Alzheimer Disease Biomarkers: Moving from CSF to Plasma for Reliable Detection of Amyloid and tau Pathology.

BACKGROUND: Development of validated biomarkers to detect early Alzheimer disease (AD) neuropathology is needed for therapeutic AD trials. Abnormal concentrations of "core" AD biomarkers, cerebrospinal fluid (CSF) amyloid beta1-42, total tau, and phosphorylated tau correlate well with neuroimaging biomarkers and autopsy findings. Nevertheless, given the limitations of established CSF and neuroimaging biomarkers, accelerated development of blood-based AD biomarkers is underway. CONTENT: Here we describe the clinical significance of CSF and plasma AD biomarkers to detect disease pathology throughout the Alzheimer continuum and correlate with imaging biomarkers. Use of the AT(N) classification by CSF and imaging biomarkers provides a more objective biologically based diagnosis of AD than clinical diagnosis alone. Significant progress in measuring CSF AD biomarkers using extensively validated highly automated assay systems has facilitated their transition from research use only to approved in vitro diagnostics tests for clinical use. We summarize development of plasma AD biomarkers as screening tools for enrollment and monitoring participants in therapeutic trials and ultimately in clinical care. Finally, we discuss the challenges for AD biomarkers use in clinical trials and precision medicine, emphasizing the possible ethnocultural differences in the levels of AD biomarkers. SUMMARY: CSF AD biomarker measurements using fully automated analytical platforms is possible. Building on this experience, validated blood-based biomarker tests are being implemented on highly automated immunoassay and mass spectrometry platforms. The progress made developing analytically and clinically validated plasma AD biomarkers within the AT(N) classification scheme can accelerate use of AD biomarkers in therapeutic trials and routine clinical practice.

Validation of Plasma Amyloid-ß 42/40 for Detecting Alzheimer Disease Amyloid Plaques.

BACKGROUND AND OBJECTIVES: To determine the diagnostic accuracy of a plasma Aß42/Aß40 assay in classifying amyloid PET status across global research studies using samples collected by multiple centers that utilize different blood collection and processing protocols. METHODS: Plasma samples (n = 465) were obtained from 3 large Alzheimer disease (AD) research cohorts in the United States (n = 182), Australia (n = 183), and Sweden (n = 100). Plasma Aß42/Aß40 was measured by a high precision immunoprecipitation mass spectrometry (IPMS) assay and compared to the reference standards of amyloid PET and CSF Aß42/Aß40. RESULTS: In the combined cohort of 465 participants, plasma Aß42/Aß40 had good concordance with amyloid PET status (receiver operating characteristic area under the curve [AUC] 0.84, 95% confidence interval [CI] 0.80-0.87); concordance improved with the inclusion of APOE ε4 carrier status (AUC 0.88, 95% CI 0.85-0.91). The AUC of plasma Aß42/Aß40 with CSF amyloid status was 0.85 (95% CI 0.78-0.91) and improved to 0.93 (95% CI 0.89-0.97) with APOE ε4 status. These findings were consistent across the 3 cohorts, despite differences in protocols. The assay performed similarly in both cognitively unimpaired and impaired individuals. DISCUSSION: Plasma Aß42/Aß40 is a robust measure for detecting amyloid plaques and can be utilized to aid in the diagnosis of AD, identify those at risk for future dementia due to AD, and improve the diversity of populations enrolled in AD research and clinical trials. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that plasma Aß42/Aß40, as measured by a high precision IPMS assay, accurately diagnoses brain amyloidosis in both cognitively unimpaired and impaired research participants.

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.

Mass spectrometry-based methods for robust measurement of Alzheimer's disease biomarkers in biological fluids.

