Clinical Chemistry 53: 859-865, 2007.
First published March 29, 2007; 10.1373/clinchem.2006.081679
(Clinical Chemistry. 2007;53:859-865.)
© 2007 American Association for Clinical Chemistry, Inc.
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Proteomics and Protein Markers |
Diagnostic Accuracy of ELISA and xMAP Technology for Analysis of Amyloid ß42 and Tau Proteins
Thierry S.M. Reijn1,2,3,
Marcel Olde Rikkert2,4,
Wieneke J.A. van Geel3,
Danielle de Jong1 and
Marcel M. Verbeek1,2,3,a
Department of1
Neurology, 2
Alzheimer Centre Nijmegen, 3
Laboratory of Pediatrics and Neurology, and 4
Department of Geriatric Medicine, Radboud University Nijmegen Medical Centre, The Netherlands.
aAddress correspondence to this author at: Department of Neurology, 830 LKN, Radboud University Nijmegen Medical Centre, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Fax 31-24-3668754; e-mail m.verbeek{at}cukz.umcn.nl.
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Abstract
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Background: Cerebrospinal fluid (CSF) concentrations of amyloid ß42 (Aß42) peptides and tau proteins may serve as biomarkers for Alzheimer disease (AD). Recently, the xMAP technology has been introduced as an alternative to ELISA for measurement of these markers.
Methods: We used xMAP assays and ELISA to analyze CSF concentrations of Aß42, total tau (t-tau), and tau phosphorylated at threonine 181 (p-tau181) in samples from 69 patients with Alzheimer disease, 26 patients with vascular dementia, and 55 controls without neurological disorders.
Results: High CV values (>28%) for the ratio of xMAP:ELISA were observed for each biomarker, indicating that a constant correction factor cannot be applied to recalculate xMAP results into ELISA results. When a combination of CSF markers was used, the sensitivity, specificity, and area under the ROC curves for xMAP assays and ELISAs were not significantly different in differentiating AD patients from vascular dementia patients and controls.
Conclusions: A constant conversion factor cannot be used successfully to recalculate results obtained with xMAP assays to those from the ELISAs. With the use of analysis of a combination of Aß42, t-tau, and p-tau in CSF, however, differentiation of clinical groups is equivalent when either xMAP technology or conventional ELISA is used.
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Introduction
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Accurate and early differentiation of Alzheimer disease (AD)1
from other dementia disorders, such as vascular dementia (VaD), dementia with Lewy bodies, and frontotemporal lobar dementia, is becoming increasingly important (1) because symptomatic drugs are specifically available for AD patients and neuroprotective drugs based on altered amyloid ß (Aß) metabolism are being developed (2).
Aß42, total tau (t-tau), and phosphorylated tau (p-tau) in cerebrospinal fluid (CSF) may serve as biomarkers in the diagnostic work-up of dementia patients. In AD patients compared with controls, CSF concentrations of t-tau and p-tau have consistently been shown to be increased and concentrations of Aß42 decreased [reviewed in (3)(4)(5)]. Measurement of CSF concentrations of t-tau and Aß42 enables variable but potentially highly accurate differentiation between AD patients and controls (sensitivity and specificity generally >80%). Based on the validity data, these CSF analyses seem better for ruling in than ruling out the AD diagnosis. CSF p-tau concentrations enhance the ability to differentiate AD from either dementia with Lewy bodies or VaD (6). In addition, CSF Aß42, t-tau, and p-tau analyses enable differentiation of patients with mild cognitive impairment (MCI) who progressively develop AD over time (up to 15% per year) from MCI patients with a stable disease course (7)(8), at a sensitivity of 95% and a specificity of 83% (9).
