EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 18 to 22 October 2010

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1 ENGLISH ONLY EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 18 to 22 October 2010 COLLABORATIVE STUDY FOR VALUE ASSIGNMENT OF THE 4 th INTERNATIONAL STANDARD FOR FACTORS II, VII, IX, X, PLASMA Elaine Gray 1,3, Helen Barson 1, Jason Hockley 2 and Peter Rigsby 2 1 Haemostasis Section, 2 Biostatistics National Institute for Biological Standards and Control Potters Bar, Hertfordshire, EN6 3QG, UK. 3 Principal Investigator: World Health Organization 2010 All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: ; fax: ; bookorders@who.int). Requests for permission to reproduce or translate WHO publications whether for sale or for noncommercial distribution should be addressed to WHO Press, at the above address (fax: ; e- mail: permissions@who.int). The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement. The mention of specific companies or of certain manufacturers products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned. Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters. All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this publication.

2 Page 2 Summary Twenty-nine laboratories from 14 different countries participated in a collaborative study to value assign the proposed 4 th International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma (09/172) against the 3 rd International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma (99/826). Local normal pooled plasmas were included to provide information on any drift of the plasma unit. Over 75% of the laboratories obtained potency estimates that had intra-laboratory geometric coefficients of variation (GCV) of less than 5% when the proposed standards were assayed against the 3 rd International Standard, indicating that the participants performed assays for factors II, VII, IX and X reproducibly and with high precision. For all four factors, inter-laboratory variability was low for estimates of the proposed 4 th International Standard against the 3rd International Standard (GCV <3%), and higher against the local plasma pools (GCV 5-11%). There were no significant differences in potency estimates from chromogenic and clotting assays and they were therefore combined to give the overall mean potencies. Potency estimates calculated relative to the local normal plasma pools were 1%, 6%, 6% and 5% lower for FII, FVII, FIX and FX respectively when compared to potencies calculated relative to the 3rd International Standard and the differences in the estimates were significant for FVII, FIX and FX. The differences in the potency estimates could not be attributed to degradation of the 3 rd International Standard, but the variability of the plasma pools could be an important contributing factor. The excellent agreement between a large number of laboratories contributed to the statistical significance of the discrepancies. It is therefore recommended that the proposed 4 th International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma (09/172) be assigned with potencies for functional activity, calculated relative to the 3 rd International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma: Factor II: Factor VII: Factor IX: Factor X: 0.89 IU/ampoule 0.99 IU/ampoule 0.86 IU/ampoule 0.89 IU/ampoule Introduction Blood coagulation factors II, VII, IX and X are vitamin K dependent proteins and are essential for haemostasis. Congenital deficiency of blood coagulation factor IX, haemophilia B, is well documented and this x-linked heritable disorder is found in 1:30,000 males worldwide with no racial preferences. Severity of bleeding correlates with plasmatic factor IX (FIX) activity. Patients with severe symptoms usually have less than 1% (0.01 IU/ml) FIX activity and moderate haemophilia B is associated with FIX levels of 1 4 %. Factor II, VII and X congenital deficiencies are rare autosomal recessive disorders. Their prevalence in the general population are approximately 1 in 2 million, 1 in 500,000, and 1 in 1 million for FII, VII and X respectively (1). Their frequency is increased times where consanguineous marriages are practised (2). Inherited combined deficiency of all factors is very rare and up to 2008, less than 30 cases have been documented (3). Acquired deficiency of vitamin K dependent clotting factors is more common and can be a result of liver disease, dietary vitamin K deficiency and overdose of vitamin K antagonists (eg warfarin). Effective diagnosis, monitoring and treatment of these disorders depend on accurate quantitative measurement of these coagulation factors. International standards for these four blood coagulation factors, value assigned in international unit (IU) have been used successfully to reduce variability in the estimation of activity within and between laboratories. The current 3 rd International Standard used by clinical laboratories,

3 Page 3 diagnostic manufacturers and therapeutics producers has been invaluable in ensuring good global agreement in measurement of these four important clotting factors. The 3rd International Standard (IS) for Factors II, VII, IX and X, Plasma (99/826) was established by the Expert Committee on Biological Standardisation (ECBS) of the World Health Organisation (WHO) in October Due to depletion of stock, it is now necessary to replace this IS and this study served to value assign a replacement IS for FII, FVII, FIX and FX against the 3 rd IS. In addition, this study also calibrated the International Society for Thrombosis and Haemostasis/Scientific and Standardisation Committee (ISTH/SSC) Secondary Coagulation Standard, Lot# 4. The results for the calibration of this secondary plasma standard will not be discussed in this report and will be detailed in a separate report for the calibration of all of the analytes in Lot#4, available in the first quarter of In order to assess the relationship between the international unit (IU) and the normal plasma unit of these four coagulation factors, locally collected normal pooled plasmas were included as one of the samples in the study. Participants Twenty-nine laboratories from 14 different countries (1 Australia, 1 Austria, 3 Canada, 1 Croatia, 2 Denmark, 3 France, 1 Germany, 1 India, 2 Italy, 1 The Netherlands, 1 Spain, 1 Sweden, 8 UK, 3 USA) agreed to participate in the study, of which 28 returned data in time for the statistical analysis. Unfortunately one laboratory (29) returned data too late to be included in the analysis, however the data from this laboratory are shown in Appendix 4. The participants included 15 clinical laboratories, 10 diagnostics manufacturers, 2 therapeutic manufacturers and 2 regulatory authorities. 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. The Candidate, NIBSC code 09/172 Eighty-five donations of platelet poor normal plasma from the Welsh Blood Service, collected in CPD-adenine and buffered with 0.05 M HEPES were pooled, distributed into glass ampoules, filled and freeze-dried according to guidelines for production of international biological standards (4; 5). Each individual plasma donation has been tested and found negative for anti- HIV 1/2, HBsAg and anti-hepatitis C. This candidate was coded as sample A in this study. The product characteristics are shown in the following table. This proposed standard is intended to be used in the in vitro diagnostics field and it relates to BS EN ISO 17511:2003 Section 5.5.

4 Page 4 NIBSC Code 09/172 Presentation Sealed, glass 5 ml DIN ampoules Number of Ampoules available 20,000 Liquid filling weight (g) Mean=1.1078; Range= CV of fill mass (%) 0.20 (n=849) Homogeneity of the fill by activity: 4 ampoules selected from the beginning, GCV p value every 5000 amp interval and the end of the fill were assayed against the 2 nd IS FII using clot-based assays. 2 independent FVII assays per ampoule were carried out. In total 24 ampoules and 48 assays per FIX factor. Effect of fill position assessed by ANOVA of log potencies. FX Mean dry weight (g, n = 5) Mean head space oxygen (%) 0.14 (n =12) Residual moisture (%) (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 Samples The following coded samples were sent to each participant: S 3rd International Standard for Factors II, VII, IX, X, Plasma (99/826), containing 0.91 IU FII, 1.00 IU FVII, 0.86 IU FIX and 0.93 IU FX per ampoule. A Proposed 4th International Standard for Factors II, VII, IX, X, Plasma (09/172) containing approximately 1.0 IU per ampoule for all four factors. P ISTH SSC Lot 3 containing 0.86 IU FII, 0.87 IU FVII, 0.94 IU FIX and 0.86 IU FX per vial. Q ISTH SSC Lot 4 containing approximately 1.0 IU FII, FVII, FIX and FX per vial. In addition, the participants were requested to collect normal pooled plasma, which was coded N in the study: N1 N2 Fresh normal plasma pools prepared locally according to the instructions in Appendix 2. Assay Methods Each participant was requested to perform their routine in-house functional method(s) for the four coagulation factors. A list of reagents, methods and instruments used by the participants is given in Appendix 3. Factor II: 24 laboratories used a prothrombin time-based clotting method with commercial thromboplastin reagents (sources were rabbit brain, human placenta or recombinant human) and

