Development of a novel, rapid assay for detection of heparin-binding defect antithrombin deficiencies: the heparin-antithrombin binding (HAB) ratio

• HAB ratio is a novel screening procedure for type II HBD AT deficiency
• HAB ratio is a rapid alternative to currently available techniques that identify HBDs
• Not all HBDs generate higher AT levels upon prolonged incubation with heparin

IntroductionQualitative and quantitative antithrombin deficiency predisposes to thrombosis, although patients with heparin-binding dysfunction have a lower incidence than other sub-types. Assays discriminating between qualitative sub-types are not widely available.Patients/MethodsExtended heparin incubation in antithrombin activity assays can overestimate levels in patients with HBDs. Plasmas from genetically proven HBD patients were assayed for antithrombin activity by factor Xa-inhibition and thrombin-inhibition at varying incubation times. Optimal pairings were assessed for generating a quantifiable discrepancy in HBDs by deriving a ratio between results from short and prolonged heparin-incubation assays respectively, the Heparin-antithrombin binding (HAB) ratio. Fourteen patients with hereditary antithrombin deficiency, including five with HBDs, were analysed.ResultsThe FXa-inhibition assay with 30s and 300 s incubations clearly identified a heterozygous p.Pro73Leu and homozygous p.Leu131Phe, giving HAB ratios of 0.24 and 0.67 respectively (reference range 0.90 – 1.01). However, three plasmas containing mutations with markedly reduced or absent heparin affinity (p.Lys146Glu, p.Gln150Pro, p.Arg79Cys) gave normal results. Nine antithrombin deficient plasmas were tested with the thrombin-inhibition assay and all generated reduced HAB ratios whilst two normal donors did not. The three available HBD plasmas generated lower values than non-HBD plasmas. The mildly reduced HAB ratios in non-HBD deficiencies may have been due to heparin cofactor II reacting with bovine thrombin during extended incubation.ConclusionsHAB ratio from FXa-inhibition assays distinguishes some but not all HBD from non-HBD antithrombins, and thrombin-inhibition assays may be diagnostically applicable with sub-type specific cut-offs.