Collaborative study on monitoring methods to determine direct thrombin inhibitors lepirudin and argatroban
2005; Elsevier BV; Volume: 3; Issue: 9 Linguagem: Inglês
10.1111/j.1538-7836.2005.01577.x
ISSN1538-7933
Autores Tópico(s)Heparin-Induced Thrombocytopenia and Thrombosis
ResumoDirect thrombin inhibitors are now used clinically for the prophylaxis and treatment of thrombosis and related cardiovascular diseases. Although all these inhibitors bind to thrombin, their modes of action are different. Routine anticoagulant monitoring tests such as the activated partial thromboplastin time (APTT) are used to estimate the activity of these inhibitors. However, no studies have been carried out to assess the suitability of these tests. An international collaborative study was carried out utilizing a panel of plasmas spiked with lepirudin and argatroban to evaluate the robustness and the sensitivity of the different monitoring methods. Pooled platelet poor plasma (PPP) was prepared from 40 donations of blood collected in CPD‐adenine, at the North London Blood Transfusion Centre, Colindale, London, UK. It was buffered with HEPES to a final concentration of 0.04 mol L−1. Nine coded plasma samples (A‐I) were prepared by spiking lepirudin (Refludan, Hoechst Marion Roussel, Germany) and argatroban (Mitsubishi‐Tokyo Pharmaceut. Inc., Tokyo, Japan) at a final concentration of 0, 0.31, 0.63, 1.25, and 2.5 μg mL−1 into the PPP. All plasmas were filled into glass sealed ampoules and then freeze‐dried according to the conditions used for International Biological Standards. Four sets of plasma samples for each method were sent to the investigators to assay one set of samples for 4 days in quadruplicate. Fourteen laboratories agreed to take part in the study, with 13 laboratories returning data for analysis. The participants included manufacturers of thrombin inhibitors, diagnostic equipment and reagent manufacturers, clinical laboratories, and academic institutes. APTT using local reagents and methods (L‐APTT) Ten laboratories carried out the APTT, using their own in‐house reagents. Seven different reagents were used (Actin FS, Thrombosil I, Pathromtin SL, Synthasil APTT, Automated APTT, STA‐PTT, STA – CK Prest 5). APTT using a common reagent (C‐APTT) The common APTT reagent used was Actin FS (Aventis Pharma, Marburg, Germany). Ten laboratories returned results for this test. Anti‐IIa Chromogenic assay was performed at 13 laboratories with the S2238 chromogenic substrate from Instrumentation Laboratory/Haemochrom Diagnostica (Essen, Germany). Ecarin clotting time– wet chemistry (Wet ECT) reagents These were donated by Dr Götz Nowak (University Jena, Germany). Ten laboratories carried out this test. ECT– dry chemistry reagent It was available on test cards, which were analyzed with the TAS analyzer (Cardiovascular Diagnostics Inc., Raleigh, NC, USA). The low ecarin reagent card is referred as the dry ECT, the higher concentration ecarin card is referred as TIM. Enzyme immunoassay (ELISA) kit It was only available for the measurement of lepirudin samples (Immuno Bind, Hirudin Elisa kit, American Diagnostica Inc., Greenwich, CT, USA, kindly supplied by its president Richard Hart). Intra‐laboratory (day‐to‐day) and inter‐laboratory variations, as represented by the % geometric coefficient of variation (%GCV), were determined to assess the reproducibility and robustness of the different methods. The APTT using local reagents (L‐APTT) showed normal ranges between 32 and 44 s in the participating laboratories. Except for one laboratory, all concentrations of lepirudin and argatroban were measured with coagulation times ranging from 88 to 177 s for lepirudin and 98–179 s with argatroban. Of the seven local reagents used in the study, one reagent, which may be related to is reagent/instrument combination, was unable to give clotting times for the highest concentration (2.5 μg mL−1) of the inhibitors. All of the local reagents were able to detect the lowest concentration (0.31 μg mL−1) of the inhibitors with 1.68 and 1.89 being the lowest APTT ratios obtained for lepirudin and argatroban samples, respectively. Despite the use of different reagents, the APTT ratios and indeed the actual clotting times were similar for the same concentration of two inhibitors. The common APTT gave normal ranges from 30 to 44 s. All concentrations of lepirudin and argatroban were detected with longest coagulation time being 164 s. For the wet ECT, the normal values ranged from 43 to 55 s. However, the long‐clotting times (some of reported clotting times for 2.5 μg mL−1 of argatroban were >800 s) induced by 0.63 and 2.5 μg mL−1 of lepirudin and argatroban were not always registered by the instruments used by the participants. The same concentration of argatroban gave longer clotting times than the corresponding lepirudin samples. Therefore, the argatroban samples resulted in higher ECT ratios than the lepirudin samples. For the dry ECT, normal ranges were reported to be between 24 and 62 s. Some laboratories had unmeasurably long‐clotting times with the highest concentrations of lepirudin and argatroban. Complete data sets were available from ten laboratories; most of them reporting coagulation times above 700 s at 2.5 μg mL−1 for both lepirudin and argatroban samples. The TIM‐card gave normal ranges between 17 and 29 s. All laboratories reported measurable coagulation times for all concentrations of lepirudin and argatroban. All 13 laboratories performed the anti‐IIa chromogenic assay. The dilution of lepirudin resulted in a steeper optical density–concentration relationship compared with the same concentration range of argatroban in all laboratories. The ELISA was determined in 10 laboratories and was only carried out on lepirudin samples. None of the laboratories was able to detect 0.31 μg mL−1 of lepirudin, which gave a similar absorbance to the negative plasma. However, all the samples containing higher concentrations of lepirudin provided measurable responses. In general, all APTT methods gave similar geometric mean APTT ratios for the same concentrations of the two inhibitors. Although the geometric mean ratios for the three ECT methods were different for the different inhibitors, the ratios were similar for the same DTI. The anti‐IIa chromogenic assay was found to be more sensitive to lepirudin, with and steeper dose–responses. Higher ratios were obtained for the lepirudin spiked plasma samples than for the argatroban samples. Some of the laboratories detected only concentrations above 0.31 μg mL−1 of argatroban by the anti‐IIa chromogenic assay. In summary, as shown in Fig. 1A and B, the ECT methods gave the steepest slopes while the APTT and the anti‐IIa methods gave flatter dose–response curves, suggesting that these methods were less sensitive than the ECTs. The APTTs, using either local or common reagents, gave the lowest intra‐laboratory variability; with the exception of one laboratory, the entire %GCVs were below 10% and the majority were <5%. The intra‐laboratory variations for ECTs were also relatively low, with the exception of a couple of laboratories; most of the %GCV results were below 10%. For the lepirudin ELISA, three laboratories found a %GCV over 10%, with the other six laboratories showing a low GCV (<6%). The anti‐IIa chromogenic assay found the highest variability within the laboratory, with the majority of the %GCV values well over 10% for both inhibitors. Comparing the performance of all the methods, the inter‐laboratory variations for measurement of lepirudin were in order of lowest to the highest: TIM < C‐APTT < Dry ECT < L‐APTT < wet ECT < anti‐IIa < ELISA. For the measurement of argatroban were in order of lowest to the highest inter‐laboratory variations: C‐APTT = dry ECT < L‐APTT < TIM < anti‐IIa < wet ECT. Activated partial thromboplastin time and the TAS‐analyzer with ECT‐cards gave the most reproducible results compared with other methods. Further studies will evaluate the validity for patient samples.
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