Analyzer-to-Analyzer Variations in Assaying Ultralow Concentrated Biomarkers Associated with Neurodegenerative Diseases Using Immunomagnetic Reduction.

By utilizing a high-temperature superconducting quantum interference device (high-Tc SQUID) magnetometer, an alternating current (AC) magnetosusceptometer, referred to as an analyzer, was developed for ultrasensitive immunoassays. The analyzer has been applied to assay biomarkers in human plasma associated with Alzheimer's disease (AD) and Parkinson's disease (PD). The involved assay methodology is the so-called immunomagnetic reduction (IMR). Such an analyzer has been approved for clinical use in Taiwan and Europe. The mass production of the analyzer is needed for clinical utilities. The issue of exploring analyzer-to-analyzer variations in the performances becomes critical. Unfortunately, there is no standard characterization to determine the variations in performances among analyzers. In this study, key characterizations, such as output signal stability, signal-to-noise ratio, measured concentrations of a control sample, etc., are proposed. In total, three analyzers are characterized in this work. The detected biomarkers include amyloid peptides, total tau protein, phosphorylated tau protein, and α-synuclein protein for AD and PD. Through one-way ANOVA for any of the characterizations among the three analyzers, it was found that there was no significant difference in any of these characterizations among the analyzers (p > 0.05). Furthermore, the three analyzers are applied to assay biomolecules for AD and PD in reference samples. High correlations (r > 0.8) in measured concentrations of any of these biomarkers in reference samples were obtained among the three analyzers. The results demonstrate that the proposed characterizations are feasible for achieving consistent performance among high-Tc SQUID-based AC magnetosusceptometers for assaying biomolecules.

Plasma Neurofilament Light Chains as Blood-Based Biomarkers for Early Diagnosis of Canine Cognitive Dysfunction Syndrome.

The number of elderly dogs is increasing significantly worldwide, and many elderly dogs develop canine cognitive dysfunction syndrome (CCDS). CCDS is the canine analog of Alzheimer's disease (AD) in humans. It is very important to develop techniques for detecting CDDS in dogs. Thus, we used the detection of neurofilament light chains (NfL) in plasma as a blood-based biomarker for the early diagnosis of canine Alzheimer's disease using immunomagnetic reduction (IMR) technology by immobilizing NfL antibodies on magnetic nanoparticles. According to the 50-point CCDS rating scale, we divided 36 dogs into 15 with CCDS and 21 without the disease. The results of our IMR assay showed that the plasma NfL levels of dogs with CCDS were significantly increased compared to normal dogs (p < 0.01). By plasma biochemical analysis, we further confirmed that the liver and renal dysfunction biomarkers of dogs with CCDS were significantly elevated compared to normal dogs (p < 0.01-0.05). On the basis of our preliminary study, we propose that IMR technology could be an ideal biosensor for detecting plasma NfL for the early diagnosis of CCDS.

Relevance of plasma biomarkers to pathologies in Alzheimer's disease, Parkinson's disease and frontotemporal dementia.

Amyloid plaques and tau tangles are pathological hallmarks of Alzheimer's disease (AD). Parkinson's disease (PD) results from the accumulation of α-synuclein. TAR DNA-binding protein (TDP-43) and total tau protein (T-Tau) play roles in FTD pathology. All of the pathological evidence was found in the biopsy. However, it is impossible to perform stein examinations in clinical practice. Assays of biomarkers in plasma would be convenient. It would be better to investigate the combinations of various biomarkers in AD, PD and FTD. Ninety-one subjects without neurodegenerative diseases, 76 patients with amnesic mild cognitive impairment (aMCI) or AD dementia, combined as AD family, were enrolled. One hundred and nine PD patients with normal cognition (PD-NC) or dementia (PDD), combined as PD family, were enrolled. Twenty-five FTD patients were enrolled for assays of plasma amyloid ß 1-40 (Aß1-40), Aß1-42, T-Tau, α-synuclein and TDP-43 using immunomagnetic reduction (IMR). The results show that Aßs and T-Tau are major domains in AD family. α-synuclein is highly dominant in PD family. FTD is closely associated with TDP-43 and T-Tau. The dominant plasma biomarkers in AD family, PD family and FTD are consistent with pathology. This implies that plasma biomarkers are promising for precise and differential assessments of AD, PD and FTD in clinical practice.

