Human Neurology 3-Plex A
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1 Human Neurology 3-Plex A SUMMARY AND EXPLANATION OF THE TEST The Human N3PA assay is a digital immunoassay for the quantitative determination of total Tau, Aβ42, and Aβ40 in human plasma and CSF. Determination in serum samples are not reported due to high variability of Aβ40 and Aβ42 in some healthy donor sample sets. This assay is for research use only and not for use in diagnostic procedures. Tau is a microtubule-stabilizing protein primarily localized in central nervous system neurons but also expressed at low levels in astrocytes and oligodendrocytes. Tau consists of six isoforms in the human brain with molecular weights of 48,000 to 67,000 daltons, depending on isoform. Aβ42 and Aβ40 are two proteolytic products from the amyloid precursor protein (APP). Beta-secretase cleavage of APP initially results in the production of an APP fragment that is further cleaved by gamma-secretase at residues 40 or 42 to generate two main forms of amyloid beta, Aβ42 and Aβ40. Amyloid beta (Aβ) peptides (including a shorter Aβ38 isoform) are produced by different cell types in the body, but the expression is particularly high in the brain. Tau and amyloid β related pathologies have been the hallmark of Alzheimer s disease. CSF and blood tau and amyloid have been tested and monitored as potential biomarkers for Alzheimer s disease, mild cognitive impairment, vascular dementia, and other neurodegenerative disorders. Reagent Kit HD- Analyzer
2 Page 2 SUMMARY AND EXPLANATION OF THE TAU TEST Tau is a microtubule-stabilizing protein primarily localized in central nervous system neurons, but also expressed at low levels in astrocytes and oligodendrocytes. Tau consists of six isoforms in the human brain, with molecular weights of 48,000 to 67,000 daltons depending on isoform. Tau elevation is observed in the cerebrospinal fluid (CSF) of patients with neurodegenerative disease and head injuries, suggesting its extracellular release during neuronal damage and a role as a biomarker with specificity for brain injury. Potential movement of elevated CSF tau across the blood-brain barrier presents a possibility that measurements of tau in blood could provide a convenient peripheral window into brain/csf status. Studies of tau in serum and plasma have been hampered by its low abundance (typically low ), and there are relatively few reports characterizing the appearance of tau in blood or evaluating the usefulness of this biomarker for brain injury assessment. Recent reports using digital immunoassay technology have shown elevation in peripheral tau associated with hypoxic brain injury, concussed hockey players, and repetitive minimal head injury in Olympic boxing. The Human Neurology 3-Plex Total Tau assay uses a combination of monoclonal antibodies that react with both normal and phosphorylated tau. With an epitope in the midregion of the molecule, the assay recognizes all tau isoforms. GENERAL CHARACTERISTICS OF THE TAU TEST Healthy donors 0000 Median [tau].43 Median [tau] AEB %CV dose Tau, 00 0 Tau, Figure : Tau immunoassay calibration curve. Four-parameter curve fit parameters are depicted. Tau, Figure 2: Dose CV profile. Diluted serum was assayed in reps of 3 over multiple days and run (96 determinations). Concentrations are uncorrected for pre-dilutions. 0. Plasma CSF Figure 3: [Tau] in EDTA plasma (n = 20) and CSF (n = 20) from healthy donors. Error bars depict mean and interquartile ranges. Table : General characteristics of Neurology 3-Plex A Tau immunoassay Calibration range 0-00 Dynamic range Lower limit of detection (2.5 SD; 3 reps x 2 estimates across 2 runs, 3 instruments, 2 lots; mean LoD 2 ) 0.09 Lower limit of quantification 3 (2 runs, 3 instruments, 2 reagent lots, mean LoQ) Spike-recovery: Plasma (tau 44 spiked into 4 plasma samples at 20 and 200, mean 4 ) 6.3% Spike-recovery: CSF (tau 4 spiked into 4 CSF samples at 400 and 4000, mean 5 ) 07.4% Linearity (high tau plasma sample fractionally admixed with low tau plasma sample, mean of 0 levels 6 ) 00.3% Dilution linearity: Plasma (spiked plasma diluted 2X serially from MRD to 6-fold w/ Diluent; mean recovery 7 ) 4.6% Dilution linearity: CSF (CSF diluted 2X serially from MRD to 28-fold w/ Diluent; mean recovery 8 ) 0.4% Inter lot CV (Pool of CVs from 6 samples 9 tested with 2 reagent lots across 2 runs x 3 instruments) 3.5% Inter instrument CV (Pool of CVs from 6 samples 9 tested with 3 instruments across 2 runs x 2 reagent lots) 4.