Prothrombin time determining reagent and methods of using and preparing the same

A process for preparing a reagent containing a thromboplastin having proteolytic activity and an ANBA chromogenic substrate for thrombin for photometric determination of prothrombin time, comprising adding an ANBA chromogenic substrate for thrombin and a thromboplastin having proteolytic activity to a buffer solution having a pH in the range of 7-8.5 to form a mixture and freeze drying the mixture, wherein the ANBA chromogenic substrate is such that it is converted by the thromboplastin, in aqueous suspension in the presence of calcium ions, to the extent of less than 0.00125 times its initial concentration per hour at 37.degree. C.

A method for the photometric determination of prothrombin time in plasma, which comprises providing a material which yields calcium ions when added to water, providing a freeze-dried mixture of a thromboplastin having proteolytic activity and an ANBA chromogenic stubstrate for thrombin which is converted by the thromboplastin, in the presence of calcium ions, to the extent of less than 0.00125 times its intial concentration per hour at 37 C., adding the material and the freeze-dried mixture to water to form a solution which contains calcium ions at a concentration of 0.5-10 mmol/l, diluting a plasma sample with the solution to form a plasma mixture, and determining the prothrombin time in the plasma sample by determining the time in which a specified extinction increase is obtained in the plasma mixture.