Reagent kit for the determination of anti-Xa activity of heparin by the optical method ("Reakhrom-heparin") according to TU 9398-032-05595541-2011 (GP-1)
∗ Reagent kit for the determination of anti-Xa activity of heparin by the chromogenic method ("Reakhrom-heparin") according to TU 21.20.23-073-05595541-2021 (GP-1/1)
∗∗ The kits are intended for monitoring the administration of heparin (including low molecular weight) by determining its anti-Xa activity in human plasma.
∗The GP-1 kit is intended for automatic, semi-automatic analyzers, as well as for manual determination of anti-Xa activity.
∗∗The GP-1/1 kit is only for automatic coagulometers. All components of this kit are packaged in bottles with screw caps. The improved chromogenic substrate included in the kit allows the use of this kit on standard protocols without additional modifications. Monitoring the administration of heparin allows preventing hemorrhagic complications and, at the same time, assessing the effectiveness of anticoagulant therapy. Method principle: The method of determining heparin activity is based on the ability of the AT III-heparin complex to neutralize activated factor Xa. Heparin activity is determined in plasma by adding an excess of AT III and factor Xa to it. In this process, factor Xa is inhibited by the AT III-heparin complex in proportion to the amount of heparin in the plasma. The remaining amount of factor Xa catalyzes the cleavage of para-nitroaniline (pNA) from the synthetic chromogenic substrate. The absorption of free pNA, determined at a wavelength of 405 nm, is inversely proportional to the heparin activity in the plasma. The process follows the scheme: AT III (excess) + heparin → AT III-heparin. AT III-heparin + Xa (excess) → AT III-heparin-Xa+Xa (remainder). Substrate-pNA + Xa (remainder) ⇒ Peptide + pNA. To construct a calibration curve, calibrator plasmas with known heparin activities, indicated in the medical device passport, are used. On the ordinate axis (Y) on a linear scale, optical density values obtained for each calibrator plasma are plotted, and on the abscissa axis (X) on a linear scale, heparin activity is plotted in anti-Xa units/ml of plasma. The correctness of the calibration curve construction is monitored using control plasmas.
Composition of the GP-1 kit: Antithrombin III, lyophilized (1 ml/vial) – 2 vials;
• Factor Xa, lyophilized (2 ml/vial) – 2 vials;
• Chromogenic substrate, lyophilized (2 ml/vial) – 2 vials;
• Concentrated buffer (5 ml/vial) – 2 vials;
• Calibrator plasmas (1 ml/vial) — 3 vials;
• Control plasmas (1 ml/vial) — 2 vials. One GP-1 kit is intended for 20 determinations with a reagent consumption of 0.2 ml using the manual method or 80 determinations with a reagent consumption of 0.05 ml using the tablet method.
The number of determinations using the Reakhrom-heparin GP-1/1 kit depends on the type of automatic coagulometer used. Composition of the GP-1/1 kit:
• Antithrombin III, lyophilized (1.0 IU/vial) – 2 vials; • Factor Xa, lyophilized (15.0 NKat/vial) – 4 vials;
• Chromogenic substrate, lyophilized (3.0 µM/vial) – 2 vials; • Imidazole buffer – 5.0 ml in a bottle – 1 vial;
• Plasma calibrator level 0, lyophilized (volume after reconstitution 1.0 ml/vial) – 1 vial;
• Plasma calibrator level 1, lyophilized (volume after reconstitution 1.0 ml/vial) – 1 vial;
• Plasma calibrator level 2, lyophilized (volume after reconstitution 1.0 ml/vial) – 1 vial;
• Control plasma level 1, lyophilized (volume after reconstitution 1.0 ml/vial) – 1 vial;
• Control plasma level 2, lyophilized (volume after reconstitution 1.0 ml/vial) – 1 vial.
The anti-Xa activity of heparin in calibrator plasmas is: Level 0 – 0 IU/ml; Level 1 – 0.4 – 0.7 IU/ml; Level 2 – 0.8 – 1.2 IU/ml. The anti-Xa activity of heparin in control plasmas is: Level 1 – 0.4 – 0.7 IU/ml; Level 2 – 0.8 – 1.2 IU/ml. Interpretation of results: When administering heparin for prophylactic purposes, its anti-Xa activity in plasma should be 0.3 – 0.7 IU/ml. When administering heparin for therapeutic purposes, its anti-Xa activity in plasma should be 0.7 – 1.2 IU/ml.
