Cytospin system

Peripheral blood (10 ml) and tissue samples were collected preoperatively and postoperatively for CAC detection. Chromosome 3 and 10 [probes for 3p22.1/3q29 (196F4) and 10q22.3/CEP10] abnormalities of peripheral blood mononuclear cells (PBMCs) of the pulmonary nodule population were qualitatively detected using the Mononuclear Cell Chromosomal Abnormality Detection Kit (Zhuhai Sanmed Biotechnology Ltd.). The assay was performed according to manufacturer’s manual and was described in previous publications (cite our own papers). In brief, PBMCs were enriched via Ficoll density gradient and deposited to microscope glass slides by Cytospin system (Thermo Fisher). The cells were subsequently fixed for 4-color FISH (Katz et al. 2010 (link), 2020 ; Yendamuri et al. 2008 ) or storage at − 20 °C. Cell nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI).
The FISH samples were digitalized by the Duet System (Allegro plus, BioView Ltd.) to visualize the chromosomal targets in Chr. 3 and 10. Signal distribution in each cell was enumerated to identify chromosome loci gain or loss. Cells with polysomy in at least two different fluorescence channels were characterized as CACs.

Cells from culture or FNA were added to slides after processing with a Cytospin system (Thermo Scientific). Cells were permeabilized and blocked in a solution of Odyssey blocking buffer (LI-COR Biosciences), 1 mg/mL Salmon Sperm DNA (Sigma Aldrich, D7656) and 25 µM poly-T blocker (24-mer), and 0.1% Triton-X 100 at RT. All fixed cells were then subjected to primary mAb–DNA-Fl labeling followed by cycling. The workflow for samples included ~30 min for collagenase treatment of samples and fixation in a lyse fix buffer, followed by cytospinning samples onto a slide (~5 min). Samples were then prepared for cycling via a 2 h pre-blocking step. Each detection cycle was carried out over a 45 min antibody incubation followed by a 30 min period for fluorophore washing and DNA strand capping. Total sample preparation times varied based on the number of proteins analyzed, but generally ranged from 4 to 8 h. The hands-on time was substantially shorter (~1 h). Irrespective of the protocol, the analysis allowed same day turn-arounds for faster therapeutic decision making, something sorely missed in current clinical practice.

Differential BAL white cell counts were completed using a variant of the Romanowsky–Giemsa stain, Diff-Quik. Superfrost™ Plus microscope slides were mounted to a Shandon cytospin system and were placed into a Cytospin 4 chamber (all Thermo Fisher) before 150 µL of BALF was added to the funnel. Samples were spun at 200 RPM for 5 min before the slide was removed from the chamber. After air drying for 5 min, the slides were fixed and stained by immersing them 6 times in methanol, 5 times in Eosin Y, 3 times in methylene blue, 5 times in a buffered solution, and 5 times in a rinse solution before being left to dry. After drying, one drop of DPX mountant (VWR International, Dublin, Ireland) was placed on each section and a cover slip (Thermo Fisher) was placed over and allowed to dry. Stained cells were visualised under light microscopy using a BX43 Olympus microscope (Olympus Life Sciences, Tokyo, Japan), and images were taken with a GXCAM-EYE-5 microscope attachment (GT Vision Ltd. Sudbury, Newmarket, UK). Differential morphological analysis was performed on the images to determine monocytes/macrophages. A total of 300 cells across at least 3 images were analysed per sample.

Cells were pelleted on to glass slides using the Cytospin system (Thermo Scientific, Paisley, UK) and fixed in acetone. Slides were stained using the Sequenza Immunostaining Center (Thermo Scientific) using 50 µL reaction volumes with reagents diluted in PBS as follows: cells were incubated with anti-T. annulata p104 vb (1C12 [13 (link)]) followed by goat anti-mouse IgG FITC conjugated antibody (Molecular Probes) then 3 nM DAPI. Slides were mounted using Fluorescent Mounting Media (Agilent Technologies, Wokingham, UK) and examined using a DMLB Fluorescent Microscope (Leica, Wetzlar, Germany).