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Set of reagents for determining the activity of antithrombin III by chromogenic method (Rechrom-ATIII) according to TU 21.20.23-062-05595541-2017. Designed for quantitative determination of antithrombin III activity in blood plasma using a chromogenic method, both manually and on automatic analyzers.

Antithrombin III (ATIII) or heparin cofactor is the main antagonist of thrombin in the formation of this enzyme in the blood. Inherited and acquired deficiency of ATIII in the blood leads to the development of disseminated intravascular coagulation and, in some cases, acute thrombosis. Reduced concentration of ATIII is observed in patients with liver diseases (cirrhosis), with nephrotic syndrome due to excretion of the inhibitor in the urine. Injections of heparin and oral contraceptives reduce the concentration of ATIII.

Method principle: The method for determining the activity of ATIII is based on the ability of ATIII to neutralize thrombin in the presence of heparin. ATIII activity is determined in plasma by adding an excess of thrombin to it. In this case, thrombin is inhibited by the ATIII-heparin complex in proportion to the amount of ATIII in the plasma. The remaining amount of thrombin catalyzes the cleavage of para-nitroaniline (pNA) from a synthetic chromogenic substrate. The absorption of free pNA, determined at 405 nm, is inversely proportional to the activity of ATIII.

The process proceeds according to the following scheme: ATIII + heparin (excess) ⇒ ATIII-heparin. ATIII-heparin + thrombin (excess) ⇒ ATIII-heparin-thrombin + thrombin (remainder). Substrate-pNA + thrombin (remainder) ⇒ Peptide + pNA.

To construct a calibration curve, a plasma calibrator with a known ATIII activity (A) is used. On the ordinate axis (Y), the optical density values obtained for each dilution of the plasma calibrator are plotted on a linear scale, and on the abscissa axis (X), the antithrombin III activity in % is plotted on a linear scale.

Using the calibration curve and the clotting time value of the test sample, the ATIII activity is determined in %.

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Set composition:

  • 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;
  • Imidazole buffer, concentrated (5 ml/vial) – 1 vial.

One kit is intended for conducting 40 determinations by the manual method, 60 determinations on Sysmex coagulometers, and 80 determinations on ACL coagulometers. The number of analyses on other automatic analyzers depends on the design of the device. Normal and pathological values of antithrombin III activity should be monitored using Control Plasma, code KM-2.