Probe-based confocal laser endomicroscopy: an evaluation of its role towards real-time, in vivo, in situ intraoperative applications

Probe-based confocal laser endomicroscopy (pCLE) is an emerging imaging tool that allows real-time in situ morphological imaging at cellular and subcellular resolution. Its ability to image morphological features of epithelial surfaces of the gastrointestinal tract, biliary tree and respiratory tree rendered differentiation of macroscopically inconspicuous neoplastic and non-neoplastic tissues possible in real-time. However, its role outwith the endoluminal environment for surgical applications has been comparatively sparsely investigated and little reported on its ability to characterise morphological features beyond endoluminal applications. This thesis aims to systematically evaluate the potential pCLE has in visualization of soft tissue morphology in applications pertaining to breast conserving surgery (BCS), parathyroid surgery and thyroid surgery; whereby morphological information regarding cavity wall margin status, tissue-specific entity and viability status of preserved parathyroid glands (PG), respectively, could potentially guide decision-making intraoperatively. The perceptions that pCLE imaging is confined to endoluminal mucosal surfaces, the inability of pCLE to perform image acquisition through sterile transparent sheaths and the inability of surgeons to interpret pCLE images were interrogated using three small feasibility studies. Firstly, in a study carried out on a live, anaesthetised, porcine model, pCLE image acquisition of morphological architecture of soft tissues of the neck e.g. thyroid, lymph nodes, adipose, skeletal and smooth muscles, were shown to be feasible in an intraoperative field and the presence dried blood on the tissue surface did not impede the consistency of morphological architecture visualization. Secondly, we demonstrated that utilization of a sterile transparent sheath did not impede pCLE image acquisition and that the quality of images obtained was comparable to that of without the sheath. Thirdly, we have shown that surgeons with little or no histopathology background were able to acquire the relevant pattern recognition skills to interpret pCLE images following a training session utilizing a validated pCLE morphological classification from colorectal lesions. Building upon these discoveries, we elucidated the potential of pCLE to image neoplastic and non-neoplastic breast morphology with the envisaged application of identifying residual cancerous foci intraoperatively, thereby guiding operative decision making based on real-time breast cavity scanning during BCS. Preliminary ex vivo analyses from 71 freshly excised, acriflavine-stained neoplastic and non-neoplastic tissues samples from 50 breast cancer patients show excellent correlation with histopathology findings. In particular, the glandular structures, adipocytes and collagen fibres of non-neoplastic breast tissues were readily visualised on pCLE images. These were distinguishable from the markedly haphazard and hypercellular architecture exhibited by invasive and non-invasive carcinoma. We developed a classification based on description of pCLE morphology unique to neoplastic and non-neoplastic breast morphology and validated this with 17 histopathologists and surgeons through a systematic pattern recognition training session based on this classification where they were subsequently subjected to objective assessment of 50 pCLE images while blinded to histopathology results. The overall mean accuracy of pCLE image interpretation for histopathologists and surgeons were 94% and 92%, respectively. The overall inter-observer agreement was ‘almost perfect’ (κ=0.81) for the former and ‘substantial’ (κ=0.77), for the latter. We explored the role of intravenous fluorescein sodium (FS) in a prospective, cross-sectional, observational study of 10 patients undergoing BCS where they received between 1.5ml to 3.5ml of intravenous bolus of 10% fluorescein sodium (FS) intraoperatively. Ex vivo analyses of FS-stained breast samples showed that dense fibrous tissue response evoked by infiltrating tumor cells were readily visualised as fluorescent regions with haphazardly arranged, amorphous-looking collagen fibres. However, the lack of nuclei visualization rendered differentiation of neoplastic from non-neoplastic tissues impossible. Nevertheless, the uniformity that FS staining imparts to all tissue layers facilitated creation of longer and meaningful pCLE mosaics. These findings could have important implications where tissue deformation could result in AH-stained layers intermittently fail to coincide with the optical slice imaged at the respective depth. The promising findings of AH-stained breast tissues were found to be potentially relevant in parathyroid surgery. Similar analyses on freshly excised AH-stained parathyroid specimens from 35 patients undergoing parathyroidectomy for primary and secondary hyperparathyroidism showed nest-like arrangements of parenchymal cells, fibrovascular septum and microfollicles of diseased PGs were readily identifiable on CE images and these were consistent with histopathological findings. Following pattern recognition training based on an in-house developed classification system, these were distinguishable from epithelial-lined thyroid follicles and polygonal-shaped adipocytes with mean accuracies of 94% and 93% for histopathologists and surgeons, respectively, and high overall inter-observer agreement, κ=0.82. Where intraoperative identification of diseased PGs presents a challenge especially in multi-glandular disease and re-operative surgery, pCLE could potentially facilitate its recognition. Finally, the role for pCLE imaging of PG vasculature was explored by means of an intraoperative clinical study utilising a sterile-transparent draped pCLE probe on 20 patients undergoing thyroid and parathyroid surgery. Utilising intravenous FS, branched-vessels including capillary networks were readily visualised. Vascular flow on viable glands was depicted by unidirectional, high velocity thrusts of dark-coloured erythrocytes within hyperfluorescent vessels or diffusely in the parenchyma whereas these were absent on non-viable (post-excision) glands. Further analysis on preserved PGs showed that absence of blood flow was found in patients who had sub-optimal post-operative parathyroid function. Given that visual assessment of tissue discolouration is not a reliable method of determining parathyroid gland viability during thyroidectomy, information regarding viability of preserved PGs decisions could potentially aid decisions pertinent to autotransplantation remains challenging. This thesis significantly expands upon the potential intraoperative applications of pCLE. Evidently, these findings are preliminary and warrant further evaluation in well-powered clinical trials. However a systematic approach to investigate the optimal trade-offs between the optical resolution requirements of tissue morphology visualization and deployability of pCLE probe holds the key to successful clinical translation. In particular, evaluation of a robust mechatronically enhanced platform equipped with the flexibility to cater for tissue surface deformation and precision mechanisms that generates accurate spatio-temporal localisation in real-time to aid intraoperative decision making constitutes the next stage of research priorities.Open Acces