Applications of Immunomagnetic Reduction Technology as a Biosensor in Therapeutic Evaluation of Chinese Herbal Medicine in Tauopathy Alleviation of an AD Drosophila Model.

Alzheimer's disease (AD) is the most common form of dementia. The most convincing biomarkers in the blood for AD are currently ß-amyloid (Aß) and Tau protein because amyloid plaques and neurofibrillary tangles are pathological hallmarks in the brains of patients with AD. The development of assay technologies in diagnosing early-stage AD is very important. The study of human AD subjects is hindered by ethical and technical limitations. Thus, many studies have therefore turned to AD animal models, such as Drosophila melanogaster, to explore AD pathology. However, AD biomarkers such as Aß and p-Tau protein in Drosophilamelanogaster occur at extremely low levels and are difficult to detect precisely. In this study, we applied the immunomagnetic reduction (IMR) technology of nanoparticles for the detection of p-Tau expressions in hTauR406W flies, an AD Drosophila model. Furthermore, we used IMR technology as a biosensor in the therapeutic evaluation of Chinese herbal medicines in hTauR406W flies with Tau-induced toxicity. To uncover the pathogenic pathway and identify therapeutic interventions of Chinese herbal medicines in Tau-induced toxicity, we modeled tauopathy in the notum of hTauR406W flies. Our IMR data showed that the selected Chinese herbal medicines can significantly reduce p-Tau expressions in hTauR406W flies. Using evidence of notal bristle quantification and Western blotting analysis, we confirmed the validity of the IMR data. Thus, we suggest that IMR can serve as a new tool for measuring tauopathy and therapeutic evaluation of Chinese herbal medicine in an AD Drosophila model.

Synuclein Motor Dysfunction Composite Scale for the Discrimination of Dementia With Lewy Bodies From Alzheimer's Disease.

Background: An abnormal increase of α-synuclein in the brain is the hallmark of dementia with Lewy bodies (DLB). However, the diagnostic power of plasma α-synuclein in DLB is not yet confirmed. Parkinsonism is highly associated with and is one of the core clinical features of DLB. We studied plasma α-synuclein and developed a novel tool that combined plasma α-synuclein level and Motor Dysfunction Questionnaire (MDQ), namely Synuclein Motor Dysfunction Composite Scale (SMDCS), for the clinical discrimination of DLB from Alzheimer's disease (AD). Methods: This cross-sectional study analyzed participants' demographical data, plasma α-synuclein level, MDQ, structured clinical history questionnaire, neuropsychological and motor function tests, and neuroimaging studies. The power of plasma α-synuclein level, MDQ, and SMDCS for discriminating DLB from non-demented controls (NC) or AD were compared. Results: Overall, 121 participants diagnosed as 58 DLB, 31 AD, and 31 NC were enrolled. Patients with DLB had significantly higher mean plasma α-synuclein level (0.24 ± 0.32 pg/ml) compared to the NC group (0.08 ± 0.05 pg/ml) and the AD group (0.08 ± 0.05 pg/ml). The DLB group demonstrated higher MDQ (2.95 ± 1.60) compared to the NC (0.42 ± 0.98) or AD (0.44 ± 0.99) groups. The sensitivity/specificity of plasma α-synuclein level, MDQ, and SMDCS for differentiating DLB from non-DLB were 0.80/0.64, 0.83/0.89, and 0.88/0.93, respectively. Conclusion: Both plasma α-synuclein and MDQ were significantly higher in patients with DLB compared to the NC or AD groups. The novel SMDCS, significantly improved accuracy for the clinical differentiation of DLB from AD or NC.

Effect of Time to Detection on the Measured Concentrations of Blood Proteins Associated with Alzheimer's Disease.

Background: For assays using immunomagnetic reduction, a reagent composed of antibody-functionalized magnetic nanoparticles is dispersed in phosphate-buffered saline solution. The real-time signals of alternating-current (ac) magnetic susceptibility, χac, of the reagent are subsequently recorded after mixing the reagent with a biofluid sample. After mixing the reagent and sample, the reduction in χac of the mixture is calculated and used to quantify the concentration of the target biomarker in the sample. The reduction does not occur immediately but rather occurs at some time after mixing. This observation implies that the time elapsed before recording the real-time signals of χac of a reagent-sample mixture needs to be investigated to ensure that the signals are fully recorded. In this work, the effect of time to detection on the measured concentrations of proteins in human plasma after mixing the reagent and sample is examined. Methods: The proteins analyzed are related to Alzheimer's disease: amyloid ß 1-40, amyloid ß 1-42, and Tau protein. The investigated times to detection after the mixing the reagent and sample are 0, 20, 30, 40, and 120 min. Results: The results show that the recording of real-time signals of χac should be conducted within 20 min after mixing the reagent and sample.

