Antemortem Plasma Research Articles

A False-positive Diagnosis of a Lethal Serotonin Syndrome Based on Postmortem Whole-blood Levels of Sertraline: How Forensic Detective Work Uses Medical Knowledge and Clinical Pharmacology to Solve Cases.

This column is the third in a 3-part series describing cases in which general medical knowledge, including psychiatric and clinical pharmacology, was instrumental in determining whether dereliction was the direct cause of damages in a malpractice suit. This case illustrates how not taking into account the following variables can result in a false-positive diagnosis of a lethal serotonin syndrome: (a) the time course of treatment, (b) the time course of symptoms, (c) the difference between antemortem plasma and postmortem whole-blood levels of highly protein bound and highly lipophilic drugs. The case also illustrates how taking those 3 variables into account led to the conclusion that there was no dereliction in the care of the patient that was the direct cause of his death, and hence, there was no medical malpractice.

Optimal blood tau species for the detection of Alzheimer’s disease neuropathology: an immunoprecipitation mass spectrometry and autopsy study

Plasma-to-autopsy studies are essential for validation of blood biomarkers and understanding their relation to Alzheimer’s disease (AD) pathology. Few such studies have been done on phosphorylated tau (p-tau) and those that exist have made limited or no comparison of the different p-tau variants. This study is the first to use immunoprecipitation mass spectrometry (IP-MS) to compare the accuracy of eight different plasma tau species in predicting autopsy-confirmed AD. The sample included 123 participants (AD = 69, non-AD = 54) from the Boston University Alzheimer’s disease Research Center who had an available ante-mortem plasma sample and donated their brain. Plasma samples proximate to death were analyzed by targeted IP-MS for six different tryptic phosphorylated (p-tau-181, 199, 202, 205, 217, 231), and two non-phosphorylated tau (195–205, 212–221) peptides. NIA-Reagan Institute criteria were used for the neuropathological diagnosis of AD. Binary logistic regressions tested the association between each plasma peptide and autopsy-confirmed AD status. Area under the receiver operating curve (AUC) statistics were generated using predicted probabilities from the logistic regression models. Odds Ratio (OR) was used to study associations between the different plasma tau species and CERAD and Braak classifications. All tau species were increased in AD compared to non-AD, but p-tau217, p-tau205 and p-tau231 showed the highest fold-changes. Plasma p-tau217 (AUC = 89.8), p-tau231 (AUC = 83.4), and p-tau205 (AUC = 81.3) all had excellent accuracy in discriminating AD from non-AD brain donors, even among those with CDR < 1). Furthermore, p-tau217, p-tau205 and p-tau231 showed the highest ORs with both CERAD (ORp-tau217 = 15.29, ORp-tau205 = 5.05 and ORp-tau231 = 3.86) and Braak staging (ORp-tau217 = 14.29, ORp-tau205 = 5.27 and ORp-tau231 = 4.02) but presented increased levels at different amyloid and tau stages determined by neuropathological examination. Our findings support plasma p-tau217 as the most promising p-tau species for detecting AD brain pathology. Plasma p-tau231 and p-tau205 may additionally function as markers for different stages of the disease.

Proteome Analysis in Plasma and Brain Reveals a Novel Plasma Biomarker Linked to Antemortem Cognitive Impairment and Postmortem Neuropathological Traits for Alzheimer Disease

AbstractBackgroundIdentification of blood‐based biomarkers that are indicative of Alzheimer’s disease (AD) pathology may help with early detection.MethodsWe generated proteome data for ∼7,000 proteins in both antemortem plasma and postmortem brain tissue from 103 participants of the Boston University Alzheimer’s Disease Research Center. All participants completed comprehensive neuropsychological evaluations and dementia severity was measured by global cognitive dementia rating (CDR) scores. A pathological AD diagnosis was established using NIA Reagan criteria. We conducted association tests with dementia (i.e., CDR ≥1 for dementia, CDR &lt;1 for control) and autopsy‐confirmed AD status as binary outcomes. Plasma proteins significantly associated with clinical or pathological AD (P&lt;0.05) were tested for association with quantitative cognitive tests and AD‐related neuropathological traits in which outcomes were rank‐transformed after adjusting for sex and either age at last antemortem exam or death.ResultsOf the 103 brain donors, 43 were diagnosed with dementia and 55 had autopsy‐confirmed AD. Five proteins (ACES, AURKB, CBARP, G45IP, and MMP‐8) measured in plasma were significantly associated with both pathological AD and dementia, while 252 plasma proteins were associated with either diagnosis (P&lt;0.05). MMP‐8 was the only protein that was significantly associated in both plasma and brain with autopsy‐confirmed AD (Pplasma = 0.005, Pbrain = 0.001) and dementia (Pplasma = 0.01, Pbrain = 9×10−5). ACES and AURKB levels in plasma and brain were significantly associated with dementia (P&lt;0.05). Levels of these. Increased MMP‐8 level in plasma was associated with decreased animals (P = 0.03) and scores (P = 0.04), which both assess verbal fluency. Dementia and pathologically confirmed AD were associated with lower AURKB and elevated ACES levels in plasma. Decreased plasma ACES level was associated with executive function (TrailsB: P = 0.01) and language (Boston Naming Test: P = 0.02) impairment, and the reduced plasma AURKB level was associated with memory impairment (Logical Memory Immediate Recall score: P = 0.03).ConclusionMMP‐8, a neutrophil collagenase and member of the matrix metalloproteinase family, is a potential blood‐based biomarker for dementia and underlying AD pathology and is associated predominantly with impaired verbal fluency.