% Typical serum/plasma sample volume (Includes dead volume; see Package Insert for details) 46 µl Minimum serum/plasma sample volume (Depends on dilution procedure; see Package Insert for details) 3.5 µl CSF sample volume (Includes dead volume; See Package Insert for details) 2.3 µl s auto-diluted 4X; note: ranges may vary between calibrator lots. 2 SD 0.04 ; range %CV, 80-20% recovery; pooled CV 9.%, mean recovery 07.3%; not corrected for pre-dilutions; 4X LoQ Recovery range %. 5 Recovery range %. 6 Recovery range %. 7 Recovery range %; minimum recommended dilution (MRD) :4. 8 Recovery range %; MRD :80. 9 Range The Human Neurology 3-Plex A assay reliably quantifies tau in plasma and CSF in healthy subjects. HD- Analyzer
3 Page 3 REPRODUCIBILITY AND REPEATABILITY CHARACTERISTICS OF THE TAU TEST Tau, Tau, Mean Tau, Figure 4: Reproducibility across reagent lots. Six native and spiked serum and plasma tau samples tested across 2 runs x 3 instruments each lot. Mean Tau, Figure 5: Reproducibility across instruments. Six native and spiked serum and plasma tau samples tested across 2 runs x 2 reagent lots each instrument. Table 2: Reproducibility precision of Neurology 3-Plex A Tau immunoassay across instruments and reagent lots. Reproducibility was determined with guidance from CLSI Protocol EP5-A. Six samples consisting of two serum panels, two plasma panels, and two tau controls were assayed in replicates of three for two runs on each of three instruments and two reagent lots. Analysis of variance (nested ANOVA) results are summarized in the following table. Mean Tau %CV, n=36 %CV, n=2 Between Lot %CV, n=2 Between Instrument %CV, n=3 Control Control Plasma Panel Serum Panel Plasma Panel Serum Panel Table 3: Repeatability precision of Neurology 3-Plex A Tau immunoassay. Repeatability was determined in a separate study from Table 2 with guidance from CLSI Protocol EP5-A. Six samples consisting of three serum panels, one plasma panel, and two tau controls were assayed in replicates of three at two separate times per day for five days using a single lot of reagents and calibrators. Analysis of variance (nested ANOVA) results are summarized in the following table. Mean Tau %CV, n=30 %CV, n=0 Between Day %CV, n=5 Control Control Plasma Panel Serum Panel Plasma Panel Serum Panel HD- Analyzer
4 Page 4 SUMMARY AND EXPLANATION OF THE Aβ42 TEST Aβ42 is a 42 amino acid proteolytic product from the amyloid precursor protein that has gained considerable attention as a biomarker correlating with Alzheimer disease (AD) onset, mild cognitive impairment, vascular dementia, and other cognitive disorders. Amyloid beta (Aβ) peptides (including the shorter Aβ38 and Aβ40 isoforms) are produced by many cell types in the body but the expression is particularly high in the brain. Accumulation of Aβ in the form of extracellular plaques is a neuropathological hallmark of AD and thought to play a central role in the neurodegenerative process. Substantial clinical validation has now been developed around disease relevance of cerebrospinal fluid (CSF) levels of Aβ42, and there follows a significant interest in measuring blood levels of this marker. Concentrations of Aβ42 in blood are over 00-fold lower than in cerebrospinal fluid, requiring high analytical sensitivity for its reliable measurement. GENERAL CHARACTERISTICS OF THE Aβ42 TEST Healthy donors 0000 Median [Aβ42]. Median [Aβ42] AEB %CV dose Aβ42, 00 0 Aβ42, Aβ42, Plasma CSF Figure : Aβ42 immunoassay calibration curve. Four-parameter curve fit parameters are depicted. Figure 2: Dose CV profile. Diluted serum was assayed in reps of 3 over multiple days and runs (53 determinations). Concentrations are uncorrected for pre-dilutions. Figure 3: [Aβ42] in EDTA plasma (n = 20) and CSF (n = 20) from healthy donors. Error bars depict median and interquartile ranges. Table : General characteristics of Neurology 3-Plex A Aβ42 immunoassay Calibration range 0-60 Dynamic range Lower limit of detection (2.5 SD; 3 reps x 2 estimates across 2 runs, 3 instruments, 2 lots; mean LoD 2 ) Lower limit of quantification 3 (2 runs, 3 instruments, 2 reagent lots, mean LoQ) 0.42 Spike-recovery: Plasma (Aβ42 spiked into 4 plasma samples at 5 and 50, mean 4 ) 63.