Alzheimer's disease (AD) is the most common form of dementia affecting 60%-70% of people afflicted with this disease. Accurate antemortem diagnosis is urgently needed for early detection of AD to enable reliable estimation of prognosis, intervention, and monitoring of the disease. The National Institute on Aging/Alzheimer's Association sponsored the 'Research Framework: towards a biological definition of AD', which recommends using different biomarkers in living persons for a biomarker-based definition of AD regardless of clinical status. Fluid biomarkers represent one of key groups of them. Since cerebrospinal fluid (CSF) is in direct contact with brain and many proteins present in the brain can be detected in CSF, this fluid has been regarded as the best biofluid in which to measure AD biomarkers. Recently, technological advancements in protein detection made possible the effective study of plasma AD biomarkers despite their significantly lower concentrations versus to that in CSF. This and other challenges that face plasma-based biomarker measurements can be overcome by using mass spectrometry. In this review, we discuss AD biomarkers which can be reliably measured in CSF and plasma using targeted mass spectrometry coupled to liquid chromatography (LC/MS/MS). We describe progress in LC/MS/MS methods' development, emphasize the challenges, and summarize major findings. We also highlight the role of mass spectrometry and progress made in the process of global standardization of the measurement of Aß42/Aß40. Finally, we briefly describe exploratory proteomics which seek to identify new biomarkers that can contribute to detection of co-pathological processes that are common in sporadic AD.

Effect of escitalopram dose and treatment duration on CSF Aß levels in healthy older adults: A controlled clinical trial.

OBJECTIVE: To determine whether treatment with escitalopram compared with placebo would lower CSF ß-amyloid 42 (Aß42) levels. RATIONALE: Serotonin signaling suppresses Aß42 in animal models of Alzheimer disease (AD) and young healthy humans. In a prospective study in older adults, we examined dose and treatment duration effects of escitalopram. METHODS: Using lumbar punctures to sample CSF levels before and after a course of escitalopram treatment, cognitively normal older adults (n = 114) were assigned to placebo, 20 mg escitalopram × 2 weeks, 20 mg escitalopram × 8 weeks, or 30 mg escitalopram × 8 weeks; CSF sampled pretreatment and posttreatment and within-subject percent change in Aß42 was used as the primary outcome in subsequent analyses. RESULTS: An overall 9.4% greater reduction in CSF Aß42 was found in escitalopram-treated compared with placebo-treated groups (p < 0.001, 95% confidence interval [CI] 4.9%-14.2%, d = 0.81). Positive baseline Aß status (CSF Aß42 levels <250 pg/mL) was associated with smaller Aß42 reduction (p = 0.006, 95% CI -16.7% to 0.5%, d = -0.52) compared with negative baseline amyloid status (CSF Aß42 levels >250 pg/mL). CONCLUSIONS: Short-term longitudinal doses of escitalopram decreased CSF Aß42 in cognitively normal older adults, the target group for AD prevention. CLINICALTRIALSGOV IDENTIFIER: NCT02161458. CLASSIFICATION OF EVIDENCE: This study provides Class II evidence that for cognitively normal older adults, escitalopram decreases CSF Aß42.

First amyloid ß1-42 certified reference material for re-calibrating commercial immunoassays.

INTRODUCTION: Reference materials based on human cerebrospinal fluid were certified for the mass concentration of amyloid beta (Aß)1-42 (Aß42 ). They are intended to be used to calibrate diagnostic assays for Aß42 . METHODS: The three certified reference materials (CRMs), ERM-DA480/IFCC, ERM-DA481/IFCC and ERM-DA482/IFCC, were prepared at three concentration levels and characterized using isotope dilution mass spectrometry methods. Roche, EUROIMMUN, and Fujirebio used the three CRMs to re-calibrate their immunoassays. RESULTS: The certified Aß42 mass concentrations in ERM-DA480/IFCC, ERM-DA481/IFCC, and ERM-DA482/IFCC are 0.45, 0.72, and 1.22 µg/L, respectively, with expanded uncertainties (k = 2) of 0.07, 0.11, and 0.18 µg/L, respectively. Before re-calibration, a good correlation (Pearson's r > 0.97), yet large biases, were observed between results from different commercial assays. After re-calibration the between-assay bias was reduced to < 5%. DISCUSSION: The Aß42 CRMs can ensure the equivalence of results between methods and across platforms for the measurement of Aß42 .

