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WHO/BS/2012.2208 ENGLISH ONLY

EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 15 to 19 October 2012

Value Assignment to the WHO 2nd International Standard for Fibrinogen Concentrate (09/242)

Sanj Raut†§, Sarah Daniels†, Michelle Hamill* and Alan Heath*

Haemostasis Section† and Biostatistics Section*, National Institute for Biological Standards

and Control, Potters Bar, Hertfordshire, EN6 3QG, UK

§Principal investigator and coordinator of project

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 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).

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WHO/BS/2012.2208

Summary Two candidate materials, (samples B and C), have been evaluated as potential replacements of the WHO 1st International Standard (IS) for Fibrinogen Concentrate, by assays relative to the current primary WHO 1st IS for Fibrinogen Concentrate (98/614), in international collaborative studies involving 27 laboratories. All laboratories were instructed to use their routine methods for assessment of both Clottable and Total Protein. For Clottable Protein estimates, laboratories used either Clauss assays or subsequent protein determination assay methods following clot removal (CLOTr) e.g. Kjeldahl, Biuret, absorbance assays. Several laboratories performed more than one assay method. ELISAs were also carried to determine Clottable Protein estimates, however, data from this method were not included in the combined overall means as they do not represent fibrinogen function. For Total Protein estimates, laboratories used protein assays such as colorimetric assays e.g. Bicinchonic Acid (BCA) protein assay, Biuret assay, BioRad protein assay or N2 determination - Kjeldahl assay or absorbance assays. Intra-laboratory variability, for both candidates, as measured by geometric coefficient of variation (GCV), was found to range from 0.7-11.6% for Clottable Protein and from 0.8 - 7.6 % for Total Protein, with GCVs from the majority of the labs below 8%. For Clottable Protein estimates, there was good agreement in potencies between laboratories using the CLOTr methods when assayed relative to the WHO 1st IS, with the lowest inter-laboratory variability for candidate sample B, where a GCV value of 3.6% and a mean potency of 10.86 mg/ml (n=10) was obtained; whilst for sample C, a GCV value of 6.0% and a mean potency of 10.04 mg/ml by CLOTr (n=5) was obtained. There was poorer agreement in potencies between laboratories using the Clauss assays when assayed relative to the WHO 1st IS, with a GCV value of 17.2% and mean potency of 12.85 mg/ml (n=24) for sample B; and a GCV value of 26.8% and mean potency of 18.15 mg/ml (n=17) for sample C. A combined mean potency value of 12.23 mg/ml was obtained for sample B (GCV=16.7%; n=34), whilst a combined mean potency value of 15.86 mg/ml was obtained for sample C (n=22; GCV=38.9%). Furthermore, there was a significant difference between Clauss assays and CLOTr methods, with the Clauss assay giving 18% (p<0.00002) & 81% (p<0.001) higher potencies for samples B and C respectively, compared to the CLOTr method. For Total Protein estimates, there was good agreement in potencies between laboratories for both candidates, with a GCV value of 4.4% and a mean potency of 15.04 mg/ml (n=9) for sample B and a GCV value of 7.7% and a mean potency of 12.10 mg/ml (n=10) for sample C. Proposal Overall, it is proposed that sample B is the favoured candidate for the following reasons:

- Better agreement in mean values obtained for Clottable Protein (Clauss assay and CLOTr method) and for Total Protein content for sample B compared to sample C.

- Lowest overall inter-laboratory variability for combined estimates (GCV=16.7% Clottable Protein; GCV=4.4% Total Protein) for sample B compared to sample C (GCV=38.9% Clottable Protein; GCV=7.7% Total Protein).

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However, due to the large discrepancy observed between the methods for Clottable Protein estimates, it would mean that combination of method potencies for value assignment for Clottable protein would cause a large discontinuity in the International Unit, which is unacceptable, particularly in relation to product labeling and testing by regulatory authorities. Furthermore, the high variability observed when using the Clauss assay for sample B (GCV=17.2%) compared to the CLOTr method (GCV=3.6%) makes the data from Clauss assay unsuitable for value assignment to the proposed 2nd IS. It is therefore proposed that candidate B (NIBSC code 09/242) be accepted as the WHO 2nd International Standard for Fibrinogen Concentrate with a Clottable Protein potency of 10.9 mg/ampoule (based on the CLOTr method) and a Total Protein potency of 15.0 mg/ampoule.

Introduction The current World Health Organization (WHO) 1st International Standard (IS) for Fibrinogen Concentrate (98/614) (Sands et al, 2000) is used in potency estimation of fibrinogen in plasma-derived therapeutic concentrates, which are primarily used in the treatment of bleeding in patients with congenital or acquired fibrinogen deficiencies (Fenger-Eriksen et al, 2008; Farriols Danés et al, 2008), or are components of Fibrin Sealants routinely used as haemostatic agents (Spotnitz et al, 2008; Achneck et al, 2010). Stocks of this standard are running low and are expected to be exhausted by end of 2012. The National Institute for Biological Standards and Control, NIBSC (a WHO Collaborating Centre) has undertaken a project to replace this standard. Following trial-fill studies on a number of therapeutic fibrinogen concentrates, two candidate materials were selected based on fill characteristics and stability of fibrinogen potency. Ten thousand (10,000) ampoules of each of the 2 candidate materials have been prepared. This project was reviewed and endorsed by the WHO Expert Committee on Biological Standardisation (ECBS) in October 2009 and by the ISTH/SSC Sub-Committee on Factor XIII and Fibrinogen at the 55th SSC meeting of the ISTH in July 2009 (Boston). Objectives of the study To assign values for Clottable Protein and Total protein to the replacement for the WHO 1st IS for Fibrinogen concentrate.

Participants Twenty seven (27) laboratories (from 12 countries) participated in the value assignment exercise and returned data for analysis. They are listed in Appendix I. The participants included 14 manufacturers, 11 academic institutes and 2 national control authorities. Laboratories were coded for the study and the order of listing in Appendix I does not necessarily correspond with the numerical codes. All raw data returned by the participants were analysed at NIBSC.

Materials Proposed 2nd IS Candidate, Sample B (09/242): the raw material for this candidate was a plasma-derived fibrinogen concentrate, prepared using precipitation and chromatography methods. All donations used to prepare this product were tested and found negative for HBsAg, anti-HIV-1 and -2, anti-HCV, HCV-RNA (plasma pools). Manufacturing of this product also included 2 viral inactivation steps, solvent detergent treatment and heat treatment at 80°C for 72

WHO/BS/2012.2208 hours. After reconstitution of the product, the concentrate material was pooled and formulated in the following buffer: 40mM Tri-Sodium Citrate.2H2O, 20mM Tris-HCl, 3% sucrose (w/v), 4mM L-arginine-HCl (pH 7.3). The formulated material was filled and freeze-dried in sealed glass ampoules at NIBSC, under conditions required for International Standards (Campbell, 1974). One ml of this material was dispensed into each of approximately 10,000 ampoules. The mean filling weight was 1.0093 g (range 1.0015 g to 1.0135 g) and the coefficient of variation (CV) was 0.27% based on 468 check-weight samples. Mean residual moisture after freeze-drying was 0.39% (CV 19.3%, n=12) and mean oxygen headspace was 0.11% (CV 55.2%, n=12). Secondary Candidate, Sample C (10/100): the raw material for this candidate was a plasma-derived fibrinogen concentrate bulk. All donations used to prepare this product were tested and found negative for HBsAg, anti-HIV-1 and -2, anti-HCV, HCV-RNA (plasma pools). The bulk concentrate powder was dissolved in the following formulation: 90mM NaCl, 20mM Tri-Sodium Citrate.2H2O, 67mM Glycine, 83mM Glucose (pH 7.0). The formulated material was filled and freeze-dried in sealed glass ampoules at NIBSC, under conditions required for International Standards (Campbell, 1974). One ml of this material was dispensed into each of approximately 10,000 ampoules. The mean filling weight was 1.0083 g (range 1.0005 g to 1.0145 g) and the coefficient of variation (CV) was 0.20% based on 489 check-weight samples. Mean residual moisture after freeze-drying was 1.22% (CV 18.1%, n=6) and mean oxygen headspace was 0.38% (CV 32.1%, n=12). Sample 1st IS - WHO 1st IS for Fibrinogen Concentrate (98/614): This primary standard was supplied by NIBSC and has the following assigned potencies: Clottable Protein: 10.4 mg/ampoule Total Protein: 15.0 mg/ampoule Materials dispatched for study Ten ampoules of each of the above materials were dispatched by NIBSC. Participants were asked not to use any additional test materials in the assays, such as any internal (in-house) standards.

Assay Methods and Study Design Participants were requested to carry out 4 independent assays for each analyte/potency parameter (i.e. 4 assays for Clottable Protein determination and 4 assays for Total Protein determination), using their normal routine methodologies, e.g. Clauss assay or clot removal method (CLOTr - where subsequent protein determination assay methods were carried out following clot removal e.g. Kjeldahl N2, Biuret, absorbance assays) for Clottable Protein determination or protein assays for Total Protein determination e.g. colorimetric, absorbance or Kjeldahl N2 assays), preferably assays by more than one method. They were requested to carry out assays relative to the current WHO 1st IS Fibrinogen Concentrate (98/614), on separate days using fresh ampoules of samples B, C and 1st IS in each assay, and according to a balanced assay design recommended in the study protocol (Appendix II) for those laboratories carrying out Clauss assays for Clottable Protein determination. A separate ampoule of each material was provided for each assay. Laboratories that used more than one method were requested to use material from the same ampoules for each method, provided this could be done within 2 hours of reconstitution. Instructions and assay design were provided in the study protocol (Appendix II). The details of the methods, instruments and reagents used by the participants are listed in Appendix III.

