EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 17 to 21 October 2016
COLLABORATIVE STUDY ON THE 2ND INTERNATIONAL STANDARD FOR BLOOD COAGULATION FACTOR XI, PLASMA (15/180): ASSIGNMENT OF
FUNCTIONAL AND ANTIGEN VALUES
Helen Wilmot1, Peter Rigsby2, Jason Hockley2 and Elaine Gray1
1Haemostasis Section and 2Biostatistics Group, National Institute for Biological Standards and Control,
Potters Bar, Hertfordshire, EN6 3QG, UK.
helen.wilmot@nibsc.org NOTE:
This document has been prepared for the purpose of inviting comments and suggestions on the proposals contained therein, which will then be considered by the Expert Committee on
Biological Standardization (ECBS). Comments MUST be received by 16 September 2016 and should be addressed to the World Health Organization, 1211 Geneva 27, Switzerland, attention:
Technologies, Standards and Norms (TSN). Comments may also be submitted electronically to the Responsible Officer: Dr C M Nübling at email: nueblingc@who.int
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Summary
The aims of this study were to assign Factor XI functional activity to the 2nd International
Standard (IS) for Blood Coagulation Factor XI, Plasma, and to additionally assign a new analyte, FXI antigen, to the same International Standard. Assignment of the functional activity value in International Units (IU) was performed by one-stage clotting assay by 29 laboratories, relative to the 1st IS. The majority of intra-laboratory GCVs were less than 5% and the inter-laboratory variability was extremely low at 1.8%, with an overall geometric mean of 0.71 IU/ampoule. The antigen value assignment was performed by 11 laboratories and was calculated relative to normal plasma pools. As is customary with new coagulation factor analytes, the amount of analyte present in 1 ml of normal plasma was taken to be 1 unit, therefore the assignment of antigen is in IU/ampoule relative to normal plasma pools. The inter-laboratory variation of the antigen assays was acceptable at 10%, with an overall geometric mean of 0.78 IU/ampoule.
Proposals for establishment:
• To establish candidate 15/180 as the 2nd International Standard for FXI, Plasma, with assigned values of:
o FXI functional activity (FXI:C) of 0.71 IU/ampoule o FXI antigen value (FXI:Ag) of 0.78 IU/ampoule
Introduction
Factor XI deficiency is generally mild and bleeding is most often associated with surgery or trauma, though bleeding phenotype can vary and is not always correlated to FXI clotting activity.
Deficiency is most common in Ashkenazi Jews (around 1 in 190 are homozygous for mutation in the F11 gene and around 1 in 8 are heterozygous), but has now been identified in a wide variety of populations. There are a number of inherited mutations that cause FXI deficiency, most of which lead to a decrease in antigen and functional activity, though some patients (around 4%) experience a decrease in functional activity only.1
The 1st International Standard (IS) for Blood Coagulation Factor XI, Plasma (04/102), was established by the Expert Committee on Biological Standardisation (ECBS) of the World Health Organisation (WHO) in October 20052. The standard is used to aid diagnosis of factor XI (FXI) deficiency and to assign potency to licensed FXI concentrates as well as control of virus
inactivated plasma products, both of which are used for treatment of patients. As with most coagulation factors and inhibitors where the International Unit (IU) is defined as the amount of activity in 1 ml of pooled normal plasma, this first IS was assigned with FXI functional activity (FXI:C) relative to local normal plasma pools only. The stock levels of the 1st IS are now near depletion and a replacement standard is required. For continuity of the IU, the replacement IS for FXI functional activity (FXI:C) was value assigned relative to the 1st IS in this collaborative study. The relationship of the IS to normal plasma was also examined. In addition, this study also aimed to establish an antigen value for FXI (FXI:Ag) for the same candidate relative to
laboratories’ local normal pooled plasma. This will enable differentiation between patients who have low functional activity but normal antigen FXI levels and patients who have both low functional and antigen FXI levels.
The replacement of the 1st IS for FXI, Plasma (FXI:C) and the FXI:Ag value assignment projects were endorsed by the WHO ECBS in October 2014.
Candidate WHO 2nd International Standard for Factor XI, Plasma (15/180)
Bulk material was purchased from the United Kingdom Blood Service in the form of plasma which had been prepared by centrifugation of whole blood collected into CPD adenine
anticoagulant. A second centrifugation step was performed to remove all cellular material and the plasma rapidly frozen at -70°C. Individual donations were tested at source and found to be negative for HBsAg, anti-HIV-1 and HIV-2 antibodies, and anti-HCV. The material was prepared for filling by thawing immediately prior to use in a 37°C waterbath and then pooled.
Glycine and HEPES were added at a final concentration of 1% w/v and 40 mM, respectively. To avoid activation of FXI by cold activation or contact with glass, plastic vessels were used and the plasma was maintained at room temperature after thawing and throughout the duration of the fill.
The product was filled into siliconized glass ampoules and freeze dried over a 5 day cycle. The finished product summary is as follows:
Code number 15/180
Presentation Sealed, siliconized glass ampoules
Number of ampoules available 6000
Date filled 01 October 2015
Mean fill mass (n=410) 1.0094 g
Precision of fill (CV of fill mass) (n=410) 0.300%
Residual moisture (n=12) 0.605%
Mean dry weight (n=3) 0.0928 g
Mean oxygen head space (n=12) 0.23 %
Storage conditions -20°C
Address of processing facility NIBSC, Potters Bar, EN6 3QG, UK
Address of custodian NIBSC, Potters Bar, EN6 3QG, UK
Activation status of the proposed 2nd IS
Non-activated partial thromboplastin time (NAPTT) is sensitive to activated clotting factors and was used to profile the activation status of the finished product. The clotting times of the
propsed 2nd IS (15/180) and the 1st IS (04/102) are shown below. A clotting time of greater than 250 s indicates that the plasma is relatively unactivated.
