Simplified assay for antithrombin III activity using chromogenic peptide substrate. Manual and automated method

Area of Science:

• Clinical Chemistry
• Hematology
• Biochemical Assays

Background:

• Accurate measurement of antithrombin III (AT) is crucial for diagnosing and managing thrombotic disorders.
• Existing manual assays for AT can be time-consuming and complex.
• Development of a simplified and rapid AT assay is needed for routine clinical practice.

Purpose of the Study:

• To describe a simplified and rapid method for the quantitative assay of antithrombin III (AT).
• To evaluate the performance of the new assay using different thrombin substrates and temperatures.
• To assess the correlation of the simplified assay with established methods and its utility in clinical settings.

Main Methods:

• A simplified assay for antithrombin III (AT) was developed using the thrombin substrate 2AcOH X H-D-CHG-Ala-Arg-pNA (Th-1) or H-D-Phe-Pip-Arg-pNA (S-2238).
• Assays were performed at 30°C or 37°C.
• The method was validated by comparing results with immunoquantification and an automated centrifugal analyzer (Cobas-Bio) in 80 patient plasma samples.

Main Results:

• The standard curve for the AT assay was linear across a broad range (12.5-150%).
• The simplified assay demonstrated high correlation (r = 0.96) with both immunoquantification and an automated method.
• The assay confirmed low AT levels in patients with hereditary deficiency, liver cirrhosis, disseminated intravascular coagulation (DIC), and during heparin treatment for thrombosis.

Conclusions:

• The described simplified AT assay is rapid, reliable, and suitable for routine clinical use.
• The assay's high correlation with existing methods validates its accuracy.
• This method effectively identifies patients with clinically significant low antithrombin III levels.