Simoa human neurology 4 plex e assay

Biomarkers were tested for 1986 individual plasma samples from the ADCC. P-tau181 concentration was measured using the Simoa P-tau181 Advantage Kit, whilst Aβ₄₀, Aβ₄₂, NfL and GFAP concentrations were measured using the Simoa Human Neurology 4-Plex E (N4PE) assay (Quanterix). The measurements were performed in one round of experiments using one batch of reagents with the analysts blinded to diagnosis and clinical data. All measurements for all five analytes were above the limit of detection of the assays. Intra-assay coefficients of variation were below 10%. These data were then matched to phenotype information. Thirty-nine samples were removed at this stage based on missing/mismatching data for age and gender or due to ID duplication, leaving 1947 individuals for further analysis. Samples were excluded for each biomarker analysis on a case-by-case basis, based on outlier thresholds calculated using Median Absolute Deviation (MAD).^{30 (link)} This method is more robust to remote outliers than the mean and SD method and copes better with skewed data due to its reliance on non-parametric measures of central tendency and variation. Pearson’s correlations between biomarkers were calculated for the 1735 samples which had no outlier measurements for any biomarker. Details of biomarker distributions are in Table 1.

We screened serum samples at chosen time points of the SARS-CoV-2 disease course for the expression of pro- and anti-inflammatory cytokines using the Simoa CorPlex Human Cytokine 10-plex Panel 1 assay (Quanterix, Billerica, MA, USA). We targeted the following analytes: IFNγ, IL1β, IL4, IL5, IL6, IL8, IL10, IL12p70, IL22 and TNFα. In addition, markers for neuroinflammation were quantified using the Simoa Human Neurology 4-Plex E assay (Quanterix) for Abeta 40 (Aβ40), Abeta 42 (Aβ42), Glial Fibrillary Acidic Protein (GFAP™) and Neurofilament light (Nf-L). Data for the 10 CorPlex were acquired and analyzed on the SP-X Imaging and Analysis System™ (Quanterix), whereas the Neurology 4-Plex E assay was analyzed on the Simoa^® HD-X Analyzer™ (Quanterix). Both kits were used according to the manufacturer’s instructions.

Blood samples were obtained by venepuncture and collected in EDTA tubes. They were centrifuged to isolate plasma, aliquoted and stored at −70°C until further analyses. Plasma assays were conducted at the UK Dementia Research Institute biomarker laboratory. Plasma samples were thawed on wet ice, centrifuged at 500× g for 5 min at 4°C. Calibrators (neat) and samples (plasma: 1:4 dilution) were measured in duplicates. The plasma assays measured were the Quanterix Simoa Human Neurology 4-Plex E assay (measuring Aβ40, Aβ42, GFAP and NfL) and the Quanterix Simoa p-tau181 measuring p-tau181 of the human tau protein. Assays were performed using the Simoa-HD1 according to the manufacturer’s protocol (Quanterix Corp, Billerica, Massachusetts, USA) (Rissin et al). All samples were analysed at the same time using the same batch of reagents. A four-parameter logistic curve fit data reduction method was used to generate a calibration curve. Two control samples of known concentration of the protein of interest (high-control and low-control) were included as quality control. The mean coefficient of variation percentage for p-tau181 was 6.29, for NfL 4.08, for Aβ42 3.06, for Aβ40 2.55 and for GFAP 4.12.