DiaPharma Group, Inc.

For in vitro diagnostic use

Intended use of the kit

A chromogenic kit for the quantitative determination of plasminogen (Plg) activity in human citrated plasma using microplate, test tube and automated methods. Addition of plasminogen-free fibrinogen to the streptokinase reagent overcomes the overestimation of plasminogen which may arise in patients with elevated levels of FDP or fibrinogen. Not sensitive to elevated concentrations of FDP or fibrinogen in plasma. Validated and documented protocols for a wide range of instruments.

Background and summary

Measurement of plasminogen levels may aid in the diagnosis of fibrinolytic (blood clotting) disorders. Plasminogen is the precursor protein which after activation by plasminogen activators such as t-PA, u-PA or streptokinase, will form plasmin. The serine protease plasmin is a potent enzyme with a wide range of physiological functions. It is important to measure plasminogen levels since abnormal plasminogen activity is associated with different clinical circumstances.

Measurement principle

The plasminogen present in the sample is activated by the addition of an excess of streptokinase (Sk) forming a plasminogen-streptokinase (Plg/Sk) complex.

Plasminogen-depleted fibrinogen is included in the streptokinase reagent in order to avoid the risk of overestimation of plasminogen in the pathological plasmas containing elevated levels of fibrinogen (Fib) and/or fibrin degradation products (FDP).

The Plg·Sk/Fib complex is determined by the rate of hydrolysis of the chromogenic substrate S-2403.

Reagents -- Composition, Packaging, Storage and Stability

• S-2403 8.4 mg 2 vials. Chromogenic substrate pyroGlu-Phe-Lys-pNA·HCl lyophilized with mannitol as bulking agent. Stability after reconstitution: 6 months at 2-8°C in the original vial.
• Streptokinase 20 000 IU/Fibrinogen 3 mg 2 vials. Contains human albumin, Tween 80 and a fibrin polymerization inhibitor. Stability after reconstitution: 1 month at 2-8°C in the original vial.

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

WARNING: Do not use reagents beyond the expiry date printed on the package label. Discard if the substrate solution appears yellow.

Reagents required but not provided:

• Deionized water, filtered through 0.22 μm or NCCLS type II water5
• Saline (0.9% NaCl)
• Calibrated human normal plasma
• Appropriate controls calibrated for plasminogen activity
• Acetic acid 20% or citric acid 2% (end-point method)

Materials required but not provided:

• Spectrophotometer 405 nm (and 490 nm for microplate procedure)
• Incubator 37°C ± 0.2°C
• Microplates* or semi-micro cuvettes
• Centrifuge, 2000 x g
• Plastic test tubes
• Stopwatch
• Vortex mixer
• Calibrated pipettes
• Linear graph paper

*NOTE: Do not use microplates intended for coating!

Number of determinations:

• 50 (test tube)
• 200 (microplate)
• up to 200 (automated)