Ashrafzadeh‐Kian, Susan

This cross-sectional study evaluates the performance of the US Food and Drug Administration–cleared phosphorylated tau 217 (p-tau217) to β-amyloid 42 ratio cut points used to detect amyloid pathology associated with Alzheimer disease.

Abstract INTRODUCTION Plasma phosphorylated tau at threonine 217 (p‐tau217) has emerged as one of the most promising blood‐based biomarkers for Alzheimer's disease (AD). While rare, heterophilic antibodies (HAb) are a persistent potential confounding factor in immunoassays. METHODS Potential HAb interference in the Lumipulse G pTau217 assay in samples exhibiting concentrations > 10 pg/mL were investigated. Samples were subjected to HAb blocking reagent (HBT), and in some cases, polyethylene glycol (PEG) precipitation and serial dilutions. RESULTS In 14 of the 15 suspected HAb cases, HBT treatment greatly reduced measured p‐tau217 concentrations. and in one of three specimens selected for further investigation, reduced Aβ42 concentrations PEG precipitation in the three selected samples also reduced p‐tau217 concentrations, while serial dilution yielded mixed effects. Control specimens were unaffected by sample treatment or dilutions. DISCUSSION HAb may result in falsely elevated p‐tau217 and p‐tau217/Aβ42 ratio. HAb interference should be considered in cases of unusually high p‐tau217 concentrations prior to clinical interpretation. Highlights Potential heterophile antibody (HAb) interference in the Lumipulse p‐tau217 immunoassay was investigated. Investigation was performed using HAb blocking reagent, and in some cases also using PEG precipitation . Patients with p‐tau217 concentrations > 10 pg/mL exhibited positive HAb interference in 14 of 15 cases. HAb interference can lead to falsely positive p‐tau217/Aβ42 ratios.

Abstract Background The polymorphic APOE gene is a susceptibility gene for Alzheimer's disease (AD). The ε4 allele is associated with increased risk for AD, particularly late‐onset disease, in a dose‐dependent manner. Additionally, homozygous ε4/ε4 individuals have increased risk of adverse events when undergoing amyloid lowering treatments. APOE genotyping is considered the gold standard for determination of ApoE4 zygosity. Recently, proteotype assays for ApoE4 have become available for high‐throughput immunoassay analyzers. These assays utilize the same specimen type as AD blood biomarkers and can potentially offer improved turnaround times compared to genotyping. In this study, we compare the performance of two automated ApoE4 proteotype research use only (RUO) immunoassays against APOE genotyping. Method EDTA‐plasma samples ( n = 65) from individuals undergoing ApoE PCR‐based genotyping for clinical purposes were evaluated. Samples were tested with the Beckman Coulter APOE ε4 RUO assay on the Access 2 and DXI 9000 instruments (Beckman Coulter, Inc), which provides algorithm calculated results for determining ApoE4 zygosity. Manufacturer generated data from 300 genotyped samples was used to calculate estimated cut‐points for ApoE4 zygosity for this study with results <0.55 classified as no E4, ≥0.55 to <4.55 as heterozygous E4, and ≥4.55 as homozygous E4. Samples were also tested with the Lumipulse G ApoE4 and Pan‐ApoE RUO assays on the Fujirebio Lumipulse G1200 analyzer. The ratio of ApoE4/Pan‐ApoE was utilized for determining zygosity with results <5% classified as no E4, ≥5% to <75% as heterozygous E4, and ≥75% as homozygous E4, as listed in the manufacturer IFU. Result APOE genotype distributions were (n, %): ε3ε4 (30, 46%), ε3ε3 (18, 28%), ε4ε4 (12, 18%), ε2ε3 (3, 5%), ε2ε4 (2, 3%). ApoE4 zygosity was categorized as no ε4 (21, 32%), heterozygous ε4 (32, 49%), or homozygous ε4 (12, 19%). The Beckman Coulter APOE ε4 RUO assay classified 65/65 (100%) patients correctly using manufacturer generated cut‐points on both instruments. The Lumipulse G ApoE4 and Pan‐ApoE RUO assays classified 61/65 (94%) patients correctly using the manufacturer listed cut‐points. Conclusion ApoE4 proteotype immunoassays may represent simplified and more accessible testing alternatives to genotyping for the assessment of ApoE4 zygosity in individuals being considered for amyloid lowering treatments.