5 Page 5 FII-deficient plasma. Three laboratories used chromogenic assays; two laboratories used Ecarinbased assays, while one laboratory employed a prothrombinase-based method. Factor VII: A one-stage clotting method was used by 26 laboratories, using thromboplastin reagents from rabbit brain, human placenta or recombinant human thromboplastin, and FVIIdeficient plasma. Chromogenic assays were used by three laboratories, two using the Chromogenix Coaset FVII kit and one using the Biophen FVII kit. Factor IX: 27 laboratories used a one-stage clotting method based on the activated partial thromboplastin time (APTT), using a variety of activators, phospholipids and FIX-deficient plasma. One laboratory used a chromogenic method (Rossix Factor IX kit). One lab (23) carried out antigen assays using the AG Affinity Biological kit. Factor X: A one-stage clotting assay was used by 26 laboratories, using a variety of thromboplastin reagents (rabbit brain, human placenta or recombinant human), and FX-deficient plasma. Two laboratories used chromogenic methods, using Russell s Viper Venom as an activator. Study Design Participants were requested to carry out four assays for each factor (FII, FVII, FIX, FX) using fresh ampoules of samples S, A, P, and Q and a normal plasma pool (N) in each assay and to assay factors VII and IX on one set of ampoules and factors II and X on a second set of ampoules. Where feasible, participants were requested to collect fresh plasma on two separate days to prepare two normal plasma pools (N). It was requested that each fresh pool was tested in the study on the day of collection and that a frozen sample of the same pool should be used in the study on a separate day, or, if possible, to use fresh plasma pools for all assays. Some laboratories that were unable to prepare the fresh plasma pools used frozen plasma pools instead. Within each assay, participants were requested to assay three dilutions of each of the samples S, A, P and Q, in replicate, according to balanced assay designs. Raw assay data were returned together with calculated estimates for samples A (proposed 4th IS), P (SSC Lot#3), Q (SSC Lot#4) and N (local normal pooled plasma) relative to sample S (3rd IS) from each individual assay. Statistical Analysis An independent statistical analysis of raw data was performed at NIBSC. Relative potency estimates were calculated by fitting a parallel-line model (6). All data were plotted and assay validity was assessed both visually and by analysis of variance. Where significant deviations from the model appeared to result from underestimation of residual error, parallelism was confirmed by using the deviations from linearity as an alternative estimate of residual error. All mean potencies given in this report are unweighted geometric mean (GM) potencies. Variability between assays and laboratories has been expressed using geometric coefficients of variation (GCV = {10 s -1} 100% where s is the standard deviation of the log transformed potency estimates). Grubbs Test (7) was applied to the log transformed laboratory mean estimates in order to detect any significant outliers. Comparisons between methods and with results from

6 Page 6 previous studies have been made by unpaired t-test of log transformed laboratory mean estimates. Factor II assays A parallel-line model comparing assay response (untransformed in laboratory 11; log transformed in laboratories 1, 4, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16a, 16b, 17a, 17b, 18b, 19, 20b, 22, 23, 24, 25, 26, 27 & 28a; with a reciprocal transformation in laboratories 2, 3 & 5) to concentration was used for analysis. Factor VII assays A parallel-line model comparing assay response (untransformed in laboratories 8, 12 & 13; log transformed in laboratories 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 14, 17b, 18a, 19, 20b, 21, 22, 23, 24, 25, 27 & 28a; square root transformed in laboratories 15, 16a, 16b, 26 & 28b) to concentration was used. Factor IX assays A parallel-line model comparing assay response (untransformed in laboratories 1, 6, 10, 14, 15, 16a, 16b, 18a, 19, 21, 22, 24, 25, 26 & 27; log transformed in laboratories 2, 3, 4, 5, 7, 8, 9, 11, 12, 13, 17a, 20a, 23 & 28a) to concentration was used. Factor X assays A parallel-line model comparing assay response (log transformed laboratories 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16a, 16b, 18b, 19, 20b, 22, 23, 24, 25, 27 & 28a; square root transformed in laboratory 26) to concentration was used. Results Assay Data Twenty eight participants returned a total of 454 assays which comprised 114 FII assays, 120 FVII assays, 115 FIX assays and 105 FX assays. Lab 29 did not return data in time to be included in the analysis and the locally calculated potency estimates are presented in Appendix 4. The individual assay results, together with the geometric mean potencies (GM) and the intra-laboratory variation expressed as %GCV are presented in Appendix 5. Assay Validity The majority of assays showed no significant (p<0.01) deviations from the fitted model. Exceptions are detailed below. Factor II assays In assays 2, 3 and 4 by laboratory 14, sample N was excluded because the response range was not comparable to the other samples, Assay 3 by laboratory 17a, was excluded as all samples were non-linear.

7 Page 7 Factor VII assays In assay 2 by laboratory 7, sample N was found to be non-linear. In assays 2, 3 and 4 by laboratory 14, sample N was excluded because the response range was not comparable to the other samples. Factor IX assays In assay 1 by laboratory 2, sample S was found to be non-linear. In assay 4 by laboratory 3, samples Q and N were found to be non-linear. In assays 1 and 3 by laboratory 13, sample N was found to be non-linear. In assays 2, 3 and 4 by laboratory 14, sample N was excluded because the response range was not comparable to the other samples. Factor X assays In assay 1 by laboratory 13, sample S was found to be non-linear. Assays 2 and 4 by laboratory 14 were excluded due to non-linearity of the samples. The proposed 4 th IS for Factors II, VII, IX and X, Plasma (sample A, 09/172) Intra- and inter- laboratory variability Estimates of intra-laboratory variability (between assays) for all four factors in sample A are given as geometric coefficients of variation (GCV) for potency estimates relative to sample S (Table 1a). Intra-laboratory variability (GCV) for estimates of sample A, relative to sample S, exceeded 5% in 5, 7, 6 and 3 laboratories for FII, FVII, FIX and FX respectively, with close to 80% of the potency estimates giving GCV lower than 5%. Intra-laboratory variability was generally higher for estimates of sample A, relative to the normal plasma pools, for all factors with GCV exceeding 5% in 12, 14, 13 and 12 laboratories for FII, FVII, FIX and FX respectively (Table 1b). Variability between laboratories for estimates of sample A relative to the 3rd IS and relative to sample N (normal plasma pools) for each factor, is shown in Tables 1a and 1b and is also summarized in Table 1c. Estimates of inter-laboratory variability (GCV) for estimates of sample A relative to the 3 rd IS were less than 3% for all four factors. However, the inter-laboratory variability (GCV) for estimates of A relative to N (combined fresh and frozen plasma pools) was greater; 5.1%, 11.2%, 8.0% and 8.0% for factors II, VII, IX and X, respectively. Potency estimates The overall potency estimates for sample A (proposed 4 th IS) were calculated relative to the assigned values for the 3 rd IS and relative to an arbitrary value of 1.0 unit per ml for the local normal pooled plasmas. Geometric mean potency estimates from each laboratory and the overall potency estimates, together with 95% confidence limits, for all four factors are shown in Tables 1a and 1b. The potency estimates relative to the 3 rd IS are also presented in histogram form in Figures 1a 1d. The histograms illustrate good agreement between laboratories for sample A relative to the 3 rd IS,

