WHO/BS/10.2145 ENGLISH ONLY
EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 18 to 22 October 2010
COLLABORATIVE STUDY FOR VALUE ASSIGNMENT OF
THE 4th INTERNATIONAL STANDARD FOR FACTORS II, VII, IX, X, PLASMA Elaine Gray1,3, Helen Barson1, Jason Hockley2 and Peter Rigsby2
1Haemostasis Section, 2Biostatistics
National Institute for Biological Standards and Control Potters Bar, Hertfordshire, EN6 3QG, UK.
3 Principal Investigator:
© World Health Organization 2010
All rights reserved. Publications of the World Health Organization can be obtained from WHO Press, World Health Organization, 20 Avenue Appia, 1211 Geneva 27, Switzerland (tel.: +41 22 791 3264; fax: +41 22 791 4857; e-mail:
bookorders@who.int). Requests for permission to reproduce or translate WHO publications – whether for sale or for noncommercial distribution – should be addressed to WHO Press, at the above address (fax: +41 22 791 4806; e- mail: permissions@who.int).
The designations employed and the presentation of the material in this publication do not imply the expression of any opinion whatsoever on the part of the World Health Organization concerning the legal status of any country, territory, city or area or of its authorities, or concerning the delimitation of its frontiers or boundaries. Dotted lines on maps represent approximate border lines for which there may not yet be full agreement.
The mention of specific companies or of certain manufacturers’ products does not imply that they are endorsed or recommended by the World Health Organization in preference to others of a similar nature that are not mentioned.
Errors and omissions excepted, the names of proprietary products are distinguished by initial capital letters.
All reasonable precautions have been taken by the World Health Organization to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either expressed or implied. The responsibility for the interpretation and use of the material lies with the reader. In no event shall the World Health Organization be liable for damages arising from its use. The named authors alone are responsible for the views expressed in this publication.
Summary
Twenty-nine laboratories from 14 different countries participated in a collaborative study to value assign the proposed 4th International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma (09/172) against the 3rd International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma (99/826). Local normal pooled plasmas were included to provide information on any drift of the “plasma” unit. Over 75% of the laboratories obtained potency estimates that had intra-laboratory geometric coefficients of variation (GCV) of less than 5%
when the proposed standards were assayed against the 3rd International Standard, indicating that the participants performed assays for factors II, VII, IX and X reproducibly and with high precision. For all four factors, inter-laboratory variability was low for estimates of the proposed 4th International Standard against the 3rd International Standard (GCV <3%), and higher against the local plasma pools (GCV 5 - 11%). There were no significant differences in potency
estimates from chromogenic and clotting assays and they were therefore combined to give the overall mean potencies. Potency estimates calculated relative to the local normal plasma pools were 1%, 6%, 6% and 5% lower for FII, FVII, FIX and FX respectively when compared to potencies calculated relative to the 3rd International Standard and the differences in the estimates were significant for FVII, FIX and FX. The differences in the potency estimates could not be attributed to degradation of the 3rd International Standard, but the variability of the plasma pools could be an important contributing factor. The excellent agreement between a large number of laboratories contributed to the statistical significance of the discrepancies. It is therefore
recommended that the proposed 4th International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma (09/172) be assigned with potencies for functional activity, calculated relative to the 3rd International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma:
Factor II: 0.89 IU/ampoule Factor VII: 0.99 IU/ampoule Factor IX: 0.86 IU/ampoule Factor X: 0.89 IU/ampoule
Introduction
Blood coagulation factors II, VII, IX and X are vitamin K dependent proteins and are essential for haemostasis. Congenital deficiency of blood coagulation factor IX, haemophilia B, is well documented and this x-linked heritable disorder is found in 1:30,000 males worldwide with no racial preferences. Severity of bleeding correlates with plasmatic factor IX (FIX) activity.
Patients with severe symptoms usually have less than 1% (0.01 IU/ml) FIX activity and
moderate haemophilia B is associated with FIX levels of 1 – 4 %. Factor II, VII and X congenital deficiencies are rare autosomal recessive disorders. Their prevalence in the general population are approximately 1 in 2 million, 1 in 500,000, and 1 in 1 million for FII, VII and X respectively (1). Their frequency is increased 10-20 times where consanguineous marriages are practised (2). Inherited combined deficiency of all factors is very rare and up to 2008, less than 30 cases have been documented (3). Acquired deficiency of vitamin K dependent clotting factors is more common and can be a result of liver disease, dietary vitamin K deficiency and overdose of vitamin K antagonists (eg warfarin). Effective diagnosis, monitoring and treatment of these disorders depend on accurate quantitative measurement of these coagulation factors.
International standards for these four blood coagulation factors, value assigned in international unit (IU) have been used successfully to reduce variability in the estimation of activity within and between laboratories. The current 3rd International Standard used by clinical laboratories,
diagnostic manufacturers and therapeutics producers has been invaluable in ensuring good global agreement in measurement of these four important clotting factors.
The 3rd International Standard (IS) for Factors II, VII, IX and X, Plasma (99/826) was
established by the Expert Committee on Biological Standardisation (ECBS) of the World Health Organisation (WHO) in October 2001. Due to depletion of stock, it is now necessary to replace this IS and this study served to value assign a replacement IS for FII, FVII, FIX and FX against the 3rd IS. In addition, this study also calibrated the International Society for Thrombosis and Haemostasis/Scientific and Standardisation Committee (ISTH/SSC) Secondary Coagulation Standard, Lot# 4. The results for the calibration of this secondary plasma standard will not be discussed in this report and will be detailed in a separate report for the calibration of all of the analytes in Lot#4, available in the first quarter of 2011. In order to assess the relationship between the international unit (IU) and the normal plasma unit of these four coagulation factors, locally collected normal pooled plasmas were included as one of the samples in the study.
Participants
Twenty-nine laboratories from 14 different countries (1 Australia, 1 Austria, 3 Canada, 1 Croatia, 2 Denmark, 3 France, 1 Germany, 1 India, 2 Italy, 1 The Netherlands, 1 Spain, 1 Sweden, 8 UK, 3 USA) agreed to participate in the study, of which 28 returned data in time for the statistical analysis. Unfortunately one laboratory (29) returned data too late to be included in the analysis, however the data from this laboratory are shown in Appendix 4. The participants included 15 clinical laboratories, 10 diagnostics manufacturers, 2 therapeutic manufacturers and 2 regulatory authorities. A list of participants is given in Appendix I at the end of this report. Each laboratory is referred to in this report by an arbitrarily assigned number, not necessarily representing the order of listing in the Appendix.
