Second-generation Elecsys cerebrospinal fluid immunoassays aid diagnosis of early Alzheimer's disease.

OBJECTIVES: Timely diagnosis of Alzheimer's disease (AD) is critical for appropriate treatment/patient management. Cerebrospinal fluid (CSF) biomarker analysis is often used to aid diagnosis. We assessed analytical performance of second-generation (Gen II) Elecsys® CSF immunoassays (Roche Diagnostics International Ltd), and adjusted existing cut-offs, to evaluate their potential utility in clinical routine. METHODS: Analytical performance was assessed using CSF samples measured with Elecsys CSF Gen II immunoassays on cobas e analyzers. Aß42 Gen I/Gen II immunoassay method comparisons were performed (Passing-Bablok regression). Cut-off values were adjusted using estimated bias in biomarker levels between BioFINDER protocol aliquots/Gen I immunoassays and Gen II protocol aliquots/immunoassays. Distribution of Gen II immunoassay values was evaluated in AD, mild cognitive impairment (MCI), and cognitively normal cohorts; percentage observations outside the measuring range were derived. RESULTS: The Gen II immunoassays demonstrated good analytical performance, including repeatability, intermediate precision, lot-to-lot agreement (Pearson's r: ≥0.999), and platform agreement (Pearson's r: ≥0.995). Aß42 Gen I/Gen II immunoassay measurements were strongly correlated (Pearson's r: 0.985-0.999). Aß42 Gen II immunoassay cut-offs were adjusted to 1,030 and 800 ng/L, and pTau181/Aß42 ratio cut-offs to 0.023 and 0.029, for Gen II and I protocols, respectively. No observations were below the lower limit of the measuring range; above the upper limit, there were none from the AD cohort, and 2.6 and 6.8% from the MCI and cognitively normal cohorts, respectively. CONCLUSIONS: Our findings suggest that the Gen II immunoassays have potential utility in clinical routine to aid diagnosis of AD.

Clinical performance and robustness evaluation of plasma amyloid-ß42/40 prescreening.

INTRODUCTION: Further evidence is needed to support the use of plasma amyloid ß (Aß) biomarkers as Alzheimer's disease prescreening tools. This study evaluated the clinical performance and robustness of plasma Aß42 /Aß40 for amyloid positivity prescreening. METHODS: Data were collected from 333 BioFINDER and 121 Alzheimer's Disease Neuroimaging Initiative study participants. Risk and predictive values versus percentile of plasma Aß42 /Aß40 evaluated the actionability of plasma Aß42 /Aß40 , and simulations modeled the impact of potential uncertainties and biases. Amyloid PET was the brain amyloidosis reference standard. RESULTS: Elecsys plasma Aß42 /Aß40 could potentially rule out amyloid pathology in populations with low-to-moderate amyloid positivity prevalence. However, simulations showed small measurement or pre-analytical errors in Aß42 and/or Aß40 cause misclassifications, impacting sensitivity or specificity. The minor fold change between amyloid PET positive and negative cases explains the biomarkers low robustness. DISCUSSION: Implementing plasma Aß42 /Aß40 for routine clinical use may pose significant challenges, with misclassification risks. HIGHLIGHTS: Plasma Aß42 /Aß40 ruled out amyloid PET positivity in a setting of low amyloid-positive prevalence. Including (pre-) analytical errors or measurement biases caused misclassifications. Plasma Aß42 /Aß40 had a low inherent dynamic range, independent of analytical method. Other blood biomarkers may be easier to implement as robust prescreening tools.

An accurate fully automated panel of plasma biomarkers for Alzheimer's disease.

