DiaPharma Group, Inc.

Intended use

A chromogenic kit for the in vtiro diagnostic photometric determination of heparin and low molecular heparin (LMW) in human plasma. The Coatest Heparin kit determines the anti-FXa activity of LMW heparin and UF heparin. Excellent reagent stability. Suitable for both large and small laboratories.

Measurement principle

Heparin is analysed as a complex with Antithrombin (AT) present in the sample. The concentration of this complex is dependent on the availability of AT. In order to obtain a more constant concentration of AT, purified AT is added to the test plasma. FXa (in excess) is neutralized in proportion to the amount of heparin, which determines the amount of [Heparin · AT] complex.The remaining amount of FXa hydrolyses the chromogenic substrate S-2222 thus liberating the chromophoric group, pNA. The color is then read photometrically at 405 nm.

Reagents -- Package, Storage and Stability

When kept at 2-8°C the sealed reagents are stable until expiry date printed on the label. Avoid contamination by microorganisms in opened vials.

1. S-2222 1 vial. Chromogenic substrate (Bz-IIe-Glu-(g-OR)-Gly-Arg-pNA·HCl) 15 mg with mannitol added as a bulking agent. Reconstitute with 20 mL sterile water to obtain a concentration of 1 mmol/L. The solution is stable for 6 months at 2-8°C.

2. Factor Xa 1 vial. Bovine Factor Xa 71 nkat. Reconstitute with 10 mL sterile water. The reconstituted Factor Xa is stable for 1 month at 2-8°C or 6 months at -20°C or below.

3. Buffer, stock solution 1 vial. Tris 0.5 mol/L, pH 8.4, 10 mL. An opened vial of stock solution is stable for 2 months at 2-8°C. Before use dilute accordingly: 1 volume of stock solution with 9 volumes of sterile water.

4. Antithrombin 1 vial. Lyophilized human Antithrombin, 10 IU. Reconstitute with 10 mL sterile water to obtain a concentration of 1 IU/mL. The reconstituted Antithrombin is stable for 1 month at 2-8°C or 6 months at -20°C or below.

5. Normal Plasma (human) 4 vials. Lyophilizied plasma. Reconstitute with 1.00 mL sterile water. The reconstituted plasma is stable for two weeks at 2-8°C or 1 month at -20°C or below

Reagents not provided

• Sterile water
• Acetic acid 20% or citric acid 2%
• NaCl 0.9% (saline)
• Heparin

Material required but not provided

• Photometer, 405 nm
• Heating device, 37°C ± 0.2°C
• Calibrated pipettes
• Plastic test tubes
• Semi-micro cuvettes
• Mixer
• Stopwatch
• Centrifuge, 2000 x g

Number of determinations:
Test tube: 100
Microplate: 400
Automated: up to 285