The Candidate, NIBSC code 09/172
Eighty-five donations of platelet poor normal plasma from the Welsh Blood Service, collected in CPD-adenine and buffered with 0.05 M HEPES were pooled, distributed into glass ampoules, filled and freeze-dried according to guidelines for production of international biological standards (4; 5). Each individual plasma donation has been tested and found negative for anti- HIV 1/2, HBsAg and anti-hepatitis C. This candidate was coded as sample A in this study. The product characteristics are shown in the following table. This proposed standard is intended to be used in the in vitro diagnostics field and it relates to BS EN ISO 17511:2003 Section 5.5.
NIBSC Code 09/172
Presentation Sealed, glass 5 ml DIN ampoules
Number of Ampoules available 20,000
Liquid filling weight (g) Mean=1.1078; Range=1.1000 – 1.1140
CV of fill mass (%) 0.20 (n=849)
GCV p value
FII 3.7 0.9777
FVII 5.8 0.6254
FIX 1.7 0.9932
Homogeneity of the fill by activity:
4 ampoules selected from the beginning, every 5000 amp interval and the end of the fill were assayed against the 2nd IS using clot-based assays. 2 independent assays per ampoule were carried out. In total 24 ampoules and 48 assays per factor. Effect of fill position assessed by
ANOVA of log potencies. FX 1.8 0.9954
Mean dry weight (g, n = 5) 0.1034
Mean head space oxygen (%) 0.14 (n =12)
Residual moisture (%) 0.225 (n = 12)
Storage temperature -20°C
Address of processing facility NIBSC, Potters Bar, EN6 3QG, UK Address of present custodian NIBSC, Potters Bar, EN6 3QG, UK
Samples
The following coded samples were sent to each participant:
S 3rd International Standard for Factors II, VII, IX, X, Plasma (99/826), containing 0.91 IU FII, 1.00 IU FVII, 0.86 IU FIX and 0.93 IU FX per ampoule.
A Proposed 4th International Standard for Factors II, VII, IX, X, Plasma (09/172) containing approximately 1.0 IU per ampoule for all four factors.
P ISTH SSC Lot 3 containing 0.86 IU FII, 0.87 IU FVII, 0.94 IU FIX and 0.86 IU FX per vial.
Q ISTH SSC Lot 4 containing approximately 1.0 IU FII, FVII, FIX and FX per vial.
In addition, the participants were requested to collect normal pooled plasma, which was coded N in the study:
N1 – N2 Fresh normal plasma pools prepared locally according to the instructions in Appendix 2.
Assay Methods
Each participant was requested to perform their routine in-house functional method(s) for the four coagulation factors. A list of reagents, methods and instruments used by the participants is given in Appendix 3.
Factor II: 24 laboratories used a prothrombin time-based clotting method with commercial thromboplastin reagents (sources were rabbit brain, human placenta or recombinant human) and
FII-deficient plasma. Three laboratories used chromogenic assays; two laboratories used Ecarin- based assays, while one laboratory employed a prothrombinase-based method.
Factor VII: A one-stage clotting method was used by 26 laboratories, using thromboplastin reagents from rabbit brain, human placenta or recombinant human thromboplastin, and FVII- deficient plasma. Chromogenic assays were used by three laboratories, two using the Chromogenix Coaset FVII kit and one using the Biophen FVII kit.
Factor IX: 27 laboratories used a one-stage clotting method based on the activated partial thromboplastin time (APTT), using a variety of activators, phospholipids and FIX-deficient plasma. One laboratory used a chromogenic method (Rossix Factor IX kit). One lab (23) carried out antigen assays using the AG Affinity Biological kit.
Factor X: A one-stage clotting assay was used by 26 laboratories, using a variety of
thromboplastin reagents (rabbit brain, human placenta or recombinant human), and FX-deficient plasma. Two laboratories used chromogenic methods, using Russell’s Viper Venom as an activator.
Study Design
Participants were requested to carry out four assays for each factor (FII, FVII, FIX, FX) using fresh ampoules of samples S, A, P, and Q and a normal plasma pool (N) in each assay and to assay factors VII and IX on one set of ampoules and factors II and X on a second set of ampoules. Where feasible, participants were requested to collect fresh plasma on two separate days to prepare two normal plasma pools (N). It was requested that each fresh pool was tested in the study on the day of collection and that a frozen sample of the same pool should be used in the study on a separate day, or, if possible, to use fresh plasma pools for all assays. Some laboratories that were unable to prepare the fresh plasma pools used frozen plasma pools instead.
Within each assay, participants were requested to assay three dilutions of each of the samples S, A, P and Q, in replicate, according to balanced assay designs.
Raw assay data were returned together with calculated estimates for samples A (proposed 4th IS), P (SSC Lot#3), Q (SSC Lot#4) and N (local normal pooled plasma) relative to sample S (3rd IS) from each individual assay.
Statistical Analysis
An independent statistical analysis of raw data was performed at NIBSC. Relative potency estimates were calculated by fitting a parallel-line model (6). All data were plotted and assay validity was assessed both visually and by analysis of variance. Where significant deviations from the model appeared to result from underestimation of residual error, parallelism was confirmed by using the deviations from linearity as an alternative estimate of residual error. All mean potencies given in this report are unweighted geometric mean (GM) potencies. Variability between assays and laboratories has been expressed using geometric coefficients of variation (GCV = {10s-1}×100% where s is the standard deviation of the log transformed potency estimates). Grubbs’ Test (7) was applied to the log transformed laboratory mean estimates in order to detect any significant outliers. Comparisons between methods and with results from
previous studies have been made by unpaired t-test of log transformed laboratory mean estimates.
Factor II assays
A parallel-line model comparing assay response (untransformed in laboratory 11; log
transformed in laboratories 1, 4, 6, 7, 8, 9, 10, 12, 13, 14, 15, 16a, 16b, 17a, 17b, 18b, 19, 20b, 22, 23, 24, 25, 26, 27 & 28a; with a reciprocal transformation in laboratories 2, 3 & 5) to concentration was used for analysis.