Mean clotting time (s) n=9 SD
15/180 300 2.26
04/102 310 6.80
The clotting time of the proposed 2nd IS was similar to that of the 1st IS and therefore is deemed to be suitable. A chromogenic assay for FXIa was also performed, however the assay kit is not designed for use with plasma and no dose response was observed. Taking single point
estimations did give an estimation of FXIa activity of around 2.3 mIU/ml for the proposed 2nd IS.
Spiking this amount of FXIa into the 1st IS and assaying by FXI clotting assay did not alter the potency estimate of the 1st IS, so it is highly unlikely that any low level FXIa which may be present in the proposed 2nd IS would affect the overall potency estimates.
Stability studies
On-bench and accelerated degradation studies have been carried out. The results of the FXI:C on-bench stability are shown below, with potencies representing 2 assays at each time-point and determined relative to a fresh ampoule of 15/180 at each time. Potency is expressed as
percentage relative to the fresh ampoule. The potency after 4 hours storage at room temperature in a capped plastic tube overlaps well with that at 0 h, indicating the material is stable for at least 4 hours when stored at room temperature.
FXI functional activity (FXI:C) Time % potency – combined
(95% confidence intervals)
0 h 97.70
(93.58-102.00)
1 h 96.83
(94.15-99.58)
2 h 96.13
(91.38-101.13)
3 h 96.99
(92.51-101.69)
4 h 98.02
(93.33-102.95)
On-bench stability studies of FXI antigen were carried out, with potencies shown below representing one assay performed with duplicate dilutions. Potency is expressed in percent, relative to time 0 (assigned to be 100%). The 95% confidence limits at 4 h overlap 100%, indicating the product is stable for at least 4 hours when stored at room temperature.
FXI antigen (FXI:Ag) Time % potency – combined
(95% confidence intervals)
1 h 116.09
(105.99-127.24)
2 h 106.48
(97.22-116.65)
3 h 103.16
(94.19-113.00)
4 h 106.57
(97.31-116.76)
Accelerated degradation studies have been performed after 1.5 and 4 months storage at low and high temperatures. The predicted loss per year for FXI:C at each temperature is shown below, based on cumulative results from both time-points. The predicted percentage loss for FXI functional activity at the normal storage temperature of -20 °C is 0.002%, showing that the material is very stable at this temperature. Degradation studies are on-going.
FXI functional activity (FXI:C) Storage
temperature (°C)
Predicted loss per year (%) (relative to
-150)
95% upper confidence limit (% loss)
-150 <0.001 <0.001
-70 <0.001 <0.001
-20 0.002 0.502
4 0.020 5.687
20 0.068 19.054
37 0.222 44.322
The predicted stability of the antigen is as shown below, though it should be noted that the data is based on one time-point only. The 95% upper confidence limits for the predicted loss for antigen are relatively high, indicating a degree of assay variability, which in turn is reflected in the predicted loss. More data is required to obtain an accurate measure of stability and this will be achieved by both accelerated and real-time monitoring.
FXI antigen (FXI:Ag) Storage
temperature (°C)
Predicted loss per year (%) (relative to
-70)
95% upper confidence limit (% loss)
-150 <0.001 <0.001
-70 <0.001 <0.001
-20 0.194 3.100
4 1.775 15.786
20 6.229 31.151
37 19.462 41.168
Participants
Twenty nine laboratories took part in the study for the FXI:C assignment, and eleven for the FXI:Ag assignment. For the functional assays, the participating laboratories were from Austria (3), Canada (1), Croatia (1), Denmark (1), France (4), Germany (4), Italy (1) The Netherlands (1), Spain (2), United Kingdom (9) and USA (2). These comprised of 8 clinical laboratories, 9 therapeutics producers, 6 diagnostics manufacturers, 5 regulatory laboratories and 1 research laboratory. All participants returned data in time for analysis, however one laboratory (lab 32) performed single point estimates only, hence was excluded from the overall analysis (these
results are shown in Appendix III, Table 3; reported potencies were approximately 6% lower than the overall geometric mean for the candidate). For the FXI antigen study, the participating laboratories were from Austria (1), Canada (1), Denmark (1), France (2), Germany (1), Spain (1) United Kingdom (2) and USA (2), comprising 2 clinical laboratories, 1 diagnostics
manufacturer, 6 plasma therapeutics producers and 2 regulatory laboratories. One other laboratory withdrew due to problems with supply of the kits from the manufacturers, as the assays could not have been completed in time for analysis. Each participating laboratory is referred to in the report by an arbitrarily assigned number and is listed in Appendix I, the order of which does not represent the assigned laboratory number.
Collaborative study design
The samples sent out for study were as follows:
S (04/102): 1st IS for Factor XI, Plasma, FXI:C potency 0.86 IU/ampoule (used for functional activity assays only)
A: (15/180): Candidate for 2nd IS for Factor XI, Plasma B: (15/180): Coded duplicate
P: Local plasma pool
Although the 2nd IS factor XI potency will be established relative to the 1st IS, the relationship between the plasma pool unit was also examined. Since the FXI antigen assignment is a new value, the unitage of the standard will be assigned relative to normal plasma pools. For these reasons, participants were asked to collect fresh local plasma pools for use both fresh and
subsequently frozen during the study. Laboratories were asked to follow a supplied protocol for collection of plasma pools (see collaborative study protocol in appendix II). NIBSC in-house studies on both FXI functional activity and FXI antigen assays have shown that there is no significant difference in results when using fresh plasma pools compared to the same pool of plasma used after freezing, therefore if participants were unable to collect fresh plasma pools, then frozen plasma could be used as an alternative. A total of 5 laboratories used fresh plasma pools for the FXI functional assays (18 assays in total), and 2 laboratories used fresh plasma pools in the FXI antigen assays (4 assays in total). Across the pools used in the study (fresh and frozen) for either functional activity or antigen value, plasma from more than 20,000 donors was used.