Identification of Necessary Plasma Biomarkers for Prediction of Alzheimer’s Disease Neuropathologic Change

AbstractBackgroundPlasma biomarkers for Alzheimer’s disease (AD) such as phosphorylated tau protein 181 (p‐tau 181), neurofilament light chain (NfL), amyloid‐β 42/40 ratio (Aβ42/40), and glial fibrillary acidic protein (GFAP) have shown high performance in the discrimination of those with clinical mild cognitive impairment (MCI) or dementia from cognitively unimpaired individuals, but few population‐based autopsy studies have evaluated the neuropathological correlates of plasma biomarkers.MethodWe utilized a population‐based prospective study of 350 participants with autopsy and antemortem plasma biomarker data. Plasma biomarkers Aβ42/40 ratio, p‐tau181, GFAP, and NfL were measured using Quanterix Simoa assays. We predicted AD neuropathological change (ADNC), Braak staging, and neuritic plaque score using these plasma biomarkers and investigated the added utility of cognitive and demographic information. We utilized a variable selection procedure in cross‐validated (CV) logistic regression models by minimizing the Bayesian Information Criterion to identify the best set of predictors for a given outcome among plasma biomarkers, demographic variables, and neuropsychological tests included in the Mayo Clinic Preclinical Alzheimer Cognitive Composite (Mayo‐PACC).ResultADNC was best predicted with plasma GFAP, NfL, p‐tau181 biomarkers along with APOE ε4 carrier status and Mayo‐PACC cognitive score (CV AUC = 0.821). Braak staging was best predicted using plasma GFAP, p‐tau181, and cognitive scores (CV AUC = 0.788). Neuritic plaque score was best predicted using plasma Aβ42/40 ratio, p‐tau181, GFAP, and NfL biomarkers (CV AUC = 0.778). Inclusion of plasma biomarkers demonstrated significantly better prediction of Braak staging (p&lt;0.01) and neuritic plaque score (p&lt;0.01), but not ADNC (p = 0.13), when compared to models that included only cognitive and demographic information. Univariate models consistently underperformed in comparison to multivariate models.ConclusionPlasma biomarkers can provide valuable predictive information about the presence of AD pathology, which can facilitate earlier recognition and diagnosis of AD. Incorporation of demographic and cognitive variables along with plasma variables in multivariable models will improve prediction accuracy of AD pathology, specifically Braak staging and ADNC.

Identifying Optimal Blood Tau Epitopes for the Detection of Alzheimer’s Disease Neuropathology: An Immunoprecipitation Mass Spectrometry and Autopsy Study