Robust assays of these biomarkers can enhance their usefulness in AD diagnosis. In most clinical studies, ELISAs have been used to quantify concentrations of Aß42, t-tau, and p-tau in CSF. However, this type of assay inherently has relatively high interassay variation (10), a problem that may be overcome by assays based on xMAP technology, which has several advantages over conventional ELISA. Compared with ELISA, xMAP technology requires less total assay time, fewer procedural steps, and a smaller sample volume. It also allows simultaneous detection of multiple analytes at the same time and has higher reproducibility than ELISA because the result of each analysis is the mean of multiple (typically 50–100) readings (11)(12). xMAP assays to quantify Aß42, t-tau, and tau phosphorylated at threonine 181 (p-tau181) in CSF and test characteristics for calibration, precision, and specificity have been recently described (13).
We performed a comparative analysis of the established ELISA techniques with the xMAP technology for the quantification of Aß42, t-tau, and p-tau181 in CSF of AD and VaD patients and controls. We also studied the clinical performance of the individual assays for differentiation of AD patients from controls or VaD patients, a use that has already been demonstrated for conventional ELISAs.
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Materials and Methods
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patients
Patients with mild to moderate AD (n = 69) and VaD (n = 26) were selected from a large database containing 260 patients with cognitive impairment or dementia of various origins (e.g., degenerative, vascular, hereditary, inflammatory, or metabolic) who visited our outpatient clinic between 1996 and 2006. Only patients with a diagnosis of probable AD or VaD, according to accepted criteria (14)(15), were included. The standard diagnostic examination protocol included a complete geriatric assessment, neurological examination, neuropsychological testing, laboratory testing, imaging of the brain, and a lumbar puncture. Sex distribution was comparable (P = 0.22; 
2 test). The AD and VaD groups did not differ significantly with respect to Mini Mental State Examination results (P = 0.44) or disease duration (P = 0.69). As controls we included 55 individuals older than 50 years without neurological disorders who visited our outpatient clinic for various reasons. CSF analysis results were within reference intervals for leukocyte and erythrocyte counts and total protein glucose, lactate, hemoglobin, and bilirubin concentrations, and CSF did not contain oligoclonal IgG bands. Age in the control group was significantly lower than in the AD and VaD groups (Table 1
).
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Table 1. Age, Mini Mental State Examination score, disease duration, and CSF markers measured with both xMAP and ELISA in AD patients, VaD patients, and controls.
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csf analysis
Lumbar punctures were performed after informed consent was obtained from the patient or the patient’s legal representative. CSF from all participants was collected in polypropylene tubes, transported at room temperature to the adjacent laboratory within 30 min, centrifuged after determination of cell number, and immediately divided into aliquots and stored at –80 °C until analysis. The concentrations (in ng/L) of t-tau, Aß42, and p-tau181 in CSF were measured with ELISA and the xMAP-based Innobia assay (all from Innogenetics NV). ELISA and xMAP analyses were independently performed in 2 separate aliquots of CSF. Therefore, availability of at least 2 aliquots per study participant was a prerequisite for study inclusion. ELISAs were routinely performed biweekly; thus samples were analyzed within 2 weeks after withdrawal. xMAP assays were performed batch-wise with stored samples. Antibodies used in the respective assays are presented in the supplemental data Table 1
(see the Data Supplement that accompanies the online version of this article at http://www.clinchem.org/content/vol53/issue5). Analysis was performed according to manufacturer specifications by 2 experienced technicians. The effects of traumatic puncture have not been systematically evaluated, but samples containing >500 erythrocytes/mL were not included.
statistical analysis
Statistical procedures were performed with GraphPad Prism and SPSS12.0 software. All data, except for CSF t-tau in the VaD group, showed gaussian distribution; therefore, Kruskal–Wallis test with Dunn’s posthoc test was used for multiple comparisons. Cutoff values, sensitivity, and specificity for biomarkers in different groups were calculated using ROC curves. For each biomarker cutoff values with the most optimal combination of sensitivity and specificity to differentiate between the 2 groups were calculated. The boundary value was constructed under the condition of equal "costs" of misclassification of patients and nonpatients. Pairwise comparison of ROC curves was performed with MedCalc (Mariakerke, Belgium). Correlation analysis was performed with the Spearman method. Bland–Altman curves were constructed for comparison between ELISA and xMAP assays (16). The CVs of the ratios of xMAP:ELISA values were calculated. Multivariate logistic regression with forward selection procedures was used to identify variables that contributed independently to differentiation of AD from either neurological control (NC) or VaD. Reference values were determined by calculating the 10th and 90th percentiles of the data obtained within the NC group.
