Working Group on Coagulation Standards

29 June 2006
Oslo Kongressenter, Norway

Co-Chairs: Jane Lenahan and Tony Hubbard

Status of Lot #2

Dr Hubbard reviewed the despatch of Lot #2 from May 2001 up to expiry at the end of June 2006. Lot #2 has been used steadily over the past 5 years and a total of 43,700 vial have been despatched to 41 different organisations in 12 different countries. Approximately 22,000 vials have been used in QA surveys by UK NEQAS and CAP. There are approximately 1100 vials of Lot #2 remaining.

Assessment of the stability of Lot #2 has been carried out using three different approaches:

• accelerated degradation study over 6 years on FVII and FVIII activity has produced robust mean predictions of loss which did not exceed 0.011% per year for vials stored at -20 °C,
• a real-time comparison of FVII and FVIII activity in vials stored at -20 °C vs vials stored at -70 °C for 6 years has indicated no relative loss in the -20 °C vials,
• a comparison of estimates for Lot #2 obtained in the original calibration exercise (in 1999) and those obtained from re-assays during the calibration of Lot #3 (in 2005) has indicated no significant differences for any of the analytes.

The overall conclusion from this data is that Lot #2 has been extremely stable during its lifetime and we should expect similar stability for Lot #3 considering the similarity of its manufacture.

Calibration of Protein C for Lot #3

Dr Gray presented the calibration for Protein C which was carried out as part of the collaborative study for the proposed WHO 2 nd IS Protein C Plasma. Combination of 25 estimates for Protein C function produced an overall mean of 0.89 IU/ml with low inter-laboratory variability (GCV) of 4.2 %. Combination of 9 estimates for Protein C antigen gave an overall mean of 0.89 IU/ml and inter-laboratory variability of only 2.2%. It was proposed that the value of 0.89 IU/vial be assigned to Lot #3 for both analytes. Calculation of estimates for Lot #3 relative to the proposed assigned values for the WHO 2 nd IS produced identical mean estimates of 0.89 IU/vial for function and antigen. This indicates that the proposed assigned values for Lot #3 will not require re-assessment when the proposed WHO 2 nd IS Protein C is established. Lot #2 was also included in the calibration exercise and there were no significant differences between the estimates obtained in the present study and the original calibration in 1999. This indicates that Lot #2 has been extremely stable over the last 6 years.

Status of Lot #3

Dr Hubbard presented details of the manufacture of Lot #3 followed by a summary of the calibration for 19 analytes which involved over 1000 separate assays and is now completed. Results from the initial stability testing, based on Factors V, VII, VIII and XI, have indicated FV to be the most labile with a mean predicted % loss per year at -20 °C of 0.184%. An expiry date of “end December 2014” for Lot #3 was proposed; this was based on the upper 95% confidence limit of loss for FV which indicated a “shelf-life” of approximately 11 years. However, based on the current despatch of Lot #2 it is likely that stocks of Lot #3 will be exhausted by 2012.

A draft label for Lot #3 was presented which recommended storage between -20 to -40 °C; this was included to discourage storage in -80 °C freezers. However, it was noted that long-term studies on Lot #2 had indicated no loss of activity at -70 °C. The Working Group agreed that the recommended storage temperature should be amended to -20 to -70 °C.

A draft “Instructions For Use” for Lot #3 was presented. This includes details of the manufacturing together with information on storage and reconstitution as well as “Caution”, “Liability” and “Safety” sections. The “Customer Feedback Form” will also be included as an integral part of the IFU. The final version will include both NIBSC and ISTH logos.

The minimum order size for Lot #3 will remain at 100 vials and shipping will continue by courier at the cost of the customer. The price of Lot #3 will be $5 per vial. Labelling and despatch of Lot #3 is scheduled to commence in July 2006.

Calibration of Lot #3 for tPA antigen

Dr Longstaff presented the results of a collaborative study to evaluate the calibration of Lot #3 (and a potential new WHO IS, 94/730) for tPA antigen. Estimates for tPA in Lots #2 and #3, relative to kit standards, were associated with large inter-laboratory variability (GCV >60%) and this was slightly improved by the re-calculation of estimates relative to a common standard , 94/730. There were clearly reproducible differences between methods. However, the inter-laboratory variability of estimates for Lot #3 could be greatly reduced if estimates were re-calculated using a correction factor for each different method. Despite the overall large inter-laboratory variability similar mean values of approximately 3 ng/ml and 25 ng/ml were obtained for Lot #3 and 94/730, respectively, in different studies. Dr Longstaff indicated that the proposal to calibrate 94/730 (and Lot #3) will be reviewed by the Fibrinolysis sub-committee. Pending this outcome the proposal to calibrate Lot #3 will be circulated to the Executive Board of the Working Group.

Data from proficiency studies and use of Lot #3 as a support tool by EQA schemes

Dr Kitchen presented data from EQA surveys on FVIII, FIX and Protein S estimation which demonstrated how inter-laboratory discrepancies can be linked to the use of different reference plasmas and particular reference plasma/method combinations. The inclusion of the SSC Standard as a test sample in EQA schemes not only provides information on the validity of the assigned values on the SSC Standard but also a check on the calibration of commercial calibrants. Tests on Lot #2 indicated significant differences between some commercial calibrant plasmas in the estimation of FV and FXI. Several EQA studies testing Lot #2 for FVIII:C have returned values higher than the assigned and this appears to be related to the failure of manufacturers to assimilate the re-alignment of the IU for FVIII:C which occurred with the establishment of the WHO 4 th IS FVIII/VWF Plasma. These data indicate that the SSC Standard is a very valuable tool in the resolution of potency estimation problems which may be related to inaccurately calibrated commercial references. It was proposed that a small amount of the SSC Standard ( Lot #3) could be made available to QA organisers for use in problem-solving. The Working Group agreed to this use in principle, however, it will be necessary to explore the feasibility of this use with regard to the number of vials required and the logistics of despatch before approval can be sought from the ISTH Executive Committee.

Lot #3 and the JCTLM database

De Gray reviewed the terms of reference for the JCTLM Working Group which will allow Lot #3 to be registered as an “internationally certified reference material” and thereby be exempt from the requirement for CE marking. Lot #2 was nominated in Cycle I of the exercise and Lot #3 will be nominated in Cycle IV. Dr Gray indicated that evidence of commutability for Lot #3 may be required for all 19 analytes in the testing of patient samples as part of this process. This would be an enormous undertaking and Dr Gray will confirm the exact requirements.