Factor VII assays
A parallel-line model comparing assay response (untransformed in laboratories 8, 12 & 13; log transformed in laboratories 1, 2, 3, 4, 5, 6, 7, 9, 10, 11, 14, 17b, 18a, 19, 20b, 21, 22, 23, 24, 25, 27 & 28a; square root transformed in laboratories 15, 16a, 16b, 26 & 28b) to concentration was used.
Factor IX assays
A parallel-line model comparing assay response (untransformed in laboratories 1, 6, 10, 14, 15, 16a, 16b, 18a, 19, 21, 22, 24, 25, 26 & 27; log transformed in laboratories 2, 3, 4, 5, 7, 8, 9, 11, 12, 13, 17a, 20a, 23 & 28a) to concentration was used.
Factor X assays
A parallel-line model comparing assay response (log transformed laboratories 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16a, 16b, 18b, 19, 20b, 22, 23, 24, 25, 27 & 28a; square root
transformed in laboratory 26) to concentration was used.
Results
Assay Data
Twenty eight participants returned a total of 454 assays which comprised 114 FII assays, 120 FVII assays, 115 FIX assays and 105 FX assays. Lab 29 did not return data in time to be included in the analysis and the locally calculated potency estimates are presented in Appendix 4. The individual assay results, together with the geometric mean potencies (GM) and the intra-laboratory variation expressed as %GCV are presented in Appendix 5.
Assay Validity
The majority of assays showed no significant (p<0.01) deviations from the fitted model.
Exceptions are detailed below.
Factor II assays
• In assays 2, 3 and 4 by laboratory 14, sample N was excluded because the response range was not comparable to the other samples,
• Assay 3 by laboratory 17a, was excluded as all samples were non-linear.
Factor VII assays
• In assay 2 by laboratory 7, sample N was found to be non-linear.
• In assays 2, 3 and 4 by laboratory 14, sample N was excluded because the response range was not comparable to the other samples.
Factor IX assays
• In assay 1 by laboratory 2, sample S was found to be non-linear.
• In assay 4 by laboratory 3, samples Q and N were found to be non-linear.
• In assays 1 and 3 by laboratory 13, sample N was found to be non-linear.
• In assays 2, 3 and 4 by laboratory 14, sample N was excluded because the response range was not comparable to the other samples.
Factor X assays
• In assay 1 by laboratory 13, sample S was found to be non-linear.
• Assays 2 and 4 by laboratory 14 were excluded due to non-linearity of the samples.
The proposed 4
thIS for Factors II, VII, IX and X, Plasma (sample A, 09/172)
Intra- and inter- laboratory variability
Estimates of intra-laboratory variability (between assays) for all four factors in sample A are given as geometric coefficients of variation (GCV) for potency estimates relative to sample S (Table 1a). Intra-laboratory variability (GCV) for estimates of sample A, relative to sample S, exceeded 5% in 5, 7, 6 and 3 laboratories for FII, FVII, FIX and FX respectively, with close to 80% of the potency estimates giving GCV lower than 5%. Intra-laboratory variability was generally higher for estimates of sample A, relative to the normal plasma pools, for all factors with GCV exceeding 5% in 12, 14, 13 and 12 laboratories for FII, FVII, FIX and FX respectively (Table 1b).
Variability between laboratories for estimates of sample A relative to the 3rd IS and relative to sample N (normal plasma pools) for each factor, is shown in Tables 1a and 1b and is also
summarized in Table 1c. Estimates of inter-laboratory variability (GCV) for estimates of sample A relative to the 3rd IS were less than 3% for all four factors. However, the inter-laboratory variability (GCV) for estimates of A relative to N (combined fresh and frozen plasma pools) was greater; 5.1%, 11.2%, 8.0% and 8.0% for factors II, VII, IX and X, respectively.
Potency estimates
The overall potency estimates for sample A (proposed 4th IS) were calculated relative to the
assigned values for the 3rd IS and relative to an arbitrary value of 1.0 unit per ml for the local normal pooled plasmas. Geometric mean potency estimates from each laboratory and the overall potency estimates, together with 95% confidence limits, for all four factors are shown in Tables 1a and 1b.
The potency estimates relative to the 3rd IS are also presented in histogram form in Figures 1a – 1d.
The histograms illustrate good agreement between laboratories for sample A relative to the 3rd IS,
for all 4 factors; with the exception of 2 outliers for FII, 1 outlier for FVII and 1 outlier for FX, all the potency estimates were within 3 - 7% of the overall mean potencies. Outlier detection indicated that the FII estimate of potency by laboratory 8 (clotting assay) was significantly higher and laboratory 18b (chromogenic assay) was significantly lower than all other FII estimates; the FVII and FX estimates by laboratory 8 (clotting assays) were significantly higher than the all other FVII and FX estimates. These results were therefore excluded from the calculations of overall mean potency estimates of 0.892, 0.987, 0.863 and 0.887 IU/ampoule for FII, FVII, FIX and FX respectively.
Comparison of the potency estimates by clotting assays with results from the chromogenic assays suggested that the ratios of clotting to chromogenic potencies for all four factors were close to 1 (Table 1d). However, there were insufficient numbers of laboratories carrying out chromogenic assays to statistically compare the potency estimates against those obtained by clotting assays, but it is clear that for all four factors, the individual potency estimates by chromogenic methods were all within the potency range by clotting assays (Table 1a and 1b).
Table 1c shows that the FVII, FIX and FX overall mean potency estimates for sample A relative to the normal pooled plasmas were significantly lower than the overall mean estimates relative to the 3rd IS by 6%, 6% and 5% for FVII (p = 0.007), FIX (p < 0.001) and FX (p = 0.004)
respectively. There was no significant difference between the mean potency estimates for FII relative to 3rd IS and the normal pooled plasmas (p = 0.316).
Locally collected normal plasma pools
Sixteen laboratories carried out assays using fresh plasma pools, prepared from a total of 273 donors. These pools were also used frozen in repeat assays. In addition, five laboratories used frozen plasma pools collected prior to the study, containing plasma from 98 volunteers. All other laboratories used commercially available frozen plasma pools (from at least 376 donors) except one (lab 21), which used lyophilised plasma. In total there were at least 747 donors.
