Clinical performance of an antibody-free assay for plasma Aß42/Aß40 to detect early alterations of Alzheimer's disease in individuals with subjective cognitive decline.

BACKGROUND: Accessible and cost-effective diagnostic tools are urgently needed to accurately quantify blood biomarkers to support early diagnosis of Alzheimer's disease (AD). In this study, we investigated the ability of plasma amyloid-beta (Aß)42/Aß40 ratio measured by an antibody-free mass-spectrometric (MS) method, ABtest-MS, to detect early pathological changes of AD. METHODS: This cohort study included data from the baseline and 2-year follow-up visits from the Fundació ACE Healthy Brain Initiative (FACEHBI) study. Plasma Aß42/Aß40 was measured with ABtest-MS and compared to 18F-Florbetaben PET as the reference standard (cutoff for early amyloid deposition of 13.5 centiloids). Cross-validation was performed in an independent DPUK-Korean cohort. Additionally, associations of plasma Aß42/Aß40 with episodic memory performance and brain atrophy were assessed. RESULTS: The FACEHBI cohort at baseline included 200 healthy individuals with subjective cognitive decline (SCD), of which 36 (18%) were Aß-PET positive. Plasma Aß42/Aß40 levels were significantly lower in Aß-PET positive individuals (median [interquartile range, IQR], 0.215 [0.203-0.236]) versus Aß-PET negative subjects (median [IQR], 0.261 [0.244-0.279]) (P < .001). Plasma Aß42/Aß40 was significantly correlated with Aß-PET levels (rho = -0.390; P < .001) and identified Aß-PET status with an area under the receiver operating characteristic curve (AUC) of 0.87 (95% confidence interval [CI], 0.80-0.93). A cutoff for the Aß42/Aß40 ratio of 0.241 (maximum Youden index) yielded a sensitivity of 86.1% and a specificity of 80.5%. These findings were cross-validated in an independent DPUK-Korean cohort (AUC 0.86 [95% CI 0.77-0.95]). Lower plasma Aß42/Aß40 ratio was associated with worse episodic memory performance and increased brain atrophy. Plasma Aß42/Aß40 at baseline predicted clinical conversion to mild cognitive impairment and longitudinal changes in amyloid deposition and brain atrophy at 2-year follow-up. CONCLUSIONS: This study suggests that plasma Aß42/Aß40, as determined by this MS-based assay, has potential value as an accurate and cost-effective tool to identify individuals in the earliest stages of AD, supporting its implementation in clinical trials, preventative strategies and clinical practice.

Characterization of pre-analytical sample handling effects on a panel of Alzheimer's disease-related blood-based biomarkers: Results from the Standardization of Alzheimer's Blood Biomarkers (SABB) working group.

INTRODUCTION: Pre-analytical sample handling might affect the results of Alzheimer's disease blood-based biomarkers. We empirically tested variations of common blood collection and handling procedures. METHODS: We created sample sets that address the effect of blood collection tube type, and of ethylene diamine tetraacetic acid plasma delayed centrifugation, centrifugation temperature, aliquot volume, delayed storage, and freeze-thawing. We measured amyloid beta (Aß)42 and 40 peptides with six assays, and Aß oligomerization-tendency (OAß), amyloid precursor protein (APP)699-711 , glial fibrillary acidic protein (GFAP), neurofilament light (NfL), total tau (t-tau), and phosphorylated tau181. RESULTS: Collection tube type resulted in different values of all assessed markers. Delayed plasma centrifugation and storage affected Aß and t-tau; t-tau was additionally affected by centrifugation temperature. The other markers were resistant to handling variations. DISCUSSION: We constructed a standardized operating procedure for plasma handling, to facilitate introduction of blood-based biomarkers into the research and clinical settings.

The global Alzheimer's Association round robin study on plasma amyloid ß methods.