INTRODUCTION: There is a great need for fully automated plasma assays that can measure amyloid beta (Aß) pathology and predict future Alzheimer's disease (AD) dementia. METHODS: Two cohorts (n = 920) were examined: Panel A+ (n = 32 cognitively unimpaired [CU], n = 106 mild cognitive impairment [MCI], and n = 89 AD) and BioFINDER-1 (n = 461 CU, n = 232 MCI). Plasma Aß42/Aß40, phosphorylated tau (p-tau)181, two p-tau217 variants, ApoE4 protein, neurofilament light, and GFAP were measured using Elecsys prototype immunoassays. RESULTS: The best biomarker for discriminating Aß-positive versus Aß-negative participants was Aß42/Aß40 (are under the curve [AUC] 0.83-0.87). Combining Aß42/Aß40, p-tau181, and ApoE4 improved the AUCs significantly (0.90 to 0.93; P< 0.01). Adding additional biomarkers had marginal effects (ΔAUC ≤0.01). In BioFINDER, p-tau181, p-tau217, and ApoE4 predicted AD dementia within 6 years in CU (AUC 0.88) and p-tau181, p-tau217, and Aß42/Aß40 in MCI (AUC 0.87). DISCUSSION: The high accuracies for Aß pathology and future AD dementia using fully automated instruments are promising for implementing plasma biomarkers in clinical trials and clinical routine.

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 .

Technical performance of a novel, fully automated electrochemiluminescence immunoassay for the quantitation of ß-amyloid (1-42) in human cerebrospinal fluid.

INTRODUCTION: Available assays for quantitation of the Alzheimer's disease (AD) biomarker amyloid-beta 1-42 (Aß [1-42]) in cerebrospinal fluid demonstrate significant variability and lack of standardization to reference measurement procedures (RMPs). We report analytical performance data for the novel Elecsys ß-amyloid (1-42) assay (Roche Diagnostics). METHODS: Lot-to-lot comparability was tested using method comparison. Performance parameters were measured according to Clinical & Laboratory Standards Institute (CLSI) guidelines. The assay was standardized to a Joint Committee for Traceability in Laboratory Medicine (JCTLM) approved RMP. RESULTS: Limit of quantitation was <11.28 pg/mL, and the assay was linear throughout the measuring range (200-1700 pg/mL). Excellent lot-to-lot comparability was observed (correlation coefficients [Pearson's r] >0.995; bias in medical decision area <2%). Repeatability coefficients of variation (CVs) were 1.0%-1.6%, intermediate CVs were 1.9%-4.0%, and intermodule CVs were 1.1%-3.9%. Estimated total reproducibility was 2.0%-5.1%. Correlation with the RMP was good (Pearson's r, 0.93). DISCUSSION: The Elecsys ß-amyloid (1-42) assay has high analytical performance that may improve biomarker-based AD diagnosis.

NGAL, L-FABP, and KIM-1 in comparison to established markers of renal dysfunction.

BACKGROUND: New urinary biomarkers like neutrophil gelatinase-associated lipocalin (NGAL), liver-type fatty acid binding protein (L-FABP), and kidney injury molecule-1 (KIM-1) open the opportunity to detect kidney injuries in early stages. Our study aimed at evaluating NGAL, L-FABP, and KIM-1 in comparison to established markers of urine protein differentiation for detection of renal dysfunction. METHODS: On the basis of the PROTIS expert system (for differentiation of glomerulo-/tubulopathy) urine and plasma samples of 263 randomly selected patients were routinely examined (urine: total protein, albumin, IgG, α1-microglobulin, creatinine, and dip stick results for leukocytes, blood, protein, glucose, pH, and nitrite; plasma: creatinine and cystatin C) followed by the analysis of the new urine biomarkers NGAL (CMIA), L-FABP (ECLIA), and KIM-1 (ELISA). RESULTS: Of the three new markers L-FABP showed the highest correlation with α1-microglobulin (r=0.76, p<0.01) and was closest associated with the degree of tubular proteinuria assessed by the PROTIS system. NGAL distinguished the PROTIS proteinuria groups with distinctive tubular proteinurias from the controls as well, but revealed a marked diagnostic influence by leukocyturia. Urinary KIM-1 revealed only a weak diagnostic value for the detection of renal injury. CONCLUSIONS: Urinary NGAL and L-FABP proved to be promising candidates for detecting injuries of the renal tubular system over a broad range of clinical conditions. L-FABP showed a better diagnostic performance and a lower interference by leukocyturia and hematuria than NGAL. Both markers may serve as sensitive tissue injury markers in addition to the established markers of renal functional impairment.