AbstractBackgroundPlasma‐to‐autopsy studies are essential for validation of plasma biomarkers for the detection of Alzheimer’s disease (AD). Few such studies have been done and those that exist have had limited or no comparison of the different tau epitopes. This plasma‐to‐autopsy study is the first to use immunoprecipitation mass spectrometry (IP‐MS) to compare the accuracy of eight different plasma tau epitopes (six p‐tau, two total tau) in predicting autopsy‐confirmed AD.MethodThe sample included 123 participants from the Boston University Alzheimer’s Disease Research Center who had an available antemortem plasma sample and donated their brain for neuropathological examination. Plasma samples proximate to death were analyzed for six different plasma p‐tau (181+199+202+205+217+231) and two total tau (t‐tau) epitopes (195‐205, 212‐221) simultaneously using IP‐MS. NIA‐Reagan Institute criteria were used for the neuropathological diagnosis of AD. Binary logistic regressions tested the association between each plasma epitope and autopsy‐confirmed AD status. Area under the receiver operating curve (AUC) statistics were generated using predicted probabilities from the logistic regression models. Analyses were repeated stratified by Clinical Dementia Rating (CDR) score (i.e., &lt;1 and ≥1) at the time of blood draw. Odds Ratio (OR) was used to study associations between the different plasma tau species and CERAD and Braak classifications.Result Table 1 shows sample characteristics. Sixty‐nine of the 123 (56.1%) brain donors had autopsy‐confirmed AD and the average interval between blood draw and death was ∼5 years. Table 2 summarizes logistic regressions and Figure 1 displays ROC curves. Plasma p‐tau217 (AUC = 89.8), p‐tau231 (AUC = 83.4), and p‐tau205 (AUC = 81.3) all had excellent accuracy in discriminating AD from non‐AD brain donors. Among those with CDR≥1, p‐tau217, p‐tau231, and p‐tau205 had outstanding discrimination and all other epitopes had excellent discrimination. Among those with CDR&lt;1, p‐tau217 (AUC = 86.3) and p‐tau231 (AUC = 80.6) still had excellent discrimination accuracy; p‐tau181, p‐tau205, and t‐tau195‐209 had acceptable discrimination. Furthermore, p‐tau217, p‐tau205 and p‐tau231 were the tau species that showed higher ORs with both CERAD (ORp‐tau217 = 15.29, ORp‐tau205 = 5.05 and ORp‐tau231 = 3.86) and Braak staging (ORp‐tau217 = 14.29, ORp‐tau205 = 5.27 and ORp‐tau231 = 4.02).ConclusionPlasma levels of p‐tau217, p‐tau231, and p‐tau205 showed the best discrimination accuracy for AD confirmed neuropathology in both mild and severe cognitive impairment.

Plasma biomarkers for prediction of Alzheimer's disease neuropathologic change.

While plasma biomarkers for Alzheimer's disease (AD) are increasingly being evaluated for clinical diagnosis and prognosis, few population-based autopsy studies have evaluated their utility in the context of predicting neuropathological changes. Our goal was to investigate the utility of clinically available plasma markers in predicting Braak staging, neuritic plaque score, Thal phase, and overall AD neuropathological change (ADNC).We utilized a population-based prospective study of 350 participants with autopsy and antemortem plasma biomarker testing using clinically available antibody assay (Quanterix) consisting of Aβ42/40 ratio, p-tau181, GFAP, and NfL. We utilized a variable selection procedure in cross-validated (CV) logistic regression models to identify the best set of plasma predictors along with demographic variables, and a subset of neuropsychological tests comprising the Mayo Clinic Preclinical Alzheimer Cognitive Composite (Mayo-PACC). ADNC was best predicted with plasma GFAP, NfL, p-tau181 biomarkers along with APOE ε4 carrier status and Mayo-PACC cognitive score (CV AUC = 0.798). Braak staging was best predicted using plasma GFAP, p-tau181, and cognitive scores (CV AUC = 0.774). Neuritic plaque score was best predicted using plasma Aβ42/40 ratio, p-tau181, GFAP, and NfL biomarkers (CV AUC = 0.770). Thal phase was best predicted using GFAP, NfL, p-tau181, APOE ε4 carrier status and Mayo-PACC cognitive score (CV AUC = 0.754). We found that GFAP and p-tau provided non-overlapping information on both neuritic plaque and Braak stage scores whereas Aβ42/40 and NfL were mainly useful for prediction of neuritic plaque scores. Separating participants by cognitive status improved predictive performance, particularly when plasma biomarkers were included. Plasma biomarkers can differentially inform about overall ADNC pathology, Braak staging, and neuritic plaque score when combined with demographics and cognitive variables and have significant utility for earlier detection of AD.

369 Differentiating Dementia with Lewy Bodies and Alzheimer’s Disease Using Extracellular Vesicles

OBJECTIVES/GOALS: Extracellular vesicles (EVs) in biofluids can reflect pathology in the brain (like alpha-synuclein protein linked to Dementia with Lewy Bodies) making them attractive disease biomarkers to distinguish disease. Here, we will investigate their potential as non-invasive biomarkers to differentiate Dementia with Lewy Bodies &amp; Alzheimer's Disease. METHODS/STUDY POPULATION: We will leverage the collection of antemortem plasma from autopsy confirmed Dementia with Lewy Bodies (DLB), Alzheimer's Disease (AD) &amp; healthy controls (HC) through the Mayo Clinic Brain Bank. We will characterize antemortem brain-derived plasma EVs (bPEVs) &amp; post-mortem brain-derived EVs (bEVs) of the same patient using nanoparticle tracking, electron microscopy, &amp; western blotting for EV-associated proteins. We will then measure levels of total &amp; phosphorylated alpha-synuclein (asyn) using AlphaLisa. We will assess EVs' ability to initiate asyn aggregation using a real-time quaking-induced conversion (RT-QuIC) assay &amp; a cell-based Fluorescence Resonance Energy Transfer (FRET) assay. Once characterized, this will allow differentiation of bpEVs in DLB, AD, or healthy controls. RESULTS/ANTICIPATED RESULTS: We hypothesize that asyn levels and seeding capacity will discriminate DLB from AD. With AlphaLisa assay, we expect higher total and phosphorylated asyn levels in DLB bpEVs and their corresponding post-mortem bEVs. Using RT-QuIC and FRET assay, we expect EVs isolated from DLB patient samples to support seeded aggregation, whereas EVs from AD and HC will not. DISCUSSION/SIGNIFICANCE: To date, no diagnostic or less invasive biomarkers can distinguish DLB from AD. The successful completion of the aims outlined in this proposal will identify characteristics of bpEVs that differentiate DLB from AD or HC and support the development of bpEVs as a non-invasive, early biomarker to diagnose patients presenting with dementia from DLB.

