EXPERT COMMITTEE ON BIOLOGICAL STANDARDIZATION Geneva, 24 to 28 October 2005
ASSIGNMENT OF POTENCY TO THE WHO 1ST INTERNATIONAL STANDARD FOR
BLOOD COAGULATION FACTOR XI IN PLASMA, HUMAN (04/102) AND CALIBRATION OF BLOOD COAGULATION FACTOR XI IN
SSC SECONDARY PLASMA STANDARD LOT #3
Elaine Gray, Stephen Thomas and Peter Rigsby* Division of Haematology and *Biostatistics Section
National Institute for Biological Standards and Control Potters Bar, Hertfordshire, UK.
SUMMARY Twenty-seven laboratories from 11 countries took part in a collaborative study to assign a potency value to a proposed World Health Organisation (WHO) 1st International Standard for Blood Coagulation Factor XI, Plasma, Human (04/102) and also to calibrate the Scientific Standardisation Committee (SSC) secondary plasma standard Lot#3 for factor XI activity. The proposed WHO 1st IS was assayed against locally collected fresh normal
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All reasonable precautions have been taken by WHO to verify the information contained in this publication. However, the published material is being distributed without warranty of any kind, either express 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
W`HO/BS/05.2017 Page 2 plasma pools (n = 42). Intra-laboratory variability (GCV) was found to range from 2.0 – 13.8 %, with the GCV for majority of laboratories being less than 10 %. Good inter-laboratory agreement (GCV = 7.2 %) was also obtained. Comparison of results from fresh and respective frozen normal plasma pools showed no significant differences between estimates from fresh and frozen plasma pools. It was therefore proposed that the potency for the proposed WHO 1st IS for FXI, Plasma be based on overall mean value obtained from assays relative to both fresh and frozen normal plasma pools (number of pools = 48, number of donors >2000) and this was found to be 0.86 IU/ampoule. Similar intra- (GCVs ranged from 2.3 – 13.0 %) and inter-laboratory variability (GCV = 7.7 %) were found for the SSC Lot#3 against the normal plasma pools. When assayed against the proposed WHO IS, the inter-laboratory variability was found to be much lower at 3.1 % It is proposed that the WHO 1st IS for FXI, Plasma be assigned with the value estimated against both fresh and frozen normal plasma pools:
0.86 IU/ampoule and that the FXI potency in SSC Lot#3 be assigned with the value estimated against the proposed WHO 1st IS for FXI, Plasma (04/102):
102 % (101 – 103 %) of the proposed WHO 1st IS for FXI, Plasma
This recommendation has been considered and approved by the Scientific and Standardization Committee (SSC) of the ISTH (International Society on Thrombosis and Haemostasis) in August 2005. 1. INTRODUCTION Factor XI deficiency is known as haemophilia C and was first described by Rosenthal et al in 1953 (1). It is inherited as an autosomal recessive bleeding disorder. Homozygotes have functional activity of less than 0.1 U/mL, while heterozygotes have activity of 0.25 – 0.7 U/mL or are within normal ranges. Factor XI deficiency is most frequently observed in Ashkenazi Jews (2) and 9.1 % of this ethnic group are carrier of either type II or type III mutations and 0.22 % is expected to have severe FXI deficiency (3). Iraqi Jews have been found to have only type II mutation with a predicted 3% heterozygosity and 0.03% homozygosity (3). Factor XI deficiency patients have also been reported in English, African-American, German, Indian, Italian, Korean, Japanese, Chinese, Portuguese, Sweden, Yugoslav. Arab and Iranian populations (4 - 10). All 3 types of mutations lead to a decrease in FXI antigens and only one patient so far has been reported to have dysfunctional FXI protein (5). There are two plasma derived concentrates available for the replacement therapy and these concentrates are calibrated against different plasma pools. Currently, a WHO international standard (IS) is not available for calibration of FXI activity and laboratories employ locally collected normal plasma pools or commercially available frozen or freeze-dried plasmas as references. From a recent UK National External Quality Assessment Service (NEQAS) for Blood Coagulation
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survey which included 259 participants who calibrated the same plasma samples against different commercial calibrants, the CV was found to be 17 % (11). Clearly, a common standard is required to improve inter-laboratory agreement for measurement of FXI activity. The main aim of this study was to assign an internationally agreed potency value in international unit (IU) to a batch of normal pooled plasma, processed according to guidelines for the production of WHO IS, relative to locally collected normal plasma pools. This standard is intended for use in the in vitro diagnostic area as described in ISO 17511 and will be available for the calibration of commercial reference plasmas, secondary and in-house standards. In accordance with value assignment of potencies for other coagulation factors and inhibitors, the potency of the 1st International Standard for Factor XI, Plasma was also estimated relative to the activity of locally collected normal plasma pools which were arbitrarily assigned a value of 1.0 unit per ml. This approach allows the IU to approximate to the population mean value for FXI functional activity and so facilitates the definition and understanding of the normal and pathological ranges. In addition, this study also gave an opportunity to calibrate the Scientific and Standardisation Committee (SSC) secondary plasma standard Lot#3 for FXI activity. The SSC Lot#3 consists of a freeze-dried pooled normal plasma in rubber-sealed glass vials and is a secondary plasma standard prepared under the auspices of the SSC/ISTH Working Group on Coagulation Standards; it is intended for use by in vitro diagnostic manufacturers as a working standard to calibrate their commercial kits. The inclusion of the SSC Lot#3 standard also allows the evaluation of commutability for the primary use of the IS in the calibration of secondary reference plasmas using different methods and reagents 2. PARTICIPANTS Twenty-seven laboratories from 11 countries (3 Australia, 2 Austria, 1 Canada, 2 France, 1 Germany, 1 Israel, 2 Italy, 1 New Zealand, 1 The Netherlands, 8 UK, 5 USA) and comprised of 4 regulatory institutes, 12 clinical laboratories, 5 plasma products therapeutics producers and 6 in-vitro diagnostic manufacturers, participated and returned data for the study. A list of participants is given in Appendix I. Each laboratory is referred to in this report by an arbitrarily assigned number, not necessarily representing the order of listing in the Appendix.
