H. Zhang1, J. Liu1, N. Zhang1, A. Jeromin2, Z.J. Lin1
1. Frontage Laboratories Inc., Exton, Pennsylvania, USA; 2. ALZpath. Inc, Carlsbad, California, USA
Corresponding Author: Zhongping John Lin, Frontage Laboratories Inc., 700 Pennsylvania Drive, Exton, Pennsylvania 19341, USA, Email: JLin@frontagelab.com
J Prev Alz Dis 2024;
Published online August 10, 2024, http://dx.doi.org/10.14283/jpad.2024.155
Abstract
BACKGROUND: Both blood (plasma and serum) and CSF tau phosphorylated at Threonine 217 (pTau217) have been shown to be able to discriminate early to mild Alzheimer’s disease (AD) from non-AD neurodegenerative disorders and healthy controls with high sensitivity and specificity.
OBJECTIVES: We report the full validation of the ALZpath ultra-sensitive pTau217 research-use only (RUO) assays for human EDTA plasma, serum, and CSF using the Quanterix Simoa HD-X platform, in support of clinical trials and early diagnosis of Alzheimer’s disease.
METHODS: The ALZpath pTau217 Simoa Advantage v2 kit from ALZpath/Quanterix was used for method development and validation of pTau217 in human plasma, serum, and CSF. All three method validations have followed the 2018 FDA BMV and 2022 ICH M10 guidelines.
RESULTS: Validation of pTau217 quantitation in human plasma, serum, and CSF demonstrated that the validated pTau217 assay has an analytical LLOQ of 0.00977 pg/mL. It is one of the most sensitive assays with a minimal required sample volume of 33.3 µl for plasma and serum, and 5 µl for CSF, and is relatively cost-effective, compared to the currently published data. The minimum required dilution (MRD) for plasma, serum, and CSF were determined. The analyte passed short-term stability tests. It was also tolerant of moderate hemolytic, and lipemic interferences in plasma and serum. pTau217 was detected in 100% of tested healthy control plasma, 90.0% of healthy control serum, 92.3% of healthy control CSF samples, and 100% of AD plasma and CSF samples. The validated assay was successfully applied in the analysis of AD samples, with data showing dramatic differences in the pTau217 levels between healthy control subjects and AD patients in both plasma and CSF.
CONCLUSION: Validation of the ALZpath pTau217 Simoa assay in human plasma, serum, and CSF demonstrates ultra sensitivity, high accuracy, and assay robustness. Together with other established and sensitive assays for pTau181, GFAP, and NfL, these assays have immediate utility for application in clinical trials and can play a role in the early diagnosis of neurodegenerative diseases.
Key words: pTau217, method validation, human plasma, serum, CSF.
Introduction
Alzheimer’s disease (AD), a progressive neurodegenerative disorder, neuropathologically characterized with β amyloid (Aβ) plaques (1-3) and neurofibrillary tau tangles (4-5), is the most common form of dementia (accounting for 60-70% of cases) (6). According to the Alzheimer’s Association 2023 Report, 6.7 million people in the United States are living with Alzheimer’s dementia and the number is projected to reach 13.8 million by 2060 (7). Additionally, more than 11 million family members and unpaid caregivers provided an estimated 18 billion hours to people with Alzheimer’s and other dementias in 2022 (8). AD disorder manifests with a progressive characteristic complex and multifactorial sequence, ranging from asymptomatic to mild cognitive impairment to severe dementia (9-10). Enormous efforts are input to the prognosis and diagnosis and in finding effective treatments for this intractable devastating disorder (11-16). Due to the progressive nature of AD, it is essential for early diagnosis/prognosis, to enhance medical decision making, and allow sufficient time to intervene in the disease process for AD patients.
The development and discovery of blood-based biomarkers with high sensitivity, specificity, and accuracy, are urgently required for early sentinel screening to detect AD ahead of the progression to characteristic dementia stages (17, 18). Promising progress has been shown in blood-based assay development in AD research, which is still constantly advancing, together with the development of various and more sensitive platforms. Two major examples are amyloid beta (Aβ42/Aβ40) PET imaging for Aβ plaques formed by misfolded amyloid β (1-2, 19), and PET with high affinity tau and neurofibrillary tangles (from paired helical filaments) formed by misfolded hyperphosphorylated tau proteins (4, 20). Other examples include CSF Aβ42/Aβ40 and CSF tau (21-24), volumetric MRI imaging (25) and machine learning (26).
