A multicentre assessment of the endogenous thrombin potential using a continuous monitoring amidolytic technique

Abstract number: P1587

Lawrie* A. S., Gray† E., Leeming‡ D., Davidson§ S. J., Purdy* G., Iampietro† R., Craig‡ S., Rigsby† P., Mackie* I. J.

§Department of Haematology, Royal Brompton Hospital, UK *Haemostasis Unit, University College London, UK; †N.I.B.S.C., UK; ‡Thrombosis Reference Centre, Manchester, UK;

We assessed between laboratory variation in potency estimates, inter- and intra-assay imprecision associated with the measurement of endogenous thrombin potential (ETP). Initial studies were undertaken using techniques which had evolved in each of the participating laboratories from the method of Wielders et al. (Thromb Haemost 1997, 77, 629–36). All laboratories used plasma defibrinated with Ancrod (Agkistrodon rhodostoma venom. Sigma-Aldrich), the source of tissue factor was either Innovin® (Dade Behring) or RecombiPlastTin® (Haemoliance®, Instrumentation Laboratory), the chromogenic substrate used was either CBS.0068 (Diagnostica Stago) or Pefachrom® TG (Pentapharm); reactions were monitored using a COBAS Mira (at two centers), a thermostatically controlled microtiter plate reader, or an ACL-300R. Samples from normal healthy subjects (n = 10), 2 patients receiving coumarin therapy (INR ~2.0 and INR ~4.0) and a further two subjects receiving treatment with unfractionated heparin (0.07 IU mL^-1 and 0.31 IU mL^-1) were assayed relative to a lyophilized normal plasma that had arbitrarily been assigned a potency of 100%. Considerable variation between centers was observed in potency estimates on samples from patients receiving heparin and coumarin therapy (INR ~2.0 ETP 28.7–56.0%; INR ~4.0 ETP 26.1–47.5%; heparin 0.07 IU mL^-1 27.5–126.2%; heparin 0.31 IU mL^-1 ETP 0–67.2%. Mean CV = 60%); however, individual laboratories using fully automated techniques achieved acceptable levels of inter- and intra-assay imprecision as assessed by the coefficient of variation (intra-assay CV < 9.5%, inter-assay CV < 12.5%). A second study to assess a similar range of samples, using a standardized assay protocol and incorporating the appraisal of two chromogenic substrates, CBS.0068 or Pefachrom® TG, demonstrated in most instances there was a markedly improved agreement in potency estimates between centers (INR ~2.0 ETP 52.9–64.7%; INR ~4.0 ETP 18.3–51.5% heparin 0.05 IU mL^-1 ETP 37.5–59.1%; heparin 0.25 IU mL^-1 ETP 8.9–18.0%; heparin 0.40 IU mL^-1 ETP 8.0–12.5%. Mean CV = 18.1%) and good correlation (r > 0.96) between results obtained using the chromogenic substrates.