The issue of commutability was considered, and sourcing of appropriate samples was
investigated. The samples would need to be plasma from Factor XI deficient patients with FXI levels of around 1-5% of normal. Due to the fact that FXI deficieny is relatively rare and that a large volume of plasma would be required for the study, it was not possible to source samples for this purpose. Commerical sources of deficient plasma were either completely deficient
(immune-depeleted) and therefore not appropriate, or were pooled plasmas, which would not address commutability.
Assay methods
Participants were asked to perform their in-house method for factor XI functional activity and antigen. Four assays for each were requested, using freshly reconstituted ampoules of samples for each assay. A balanced order of testing was suggested (for details of assay design refer to collaborative study protocols in appendix II). All participants assaying for factor XI functional activity employed the one-stage clotting method using an activated partial thromboplastin time (APTT) reagent. In total, 13 different APTT reagents were used across a variety of instruments and sources of deficient plasma. For antigen assays, 5 different kits or paired antibody sets were used. A list of reagents and details of local plasma pools used is given in Tables 1 and 2.
Statistical Analysis
An independent statistical analysis of raw data was performed at NIBSC. Relative potency estimates were calculated by fitting a parallel-line model3 based on a linear section of the response range using a minimum of three dilutions for all samples. Non-linearity and non- parallelism were considered in the assessment of assay validity. All data were plotted and a visual assessment was used to determine linearity. Non-parallelism was assessed by calculation of the ratio of fitted slopes for the test and reference samples under consideration. A log10
transformation of the assay response was used for the analysis of the factor XI antigen assays.
No transformation was necessary for the factor XI functional assays.
For the factor XI functional assays, candidates A and B (coded duplicates) were analysed and potency was assigned relative to the current IS (coded S). Functional activity of these coded duplicates were also analysed relative to local plasma pools (assuming potency value of 1 u/ml) and compared with potencies obtained against the current IS. For the factor XI antigen assays, coded samples A and B were analysed relative to the plasma pool sample (assuming potency value of 1 u/ml) in each assay to provide a relative potency estimate.
All mean potencies given in this report are unweighted geometric mean (GM) potencies.
Variability between assays and laboratories has been expressed using geometric coefficients of variation (GCV = {10s-1}×100% where s is the standard deviation of the log10 transformed potency estimates).
Grubbs’ Test4 was applied to the log transformed laboratory mean estimates in order to detect any significant outliers. Comparisons between methods were made by appropriate t-tests of log transformed laboratory mean estimates.
Parallelism of dose-response slopes was evaluated by setting an acceptable range of values for the ratio of fitted slopes for the test and reference samples. To determine this range for the factor XI functional assays a parametric tolerance interval (95% confidence, 95% coverage) was
calculated using the ratio of the fitted slopes of coded duplicate samples A and B, and the more extreme of the upper and lower bounds used with its reciprocal value to set symmetrical limits on the acceptable ratio of slopes around 1. This result was an acceptable ratio of 0.91 to 1.10.
The same calculation method was used for the factor XI antigen assays, with a result of 0.79 to 1.27, and it was decided to use limits of 0.80 to 1.25 to demonstrate acceptable parallelism in these assays. This narrower range was selected because it is generally considered to be the widest range acceptable for these types of assays.
Assay validity FXI functional assays
All laboratories performed 4 assays using one-stage clotting assay, with a total of thirteen different APTT reagents used across 140 assays. Where a laboratory had performed more than one set of assays using a different APTT reagent or coagulometer, these are designated as a, b etc. In the case of labs 26 and 31, a and b refer to the two different sources of plasma pools used within the same assays therefore these labs have one set of results relative to S, but two results relative to P (one set relative to each plasma pool). Assay 3 from Lab 17 was excluded from the analysis due to poor replicates and, for the analysis relative to S, all of the assays from Lab 17 were excluded because the results were found to be outliers. In addition, for the whole study, a total of 8 assays for A against S, 6 for B against S, 19 for A against P and 13 for B against P were excluded from the analysis for non-parallelism.
FXI antigen assays
With the exception of Lab 26, which performed 6 assays, all laboratories performed 4 antigen assays for FXI antigen. In the case of lab 26, a and b refer to the two different sources of plasma pools used within the same assays, therefore lab 26 has two set of results for FXI antigen against P. Of the 52 assays performed, 4 assays of B against P were excluded for non-parallelism. No assays for A against P were excluded for non-parallelism. One assay from Lab 24 was
withdrawn by the laboratory.
RESULTS AND DISCUSSION FXI functional assays
Analysis relative to S, the 1st IS for FXI, Plasma
Results for samples A and B were analysed relative to sample S, the 1st IS for FXI, and the individual assay results together with the laboratory geometric mean and GCV are shown in Tables 3 and 4. For comparison, the laboratories’ own calculated results relative to S are shown, alongside the centrally calculated results, in Appendix III, Table 1. On the whole, the
laboratories’ own calculated data were similar to NIBSC’s calculated results, indicating that the laboratories were able to analyse the data appropriately. The laboratories were also able to perform precise assays, with the majority of intra-laboratory GCVs being less than 5% for A or B. The overall potency of A compared to S was 0.71 IU/ampoule, with a GCV of 1.60%. For B, it was also 0.71 IU/ampoule, with a GCV of 2.40%. The good agreement of potencies for
sample A and B, the coded duplicates, showed that there was no assay design bias and that the
laboratories were able to measure FXI:C accurately. As samples A and B were coded duplicates, the results for both A and B vs S were combined to give an overall estimate (AB vs S). Each laboratory’s geometric mean and GCV are shown in Table 5. The overall result of AB vs S was 0.71 IU/ampoule, the same as for A or B individually against S, and the inter-laboratory GCV was 1.80%. Results from Lab 17 were detected as statistical outliers and were excluded from the overall calculation of potency and inter-laboratory GCV. The excellent agreement between the laboratories as shown by the close distribution of laboratory geometric means is demonstrated in Figure 1.
