GCV= 6.4%, n = 68

Outliers: clotting, Lab15; Chromogenic ,

Lab 21

GM= 8.9 IU/ml; GCV= 16.9%, n = 68

Outliers: clotting, Lab15; Chromogenic ,

Lab 21

WHO/BS/2015.2261

Figure 5: Laboratories’ potency estimates for samples A, B, C and D relative to S, the 4th

IS

for FIX concentrate, obtained using different APTT reagents

Potency range: 7.6 – 12.4 IU/ml




WHO/BS/2015.2261

Figure 6: Histogram showing estimated potency of sample P relative to sample S, the 4th

IS



Figure 7: Histogram showing estimated potency of sample A relative to sample D,

assuming potency of 9.4 IU/ml. Each box denotes overall geometric mean potency (GM)

from one laboratory. For comparison - Inset: histogram of sample A relative to 4th

IS for

FIX concentrate.

Table 12: Accelerated Degradation Study

GM= 0.88 IU/ml; GCV= 15.7%, n = 68

Outliers: clotting, Lab15; Chromogenic, Lab 21

GM= 9.9 IU/ml; GCV= 5.7%, n = 68

Outliers: clotting, Lab15; Chromogenic, Lab 21

WHO/BS/2015.2261

Sample A, 07/142

(7.5 years)

Sample B, 14/148

(6 and 8 months)

Sample C, 14/162

(6 months)

Sample D, 14/180

(6 months)

Temp °C Potency

Loss

%/year

95%

Upper

CL %Loss

/year

Potency

Loss

%/year

95%

Upper

CL %Loss

/year

Potency

Loss

%/year

95%

Upper

CL %Loss

/year

Potency

Loss

%/year

95%

Upper

CL %Loss

/year

-20 0.000 0.000 0.000 0.000 0.001 0.002 0.000 0.000

+4 0.002 0.005 0.018 0.028 0.068 0.160 0.000 0.001

+20 0.105 0.164 0.547 0.715 1.088 2.031 0.003 0.186

+37 3.745 4.584 12.884 14.315 14.136 19.614 0.431 13.184

Table 13: Mean NAPTT clotting times carried out in accordance with EP method for

activated coagulation factors. Dilutions carried out based on estimated potency against

sample S by clotting assays (Table 3A)

Mean Blank (s)

N = 4

Mean 1/10 (s)

N = 2

Mean 1/100 (s)

N = 2

S 316 238 269

A 315 232 272

B 315 243 275

C 312 245 271

D 288 239 273

Table 14: Estimated level of activated factor IX (FIXa) (by chromogenic assays, n = 2,

against the 1st International Standard for FIXa) in the 4

th IS for FIX, concentrate (S) and

the candidates.

FIXa mIU/ampoule

(95% CL)

IU FIXa/1000

IU FIX*

S 3.5

(2.9 – 4.1) 0.44

A 12.3

(11..8 – 12.9) 1.26

B 4.3

(3.9 – 4.8) 0.41

C 3.7

(3.4 – 4.0) 0.43

D 2.3

(2.0 – 2.6) 0.24

*Based on estimated FIX against sample S (clotting)

WHO/BS/2015.2261

Appendix I: List of Participants

Kirsten Villadsen, Aarhus Universitets Hospital, Denmark

Denise Foulon, Affinity Biologicals, Canada

Stephanie Eichmeir, Christoph Kefeder & Heidemarie Schindl, AGES MEA OMCL, Austria

Natalija Kerestes, Baxter AG (IBC/Coagulation & Biochemical Methods), Austria

Peter Gaertner, Baxter AG (QC Vienna), Austria

Martin Blum, Baxter AG, Austria

Herbert Gritsch, Baxter Innovations GmbH, Austria

Amanda Blande, Bio Products Laboratory (QC), UK

Ruth Archer & Sarah Kingsland, Bio Products Laboratory (R&D), UK

Buyue Yang, Jurg Sommer & Sara Bardan, Biogen (Cambridge Campus), USA

Maureen Shreve, Biogen Idec, USA

Kerstin Dohme, Biotest AG (QC), Germany

Steffen Kistner, Karin Fuchs & Jens Daufenbach, Biotest AG, Germany

Yideng Liang, Tseday Tegegn & Mikhail Ovanesov, CBER/FDA, USA

Wendy Hollon & Meera Chitlur, Children's Hospital of Michigan, USA

Annette Feusser, CSL Behring, Germany

Klara Howard & Martina Treutlein, CSL Behring, Germany

Nathalie Barat, François Nicham & Jérôme Beltran, Diagnostica Stago (R&D Department), France