W`HO/BS/05.2017 Page 4 3. SAMPLES 3.1 Candidate WHO 1st International Standard Factor XI, Plasma (04/102) Bulk Material The Proposed WHO 1st IS was prepared from a plasma pool derived from 19 normal healthy donors (United Kingdom Blood Service, North London Blood Transfusion Centre). Blood was collected using conventional venepuncture into CPD-adenine anticoagulant at a nominal ratio of 63 ml anticoagulant to 450 ml whole blood. The donations underwent leuko-filtration followed by two centrifugation steps after which the plasma was frozen rapidly and stored at -70 oC until the day of ampoule filling. Individual donations were tested and found negative for HBsAg, antibodies to HIV-1 and -2 and antibodies to HCV. The donations were also tested as mini-pools and found negative for the presence of HCV RNA using a PCR technique. On the morning of the fill the plasma units were thawed in a waterbath at 37 oC and pooled. Glycine and a buffering agent HEPES (N-[2-Hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid) and were added to the pooled plasma at a final concentration of 1 % w:v and 40 mmol/L respectively. To avoid activation of FXI, polyethylene vessels were used for storage and transport of the pooled plasma. The pooled plasma was also maintained at room temperature after thawing and throughout filling of the material into siliconised glass ampoules. The final ampouled material was produced as described by Campbell (12) and the finished product summary is as follows:
Code number 04/102
Presentation Sealed, siliconized glass 5 ml DIN ampoules
Number of ampoules available 3000
Date filled 15 April 2004
Precision of fill – CV of fill mass (%, n = 57) 0.25
Residual moisture after lyophilisation and secondary desiccation (%, n = 6) 0.0489
Mean dry weight (g, n=6) 0.1004
Storage conditions -20 °C
Address of processing facility NIBSC, Potters Bar, EN6 3QG, UK
Address of present custodian NIBSC, Potters Bar, EN6 3QG, UK
WHO/BS/05.2017 Page 5
Activation status of the proposed WHO IS The non-activated partial thromboplastin time (NAPTT) is known to be sensitive to activated clotting factors especially factor XIa and so it was used to assess the activation status of the finished product. Clotting times longer than 250s indicate the samples to be relatively unactivated. The NAPTT obtained for the proposed WHO IS and other freeze-dried plasma preparations were compared to the 2 NIBSC frozen normal plasma pools (Frozen pool a and Frozen pool b), each pool consisted of 8 donors.:
Mean (n=6) SD 04/102, Proposed WHO IS 298 22
99/826, 3rd IS for FII, VII, IX and X, Plasma 242 18
SSC Lot#2 470 46
SSC Lot#3 298 22
Frozen pool a 389 40
Frozen pool b 361 29 Although the mean NAPTT for the proposed WHO IS for FXI, 04/102 was not as long as the frozen normal plasma pool, the plasma is relatively unactivated and deemed to be a suitable reference for FXI activity. The SSC Lot#3 gave similar results to the proposed IS. 3.2 SSC/ISTH Secondary Coagulation Standard Lot #3 The SSC/ISTH Secondary Coagulation Standard Lot #3 (SSC Lot #3) was prepared by a commercial manufacturer from a pool of 55 litres of normal plasma collected using apheresis. SSC Lot #3 consists of rubber-sealed, screw-capped vials each containing 1 ml of pooled normal plasma, freeze-dried. 3.3 Coding of the samples Coded samples included in the study were: A - Proposed WHO 1st IS Factor XI Plasma, (04/102) B - SSC/ISTH Secondary Coagulation Standard Lot #3 N1, N2 - fresh normal plasma pools prepared locally F1, F2 - frozen normal plasma pools prepared locally H - in-house standard if available
W`HO/BS/05.2017 Page 6 As the quality of the local normal plasma pool is very important for accurate estimation of FXI activity, the participants were requested to prepare both the fresh and frozen plasma pools strictly according to the protocol provided (Appendix II). In total, 42 fresh pools with a total of 351 donors (176 male and 175 female) were collected by the participants for the study. Four labs used pre-prepared frozen pools with a total number of greater than 2000 donors. 4. COLLABORATIVE STUDY