Several of the laboratories used Actin FS, SynthASil or APTT-SP as the APTT reagent in their assays. To determine if these reagents produced any method bias, the AB vs S geometric means of these laboratories were combined and compared to the overall geometric mean. Table 6 shows that the results for Actin FS (0.70 IU/ampoule; 5 laboratories), SynthASil (0.71
IU/ampoule; 7 laboratories) and APTT-SP (0.72 IU/ampoule; 5 laboratories) compared well with the overall geometric mean of 0.71 IU/ampoule for AB, indicating no method bias. The overall geometric mean of the other APTT reagents (excluding Actin FS, SynthASil and APTT-SP) was 0.71 IU/ampoule, with 95% confidence limits of 0.701-0.716 IU/ampoule.
Analysis relative to plasma pools (P)
Participants were asked to collect two plasma pools (P1 and P2) for use both as fresh and frozen pools in the assays. Where participants were unable to collect fresh plasma pools, it was
requested that two different batches of local frozen plasma pools were used. In order to assess the relationship with the plasma unit (where the amount of FXI in 1 ml of plasma is taken to be 1 u/ml), the data were analysed relative to P. The assay results for A and B vs P for each
laboratory are shown in Tables 7 and 8, together with the laboratory geometric means and intra- laboratory variation (GCV). The intra-laboratory variations were higher than that of the data compared to S, most likely due to the differences between the batches of plasma pools used within each laboratory. However, the majority of the results still had a GCV of less than 10%, showing that the laboratories were performing the assays with good precision. The overall geometric mean for sample A or B relative to P was 0.72 u/ampoule, agreeing well with the potency of 0.71 u/ampoule relative to S. As before, the results for A and B relative to P were combined, and the overall results shown in Table 9 and Figure 2. The inter-laboratory variation was higher than for AB vs S, at 7.20% overall, but still below 10%. The overall geometric mean for AB vs P was 0.72 u/ampoule, compared to 0.71 IU/ampoule relative to S. The results for AB vs S and AB vs P were compared using a paired two-tailed t-test and the result showed no
significant difference between the two values (p=0.215), indicating that a good relationship between the IU and the plasma unit had been maintained. A comparison of assays performed using fresh plasma pools to assays where frozen plasma pools were used showed there was no difference in the results between fresh and frozen pools (p=0.704).
FXI antigen assays
Participants were asked to collect two plasma pools (P1 and P2) for use both as fresh and frozen pools in the assays. Where participants were unable to collect fresh plasma pools, it was
requested that two different batches of local frozen plasma pools were used. As there is no current International Standard for FXI antigen, the units of FXI antigen in 1 ml of plasma pool was taken to be 1, and the potencies of A and B calculated relative to the laboratories’ local plasma pool. The individual assay results for samples A and B, together with the laboratory geometric mean and GCV are shown in Tables 10 and 11. The laboratories own calculated results are shown in Appendix III, Table 2, alongside the centrally calculated results. As with the FXI:C results, in general, the locally calculated data were similar to those obtained by NIBSC. The overall geometric mean for A against P was 0.78 u/ampoule, compared to 0.79 u/ampoule for B against P. The inter-laboratory variation was 10% for A and B, showing acceptable agreement between laboratories. Overall, the intra-laboratory variations ranged between 0.5 and 18%, with most laboratories having a GCV of less than 10%. The variation of these assays would be expected to be higher than that of the FXI functional assays, by virtue of the fact that the calculations are relative to the local plasma pools, which themselves have inherent variability. Due to the low number of assays performed using fresh plasma pools (5) it was not possible to compare fresh and frozen plasma pools.
Since A and B were coded duplicates, the results were combined to give an overall result for AB against P. The results are shown in Table 12. The overall geometric mean was 0.78 u/ampoule, in good agreement with the results for A or B separately against P. Again, the variability of the assays was acceptable, at 10% overall. No statistical outliers were detected. The individual assay results from each lab are shown graphically in Figure 3, and the laboratory geometric means are shown plotted in Figure 4. These graphs show a fairly even distribution of results around the overall geometric mean.
Of the 12 laboratories that took part, 5 used an antigen kit from Affinity Biologicals. The geometric means for AB against P of these laboratories were combined and resulted in a geometric mean of 0.72 u/ampoule (with 95% confidence limits of 0.695-0.749 u/ampoule).
This is slightly lower than the overall result for AB against P (0.78 u/ampoule). The result for the other laboratories not using the Affinity Biologicals kit was 0.82 u/ampoule, with 95%
confidence limits of 0.754-0.899 u/ampoule, which overlap the overall geometric mean of AB vs P, but not that of the laboratories using the Affinity Biologicals kit. A comparison of the two groupings was made using the unpaired two-tailed t-test and the difference was found to be significant (p<0.05). The difference suggests that the Affinity Biologicals kit may yield lower results than other kits, however this could also be a reflection of the different plasma pools used within each laboratory. Due to the small number of laboratories taking part in the study and the fact that the two groupings had 95% confidence limits that were extremely close to being overlapping, the consensus geometric mean was taken as the antigen value for the candidate preparation. Any effect of minor method bias on the results is likely to be small.
RECOMMENDATIONS
The recommendations to the Expert Committee for Biological Standardization (ECBS) of the World Health Organization (WHO) are:
• To establish candidate 15/180 as the 2nd International Standard for FXI, Plasma, with assigned values of:
o FXI functional activity (FXI:C) of 0.71 IU/ampoule o FXI antigen value (FXI:Ag) of 0.78 IU/ampoule A draft Instruction For Use (IFU) is shown in Appendix IV.
The collaborative study has been reviewed by the Participants and the Experts nominated by the Scientific and Standardisation Committee (SSC) of the International Society on Thrombosis and Haemostasis and their comments and the NIBSC corresponding responses are shown below. All participants agreed with the recommendation and the SSC has also endorsed the proposal at the Business Meeting of the 62nd Annual SSC meeting, June 2016 to go forward for establishment by the ECBS.