Comparison of fresh and frozen local pools (Table 2a – 2d) tested against sample S, the 3rd IS indicated that there was no significant difference for all four factors (FII, p = 0.866; FVII, p = 0.735; FIX, p = 0.985; FX, p = 0.724 in a paired t-test (Table 2e)) in laboratories that used both locally collected fresh and frozen plasma. The results from lab 21 that used lyophilised plasma were not included in the overall potency estimates for the normal pooled plasmas. The
combined potencies of 1.020, 1.066, 1.069 and 1.050 IU/ml for FII, FVII, FIX, and FX, respectively, were therefore presented (Table 2e). Histograms of individual laboratory mean potency as a percentage of the overall mean potency for all four factors (Figures 2a – 2d) show wider ranges of potency than were found with the other samples in the study, but the agreement between laboratories is reasonable, with inter-laboratory GCV being 6.1%, 10.0%, 7.7% and 7.7% for FII, FVII, FIX and FX respectively (Table 4e).
Antigen measurement for factor IX
Laboratory 23 also carried out FIX antigen measurements and as there is no assigned antigen value for the international standard, the antigen estimates were calculated relative to the normal pooled plasma from this laboratory. Four different frozen pools, each prepared with 8 individual donors, were used in the four separate assays. Table 3 shows the individual assay results together with the geometric mean potencies and the intra-laboratory variation expressed as GCV. The geometric mean potency for samples S (3rd IS) and A (proposed 4th IS) relative to the plasma pools was found to be 0.864 and 0.872 u/ampoule.
Stability study
Accelerated degradation study (8) of the proposed IS, 09/172 was carried out at NIBSC using specific clot-based assays with APTT reagent for FIX and thromboplastin for FII, VII and X. At present, the data from 2 time points (3 and 6 months storage) were insufficient to give a robust prediction of the percentage loss of activity for the four factors. However, the potencies
estimated by clotting assays of the -20°C and the +4 samples after 6 months storage showed that there is no significant loss in potency compared to the -70°C samples (Table 4a; 95% confidence limits for relative potencies include 1.0). This candidate has been prepared in the same manner as the 3rd IS, 99/826 and as shown in Table 4b, for 99/826, there were insignificant loss of activity in the -20°C samples after 8 years storage by comparison with the -150°C material. It is expected that the proposed 4th IS, 09/172 would be as stable as the 3rd IS. Continual real time degradation study of the –20°C against ampoules stored at –150°C and further accelerated degradation study at elevated temperatures will be carried out to monitor the stability of the replacement standard.
Discussion
The primary aim of this study was to value assign a replacement International Standard for Factors II, VII, IX, X, Plasma. It also provided an opportunity to re-examine the continuity of the “plasma unit” for factors II, VII, VII and X by comparison with local normal pooled plasmas.
The majority of the laboratories employed clot-based methods for all four factors. Although statistical assessment cannot be made on differences between potency estimates obtained using chromogenic and clotting assays, the values obtained by chromogenic procedures were all within the potency ranges of the clotting methods. Therefore, the potencies obtained by chromogenic and clotting methods against the 3rd IS or normal plasma pools were combined. It was not possible to investigate the effect of different types of methods on potency estimation since most laboratories employed Prothrombin time based assays for FII, FVII and FX and APTT type of assays for FIX.
There were no observable differences in potencies obtained by the different instruments and reagents for all four factors. Laboratory 23 also carried out antigen assays for FIX and the
calculated data are for information only and have not been included in the overall estimated potency for FIX.
The ratio of FVII potency estimates from the clotting and chromogenic methods can give an indication of the activation status of FVII (9), e.g. a clotting/chromogenic ratio >1.0 indicates that FVII in the test sample is relatively more activated than the reference standard. In the calibration of the 3rd IS against the 2nd IS, a clotting/chromogenic ratio of 0.92 was obtained for the 3rd IS; an indication that the 3rd IS was not activated. In the current study, when sample A, the proposed 4th IS was assayed against the 3rd IS, the clotting/chromogenic FVII potency ratio of 1.03 (Table 1d) was obtained, thus suggesting that the proposed 4th IS is relatively unactivated. Although it is not yet established that the clotting/chromogenic ratios of other coagulation factors can be used as indicators of activation, Table 1d shows that ratios close to 1 were also estimated for FII, FIX and FX.
For all four factors, no significant differences were found between potency estimates calculated relative to the fresh or frozen local plasma pools and it was therefore valid to combine potencies against all plasma pools (Table 2e).
With a few exceptions, intra-laboratory (between assay) GCV were below 5% when the proposed 4th IS was assayed against the 3rd IS, indicating good reproducibility and robustness of the assays for all four factors. When the proposed 4th IS was assayed against the local plasma pools, the intra- laboratory variability was generally higher. This is probably due to the variability in the plasma pools rather than the poor reproducibility of the assays.
Agreement between laboratories was excellent when the proposed 4th IS was assayed against the 3rd IS; the inter-laboratory variability, expressed as GCV, was less than 3%. Greater variability was found for estimates of the proposed 4th IS when assayed against the normal plasma pools. With the exception of FVII, that yielded GCV of just over 11%, the inter-laboratory variability for FII, FIX and FX estimates relative to both fresh and frozen pools combined did not exceed 8% (Table 1c).
As expected, there was higher inter-laboratory variability for estimates of local normal pooled plasmas compared with the other three lyophilised plasma samples, with the GCV being more than 6% for all 4 factors.
With the exception of FII, there were significant differences in the FVII, FIX and FX overall mean potency estimates for the proposed 4th IS when assayed against the 3rd IS and the local normal plasma pools (Table 1c). Mean potency estimates calculated relative to the normal plasma pools were 1%, 6%, 6% and 5% lower for FII, FVII, FIX and FX, respectively, compared to potencies estimated relative to the 3rd IS. One reason for this discrepancy could be the
degradation of the 3rd IS during its lifetime, but the accelerated degradation study of the 3rd IS predicted <0.5% of loss of activity per year for all four factors during its storage at -20°C.
