The study was part of research project IG 2020 ID 24501, supported by the Italian Association for Cancer Research (AIRC) and approved by the Ethical Committee of the Regional Health District “Area Vasta Emilia Nord” (AVEN, http://www.ausl.pc.it/comitato_etico/), protocol #2021/0081925. Fasting (12 h) blood specimens were collected before surgery in lavender-top blood tubes (K2-EDTA) at a quaternary skull base and neurotologic center (Gruppo Otologico Clinic, Piacenza, Italy), from 56 patients affected with HNPGL (59 samples, as 3 patients were sampled at two metachronous surgical stages), 10 patients with acoustic neuroma (AN), and 2 with cholesteatoma (CH). Fasting blood samples from 24 healthy age-matched controls (HCs) were donated by consenting in-house staff declaring no specific diseases and regularly controlled by institutional health check (Table S2). Informed consent was obtained from all recruited subjects. All the HNPGL plasma samples, except 4 from tympanic HNPGLs, derived from patients who had been embolized approximately 72 h before blood sampling. Blood samples were maintained at ambient temperature, plasma was prepared within 24 h by centrifugation (10 min at 1000 x g speed at room temperature), aliquoted in sterile 1.5 ml Eppendorf tubes and stored at -80 °C until testing. 21 HNPGL patients were tested on both plasma and whole dried venous blood (DVB) samples. The latter were obtained by spotting 50 μL (~1 drop) of fresh blood onto untouched Whatman 903 (W-903) filter paper discs. Spotted filters were dried for 2–3 h under hood at room temperature and saved in sterile envelopes at room temperature until testing. Twenty-four healthy volunteers were selected as controls. Nine (9) individual DVB specimens were obtained as described above from healthy controls. In parallel, samples of dried capillary blood (DCB) obtained by spotting few drops of capillary blood onto W-903 filter paper were donated from 16 healthy controls. Age and sex distribution of the healthy controls (HCs) and of the HNPGL and acoustic neuroma/cholesteatoma (AN/CH) patients are reported in Table S3; essential clinicopathologic and genetic characteristics of the HNPGL patients, including tumor localization, Shamblin class (used here for both carotid body and vagal PGLs according to Sanna M. et al.) [9 , 56 , 57 ], Sanna’s modified Fisch class (for tympanic/tympanojugular PGLs) [10 , 58 (link)] and SDHx germline mutation status, are listed in Table S4.
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