Analytical and Clinical Performance of Amyloid-Beta Peptides Measurements in CSF of ADNIGO/2 Participants by an LC-MS/MS Reference Method.

BACKGROUND: Cerebrospinal fluid (CSF) amyloid-ß1-42 (Aß42) reliably detects brain amyloidosis based on its high concordance with plaque burden at autopsy and with amyloid positron emission tomography (PET) ligand retention observed in several studies. Low CSF Aß42 concentrations in normal aging and dementia are associated with the presence of fibrillary Aß across brain regions detected by amyloid PET imaging. METHODS: An LC-MS/MS reference method for Aß42, modified by adding Aß40 and Aß38 peptides to calibrators, was used to analyze 1445 CSF samples from ADNIGO/2 participants. Seventy runs were completed using 2 different lots of calibrators. For preparation of Aß42 calibrators and controls spiking solution, reference Aß42 standard with certified concentration was obtained from EC-JRC-IRMM (Belgium). Aß40 and Aß38 standards were purchased from rPeptide. Aß42 calibrators' accuracy was established using CSF-based Aß42 Certified Reference Materials (CRM). RESULTS: CRM-adjusted Aß42 calibrator concentrations were calculated using the regression equation Y (CRM-adjusted) = 0.89X (calibrators) + 32.6. Control samples and CSF pools yielded imprecision ranging from 6.5 to 10.2% (Aß42) and 2.2 to 7.0% (Aß40). None of the CSF pools showed statistically significant differences in Aß42 concentrations across 2 different calibrator lots. Comparison of Aß42 with Aß42/Aß40 showed that the ratio improved concordance with concurrent [18F]-florbetapir PET as a measure of fibrillar Aß (n = 766) from 81 to 88%. CONCLUSIONS: Long-term performance assessment substantiates our modified LC-MS/MS reference method for 3 Aß peptides. The improved diagnostic performance of the CSF ratio Aß42/Aß40 suggests that Aß42 and Aß40 should be measured together and supports the need for an Aß40 CRM.

Detection of Alzheimer Disease Pathology in Patients Using Biochemical Biomarkers: Prospects and Challenges for Use in Clinical Practice.

BACKGROUND: Thirty-four years ago, amyloid-ß 1-42 peptide was identified in amyloid plaques from brain tissue obtained from patients with Alzheimer disease (AD) and Down syndrome. This finding led to development of immunoassays for this marker of amyloid plaque burden in cerebrospinal fluid (CSF) approximately 10 years later. Subsequently, research immunoassays were developed for total τ protein and τ phosphorylated at the threonine 181 position. Subsequent studies documented the clinical utility of these biomarkers of amyloid plaque burden or τ tangle pathology in cohorts of living patients. CONTENT: We describe the following: (a) clinical utility of AD biomarkers; (b) measurement challenges, including development of mass spectrometry-based reference methods and automated immunoassays; (c) development of "appropriate use criteria" (AUC) guidelines for safe/appropriate use of CSF testing for diagnosis of AD developed by neurologists, a neuroethicist, and laboratorians; (d) a framework, sponsored by the National Institute of Aging-Alzheimer's Association (NIA-AA), that defines AD on the basis of CSF and imaging methods for detecting amyloid plaque burden, τ tangle pathology, and neurodegeneration. This framework's purpose was investigative but has important implications for future clinical practice; (e) recognition of copathologies in AD patients and challenges for developing methods to detect these in living patients. SUMMARY: The field can expect availability of validated research tools for detection of AD pathology that support clinical treatment trials of disease-modifying agents and, ultimately, use in clinical practice. Validated methods are becoming available for CSF testing; emergence of validated methods for AD biomarkers in plasma can be expected in the next few years.

Derivation of cutoffs for the Elecsys® amyloid ß (1-42) assay in Alzheimer's disease.