Specific associations between plasma biomarkers and postmortem amyloid plaque and tau tangle loads

Several promising plasma biomarkers for Alzheimer's disease have been recently developed, but their neuropathological correlates have not yet been fully determined. To investigate and compare independent associations between multiple plasma biomarkers (p‐tau181, p‐tau217, p‐tau231, Aβ42/40, GFAP, and NfL) and neuropathologic measures of amyloid and tau, we included 105 participants from the Arizona Study of Aging and Neurodegenerative Disorders (AZSAND) with antemortem plasma samples and a postmortem neuropathological exam, 48 of whom had longitudinal p‐tau217 and p‐tau181. When simultaneously including plaque and tangle loads, the Aβ42/40 ratio and p‐tau231 were only associated with plaques (ρAβ42/40[95%CI] = −0.53[−0.65, −0.35], ρp‐tau231[95%CI] = 0.28[0.10, 0.43]), GFAP was only associated with tangles (ρGFAP[95%CI] = 0.39[0.17, 0.57]), and p‐tau217 and p‐tau181 were associated with both plaques (ρp‐tau217[95%CI] = 0.40[0.21, 0.56], ρp‐tau181[95%CI] = 0.36[0.15, 0.50]) and tangles (ρp‐tau217[95%CI] = 0.52[0.34, 0.66]; ρp‐tau181[95%CI] = 0.36[0.17, 0.52]). A model combining p‐tau217 and the Aβ42/40 ratio showed the highest accuracy for predicting the presence of Alzheimer's disease neuropathological change (ADNC, AUC[95%CI] = 0.89[0.82, 0.96]) and plaque load (R2 = 0.55), while p‐tau217 alone was optimal for predicting tangle load (R2 = 0.45). Our results suggest that high‐performing assays of plasma p‐tau217 and Aβ42/40 might be an optimal combination to assess Alzheimer's‐related pathology in vivo.

Global neuropathologic severity of Alzheimer’s disease and locus coeruleus vulnerability influences plasma phosphorylated tau levels

BackgroundAdvances in ultrasensitive detection of phosphorylated tau (p-tau) in plasma has enabled the use of blood tests to measure Alzheimer’s disease (AD) biomarker changes. Examination of postmortem brains of participants with antemortem plasma p-tau levels remains critical to understanding comorbid and AD-specific contribution to these biomarker changes.MethodsWe analyzed 35 population-based Mayo Clinic Study of Aging participants with plasma p-tau at threonine 181 and threonine 217 (p-tau181, p-tau217) available within 3 years of death. Autopsied participants included cognitively unimpaired, mild cognitive impairment, AD dementia, and non-AD neurodegenerative disorders. Global neuropathologic scales of tau, amyloid-β, TDP-43, and cerebrovascular disease were examined. Regional digital pathology measures of tau (phosphorylated threonine 181 and 217 [pT181, pT217]) and amyloid-β (6F/3D) were quantified in hippocampus and parietal cortex. Neurotransmitter hubs reported to influence development of tangles (nucleus basalis of Meynert) and amyloid-β plaques (locus coeruleus) were evaluated.ResultsThe strongest regional associations were with parietal cortex for tau burden (p-tau181 R = 0.55, p = 0.003; p-tau217 R = 0.66, p < 0.001) and amyloid-β burden (p-tau181 R = 0.59, p < 0.001; p-tau217 R = 0.71, p < 0.001). Linear regression analysis of global neuropathologic scales explained 31% of variability in plasma p-tau181 (Adj. R2 = 0.31) and 59% in plasma p-tau217 (Adj. R2 = 0.59). Neither TDP-43 nor cerebrovascular disease global scales independently contributed to variability. Global scales of tau pathology (β-coefficient = 0.060, p = 0.016) and amyloid-β pathology (β-coefficient = 0.080, p < 0.001) independently predicted plasma p-tau217 when modeled together with co-pathologies, but only amyloid-β (β-coefficient = 0.33, p = 0.021) significantly predicted plasma p-tau181. While nucleus basalis of Meynert neuron count/mm2 was not associated with plasma p-tau levels, a lower locus coeruleus neuron count/mm2 was associated with higher plasma p-tau181 (R = -0.50, p = 0.007) and higher plasma p-tau217 (R = -0.55, p = 0.002). Cognitive scores (Adj. R2 = 0.25–0.32) were predicted by the global tau scale, but not by the global amyloid-β scale or plasma p-tau when modeled simultaneously.ConclusionsHigher soluble plasma p-tau levels may be the result of an intersection between insoluble deposits of amyloid-β and tau accumulation in brain, and may be associated with locus coeruleus degeneration.

