Diagnostic performance of automated plasma amyloid-β assays combined with pre-analytical immunoprecipitation

Abstract

Background: Measurements of the amyloid-β (Aβ) 42/40 ratio in blood plasma may support the early diagnosis of Alzheimer's disease and aid in the selection of suitable participants in clinical trials. Here, we compared the diagnostic performance of fully automated prototype plasma Aβ42/40 assays with and without pre-analytical sample workup by immunoprecipitation.

Methods: A pre-selected clinical sample comprising 42 subjects with normal and 38 subjects with low cerebrospinal fluid (CSF) Aβ42/40 ratios was studied. The plasma Aβ42/40 ratios were determined with fully automated prototype Elecsys® immunoassays (Roche Diagnostics GmbH, Penzberg, Germany) by direct measurements in EDTA plasma or after pre-analytical Aβ immunoprecipitation. The diagnostic performance for the detection of abnormal CSF Aβ42/40 was analyzed by receiver operating characteristic (ROC) analysis. In an additional post hoc analysis, a biomarker-supported clinical diagnosis was used as a second endpoint.

Results: Pre-analytical immunoprecipitation resulted in a significant increase in the area under the ROC curve (AUC) from 0.73 to 0.88 (p = 0.01547) for identifying subjects with abnormal CSF Aβ42/40. A similar improvement in the diagnostic performance by pre-analytical immunoprecipitation was also observed when a biomarker-supported clinical diagnosis was used as a second endpoint (AUC increase from 0.77 to 0.92, p = 0.01576).

Conclusions: Our preliminary observations indicate that pre-analytical Aβ immunoprecipitation can improve the diagnostic performance of plasma Aβ assays for detecting brain amyloid pathology. The findings may aid in the further development of blood-based immunoassays for Alzheimer's disease ultimately suitable for screening and routine use.

Keywords: Alzheimer’s disease; Biomarker assay; Immunoprecipitation; Plasma Amyloid-β 42/40; Pre-analytical sample workup.

Fig. 1

Schematic representation of the pre-analytical process before automated testing of IP-eluates from blood plasma. Aβ, amyloid-β; IP, immunoprecipitation

Fig. 2

Correlations between direct plasma Aβ measurements on Elecsys and measurements after immunoprecipitation (IP). The measured concentrations of A Aβ40, B Aβ42, and C the Aβ42/40 ratio in diluted IP-eluates (Elecsys IP-eluate, Y) are plotted against the corresponding measurements in plasma (Elecsys plasma, X). Points are colored by diagnostic group (red = Aβ+, blue = Aβ−). The diagonal dashed lines correspond to the identity lines. Spearman correlation coefficients and p-values are indicated. Aβ, amyloid-β; Aβ+, Aβ-positive: CSF Aβ42/40 ≤ 0.050; Aβ−, Aβ-negative: CSF Aβ42/40 > 0.050

Fig. 3

Receiver operating characteristic (ROC) curves for Elecsys measurements without or with pre-analytical immunoprecipitation (IP). ROC curves for the classification of the study participants into the diagnostic groups Aβ-positive (Aβ+) and Aβ-negative (Aβ−) were calculated for Elecsys measurements in plasma or IP-eluates. 95% confidence intervals were calculated using the DeLong approach and are indicated in brackets. Aβ, amyloid-β; Aβ+, Aβ-positive: CSF Aβ42/40 ≤ 0.050; Aβ−, Aβ-negative: CSF Aβ42/40 > 0.050; IP, immunoprecipitation

Fig. 4

Correlation of plasma Aβ42/40 measurements after pre-analytical Aβ immunoprecipitation on two different immunoassay platforms. Following magnetic bead immunoprecipitation (IP) from EDTA blood plasma, Aβ42/40 was quantified in diluted IP-eluates with Elecsys (5-fold dilution) and MSD (6-fold dilution). Data points are colored by diagnostic group (red = Aβ+, blue = Aβ−). The diagonal dashed line corresponds to the identity line. Aβ, amyloid-β; Aβ+, Aβ-positive: CSF Aβ42/40 ≤ 0.050; Aβ−, Aβ-negative: CSF Aβ42/40 > 0.050; MSD, Meso Scale Discovery V-PLEX Aβ panel 1 (6E10) assay kit

Fig. 5

ROC curves for Aβ− vs. Aβ+ by logistic regression with single and bivariate models. Logistic regression receiver operating characteristic (ROC) analysis with leave-10-out cross-validation for single marker (blue lines) and bivariate models (including the ApoE4 allele frequency, black lines) for the Aβ42/Aβ40 ratio measured A on Elecsys in plasma without pre-analytical workup, B on Elecsys with pre-analytical Aβ immunoprecipitation (IP-eluate), and C on Meso Scale Discovery (MSD) V-PLEX Aβ panel 1 (6E10) assay with pre-analytical Aβ IP (IP-eluate). Aβ, amyloid-β; Aβ+, Aβ-positive: CSF Aβ42/40 ≤ 0.050; Aβ−, Aβ-negative: CSF Aβ42/40 > 0.050

Fig. 6

Correlations between CSF Aβ values and plasma Aβ measurements by three different assays. The cerebrospinal fluid (CSF) concentrations of Aβ40 (A, B, C), Aβ42 (D, E, F) and the CSF Aβ42/Aβ40 ratio (G, H, I) are plotted against the corresponding plasma values measured with Elecsys in plasma (A, D, G), Elecsys in IP-eluates (B, E, H) and MSD in IP-eluates (C, F, I). Spearman correlation coefficients and p-values are indicated. Data points are colored by diagnostic group (red = Aβ+, blue = Aβ−). Aβ, amyloid-β; Aβ+, Aβ-positive: CSF Aβ42/40 ≤ 0.050; Aβ−, Aβ-negative: CSF Aβ42/40 > 0.050; IP, immunoprecipitation; MSD, Meso Scale Discovery V-PLEX Aβ panel 1 (6E10) assay kit