Neurotherapy of Yi-Gan-San, a Traditional Herbal Medicine, in an Alzheimer's Disease Model of Drosophila melanogaster by Alleviating Aß42 Expression.

Alzheimer's disease (AD), a main cause of dementia, is the most common neurodegenerative disease that is related to the abnormal accumulation of amyloid ß (Aß) proteins. Yi-Gan-San (YGS), a traditional herbal medicine, has been used for the management of neurodegenerative disorders and for the treatment of neurosis, insomnia and dementia. The aim of this study was to examine antioxidant capacity and cytotoxicity of YGS treatment by using 2,2-Diphenyl-1-picrylhydrazyl (DPPH) and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assays in vitro. We explored neuroprotective effects of YGS treatment in alleviating Aß neurotoxicity of Drosophila melanogaster in vivo by comparing survival rate, climbing index, and Aß expressions through retinal green fluorescent protein (GFP) expression, highly sensitive immunomagnetic reduction (IMR) and Western blotting assays. In the in vitro study, our results showed that scavenging activities of free radical and SH-SY5Y nerve cell viability were increased significantly (p < 0.01-0.05). In the in vivo study, Aß42-expressing flies (Aß42-GFP flies) and their WT flies (mCD8-GFP flies) were used as an animal model to examine the neurotherapeutic effects of YGS treatment. Our results showed that, in comparison with those Aß42 flies under sham treatments, Aß42 flies under YGS treatments showed a greater survival rate, better climbing speed, and lower Aß42 aggregation in Drosophila brain tissue (p < 0.01). Our findings suggest that YGS should have a beneficial alternative therapy for AD and dementia via alleviating Aß neurotoxicity in the brain tissue.

Evidence of plasma biomarkers indicating high risk of dementia in cognitively normal subjects.

Subjects with comorbidities are at risk for neurodegeneration. There is a lack of a direct relationship between comorbidities and neurodegeneration. In this study, immunomagnetic reduction (IMR) assays were utilized to assay plasma Aß1-42 and total tau protein (T-Tau) levels in poststroke (PS, n = 27), family history of Alzheimer's disease (ADFH, n = 35), diabetes (n = 21), end-stage renal disease (ESRD, n = 41), obstructive sleep apnea (OSA, n = 20), Alzheimer's disease (AD, n = 65). Thirty-seven healthy controls (HCs) were enrolled. The measured concentrations of plasma Aß1-42 were 14.26 ± 1.42, 15.43 ± 1.76, 15.52 ± 1.60, 16.15 ± 1.05, 16.52 ± 0.59, 15.97 ± 0.54 and 20.06 ± 3.09 pg/mL in HC, PS, ADFH, diabetes, ESRD, OSA and AD groups, respectively. The corresponding concentrations of plasma T-Tau were 15.13 ± 3.62, 19.29 ± 8.01, 17.93 ± 6.26, 19.74 ± 2.92, 21.54 ± 2.72, 20.17 ± 2.77 and 41.24 ± 14.64 pg/mL. The plasma levels of Aß1-42 and T-Tau in were significantly higher in the PS, ADFH, diabetes, ESRD and OSA groups than controls (Aß1-42 in PS: 15.43 ± 1.76 pg/mL vs. 14.26 ± 1.42 pg/mL, p < 0.005; T-Tau in PS: 19.29 ± 8.01 vs. 15.13 ± 3.62 pg/mL, p < 0.005, Aß1-42 in ADFH: 15.52 ± 1.60 pg/mL vs. 14.26 ± 1.42 pg/mL, p < 0.001; T-Tau in ADFH: 17.93 ± 6.26 vs. 15.13 ± 3.62 pg/mL, p < 0.005, Aß1-42 in diabetes: 16.15 ± 1.05 pg/mL vs. 14.26 ± 1.42 pg/mL, p < 0.001; T-Tau in diabetes: 19.74 ± 2.92 vs. 15.13 ± 3.62 pg/mL, p < 0.001, Aß1-42 in ESRD: 16.52 ± 0.59 pg/mL vs. 14.26 ± 1.42 pg/mL, p < 0.001; T-Tau in ESRD: 21.54 ± 2.72 vs. 15.13 ± 3.62 pg/mL, p < 0.001, Aß1-42 in OSA: 15.97 ± 0.54 pg/mL vs. 14.26 ± 1.42 pg/mL, p < 0.001; T-Tau in OSA: 20.17 ± 2.77 vs. 15.13 ± 3.62 pg/mL, p < 0.001). This evidence indicates the high risk for dementia in these groups from the perspective of plasma biomarkers.