Plasma Biomarkers of Amyloid and Neurodegeneration predictive of Neuropathologic Scales of Cerebrovascular Disease

AbstractBackgroundBlood‐based biomarkers of amyloid and neurodegeneration are increasingly used in clinical trials and entering clinical use. Differentially mapping the plasma markers to post‐mortem neuropathology in a community‐based sample is critical for interpretation.MethodWe identified 64 Mayo Clinic Study of Aging (MCSA) participants with antemortem plasma markers (Aβ42, Aβ40, NfL, T‐tau, Aβ42/40 ratio) measured on the Quanterix Simoa HD‐1 analyzer and with postmortem neuropathologic assessments of Alzheimer’s pathology (Braak tangle stage, Thal phase, neuritic plaque burden) and cerebrovascular pathology (Strozyk scale focused on macrovascular brain changes and Kalaria scale focused on detailed micro‐ and macro‐ vascular brain changes). Weighted linear regression models with adjustment for time between plasma markers and death were conducted with exact permutation tests to evaluate the relationships between antemortem plasma and postmortem neuropathology markers.ResultHigher plasma NfL levels and Aβ42/40 ratio were associated with both cerebrovascular neuropathologic scales (p&lt;0.05) but were not associated with Braak neurofibrillary tangle stage, neuritic plaque score, or Thal stage (p&gt;0.5). Plasma T‐tau was not associated with any pathological assessment. Plasma Aβ42/40 and NfL explained up to 18% of the variability in cerebrovascular neuropathologic scales.ConclusionIn this community sample of participants predominantly without dementia, plasma markers for amyloid (Aβ42/40) and neurodegeneration (NfL) were associated with cerebrovascular neuropathology at autopsy. NfL is a non‐specific marker of brain injury, therefore, its association with cerebrovascular neuropathology was in the expected direction. The association between greater cerebrovascular neuropathology and elevated Aβ42/40 requires further investigation. These findings provide evidence that cerebrovascular disease may significantly influence levels of plasma biomarkers proposed for Alzheimer’s disease. This information will be important to consider for accurate clinical interpretation of the proposed biomarkers.

Association of plasma biomarkers of amyloid and neurodegeneration with cerebrovascular disease and Alzheimer's disease

The objective of this study was to determine the differential mapping of plasma biomarkers to postmortem neuropathology measures. We identified 64 participants in a population-based study with antemortem plasma markers (amyloid-β [Aβ] x-42, Aβx-40, neurofilament light [NfL], and total tau [T-tau]) who also had neuropathologic assessments of Alzheimer's and cerebrovascular pathology. We conducted weighted linear-regression models to evaluate relationships between plasma measures and neuropathology. Higher plasma NfL and Aβ42/40 ratio were associated with cerebrovascular neuropathologic scales (p < 0.05) but not with Braak stage, neuritic plaque score, or Thal phase. Plasma Aβ42/40 and NfL explained up to 18% of the variability in cerebrovascular neuropathologic scales. In participants predominantly with modest levels of Alzheimer's pathologic change, biomarkers of amyloid and neurodegeneration were associated with cerebrovascular neuropathology. NfL is a non-specific marker of brain injury, therefore its association with cerebrovascular neuropathology was expected. The association between elevated Aβ42/40 and cerebrovascular disease pathology needs further investigation but could be due to the use of less specific amyloid-β assays (x-40, x-42).

Ante-mortem plasma phosphorylated tau (181) predicts Alzheimer's disease neuropathology and regional tau at autopsy.