4.1 Assay Design: Details of the assay design and raw data analysis were as stated in the protocol which is attached as Appendix II. Briefly, each participant was requested to carry out 4 independent assays on 4 sets of samples and to follow the suggested balanced assay designs as described in the study protocol. 4.2 Assay Methods: Each participant was requested to perform their routine in-house method for factor XI. With the exception of Lab 23 that employed a chromogenic assay, all participants estimated FXI functional activity by the APTT. Eleven APTT reagents from 6 different manufacturers and substrate FXI deficient plasma from 9 different plasma providers were used. The details of the instruments and reagents used by the participants are listed in Table 1. 4.3 Statistical Analysis: All assays were analysed using the principles of multiple parallel line bioassay (13) comparing transformed assay response to log concentration. An analysis of variance gives an assessment of the linearity and parallelism of the response lines. All results were also plotted and the validity of the assays was assessed both visually and by analysis of variance, with tests for deviations from linearity and parallelism being performed at the 1 % level of significance. For the majority of assays, the untransformed assay responses were found to give the best linearity with log concentration. For one laboratory, a log transformation of the responses was found to be more appropriate. For another laboratory, responses were transformed to percentages relative to the estimated upper and lower limits of the dose-response curve and weighted regression of logit response on log concentration was used. All mean potencies given in this report are unweighted geometric mean potencies. Corresponding 95 % confidence limits (CL) have also been calculated. Variability between assays (intra-laboratory variability) and laboratories (inter-laboratory variability) have been expressed using geometric coefficients of variation (% GCV) (14). Detection of outliers was performed using Duncan’s Multiple Range Test (15).
WHO/BS/05.2017 Page 7
5 RESULTS 5.1 Assay data and assay validity: Except for Labs 17 and 24, which returned only 2 assays each, all other laboratories returned 4 assays as requested. A total of 104 sets of assay results were returned for analysis, 100 APTT assays and 4 chromogenic assays. It was possible to assess linearity and parallelism in almost all laboratories. Exceptions were Labs 10 and 23, where only a single response at each dilution was provided for all samples. The majority of assays passed all validity criteria. Deviations from linearity and/or parallelism were detected in 13 out of 104 assays. In most of these cases, these deviations were probably due to inadequate replication in the assay design. In the first two assays by laboratory 25, samples A, B and N were all non-parallel to one another, so no results are given for these assays. In the second two assays by this laboratory, sample A was non-parallel to N, so only results for sample B are shown. In two assays by laboratory 19, sample A was non-linear or non-parallel to B and N, so no results for sample A are shown here. Following visual assessment, all other assays were included. 5.2 Potency estimates for the proposed WHO 1st IS (A) relative to normal pools (N1, N2, F1, F2) Twenty laboratories collected local normal plasma pools and prepared fresh and respective frozen pools according to the study protocol (Appendix II), one laboratory (Lab 20) collected 2 fresh pools on 2 separate days, but did not prepare and use the respective frozen pools. The other 6 laboratories used pre-prepared local frozen pools or commercially available freeze-dried pooled plasma. In order to determine whether results from assays using frozen plasma pools should be included in the assignment of potency for the proposed WHO standard, initial analyses were carried out on results from the 20 laboratories using freshly collected and respective frozen pools. Detailed values of individual laboratory mean potencies of sample A, relative to local normal plasma pools, N1, N2, F1 and F2 for these 20 laboratories are listed in Table 2a and Figure 1 together with 95 % confidence limits and intra-laboratory (between assays) variation expressed as % GCV. Due to non-parallelism, results for sample A from Lab 25 were not included for analysis. Assuming the potency of the normal plasma pools to be 1 U/mL, the mean estimates from individual laboratories were calculated to range from 0.77 to 0.98 U/ampoule. The intra-laboratory variability was good, % GCV ranging from 2.0 to 13.8 (3 labs >10 %, 8 labs 5 – 10 %, 8 labs <5 %). No outliers were detected. The overall mean potency was estimated to be 0.85 U/ampoule and the inter-laboratory variability (GCV) was found to 6.6 %.
