WHO/BS/2012.2206 ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 15 to 19 October 2012
Evaluation of the proposed WHO 1
stReference Reagent for Activated Blood Coagulation Factor XI (FXIa), Human
Elaine Gray1,3, Helen Wilmot1, John Hogwood1 and Peter Rigsby2
1Haemostasis Section, 2Biostatistics Section,3Principal Investigator National Institute for Biological Standards and Control
Potters Bar, Hertfordshire, EN6 3QG, UK
This document has been prepared for the purpose of inviting comments and suggestions on the proposals contained therein, which will then be considered by the Expert Committee on Biological Standardization (ECBS). Comments MUST be received by 01 October 2012 and
should be submitted electronically to the Responsible Officer: Dr Ana Padilla at email:
[email protected], with a copy to Dr David Wood at email: [email protected].
© World Health Organization 2012
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Summary
The proposed 1st International Reference Reagent for Activated Blood Coagulation Factor XI, Human, is a purified preparation of human activated blood coagulation factor XI (FXIa) which has been ampouled and lyophilised in accordance with guidelines for production of international standards. This batch of reference reagent has been assigned an arbitrary unitage of 10 u/ampoule and it is intended for use in the quantification of FXIa. The homogeneity of the batch is evidenced by the reasonably low coefficient of variation of the fill and the low inter-laboratory variability observed between the potency estimates of the coded duplicate, sample A. This study also showed this reagent is suitable for the measurement of FXIa present in IVIG products.
It is therefore recommended that 11/236 be established as the 1st International Reference Reagent for Activated Blood Coagulation Factor XIa (FXIa), Human, with an assigned value of 10 u/ampoule.
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) and the United States Food and Drug Administration (US FDA), 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 procoagulant activity. The European Pharmacopoeial 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 have 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 recognized 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 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 is made to produce an International Reference Reagent (IRR) for Blood Coagulation Factor XIa, Human. It is envisaged that this IRR will be replaced by an International Standard for FXIa in 2 to 3 years. This proposed 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 proposed international reference reagent as a standard for measurement of FXIa in IVIG products.
Participants
Eleven laboratories were invited to take part, with six participants returning data in time for inclusion in this report. Results from other participants will be fully evaluated and a summary report will be sent to the participants and, if required, to ECBS in October next year (2013).
The participants included one diagnostics manufacturer, six therapeutic manufacturers and four regulatory authorities. A list of participants having returned data thus far 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 Candidate, NIBSC code 11/236
The bulk for the candidate 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 then distributed in glass ampoules, filled and freeze-dried according to guidelines for production of international standards1. The product characteristics are listed in the following table.
NIBSC Code 11/236
Presentation Sealed, glass 5 ml DIN ampoules
Filling date 24th May 2012
Number of Ampoules available 2500
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 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
Samples
The following coded samples were sent to the participants
CODE PREPARATION
S Proposed 1st International 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.
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.
Study Design
This was a two part 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 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).
Raw assay data were returned, together with calculated estimates for all of the samples relative to sample S (the proposed IRR, 11/236) from each individual assay.
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.
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 proposed IRR, were calculated by parallel-line analysis2 of log transformed assay response against log concentration and were calculated 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 = {10s-1}×100% where s is the standard deviation of the log10 transformed potencies).
Laboratory two 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.
Results and Discussion
The main aim of this study was to evaluate the performance of the proposed IRR 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 proposed IRR 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 1st 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. With the exception of lab 3b, 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 3b employed a fluorogenic substrate that directly interacts with FXIa. However, this fluorogenic substrate is not specific for FXIa and results can be influenced by the presence of kallikrein and other proteases. This partly explains the higher activity estimated for samples B and E (figures 2 and 4) by comparison with potencies reported by the other participants.
Sample A was a coded duplicate of sample S, the proposed IRR. As the IRR has an arbitrary unitage of 10 u/ampoule, the potency estimates for sample A should be close to this assigned value. Figure 1 and table 6 show clearly that the range of potency estimates (8.01 – 11.71 u/ampoule) for sample A calculated relative to sample S was close to 10 u/ampoule, with a %GCV less than 10%. The between assay agreement was better in some laboratories than others, with intra-laboratory GCV ranging from 2.14 -14.80% (Tables 1 -5).
