WHO/BS/2013.2230 Addendum 2 ENGLISH ONLY

(1)

WHO/BS/2013.2230 Addendum 2 ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 21 to 25 October 2013

Final report on the evaluation of the WHO Reference Reagent for Activated Blood Coagulation Factor XI (FXIa), Human

Helen Wilmot^1,3, John Hogwood¹,Peter Rigsby²and Elaine Gray¹

1Haemostasis Section, ²Biostatistics Section, National Institute for Biological Standards and Control

Potters Bar, Hertfordshire, EN6 3QG, UK

3Principal Investigator

© World Health Organization 2013

All rights reserved. Publications of the World Health Organization are available on the WHO web site (www.who.int) or can be purchased from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel: +41 22 791 3264; fax:

+41 22 791 4857; e-mail: [email protected]). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press through the WHO web site (http://www.who.int/about/

licensing/copyright_form/en/index.html). 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)

Summary

Twelve laboratories from nine different countries participated in a “fit for purpose” collaborative study on the Reference Reagent for Activated Blood Coagulation Factor XI (FXIa), 11/236. The study had two parts; Part A being a fit for purpose study to examine whether the material could serve as a reference for FXIa in IVIG and Part B examining the effect of different IVIG matrices on the performance of the material in the assays.

Overall in Part A, intra-laboratory variability was quite high, however, most laboratories were able to quantify FXIa in IVIG samples, including those from different manufacturers. A coded duplicate of the reference material was included in this part of the study and it was reassuring to note that although the intra-lab variability as expressed by GCV was ranged from 2.15% to 17.23%, the inter-lab variability was low (GCV 5.55%) for this sample. In Part B, participants were asked to spike the reference material into their own IVIG samples at 3 different dilutions.

The recovery of the spiked FXIa was then measured. The inter- and intra-laboratory variation of labs using FXIa-specific assays was low (around 10%) and these labs were able to recover close to the expected amount of FXIa in the samples.

Overall the results support the Reference Reagent for FXIa as a reference reagent for different IVIG preparations and very well as a reagent to create IVIG matrix-specific references within each laboratory. It also serves to unite the FXIa units used by different laboratories, as previously there was no common unit for measurement of FXIa.

The FXIa reference material was assigned as the WHO Reference Reagent for FXIa, with an arbitrary unitage of 10 units per ampoule in October 2012. This report provides the full results from the fit for purpose study carried out in 2012, which were not available in time for the ECBS report submission deadline in 2012.

Introduction

Between 2008 and 2010, a cluster of thrombotic events was associated with the administration of intravenous immunoglobulins (IVIGs). Following investigation by the European Official

Medicines Control Laboratories (OMCLs), European Medicines Agency (EMA), the United States Food and Drug Administration (US FDA) and the manufacturer, the thrombotic cause was identified as factor XIa, though other procoagulant components were not completely ruled out.

Subsequently, IVIG products from one manufacturer were suspended. IVIGs from other

manufacturers were also investigated, some of which were found to contain similar procoagulant activity. The European Pharmacopoeia monograph for Human Normal Immunoglobulin for Intravenous Administration was revised with effect from January 2012 and now requires step(s) in the production method that have been shown to remove thrombosis generating agents and that the product does not exhibit thrombogenic (procoagulant) activity. The European regulators requested the manufacturers submit batch data by July 2012 that supports the removal or absence of procoagulant activity from their products. An international collaborative study carried out by NIBSC in 2011 indicated that there is an urgent need to standardize assay methods for

procoagulant activity in IVIG products. It was recognised that although FXIa is one of the major procoagulant components, it would be important to have orthogonal methods to detect

thrombogenicity. The assay methods being considered are Non-Activated Partial

Thromboplastin Time (NAPTT), Thrombin Generation Test (TGT) and FXIa assays. Currently, there are no common qualitative or quantitative reference materials or system suitability controls available to develop assay methods and harmonise the assay results for these tests. It was agreed by the stakeholders (OMCLs, regulatory bodies, WHO Collaborating Centres (Paul Ehrlich Institut (PEI), Center for Biologics Evaluation and Research (CBER), NIBSC) and

(3)

manufacturers) that in order to ensure the safety of IVIG products, internationally harmonised reference materials should be developed for these tests. As FXIa is the major procoagulant component, a proposal has been made to produce an international standard for FXIa. However to produce a new well characterised international standard will take 2 to 3 years and therefore an interim material is required to cover this period. A proposal was made to produce an Reference Reagent (RR) for Blood Coagulation Factor XIa, Human. It is envisaged that this RR will be replaced by an International Standard for FXIa in 2 to 3 years. This interim reference reagent is assigned with an arbitrary unit of 10 u/ampoule by NIBSC and this study serves as a “fit for purpose” study to assess the suitability of the Reference Reagent as a standard for measurement of FXIa in IVIG products.

A preliminary report¹ with partial analysis of the collaborative study was submitted to ECBS in October 2012 and the ECBS endorsed the establishment of this candidate as the Reference Reagent for Activated Blood Coagulation Factor XI (FXIa), Human with the proviso that a report with full analysis of all the results and stability data should be presented to the ECBS in October 2013.

This report covers the analysis of data submitted by all the participants, together with accelerated degradation and on–bench stability results.

Participants

Twelve laboratories from 9 different countries (1 Austria, 1 Belgium, 1 Canada, 1 France, 1 Germany, 1 Spain, 1 Sweden, 2 UK, 3 USA) agreed to participate in the study. The participants included 3 diagnostics manufacturers, 5 therapeutic manufacturers and 4 regulatory authorities.