Indeed, assays of the –20°C against the -150°C samples in 2010 after 8 years storage showed a loss of <3% in the FVII activity, while no loss of activity for FII, FIX and FX was detected (Table 4b). Other possible reasons for the drift are the differences in the collection methods of the local pools, the bulk plasma pools used for the filling of the standards, and the geographical spread of the different local pools collected for assays against the IS. There are a large number of laboratories and the low level of variability between them (inter-laboratory GCV of less than 3% for all four factors) also contributes to the statistical significance of this discrepancy.
Proposal to the Participants
There are two main considerations in deciding on the assigned potencies, a) continuity of the IU between the 3rd IS and the 4th IS and, b) maintaining the link between the IU and the "plasma unit".
Significant differences were found for three of the four factors between the potencies estimated relative to the 3rd IS and the normal plasma pools. In the past, when similar discrepancies were found as in the calibration of the 4th IS Factor VIII/von Willebrand Factor, Plasma (97/586) and the 3rd IS for Blood Coagulation Factors II, VII, IX and X, Plasma (99/826), it was agreed that the mean of values relative to the previous International Standard and the normal plasma pools should be assigned. However, as shown by the results of this study, this approach did not prevent further
“drift” of the plasma unit. In addition, the differences in potencies may be due to the extremely low variability of the assay results and therefore the “drift” may be a statistical artefact. Since it is of paramount importance to have continuity of the international unit, it is proposed that the proposed 4th IS be value assigned by potencies obtained against the 3rd IS only. The proposed overall mean potencies for functional activity for the four factors are: FII – 0.89 IU/ampoule; FVII – 0.99 IU/ampoule; FIX – 0.86 IU/ampoule and FX – 0.89 IU/ampoule.
Responses from participants and the experts nominated by the SSC/ISTH Plasma Coagulation Inhibitors Sub-Committee
All participants agreed with the proposals. There were no comments in relation to the analysis or interpretation of the data in the study.
The report has also been circulated to 12 experts nominated by the SSC/ISTH FVIII/FIX Sub- committee and all have agreed with the proposed assigned values for the proposed 4th IS for Blood Coagulation Factors II, VII, IX and X, Plasma. The proposal to accept the preparation coded 09/172 as the WHO 4th IS for Blood Coagulation Factors II, VII, IX and X, Plasma with the recommended assigned values was discussed at the WHO-ISTH Liaison Group Meeting and subsequently endorsed at the SSC Business Meeting, held in Cairo, Egypt on 25 May 2010.
Proposal and recommendation to the ECBS
Sample A, 09/172, be the 4th International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma with the following assigned values for functional activity relative to the 3rd International Standard for Blood Coagulation Factors II, VII, IX and X, Plasma:
Factor II: 0.89 IU/ampoule Factor VII: 0.99 IU/ampoule Factor IX: 0.86 IU/ampoule Factor X: 0.89 IU/ampoule
The Instruction for Use for the proposed Standard, 09/172 is illustrated in Appendix 5.
References
1. Mannucci PM, Duga S, Peyvandi F. Recessively inherited coagulation disorders. Blood 2004; 104:1243-52.
2. Peyvandi F, Duga S, Akhavan S, Mannucci PM. Rare coagulation deficiencies.
Haemophilia 2002; 8: 308-32
3. Weston BW, Monahan PE. Familial deficiency of vitamin K-dependent clotting factors.
Haemophilia 2008; 14: 1209–13
4. Campbell PJ. International biological standards and reference preparations. 1.
Preparation and presentation of materials to serve as standards and reference preparations. J Biol Standardisation 1974; 2: 249-67
5. Recommendations for the preparation, characterization and establishment of international and other biological reference standards (revised 2004). In: WHO TRS, No. 932, 2006, Annex 2. pp.114-119 (section A.7)
6. Finney DJ. Statistical Method in Biological Assay. 3rd Edition. London: Charles Griffin 1978
7. Grubbs F. Procedures for Detecting Outlying Observations in Samples. Technometrics, 1969; 11: 1-21
