The study participants were recruited from the Department of Endocrinology and Metabolic Diseases, Polish Mother’s Memorial Hospital – Research Institute in Lodz. Prior to the enrolment, all of the participants signed an informed consent, according to the study protocol approved by the local Ethics Committee. The study group included 24 patients (19 women and 5 men, age 50,6 ± 12,75 years, mean ± SD) thyroidectomized because of differentiated thyroid carcinoma (papillary thyroid carcinoma, n = 22 or follicular thyroid carcinoma, n = 2). Total thyroidectomies were performed earlier [mean time period before our study 7,17 ± 5,2 years (mean ± SD; range: 1–20 years)]. The patients with metastases, immunological or metabolic disorders (i.e. diabetes mellitus), as well as patients with clinical or laboratory signs of ongoing inflammatory processes were excluded from the study. At the time of study participation, the patients received rhTSH (Thyrogen, Genzyme Corporation; 0.9 mg i.m., followed by second 0.9 mg i.m. injection 24 hours later) as a routine control of potential thyroid cancer activity.
Peripheral blood samples were collected between 08.00 and 09.00 AM after an overnight fast. Venous blood was obtained by clean venipuncture (needle gauge 19), avoiding slow flowing draws and/or traumatic venipunctures. The blood samples were collected from the same patient (n = 24) at two (2) consecutive time points: (i) directly before the commencement of rhTSH administration and (ii) five (5) days after first rhTSH injection.
Free triiodothyronine (FT3), free thyroxine (FT4) and TSH concentrations were measured by the immunoradiometric (IRMA) method with appropriate kits (BRAHMS, Berlin, Germany; range normal values: TSH: 0.27–4.2 mIU/L; FT3: 2,6–4,4 pg/mL; FT4: 0,93–1,7 ng/dL). The concentration of thyroglobulin (Tg) was assessed with Elecsys Tg reagent kit and Cobas e 411 analyser (Roche Diagnostic Mannheim, Germany) and the concentration of Tg antibody was measured by the electrochemiluminescence (ECLIA) method with appropriate kits (Roche Diagnostic Mannheim, Germany, normal value: Tg antibody & 115 IU/ml) and equipment (Modular Analytics E170 - Roche Diagnostic).
Fluorescence-activated cell sorting (FACS) analysis
Whole blood samples obtained from study participants were assessed on the same day by flow cytometry, using a FACSCanto II® cytometer and FACSDiva® software (BD Biosciences, San Jose, CA, USA). Peripheral blood DCs subsets can be recognized on the basis of surface expression pattern of blood dendritic cell antigens (BDCAs). Two distinct populations of mDCs are characterized by expression of: BDCA1 (which has been shown to be identical to CD1c) – mDC1, or CD141/BDCA3 – mDC2. In contrast, BDCA2 and BDCA4 are specific for blood pDCs
[10–12]. Monoclonal antibodies (mAb) specific for BDCA antigens were purchased from Miltenyi Biotec (Bergisch Gladbach, Germany). All remaining mAb and appropriate isotype controls were purchased from BD Biosciences Pharmingen (San Jose, CA, USA). The staining of peripheral blood DCs subtypes was performed as described earlier
[13, 14]. Shortly, the BDCA2+ plasmacytoid DCs and the BDCA3+ myeloid DCs subtypes were recognized by staining with allophycocyanin (APC) conjugated antibodies specific for BDCA2 (AC144, mouse IgG1), and BDCA3 (AD5-14H12, mouse IgG1), respectively. The CD1c/BDCA1+ subtype was recognized as a population positive for anti-BDCA1-APC (AD5-8E7, mouse IgG2a) and negative for anti-CD19-PerCP-Cy5.5 (HIB19, mouse IgG1) staining. All DCs populations were counterstained with phycoerythrin (PE) conjugated mAb: anti-CD11c (B-ly6, mouse IgG1) and anti-CD86 (2331/FUN-1, mouse IgG1). After the incubation with mAb erythrocytes were lysed (15 minutes at room temperature) with FACS Lysing Solution (BD Biosciences, San Jose, CA, USA). The leukocyte fraction was then washed twice with cold phosphate buffered saline (PBS), counted and suspended in PBS for FACS analysis. To avoid an unspecific antibody-binding, a FcR-blocking reagent (Miltenyi Biotec, Bergisch Gladbach, Germany) was applied in all analyses. DCs subpopulations were analyzed in a blinded way and the results were expressed as a percentage of peripheral blood mononuclear cells (PBMC) fraction.
Statistical analysis was performed using the Prism 5.0 statistical software (GraphPad, La Jolla, CA, USA). The normality of distribution was assessed utilizing the Shaphiro-Wilk test and the differences in peripheral blood cell populations were analyzed with the Wilcoxon matched-pairs signed rank test. In all the analyses, results were considered statistically significant when p & 0.05.