8 Page 8 for all 4 factors; with the exception of 2 outliers for FII, 1 outlier for FVII and 1 outlier for FX, all the potency estimates were within 3-7% of the overall mean potencies. Outlier detection indicated that the FII estimate of potency by laboratory 8 (clotting assay) was significantly higher and laboratory 18b (chromogenic assay) was significantly lower than all other FII estimates; the FVII and FX estimates by laboratory 8 (clotting assays) were significantly higher than the all other FVII and FX estimates. These results were therefore excluded from the calculations of overall mean potency estimates of 0.892, 0.987, and IU/ampoule for FII, FVII, FIX and FX respectively. Comparison of the potency estimates by clotting assays with results from the chromogenic assays suggested that the ratios of clotting to chromogenic potencies for all four factors were close to 1 (Table 1d). However, there were insufficient numbers of laboratories carrying out chromogenic assays to statistically compare the potency estimates against those obtained by clotting assays, but it is clear that for all four factors, the individual potency estimates by chromogenic methods were all within the potency range by clotting assays (Table 1a and 1b). Table 1c shows that the FVII, FIX and FX overall mean potency estimates for sample A relative to the normal pooled plasmas were significantly lower than the overall mean estimates relative to the 3rd IS by 6%, 6% and 5% for FVII (p = 0.007), FIX (p < 0.001) and FX (p = 0.004) respectively. There was no significant difference between the mean potency estimates for FII relative to 3 rd IS and the normal pooled plasmas (p = 0.316). Locally collected normal plasma pools Sixteen laboratories carried out assays using fresh plasma pools, prepared from a total of 273 donors. These pools were also used frozen in repeat assays. In addition, five laboratories used frozen plasma pools collected prior to the study, containing plasma from 98 volunteers. All other laboratories used commercially available frozen plasma pools (from at least 376 donors) except one (lab 21), which used lyophilised plasma. In total there were at least 747 donors. Comparison of fresh and frozen local pools (Table 2a 2d) tested against sample S, the 3 rd IS indicated that there was no significant difference for all four factors (FII, p = 0.866; FVII, p = 0.735; FIX, p = 0.985; FX, p = in a paired t-test (Table 2e)) in laboratories that used both locally collected fresh and frozen plasma. The results from lab 21 that used lyophilised plasma were not included in the overall potency estimates for the normal pooled plasmas. The combined potencies of 1.020, 1.066, and IU/ml for FII, FVII, FIX, and FX, respectively, were therefore presented (Table 2e). Histograms of individual laboratory mean potency as a percentage of the overall mean potency for all four factors (Figures 2a 2d) show wider ranges of potency than were found with the other samples in the study, but the agreement between laboratories is reasonable, with inter-laboratory GCV being 6.1%, 10.0%, 7.7% and 7.7% for FII, FVII, FIX and FX respectively (Table 4e). Antigen measurement for factor IX Laboratory 23 also carried out FIX antigen measurements and as there is no assigned antigen value for the international standard, the antigen estimates were calculated relative to the normal pooled plasma from this laboratory. Four different frozen pools, each prepared with 8 individual donors, were used in the four separate assays. Table 3 shows the individual assay results together with the geometric mean potencies and the intra-laboratory variation expressed as GCV. The geometric mean potency for samples S (3 rd IS) and A (proposed 4 th IS) relative to the plasma pools was found to be and u/ampoule.

9 Page 9 Stability study Accelerated degradation study (8) of the proposed IS, 09/172 was carried out at NIBSC using specific clot-based assays with APTT reagent for FIX and thromboplastin for FII, VII and X. At present, the data from 2 time points (3 and 6 months storage) were insufficient to give a robust prediction of the percentage loss of activity for the four factors. However, the potencies estimated by clotting assays of the -20 C and the +4 samples after 6 months storage showed that there is no significant loss in potency compared to the -70 C samples (Table 4a; 95% confidence limits for relative potencies include 1.0). This candidate has been prepared in the same manner as the 3 rd IS, 99/826 and as shown in Table 4b, for 99/826, there were insignificant loss of activity in the -20 C samples after 8 years storage by comparison with the -150 C material. It is expected that the proposed 4 th IS, 09/172 would be as stable as the 3 rd IS. Continual real time degradation study of the 20 C against ampoules stored at 150 C and further accelerated degradation study at elevated temperatures will be carried out to monitor the stability of the replacement standard. Discussion The primary aim of this study was to value assign a replacement International Standard for Factors II, VII, IX, X, Plasma. It also provided an opportunity to re-examine the continuity of the plasma unit for factors II, VII, VII and X by comparison with local normal pooled plasmas. The majority of the laboratories employed clot-based methods for all four factors. Although statistical assessment cannot be made on differences between potency estimates obtained using chromogenic and clotting assays, the values obtained by chromogenic procedures were all within the potency ranges of the clotting methods. Therefore, the potencies obtained by chromogenic and clotting methods against the 3rd IS or normal plasma pools were combined. It was not possible to investigate the effect of different types of methods on potency estimation since most laboratories employed Prothrombin time based assays for FII, FVII and FX and APTT type of assays for FIX. There were no observable differences in potencies obtained by the different instruments and reagents for all four factors. Laboratory 23 also carried out antigen assays for FIX and the calculated data are for information only and have not been included in the overall estimated potency for FIX. The ratio of FVII potency estimates from the clotting and chromogenic methods can give an indication of the activation status of FVII (9), e.g. a clotting/chromogenic ratio >1.0 indicates that FVII in the test sample is relatively more activated than the reference standard. In the calibration of the 3rd IS against the 2nd IS, a clotting/chromogenic ratio of 0.92 was obtained for the 3 rd IS; an indication that the 3 rd IS was not activated. In the current study, when sample A, the proposed 4 th IS was assayed against the 3 rd IS, the clotting/chromogenic FVII potency ratio of 1.03 (Table 1d) was obtained, thus suggesting that the proposed 4 th IS is relatively unactivated. Although it is not yet established that the clotting/chromogenic ratios of other coagulation factors can be used as indicators of activation, Table 1d shows that ratios close to 1 were also estimated for FII, FIX and FX. For all four factors, no significant differences were found between potency estimates calculated relative to the fresh or frozen local plasma pools and it was therefore valid to combine potencies against all plasma pools (Table 2e).

10 Page 10 With a few exceptions, intra-laboratory (between assay) GCV were below 5% when the proposed 4th IS was assayed against the 3 rd IS, indicating good reproducibility and robustness of the assays for all four factors. When the proposed 4th IS was assayed against the local plasma pools, the intralaboratory variability was generally higher. This is probably due to the variability in the plasma pools rather than the poor reproducibility of the assays. Agreement between laboratories was excellent when the proposed 4 th IS was assayed against the 3 rd IS; the inter-laboratory variability, expressed as GCV, was less than 3%. Greater variability was found for estimates of the proposed 4 th IS when assayed against the normal plasma pools. With the exception of FVII, that yielded GCV of just over 11%, the inter-laboratory variability for FII, FIX and FX estimates relative to both fresh and frozen pools combined did not exceed 8% (Table 1c). As expected, there was higher inter-laboratory variability for estimates of local normal pooled plasmas compared with the other three lyophilised plasma samples, with the GCV being more than 6% for all 4 factors. With the exception of FII, there were significant differences in the FVII, FIX and FX overall mean potency estimates for the proposed 4th IS when assayed against the 3rd IS and the local normal plasma pools (Table 1c). Mean potency estimates calculated relative to the normal plasma pools were 1%, 6%, 6% and 5% lower for FII, FVII, FIX and FX, respectively, compared to potencies estimated relative to the 3rd IS. One reason for this discrepancy could be the degradation of the 3rd IS during its lifetime, but the accelerated degradation study of the 3 rd IS predicted <0.5% of loss of activity per year for all four factors during its storage at -20 C. Indeed, assays of the 20 C against the -150 C samples in 2010 after 8 years storage showed a loss of <3% in the FVII activity, while no loss of activity for FII, FIX and FX was detected (Table 4b). Other possible reasons for the drift are the differences in the collection methods of the local pools, the bulk plasma pools used for the filling of the standards, and the geographical spread of the different local pools collected for assays against the IS. There are a large number of laboratories and the low level of variability between them (inter-laboratory GCV of less than 3% for all four factors) also contributes to the statistical significance of this discrepancy. Proposal to the Participants There are two main considerations in deciding on the assigned potencies, a) continuity of the IU between the 3rd IS and the 4th IS and, b) maintaining the link between the IU and the "plasma unit". Significant differences were found for three of the four factors between the potencies estimated relative to the 3rd IS and the normal plasma pools. In the past, when similar discrepancies were found as in the calibration of the 4th IS Factor VIII/von Willebrand Factor, Plasma (97/586) and the 3 rd IS for Blood Coagulation Factors II, VII, IX and X, Plasma (99/826), it was agreed that the mean of values relative to the previous International Standard and the normal plasma pools should be assigned. However, as shown by the results of this study, this approach did not prevent further drift of the plasma unit. In addition, the differences in potencies may be due to the extremely low variability of the assay results and therefore the drift may be a statistical artefact. Since it is of paramount importance to have continuity of the international unit, it is proposed that the proposed 4 th IS be value assigned by potencies obtained against the 3 rd IS only. The proposed overall mean potencies for functional activity for the four factors are: FII 0.89 IU/ampoule; FVII 0.99 IU/ampoule; FIX 0.86 IU/ampoule and FX 0.89 IU/ampoule.