A list of participants is given in Appendix 1 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 WHO Reference Reagent for Factor XIa, NIBSC code 11/236

The bulk for the RR was purchased and the certificate of analysis from the vendor indicated that it had a specific activity of 606 U/mg, based on the activity in factor XI clotting assays, where 1 unit equals FXI activity in 1 ml of normal plasma. The starting material was certified by the manufacturer as being negative for anti-HIV1/2, HBsAg and hepatitis C. The material was prepared by activating purified human FXI with FXIIa and subsequently purified to homogeneity by a combination of affinity chromographic methods. The single batch of material was diluted at NIBSC in 50 mM Tris, 150 mM NaCl, 5 mg/ml trehalose and 0.5% human serum albumin. The material was distributed in glass ampoules, filled and freeze-dried according to guidelines for production of international standards. The product characteristics are listed in the following table.

(4)

NIBSC Code 11/236

Presentation Sealed, glass 5 ml DIN ampoules

Filling date 24^th May 2012

Number of Ampoules available 2775

Liquid filling weight (g) (n=587, measurements taken from all

3 pumps throughout the duration of the fill) 1.0082

CV of fill mass (%) 0.3069

Homogeneity of the fill by activity: 3 ampoules selected from the beginning of the fill and the end of the fill and were assayed against an in-house FXIa standard using an in-house method (adapted Biophen FIXa kit). 2 assays per ampoule were carried out. Effect of fill position was assessed by ANOVA of log potencies.

GCV p

FXIa 0.701 0.574

Mean dry weight (g) (n=6) 0.0260 (CV 0.95%)

Mean head space oxygen (%) (n=12) 0.53 (CV 37.13%)

Residual moisture (%) (n=11) 0.189 (CV 16.06%)

Storage temperature -20°C

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

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

Study design

This was a two part study.

PART A: A “fit for purpose” study for the WHO reference reagent for FXIa (11/236) assessing the performance in FXIa quantitative assays and the ability to serve as a reference reagent for FXIa in IVIG samples with various levels of FXIa activity.

PART B: An optional study for the participants with an aim to investigate the effect of different IVIG matrices on the validity of the assays. In-house IVIG preparations were required for this part.

Two groups of participants were recruited. The first group of participants was requested to carry out both Part A and Part B of the study, while a second group of participants carried out Part B only. Participants were requested to carry out four assays for FXIa using fresh ampoules of all the samples provided in each assay. Within each assay, participants were requested to assay at least three dilutions of each of the samples in replicate, according to balanced assay designs (study protocols shown in Appendix 2).

Participants were requested to return raw data together with calculated estimates for all of the samples relative to sample S (the IRR, 11/236) from each individual assay.

Samples Part A:

S: WHO Reference Reagent for Factor XIa (11/236) – 10 units/ampoule - 6 ampoules supplied.

A: Factor XIa preparation- 10 units/ampoule – 4 ampoules supplied

B: IVIG preparation containing high procoagulant activity, 5% protein (10/224) - 4 ampoules supplied.

(5)

C: IVIG preparation containing moderate procoagulant activity, 5% protein (10/222) - 4 ampoules supplied.

D: IVIG preparation containing low procoagulant activity, 5% protein (10/220) - 4 ampoules supplied.

E: IVIG preparation containing moderate procoagulant activity, 5% protein (10/282) - 4 ampoules supplied.

Part B:

S: WHO Reference Reagent for Factor XIa (11/236) – 10 units/ampoule - 6 ampoules supplied.

Assay methods

Each participant was requested to perform their routine in-house functional method(s) for FXIa.

Some laboratories performed more than one method and in this case the data from each method were treated as separate sets of results and referred to as Lab 3a and Lab3b, for example. A list of reagents, methods and instruments, together with their in-house FXIa standard and IVIGs used by the participants is given in Appendix 3. For Part A, Lab 1, 2, 3a and 4 carried out functional activity assays that were based on the conversion of FIX to FIXa. Lab 6a added a FXI/FXIa immune-capture step before detection of FXIa by chromogenic substrate. These assays are referred to in the subsequent Results and Discussion section as “specific tests”. Labs 3b and 5 carried out chromogenic substrate assay methods that could be influenced by other proteases and these are referred to as “other tests”. For part B, 4 laboratories used FXIa-specific chromogenic kits, which are referred to as “specific tests”. One lab (10) carried out a FXI clotting assay and Lab 12a used a fluorogenic substrate assay. Finally, two laboratories carried out ELISAs for FXIa.

Statistical analysis

An independent statistical analysis of raw data was performed at NIBSC. Potency estimates of the test samples in the study, relative to the RR, were calculated by parallel-line analysis² of log transformed assay response against log concentration, independently for each test sample included in each assay. Assay validity was assessed by analysis of variance and any deviations from linearity and parallelism were considered significant at the 1% level (p<0.01). Where significant deviations from the model appeared to result from underestimation of residual error, linearity was assessed by visual inspection of the plotted data and non-parallelism was assessed using deviations from linearity as an alternative residual error. Any assays rejected for

deviations from linearity or parallelism are indicated in the tables of results. Results from all valid assays were combined to generate unweighted geometric mean potencies for each laboratory and these laboratory means were used to calculate overall unweighted geometric mean 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 log₁₀ transformed potencies).

Laboratory 2 performed each assay using two microtitre plates, with all samples present on both plates. The data from each plate were therefore analysed separately, and the results from the two plates combined to give an overall potency estimate for each assay. Data from this laboratory were found not to lie in the linear portion of the dose-response curve, therefore the data from the highest dilution was excluded and the data were assessed for parallelism using two dilutions only.

(6)

Laboratory 6a returned single data points for each dilution, therefore the assays were assessed by using deviations from linearity as the estimate of residual error. Laboratory 12a returned data from single dilutions only for all samples other than S, therefore the results could not be analysed by parallel line bioassay. The results presented are therefore the laboratory’s own calculated data.

Data from part B, the spiking experiments, were evaluated by plotting the observed log potencies against their expected values and also assessing the differences between observed and expected values.