Blood-based biomarkers such as tau phosphorylated at threonine 181 (phosphorylated-tau181) represent an accessible, cost-effective and scalable approach for the in vivo detection of Alzheimer's disease pathophysiology. Plasma-pathological correlation studies are needed to validate plasma phosphorylated-tau181 as an accurate and reliable biomarker of Alzheimer's disease neuropathological changes. This plasma-to-autopsy correlation study included participants from the Boston University Alzheimer's Disease Research Center who had a plasma sample analysed for phosphorylated-tau181 between 2008 and 2018 and donated their brain for neuropathological examination. Plasma phosphorelated-tau181 was measured with single molecule array technology. Of 103 participants, 62 (60.2%) had autopsy-confirmed Alzheimer's disease. Average time between blood draw and death was 5.6 years (standard deviation = 3.1 years). Multivariable analyses showed higher plasma phosphorylated-tau181 concentrations were associated with increased odds for having autopsy-confirmed Alzheimer's disease [AUC = 0.82, OR = 1.07, 95% CI = 1.03-1.11, P < 0.01; phosphorylated-tau standardized (z-transformed): OR = 2.98, 95% CI = 1.50-5.93, P < 0.01]. Higher plasma phosphorylated-tau181 levels were associated with increased odds for having a higher Braak stage (OR = 1.06, 95% CI = 1.02-1.09, P < 0.01) and more severe phosphorylated-tau across six cortical and subcortical brain regions (ORs = 1.03-1.06, P < 0.05). The association between plasma phosphorylated-tau181 and Alzheimer's disease was strongest in those who were demented at time of blood draw (OR = 1.25, 95%CI = 1.02-1.53), but an effect existed among the non-demented (OR = 1.05, 95% CI = 1.01-1.10). There was higher discrimination accuracy for Alzheimer's disease when blood draw occurred in years closer to death; however, higher plasma phosphorylated-tau181 levels were associated with Alzheimer's disease even when blood draw occurred >5 years from death. Ante-mortem plasma phosphorylated-tau181 concentrations were associated with Alzheimer's disease neuropathology and accurately differentiated brain donors with and without autopsy-confirmed Alzheimer's disease. These findings support plasma phosphorylated-tau181 as a scalable biomarker for the detection of Alzheimer's disease.

Neuronal-Derived EV Biomarkers Track Cognitive Decline in Alzheimer’s Disease

The hallmarks of Alzheimer’s disease (AD) pathology are senile plaques containing amyloid-beta (Aβ) and neurofibrillary tangles containing hyperphosphorylated tau. Additional pathologies often co-exist, whereas multiple pathogenic mechanisms are involved in AD, especially synaptic degeneration, which necessitate the need for synaptic integrity-related biomarkers alongside Aβ- and tau-related biomarkers. Plasma neuron-derived Extracellular Vesicles EVs (NDEVs) provide biomarkers related to Aβ and tau and synaptic degeneration. Here, to further establish the latter as a “liquid biopsy” for AD, we examined their relationship with ante-mortem cognition in pathologically-confirmed AD cases. We immunoprecipitated NDEVs by targeting neuronal marker L1CAM from ante-mortem plasma samples from 61 autopsy-confirmed cases of pure AD or AD with additional pathologies and measured Aβ42, p181-Tau, total Tau, synaptophysin, synaptopodin and three canonical EV markers, CD63, CD81 and CD9. Higher NDEV Aβ42 levels were consistently associated with better cognitive status, memory, fluency, working memory and executive function. Higher levels of NDEV synaptic integrity-related biomarkers were associated with better performance on executive function tasks. Our findings motivate the hypothesis that releasing Aβ42-laden NDEVs may be an adaptive mechanism in AD.

Femoral blood concentrations of the designer benzodiazepine etizolam in post-mortem cases.

Etizolam is a thienodiazepine that although licensed for clinical usage in Japan, India and South Korea is commonly abused and detected in post-mortem cases around the world. To date, there are limited data in the literature to allow for the interpretation of blood concentrations of etizolam in post-mortem cases. A liquid chromatography with tandem mass spectrometry method was used to quantitate etizolam concentrations in 28 post-mortem cases where etizolam was detected. The median concentration of etizolam in femoral blood was 8.5 ng/mL (range 1.0-172.0 ng/mL; n = 24); in antemortem plasma, the etizolam concentration range was 4-44 ng/mL (n = 4). The mean age of the individuals abusing etizolam was 38.5 ± 8.4 years (median 39 years), with the majority being male (86%). In all of the cases, multiple drugs were detected, with the most common being pregabalin (61%) followed by morphine/heroin (54%), diazepam (54%) and benzoylecgonine (21%), illustrating the increasing problem of poly-substance use in drug abusers. The cause of death in the cases in which etizolam was detected was multi-drug toxicity in 87.5% of the cases, with 12.5% unrelated to drug use (hangings and blunt-force trauma). These data will further help forensic practitioners with the interpretation of post-mortem etizolam concentrations.