Samples C and D have relatively low procoagulant activity. None of the assay methods were able to detect any FXIa activity in sample D. Lab 2 and 3b failed to measure any activity in sample C. For Lab 2, although there was a dose-response for sample C, it was found to be non- parallel to sample S. The reason for this is unknown. It is unlikely that this was due to matrix effect as sample B, C and D are produced from the same product. For Lab 3b, the assay method was not sufficiently sensitive to give a dose-response for sample C. The inter-laboratory variability as expressed by GCV was over 160%. This high variability may be partly due to the 3 fold difference in estimates from Lab 4 by comparison with results from Lab 1 and Lab 3
(Figure 3). However, it is encouraging that the within-laboratory GCV, in Lab 4 at least, was less than 4% (Table 5).
With the exception of Lab 2, all other laboratories were to able measure FXIa in sample E (Figure 4). Taking all the assays carried out on sample E by the participants, over 70% of assays gave statistically valid comparisons between samples S and E (Tables 1 – 5). Sample E was made from a different IVIG product to samples B, C and D suggesting that sample S, the proposed IRR, would be a suitable FXIa calibrant for different IVIG preparations. This is confirmed by data provided for recovery of the FXIa spiked into the different IVIG products. A range of IVIG products with 5, 10 and 20% protein concentrations and different excipients was used by the participants. Lab 1, Lab 2, Lab 3a and Lab 3b spiked the IVIGs of their choice with sample S at 1/100 which should yield 0.1 u/ml of FXIa. Figure 5 shows that with the exception of Lab 3b, the other three participants were able to recover the spiked FXIa. There were only 2 invalid assays due to non-parallelism (Lab 3a, Table 3). The intra-lab variability was low in Lab 1, but higher in Lab 2 and Lab3a. The reason(s) for the higher variability is not known, but it could be the effect of the excipient or the higher protein concentration of the IVIG employed by Lab 3a. Lab 5 and Lab 6 spiked sample S at 3 different concentrations: 1/50, 1/100 and 1/200, giving expected final FXIa concentrations of 0.2, 0.1 and 0.05 u/ml of IVIGs. All the assays were statistically valid, with excellent recovery of the spiked FXIa (Figures 6 and 7).
Overall sample S, the proposed IRR, performed reasonably well as a reference reagent for FXIa quantification assays in different laboratories. The homogeneity of the batch is evidenced by the reasonably low coefficient of variation of the fill and the low variability as expressed by %GCV for the potency estimates of the coded duplicate, sample A. The good recovery and statistically valid assays in the spiking experiments carried out by different laboratories using different IVIG products also indicates the suitability of this reference reagent for measurement of FXIa present in IVIG products. Further results are expected from other participants and the final analysis of this study with results from all participants will be reported to participants later this year and if necessary to ECBS in 2013.
Stability study
An accelerated degradation study of 11/236 has been initiated. The first time-point (3 months storage) for potency testing predicted that there will be no significant loss of activity when the ampoules are stored at -20°C (Table 7) suggesting that this reference reagent will be stable till an international standard for FXIa becomes available. Accelerated degradation and real time monitoring will continue for this reference reagent.
Proposal and Recommendation to the ECBS
Sample S, 11/236, be the WHO 1st International Reference Reagent for Activated Factor XI, Human (FXIa), with the following arbitrary unit:
Factor XIa: 10 u/ampoule
The Instruction for Use for the proposed Standard, 11/236 is illustrated in Appendix 4.
References
1. 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.114-119 (section A.7)
2. Finney DJ. Statistical Method in Biological Assay. 3rd Edition. London: Charles Griffin 1978.
Acknowledgements
We would like to thank the participants of the study.