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Statistical Analysis All assays were analysed as multiple parallel line bioassays comparing log response to log concentration (Finney, 1978). All assay data were plotted, and in some cases individual data points were excluded from extreme ends of the dose-response curve to improve the fit of the parallel line model to the data. The statistical validity of the assays was assessed by the usual ANOVA tests for parallelism and linearity and by visual inspection of the plotted dose-response curves. For each assay the potency estimates of samples B & C were calculated relative to the concurrently tested 1st IS Fibrinogen Concentrate (98/614). Combined potency estimates for each laboratory were obtained by taking unweighted geometric means of results from all assays. Overall combined estimates were obtained by taking unweighted geometric means of the mean results from the different laboratories. Where a laboratory performed more than one assay method, the results for each method were analysed as if from separate laboratories. Intra- and inter-laboratory variability is expressed as the percentage geometric coefficient of variation (%GCV) (Kirkwood, 1979). The mean potency estimates calculated by the participating laboratories are also presented (Appendix IV). Grubbs test (Grubbs, 1969) was used to assess any outlying laboratory mean potency estimates. For the laboratories measuring clottable protein, differences in potency estimates between assay methods (Clauss assay or CLOTr methods) were assessed by 2-sample student’s t-tests (Altman, 1991) (where the distribution of potency estimates appeared to be normally distributed) or by Mann-Whitney tests (Altman, 1991).

Results Data received Clottable Protein: Results were received from 26 laboratories and comprised a total of 34 data sets for sample B (25 data sets from Main Study; 10 data sets from Field Study) & 22 data sets (Main Study) for sample C. Laboratories used either a Clauss assay (24 data sets for sample B; 17 data sets for sample C), or a CLOTr method (10 data sets for sample B; 5 data sets for sample C). In addition 2 laboratories submitted data from ELISAs (2 data sets) for samples B and C. Several laboratories performed more than one assay method (laboratories 3, 10, 14, 20 & 26) or more than one variation of the same method (laboratories 3, 7, & 18). Laboratories 7 and 18 each performed two different Clauss assays (see Appendix III), with different kits and different thrombin concentrations respectively. They were analysed separately and coded: 7A (Multifibrin U), 7B (rFibrinogen in-house assay); 18A (STA-Fib 2) and 18B (STA-Fibrinogen). Laboratory 3 performed two different versions of the CLOTr method (see Appendix III). Total Protein: Data were received from 9 laboratories with a total of 10 datasets for both candidates (see Appendix III). Laboratories used protein assays such as colorimetric assays e.g. Bicinchonic Acid (BCA) protein assay (1 data set), Biuret assay (4 data sets), BioRad protein assay (1 data set), N2 determination - Kjeldahl assay (2 data sets) or absorbance assays (2 data

WHO/BS/2012.2208 sets). Laboratory 26 provided two different methods and were analysed separately as 26A (Kjeldahl) and 26B (Biuret). Assay Validity All assays were found to be valid for linearity and parallelism except the exclusions detailed below. For laboratory 9, clotting times appeared to be very low for all dilutions and so the data were excluded. Data for laboratory 19 showed a lack of dose-response and the data were excluded from subsequent analyses. Some data from individual samples were also excluded. Sample B was excluded from laboratory 7B and sample C from laboratory 23 due to non-parallelism. Sample C was also excluded from laboratory 20A (clottable protein) due to a lack of dose-response. Clottable Protein Estimates Relative to the 1st IS Fibrinogen Concentrate (98/614) The laboratory geometric mean estimates of potency, the intra-laboratory %GCVs, together with the overall geometric mean and inter-laboratory %GCVs, relative to the current 1st IS, for the Clauss assays, CLOTr methods & ELISAs are shown in Table 1. The table also shows the combined overall means and the inter-laboratory %GCVs. Data from ELISAs, although analysed in the study, were not included in the combined overall mean values as they do not represent fibrinogen function. The potency estimates are also shown in the form of stacking histograms (Figures 1-4). Each box represents the laboratory mean, expressed as a percentage of the overall geometric mean (excluding ELISA data) calculated from results from Clauss assays and CLOTr methods combined. The boxes are labelled with the laboratory code number. The potency estimates calculated by the participating laboratories are shown in Appendix IV. Proposed 2nd IS, Sample B: Estimates of Clottable Protein for the proposed WHO 2nd IS, sample B (relative to the 1st IS) are shown in Table 1. From the histogram (Figure 1) and Table 1, it can be seen that, there is poor agreement between assay methods. Combination of estimates from all assays (CLOTr & Clauss) gave an inter-laboratory GCV of 16.7%, with an overall combined geometric mean potency of 12.23 mg/ml (n=34) (excluding ELISA data). The Clauss assays gave higher results compared to the CLOTr methods, with the overall means being 12.85 mg/ml (GCV=17.2%; n=24) and 10.86 mg/ml (GCV=3.6%; n=10), for Clauss assays and CLOTr methods, respectively. This difference in potency (by 18%) between Clauss assays and CLOTr methods was found to be statistically significant (p=0.00002). The geometric mean potency for the ELISA data was 12.16 mg/ml (GCV=15.1%; n=2). No individual laboratories were found to be outliers. Intra-laboratory GCVs ranged from 0.7-10.0% (n=24) for the Clauss assays and 0.8-11.0% (n=10) for the CLOTr methods and from 1.05-18.5% (n=2) for the ELISAs. Sample C: From the histogram (Figure 2) and Table 1, it can be seen that for sample C, when potency is expressed relative to the current 1st IS, there is even poorer agreement between assay methods. Furthermore, when potency is expressed relative to the current 1st IS, agreement between laboratories for sample C is poorer than for sample B. The overall inter-laboratory

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GCV is 38.9% with a combined geometric mean potency of 15.86 mg/ml (n=22) (excluding ELISA data). Once again Clauss assays gave higher potencies compared to CLOTr methods with geometric mean potencies of 18.15 mg/ml (GCV=26.8%; n=17) and 10.04 mg/ml (GCV=6.0%; n=5) for Clauss assays and CLOTr methods, respectively. This difference in potency (by 81%) between Clauss assays and CLOTr methods was found to be statistically significant (p=0.001). The geometric mean potency for the ELISA data was 10.62 mg/ml (GCV=12.7%; n=2). No individual laboratory was found to be an outlier. Intra-laboratory GCVs ranged from 0.7-11.6% (n=17) for the Clauss assays, 2.4-11.3% (n=5) for the CLOTr methods and from 4.04-13.8% (n=2) for the ELISAs. Clottable Protein Potency Estimates for the proposed WHO 2nd IS (Sample B) using Absolute methods (Kjeldahl and Weighing methods) Three laboratories also carried out absolute measurements [Kjeldahl and Weighing (Ingram, 1952) methods] for Clottable Protein (Table 2). Mean individual laboratory estimates for proposed 2nd IS (sample B) of 10.34 mg/ml (intra-laboratory GCV=1.2%) and 11.35 mg/ml (intra-laboratory GCV=0.3%) were obtained from Laboratories 15 and 30 respectively for the Kjeldahl method and estimate of 11.22 mg/ml (intra-laboratory GCV=2.9%) obtained from Laboratory 26 for the Weighing method (Ingram, 1952), giving an overall mean estimate of 10.96 mg/ml (inter-laboratory GCV=5.9%; n=3). Total Protein Estimates Relative to the 1st IS Fibrinogen Concentrate The laboratory geometric mean estimates of potency, relative to the current 1st IS, the intra-laboratory %GCVs, the overall geometric mean and inter-laboratory %GCVs, are shown in Table 3. The potency estimates are also shown in the form of stacking histograms (Figures 3-4). Each box represents the laboratory mean, expressed as a percentage of the overall geometric mean. The potency estimates calculated by the participating laboratories are shown in Appendix IV. Proposed 2nd IS, Sample B: From the histogram (Figure 3) and Table 3, it can be seen that there is good agreement between laboratories for Total Protein determination, except for the result from laboratory 26A, which was found to be an outlier using the Grubbs test. The overall inter-laboratory GCV is 4.4% with an overall geometric mean potency of 15.04 mg/ml (excluding outlier laboratory 26; n=9). Intra-laboratory GCVs ranged from 0.8 - 7.0% (n=9) for sample B. Sample C: From the histogram (Figure 4) and Table 3, it can be seen that, when potency is expressed relative to the current 1st IS, there is slightly poorer agreement between laboratories (although still good) for sample C than for sample B. The overall inter-laboratory GCV is 7.7% and the overall geometric mean potency is 12.10 mg/ml (n=10). No individual laboratory was found to be an outlier. Intra-laboratory GCVs ranged from 1.2 - 7.6% (n=10) for sample C. Stability Studies

Accelerated degradation study Investigations to assess the long-term stability of the candidate preparations (samples B & C) were carried out through accelerated degradation studies which allow the prediction of