Celine Aime, Juliette Grenet, Isabelle Regairaz & Jessica Lelansans, Diagnostica Stago, France

Joy Bosica & Derek Toth, Emergent Biosolutions, Canada

Dot Adcock, Stephanie Stubert & Mike Taylor, Esoterix Coagulation, Laboratory Corporation of

America Holdings, USA

Brigitte Wild & Chantal Raphalen, European Directorate for the Quality of Medicines and

Healthcare, France

Sharon Flan & Barb Young, George King BioMedical, USA

Will Stevens & Albert Cheung, Health Canada, Canada

Joanne McGrath, Hemotasis Reference Laboratory, Canada

Jean Amiral, Hyphen Biomed, France

Luis Soares & Mjoao Portela, Infarmed (DCQ), Portugal

Nuria Hosta, Instituto Grifols, Spain

Stefania Menga, Paola Rossi & Nicola Luchesi, Kedrion, Italy

Claudia van Rijn & Jeroen Eikenboom, Leiden University Medical Center, Netherlands

Francois Hemery, LFB, France

Chi Young Ahn, Ki Kyung Jung & Sang Mi Park, Ministry of Food and Drug Safety, Korea

Stella Williams & Luis Figerido, National Institute for Biological Standards and Control, UK

Wang Quigzhou, National Institute for Food and Drug Control China

Madoka Kuramitsu, National Institute of Infectious Diseases, Japan

Anne Fuglsang Barslund, Novo Nordisk A/S, Denmark

Vibeke Sønder, Novo Nordisk Park, Denmark

WHO/BS/2015.2261

Monika Stadler, Katharina Pock, Susanna Huber and Sandra Janisch, Octapharma Pharmazeutika

Produktions GesmBH, Austria

Peter Baker, Oxford Haemophilia and Thrombosis Centre, UK

Andreas Hunfeld, Sylvia Rosenkranz & Andrea Schroda, Paul-Ehrlich-Institut, Germany

Yolanda Samino Garcia, Jose Alberto Martinez-Marin, Juan Solis-Fernandez, Esther Otero

Rodriguez, Emma Rodriguez Vignote, Helena Holguin Asensio & Pedro Paredes-Martinez, Pfizer

Algete (Wyeth Farma SA), Spain

Robin Verhaar, RIVM-GZB, Netherlands

Pia Bryngelhed & Steffen Rosen, Rossix AB, Sweden

Anne Riddell, Royal Free Hospital, UK

Annette Bowyer, Royal Hallamshire Hospital, UK

Jeannette Rentenaar & Carel Eckmann, Sanquin Blood Supply Foundation, Netherlands

Regina Gebauer & Michael Timme, Siemens Healthcare Diagnostics Products GmbH, Germany

Katja Boegli, Michael Gilgen, Alice Leuenberger, Swissmedic, Switzerland

Samuel Ling, Alison Jones, Lu Liu & Chong Loh, Therapeutics Goods Administration (OLSS

Biochemistry), Australia

Renata Zadro, University Hospital Center Zagreb, Croatia

WHO/BS/2015.2261

Appendix II – Study Protocol

Collaborative study to value assign potencies to International Standard for Factor IX

Aims of study:

1. To value assign the 5th

International Standard for Blood Coagulation Factor IX,

Concentrate/European Pharmacopoeia (EP) Factor IX, BRP

2. To value assign an International Standard for Recombinant Factor IX/EP Recombinant

Factor IX, BRP

3. To value assign Factor IX antigen to the 4th


Factor II, VII, IX, X, Plasma

Samples

S – the 4th

I.S. Factor IX, 07/182 potency 7.9 IU/ampoule

P – the 4th

I.S. Blood Coagulation Factor II, VII, IX, X, Plasma, potency 0.86 IU/ampoule

A – candidate sample, potency 8 – 10 IU/ampoule

B – test sample, potency 10 – 12 IU/ampoule

C – candidate sample, potency 8 – 10 IU/ampoule

D – candidate sample, potency 8 – 10 IU/ampoule

Pr – purple capped samples (sent on dry ice) – store at -80°C, and thaw in 37°C waterbath

The samples should be handled as follows:

1. Store all unopened ampoules below -20°C.

2. Allow ampoules to warm to room temperature (~15 minutes) prior to reconstitution.

3. Open ampoule after ensuring all the contents are in the lower half and reconstitute with

1.0 ml distilled water. Allow the ampoule to stand for 10 minutes at room temperature

and aid reconstitution by gentle swirling, ensuring the contents has completely dissolved.

Transfer the entire contents to a plastic tube.

4. Once reconstituted, the materials should be kept on melting ice and assayed within 3

hours.

