Effect of blood collection tube containing protease inhibitors on the pre-analytical stability of Alzheimer’s disease plasma biomarkers

INTRODUCTION The reliability of plasma Alzheimer’s disease (AD) biomarkers can be compromised by protease-induced degradation. This limits the feasibility of conducting plasma biomarker studies in environments that lack the capacity for immediate processing and appropriate storage of blood samples. We hypothesized that blood collection tube supplementation with protease inhibitors can improve the stability of plasma biomarkers at room temperatures (RT). This study conducted a comparative analysis of blood biomarker stability in traditional ethylenediaminetetraacetic acid (EDTA) tubes versus BD™ P100 collection tubes, the latter being coated with a protease inhibitor cocktail. The stability of six plasma AD biomarkers was evaluated over time under RT conditions.

We evaluated three experimental approaches. In Approach 1, pooled plasma samples underwent storage at RT for up to 96 hours. In Approach 2, plasma samples isolated upfront from whole blood collected into EDTA or P100 tubes were stored at RT for 0h or 24h before biomarker measurements. In Approach 3, whole blood samples were collected into paired EDTA or P100 tubes, followed by storage at RT for 0h or 24h before isolating the plasma for analyses. Biomarkers were measured with Single Molecule Array (Simoa) and immunoprecipitation-mass spectrometry (IP-MS) assays.

Both the IP-MS and Simoa methods revealed that the use of P100 tubes significantly improved the stability of Aβ42 and Aβ40 across all approaches. Additionally, the Aβ42/Aβ40 ratio levels were significantly stabilized only in the IP-MS assay in Approach 3. No significant differences were observed in the levels of plasma p-tau181, GFAP, and NfL for samples collected using either tube type in any of the approaches.

Supplementation of blood collection tubes with protease inhibitors could reduce the protease-induced degradation of plasma Aβ42 and Aβ40, and the Aβ ratio for IP-MS assay. This has crucial implications for preanalytical procedures, particularly in resource-limited settings. Competing Interest Statement The authors have declared no competing interest. Funding Statement This study was supported by the Joseph A. Massaro Alzheimers Research Fund of The Pittsburgh Foundation (#AD2018-97918) and the NIH (P30 AG066468). TKK was supported by the NIH (1 R01 AG083874-01, 1 U24 AG082930-01, 1 RF1 AG052525-01A1, 5 P30 AG066468-04, 5 R01 AG053952-05, 3 R01 MH121619-04S1, 5 R37 AG023651-18, 2 RF1 AG025516-12A1, 5 R01 AG073267-02, 2 R01 MH108509-06, 5 R01 AG075336-02, 5 R01 AG072641-02, 2 P01 AG025204-16), the Swedish Research Council (Vetenskapradet; #2021-03244), the Alzheimers Association (#AARF-21-850325), the Swedish Alzheimer Foundation (Alzheimerfonden), the Aina (Ann) Wallstroms and Mary-Ann Sjobloms stiftelsen, and the Emil och Wera Cornells stiftelsen. Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The ADRC study was approved by the University of Pittsburgh Institutional Review Board (MOD19110245-023). I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Footnotes ↵# Joint senior authors Data Availability All data produced in the present study are available upon reasonable request to the authors