WHO/BS/2012.2208 degradation rates for ampoules stored at low temperatures (e.g. -20°C) based on the observed loss in samples stored at elevated temperatures (e.g. +4, +20, +37, +45 °C) (Kirkwood and Tydeman, 1984). This is an indirect method used to determine rate of loss based on the relationship between reaction rates and temperature given by the Arrhenius equation and where a first order reaction rate is assumed (Kirkwood, 1977). Ampoules of the candidate preparations (samples B & C) were placed into elevated temperature storage in May 2010. They were assayed concurrently with samples stored at the usual storage temperature of -20°C. Three laboratories assayed samples that had been stored for 12 months. Each laboratory carried out 3 repeat assays for both Clottable Protein and Total Protein. Results of all assays from individual laboratories were combined, to give the figures shown in Tables 4 & 5, for Clottable Protein and Total Protein respectively, where the residual potencies of samples stored at elevated temperature are expressed as a % relative to the -20°C sample. Clottable Protein: There was no observed drop in potency when sample C was stored for 12 months at +4°C & +20°C relative to the baseline -20°C sample. The Arrhenius model for accelerated degradation was applied to obtain predictions of the expected loss in potency over time at different temperatures. Results in Table 4 show that the sample C is stable for long periods at -20°C with a mean predicted % loss per year of 0.001. It also appears to be stable for shorter periods at higher temperatures with a predicted % loss per month of 0.005, 0.09 and 1.3 when stored at +4°C, +20°C and +37°C respectively. For candidate sample B, there was no observed loss in potency after storage for 12 months at any of the higher temperatures relative to the baseline -20°C sample. It was therefore not possible to obtain a predicted % loss per year from the data, although the observed potencies show that the preparation is stable for long term storage at -20°C and short periods at +20°C and +37°C. Furthermore, both samples B & C stored at -20°C, +20°C, +37°C and +45°C were also assessed after a 10 month period at NIBSC, using the CLOTr method, where results were obtained as direct OD ratios of the higher temperatures relative to -20°C sample (see Table 4). Mean potency values (relative to the -20°C sample) of 97%, 100% & 101% for sample B and of 100%, 100% & 106% for sample C, were obtained when stored at +20°C, +37°C and +45°C respectively. Total Protein: For candidate samples B & C the residual relative potencies for samples stored at elevated temperatures for 10 months or 12 months differed by no more than 5% from samples stored at -20°C. There was insufficient degradation to allow calculations of predicted loss (see Table 5). Overall, these figures represent very good stability particularly for the candidate sample B, and indicate that sample B would be suitable to serve as a WHO International Standard. However, the accelerated degradation studies should be continued and the stability confirmed with further assessments after 3 and 5 years storage. Furthermore, as with all WHO International Standards, the proposed IS will undergo real-time stability monitoring throughout the life time of the standard. Stability of proposed 2nd IS (sample B - 09/242) after reconstitution Although the Instructions for Use will recommend that assays are performed as soon as possible after reconstitution it is useful to indicate a suitable period of use. In common with previous WHO Concentrate Standards for blood coagulation factors it is recommended that the standard is transferred, after reconstitution, to a plastic tube. Recommendations for the storage after reconstitution have been limited to the period of storage on melting ice since local ambient

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temperature can vary considerably. The mean Clottable Protein results from three separate tests performed at NIBSC, indicated that 96%, 94% and 104% of the starting potency of Fibrinogen was retained for the freshly reconstituted proposed standard when stored on melting ice in plastic tubes, for 2, 4 and 6 hours, respectively. Based on this, a conservative estimate of a 3 hour maximum storage period is recommended, which is sufficient for numerous assays to be performed. The use of frozen aliquots of the proposed 2nd IS for the estimation of Fibrinogen potency is not recommended. Effect of Proposed & Alternative Assigned Values on the Clottable Protein Potency Estimates for Fibrinogen Concentrate Products In order to assess the impact on product potency if value assignment of the WHO 2nd IS was based on combined data from both Clauss assays and CLOTr methods, a field study involving 8 laboratories (consisting of manufacturers and regulators) was carried out where actual products were assayed for Clottable Protein estimates relative to the proposed 2nd IS (sample B – 09/242) and compared to assays relative to the current 1st IS (98/614), see Appendix V. Results from assays on different Fibrinogen Concentrate products are summarised in Table 6, where data obtained tended to give 2-9% higher potencies for Clauss Assays when assayed relative to the current 1st IS compared to assays relative to the proposed 2nd IS sample B (with an assigned potency based on Combined Method Mean (12.1 mg/ml). For the CLOTr method, between 8-14% lower potencies were obtained for assays relative to the current 1st IS than when compared to assays relative to the proposed 2nd IS (with an assigned potency based on Combined Method Mean), see Table 6. This represented between 10-23% shift in potency. However, minimal discrepancy (i.e. minimal shift in potency) was obtained for CLOTr method when assayed relative to the proposed 2nd IS (sample B) with a proposed assigned value of 10.9 mg/ml (based on CLOTr potency) whilst a greater shift in potency is observed with the Clauss Assay (see Table 6).

Discussion The current WHO 1st IS for Fibrinogen Concentrate (98/614) was established by the WHO/ECBS (Expert Committee on Biological Standardization) in November 2000 with an assigned Clottable Protein potency of 10.4 mg/ampoule and assigned Total Protein potency of 15.0 mg/ampoule (Sands et al, 2000). Stocks of this standard are running low and are expected to be depleted by end of 2012. As such, international collaborative studies were undertaken by NIBSC to calibrate the replacement of the above standard. The WHO 1st IS (98/614) was previously calibrated for Clottable Protein potency using predominantly CLOTr methods (although smaller number of data sets from Clauss assays were also included), against laboratories’ own in-house fibrinogen standards and following their own routine methodologies (Sands et al, 2000). For the sake of harmonisation and continuity of the unitage, assays on replacement candidate materials in this study were similarly carried out using methods including Clauss assays as well as CLOTr methods but against the current WHO 1st IS for Fibrinogen Concentrate (98/614) (sample 1st IS). However, data from ELISAs, which measure protein concentration rather than fibrinogen function, were excluded from the overall combined geometric mean values for the Clottable Protein parameter. The Total Protein content for the WHO 1st IS (98/614) was previously calibrated using a variety of methods (Sands et al, 2000) e.g. optical density/spectrophotometery measurements, protein N2

WHO/BS/2012.2208 determinations and dye binding assays. The Total Protein content for the replacement candidates was similarly determined. This report presents data from assays of 2 fibrinogen candidate preparations, (samples B and C) with the emphasis primarily on value assignment to the replacement for the WHO 1st IS for Fibrinogen Concentrate (98/614). Intra-laboratory variability The variability of assays within laboratories differed considerably for both candidates, with GCV’s ranging from 0.7-11.6% for Clottable Protein and from 0.8 - 7.6 % for Total Protein, with intra-laboratory GCVs from the majority of the labs below 8% (Tables 1 & 3). There was no obvious trend for one method to give better or worse inter-assay variability; lower intra-laboratory GCV’s were associated with particular laboratories rather than particular methods, indicating that internal quality control procedures within laboratories are probably the most important factor in determining reproducibility of fibrinogen (Clottable & Total Protein) assays, rather than the method used. Inter-laboratory variability Inter-laboratory variability for Clottable and Total Protein estimates for both samples by the different methods is given in Tables 1 & 3. Clottable Protein: Clauss assays vs CLOTr methods For Clottable Protein estimates relative to the 1st IS, there was very good agreement between laboratories (for both candidate materials) for the CLOTr methods with GCV values of 3.6% and 6.0% for samples B & C respectively. There was much poorer agreement between laboratories for the Clauss assays with higher GCV values of 17.2% and 26.8% for samples B & C respectively. This resulted in high overall GCV values of 16.7% and 38.9% for samples B & C respectively (see Table 1). These results indicate that Clauss assays are highly variable when measuring fibrinogen concentrates and caution should be exercised when doing so, in particular when assigning Clottable Protein potencies for lot release for fibrinogen concentrate products, for which the Clauss assay should at least be validated against a CLOTr method. Total Protein For Total Protein estimates relative to the 1st IS, there was very good agreement between laboratories for both candidate samples, with overall method GCVs of 4.4% and 7.7% for candidate samples B and C respectively (see Table 3). Potency estimates of candidate materials Mean potency estimates by the two methods (Clottable Protein & Total Protein) for the 2 candidate samples relative to the current WHO 1st IS for Fibrinogen Concentrate (98/614) are given in Tables 1 & 3. For Total Protein, overall mean potencies of 15.04 mg/ampoule and 12.10 mg/ampoule were obtained for samples B and C respectively.

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For Clottable Protein, when assayed relative to the the current WHO 1st IS for Fibrinogen Concentrate (98/614), the Clauss assay gave a significantly higher potency compared to the CLOTr methods, by 18% and 81%, for samples B & C respectively. In the study to calibrate the current WHO 1st IS for Fibrinogen Concentrate (98/614) (Sands et al, 2000), there was no significant method discrepancy between Clauss assay and the CLOTr (clot removal) methods. This may be due to the fact that Clauss assays represented only 5 data sets (25% of total data sets) for the overall combined estimate in the calibration of the current WHO 1st IS for Fibrinogen Concentrate (98/614) (Sands et al, 2000), whereas in this study Clauss assays represent 24 data sets (71% of total data sets) for sample B and 17 datasets (71% of total data sets) for sample C. The higher variability, observed for the Clauss assays in this study, has therefore had a significant influence on the overall GCV values for samples B & C. It was also interesting to note that for sample C questionable results were obtained by Clauss assay with a mean Clauss potency of 18.15 mg/ml (Table 1). This value was actually greater than the mean Total Protein content of 12.1 mg/ml for sample C (Table 3), whereas the mean CLOTr value for sample C (10.04 mg/ml) (Table 1) was in the expected range. Overall, taking all the data into consideration, it is proposed that sample B is the favoured candidate over sample C, for the following reasons:

- Better agreement in mean (and combined) values obtained for Clottable Protein (Clauss assay and CLOTr method) and for Total Protein content for sample B compared to sample C.

- Lowest overall inter-laboratory variability for combined estimates (GCV=16.7%

Clottable Protein; GCV=4.4% Total Protein) for sample B compared to sample C (GCV=38.9% Clottable Protein; GCV=7.7% Total Protein).

- Lower discrepancy between Clauss Assay and the CLOTr method for the Clottable

Protein determination for sample B compared to sample C.

- Lower mean residual moisture after freeze-drying for sample B (0.39%) compared to sample C (1.22%) indicating the possibility of a more stable candidate long-term.

However, the large discrepancy between CLOTr method and Clauss assay would mean that combination of method potencies for value assignment for Clottable protein would cause a large discontinuity in the International Unit, particularly in relation to product labeling and testing by manufacturers and national control laboratories. Case for Assigning Clottable Protein Potency Using the CLOTr method In the interest of continuity it was proposed that the candidate sample B be assigned the consensus mean value from the CLOTr methods for the following reasons:

• The value assigned to the WHO 2nd IS should reflect the methods used by manufacturers & national control laboratories, primarily the CLOTr methods.