Design of study – functional activity (FIX:C):

Please read this protocol thoroughly before commencing the testing of samples. If there are

any queries please contact John Hogwood (email address at the end of the protocol)

WHO/BS/2015.2261

Samples: Samples A, B, C, D, P and S are coded ampoules dispatched from NIBSC. Four

ampoules of each sample have been provided. For laboratories performing the functional and

antigen methods, an extra set of 4 ampoules will be provided.

All participants should carry out 4 independent assays using their routine method for measuring

factor IX, an independent assay being defined as a completely fresh set of dilutions. For each

sample at least 3 dilutions should be used, with each sample tested in replicate. The following is

an example assay design for testing all samples:

Assay design (7/14 place)

Assay 1 S1 A1 B1 C1 D1 P1 Pr1 Pr2 P1 D1 C1 B1 A1 S1

Assay 2 A2 B2 C2 P1 Pr1 P2 S2 S2 P2 D2 Pr2 C2 B2 A2

Assay 3 C3 Pr1 D3 S3 B3 P3 A3 A3 P3 B3 S3 D3 Pr2 C3

Assay 4 P4 D4 Pr1 C4 B4 A4 S4 S4 A4 B4 C4 Pr2 D4 P4

If the above design cannot be carried out in your laboratory, please contact John Hogwood

indicating the number of samples/dilutions that can be tested, and an alternative assay design will

be provided.

The calibration of all samples for functional potency should be against the 4th

International

Standard for FIX, Concentrate. Record clotting times or absorbances and calculated potencies in

the results sheet provide by email with this protocol.

Design of study – antigen (FIX:Ag):

Samples: Samples A, B, C, D, P, S and frozen Pr (Purple capped)are coded ampoules

dispatched from NIBSC. Four ampoules of each sample have been provided. For laboratories

performing the functional and antigen methods, an extra set of 4 ampoules will be provided.

Additional samples required

L1 – locally collected normal pool (day 1)

L2 – locally collected normal pool (day 2)

Please see appendix for pool suggested preparation.

All participants should carry out 4 independent assays for each method used, an independent

assay being defined as a completely fresh set of dilutions. For each sample at least 3 dilutions

should be used, with each sample tested in replicate.

For the assignment of antigen value please used the following design

Assay design (8/16place)

WHO/BS/2015.2261

Assay 1 S1 A1 B1 C1 D1 P1 Pr1 L1 L1 Pr2 P1 D1 C1 B1 A1 S1

Assay 2 A2 B2 C2 D2 L2 Pr1 P2 S2 S2 P2 Pr2 L2 D2 C2 B2 A2

Assay 3 C3 Pr1 L1* D3 S3 B3 P3 A3 A3 P3 B3 S3 D3 L1* Pr2 C3

Assay 4 L2* P4 D4 Pr1 C4 B4 A4 S4 S4 A4 B4 C4 Pr2 D4 P4 L2*

Where L1* is frozen pool of L1 and L2* is frozen pool of L2

It is appreciated that not all laboratories will be able to prepare plasma pools for testing. If frozen

pooled plasma is used, please use more than one batch, and provide details in the worksheet for

the return of results.

If the above design cannot be carried out in your laboratory, please John Hogwood indicating the

number of samples/dilutions that can be tested, and an alternative assay design will be provided.

The calibration of all samples should be against the locally collected pools (fresh or frozen)

assigned with an arbitrary 1 unit/ml. Record absorbances and calculated potencies in the results

sheet provide by email with this protocol. If a local standard or control is included in the assay

please return this data included the expected/assigned value to the material.

Return of data:

If there are any questions or you require additional samples please contact via the email below.

Raw data and estimated potencies should be recorded on the appropriate result sheets, and

returned by email to John Hogwood: [email protected] before 13 February 2015.

mailto:[email protected]

WHO/BS/2015.2261

Appendix: PREPARATION OF FRESH NORMAL PLASMA POOLS

Collect fresh normal plasma as described below, on two separate days, giving pools N1 and N2.

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 women who are pregnant or 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 4 oC 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. Keep the plasma pool

in a plastic stoppered tube at 4 oC during the assay session. Snap-freeze aliquots of each pool

(L1*, L2*) for assays 2 and 4.