Results and discussion Part A

The main aim of this study was to evaluate the performance of the RR for FXIa as a reference material for measurement of FXIa in IVIG products. This is the first reference material for FXIa.

The collaborative study carried out in 2011/2012 showed the dynamic ranges of the assays and the amount of FXIa detected in the IVIG products. Based on this information, the RR has been assigned an arbitrary unitage of 10 u/ampoule. This unitage has no relationship with the International Unit of Factor XI assigned to the 1^st International Standard for Blood Coagulation Factor XI, Plasma, 04/102. As this is quantitative measurement, it was important that the participants carried out quantitative methods for this study. The individual assay results from each lab are shown in Appendix 4a. With the exception of labs 3b, 5 and 6a, all laboratories returned results obtained using functional activity assays that were based on the activation of FIX, with FXa generation as the final measurement readout. Lab 6a did not use an assay based on FXa generation, but employed an antibody capture step (anti-FXI Ab) to make the assay specific, followed by use of a chromogenic substrate (S2288). Lab 3b used a fluorogenic substrate that directly interacts with FXIa. However, this substrate is not specific for FXIa and results can be influenced by the presence of kallikrein and other proteases. Similarly, lab 5 used a chromogenic substrate, S2366, which is also not specific for FXIa and can be influenced by other proteases.

Tables 1 to 4 show the summarised data for samples A, B, C and E. Sample D was a low procoagulant activity IVIG sample and none of the laboratories reported quantifiable levels of FXIa in this sample. Each table shows the data from the specific assays (Labs 1, 2, 3a, 4 and 6a), together with the data from the other assays (Labs 3b and 5) and finally the combined data.

Sample A (Table 1) was a coded duplicate of sample S, the RR, and the results therefore provide a good indication of the precision and accuracy within labs. As the RR has an arbitrary unitage of 10 u/ampoule, the potency estimates for sample A should be close to this assigned value.

Table 1 and Figure 1 show clearly that the range of potency estimates for sample A calculated relative to sample S was around 10 u/amp, with a geometric mean of 10.03 u/amp and a GCV less than 5%. Therefore, overall agreement was good. However, the intra-lab variability could be improved upon, with only one of the labs performing specific tests having a GCV of less than 10% (Lab 4; 2.15%) and the range of potency estimates from 8.01 to 12.94 u/amp in individual assays.

Samples B and E were high procoagulant activity IVIG samples from different manufacturers.

Most laboratories were able to quantify FXIa in both samples (Tables 2 and 4 and Figures 2 and 4) and generated statistically valid assays, suggesting that sample S, the RR, would be a suitable calibrant for different IVIG preparations. For sample B, all the laboratories carrying out specific tests for FXIa were able to quantify FXIa, though the ranges of overall geometric means was quite wide (0.32 to 1.31 u/amp) and the %GCV was high overall. Apart from Lab 4, with a GCV

(7)

of 1.8%, the %GCVs from within each laboratory were high (15.24 to 39.69), however it is encouraging that labs using similar methods (1, 2, 3a and 4) reported similar results (between 0.32 and 0.85 u/amp). For sample E, Labs 2 and 6a were not able to quantify FXIa. For Lab 6a the responses for sample E were outside the range of the standard curve. For Lab 2 the results were not parallel to the standard and this could be as a result of the doses for the standard and sample not lying in the most linear part of the response. The labs that could quantify FXIa were in relatively close agreement with each other, giving an overall value of 0.14 u/amp. Assays carried out by Labs 3b and 5 on samples B and E were not specific for FXIa and in general the results were found to be higher than those obtained using specific tests. Other proteases present in the IVIG samples could have contributed to these overall values.

Samples C and D were produced from the same IVIG product as sample B. None of the labs were able to detect FXIa in sample D, which was a low procoagulant activity sample. Sample C had intermediate activity and only labs 1, 3a and 4 were able to quantify the FXIa content (Table 3 and Fig. 3). Lab 2 did show a dose response for sample C however the results were not

parallel to the standard. The reason for this is unknown but it is unlikely that this was due to matrix effect as sample B, C and D were all produced from the same product. The inter- lab %GCV for labs 1, 3a and 4 was very high at 165% due to the results from Lab 4 being around 3 fold higher than for Labs 1 and 3a.

Overall, the intra-laboratory variability was quite high for the IVIG samples, with only one laboratory consistently having a %GCV of <5. This could be partly due to the inexperience of the laboratories in carrying out these assays. However, it is encouraging that most laboratories were able to quantify FXIa in the high procoagulant activity samples and since these were from different manufacturers it does suggest that the FXIa RR can serve as a reference material for a range of IVIG preparations.

Part B

Part B of the study was designed to assess how well the RR would perform as a reference in different IVIG preparations. For this part of the study, participants were asked to spike sample S into their own IVIG preparation at 1/50, 1/100 and 1/200 dilutions. The recovery of the FXIa from the spiked samples against the purified reference gives an indication of how the reference may be affected by the different IVIG matrices. Labs 7, 8, 9, 10, 11 and 12 participated in this part of the study. Lab 10 used the FXI clotting assay and spiked with FXIa dilutions of 1/5, 1/10 and 1/20. The individual assay results from each laboratory are shown in Appendix 4b. Labs 1, 2, 3a and 3b from part A also participated, but were asked to spike at FXIa at a single dilution of 1/100. Lab 6a also participated, spiking at a dilution of 1/10. On the whole, the %GCV was good, with most labs having intra-laboratory variability of less than 10%. Table 5 shows the summarised data from all labs for sample X (1/50 dilution of FXIa into IVG), Y (1/100 dilution) and Z (1/200 dilution). Although the inter-lab variation was high (up to 147%), the variation was reduced to around 10% when only FXIa specific assays were used in the calculations. Also when FXIa specific assays were used, the overall geometric mean for recovery of the spiked FXIa was close to that expected for each dilution.