11 Page 11 Responses from participants and the experts nominated by the SSC/ISTH Plasma Coagulation Inhibitors Sub-Committee All participants agreed with the proposals. There were no comments in relation to the analysis or interpretation of the data in the study. The report has also been circulated to 12 experts nominated by the SSC/ISTH FVIII/FIX Subcommittee and all have agreed with the proposed assigned values for the proposed 4 th IS for Blood Coagulation Factors II, VII, IX and X, Plasma. The proposal to accept the preparation coded 09/172 as the WHO 4 th IS for Blood Coagulation Factors II, VII, IX and X, Plasma with the recommended assigned values was discussed at the WHO-ISTH Liaison Group Meeting and subsequently endorsed at the SSC Business Meeting, held in Cairo, Egypt on 25 May Proposal and recommendation to the ECBS Sample A, 09/172, be the 4 th International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma with the following assigned values for functional activity relative to the 3 rd International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma: Factor II: Factor VII: Factor IX: Factor X: 0.89 IU/ampoule 0.99 IU/ampoule 0.86 IU/ampoule 0.89 IU/ampoule The Instruction for Use for the proposed Standard, 09/172 is illustrated in Appendix 5. References 1. Mannucci PM, Duga S, Peyvandi F. Recessively inherited coagulation disorders. Blood 2004; 104: Peyvandi F, Duga S, Akhavan S, Mannucci PM. Rare coagulation deficiencies. Haemophilia 2002; 8: Weston BW, Monahan PE. Familial deficiency of vitamin K-dependent clotting factors. Haemophilia 2008; 14: Campbell PJ. International biological standards and reference preparations. 1. Preparation and presentation of materials to serve as standards and reference preparations. J Biol Standardisation 1974; 2: Recommendations for the preparation, characterization and establishment of international and other biological reference standards (revised 2004). In: WHO TRS, No. 932, 2006, Annex 2. pp (section A.7) 6. Finney DJ. Statistical Method in Biological Assay. 3rd Edition. London: Charles Griffin 1978

12 Page Grubbs F. Procedures for Detecting Outlying Observations in Samples. Technometrics, 1969; 11: Kirkwood T.B.L. (1977). Predicting the stability of biological standards and products. Biometrics, 33: Osterud B. How to measure factor VII and factor VII activation. Haemostasis, 1983; 13(3):161-8 Acknowledgments We would like to thank the participants of the study and the support of the FVIII/FIX Subcommittee of the SSC/ISTH.

13 Page 13 Table 1a: Potency estimates, intra- and inter-laboratory GCV for factors II, VII, IX and X in sample A relative to sample S, the 3 rd IS for Factors II, VII, IX and X, Plasma Assay Method Lab FII FVII FIX FX IU/amp GCV IU/amp GCV IU/amp GCV IU/amp GCV (n=4) 1.1% (n =4) 1.6% (n=4) 2.3% (n=4) 2.0% (n=4) 3.9% (n=4) 0.4% 0.871(n=3) 2.0% (n=4) 2.5% (n=4) 3.3% (n=4) 2.6% (n=4) 8.0% (n=4) 2.3% (n=4) 1.1% (n=4) 2.4% (n=4) 1.7% (n=4) 2.8% (n=4) 0.9% (n=4) 2.6% (n=4) 2.3% (n=4) 1.1% (n=4) 5.6% (n=4) 6.3% (n=4) 3.7% (n=4) 3.6% (n=4) 2.2% (n=4) 6.2% (n=4) 3.2% (n=4) 1.8% (n=4) 3.0% (n=4) 10.5% (n=4) 4.4% (n=4) 5.2% (n=4) 1.8% (n=4) 3.1% (n=4) 2.7% 0.888(n=4) 2.7% (n=4) 2.0% (n=4) 1.8% (n=4) 0.8% (n=4) 1.3% (n=4) 10.5% (n=4) 2.7% (n=4) 3.5% (n=4) 7.3% (n=4) 3.8% (n=4) 6.7% (n=4) 9.5% (n=4) 3.3% (n=4) 2.7% (n=4) 3.0% (n=4) 10.9% (n=3) 2.1% CLO (n=4) 11.3% (n=4) 9.0% (n=4) 6.7% (n=2) N/A (n=4) 4.2% (n=4) 8.1% (n=4) 10.8% (n=4) 6.1% 16a (n=4) 3.9% (n=4) 2.6% (n=4) 2.5% (n=4) 3.2% 16b (n=3) 1.7% (n=4) 2.8% (n=4) 2.0% (n=4) 2.4% 18a NT (n=4) 1.8% (n=4) 1.2% NT (n=4) 1.1% (n=4) 2.2% (n=4) 1.7% (n=4) 4.1% 20a NT - NT (n=4) 3.9% NT - 21 NT (n=4) 2.0% (n=4) 0.8% NT (n=4) 3.9% (n=4) 2.6% (n=4) 3.7% (n=4) 4.2% (n=4) 1.3% (n=4) 1.1% (n=4) 2.8% (n=4) 1.3% (n=4) 4.4% (n=4) 4.2% (n=4) 1.0% (n=4) 3.5% (n=4) 1.5% (n=4) 4.0% (n=4) 3.1% (n=4) 1.2% (n=4) 2.9% (n=4) 2.6% (n=4) 0.6% (n=4) 1.4% (n=4) 5.9% (n=4) 3.3% (n=4) 3.6% (n=4) 2.8% 28a (n=4) 4.0% (n=4) 3.3% (n=4) 5.6% (n=4) 2.4% 17a (n=3) 3.0% NT (n=4) 2.2% NT - CH 17b (n=4) 0.9% (n=4) 8.4% NT - NT - 18b (n=4) 6.2% NT - NT (n=4) 3.0% 20b (n=4) 1.5% (n=4) 3.8% NT (n=4) 2.4% 28b NT (n=4) 1.4% NT - NT - Overall GM (n=27) (n=29) (n=29) (n=26) 95% CL Between Lab GCV 1.2% 2.9% 2.8% 1.5% n: no of assays; NT: not tested; CLO: clotting assay; CH: chromogenic assay; N/A: not applicable; GM: geometric mean; GCV: geometric coefficient of variation; CL: confidence limits. Shaded boxes indicate outliers and are excluded from overall GM.

