Background The last few years have seen major advances in blood biomarkers for Alzheimer's Disease (AD) with the development of ultrasensitive immunoassays, promising to transform how we diagnose, prognose, and track progression of neurodegenerative dementias. Methods We evaluated a panel of four novel ultrasensitive electrochemiluminescence (ECL) immunoassays against presumed CNS derived proteins of interest in AD in plasma [phosphorylated-Tau181 (pTau181), total Tau (tTau), neurofilament light (NfL), and glial fibrillary acidic protein (GFAP)]. Two sets of banked plasma samples from the Massachusetts Alzheimer's Disease Research Center's longitudinal cohort study were examined: A longitudinal prognostic sample ( n = 85) consisting of individuals with mild cognitive impairment (MCI) and 4 years of follow-up and a cross-sectional sample ( n = 238) consisting of individuals with AD, other neurodegenerative diseases (OND), and normal cognition (CN). Results Participants with MCI who progressed to dementia due to probable AD during follow-up had higher baseline plasma concentrations of pTau181, NfL, and GFAP compared to non-progressors. The best prognostic discrimination was observed with pTau181 (AUC = 0.83, 1.7-fold increase) and GFAP (AUC = 0.83, 1.6-fold increase). Participants with autopsy- and/or biomarker verified AD had higher plasma levels of pTau181, tTau and GFAP compared to CN and OND, while NfL was elevated in AD and further increased in OND. The best diagnostic discrimination was observed with pTau181 (AD vs CN: AUC = 0.90, 2-fold increase; AD vs. OND: AUC = 0.84, 1.5-fold increase) but tTau, NfL, and GFAP also showed good discrimination between AD and CN (AUC = 0.81–0.85; 1.5–2.2 fold increase). Conclusions These new ultrasensitive ECL plasma assays for pTau181, tTau, NfL, and GFAP demonstrated diagnostic utility for detection of AD. Moreover, the absolute baseline plasma levels of pTau181 and GFAP reflect cognitive decline over the next 4 years, providing prognostic information that may have utility in both clinical practice and clinical trial populations.

Normal aging is associated with decreased functional connectivity (FC) in the brain’s default mode network (DMN). Additionally, cerebral burden of both amyloid-β (Aβ) and tau proteins further contribute to decreased DMN FC in the course of the normal aging process (Wales and Leung, 2021). However, the current literature is sparse and unclear as to the independent actions of Aβand tau on FC of DMN, with tau being associated with general hypoconnectivity (Wales and Leung, 2021). The current pilot study examines resting state fMRI (rsfMRI) data in conjunction with Alzheimer’s Amyloid/Tau/Neurodegeneration (ATN) biofluid biomarkers in healthy individuals, using the isthmus cingulate of the retrosplenial cortex as seed for FC analysis of the DMN. rsfMRI scans are collected in the LifeSPAN Biobanking project [ongoing]. Scans are preprocessed utilizing standard FreeSurfer pipeline. Standard parcellations are assigned using the Desikan-Killiany atlas. In initial analyses, isthmus cingulate served as the seed for DMN analysis using the FSFAST processing stream within FreeSurfer. ATN biomarkers are obtained from cerebrospinal fluid (CSF) and the following biomarker ratios were computed: Aβ 42 /Aβ 40 (Aβ ratio) and phosphorylated tau-181 (pTau181) / total Tau (tTau) (pTau181/tTau ratio). In the initial 14 participants (aged 26-82 years) [Table 1], General Linear Models (GLMs), adjusted for age, were utilized to evaluate the associations between DMN FC and the following AD biomarkers: Aβ 40 , Aβ 42 , Aβ ratio, tTau, pTau181, and pTau181/tTau ratio [Table 2; Figure 1]. After correcting for multiple comparisons (p<0.05), significant association between tTau, and the FC between the DMN and the right inferior temporal region was observed [Table 3]. The pilot analysis indicates at least tTau has an association with DMN FC, but sample size precludes conclusion or interpretation. Preliminary data obtained in this exploratory analysis provides a framework for examining the effect of AD biomarkers on the DMN and other functional networks. Future research will incorporate synaptic, inflammatory and other markers into similar statistical models and report preliminary associations. Reference Wales R, Leung H-C. The Effects of Amyloid and Tau on Functional Network Connectivity in Older Populations. Brain. 2021;142(8):2483-2491. doi:10.1093/brain/awz162