W`HO/BS/05.2017 Page 8 Estimates relative to the frozen pooled plasma were compared to potency values obtained using the respective fresh pool and the results are expressed as a ratio of frozen over normal pool (Table 2b). A ratio close to 1 indicates that there is little difference between the results from assays using fresh or frozen pools. As Lab 20 did not use the respective fresh and frozen pools, their results could not be used for analysis of fresh vs frozen but the data was included in subsequent analysis of the overall mean potency. The overall potency ratios for both F1/N1 and F2/N1 were 1.00 with 95 % CL of 0.98 to 1.03. This indicates that there is no significant difference between results obtained against fresh or frozen plasmas. This was confirmed by paired t-test (p = 0.95). Further analysis was therefore performed including all assays using frozen plasma pools (Table 2c). Combination of all the results against all fresh and frozen pools gave an overall mean potency of 0.86 U/ampoule for the proposed WHO 1st IS (Sample A). The 95 % CL were 0.83 - 0.88 U/ampoule. Agreement between laboratories was good with an inter-laboratory variability (GCV) of 7.2 %. 5.3 Potency estimates for SSC Lot#3 (Sample B) relative to normal pools (N1, N2, F1, F2) and the proposed WHO 1st IS The individual laboratory geometric mean FXI potencies for SSC Lot#3 together with the intra-laboratory variability listed as % GCV for the 20 laboratories that collected fresh normal plasma pools are shown in Table 3a and Figure 2. Assuming the potency of the normal plasma pools to be 1 U/mL, the mean estimates from individual laboratories were calculated to range from 0.75 to 1.04 U/vial. The intra-laboratory variability was good, % GCV ranging from 2.3 to 13.0 (2 labs >10 %, 4 labs 5 – 10 %, 13 labs <5 %). No outliers were detected. The overall mean potency was estimated to be 0.86 U/vial and the inter-laboratory variability (GCV) was found to be 7.9 %. As with Sample A, the mean potency ratio for Sample B obtained for frozen relative to fresh plasma pool was calculated to be 1 (Table 3b). Analysis of data including all fresh and frozen plasma pools assays gave an overall mean potency estimate of 0.87 U/vial, with a 95 % CL of 0.84 to 0.89 U/vial and a GCV (inter-laboratory variability) of 7.7 % (Table 3c). Potency measurements of SSC Lot#3 estimated relative to a nominal value of 100 % assigned to the proposed WHO 1st IS are shown Table 3d and Figure 3. Intra-laboratory variability (GCV) ranged from 1.2 to 12.9 %, with 19 laboratories obtaining GCV <5 %. Overall mean potency estimate was found to be 102 % (range = 95 to 107 %; 95 % CL = 101 to 103 %) and excellent inter-laboratory agreement was indicated by a low GCV of 3.1 %. 5.4 Potency estimates of in-house standards relative to normal pools (N1, N2, F1, F2) Thirteen laboratories included their in-house standards in the study and details of the source of the in-house standard are shown in Table 4a. Calculated results for individual laboratory against known assigned potencies of the in-house standards are shown in Table 4b. Assuming the normal plasma pools to be 1 U/mL, the in-house standards estimated values ranged from 0.81 to 1.04 U/mL. For the majority of the in-house
WHO/BS/05.2017 Page 9
standards, the estimated values from this study were found to be different from their assigned value and the differences were up to 22 %. 5.5 Variability of the local normal plasma pools Estimates of the FXI potencies in the different local normal plasma pools were recalculated relative to the proposed WHO 1st IS using the proposed assigned value of 0.86 IU/ampoule. Table 5 shows that the estimates of the local pools ranged from 0.86 – 1.12 IU/mL with a mean of 1.00 IU/mL. 6 STABILITY STUDIES 6.1 Accelerated degradation study Stability of the Proposed WHO 1st IS has been assessed in an accelerated degradation study which involves the potency estimation of ampoules stored at elevated temperatures (4, 20, 37, 45 oC) relative to ampoules stored at -150 oC. The study was carried out in one laboratory (NIBSC), using a one-stage assay based on the APTT. The observed relative loss of potency is analysed using the Arrhenius equation in order to provide a prediction of loss per year for ampoules stored at various temperatures (16). Estimates on % predicted loss of activity per year based on activities assessed at 3 different time points over a period of 11 months storage are as follows:
Storage temperature (°°°°C)
% Potency relative to sample
stored at -150 °°°°C (11 months)
Predicted loss per year (%)
-20 - 0
+4 - 0.034
+20 96.3 0.747
+37 81.8 13.327
+45 68.6 -
There was no predicted loss of activity for samples stored at -20oC, the storage temperature of the proposed IS, thus indicating that the preparation is exceedingly stable and suitable for long term use as an International Standard. The predicted loss for samples stored at +20oC is 0.7% per year and this supports the shipment of ampoules at ambient temperature. The accelerated degradation study and real time monitoring will continue for the lifetime of the standard.