INTRODUCTION: An Elecsys® Amyloid ß (Aß [1-42]) immunoassay cutoff for classification of patients with Alzheimer's disease was investigated. METHODS: Cerebrospinal fluid samples collected from patients with mild-to-moderate Alzheimer's disease were analyzed by Elecsys® immunoassays: (1) Aß (1-42), (2) total tau, and (3) phosphorylated tau. Cutoffs (Aß [1-42] and ratios with tau) were estimated by method comparison between AlzBio3 (n = 206), mixture modeling (n = 216), and concordance with florbetapir F 18 imaging-based classification (n = 75). RESULTS: A 1065-pg/mL (95% confidence interval: 985-1153) Elecsys® Aß (1-42) cutoff provided 94% overall percentage agreement with AlzBio3. Comparable cutoff estimates (95% confidence interval) were derived from mixture modeling (equally weighted: 1017 [949-1205] pg/mL; prevalence weighted: 1172 [1081-1344] pg/mL) and concordance with florbetapir F 18 imaging (visual read: 1198 [998-1591] pg/mL; automated: 1198 [1051-1638] pg/mL). DISCUSSION: Based on three approaches, a 1100-pg/mL Elecsys® Aß (1-42) cutoff is suitable for clinical trials with similar populations and preanalytical handling.

CSF Aß1-42 - an excellent but complicated Alzheimer's biomarker - a route to standardisation.

The 42 amino acid form of amyloid ß (Aß1-42) in cerebrospinal fluid (CSF) has been widely accepted as a central biomarker for Alzheimer's disease. Several immunoassays for CSF Aß1-42 are commercially available, but can suffer from between laboratory and batch-to-batch variability as well as lack of standardisation across assays. As a consequence, no general cut-off values have been established for a specific context of use (e.g., clinical diagnostics) and selection of individuals for enrolment in clinical trials (patient stratification) remains challenging. The International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) has initiated a working group for CSF proteins (WG-CSF) to facilitate standardisation of CSF Aß1-42 measurement results. The efforts of the IFCC WG-CSF include the development of certified reference materials (CRMs) and reference measurement procedures (RMPs) for key biomarkers. Two candidate RMPs for quantification of Aß1-42 in CSF based on liquid chromatography tandem mass spectrometry have been developed and tested in two ring trials. Furthermore, two commutability studies including native CSF pools, artificial CSF and spiked materials have been completed. On the basis of these studies, human CSF pools containing only endogenous Aß1-42 at three concentrations were selected as the format for future CRMs that are now being processed.

Active Cigarette Smoking in Cognitively-Normal Elders and Probable Alzheimer's Disease is Associated with Elevated Cerebrospinal Fluid Oxidative Stress Biomarkers.

Neurodegenerative diseases and chronic cigarette smoking are associated with increased cerebral oxidative stress (OxS). Elevated F2-isoprostane levels in biological fluid is a recognized marker of OxS. This study assessed the association of active cigarette smoking with F2-isoprostane in concentrations in cognitively-normal elders (CN), and those with mild cognitive impairment (MCI) and probable Alzheimer's disease (AD). Smoking and non-smoking CN (n = 83), MCI (n = 164), and probable AD (n = 101) were compared on cerebrospinal fluid (CSF) iPF2α-III and 8,12, iso-iPF2α-VI F2-isoprostane concentrations. Associations between F2-isoprostane levels and hippocampal volumes were also evaluated. In CN and AD, smokers had higher iPF2α-III concentration; overall, smoking AD showed the highest iPF2α-III concentration across groups. Smoking and non-smoking MCI did not differ on iPF2α-III concentration. No group differences were apparent on 8,12, iso-iPF2α-VI concentration, but across AD, higher 8,12, iso-iPF2α-VI level was related to smaller left and total hippocampal volumes. Results indicate that active cigarette smoking in CN and probable AD is associated with increased central nervous system OxS. Further investigation of factors mediating/moderating the absence of smoking effects on CSF F2-isoprostane levels in MCI is warranted. In AD, increasing magnitude of OxS appeared to be related to smaller hippocampal volume. This study contributes additional novel information to the mounting body of evidence that cigarette smoking is associated with adverse effects on the human central nervous system across the lifespan.

Round robin test on quantification of amyloid-ß 1-42 in cerebrospinal fluid by mass spectrometry.