Abstract Background Clinically available plasma p ‐tau217 assays may be utilized in the determination of eligibility for amyloid‐lowering therapies. Our institution recently introduced a plasma p ‐tau217 assay in clinical practice using a two‐cutpoint approach that optimizes the accuracy while minimizing false positive risk and categorizing ≤20% of results as intermediate. In this study, we prospectively evaluate the performance of a plasma p ‐tau217 assay for detection of amyloid pathology using these predefined cutpoints. Method Patients recruited were being considered for lecanemab therapy at a tertiary care subspecialty clinic. Plasma p ‐tau217 was measured using the clinically available Fujirebio Lumipulse p ‐tau217 assay at Mayo Clinic, and results (pg/mL) were classified as either negative (≤0.185), intermediate (0.186–0.324) or positive (≥0.325). Amyloid pathology was determined by amyloid‐PET and/or CSF p ‐tau181/Aβ42. Result Ninety‐three patients were recruited between November 2023 and December 2024. Of these, 49 underwent amyloid‐PET, 4 had CSF p ‐tau181/Aβ42 measurements, and 40 had both PET and CSF results (with 3/40 discordant cases), for use in the assessment of amyloid pathology. The three discordant cases were considered amyloid positive for purposes of plasma p ‐tau217 comparison. Overall amyloid positivity was 90%. Plasma p ‐tau217 results were positive in 83% (77/93), negative in 6% (6/93), and intermediate in 11% (10/93) of patients. An elevated p ‐tau217 predicted the presence of amyloid positivity with a diagnostic accuracy of 96% (95% CI 90% − 99%). In the intermediate range, 80% of patients were positive for amyloid pathology. In patients with a definitive p ‐tau217 result (positive or negative), the positive and negative predictive values were 97% and 83%. There were three patients with discordant results: one PET+/p‐tau217‐, one PET‐/p‐tau217+, and one CSF‐/p‐tau217+. Clinical presentation of these discordant cases was examined, and relevant findings will be discussed. Conclusion The Lumipulse plasma p ‐tau217 assay exhibited acceptable diagnostic accuracy for the determination of amyloid pathology when compared to either amyloid‐PET or CSF p ‐tau181/Aβ42. This prospective study confirms the utility a two‐cutpoint approach for interpreting plasma p ‐tau217 for the assessment of amyloid pathology eligibility for amyloid‐lowering therapies in a heterogenous clinical cohort.

Abstract Background Anti‐amyloid therapy trials have reported changes in some Alzheimer's disease (AD) blood biomarkers (BBMs) suggesting that these BBMs may be useful minimally invasive measures of amyloid clearance and treatment outcomes. However, a better understanding of how AD BBMs changes at the individual level reflect treatment response in relation to other relevant clinical endpoints is necessary before they can be adopted for treatment monitoring. Here, several AD BBMs were assessed longitudinally in patients undergoing lecanemab therapy. Method Patients undergoing lecanemab therapy at Mayo Clinic, Rochester, MN were recruited to participate in this study. Patients were asked to provide an EDTA‐plasma sample before treatment initiation during the first infusion visit and thereafter at 3‐, 6‐, 12‐, and 18 months post‐treatment. p ‐tau217, p ‐tau181, GFAP, NfL, Aβ42, Aβ40, Aβ42/40, and p ‐tau217/Aβ42 measurements were obtained using Fujirebio Lumipulse assays. Biomarker median differences were assessed between all time points. Patients were grouped into three categories: a decreased biomarker (median decrease of at least 10%), an increased biomarker (median increase of at least 10%), and a stable biomarker (median change < +/−10%.) Biomarker data up to 6 months after treatment is presented. Result Of forty‐one patients enrolled since November 2023, twenty had time points up to 6 months. None of the biomarkers showed a statistically significant median change from baseline at 3 months ( p >0.05). At 6 months, only Aβ42/40 showed a statistically significant median change (decrease) between 0 and 6 months ( n = 20, p = 0.0058). Table 1 shows patients grouped by median biomarker changes from baseline to 6 months. GFAP, p ‐tau217, and Aβ42/40 exhibited the greatest number of patients with decreased biomarker concentrations (80%, 60%, and 60% of patients with median decreases of ‐25%, ‐26%, and ‐18% respectively), followed by p ‐tau181 (50% of patients; median decrease ‐22%), p ‐tau217/Aβ42 (45% of patients; median decrease ‐40%) and Aβ42 (45% of patients; median decrease ‐18%). None of the patients showed a decreased NfL or Aβ40 concentration. Conclusion Preliminarily analysis revealed that GFAP, p ‐tau217, and Aβ42/40 had the most frequent post‐lecanemab biomarker decreases. Additional sample collection is ongoing to assess the association of AD BBM changes with Amyloid PET over time.

