SCIENTIFIC SUBCOMMITTEE SESSION
7 July 2007 Palexpo, Geneva, Switzerland

Working Group on Coagulation Standards

Chair: A. Hubbard ( UK)

Review of Lot #3 (A Hubbard, NIBSC, UK)

Background details for Lot #3 were presented to the WG (source and filling, assigned values, despatch conditions). The SSC/ISTH Secondary Coagulation Standard Lot #3 became available in July 2006, following expiry of Lot #2, and 12,990 vials have since been despatched. The majority of Lot #3 was despatched to manufacturers (7480 vials to 21 different companies in 9 different countries) and a lesser but still considerable amount was ordered by EQA schemes (total of 4900 vials to UK NEQAS and the College of American Pathologists). A relatively small amount was used in calibration exercises and research projects (610 vials). The use in the first year exceeded the annual average for Lot #2 (approx 9,000 vials) and will have a significant impact on the lifetime of Lot #3 if it continues at this rate. Representatives from the EQA scheme UK NEQAS indicated that they will require less vials over the coming year. At least 24 months, but preferably 30 months, notice will be needed to complete the processes of tender and calibration for a replacement to Lot #3. The stability of Lot #3 is under continuous assessment through an accelerated degradation study. Results from testing performed by NIBSC and the Royal Hallamshire Hospital, Sheffield have indicated that Lot #3 is extremely stable with predicted losses per year at -20 ^oC of less than 0.1% for coagulation factors V, VII, VIII and XI.

Calibration of Lot #3 for t-PA antigen and PAI-1 antigen (C Longstaff, NIBSC, UK)

The results of an international collaborative study to calibrate the proposed 1 st International Standard t-PA antigen (94/730) were presented; this study also included the calibration of Lot #3. A mean value of 25 ng/ml (n=12) was obtained for 94/730 (spiked plasma) and a value of 3.0 ng/ml (n=14) for Lot #3 (normal plasma). The study participants and the Fibrinolysis sub-committee have agreed that the mean values should be assigned to 94/730 and Lot #3. The proposed IS (94/730) will be submitted to the Expert Committee on Biological Standardisation of WHO for establishment in October 2007. Following the establishment of 94/730 the proposal to assign a value of 3.0 ng/ml to Lot #3 will be circulated to the Executive Board and to SSC. It was noted that the level of t-PA antigen in Lot #3 was close to the lower limit of detection for some methods and this may compromise its use as a reference standard.

Estimates for PAI-1 antigen in Lot #3 from a study in 2005 and a more recent study (2007) were presented. The recent study included 7 different methods and also included 3 freshly collected plasma pools (low, medium, high) as well as a second freeze-dried, pooled, normal plasma (06/053). Lot #3 and 06/053 were found to have higher PAI-Ag content than the 3 fresh pools and there was considerable inter-laboratory variability for all samples (GCV range 49 – 78%; Lot #3 GCV 53%). After harmonisation of the individual laboratory results, relative to a consensus mean regression curve, the inter-laboratory variability was reduced to a GCV range of 6 – 35 % (Lot #3 GCV 14%). There was some discussion over the definition of the “normal” level for PAI-1 antigen. In the absence of an International Standard it was considered premature to assign a value for PAI-1 antigen to Lot #3.

Experience of EQA schemes with Lot #3

UK NEQAS (S Kitchen, UK)

Lot #3 was distributed as an anonymous sample in 2 surveys in 2006 for the testing of analytes FII, FV, FVII, FXI, Protein C, Protein S and Antithrombin. Median FV and FXI estimates differed from the assigned values by 6 – 21 % and 0 – 14 % respectively (depending on reference plasma) and this probably indicates that the recently established International Standards for FV and FXI have not been used for assignment to commercial calibrant plasmas. It is expected that this deviation should be reduced in the future as the IS and Lot #3 are used. Median FII estimates were close to the assigned value (-3 to +4 % depending on reference plasma). Median estimates for FVIII:C relative to 3 reference plasmas were close to the assigned value whereas estimates relative to 2 others differed by 12 and 20 %. Similar discrepancies were also found with other test samples and investigations concluded that this was not caused by mis-calibration of Lot #3. One of the manufacturers has since re-labeled the FVIII:C content of their reference plasma. Median estimates for Antithrombin, by method, recovered the assigned value (difference -1%). Median free Protein S antigen estimates differed by -1 to +8 % of the assigned value depending on method. Overall median Protein S activity differed by 3% from the assigned value and by less than 10% for 3 individual methods; however, one method differed by 19% and was a cause for concern. The median estimates for Protein C activity (chromogenic) differed from the assigned value by 4% overall and by 10% or less depending on the reference plasma used. Out of 168 centres performing a full phenotypic thrombophilia screen 85.5% reported that Lot #3 was normal.