14 Page 14 Table 1b: Potency estimates, intra- and inter-laboratory GCV for factors II, VII, IX and X in sample A relative to sample N, the local normal pooled plasma (assumed 1 u/ml) Assay Method Lab FII FVII FIX FX IU/amp GCV IU/amp GCV IU/amp GCV IU/amp GCV (n=4) 3.3% (n=4) 11.1% (n=4) 6.0% (n=4) 2.9% (n=4) 4.9% (n=4) 15.1% (n=3) 4.3% (n=4) 4.4% (n=4) 2.5% (n=4) 10.4% (n=3) 6.2% (n=4) 4.9% (n=4) 2.1% (n=4) 2.7% (n=4) 3.8% (n=4) 1.3% (n=4) 1.7% (n=4) 4.4% (n=4) 3.7% (n=4) 2.7% (n=4) 4.4% (n=4) 8.7% (n=4) 9.1% (n=4) 6.4% (n=4) 3.3% (n=3) 12.1% (n=4) 7.3% (n=4) 4.8% (n=4) 2.5% (n=4) 3.1% (n=4) 3.3% (n=4) 3.6% (n=4) 3.2% (n=4) 3.4% (n=4) 5.1% (n=4) 3.3% (n=4) 10.7% (n=4) 4.3% (n=4) 3.3% (n=4) 3.3% (n=4) 10.3% (n=4) 3.4% (n=4) 5.7% (n=4) 11.8% (n=4) 1.6% (n=4) 6.1% (n=4) 4.5% (n=4) 1.8% (n=4) 2.4% (n=4) 2.2% (n=2) N/A (n=3) 5.3% CLO (n=1) N/A (n=1) N/A (n=1) N/A (n=2) N/A (n=4) 2.0% (n=4) 9.0% (n=4) 15.3% (n=4) 9.8% 16a (n=4) 6.3% (n=4) 5.4% (n=4) 3.7% (n=4) 4.7% 16b (n=4) 5.5% (n=4) 3.0% (n=4) 4.6% (n=4) 5.6% 18a NT (n=4) 9.8% (n=4) 8.8% (n=4) 9.4% (n=4) 5.1% (n=4) 2.8% (n=4) 2.4% (n=4) 2.9% 20a NT - NT (n=4) 12.3% NT - 21 NT (n=4) 1.0% (n=4) 1.1% NT (n=4) 6.2% (n=4) 4.9% (n=4) 5.1% (n=4) 7.2% (n=4) 5.2% (n=4) 8.5% (n=4) 12.9% (n=4) 7.4% (n=4) 5.4% (n=4) 9.6% (n=4) 4.9% (n=4) 5.5% (n=4) 8.0% (n=4) 6.0% (n=4) 2.2% (n=4) 8.0% (n=4) 3.7% (n=4) 3.2% (n=4) 5.8% (n=4) 4.3% (n=4) 5.2% (n=4) 11.4% (n=4) 5.8% (n=4) 4.1% 28a (n=4) 3.5% (n=4) 2.6% (n=4) 2.8% (n=4) 4.0% 17a NT - NT - NT - NT - CH 17b NT - NT - NT - NT - 18b (n=4) 15.7% NT - NT (n=4) 7.0% 20b (n=4) 10.0% (n=4) 7.3% NT (n=4) 10.9% 28b NT (n=4) 3.1% NT - NT - Overall GM (n=26) (n=29) (n=28) (n=26) 95% CL Between Lab GCV 5.1% 11.2% 8.0% 8.0% n: no of assays; NT: not tested; CLO: clotting assay; CH: chromogenic assay; N/A: not applicable; GM: geometric mean; GCV: geometric coefficient of variation; CL: confidence limits. Shaded boxes indicate outliers and are excluded from overall GM.

15 Page 15 Table 1c: Summary of potency estimates, 95% confidence limits (CL), inter-laboratory variation (GCV) for sample A against the 3 rd IS and the normal pooled plasmas Vs sample S, 3 rd IS Potency estimates (95% CL) IU/ampoule Interlaboratory GCV Vs sample N, normal pooled plasma Interlaboratory Potency estimates (95% CL) IU/ampoule GCV Difference between potencies t-test, p value FII ( ) 1.2 % ( ) 5.1% 1.4% FVII ( ) 2.9% ( ) 11.2% 5.5% FIX ( ) 2.8% ( ) 8.0% 6.0% <0.001 FX ( ) 1.5% ( ) 8.0% 4.5% Table 1d: Ratios of clotting to chromogenic potency estimates for sample A relative to the 3 rd IS Potency estimates (IU/ampoule) Clotting Chromogenic Ratio Clotting/Chromogenic FII (n = 24) (n = 3) FVII (n = 26) (n = 3) FIX (n = 28) (n = 1) FX (n = 24) (n = 2) 0.987

16 Page 16 Table 2a: Comparison of FII potency estimates for fresh and frozen local pooled plasmas relative to the 3 rd IS Lab Method Assay 1 Assay 2 Assay 3 Assay 4 GM Fresh Frozen All GCV % % % % % % % % % % % % % CLO N/A % 16a % 16b % 18a % % % % % % % % % 20b CH % N/A: not applicable; CLO: clotting assay; CH: chromogenic assay; GM: geometric mean; GCV: geometric coefficient of variation. Bold italic text indicates fresh plasma data.

17 Page 17 Table 2b: Comparison of FVII potency estimates for fresh and frozen local pooled plasmas relative to the 3 rd IS Lab Method Assay 1 Assay 2 Assay 3 Assay 4 GM Fresh Frozen All GCV % % % % % % % % % % % % % 14 CLO N/A % 16a % 16b % 18a % % % % % % % % % 28a % 20b % CH 28b % N/A: not applicable; CLO: clotting assay; CH: chromogenic assay; GM: geometric mean; GCV: geometric coefficient of variation. Bold italic text indicates fresh plasma data. Shaded boxes indicate exclusion from overall potency estimate due to the use of lyophilised plasma.

18 Page 18 Table 2c: Comparison of FIX potency estimates for fresh and frozen local pooled plasmas relative to the 3 rd IS Lab Method Assay 1 Assay 2 Assay 3 Assay 4 GM Fresh Frozen All GCV % % % % % % % % % % % % N/A N/A CLO % 16a % 16b % 18a % % 20a % % % % % % % % 28a % N/A: not applicable; CLO: clotting assay; CH: chromogenic assay; GM: geometric mean; GCV: geometric coefficient of variation. Bold italic text indicates fresh plasma data. Shaded boxes indicate exclusion from overall potency estimate due to the use of lyophilised plasma.

19 Page 19 Table 2d: Comparison of FX potency estimates for fresh and frozen local pooled plasmas relative to the 3 rd IS Lab Method Assay 1 Assay 2 Assay 3 Assay 4 GM Fresh Frozen All GCV % % % % % % % % % % % % 13 CLO % N/A N/A % 16a % 16b % % % % % % % % 28a % 18b % CH 20b % N/A: not applicable; CLO: clotting assay; CH: chromogenic assay; GM: geometric mean; GCV: geometric coefficient of variation. Bold italic text indicates fresh plasma data. Shaded boxes represent outliers. Table 2e: Summary of geometric mean potencies and inter-laboratory variation, expressed as GCV for fresh and frozen pooled plasmas relative to the 3 rd IS Fresh plasma GM IU/ml Frozen plasma GM IU/ml FII FVII FIX FX All GM (95% CL) IU/ml ( ) ( ) ( ) ( ) Between laboratory GCV t-test, fresh vs frozen p value 6.1% % % % 0.724

20 Page 20 Table 3: Summary of FIX antigen results from Laboratory 23 relative to sample N, the local normal pooled plasma (assumed 1 u/ml) Samples u/ampoule Assay 1 Assay 2 Assay 3 Assay 4 GM GCV S, 3 rd IS % A % GM: geometric mean; GCV: geometric coefficient of variation Table 4a: Accelerated degradation study: Potencies with 95% confidence limits relative to - 70 C samples after 6 months storage at elevated temperatures Factor -20 C +4 C +20 C +37 C II *1.016 *0.996 * ( ) ( ) ( ) ( ) VII *0.994 * ( ) ( ) ( ) ( ) IX *1.020 * ( ) ( ) ( ) Non-parallel X *1.000 * ( ) ( ) ( ) ( ) * no significant difference in potency compared to -70 C samples Table 4b: Real-Time stability monitoring and accelerated degradation study (8 years storage, 4 time points) for the 3 rd IS, 99/826, relative to the -150 C samples Predicted Potency % loss/year for -20 C samples 95% upper confidence limit for % loss/year FII FVII FIX FX April 2010 Real time monitoring Relative potency of -20 C vs -150 C ( ) ( ) ( ) ( )

