David C. Kressin

The effect of time and temperature variables on routine coagulation tests.

This study evaluates the effects of time and temperature variables on routine coagulation assays [Prothrombin Time test and Activated Partial Thromboplastin Time (APTT) test]. Four different groups were studied: healthy volunteers, hospitalized patients not receiving anticoagulants, patients receiving oral anticoagulant therapy and patients receiving unfractionated heparin therapy. Samples were subjected to one of four conditions: (1) centrifuged immediately and stored at room temperature (20–22°C); (2) centrifuged immediately and stored on ice (4°C); (3) stored as whole blood without centrifugation, at room temperature and (4) stored without centrifugation, on ice. Coagulation tests were performed as soon as possible after phlebotomy and at specified times up to 24 h. Our data demonstrate that prothrombin time results are stable for up to 24 h, remaining constant regardless of storage conditions. APTT assays are stable for up to 8 h, except for patients receiving unfractionated heparin therapy. Heparinized samples, when stored uncentrifuged at room temperature, demonstrate a clinically significant shortening of the APTT and individual samples demonstrate a greater than 50% decrease in ex-vivo heparin levels at 4 h.

Inhibition of thrombin by iopromide in vitro.

Iopromide is a nonionic, iodinated, monomeric, radiographic contrast agent used in various indications, including coronary angiography and visceral and peripheral arteriography. Nonionic contrast media have been postulated to increase thrombogenicity when compared with ionic contrast media. The goal of this study was to characterize the interaction of iopromide with thrombin, specifically to determine the rate, extent, specificity, and reversibility of the thrombin inhibition by iopromide, the integrity of the thrombin–iopromide complex, and the inhibitory potency of iopromide using a validated assay methodology. Iopromide was mixed with purified thrombin or pooled serum from healthy male and female donors. The final concentrations of iopromide in the presence of estimated physiologic concentrations of thrombin (1 nmol/L) were 0–184 mmol/L. After incubation for defined time intervals, the activity of thrombin was determined by adding substrate and measuring the absorbance of the generated chromophores at 405 nm. The possible inhibition of the protease trypsin by iopromide was investigated to evaluate the specificity of thrombin inhibition by iopromide. Iopromide was compared with Thromstop, a known thrombin inhibitor, to assess the relative potency of iopromide. The inhibition of thrombin by iopromide was immediate, rapidly reversible, and proportionate to the iopromide concentrations. The minimum inhibitory concentration of iopromide was 50 mmol/L. At the highest iopromide concentration tested, 184 mmol/L, the mean inhibition of thrombin activity was 44.5%. The mean concentration of iopromide associated with a 50% inhibition was 206 mmol/L. The inhibitory potency of iopromide was 4 ×106 times smaller than that of Thromstop. The inhibition of thrombin by iopromide is specific, because trypsin was not inhibited by iopromide. The results indicate that in vitro iopromide at clinically relevant concentrations partially inhibits thrombin activity. However, the in vitro model used does not consider other factors that may be relevant for the overall coagulation response in vivo.