% Spike-recovery: CSF (Aβ42 spiked into 4 CSF samples at 400 and 4000, mean 5 ) 6.7% Linearity (high tau plasma sample fractionally admixed with low tau plasma sample, mean of 0 levels 6 ) 93.0% Dilution linearity: Plasma (spiked plasma diluted 2X serially from MRD to 6-fold w/ Diluent; mean recovery 7 ) 92.6% Dilution linearity: CSF (CSF diluted 2X serially from MRD to 28-fold w/ Diluent; mean recovery 8 ) 98.3% Inter lot CV (Pool of CVs from 6 samples 9 tested with 2 reagent lots across 2 runs x 3 instruments) 3.5% Inter instrument CV (Pool of CVs from 6 samples 9 tested with 3 instruments across 2 runs x 2 reagent lots) 4.% Typical serum/plasma sample volume (Includes dead volume; see Package Insert for details) 46 µl Minimum serum/plasma sample volume (Depends on dilution procedure; see Package Insert for details) 3.5 µl CSF sample volume (Includes dead volume; See Package Insert for details) 2.3 µl s auto-diluted 4X; note: ranges may vary between calibrator lots 2 SD ; range %CV, 80-20% recovery; pooled CV 5.6%, mean recovery 08.7%; not corrected for pre-dilutions; 4X LoQ Recovery range %. 5 Recovery range %. 6 Recovery range %. 7 Recovery range %; minimum recommended dilution (MRD) :4. 8 Recovery range %; MRD :80. 9 Range The Human Neurology 3-Plex A assay reliably quantifies Aβ42 in plasma and CSF in healthy subjects. HD- Analyzer
5 Page 5 REPRODUCIBILITY AND REPEATABILITY CHARACTERISTICS OF THE Aβ42 TEST Aβ42, Aβ42, Mean Aβ42, Figure 4: Reproducibility across reagent lots. Six native and spiked serum and plasma Aβ42 samples tested across 2 runs x 3 instruments each lot. Mean Aβ42, Figure 5: Reproducibility across instruments. Six native and spiked serum and plasma Aβ42 samples tested across 2 runs x 2 reagent lots each instrument. Table 2: Reproducibility precision of Neurology 3-Plex A Aβ42 immunoassay across instruments and reagent lots. Reproducibility was determined with guidance from CLSI Protocol EP5-A. Six samples consisting of two serum panels, two plasma panels, and two Aβ42 controls were assayed in replicates of three for two runs on each of three instruments and two reagent lots. Analysis of variance (nested ANOVA) results are summarized in the following table. Mean Aβ42 %CV, n=36 %CV, n=2 Between Lot %CV, n=2 Between Instrument %CV, n=3 Control Control Plasma Panel Serum Panel Plasma Panel Serum Panel Table 3: Repeatability precision of Neurology 3-Plex A Aβ42 immunoassay. Repeatability was determined in a separate study from Table 2 with guidance from CLSI Protocol EP5-A. Six samples consisting of three serum panels, one plasma panel, and two Aβ42 controls were assayed in replicates of three at two separate times per day for five days using a single lot of reagents and calibrators. Analysis of variance (nested ANOVA) results are summarized in the following table. Mean Aβ42 %CV, n=30 %CV, n=0 Between Day %CV, n=5 Control Control Plasma Panel Serum Panel Plasma Panel Serum Panel HD- Analyzer
6 Page 6 SUMMARY AND EXPLANATION OF THE Aβ40 TEST Aβ40 is a 40 amino acid proteolytic product from the amyloid precursor protein (APP) that has gained attention as a biomarker correlating with Alzheimer disease (AD) onset, mild cognitive impairment, vascular dementia, and other cognitive disorders. Beta-secretase cleavage of APP initially results in the production of an APP fragment that is further cleaved by gamma-secretase at residues to generate two main forms of amyloid beta, Aβ40 and Aβ42. Amyloid beta (Aβ) peptides (including a shorter Aβ38 isoform) are produced by different cell types in the body, but the expression is particularly high in the brain. Accumulation of Aβ in the form of extracellular plaques is a neuropathological