W`HO/BS/05.2017 Page 10 6.2 Stability 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. The stability of coagulation factors in plasma standards, after reconstitution, is mainly affected by two components - the surface of the container and the storage temperature. Unlike other WHO IS for blood coagulation factors it is recommended that upon reconstitution, the standard should either be transferred to a plastic tube or retained in the siliconised ampoule at room temperature (18 - 22 oC) in order to prevent cold activation of FXI. Results from NIBSC indicated no significant change in of FXI clotting activity when the reconstituted material was stored at room temperature in the siliconised ampoules for over 2 hours. However, users will be advised that local validation will be necessary if the reconstituted standard is stored under different conditions. The use of frozen aliquots of the Proposed WHO International Standard cannot be recommended since the effect of freezing and thawing, under local conditions, on the FXI activity is unpredictable. 7 DISCUSSION The main aims of this study were to assign and calibrate the functional activity value to the proposed WHO International Standard for Blood Coagulation Factor XI, Plasma, Human (04/102) and to the SSC Secondary Coagulation Factor Plasma Standard Lot#3. Following the tradition of assignment of potencies to the first international standards for other coagulation factors and inhibitors, locally collected normal plasma pools were also used as the reference for the assignment of potency to the 1st IS for plasma FXI. As FXI is known to activate easily, the participants of the study were requested to process their local normal plasma pools according to a protocol recommended by the members of the SSC Subcommittee for Plasma Kallikrein-Kinin System. The protocol was designed to minimise activation and specified that the normal pools had to be assayed within one hour of collection. This meant that it was difficult for the participants to prepare the fresh pools of more than 8 donors and that fresh frozen pools had to be used for part of the study. As the effect of freeze-thawing on FXI functional activity was not clear, the estimates of FXI activity in the proposed WHO 1st IS relative to the fresh and the respective frozen pools were compared to establish whether there was significant loss or activation during the freeze-thawing process. The comparative results (Tables 2b and 3b) show clearly that there was no significant difference between the overall potency estimates of the proposed WHO 1st IS and the SSC Lot#3 relative to the fresh or the respective frozen pools and the potency ratio of frozen against fresh pools was found to be 1.00 for both the proposed IS and the SSC plasma standard. Therefore, results from assays using both fresh and frozen pools were used for the calculation of the combined overall potency estimate for the proposed WHO 1st IS.
WHO/BS/05.2017 Page 11
Relative to the local normal plasma pools, the intra-laboratory (between assay) variability was low, with the majority of the GCVs being less than 10 % (Tables 2c and 3c). There was also good inter-laboratory agreement - the inter-laboratory variability, expressed as GCV, being 7.2 and 7.7 % for the proposed IS and the SSC Lot#3 respectively. This inter-laboratory variability was much lower than the 17 % found by the UK NEQAS for Blood Coagulation in one of their recent exercises (November 2004) when different local pooled plasmas and commercial plasmas were used as references (1). The present study has also shown clearly that assays of test samples against different local normal pools or commercially available reference materials can yield up to a 22 % difference in activity value for the same test sample (Tables 4 and 5). As demonstrated by the GCV of 3.1 % obtained from the calibration of the SSC Lot#3 against the proposed WHO 1st IS (Table 3d) in the current study, the availability of a common standard such as the WHO IS will help to improve inter-laboratory agreement. There was no clear relationship between the APTT reagents and/or the substrate deficient plasmas and the potencies estimated. As only one laboratory employed a chromogenic method, it was not possible to determine whether there was any bias towards one type of method. 8 CONCLUSION AND RECOMMENDATION Proposed FXI potency for the WHO 1st IS (04/102) It is proposed that the WHO 1st IS for FXI, Plasma be assigned with the value estimated against both fresh and frozen normal plasma pools:
0.86 IU/ampoule
Nineteen out of the 27 participants have agreed with the proposal. The other 8 have yet to respond. This recommendation has been considered and approved by the Scientific and Standardization Committee (SSC) of the ISTH (International Society on Thrombosis and Haemostasis) in August 2005.
W`HO/BS/05.2017 Page 12 REFERENCES: 1. Rosenthal R, Dreskin O, Rosenthal N. New hemophilia-like disease caused by
deficiency of a third plasma thromboplastin factor. Proc Soc Exp Biol Med. 1953; 82: 171-174
2. Seligsohn U. High gene frequency of factor XI (PTA) deficiency in Ashkenazi Jews. Blood. 1978; 516:1223 – 1228
3. Shpilberg O, Peretz H, Zivelin Ad, et al. One of the two common mutations causing factor XI deficiency in Ashkenazi Jews (type II) is also prevalent in Iraqi Jews, who represent the ancient gene pool of Jews. Blood. 1995; 85: 429 – 432
4. Saito H, Ratnoff OD, Bouma BN, Seligshon U. Failure to detect variant (CRM+) plasma thromboplastin antecedent deficiency: a study of 125 patients in several ethnic background. J Lab Clin Med. 1985; 106: 718-722
5. Zivelin A, Ogawa, T, Bulvik S, et al. Severe factor XI deficiency caused by a Gly 555 to Glu mutation (factor XI – Glu555): a cross reactive material positive variant defect in factor XI activation. J Thromb Haemost. 2004
6. Imanaka Y, Lal K, Nishimura T, et al. Identification of two novel mutations in non-Jewish factor XI deficiency. Br J Haematol. 1995; 90:916 -920
7. Wistinghausen B, Reischer A, Oddous C et al. Severe factor XI deficiency in an Arab family associated with a novel mutation in exon 11. Br J Haematol. 1997; 99: 575-577
8. Martincic D, Zimmerman SA, Ware RE, et al. Identification of mutations and polymorphisms in factor XI genes of an African American family by dideoxyfingerprinting. Blood. 1998; 92: 3309-3317.
