Assessment of a Serum Assay for Quantification of Abdominal Aortic Calcification
Intimal vascular calcification is an important marker of atherosclerosis, and its severity is an independent risk factor for cardiovascular events.1 Measurement of coronary or aortic calcification requires CT-based imaging and elaborate data analysis to ensure accuracy.2 A blood assay which could quantify the severity of vascular calcification would be less expensive, avoid exposure to radiation, and be more accessible than present imaging based methods. Osteoprotegerin (OPG) and osteopontin (OPN) are present in human serum and have been implicated in vascular calcification.3,4 In this prospective study we assessed the value of a number of serum assays for OPG and OPN in determining abdominal aortic calcification. Firstly, we investigated the assay characteristics and reproducibility of 5 ELISAs using a subset of patients. Based on the findings of these assessments, we selected 3 assays for full evaluation in the entire cohort.
One hundred and nine patients with peripheral vascular disease had fasting serum obtained the morning before measurement of infrarenal abdominal aortic calcification. We assessed a variety of different commercial ELISAs (details are given in the supplemental Methods, available online at http://atvb.ahajournals.org).
Assessment of Assay Characteristics
We identified a large number of commercial ELISAs for OPG with varying reported assay characteristics (see supplemental Methods). We initially compared the OPG ELISAs in terms of recognized assay requirements.5 The calibration curves and linearity of the assays were excellent (supplemental Table I). Concordance correlation coefficients for intra- and inter-assay reproducibility were good but most reproducible for the DuoSet assay (supplemental Table II). Each OPG assay uses a different form of OPG as a reference standard (supplemental Figure I). As a measure of accuracy we assessed the ability of the different assays to recognize added OPG spiked into serum. The mean recovery was 57±7.5%, 83±5.0%, and 58±9.0% for the Duoset, Biovendor, and Biomedica ELISAs, respectively (n=8 for each assay). The physiological ligand RANKL had little effect on the assay results (see supplemental Results). Two assays for serum OPN were assessed (Quantikine R&D Systems and IBL Immuno-Biological Laboratories). We found similar assay characteristics for these kits (mean recovery 90±4.5% and 88.3±9.1%, intra- and inter-assay concordance correlation coefficient 0.990 to 0.994).
Comparison of Serum OPG and OPN in Determining Aortic Calcification
We assessed the ability of serum OPG assessed with both BioVendor and DuoSet ELISAs and OPN measured with the R&D quantikine kit to predict abdominal aortic calcification. The characteristics of the patients entered into the study are shown in supplemental Table III. The mean volume of infrarenal abdominal aortic calcification was 153.7±142.5 cm2 (range 4 to 5650 cm2). Serum OPG measured by DuoSet (r=0.26, P=0.006) and BioVendor assays (r=0.27, P=0.004) but not serum OPN (r=0.03, P=0.76) was correlated with aortic calcification (Figure, a). Patients were divided into quartiles based on volume of aortic calcification. Serum OPG but not OPN was related to calcification quartile (Figure, b). Receiver operating characteristic curves demonstrated that a cut-off value of OPG of 72 pM (DuoSet; sensitivity 85%, specificity 55%, negative predictive value 92% based on prevalence of 25%, area under the curve 0.68, 95% CI 0.58 to 0.79, P=0.004) and 8.49 pM (Biovendor; sensitivity 56%, specificity 76%, negative predictive value 84% based on prevalence of 25%, area under the curve 0.67, 95% CI 0.56 to 0.79, P=0.007) were the most accurate in determining the highest quartile of aortic calcification (Figure, c).
Comparison of the DuoSet and BioVendor ELISA
The serum OPG concentrations measured by the two OPG ELISAs were noted to be very different with limited concordance between values (Table). To further investigate these differences the 109 serum samples and opposing manufacturer’s standards were assessed in each assay. These results allowed the calculation of the concentration of the serum samples by reference to the two different standards in each assay separately. Using the supplied standards to calculate results the mean (±standard deviation), OPG concentrations for the samples were 78.4±32.4 and 7.59±2.75 pM in the Duoset and Biovendor ELISAs, respectively. The difference in values were markedly reduced by the use of similar standards in the different ELISA plates; eg, using Biovendor standards to read the Duoset and Biovendor ELISAs resulted in mean serum OPG concentrations of 9.73±5.86 and 7.59±2.75, respectively (Table).
A number of previous studies have assessed the association of serum calcification markers, such as osteoprotegerin and osteopontin, with abdominal aortic calcification.1,3,4,6–8 In many of these studies the reproducibility and accuracy of the assessment methods has not been quantitated, with a number of investigators relying on subjective techniques such as plain X-ray.3 Other studies have only involved small numbers of patients4,6,8 or were reserved to those patients with renal failure.7 In this study we used a carefully validated CT protocol to accurately measure the volume of infrarenal aortic calcification and, in addition, thoroughly assessed the assay characteristics for our calcification markers (supplemental Tables I and II).2 We found an association between serum concentrations of OPG but not OPN and severity of infrarenal abdominal aortic calcification (Figure). The association suggests the potential value of serum assays for OPG in predicting aortic calcification and cardiovascular risk. Indeed, serum concentrations of OPG have been related to cardiovascular events.9 However, our study also emphasized that at present the available commercial ELISAs for OPG have a number of differences with marked variation in calculated serum concentrations with different assays (Table). These variations appear largely attributable to differences in the standards used in the ELISAs (Table; supplemental Figure I). Which assay gives values closest to true serum OPG requires further assessment, but the greater similarity of the DuoSet standard to full-length OPG suggests this ELISA may be more representative. Further improvements in OPG assays may lead to better sensitivity and specificity in detecting significant aortic calcification and allow the assay to become clinically useful. The relatively low cost of the DuoSet ELISA supports the use of this assay in large screening studies.
Sources of Funding
This project is supported by grant numbers RO1 HL080010-01 from the National Institute of Health, USA, and 279408 from the National Health and Medical Research Council Australia.
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