Intended use

A chromogenic kit or the quantitative determination of unfractionated heparin (UF Heparin) or low molecular weight heparin (LMW Heparin) in human citrated plasma using automated and microplate methods.

Background and summary

Heparin is the most frequently used antithrombotic therapeutic.

The biological activity of this sulfated glycosaminoglycan resides in its ability to accelerate (up to 2000-fold) the inhibitory effect of antithrombin (AT) on the coagulation proteases.

The amount of LMW Heparin or UF Heparin is determined from the anti-FXa activity expressed by the [AT · Heparin] complex formed in plasma.

Measuring principle

Factor Xa (FXa) is added to a mixture of undiluted plasma and the chromogenic substrate S-2732.

When Heparin and AT are complexed, two competing reactions occur simultaneously:

1. Inhibition of FXa by the [AT · Heparin] complex.
2. Reaction of FXa with S-2732 resulting in cleavage of pNA. The pNA release measured at 405 nm is inversely proportional to the heparin level in the sample. In order to reduce the influence from heparin antagonists, such as platelet factor 4 (PF4), dextran sulfate is included in the reaction mixture.

Reagents -- Packaging, Storage and Stability

The sealed reagents are stable at 2-8°C until the expiry date printed on the label.

1. S-2732, 15 mg 2 vials. Chromogenic substrate, Suc-Ile-Glu(γ-pip)-Gly-Arg-pNA·HCl lyophilized with detergent and mannitol as bulking agent. Stability after reconstitution: 3 months at +2-8°C in the original vial. Avoid exposure to light. Discard the substrate solution if it appears yellow. Avoid contamination by microorganisms.
2. Factor Xa, 35 nkat 2 vials. Lyophilized bovine FXa containing Tris buffer, EDTA, NaCl, dextran sulfate and bovine serum albumin. Stability after reconstitution: 3 months at +2-8°C in the original vial.

WARNING: Do not use reagents beyond the expiry date printed on the package label.

Reagent preparation:

For the microplate method reconstitute REAGENTS 1 and 2 with 5.0 mL of water (see REAGENTS 3). Replace the stoppers and swirl gently. Make sure of the complete reconstitution of the product. Keep reagent at 15-25°C for 10-30 min and invert before use.

NOTE: Other reagent reconstitution volumes may apply for automated methods. (See section: INSTRUMENT APPLICATIONS). The reagents are not interchangeable between lots.

Reagents required but not provided:

• 3. Deionized water filtered through 0.22 mm or NCCLS type II water.5
• 4. Acetic acid 20% or citric acid 2% (end-point method).
• 5. Saline (0.9% NaCl).
• 6. Human normal plasma.
• 7. Calibrator plasma for LMW Heparin and/or UF Heparin calibrated against International Standards.
• 8. Controls for LMW Heparin and/or UF Heparin activity.

NOTE: Antithrombin reagent and tris buffer is required for the ACL Hundred/Thousand Series method (the assay is run as a two stage method with the addition of antithrombin). See the instrument Application Sheet for specific information.

Materials required but not provided:

• Spectrophotometer 405 nm (and 490 nm for the microplate procedure)
• Incubator 37°C
• Microplates*
• Centrifuge, 2000 x g
• Plastic test tubes
• Stopwatch
• Vortex mixer
• Calibrated pipettes
• Linear graph paper

*NOTE: Do not use microplates intended for coating