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Clinical Chemistry and Laboratory Medicine (CCLM)

Published in Association with the European Federation of Clinical Chemistry and Laboratory Medicine (EFLM)

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Ed. by Gillery, Philippe / Lackner, Karl J. / Lippi, Giuseppe / Melichar, Bohuslav / Payne, Deborah A. / Schlattmann, Peter / Tate, Jillian R.

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Modified phosphatidylserine-dependent antithrombin ELISA enables identification of patients negative for other antiphospholipid antibodies and also detects low avidity antibodies

1 / Aleš Ambrožič1 / Saša Čučnik1 / Tanja Kveder1 / Blaž Rozman1 / Borut Božič1, 2

1Department of Rheumatology, University Medical Centre, Ljubljana, Slovenia

2Faculty of Pharmacy, Chair for Clinical Biochemistry, University of Ljubljana, Ljubljana, Slovenia

Corresponding author: Polona Žigon, MSc, Immunology Laboratory, Department of Rheumatology, University Medical Centre Ljubljana, Vodnikova 62, 1000 Ljubljana, Slovenia Phone: +386 1 522 54 79, Fax: +386 1 519 53 38

Citation Information: Clinical Chemistry and Laboratory Medicine. Volume 49, Issue 6, Pages 1011–1018, ISSN (Online) 1437-4331, ISSN (Print) 1434-6621, DOI: 10.1515/CCLM.2011.162, May 2011

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Background: Two approaches for detecting anti-prothrombin antibodies have been described. The first detects antibodies against prothrombin alone and the second, phos-phatidylserine-dependent antiprothrombin antibodies. The latter more often correlate with clinical manifestations of antiphospholipid syndrome and with lupus anticoagulant activity.

Methods: In order to increase the capacity of antibody binding, we modified the previously described phosphatidylser-ine-dependent antiprothrombin ELISA and determined their avidity. We examined 203 patients with systemic autoimmune diseases and 222 blood donors.

Results: Our modification resulted in a greater intensity of antibody binding to prothrombin on phosphatidylserine-coated plate surfaces compared to the previously described method. By changing ELISA conditions, we were able to detect with one assay the two, presumably different, populations of antiprothrombin antibodies. Diagnostic specificities of both ELISAs for antiphospholipid syndrome were similar (92.5% vs. 93.1%), while the sensitivity of the modified phosphatidylserine-dependent antiprothrombin ELISA was significantly higher than the anti-prothrombin alone ELISA (59% vs. 25%). Low avidity antiprothrombin antibodies were only detected in the modified phosphatidylserine-dependent antiprothrombin ELISA. Four percent of patients with positive phosphatidylserine-dependent antiprothrombin antibodies, showing clinical manifestations of antiphospholipid syndrome, were negative for all other antiphospholipid antibodies. The risk for antiphospholipid syndrome increased with the number of antiphospholipid antibody positivity.

Conclusions: We conclude that antibodies detected with a modified phosphatidylserine-dependent antiprothrombin ELISA could improve the diagnosis of antiphospholipid syndrome by offering additional information on the risk for thrombosis, especially in patients negative for other antiphospholipid antibodies.

Keywords: antiphospholipid antibody; antiphospholipid syndrome; antiprothrombin antibodies; enzyme immunoassay; phosphatidylserine-dependent antiprothrombin antibodies

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