Attendance fluctuated between 80-120 persons.

Blood collection and handling procedures (Moderator: Dr. I. Walker)

Dr. I. Walker summarized the items that were rather well established and focused on (a) residual items that should be considered and were not well documented, (b) a future report aiming at defining minimal requirements, taking into account haemostasis assays as broad as possible (fibrinolysis, coagulation, platelets). She questioned whether the SSC should take over the recommendation of ICSH to use uniformly one citrate concentration being 0,109 mol/l. Dr. J Conard introduced the issue of influence of menstrual cycle. Dr. B. Polack summarized critical items of quality control of the increasingly used CTAD tubes and identified the absence of external quality control on such specialized tubes. Mr. P. Meijer showed data that the low pH in Stabilyte tubes might interfere with the assay system by lowering the pH. Dr. M. Stegnar identified the nearly complete absence of data on stability of analytes upon storage of plasma and showed some preliminary data suggesting the importance of this item.

It was decided that Dr. Walker should proceed to prepare a proposal for minimal requirements, as above, which should be circulated among other subcommittees to provide a broad basis for discussion of the proposal next year. In addition, a report form accompanying blood collection will be drafted for discussion (Drs. Walker and Kluft).

In addition, subgroups will work on filling in gaps in knowledge that are considered of importance. Dr. Conard with the assistance of presently known volunteers, Drs. Stegnar, Kluft, Jespersen, van de Ende and Douglas, will produce a report on fluctuations of haemostasis factors during the menstrual cycle. A practical approach of recording the time of the last menses will be included in the aforementioned report form.

Dr. Stegnar, with the assistance of Drs. Gaffney, Douglas and Kluft, will further evaluate how studies on stability of stored samples should be organized. Dr. Kluft will submit a request to selected scientists, both within and outside the fibrinolysis field, to ask for data they might have on stability of stored samples. This matter was considered urgent in view of the use of the SSC secondary standard, and Dr. Jespersen suggested that specific attention be paid in this respect to lyophilization and other potential matrix problems.

SSC Secondary Standard: Report on assignment of a value for t-PA and PAI-1 antigen (reported by Drs. Gram and de Maat)

Using the NIBSC standard as a calibrator for the methods, values were assigned to the SSC secondary standard. Five companies with commercially available kits reacted very positively and provided free reagents. Unfortunately, the results between the methods used disagreed strongly by a factor of three to five for t-PA antigen and by a factor of two for PAI-1 antigen. No value could therefore be assigned. It was decided that the assistance of the company scientists would be requested to find the reason for the discrepancies and a renewed discussion about establishment of a reference method might be needed.

The assignment for plasminogen and antiplasmin was delayed to await the introduction of a new NIBSC batch of the required standards and is expected to be available next year. Dr. Gaffney reported to have ampouled a new plasmin standard (97/536) to succeed 77/588 and also a new glu plasminogen standard to replace the British standard 78/646. It was decided to seek WHO approval for the glu-plasminogen standard, as well.

Assignment of a value to the t-PA antigen plasma standard (reported by Dr. Gaffney)

The subcommittee agreed with the proposal of the assigned value on the new plasma t-PA antigen standard (94/730). Dr. Gaffney will propose this to the WHO. It was further agreed that information on the variation in detected t-PA antigen in the assignment study and information about the added amount of t-PA should be accessible for users.

Assignment of an antigen value to the PAI-1 standard for activity (reported by Dr. Gaffney)

The activity standard 92/564 established by the WHO showed in a collaborative study a variation in results too wide to be acceptable. The subcommittee agreed with Dr. Gaffney's suggestion not to proceed to WHO.

Update on the scu-PA standards and methods of assay (reported by Dr. Gaffney)

New batches of non-glycosilated (NG)(95/564) and glycosilated (G) (95/668) standards for scu-PA show excellent stability at six months (20¼ and 37¼ C). Measurement (after activation) directly on chromogenic substrate shows virtually identical contents of both preparations in agreement with amino-acid analysis. Expression in SI units (katal) is now possible. It was decided that this was now the preferred method for comparison with other preparations. The previous discrepancy with tests of in vitro biological activity was strongly reduced by introduction of a clot lysis assay using exogenous lysis instead of endogenous lysis. The warning was issued not to confuse in vitro resemblance and standardization of NG and G with thrombolytic efficacy which might depend on other factors as well.

Mr. Meijer assisted by Drs. Hanns, Christensen, Wiman and Kluft had prepared a report and a set of criteria for specificity and proposed methods of testing. Dr. Booth, assisted by Drs. Chandler, Ersdal, Rijken, Kinnby and Stegnar, did so for t-PA antigen. The subcommittee added suggestions and finally approved the criteria and methods for both analytes. Both groups will prepare a full report for publication, which will be reviewed by the PGM before further processing within SSC.

The working group on Scu-PA (Drs. Binder, Dooijewaard, Gunzler, Benraad) issued an interim report and will report next year.

In view of the assignment of the SSC secondary standard, it was decided to form a new working group for plasminogen which will report next year (chairman and participants to be invited).

Dr. M. de Maat, on behalf of a working group (plus Drs. F. Green and N. Sala) on genetic methodology formed within the frame-work of ETRO (Population genetics of hemostatic risk factors for arterial vascular disease), summarized the nomenclature that should be used for intron and exon mutations in genes of interest for vascular diseases when the recommendations of Beaudet and Tsue (Human Mutation 1993; 2: 245-8) were followed. It was felt it would be of great help to publish a table of old versus new "names" and to explain the principles. It was decided to submit this approach as a proposal to the SSC before a report was made and ask for more general support and approval of this effort

Dr. M. de Maat, on behalf of the working group mentioned above, summarized measures that should be taken to assure quality of genetic methods. Measures are different for different methods. Publication of quality control techniques that are known to expert laboratories might be useful to support the dissemination of genetic methods to many new laboratories. It was decided that Dr. de Maat would arrange a collaborative study, including distribution of samples among interested laboratories, to document the magnitude of the problem. This will be reported next year.

In addition to what has been mentioned above, it was suggested to pay attention to standardization of D-dimer and to explore the developments around TAFI.

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