Diagnostic performance of automated plasma amyloid-ß assays combined with pre-analytical immunoprecipitation.

BACKGROUND: Measurements of the amyloid-ß (Aß) 42/40 ratio in blood plasma may support the early diagnosis of Alzheimer's disease and aid in the selection of suitable participants in clinical trials. Here, we compared the diagnostic performance of fully automated prototype plasma Aß42/40 assays with and without pre-analytical sample workup by immunoprecipitation. METHODS: A pre-selected clinical sample comprising 42 subjects with normal and 38 subjects with low cerebrospinal fluid (CSF) Aß42/40 ratios was studied. The plasma Aß42/40 ratios were determined with fully automated prototype Elecsys® immunoassays (Roche Diagnostics GmbH, Penzberg, Germany) by direct measurements in EDTA plasma or after pre-analytical Aß immunoprecipitation. The diagnostic performance for the detection of abnormal CSF Aß42/40 was analyzed by receiver operating characteristic (ROC) analysis. In an additional post hoc analysis, a biomarker-supported clinical diagnosis was used as a second endpoint. RESULTS: Pre-analytical immunoprecipitation resulted in a significant increase in the area under the ROC curve (AUC) from 0.73 to 0.88 (p = 0.01547) for identifying subjects with abnormal CSF Aß42/40. A similar improvement in the diagnostic performance by pre-analytical immunoprecipitation was also observed when a biomarker-supported clinical diagnosis was used as a second endpoint (AUC increase from 0.77 to 0.92, p = 0.01576). CONCLUSIONS: Our preliminary observations indicate that pre-analytical Aß immunoprecipitation can improve the diagnostic performance of plasma Aß assays for detecting brain amyloid pathology. The findings may aid in the further development of blood-based immunoassays for Alzheimer's disease ultimately suitable for screening and routine use.

Pre-analytical protocol for measuring Alzheimer's disease biomarkers in fresh CSF.

INTRODUCTION: We aimed to establish a standardized, routine-use pre-analytical protocol for measuring Alzheimer's disease (AD) biomarkers in cerebrospinal fluid (CSF). METHODS: The effect of pre-analytical factors (sample collection/handling/storage/transportation) on biomarker levels was assessed using freshly collected CSF. Tube type/sterilization was assessed using previously frozen samples. A low-bind false-bottom tube (FBT, Sarstedt) was used for all experiments, except tube types/sterilization experiments. Biomarkers were measured using Elecsys CSF assays. RESULTS: Amyloid beta (Aß)1-42 levels varied by tube type, using a low-bind FBT reduced variation. Aß1-42 levels were higher with no mixing versus roller/inversion mixing. Aß1-42 levels were lower with horizontal versus upright transportation; this was resolved by maximal tube filling and storage at 2°C to 8°C. Aß1-40 levels were less strongly affected. Phospho-tau and total-tau levels were largely unaffected. DISCUSSION: We propose an easy-to-use, standardized, routine-use pre-analytical protocol, using low-bind FBTs, for measuring AD CSF biomarkers in clinical practice.

Elecsys CSF biomarker immunoassays demonstrate concordance with amyloid-PET imaging.

