An international collaborative study to investigate a proposed reference method for the determination of potency measurements of fibrinolytics in absolute units.

Abstract

Traditionally, WHO International Standards (IS) have been calibrated in International Units (IU) by consensus following an international collaborative study. In the area of coagulation and fibrinolysis standards it is also common for laboratories involved in such studies to perform their own in-house methods, although guidelines may be defined to include recommendations of replication and randomization of sample testing to improve the robustness of the study. The historic basis of this approach has been to develop a common reference standard to facilitate comparisons of results for the relative potency of standard and test preparations in laboratories using different methods [1]. However, this approach has been criticized and suggestions for improvements have been made which would bring the standardization of biologicals more in line with other calibrators used in medicinal chemistry. Guidelines for the introduction of a metrologically sound approach to standardization have been detailed elsewhere [2]. General goals include standardization of methods and the introduction of a hierarchy of reference materials and procedures, each with an assigned uncertainty, to provide a system of metrological traceability where testing of routine samples can ultimately be traced back to a primary calibrator and primary reference method that are defined in SI units [3]. We have previously published a proposal for a reference method developed to measure the potency of thrombolytic products (plasminogen activators) [4] that did allow the calculation of activity in enzyme units (moles of product per second in the defined method), which could theoretically be converted into katals. The katal (mole per second) is the coherent derived SI unit of measurement for enzyme activity and is at the top of the hierarchy for the catalytic concentration of an enzyme [5]. Thus it would be possible theoretically to define the concentration of an IS not only in IU but also by absolute SI units. With the encouragement of the Fibrinolysis SSC, an international collaborative study was organized in which laboratories expert in fibrinolysis methods were recruited to perform the defined method [4] using the current IS for urokinase (uPA, 87/594), tissue plasminogen activator, (tPA, 98/714) and streptokinase (SK, 00/464). The study was planned as far as possible to remove possible sources of variation. All necessary reagents were provided, including IS, plasminogen substrate (NIBSC reagent 97/534), and chromogenic substrate for plasmin CS-41(03) (Hyphen BioMed, France). In addition, thrombin (NIBSC reagent 01/578) and fibrinogen concentrate (1st IS 98/614) were provided in order to make clots as consistently as possible in all laboratories. Plasmin (3rd IS, 97/ 536) was also provided to all laboratories to perform a series of assays in which a range of concentrations of chromogenic substrate was hydrolysed completely in order to calculate an extinction value for p-nitroaniline for each laboratory that was specific to the equipment they used. This value was critical to calculate the molar concentration of p-nitroaniline released during the plasminogen activation reaction, which in turn allows the molar concentration and rate of plasmin generation to be calculated and thus express the activity of the plasminogen activator in SI units. The only materials provided by the laboratories in the study were Tris buffer and microtitre plates. A detailed collaborative study protocol was agreed in conjunction with participating laboratories and other outside interested parties over a series of months ahead of the practical phase of the study. Twelve participants contributed a total of 36 assays, each of which included the three activators, uPA, tPA and SK at four doses, in quadruplicate for each point. Raw data of absorbance vs. time were returned to NIBSC for analysis, where rates of plasminogen Correspondence: C. Longstaff, Haemostasis Section, National Institute for Biological Standards and Control, South Mimms, Herts, EN6 3QG, UK. Tel.: +44 1707 641253; fax: +44 1707 641050; e-mail: clongstaff@ nibsc.ac.uk