8. Kirkwood T.B.L. (1977). Predicting the stability of biological standards and products. Biometrics, 33: 736-742.
9. Osterud B. How to measure factor VII and factor VII activation. Haemostasis, 1983;
13(3):161-8
Acknowledgments
We would like to thank the participants of the study and the support of the FVIII/FIX Subcommittee of the SSC/ISTH.
Table 1a: Potency estimates, intra- and inter-laboratory GCV for factors II, VII, IX and X in sample A relative to sample S, the 3rd IS for Factors II, VII, IX and X, Plasma
FII FVII FIX FX
Assay Method Lab
IU/amp GCV IU/amp GCV IU/amp GCV IU/amp GCV
01 0.886 (n=4) 1.1% 0.977 (n =4) 1.6% 0.845 (n=4) 2.3% 0.878 (n=4) 2.0%
02 0.903 (n=4) 3.9% 0.982 (n=4) 0.4% 0.871(n=3) 2.0% 0.896 (n=4) 2.5%
03 0.892 (n=4) 3.3% 1.017 (n=4) 2.6% 0.836 (n=4) 8.0% 0.879 (n=4) 2.3%
04 0.893 (n=4) 1.1% 0.976 (n=4) 2.4% 0.844 (n=4) 1.7% 0.876 (n=4) 2.8%
05 0.891 (n=4) 0.9% 1.000 (n=4) 2.6% 0.846 (n=4) 2.3% 0.883 (n=4) 1.1%
06 0.889 (n=4) 5.6% 0.976 (n=4) 6.3% 0.874 (n=4) 3.7% 0.861 (n=4) 3.6%
07 0.885 (n=4) 2.2% 0.994 (n=4) 6.2% 0.883 (n=4) 3.2% 0.866 (n=4) 1.8%
08 0.961 (n=4) 3.0% 1.104 (n=4) 10.5% 0.923 (n=4) 4.4% 0.946 (n=4) 5.2%
09 0.902 (n=4) 1.8% 0.945 (n=4) 3.1% 0.840 (n=4) 2.7% 0.888(n=4) 2.7%
10 0.904 (n=4) 2.0% 1.015 (n=4) 1.8% 0.919 (n=4) 0.8% 0.895 (n=4) 1.3%
11 0.902 (n=4) 10.5% 0.964 (n=4) 2.7% 0.836 (n=4) 3.5% 0.894 (n=4) 7.3%
12 0.873 (n=4) 3.8% 0.964 (n=4) 6.7% 0.906 (n=4) 9.5% 0.871 (n=4) 3.3%
13 0.900 (n=4) 2.7% 0.964 (n=4) 3.0% 0.885 (n=4) 10.9% 0.897 (n=3) 2.1%
14 0.915 (n=4) 11.3% 1.009 (n=4) 9.0% 0.888 (n=4) 6.7% 0.870 (n=2) N/A 15 0.886 (n=4) 4.2% 0.919 (n=4) 8.1% 0.858 (n=4) 10.8% 0.886 (n=4) 6.1%
16a 0.895 (n=4) 3.9% 1.006 (n=4) 2.6% 0.841 (n=4) 2.5% 0.902 (n=4) 3.2%
16b 0.894 (n=3) 1.7% 0.981 (n=4) 2.8% 0.838 (n=4) 2.0% 0.884 (n=4) 2.4%
18a NT - 1.000 (n=4) 1.8% 0.850 (n=4) 1.2% NT -
19 0.905 (n=4) 1.1% 1.022 (n=4) 2.2% 0.843 (n=4) 1.7% 0.912 (n=4) 4.1%
20a NT - NT - 0.847 (n=4) 3.9% NT -
21 NT - 0.990 (n=4) 2.0% 0.847 (n=4) 0.8% NT -
22 0.907(n=4) 3.9% 1.023 (n=4) 2.6% 0.865 (n=4) 3.7% 0.911 (n=4) 4.2%
23 0.870 (n=4) 1.3% 1.003 (n=4) 1.1% 0.858 (n=4) 2.8% 0.887 (n=4) 1.3%
24 0.893 (n=4) 4.4% 0.984 (n=4) 4.2% 0.867 (n=4) 1.0% 0.890 (n=4) 3.5%
25 0.883 (n=4) 1.5% 1.006 (n=4) 4.0% 0.856 (n=4) 3.1% 0.886 (n=4) 1.2%
26 0.876 (n=4) 2.9% 0.979 (n=4) 2.6% 0.846 (n=4) 0.6% 0.898 (n=4) 1.4%
27 0.892 (n=4) 5.9% 1.007 (n=4) 3.3% 0.863 (n=4) 3.6% 0.878 (n=4) 2.8%
CLO
28a 0.893 (n=4) 4.0% 1.042 (n=4) 3.3% 0.884 (n=4) 5.6% 0.889 (n=4) 2.4%
17a 0.887 (n=3) 3.0% NT - 0.888 (n=4) 2.2% NT -
17b 0.891 (n=4) 0.9% 1.001 (n=4) 8.4% NT - NT -
18b 0.854 (n=4) 6.2% NT - NT - 0.912 (n=4) 3.0%
20b 0.890 (n=4) 1.5% 0.939 (n=4) 3.8% NT - 0.884 (n=4) 2.4%
CH
28b NT - 0.956 (n=4) 1.4% NT - NT -
Overall GM 0.892 (n=27) 0.987 (n=29) 0.863 (n=29) 0.887 (n=26) 95% CL 0.888 – 0.897 0.977 – 0.998 0.854 – 0.873 0.882 – 0.893 Between Lab
GCV 1.2% 2.9% 2.8% 1.5%
n: no of assays; NT: not tested; CLO: clotting assay; CH: chromogenic assay; N/A: not applicable; GM: geometric mean; GCV: geometric coefficient of variation; CL: confidence limits.
Shaded boxes indicate outliers and are excluded from overall GM.
Table 1b: Potency estimates, intra- and inter-laboratory GCV for factors II, VII, IX and X in sample A relative to sample N, the local normal pooled plasma (assumed 1 u/ml)
FII FVII FIX FX
Assay Method Lab
IU/amp GCV IU/amp GCV IU/amp GCV IU/amp GCV
01 0.886 (n=4) 3.3% 1.027 (n=4) 11.1% 0.795 (n=4) 6.0% 0.845 (n=4) 2.9%
02 0.900 (n=4) 4.9% 0.795 (n=4) 15.1% 0.684 (n=3) 4.3% 0.860 (n=4) 4.4%
03 0.800 (n=4) 2.5% 1.075 (n=4) 10.4% 0.749 (n=3) 6.2% 0.760 (n=4) 4.9%
04 0.848 (n=4) 2.1% 0.994 (n=4) 2.7% 0.808 (n=4) 3.8% 0.856 (n=4) 1.3%
05 0.848 (n=4) 1.7% 0.975 (n=4) 4.4% 0.795 (n=4) 3.7% 0.850 (n=4) 2.7%
06 0.815 (n=4) 4.4% 0.843 (n=4) 8.7% 0.815 (n=4) 9.1% 0.723 (n=4) 6.4%
07 0.880 (n=4) 3.3% 0.965 (n=3) 12.1% 0.794 (n=4) 7.3% 0.799 (n=4) 4.8%
08 0.852 (n=4) 2.5% 0.872 (n=4) 3.1% 0.816 (n=4) 3.3% 0.773 (n=4) 3.6%
09 0.927 (n=4) 3.2% 0.846 (n=4) 3.4% 0.820 (n=4) 5.1% 0.949 (n=4) 3.3%
10 0.944 (n=4) 10.7% 1.048 (n=4) 4.3% 0.958 (n=4) 3.3% 0.970 (n=4) 3.3%
11 0.865 (n=4) 10.3% 0.752 (n=4) 3.4% 0.730 (n=4) 5.7% 0.752 (n=4) 11.8%
12 0.863 (n=4) 1.6% 0.779 (n=4) 6.1% 0.766 (n=4) 4.5% 0.800 (n=4) 1.8%