INTRODUCTION: Cerebrospinal fluid (CSF) amyloid-ß 1-42 (Aß42) is an important biomarker for Alzheimer's disease, both in diagnostics and to monitor disease-modifying therapies. However, there is a great need for standardization of methods used for quantification. To overcome problems associated with immunoassays, liquid chromatography-tandem mass spectrometry (LC-MS/MS) has emerged as a critical orthogonal alternative. METHODS: We compared results for CSF Aß42 quantification in a round robin study performed in four laboratories using similar sample preparation methods and LC-MS instrumentation. RESULTS: The LC-MS results showed excellent correlation between laboratories (r(2) >0.98), high analytical precision, and good correlation with enzyme-linked immunosorbent assay (r(2) >0.85). The use of a common reference sample further decreased interlaboratory variation. DISCUSSION: Our results indicate that LC-MS is suitable for absolute quantification of Aß42 in CSF and highlight the importance of developing a certified reference material.

The Alzheimer's Disease Neuroimaging Initiative 2 Biomarker Core: A review of progress and plans.

INTRODUCTION: We describe Alzheimer's Disease Neuroimaging Initiative (ADNI) Biomarker Core progress including: the Biobank; cerebrospinal fluid (CSF) amyloid beta (Aß1-42), t-tau, and p-tau181 analytical performance, definition of Alzheimer's disease (AD) profile for plaque, and tangle burden detection and increased risk for progression to AD; AD disease heterogeneity; progress in standardization; and new studies using ADNI biofluids. METHODS: Review publications authored or coauthored by ADNI Biomarker core faculty and selected non-ADNI studies to deepen the understanding and interpretation of CSF Aß1-42, t-tau, and p-tau181 data. RESULTS: CSF AD biomarker measurements with the qualified AlzBio3 immunoassay detects neuropathologic AD hallmarks in preclinical and prodromal disease stages, based on CSF studies in non-ADNI living subjects followed by the autopsy confirmation of AD. Collaboration across ADNI cores generated the temporal ordering model of AD biomarkers varying across individuals because of genetic/environmental factors that increase/decrease resilience to AD pathologies. DISCUSSION: Further studies will refine this model and enable the use of biomarkers studied in ADNI clinically and in disease-modifying therapeutic trials.

History of cigarette smoking in cognitively-normal elders is associated with elevated cerebrospinal fluid biomarkers of oxidative stress.

BACKGROUND: Cigarette smoking in adults is associated with abnormalities in brain neurobiology. Smoking-induced central nervous system oxidative stress (OxS) is a potential mechanism associated with these abnormalities. The goal of this study was to compare cognitively-normal elders on cerebrospinal fluid (CSF) levels of F2-isoprostane biomarkers of OxS. METHODS: Elders with a lifetime history of smoking (smokers; n=50; 75±5 years of age; 34±28 pack-years; approximately 12% were actively smoking at the time of study) were compared to never-smokers (n=61; 76±6 years of age) on CSF iPF2α-III and 8,12, iso-iPF2α-VI F2-isoprostanes levels. F2-isoprostanes levels were quantitated with HPLC-atmospheric pressure chemical ionization-tandem mass spectrometry. Associations between F2-isoprostanes levels, hippocampal volumes, and cigarette exposure measures were also evaluated. RESULTS: Smokers showed higher iPF2α-III level than never-smokers. An age×smoking status interaction was observed for 8,12, iso-iPF2α-VI, where smokers demonstrate a significantly greater concentration with increasing age than never-smokers. In smokers only, higher 8,12, iso-iPF2α-VI concentration was associated with smaller hippocampal volume, and greater iPF2α-III level was related to greater pack years. CONCLUSIONS: This is the first study to demonstrate that a history of cigarette smoking in cognitively-normal elders was associated with significantly elevated CSF F2-isoprostane levels and greater age-related increases in F2-isoprostanes, and that higher F2-isoprostane levels in smokers were related to smaller hippocampal volume. These findings provide additional novel evidence that a history of chronic smoking during adulthood is associated with adverse effects on the human brain that are potentially enduring even with extended smoking cessation.