Development of a 36-Channel Instrument for Assaying Biomarkers of Ultralow Concentrations Utilizing Immunomagnetic Reduction.

With the demands of the high-throughput assay of biomarkers of ultralow concentrations in clinics, a 36-channel instrument utilizing immunomagnetic reduction (IMR) has been developed. The instrument involves the use of a high-T c superconducting-quantum-interference-device (SQUID) magnetometer to detect the signals due to the associations between target biomarker molecules and the antibody-functionalized magnetic nanoparticles in the reagent of IMR. In addition to illustrating the design and the measurements of the instrument, the assay characterizations for eight kinds of biomarkers related to neurodegenerative disease are investigated. Furthermore, the assay results among three independent instruments were compared. For an instrument, the channel-to-channel variations in measured concentrations of biomarkers are within a range of 2.09 to 5.62%. The assay accuracy was found to be from 99 to 103.7%. The p values in measured concentrations for any of the tested biomarkers were higher than 0.05 among the three instruments. The results demonstrate high throughput, high stability, and high consistency for the SQUID-IMR instruments.

Assessment of High Risk for Alzheimer's Disease Using Plasma Biomarkers in Subjects with Normal Cognition in Taiwan: A Preliminary Study.

BACKGROUND: In Alzheimer's disease (AD), cognitive impairment begins 10-15 years later than neurodegeneration in the brain. Plasma biomarkers are promising candidates for assessing neurodegeneration in people with normal cognition. It has been reported that subjects with the concentration of plasma amyloid-ß 1-42×total tau protein higher than 455 pg2/ml2 are assessed as having a high risk of amnesic mild impairment or AD, denoted as high risk of AD (HRAD). OBJECTIVE: The prevalence of high-risk for dementia in cognitively normal controls is explored by assaying plasma biomarkers. METHODS: 422 subjects with normal cognition were enrolled around Taiwan. Plasma Aß1-40, Aß1-42, and T-Tau levels were assayed using immunomagnetic reduction to assess the risk of dementia. RESULTS: The results showed that 4.6% of young adults (age: 20-44 years), 8.5% of middle-aged adults (age: 45-64 years), and 7.3% of elderly adults (age: 65-90 years) had HRAD. The percentage of individuals with HRAD dramatically increased in middle-aged and elderly adults compared to young adults. CONCLUSION: The percentage of HRAD in cognitively normal subjects are approximately 10%, which reveals that the potentially public-health problem of AD in normal population. Although the subject having abnormal levels of Aß or tau is not definitely going on to develop cognitive declines or AD, the risk of suffering cognitive impairment in future is relatively high. Suitable managements are suggested for these high-risk cognitively normal population. Worth noting, attention should be paid to preventing cognitive impairment due to AD, not only in elderly adults but also middle-aged adults.

Assessing Plasma Levels of α-Synuclein and Neurofilament Light Chain by Different Blood Preparation Methods.

The potential biomarkers of Parkinson's disease are α-synuclein and neurofilament light chain (NFL). However, inconsistent preanalytical preparation of plasma could lead to variations in levels of these biomarkers. Different types of potassium salts of EDTA and different centrifugation temperatures during plasma preparation may affect the results of α-synuclein and NFL measurements. In this study, we prepared plasma from eight patients with Parkinson's disease (PD) and seven healthy controls (HCs) by using di- and tri-potassium (K2- and K3-) EDTA tubes and recruited a separated cohort with 42 PD patients and 40 HCs for plasma samples prepared from whole blood by centrifugation at room temperature and 4°C, respectively, in K2-EDTA tubes. The plasma levels of α-synuclein and NFL in K2- and K3-EDTA were similar. However, the levels of α-synuclein in the plasma prepared at 4°C (101.57 ± 43.43 fg/ml) were significantly lower compared with those at room temperature (181.23 ± 196.31 fg/ml, P < 0.001). Room temperature preparation demonstrated elevated plasma levels of α-synuclein in PD patients (256.6 ± 50.2 fg/ml) compared with the HCs (102.1 ± 0.66 fg/ml, P < 0.001), whereas this increase in PD was not present by preparation at 4°C. Both plasma preparations at room temperature and 4°C demonstrated consistent results of NFL, which are increased in PD patients compared with HCs. Our findings confirmed that K2- and K3-EDTA tubes were interchangeable for analyzing plasma levels of α-synuclein and NFL. Centrifugation at 4°C during plasma preparation generates considerable reduction and variation of α-synuclein level that might hinder the detection of α-synuclein level changes in PD.

