Plasma Coagulation Inhibitors
Friday, 30 June 2006
Oslo Kongressenter, Norway
Chair: Elaine Gray, UK
Co-Chairs: F. Bernardi, Italy; K. Suzuki, Japan; H.C. Whinna, USA
WHO International Standards
Chair: HC Whinna
Proposed international standards for Protein C. E Gray
Twenty laboratories from 10 countries took part in a collaborative study to assign potency values to 2 proposed World Health Organisation (WHO) international standards: the 2nd International Standard for Protein C, Plasma, Human (02/342) and the 1st International Standard for Protein C, Concentrate, Human ( 04/252) and also to calibrate the Scientific Standardisation Committee (SSC) secondary plasma standard Lot#3 for Protein C functional activity and antigen. The proposed candidates were assayed against the 1st International Standard for Protein C, Plasma, Human (86/622) and locally collected normal plasma pools (n = 38). Intra-laboratory variability (GCV) was found to range from 0.3 – 21.3%, with the GCV for the majority of laboratories being less than 10%. Good inter-laboratory agreement, with the majority of the GCV being less than 10% (GCV range = 1.4 – 15.6 %) was also obtained. Comparison of results against local plasma pools with results against the 1st IS for Protein C, Plasma, Human, 86/622 showed significant differences between estimates. Considering the demonstrated stability of the 1st IS for Protein C, the more likely reason for the discrepancy is the change in Protein C levels in normal plasma pools over time. In order to preserve the continuity of the international unit, it was therefore proposed that the potency values for the proposed WHO 2nd IS for Protein C, Plasma, Human 02/342, the proposed 1st International Standard for Protein C, Concentrate, Human, 04/252, the SSC Lot#3 be based on overall mean value obtained from assays relative to the 1st International standard for Protein C, Plasma, Human, 86/622 only. All participants agreed with the proposal for the assignment of potencies to the candidate 2 nd IS for Protein C, Plasma. Nineteen out of the 20 participants agreed with the recommendation for the Protein C, Concentrate. However, due to discrepancy of performance of the candidate and their in-house material, one participant would like the proposed standard to be labelled with both the clotting and chromogenic values. The recommendation to establish the concentrate standard will be deferred until results from further study is available to resolve this issue.
Proposed International Standard for Protein S, Plasma. T Hubbard
Twenty laboratories from 11 countries have participated in the collaborative study to calibrate the proposed WHO 2nd IS Protein S Plasma (03/228) for total antigen, free antigen and function. Estimates of intra-laboratory variability were acceptably low with geometric coefficients of variation (GCV) below 10 % for 80/96 data sets. Only 5 out of a total of 189 assays were excluded from the analysis. The inter-laboratory variability (GCV) for estimates relative to the WHO 1st IS (range 3.33 - 9.02 %) was lower for each parameter compared to estimates relative to the local normal plasma pools (range 7.88 - 13.16 %). Largest inter-laboratory variability was found for the estimates of free antigen, particularly those incorporating PEG precipitation steps. There were no significant differences between the estimates calculated relative to the WHO 1st IS and the local normal pools for any of the three parameters and the overall mean estimates were extremely close (total antigen 0.83 vs 0.82; free antigen 0.81 vs 0.81; function 0.77 vs 0.75 respectively). These results are very encouraging in that they have confirmed the original definition of the IU and are also consistent with the stability of the WHO 1st IS and its suitability for the calibration of the proposed WHO 2nd IS. It is proposed that the WHO 2nd IS Protein S Plasma (03/228) be assigned the mean estimates calculated relative to the WHO 1st IS as follows:
Total antigen 0.83 IU/ampoule; Free antigen 0.81 IU/ampoule; Function 0.77 IU/ampoule
All 20 participants have agreed to the proposed potencies and it is planned to submit the calibration to WHO ECBS in October for formal establishment.
Replacement of 2nd International Standard for Antithrombin, Concentrate. E Gray
The stock level of the 2nd International Standard for Antithrombin, Concentrates is running low and has to be replaced within the next 18 months. There is a call for donation of candidate materials and participants for the forthcoming collaborative study which will be initiated in November 2006.
Reference Materials and Methods for Antithrombin
Update on activities of theISTH/IFCCJoint Committee on Standardisation of Coagulation tests (C-SCT): Working Party on reference materials and methods for antithrombin: Pilot study on primary reference methods for antithrombin. E Gray/CM Jackson
A pilot study has now been initiated to evaluate a primary method for antithrombin activity. This is based on the titration of antithrombin activity against factor Xa. Purified antithrombin and human and bovine factor Xa are now being sourced as critical reagents for this assay.
Joint session with the Control of Anticoagulation
Global Coagulation/Haemostatic Tests
Chair: HC Whinna, Dr van den Besselaar
Report on the international collaborative study on fluorogenic methods for Thrombin generation test. E Gray on behalf of the Working Party on Thrombin Generation Tests
An international collaborative study involving 39 laboratories was carried out to investigate the sources of variability in thrombin generation tests. It was concluded that the concentration and source of trigger (tissue factor and/phospholipids) were the major determinant of intra- and inter-laboratory variability. Pre-analytical variables also influence the comparability of the test. By normalising the results against a “reference” plasma, the variability could be reduced. The Working Party (WP) on Thrombin Generation Tests therefore propose to develop and evaluate a reference plasma for thrombin generation test and to assess the concentrations and sources of tissue factor for use in the study of different clinical plasma samples. The WP would like to work with the experts in the FVIII/FIX subcommittee to define TGT protocols for the testing of haemophilia plasma.