Qualification of a surrogate matrix-based absolute quantification method for amyloid-ß42 in human cerebrospinal fluid using 2D UPLC-tandem mass spectrometry.

The primary aims of this work were to: 1) establish a calibrator surrogate matrix for quantification of amyloid-ß (Aß)42 in human cerebrospinal fluid (CSF) and preparation of quality control samples for LC-MS-MS methodology, 2) validate analytical performance of the assay, and 3) evaluate its diagnostic utility and compare it with the AlzBio3 immunoassay. The analytical methodology was based on a 2D-UPLC-MS-MS platform. Sample pretreatment used 5 M guanidine hydrochloride and extraction on µElution SPE columns as previously described. A column cleaning procedure involved gradual removal of aqueous solvents by acetonitrile assured consistent long-term chromatography performance. Receiver-operator characteristic (ROC) curve and correlation analyses evaluated the diagnostic utility of UPLC-MS-MS compared to AlzBio3 immunoassay for detection of Alzheimer's disease (AD). The surrogate matrix, artificial CSF containing 4 mg/mL of BSA, provides linear and reproducible calibration comparable to human pooled CSF as calibration matrix. Appropriate cleaning of the trapping and analytical columns provided every-day, trouble-free runs. Analyses of CSF Aß42 showed that UPLC-MS-MS distinguished neuropathologically-diagnosed AD subjects from healthy controls with at least equivalent diagnostic utility to AlzBio3. Comparison of ROC curves for these two assays showed no statistically significant difference (p = 0.2229). Linear regression analysis of Aß42 concentrations measured by this mass spectrometry-based method compared to the AlzBio3 immunoassay showed significantly higher but highly correlated results. In conclusion, the newly established surrogate matrix for 2D-UPLC-MS-MS measurement of Aß42 provides selective, reproducible, and accurate results. The documented analytical performance and diagnostic performance for AD versus controls supports consideration as a candidate reference method.

Clinical utility and analytical challenges in measurement of cerebrospinal fluid amyloid-ß(1-42) and τ proteins as Alzheimer disease biomarkers.

BACKGROUND: Over the past 2 decades, clinical studies have provided evidence that cerebrospinal fluid (CSF) amyloid ß(1-42) (Aß(1-42)), total τ (t-τ), and τ phosphorylated at Thr181 (p-τ(181)) are reliable biochemical markers of Alzheimer disease (AD) neuropathology. CONTENT: In this review, we summarize the clinical performance and describe the major challenges for the analytical performance of the most widely used immunoassay platforms [based on ELISA or microbead-based multianalyte profiling (xMAP) technology] for the measurement of CSF AD biomarkers (Aß(1-42), t-τ, and p-τ(181)). With foundational immunoassay data providing the diagnostic and prognostic values of CSF AD biomarkers, the newly revised criteria for the diagnosis of AD include CSF AD biomarkers for use in research settings. In addition, it has been suggested that the selection of AD patients at the predementia stage by use of CSF AD biomarkers can improve the statistical power of clinical trial design. Owing to the lack of a replenishable and commutable human CSF-based standardized reference material (SRM) and significant differences across different immunoassay platforms, the diagnostic-prognostic cutpoints of CSF AD biomarker concentrations are not universal at this time. These challenges can be effectively met in the future, however, through collaborative ongoing standardization efforts to minimize the sources of analytical variability and to develop reference methods and SRMs. SUMMARY: Measurements of CSF Aß(1-42), t-τ, and p-τ(181) with analytically qualified immunoassays reliably reflect the neuropathologic hallmarks of AD in patients at the early predementia stage of the disease and even in presymptomatic patients. Thus these CSF biomarker tests are useful for early diagnosis of AD, prediction of disease progression, and efficient design of drug intervention clinical trials.

Improved protocol for measurement of plasma ß-amyloid in longitudinal evaluation of Alzheimer's Disease Neuroimaging Initiative study patients.