Figures 5 to 10 show the data from Labs 7 to 12a, with observed log potencies plotted against their expected values (a), and also the difference (observed minus expected) against the expected values (b). These figures can be used to assess how the assay results vary depending on the dilution used and also how the recovery of the spiked FXIa compares to that expected. Overall, the laboratories’ results did not vary much according to dilution (figures 5-10 (a)) and the difference between expected and observed was close to zero (Figures 5-10 (b)). Regression

(8)

analysis of the data (from figures (a) in each case) was significant for laboratories 10 and 12a only (p<0.001 in both cases), suggesting that the recovery of activity in the spiked samples was not as expected for these assays.

The results from Labs 9 and 10 (Figs. 7 and 8) indicate that there was a trend towards more variation in the potency estimates as the dilution factor increased. This could be due to the way in which the dilutions were prepared or due to the dynamic range of the assays. Since Lab 9 used the same assay kit as Labs 8 and 11, in this case it is probably due to the preparation of the dilutions. Lab 10 used the FXI clotting assay, spiking IVIG at 1/5, 1/10 and 1/20 dilutions. For this lab the assay variability appeared to increase with higher dilutions but it is interesting to note that the recovery was always less than expected. Lab 12a used a non-specific assay for FXIa, based on cleavage of chromogenic substrate SN13a. The data shown for Lab 12a is the laboratory’s own calculated data. Here, the assay variability did not appear to be affected by dilution, however there was an inhibitory effect on activity at lower dilutions (Figure 10 (b)) because the difference between observed and expected potency was closest to zero at the 1/200 dilutions. The inhibitory effect could either be a result of the chromogenic substrate binding competitively to a non-active substance, or a result of a component of the IVIG preparation inhibiting the FXIa activity at lower dilutions. Some inhibition of activity was also seen in data from Lab 6a (Appendix 4a, Table 7), who spiked FXIa at a 1/10 dilution into IVIG. In this case only around 75% recovery was observed.

Several different IVIG preparations were used in this part of the study. The protein content and excipient for each preparation are detailed in the table in Appendix 3. The protein content varied from 5 to 20% and the excipients used included glycine, maltose, sorbitol and L-proline. The fact that all labs using specific FXIa assays had good recovery of FXIa when spiked at 1/50, 1/100 or 1/200 shows that the matrices of different IVIG preparations have minimal influence over the activity of FXIa in these assays and therefore this reference reagent will work well as a reference material when spiked into various different IVIG preparations. This suggests that using the reference material in this way provides an excellent reference material for a particular IVIG product.

Two laboratories carried out ELISAs for FXI/FXIa. The data are presented in Tables 6 and 7 and represents that laboratory’s own calculated data. In the case of Lab 6b, the values are calculated relative to the laboratory’s own in-house standard and thus cannot be directly

compared to the activity data from Lab 6a presented in Appendix 3. However, it was interesting to note that samples C and E appeared to have similar antigen values despite having quite different activities (Tables 3 and 4) and that samples B, C and D had decreasing amounts of antigen, which aligns well with the different overall potencies reported (Tables 2 and 3; no activity reported for sample D). Lab 12b used S as the standard in their assay and also included S as a duplicate sample (Table 8). Comparing the ELISA data to the data from the functional assay (Appendix 4b; Table 6), the overall antigen values agree with the overall potency estimates, with the exception of X (IVIG spiked with 1/50 FXIa), where the antigen value was twice that of the potency estimate. However, as discussed previously, the functional assay appeared to show inhibition, so that probably explains the observed differences.

Supplementary data provided by Lab 3c

Laboratory 3 carried out the thrombin generation test on the samples, with the value estimated taken against their in-house standard for FXIa. The results are tabulated in Appendix 5. The data reported is the peak thrombin (nM) that each sample generated in congenital factor XI deficient plasma. The returned results have a GCV range from 19.8% to 42.6%. Re-analysis of

(9)

samples against S using parallel line analysis reduced the GCV range to 13.0 to 28.7%. An internal IVIG sample included did not give measurable peak thrombin, but when spiked with 1/100 sample S a peak thrombin level was observed which gave a high GCV 89.3% which was not improved when recalculated against S, GCV 141.2%.

Conclusion

Overall sample S, the WHO RR, performed well as a reference reagent for FXIa quantification in different preparations of IVIG in different laboratories and the excellent recovery in the spiking experiments also indicates the suitability of this reference reagent for measurement of FXIa present in IVIG products. The reference material also serves to resolve the difference in the unitage of FXIa used throughout different labs, as currently units vary between mass units and activity units.

The reference material (11/236) was established as the WHO Reference Reagent for Activated Coagulation Factor XI, Human, in October 2012. Over 1000 ampoules of this RR have been issued and feedback from some of the Official Medicine control Laboratories (OMCLs) and manufacturers for IVIG products indicated that this RR is helpful in the harmonization of results from procoagulant activity assays generated within and between laboratories. The data from this study and the usage rate of this RR support the need for an international standard for FXIa.

Stability studies

On-bench and accelerated degradation studies have been carried out. The results of the on-bench stability are shown below, with potencies representing 2 assays at each time-point and

determined relative to a fresh ampoule of 11/236 at each time. The potency after 3 hours storage on ice overlaps well with that at 0 h, indicating the material is stable for at least 3 hours when kept on ice.

Time on ice

Potency u/ml – combined (95% confidence intervals)

0 h 9.77

(9.40-10.15)

1 h 9.94

(9.51-10.39)

2 h 9.40

(9.22-9.59)

3 h 9.80

(9.08-10.59)

Accelerated degradation studies have been performed after 3 and 9 months storage at low and high temperatures. The predicted loss per year at each temperature is shown below, based on cumulative results from both time-points. The predicted percentage loss at the normal storage temperature of -20 °C is 0.001%, showing that the material is very stable at this temperature.