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Results
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Interassay CVs of the assays were 3.8%–8.4% (see Table 2
in the online Data Supplement). Dynamic ranges of the ELISA and xMAP assays are presented in Table 3 of the online Data Supplement.
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Table 2. Clinical performance of CSF analyses (both xMAP and ELISA) in the differentiation of AD patients from VaD patients and controls.
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The results of the CSF analysis of Aß42, t-tau, and p-tau181 by ELISA and xMAP in the 3 patient groups are depicted in Table 1
. The distribution of results in the NC group (10th and 90th percentiles to define reference ranges) for each of the 3 biomarkers are presented in Table 4 of the online Data Supplement. The mean (SD) xMAP:ELISA ratios (for all patients) were 0.48 (0.13) for Aß42, 0.19 (0.057) for t-tau, and 0.74 (0.22) for p-tau181. CVs of the xMAP:ELISA ratios were as follows: Aß42, 28%; t-tau, 30%; p-tau181, 30%. The xMAP:ELISA ratio for each parameter was plotted against the mean of the 2 methods, according to Bland and Altman (Fig. 1
) (16). To recalculate xMAP data into concentrations obtained with the ELISAs over a broad concentration range, we felt that a deviation of the mean ratio of 7.6% (Aß42 and p-tau) or 9.4% (t-tau) would be acceptable (based on the following calculation: the difference should be smaller than one fourth of the SD of the population values (17), which is equivalent to one sixteenth of the 95% reference interval). The observed ratio is clearly higher for all proteins. Correlation between the xMAP and ELISA analyses of Aß42 was relatively low in the NC (r = 0.57) and AD groups (r = 0.51), whereas it was higher in the VaD group (r = 0.88) and in the total patient population (r = 0.81); see also Fig. 2
. Correlation between the 2 assays for t-tau was generally high: r = 0.77 (NC), r = 0.87 (AD), r = 0.92 (VaD), and r = 0.93 (total group). However, correlation between the 2 p-tau181 assays was lower: r = 0.62 (NC), r = 0.82 (AD), r = 0.71 (VaD), and r = 0.88 (total group). All correlations were highly significant (P <0.0001).
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Figure 1. Bland–Altman plots of Aß42 (A), t-tau (B) and p-tau181 (C) concentrations measured by xMAP and ELISA.
CSF samples were analyzed both by xMAP and ELISA assays; concentrations are all in ng/L. At the x-axis the average of both measurements is depicted, at the y-axis the ratio between xMAP and ELISA measurements is depicted.
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Figure 2. Correlation between xMAP and ELISA analyses of Aß42 (A), t-tau (B) and p-tau181 (C) for the entire study population.
CSF samples were analyzed both by xMAP and ELISA assays; concentrations are all in ng/L. Different symbols refer to the 3 patient groups (AD, Alzheimer disease; VaD, vascular dementia; NC, normal controls). Equations from the linear regression analysis are as follows. Aß42 [xMAP] = 116 (95% CI: 50–182) + 1.76 (95% CI: 1.5–2.0) x Aß42 [ELISA] (mean standardized residual: 0.0; SD: 1.0); t-tau [xMAP] = 3.0 (95% CI:–33–39) + 5.7 (95% CI: 5.4–6.0) x t-tau [ELISA] (mean standardized residual: 0.00; SD: 1.0); p-tau [xMAP] = 6.9 (95% CI: 0.78–13) + 1.24 (95% CI: 1.2–1.3) x p-tau [ELISA] (mean standardized residual: 0.0; SD: 1.0).
