Reducing the environmental impact of surgery on a global scale: systematic review and co-prioritization with healthcare workers in 132 countries

Abstract Background Healthcare cannot achieve net-zero carbon without addressing operating theatres. The aim of this study was to prioritize feasible interventions to reduce the environmental impact of operating theatres. Methods This study adopted a four-phase Delphi consensus co-prioritization methodology. In phase 1, a systematic review of published interventions and global consultation of perioperative healthcare professionals were used to longlist interventions. In phase 2, iterative thematic analysis consolidated comparable interventions into a shortlist. In phase 3, the shortlist was co-prioritized based on patient and clinician views on acceptability, feasibility, and safety. In phase 4, ranked lists of interventions were presented by their relevance to high-income countries and low–middle-income countries. Results In phase 1, 43 interventions were identified, which had low uptake in practice according to 3042 professionals globally. In phase 2, a shortlist of 15 intervention domains was generated. In phase 3, interventions were deemed acceptable for more than 90 per cent of patients except for reducing general anaesthesia (84 per cent) and re-sterilization of ‘single-use’ consumables (86 per cent). In phase 4, the top three shortlisted interventions for high-income countries were: introducing recycling; reducing use of anaesthetic gases; and appropriate clinical waste processing. In phase 4, the top three shortlisted interventions for low–middle-income countries were: introducing reusable surgical devices; reducing use of consumables; and reducing the use of general anaesthesia. Conclusion This is a step toward environmentally sustainable operating environments with actionable interventions applicable to both high– and low–middle–income countries

EpCAM-Independent Enrichment of Circulating Tumor Cells in Metastatic Breast Cancer.

Circulating tumor cells (CTCs) are the potential precursors of metastatic disease. Most assays established for the enumeration of CTCs so far-including the gold standard CellSearch-rely on the expression of the cell surface marker epithelial cell adhesion molecule (EpCAM). But, these approaches may not detect CTCs that express no/low levels of EpCAM, e.g. by undergoing epithelial-to-mesenchymal transition (EMT). Here we present an enrichment strategy combining different antibodies specific for surface proteins and extracellular matrix (ECM) components to capture an EpCAMlow/neg cell line and EpCAMneg CTCs from blood samples of breast cancer patients depleted for EpCAM-positive cells. The expression of respective proteins (Trop2, CD49f, c-Met, CK8, CD44, ADAM8, CD146, TEM8, CD47) was verified by immunofluorescence on EpCAMpos (e.g. MCF7, SKBR3) and EpCAMlow/neg (MDA-MB-231) breast cancer cell lines. To test antibodies and ECM proteins (e.g. hyaluronic acid (HA), collagen I, laminin) for capturing EpCAMneg cells, the capture molecules were first spotted in a single- and multi-array format onto aldehyde-coated glass slides. Tumor cell adhesion of EpCAMpos/neg cell lines was then determined and visualized by Coomassie/MitoTracker staining. In consequence, marginal binding of EpCAMlow/neg MDA-MB-231 cells to EpCAM-antibodies could be observed. However, efficient adhesion/capturing of EpCAMlow/neg cells could be achieved via HA and immobilized antibodies against CD49f and Trop2. Optimal capture conditions were then applied to immunomagnetic beads to detect EpCAMneg CTCs from clinical samples. Captured CTCs were verified/quantified by immunofluorescence staining for anti-pan-Cytokeratin (CK)-FITC/anti-CD45 AF647/DAPI. In total, in 20 out of 29 EpCAM-depleted fractions (69%) from 25 metastatic breast cancer patients additional EpCAMneg CTCs could be identified [range of 1-24 CTCs per sample] applying Trop2, CD49f, c-Met, CK8 and/or HA magnetic enrichment. EpCAMneg dual-positive (CKpos/CD45pos) cells could be traced in 28 out of 29 samples [range 1-480]. By single-cell array-based comparative genomic hybridization we were able to demonstrate the malignant nature of one EpCAMneg subpopulation. In conclusion, we established a novel enhanced CTC enrichment strategy to capture EpCAMneg CTCs from clinical blood samples by targeting various cell surface antigens with antibody mixtures and ECM components

Adhesion of EpCAM^pos/neg cells on multi-marker arrays.

<p>2x10⁵ EpCAM^pos (cell pool of MCF7, SKBR3, HCC1500, ZR-75-1, TMX2-28) (A) and EpCAM^low/neg cells (MDA-MB-231) (B) were either stained with 1 μM MitoTracker Green FM (A) or MitoTracker Orange CM (B) and incubated for cell adhesion experiments on NEXTERION slides AL, coated with different antibodies and ECM molecules, alone and in combination (0.1 mg/ml each). The labeling above the single spots indicates respective capture molecules (Iso = isotype control, ms = mouse, Lam = laminin, Col = collagen, HA = hyaluronic acid, rt = rat, T = Trop2, EpC = EpCAM, 49f = CD49f, rb = rabbit). (C) Overlay image of (A) and (B); scale bars (white) = 500 μm, 20x magnification. (D) Array layout (5x5 mm) with 36 spots (spot diameter = 500 μm; pitch = 800 μm) printed on NEXTERION slides AL.</p

Representative images of potential CTCs (CK^pos/CD45^neg) and double positive (CK^pos/CD45^pos) events enriched from EpCAM-depleted patient samples.

<p>Immunofluorescence staining of (A) CTCs (top: #8, Adem-c-Met; bottom: #25, Dyna-CD49f) and (B) CK^pos/CD45^pos events (top: #8, Adem-c-Met; bottom: #13, Adem-Trop2) enriched after EpCAM-depletion are shown. Adem-/Dynabeads captured cells were stained for DAPI (blue), pan-CK (green) and CD45 (yellow); 40x magnification.</p

Adhesion of EpCAM^pos/neg cells on manually spotted arrays.

<p>2.5x10⁴ EpCAM^pos cells (pool of MCF7, SKBR3, HCC1500 and ZR-75-1; upper panel) and EpCAM^low/_neg cells (MDA-MB-231; lower panel) were incubated for cell adhesion experiments on glass substrates (NEXTERION slides AL) manually coated with anti-EpCAM [Ber-EP4], anti-Trop2, anti-CD49f (0.1 mg/ml each), collagen I (Col), hyaluronic acid (HA) and laminin (Lam) (0.2 mg/ml each). Cell adhesion was visualized by Coomassie; 20x magnification.</p

Enrichment of EpCAM^pos/neg cells with Dynabeads.

<p>EpCAM^pos (upper panel; MCF7, SKBR3, T47D, HCC1500 and ZR-75-1) and EpCAM^low/neg (lower panel; MDA-MB-231) cells were captured with Dynabeads coupled with antibodies for EpCAM, Trop2 and CD49f (1 μg ab/2.5x10⁵ cells/1x10⁷ beads). No cells bound to uncoated Dynabeads (NT = no target). Cells were imaged after a DAPI stain and are depicted as brightfield/DAPI merge; 10x magnification.</p