Degradation studies are on-going.

(10)

Storage temperature

(°C)

Predicted loss per year (%) (relative to -150)

-150 <0.001

-70 <0.001

-20 0.001

4 0.067

20 1.089

37 14.302

A statement on the stability of this material has been inserted into the instructions for use (see appendix 6).

Participant responses

All participants were sent the report for comment and all agreed with the proposal to establish 11/236 as the Reference Reagent for FXIa. Two participants suggested that a different unitage could be used (one suggesting 1 unit/ampoule, the other 100 unit/ampoule), however neither objected to 10 units per ampoule. NIBSC explained that at the start of the study the FXIa content of various IVIG products was unknown, hence 10 units was selected to give a wide enough scope to cover a range of eventualities. It was also important to distinguish the FXIa unit from that of the FXI plasma unit to avoid confusion. One participant also asked for comment on the variability of the results for the reference reagent, to which NIBSC responded that the coded duplicates (samples S and A) showed that variability was consistent with other collaborative studies and that the homogeneity of the batch was good.

Acknowledgments

The authors would like to thank the participants of the collaborative study, many of whom completed the testing under tight timescales.

References

1. Elaine Gray, Helen Wilmot, John Hogwood and Peter Rigsby. Evaluation of the proposed WHO 1st Reference Reagent for Activated Blood Coagulation Factor XI (FXIa), Human.

WHO/BS/2012.2206

2. Finney DJ. Statistical Methods in Biological Assay. 3rd Edition. London: Charles Griffin 1978.

(11)

Table 1: Overall results for Sample A, a coded duplicate of Sample S

Sample A GM 95% CL %GCV

Specific Tests

Lab 1 10.25 8.71- 12.05 10.71

Lab 2 9.38 7.28-12.09 17.23

Lab 3a 10.38 8.90- 12.10 10.12

Lab 4 9.62 9.30- 9.95 2.15

Lab 6a 10.67 8.48-13.41 15.45

Overall GM

(u/amp) 10.05 9.40-10.75 5.55

Range (u/amp) 8.01-12.94

Other Tests Lab 3b 9.82 9.57- 10.08 2.48

Lab 5 10.16 10.55-10.81 3.98

Overall GM

(u/amp) 9.82 NA NA

Range (u/amp) 9.56-10.51

All Tests Overall GM

(u/amp) 10.03 9.62-10.46 4.64

Range (u/amp) 8.01-12.94

GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NA: not applicable

Table 2: Overall results for Sample B, a high procoagulant activity IVIG

Sample B GM 95% CL %GCV

Specific Tests

Lab 1 0.37 - -

Lab 2 0.32 0.26-0.40 15.24

Lab 3a 0.53 0.38-0.76 39.69

Lab 4 0.85 0.82-0.89 1.80

Lab 6a 1.31 0.96-1.79 21.52

Overall GM

(u/amp) 0.59 0.28-1.22 79.66

Range (u/amp) 0.27-1.66

Other Tests Lab 3b 1.26 0.97 - 1.62 17.44

Lab 5 - - -

Overall GM

(u/amp) NA NA NA

Range (u/amp) 1.16-1.60

(u/amp) 0.67 0.35-1.26 83.98

Range (u/amp) 0.27-1.66

GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NA: not applicable

(12)

Table 3: Overall results for Sample C, an intermediate procoagulant activity IVIG

Sample C GM 95% CL %GCV

Specific Tests

Lab 1 0.02 0.004-0.100 88.57

Lab 2 NP - -

Lab 3a 0.05 0.04 - 0.07 31.68

Lab 4 0.14 0.13-0.15 3.77

Lab 6a LOQ - -

Overall GM

(u/amp) 0.052 0.004-0.583 164.72

Range (u/amp) 0.01-0.14

Other Tests Lab 3b ODR NA NA

Lab 5 LOQ NA NA

Overall GM

(u/amp) NA NA NA

Range (u/amp) NA

(u/amp) 0.052 0.29-1.19 164.72

Range (u/amp) 0.01-0.14

GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NP: Not parallel; LOQ: Below limit of quantification; ODR: outside detection range;

NA: not applicable

Table 4: Overall results for Sample E, a high procoagulant activity IVIG

Sample E GM 95% CL %GCV

Specific Tests

Lab 1 0.08 0.057- 0.1018 15.62

Lab 2 NP - -

Lab 3a 0.14 0.11 - 0.18 16.569

Lab 4 0.23 0.23 - 0.24 2.49

Lab 6a LOQ - -

Overall GM

(u/amp) 0.137 0.07-0.51 69.61

Range (u/amp) 0.07-0.24

Other Tests Lab 3b 1.17 0.97-1.41 12.49

Lab 5 4.30 3.87-4.81 7.05

Overall GM

(u/amp) NA NA NA

Range (u/amp) 0.99-4.72

(u/amp) 0.419 0.05-3.22 416.35

Range (u/amp) 0.07-4.72

GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NP: Not parallel; LOQ: Below limit of quantification; NA: not applicable

(13)

Figure 1: Potency estimates from each laboratory for Sample A. The geometric mean from each laboratory is indicated by the horizontal line.

Figure 2: Potency estimates from each laboratory for Sample B. The geometric mean from each laboratory is indicated by the horizontal line.

(14)

Figure 3: Potency estimates from each laboratory for Sample C. The geometric mean from each laboratory is indicated by the horizontal line.

Figure 4: Potency estimates from each laboratory for Sample E. The geometric mean from each laboratory is indicated by the horizontal line.