13 0.876 (n=4) 2.4% 0.845 (n=4) 2.2% 0.798 (n=2) N/A 0.795 (n=3) 5.3%
14 0.721 (n=1) N/A 0.835 (n=1) N/A 0.749 (n=1) N/A 0.791 (n=2) N/A 15 0.973 (n=4) 2.0% 1.030 (n=4) 9.0% 0.863 (n=4) 15.3% 1.127 (n=4) 9.8%
16a 0.912 (n=4) 6.3% 0.972 (n=4) 5.4% 0.869 (n=4) 3.7% 0.878 (n=4) 4.7%
16b 0.928 (n=4) 5.5% 0.959 (n=4) 3.0% 0.825 (n=4) 4.6% 0.903 (n=4) 5.6%
18a NT - 0.912 (n=4) 9.8% 0.749 (n=4) 8.8% 0.915 (n=4) 9.4%
19 0.904 (n=4) 5.1% 1.078 (n=4) 2.8% 0.893 (n=4) 2.4% 0.845 (n=4) 2.9%
20a NT - NT - 0.803 (n=4) 12.3% NT -
21 NT - 1.110 (n=4) 1.0% 0.936 (n=4) 1.1% NT -
22 0.825 (n=4) 6.2% 1.012 (n=4) 4.9% 0.891 (n=4) 5.1% 0.873 (n=4) 7.2%
23 0.838 (n=4) 5.2% 0.940 (n=4) 8.5% 0.744 (n=4) 12.9% 0.807 (n=4) 7.4%
24 0.880 (n=4) 5.4% 0.980 (n=4) 9.6% 0.820 (n=4) 4.9% 0.830 (n=4) 5.5%
25 0.859 (n=4) 8.0% 0.987 (n=4) 6.0% 0.789 (n=4) 2.2% 0.848 (n=4) 8.0%
26 0.931 (n=4) 3.7% 0.943 (n=4) 3.2% 0.824 (n=4) 5.8% 0.887 (n=4) 4.3%
27 0.856 (n=4) 5.2% 1.032 (n=4) 11.4% 0.812 (n=4) 5.8% 0.849 (n=4) 4.1%
CLO
28a 0.900 (n=4) 3.5% 0.928 (n=4) 2.6% 0.909 (n=4) 2.8% 0.916 (n=4) 4.0%
17a NT - NT - NT - NT -
17b NT - NT - NT - NT -
18b 0.843 (n=4) 15.7% NT - NT - 0.924 (n=4) 7.0%
20b 0.946 (n=4) 10.0% 0.843 (n=4) 7.3% NT - 0.938 (n=4) 10.9%
CH
28b NT - 0.819 (n=4) 3.1% NT - NT -
Overall GM 0.880 (n=26) 0.933 (n=29) 0.812 (n=28) 0.847 (n=26) 95% CL 0.862 – 0.897 0.896 – 0.971 0.788 – 0.837 0.821 – 0.874 Between Lab
GCV 5.1% 11.2% 8.0% 8.0%
n: no of assays; NT: not tested; CLO: clotting assay; CH: chromogenic assay; N/A: not applicable; GM: geometric mean; GCV: geometric coefficient of variation; CL: confidence limits.
Shaded boxes indicate outliers and are excluded from overall GM.
Table 1c: Summary of potency estimates, 95% confidence limits (CL), inter-laboratory variation (GCV) for sample A against the 3rd IS and the normal pooled plasmas
Vs sample S, 3rd IS Vs sample N, normal pooled plasma Potency estimates
(95% CL) IU/ampoule
Inter- laboratory
GCV
Potency estimates (95% CL) IU/ampoule
Inter- laboratory
GCV
Difference between potencies
t-test, p value FII 0.892 (0.888 – 0.897) 1.2 % 0.880 (0.862 – 0.897) 5.1% 1.4% 0.316 FVII 0.987 (0.977 – 0.998) 2.9% 0.933 (0.896 – 0.971) 11.2% 5.5% 0.007 FIX 0.863 (0.854 – 0.873) 2.8% 0.812 (0.788 – 0.837) 8.0% 6.0% <0.001
FX 0.887 (0.882 – 0.893) 1.5% 0.847 (0.821 – 0.874) 8.0% 4.5% 0.004
Table 1d: Ratios of clotting to chromogenic potency estimates for sample A relative to the 3rd IS
Potency estimates (IU/ampoule)
Clotting Chromogenic Ratio Clotting/Chromogenic
FII 0.893 (n = 24) 0.889 (n = 3) 1.004
FVII 0.990 (n = 26) 0.965 (n = 3) 1.026
FIX 0.862 (n = 28) 0.888 (n = 1) 0.971
FX 0.886 (n = 24) 0.898 (n = 2) 0.987
Table 2a: Comparison of FII potency estimates for fresh and frozen local pooled plasmas relative to the 3rd IS
Lab Method Assay 1 Assay 2 Assay 3 Assay 4 GM
Fresh Frozen All GCV
1 1.066 0.980 0.966 0.995 - 1.001 1.001 4.4%
2 0.940 1.060 1.061 0.957 - 1.003 1.003 6.7%
3 1.115 1.112 1.135 1.098 1.125 1.105 1.115 1.4%
4 1.048 1.040 1.068 1.056 - 1.053 1.053 1.2%
5 1.050 1.030 1.059 1.063 - 1.050 1.050 1.4%
6 1.041 1.120 1.060 1.148 1.050 1.134 1.091 4.7%
7 0.978 0.995 1.043 1.007 - 1.005 1.005 2.7%
8 1.148 1.113 1.114 1.137 1.131 1.125 1.128 1.6%
9 0.961 1.009 0.974 0.946 0.967 0.977 0.972 2.8%
10 0.896 0.888 1.050 1.008 0.970 0.946 0.958 8.8%
11 0.969 1.226 1.003 0.991 0.986 1.102 1.042 11.5%
12 0.997 1.033 0.969 1.047 - 1.011 1.011 3.6%
13 1.078 1.056 0.977 1.002 - 1.027 1.027 4.7%
14 1.177 - - - 1.177 - 1.177 N/A
15 0.928 0.922 0.915 0.879 - 0.911 0.911 2.5%
16a 0.942 0.959 0.999 1.028 0.970 0.993 0.981 4.0%
16b 0.931 0.905 1.030 0.990 0.979 0.947 0.963 6.1%
18a 1.302 0.893 1.020 0.890 1.152 0.891 1.014 19.6%
19 1.051 1.042 0.977 0.940 1.013 0.990 1.002 5.5%
22 1.148 1.108 1.071 1.072 1.109 1.090 1.099 3.4%
23 0.995 1.016 1.133 1.018 - 1.039 1.039 6.0%
24 0.955 1.008 1.040 1.057 1.040 1.006 1.014 4.6%
25 0.972 0.966 1.080 1.097 1.025 1.029 1.027 7.0%
26 1.009 0.962 0.885 0.914 - 0.941 0.941 6.0%
27 0.996 1.057 1.051 1.065 1.023 1.061 1.042 3.1%
28
CLO
0.941 1.016 0.974 1.043 0.957 1.029 0.993 4.7%
20b CH 0.854 0.896 1.011 1.010 0.929 0.951 0.940 8.9%
N/A: not applicable; CLO: clotting assay; CH: chromogenic assay; GM: geometric mean; GCV:
geometric coefficient of variation.