Table 1: Lab 1 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the proposed IRR for FXIa
Samples
u/ml
%GCV Assay 1 Assay 2 Assay 3 Assay 4 GM 95% CL
A 11.71 9.15 10.27 10.04 10.25 8.70 - 12.10 10.70
B NP NP 0.37 0.38 0.37 - -
C 0.01 NP 0.03 0.03 0.02 0.004 - 0.1 88.60
D LOQ LOQ LOQ LOQ - - -
E NP 0.07 0.09 0.09 0.08 0.069 - 0.103 13.40
IHXIa 0.04 NP 0.04 0.04 0.04 - -
IVIG LOD LOD LOD LOD - - -
IVIG+XIa 0.10 0.11 0.11 0.11 0.11 0.09 - 0.12 7.90 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NP: Not parallel;
LOQ: Below limit of quantification; LOD: Below limit of detection; IHXIa: in-house FXIa preparation;
IVIG+XIa: IVIG spiked with 1/100 of sample S
Table 2: Lab 2 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the proposed IRR for FXIa
Samples
u/ml
%GCV Assay 1 Assay 2 Assay 3 Assay 4 GM 95% CL
A 8.59 11.50 8.01 9.79 9.38 7.90 - 11.14 14.80 B 0.27 0.31 0.33 0.38 0.32 0.28 - 0.37 12.98
C NP NP NP NP - - -
D LOQ LOQ LOQ LOQ - - -
E NP NP NP NP - - -
IHXIa 0.29 0.35 0.25 0.32 0.30 0.26 - 0.35 13.20
IVIG LOQ LOQ LOQ LOQ - - -
IVIG+XIa 0.09 0.11 0.14 0.16 0.12 0.09 - 0.16 24.60 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NP: Not parallel;
LOQ: Below limit of quantification; IHXIa: in-house FXIa preparation; IVIG+XIa: IVIG spiked with 1/100 of sample S
Table 3: Lab 3a - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the proposed IRR for FXIa
Samples
u/ml
%GCV Assay
1
Assay 2
Assay 3
Assay 4
Assay 5
Assay 6
Assay 7
Assay
8 GM 95% CL
A 11.47 10.33 10.74 VR NP NL 9.12 NP 10.38 8.90 - 12.10 10.12
B 0.75 0.57 0.59 0.28 NP NP 0.59 0.56 0.53 0.37 - 0.76 40.80
C 0.06 0.04 0.05 VR NL 0.09 0.05 0.05 0.05 0.04 - 0.07 34.50
D LOQ LOQ LOQ LOQ LOQ LOQ LOQ LOQ LOQ - -
E 0.18 0.13 0.13 VR NL NL 0.14 NL 0.14 0.11 - 0.18 16.58
IVIG LOQ LOQ LOQ LOQ LOQ LOQ LOQ LOQ LOQ - -
IVIG+XIa 0.26 0.17 0.09 0.28 NP NP 0.08 0.06 0.13 0.06 - 0.26 95.2 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; NP: Not parallel;
NL: not linear; VR: highly variable results; LOQ: Below limit of quantification; IHXIa: in-house FXIa preparation;
IVIG+XIa: IVIG spiked with 1/100 of sample S
Table 4: Lab 3b - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the proposed IRR for FXIa
Samples
u/ml
%GCV Assay 1 Assay 2 Assay 3 Assay 4 Assay 5 Assay 6 Assay 7 Assay 8 GM 95% CL
A NT NT 9.78 9.65 9.72 9.56 10.17 10.07 9.82 9.58 - 10.08 2.45 B NT NT 1.60 1.16 ODR 1.16 1.16 ODR 1.26 0.97 - 1.63 17.5
C NT NT ODR ODR ODR ODR ODR ODR - - -
D NT NT ODR ODR ODR ODR ODR ODR - - -
E NT NT 1.28 1.178 NL 0.99 NL 1.26 1.17 0.97 - 1.41 12.3
IVIG NT NT ODR ODR ODR ODR ODR ODR - - -
IVIG+XIa NT NT ODR ODR ODR ODR ODR ODR - - -
NT: not tested; GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation;
NL: not linear; ODR: Outside detection range; IHXIa: in-house FXIa preparation; IVIG+XIa: IVIG spiked with 1/100 of sample S
Table 5: Lab 4 - Potency estimates and intra-laboratory GCV for test samples relative to sample S, the proposed IRR for FXIa
Samples
u/ml
%GCV Assay 1 Assay 2 Assay 3 Assay 4 GM 95% CL
A 9.34 9.82 9.69 9.64 9.62 9.30 - 9.95 2.14
B - 0.85 0.84 0.87 0.85 0.82 - 0.89 1.80
C 0.13 0.14 0.14 0.14 0.14 0.13 - 0.15 3.80
D LOD LOD LOD LOD - - -
E 0.23 0.24 0.23 0.24 0.23 0.23 - 0.24 2.50
IHXIa NT 775.32 806.25 843.14 807.76 727.80 - 896.51 4.30 GM: geometric mean; 95% CL: 95% confidence limits; %GCV: geometric coefficient of variation; LOQ: Below limit of quantification; NT: not tested; IHXIa: in-house FXIa preparation
