Procarboxypeptidase U / TAFI
Dr. D. Hendriks provided an overview on the nomenclature, on the current assays and on the function of this enzyme in the fibrinolytic system. This was followed by the presentation of a newly developed chromogenic substrate based activity assay that, in contrast to existing functional assays, might allow to analyse large numbers of samples. Dr. Hendriks briefly mentioned that at a recent wet workshop in Leiden, three commercial assays ( one for activity, two for antigen) had been evaluated. Since only a limited number of people (often unexperienced) participated and only a limited number of samples were included, no firm conclusion could be drawn. Dr. Mosnier reported the results of their study on the measurement of TAFI levels in 25 normal plasma samples using 5 different assays (3 antigen and 2 activity assays). As presented at the 2000 Maastricht SSC meeting, the TAFI levels as measured by the 5 assays, showed good correlation. However, additional analysis of the results indicated that there might be a systematic variation in the TAFI levels (related to the use of the commercially available sheep anti-TAFI polyclonal antibody) that needs further investigation. The meeting agreed with the organisation of a collaborative interlaboratory study on available proCPU/TAFI assays. Laboratories interested in participating were requested to sign up at the end of the session.

Standards for proteins of the fibrinolytic system
Dr. Longstaff reported that at the WHO/ECBS meeting in October 2000 it was agreed that the recommendations of the SSC be adopted to change the name of the new IS for tPA (98/714) FROM: Alteplase (recombinant tissue plasminogen activator), 1st IS, TO: Tissue plasminogen activator, human, recombinant, 3rd IS.

Dr. Longstaff presented the results of a collaborative study that was organised in 2000/2001, involving 16 labs, to replace the 2nd IS for Streptokinase. The study included 4 coded Streptokinase samples and participants were asked to perform one or more of the 2 methods provided, or their own in-house method. At the end of the study there was universal agreement by participants with the two proposals arising from the study, 1) on potency and identity of the new standard; and 2) on methodology. 1) It was agreed that preparation 00/464 should be recommended to the WHO as the 3rd IS for Streptokinase with a potency of 1030 IU/ampoule. 2) It was agreed that a chromogenic assay without fibrin (one of the methods provided in the study) would make a suitable reference method for Streptokinase activity determinations. It is likely that this method will be adopted as the reference method of the European Pharmacopoeia. Prior to the SSC meeting the report of this study had also been sent to various members of the subcommittee for comments. Eleven responded and all approved the report. The report was subsequently approved by the meeting.

D-dimer
Dr. Nesheim briefly discussed the structure of fibrinogen, the polymerization of fibrin momomers, as well as the subsequent cleaveage of fibrin by plasmin to form a family of fibrin degradation products. This was followed by a brief presentation of results wherein the products released from a Factor XIIIa cross-linked clot perfused with dilute plasmin were characterized with respect to their average molecular weight, their molecular weight distribution, and their chain composition. Because the fragments are soluble and well characterized with respect to absolute concentration, chain composition, molecular weight and D-dimer content, they would be excellent candidates for D-dimer standards by which to calibrate and compare various assays.

Dr. C.-E. Dempfle presented the results of the FACT-3 trial (Fibrin Assay Comparison Trial) in which 23 samples ( including normal samples as well as samples from different pathologies) were subjected to analysis by 22 commercially available assays. A wide range of values was observed when comparing the results obtained with the various kits; however, a good correlation between the methods was obtained. The latter allowed the generation of conversion factors by which the data from each respective assay could be normalized, resulting in a harmonization of the data obtained from all DVT and most of the DIC samples. For some particular assays this procedure appeared not to be applicable for the data obtained with normal samples. Larger studies will be needed to confirm the general applicability of these conversion factors. The study also demonstrated that a harmonization of the data obtained with the different assays could also be achieved by using a common calibrator consisting of a high-molecular weight, partially plasmin degraded, cross-linked fibrin preparation.

Dr. P. Meijer presented data of the "D-dimer comparison trial," a joint project of the ECAT Foundation and INSTAND performed within the framework of their external quality assessment programmes. The scope of this project was to evaluate the analytical performance of quantitative D-Dimer assays in daily laboratory routine and was focussed at the inter-laboratory variability per assay and the difference in outcome between different methods. A set of 7 samples generated by mixing pooled normal plasma with pooled patient plasma containing elevated D-dimer levels, at different ratios to about 450 laboratories. Preliminary data analysis on the results of about 200 participants from the ECAT Foundation was discussed. The mean inter-laboratory CV ranged from 5 ó 80%. The ratio between the lowest and highest mean value of different methods on different D-Dimer levels ranged from 3 ó 14. Dr. P. Meijer concluded that, with respect to harmonisation of D-Dimer methods, not only the standardisation of the calibration but also the difference in sensitivity should be taken into account.

Dr. Kitchen presented data of recent studies (September 1999 and November 2000) in which two test samples were distributed to more than 300 centres. Latex agglutination was used by 165-215 centres. During this period the use of quantitative (automated and ELISA) methods increased from 40 to 110. Each sample was a lyophilised pool of plasmas from hospitalised patients with elevated D-dimer. Results were grouped by technique and the median D-dimer with different latex agglutination methods varied form 400 to 2000 ng/ml. This was similar for both samples. For one automated assay the median result was 359 ng/ml (range 250-430) with a CV of 15%, for another method the median was 3030 ng/ml (range 1800-3200). For one automated method the CV of results was 54%. For one method the median test result was only 20% above the upper limit of normality. For others the median test result was more than 6-fold higher than the upper limit of normal. It was concluded that D-dimer assays in routine use vary widely in the results obtained, in the discrimination between normal and abnormal, and in their precision . Improved standardization is required.

Matrix Metalloproteinases
The matrix metalloproteases (MMPs) form a family of over twenty closely related zinc dependent proteases. MMPs are involved in the degradation of many components of the extracellular matrix. MMPs are believed to be involved in various (disease) processes involving matrix remodelling like rheumatoid arthritis, scar formation and invasion and metastasis of tumor cells. Increasing evidence for a role of MMPs in fibrinolysis and cardiovascular disease is accumulating, i.e. MMPs can be activated by plasmin as well as by thrombin and activated MMPs may induce activation of platelets. Dr. J. Verheijen gave an overview on assays for quantitation of MMP activity and antigen. Quantitation methods include: zymography, degradation of matrix components, immunological methods like ELISA and fluorogenic or chromogenic methods involving synthetic peptide or protein substrates. The specificity of the methods is often poorly documented. In biological samples MMPs occur in multiple forms, i.e., free active enzyme, inactive pro-enzyme, active or inactive complexes with protein inhibitors and membrane- or matrix-bound forms. The extensive structural and functional similarity between the various MMPs, the occurrence of multiple forms and the lack of standard preparations and reference methods complicate a reliable quantitation of these enzymes and make interlaboratory comparison of results very difficult. In vitro and in vivo methods for the investigation of MMPs were subsequently discussed by Dr. Z. Galis. Her studies, involving a variety of experimental approaches ( in vitro, in vivo, including particular knock-out animal models), clearly demonstrated the role of upregulation of gene expression of various MMPs in atherosclerotic disease, vascular remodeling and angiogenesis. It could also be concluded that MMPs play an important role in the degradation of fibrin matrices during angiogenesis.

Topics for 2002
Attendees were encouraged to communicate with the co-chairpersons. At the 2002 meeting progress on proCPU/TAFI measurements and D-dimer standardization should be included as well as the topic "Standards" .

The meeting was attended by 130-140 people including all the co-chairs. The meeting was closed at 4.55 pm.