Discriminative Accuracy of Plasma Phospho-tau217 for Alzheimer Disease vs Other Neurodegenerative Disorders

There are limitations in current diagnostic testing approaches for Alzheimer disease (AD). To examine plasma tau phosphorylated at threonine 217 (P-tau217) as a diagnostic biomarker for AD. Three cross-sectional cohorts: an Arizona-based neuropathology cohort (cohort 1), including 34 participants with AD and 47 without AD (dates of enrollment, May 2007-January 2019); the Swedish BioFINDER-2 cohort (cohort 2), including cognitively unimpaired participants (n = 301) and clinically diagnosed patients with mild cognitive impairment (MCI) (n = 178), AD dementia (n = 121), and other neurodegenerative diseases (n = 99) (April 2017-September 2019); and a Colombian autosomal-dominant AD kindred (cohort 3), including 365 PSEN1 E280A mutation carriers and 257 mutation noncarriers (December 2013-February 2017). Plasma P-tau217. Primary outcome was the discriminative accuracy of plasma P-tau217 for AD (clinical or neuropathological diagnosis). Secondary outcome was the association with tau pathology (determined using neuropathology or positron emission tomography [PET]). Mean age was 83.5 (SD, 8.5) years in cohort 1, 69.1 (SD, 10.3) years in cohort 2, and 35.8 (SD, 10.7) years in cohort 3; 38% were women in cohort 1, 51% in cohort 2, and 57% in cohort 3. In cohort 1, antemortem plasma P-tau217 differentiated neuropathologically defined AD from non-AD (area under the curve [AUC], 0.89 [95% CI, 0.81-0.97]) with significantly higher accuracy than plasma P-tau181 and neurofilament light chain (NfL) (AUC range, 0.50-0.72; P < .05). The discriminative accuracy of plasma P-tau217 in cohort 2 for clinical AD dementia vs other neurodegenerative diseases (AUC, 0.96 [95% CI, 0.93-0.98]) was significantly higher than plasma P-tau181, plasma NfL, and MRI measures (AUC range, 0.50-0.81; P < .001) but not significantly different compared with cerebrospinal fluid (CSF) P-tau217, CSF P-tau181, and tau-PET (AUC range, 0.90-0.99; P > .15). In cohort 3, plasma P-tau217 levels were significantly greater among PSEN1 mutation carriers, compared with noncarriers, from approximately 25 years and older, which is 20 years prior to estimated onset of MCI among mutation carriers. Plasma P-tau217 levels correlated with tau tangles in participants with (Spearman ρ = 0.64; P < .001), but not without (Spearman ρ = 0.15; P = .33), β-amyloid plaques in cohort 1. In cohort 2, plasma P-tau217 discriminated abnormal vs normal tau-PET scans (AUC, 0.93 [95% CI, 0.91-0.96]) with significantly higher accuracy than plasma P-tau181, plasma NfL, CSF P-tau181, CSF Aβ42:Aβ40 ratio, and MRI measures (AUC range, 0.67-0.90; P < .05), but its performance was not significantly different compared with CSF P-tau217 (AUC, 0.96; P = .22). Among 1402 participants from 3 selected cohorts, plasma P-tau217 discriminated AD from other neurodegenerative diseases, with significantly higher accuracy than established plasma- and MRI-based biomarkers, and its performance was not significantly different from key CSF- or PET-based measures. Further research is needed to optimize the assay, validate the findings in unselected and diverse populations, and determine its potential role in clinical care.

Plasma soluble CD163 is associated with postmortem brain pathology in human immunodeficiency virus infection.

Higher plasma soluble cluster of differentiation (CD)163 (sCD163), shed by monocytes and macrophages, correlates with neurocognitive impairment in HIV infection. We hypothesized that higher antemortem plasma or cerebrospinal fluid (CSF) sCD163 would be associated with greater postmortem neurodegeneration and/or microgliosis. Retrospective, postmortem observational study. We measured sCD163 levels in antemortem plasma (n = 54) and CSF (n = 32) samples from 74 HIV-seropositive participants (median 5 months before death) who donated their brains to research at autopsy. Postmortem, we quantified markers of synaptodendritic damage (microtubule-associated protein 2, synaptophysin), microgliosis [human leukocyte antigen DR (HLA-DR), ionized calcium-binding adaptor molecule 1], astrocytosis (glial fibrillary acidic protein), and impaired protein clearance (β-amyloid) in frontal cortex, hippocampus, putamen, and internal capsule. Multivariable least-squares regression was used to evaluate the association between plasma or CSF sCD163 and histological measures, correcting for multiple comparisons. Higher plasma sCD163 was associated with lower microtubule-associated protein 2 in frontal cortex [B = -0.23, 95% confidence interval (CI) -0.41 to -0.06, P = 0.04], putamen (B = 0.32, 95% CI -0.52 to -0.12, P = 0.02), and hippocampus (B = -0.23, 95% CI -0.35 to -0.10, P = 0.01), and with lower synaptophysin in hippocampus (B = -0.25, 95% CI -0.42 to -0.03, P = 0.02) but not putamen or frontal cortex (P > 0.05). Higher plasma sCD163 was associated with higher HLA-DR in putamen (B = 0.17, 95% CI 0.08 to 0.26, P = 0.008). CSF sCD163 was not associated with any histological measure (P > 0.05). Higher plasma sCD163 in life is associated with greater synaptodendritic damage and microglial activation in cortical and subcortical brain regions.