9. Sato E, Kawamata N, Kato A, Oshimi K. A novel mutation that leads to a congenital factor XI deficiency in a Japanese family. Am J Hematol. 2000; 63:165 -169.
10. Wu WM, Wang HL. Wang XF, et al. Identification of two novel factor XI nonsense mutations Trp228stop and Trp383stop in a Chinese pedigree of congenital factor XI deficiency. Zhonghua Xue Ye Xue Za Zhi. 2003; 24:126 – 128
11. Steve Kitchen. UK NEQAS for Blood Coagulation. Personal communication. 12. 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
13. Finney DJ. Statistical methods in biological assay. 3rd edn London: Charles Griffin 1978
14. Kirkwood,TBL. Geometric means and measures of dispersion. Biometrics 1979; 35: 908-909
15. Duncan, DB. T-Tests and intervals for comparisons suggested by the data. Biometrics. 1975, 31, 339 – 359
16. Kirkwood TBL, Tydeman M. Design and analysis of accelerated degradation tests for the stability of biological standards II. A flexible computer program for data analysis. J Biol Standardisatioin. 1984. 12: 207 -214
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9 ACKNOWLEDGEMENTS We would like to acknowledge the participants who made this collaborative study possible, the support of the International Society on Thrombosis and Haemostasis/Scientific and Standardisation Committee for Plasma Kallikrein-Kinin System and the Working Group on Coagulation Standards and the UK NEQAS for Blood Coagulation for sharing information on their survey results.
W`HO/BS/05.2017 Page 14 Table 1a: Methods and reagents used by the participants in the study
Table 2a: Potency estimates for FXI (U/ampoule) for sample A, the proposed WHO 1st IS FXI, plasma (04/102) relative to normal fresh and respective frozen plasma pools
W`HO/BS/05.2017 Page 16 Table 2b: Ratios of potency estimates for Sample A, the proposed WHO 1st IS FXI, plasma (04/102) relative to normal fresh and respective frozen plasma pools
Table 2c: Potency estimates for FXI (U/ampoule) for sample A, the proposed WHO 1st IS FXI, plasma (04/102) relative to normal fresh and all frozen plasma pools
W`HO/BS/05.2017 Page 18 Table 3a: Potency estimates for FXI (U/vial) for sample B, the SSC secondary coagulation factor plasma standard Lot#3, relative to normal fresh and respective frozen plasma pools
Table 3b: Ratios of potency estimates for Sample B, the SSC secondary coagulation factor plasma standard Lot#3, relative to normal fresh and respective frozen plasma pools
W`HO/BS/05.2017 Page 20 Table 3c: Potency estimates for FXI (U/vial) for sample B, the SSC secondary coagulation factor plasma standard Lot#3 relative to normal fresh and all frozen plasma pools
Table 3d: Estimates of FXI potency of Sample B, the SSC secondary coagulation factor plasma standard, expressed as % of Sample A, the proposed WHO 1st IS for FXI, Plasma
W`HO/BS/05.2017 Page 22 Table 4a: Sources of in-house standards
Lab Source of In House Standard
1 Dade Behring Standard Human Plasma 2 Helena Specialty Assayed Reference Plasma 3 Technoclone Coagulation Reference 4 Technoclone Coagulation Reference 5 In house plasma pool 9 MDA Verify Ref Plasma
11 Technoclone Coagulation Reference 15 Dade Behring Standard Human Plasma 16 Dade Behring Standard Human Plasma 18 Diagnostica Stago Unicalibrator 22 Diagnostica Stago Unicalibrator 24 MDA Verify reference plasma 27 MDA Verify reference plasma
WHO/BS/05.2017 Page 23
Table 4b: Potency estimates of in-house standards relative to the normal plasma pools
Assay
Lab 1 2 3 4
Geometric Mean GCV(%) Assigned
Potency % difference
estimated/assigned
1 1.10 0.94 1.05 0.91 1.00 9.8 0.93 +7.5
2 0.93 0.94 0.91 0.88 0.91 2.8 -
3 0.83 0.83 0.83 0.82 0.83 0.5 1.04 -20.2
4 0.80 0.80 0.84 0.80 0.81 2.3 1.04 -22.1
5 0.94 0.98 0.95 0.97 0.96 1.5 - -
9 0.87 0.82 0.91 0.79 0.85 6.5 - -
11 1.06 1.04 1.01 1.04 1.04 2.1 1.04 0
15 1.15 0.93 1.09 0.95 1.03 11.1 - -
16 0.83 0.88 0.82 0.95 0.87 6.9 - -
18 1.01 0.84 0.96 0.80 0.90 11.7 1.00 -10.0
22 1.09 1.04 1.07 0.98 1.04 4.9 1.00 +4.0
24 0.94 1.00 - - 0.97 - 1.00 -3.0
27 1.14 0.90 1.05 0.85 0.98 14.4 - -
W`HO/BS/05.2017 Page 24 Table 5: Estimates of normal plasma pools in IU/mL relative to Sample A, the proposed WHO 1st IS for FXI, Plasma, assuming an assigned value of 0.86 IU/ampoule
Fig 1: Mean laboratory potency estimates for Sample A, the proposed WHO 1st IS for FXI, Plasma relative to the normal plasma pools, expressed as % of the overall mean value
Num
ber o
f lab
s
0
1
2
3
4
5
6
7
8
9
10
Potency (as % of overall mean estimate)