Bold italic text indicates fresh plasma data.
Table 2b: Comparison of FVII potency estimates for fresh and frozen local pooled plasmas relative to the 3rd IS
Lab Method Assay 1 Assay 2 Assay 3 Assay 4 GM
Fresh Frozen All GCV
1 0.803 0.999 1.005 1.015 - 0.951 0.951 11.9%
2 1.393 1.341 1.014 1.228 - 1.235 1.235 15.2%
3 1.052 1.005 0.837 0.906 0.938 0.954 0.946 10.8%
4 0.971 0.999 0.963 0.997 - 0.982 0.982 1.9%
5 1.122 0.997 0.986 1.004 - 1.026 1.026 6.2%
6 1.021 1.195 1.181 1.248 1.098 1.221 1.158 9.1%
7 1.171 - 0.938 1.010 - 1.035 1.035 12.0%
8 1.462 1.188 1.234 1.195 1.343 1.191 1.265 10.3%
9 1.118 1.194 1.059 1.100 1.088 1.146 1.117 5.1%
10 0.964 0.945 0.959 1.008 0.961 0.976 0.969 2.8%
11 1.200 1.308 1.309 1.312 1.253 1.310 1.281 4.5%
12 1.171 1.267 1.291 1.228 - 1.238 1.238 4.4%
13 1.190 1.160 1.090 1.125 - 1.141 1.141 3.9%
14 1.119 - - . 1.119 - 1.119 N/A
15 1.024 0.908 0.807 0.844 - 0.892 0.892 11.0%
16a 1.104 1.062 1.007 0.968 1.054 1.014 1.034 6.0%
16b 1.083 1.037 1.002 0.973 1.042 1.004 1.023 4.7%
18a 1.031 0.990 1.184 1.197 1.105 1.089 1.097 10.1%
19 0.938 0.978 0.925 0.953 0.931 0.965 0.948 2.4%
21 0.912 0.888 0.891 0.878 - - 0.892 1.6%
22 1.058 0.979 1.034 0.976 1.046 0.977 1.011 4.1%
23 0.982 1.095 1.163 1.037 - 1.067 1.067 7.5%
24 1.065 1.042 0.945 0.971 0.945 1.025 1.005 5.8%
25 0.996 0.967 1.020 1.097 1.008 1.030 1.019 5.6%
26 1.097 0.967 1.036 1.058 - 1.038 1.039 5.5%
27 0.860 0.877 1.073 1.119 0.961 0.991 0.976 14.5%
28a
CLO
1.115 1.119 1.130 1.129 1.122 1.124 1.123 0.7%
20b 1.082 1.044 1.235 1.105 1.156 1.074 1.114 7.5%
28b CH
1.154 1.211 1.148 1.158 1.151 1.184 1.167 2.5%
N/A: not applicable; CLO: clotting assay; CH: chromogenic assay; GM: geometric mean; GCV:
geometric coefficient of variation.
Bold italic text indicates fresh plasma data.
Shaded boxes indicate exclusion from overall potency estimate due to the use of lyophilised plasma.
Table 2c: Comparison of FIX potency estimates for fresh and frozen local pooled plasmas relative to the 3rd IS
Lab Method Assay 1 Assay 2 Assay 3 Assay 4 GM
Fresh Frozen All GCV
1 1.138 1.068 0.965 1.089 - 1.063 1.063 7.2%
2 - 1.300 1.206 1.316 - 1.273 1.273 4.9%
3 1.066 1.109 1.106 - 1.086 1.109 1.093 2.2%
4 1.014 1.015 1.021 1.133 - 1.045 1.045 5.6%
5 1.053 1.135 1.041 1.033 - 1.065 1.065 4.4%
6 1.062 1.037 1.190 1.010 1.124 1.023 1.073 7.5%
7 1.172 1.215 1.032 1.045 - 1.113 1.113 8.5%
8 1.255 1.057 1.084 1.135 1.166 1.095 1.130 7.9%
9 1.000 1.014 1.063 1.021 1.031 1.017 1.024 2.7%
10 0.967 0.914 0.984 0.976 0.975 0.944 0.960 3.4%
11 1.093 1.148 1.160 1.185 1.126 1.166 1.146 3.5%
12 0.995 1.282 1.269 1.207 - 1.182 1.182 12.5%
13 - 1.154 - 1.246 - 1.199 1.199 N/A
14 1.138 - - - 1.138 - 1.138 N/A
15 1.062 0.949 0.997 0.975 - 0.995 0.995 4.9%
16a 0.947 0.956 0.973 0.992 0.960 0.974 0.967 2.1%
16b 0.991 0.998 1.021 1.053 1.006 1.025 1.015 2.8%
18a 1.038 1.069 1.202 1.246 1.117 1.154 1.135 9.2%
19 0.977 0.944 0.927 0.931 0.952 0.937 0.945 2.4%
20a 0.969 0.967 1.190 1.109 1.074 1.036 1.055 10.8%
21 0.930 0.899 0.904 0.887 - - 0.905 2.0%
22 1.006 0.956 0.972 0.947 0.989 0.951 0.970 2.7%
23 1.034 1.237 1.273 1.082 - 1.152 1.152 10.6%
24 1.092 1.082 1.059 0.999 1.059 1.057 1.057 4.1%
25 1.115 1.066 1.055 1.108 1.085 1.087 1.086 2.8%
26 1.088 1.054 0.950 1.020 - 1.027 1.027 6.0%
27 1.019 1.111 1.000 1.123 1.009 1.117 1.062 6.1%
28a
CLO
0.937 1.011 0.946 0.998 0.941 1.004 0.972 3.9%
N/A: not applicable; CLO: clotting assay; CH: chromogenic assay; GM: geometric mean; GCV:
geometric coefficient of variation.
Bold italic text indicates fresh plasma data.
Shaded boxes indicate exclusion from overall potency estimate due to the use of lyophilised plasma.
