Cobas e601 module

The serum tubes were centrifuged, aliquoted, and frozen within four hours of the blood draw. The EDTA tubes with whole-blood samples were kept on ice, aliquoted, and frozen at − 80 °C within two hours of the blood draw. Confirmatory serology was performed based on the detection of anti-SARS-CoV-2 antibody against nucleocapsid, as measured by the Roche Cobas e601 module (Roche Diagnostics GmbH, Mannheim, Germany).

Fasting peripheral blood was collected in tubes containing ethylenediaminetetraacetic acid before and after NACT. The serum was separated by centrifugation at 3000 rpm for 10 min at room temperature, aliquoted, and stored at − 80 °C. The HMGB1 concentration was measured using the Sandwich ELISA of Shino-Test (Tokyo, Japan). The samples were added to the wells of microtiter dishes coated with anti-HMGB1 antibody. After incubation for 24 h at 4 °C, the plates were washed and incubated with a second enzyme-labelled antibody for 1 h at room temperature. A colored solution was added for 30 min, and the HMGB1 concentration was spectrophotometrically determined at 450 nm using a standard curve prepared with the kit. CEA, CK19, CA15-3, and E-cadherin levels were measured by enzymatic chemiluminescent immunoassay on an automatic immunoassay analyzer (Cobas e601 module, Roche, Sweden) using enzyme-linked immunosorbent assay (ELISA) kits from CanAg Diagnostics, Sweden and Roche Diagnostics, Sweden, according to the manufacturer’s instructions. Th17, CD4⁺CD25⁺ Tregs, and Treg cells were assessed using a flow cytometer (FACSymphony A1, BD Biosciences, USA). All samples were assayed in triplicates.

Roche Elecsys Anti-SARS-CoV-2 S electrochemiluminescence immunoassay (ECLIA) for the in vitro quantitative determination of antibodies (including IgG) against spike RBD of SARS-CoV-2 in human serum was performed on Roche Cobas e 601 module. According to the manufacturer, the correlation test between Roche Elecsys Anti - SARS-CoV-2 S units per mL and WHO International Standards for anti-SARS-CoV-2 immunoglobulins showed an excellent correlation (r² = 0.9992, slope = 0.972, intercept = 0.0072), thus allowing to consider specific Roche Elecsys Anti-SARS-CoV-2 S U/mL units equivalent to WHO International Standard BAU/mL (Binding Arbitrary Units per mL). Measuring range spanned from 0.4 BAU/mL to 2500.0 BAU/mL; values higher than 0.8 BAU/mL were considered positive.

The HPT axis test indicators include thyroid-stimulating hormone (TSH; normal value: 0.27–4.2 mU/L), triiodothyronine (T3; normal value: 1.3–3.1 mmol/L), thyroxine (T4; normal values: 62.0–164.0 mmol/L), free triiodothyronine (FT3; normal value: 3.6–7.5 pmol/L), and free thyroxine (FT4; normal value: 12.0–22.0 pmol/L). The HPA axis test indicators include ACTH (normal value: 5.0–78.0 ng/L) and 8:00 A.M. cortisol (PTC; normal value: 147.3–609.3 mmol/L). Fasting venous blood was taken by drawing 4 mL of cubital venous blood at 8 A.M. after overnight fasting. All analyses were performed using a Roche Cobas e601 module (Roche, Basel, Switzerland) via electrochemiluminescence. All reagents and calibrations were performed according to manufacturer instructions.

Quantitative evaluation of SARS-CoV-2 IgG anti-spike (S) glycoprotein antibodies was performed by the Liaison^® SARS-CoV-2 TrimericS IgG chemiluminescent immuno-assay (CLIA) on the Liaison XL (Diasorin^® S.P.A., Saluggia, Italy), according to the manufacturer’s instructions. The cut-off for positivity was 33.8 binding activity units (BAU)/mL. This assay showed an optimal correlation with the micro-neutralization test (negative and positive agreement of 100% and 96.9%, respectively) and was standardized against the WHO internal standard [42 (link)]. Regarding analytical performance, high sensitivity (98.7%) together with high specificity (99.5%) ensure accurate results. Samples containing levels of IgG anti-S antibodies above the measurement range (>2080 BAU/mL) were further diluted 1:10 using LIAISON^® TrimericS IgG Diluent. In order to exclude previous SARS-CoV-2 asymptomatic infection during the overall period considered, the anti-N response was determined using the Roche Elecsys^® Anti-SARS-CoV-2 electro-chemiluminescence immuno-assay (ECLIA) on the Cobas e 601 module (Roche^®, Mannheim, Germany).

AMH assay was performed using electrochemiluminescence technology on cobas e 601 module, Roche using Elecsys AMH kit. Results were expressed in ng/mL. Intraassay and interassay coefficient of variation were 1.7% & 3.5%, respectively.