Kinetic Clotting Test

Method For Testing Thromboresistant Surface Properties - Kinetic Clotting Test... 

A simple in vitro screening test was developed in our laboratory and named the "kinetic clotting test"(6). The kinetic method is... 

Sakai H, Hisamoto S, Fukutomi I, et al. Detection of lipopolysaccharide in hemoglobin-vesicles by Limulus amebocyte lysate test with kinetic-turbidimetric gel clotting analysis and pretreatment of surfactant. J Pharm Sci 2004 93 310. 

Bacterial endotoxin kinetic test or gel clot limit standard test method (provide reference number)... 

The PPC, required by the FDA Guideline, is a sample of the test material that contains a concentration of endotoxin that is double the labeled sensitivity of the LAL reagent.The PPC must be tested with each sample in a Limits test or kinetic LAL assay to ensure that a result is valid and free of interference. The most accurate and reliable technique is the hot spike method that requires adding 10 pi of endotoxin standard to the reaction vessel (tube or well) before addition of LAL. The gel clot method requires the addition of 10 pi of 20 A to the reaction mixture. Kinetic methods require the addition of 10 pi of a standard, 10 times greater than the spike concentration, to the wells or tubes designated as PPCs. Only inhibition is seen in gel-clot Limits tests, whereas both inhibition and enhancement are seen in kinetic LAL methods. 

The results of endotoxin tests for in-process solutions, bulk materials, and finished parenteral products should be reported in the same units as those assigned to the product. Two factors determine the sensitivity of a BET. For infusion solutions and device extracts, the gel-clot sensitivity or the lowest point on the standard curve (lambda for kinetic LAL) and the amount of dilution determine test sensitivity.For products that have an endotoxin limit in EU/mg, the choice of lambda and the concentration of the test material determine sensitivity. The formula for product-specific sensitivity (PSS) is a convenient way to calculate the sensitivity of a BET for this type of product, where ... 

The samples of products are incubated with Limulus amoebocyte lysate at 37°C. If endotoxins are present a solid gel forms, indicating the presence of endotoxins. The British Pharmacopoeia (2002) describes six separate methodologies for the test for endotoxin. These are (A) gel-clot limit test (B) gel-clot semi quantitative (C) turbidimetric kinetic method (D) chromogenic kinetic method (E) chro-mogenic end-point method and (F) turbidimetric end-point method. There are checks for interfering factors. Any validated method may be used, but the gel-clot method is the referee test in the case of dispute. Coloured products cannot be tested by... 

Other LAL methods are available based on turbidimetry and colorimetry. Reaction mixtures become turbid as gels, or clots form between LAL and bacterial endotoxin. Turbidimetry (sensitive to 0.(X)1 EU/mL) is more sensitive for detecting bacterial endotoxin than gel clot assays (sensitive to 0.03 EU/mL) because turbidity is discernible at low concentrations of endotoxin at which finn gels do not form. The rate at which turbidity increases is proportional to the concentration of endotoxin in the material under test. This principle may be applied in end-point or kinetic assays. 

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