Early Detection of Alzheimer’s Disease-Related Pathology Using a Multi-Disease Diagnostic Platform Employing Autoantibodies as Blood-Based Biomarkers

Background: Evidence for the universal presence of IgG autoantibodies in blood and their potential utility for the diagnosis of Alzheimer’s disease (AD) and other neurodegenerative diseases has been extensively demonstrated by our laboratory. The fact that AD-related neuropathological changes in the brain can begin more than a decade before tell-tale symptoms emerge has made it difficult to develop diagnostic tests useful for detecting the earliest stages of AD pathogenesis. Objective: To determine the utility of a panel of autoantibodies for detecting the presence of AD-related pathology along the early AD continuum, including at pre-symptomatic [an average of 4 years before the transition to mild cognitive impairment (MCI)/AD)], prodromal AD (MCI), and mild-moderate AD stages. Methods: A total of 328 serum samples from multiple cohorts, including ADNI subjects with confirmed pre-symptomatic, prodromal, and mild-moderate AD, were screened using Luminex xMAP® technology to predict the probability of the presence of AD-related pathology. A panel of eight autoantibodies with age as a covariate was evaluated using randomForest and receiver operating characteristic (ROC) curves. Results: Autoantibody biomarkers alone predicted the probability of the presence of AD-related pathology with 81.0% accuracy and an area under the curve (AUC) of 0.84 (95% CI = 0.78–0.91). Inclusion of age as a parameter to the model improved the AUC (0.96; 95% CI = 0.93–0.99) and overall accuracy (93.0%). Conclusion: Blood-based autoantibodies can be used as an accurate, non-invasive, inexpensive, and widely accessible diagnostic screener for detecting AD-related pathology at pre-symptomatic and prodromal AD stages that could aid clinicians in diagnosing AD.

of 0.5; and 4) general cognitive and functional performance sufficiently preserved such that a diagnosis of dementia could not be made by the site physician at the time of screening. Finally, mild AD was diagnosed with a MMSE between 20 to 26, CDR of 0.5 or 1.0, and meeting NINCDS/ADRDA criteria for probable AD.
Since the inception of ADNI, there have been modifications to the diagnostic criteria. Per ADNI 2 criteria, normal controls maintained the same diagnostic criteria as in ADNI 1, with the addition of performing within normative expectations on Logical Memory II subscale (Story A Delayed Paragraph) from the WMS-R (≥9 for 16 or more years of education, ≥5 for 8 to 15 years of education, and ≥3 for 0 to 7 years of education). MCI and AD diagnostic criteria remained similar to ADNI 1 criteria. The protocols for full criteria of each ADNI phase are described elsewhere (https://adni.loni.usc.edu/methods/documents/). Thirty-three AD samples were also collected from the New Jersey Institute of Success Aging Memory and Aging Program (MAP). All patients underwent an extensive neuropsychological evaluation, were examined by a social worker and a board-certified geriatric psychiatrist, and were studied with brain MRI scans and blood serum tests. Exclusion criteria included a history of head injury, substance abuse, and major psychiatric disorders including major depression, epilepsy, B12, folate, or thyroid deficiency. This study was approved by the Rowan University institutional review board with consent obtained consistent with the Declaration of Helsinki.
The neuropsychological protocol used to classify groups included three domains of cognition: executive functions, language, and verbal memory. All test scores were expressed as z-scores derived from previously published normative data. Executive functioning measures included the Boston Revision of the Wechsler Memory Scale Mental Control subtest [4], letter fluency test ('FAS') [5,6], and Trail Making Test-Part B [5,7]. Language measures included the Boston Naming Test [5,8], 'animal' fluency test [5], and WAIS-III Similarities subtest [9].
Finally, verbal memory included the CVLT-short form total learning trials, delayed free recall, and delayed recognition discriminability index [10]. All patients had an informant and presented with subjective cognitive concerns (patient or informant). Using 3-cluster classification statistical methods, a normal group and two MCI groups (amnestic-dominant and mixed-group) were generated. These MCI subtypes were then collated into one MCI group. The normal group was generally free of cognitive impairment. Comparatively, the MCI group presented with evidence of cognitive impairment relative to age and education. Of note, both the normal and MCI groups had preserved functional abilities per responses using the Lawton and Brody Scale [11]. AD was diagnosed in the presence of objective cognitive impairment and functional decline. Dementia was diagnosed via consensus of neuropsychologist, psychiatrist, and social worker. This study was approved by the Rowan University Institutional Review Board.
Individual serum pre-processing information ADNI Blood was collected in a red-top tube (BD 367820) and allowed to sit for at least 30 minutes at room temperature to clot. The tube is then centrifuged at 3,000 rpm for 15 min. Serum is pipetted off the clot into a prelabeled 13 ml plastic transfer tube, and then immediately capped and frozen at -80°C.

Durin Technologies Inc.
Blood was collected in a red-top tube (BD 367820) and allowed to sit for at least 30 min to clot. The tube is then centrifuged at 3,000 rpm for 15 min. Serum is transferred by pipetting off the clot into prelabeled cryovials, and then immediately capped and frozen at -80°C.

Reprocell
Blood was collected in a red-top tube (BD 367820) and allowed to sit for at least 30 min to clot. The tube is then centrifuged at 2,000 rpm for 10 min. Serum is transferred by pipetting off the clot into prelabeled cryovials, and then immediately capped and frozen at -80°C.

Parkinson's Study Group
Blood was collected in a red-top tube and allowed to sit for at least 30 min to clot. The tube is then centrifuged. Serum is transferred by pipetting off the clot into two 7 mL plastic vials, and then immediately capped and frozen at -80°C.
Blood was collected in a serum separator tube (SST; red tiger top) (BD 367985) and allowed to sit for at least 30 min to clot. The tube is then centrifuged at 1,300 rpm -2,000 rpm for 10-15 min. Serum is transferred by pipetting off the clot into 1 mL cryovials, and then immediately capped and frozen at -20°C or cooler.