Monitoring endogenous thrombin generation in healthy indiduals and patients after a first acute myocardial infarction ( Clinical validation of the Calibrated Automated Thrombogram) R van Oerle/ H Spronk
Several studies have shown a persistent hypercoagulable state following and acute chest syndrome, by coagulation activation markers. Theoretically, quantification of endogenous thrombin generation potential (ETP) may offer a more detailed analysis of the intrinsic properties of an individual’s plasma with regard to hypercoagulability, but none of the commercial methods have been rigorously validated in normal individuals as well as in patients. We performed a series of pre-clinical standardization studies of the calibrated automated thrombogram in normal volunteers and applied this method in consecutive patients with a first acute myocardial infarction (AMI).
Thrombin generation studied in healthy volunteers (n=139) showed significant differences in peak height, time to peak and time to tail between males and females, whereas lag time and endogenous thrombin potential (ETP) were comparable. Over a three month period repeated measurements showed unaltered thrombin generation.
Thrombin generation was studied in 55 patients after a first AMI on admission, after 4 days, 3 and 6 months. On admission, patients showed increased thrombin generation: ETP-ratios were elevated compared to healthy persons (1.238, SD:0.264 vs. controls 1.041, SD:0.155, 95%CI:1.015-1.067) and peak values were (1.479, SD:0.344 vs. controls 1.007, SD:0.177, 95%CI:0.977-1.037). Antithrombotic treatment with low molecular weight heparin (LMWH) dose dependently suppressed ETP and peak height (r²=0.772, p<0.0001 and r²=0.808, p<0.0001, respectively). ETP and peak height remained elevated as compared to normal persons at 3 and 6 months and showed time variation effects in contrast to normals. In conclusion, preclinical studies confirm the reproducibility and stability in time of CAT-analysis and the method is suitable for detecting hypercoagulability in patients after a first AMI.
International multi-centre assessment of the calibrated automated thrombogram thrombin generation assay. Y Dargaud
The objective of this study was to assess inter-laboratory variations of the Calibrated Automated Thrombogram (CAT) results and also intra- and inter-assay imprecision of the test within 5 European centres with proven experience in TG measurements. A large variability of ETP results between centres were found when the centre were using different sources and concentrations of tissue factor and phospholipids. Results are incomparable and multi-centre clinical studies can not be designed. When a standardized protocol was used by all the centres the variability could be limited. Under these test conditions, contact factor inhibition improved the intra-assay CV in all centres. There is a need for experienced operators and for the use of the same version of the software will help to reduce variability. These results emphasize the requirement for a standardized protocol using standard reagents before organizing multi-centre clinical trials and before a wider application of CAT in clinical laboratories.
A new global assay with small amounts of recombinant tissue factor and tissue- plasminogen activator providing novel parameters to determine the overall hemostatic potential. S He/M Blombäck
Dr He described an assay for the Overall Haemostatic Potential with plasma containing recombinant tissue factor, t-PA and phospholipid. The expected findings from the commercial plasmas with coagulant deficiencies and samples with high activity of PAI-1 indicate that the new method can detect physiologically relevant actions, as regards fibrinogen clotting and fibrin digestion regulated by thrombin generation and plasminogen activation respectively. Increased coagulation and decreased fibrinolysis, as well as the induced changes by heparin/a thrombin inhibitor in the thrombotic cases suggest that the new approach may determine hypercoagulation and monitor anticoagulating therapies. Decreased overall haemostatic potential based on low levels of coagulation and high level of fibrinolysis was also found in the haemophiliacs, showing a possibility that a criteria in addition to the FVIII/FIX concentrations may be created according to the assay results for better selecting patients who really need the regular prophylactic treatment.Ongoing studies will further explore haemostatic disturbances in more clinical materials, to confirm whether this simple approach can become a laboratory tool in clinical routine.
Methods recording dynamics of fibrin formation. B. Sørensn/J. Ingerslev
Traditional plasma coagulation analyses, such as the PT and APTT usually only provide information of the early start of clot formation. However, following the initiation of clot formation, there is a rate specific dynamic development of the clot. During recent years thrombelastometry has been used extensively to visualise the dynamic properties of continunous whole blood clot formation. In our center a thrombelastographic model, employing minute amounts of tissue factor as activator, has been explored to demonstrate phenotype heterogeneity of patients with severe haemophilia, dose titration response to rFVIII, rFVIIa, aPCC in haemophilia, evaluation of anticoagulants and modalities for reversal, detection of tarumatic and dilutional coagulopathy and methods for reversal as well as detection of hypercoagulation. Continuous profiles of plasma clotting can be obtained from several coagulation instruments. Adopting simple signal processing, including differentiation and filtration, dynamic profiles and parameters of e.g. APTT plasma clotting analysis can be generated. This presentation summarize the application of dynamic APTT clotting parameters in patients with haemophilia as well as patients with an episode of verified venous thromboembolism. In summary, the maximum velocity of APTT induced plasma clotting reflects more heteroneity amongst patients with sevevere haemophilia A (FVIII:C < 0.01) than standard APTT clotting times. Individualized in vitro rFVIII spiking experiments may serve as an additional laboratory tool for selecting appropriate dose regimens. In patients with a history of verified venous thrombosis, our data suggest that the maximum velocity of APTT plasma clotting represent a stronger predictor for hypercoagulation than standard APTT measures. Ongoing prospective studies aim at evaluating the clinical correlation and feasibility