Normal Pressure Hydrocephalus (NPH) is a neurological condition where there is enlarged ventricular size with normal opening pressure at lumbar puncture. Etiology is uncertain but it is presumed to occur from an imbalance in the production and/or absorption of cerebrospinal fluid (CSF) leading to expansion of the ventricles. Clinical features include gait dysfunction, cognitive impairments, bladder incontinence. Diagnosis is challenging, given a large overlap in clinical and neuroradiological features and co-morbidity with other more common diseases of aging, especially Alzheimer’s disease (AD) and Lewy body diseases. CSF amyloid and tau biomarkers can be helpful to identify AD pathology but do not exclude concurrent NPH. Currently, there are no biofluid biomarkers to assist in diagnosis of NPH. Here we describe a proteomic discovery pilot to identify candidate protein biomarkers associated with NPH. 176 banked CSF samples from the MassGeneral Institute for Neurodegenerative Disease biorepository were separated into three groups: NPH (n=30), AD (n=65), and cognitively unimpaired neurological controls (CU-N; n=81). Diagnoses were established based on comprehensive chart review and CSF AD biomarkers. For proteomic analysis, samples were prepared in batches and analyzed by LC-MS/MS on an Orbitrap Fusion using a data-independent acquisition method with non-overlapping windows from 400-1000 m/z. Batches were run sequentially, and raw files were searched per batch using Scaffold-DIA and a custom data-dependent generated library. Five proteins present in hemoglobulin (HBD and HBB), immunoglobulins (LV39 and KV105), and fibrinogen (FIBA) were increased in NPH compared to AD (Benjamini-Hochberg adjusted p-value pAdj <0.05). CSF erythrocyte counts were undetectable in all but one NPH subjects suggesting that a traumatic LP was not the cause of the increased levels. Two neuron associated proteins (VGF and NPTXR) were detected at lower levels in NPH compared to CU-N subjects (pAdj<0.0001). Alpha-1-antichymotrypsin (AACT), was detected at high concentrations both in NPH and AD compared to CU-N subjects (pAdj<0.02). Proteomic analysis in NPH may identify novel pathological processes involved in the disease process and aid in the development of biomarkers in NPH.

Novel biomarkers are necessary for improving differential diagnosis of Alzheimer’s Disease (AD), disease monitoring, and treatment personalization. Core cerebrospinal fluid (CSF) AD biomarkers: amyloid-ß (Aß), total-tau (T-tau), and phosphorylated-tau (P-tau181) are strong markers of the presence of amyloid pathology, but are not suitable for measuring disease progression or drug response in a clinical trial . In this context, blood-based biomarkers are increasingly desirable, as plasma is less invasive than CSF, enabling short-term repeated sampling. Technical performance of Olink Proteomic’s multiplex proximity extension assay was evaluated using plasma samples from the MADRC longitudinal cohort. Over 400 analytes were measured on five off-the-shelf panels. Inter-plate and intra-plate coefficient of variations (CVs) were calculated from 3 samples run in duplicate on each plate. ANOVA was used to assess proportion of technical versus biological sources of variance. Multi-protein investigations require consideration of multiple testing. Power calculations were performed using baseline samples from n=34 Controls and n=20 Dementia-AD subjects to demonstrate optimal sample size for Olink studies. The majority of analyte mean CVs fell within the acceptable range for inter and intra-plate measurements (<15%). Higher CVs were generally related to lower analyte abundance. Most analytes were relatively stable (Biotemporal CV < 15%) in control individuals year to year. ANOVA determined that the greatest source of variation in plasma was due to biological inter-individual variability, as opposed to technical variation for all but 73 proteins. With regards to experimental power in a 450 protein experiment, a high effect size protein such as NfL required fewer than 50 samples per group to achieve confidence in observed significant differences. In contrast, the moderate effect size MCP-1 required just under 500 samples per group when 450 proteins are measured. This decreased to n=400 if only 100 proteins are measured, and n=170 if MCP-1 is measured alone. Olink technology is technically robust and reliable, with biological factors being the primary source of variation for most proteins in plasma. Particularly for medium effect size proteins, sample size is an important consideration when planning experiments using this technology.

Fujirebio’s Lumipulse G1200 is designed to perform fully-automated, rapid, random access immunoassays for biomarker quantification in biofluids. The Lumipulse instrument was used for quantification of the core Alzheimer’s Disease (AD) cerebrospinal fluid (CSF) biomarkers for Amyloid, Tau, and neurodegeneration (ATN): amyloid β 1–40 (Aβ40), amyloid β 1–42 (Aβ42), phosphorylated Tau 181 (pTau181), and total Tau (tTau). This study evaluated the reliability of the Lumipulse assays by measuring intra- and inter-batch coefficients of variation (CV), and the correlation between Lumipulse values and the clinically used Athena ADmark Aβ42, pTau181 and tTau assays. 423 CSF samples collected from diagnostic lumbar punctures between 2015 and 2020 were obtained from the MIND biorepository. Aβ40, Aβ42, tTau, and pTau were measured using the Lumipulse G1200 instrument in batches of 50-60 samples. We tested 60 samples in duplicate to compare the intrabatch stability using the CVs between each pair. Each batch was run with a new set of calibrator controls at the start of each assay to ensure stability for each biomarker run. Finally, we correlated values from the Lumipulse with results from the clinical evaluation using the Athena ADmark test. We observed low variability in calibrator and control signals between batches with an overall calibrator signal CV of <5% for Aβ40 and tTau, and <10% for Aβ42 and pTau. Control signal CV values for all analytes were <5%. The mean duplicate CV across all batches was similarly stable at <10% for Aβ40, Aβ42, and tTau, and <5% for pTau. Subjects with CV >20% were seen <5% of the time. The correlations between Lumipulse and clinical ADmark results were high for pTau and tTau (R 2 >0.8 for both analytes), but lower for Aβ42 (R 2 ∼0.5). Our results demonstrated that the Lumipulse G1200 is a reliable, easy to use platform for ATN quantification with low sample to sample variability, and generally high correlation to established clinical standards. Inconsistent preanalytical sample handling of clinical send-outs for Athena ADmark Aβ42 likely resulted in a poor correlation with Lumipulse G1200. The performance of the calibrators and controls proved that the standardization in each batch was consistent.