• Fibrinogen Concentrate product field study shows minimal shift in potency when assayed relative to the proposed 2nd IS with a proposed assigned value based on CLOTr method.

• Data from laboratories using CLOTr method is less variable (GCV < 4%) compared to data from laboratories using Clauss Assay (GCV~17%).

WHO/BS/2012.2208

• CLOTr potency value (10.9 mg/ml) is supported by good agreement with additional data obtained from absolute measurements for Clottable Protein (Kjeldahl & Weighing methods: mean potency=10.96 mg/ml).

• Best continuity for product labelling by CLOTr method (the 1st IS was value assigned with data from CLOTr methods primarily).

Proposal: It is therefore proposed that candidate preparation sample B (NIBSC code 09/242) be accepted as the 2nd International Standard for Fibrinogen concentrate with assigned potencies of: Clottable Protein - 10.9 mg/ampoule Total Protein - 15.0 mg/ampoule

Comments from participants and the ISTH/SSC Factor XIII and Fibrinogen Subcommittee Responses were received from all the participants (27 laboratories), all of whom agreed that the candidate material Sample B (09/242) is suitable to serve as the WHO 2nd IS Fibrinogen Concentrate. 27/27 laboratories agreed with assignment for Total Protein potency of 15.0 mg/ampoule. 26/27 laboratories agreed with assignment for Clottable Protein potency of 10.9 mg/ampoule. There were the following comments with respect to the objection from one participant, together with response from NIBSC: Comments: We do not agree to change candidate B (09/242) value to 10.9 mg of fibrinogen obtained by using the CLOT method, since most laboratories use the von Clauss method to determine the Clottable protein potency. We consider that the method of the Eur. Ph. is difficult to perform in the laboratory and we don´t know if it is possible its automation. Do you know if it is possible to automate? In the first place, we should test CLOT method with our products and, in case to change it, to validate the method for them and check its consistence. Response from NIBSC: The laboratory concerned was informed that the variability for the proposed 2nd IS (sample B - 09/242) was exceptionally high using the Clauss assay (GCVs= 17.2%) compared to the CLOTr method (GCV< 4%) and together with the large discrepancy (~18%) in Clottable Protein potency between the two methods made value assignment to the proposed 2nd IS (09/242) based on a combined method mean unacceptable. In order to maintain continuity of the fibrinogen unit, value assignment for Clottable Protein potency was therefore proposed based on CLOTr method, the data from which was also supported by absolute methods such as Kjeldahl method. They were informed that the Clauss assays could still be used for labelling of products as long as it was validated against a CLOTr method. They were also reminded that any issues relating to the European Pharmacopoeia should be addressed to the EMA/EDQM. In addition, responses have also been sought from experts associated with the Factor XIII/Fibrinogen sub-committee of the ISTH/SSC. Responses from 8 experts in the field were received and all were in favour of the proposal.

WHO/BS/2012.2208

The proposal to accept candidate sample B (coded 09/242) as the WHO 2nd IS Fibrinogen Concentrate with assigned potencies of: Clottable Protein - 10.9 mg/ampoule Total Protein - 15.0 mg/ampoule was presented to the WHO-ISTH Standards Liaison Group and the Factor XIII and Fibrinogen Subcommittee at the ISTH/SSC meeting and the recommendation endorsed at the SSC Annual Business Meeting, held in Liverpool UK, on 30th June 2012.

References Achneck HE, Sileshi B, Jamiolkowski RM, Albala DM, Shapiro ML, Lawson JH (2010). A comprehensive review of topical hemostatic agents: efficacy and recommendations for use. Ann Surg 251(2);217. Altman DG (1991), Practical Statistics for Medical Research, 1st Edition, Chapman and Hall. London. Campbell PJ (1974). International biological standards and reference preparations. 1. Preparation and presentation of materials to serve as standards and reference preparations. J Biol Standardisation 2; 249-67. Farriols Danés A, Gallur Cuenca L, Rodríguez Bueno S, Mendarte Barrenechea L, Bruno Montoro Ronsano J (2008). Efficacy and tolerability of human fibrinogen concentrate administration to patients with acquired fibrinogen deficiency and active or in high-risk severe bleeding. Vox Sang 94; 221-6. Fenger-Eriksen C, Lindberg-Larsen M, Christensen AQ, Ingerslev J and Sørensen B (2008). Fibrinogen concentrate substitution therapy in patients with massive haemorrhage and low plasma fibrinogen concentrations. BJA 101; 769-73.

Finney DJ (1978). Statistical methods in biological assay. 3rd edition Charles Griffin. London.

Grubbs F (1969). Procedures for detecting outlying observations in samples. Technometrics 11; 1-21. Ingram GIC (1952). The determination of plasma fibrinogen by the clot method. Biochem J 51(5); 583-5.

Kirkwood, TBL (1977). Predicting the stability of biological standards and products. Biometrics 33 (4); 736-42. Kirkwood TBL (1979). Geometric means and measures of dispersion. Biometrics 35; 908-9.

Kirkwood TBL, Tydeman MS (1984). Design and analysis of accelerated degradation tests for the stability of biological standards II. A flexible computer program for data analysis. J Biol Standardisation 12; 207-14.

WHO/BS/2012.2208

Sands D, Whitton C, Barrowcliffe TW (2000). Collaborative Study to establish an international standard for fibrinogen concentrate using clottable and total protein methods. WHO/ECBS Report. WHO BS Document BS/00.1927.

Spotnitz WD, Burks S (2008). Hemostats, sealants, and adhesives: components of the surgical toolbox. Transfusion 48(7):1502.

Acknowledgements The contributions of all the participants in the study are gratefully acknowledged (Appendix I). We are grateful to our colleagues in the Standards Division (Paul Jefferson and his team in SPD and Paul Matejtschuk and his team at TDI) NIBSC, for ampouling and processing the candidate and trial preparations and for the dispatch of collaborative study samples to participants. We are grateful to Baxter Healthcare (Austria) and BPL (UK) for their kind donation of materials for the study. We further like to thank the ISTH/SSC Fibrinogen and Factor XIII Subcommittee (current Chair - Professor Hans-Peter Kohler; previous Chair - Professor Moniek de Maat) for their guidance.

WHO/BS/2012.2208

Table 1. Clottable Protein mean laboratory estimates for the proposed WHO 2nd IS (sample B) and sample C relative to WHO 1st IS Fibrinogen Concentrate (sample 1st IS - Clottable Protein: 10.4 mg/ml), together with estimates of intra-laboratory variability (GCV%) for individual laboratories and inter-laboratory variability for method estimates and combined estimates.

Lab No.

Proposed 2nd IS, Sample B

(09/242) [Data from Main & Field Studies]

Sample C (10/100)

[Data from Main Study] No of

Assays (n)

Geometric Mean

(mg/ml)

Intra-Lab

GCV%

No of Assays

(n)

Geometric Mean (mg/ml)

Intra-Lab

GCV%

Clauss Assay

Data from Main Study

1 4 13.48 3.5 4 16.99 1.6 2 4 12.68 1.5 4 16.30 1.5 6 4 11.98 1.9 4 15.71 2.3

7A 4 11.21 1.7 4 15.65 11.6 7B - - - 4 15.58 0.7 8 4 13.10 3.7 4 19.72 5.9

10A 4 10.31 3.5 4 - - 11 4 14.76 7.3 4 25.01 10.9 13 4 13.62 4.6 4 17.02 2.6

14A 4 14.96 5.2 4 24.18 7.5 17 4 12.62 4.5 4 16.53 3.7

18A 4 12.53 2.0 4 14.63 3.2 18B 4 12.17 0.7 4 15.52 1.2 20A 4 16.08 10.0 4 - - 21 4 14.54 3.2 4 25.90 4.7 22 4 15.44 8.0 4 28.12 5.1 23 4 15.62 9.1 4 - - 25 5 9.97 2.8 5 14.90 4.5

26A 4 12.13 6.5 4 12.59 3.3 27A 4 15.53 3.3 4 22.82 6.7

Data from Field Study

3B 4 12.38 1.2 - - - 26C 4 12.92 2.7 - - - 27B 4 13.13 2.7 - - - 28 2 8.11 6.0 - - - 31 4 12.70 9.7 - - -

Geometric Mean = 12.85 (n=24)

Inter-Lab GCV = 17.2%



CLOTr Method


3A 4 10.36 3.4 4 9.54 11.3 5A 4 11.13 11.0 4 9.74 3.1 10B 4 10.58 0.8 4 9.59 2.4 15 4 11.08 2.7 4 10.67 4.0

26B 4 11.62 6.7 4 10.71 7.1


3C 4 10.45 1.3 - - - 5B 4 11.13 3.4 - - -

10C 4 10.66 1.0 - - - 29 4 10.71 1.2 - - - 30 4 10.94 1.8 - - -





ELISA


14B 4 13.43 18.5 4 11.56 13.8 20B 4 11.01 1.05 4 9.76 4.04





Combined Geometric Mean = 12.23 (n=34)

Inter-Lab GCV = 16.7% (Excluding ELISA Data)

Combined Geometric Mean = 15.86 (n=22)

Inter-Lab GCV = 38.9% (Excluding ELISA Data)

WHO/BS/2012.2208 Table 2. Mean laboratory estimates for the proposed WHO 2nd IS (sample B) using absolute measurements (Kjeldahl method; Weighing method) for Clottable Protein, together with estimates of intra-laboratory variability (GCV%) for individual laboratories and inter-laboratory variability for overall mean estimates.

Lab No.


