Architect immunoassay

Peripheral blood samples (Li-Heparin coated tubes) were collected from 63 healthy controls (HC) and 227 MS patients in collaboration with the University Biobank Limburg (UBiLim). CMV and Epstein-Barr virus (EBV) status and titers (CMV IgG and EBV EBNA IgG) were determined in serum samples via Vidas ELFA (bioMérieux, Marcy l’Etoile, France) and Architect immunoassay (Abbott, Illinois, USA). Clinical data are presented in Table 1; there were no significant differences between CMV positive or negative donors, neither in MS patients nor in healthy controls.Table 1

Study subjects for CD4⁺CD28^null T cell analysis.

	MS patients		Healthy controls
	CMV+	CMV−	CMV+	CMV−
Number	100	127	24	39
Age (y)	47 ± 13	44 ± 14	32 ± 9	32 ± 10
Male/Female (ratio)	26/74 (0.35)	35/92 (0.38)	7/17 (0.41)	14/25 (0.56)
EBV serostatus (−/border/+)	0/2/64	2/0/87	NA
Disease duration range	1 mo–40 y	0 mo–37 y	NA
EDSS range	0–7	0–7.5	NA
Disease type			NA
CIS	5	6
RR-MS	63	78
CP-MS	32	43
Treatment#			NA
No treatment	46	56
IFNβ	25	47
Glatiramer acetate	15	10
Natalizumab	9	6
Alemtuzumab	2	4
Teriflunomide	/	3
Dimethyl fumarate	2	/
Methotrexate	1	1

^#Within 3 months before blood collection.

MS, multiple sclerosis; EDSS, expanded disability status scale; CIS, clinically isolated syndrome; RR, relapsing remitting; CP, chronic progressive; IFNβ, interferon beta; CMV, cytomegalovirus; NA, not applicable.

Random plasma C-peptide was measured using an Abbott Architect immunoassay. In-house studies have demonstrated a CV of 7% at 7 pmol/l and of 15% at 4 pmol/l. Based on these data, we report 4 pmol/l as the limit of quantification in this study. HbA_1c was measured by ion-exchange high performance liquid chromatography using the Arkray Adams A1c automated platform (A. Menarini Diagnostics, Winnersh, UK).

Whole blood samples were collected in CPT tubes from 274 of the 320 learners. Plasma and peripheral blood mononuclear cells (PBMCs) were isolated by centrifugation. The presence of nucleocapsid protein (NP)-specific IgG was determined with the Abbott Architect immunoassay. IgM and IgG antibody titers against the NP and the receptor-binding domain (RBD) of the spike protein were measured using standard ELISA⁶ . Specific SARS-CoV-2 neutralizing antibodies were measured using focus reduction neutralizing assays⁶ . IFN-γ producing T cells following stimulation with overlapping peptide pools were determined using mAB ELISpot plates and PBMCs from 34 SARS-CoV-2 seropositive samples and 34 age- and sex-matched uninfected controls from enrolled participants⁷ . Circulating immune and inflammatory mediators (e.g., TNF-α, IL-6) were also measured using the Human 45-Plex kit from R&D⁷ . Immunophenotyping to identify innate and adaptive cells was done using flow cytometry in 15 SARS-CoV-2 seropositive samples and 15 age- and sex-matched controls⁷ . As an added benefit to the risk of phlebotomy, learners were offered a non-fasting lipid screening, measured by enzymatic reflectance spectrophotometry. This screening test is highly recommended for children and adolescents by the American Academy of Pediatrics but underutilized^{8 (link)}.

CPT tubes were processed for plasma up to 16 hours after collection, but typically within 8 hours of collection. Plasma collected from participants at in-person study visits and serum self-collected by participants via Tasso devices were both tested via ELISA, using the receptor binding domain (RBD) of the SARS-CoV-2 spike protein to detect total SARS-CoV-2 immunoglobulin (Ig) in plasma [23 (link)]. Specimens were also tested via the Abbott Architect Immunoassay, using the nucleocapsid protein to detect total SARS-CoV-2 IgG in plasma [24 (link)].

Peripheral blood samples were collected from each participant before any HCC treatment. Serum samples were separated from the blood samples by centrifugation at 700g for 10 min. Serum samples were subsequently aliquoted and frozen at -80°C until testing. The sample storage facilities and conditions were standardized at each study site. Serum samples were sent to the Abbott testing centers on a regular basis, and liquid nitrogen was used during transportation. AFP and PIVKA-II serum concentrations were measured with the commercially available ARCHITECT immunoassay per the defined protocol (Abbott Diagnostics). Serum measurements of AFP-L3% were determined utilizing the Fujirebio assay (Fujirebio Diagnostics). The lower limits of detection for AFP, PIVKA-II, and AFP-L3% assays were 0.6 ng/mL, 5.0 mAU/mL, and 0.5%, respectively. The technicians performing the laboratory tests were blinded to the diagnosis of the participants. No adverse events related to serum sample collection were observed.