Minimal requirements for antiphospholipid antibodies ELISAs proposed by the European Forum on antiphospholipid antibodies
Introduction
During the last 20 years, the wide application of antiphospholipid (aPL) ELISAs and in particular of anticardiolipin (aCL) antibody assays was determinant in the definition of the antiphospholipid antibody syndrome (APS) and of its pathogenic mechanisms, mainly consisting of antibody-mediated thrombosis. Consequently, aCL ELISA was introduced as one of the laboratory classification criteria of the syndrome [1].
Despite its recognized practical importance, and several standardization workshops [2], [3], aCL immunoassay, 20 years after the publication of the first paper, still shows a high inter-laboratory variability [4], [5], [6]. The reasons for this poor performance can be linked to the difference among the protocols applied by either different companies producing commercial kits or workers performing homemade assays [4], [5].
A limited number of studies focused on anti-beta2 glycoprotein I (anti-β2GPI) ELISA, but a significant variability was shown also in the results of this test [7], that still lacks accepted rules in the reporting of results.
Consequently, it is not surprising that large metanalyses recently performed were not able to give a clear clinical significance to aCL and anti-β2GPI immunoassays, underlying the need for standardization or harmonization of the methods [8], [9].
After 3 years of common work within the European APL Forum, we showed that the use of a common protocol and of common standard preparations can improve the reliability of aCL assay; however, this effort did not have any practical consequences, because no improvement was achieved in the routine performance of these tests.
Section snippets
Identification of European APL forum minimal requirements
In preparation of the IV European Forum Meeting (London, 16–17 January), the Standardization Committee members actively collaborated, by mail exchanges, to identify methodological improvements that could be applied to aPL ELISAs on a large scale. The conclusion of this discussion was presented at the Forum Meeting and approved by the delegates, underlining their concern particularly for the publication of clinical studies.
Taking into account the studies performed during the previous years, the
HCAL and EY2C9 use as calibrators
To validate the efficacy of HCAL and EY2C9, suggested by our previous study [5], we undertook a collaborative work on European scale involving 36 laboratories. All the participants received five serum samples and were asked to study them by their routine aCL and anti-β2GPI assay. The samples were previously selected and analysed in three laboratories (Marseille, Geneva and Brescia) involved in the European Forum Standardization Committee. One sample was from a normal subject and four from APS
Interactive Forum-companies workshop
During the IV APL European Forum Meeting (London, 16–17 January 2004), the minimal requirements were presented to the few companies producing kits for the two assays that were present there. Acting on a proposition from Marie-Claire Boffa, we decided to share our ideas with all Companies producing aCL and anti-β2GPI immunoassays, that we could reach. With this intent, the Standardization Committee invited 17 companies to an interactive workshop, that was held on the 3rd of May 2004 and
Conclusions
In the area of aPL ELISAs standardization, the Standardization Committee of the APL European Forum is focusing on a realistic possible improvement in the assays performance. Our efforts are concentrating on two distinct aspects, both critical in the general improvement of the knowledge in the area and in the patients’ classification. The first one underlines the “minimal requirements” of aCL and anti-β2GPI immunoassays necessary to get comparable results in clinical studies. It appears
Acknowledgments
The authors want to thank Takao Koike for generously providing HCAL and EY2C9 to the Forum; all the companies attending the Interactive Workshop, for their prompt answers and their interest; Franco Franceschini, Robert Roubey and Yehuda Shoenfeld for their helpful discussion.
In addition, we want to acknowledge the Chairmen of the International Institutions devoted to the Autoantibody Tests Standardization: Marvin Fritzler (AF/IUIS/CDC/WHO Committee for Standardization of the Autoantibody
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