Neurodegeneration and Vascular Burden on Cognition After Midlife: A Plasma and Neuroimaging Biomarker Study.

Background and Objectives: Neurodegeneration and vascular burden are the two most common causes of post-stroke cognitive impairment. However, the interrelationship between the plasma beta-amyloid (Aß) and tau protein, cortical atrophy and brain amyloid accumulation on PET imaging in stroke patients is undetermined. We aimed to explore: (1) the relationships of cortical thickness and amyloid burden on PET with plasma Aß40, Aß42, tau protein and their composite scores in stroke patients; and (2) the associations of post-stroke cognitive presentations with these plasma and neuroimaging biomarkers. Methods: The prospective project recruited first-ever ischemic stroke patients around 3 months after stroke onset. The plasma Aß40, Aß42, and total tau protein were measured with the immunomagnetic reduction method. Cortical thickness was evaluated on MRI, and cortical amyloid plaque deposition was evaluated by 18F-florbetapir PET. Cognition was evaluated with Mini-Mental State Examination (MMSE), Geriatric Depression Scale (GDS), Dementia Rating Scale-2 (DRS-2). Results: The study recruited 24 stroke patients and 13 normal controls. The plasma tau and tau*Aß42 levels were correlated with mean cortical thickness after age adjustment. The Aß42/Aß40 ratio was correlated with global cortical 18F-florbetapir uptake value. The DRS-2 and GDS scores were associated with mean cortical thickness and plasma biomarkers, including Aß42/Aß40, tau, tau*Aß42, tau/Aß42, and tau/Aß40 levels, in stroke patients. Conclusion: Plasma Aß, tau, and their composite scores were associated with cognitive performance 3 months after stroke, and these plasma biomarkers were correlated with corresponding imaging biomarkers of neurodegeneration. Further longitudinal studies with a larger sample size are warranted to replicate the study results.

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.

Investigation of the Number of Tests Required for Assaying Plasma Biomarkers Associated with Alzheimer's Disease Using Immunomagnetic Reduction.

INTRODUCTION: Concentrations of plasma biomarkers associated with Alzheimer's disease have been reported to be as low as several tens of picograms/milliliter (pg/ml). However, in assays measuring these biomarkers, it is likely that repeated measurements are necessary to obtain reliable values. METHODS: We performed assays as a single test or as duplicate, quadruplicate, fivefold and tenfold repeated tests, on samples spiked with different concentrations of amyloid ß 1-40 (Aß1-40; 1-1000 pg/ml), Aß1-42 (1-30,000 pg/ml) and total Tau protein (T-Tau; 0.1-1000 pg/ml), with the aim to to calculate the coefficients of variation (CVs). RESULTS: The results demonstrated common changes in the CVs with changes in the number of tests for a given sample: the CVs decreased with increases in the number of tests from one to ten. All CV values were distributed within the range of 0.35 to 15.5%; as such, the CV values were all lower than the acceptable value of 20%. CONCLUSION: Based on this study, a single assay of Aß1-40, Aß1-42 and T-Tau, respectively, provides reliable results in terms of the measurement of that plasma biomarker.

Synergistic Association between Plasma Aß1-42 and p-tau in Alzheimer's Disease but Not in Parkinson's Disease or Frontotemporal Dementia.