BACKGROUND: ß-amyloid (Aß) positron emission tomography (PET) imaging is currently the only Food and Drug Administration-approved method to support clinical diagnosis of Alzheimer's disease (AD). However, numerous research studies support the use of cerebrospinal fluid (CSF) biomarkers, as a cost-efficient, quick and equally valid method to define AD pathology. METHODS: Using automated Elecsys® assays (Roche Diagnostics) for Aß (1-42) (Aß42), Aß (1-40) (Aß40), total tau (tTau) and phosphorylated tau (181P) (pTau), we examined CSF samples from 202 participants of the Australian Imaging, Biomarkers and Lifestyle (AIBL) study of ageing cohort, to demonstrate the concordance with pathological AD via PET imaging. RESULTS: Ratios Aß42/Aß40, tTau/Aß42 and pTau/Aß42 had higher receiver operator characteristic-area under the curve (all 0.94), and greater concordance with Aß-PET (overall percentage agreement ~ 90%), compared with individual biomarkers. CONCLUSION: Strong concordance between CSF biomarkers and Aß-PET status was observed overall, including for cognitively normal participants, further strengthening the association between these markers of AD neuropathological burden for both developmental research studies and for use in clinical trials.

Method comparison study of the Elecsys® ß-Amyloid (1-42) CSF assay versus comparator assays and LC-MS/MS.

BACKGROUND: Alzheimer's disease (AD) biomarkers, such as cerebrospinal fluid (CSF) amyloid-ß (1-42; Aß42), can provide high diagnostic accuracy. Several immunoassays are available for Aß42 quantitation, but standardisation across assays remains an issue. We compared the Elecsys® ß-Amyloid (1-42) CSF assay with three assays and two liquid chromatography tandem mass spectrometry (LC-MS/MS) methods. METHODS: Three method comparison studies evaluated the correlation between the Elecsys® ß-Amyloid (1-42) CSF assay versus: INNOTEST® ß-AMYLOID(1-42) (860 samples) and the Roche Diagnostics-developed LC-MS/MS method (250 samples); INNO-BIA AlzBio3 and the University of Pennsylvania (UPenn)-developed LC-MS/MS method (250 samples); and ADx-EUROIMMUN Beta-Amyloid (1-42) enzyme-linked immunosorbent assay (ELISA) (49 samples). RESULTS: High correlation was demonstrated between Elecsys® ß-Amyloid (1-42) CSF and comparator assays: INNOTEST® ß-AMYLOID(1-42) (Spearman's ρ, 0.954); INNO-BIA AlzBio3 (Spearman's ρ, 0.864); ADx-EUROIMMUN Beta-Amyloid (1-42) ELISA (Pearson's r, 0.925). Elecsys® assay and LC-MS/MS measurements were highly correlated: Pearson's r, 0.949 (Roche Diagnostics-developed method) and 0.943 (UPenn-developed method). CONCLUSION: Findings from this multicentre evaluation further support use of the Elecsys® ß-Amyloid (1-42) CSF assay to aid AD diagnosis. CSF-based certified reference materials should improve agreement across assays and mass spectrometry-based methods, which is essential to establish a global uniform CSF Aß42 cut-off to detect amyloid pathology.

Elecsys® Total-Tau and Phospho-Tau (181P) CSF assays: Analytical performance of the novel, fully automated immunoassays for quantification of tau proteins in human cerebrospinal fluid.

BACKGROUND: Total tau (tTau) and phosphorylated 181P tau (pTau) are supportive diagnostic cerebrospinal fluid (CSF) biomarkers for Alzheimer's disease. Manual CSF tau assays are limited by lot-to-lot and between-laboratory variability and long incubation/turnaround times. Elecsys® Total-Tau CSF and Phospho-Tau (181P) CSF immunoassays were developed for fully automated cobas e analyzers, allowing broader access in clinical practice and trials. METHODS: Analytical performance, reproducibility, method comparisons with commercially available assays, and lot-to-lot and platform comparability (cobas e 601/411) of the Elecsys® CSF assays were assessed. Tau distributions and concentration ranges were evaluated in CSF samples from two clinical cohorts. RESULTS: Both assays showed high sensitivity (limit of quantitation [LoQ]: 63 pg/mL [tTau]; 4 pg/mL [pTau]) and linearity over the measuring range (80-1300 pg/mL; 8-120 pg/mL), which covered the entire concentration range measured in clinical samples. Lot-to-lot and platform comparability demonstrated good consistency (Pearson's r: 0.998; 1.000). Multicenter evaluation coefficients of variation (CVs): repeatability, < 1.8%; intermediate precision, < 2.8%; between-laboratory variability, < 2.7% (both assays); and total reproducibility, < 6.7% (tTau) and < 4.7% (pTau). Elecsys® CSF assays demonstrated good correlation with commercially available tau assays. CONCLUSIONS: Elecsys® Total-Tau CSF and Phospho-Tau (181P) CSF assays demonstrate good analytical performance with clinically relevant measuring ranges; data support their use in clinical trials and practice.