21 Page 21 Figure 1a: Laboratory potency estimates as % of the overall mean potency (including outliers) for FII in sample A, the proposed 4th IS Factors II, VII, IX, X, plasma relative to sample S, the 3rd IS Factors II, VII, IX, X, plasma, (99/826). The number in the square denotes the laboratory code. 22 Number of Laboratories a 20b 17b 17a 16b 16a 18b Laboratory Mean Potency as % of Overall Mean GM = 0.89 IU/amp GCV = 1.2% Outliers = Labs 8,18b Clotting Assays Chromogenic Assays Figure 1b: Laboratory potency estimates as % of the overall mean potency (including outliers) for FVII in sample A, the proposed 4th IS Factors II, VII, IX, X, plasma relative to sample S, the 3rd IS Factors II, VII, IX, X, plasma, (99/826). The number in the square denotes the laboratory code GM = 0.99 IU/amp GCV = 2.9% Outlier = Labs 8 Number of Laboratories a 17b 8 16b 16a 28b b a Laboratory Mean Potency as % of Overall Mean Clotting Assays Chromogenic Assays

22 Page 22 Figure 1c: Laboratory potency estimates as % of the overall mean potency (including outliers) for FIX in sample A, the proposed 4th IS Factors II, VII, IX, X, plasma relative to sample S, the 3rd IS Factors II, VII, IX, X, plasma, (99/826). The number in the square denotes the laboratory code GM = 0.86 IU/amp GCV = 2.8% 16 Number of Laboratories 14 20a 12 18a 16b 10 16a a a Laboratory Mean Potency as % of Overall Mean Clotting Assays Chromogenic Assays Figure 1d: Laboratory potency estimates as % of the overall mean potency (including outliers) for FX in sample A, the proposed 4th IS Factors II, VII, IX, X, plasma relative to sample S, the 3rd IS Factors II, VII, IX, X, plasma, (99/826). The number in the square denotes the laboratory code GM = 0.89 IU/amp GCV = 1.5% Outlier = Labs 8 Number of Laboratories 28a 14 20b 16b b a Laboratory Mean Potency as % of Overall Mean Clotting Assays Chromogenic Assays

23 Page 23 Figure 2a: Laboratory potency estimates as % of the overall mean potency (including outliers) for FII in sample N (local pooled plasma) relative to sample S, the 3rd IS Factors II, VII, IX, X, plasma, (99/826). The number in the square denotes the laboratory code GM = 1.02 IU/amp GCV = 6.1% Number of Laboratories a 18b 4 16b a b Laboratory Mean Potency as % of Overall Mean Clotting Assays Chromogenic Assays Figure 2b: Laboratory potency estimates as % of the overall mean potency (including outliers) for FVII in sample N (local pooled plasma) relative to sample S, the 3rd IS Factors II, VII, IX, X, plasma, (99/826). The number in the square denotes the laboratory code Number of Laboratories GM = 1.07 IU/amp GCV = 10.0% Lab 21 lyophilised plasma excluded 6 16b 4 16a 28a b b a Laboratory Mean Potency as % of Overall Mean Clotting Assays Chromogenic Assays

24 Page 24 Figure 2c: Laboratory potency estimates as % of the overall mean potency (including outliers) for FIX in sample N (local pooled plasma) relative to sample S, the 3rd IS Factors II, VII, IX, X, plasma, (99/826). The number in the square denotes the laboratory code Number of Laboratories GM = 1.07 IU/amp GCV = 7.7% Lab 21 lyophilised plasma excluded 6 20a 27 18a a 28a b Laboratory Mean Potency as % of Overall Mean Clotting Assays Chromogenic Assays Figure 2d: Laboratory potency estimates as % of the overall mean potency (including outliers) for FX in sample N (local pooled plasma) relative to sample S, the 3rd IS Factors II, VII, IX, X, plasma, (99/826). The number in the square denotes the laboratory code Number of Laboratories b 10 18b 16b 26 16a a Laboratory Mean Potency as % of Overall Mean GM = 1.05 IU/amp GCV = 7.7% Outlier = Lab 15 Clotting Assays Chromogenic Assays

25 Page 25 Appendix 1: List of participants Renata Zadro, Clinical Hospital Center, Zagreb, Croatia J.M.Rentenaar, Sanquin Blood Supply Foundation, Amsterdam, The Netherlands Kampta Sukhu, Oxford Haemophilia and Thrombosis Centre, Headington, UK Jørgen Jespersen and Johannes Sidelmann, Unit for Thrombosis Research, University of Southern Denmark, Esbjerg, Denmark Sukesh Nair, Christian Medical College, Vellore, India Michael Timme, Siemens Healthcare Diagnostics Products GmbH, Marburg, Germany Roger Luddington, Addenbrooke's Hospital, Cambridge, UK Marc Boylan, Pecision Biologic Inc, Dartmouth, NS, Canada Rossella Bader, Bianchi Bonomi Hemophilia and Thrombosis Center, Mangiagalli and Regina Elena Foundation, 9-Milan, Italy Caludine Caron, Laboratoire d'hématologie Hémostase, Lille Cedex, France Kirsten Villadsen and Anne-Mette Hvas, Center for Haemophillia and Thrombosis, Skejby, Denmark Christopher Ludlam and Lorna Germain, Department of Haematology, Edinburgh, UK Jean Amiral, Hyphen Biomed, Neuville sur Oise, France Dot Adcock and Laurie Desjardin, Esoterix Coagulation, Englewood, CO, USA Anne Riddell, Haemophillia Centre and Thrombosis Unit, London, UK Mariona Bono, Diagnostic Grifols, Barcelona, Spain Kathleen Trumbull and Wendy Olend, Instrumentation Laboratory, Bedford, MA, USA Barbara Kerbl, Technoclone GmbH, Vienna, Austria Denise Foulon and Hugh Hoogendoorn, Affinity Biologicals Inc., Ancaster, ON, Canada Joanne McGrath, Haemostasis Reference Laboratory, Hamilton, Ontario, Canada Helen Barson and Malinee Beeharry, NIBSC, Potters Bar, UK Nathalie Barat and Francois Nicham, Diagnostica Stago, Gennevilliers Cedex, France Jim Conkie and Grainne Hickman, Glasgow Royal Infirmary, Glasgow, UK Steffen Rosen, Rossix, Molndal, Sweden Annette Bowyer, Royal Hallamshire Hospital, Sheffield, UK Lisa Saminaden, Royal Liverpool Hospital, Liverpool, UK Judith Gillissen and Kathryn Bradley, George King Bio-medical, Inc., Kansas, USA Chong Loh, Allison Jones and Tursum Kerim, Therapeutic Goods Administration, Symonston ACT 2609, Australia Nicola Lucchesi and David Gambelli, Kedrion S.p.a, Galliano (Lucca), Italy

26 Page 26 Appendix 2: Protocol for collaborative study CALIBRATION OF PROPOSED 4th INTERNATIONAL STANDARD FACTORS II, VII, IX, X, PLASMA (09/172) and SSC lot 4. CS401 STUDY PROTOCOL 1 SAMPLES FOR ASSAY CODE S PREPARATION 3rd International Standard for Factors II, VII, IX, X, plasma (99/826), containing 0.91 IU FII, 1.00 IU FVII, 0.86 IU FIX and 0.93 IU FX per ampoule - 8 ampoules are supplied A Proposed 4th International Standard for Factors II, VII, IX, X, plasma (09/172), containing approximately 1.0 IU per ampoule for all four factors - 8 ampoules are supplied P Q N1 N2 ISTH SSC Lot 3 containing 0.86 IU FII, 0.87 IU FVII, 0.94 IU FIX and 0.86 IU FX per ampoule - 8 ampoules supplied. ISTH SSC Lot 4 containing approximately 1.0 IU/ml FII, FVII, FIX and FX per ampoule - 8 ampoules supplied. Fresh normal plasma pools prepared locally according to the instructions in Appendix one. 2 STORAGE AND RECONSTITUTION OF AMPOULES/ VIALS OF S, A, P AND Q Store all unopened ampoules/vials at -20 o C or below. Ampoules/vials should be allowed to warm to room temperature before reconstitution. Directions for opening DIN ampoules DIN ampoules have an easy-open coloured stress point, where the narrow ampoule stem joins the wider ampoule body. Tap the ampoule gently to collect the material at the bottom (labelled) end. Ensure that the disposable ampoule safety breaker provided is pushed down on the stem of the ampoule and against the shoulder of the ampoule body. Hold the body of the ampoule in one hand and the disposable ampoule breaker covering the ampoule stem between the thumb and first finger of the other hand. Apply a bending force to open the ampoule at the coloured stress point, primarily using the hand holding the plastic collar. Care should be taken to avoid cuts and projectile glass fragments that might enter the eyes, for example, by the use of suitable gloves and an eye shield. Take care that no material is lost from the ampoule and no glass falls into the ampoule. Within the ampoule is dry nitrogen gas at slightly less than atmospheric pressure. A new disposable ampoule breaker is provided with each DIN ampoule. Directions for opening screw cap vials Vials have a screw cap and stopper. The cap should be removed by turning anti-clockwise, please note on removal of the cap, the stopper may remain in the vial or be removed with the cap. Care should be taken on removal of cap to prevent the contents escaping.