Participants’ Review
All participants were sent a copy of the report along with a questionnaire asking for their
agreement or otherwise with the recommendations to establish candidate 15/180 as the 2nd IS for Factor XI, Plasma, with a FXI:C value of 0.71 IU/ampoule and a FXI:Ag value of 0.78
IU/ampoule. The participants were requested to respond within two weeks if they had any comments or objections to the recommendations, otherwise a null response would be taken as agreement with no comments. Twenty-two of the participants chose to respond, all giving their agreement to the recommendations. Any comments were related to the names or affiliations of the participants and one requested a minor change to Figure 2, all of which have been completed.
SSC Experts’ Review
The proposals and recommendations presented in this report were approved at the SSC Board Meeting in Montpellier in May 2016. Nine responses from the expert review were received and all endorsed the project. Some minor typographical errors were highlighted; all of which have been corrected. Other comments and NIBSC responses are shown below.
Reviewer A:
“The number of data is impressive and the study design is the best as possible.
I have only some minor remarks Table 1:
In column 4 the company/source of the deficient plasma is mentioned.
Is this also the company of the APTT reagent? This information is missing in the table.
For the deficient plasma much more important information is the properties of the deficient plasma used; is it immune depleted plasma or congenital deficient plasma. It is interesting if there is a difference in result. If not this could also be mentioned as a remark.
Appendix III Table 1:
The calculated result by the own laboratory or by the NIBSC calculation is not always the same.
Therefore in the development of an international standard not only the comparison of the rough measured data is important, but also to teach the laboratories the method to extract the final result.”
NIBSC response:
The source of APTT reagent has been added to Table 1.
For the deficient plasma, 6 laboratories used plasma from deficient donors (congenital) and there was no difference in potency from these labs (average 0.70 IU/ampoule with 95% confidence limits of 0.68-0.73 IU/ampoule) compared to the overall assigned potency.
On occasion there are some discrepancies between the laboratory reported results and the NIBSC calculated results. In these instances the participating laboratory is contacted and possible
reasons for the discrepancies are discussed. Usually the discrepancies are identified as being caused by a reporting error in the raw data. The laboratories taking part in such studies are usually expert laboratories that are adept at using appropriate data analysis methods.
Reviewer B:
There are two issues the authors need to address.
1) Please justify why the blood for this study was centrifuged at 4 degrees Celsius? This fact can result in C1 inhibitor inactivation (Weiss R et al. Blood 68:239,1986) with increased in vitro FXII contact activation on the tube surface resulting in prior activation and lowering of zymogen FXI in the ampules. Perhaps that is the reason the level of FXI is only 0.71 U/ml in the 2nd ampuole. It also leads to cold activation of FVII and FIX by FXIIa (Seligsohn U et al. Thromb Res. 13:1049, 1978).
2) Should the results be expressed as “u/ml” or “U/ml”. What is the international standard.
NIBSC response:
1. From the pre-study questionnaires it was identified that only a few laboratories were able to collect fresh plasma pools for the study (5 labs). The decision was taken to
recommend centrifugation at 4°C in order to maintain consistency with frozen plasma pools which are routinely prepared by centrifugation in this manner. The potency of FXI:C is assigned relative to the 1st IS and not the plasma pools, so the centrifugation of local plasma pools at 4°C would not impact the overall assigned potency of FXI:C. The bulk material itself was maintained at room temperature throughout the duration of the fill in order to avoid cold activation and in-house studies using NAPTT and FXIa assays indicated minimal activation of the product had occurred.
2. The results for FXI:C are expressed u/ampoule when referring to results calculated
relative to plasma pools because it is an accepted assumption that 1 ml of plasma contains 1 unit, rather than being an official unitage. The FXI:Ag results are expressed u/ampoule throughout the report as there is no International Standard for FXI:Ag. The
recommendations refer to the assignment of units in IU/ampoule because this is the terminology that would be used on establishment of the standard.
Reviewer C:
“A well designed study from an extensive number of laboratories yielding a conclusive agreement on the potency of the FXI standard in terms of both activity and antigen. My only slight concern is the large intra-laboratory variation of a couple of the reported laboratories particularly number 27. However, the data from the remaining laboratories is sufficiently tight to support the proposal.”
NIBSC response:
A small number of laboratories do have intra-laboratory variations that are higher than others;
however, it is the geometric mean from each laboratory that is used to calculate the overall potency. The inter-laboratory agreement for FXI:C is excellent with a GCV of 1.8% and for FXI:Ag is good at 10%. In this case, intra-laboratory variation has minimal impact on the overall potency estimate and, in some cases, could be a reflection of the differences in the two plasma pools used by each laboratory and/or an indication of the performance of the laboratory.
Reviewer D:
This is a carefully performed study and the recommendations of the Expert Committee for Biological Standardization of the World Health Organization are sound.
Reviewer E:
Study & analyses appear to have been performed in a careful manner and the conclusions are well supported by the study results.