(09/242)

No of Assays

(n)

Geometric

Mean (mg/ml)

Intra-Lab

GCV%

Absolute Method

15 4 10.34 1.2 Kjeldahl 26 4 11.22 2.9 Weight 30 4 11.35 0.3 Kjeldahl

Overall Geometric Mean =10.96 (n=3)


Table 3. Total Protein mean laboratory estimates for the proposed WHO 2nd IS (sample B) and sample C relative to WHO 1st IS Fibrinogen Concentrate (sample 1st IS - assigned Total Protein potency: 15.0 mg/ml), together with estimates of intra-laboratory variability (GCV%) for individual laboratories and inter-laboratory variability for overall mean estimates.

Lab No.


(09/242)

Sample C (10/100)

No of Assays

(n)

Geometric Mean

(mg/ml)

Intra-Lab

GCV%

No of Assays

(n)

Geometric Mean (mg/ml)

Intra-Lab

GCV% 1 4 14.79 2.8 4 11.50 2.9 3 4 15.43 3.1 4 13.91 7.6 5 4 13.82 7.0 4 11.69 1.3 10 4 14.72 0.9 4 11.10 2.1 15 4 14.66 0.8 4 11.38 1.7 20 4 15.99 5.4 4 12.65 4.6 25 4 15.22 1.3 4 12.08 1.3

26A 4 29.64 2.4 4 13.39 1.2 26B 4 15.09 3.2 4 12.19 2.7 27 4 15.71 4.6 4 11.46 5.1

Overall Geometric Mean =15.04 (n=9)

Inter-Lab GCV = 4.4% (Excluding outlier Lab 26A)

Overall Geometric Mean = 12.10 (n=10)


WHO/BS/2012.2208

Table 4. Clottable Protein results from the accelerated degradation study on the candidate materials samples B & C for the proposed WHO 2nd IS for fibrinogen concentrate. Candidate Materials

Lab ID

Mean residual potencies after storage

(% vs -20°C ampoules) and [95% confidence limits]

§Mean predicted % loss

per year at -20°C

+4°C

+20°C

+37°C

+45°C

Sample B (09/242)

A†

B†

C†

----------

B*

101

[98.9 - 102.4]

101 [98.7 - 102.4]

99

[94.7 - 104.3]

----------

nt

99

[97.5 - 100.9]

103 [100.9 - 104.6]

101

[100.0 - 101.6]

----------

97 [83.9 - 110.1]

101

[99.4 - 102.9]

103 [100.7 - 104.4]

101

[97.6 - 104.0]

----------

100 [94.1 - 106.6]

103

[101.3 - 104.9]

106 [104.2 - 108.0]

101

[101.1 - 102.7]

----------

101 [93.1 - 108.3]

No observed loss in potency after storage

up to 12 months

--------------------------- -

Sample C (10/100)

A†

B†

C†

----------

B*

97

[87.6 - 106.5]

98 [96.5 - 100.4]

101

[99.1 - 102.0]

----------

nt

97

[94.9 - 99.6]

96 [93.9 - 97.6]

99

[97.2 - 100.1]

----------

100 [87.3 – 112.7]

87

[84.4 - 88.7]

86 [84.2 - 97.6]

90

[83.1 - 98.0]

----------

100 [80.4 – 118.6]

62

[59.9 - 64.3]

60 [58.3 - 60.9]

-

----------

106 [106.0 – 106.0]

0.001%

--------------------------- -

† tested after storage for 12 months - results are the mean values from 3 independent Clauss assays; * tested after storage for 10 months - results are the mean values from 3 independent CLOTr method; § results are based on limited data and further stability studies are ongoing. nt: not tested

WHO/BS/2012.2208 Table 5. Total Protein results from the accelerated degradation study on the candidate materials samples B & C for the proposed WHO 2nd IS for fibrinogen concentrate. Candidate Materials

Lab ID

Mean residual potencies after storage

(% vs -20°C ampoules) and [95% confidence limits]

§Mean predicted % loss per year at -

20°C

+4°C

+20°C

+37°C

+45°C

Sample B (09/242)

B†

C†

----------

B*

100

[96.8 - 102.5]

104 [100.7 - 107.3]

----------

nt

100

[97.5 - 102.5]

103 [100.1 - 105.9]

----------

97

[84.3 - 109.7]

100

[100.0 - 100.0]

105 [102.6 - 107.4]

----------

100

[80.4 - 118.6]

100

[98.9 - 101.8]

101 [98.4 - 103.6]

----------

102

[51.2 - 152.8]

No observed loss in potency after

storage up to 12 months

------------------------

-

Sample C (10/100)

B†

C†

----------

B*

100

[98.9 - 101.8]

97 [96.6 - 97.4]

----------

nt

99

[99.0 - 99.0]

98 [96.5 - 99.5]

----------

98

[91.5 - 103.9]

101

[98.5 - 103.5]

98 [96.4 - 99.6]

----------

101

[88.3 - 113.7]

113

[106.6 - 118.1]

99 [98.0 - 100.0]

----------

112

[79.7 - 143.3]

No observed loss in potency after

storage up to 12 months

------------------------

-

† tested after storage for 12 months - results are the mean values from 3 independent assays * tested after storage for 10 months - results are the mean values from 3 independent assays §Results are based on limited data and further stability studies are ongoing. nt: not tested

WHO/BS/2012.2208

Table 6. Summary table showing the % shift (change) in potency of Fibrinogen Concentrate Products. Comparison of Clottable Protein potencies relative to the current 1st IS (98/614 -assigned potency 10.4 mg/ml) and the proposed 2nd IS, sample B (09/242 - with various assigned potency options).

Clauss Assay

(4 Laboratories)14‐21% 2‐9% 2‐4%

CLOTr Method

(4 Laboratories)2‐4% 8‐14% 17‐19%

% Change in Clottable Protein Potency of Fibrinogen Products when

comparing the current WHO 1st IS vs proposed WHO 2nd IS

Proposed 2nd IS

assigned value based on

CLOTr Method:

10.9 mg/ml

Alternative 2nd IS


Combined Method Mean:

12.1 mg/ml

Alternative 2nd IS


Clauss Assay:

12.9 mg/ml

WHO/BS/2012.2208 Figure 1. Stacking histogram showing Clottable Protein mean laboratory potency estimates (as a % of Overall Mean) for the proposed 2nd IS sample B relative to the 1st IS Fibrinogen Concentrate (98/614). Each box has a laboratory code number and represents the laboratory mean, expressed as a percentage of the overall combined geometric mean. Results are from Clauss Assays, CLOTr methods and ELISAs.

Nu

mb

er

of L

ab

ora

torie

s

0

2

4

6

8

10

Potencies of Sample B vs 1st IS (98/614) as % of Overall Potency

50 75 100 125 150

28 25 10A

10B

10C

3A

3C

15

20B

29

30

5A

5B

7A

26B 18A

18B

26A

3B

6

17

2

26C

27B

31

8

1

13

14B

11

14A

21

22

27A

20A

23

Clauss Assay

CLOTr

ELISA

WHO/BS/2012.2208

Figure 2. Stacking histogram showing Clottable Protein mean laboratory potency estimates (as a % of Overall Mean) for sample C relative to the 1st IS Fibrinogen Concentrate (98/614). Each box has a laboratory code number and represents the laboratory mean, expressed as a percentage of the overall combined geometric mean. Results are from Clauss Assays, CLOTr methods and ELISAs.

Clauss Assay

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Potencies of Sample C vs 1st IS (98/614) as % of Overall Mean

50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

10B

20B

3

5

14B

15

26B

26A 18A

25

17

18B

2

6

7A

7B

1

13

8 27 14A 11

21

22

Num

ber o

f Lab

orat

orie

s

WHO/BS/2012.2208 Figure 3. Stacking histogram showing Total Protein mean laboratory potency estimates (as a % of Overall Mean) for the proposed 2nd IS sample B relative to the 1st IS. Each box has a laboratory code number and represents the laboratory mean, expressed as a percentage of the overall geometric mean.

Fibrinogen Concentrate Total Protein

Potencies of Sample B relative to 98/614 as % of overall mean (15.04)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Potencies of Sample B v 98/614

50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200 210

5 1

10

15

25

26B

27

3

20 26A

WHO/BS/2012.2208

Figure 4. Stacking histogram showing Total Protein mean laboratory potency estimates (as a % of Overall Mean) for sample C relative to the 1st IS. Each box has a laboratory code number and represents the laboratory mean, expressed as a percentage of the overall geometric mean.

Fibrinogen Concentrate Total Protein

Potencies of Sample C relative to 98/614 as % of overall mean (12.10)

0

1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

Potencies of Sample C v 98/614

50 60 70 80 90 100 110 120 130 140 150 160 170 180 190 200

10

15

27

1

20

25

26B

5

26A

3

WHO/BS/2012.2208

APPENDIX I PARTICIPATING LABORATORIES Robert Ariëns, Daniel Whalley, LIGHT Laboratories, Leeds University, Leeds, UK.

Rossella Bader, Bianchi Bonomi Hemophilia and Thrombosis Center, University of Milan, Milan, ITALY.

Nathalie Barat, François Nicham, Diagnostica Stago R&D, Gennevilliers Cedex, FRANCE.

Chris Backhouse, Scott Pearson, Viv Sung, Lisa Evans, Shane Norton, Chemistry Section, Bio Products Laboratory,

Elstree, UK.

Mariona Bono, R&D Reagents, Haemostasis Department, Diagnostic Grifols, Barcelona, SPAIN.

Sossio Costanzo, Alessandro Starace, Chemical Laboratory and Biochemical Laboratory, Kedrion, S. Antimo (NA),

ITALY*.

Terrie Emson, Chris Charlesworth, Lincoln County Hospital, Lincoln, UK.

Denise Foulon, Affinity Biologicals Inc., Ancaster, CANADA.

Peter Gärtner, Baxter QC, Vienna, AUSTRIA.