INTRODUCTION: Blood-based assays to measure brain amyloid beta (Aß) deposition are an attractive alternative to the cerebrospinal fluid (CSF)-based assays currently used in clinical settings. In this study, we examined different blood-based assays to measure Aß and how they compare among centers and assays. METHODS: Aliquots from 81 plasma samples were distributed to 10 participating centers. Seven immunological assays and four mass-spectrometric methods were used to measure plasma Aß concentrations. RESULTS: Correlations were weak for Aß42 while Aß40 correlations were stronger. The ratio Aß42/Aß40 did not improve the correlations and showed weak correlations. DISCUSSION: The poor correlations for Aß42 in plasma might have several potential explanations, such as the high levels of plasma proteins (compared to CSF), sensitivity to pre-analytical sample handling and specificity, and cross-reactivity of different antibodies. Different methods might also measure different pools of plasma Aß42. We, however, hypothesize that greater correlations might be seen in future studies because many of the methods have been refined during completion of this study.

Longitudinal evaluation of the natural history of amyloid-ß in plasma and brain.

Plasma amyloid-ß peptide concentration has recently been shown to have high accuracy to predict amyloid-ß plaque burden in the brain. These amyloid-ß plasma markers will allow wider screening of the population and simplify and reduce screening costs for therapeutic trials in Alzheimer's disease. The aim of this study was to determine how longitudinal changes in blood amyloid-ß track with changes in brain amyloid-ß. Australian Imaging, Biomarker and Lifestyle study participants with a minimum of two assessments were evaluated (111 cognitively normal, 7 mild cognitively impaired, 15 participants with Alzheimer's disease). Amyloid-ß burden in the brain was evaluated through PET and was expressed in Centiloids. Total protein amyloid-ß 42/40 plasma ratios were determined using ABtest® assays. We applied our method for obtaining natural history trajectories from short term data to measures of total protein amyloid-ß 42/40 plasma ratios and PET amyloid-ß. The natural history trajectory of total protein amyloid-ß 42/40 plasma ratios appears to approximately mirror that of PET amyloid-ß, with both spanning decades. Rates of change of 7.9% and 8.8%, were observed for total protein amyloid-ß 42/40 plasma ratios and PET amyloid-ß, respectively. The trajectory of plasma amyloid-ß preceded that of brain amyloid-ß by a median value of 6 years (significant at 88% confidence interval). These findings, showing the tight association between changes in plasma and brain amyloid-ß, support the use of plasma total protein amyloid-ß 42/40 plasma ratios as a surrogate marker of brain amyloid-ß. Also, that plasma total protein amyloid-ß 42/40 plasma ratios has potential utility in monitoring trial participants, and as an outcome measure.

Total Aß42/Aß40 ratio in plasma predicts amyloid-PET status, independent of clinical AD diagnosis.

OBJECTIVE: To explore whether the plasma total ß-amyloid (Aß) Aß42/Aß40 ratio is a reliable predictor of the amyloid-PET status by exploring the association between these 2 variables in a subset of the Australian Imaging, Biomarkers and Lifestyle (AIBL) study of aging cohort. METHODS: Taking plasma samples at 3 separate time points, month 18 (n = 176), month 36 (n = 169), and month 54 (n = 135), we assessed the total Aß42/Aß40 ratio in plasma (TP42/40) with regard to neocortical Aß burden via PET standardized uptake value ratio (SUVR) and investigated both association with Aß-PET status and correlation (and agreement) with SUVR. RESULTS: The TP42/40 plasma ratio was significantly reduced in amyloid-PET-positive participants at all time points (p < 0.0001). Adjusting for covariates age, gender, APOE ε4 allele status, and clinical classification clearly affects the significance, with p values reduced and only comparisons at 54 months retaining significance (p = 0.006). Correlations with SUVR were similar across each time point, with Spearman ρ reaching -0.64 (p < 0.0001). Area under the curve values were highly reproducible over time points, with values ranging from 0.880 at 36 months to 0.913 at 54 months. In assessments of the healthy control group only, the same relationships were found. CONCLUSIONS: The current study demonstrates reproducibility of the plasma assay to discriminate between amyloid-PET positive and negative over 3 time points, which can help to substantially reducing the screening rate of failure for clinical trials targeting preclinical or prodromal disease. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that plasma total Aß42/Aß40 ratio is associated with neocortical amyloid burden as measured by PET SUVR.

