Chemiluminescent microparticle immunoassay

Blood samples were collected immediately after recruitment in the clinic or admission to the ward. A maximum of 4 mL of venous blood was collected in a plain tube and centrifuged immediately. The serum was aliquoted and stored frozen at −80 °C until analysis. Serum AFP levels were measured by a commercially available Chemiluminescent Microparticle Immunoassay (Architect, Abbott Diagnostic, Abbott Park, IL, USA). Serum PIVKA-II levels were measured by Chemiluminescent Microparticle Immunoassay (Architect, Abbott Diagnostic, Abbott Park, IL, USA) according to the manufacturer’s instructions.

Total testosterone serum levels were measured by Chemiluminescent Microparticle Immunoassay (Architect, Abbott, Dundee, UK), with inter- and intra-assay coefficients of variation (CV) of 5.2 and 5.1%, respectively. FSH and LH were measured by Chemiluminescent Microparticle Immunoassay (Architect, Abbott, Longford, Ireland) with inter- and intra-assay CV of 4.1 and 3.1% for LH, and 4.6 and 4.2% for FSH, respectively. PRL was measured by Chemiluminescent Immunoassay (Beckman Coulter, Brea, CA, USA) with inter- and intra-assay CV of 4.2 and 1.6%, respectively.
The laboratory reference ranges were 2.2–8.7 ng/dL for testosterone, 1–9 IU/L for LH, 1–12 IU/L for FSH and 3–13 ng/mL for PRL. The assay methods and kits used did not change over the years for all hormones considered.

Plasma specimens were collected from patients admitted to the Urology Clinic of the Clinical Emergency County Hospital in Timisoara, Romania. All patients had undergone trans-rectal biopsies for histopathological PCa diagnoses. All patients’ clinical data are summarized in Table 1 and extensively presented in Table S1, Supplementary Materials. Cancer-free control samples were collected from subjects with no prostate pathology from the same institution. All controls had normal Prostate-Specific Antigen (PSA) levels (<4 ng/mL) verified by chemiluminescent microparticle immunoassay (Abbott Diagnostics, Lake Forest, IL, USA).
All subjects provided informed consent for use of their biological samples and the study was approved by the Ethics Committees of the participating institutions (the Clinical Emergency County Hospital in Timisoara, code no. 71/05.08.2014 and the Victor Babes University of Medicine and Pharmacy Timisoara, code no. 9/13.05.2014 extended by code no. 33_2017).
Venous blood was collected in EDTA-treated blood collection tubes and was immediately centrifuged for 15 min at 2000× g for plasma separation which was subsequently frozen at −80 °C.

This study comprised a total number of 71 subjects (48 PCa patients and 23 healthy controls). Blood was collected from each individual at the Urology Clinic of the Clinical Emergency County Hospital in Timisoara, Romania, following their agreement to participate in the study (all the subjects filled in a written IRB informed consent for the use of their biological specimens). The study was approved by the Ethics Committee of the participating institutions, in accordance with the 1964 Declaration of Helsinki and its later amendments, venous blood was collected in specific EDTA collection tubes; then, the plasma was separated and kept at −80 °C until further use. The patients were diagnosed with prostate adenocarcinoma following transrectal biopsies for histopathological diagnosis of PCa. The control volunteers had no prostate disease and normal PSA values (<4 ng/mL), verified by chemiluminescent microparticle immunoassay (Abbott Diagnostics, Chicago, IL, USA), and their blood samples were drawn at the same clinic.

Total homocysteine was assayed in plasma with a chemiluminescent microparticle immunoassay (Abbott Diagnostics, IL, USA). Plasma was diluted 1:10 or 1:5 with Multi-Assay Manual Diluent No. 7D82-50 (Abbott Diagnostics) prior to analysis by Architect i2000SR Analyzer (Abbott Diagnostics) at PathWest (WA, Australia).