80 90 100 110 120 130
10 9
13
3
4
6
26
24
15
22
5
16
20
2 8
17
21
27
11
12
14 23 1 19
W`HO/BS/05.2017 Page 26 Fig 2: Mean laboratory potency FXI estimates for Sample B, SSC Secondary Coagulation Plasma Standard Lot#3, relative to the normal plasma pools, expressed as % of the overall mean value
Num
ber o
f lab
s
0
1
2
3
4
5
6
7
8
9
10
Potency (as % of overall mean estimate)
80 90 100 110 120 130
25 9 3
4
6
10
13
22
15
16
26
5
14
24
2
21
27
20
8
12
11
17
19
23 1
WHO/BS/05.2017 Page 27
Fig 3: Mean laboratory potency FXI estimates for Sample B, SSC Secondary Coagulation Plasma Standard Lot#3, relative to Sample A, the proposed WHO 1st IS for FXI, Plasma, expressed as % of the overall mean value
Num
ber o
f lab
s
0
1
2
3
4
5
6
7
8
9
10
Potency (as % of overall mean estimate)
80 90 100 110 120 130
14 19
22
3
4
9
15
16
18
5
6
11
12
17
21
23
26
27
2
8
1
7
10
13
20
24
W`HO/BS/05.2017 Page 28 Appendix I: List of Participants Dr Rosella Bader, Angelo Bianchi Bonomi, Milano, Italy Dr Assunta Bellisario & Dr Alessandro Starace, Hardis S.p.A. – Group Kedrion, S. Antimo, Naples, Italy Ms Angela Birchall & Mrs Anne Abrahams, Bio Products Laboratory, Elstree, UK Dr Paula Bolton-Maggs & Ms Doreen Shanks, Manchester Royal Infirmary, Manchester, UK Dr Stephen Duff & Mr Marc Boylan, Precision BioLogic, Dartmouth, Canada Ms Elizabeth Duncan & Ms Cheryl Casey, Institute of Medical and Veterinary Science, Adelaide, Australia Mr Didier Elminger, Octapharma SA, Lingolsheim, France Dr Ty Fanella, DiaPharma Group Inc., West Chester, USA Dr David Gailani & Dr Gail Baxter, Vanderbilt University, Nashville, USA Dr Judith Gillissen & Dr Kathryn Bradley, George King Biomedical, Overland Park, USA Dr Paul Harper & Dr Lee Glogoski, Auckland City Hospital, Grafton, New Zealand Dr Andreas Hunfeld & Mrs Sylvia Rosenkranz, Paul-Ehrlich-Institut, Langen, Germany Ms Leesa Ivey, Fremantle Hospital, Western Australia Dr Wynne Jones & Dr Simon Harris, Kent & Canterbury Hospital, Canterbury, UK Dr Barbara Kerbl, Technoclone GmbH, Vienna, Austria Dr Steven Kitchen & Ms Annette Bowyer, Royal Hallamshire Hospital, Sheffield, UK Dr Daniel Lawson & Dr Paul Braun, bioMérieux, Durham, USA Ms Lydia Lim & Mr Michael Taylor, St Vincent's Hospital, Mebourne, Australia Dr Ian Mackie & Mr Andrew Lawrie, Haematology Department, UCL, London, UK Dr Koen Mertens & Dr Carel Eckmann, Sanquin Blood Supply Foundation, Amsterdam, The Netherlands
WHO/BS/05.2017 Page 29
Dr Guy Rautmann, European Pharmacopoeia, Strasbourg, France Professor Uri Seligsohn, Dr Ariella Zivelin & Dr Ilia Tamarin, Chaim Sheba Medical Center, Tel Hashomer, Israel Mr Kampta Sukhu, Churchill Hospital, Oxford, UK Dr Stephen Thomas, NIBSC, South Mimms, UK Dr Kathleen Trumbull & Dr Barbara Phillips, Instrumentation Laboratory, Lexington, USA Dr Josef Weinberger & Dr Dagmar Krause, Octapharma Pharmazeutika Produktionsges.m.b.H, Vienna, Austria Ms Lynne Weller, NIBSC, South Mimms, UK
W`HO/BS/05.2017 Page 30 Appendix II – Study Protocol INTERNATIONAL COLLABORATIVE STUDY ON THE 1ST INTERNATIONAL STANDARD FOR FACTOR XI AND CALIBRATION OF FXI VALUE FOR SSC
LOT#3
PROTOCOL FOR THE COLLABORATIVE STUDY (CS278) 1 SAMPLES INCLUDED IN THE ASSAYS A - Proposed 1st IS Factor XI Plasma - provided B - SSC/ISTH Secondary Coagulation Standard Lot #3 - provided N1, N2 - fresh normal plasma pools prepared locally (see section 5) F1, F2 - frozen normal plasma pools prepared locally (see section 5) H - in-house standard if available 2 STORAGE AND RECONSTITUTION OF SAMPLES A AND B Store the unopened ampoules and vials of A and B at -20 oC or below. Allow the ampoules and vials to warm to room temperature before reconstitution. Tap gently to ensure that all of the contents are in the lower part of the ampoules and vials. Reconstitute by adding 1.0 ml of distilled water. Dissolve the contents with gentle agitation at room temperature. When reconstitution is complete transfer the entire contents to stoppered polypropylene tubes and store at room temperature during the assay period. 3 GENERAL PLAN OF THE STUDY You are requested to carry out 4 independent assays by your routine method using fresh ampoules and vials for each assay. For each independent assay, estimates of FXI functional activity must be carried out on freshly reconstituted ampoules/vials. Two independent assays must be carried out using fresh normal plasma pools (N1 and N2) and 2 independent assays using the corresponding frozen pools (F1 and F2).