(15)

Table 5: Summary of results from FXIa spiked in IVIG

Assay results in u/ml IVIG + 1/50 FXIa

(X)

IVIG + 1/100 FXIa (Y)

IVIG + 1/200 FXIa (Z)

Expected recovery 0.200 0.100 0.050

Lab 1 - 0.11 -

Lab 2 - 0.12 -

Lab 3a - 0.13 -

Lab 7 0.200 0.099 0.050

Lab 8 0.198 0.098 0.053

Lab 9 0.219 0.097 0.046

Lab 11 0.197 0.103 0.055

Lab 12a* 0.027 0.075 0.051

GM (overall) 0.135 0.102 0.050

%GCV (overall) 146.92 17.92 6.98

95% CL (overall) 0.044-0.417 0.089-0.117 0.046-0.055

GM (specific assays) 0.203 0.107 0.050

%GCV (specific assays) 5.12 11.95 8.1

95% CL (specific

assays) 0.187-0.220 0.096-0.119 0.044-0.057

* Non-specific assay. GM: geometric mean; %GCV: geometric coefficient of variation; 95% CL:

95% confidence limits

Lab 10 used dilutions of 1/5, 1/10 and 1/20 and therefore the data is not shown above. Lab 3b took part (1/100 dilution only) but the results were outside the detection range and are therefore not shown.

(16)

(a)

(b)

Figure 5: Plots of (a) expected versus observed log u/ml and (b) difference versus expected log u/ml for IVIG samples spiked with 0.2, 0.1 and 0.05 u/ml FXIa (Lab 7)

y = 0.9975x - 0.0029 R² = 0.9974

-1.6 -1.2 -0.8 -0.4 0

log u/ml observed

log u/ml expected

Lab 7

-0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5

-1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0

obs-exp (log u/ml)

Lab 7

(17)

(a)

(b)

y = 0.9388x - 0.0538 R² = 0.988

-1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0

log u/ml observed

Lab 8

-0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5

-1.4obs-exp -1.2 -1 -0.8 -0.6 -0.4 -0.2 0

Lab 8

(18)

(a)

(b)

y = 1.1201x + 0.1188 R² = 0.9712

-1.6 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0

log u/ml observed

Lab 9

-0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5

-1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0

obs-exp

Lab 9

(19)

(a)

(b)

Figure 8: Plots of (a) expected versus observed log u/ml and (b) difference versus expected log u/ml for IVIG samples spiked with 0.2, 0.1 and 0.05 u/ml FXIa. Please note lab 10 carried out FXI clotting assay with spiked FXIa at 1/5, 1/10 and 1/20 (Lab 10)

y = 1.1054x - 0.0785 R² = 0.9816

-0.6 -0.4 -0.2 0 0.2 0.4

-0.4 -0.2 0 0.2 0.4

log u/ml observed

Lab 10

-0.5 -0.3 -0.1 0.1 0.3 0.5

-0.4obs-exp -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4

Lab 10

(20)

(a)

(b)

y = 0.9158x - 0.066 R² = 0.9699

-1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0

log u/ml observed

Lab 11

-0.5 -0.3 -0.1 0.1 0.3 0.5

-1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0

obs-exp

Lab 11

(21)

(a)

(b)

Figure 10: Plots of (a) expected versus observed log u/ml and (b) difference versus expected log u/ml for IVIG samples spiked with 0.2, 0.1 and 0.05 u/ml FXIa. Data represents the laboratory’s own calculated data (Lab 12a)

y = 0.4719x - 0.8008 R² = 0.9672

-1.6 -1.4 -1.2 -1 -0.8 -0.6 -0.4 -0.2 0

log u/ml observed

Lab 12

-0.5 -0.4 -0.3 -0.2 -0.1 0 0.1 0.2 0.3 0.4 0.5

-1.4obs-exp -1.2 -1 -0.8 -0.6 -0.4 -0.2 0

Log u/ml expected

Lab 12

(22)

Table 6: Lab 6b ELISA data (relative to in-house FXIa standard) FXI total antigen (ng/ml)

Sample mean S.D.

S 2011.2 141.5

A 2119.1 108.0

B 1804.1 51.0

C 647.2 35.7

D 523.6 71.3

E 694.9 74.8

IVIG 405.8 30.1

S.D.: standard deviation

Table 7: Lab 12b ELISA data (relative to S) Sample

u/amp

%GCV Assay 1 Assay 2 Assay 3 Assay 4 GM 95% CL

W 0.02 0.04 0.02 0.05 0.029 0.014-0.063 60.55

X 0.164 0.218 0.096 0.174 0.156 0.089-0.271 41.54 Y 0.078 0.110 0.055 0.051 0.070 0.039-0.122 42.37 Z 0.045 0.075 0.035 0.077 0.054 0.029-0.101 47.55 IHIXa 0.35 0.37 0.31 0.35 0.344 0.305-0.387 7.75 S 11.68 12.03 9.01 10.00 10.61 8.55-13.16 14.53 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; IHXIa: in-house FXIa preparation (different preparation to that used in the functional assay in Appendix 4).

(23)

Appendix 1: Participants

Michael Etscheid and Nathalie Beer, Paul Ehrlich Institute, Lange, Germany

Tenzin W. Lhakhang, Richard Jenny and Ryan Dorfman, Haematologic Technologies, Inc., Vermont, USA

Berta Pons and Marta José, Instituto Grifols SA, Barcelona, Spain

Yideng Liang, Samuel Woodle and Mikhail Ovanesov, CBER/FDA, Bethesda, USA Helen Wilmot, NIBSC, Potters Bar, UK

Steffen Rosén, Pia Bryngelhed and Per Rosén, Rossix, Molndal, Sweden

Rebecca Scott, Tara Dolan, Myfanwy Owen, Martyn Paddick and John More, Bio Products Laboratory, Elstree, UK

Geoffrey Pot, Iris Timmermans and Peter Turecek, Baxter Bioscience, Lessines, Belguim Dagmar Krause, Martina Schwarz, Gerda Wiry, Andrea Buchacher, Octapharma Pharmazeutika Produktions GesmbH, Vienna, Austria

Russell Pronyk and Derek Toth, Cangene Corporation, Winnipeg, Canada

Glenn Stevenson, Melanie Williams and Pete Vandeberg, Grifols Therapeutics Inc., Raleigh, USA

Valérie Lievre and Laurent Fleury, Agence nationale de sécurité du médicament et des produits de santé, Saint-Denis cedex, France

(24)

Appendix 2: Protocols for Collaborative Study

Collaborative Study on the proposed

1st WHO Reference Reagent for Factor XIa.

