This study was designed as a prospective observational trial and was approved by the Ethical Committee of Motol University Hospital. Only patients with signed informed consent and fulfilling inclusion criteria (1. ESID / ICON diagnostic criteria for CVID (3, 4), at least 3-month interval of follow up and exposition to the regular immunoglobulin replacement therapy) and exclusion criteria (previous exposition to the immunosupressive/corticosteroid therapy and/or the diagnosis of AIT or T1D) were enrolled and followed for up to 2 years.
The antibodies were detected in patients´ sera using various laboratory methods including indirect immunofluorescence (ASP 1200, Werfen, Barcelona, Spain): anti-nuclear antibodies (ANA;Kallestad HEp-2 Cell Line Substrate Kit, Bio-Rad, Hercules, CA, USA)), double-stranded (ds) DNA (NOVA Lite dsDNA Crithidia luciliae Kit, Werfen, Barcelona, Spain); enzyme-linked immuno sorbent assay, ELISA (QUANTA-Lyser 3000, Inova Diagnostics, San Diego, CA, USA): IgG rheumatoid factor – (RF; QUANTA Lite RF IgG, Werfen, Barcelona, Spain), anti-cardiolipin (ACLA; Anti-Cardiolipin IgG Kit, Orgentec, Chicago, IL, USA), anti-myeloperoxidase (MPO; Anti-MPO Kit, Orgentec, Chicago, IL, USA) , anti-proteinase 3 (PR3; Anti-PR3 Kit, Orgentec, Chicago, IL, USA), extractable nuclear antigens (ENAs, QUANTA Lite ENA 6 Kit, Werfen, Barcelona, Spain) - anti-Smith antigen (Sm), anti-ribonucleoprotein (RNP), anti-SS-A (Ro), anti-SS-B (La), anti-DNA topoisomerase I (Scl-70), anti-histidyl transfer RNA synthetase (Jo-1); chemiluminescent immunoassay (CLIA; Cobas e601, Roche, Mannheim, Germany ): anti-thyroid peroxidase (aTPO; Anti-TPO Kit, Roche, Mannheim, Germany), anti-thyroglobulin (aTG; Anti-TG Kit, Roche, Mannheim, Germany); western blot (WB; D-tek, Mons, Belgium) - anti-F-actin, anti-soluble liver antigen (SLA), anti-liver/kidney microsome type 1 (LKM1), anti-liver cytosolic antigen type 1 (LC1), anti-mitochondrial M2 (AMAM2); radioimmunoassay (RIA; Berthold LB2111, Bad Wildbad, Germany ) – anti-glutamic acid decarboxylase (GAD; diagnostic kitMediPan, Berlin, Germany), anti-insulin (IAA; diagnostic kit MediPan, Berlin, Germany), anti-tyrosine phosphatase (IA2; diagnostic kit MediPan, Berlin, Germany). The same spectrum of autoantibodies was also asssessed in the immunoglobulin therapeutics used for IRT. All immunoglobulin solutions were diluted to 1% concentration equal to 10g/L before the analysis. The dilution was performed with 5% solution of bovine serum albumin (Bovine Serum Albumin lyophilized IgG-free powder, Merck, Darmstadt, Germany) and 0.09% solution of natrium chloride (Natrium Chloratum, Biotika Solutio Isotonica, Biotika, Prague, Czech Republic). Four immunoglobulin therapeutics administered to the enrolled patients for IRT were analyzed – 2 for intravenous administration (10% IVIG – I, CSL Behring, Marburg, Germany; 10% IVIG – II, Baxalta Innovations, Vienna, Austria) and 2 for subcutaneous administration (20% SCIG, CSL Behring, Marburg, Germany; 16.5% SCIG Octapharma, Anderlecht, Belgium). Two different batches from each therapeutics were assessed, all the analyzed batches were also used for IRT in the enrolled patients.
Apart from the spectrum of autoantibodies the parameters of glucose and inzulin metabolism – fasting serum concentration of C-peptide (C-peptide Kit, Roche, Mannheim, Germany) measured by CLIA (Cobas e601, Roche, Mannheim, Germany) and glycosylated hemoglobin A1c (g-Hgb; Capillarys Hb A1c Kit, Sebia, France) using capillary electrophoresis(Capillarys 2 Analyzer, Sebia, France); thyroid gland function – free thyroxine (fT4; ADVIA Centaur FT4 assay), thyroid stimulating hormone (TSH; ADVIA Centaur FT4 assay) assessed by CLIA on ADVIA Centaur XPT Systems (Siemens, Tarrytown, NY). and thyroid gland ultrasonography (USG; Toshiba Nemio MX, Tokyo, Japan) were performed at screening and then in a year-long intervals and at the end of the study. Clinical follow-up visits were conducted in 3-month intervals with IgG serum levels assessment using nephelometry (IMMAGE 800, Beckman Coulter, Indianapolis, IN, USA) and Human Serum IgG Kit (Beckman Coulter, Indianapolis, IN, USA).
The selected laboratory parameters were also compared to a cohort of 40 newly diagnosed T1D and 50 AIT patients.
Mean values and standard deviations (SD) were calculated. Two-Sample T test was used for unpaired parametric, Paired T test for paired parametric, and Kruskal-Wallis for multiple non-parametric data set. The differences were statistically significant when p value was ≤0.05). Statistical analysis was perfomed in Minitab, version 17.1 (Minitab Inc., State College, PA, USA).