Assay number
Normal pool Ampoules of A and B
1 N1 1 2 N2 2 3 F1 3 4 F2 4
WHO/BS/05.2017 Page 31
4 ASSAY DESIGN All three preparations (A, B, N or F) are included in each of the 4 assays. A minimum of 3 dilutions of each preparation should be tested, in replicate, within each assay. Please follow a balanced assay design such as the optimal 18-place assay described below. In the following design, each letter refers to a separate set of three or more dilutions where A, A' and B, B' etc. refer to fresh sets of dilutions (replicates) made from the same ampoule. Design for 18-place assay – (not including in-house standard, H) Assay 1 A B N1 N1' B' A' Assay 2 B N2 A A' N2' B' Assay 3 B A F1 F1' A' B' Assay 4 F2 B A A' B' F2' Design for 24-place assay – (including in-house standard, H) Assay 1 A B N1 H H’ N1' B' A' Assay 2 B N2 H A A' H’ N2' B' Assay 3 H A B F1 F1’ B’ A’ H’ Assay 4 F2 H A B B’ A’ H’ F2’ 5 COLLECTION AND PREPARATION OF FRESH AND FROZEN NORMAL PLASMA Two fresh normal plasma pools N1 and N2 should be collected on 2 separate days. Aliquots of each of the fresh pools should be frozen as soon as the fresh pool has been prepared, to produce frozen plasma pools F1 and F2. Hence 2 assays will be carried out using fresh pools and 2 assays will be performed using the corresponding frozen pools. Donors: Normal healthy volunteers, excluding pregnant women and women taking oral contraceptives. Take blood from no less than 8 different donors but as many as possible for each pool; ideally from an equal number of males and females. The aim is to have as many different donors as possible from each laboratory. Anticoagulant: Venous blood should be collected with minimal stasis using a 19 or 21 gauge needle/butterfly. Collect 9 volumes of blood into siliconised glass or polypropylene containers (vacutainers are acceptable) containing one volume of 0.109 mol/L tri-sodium citrate or a mixture of tri-sodium citrate and citric acid with a total citrate concentration of 0.109 mol/L. If using vacutainers, 0.105 mol/L tri-sodium citrate is acceptable. Mix 5 – 6 times by gentle inversion. Collected blood should be stored at
W`HO/BS/05.2017 Page 32 room temperature (18 – 20 °C) until centrifugation and should not be stored for more than 1 hour before centrifugation. Centrifugation: Spin each individual donation at room temperature, 2000 g for 15 minutes. Remove plasma using a plastic pipette into polypropylene/siliconised tubes and centrifuge the plasma again at room temperature 2000 g for 15 minutes. Pool the plasma from the individual donors. Storage: For the fresh pools, N1 and N2, keep the plasma pool in stoppered polypropylene/siliconised tubes at room temperature and assay within an hour of preparation. Freeze aliquots of each pool to prepare F1 and F2 for subsequent assays. Assays using fresh frozen pools F1 and F2: On day of assay, thaw frozen plasma pool at 37 °C, mix gently and assay immediately. 6 RESULTS Please return your raw assay data on the enclosed results sheets to allow analysis at NIBSC. Please ensure that your results are presented as true raw data (eg. clotting time, optical density) rather than as % or units relative to an in-house standard. You are also invited to calculate your own estimates for A and B relative to N1, N2, F1 or F2 (see enclosed results sheet). Please return the results sheets and questionnaire to: Dr Stephen Thomas, Division of Haematology, NIBSC, Blanche Lane, South Mimms, Potters Bar, Herts, EN6 3QG United Kingdom. Fax: +44 (0) 1707 646730 E-mail: [email protected]