BACKGROUND: The interassay variability and inconsistency of plasma ß-amyloid (Aß) measurements among centers are major factors precluding the interpretation of results and a substantial obstacle in the meta-analysis across studies of this biomarker. The goal of this investigation was to address these problems by improving the performance of the bioanalytical method. METHODS: We used the Luminex immunoassay platform with a multiplex microsphere-based reagent kit from Innogenetics. A robotic pipetting system was used to perform crucial steps of the procedure. The performance of this method was evaluated using two kit control samples and two quality control plasma samples from volunteer donors, and by retesting previously assayed patient samples in each run. This setup was applied to process 2454 patient plasma samples from the Alzheimer's Disease Neuroimaging Initiative study biofluid repository. We have additionally evaluated the correlations between our results and cerebrospinal fluid (CSF) biomarker data using mixed-effects modeling. RESULTS: The average precision values of the kit controls were 8.3% for Aß(1-40) and 4.0% for Aß(1-42), whereas the values for the plasma quality controls were 6.4% for Aß(1-40) and 4.8% for Aß(1-42). From the test-retest evaluation, the average precision was 7.2% for Aß(1-40) and 4.5% for Aß(1-42). The range of final plasma results for Alzheimer's Disease Neuroimaging Initiative patients was 13 to 372 pg/mL (median: 164 pg/mL) for Aß(1-40) and 3.5 to 103 pg/mL (median: 39.3 pg/mL) for Aß(1-42). We found that sample collection parameters (blood volume and time to freeze) have a small, but significant, influence on the result. No significant difference was found between plasma Aß levels for patients with Alzheimer's disease and healthy control subjects. We have determined multiple significant correlations of plasma Aß(1-42) levels with CSF biomarkers. The relatively strongest, although modest, correlation was found between plasma Aß(1-42) levels and CSF p-tau(181)/Aß(1-42) ratio in patients with mild cognitive impairment. Plasma Aß(1-40) correlations with CSF biomarkers were weaker and diminished completely when we used longitudinal data. No significant correlations were found for the plasma Aß(1-42)/Aß(1-40) ratio. CONCLUSIONS: The precision of our robotized method represents a substantial improvement over results reported in the literature. Multiple significant correlations between plasma and CSF biomarkers were found. Although these correlations are not strong enough to support the use of plasma Aß measurement as a diagnostic screening test, plasma Aß(1-42) levels are well suited for use as a pharmacodynamic marker.

Evaluation of performance of new, isotopically labeled internal standard ([13c2d4]RAD001) for everolimus using a novel high-performance liquid chromatography tandem mass spectrometry method.

INTRODUCTION: Everolimus was approved for the prevention of organ rejection of kidney transplants in adult patients in April 2010 by the US Food and Drug Administration. Therapeutic drug monitoring of everolimus is recommended for all patients receiving this immunosuppressant. The goal of this study was to improve and revalidate our previously published high-performance liquid chromatography tandem mass spectrometry method for the measurement of everolimus and to assess the performance of the recently introduced isotopically labeled internal standard (IS). METHOD: For this method, the following innovative changes were incorporated: (1) sample preparation includes the addition of water to hemolyze red blood cells; (2) a separate extraction column is added to the system; (3) different settings of the switching valve are used to minimize dead volume; (4) a more sensitive mass spectrometer is used (API 5000) for drug detection; (5) isotopically labeled everolimus is used as the IS. RESULTS: The addition of water to blood before adding ZnSO4 with methanol improves absolute recovery of everolimus from 77.3% ± 6.18% to 82.3% ± 6.3%. The use of the more sensitive mass spectrometer permitted the use of a smaller sample volume and lowered the lower limit of quantification from 1.0 to 0.5 ng/mL. Between-day precision for quality control samples is below 9%, and the accuracy ranged from 94.8% to 106.4%. Neither carryover nor matrix effects were observed. A comparison study showed very good agreement between the results obtained by our laboratory and those obtained by a reference laboratory (r = 0.93). The performance of the new IS, [13c2d4] RAD001, was compared with that of SDZ RAD 223-756. Everolimus concentration results obtained using the isotopically labeled IS agreed more closely with the results from the reference laboratory (using 13c2d4) as compared with everolimus concentration results obtained using the analog (SDZ RAD 223-756). CONCLUSIONS: This revised methodology together with the comparison study data proved that our novel method for the measurement of everolimus is sensitive, precise, reliable, and robust.