Plasma amyloid beta levels are associated with cerebral amyloid and tau deposition.

INTRODUCTION: We investigated the relationship of plasma amyloid beta (Aß) with cerebral deposition of Aß and tau on positron emission tomography (PET). METHODS: Forty-four participants (18 cognitively normal older adults [CN], 10 mild cognitive impairment, 16 Alzheimer's disease [AD]) underwent amyloid PET and a blood draw. Free and total plasma Aß40 and Aß42 were assessed using a validated assay. Thirty-seven participants (17 CN, 8 mild cognitive impairment, 12 AD) also underwent a [18F]flortaucipir scan. Scans were preprocessed by standard techniques, and mean global and regional amyloid and tau values were extracted. Free Aß42/Aß40 (Aß F42:F40) and total Aß42/Aß40 (Aß T42:T40) were evaluated for differences by diagnosis and relation to PET Aß positivity. Relationships between these measures and cerebral Aß and tau on both regional and voxel-wise basis were also evaluated. RESULTS: Lower Aß T42:T40 was associated with diagnosis and PET Aß positivity. Lower plasma Aß T42:T40 ratios predicted cerebral Aß positivity, both across the full sample and in CN only. Finally, lower plasma Aß T42:T40 ratios were associated with increased cortical Aß and tau in AD-related regions on both regional and voxel-wise analyses. DISCUSSION: Plasma Aß measures may be useful biomarkers for predicting cerebral Aß and tau. Additional studies in larger samples are warranted.

Blood Markers in Healthy-Aged Nonagenarians: A Combination of High Telomere Length and Low Amyloidß Are Strongly Associated With Healthy Aging in the Oldest Old.

Many factors may converge in healthy aging in the oldest old, but their association and predictive power on healthy or functionally impaired aging has yet to be demonstrated. By detecting healthy aging and in turn, poor aging, we could take action to prevent chronic diseases associated with age. We conducted a pilot study comparing results of a set of markers (peripheral blood mononuclear cell or PBMC telomere length, circulating Aß peptides, anti-Aß antibodies, and ApoE status) previously associated with poor aging or cognitive deterioration, and their combinations, in a cohort of "neurologically healthy" (both motor and cognitive) nonagenarians (n = 20) and functionally impaired, institutionalized nonagenarians (n = 38) recruited between 2014 and 2015. We recruited 58 nonagenarians (41 women, 70.7%; mean age: 92.37 years in the neurologically healthy group vs. 94.13 years in the functionally impaired group). Healthy nonagenarians had significantly higher mean PBMC telomere lengths (mean = 7, p = 0.001), this being inversely correlated with functional impairment, and lower circulating Aß40 (total in plasma fraction or TP and free in plasma fraction or FP), Aß42 (TP and FP) and Aß17 (FP) levels (FP40 131.35, p = 0.004; TP40 299.10, p = 0.007; FP42 6.29, p = 0.009; TP42 22.53, p = 0.019; FP17 1.32 p = 0.001; TP17 4.47, p = 0.3), after adjusting by age. Although healthy nonagenarians had higher anti-Aß40 antibody levels (net adsorbed signal or NAS ± SD: 0.211 ± 0.107), the number of participants that pass the threshold (NAS > 3) to be considered as positive did not show such a strong association. There was no association with ApoE status. Additionally, we propose a "Composite Neurologically Healthy Aging Score" combining TP40 and mean PBMC telomere length, the strongest correlation of measured biomarkers with neurologically healthy status in nonagenarians (AUC = 0.904).

Plasma amyloid ß 42/40 ratios as biomarkers for amyloid ß cerebral deposition in cognitively normal individuals.

