Minimal requirements for antiphospholipid antibodies ELISAs proposed by the European Forum on antiphospholipid antibodies

https://doi.org/10.1016/j.thromres.2004.06.035Get rights and content

Abstract

Antiphospholipid ELISAs are part of the Antiphospholipid Antibodies Syndrome classification criteria, having the same diagnostic value as lupus anticoagulant. However, sometimes their results appear scarcely meaningful especially when wide metanalyses studies are performed, probably because of their well-known inter-laboratory variability. The application of a common protocol was shown to improve the test reproducibility, but this observation did not have any influence on the routine performances.

After discussion among experts at the European level, we identified four conditions named “minimal requirements” considered useful to decrease the inter-laboratory variability:

  • (1)

    to run the samples in duplicate;

  • (2)

    to determine the cut off level in each laboratory analysing at least 50 samples from normal subjects, possibly age- and sex-matched with the patient population usually attending the Centre;

  • (3)

    to calculate the cut-off level in percentiles;

  • (4)

    to use stable external controls in the test.

A collaborative study involving 36 European centres proved that the use of monoclonal anti-beta2 glycoprotein I antibodies, HCAL (IgG) and EY2C9 (IgM) as standards, can help to reduce the inter-laboratory coefficient of variation both in anticardiolipin (aCL) and anti-β2GPI (anti-beta2 glycoprotein I) ELISA. Therefore, we propose HCAL and EY2C9 as external controls, but other monoclonal or polyclonal preparations may be considered.

During an interactive workshop held last May in Italy, 16 companies producing these tests agreed to consider the introduction of the “requirements” in their products. We suggest to adopt these “requirements” particularly in clinical studies, in order to compare more easily the literature data.

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

References (15)

There are more references available in the full text version of this article.

Cited by (142)

  • Clinical value of anti-domain I-β2Glycoprotein 1 antibodies in antiphospholipid antibody carriers. A single centre, prospective observational follow-up study

    2018, Clinica Chimica Acta
    Citation Excerpt :

    Antibody levels higher than 20 chemiluminescence units (CU) were considered positive. IgG/IgM a-β2GPI and IgG/IgM aCL were determined using homemade ELISA methods following the European Forum on aPL antibody recommendations [12, 13], as described elsewhere [14]. Cut-off values for medium–high levels of IgG/IgM a-β2GPI and IgG/IgM aCL antibodies were calculated as greater than the 99th percentile of sera from 120 healthy blood donors matched for age and sex with the study population [15].

  • Beyond thrombosis: Anti-β2GPI domain 1 antibodies identify late pregnancy morbidity in anti-phospholipid syndrome

    2018, Journal of Autoimmunity
    Citation Excerpt :

    Clinical and laboratory characteristics of included women are detailed in Table 1. All women were first investigated using criteria aPL assays: LA was performed according to international guidelines [31]; aCL and anti-β2GPI IgG/IgM were detected by internationally validated in-house ELISA methods [32,33], routinely used in clinical practice. The threshold values to define medium-high positivity for ELISA assays were set upon the 99th percentile of a control population of 100 healthy donors, according to Miyakis [1].

View all citing articles on Scopus
View full text