Article pubs.acs.org/ac
Heterogeneity between Diagnostic Tests for IgA anti-Beta2 Glycoprotein I: Explaining the Controversy in Studies of Association with Vascular Pathology José A. Martínez-Flores,†,∇ Manuel Serrano,‡,∇ Javier Alfaro,† Sergio Mora,† Estela Paz-Artal,†,§ José M. Morales,‡ and Antonio Serrano*,†,§ †
Department of Immunology and ‡Department of Nephrology, Instituto de Investigación Hospital Universitario, 12 de Octubre, Madrid, Spain § Section of Immunology, Universidad San Pablo-CEU, Madrid, Spain ABSTRACT: IgA antibeta 2 Glycoprotein I (β2GPI) antibodies test can identify some patients with antiphospholipid syndrome (APS) that are negative for other isotypes. Controversy exists because some studies have reported a strong association of these antibodies with vascular disease, while others have not confirmed this observation. Our hypothesis is that these contradictory results may be due to differences among commercial diagnostic kits. To answer this question, we have compared the results obtained with several of the most commonly used commercial IgA anti β2GPI antibodies (aβ2GPI) diagnostic assays on specimens from individuals suspected of having APS. Sera from 69 patients (37 positive and 32 negative for IgA aβ2GPI) were analyzed with seven different commercial ELISA kits for IgA aβ2GPI, following instructions and cutoffs provided by the manufacturer. Our results showed important differences in the sensitivity and specificity of the different assays. Two of the seven kits tested had a sensitivity level below 65% for IgA aβ2GPI, and three showed levels of specificity lower than 80%. Some commercial kits to detect IgA aβ2GPI are suboptimal. Variability between kits may account for the discrepancy in results obtained and for the lack of consensus concerning their clinical significance. It is important that the scientific community work to standardize assay performance so that the true clinical significance of this important clinical marker can be clearly established.
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these studies may account for some of the conflicting observations. The aim of this study is to compare the sensitivity and specificity performance of different commercial ELISA kits for the detection of IgA aβ2GPI in a cohort of patients with clinical compatible with APS randomly chosen disease controls.
ntiphospholipid syndrome (APS) is a prothrombotic disorder1 that usually occurs in the presence of antibodies against phospholipids (PL), PL-binding proteins, or complexes formed by both molecules.2 The most commonly detected subgroup of antibodies in APS are those directed against lupus anticoagulant (LA), cardiolipin (CL), and beta 2-glycoprotein I (β2GPI).3 Most of the APS autoantibodies are not directly targeted to isolated PL, but require the association of the PL with a protein co-factor β2GPI.4 These autoantibodies attach to the β2GPI-PL-complex located on the cellular membrane of platelets, monocites, and cells involved in the coagulation cascade.4 The interference with the endothelial membrane dynamics and coagulation factors seems to be responsible of the hypercoagulability observed in APS patients.5 Whereas only autoantibodies for G and M immunoglobulin isotypes are considered relevant for APS diagnosis at present, some articles have been reported regarding the importance of IgA isotype antibodies and vascular pathology.6−8 Despite these observations, many clinicians question the relevance of IgA antibodies because some publications show no association.9 The lack of standards for the IgA anti β2GPI antibodies (aβ2GPI) detection and the disparity among results suggest that significant differences between the methodologies used in © 2013 American Chemical Society
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MATERIALS AND METHODS Frozen samples from 69 patients were randomly chosen from our sera library of positive and negative controls for IgA aβ2GPI. Sera proceed from patients suspected of having APS that were submitted for aPL testing. We classified the sera as positive (n = 37) or negative (n = 32) using Kit Number 1 (K1; QUANTA Lite IgA β2GPI from INOVA Diagnostics, San Diego, CA, USA). Sera with IgA aβ2GPI levels greater than or equal to 24 units (20% above the cutoff suggested by the manufacturer, 20 units) were considered positive. All the IgA aβ2GPI positive sera were Received: October 4, 2013 Accepted: November 19, 2013 Published: November 19, 2013 12093
dx.doi.org/10.1021/ac403194t | Anal. Chem. 2013, 85, 12093−12098
Analytical Chemistry
Article
Table 1. Results of the Sera Tested by Manufacturers Using the Cutoff Set by the Kit and Compared to That of Kit K1 To Evaluate the Sensitivity and Specificitya
a
kit
cutoff (U/mL)
true positives
true negatives
K2 K3 K4 K5 K6 K7
18 8 20 15 4 20
30 33 0 35 37 13
32 31 31 31 8 24
false false positives negatives 0 1 1 1 24 8
7 4 37 2 0 24
positive predictive value (%)
negative predictive value (%)
sensitivity (%)
specificity (%)
kappa value
kappa 95% CI
81 89 0 95 100 35
100 97 97 97 25 75
100 97 0 97 61 62
82 89 46 94 100 50
0.799 0.855 −0.029 0.913 0.263 0.098
0.658 to 0.940 0.733 to 0.977 −0.248 to 0.190 0.816 to 1.009 0.025 to 0.501 −0.131 to 0.327
Kits K2, K3, and K5 show good sensitivity and Kits K2, K3, K5, and K6 show good specificity. Kits K2, K3, and K5 have kappa values over 0.7.
into two groups; the first one, which is composed of kits K2, K3, and K5 showed kappa index values higher than 0.75 over the reference kit, with sensitivity, specificity, and positive and negative predictive values above 85%. The second group, comprised of kits K4, K6, and K7, had kappa values below 0.3 and the worst figures for sensitivity, specificity, and positive and negative predictive values (Table 1). All discordant results were repeated and confirmed. Analyzing low, medium, and high positive groups suggest that discrepancies in the detection of positives among kits K2, K3, K5 and the reference kit was the failure to detect low positive results (Figure 1). Kit K4 is not able to detect any positives, and Kit K6 identifies all the positive sera (Figure 1), but was unable to discriminate negatives (see Table 1).
negative for aCL IgG, IgA and IgM and aβ2GPI IgG, and IgM using QUANTA Lite IgG β2GPI, IgM β2GPI, aCL IgG, aCL, IgA, and aCL IgM from INOVA Diagnostics, San Diego, CA, USA. Clinical characteristics of 37 IgA aβ2GPI positive patients were as follows: venous thrombosis (n = 13), arterial thrombosis and stroke (n = 3), recurrent pregnancy loss (n = 2), pulmonary thrombosis (n = 8), suspected APS without other clinical data (n = 8), and hemodialysis patients (N = 3). The 32 negative control sera consisted of 15 sera from anonymous blood donors and 17 sera from patients without clinical APS who (i) requested an aPL study in the context of routine health examinations and (ii) tested negative for aCL and aβ2GPI IgG, IgA, and IgM. The IgA aβ2GPI values in this group were