INTRODUCTION: Plasma amyloid ß (Aß) peptides have been previously studied as candidate biomarkers to increase recruitment efficiency in secondary prevention clinical trials for Alzheimer's disease. METHODS: Free and total Aß42/40 plasma ratios (FP42/40 and TP42/40, respectively) were determined using ABtest assays in cognitively normal subjects from the Australian Imaging, Biomarker and Lifestyle Flagship Study. This population was followed-up for 72 months and their cortical Aß burden was assessed with positron emission tomography. RESULTS: Cross-sectional and longitudinal analyses showed an inverse association of Aß42/40 plasma ratios and cortical Aß burden. Optimized as a screening tool, TP42/40 reached 81% positive predictive value of high cortical Aß burden, which represents 110% increase over the population prevalence of cortical Aß positivity. DISCUSSION: These findings support the use of plasma Aß42/40 ratios as surrogate biomarkers of cortical Aß deposition and enrichment tools, reducing the number of subjects submitted to invasive tests and, consequently, recruitment costs in clinical trials targeting cognitively normal individuals.

Validation of Immunoassay-Based Tools for the Comprehensive Quantification of Aß40 and Aß42 Peptides in Plasma.

Recent advances in neuroimaging and cerebrospinal fluid (CSF) biomarker assays have provided evidence of a long preclinical stage of Alzheimer's disease (AD). This period is being increasingly targeted for secondary prevention trials of new therapies. In this context, the interest of a noninvasive, cost-effective amyloid-ß (Aß) blood-based test does not need to be overstated. Nevertheless, a thorough validation of these bioanalytical methods should be performed as a prerequisite for confident interpretation of clinical results. The aim of this study was to validate ELISA sandwich colorimetric ABtest40 and ABtest42 for the quantification of Aß40 and Aß42 in human plasma. The validation parameters assessed included precision, accuracy, sensitivity, specificity, recovery, and dilution linearity. ABtest40 and ABtest42 proved to be specific for their target peptide using Aß peptides with sequence similar to the target. Mean relative error in the quantification was found to be below 7.5% for both assays, with high intra-assay, inter-assay, and inter-batch precision (CV <9.0% on average). Sensitivity was assessed by determination of the limit of quantification fulfilling precision and accuracy criteria; it was established at 7.60 pg/ml and 3.60 pg/ml for ABtest40 and ABtest42, respectively. Plasma dilution linearity was demonstrated in PBS; however, dilution in a proprietary formulated buffer significantly increased the recovery of both Aß40 and Aß42 masked by matrix interactions, allowing a more comprehensive assessment of the free and total peptide levels in the plasma. In conclusion, both assays were successfully validated as tools for the quantification Aß40 and Aß42 in plasma.

Outstanding Phenotypic Differences in the Profile of Amyloid-ß between Tg2576 and APPswe/PS1dE9 Transgenic Mouse Models of Alzheimer's Disease.

APPswe/PS1dE9 and Tg2576 are very common transgenic mouse models of Alzheimer's disease (AD), used in many laboratories as tools to research the mechanistic process leading to the disease. In order to augment our knowledge about the amyloid-ß (Aß) isoforms present in both transgenic mouse models, we have developed two chromatographic methods, one acidic and the other basic, for the characterization of the Aß species produced in the brains of the two transgenic mouse models. After immunoprecipitation and micro-liquid chromatography-electrospray ionization mass spectrometry/mass spectrometry, 10 species of Aß, surprisingly all of human origin, were detected in the brain of Tg2576 mouse, whereas 39 species, of both murine and human origin, were detected in the brain of the APP/PS1 mouse. To the best of our knowledge, this is the first study showing the identification of such a high number of Aß species in the brain of the APP/PS1 transgenic mouse, whereas, in contrast, a much lower number of Aß species were identified in the Tg2576 mouse. Therefore, this study brings to light a relevant phenotypic difference between these two popular mice models of AD.

Optimized protocol for amyloid-ß extraction from the brain.

Brain levels of amyloid-ß (Aß) are frequently assessed in transgenic mice models of Alzheimer's disease. The procedure involves tissue sample homogenization using different buffers in a sequential process. No attempt has been made to assess if these procedures are able to extract the total amount of Aß present in the samples. Here we present data suggesting that standard protocols can lead to a dramatic underestimation of the Aß content. Results show that higher extraction buffer volumes and at least two repetitions of the soluble and membrane-bound extraction steps are necessary for a more accurate estimation of the Aß content in brain tissues.