hallmark of AD and believed to play a central role in the neurodegenerative process. Aβ40 is the major amyloid component in these plaques and is thought to be an initiating factor of AD plaques. In healthy and disease states Aβ40 is the most abundant form of the amyloid peptides in both cerebrospinal fluid (CSF) and plasma (0 20X higher than Aβ42). Recent studies suggest that a decrease in the ratio of Aβ40/Aβ42 may indicate AD progression. GENERAL CHARACTERISTICS OF THE Aβ40 TEST Healthy donors Median [Aβ40] 209 Median [Aβ40] 6898 AEB %CV dose Aβ40, Aβ40, Figure : Aβ40 immunoassay calibration curve. Four-parameter curve fit parameters are depicted. Aβ40, Figure 2: Dose CV profile. Diluted serum was assayed in reps of 3 over multiple days and runs (8 determinations). Concentrations are uncorrected for pre-dilutions. 00 Plasma CSF Figure 3: [Aβ40] in EDTA plasma (n = 20) and CSF (n = 20) from healthy donors. Error bars depict median and interquartile ranges. Table : General characteristics of Neurology 3-Plex A Aβ40 immunoassay Calibration range 0-40 Dynamic range Lower limit of detection (2.5 SD; 3 reps x 2 estimates across 2 runs, 3 instruments, 2 lots; mean LoD 2 ) 0.96 Lower limit of quantification 3 (2 runs, 3 instruments, 2 reagent lots, mean LoQ) Linearity (high Aβ40 serum sample fractionally admixed with low tau serum sample, mean of 0 levels 4 ) 96.0% Dilution linearity: Plasma (spiked plasma diluted 2X serially from MRD to 6-fold w/ Diluent; mean recovery 5 ) 04.9% Dilution linearity: CSF (CSF diluted 2X serially from MRD to 28-fold w/ Diluent; mean recovery 6 ) 83.0% Inter lot CV (Pool of CVs from 6 samples 7 tested with 2 reagent lots across 2 runs x 3 instruments) 3.5% Inter instrument CV (Pool of CVs from 6 samples 7 tested with 3 instruments across 2 runs x 2 reagent lots) 4.% Typical serum/plasma sample volume (Includes dead volume; see Package Insert for details) 46 µl Minimum serum/plasma sample volume (Depends on dilution procedure; see Package Insert for details) 3.5 µl CSF sample volume (Includes dead volume; See Package Insert for details) 2.3 µl s auto-diluted 4X; note: ranges may vary between calibrator lots 2 SD ; range %CV, 80-20% recovery; pooled CV 2.5%, mean recovery 07.0%; not corrected for pre-dilutions; 4X LoQ Recovery range %. 5 Recovery range ; minimum recommended dilution (MRD) :4 6 Recovery range %; MRD :80. 7 Range The Human Neurology 3-Plex A assay reliably quantifies Aβ40 in plasma and CSF in healthy subjects. HD- Analyzer
7 Page 7 REPRODUCIBILITY AND REPEATABILITY CHARACTERISTICS OF THE Aβ40 TEST Aβ40, Aβ40, Mean Aβ40, Figure 4: Reproducibility across reagent lots. Six native and spiked serum and plasma Aβ40 samples tested across 2 runs x 3 instruments each lot. Mean Aβ40, Figure 5: Reproducibility across instruments. Six native and spiked serum and plasma Aβ40 samples tested across 2 runs x 2 reagent lots each instrument. Table 2: Reproducibility precision of Neurology 3-Plex A Aβ40 immunoassay across instruments and reagent lots. Reproducibility was determined with guidance from CLSI Protocol EP5-A. Six samples consisting of two serum panels, two plasma panels, and two Aβ40 controls were assayed in replicates of three for two runs on each of three instruments and two reagent lots. Analysis of variance (nested ANOVA) results are summarized in the following table. Mean Aβ40 %CV, n=36 %CV, n=2 Between Lot %CV, n=2 Between Instrument %CV, n=3 Control Control Plasma Panel Serum Panel Plasma Panel Serum Panel Table 3: Repeatability precision of Neurology 3-Plex A Aβ40 immunoassay. Repeatability was determined in a separate study from Table 2 with guidance from CLSI Protocol EP5-A. Six samples consisting of three serum panels, one plasma panel, and two Aβ40 controls were assayed in replicates of three at two separate times per day for five days using a single lot of reagents and calibrators. Analysis of variance (nested ANOVA) results are summarized in the following table. Mean Aβ40 %CV, n=30 %CV, n=0 Between Day %CV, n=5 Control Control Plasma Panel Serum Panel Plasma Panel Serum Panel HD- Analyzer
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