Nuria Hosta, Marta Gensana, Instituto Grifols, Barcelona, SPAIN.*

Kevin Horner, Steve Kitchen, Royal Hallamshire Hospital, Sheffield, UK.

Andreas Hunfeld, Renate Nawrot, Paul-Ehrlich Institut, Langen, Germany*.

David Keeling, Kampta Sukhu, Oxford Haemophilia and Thrombosis Centre, Oxford, UK† .

Sarah Kingsland, Ruth Archer, Hector Bolsa, Bio Products Laboratory, Elstree, UK*† .

Steffen Kistner, Karin Fuchs, Biotest AG, Dreieich, GERMANY*.

Wolfgang Korte, Karin Jung, Institut für Klinische Chemie und Hämatologie, St. Gallen, SWITZERLAND.

Andrew Lawrie, Haemostasis Research Unit, University College London, London UK.

Andrea Lichte, Dieter Koch, Siemens Healthcare Diagnostics Products GmbH, Marburg, GERMANY.

Moniek P.M. de Maat, Erasmus University Medical Centre, Rotterdam, THE NETHERLANDS.

Joost C.M. Meijers, Academic Medical Center, University of Amsterdam, THE NETHERLANDS.

Catherine Michalski, Aline Laulan, Ingrid Zede, LFB Biomédicaments, Lille-Cedex, FRANCE*.

Sukesh C Nair, Christian Medical College Hospital, Vellore, INDIA.

Neri Philosof, Anda Granot, OMRIX, Kiryat Uno, ISRAEL*.

Marlien Pieters, North West University, Potchefstroom, SOUTH AFRICA.

Sanj Raut, Sarah Daniels, NIBSC, Hertfordshire, UK*† .

Pierre-Harald Schmidt, Spectroscopy & Elementary Analysis, CSL Behring, Marburg, GERMANY*.

Christelle Thouvenin, Stago, Laboratoire Etudes et Essais, Franconville, FRANCE.

* Laboratories that participated in the Field Study. † Laboratories that participated in a separate Stability Study.

WHO/BS/2012.2208

APPENDIX II

INTERNATIONAL COLLABORATIVE STUDY TO CALIBRATE THE WHO 2nd

INTERNATIONAL STANDARD FOR FIBRINOGEN CONCENTRATE:

VALUE ASSIGNMENT FOR ANALYTE: FIBRINOGEN

PROTOCOL & ASSAY INSTRUCTIONS

1. AIMS OF STUDY

To calibrate the proposed WHO 2nd International Standard (IS) for Fibrinogen Concentrate and

value assign for Fibrinogen Potency (Clottable and Total Protein).

2. MATERIALS

There are 3 test materials provided (10 ampoules of each):

CODE

1st IS WHO 1st IS Fibrinogen Concentrate (98/614), assigned potencies:

- Clottable Protein = 10.4 mg/ampoule - Total Protein = 15.0 mg/ampoule

B WHO IS Candidate Fibrinogen Concentrate B (09/242):

- Clottable Protein ~ 8-12 mg/ampoule - Total Protein ~ 10-15 mg/ampoule

C WHO IS Candidate Fibrinogen Concentrate C (10/100):

- Clottable Protein ~ 8-12 mg/ampoule - Total Protein ~ 10-15 mg/ampoule

3. RECONSTITUTION AND STORAGE

1. On receipt, store all samples (unopened ampoules) at -20oC.

2. On the day of assay, bring ampoules to room/ambient temperature.

3. Reconstitute at room/ambient temperature with 1.0 mL of distilled H2O or water for

injection (WFI) and allow contents to solubilise for 10-15 minutes at room/ambient

temperature.

4. Mix gently and thoroughly to ensure complete solubilisation and transfer entire contents of

ampoule to a plastic tube.

5. Keep samples at room temperature during assays.

WHO/BS/2012.2208 4. NUMBER OF ASSAYS AND AMPOULES

A minimum of four independent assays are requested for each method (i.e. 4 for Clottable

Protein and 4 for Total Protein) from each laboratory, preferably carried out over separate days

rather than all on the same day, in accordance with your routine methodology. Although 10

ampoules of each test material are provided, only 8 ampoules of each are needed for both methods.

A fresh separate ampoule of each material should be used for each potency assay (i.e. 4 ampoules

for Clottable Protein assays and 4 ampoules for Total Protein assays). Some laboratories may

carry out only the Clottable Protein assays or only the Total Protein assays, in which case there

will be 6 spare ampoules of each sample.

If you are using more than one Clottable Protein method or more than one Total Protein method,

material from the same ampoules should be used for each specific method, provided this can be

done within 2 hours of reconstitution. If this is impossible, please contact us. Alternatively, the

spare ampoules can be used for this purpose.

5. ASSAY METHODS

Laboratories are asked to use their routine assay method for the measurement of both Clottable

Protein (e.g. Clauss Assay) and Total Protein (e.g. Kjeldahl assay) concentrations, for those

laboratories that can perform both. Please indicate potency method used on the Result Sheets.

6. CLOTTABLE PROTEIN: CLAUSS ASSAYS - DESIGN AND ANALYSIS Assay Dilution of Samples

All working dilutions of each sample should be carried out using routine buffers. At least three

dilutions should be made. After making all assay dilutions, discard reconstituted material and make

up a fresh ampoule for the next assay.

Assays using Parallel Line Analysis

A balanced order of testing should be adopted wherever possible (see Assay Design below).

In the following assay design, each letter represents a fresh set of 3 or more different (e.g. 1/5, 1/10,

1/20) dilutions. Where a letter is repeated twice within an assay, a replicate fresh set of dilutions

from the same ampoule must be made. Remember to use a fresh set of ampoules for each assay.

Please perform assays using the following design only. If this is not possible or you would like

to use a different design please contact us.

WHO/BS/2012.2208

Assay Design for Clauss Assay

6 Samples per Assay (4 independent estimates for B and C in total per method) Assay No Order of Testing

_____________________________________________________________________ 1 1st IS Bamp 1 Camp 1 Camp 1 Bamp 1 1st IS

2 Camp 2 1st IS Bamp 2 Bamp 2 1st IS Camp 2 3 Bamp 3 Camp 3 1st IS 1st IS Camp 3 Bamp3

4 1st IS Bamp 4 Camp4 Camp 4 Bamp 4 1st IS _____________________________________________________________________ Key to Samples:

B - WHO IS Candidate (09/242); C - WHO IS Candidate (10/100); 1st IS - WHO 1st IS

Fibrinogen Concentrate (98/614).

Assays involving Interpolation to a Standard Curve

Sample 1st IS should be used to construct a standard curve preferably using at least two

independent sets of dilutions (replicates). Samples B and C should be assayed using at least two

and preferably three different dilutions tested in replicate.

Other Methods

Please carry out a minimum of four independent assays using your normal routine methodology

for determination of Clottable Protein concentration.

For clot dissolution/supernatant + E280 measurement for determination of Clottable Protein

concentration, please record Extinction Coefficient(s) used on the result sheet template provided

(page 8).

7. TOTAL PROTEIN (e.g. Biuret, Gravimetric, Dye binding/ODs, E280, Kjeldahl or

Immunological methods)

Please carry out a minimum of four independent assays using your normal routine methodology.

For E280 measurement for determination of Total Protein concentration, please record

Extinction Coefficient(s) used on the Result Sheet 3 provided (page 7).

WHO/BS/2012.2208 8. SUBMISSION OF ASSAY RESULTS AND METHODOLOGICAL DETAIL Please record your results and all raw data (i.e. OD values or clotting times etc) including all details

of dilutions performed, on the appropriate accompanying Results Sheet templates (pages 5, 7 & 9).

Please retain your raw assay data for possible future reference.

CLOTTABLE PROTEIN ASSAYS

In addition, for laboratories carrying out CLAUSS assays, you are invited to return your own

calculations for potency estimates of Fibrinogen Clottable Protein (using Result Sheet 2, page 6)

for samples B & C against the current WHO 1st IS Fibrinogen Concentrate (98/614) 1st IS, with

the latter having a Clottable Protein assigned potency of 10.4 mg/ampoule.

For laboratories carrying out other methods, you are invited to return your own calculations for

potency estimates (Fibrinogen Clottable Protein) for samples B, C and 1st IS (WHO 1st IS

Fibrinogen Concentrate - 98/614, using Results Sheet 4 (page 8).

TOTAL PROTEIN ASSAYS

For laboratories carrying out Total Protein determination, you are invited to return your own

calculations for potency estimates (Total Protein) for samples B, C and 1st IS (WHO 1st IS

Fibrinogen Concentrate - 98/614, using Results Sheet 6 (page 10).

Please also complete the attached questionnaire and provide details of your assays (page 9).

PLEASE SEND YOUR RESULTS ELECTRONICALLY

BY 30th OCTOBER 2010

TO:

Dr S. Raut

E-mail: [email protected]

WHO/BS/2012.2208

EXAMPLE

STUDY TO CALIBRATE THE WHO 2nd INTERNATIONAL STANDARD FOR FIBRINOGEN CONCENTRATE

RESULTS SHEET 1: RAW DATA

Laboratory: Date:_________________

Potency Method: CLOTTABLE Protein - CLAUSS ASSAY Assay No.:

Preparation

(Code No.)

Working Dilutions:

Replicate Response (s)

Clotting Time (Sec)

WHO IS Candidate Concentrate B

(09/242)

Bampoule 1

e.g. 1/5

1/10

1/20

WHO IS Candidate

Fibrinogen Concentrate C (10/100)

Campoule 1

e.g. 1/5

1/10

1/20

WHO 1st IS Fibrinogen Concentrate

(98/614)

1st IS

e.g. 1/5

1/10

1/20

For laboratories performing Interpolation to a Standard Curve, please give following details: Standard Used: Dilution Range of Standard

Replicate

Response (s)

WHO/BS/2012.2208 EXAMPLE


RESULTS SHEET 2: LABORATORY’s OWN CALCULATED POTENCIES

Laboratory:

Potency Method: CLOTTABLE Protein - CLAUSS ASSAY Date:_________________

Method of Calculation: Parallel Line/other?