Table 1: List of APTT reagent, incubation time, FXI deficient plasma, coagulometer, and plasma pool used by each participant for FXI functional assays
Lab APTT reagent (manufacturer)
Incubation time
Deficient plasma source
Coagulometer Plasma pool (no.
donors) 1 Actin FS
(Siemens)
180 s Siemens BCS-XP Local frozen (450)
2 SynthASil (IL)
180 s IL ACL TOP 700 Local fresh (P1-13,
P2-14) 3 Actin FS
(Siemens)
180 s Dade Sysmex CS5100 Local frozen (6, 20, 10, 20)
4 Cephen (Hyphen BioMed)
240 s Hyphen BioMed STAR Commercial frozen
(P1-20, P2-20) 5a Pathromtin SL
(Siemens)
120 s Siemens BCS XP Local fresh (P1-10,
P2-10) 5b Actin FSL
(Siemens)
180 s Siemens BCS XP Local fresh (P1-10,
P2-10) 5c Pathromtin SL
(Siemens)
180 s Siemens CA-1500 Local fresh (P1-10,
P2-10) 5d Actin FS
(Siemens)
180 s Siemens CA-1500 Local fresh (P1-10,
P2-10)
8 APTT-SP
(Werfen)
165 s Helena Biosciences ACL 200/3000 Plus
Local frozen (P1- 1000, P2-1000) 9 Cephascreen
(Stago)
300 s Stago BCS-XP Commercial frozen
(P1-20, P2-10) 10 SynthASil
(IL)
180 s IL ACL TOP 500 Local fresh (P1-8,
P2-9) 11 SynthASil
(IL)
200 s American Diagnostica
ACL TOP 500 Commercial frozen (P1-20, P2-20) 12 Actin FSL
(Siemens)
180 s Siemens BCS-XP Commercial frozen
(20) 13 DG-APTT
Synth (Diganostic Grifols)
300 s Diagnostic Grifols Q Hemostasis Analyzer
Local frozen (P1- 21, P2-21)
14 APTT-SP (IL)
360 s Precision Biologic (frozen)
STA-R Evolution
Local frozen (1800) 16 APTT-SP
(IL)
300 s Diagnostic Grifols ACL Elite Pro Local frozen (P1- 4500, P2-4500, P3- 4500, P4-4500) 17 CK Prest
(Stago)
240 s Affinity Biologicals (frozen)
STA-R Evolution
Local frozen (P1- 20, P2-20) 18 Pathromtin SL
(Siemens)
120 s Siemens BCS-XP Commercial
(P1-not specified, P2-20, frozen) 19 CK Prest
(Stago)
240 s Diagnostica Stago STAR Commercial lyophilised (1)
20 SynthASil (IL)
180 s Technoclone ACL TOP 700 Commercial frozen (P1-20, P2-20) 21 Actin FSL
(Siemens)
180 s Siemens Sysmex
CA7000
Local frozen (35) 22 SynthASil
(IL)
180 s IL ACL TOP 500 Fresh (P1-6, P2-6)
23 SynthASil (IL)
200 s IL ACL TOP 700 Fresh (p1-8, P2-8)
24 APTT-SP (IL)
300 s Stago ACL TOP 500 Fresh (P1-10, P2-
10) 25a Dapttin
(Technoclone)
240 s Technoclone Ceveron Alpha Frozen (P1-12, P2- 12)
25b Siron LS (Technoclone)
240 s Technoclone Ceveron Alpha Frozen (P1-12, P2- 12)
26a SynthAFax (IL)
300 s Affinity Biologicals
Elite Pro Commercial frozen (20)
26b SynthAFax (IL)
300 s Affinity Biologicals
Elite Pro Commercial frozen (30)
27a CK Prest (Stago)
240 s Stago STA-R Local frozen (P1-
20, P2-55) 27b Cephascreen
(Stago)
240 s Stago STA-R Local frozen (P1-
20, P2-55) 27c PTT A
(Stago)
240 s Stago STA-R Local frozen (P1-
20, P2-55) 27d CK Prest
(Stago)
240 s Stago
(ImmunoDeficient)
STA-R Local frozen (P1- 20, P2-55) 28 APTT-SP
(IL)
300 s IL ACL 9000 Fresh (P1-8, P2-8)
29 Actin FS (Siemens)
180 s Diagnostica Stago BCS-XP Fresh (P1-8, P2-8) 30 Actin FS
(Siemens)
180 s Precision Biologic (frozen)
Sysmex CS5100 Commercial frozen (P1-20) and local frozen (P2-not specified) 31a SynthASil
(IL)
200 s Haematologic Technologies (frozen)
ACL TOP 500 Local frozen (1134)
31b SynthASil (IL)
200 s Haematologic Technologies (frozen)
ACL TOP 500 Local frozen (388)
Table 2: List of FXI antigen kit/paired antibodies and local plasma pool used by each laboratory
Lab Antibody/kit source Plasma pool (no. donors) Lab 6 Cedarlane paired
antibodies
Commerical frozen (P1-20, P2-20) Lab 7 Cedarlane Lyophilised (donors not specified) Lab 14 Coachrom paired
antibodies
Fresh (P1-1800, P2-1800)
Lab 15 Molecular Innovation kit Commercial frozen (p1-10, P2-10, P3- 10, P4-10)
Lab 16 Dunn Labortechnick kit Local frozen (P1-4500, P2-4500, P3- 4500, P4-4500)
Lab 17 VisuLize ELISA Kit, Affinity Biologicals
Local frozen (P1-20, P2-20) Lab 18 CoaChrom paired
antibodies
Commercial frozen (P1-not specified, P2-20)
Lab 24 VisuLize ELISA Kit, Affinity Biologicals
Fresh (P1-10, P2-10) Lab 26a VisuLize ELISA Kit,
Affinity Biologicals
Commercial frozen (single batch-20) Lab 26b VisuLize ELISA Kit,
Affinity Biologicals
Commercial frozen (single batch-30) Lab 28 VisuLize ELISA Kit,
Affinity Biologicals
Local frozen (P1-8, P2-8)
Table 3: Individual assay results for FXI functional activity in IU/ampoule for Sample A against Sample S, the 1st IS for FXI, Plasma. Each laboratory’s geometric mean is reported and intra- laboratory variation is shown as GCV (%). Lab 17 (shaded) was excluded from the overall analysis as an outlier. The overall geometric mean and inter-laboratory GCV is also shown.
Figures in brackets indicate the 95% confidence limits. NP: non-parallel.