WHO/BS/2015.2261

Appendix III: Individual assay results

Table S1 – Functional Activity by clotting: Potency values (IU/ampoule) calculated relative

to S, 4th

IS Factor IX Concentrate for each sample within each assay set returned

Lab Method Sample Assay 1 Assay 2 Assay 3 Assay 4 Assay 5

01 Actin-FSL

A 9.1 10.0 9.9 10.4 B 10.7 9.9 10.8 11.7 C 9.7 9.4 9.7 10.3 D 10.0 9.0 9.4 9.3 P 0.88 0.87 0.93 0.91

02 Actin-FS

A 12.7 11.9 12.1 12.6 B 10.9 9.8 10.1 11.2 C 9.3 8.4 9.0 9.2 D 12.5 11.0 11.8 12.5 P 0.84 0.74 0.73 0.80 Purple 10.3 8.9 9.4 9.8

02 APTT-SP

A 9.3 9.2 9.8 9.6 B 9.8 10.6 10.5 11.1 C 9.2 8.7 8.4 8.8 D 9.6 8.8 8.5 8.8 P 0.93 0.88 0.89 0.90 Purple 7.8 8.8 7.6 9.0

02 Cephascreen

A 12.2 11.8 12.4 13.1 B 10.9 10.9 10.1 11.2 C 8.8 8.8 8.4 9.2 D 11.5 11.2 11.0 12.2 P 0.81 0.82 0.77 0.88 Purple 9.5 10.2 10.1 11.3

02 Dapttin

A 10.8 12.3 11.2 10.7 B 11.0 11.0 nl 10.9 C 9.5 9.4 8.9 9.0 D nl nl 10.1 10.0 P 0.86 nl 0.87 0.82 Purple 8.8 9.2 10.1 nl

02 Synthasil

A 11.6 10.9 10.7 10.5 B 11.0 10.6 9.4 10.7 C 9.2 9.0 8.5 8.9 D 10.9 10.2 10.0 11.2 P 0.82 0.77 0.76 0.76 Purple 8.8 9.6 9.0 9.6

03 C.K. Prest

A 8.8 10.0 8.4 8.9 B 11.8 11.2 10.4 10.3 C 8.3 9.4 8.7 8.6 D 8.2 8.9 7.7 8.7 P 0.71 0.68 0.64 0.67

04 Clotting

A 8.0 7.9 8.9 7.7 B 10.1 10.4 11.2 10.4 C 8.3 8.3 9.5 8.2 D 7.5 7.4 7.9 7.5 P 0.77 nl 0.88 0.80

05 Pathromtin

A 9.6 9.3 9.4 9.6 B 11.1 10.9 11.1 11.1 C 8.9 8.6 8.8 9.0 D 8.3 8.4 8.6 8.7 P 1.14 1.14 1.16 1.16

06 ActinFS

A 11.4 12.1 13.3 12.8 B 10.5 10.8 10.3 9.9 C 9.1 9.1 10.1 8.7 D 9.5 12.1 13.7 12.0

WHO/BS/2015.2261


P 0.78 0.77 0.90 0.77

07 PTT

A 9.9 10.7 10.6 10.3 B 10.1 8.3 13.4 10.3 C 9.9 np np 8.6 D 10.5 np 15.5 13.2 P 0.97 np 1.28 1.08

08 ActinFSL-BSCXP

A 9.2 9.4 9.1 9.3 B 10.0 10.5 9.8 10.7 C 8.2 8.2 8.2 9.0 D 9.3 9.0 8.7 9.6 P 0.80 0.83 0.77 0.84

08 ActinFSL-CA-1500

A 10.4 10.0 9.7 9.8 B 10.4 10.2 10.0 10.4 C 9.2 8.2 8.9 8.9 D 10.4 9.9 10.1 10.2 P 0.79 0.78 0.75 0.76

08 Pathromtin-BSCXP

A 8.8 9.2 9.2 9.4 B 9.8 10.3 10.5 10.9 C 7.9 8.5 8.8 9.1 D 8.7 8.1 8.8 9.2 P 0.80 0.88 0.86 0.88

08 Pathromtin-CA-1500

A 8.8 8.2 8.5 9.2 B 10.2 9.6 10.4 10.8 C 8.9 7.8 8.8 9.4 D 8.7 8.3 8.7 8.9 P 0.78 0.76 0.78 0.82

09 ActinFS

A 10.0 9.0 9.2 8.7 B 10.7 11.2 10.5 10.7 C 8.9 9.2 9.1 8.4 D 8.7 9.1 8.4 8.8 P 0.91 0.91 0.86 0.85

10 ActinFSL

A 10.2 8.4 10.7 10.3 B 11.7 8.4 9.5 8.2 C 8.5 7.4 8.1 7.7 D 10.7 8.7 9.9 9.6 P 1.19 0.97 1.14 1.08

11 CK-Prest

A 10.0 9.7 10.2 10.6 B 10.3 10.2 10.1 10.9 C 8.2 8.7 8.7 9.1 D 8.7 9.8 9.6 9.8 P 0.98 1.03 1.02 1.05

13 TriniClot

A 10.2 9.9 10.5 10.5 B 11.0 10.8 12.3 11.5 C 8.1 8.0 8.6 7.9 D np 8.6 np 9.6 P 1.22 np np np

