Collection and Processing of Unstimulated Saliva

Unstimulated mixed saliva was collected from all patients between 9:00 and 11:00 a.m., with a Salivette cotton roll (Sarstedt, Nümbrecht, Germany) as described previously [15 (link)]. One hour before sampling, ASD and TD patients refrained from eating, drinking, mouth rinsing, and teeth brushing. Salivette rolls were chewed for 5 min and then placed in special centrifugation tubes. Each sample was refrigerated or stored on ice until arrival at the laboratory, which was no longer than 45 min after the sample had been collected. All the samples were first weighed and then centrifuged at 4500× g for 10 min at 4 °C. The supernatant from whole saliva samples obtained by passive flow and saliva samples from the Salivettes were transferred to 1.5 mL Eppendorf tubes and stored at −80 °C for a maximum of three months until analysis. In all cases, the minimum volume of saliva obtained was 1 mL.