Xanthochromia of the skull bone associated with HbA1c

The color of the surface of 105 skull bones (part of the parietal bone) was determined using a portable spectral colorimeter (spectro color®). By this means it was possible to characterize the color objectively according to the L*a*b* color system defined by the “International Commission de l’Eclairage” (CIE). Biochemical markers of carbohydrate metabolism, HbA1c from venous blood, and glucose/lactate concentrations from vitreous humor, were also determined, for assessment of the ante-mortem plasma glucose concentration using Traub's sum formula. As biochemical markers for lipid metabolism disorder, cholesterol, triglycerides, high density lipoprotein (HDL), low density lipoprotein (LDL) and very low density lipoprotein (VLDL) were all determined from venous blood. There is a significant correlation of bone yellowing with HbA1c (p<0.001) and age (p<0.001). The literature asserts a significant correlation between diabetic condition and yellowing of the skull bone. Despite efforts to find the substance responsible for the yellowing of the bone in chronic metabolism disorder, no significant correlation was found between bone color and lipoproteins/bone extracted lipid acids.

Postmortem Increase of Flecainide Level in Cardiac Blood

We encountered a case of fatal congestive heart failure that occurred under the influence of flecainide. In this case, an extreme postmortem increase in the flecainide level was identified in cardiac blood. The patient had been administered 400 mg/day of flecainide for seven days before death. Antemortem plasma obtained 13 h before death showed a flecainide concentration of 2.5 mg/L and a pH of 7.4. In comparison, centrifuged supernatants of postmortem right and left cardiac blood contained flecainide concentrations of 13.8 and 44.2 mg/L, respectively, with pH of 5.5 in both samples. This increase in blood flecainide concentration was attributed to postmortem redistribution, as about 18 h had passed between the last intake of flecainide and death.

Relationship of antiretroviral treatment to postmortem brain tissue viral load in human immunodeficiency virus–infected patients

Human immunodeficiency virus (HIV)-1 invades the central nervous system (CNS) soon after infection and is partially protected there from host immunity and antiretroviral drugs (ARVs). Sanctuary from highly active antiretroviral therapy (HAART) in the CNS could result in ongoing viral replication, promoting the development of drug resistance and neurological disease. Despite the importance of these risks, no previous study has directly assessed HAART's effects on brain tissue viral load (VL). The authors evaluated 61 HIV-infected individuals for whom both histories of HAART treatment and postmortem brain tissue VL measurements were available. Two groups were defined based on HAART use in the 3 months prior to death: HAART(+) subjects had received HAART, and HAART(-) subjects had not received HAART. HIV RNA was quantified in postmortem brain tissue (log10 copies/10 microg total tissue RNA) and antemortem plasma (log10 copies/ml) by reverse transcriptase-polymerase chain reaction (RT-PCR). Brain tissue VLs were significantly lower among HAART(+) subjects compared to HAART(-) subjects (median 2.6 versus 4.1; P= .0007). These findings suggest that despite the limited CNS penetration of many antiretroviral medications, HAART is at least partially effective in suppressing CNS viral replication. Because some HAART regimens may be better than others in this regard, regimen selection strategies could be used to impede CNS viral activity, limit neuronal dysfunction, and prevent or treat clinical neurocognitive disorders in HIV-infected patients. Furthermore, such strategies might help to prevent the development of ARV resistance.

Death Following Colchicine Poisoning

A 45-year-old male was admitted to hospital after 2 to 3 days of vomiting, nausea, and diarrhea following an apparent overdose of colchicine tables. During hospitalization his white blood cell count fell dramatically. At death, 33 h following initial hospitalization, pleural effusion with bilateral bronchopneumonia was evident, together with numerous bacterial colonies and marked hypocellularity of bone marrow and reduced megakaryocytes, erythroid, and myeloid cells. The most striking histological findings were numerous metaphasic mitotic figures in gastric and small bowel epithelia. Colchicine was detected, confirmed by high pressure liquid chromatography with photodiode array detection, and quantitated in antemortem plasma collected 3.3 h following hospitalization and in postmortem blood and bile. Colchicine was not detected in liver, vitreous humor, or stomach contents.