Test

Preparation

Assay Date

Fibrinogen CLOTTABLE Protein (mg/mL)

vs 1st IS

WHO IS Candidate

Fibrinogen Concentrate B

(09/242)

B

Estimate 1 Estimate 2


MEAN Estimate

WHO IS Candidate

Fibrinogen Concentrate C

(10/100)

C



MEAN Estimate

WHO/BS/2012.2208

EXAMPLE


RESULTS SHEET 3: RAW DATA for OTHER METHODS

Laboratory: Date:__________

Potency Method: CLOTTABLE Protein: Assay No.:

(e.g. Clot Dissolution/Supernatant + Gravimetric, Biuret, Dye binding/ODs, E280, Kjeldahl)

Preparation (Code No.)

Total Dilution Factor

Replicate Responses

e.g. ODs at 280nm e.g. ODs at 315nm


(09/242)

Bampoule 1

?

?

?

?

WHO IS Candidate Fibrinogen Concentrate C

(10/100)

Campoule 1

?

?

?

?


(98/614)

1st ISampoule 1

?

?

?

?


Replicate

Response (s)



RESULTS SHEET 4:

LABORATORY’s OWN CALCULATED POTENCIES (OTHER METHODS) Laboratory:

Potency Method: CLOTTABLE Protein: Date:__________

(e.g. Clot Dissolution/Supernatant + Gravimetric, Biuret, Dye binding/ODs, E280, Kjeldahl)

Method of Calculation: e.g. Extinction Coefficient/other?

Test Preparation

Assay Date

Fibrinogen CLOTTABLE Protein (mg/mL)*

WHO IS Candidate Fibrinogen

Concentrate B (09/242)

B



MEAN Estimate

WHO IS Candidate


(10/100)

C



MEAN Estimate

WHO 1st IS Fibrinogen

Concentrate (98/614)

1st IS



MEAN Estimate

*For participants using E280 measurement for determining CLOTTABLE Protein

concentration, please give Extinction Coefficient used: ________________________

WHO/BS/2012.2208

EXAMPLE


RESULTS SHEET 5: RAW DATA for TOTAL PROTEIN Determination

Laboratory: Date:___________

Potency Method: TOTAL Protein Assay No.:

(e.g. Biuret, Gravimetric, Dye binding/ODs, E280, Kjeldahl or Immunological assays)

Preparation (Code No.)

Total Dilution Factor

Replicate Responses

e.g. ODs at 280nm e.g. ODs at 315nm


(09/242)

Bampoule 1

?

?

?

?

WHO IS Candidate Fibrinogen Concentrate C

(10/100)

Campoule 1

?

?

?

?


(98/614)

1st ISampoule 1

?

?

?

?


Replicate

Response (s)



RESULTS SHEET 6:

LABORATORY’s OWN CALCULATED POTENCIES (TOTAL PROTEIN) Laboratory:

Potency Method: TOTAL Protein: Date:____________

(e.g. Biuret, Gravimetric, Dye binding/ODs, E280, Kjeldahl or Immunological assays)

Method of Calculation: e.g. Extinction Coefficient/other?

Test

Preparation

Assay Date

Fibrinogen CLOTTABLE Protein (mg/mL)*

WHO IS Candidate

Fibrinogen Concentrate B

(09/242)

B



MEAN Estimate

WHO IS Candidate


(10/100)

C



MEAN Estimate

WHO 1st IS Fibrinogen

Concentrate (98/614)

1st IS



MEAN Estimate

*For participants using E280 measurement for determining TOTAL Protein concentration,

please give Extinction Coefficient used: ________________________

WHO/BS/2012.2208


QUESTIONNAIRE

NAME AND LABORATORY:

1. Method Details:

2. Clottable Protein Assay:

- Source of Thrombin reagent

- Instrumentation/analyser

3. Total Protein Assay:

- Critical reagents

- Instrumentation/analyser

4. Dilution Buffer:

- Buffer details including source

5. Please record any deviation from your routine method used:

6. Comments on any aspects of the study:

WHO/BS/2012.2208

APPENDIX III Participants methods and reagents used: (A) Clottable Protein Lab Method Method details Source of Thrombin Reagent Dilution: Buffer Instrumentation

1 Clauss Assay - - Factor diluent (IL) ACL 200 (IL) 2 Clauss Assay - STA® - Fibrinogen 5/lyophilized

human calcium thrombin (~80 NIH units/ml)

STA® - Owren-Koller buffer pH~7.4

STA® Analyzer: STAR-R®

3A CLOTr Clot removal + Biuret assay of supernatant

Bovine Thrombin – 50NIH units (Diagnostic Reagents Ltd)

Saline Unicam 300 series UV/VIS spectrophotometer (absorbance measured @ 540nm)

3B Clauss Assay - Thrombin Reagent - Diagnostica Stago Fibrinogen 5

Veronal Buffered Saline

ST4 Coagulometer L10284 (Diagnostica Stago )

3C CLOTr Clot removal + OD of dissolved clot

Bovine Thrombin (Diagnostica Stago)

Clot Solvent (0.15M NaCl, 7M Urea, 0.2M NaOH)

Unicam 300 series UV/VIS spectrophotometer (absorbance measured @ 280nm)

5 CLOTr Clot removal + Biuret assay of supernatant

IIa component of Beriplast (CSL Behring)

Saline Spectramax microplate reader (Molecular Devices) (absorbance measured @ 550nm)

6 Clauss Assay - Stago Thrombin Owren-Koller buffer (Diagnostiga Stago)

STAR-R Evolution (Diagnostica Stago)

7A Clauss Assay - Bovine Thrombin (Siemens Multifibren U)

Saline 0.85% Siemens BCS XP

7B Clauss Assay - Bovine Thrombin (rFibrinogen, Siemens In-House assay)

Owren-Veronal buffer (Dade)

Siemens BCS

8A Clauss Assay - Bovine Thrombin HemosIL Saline + BSA 1% ACL TOP Analyser (IL) 9 Clauss Assay - Bovine Thrombin (Siemens) Owren-Veronal buffer Siemens CA7000 Analyser 10A Clauss Assay - Bovine Thrombin (Diagnostica

Stago) 0.05M Imidazole buffer, pH 7.3

ACL TOP 500 Analyser (IL)

10B& C

CLOTr Clot removal + OD of dissolved clot

Bovine Thrombin (Diagnostica Stago)


Cecil spectrophotometer (absorbance measured @ 280nm)

11 Clauss Assay - Bovine Thrombin - 35 NIH units/ml (IL)

Imidazole buffer ACL TOP Analyser (IL)

13 Clauss Assay - Thrombin Reagent - Diagnostica Stago STA Fibrinogen 5 Assay

Owren-Koller buffer (Diagnostiga Stago)

STA Compact

14A Clauss Assay - Thrombin Reagent - Thromborel S (Siemens)

Owren’s buffer Sysmex 1500 Analyzer

14B ELISA Ant-human fibrinogen Ab (DAKO)

- PBS + 0.05% Tween20 -

15 CLOTr Clot removal + Protein (N2) determination of dissolved clot (Kjeldahl)

Thrombin, 300IU/ml (Baxter S&R) Citrate NaCl buffer (5.4g/L Citrate;7.5g/L NaCl)

Steam Distillation Apparatus Titration Unit Digestion Unit

17 Clauss Assay - Thrombin Reagent - Diagnostica Stago Fibrinogen 5

Veronal Buffered Saline

ST4 Coagulometer L10284 (Diagnostica Stago )

18A Clauss Assay STA-Fib 2 assay Titrated Human Calcium Thrombin (40 NIH units/ml), pH 7.35

STA-Owren-Koller buffer STA-R

18B Clauss Assay STA-Fibrinogen assay Titrated Human Calcium Thrombin (80 NIH units/ml), pH 7.35

STA-Owren-Koller buffer STA-R

19 Clauss Assay - Thrombin (Sigma) Veronal buffer & Imidazole buffer

Amelung KC10

20A Clauss Assay - Thrombin: HemosIL Fibrinogen-C reagent (IL)

Saline Solution (HemosIL Factor Diluent)

ACL TOP 700 LAS

20B ELISA Ant-human fibrinogen Ab (Siemens)

- Phosphate Buffer containing NaCl (N Diluent - Siemens)

Siemens BNII Analyser

21 Clauss Assay - Thrombin (Dade Behring) - Sysmex CS2100i 22 Clauss Assay - Thrombin Reagent (Siemens) (Siemens) Sysmex CS-2000i 23 Clauss Assay - IL Thrombin reagent (FibC kit -

IL) IL Factor Diluent ACL TOP (IL)

25 Clauss Assay - Human Liquid Thrombin FIB L Human (Diagnostic Grifols)

Owren buffer (Diagnostic Grifols)

Amax, Mechanical

26A Clauss Assay - Fibri-prest (Stago) Owren-Koller buffer BCS-XP (Behring) 26B CLOTr Clot removal + Drying

and Weighing Method Helena Thrombin Reagent 0.15M NaCl + 0.03M CaCl Precision Balance

27A& B

Clauss Assay - Bovine Thrombin (IL) 0.9% NaCl solution (w/v) ACL Elite Pro (IL)

28 Clauss Assay - Thrombin Reagent (Siemens) Siemens Kit (Siemens) Siemens BCS XP 29 CLOTr Clot removal + OD of

dissolved clot Thrombin Reagent (In-house) Clot Solvent (0.15M NaCl,

7M Urea, 0.2M NaOH) + PBS

Evolution 300 spectrophotometer (absorbance measured @ 280nm)

30 CLOTr Clot removal + Protein (N2) determination of dissolved clot (Kjeldahl)

Thrombin Reagent (In-house) - Steam Distillation Apparatus Titration Unit Digestion Unit

31 Clauss Assay - Thrombin Reagent (Helena Bioscience)

Owren buffer (Helena BioScience.)

