What is the role of functional antithrombin activity assay in diagnosing and managing antithrombin deficiency?

Role of Functional Antithrombin Activity Assay in Diagnosing and Managing Antithrombin Deficiency

Functional antithrombin activity assay is the recommended initial test for diagnosing antithrombin deficiency, as it can detect both quantitative (type I) and qualitative (type II) deficiencies that may increase risk of venous thromboembolism. 1

Types of Functional Antithrombin Assays

• Two main types of functional assays are available: anti-FXa (factor Xa)-based and anti-FIIa (thrombin)-based chromogenic substrate assays 2
• Anti-FXa assays are superior for detecting heparin-binding site defects (type IIb), which are the most common type II deficiencies 3
• Anti-FIIa assays may miss type II heparin-binding site defects, as heterozygotes often show activity within the reference interval despite having the deficiency 3
• In homozygous heparin-binding site mutations, anti-FIIa activities range from 48% to 80%, while anti-FXa activities are significantly lower (9% to 25%), making anti-FXa assays more sensitive 3

Diagnostic Algorithm for Antithrombin Deficiency

1. Initial Testing:

   • Begin with a functional antithrombin activity assay 1, 4
   • If activity is low, measure antithrombin antigen levels to differentiate between type I and type II deficiencies 5
   • Type I deficiency: both activity and antigen levels are reduced 6
   • Type II deficiency: reduced activity with normal antigen levels 6

2. Confirmation Process:

   • Repeat testing on a separate sample to confirm deficiency 6
   • Rule out acquired causes of antithrombin deficiency (more common than hereditary) 1
   • For suspected type II defects, compare results from both anti-FXa and anti-FIIa assays 3

3. Further Characterization (if clinically indicated):

   • Assay antithrombin in the absence of heparin 5
   • Electrophoresis to investigate heparin binding ability 5
   • Gene sequencing to identify specific mutations 5

Clinical Significance and Management Implications

• Antithrombin deficiency significantly increases risk of venous thromboembolism (VTE) 6
• Type II defects have varying thrombotic risks: type IIa (thrombin-binding site defects) are more thrombogenic than type IIb (heparin-binding site defects) 6
• Functional assays help guide anticoagulation therapy decisions for patients with confirmed deficiency 4
• Antithrombin activity measurement is useful in predicting morbidity and mortality in sepsis-associated DIC (disseminated intravascular coagulation) 4
• In septic DIC, antithrombin activity correlates with disease severity: ~80% in septic patients without organ dysfunction, ~60% with organ dysfunction, and ~40% in patients with DIC 4

Important Technical Considerations

• Sample preparation is critical: venous blood should be collected in 3.2% trisodium citrate and double-centrifuged to ensure residual platelet count <10.0 × 10^9/L 4
• Testing should be performed within 4 hours of collection or plasma should be frozen appropriately 4
• Direct thrombin inhibitors (like lepirudin) can cause false overestimation of antithrombin activity in thrombin-based assays but not in FXa-based assays 2
• Reducing incubation time of sample with enzyme/heparin reagent may increase sensitivity to type II defects 5

Common Pitfalls and Limitations

• No single functional assay can detect all forms of antithrombin deficiency 5
• Thrombin-based assays may overestimate antithrombin activity due to sensitivity toward heparin cofactor II 2
• Thrombin-based assays should not be used in patients on direct thrombin inhibitor therapy 2
• High-dose heparin treatment can affect results of thrombin-based assays 2
• Icterus, hemolysis, and lipemia can interfere with assay results, especially in undiluted plasma 4