Clot activator

During each blood draw, two aliquots of the whole blood were taken: one with a volume of 4 mL to a tube with a clot activator (Greiner Bio-One, Kremsmünster, Austria) for measurement of IgG antibodies and the second with a volume of 9 mL to a tube containing lithium heparin (Greiner Bio-One, Kremsmünster, Austria) for determination of IFN-γ concentration.
All analyses were conducted in the medical laboratory of the Silesian Park of Medical Technology Kardio-Med Silesia in Zabrze (Kardio-Med Silesia), accredited by the Polish Center of Accreditation (PCA). The study was conducted in accordance with the Helsinki Declaration. Furthermore, the study protocol was approved by the Bioethics Committee of the Medical University of Silesia in Katowice (No.: PCN/0022/KB1/50/II/20/21).

After semen collection and analysis, blood samples were drawn from the cephalic vein, transferred into serum gel tubes with clot activator (Greiner Bio-one, Kremsmünster, Austria), and placed at 4 °C for 30 min to allow clot formation. Tubes were then centrifuged at 2000× g for 5 min and sera were immediately stored at −80 °C until analysis in an external specialized laboratory (Algemeen Medisch Laboratorium, Sonic Healthcare Benelux, Antwerp, Belgium). Serum AMH levels were quantified by electrochemiluminescence immunoassay (ECLIA) using the Elecsys AMH Plus immunoassay on the cobas e411 analyzer (Roche Diagnostics International Ltd., Rotkreuz, Switzerland). Results were determined via a calibration curve generated by 2-point calibration and a master curve provided by the manufacturer. The analyzer automatically provided the AMH concentration and controls were performed for each analysis. Samples with AMH concentrations above the measuring range (>23 mg/L) were diluted to obtain a definite value. The intra- and inter-assay precision were ≤1.3 and ≤4.1%, respectively. The limits of blank, quantitation, and detection were 0.007 ng/mL, 0.030 µg/L, and 0.010 µg/L, respectively. The method was standardized against the Beckman Coulter AMH Gen II ELISA assay.

Peripheral blood samples were collected from the antecubital vein into a single VACUETTE polypropylene tube containing a serum separator and clot activator (Greiner-Bio One GmbH, Kremsmunster, Austria). Fasting PMF patients and controls were allowed to rest for at least 15 min before carrying out blood withdrawal, with the procedure performed between 8.00 and 9.00 a.m. To separate serum, blood withdrawals were kept for 30 min at room temperature and then centrifuged at 1890× g for 10 min. Serum samples were transferred into a new tube, and an aliquot of 500 μL was used for the subsequent organic solvent deproteinization [26 (link)]. Briefly, proteins were removed by the addition of 1 mL of ice-cold far UV, HPLC-grade acetonitrile to 0.5 mL of serum. After vigorous vortexing for 90 s, samples were centrifuged at 20,890× g for 10 min at 4 °C, supernatants were collected and transferred to a new tube, supplemented with 3 mL chloroform, vigorously vortexed for 120 s, and again centrifuged at 20,890× g for 10 min at 4 °C. The upper aqueous phase was collected and again extracted with chloroform to remove the organic solvent (acetonitrile). The resulting aqueous phase, free of proteins, was ready for the high-performance liquid chromatographic (HPLC) analyses of hydrophilic, low-molecular-weight metabolites.