STA Compact Coagulometer

WHO/BS/2012.2208

(B) Total Protein

Lab Method Dilution: Buffer Instrumentation 1 BCA Protein Assay Factor diluent (IL);

BCA Method (Sigma BCA-1) Spectrophotometer (absorbance measured @ 565nm)

3 Biuret Assay Saline Unicam 300 series UV/VIS spectrophotometer (absorbance measured @ 540nm)

5 Biuret Assay Saline Spectramax microplate reader (Molecular Devices) (absorbance measured @ 550nm)

10 Protein Absorbance Assay


Cecil spectrophotometer (absorbance measured @ 280nm)

15 Kjeldahl method Sulphuric Acid digestion for N2 determination

-

20 Synchron M-TP Microprotein Assay

Factor Diluent (HemosIL) UniCel DxC 800 (Beckman/Coulter)

25 Biuret Assay Physiological Saline Spectramax microplate reader (Molecular Devices) 26A Kjeldahl Method Sulpuric Acid digestion for N2

determination Buchi AutoKjeldahl Unit K370

26B Biuret Assay - Spectrophotometer (Varian Cary) + Hamilton Diluter 27 BioRad Protein Assay 0.9% NaCl solution (w/v) Spectrophotometers UV-VIS models: UV-1700 & UV-1603 (Shimadzu)

WHO/BS/2012.2208

APPENDIX IV

Laboratories’ Own Calculations Clottable Protein Potency relative to IS (10.4 mg/ml)

Lab No.


(09/242) [Data from Main & Field Studies]

Sample C (10/100)

[Data from Main Study] No of

Assays (n)

Geometric Mean

(mg/ml)

Intra-Lab

GCV%

No of Assays

(n)

Geometric Mean

(mg/ml)

Intra-Lab

GCV%

Clauss Assay


1 4 ‐ 4 ‐ 2 4 12.70 4 16.40 6 4 12.00 4 15.90

7A 4 10.12 4 14.01 7B - 11.34 4 14.03 8 4 13.25 4 16.65 9 4 . 4 .

10A 4 13.37 4 . 11 4 15.59 4 27.12 13 4 13.70 4 17.00

14A 4 ‐ 4 ‐ 17 4 13.26 4 17.22

18A 4 12.60 4 14.78 18B 4 12.27 4 15.83 19 4 11.84 4 36.57

20A 4 - 4 ‐ 21 4 14.09 4 23.7 22 4 14.90 4 23.47 23 4 13.94 4 19.73 25 5 9.92 5 14.68

26A 4 12.50 4 12.40 27A 4 15.50 4 .


3B 4 12.38 - - 26C 4 12.90 - - 27B 4 13.15 - - 28 2 - - - 31 4 12.95 - -

CLOTr Method


3A 4 ‐ 4 - 5A 4 11.4 4 9.6 10B 4 10.58 4 9.59 15 4 10.34 4 9.96

26B 4 11.30 4 10.40


3C 4 10.46 - - 5B 4 11.14 - -

10C 4 10.70 - - 29 4 10.75 - - 30 4 11.36 - -

ELISA


14B 4 - 4 - 20B 4 - 4 -

WHO/BS/2012.2208

Laboratories’ Own Calculations

Total Protein Potency relative to IS (15.0 mg/ml)

Lab No.

Sample B

Sample C

No of Assays

(n)

Laboratory’s Own Mean (mg/ml)

No of Assays

(n)

Laboratory’s Own Mean (mg/ml)

1 4 - 4 ‐ 3 4 ‐ 4 - 5 4 15.5 4 12.8

10 4 14.72 4 11.10 15 4 14.42 4 11.19 20 4 ‐ 4 ‐ 25 4 15.34 4 12.00

26A 4 29.19 4 13.18 26B 4 16.10 4 13.0 27 4 15.90 4 11.60

WHO/BS/2012.2208

APPENDIX V Field Study

Table A. Field Study data on Fibrinogen Products measured using CLOTr Methods. Fibrinogen (Fg) Clottable Protein potencies are calculated relative to the 1st IS and the proposed WHO 2nd IS (sample B), with proposed and alternative assigned potencies. Potency Ratios of assays relative to the 1st IS and the proposed WHO 2nd IS (sample B), with proposed and alternative assigned potencies, are also shown.

Fibrinogen

Products

(n= No of

assays)

Mean Fg Potency

vs 1st IS (98/614)

(assigned Potency

10.4 mg/ml)

Mean Fg Potency

vs B (09/242)

(10.9 mg/ml)

Potency Ratio:

Assays vs 1st IS/B

(B=10.9 mg/ml)

Mean Fg Potency

vs B (09/242)

(12.1 mg/ml)

Potency Ratio:

Assays vs 1st IS/B

(B=12.1 mg/ml)

Mean Fg Potency

vs B (09/242)

(12.9mg/ml)

Potency Ratio:

Assays vs 1st IS/B

(B=12.9 mg/ml)

R1 (n=4) 47.87 49.93 0.96 55.43 0.86 59.09 0.81

S1 (n=4) 55.07 57.45 0.96 63.77 0.86 67.99 0.81

T1 (n=4) 49.79 51.93 0.96 57.65 0.86 61.46 0.81

R2 (n=4) 20.94 20.49 1.02 22.75 0.92 24.25 0.86

S2 (n=4) 88.26 86.40 1.02 95.91 0.92 102.25 0.86

T2 (n=4) 96.47 94.43 1.02 104.83 0.92 111.76 0.86

R3 (n=4) 77.70 79.46 0.98 88.21 0.88 94.04 0.83

S3 (n=4) 78.13 79.91 0.98 88.71 0.88 94.58 0.83

T3 (n=4) 75.76 77.48 0.98 86.01 0.88 91.70 0.83

R4 (n=4) 73.80 75.22 0.98 83.50 0.88 89.02 0.83

S4 (n=4) 76.50 77.92 0.98 86.50 0.88 92.22 0.83

T4 (n=4) 76.80 77.74 0.99 86.30 0.89 92.01 0.83

2‐4% 8‐14% 17‐19%

Manufacturer

Lab 3

OMCL Lab 5

OMCL Lab 10

Manufacturer

Lab 29

% Shift (range)

in potency

relative to B

Lab No.

CLOTr Potency relative to the proposed WHO 2nd IS, sample B (with various assigned potency options)

Proposed Assigned value based on

CLOTr Method ‐ 10.9 mg/ml

Alternative Assigned value based on

Combined Method Mean ‐ 12.1 mg/ml


Clauss Assay ‐ 12.9 mg/ml

R, S & T represents different batches of products from each manufacturer.

WHO/BS/2012.2208

Table B. Field Study data on Fibrinogen Products measured using Clauss Assays. Fibrinogen (Fg) Clottable Protein potencies are calculated relative to the 1st IS and the proposed WHO 2nd IS (sample B) with proposed and alternative assigned potencies. Potency Ratios of assays relative to the 1st IS and the proposed WHO 2nd IS (sample B), with proposed and alternative assigned potencies, are also shown.

Fibrinogen

Products

(n= No of

assays)

Mean Fg Potency

vs 1st IS (98/614)

(assigned Potency

10.4 mg/ml)

Mean Fg Potency

vs B (09/242)

(10.9 mg/ml)

Potency Ratio:

Assays vs 1st IS/B

(B=10.9 mg/ml)

Mean Fg Potency

vs B (09/242)

(12.1 mg/ml)

Potency Ratio:

Assays vs 1st IS/B

(B=12.1 mg/ml)

Mean Fg Potency

vs B (09/242)

(12.9mg/ml)

Potency Ratio:

Assays vs 1st IS/B

(B=12.9 mg/ml)

R5 (n=4) 57.01 50.19 1.14 55.71 1.02 59.39 0.96

S5 (n=4) 50.21 44.20 1.14 49.07 1.02 52.31 0.96

T5 (n=4) 57.39 50.52 1.14 56.08 1.02 59.79 0.96

R6 (n=4) 15.66 13.22 1.19 14.67 1.07 15.64 1.00

S6 (n=4) 14.65 12.36 1.19 13.72 1.07 14.63 1.00

T6 (n=4) 15.02 12.67 1.19 14.07 1.07 15.00 1.00

R7 (n=4) 17.17 14.25 1.20 15.82 1.09 16.87 1.02

S7 (n=4) 71.31 59.18 1.21 65.69 1.09 70.03 1.02

T7 (n=4) 78.56 65.19 1.21 72.37 1.09 77.15 1.02

R8 (n=4) 62.87 53.97 1.16 59.91 1.05 63.87 0.98

S8 (n=4) 54.41 46.71 1.16 51.85 1.05 55.28 0.98

T8 (n=4) 59.29 50.90 1.16 56.50 1.05 60.24 0.98

14‐21% 2‐9% 2‐4%

Manufacturer

Lab 3

Manufacturer

Lab 26

Manufacturer

Lab 27

Manufacturer

Lab 31

% Shift (range)

in potency

relative to B

Lab No.

Clauss Potency relative to the proposed WHO 2nd IS, sample B (with various assigned potency options)

Proposed Assigned value based

on CLOTr Method ‐ 10.9 mg/ml


Combined Method Mean ‐ 12.1 mg/ml


Clauss Assay ‐ 12.9 mg/ml

R,S & T represents different batches of products from each Laboratory.

WHO/BS/2012.2208 APPENDIX VI Draft Instruction for Use form for the proposed WHO 2nd International Standard for Fibrinogen concentrate (09/242).

WHO/BS/2012.2208

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