Abstract Background Alzheimer’s disease (AD) cerebrospinal fluid (CSF) biomarkers, including the Aß42/40 and p-tau181/Aß42 ratios, are used in clinical practice to detect the presence of amyloid pathology for the diagnosis of AD. However, in some instances, discrepancies between Aß42/40 and p-tau181/Aß42 are observed. Such discrepancies may introduce uncertainties in the clinical management of patients presenting with cognitive decline. Identifying the underlying causes of these discrepancies and determining which biomarker abnormalities better predict disease progression is crucial to enhancing confidence in the use of these CSF biomarkers for the diagnosis of AD. This study aims to investigate the discordance between the Aß42/40 and p-tau181/Aß42 ratios, evaluate the frequency of these discrepancies, and identify potential causes. Methods Data from 958 individuals who had both CSF Aß42/40 and p-tau181/Aß42 tested at Mayo Clinic Laboratories between 2022 and 2024 and where the tests were ordered in CSF collected during the same lumbar puncture procedure were included. Cases with an inadequate sample collection, which could cause a false decrease in Aß42 concentrations, were excluded from the analysis. Aß42/40 was measured using the Fujirebio LUMIPULSE assays and reported as negative, likely positive, or positive. For this analysis, likely positive and positive results were combined and considered as positive (&lt;0.072). P-tau181/Aß42 was measured using the Roche Elecsys assays and above 0.028 was considered positive. Statistical analyses were conducted using R Studio. Results Aß42/40 was positive in 69.1% of cases (n=661), while p-tau181/Aß42 was positive in 62.1% (n=594). There were 8.0% discordant cases (n=77). Most frequently, the discordant cases had an abnormal Aß42/40 with a normal p-tau181/Aß42 (Aß42/40+/p-tau181/Aß42-; n=72, 94%). Compared to the concordant Aß42/40+/p-tau181/Aß42+ group, the discordant Aß42/40+/p-tau181/Aß42- group showed significantly higher Aß42 concentrations (p &lt;0.001, median [pg/mL]: 878.5 vs 587.0, respectively); whereas p-tau181 concentrations were significantly lower (p &lt;0.001, median [pg/mL]: 19.3 vs 32.1, respectively). Among the Aß42/40+/p-tau181/Aß42- group, 30 cases exhibited abnormally low Aß42 and normal p-tau181, possibly reflecting early-stage AD. There were 41 cases with a normal Aß42 concentration despite an abnormal Aß42/40, possibly reflecting an overall higher production of Aß peptides resulting in an increased Aß40 peptide (median [pg/mL]: 12627 compared to 9487 in the low Aß42 and normal p-tau181 cases (n=30); p &lt;0.001). Of these 41, 19 showed normal Aß42 but elevated p-tau181 concentrations where the use of Aß42/40 compensated for the overall higher production of Aß peptides, resulting in an abnormal ratio. In contrast, the p-tau181/Aß42 ratio was not able to normalize for these differences, leading to a normal ratio. Conclusion While overall concordance was 92.0%, our findings suggest that discrepancies between CSF Aß42/40 and p-Tau181/Aß42 are more often due to the presence of an abnormal Aß42/40 and a normal p-Tau181/Aß42. Two potential explanations for these discrepancies are early-stage AD or higher production of Aß peptides in some individuals. In the latter, differences in amyloid production would be normalized by ratios of the same biomarker (i.e. Aß42/40) but not by ratios of different biomarkers (i.e. p-Tau181/Aß42).

AbstractINTRODUCTIONWe compare the stability of phosphorylated tau (p‐tau)217, amyloid beta (Aβ)42, Aβ40, Aβ42/40, and p‐tau217/Aβ42 at different storage temperatures.METHODSTen ethylenediaminetetraacetic acid–plasma sample aliquots stored at frozen, refrigerated, and ambient temperatures were tested at various timepoints up to 30 days. The mean percent change from baseline was calculated.RESULTSAβ42 and Aβ40 concentrations decreased by >15% after 6 hours at ambient temperature and after 24 hours refrigerated, while p‐tau217 remained stable over 72 hours with < 10% deviation from baseline at either storage temperature. The Aβ42/40 ratio remained relatively constant as each analyte concentration decreased concurrently, while the p‐tau217/Aβ42 ratio deviated from baseline over time. All biomarkers were stable up to 30 days frozen.DISCUSSIONDifferences in the stability of Aβ42 and p‐tau217 may lead to altered p‐tau217/Aβ42 ratio results if samples are not handled properly during the pre‐analytical testing phase. Ideally, samples should be frozen promptly and sent to the laboratory frozen.Highlights Plasma phosphorylated tau (p‐tau)217, amyloid beta (Aβ)42, Aβ40, Aβ42/40, and p‐tau217/Aβ42 stability was assessed. P‐tau217 was stable, but Aβ42 and Aβ40 started decreasing by 6 hours at ambient temperature. The analyte stability differences led to falsely increased p‐tau217/Aβ42 ratios. Increases > 15% were seen in p‐tau217/Aβ42 after 6 hours at ambient temperature or 24 hours at 4°C.