CS487

Study Protocol A

1 INTRODUCTION

Following the reports of thromboembolic events associated with the clinical use of IVIG, investigations indicated that the procoagulant component could be FXIa. Currently a reference material is not available to aid development of assay methods and to improve intra- and inter- laboratory agreement on the measurement of FXIa. A reference standard for FXIa is urgently needed to ensure harmonisation of measurement of FXIa in IVIG products. NIBSC has produced a FXIa reference reagent as an interim measure prior to the development of an

International Standard. This interim reference reagent will be assigned with an arbitrary unit by NIBSC and this study serves as a “fit for purpose” study to assess the suitability of the proposed Reference Reagent as a standard for measurement of FXIa in IVIG products.

There are two parts to the study:

PART A: A “fit for purpose” study for the proposed WHO reference reagent for XIa (11/236) assessing the performance in FXIa quantitative assays and the ability to serve as a reference reagent for FXIa in IVIG samples with various levels of FXIa activity.

PART B: An optional study to investigate the effect of different IVIG matrices on the validity of the assays. For this part, in-house IVIG preparations must be available for use.

Part B is optional; however we would like to include as many participants as possible to take part. Part B (if chosen) must be carried out at the same time (within the same assays) as Part A.

2 SAMPLES FOR ASSAY – PART A

CODE PREPARATION

S Proposed 1st Reference Reagent for Factor XIa (11/236) – 10 units/ampoule - 6

ampoules supplied. Two extra ampoules are provided for pre-optimisation of your established method(s) to establish the working dilutions necessary for optimal performance.

A Factor XIa preparation- 10 units/ampoule – 4 ampoules supplied

B IVIG preparation containing high procoagulant activity, 5% protein (10/224) - 4 ampoules supplied.

C IVIG preparation containing moderate procoagulant activity, 5% protein (10/222) - 4 ampoules supplied.

(25)

D IVIG preparation containing low procoagulant activity, 5% protein (10/220) - 4 ampoules supplied.

E IVIG preparation containing moderate procoagulant activity, 5% protein (10/282) - 4 ampoules supplied.

IHXIa Participants’ own in-house FXIa standard, if used routinely SAMPLES FOR ASSAY – PART B

Your chosen IVIG sample (fresh sample each day)

Your chosen IVIG sample spiked at 1/100 with sample S (prepared fresh each day)

For Part B, please include an IVIG sample (same batch in each assay), both spiked and unspiked with a 1/100 dilution of sample S before dilution for the assay. For example, if the assay

requires 1/10, 1/20 and 1/40 working dilutions, please spike the undiluted sample 1/100 with sample S (11/236), before performing the 1/10, 1/20 and 1/40 dilutions. Please use the same ampoule of S as the standard in your assay.

3 STORAGE AND RECONSTITUTION OF AMPOULES OF S, A, B, C, D AND E Store all unopened ampoules at -20^oC or below. Ampoules 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.

Reconstitute the ampoule contents by adding 1 ml of distilled water. Allow the ampoule to stand for 10 minutes at room temperature and aid reconstitution by gentle swirling. Transfer contents to a plastic tube and store at 4 ^oC prior to the assays.

4 ASSAY DESIGN

PART A

Two extra ampoules of S are provided; please use these to determine the most appropriate dilutions for sample S for use in your in-house assay method before beginning the study.

(26)

Once the appropriate dilutions of sample S have been determined, assays for factor XIa should be carried out on each of the 4 sets. Please use your own in-house method. Four ampoules of each sample are provided for this. Each set should be tested on a different day (see schedule below). A balanced order of testing should be used. Please include your own in-house XIa reference, if available.

Day 1, ampoule set 1

S¹ A¹ B¹ C

1

D

1 E¹ IHXIa

1

IHXIa

2 E² D

2

C

2 B² A² S²

IHXI

a¹ S¹ A¹ B

1

C

1

D

1 E¹ E² D

2

C

2

B

2 A² S² IHXIa

2

E¹ IHXI

a¹ S¹ A

1

B

1

C

1 D¹ D² C

2

B

2

A

2 S² IHXIa

2 E²

D¹ E¹ IHXIa

1 S¹ A

1 B

1 C¹ C² B

2 A

2 S² IHXIa

2 E² D²

Each letter refers to a set of three different dilutions (e.g. 1/10, 1/20, 1/40) and S1, S2 and A1, A2 etc. refer to separate sets of dilutions (replicates) made independently from the same ampoule.

IHXIa refers to your own in-house reference for XIa. 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, B, C, D, E). The assays should be completed within two hours of reconstitution. It is preferable for the whole study to be carried out over four days.

PART B – to be carried out at the same time as Part A

For Part B of the collaborative study, please assay your chosen IVIG product (a fresh sample per day) within the same assays as above. The samples should be assayed both with and without XIa (sample S) spiked into the sample at a 1/100 dilution. This spiking should be carried out on undiluted material and then the sample diluted for assay as per your normal method. Please use the same ampoule of S as the standard in your assay.

The testing schedule is shown on the following page.