Simultaneous HPLC-MS-MS quantification of 8-iso-PGF(2alpha) and 8,12-iso-iPF(2alpha) in CSF and brain tissue samples with on-line cleanup.

Quantitation of isoprostanes such as 8-iso-PGF(2alpha) and 8,12-iso-iPF(2alpha)-VI in biological fluids has been proposed as a reliable test of oxidant stress and inflammation in a variety of disorders. This paper presents a liquid chromatography method with tandem mass spectrometry detection for the simultaneous analysis of these two isoprostanes in human CSF and brain tissue samples. An API 5000 triple quadrupole instrument (AB Sciex, Foster City, CA, USA) with an APCI ion source was used in this study. Aliquots of CSF samples (0.25mL) were treated with a methanol:zinc sulfate mixture followed by on-line cleanup on an extraction column (Validated-C(18)) with 0.1% formic acid. The brain tissue samples were homogenized and lipids were extracted using Folch solution. Solid-phase extraction columns (C(18)) were used for the purification of the brain isoprostane fraction. Chromatographic separation was achieved using an analytical column (Synergi C(18) HydroRP) with 0.1% formic acid in water and a mixture of methanol:acetonitrile under isocratic conditions. The mass spectrometer was operated in the MRM scan and negative ion mode. The quadrupoles were set to detect the molecular ions [M-H](-) and high mass fragments of isoprostanes: m/z 353-->193amu (8-iso-PGF(2alpha)) and m/z 353-->115amu (8,12-iso-iPF(2alpha)-VI) and their deuterated internal standards: m/z 357-->197amu (8-iso-PGF(2alpha)-d(4)) and m/z 364-->115amu (8,12-iso-iPF(2alpha)-VI-d(11)). The lower limit of quantification was 2.5pg/mL for 8-iso-PGF(2alpha) and 5.0pg/mL for 8,12-iso-PF(2alpha)-VI for the CSF method and 10.0pg/0.1g of tissue and 30.0pg/0.1g of tissue for 8-iso-PGF(2alpha) and 8,12-iso-iPF(2alpha)-VI, respectively, for the brain tissue method. No ion suppression or enhancement of the detection of 8-isoPGF(2alpha), 8,12-isoPF(2alpha)-VI or both internal standards was found.

Update on the biomarker core of the Alzheimer's Disease Neuroimaging Initiative subjects.

Here, we review progress by the Penn Biomarker Core in the Alzheimer's Disease Neuroimaging Initiative (ADNI) toward developing a pathological cerebrospinal fluid (CSF) and plasma biomarker signature for mild Alzheimer's disease (AD) as well as a biomarker profile that predicts conversion of mild cognitive impairment (MCI) and/or normal control subjects to AD. The Penn Biomarker Core also collaborated with other ADNI Cores to integrate data across ADNI to temporally order changes in clinical measures, imaging data, and chemical biomarkers that serve as mileposts and predictors of the conversion of normal control to MCI as well as MCI to AD, and the progression of AD. Initial CSF studies by the ADNI Biomarker Core revealed a pathological CSF biomarker signature of AD defined by the combination of Abeta1-42 and total tau (T-tau) that effectively delineates mild AD in the large multisite prospective clinical investigation conducted in ADNI. This signature appears to predict conversion from MCI to AD. Data fusion efforts across ADNI Cores generated a model for the temporal ordering of AD biomarkers which suggests that Abeta amyloid biomarkers become abnormal first, followed by changes in neurodegenerative biomarkers (CSF tau, F-18 fluorodeoxyglucose-positron emission tomography, magnetic resonance imaging) with the onset of clinical symptoms. The timing of these changes varies in individual patients due to genetic and environmental factors that increase or decrease an individual's resilience in response to progressive accumulations of AD pathologies. Further studies in ADNI will refine this model and render the biomarkers studied in ADNI more applicable to routine diagnosis and to clinical trials of disease modifying therapies.