Preservcyt

A Dacron^® cytobrush moistened with tap water was used to sample the anal canal, and was rinsed in a vial containing 20 ml of PreservCyt (Hologic, Inc., Marlborough, MA, United States) fixative medium. The Bethesda System (TBS 2001) criteria were used for cytology reporting [11 ]. Cytological results were classified as unsatisfactory, negative, atypical squamous cells of undetermined significance (ASCUS), low-grade squamous intraepithelial lesion (LSIL), or HSIL. The same ThinPrep slide was used for p16/Ki-67 immunostaining after the cytologic evaluation with the CINtec PLUS Kit (Roche MTM Laboratories). A case was considered positive if one or more squamous epithelial cell(s) stained both with a brown cytoplasmic stain (p16) and a red nuclear stain (Ki-67) irrespective of any morphologic abnormalities. The number of cells that were positively stained with both markers was recorded. All samples were stained with p16/Ki-67 regardless of slide cellularity.

The PIPAVIR project was a study conducted between August 2012 and August 2015, aiming at the development and initial clinical assessment of a novel ELISA hrHPVE7 oncoprotein detection method for cervical cancer screening. The study design has been described in detail previously [9 (link)]. Briefly, participants were non-pregnant women aged 30–60, without a history of cervical intraepithelial neoplasia (CIN), who visited the Family Planning Centre, Hippokratio Hospital of Thessaloniki, Greece and the Department of Gynecology and Obstetrics in Im Mare Klinikum, Kiel, Germany and gave their written informed consent to participate. Subsequently, a cervicovaginal sample, used for Thinprep cytology, HPV DNA genotyping and hrHPVE7 protein detection was taken and women with a positive cytology or hrHPV DNA result were referred to colposcopy followed by biopsy and/or endocervical curettage (ECC), when needed. Sampling was performed using the CervexBrush (Rovers Medical Devices, Lekstraat 10, NL-5347, KV Oss, The Netherlands) and the Cytobrush (CooperSurgical, Inc., 95 Corporate Drive, Trumbull, CT, USA) according to the manufacturer’s instructions. After sampling both brushes were immersed in a vial containing collection fluid (PreservCyt, Hologic, Bedford, MA, USA).

Study participants were represented by MSM aged ≥18 years, attending the center for Sexually Transmitted Infections (STIs) and HIV of the San Gallicano Dermatological Institute (Rome, Italy) for the Surveillance Program of Anal Intraepithelial Neoplasia (SAIN project) [33 (link)]. This project aimed to assess the prevalence, incidence, and determinants of anal HPV infection and anal lesions in HIV-infected and uninfected MSM. The participants were subjected to anal cytological sampling by means of a sterile Dacron swab as previously detailed [34 (link)]. Briefly, the collected cells were suspended in PreservCyt (Hologic), and starting from 2014, the residual sample remaining after HPV DNA testing and the anal Pap test was stored at +4 °C. For the purposes of the present study, these specimens were evaluated based on the following criteria: 1. Sample adequate for morphological interpretation; 2. results available for HPV DNA testing; 3. residual sample sufficient to perform the assays of interest for this study. Among the 480 samples with the inclusion criteria, 150 were selected in order to homogeneously represent all the cytological categories. No cases with a cancer diagnosis were available.

The PD-L1 assessment in Lund has been previously described in detail [7 (link)]. In brief, biopsied tissue was normally used for PD-L1 testing, but in cases with no or an inadequate biopsy, a cytological specimen was used if available. Also, extra cell blocks were sometimes constructed in the clinical setting for parallel PD-L1 staining during 2017–2019 even if an adequate biopsy existed.
Cytological material for IHC was fixed in CytoLyt^® (Hologic, Marlborough, MA, USA) for a few hours (except for pleural effusions that were only rapidly washed) followed by PreservCyt^® for typically 1–3 days before Cellient™ automated cell block preparation (Hologic, Marlborough, MA, USA). Bronchial cytology, including aspirations from lymph nodes, was sometimes mixed in cell blocks to increase the number of tumor cells.
Regardless of material, PD-L1 was assessed using the 22C3 assay (Agilent/pharmDx, Santa Clara, CA, USA) with staining on a Ventana Benchmark Ultra (Ventana Medical Systems Inc., Tucson, AZ, USA) using the OptiView visualization system. Control tissue (tonsil and placenta) was routinely used on each slide. PD-L1 was evaluated in the clinical diagnostic setting by several different pathologists working with thoracic pathology and PD-L1 daily.