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Reagent Kit for the Determination of Antithrombin III Activity by Chromogenic Method (Reakhrom-ATIII) according to TU 21.20.23-062-05595541-2017

Purpose: Designed for the quantitative determination of antithrombin III activity in blood plasma using a chromogenic method, both manually and with automated analyzers.

Description: Antithrombin III (ATIII) or heparin cofactor is the primary antagonist of thrombin during its formation in the blood. Both inherited and acquired deficiencies of ATIII in the blood lead to the development of disseminated intravascular coagulation and, in some cases, acute thrombosis. Reduced ATIII concentration is observed in patients with liver diseases (such as cirrhosis) and nephrotic syndrome due to inhibitor excretion in the urine. Heparin injections and oral contraceptives also reduce ATIII levels.

Method Principle: The method for determining ATIII activity is based on the ability of ATIII to neutralize thrombin in the presence of heparin. ATIII activity is measured in plasma by adding an excess of thrombin. This results in thrombin inhibition by the ATIII-heparin complex, proportional to the amount of ATIII in the plasma. The remaining thrombin catalyzes the cleavage of para-nitroaniline (pNA) from a synthetic chromogenic substrate. The absorption of free pNA, measured at 405 nm, is inversely proportional to ATIII activity.

Process Flow:

  • ATIII + excess heparin ⇒ ATIII-heparin complex.
  • ATIII-heparin complex + excess thrombin ⇒ ATIII-heparin-thrombin complex + remaining thrombin.
  • Substrate-pNA + remaining thrombin ⇒ Peptide + pNA.

For calibration, a plasma calibrator with a known ATIII activity (A) is used. Optical density values are plotted on the Y-axis on a linear scale for each dilution of the plasma calibrator, and ATIII activity in percentages is plotted on the X-axis on a linear scale.

Using the calibration curve and the coagulation time value of the sample under investigation, ATIII activity is determined in percentages.

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Kit Components:

  • Human thrombin with heparin, lyophilized (20 IU/vial) – 2 vials;
  • Plasma calibrator, lyophilized (1 ml/vial) – 1 vial;
  • Chromogenic substrate, lyophilized (4 μM/vial) – 2 vials;
  • Concentrated imidazole buffer (5 ml/vial) – 1 vial.

One kit is intended for conducting 40 manual determinations, 60 determinations on Sysmex coagulometers, and 80 determinations on ACL coagulometers. The number of analyses on other automated analyzers depends on the design of the device.

Normal and pathological values of antithrombin III activity should be monitored using a Control Plasma, code KM-2.