Sample A vs Sample S Lab Assay
1
Assay 2
Assay 3
Assay 4
Geometric mean (IU/amp)
GCV
1 0.70 0.66 0.70 0.68 0.68 3.10%
2 0.74 0.75 0.73 0.72 0.74 2.10%
3 0.70 0.66 0.73 0.71 0.70 4.80%
4 0.70 0.72 0.74 0.70 0.71 2.70%
5a 0.71 0.70 0.73 0.74 0.72 2.70%
5b 0.70 0.71 0.73 0.74 0.72 2.50%
5c 0.71 0.70 0.70 0.72 0.71 1.50%
5d 0.73 0.72 0.70 0.70 0.71 2.00%
8 0.70 0.73 0.71 0.73 0.72 2.10%
9 0.69 0.70 0.74 0.69 0.71 3.30%
10 0.71 0.68 0.71 0.75 0.71 3.90%
11 0.69 0.70 0.71 0.70 0.70 1.10%
12 0.74 0.71 0.70 0.73 0.72 2.50%
13 0.72 0.70 0.69 0.72 0.71 2.00%
14 0.75 0.73 0.69 0.72 0.72 3.80%
16 0.70 NP 0.70 0.71 0.70 0.55%
17 0.74 0.80 - 0.81 0.78 4.80%
18 0.73 0.73 0.72 0.71 0.72 1.10%
19 0.70 0.72 0.70 0.72 0.71 1.40%
20 0.70 0.75 0.67 0.70 0.70 4.70%
21 0.71 0.73 0.70 0.69 0.71 2.40%
22 0.73 0.69 0.70 0.74 0.72 3.50%
23 0.78 0.71 0.71 0.71 0.73 5.10%
24 0.69 0.75 0.73 0.70 0.72 3.70%
25a 0.66 NP NP 0.77 0.71 -
25b 0.73 0.70 0.73 NP 0.72 2.50%
26 NP 0.74 0.73 NP 0.73 -
27a NP 0.70 0.70 NP 0.70 -
27b 0.68 0.69 0.74 0.87 0.74 12.00%
27c 0.70 0.65 0.69 0.79 0.70 8.10%
27d 0.74 0.68 0.72 0.71 0.71 3.20%
28 0.68 0.73 0.70 0.71 0.71 3.10%
29 0.71 0.70 0.70 0.71 0.70 1.20%
30 0.70 0.71 0.72 0.73 0.71 1.60%
31 0.70 0.72 0.69 0.70 0.70 2.10%
Overall result for A vs S 0.71 (0.708-0.719)
2.30%
Overall result for A vs S, excluding outliers 0.71 (0.708-0.716)
1.60%
Table 4: Individual assay results for FXI functional activity in IU/ampoule for Sample B against Sample S, the 1st IS for FXI, Plasma. Each laboratory’s geometric mean is reported and intra- laboratory variation is shown as GCV (%). Lab 17 (shaded) was excluded from the overall analysis as an outlier. The overall geometric mean and inter-laboratory GCV is also shown.
Figures in brackets indicate the 95% confidence limits. NP: non-parallel.
Sample B vs Sample S Lab Assay
1
Assay 2
Assay 3
Assay 4
Geometric mean (IU/amp)
GCV
1 0.71 0.65 0.68 0.68 0.68 3.90%
2 0.70 0.72 0.73 0.71 0.72 1.50%
3 0.70 0.670 0.76 0.71 0.71 3.80%
4 0.70 0.72 0.73 0.71 0.71 1.60%
5a 0.69 0.71 0.74 0.75 0.72 3.80%
5b 0.69 0.71 0.70 0.73 0.71 2.50%
5c 0.72 0.70 0.67 0.72 0.70 3.30%
5d 0.72 0.72 0.69 0.70 0.71 2.40%
8 0.73 0.73 0.73 0.76 0.74 1.70%
9 0.72 0.68 0.71 0.71 0.70 2.50%
10 0.75 0.68 0.71 0.74 0.72 4.70%
11 0.70 0.70 0.70 0.70 0.70 0.15%
12 0.73 0.73 0.70 0.70 0.72 2.20%
13 0.74 0.71 0.69 0.72 0.72 2.90%
14 0.77 0.71 0.70 0.68 0.72 5.50%
16 0.73 0.73 0.72 0.71 0.72 1.70%
17 0.74 0.79 - 0.80 0.77 4.60%
18 0.72 0.71 0.68 0.72 0.71 2.90%
19 0.73 0.70 0.68 0.72 0.71 3.00%
20 0.67 0.74 0.68 0.68 0.69 4.60%
21 0.74 0.74 0.69 0.71 0.72 3.70%
22 0.75 0.73 0.70 0.72 0.73 3.00%
23 0.70 0.71 0.72 0.69 0.70 1.50%
24 0.72 0.74 0.71 0.73 0.72 1.60%
25a 0.73 0.65 0.58 0.80 0.69 15.00%
25b NP 0.72 0.75 0.72 0.73 2.10%
26 NP 0.71 0.76 NP 0.74 -
27a NP 0.70 0.71 NP 0.70 -
27b 0.65 0.69 0.63 NP 0.66 4.20%
27c 0.67 0.62 0.61 0.85 0.68 16.00%
27d 0.73 0.67 0.70 0.71 0.70 3.60%
28 0.69 0.72 0.72 0.70 0.71 2.50%
29 0.70 0.69 0.68 0.70 0.69 0.86%
30 0.71 0.71 0.70 0.72 0.71 0.99%
31 0.71 0.72 0.68 0.71 0.71 2.80%
Overall result for B vs S 0.71 (0.703-0.717)
2.80%
Overall result for B vs S, excluding outliers 0.71 (0.702-0.714)
2.40%
Table 5: The geometric mean for each laboratory (IU/ampoule) for samples A and B (coded duplicates) combined and analysed relative to sample S, the 1st IS for FXI, Plasma. Intra- laboratory variation is shown as GCV (%). Lab 17 (shaded) was excluded from the overall analysis as an outlier. The overall geometric mean and inter-laboratory GCV is also shown.