Beta-amyloid (Aß1-42) triggers the phosphorylation of tau protein in Alzheimer's disease (AD), but the relationship between phosphorylated tau (p-tau) and Aß1-42 in the blood is not elucidated. We investigated the association in individuals with AD (n = 62, including amnesic mild cognitive impairment and dementia), Parkinson's disease (n = 30), frontotemporal dementia (n = 25), and cognitively unimpaired controls (n = 41) using immunomagnetic reduction assays to measure plasma Aß1-42 and p-tau181 concentrations. Correlation and regression analyses were performed to examine the relation between plasma levels, demographic factors, and clinical severity. Both plasma Aß1-42 and p-tau concentrations were significantly higher in AD and frontotemporal dementia than in the controls and Parkinson's disease. A significant positive association was found between plasma p-tau and Aß1-42 in controls (r = 0.579, P < 0.001) and AD (r = 0.699, P < 0.001) but not in frontotemporal dementia or Parkinson's disease. Plasma p-tau was significantly associated with clinical severity in the AD in terms of scores of clinical dementia rating (r = 0.288, P = 0.025) and mini-mental state examination (r = -0.253, P = 0.049). Regression analysis showed that plasma Aß1-42 levels explain approximately 47.7% of the plasma p-tau levels in the AD after controlling age, gender, and clinical severity. While in non-AD participants, the clinical dementia rating explained about 47.5% of the plasma p-tau levels. The disease-specific association between plasma Aß1-42 and p-tau levels in AD implies a possible synergic effect in mechanisms involving these two pathological proteins' genesis.

Plasma Total α-Synuclein and Neurofilament Light Chain: Clinical Validation for Discriminating Parkinson's Disease from Normal Control.

BACKGROUND: A previously published paper (referred to as the original cohort) showed that using a cutoff value of 116.1 fg/mL for the plasma total α-synuclein concentrations could discriminate Parkinson's disease (PD) patients from normal controls (NCs). In this study, another independent cohort (referred to as the validation cohort) was recruited to validate the agreement between the clinical diagnosis and the use of plasma total α-synuclein to identify PD patients. In addition to total α-synuclein, plasma neurofilament light chain (NfL) in the validation cohort was detected. METHODS: Seventy PD patients and 33 NCs were enrolled in the validation cohort. A clinical diagnosis and the immunomagnetic reduction (IMR) assay for plasma total α-synuclein were performed for each participant. Thirty-three of 70 PD patients and 23 of 33 NCs were subjected to the plasma NfL assay via IMR. RESULTS: The positive, negative, and overall percentages of agreement between the clinical diagnosis and plasma total α-synuclein diagnosis determined based on 116.1 fg/mL as the cutoff value were found to be 0.943, 0.818, and 0.903, respectively. The PD patients and NCs showed plasma NfL levels of 8.38 ± 4.19 pg/mL and 17.6 ± 7.95 pg/mL (p < 0.001), respectively. The cutoff value of the plasma NfL level used to differentiate PD patients from NCs was 12.8 pg/mL, with sensitivity and specificity values of 0.788 and 0.870, respectively. CONCLUSION: The results demonstrate the usefulness of the plasma total α-synuclein concentration to discriminate PD patients from NCs and reveal the elevation of the plasma NfL level in PD patients.

Immunomagnetic Reduction Detects Plasma Aß1-42 Levels as a Potential Dominant Indicator Predicting Cognitive Decline.

Although the concentrations of Alzheimer's disease (AD) biomarkers Aß1-40, Aß1-42 and tau protein are very low in human plasma, ultrasensitive assays such as immunomagnetic reduction (IMR) are able to precisely quantify them. Review articles have described the detailed working mechanism of IMR and revealed the feasibility of detecting early-stage AD by assaying these plasma biomarkers with IMR. In this review, we aimed to compare the significance of these plasma biomarkers in predicting cognitive decline in patients with Down syndrome, stroke, or amnestic mild cognitive impairment based on findings in the literature. We found that plasma Aß1-42 might play the predominant role in predicting cognitive decline in these patients.

Effect of Times to Blood Processing on the Stability of Blood Proteins Associated with Dementia.