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For the differentiation between AD and NC, ROC analysis revealed that the t-tau ELISA performed better than the xMAP assay, whereas the reverse was true for Aß42 analysis (see Table 2
and Fig. 3
). For the p-tau181 analysis, the ELISA and xMAP assays performed similarly. Of the 3 ELISAs, the t-tau assay had the highest area under the curve (AUC), but the difference with the other assays did not reach significance; of the 3 xMAP assays, Aß42 had the highest AUC, but not significantly higher than the 2 other assays. For the most optimal differentiation between AD and controls, the combination of t-tau and Aß42 analyses was selected by multivariate logistic regression analysis. For the ELISAs, this model had a significantly higher AUC (P <0.05) than the Aß42 or p-tau181 assay, but not compared with the t-tau assay (P = 0.08). For the xMAP assays, the model had a significantly higher AUC (P <0.01) than the t-tau or p-tau181 assay, but not compared with the Aß42 assay (P = 0.20). With both the xMAP and ELISA assays, these mathematical models resulted in sensitivities and specificities >94% and AUCs >0.98 (Table 2
). The Aß42:p-tau181 ratio resulted in sensitivities and specificities >91% and AUCs >0.96 (Table 2
), but had a clinical performance comparable to the combined t-tau and Aß42 analyses with similar AUCs (P >0.05).
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Figure 3. ROC analysis of diagnostic properties of Aß42 (A), t-tau (B) and p-tau181 (C) concentrations measured by xMAP and ELISA for the differentiation of Alzheimer disease from normal controls, demonstrating the different characteristics of the xMAP and ELISA assays.
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For the differentiation between AD and VaD, ROC analysis revealed that the Aß42 ELISA performed better than the xMAP assay, whereas the reverse was true for p-tau181 analysis, and the t-tau assays performed comparably (see Table 2
and Fig. 3
). Previously, we demonstrated that the Aß42:p-tau181 ratio is a good discriminator between AD and VaD (6). For the differentiation between AD and VaD a combination of Aß42 and p-tau181, expressed either as the Aß42:p-tau181 ratio or combined into a mathematical model (see Table 2
) resulted in a higher specificity, sensitivity, AUC (P <0.05), and likelihood ratio than for Aß42 and t-tau, but not for p-tau (both xMAP and ELISA) (Table 2
, Fig. 4
). However, AUC obtained from either the Aß42:p-tau181 ratio or the mathematical model were comparable (P >0.05).
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Figure 4. ROC analysis of diagnostic properties of combinations of Aß42 and p-tau181 concentrations measured by ELISA (A) and xMAP (B) for the differentiation of AD from VaD.
Two combinations are shown: the Aß42/p-tau181 ratio and the mathematical model (as described in Table 2
).
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Discussion
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Comparison between the xMAP and ELISA methods for the quantification of Aß42, t-tau, and p-tau181 in CSF revealed the following: (a) high CV values (>28%) for the xMAP:ELISA ratio were observed for each individual parameter, indicating that a single correction factor cannot be applied to recalculate xMAP results into ELISA results; (b) the results of the Bland–Altman plot also indicated that differences between the xMAP and ELISA tests were too large to be clinically acceptable; and (c) the previous 2 observations suggest that replacement of the ELISA assays by xMAP assays should be accompanied by complete method validation, including establishment of reference intervals specific for this assay (see Results section) and different from those used for the ELISAs (18).
