This study was planned prospectively. With ethical approvel, patients who were followed by bilateral OME at least for three months or one-sided OME at least for 9 months were enrolled to the study. Patients with craniofacial anomaly, cleft palate, had history of previous ear surgery were excluded. They were operated as paracentesis and ventilation tube implementation (VTI). An informed consent form was obtained from them and their parents.
Operation: Surgical indication was made according to disease duration and/or hearing loss. Patients were operated under general anesthesia. Paracentesis was made from posteroinferior quadrant of tympanic membrane. Glu material was trapped in the suction tube and tranferred to glass tubes immediately (Figure 1). For this, glass syringe and 0.9 % serum physiologic were used. Samples were sent from right and left ear seperately. Microplastics determination was made in Enviromental Engineering Labaratory of Faculty of Engineering.
Microplastics Determination Method: It applied according to Prada et. al report (11). Fragments, except blood clots, were washed with a jet of filtered distilled water inside the laminar flow hood to remove any surface contamination and all samples were weighed inside closed glass flasks to the nearest 0.1 mg (Sartorius, Entris, Germany). A method for preparing biological samples for Nile Red staining, previously developed and tested, was followed \[16\]: (i) incubation for 24 h at 60 °C (Oasis™ Benchtop IR CO2 Incubator, Caron, OH, USA) in 30 mL of 10% KOH (w/v, ≥85%, Labchem, PA, USA); (ii) at 24 h, the temperature was raised to boiling point and the solution immediately filtered on glass fiber filter membrane (1.2 µm pore, Whatman® GF/C, Little Chalfont, Buckinghamshire, UK) mounted in a vacuum glass filtration system; (iii) 100 mL of boiling filtered distilled water was filtered to remove soaps; (iv) 10 mL of acetone was added to the cup for 10 min; (v) an additional 10 mL of acetone was filtered to remove lipophilic matter; (vi) 0.5-1 mL of 0.01 mg mL-1 of Nile Red (microscopy grade, Sigma-Aldrich, St. Louis, MO, USA) was added to the cup for 5 min; (vii) 50 mL of distilled water was filtered; and (viii) stored in glass Petri dishes kept inside cardboard boxes for a week to dry at room temperature (20 °C).
Contamination Control Measures Strict contamination control measures were conducted in all phases, namely by: (i) wearing cotton lab coats and clean gloves; (ii) working in the laminar flow hood in a clean room, maintained by cleaning with ethanol in paper towels followed by cleaning with a duster that attracts and traps dust particles and paper fibers; (iii) filtering all solutions (1.2 µm pore, Whatman® GF/C); (iv) using glass and metal materials; (v) decontaminating glass materials by submerging them in 10% HNO3 for at least 30 min and rinsing with distilled water; (vi) additionally cleaning glass Petri dishes with a N2 air jet; (vii) burning glass microfiber filter membranes at 450 °C for 3 h; (viii) cleaning the cup of the filtration system between samples by dipping it for 15 s in a solution (first batch: acetone, second batch: 30% HNO3) followed by dipping in distilled water; (ix) keeping sample containers closed as much as possible with glass lids, caps, or aluminum foil; and (x) conducting procedural blanks (solutions without samples exposed to sample preparation procedures) for each batch. Each samples were tested by three times for verification. In addition, microplastics were evaluated in serum psyisologic to prevent contamination.