Table 6: Overall potency estimate, 95% confidence limits, range, inter-laboratory GCV for Sample A relative to Sample S, the proposed IRR for FXIa
GM u/ampoule 9.88
95% Confidence limits 9.49 - 10.28 Range u/ampoule 8.01 – 11.71
%GCV 9.36 n 22
Table 7: Accelerated Degradation Study – after 3 months storage at elevated temperatures
Storage temperature
Predicted % potency loss per year
95% Upper confidence limits of potency loss per year
-70°C 0.00 0.00
-20°C 0.00 0.01
4°C 0.17 0.49
20°C 2.08 4.42
37°C 20.01 28.92
Figure 1: Laboratory potency estimates, individual assays for FXIa in sample A, relative to sample S, the proposed IRR for FXIa, 11/236.
Lab4 Lab3b
Lab3a Lab2
Lab1 12
11
10
9
8
u/ampoule
Individual Assay Potency Estimates for Sample A
Range = 8.01 - 11.71 u/ampoule GCV = 9.36% (all estimates, n = 22)
Figure 2: Laboratory potency estimates, individual assays for FXIa in sample B, relative to sample S, the proposed IRR for FXIa, 11/236.
Lab4 Lab3b
Lab3a Lab2
Lab1 1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
u/ampoule
Inter-lab GCV = 77.3%; excl Lab 3b = 54.6%
GM = 0.60; excl Lab 3b = 0.49
Individual Assay Potency Estimates for Sample B
Figure 3: Laboratory potency estimates, individual assays for FXIa in sample C, relative to sample S, the proposed IRR for FXIa, 11/236.
Lab4 Lab3b
Lab3a Lab2
Lab1 0.14
0.12
0.10
0.08
0.06
0.04
0.02
0.00
u/ampoule
Individual Assay Potency Estimates for Sample C
Inter-lab GCV = 164.6%
GM = 0.057
Below the limits of detection for Lab 2 and Lab3b
Figure 4: Laboratory potency estimates, individual assays for FXIa in sample E, relative to sample S, the proposed IRR for FXIa, 11/236.
Lab4 Lab3b
Lab3a Lab2
Lab1 1.4
1.2 1.0 0.8 0.6 0.4 0.2 0.0
u/ampoule
Individual assay potency estimates for sample E
Inter-lab GCV = 217.9%; excl Lab3b = 69.6%
GM = 0.26 u/ampoule; excl Lab3b = 0.15 u/ampoule Below limits of detection for Lab 2
Figure 5: Laboratory potency estimates, individual assays for FXIa in IVIG spiked with FXIa, relative to sample S, the proposed IRR for FXIa, 11/236
Lab3b Lab3a
Lab2 Lab1
0.30
0.25
0.20
0.15
0.10
0.05
0.00
u/ml
Inter-lab GCV = 55.6%
GM = 0.12 u/ml
Below limits of detection for Lab3b
Individual Assay Potency Estimates for FXIa spiked IVIG
Expected potency = 0.1 u/ml
Figure 6: Lab 5 - Laboratory potency estimates, individual assays for FXIa in IVIG spiked with FXIa, relative to sample S, the proposed IRR for FXIa, 11/236
1/200 1/100
1/50 0.225
0.200
0.175
0.150
0.125
0.100
0.075
0.050
u/ml
Individual assay potency estimates for FXIa spiked in IVIG
Figure 7: Lab 6 - Laboratory potency estimates, individual assays for FXIa in IVIG spiked with FXIa, relative to sample S, the proposed IRR for FXIa, 11/236
1/200 1/100
1/50 0.200
0.175
0.150
0.125
0.100
0.075
0.050
u/ml
Individual assay potency estimates for FXIa spiked in IVIG
Appendix 1: List of Participants
Yideng Liang, Samuel Woodle and Mikhail Ovanesov, CBER/FDA, Bethesda, USA Helen Wilmot, NIBSC, Potters Bar, UK
Marta Jose, Instituto Grifols SA, Barcelona, Spain
Steffen Rosen and Pia Bryngelhed, Rossix, Molndal, Sweden
Geoffrey Pot, Iris Timmermans and PeterTurecek, Baxter Bioscience, Lessines, Belguim Dagmar Krause, Martina Schwarz, Gerda Wiry, Andrea Buchacher, Octapharma Pharmazeutika Produktions GesmbH, Vienaa, Austria
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 international 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 International 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.