College of American Pathologists (M Cunningham, USA)

Lot #3 was included in a CAP Coagulation Education Survey in 2007 where analytes are measured at the discretion of the participating laboratories. A total of 33 analytes were measured. Mean estimates differed by less than 5% from the assigned value for most analytes. A larger difference (7.1 %) for FV may indicate that the assigned values to the IS and Lot #3 have not been fully assimilated by the manufacturers. Larger overall mean differences from the assigned values (13% and -16%) were also observed for Protein C function and VWF:CB respectively; in the latter case it should be noted that the mean was derived from only 3 estimates. Numerous un-labeled analytes were also measured and these were generally associated with the highest inter-laboratory variability (eg. Prothrombin fragment 1.2, CV 201%; Thrombin-Antithrombin complex, CV 196%). It was noted that this information could be extremely useful to other sub-committees as an aid in the prioritisation of future standardisation needs.

Manufacturer’s perspective
Use of International Standards and SSC Plasmas at Instrumentation Laboratory
(K Trumbull, Instrumentation Laboratory, Lexington, MA, USA)

This presentation described how four departments used International Standards and the SSC Plasma standard. House standards are calibrated directly against the relevant WHO IS with inclusion of Lot #3 to verify the assignment. Different Lots of control plasmas are calibrated relative to the House standard with inclusion of Lot #3 for verification. Lot #3 is also included in the development/verification of new or modified reagents or parameters in specific instrument systems. Lot #3 may also be used in complaint investigations which could be related to shipping stress or incorrect use of products.

Additional uses for Lot #3 (A Hubbard, NIBSC, UK)

Three additional uses for Lot #3, approved by the Executive Board, were presented.

• Lot #3 will be included in a small multi-center study to assess the inter-laboratory variability of VWF propeptide estimation and to also provide a common source of calibration for local references with the objective of improving harmonisation of results including normal and pathological reference ranges.
• Lot #3 has been included in a large multi-centre study on the standardisation of the thrombin generation test (TGT). Although Lot #3 is not proposed as a TGT reference preparation its characterisation in the study could be useful in ensuring continuity between different lots of TGT reference plasmas. Additionally Lot #3 has been used to characterise the proposed TGT reference plasmas by measuring numerous analytes.
• It has been agreed that Lot #3 can be used by EQA schemes for trouble-shooting purposes, specifically the investigation of anomalous results from laboratories identified through EQA surveys. EQA schemes will purchase Lot #3 for this purpose and be responsible for the despatch to laboratories. This use will be limited to a maximum of 1,000 vials of Lot #3 in the first year and will be reviewed at the next WG meeting in July 2008.

Lot #3 and the JCTLM database (E Gray, NIBSC, UK)

As an internationally certified reference material Lot #3 is exempt from the need for “CE marking”. In order to maintain this status it is necessary to include Lot #3 on the JCTLM (Joint Committee on Traceability in Laboratory Medicine) database of higher order reference materials. Lot #3 has been submitted for inclusion and this will reviewed in October 2007. There is also a requirement to demonstrate commutability for Lot #3 in the measurement of analytes in patient samples. It is hoped that this can be addressed by reference to the inclusion of Lot #3 in EQA surveys.

Thrombin Generation Test Reference plasmas (E Gray, NIBSC, UK)

A large multi-centre study on the Thrombin Generation Test has demonstrated that inter-laboratory variability can be considerably reduced when results are calculated relative to a common reference plasma. This study will lead to a proposal to establish a reference plasma for the TGT, possibly as a SSC reference. Subject to this approval the WG on Coagulation Standards may be requested to act as “curator” for the reference plasma and to oversee the storage, despatch and stability requirements.