A new activated factor X-based clotting method with improved specificity for procoagulant phospholipid.

Area of Science:

• Hematology
• Clinical Chemistry
• Biochemistry

Background:

• Procoagulant phospholipid (PPL) plays a critical role in hemostasis.
• Accurate measurement of PPL activity is essential for diagnosing and managing bleeding disorders.
• Existing methods for PPL assessment have limitations in sensitivity and specificity.

Purpose of the Study:

• To develop and validate an improved activated factor X-based clotting method for quantifying PPL activity.
• To assess the performance of this new assay across various sample types and anticoagulants.
• To evaluate the assay's utility in detecting PPL abnormalities.

Main Methods:

• A dilute activated factor X-activated clotting time (XACT) assay was developed using phospholipid-free porcine plasma.
• The XACT assay measures the rate-limiting effect of endogenous PPL on clotting time.
• Assay performance was evaluated with various anticoagulants, plasma abnormalities, and compared with flow cytometry for microparticle counts.

Main Results:

• The XACT assay demonstrated high sensitivity for PPL, detecting down to 50 ng/ml synthetic phospholipid.
• XACT results were significantly shortened by activated platelets and platelet microparticles.
• The assay showed resistance to heparin and most lupus anticoagulants but was not prolonged by clotting factor deficiencies.
• XACT was more stable in ethylenediamine tetra-acetate (EDTA) anticoagulated samples compared to citrated samples.
• Whole blood samples showed shorter XACT times than plasma, suggesting PPL expression by larger cellular aggregates.

Conclusions:

• The improved XACT assay is a sensitive and robust method for measuring PPL activity.
• EDTA anticoagulation is preferred for ex vivo assessment of PPL expression.
• The assay has potential for diagnosing PPL-related hemostatic abnormalities.
• Further studies are warranted to explore PPL expression in various clinical conditions.