Hd x analyzer

Serum samples were collected through venipuncture. Samples were allowed to clot at room temperature, subsequently centrifuged at 1,800 g for 10 min, and stored at −80°C in polypropylene tubes. Samples were thawed at room temperature and centrifuged at 10,000 g for 10 min. GFAP and NfL concentrations of ETPKU patients and pediatric HC were quantified with the Simoa^® Human Neurology 2-Plex B Kit (Quanterix) on a HD-1 analyzer^TM (Quanterix) according to manufacturer's instructions. GFAP and NfL concentrations in adult HC were quantified with the Simoa^® Human Neurology 4-Plex B Kit (Quanterix) on a HD-X analyzer^TM (Quanterix). All samples were measured in duplicates by aspirating two aliquots from a single well. The mean concentration of the two measurements was used for the analysis. In one patient, only one measurement could be obtained for each protein of interest. Given the low intra-sample variation between duplicate readings for the remaining samples in NfL and GFAP, these values were included in the analysis. The intra-assay coefficient of variation (CV) was calculated between duplicate readings for each protein. The mean CVs was 6.1% for GFAP and 7.1% for NfL. Samples with CVs above 20% were excluded from further analysis (GFAP n = 1, NfL n = 2). The inter-assay CV of two quality control samples over the two runs was on average 5.6% CV for GFAP and 1.9% CV for NfL.

Venipuncture blood was collected within 48 h of injury in plastic dipotassium ethylenediaminetetraacetic acid tubes, immediately placed on ice, centrifuged (15 min, 1,500g, room temperature), and stored at −80°C until analysis. Plasma protein quantifications were analyzed using the Single Molecule Array p-tau181 V2 Advantage kit (Quanterix, Lexington, MA) for measurement of p-tau181 protein on HD-x Analyzer^TM. The samples were processed according to the manufacturer's instructions. Researchers were blind to the sample's demographic and clinical information. Samples that reported below the lower limits of quantification (0.338 pg/ml) and had a coefficient of variation higher than 20% were excluded from the analysis. The protein quantification in both cohorts was within the assay range.

Plasma samples were diluted 4000-fold, and the total IgG and IgM levels against the SARS-CoV-2 spike protein were measured using a custom Single Molecule Array (Simoa) assay as described [29 (link)] on an automated HD-X Analyzer (Quanterix, Billerica, MA, USA), to provide a quantitative reference for anti-spike antibody titers in the plasma samples. Normalized mean Average Enzymes per Bead (AEB) levels were calculated using a standard set of calibrators produced by serially diluting a large volume of plasma from seroconverted individuals. Antibody concentrations were estimated using a calibration curve of recombinant anti-SARS-CoV-2 antibodies [30 (link)].

The concentration of IFNα protein levels in plasma diluted 1:1 in PBS was quantified with Single molecule array (Simoa) digital ELISA on a HD-X Analyzer (Quanterix, Billerica, MA). To prevent false positive results the Simoa assay contained an inhibitor for heterophilic antibodies. If the concentration in a sample was below the lower limit of quantification (70 fg/ml) its value was adjusted to 35 fg/ml. IFNα positivity was defined as protein levels ≥ 136 fg/ml, representing three standard deviations above mean IFNα protein concentration among healthy blood donors [30 (link)].

Serum concentrations of neurofilament light chain (NF-L) and glial fibrillary acidic protein (GFAP) in controls and ischemic stroke patients were analyzed at the Department of Biochemistry and Immunology, Lillebaelt Hospital, Vejle being accredited by Danish Accreditation Fund (DANAK) according to the ISO 15189:2012 standard that specifies requirements for quality and competence in medical laboratories. Both NF-L and GFAP were measured blinded to clinical data by single molecule array technology (Simoa, HD-X Analyzer (Quanterix, Billerica, MA, USA)) [40 (link)], using the commercially available 2-Plex assay for the quantitative determination of NF-L and GFAP in human serum (Quanterix). Quality control was performed using five internal controls in each run. Internal controls were prepared from commercially available control material provided by the manufacturer in addition to an in-house prepared serum pool. The in-house serum pool was used as an internal control and included in each run for evaluating and monitoring assay performance over time. The total analytical variation for the included controls were 10–16% total analytical CV for NF-L and 8–14% total analytical CV for GFAP. Lower limits of detection for NF-L and GFAP were 0.038 and 0.211 pg/mL, respectively, whereas the lower levels of quantification were 0.174 and 0.686 pg/mL, respectively.