More recently, blood-based biomarkers have been extensively and intensively explored for AD diagnosis and prognosis. Moreover, there are cost-effective advantages of blood draws compared with existing PET and CSF diagnostic aids, which include being minimally invasive with greater convenience for the patient and provider. These advantages also enable wider availability to patients of diversified ethnicity and incomes. Promising blood-based AD biomarkers are Aβ40/42, neurofilament light chain (NfL), glial fibrillary acidic protein (GFAP), total tau, and the hyperphosphorylated tau including pTau181, pTau217, pTau231 and pTau235, which have been frequently selected as biomarkers for Alzheimer disease (27-33). One or more of the biomarker assay platforms, such as IP-LC/MS, ELISA, MSD, and Simoa, have been tested with varying success. Other biomarkers, such as α- and β-synuclein, clusterin, BACE1, TREM2, YKL-40, IP-10, neurogranin, synaptotagmin, TDP-43, ferritin, VILIP-1, VAMP-2, SNAP-25, may be potential biomarkers of clinical progression, however they have not been established as measures in blood yet (34-37).
Among these biomarkers, pTau217 was recently discovered as a prominent AD blood-based biomarker in which it can discriminatively predict AD development decades ahead of symptom onset, diagnosis of which was once achieved only by postmortem examination (38-42). The dramatic early elevation of pTau217 is shown to be detected much earlier (two decades) than pTau-181 and other biomarkers in distinguishing AD from non-AD neurodegenerative diseases (35). Additionally, AD-related pTau217 shows a greater separation between AD patients and healthy individuals, thus potentially serving as one of the best biomarkers for clinical trial patient enrichment and companion diagnosis of neurodegeneration, specifically for Alzheimer’s disease. Here we report the full-validation results of the quantitation of pTau217 on the Quanterix Simoa HD-X platform. Results demonstrate that the ALZpath pTau217 assay is ultrasensitive with a very broad dynamic range (from 0.00977 pg/mL to 10 pg/mL). The assay demonstrates a very robust and reproducible detectability for the quantitation of pTau217 in human plasma, serum, and CSF samples.
Methods
Commercial healthy and AD-diseased human plasma (K2EDTA), serum, and CSF samples were purchased from BioIVT. Commercial ALZpath pTau217 Simoa Advantage v2 Kits (Cat No: 104371) were used for method validation of pTau217 quantitation in human plasma, serum, and CSF. Quanterix Simoa HD-X was used as the platform for pTau217 method development and validation.
Calibration standards and QC samples: calibration standards were prepared with kit-provided antigens in assay buffer. QC samples were prepared in buffer (LLOQ, ULOQ and HQC) and in authentic matrix human plasma, serum, and CSF (MQC and LQC) with endogenous analyte. Watson LIMS 7.6 and Microsoft excel were used for data analysis.
All the three method validation procedures were designed and conducted following the 2018 FDA BMV (40) and 2022 ICH M10 (41) guidelines, and SOPs at Frontage Laboratories.
Results
pTau217 Assay with ALZpath pTau217 v2 kit is ultra-sensitive for quantitation of the analyte pTau217 in human plasma, serum, and CSF.
Validation of pTau217 quantitation in human plasma, serum, and CSF was demonstrated, through one intra- and five inter-, for a total of six Accuracy & Precision runs, conducted by two independent trained analysts (Fig. 1, Table 1 and 2, Table s1 to s8). Results demonstrated that the validated pTau217 assay has an analytical LLOQ of 0.00977 pg/mL, with very high precision and accuracy (the %CV and %RE of all tested standards and QCs were within single digits). Thus, the ALZpath pTau217 v2 kit is one of the most sensitive pTau217 assays, with a minimal required sample volume of 33.3 µl for human plasma and serum, and 5 µl for human CSF. The MRDs were determined as 1:3 for plasma and serum, and 1:20 for CSF (healthy human plasma, serum and CSF samples spiked with the analyte pTau217). Human plasma and CSF passed all the 5-serial dilution linearity tests, and human serum passed dilution to 1:12 (Table s1 to s3, Table s9 to s11).
Figure 1. Representative Standard Curve for pTau217 with Simoa HD-X Platform
Table 1. Precision & Accuracy All Standards in pTau217 Validation
Table 2. Intra-run and Inter-run Accuracy and Precision
pTau217 Assay with ALZpath pTau217 v2 kit is robust and passed Short-Term Stability tests, one-month Long-Term Stability test, as well as lipemic and hemolysis interference tests.
The analyte pTau217 passed the Short-Term Stability tests. As shown in Table s12 to s14, the pTau217 test passed the criteria (2/3 stressed QC samples at each tested QC levels with % RE within ±20% and %CV ≤20%, in comparison to the unstressed QC samples) of storing biosamples at room temperature for 6 hours, 4°C refrigeration for 24 hours, and up to 5 freeze/thaw (between -70°C and room temperature) cycles prior to testing. It also passed the one-month Long-Term Stability test at -70°C before the preparation of this manuscript (Table s15-s17) (six-month and one-year Long-Term Stability tests will be performed later). Interference tests for human plasma and serum demonstrated tolerance for moderate hemolytic and lipemic interferences in plasma and serum: no interference was observed for the determination of pTau217 concentration in human plasma and serum in the presence of 1% and 3% hemolysis, and 150 and 300 mg/dL of lipid (Table s18 to s19).
pTau217 Assay with ALZpath pTau217 v2 kit can readily detect the analyte pTau217 in human plasma, serum, and CSF samples.
pTau217 was detected in 100% of tested normal plasma, 90.0% of normal serum and 92.3% of normal CSF samples, and 100% of AD plasma and CSF samples (Table 3). The validated assay was successfully applied in the sample analysis with data showing in the pTau217 levels between normal subjects and AD patients in both plasma and CSF.