Explanations for these discrepant findings comprise the use of different combinations of antibodies in the respective assays, the different interaction with antigen of antibodies coated on a microsphere (xMAP) vs antibodies coated on microtiter plate, and differences in assay procedures. Other studies that compared xMAP and ELISA assays reached conclusions similar to ours. Correlations between the 2 assay types were generally (but not always) good (19)(20)(21), absolute concentrations may vary considerably between the 2 assay types (19)(20), and specific reference values should be defined for each type of assay (19). Furthermore, from several studies it has become clear that appropriate comparison of the 2 techniques requires the use of identical antibody pairs (19), which is, in our case, not true for the Aß42 and t-tau assays, whereas in the p-tau181 assays, capture and detection antibodies were switched.
Our conclusions are not in line with those reached in a previous study (13), which suggested that a constant correction factor could be used to convert results obtained with the xMAP assays to ELISA values, with some restriction for the p-tau181 assay. Unfortunately, although the procedures to calculate the conversion factor were outlined, this factor was not given either in this paper or in a follow-up publication (9). Therefore, application of this conversion factor to our data set was not possible.
In agreement with multiple other studies (1)(4)(5), we observed that Aß42 concentrations were significantly decreased in AD vs NC, whereas t-tau and p-tau181 concentrations in AD were significantly increased, both in the conventional ELISA and in the xMAP assays. For differentiation between AD and NC, a combination of parameters, either in a mathematical model with Aß42 and t-tau or as the Aß42:p-tau181 ratio, the ELISA and xMAP assays had a comparable performance with very similar sensitivity, specificity, and AUC to differentiate the 2 groups. Thus, despite the observed differences in the individual assays described above, the identification of AD patients using the commonly used combination of biomarkers can be equally achieved with the xMAP as with the ELISA assays. The multiplex format of the xMAP assay is a major advantage for performing such combined analyses, because the 3 parameters can be simultaneously assayed in the same sample, although analysis of 2 of the 3 parameters (e.g., Aß42 in combination with either t-tau or p-tau181) may be sufficient.
The conventional boundaries between AD and VaD have become blurred by the recognition that several vascular factors, including diabetes, atherosclerosis, and hypertension, may contribute to the pathogenesis of AD (22)(23). Currently used clinical diagnostic criteria for AD and VaD have disappointing sensitivity and specificity, often leading to a diagnosis of "mixed dementia", with clinical features of AD but with multiple vascular lesions on brain images or cardiovascular risk factors (24)(25). CSF Aß42, t-tau, and p-tau181 concentrations in VaD as measured by ELISA were comparable to those in previously published reports (6)(26)(27). Combined analysis of Aß42 and p-tau181, either with xMAP or ELISA, can be used to make a clinical differentiation between AD and VaD with high sensitivity, specificity, and AUC. CSF analysis may thus aid in the clinical differentiation between AD and VaD and may help to elucidate to what degree AD-like pathology (Aß deposition in senile plaques and tau accumulation in tangles) may contribute to the clinical picture.
In conclusion, quantification of Aß42, t-tau, and p-tau181 in CSF by using the xMAP technology may replace the use of conventional ELISA formats, but results from either assay type cannot be simply recalculated with a conversion factor. CSF analysis by using the xMAP assays enabled differentiation of AD patients from VaD patients and controls with high sensitivity, specificity, and AUC, but required the definition of new reference intervals.
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Acknowledgments
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Grant/funding support: Supported in part by grants from Zon-MW Innovational Research (no. 917.46.331) and the "Hersenstichting Nederland" to M.M.V.
Financial disclosures: None declared.
Acknowledgements: We thank the technicians of the Laboratory of Pediatrics and Neurology for CSF analysis, Dr. R. Esselink for assistance in lumbar punctures, and Dr. M. Munneke for statistical advice.
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Footnotes
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1 Nonstandard abbreviations:AD, Alzheimer disease; VaD, vascular dementia; Aß42, amyloid ß42; t-tau, total tau; p-tau, phosphorylated tau; CSF, cerebrospinal fluid; MCI, mild cognitive impairment; p-tau181, tau phosphorylated at threonine 181; AUC, area under the curve; and NC, neurological control. 
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Abstract
Introduction