The ATN framework is used to help categorize research subjects as being on the Alzheimer’s disease (AD) spectrum. Analytes commonly used in biofluids to determine ATN status were measured across three platforms to examine the reliability of each technology and inter-platform variability. Cerebrospinal fluid (CSF) samples were obtained from 734 patients undergoing diagnostic lumbar punctures at the MGH Neurology Department. Amyloid β 1–40 (Aβ40), Aβ42, total Tau (tTau), and phosphorylated Tau 181 (pTau 181) were measured in all subjects using EuroImmun ELISA kits on a TECAN FreedomEVO liquid handler. 423 of the subjects also had all four biomarkers measured on a Fujirebio Lumipulse G1200 instrument and 164 had levels of Aβ40 and Aβ42 measured on the Quanterix HD-X platform. 302 of the subjects had Aβ42, tTau, and pTau 181 quantified by Athena ADmark assays as part of their clinical evaluation. Coefficient of Variations (CVs) were calculated, and data was correlated between platforms to determine inter-platform variability. The average CV for each analyte was <5% for samples analyzed on the EuroImmun/TECAN platform and <10% on the Fujirebio Lumipulse and Quanterix HD-X. CVs >20% were not seen in subjects with detectable analyte levels on the EuroImmun/TECAN or Quanterix platforms and the incidence was <5% on the Fujirebio platform. Tight correlations were observed between levels measured on the EuroImmun/TECAN and Fujirebio platforms (R 2 >0.8), while lower correlations were observed between the EuroImmun/TECAN and Quanterix platforms (R 2 =0.4-0.5 for Aβ40 and Aβ42). The lowest correlation was observed comparing Aβ42 analyzed using EuroImmun/TECAN and Athena ADmark (). ICC values were >0.7 for all analytes compared between EuroImmun/TECAN and Fujirebio platforms. We observed good reliability using all three platforms. to conserve sample. ATN values showed strong correlations between the EuroImmun/TECAN and the Fujirebio platforms and produce . The poor correlation for the Athena ADmark Aβ42 is likely a result of poor control of pre analytical factors in the samples sent for clinical testing and highlights the importance for proper sample handling.

The ATN Framework was proposed to better classify subjects on the Alzheimer’s Disease (AD) spectrum for research purposes. The role of the MassGeneral Institute for Neurodegenerative Disease (MIND) Tissue bank is to collect cerebrospinal fluid (CSF), plasma, and associated clinical data from a diverse neurological subject population. Ab40, Ab42, p-tau 181, and t-tau were quantified in CSF samples from the MIND Bank and classified according to the ATN framework to assess the utility of this research framework in a clinical trial relevant, non-cohort setting. 750 CSF samples were collected from clinically indicated lumbar punctures at the Massachusetts General Hospital Outpatient and Inpatient Neurology Services. The CSF samples were analyzed using Eurolmmun ELISA assays on a semi-automated TECAN FreedomEVO liquid handler to obtain ATN values. To optimize sensitivity and specificity we plotted these value across varying thresholds, and selected the value at which both were maximized. Assessment of cognitive impairment and neurological diagnosis was performed by chart review by an experienced neurologist. After sliding threshold analysis of clear cognitively unimpaired versus AD-Dementia cases, a threshold value for Ab42/40 Ratio (0.082, 92% sensitivity, 91% specificity), pTau (41.8 pg/mL, 91% sensitivity, 91% specificity), and tTau (280 pg/mL, 88% sensitivity, 88% specificity) was determined. 25% of the MIND bank was classified as likely AD from chart review and a similar proportion was biofluid A+T+N+. The overlap of these two subgroups was extensive but not complete. Out of the 188 A+T+N+ subjects, 152 presented clinically with a form of AD, 6 had other neurodegenerative diseases, 23 had other forms of cognitive impairment, and 7 were cognitively unimpaired. 8 of the 419 A-T-N- subjects had a clinical diagnosis of AD or mixed AD. The ATN framework can be used to provide an extra level of certainty with regards to diagnosis of likely AD in a clinical trial relevant subject population. Even in this population with clinical indication for LP, there is a small number of resilient cognitively unimpaired individuals that have AD positive biomarkers but no cognitive impairment.