Table 3. pTau217 Detectability in Human Plasma, Serum, and CSF
N.D.: not determined.
Discussion
With the urgent needs for early detection of Alzheimer’s disease, especially for the accurate and specific prediction of this devastating neurodegenerative disorder, in the aim for the early screening and recruitment stratification of preclinical and prodromal tests, biomarkers and the related assays and platforms are vigorously developed and evolving with challenges and varied success. Blood-based biomarkers are increasingly the top focuses for development, due to their ease of use and availability. Blood-based biomarkers are less invasive, cost-effective, and comparatively efficient when compared to the much more expensive and invasive PET and CSF testing for AD diagnosis and patient monitoring.
The method offers a wide dynamic range and assay sensitivity at fg/mL levels, enabling the detection of pTau217 in both normal and diseased human blood and CSF samples. This assay has been successfully developed and fully validated with ultra sensitivity, high accuracy, and great assay robustness at Frontage Laboratories. In addition, the kit lot-to-lot variability was evaluated over 3 ALZpath pTau217 kit lots by the vendor and the test results did meet the acceptance criteria of % Difference for all samples within +/- 15% (data not shown). Given these appealing qualities for clinical applications, the ALZpath pTau217 assay presents as one of the best blood-based AD biomarkers diagnostic supporting kits that can be immediately applied to clinical research and clinical trials.
In addition, blood tests of the sensitivity and robustness presented here for the ALZpath pTau217 have the potential to revolutionize AD clinical workflows where they can be effectively implemented to prescreen AD patients to enable effective stratification at all AD stages for clinical trials and patient care, given the standardization of baselines for AD biomarkers, including three relevant guidelines: Diagnostic and Statistical Manual of Mental Disorders (DSM-5TR) in 2022; the National Institute on Aging-Alzheimer’s Association (NIA-AA) definition as revised in 2011; and the International Working Group criteria as revised in 2010 (10). The ALZpath pTau217 assay has additionally been tested in independent cohorts by other laboratories and the reference ranges and clinical cutoffs were calculated. These independent evaluations demonstrated that ALZpath plasma pTau217 presented significant correlations to amyloid PET and tau PET results while also establishing very high discriminative accuracy in differentiating Aβ+ from Aβ- (47-49). The ALZpath assay has been developed on the semi-automated single-molecule assay (Simoa) platform, branded as ALZpath Dx, and launched for clinical use as a robust and scalable plasma-based ultra-sensitive laboratory-developed test (LDT) (50). It is also currently being established by evaluation in independent clinical cohorts with multiple co-morbidities to advance to an in-vitro diagnostic (IVD) test platform. This will greatly facilitate clinical screening and resultant disease stratification in AD services.
To test the applicability for AD screening in human sample analysis, we did a small-scale sample screening. As shown in Table 3, the mean concentrations for the plasma sample in the healthy and AD patients were 0.235 and 0.697 pg/mL, respectively, with a marked difference (about 3-fold) between the healthy and AD patient plasma samples. More dramatically in human CSF samples, the mean concentrations of the healthy and AD patients were 5.64 and 46.8 pg/mL, respectively, with a difference of over 8-fold between the control and AD patient CSF samples (see Figure 2). However, due to the very limited sample sizes in the current assay test, the clinical utility of this assay needs further evaluation through the testing of larger sample sizes in more complex and real-world clinical populations. For example, the release of some recent clinical trial results using the ALZpath pTau217 assay, will provide further insights into the usefulness and applicability of this assay.
Figure 2. AD and Control pTau217 Obtained from Plasma (A) and CSF (B) in pg/mL
In conclusion, the current fully validated assay quantitating pTau217 in human plasma, serum, and CSF, together with already established sensitive pTau181, GFAP, and NfL assays, can be utilized as a tool for application in clinical trials and the early diagnosis of neurodegenerative diseases.
Acknowledgements: Frontage Laboratories provided the funding for this study. We thank Frontage Biomarker team members and all PM, QC and QA staff who provided support for this study.
Funding: There is no external funding for this study. This study was funded internally by Frontage Laboratories.
Conflict of Interest Disclosure: Andreas Jeromin is an employee of ALZpath, Inc, and has stock options. All the other authors are employees of Frontage Laboratories Inc. and declare no conflict of interests for this study.
Ethical standards: Approval from the institutional review board and ethics committee was obtained for this study.
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