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Licensed Unlicensed Requires Authentication Published by De Gruyter June 26, 2014

Progressive chromogenic anti-factor Xa assay and its use in the classification of antithrombin deficiencies

  • Bettina Kovács , Zsuzsanna Bereczky , Anna Selmeczi , Réka Gindele , Zsolt Oláh , Adrienne Kerényi , Zoltán Boda and László Muszbek EMAIL logo


Background: Antithrombin (AT) is a slow-acting progressive inhibitor of activated clotting factors, particularly thrombin and activated factor X (FXa). However, the presence of heparin or heparan sulfate accelerates its effect by several magnitudes. AT deficiency, a severe thrombophilia, is classified as type I (quantitative) and type II (qualitative) deficiency. In the latter case mutations may influence the reactive site, the heparin binding-site (HBS) and exert pleiotropic effect. Heterozygous type II-HBS deficiency is a less severe thrombophilia than other heterozygous subtypes. However, as opposed to other subtypes, it also exists in homozygous form which represents a very high risk of venous thromboembolism.

Methods: A modified anti-FXa chromogenic AT assay was developed which determines both the progressive (p) and the heparin cofactor (hc) activities, in parallel. The method was evaluated and reference intervals were established. The usefulness of the assay in detecting type II-HBS AT deficiency was tested on 78 AT deficient patients including 51 type II-HBS heterozygotes and 18 homozygotes.

Results: Both p-anti-FXa and hc-anti-FXa assays showed excellent reproducibility and were not influenced by high concentrations of triglyceride, bilirubin and hemoglobin. Reference intervals for p-anti-FXa and hc-anti-FXa AT activities were 84%–117% and 81%–117%, respectively. Type II-HBS deficient patients demonstrated low (heterozygotes) or very low (homozygotes) hc-anti-FXa activity with normal or slightly decreased p-anti-FXa activity. The p/hc ratio clearly distinguished wild type controls, type II-HBS heterozygotes and homozygotes.

Conclusions: Concomitant determination of p-anti-FXa and hc-anti-FXa activities provides a reliable, clinically important diagnosis of type II-HBS AT deficiency and distinguishes between homozygotes and heterozygotes.

Corresponding author: László Muszbek, MD, PhD, Clinical Research Center and Vascular Biology, Thrombosis and Hemostasis Research Group of the Hungarian Academy of Sciences, University of Debrecen, 98 Nagyerdei Körút, 4032 Debrecen, Hungary, Phone: +36 52 431956, Fax: +36 52 340011, E-mail:


This study was supported by grants from the Hungarian National Research Fund (OTKA K109543 and PD101120), the National Development Agency (TÁMOP 4.2.2.A-11/1/KONV-2012-0045), from the Hungarian Academy of Science (MTA11003, TKI227). The authors thank Gizella Haramura for skillful technical assistance.

Author contributions: All the authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.

Financial support: None declared.

Employment or leadership: None declared.

Honorarium: None declared.

Competing interests: The funding organization(s) played no role in the study design; in the collection, analysis, and interpretation of data; in the writing of the report; or in the decision to submit the report for publication.


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Received: 2014-3-6
Accepted: 2014-6-5
Published Online: 2014-6-26
Published in Print: 2014-12-1

©2014 by De Gruyter

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