14 ActinFS

A 11.4 11.9 11.5 11.0 B 10.5 10.5 10.7 10.3 C 9.1 9.2 9.1 8.5 D 11.3 11.6 11.1 10.7 P 0.86 0.91 0.86 0.86

15 PTT

A 7.7 7.8 np nl B 8.8 8.4 nl 8.4 C 5.8 6.4 5.9 6.2 D 6.8 6.4 nl nl P 0.80 0.81 0.99 0.85

16 Actin

A 9.7 10.7 10.5 10.0 B 10.0 10.1 10.9 10.8 C 8.4 8.8 8.4 9.1 D 9.1 9.4 9.7 9.7

WHO/BS/2015.2261


P 0.95 0.88 0.94 1.01

17 APTT

A 9.5 9.1 9.6 9.7 B 10.5 9.9 11.2 10.3 C 8.5 8.2 8.5 8.8 D 9.3 9.1 8.9 9.1 P 1.03 1.05 1.06 1.08

18 ActinFS

A 10.1 10.2 10.5 9.6 B 10.7 9.7 10.4 10.9 C 8.6 8.4 9.3 9.3 D 9.3 10.1 10.0 9.8 P 0.9 0.9 1.0 0.9 BRP 0.88 0.92 0.99 0.89

19 Trinity

A 10.1 10.1 10.0 10.4 B 10.4 10.4 11.0 11.3 C 9.1 9.2 9.2 9.4 D 9.6 9.1 9.1 9.3 P 0.80 0.81 0.80 0.81

20 ActinFSL

A 10.9 11.6 10.7 nl B 11.0 11.2 10.7 12.2 C 8.9 9.5 9.4 8.9 D 10.4 11.0 10.8 11.0 P 0.78 0.76 0.75 0.89

22 SynthaSil

A 11.0 10.1 10.9 10.3 B 10.8 10.3 12.6 10.2 C 9.0 8.6 9.0 8.8 D 10.3 10.1 13.1 10.4 P 0.91 0.90 0.90 0.94

23 APTT-SP

A 10.5 10.5 12.4 10.8 B 10.7 9.8 11.1 10.1 C 9.4 8.5 nl 7.7 D 11.0 9.4 11.6 9.9 P nl 0.90 1.09 0.84

24 PTT-A

A 11.5 8.7 9.9 10.8 B 11.6 8.9 10.2 10.8 C 9.8 8.1 8.6 7.8 D 10.8 9.1 10.2 10.3 P 0.93 0.81 0.84 0.79

25 Pathromtin SL

A 9.1 9.9 9.5 9.2 B 10.2 10.6 10.4 10.3 C 8.5 8.9 8.4 8.3 D 8.3 8.9 9.0 8.8 P 0.95 1.06 0.98 0.95

27 APTT-SP

A 10.7 11.0 10.4 10.3 B 10.8 11.1 10.5 10.8 C 9.1 9.0 8.4 9.1 D 10.3 10.2 9.5 9.8 P 0.87 0.83 0.82 0.84

28 Cephascreen

A 9.8

B 10.3

C 7.3

D 9.0

P 1.07

28 CK-Prest

A 9.8 10.2

B 10.9 10.4

C 7.4 8.3

D 7.9 9.3

P 1.5 1.08

28 PTT-A A 10.8

B 11.2

C 9.4

WHO/BS/2015.2261


D 11.0

P 1.17

29 Actin

A 9.6 8.8 8.9 7.9 B 10.1 10.3 10.1 10.7 C 8.7 8.6 8.2 8.9 D 9.5 8.6 8.6 7.3 P 0.82 0.78 0.80 0.87

30 APTT Synth Grifols

A 9.4 9.6 9.8 9.9 B 10.6 10.4 10.8 10.7 C 8.7 8.6 8.7 8.6 D 9.2 9.1 9.2 9.2 P 0.79 0.81 0.83 0.84

31 Cephascreen

A 8.4 8.3 9.8 11.4 B 9.9 9.9 np 14.7 C 9.2 7.7 np 8.9 D 8.8 8.3 np 10.5 P 1.19 1.05 1.27 1.30

31 CK-Prest

A 8.8 9.2 9.6 10.4 B 9.1 9.9 10.5 10.2 C 6.6 7.3 7.4 nl D 6.7 8.2 7.9 9.2 P 0.91 0.93 1.00 0.98