Comparison of Plasma and Urine Biomarker Performance in Acute Kidney Injury.

BACKGROUND: New renal biomarkers measured in urine promise to increase specificity for risk stratification and early diagnosis of acute kidney injury (AKI) but concomitantly may be altered by urine concentration effects and chronic renal insufficiency. This study therefore directly compared the performance of AKI biomarkers in urine and plasma. METHODS: This single-center, prospective cohort study included 110 unselected adults undergoing cardiac surgery with cardiopulmonary bypass between 2009 and 2010. Plasma and/or urine concentrations of creatinine, cystatin C, neutrophil gelatinase-associated lipocalin (NGAL), liver fatty acid-binding protein (L-FABP), kidney injury molecule 1 (KIM1), and albumin as well as 15 additional biomarkers in plasma and urine were measured during the perioperative period. The primary outcome was AKI defined by AKIN serum creatinine criteria within 72 hours after surgery. RESULTS: Biomarkers in plasma showed markedly better discriminative performance for preoperative risk stratification and early postoperative (within 24h after surgery) detection of AKI than urine biomarkers. Discriminative power of urine biomarkers improved when concentrations were normalized to urinary creatinine, but urine biomarkers had still lower AUC values than plasma biomarkers. Best diagnostic performance 4h after surgery had plasma NGAL (AUC 0.83), cystatin C (0.76), MIG (0.74), and L-FAPB (0.73). Combinations of multiple biomarkers did not improve their diagnostic power. Preoperative clinical scoring systems (EuroSCORE and Cleveland Clinic Foundation Score) predicted the risk for AKI (AUC 0.76 and 0.71) and were not inferior to biomarkers. Preexisting chronic kidney disease limited the diagnostic performance of both plasma and urine biomarkers. CONCLUSIONS: In our cohort plasma biomarkers had higher discriminative power for risk stratification and early diagnosis of AKI than urine biomarkers. For preoperative risk stratification of AKI clinical models showed similar discriminative performance to biomarkers. The discriminative performance of both plasma and urine biomarkers was reduced by preexisting chronic kidney disease.

Trim14 overexpression causes the same transcriptional changes in mouse embryonic stem cells and human HEK293 cells.

The trim14 (pub, KIAA0129) gene encodes the TRIM14 protein which is a member of the tripartite motif (TRIM) family. Previously, we revealed high expression levels of trim14 in HIV- or SIV-associated lymphomas and demonstrated the influence of trim14 on mesodermal differentiation of mouse embryonic stem cells (mESC). In the present work, to elucidate the role of trim14 in normal and pathological processes in the cell, we used two different types of cells transfected with trim14: mESC and human HEK293. Using subtractive hybridization and real-time PCR, we found a number of genes which expression was elevated in trim14-transfected mESC: hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), hlx1, hbp1, junb, and pdgfrb. A further analysis of the trim14-transfected mESC at the initial stage of differentiation (embryoid bodies (EB) formation) showed essential changes in the expression of these upregulated genes. The transfection of trim14 into HEK293 also induced an enhanced expression of the several genes upregulated in trim14-transfected mESC (hsp90ab1, prr13, pu.1, tnfrsf13c (baff-r), tnfrsf13b (taci), and hlx1). Summarizing, we found similar genes that participated in trim14-directed processes both in mESC and in HEK293. These results demonstrate the presence of the similar mechanism of trim14 gene action in different types of mammalian cells.