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 -20oC 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.
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 C1 D1 E1 IHXIa1 IHXIa2 E2 D2 C2 B2 A2 S2
Day 2, ampoule set 2
IHXIa1 S1 A1 B1 C1 D1 E1 E2 D2 C2 B2 A2 S2 IHXIa2
Day 3, ampoule set 3
E1 IHXIa1 S1 A1 B1 C1 D1 D2 C2 B2 A2 S2 IHXIa2 E2 Day 4,
ampoule set 4
D1 E1 IHXIa1 S1 A1 B1 C1 C2 B2 A2 S2 IHXIa2 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 13th July 2012 to:
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 IHXIa2 E2 D2 C2 B2 A2 S2 Day 2,
Ampoule set 2
IVIG+
XIa1 S1 A1 B1 C1 D1 E1 IHXIa1 IVIG1 IVIG2 IHXIa2 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
IHXIa1 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.
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 international 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 1st 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.
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 -20oC 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.
Day 1, ampoule set 1
S1 W1 X1 Y1 Z1 IHXIa1 IHXIa2 Z2 Y2 X2 W2 S2 Day 2,
ampoule set 2
IHXIa1 S1 W1 X1 Y1 Z1 Z2 Y2 W1 X1 S2 IHXIa2
Day 3, ampoule set 3
Z1 IHXIa1 S1 W1 X1 Y1 Y2 X2 W2 S2 IHXIa2 Z2
Day 4, ampoule set 4
Y1 Z1 IHXIa1 S1 W1 X1 W1 X1 S2 IHXIa2 Z2 Y2
Each letter refers to a set of three different dilutions (e.g. 1/10, 1/20, 1/40) and S1, S2 and 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 20th July 2012 to:
Appendix 3: Reagents, Methods and Instruments used by the Participants
Lab
number Method Machine In-house XIa reference standard
Protein content and excipient of IVIG 1 Biophen FXIa kit Plate reader CAL from Biophen kit 10%; glycine
2
In-house method based on addition of recombinant FIX to
Biophen FIXa kit
MTP-reader temperature
controlled 5% IVIG 5%; Maltose
3a Biophen FXIa kit BioTek Synergy H4
Hybrid Plate Reader Purified FXIa (HTI) 20%; L-proline, polysorbate 80
3b
In-house method based on mixing samples
50%/50% with fluorogenic substrate SN-13a in a flat-bottom
half-area microplate
BioTek Synergy H4
Hybrid Plate Reader Purified FXIa (HTI) 20%; L-proline, polysorbate 80
4
In-house method based on addition of plasma derived FIX to Biophen
FIXa kit
ACL TOP 500 Purified FXIa (ERL) Part A participant
5 Biophen FXIa kit Plate reader CAL from Biophen kit 10%; Sorbitol 6 Rossix Rox Factor XIa Thermomax Plate reader In-house purified FXIa
calibrator 5%; Maltose
Append Reagen
dix 4: Draf nt for Activ
ft Instructi vated Blood
ion for Use d Coagulati
(IFU) for t ion Factor X
the Propos XI (FXIa),
ed 1st Inter Human, 1
rnational R 1/236
Reference