32 CK Prest

A 9.8 11.0 9.5 9.8 B 10.8 11.5 10.7 10.2 C 8.9 9.4 7.3 8.6 D 9.9 9.5 8.6 9.3 P 1.08 1.18 1.10 1.03

33 SynthaFax

A 7.7 6.6 8.0 8.3 7.8 B 10.3 9.5 10.6 11.9 14.6 C 9.3 7.7 10.0 9.1 11.7

D 6.6 6.5 7.0 8.2 7.9

P 0.94 0.91 0.99 0.99 1.2

34 Pathromtin SL

A 11.2 10.2 9.0 10.0 B 12.0 11.2 10.2 10.2 C 9.9 nl 8.7 8.2 D 9.2 9.4 8.7 8.8 P 0.99 1.00 0.79 0.90

35 Actin

A 10.3 10.4 10.3 9.8 B 10.5 10.4 10.6 10.2 C 8.8 8.7 8.5 8.9 D 10.2 10.4 10.1 9.8 P 0.90 0.87 0.84 0.91

36 Dapttin

A 7.9 8.5 8.1 7.7 B 10.6 11.2 10.7 10.0 C 9.0 9.2 9.2 8.8 D 7.5 8.0 7.6 7.2 P 0.96 1.02 0.93 0.91 Purple 7.9 7.7 7.8 7.5

37 Cephen

A 10.1 9.9 9.7 9.8 B 10.6 10.6 10.5 10.3 C 8.9 9.2 8.8 9.0 D 9.6 9.7 9.5 9.6 P 0.85 0.87 0.82 0.83 Purple 9.1 9.1 9.3 8.8

39 APTT-SP

A 9.2 8.9 9.0 9.5 B 10.6 10.5 10.3 10.1 C 9.1 9.2 8.5 9.8 D 9.7 9.2 8.8 8.9 P np 0.87 0.92 0.82 40 Actin-FS A 10.0 9.2 10.3 9.9

WHO/BS/2015.2261


B 10.7 9.5 9.6 9.1 C 9.2 8.6 9.3 9.7 D 10.1 8.4 9.5 10.3 P 0.86 0.73 0.84 0.80

41 Actin-FSL

A 10.1 10.0 9.7 10.7 B 10.7 10.1 10.8 10.8 C 8.3 8.0 8.1 8.3 D 9.0 np 9.6 9.5 P nl 1.31 np np

42 SynthaFax

A 7.5 7.3 6.6 9.0 B 11.2 10.4 8.0 12.1 C 9.6 9.4 10.3 13.4 D 7.1 7.1 6.1 10.1

43 APTT-SP

A 10.0 9.7 9.5 9.5 B 10.5 10.7 10.5 10.1 C 9.1 8.7 9.2 9.1 D 7.7 9.5 9.9 9.2 P 0.94 0.83 0.89 0.87

44 SynthaSil

A 10.4 10.1 10.6 10.8 B 10.6 10.2 10.5 11.1 C 8.9 9.1 8.8 9.3 D 9.5 8.3 10.5 10.3 P np 0.84 0.81 0.87 Purple 9.0 9.0 9.0 9.2

45 Actin-FS

A 8.6 8.6 8.8 10.1 B 9.4 9.1 9.9 10.5 C 7.9 8.8 9.2 8.9 D 8.5 8.9 15.5 9.8 P 0.69 0.74 0.72 0.66 Purple 7.7 8.7 9.0 8.6

46 Actin-FSL

A 9.4 9.0 8.7 8.7 B 10.5 10.5 10.1 10.5 C 8.5 8.8 7.9 nl D 8.5 8.4 8.4 8.4 P 0.69 0.70 0.70 0.74

47 ActinFS

A 11.1 11.2 10.8 10.7 B 10.8 10.6 10.2 10.3 C 9.7 9.5 9.6 9.7 D 10.7 10.5 10.3 10.4 P 0.72 0.76 0.75 0.74

48 SynthaSil

A 9.1 10.1 9.9 9.9 B 9.8 10.1 10.5 10.5 C 8.0 8.6 8.8 9.1 D 9.1 9.5 9.5 9.9 P 0.93 1.05 1.02 1.00 Purple 9.2 9.4 9.0 8.9

49 Cephen

A 11.2 10.9 10.8 10.9 B 10.6 10.6 10.4 10.4 C np 9.2 9.0 9.1 D 10.0 10.3 10.2 10.1 P 0.76 0.83 0.81 0.79 nl = non-linear, np = non-parallel