Figures in brackets indicate the 95% confidence limits.
Samples AB vs Sample S
Lab Geometric mean
(IU/amp)
GCV
1 0.68 3.40%
2 0.73 1.40%
3 0.71 4.00%
4 0.71 2.10%
5a 0.72 3.20%
5b 0.71 2.30%
5c 0.71 2.40%
5d 0.71 2.20%
8 0.73 1.70%
9 0.70 2.10%
10 0.71 4.00%
11 0.70 0.54%
12 0.72 1.90%
13 0.71 2.30%
14 0.72 4.40%
16 0.71 1.60%
17 0.78 4.70%
18 0.72 1.60%
19 0.71 1.80%
20 0.70 4.30%
21 0.71 2.90%
22 0.72 2.70%
23 0.72 2.30%
24 0.72 2.30%
25a 0.67 13.00%
25b 0.72 1.60%
26 0.74 -
27a 0.70 -
27b 0.72 13.00%
27c 0.69 12.00%
27d 0.71 3.30%
28 0.71 2.60%
29 0.70 0.96%
30 0.71 1.10%
31 0.70 2.30%
Overall results for AB vs S 0.71 (0.706-0.718)
2.30%
Overall results for AB vs S, excluding outliers
0.71 (0.706-0.715)
1.80%
Figure 1: Histogram showing each laboratory’s geometric mean for FXI:C of samples A and B combined, relative to sample S, the 1st IS for FXI, Plasma. Outliers are shown in red. The overall geometric mean was 0.71 IU/ampoule with a GCV of 1.8%.
Table 6: Laboratory geometric means for AB vs S for APTT reagents Actin FS, SynthASil and APTT-SP, their overall geometric mean and comparison to overall results for AB vs S
Lab number
(geometric mean of AB vs S, IU/amp)
Overall geometric mean, IU/amp
(95% CL)
Overall geometric mean of AB vs S (all APTT), IU/amp (95%
CL) Actin FS
(n=5)
1 (0.68)
3 (0.71)
5d (0.71)
29 (0.70)
30
(0.71) - - 0.70
(0.685-0.718)
0.71 (0.706-0.715) SynthASil
(n=7)
2 (0.73)
10 (0.71)
11 (0.70)
20 (0.70)
22 (0.72)
23 (0.72)
31 (0.70)
0.71 (0.700-0.722) APTT-SP
(n=5)
8 (0.73)
14 (0.72)
16 (0.71)
24 (0.72)
28
(0.71) - - 0.72
(0.707-0.728)
CL: Confidence limits
Number of Laboratories
0 2 4 6 8 10 12 14 16 18 20
Potency (IU/ampoule)
0.5 0.6 0.7 0.8 0.9 1.0
1 25a
3 9 11 13 19 20 28 29 30 31 27a 27c 27d 5c 5d
2 4 8 10 12 14 16 18 21 22 23 24 26 25b 27b 5a 5b
17
Table 7: Individual assay results for FXI functional activity for Sample A against Sample P, each laboratory’s local plasma pool. Each laboratory’s geometric mean is reported and intra- laboratory variation is shown as GCV (%). The overall geometric mean and inter-laboratory GCV for A against P is also shown. Figures in brackets indicate the 95% confidence limits. NP:
Non-parallel
Sample A vs Sample P Lab Assay
1
Assay 2
Assay 3
Assay 4
Geometric mean (u/amp)
GCV
1 0.75 0.62 0.71 0.65 0.68 8.90%
2 0.64 NP 0.72 NP 0.68 -
3 0.71 0.70 0.65 0.70 0.69 4.10%
4 0.85 0.83 0.75 NP 0.81 6.90%
5a 0.67 NP 0.67 0.68 0.67 1.20%
5b 0.71 0.71 0.73 0.74 0.72 2.50%
5c 0.66 0.67 0.66 0.68 0.67 1.70%
5d 0.64 0.67 0.67 0.64 0.65 2.20%
8 0.75 0.75 NP 0.71 0.74 3.70%
9 0.70 NP 0.84 0.80 0.78 10.00%
10 0.64 0.63 0.69 0.68 0.66 4.60%
11 0.81 0.81 0.76 0.74 0.78 4.50%
12 0.76 0.74 0.73 0.75 0.74 1.80%
13 0.64 0.70 0.62 0.69 0.66 6.10%
14 0.78 0.79 0.81 0.82 0.80 2.50%
16 0.74 0.79 NP 0.82 0.78 5.20%
17 0.72 0.70 - 0.72 0.71 1.40%
18 0.81 0.87 0.81 NP 0.83 4.00%
19 0.73 0.72 0.76 NP 0.74 3.20%
20 0.74 0.77 0.73 0.81 0.76 5.10%
21 0.69 0.76 0.71 0.68 0.71 4.90%
22 0.69 0.68 0.70 0.72 0.70 2.50%
23 0.77 0.67 0.70 0.70 0.71 5.80%
24 0.64 0.68 0.69 0.69 0.68 3.70%
25a NP 0.80 0.78 NP 0.79 -
25b NP 0.78 0.81 NP 0.80 -
26a NP NP 0.68 NP 0.68 -
26b 0.70 0.78 0.70 0.71 0.72 5.60%
27a NP 0.72 0.71 0.71 0.71 1.20%
27b 0.76 NP 0.76 NP 0.76 -
27c 0.75 NP 0.67 0.76 0.73 6.90%
27d 0.73 0.72 0.69 0.70 0.71 3.00%
28 0.64 0.67 0.68 0.64 0.66 3.20%
29 0.70 0.67 0.75 0.73 0.71 4.90%
30 0.69 0.72 0.65 0.66 0.68 4.70%
31a 0.75 0.78 0.77 0.76 0.77 1.90%
31b 0.72 0.76 0.74 0.72 0.73 2.50%
Overall result for A vs P 0.72 (0.706-0.738)
6.90%
