DiaPharma Group, Inc.

Intended use

A chromogenic kit for the in vitro photometric determination of heparin and low molecular heparin (LMW) activity in human plasma. One-stage assay mainly intended for non-automated laboratories. The Coatest LMW Heparin/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

LMW Heparin (e.g. Fragmin) or Heparin is analyzed as a complex with Antithrombin (AT) present in the plasma sample. Activated Factor X (FXa) is added to a mixture of plasma sample and the chromogenic peptide substrate S-2732 in a buffer. Two competing reactions then start. One is the inhibition of FXa by the [LMW Heparin · AT] complex, the other is the FXa catalyzed release of pNA from the substrate. After a certain period of time most of the FXa is inhibited and the release of pNA has essentially declined. Further release of pNA is stopped by the addition of acetic acid and the absorbance at 405 nm is measured. The correlation between absorbance (A405) and LMW Heparin/Heparin activity is linear in the 0.1-1.0 IU/mL range when plotted in a log-lin scale.

Reagents -- Package, Storage and Stability

The sealed reagents are stable at 2-8°C until the expiration date printed on the label. Avoid contamination by microorganisms of the reagent.

1. S-2732 1 vial. Chromogenic substrate (Suc-Ile-Glu(g-Pip)-Gly-Arg·pNA), 6 mg, with mannitol as bulking agent. Reconstitute with 2.6 mL water to a concentration of 2.9 mmol/L. The solution is stable for 6 months at 2-8°C.

2. Factor Xa 1 vial. Bovine Factor Xa, 13 nkat. Reconstitute with 10.4 mL water. The solution is stable for one month at 2-8°C.

3. Buffer, 20 mL 1 vial. Tris 50 mmol/L, EDTA 7.5 mmol/L, pH 8.4, I=0.2. Once opened, the buffer solution is stable for two months at 2-8°C.

4. LMW Heparin Standard, 1mL 1 vial. A LMW Heparin (Fragmin) standard, 100 IU/mL. (Calibrated against the International Standard for LMW Heparin, established by the WHO, using an anti-Factor Xa method). Once opened, the solution is stable for 6 months at 2-8°C.

Reagents required but not provided

• Deionized water, filtered through 0.22 μm or NCCLS type II water.
• Saline (0.9% NaCl).
• Pooled normal human plasma taken on ice and prepared according to "SPECIMEN COLLECTION." A lyophilized preparation is available from Chromogenix AB or subsidiaries.
• Stopper solution: acetic acid 20% or monosodium citrate 20%.

Materials required but not provided

• Photometer, 405 nm
• Semi-micro cuvette (1cm)
• Centrifuge, 2000-4000 x g
• Stopwatch
• Disposable plastic tubes
• Pipettes, calibrated

Number of determinations
Test tube method: 50
Microplate method: 125