27 Page 27 Reconstitute the ampoule/vial contents by adding 1 ml of distilled water. Allow the ampoule/vial to stand for 10 minutes at room temperature and aid reconstitution by gentle swirling. Transfer contents to a plastic tube and store at 4 o C prior to the assays. 3 OUTLINE OF STUDY If possible, please collect two sets of normal pooled plasma (see appendix one) for use fresh and then subsequently frozen. A total of four assays should be carried out for each factor (II, VII, IX, X) using fresh ampoules of S, A, P and Q, and normal plasma pool in each assay. Please perform Assay 1 on freshly prepared normal plasma pool (N1), Assay 2 on frozen plasma pool N1 (NF1), Assay 3 on a second freshly prepared normal plasma pool (N2) and Assay 4 on frozen plasma pool N2 (NF2). If freshly prepared normal pooled plasma cannot be collected, please use different batches of frozen normal pooled plasma as a substitute. Please assay FVII and FIX on one set of ampoules and FII and FX on a second set of ampoules. If you are using more than one assay method for a factor e.g. FVII clotting and chromogenic, please carry out the clotting assays first since these results will be used to calculate the assigned potency. The study design is summarised in the following table. Assay number for each Ampoule set Plasma pool no factor FVII, FIX assays FII, FX assays N NF N NF2 4 ASSAY DESIGN A balanced order of testing should be followed, for example: Assay 1 S A P Q N1 N1' Q P A' S' Assay 2* NF1 S A P Q Q' P A S NF1 Assay 3 Q N2 S A P P' A S N2 Q Assay 4* P Q NF2 S A' A' S NF2 Q P *Using frozen plasma where each letter refers to a set of three different dilutions (eg. 1/10, 1/30, 1/100) and A, A' and S, S' etc. refer to separate sets of dilutions (replicates) made independently from the same ampoule. The range of dilutions should be chosen to lie on the most linear portion of the dose-response relationship. The same range of dilutions should be used for all three materials (S, A, P, Q, N1, N2). The assays should be completed within two hours of reconstitution. It is preferable for the whole study to be carried out over four days with a fresh plasma pool prepared on two of the days, and then frozen for use in a second assay on a different day.

28 Page 28 5 USE OF FROZEN PLASMA POOLS A small number of laboratories who are unable to prepare the fresh plasma pools (N1 N2) may use frozen plasma pools instead. Ideally different batches should be used. Please enter the details of the preparations of frozen pool in the result forms. 6 RESULTS Raw data (e.g. clotting times) should be recorded on results sheets. You are also invited to calculate the relative potencies of A, P and Q vs S from your own assay results using the assigned potencies of S given in section 1. Please return your raw data and calculated potency estimates by 11 January 2010 to: elaine.@nibsc.hpa.org.uk Dr H Barson, Biotherapeutics Group, NIBSC, Blanche Lane, South Mimms, Potters Bar, Herts. EN6 3QG United Kingdom. Fax: +44 (0) APPENDIX ONE (CS401) 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 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 M). Ratio of 9 volumes blood to 1 volume of anticoagulant. Centrifugation Blood should be centrifuged at 4 o C 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 o C during the assay session. Snap-freeze aliquots of each pool (NF1, NF2) for assays 2 and 4.

29 Page 29 Appendix 3: Reagents, Methods and Instruments used by the Participants A. Factor II Lab number Chrom/ clotting? 1 STA-R Machine Factor II reagent Deficient plasma Neoplastin R thromboplastin Cryocheck 2 Q hemostasis analyser rabbit brain thromboplastin DG immunodepleted 3 ACL TOP recombiplastin (IL) Siemens (Dade) 4 ACL TOP PT (recombiplastin - IL) IL 5 CA1500 Siemens Innovin Siemens 6 Siemens BCS Siemens Innovin Precision Biologic 7 STA compact IL recombiplastin 2G Precision Biologic 8 STA compact HemosIL PT-Fib Precision Biologic 9 STA-R STA-Neoplastine CI+ STA 10 ACL TOP Recombiplastin - IL HaemosIL 11 ACL 9000 Recombiplastin - IL IL 12 ACL fra ILS Neoplastin C1 Plus Cryocheck 13 STAR / KC10 for FIX assay 1 Neoplastin CL5 (Stago) Hyphen 14 clotting ACL TOP 700 Recombiplastin - IL IL 15 KC10 Technoplastin HIS Technoclone 16a BCS Innovin PT-reagent Siemens 16b CA-1500 Thromborel S PT Siemens 19 ACL TOP cts Recombiplastin IL 22 Behring Coagulation System - BCS XP Innovin Siemens 23 CA7000 Innovin Siemens Siemens Cryocheck Precision Biologic 24 ACL TOP Recombiplastin - IL IL 25 ACL TOP Recombiplastin IL 26 Trinity MDA II Thromborel S Dade Behring Precision Biologic 27 MDA II Triniclot Precision Biologic 28 ACL TOP 500 Recombiplastin - IL Precision Biologic 29 STA compact Triniclot PT Excel George King Biomedical 17a manual Ecarin - Pentapharm N/A 17b manual in-house PTase N/A 18b 20b chromogenic N/A = not applicable Biomek 2000 (Beckmann) Echis Carinatus venom (Pentapharm) N/A ACL TOP Ecarin Sigma N/A

30 Page 30 B. Factor VII Lab number Chrom/ clotting? Machine Factor VII reagent Deficient plasma 1 STA-R PT Cryocheck 2 Q hemostasis analyser rabbit brain thromboplastin DG immunodepleted 3 ACL TOP recombiplastin (IL) Siemens (Dade) 4 ACL TOP PT (recombiplastin - IL) Diagen 5 CA1500 Siemens Innovin Siemens 6 Siemens BCS Siemens Innovin Precision Biologic 7 STA compact IL recombiplastin 2G Precision Biologic 8 STA compact HemosIL PT-Fib Precision Biologic 9 STA-R STA-Neoplastine CI+ STA 10 ACL TOP Recombiplastin - IL HaemosIL 11 ACL 9000 Recombiplastin - IL IL 12 ACL fra ILS Neoplastin C1 Plus Cryocheck 13 STAR / KC10 for FIX assay 1 Neoplastin CL5 (Stago) Hyphen 14 ACL TOP 700 Recombiplastin - IL IL 15 clotting KC10 Technoplastin HIS Technoclone 16a BCS Innovin PT-reagent Siemens 16b CA-1500 Thromborel S PT Siemens 18a Sysmex CA-7000 (Siemens) Innovin - Siemens Siemens 19 ACL TOP cts Recombiplastin IL 21 BCS Thromborel S Dade Behring Helena 22 Behring Coagulation System - BCS XP Innovin Siemens Siemens 23 CA7000 Innovin Siemens Cryocheck Precision Biologic 24 ACL TOP Recombiplastin - IL IL 25 ACL TOP Recombiplastin IL 26 Trinity MDA II Thromborel S Dade Behring Precision Biologic 27 MDA II Triniclot Precision Biologic 28a ACL TOP 500 Dade Behring Innovin Diagen 29 STA compact Hemoliance brain George King Biomedical thromboplastin 17b Manual Coaset FVII - Chromogenix N/A 20b chromogenic ACL TOP Coaset FVII Chromogenix N/A 28b ACL TOP Biophen FVII kit N/A N/A = not applicable

