Evidence-based standardized sample handling protocol for accurate blood-based Alzheimer's disease biomarker measurement: Results and consensus of the Global Biomarker Standardization Consortium

Abstract

Introduction: Blood-based biomarkers (BBMs) have revolutionized Alzheimer's disease diagnosis and monitoring. Their pre-analytical stability requires scrutiny. This study assessed pre-analytical effects to inform a standardized sample handling protocol.

Methods: Assessed pre-analytical variations included collection tube type, hemolysis, centrifugation settings, centrifugation/storage delays, tube transfers, and freeze-thawing (n = 15/experiment). Phosphorylated tau (pTau) isoforms were measured with Simoa, Lumipulse, MesoScale Discovery, and immunoprecipitation-mass spectrometry. Amyloid-beta (Aβ42, Aβ40), glial fibrillary acidic protein (GFAP), and neurofilament light (NfL) protein were measured with Simoa.

Results: All assessed BBM levels varied by over 10% by collection tube type. Aβ peptides were the most sensitive, and their levels declined >by more than 10% under storage and centrifugation delays, more steeply at room temperature (RT) compared with 2°C to 8°C. NfL and GFAP levels increased by more than 10% upon RT/-20°C storage. pTau isoforms demonstrated stability across most pre-analytical variations.

Discussion: We established an evidence-based handling protocol to ensure reliable sample handling for neurological BBMs upon adoption in clinics, trials, and research.

Highlights: Sample handling protocols can mitigate pre-analytical effects on BBM results. We developed an evidence-based, expert-consensus plasma sample handling protocol. Primary collection tube and delays to centrifuging or freezing impact AD BBMs. Plasma pTau217 is highly resistant to pre-analytical sample handling variations. Plasma Aβ42 and Aβ40 were most sensitive to pre-analytical variations.

Keywords: amyloid beta; glial fibrillary acidic protein; neurofilament light; phosphorylated tau; plasma biomarkers; pre‐analytical variability; pre‐analytics; sample handling; stability.

FIGURE 1

Overview of the 11 pre‐analytical experiments (n = 15/experiment) performed on fresh blood samples covering the complete blood processing workflow, including sample freeze‐thawing. Each pre‐analytical experiment has a reference condition and two to four experimental pre‐analytical variations. All experiments were performed on K₂EDTA plasma samples, except experiment 1, which varied the primary blood collection tube. The hemolysis experiment was based on Delgado's method (strategy 1). Note, experiment 11, freeze‐thaw cycles, had only n = 5 for condition +4FT. FT, freeze‐thaw; Hb, hemoglobin; hr, hour; K₂EDTA, dipotassium ethylenediaminetetraacetic acid; Li, lithium; Na, sodium; RT, room temperature.

FIGURE 2

Recovery% plots visualizing the effect of 11 pre‐analytical variations on pTau181 and pTau217 levels as measured by Lumipulse, Simoa, MSD, and LC‐MS and npTau217 levels, as well as pTau217/npTau217 (%pTau217) ratio as measured by LC‐MS. Levels measured in the experimental condition were normalized to levels measured in the reference conditions, i.e., centrifugation of K₂EDTA blood tubes at 1800 × g at RT after standing at RT for 30 min, followed by plasma separation and aliquoting in low binding polypropylene screw‐capped storage tubes and storage at −80°C without any delays. Horizontal dashed lines at 90% and 110% represent an allowable 10% change in experimental conditions compared to the reference condition. Box plots represent the median and interquartile range (IQR), whiskers represent the extreme data points that are within 1.5 times the IQR. LC‐MS serum measurements were below the limit of detection for most samples (data not shown). EDTA, ethylene diamine tetraacetic acid; FT, freeze‐thaw; LC‐MS, liquid chromatography‐mass spectrometry; Li‐heparin, lithium‐heparin; MSD, Meso Scale Discovery; Na‐citrate, sodium‐citrate; npTau, non‐phosphorylated tau; pTau, phosphorylated tau; RT, room temperature; Simoa, Single molecule array.

FIGURE 3

Recovery% plots visualizing the effect of 11 pre‐analytical variations on the levels of Aβ42, Aβ40, NfL, GFAP, and Aβ42/40 and pTau217/Aβ42 ratios as measured with Simoa HDX (pTau217 in the ratio, measured with Lumipulse). Levels measured in experimental conditions were normalized to levels measured in the reference conditions, i.e., centrifugation of K₂EDTA blood tubes at 1800 × g at RT after standing at RT for 30 min, followed by plasma separation and aliquoting in low binding polypropylene screw‐capped storage tubes and storage at −80°C without any delays. Horizontal dashed lines at 90% and 110% represent an allowable 10% change in the experimental conditions compared to the reference condition. Box plots represent the median and interquartile range (IQR), whiskers represent the extreme data points that are within 1.5 times the IQR. Aβ42, beta amyloid protein (1‐42); Aβ40, beta amyloid protein (1‐40); EDTA, ethylene diamine tetraacetic acid; FT, freeze‐thaw; GFAP, glial fibrillary acidic protein; Li‐heparin, lithium‐heparin; Na‐citrate, sodium‐citrate; NfL, neurofilament light chain; RT, room temperature; Simoa, Single molecule array.

FIGURE 4

Evidence‐based sample handling protocol for accurate neurological blood‐based biomarker (BBMs) measurement. The flowchart is tailored to mitigate any pre‐analytical effect on the most sensitive neurological biomarker. When measuring a specific marker, more flexibility at certain steps might be possible, which can be derived from the “allowed deviations” column. Of note, the advised, optimal sample handling protocol includes a total processing time of less than 2 h. Hemolysis degree can be measured or visually inspected using the color grade presented in the figure or by comparing it to the commonly used hemolysis palettes. EDTA, ethylene diamine tetraacetic acid; FT, freeze‐thaw; Hb, hemoglobin; max, maximum; RT, room temperature.