AbstractBackgroundNeurofilament Light Chain (NfL) is a blood biomarker of axonal injury and neurodegeneration that can be used in a variety of neurological disorders. Despite the potential clinical use of plasma NfL across multiple neurological disorders, there is increasing evidence that underlying comorbidities such as renal impairment associated with chronic kidney disease (CKD) and cardiovascular diseases can increase NfL concentrations. The objective of this study was to determine the relationship between plasma NfL concentrations and renal function (CKD staging) in individuals without known neurological conditions.MethodsIndividuals (n=62) seen in an outpatient setting at Mayo Clinic who had various stages of renal impairment (stages 1‐5 by CKD‐EPI 2021 estimated glomerular filtration rate (eGFR) equation) and did not have co‐existing conditions that are known to elevate plasma NfL were retrospectively identified through electronic medical record review. EDTA plasma from these patients collected within +/‐ 30 days of the eGFR determination was identified and stored frozen (‐80°C) until the time of testing. Samples were tested using the Simoa® NF‐light™ V2 Advantage Kit (Quanterix). NfL concentrations in pg/mL were used to calculate age‐adjusted Z‐scores (https://shiny.dkfbasel.ch/baselnflreference/) using an online calculator. Correlation analysis was performed to assess associations of continuous eGFR (mL/min/ 1.73 m2) with plasma NfL age‐adjusted Z‐scores.ResultsA strong negative correlation between NfL Z‐score and eGFR was observed (Spearman’s ρ= ‐0.806). Mean NfL Z‐scores (ranges) in each CKD stage were ‐0.4(‐2.3‐1.4) for stage 1 (N=9), 0.9(‐0.8‐1.9) for stage 2 (N=10), 1.9(‐1.2‐3.4) for stage 3 (N=29), 2.9(2.7‐3.5) for stage 4 (N=6), and 3.2(1.6‐3.7) for stage 5 (N=8); with all groups showing significant differences (P>0.05) except between groups 4 and 5 (P=0.09).ConclusionsPlasma NfL concentrations were increased in individuals with a decreased eGFR. Kidney impairment is an important potential confounding factor to consider when utilizing NfL as a biomarker of neurodegeneration in the clinical setting.

Abstract Background Vasoactive intestinal peptide (VIP), a potent vasodilator, is produced by neuronal cells located in the central nervous system, the digestive, respiratory, and urogenital tracts, and the exocrine, thyroid, and adrenal glands. Increased circulating VIP is clinically useful to detect VIP-producing tumors in patients with chronic diarrheal diseases. Objective Develop a sensitive replacement radioimmunoassay (RIA) for VIP quantitation in human plasma due to the diminished supply of the antibody used in the previous in-house method. Method Patient VIP in EDTA plasma competes with labeled (125I) VIP for a limited number of primary rabbit polyclonal antibody (BMA Biomedicals, Augst, Switzerland) binding sites. Antibody-bound VIP is then complexed with a goat anti-rabbit secondary antibody and precipitated out of the solution with the use of polyethylene glycol and centrifugation. After the supernatant is discarded, the 125I signal from the pellet is counted on the Wizard2 gamma counter (Perkin Elmer, Waltham, MA). The measured signal is inversely proportional to the amount of VIP present in the patient’s sample. Method validation of the new assay included determination of imprecision (pooled human EDTA plasma), limits of detection and quantification, analytical measurement range (AMR), accuracy by spike recovery, interferences, cross-reactivity, stability, reference interval determination, and agreement with the current Mayo laboratory-developed assay. Results Stability studies on freshly collected EDTA plasma showed VIP is stable for 3 days ambient, 7 days refrigerated, and 14 days frozen. Intra-assay imprecision studies showed 8.3, 2.3, and 1.3%CV for mean VIP concentrations of 41, 144, and 304 pg/mL. Inter-assay imprecision was 12.9, 7.6, 5.2, and 7.5%CV for mean VIP concentrations of 30, 81, 180, and 308 pg/mL. The assay limit of detection was 29 pg/mL. The limit of quantitation was 30 pg/mL (%CV = 13%) and was established via precision profile. The AMR was 30–500 pg/mL (slope of 0.98, intercept of 3.0 pg/mL, and R2 of 0.97). The mean % spike recovery using a different preparation lot of calibrator material was 89% (range 83%–93%). The assay was not affected by up to 1000 mg/dL hemoglobin, 1000 mg/dL triglycerides, or 10 mg/dL bilirubin. Peptide histidine methionine 27, neurotensin, gastric inhibitory peptide, motilin, secretin, and glucagon do not cross-react in the assay. The 97.5th reference limit was &lt;86 pg/mL (95% CI 81–90, n = 127). Positive percent agreement (PPA), negative percent agreement (NPA), and overall percent agreement (OPA) with the current Mayo VIP assay were 97, 82, and 90% respectively. Conclusion We have developed a replacement VIP RIA. This assay has a lower limit of quantitation and a broader AMR than the current assay. Concordance with the current assay was acceptable.