31 Page 31 C. Factor IX Lab number Chrom/ clotting? Machine Factor IX reagent Deficient plasma 1 STA-R APTT - Cephascreen - Stago Cryocheck 2 Q hemostasis analyser APTT (rabbit brain PL and ellagic acid) DG immunodepleted 3 ACL TOP APTT (synthasil - IL) IL 4 ACL TOP APTT (synthasil - IL) Technoclone 5 CA1500 Siemens APTT - Trinity - Triniclot APTT Siemens 6 Siemens BCS Siemens Actin FSL Precision Biologic 7 STA compact IL SP Precision Biologic 8 STA compact HemosIL APTT-SP Precision Biologic 9 STA-R STA-CK Prest STA 10 ACL TOP APTT - Synthasil (IL) HaemosIL 11 ACL 9000 APTT - IL IL 12 ACL fra ILS triniclot T1203A Cryocheck 13 STAR / KC10 for FIX assay 1 Cephen APTT (Hyphen) On KC10: CK Prest(Stago) Hyphen 14 ACL TOP 700 HemosIL APTT-SP - IL IL 15 clotting KC10 APTT Dapttin TC Technoclone 16a BCS Actin FSL APTT Siemens 16b CA-1500 Actin FS aptt Siemens 18a Sysmex CA-7000 (Siemens) Actin FSL (Siemens) Siemens 19 ACL TOP cts SynathasIL APTT IL 20a ACL Advance Actin FS Siemens Siemens 21 BCS APTT - Dade Behring Dade Behring 22 Behring Coagulation System - BCS XP Actin FS - Siemens Siemens 23 CA7000 Actin FS Cryocheck Precision Biologic 24 ACL TOP APTT Synthasil - IL IL 25 ACL TOP Synthasil APTT IL 26 Trinity MDA II Trinity Auto APTT Precision Biologic 27 MDA II Platelin LS Technoclone 28a ACL TOP 500 IL APTT Technoclone 29 STA compact Triniclot Auto APTT George Kin Biomedical 17a chromogenic Manual Rossix N/A N/A= not applicable

32 Page 32 D. Factor X Lab number Chrom/ clotting? Machine Factor X reagent Deficient plasma 1 STA-R PT Cryocheck 2 Q hemostasis analyser rabbit brain thromboplastin DG immunodepleted 3 ACL TOP recombiplastin (IL) Siemens (Dade) 4 ACL TOP PT (recombiplastin - IL) IL 5 CA1500 Siemens Innovin Siemens 6 Siemens BCS Siemens Innovin Precision Biologic 7 STA compact IL recombiplastin 2G Precision Biologic 8 STA compact HemosIL PT-Fib Precision Biologic 9 STA-R STA-Neoplastine CI+ STA 10 ACL TOP Recombiplastin - IL HaemosIL 11 ACL 9000 Recombiplastin - IL IL 12 ACL fra ILS Neoplastin C1 Plus Cryocheck 13 STAR / KC10 for FIX assay 1 Neoplastin CL5 (Stago) Hyphen 14 clotting ACL TOP 700 Recombiplastin - IL IL 15 KC10 Technoplastin HIS Technoclone 16a BCS Innovin PT-reagent Siemens 16b CA-1500 Thromborel S PT Siemens 19 ACL TOP cts Recombiplastin IL 22 Behring Coagulation System - BCS XP Innovin Siemens Siemens 23 CA7000 Innovin Siemens Cryocheck Precision Biologic 24 ACL TOP Recombiplastin - IL IL 25 ACL TOP Recombiplastin IL 26 Trinity MDA II Thromborel S Dade Behring Precision Biologic 27 MDA II Triniclot Precision Biologic 28a ACL TOP 500 Recombiplastin - IL Precision Biologic 29 STA compact Triniclot PT Excel George King Biomedical Russels viper venom 18b Biomek 2000 (Beckmann) N/A (Pentapharm) chromogenic Russels viper Venom 20b ACL TOP N/A (Pentapharm) N/A = not applicable

33 Page 33 Appendix 4: Potency estimates for each factor from Laboratory 29. Results are reported as IU/ml and each ampoule of sample A were reconstituted with 1 ml of water. Data were generated using linear regression and arithmetic mean. FII potency (IU/ml) Sample Assay 1 Assay 2 Assay 3 Assay 4 Mean CV A % Plasma (N) % FVII potency (IU/ml) Sample Assay 1 Assay 2 Assay 3 Assay 4 Mean CV A % Plasma (N) % FIX potency (IU/ml) Sample Assay 1 Assay 2 Assay 3 Assay 4 Mean CV A % Plasma (N) % FX potency (IU/ml) Sample Assay 1 Assay 2 Assay 3 Assay 4 Mean CV A % Plasma (N) % CV: coefficient of variation

34 Page 34 Appendix 5: Individual potency estimates and geometric means for each laboratory. A. FII, A relative to S Lab Assay 1 Assay 2 Assay 3 Assay 4 GM GCV % % % % % % % % % % % % % % % 16a % 16b % 17a % 17b % 18b % % 20b % % % % % % % 28a % Shaded boxes represent outliers Hatched boxes indicate chromogenic assays GM: geometric mean; GCV: geometric coefficient of variation

35 Page 35 B. FII, A relative to N Lab Assay 1 Assay 2 Assay 3 Assay 4 GM GCV % % % % % % % % % % % % % N/A % 16a % 16b % 18b % % 20b % % % % % % % 28a % Shaded boxes represent outliers Hatched boxes indicate chromogenic assays GM: geometric mean; GCV: geometric coefficient of variation

36 Page 36 C. FVII, A relative to S Lab Assay 1 Assay 2 Assay 3 Assay 4 GM GCV % % % % % % % % % % % % % % % 16a % 16b % 17b % 18a % % 20b % % % % % % % % 28a % 28b % Shaded boxes represent outliers Hatched boxes indicate chromogenic assays GM: geometric mean; GCV: geometric coefficient of variation

37 Page 37 D. FVII, A relative to N Lab Assay 1 Assay 2 Assay 3 Assay 4 GM GCV % % % % % % % % % % % % % N/A % 16a % 16b % 18a % % 20b % % % % % % % % 28a % 28b % Hatched boxes indicate chromogenic assays GM: geometric mean; GCV: geometric coefficient of variation

38 Page 38 E. FIX, A relative to S Lab Assay 1 Assay 2 Assay 3 Assay 4 GM GCV % % % % % % % % % % % % % % % 16a % 16b % 17a % 18a % % 20a % % % % % % % % 28a % Hatched boxes indicate chromogenic assays GM: geometric mean; GCV: geometric coefficient of variation

39 Page 39 F. FIX, A relative to N Lab Assay 1 Assay 2 Assay 3 Assay 4 GM GCV % % % % % % % % % % % % N/A N/A % 16a % 16b % 18a % % 20a % % % % % % % % 28a % GM: geometric mean; GCV: geometric coefficient of variation

40 Page 40 G. FX, A relative to S Lab Assay 1 Assay 2 Assay 3 Assay 4 GM GCV % % % % % % % % % % % % % N/A % 16a % 16b % 18b % % 20b % % % % % % % 28a % Shaded boxes represent outliers Hatched boxes indicate chromogenic assays GM: geometric mean; GCV: geometric coefficient of variation

41 Page 41 H. FX, A relative to N Lab Assay 1 Assay 2 Assay 3 Assay 4 GM GCV % % % % % % % % % % % % % N/A % 16a % 16b % 18b % % 20b % % % % % % % 28a % Shaded boxes represent outliers Hatched boxes indicate chromogenic assays GM: geometric mean; GCV: geometric coefficient of variation

42 Page 42 Appendix 6: Draft Instruction for Use (IFU) for the Proposed 4 th International Standard for Blood Coagulation Factors II, VII, IX, X, Plasma 09/172