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

2007; Elsevier BV; Volume: 5; Issue: 2 Linguagem: Inglês

10.1111/j.1538-7836.2007.02299.x

1538-7933

Colin Longstaff, Colin Whitton, Craig Thelwell, Danielle Belgrave,

Antiplatelet Therapy and Cardiovascular Diseases

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 activation were calculated from transformed plots of absorbance vs. time squared for the initial linear phase of the reaction (before substrate depletion). Any anomalous data were removed before final analysis. Km and kcat values for plasmin (using the 3rd IS, 97/536) on CS-41(03) were determined at NIBSC following active site titration of plasmin (giving a value of 1.62 μm as the concentration of active plasmin in the 3rd IS). Extinction values from each laboratory for hydrolysed CS-41(03) ranged between 2377 and 4003 for a 1 m solution (mean 3290, SD 488, n = 25), which shows the importance of local determination of this parameter. This is equivalent to a molar extinction coefficient of 10520 for p-nitroaniline in a 1 cm light path, in line with published estimates [6]. A simple log-log plot of all the raw data is shown in Fig. 1A for all activators from all assays. The stipulated dilution ranges of activators were deliberately chosen so the rates of plasminogen activation would overlap; however, it is immediately apparent that the absolute rates measured within and between laboratories varied widely. If the mean values are taken for all measurements, the results show that overall the assay was working for each activator, as illustrated by the linearity and overlap of rates in Fig. 1B. Nevertheless, the measures of dispersion were disappointing with % GCV of 39, 42 and 30 (at best, removing statistical outliers) for uPA, tPA and SK, respectively. Furthermore, the situation was little improved if SK was used as a reference preparation in each assay and data were recalculated relative to this reference (% GCV of uPA and tPA was 34 and 35%, respectively). Log-log plots of raw data of plasminogen activation rate versus dose from all assays. Data shown in 1A are activation rates for uPA (triangles), tPA (circles) and SK (squares) in the defined assay method from all laboratories for the three assays requested. In B the overall mean values are calculated and shown with standard deviations for all data. In conclusion, the significant variability observed in this study highlights the difficulties associated with determining absolute enzyme activities when laboratories are asked to perform a detailed yet unfamiliar method. These observations were disappointing following successful application at NIBSC of this method in two earlier collaborative studies when developing IS for tPA [7] and SK [8]. The method was shown to be both accurate and precise. Hence, a major problem seems to be to expect laboratories to execute a difficult method three times only without training or practice. Indeed, these difficulties are anticipated in descriptions of metrological traceability where only specialist Accredited Reference Measurement Laboratories are charged with the highest level roles of defining primary reference materials and methods [3]. Therefore the implementation of this kind of metrological system requires three components: materials, methods and laboratories. Despite the advantages of standardization in SI units and metrological traceability, it will likely be necessary to continue to assign values, in the fibrinolysis area at least, in the traditional way using specialist fibrinolysis laboratories and consensus values and allowing local methods in the absence of time and resources necessary to establish a network of trained, specialist accredited standardization laboratories. We are extremely grateful to all those who contributed suggestions and to those laboratories that agreed to take part: G. Rautmann, EDQM, Strasbourg, France; R. Aisina and L. Moukhametova, Moscow State University, Russia; J. Nurzynksi and C. Loh, TGA, Australia; Y. Martinez Cuellar, CIGB, Cuba; N. Moore and N. Booth, University of Aberdeen, UK; M. Rapp, Aventis Behring, Marburg, Germany; P. Declerck, University of Leuven, Belgium; C. Whitton, NIBSC, UK; J. Sidelmann, Ribe County Hospital, Denmark; P. Hermentin, Aventis Behring, Marburg, Germany; S. Agerholm, Danish Medicines Agency, Denmark; K. Kolev, Semmelweis University, Hungary; W. Kliche, I. Paal and R. Wagner, Boheringer Ingelheim, Germany; and D. Vijayarangam and S. Babu, Shantha Biotechnics, India. The authors state that they have no conflict of interest.