WHO/BS/2015.2261

Table S2 – Functional Activity by chromogenic: Potency values (IU/ampoule) calculated

relative to S, 4th

IS Factor IX Concentrate for each sample within each assay set returned

Lab Method Sample Assay 1 Assay 2 Assay 3 Assay 4

02 Hyphen

A 7.0 8.2 6.5 6.5 B 10.2 12.0 10.5 6.3 C 9.4 10.3 8.1 8.2

D 6.1 7.7 6.2 6.3 P 0.75 0.86 0.76 0.72

Purple 6.7 9.2 6.4 10.1

02 Rossix

A 8.2 7.8 8.2 7.3 B 11.2 11.1 11.6 10.1

C 8.1 9.2 9.2 8.0 D 7.5 7.6 7.5 6.8

P 0.84 0.83 0.79 0.76

Purple 7.3 7.0 7.8 6.9

04 Rossix

A 7.9 7.7 8.7 7.3 B 10.7 10.0 11.4 10.4 C 9.0 8.5 9.6 8.5

D 7.7 7.1 8.0 7.3 P 0.87 0.81 0.89 0.82

06 Rossix

A 8.8 7.2 9.8 8.4 B 10.8 9.6 12.6 10.2 C 9.7 8.8 8.9 8.4

D 7.0 7.1 8.3 7.6 P np 0.79 0.88 0.82

12 Hyphen

A 7.5 7.3 6.8 7.2 B 9.5 11.0 9.9 10.0 C 8.2 9.2 8.2 8.5

D 6.3 7.2 6.6 6.8 P 0.77 0.83 0.78 0.77

12 Rossix

A 8.2 7.1 7.3 7.6 B 10.4 10.0 10.3 nt

C 9.0 8.6 9.2 8.3

D 7.5 7.6 8.2 6.8 P 0.81 0.76 0.81 0.69

21 Hyphen

A 9.3 10.5 9.0 8.6 B 14.1 14.3 13.4 13.1

C 12.0 10.3 11.5 11.9

D 8.7 8.4 8.0 nl P 0.96 0.91 0.91 0.91

26 Rossix

A 9.1 8.3 9.4 8.5 B 10.2 10.2 10.8 10.5

C 8.9 8.8 9.0 8.6 D 8.3 9.0 8.7 8.3

P 0.91 0.89 0.85 0.78

33 Hyphen

A 8.3 8.8 8.0 8.9 B 11.6 12.1 11.4 12.3

C 9.8 10.4 9.7 10.1 D 8.2 8.6 7.9 8.7

P 0.89 0.95 0.93 0.96

36 Hyphen

A 6.9 7.6 7.8 7.9 B 9.6 10.4 10.9 11.0

C 9.0 8.7 9.5 9.3 D 6.8 7.0 7.5 7.1

P 0.83 0.77 0.83 0.79 Purple 6.6 7.1 7.0 7.3

40 Rossix A 8.1 S nl 8.8 8.0 B 10.6 S nl 10.8 9.9 C 8.9 S nl 10.3 11.2

WHO/BS/2015.2261

D 7.1 S nl 8.0 8.6 P 0.76 S nl 0.87 0.87

41 Rossix

A 8.1 8.6 7.2 7.9 B 10.4 10.2 10.4 10.1

C 8.7 8.6 9.2 8.2 D 7.3 7.4 7.4 7.3

P nl 0.91 0.88 0.89

43 Hyphen

A 6.6 np 7.6 8.4 B 8.9 8.6 10.5 10.5

C 7.8 np 8.4 9.0 D 5.8 np 7.7 8.5

P 0.67 np 0.79 nt

48 Hyphen

A 6.5 7.2 6.5

B 10.2 11.0 9.6

C 8.8 9.4 8.2

D 6.2 6.5 6.3

P 0.68 0.76 0.70

Purple 6.1 6.6 6.3

49 Hyphen

A 7.0 6.8 6.3 6.8

B 10.9 10.0 10.1 10.7 C 8.5 8.6 8.7 8.9

D 6.0 6.4 5.9 6.4 P 0.72 0.78 0.70 0.78

nl = non-linear, np = non-parallel; nt = not tested

WHO/BS/2015.2261

Table S3 – Antigen activity: Potency values (u/ampoule) calculated relative to local normal

plasma pool (assumes value of 1 u/ml) for each sample within each assay set returned