BACKGROUND: The stability of proteins in the collecting tubes after blood draw is critical to the measured concentrations of the proteins. Although the guidelines issued by the Clinical and Laboratory Standards Institute (CLSI) suggest centrifugation should take place within 2 h of drawing blood, it is very difficult to follow these guidelines in hospitals or clinics. It is necessary to study the effect of times to blood processing on the stability of the proteins of interest. METHODS: In this work, the plasma proteins of interest were those relevant to dementia, such as amyloid ß 1-40 (Aß1-40), Aß1-42, Tau protein (Tau), and α-synuclein. The times to blood processing after blood draw ranged from 0.5 to 8 h. The storage temperatures of blood were room temperature (approx. 25°C) and 30°C. After storage, blood samples were centrifuged at room temperature to obtain plasma samples. Ultrasensitive immunomagnetic reduction was applied to assay these proteins in the plasma. RESULTS: The levels of plasma Aß1-40, Tau, and α-synuclein did not significantly change until 8 h after blood draw when stored at room temperature. Plasma Aß1-42 levels did not change significantly after 8 h of storage at room temperature before blood processing. Higher storage temperatures, such as 30°C, for blood samples accelerated the significant variations in the measured concentrations of Aß1-40, Tau, and α-synuclein in plasma. CONCLUSION: According to these results, for clinical practice, it is suggested that blood samples be stored at room temperature for no longer than 4.5 h after blood draw until centrifugation for the assay of dementia biomarkers in plasma.

Development of an assay of plasma neurofilament light chain utilizing immunomagnetic reduction technology.

Axonal damage leads to the release of neurofilament light chain (NFL), which enters the CSF or blood. In this work, an assay kit for plasma NFL utilizing immunomagnetic reduction (IMR) was developed. Antibodies against NFL were immobilized on magnetic nanoparticles to develop an IMR NFL kit. The preclinical properties, such as the standard curve, limit of detection (LoD), and dynamic range, were characterized. Thirty-one normal controls (NC), fifty-two patients with Parkinson's disease (PD) or PD dementia (PDD) and thirty-one patients with Alzheimer's disease (AD) were enrolled in the study evaluating the plasma NFL assay using an IMR kit. T-tests and receiver operating characteristic (ROC) curve analysis were performed to investigate the capability for discrimination among the clinical groups according to plasma NFL levels. The LoD of the NFL assay using the IMR kit was found to be 0.18 fg/ml. The dynamic range of the NFL assay reached 1000 pg/ml. The NC group showed a plasma NFL level of 7.70 ± 4.00 pg/ml, which is significantly lower than that of the PD/PDD (15.85 ± 7.82 pg/ml, p < 0.001) and AD (19.24 ± 8.99 pg/ml, p < 0.001) groups. A significant difference in plasma NFL levels was determined between the PD and AD groups (p < 0.01). Through ROC curve analysis, the cut-off value of the plasma NFL concentration for differentiating NCs from dementia patients (AD and PD/PDD) was found to be 12.71 pg/ml, with a clinical sensitivity and specificity of 73.5% and 90.3%, respectively. The AUC was 0.868. Furthermore, the cut-off value of the plasma NFL concentration for discriminating AD from PD/PDD was found to be 18.02 pg/ml, with a clinical sensitivity and specificity of 61.3% and 65.4%, respectively. The AUC was 0.630. An ultrasensitive assay for measuring plasma NFL utilizing IMR technology was developed. Clear differences in plasma NFL concentrations were observed among NCs and PD and AD patients. These results imply that the determination of plasma NFL is promising not only for screening dementia but also for differential diagnosis.

Plasma pyroglutamate-modified amyloid beta differentiates amyloid pathology.

INTRODUCTION: Pyroglutamate-modified amyloid ß (AßpE3) could be a biomarker for Aß plaque pathology in the brain. An ultra-high-sensitive assay is needed for detecting AßpE3-40. METHODS: Immunomagnetic reduction was used for quantification of AßpE3-40 in plasma from 46 participants. The concentrations of AßpE3-40 of these subjects were compared with 18F-florbetapir positron emission tomography (PET) images. RESULTS: AßpE3-40 concentration was 44.1 ± 28.2 fg/mL in PET- (n = 28) and 91.6 ± 54.6 fg/mL in PET+ (n = 18; P < .05). The cutoff value of AßpE3-40 for discriminating PET- from PET+ was 55.5 fg/mL, resulting in a sensitivity of 83.3%, a specificity of 71.4%. The concentration of AßpE3-40 showed a moderate correlation (r = 0.437) with PET standardized uptake value ratio. DISCUSSION: We did not enroll pre-clinical AD subject with normal cognition but Aß PET+. It would be an important issue to explore the feasibility of using AßpE3-40 for screening pre-clinical subjects. CONCLUSION: These results reveal the feasibility of detecting Aß pathology using quantification of a plaque-derived Aß molecule in plasma.