5 RESULTS

Raw data (e.g. absorbance) should be recorded on the results sheet. Please return your raw data and calculated potency estimates by 13^th July 2012 to:

[email protected]

(27)

Testing schedule if taking part in both parts A and B:

Day 1, Ampoule set 1

S¹ A¹ B¹ C¹ D¹ E¹ IHXIa¹ IVIG¹ IVIG+

XIa¹

IVIG+

XIa² IVIG² IHXIa

2 E² D² C² B² A² S²

Day 2, Ampoule set 2

IVIG+

XIa¹ S¹ A¹ B¹ C¹ D¹ E¹ IHXIa

1 IVIG¹ IVIG² IHXIa

2 E² D² C² B² A² S² IVIG+

XIa² Day 3,

Ampoule set 3

IVIG¹ IVIG+

XIa¹ S¹ A¹ B¹ C¹ D¹ E¹ IHXIa¹ IHXIa² E² D² C² B² A² S² IVIG+

XIa² IVIG² Day 4,

Ampoule set 4

IHXIa

1 IVIG¹ IVIG+

XIa¹ S¹ A¹ B¹ C¹ D¹ E¹ E² D² C² B² A² S² IVIG+

XIa² IVIG² IHXIa²

Each letter refers to a set of three different dilutions (e.g. 1/10, 1/20, 1/40) and S1, S2 and A1, A2 etc. refer to separate sets of dilutions (replicates) made independently from the same ampoule. IHXIa refers to your own in-house reference for XIa; IVIG refers to your in-house IVIG preparation and IVIG+XIa refers to your in-house IVIG preparation spiked 1/100 with sample S (XIa). The range of dilutions should be chosen to lie on the most linear portion of the dose-response relationship.

(28)

Collaborative Study on the proposed 1st WHO Reference Reagent for Factor XIa.

CS487

Study Protocol (B)

1 INTRODUCTION

Following the reports of thromboembolic events associated with the clinical use of IVIG, investigations indicated that the procoagulant component could be FXIa. Currently a

reference material is not available to aid development of assay methods and to improve intra- and inter-laboratory agreement on the measurement of FXIa. A reference standard for FXIa is urgently needed to ensure harmonisation of measurement of FXIa in IVIG products.

NIBSC has produced a FXIa reference reagent as an interim measure prior to the

development of an International Standard. This interim reference reagent will be assigned with an arbitrary unit by NIBSC and this study serves as a “fit for purpose” study to assess the suitability of the proposed Reference Reagent as a standard for measurement of FXIa in IVIG products.

2. AIM OF STUDY

To investigate the effect of different IVIG matrices on the validity of quantitative FXIa assays. For this study, in-house IVIG preparations must be available for use.

3 SAMPLES FOR ASSAY

Please use the same batch of IVIG product throughout the whole study Your chosen IVIG sample (fresh sample each day)

Your same chosen IVIG sample spiked at 1/50, 1/100 and 1/200 with sample S (prepared fresh each day)

Your in-house FXIa standard

Please assay your chosen IVIG product (a fresh sample per day). The samples should be assayed both with and without XIa (sample S) spiked into the sample at a 1/50, 1/100 and 1/200 dilution. This spiking should be carried out on undiluted material and then the sample diluted for assay as per your normal method. For example, if the assay requires 1/10, 1/20 and 1/40 working dilutions, please spike the undiluted sample 1/50, 1/100 or 1/200 with sample S (11/236), before performing the 1/10, 1/20 and 1/40 dilutions. Please use the same ampoule of S for spiking as the standard in your assay.

CODE PREPARATION

S (11/236) Proposed 1^st WHO Reference Reagent for Factor XIa (11/236) – 10 units/ampoule - 6 ampoules supplied. Two extra ampoules are provided for pre-

optimisation of your established method(s) to establish the working dilutions necessary for optimal performance.

(29)

W Your chosen IVIG product

X Spike sample S at 1 in 50 dilution to your chosen IVIG product Y Spike sample S at 1 in 100 dilution to your chosen IVIG product.

Z Spike sample S at 1 in 200 dilution to your chosen IVIG product.

IHXIa Participants’ own in-house FXIa standard, if used routinely

4 STORAGE AND RECONSTITUTION OF AMPOULES OF S

Store all unopened ampoules at -20^oC or below. Ampoules 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.

Reconstitute the ampoule contents by adding 1 ml of distilled water. Allow the ampoule to stand for 10 minutes at room temperature and aid reconstitution by gentle swirling. Transfer contents to a plastic tube and store at 4 ^oC prior to the assays.

5 ASSAY DESIGN

Two extra ampoules of S are provided; please use these to determine the most appropriate dilutions for sample S for use in your in-house assay method before beginning the study.

Once the appropriate dilutions of sample S have been determined, assays for factor XIa should be carried out on each of the 4 sets. Please use your own in-house method. Four ampoules of each sample are provided for this. Each set should be tested on a different day (see schedule below). A balanced order of testing should be used. Please include your own in-house XIa reference, if available.

(30)

S¹ W¹ X¹ Y¹ Z¹ IHXI

a¹

IHXI

a² Z² Y² X² W² S²

IHXI

a¹ S¹ W¹ X¹ Y¹ Z¹ Z² Y² W¹ X¹ S² IHXI

a² Day 3,

ampoule set 3

Z¹ IHXI

a¹ S¹ W¹ X¹ Y¹ Y² X² W² S² IHXI

a² Z² Day 4,

ampoule set 4

Y¹ Z¹ IHXI

a¹ S¹ W¹ X¹ W¹ X¹ S² IHXI

a² Z² Y²

Each letter refers to a set of three different dilutions (e.g. 1/10, 1/20, 1/40) and S¹, S²and W¹, W² etc. refer to separate sets of dilutions (replicates) made independently from the same ampoule. IHXIa refers to your own in-house reference for XIa. 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 samples. The assays should be completed within two hours of reconstitution. It is preferable for the whole study to be carried out over four days.

6 RESULTS

Raw data (e.g. absorbance) should be recorded on the results sheet. Please return your raw data and calculated potency estimates by 20^th July 2012 to:

[email protected]