02 Asserachrom

A 8.8 8.2 8.3 7.4 B 15.5 12.2 11.5 11.0 C 10.8 10.3 12.5 9.2

D 7.5 6.7 7.6 6.8 P 0.96 0.95 0.89 0.96

Purple 8.8 8.2 8.0 7.0

S 11.4 11.1 11.0 9.2

02 Visulize

A 6.7 np 7.1 7.2 B 12.0 13.3 12.2 12.3 C 10.8 10.9 9.4 10.7

D 5.8 np 6.0 6.4

P 0.99 0.98 0.95 1.19 Purple 6.4 6.7 6.6 6.5

S 10.8 12.7 10.6 11.7

02 Zymutest

A 9.8 9.0 9.7 9.7 B 11.1 10.5 10.6 10.9 C 9.1 8.8 9.5 9.2

D 8.3 7.7 8.0 8.1

P 0.99 0.90 0.98 1.08 Purple 9.0 8.2 8.5 8.7

S 9.7 9.6 9.4 8.9

10 Visulize

A 6.1 6.4 5.8 5.9 B 10.9 10.9 10.7 10.4

C 9.4 9.4 8.7 9.0 D 5.5 5.6 5.6 5.2

P 0.85 0.90 0.87 0.88 S 9.9 10.0 9.3 9.4

11 Visulize

A 6.4 6.1 5.5 6.6 B 10.9 10.4 9.8 11.4

C 9.6 8.5 8.3 9.7

D 5.6 5.3 4.8 5.2 P 0.94 0.81 0.78 0.86

S 10.5 9.0 8.5 10.0

21 Cedarlane

A 3.7 2.9 3.3 3.7 B 13.9 8.9 8.9 11.2

C 9.3 7.5 np 9.3 D 2.9 2.4 3.1 3.3

P 0.99 0.67 0.78 0.84 S 13.2 9.1 10.8 12.4

23 AssayPro

A 6.4 8.1 6.4 np B 12.5 12.6 10.8 10.1

C 10.5 11.8 np 8.9

D np 7.2 5.5 np P 0.80 1.33 0.84 0.83

S np 11.2 np 8.6

24 Asserachrom

A 8.3 8.4 7.0 7.9 B 12.5 11.6 10.4 11.0

C 10.2 9.7 9.2 9.3 D 7.0 6.3 5.5 6.2

P 0.87 0.87 0.83 0.93 S 10.6 8.6 9.7 9.9

28 Asserachrom

A 7.0 8.5 6.5 7.0 B 9.7 11.3 9.3 10.9

C 8.7 10.5 8.0 8.1

D 5.8 7.2 5.4 5.8 P 0.82 0.90 0.76 0.85

WHO/BS/2015.2261


S 8.9 10.7 8.5 9.1

31 Asserachrom

A 7.4 7.6 7.0 8.6 B 10.9 11.3 9.6 12.2 C 9.4 9.6 8.1 10.3

D 6.4 6.5 5.9 7.0 P 0.87 0.90 0.80 0.96

S 9.4 10.1 8.7 10.9

33 Visulize

A 5.5 5.3 5.3 4.9 B 10.6 8.4 9.0 nl

C 9.3 9.6 9.1 nl D np 4.7 4.5 4.5

P 0.97 0.89 1.01 0.96 S 9.5 9.3 9.5 nl

34 Asserachrom

A 7.8 8.2 np 8.1 B 12.0 12.0 12.2 11.7 C 10.5 11.0 10.7 10.0

D 6.4 6.7 6.5 6.5 P 0.95 0.95 0.98 0.95

S 10.1 10.6 10.8 10.8

38 Affinity

BIologicals

A 6.9 6.9 7.1 6.5 B 12.4 12.2 12.3 12.5

C 10.7 10.4 10.0 10.5 D 5.9 5.9 5.9 5.6

P 0.96 0.98 1.00 0.95 S 12.3 11.7 11.4 11.6

40 Asserachrom

A 9.9 nl L nl L nl B 12.8 11.9 L nl L nl C 11.6 10.8 L nl L nl

D 8.44 6.94 L nl L nl P nl 0.96 L nl L nl

S np 9.5 L nl L nl

43 Asserachrom

A 5.9 6.9 2 points 6.6 B 9.2 np np np

C 8.5 9.2 8.7 9.6 D 3.8 6.1 5.4 nl

P 0.75 0.91 0.75 0.75 S 8.5 10.4 9.2 9.7

48 Visulize

A 8.2 nl B 11.7 10.4 C 10.3 9.6

D np 3.6 P 0.76 0.91

S 12.7 10.5

49 Zymutest

A np 8.8 8.4 10.0 B np 10.2 9.7 10.6

C np 7.9 8.9 9.4 D np 7.4 7.3 8.6

P np 0.85 0.81 0.99

S np 8.0 8.9 9.8

nl = non-linear, np = non-parallel;

WHO/BS/2015.2261

Appendix IV: Draft Instruction for Use

WHO/BS/2015.2261

who/bs/2015.2261 english only expert committee on

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