Line-field confocal optical coherence tomography in dermato-oncology: A literature review towards harmonized histopathology-integrated terminology.

Non-invasive diagnostics like line-field confocal optical coherence tomography (LC-OCT) are being implemented in dermato-oncology. However, unification of terminology in LC-OCT is lacking. By reviewing the LC-OCT literature in the field of dermato-oncology, this study aimed to develop a unified terminological glossary integrated with traditional histopathology. A PRISMA-guided literature-search was conducted for English-language publications on LC-OCT of actinic keratosis (AK), keratinocyte carcinoma (KC), and malignant melanoma (MM). Study characteristics and terminology were compiled. To harmonize LC-OCT terminology and integrate with histopathology, synonymous terms for image features of AK, KC, and MM were merged by two authors, organized by skin layer and lesion-type. A subset of key LC-OCT image-markers with histopathological correlates that in combination were typical of AK, squamous cell carcinoma in situ (SCCis), invasive squamous cell carcinoma (SCC), basal cell carcinoma (BCC), and MM in traditional histopathology, were selected from the glossary by an experienced dermatopathologist. Seventeen observational studies of AK (7 studies), KC (13 studies), MM (7 studies) utilizing LC-OCT were included, with 117 terms describing either AK, KC, or MM. These were merged to produce 45 merged-terms (61.5% reduction); 5 assigned to the stratum corneum (SC), 23 to the viable epidermis, 2 to dermo-epidermal junction (DEJ) and 15 to the dermis. For each lesion, mandatory key image-markers were a well-defined DEJ and presence of mild/moderate but not severe epidermal dysplasia for AK, severe epidermal dysplasia and well-defined DEJ for SCCis, interrupted DEJ and/or dermal broad infiltrative strands for invasive SCC, dermal lobules connected and/or unconnected to the epidermis for BCC, as well as single atypical melanocytes and/or nest of atypical melanocytes in the epidermis or dermis for MM. This review compiles evidence on LC-OCT in dermato-oncology, providing a harmonized histopathology-integrated terminology and key image-markers for each lesion. Further evaluation is required to determine the clinical value of these findings.

Role of reflectance confocal microscopy for in vivo investigation of oral disorders: White, red and pigmented lesions.

In vivo reflectance confocal microscopy (RCM) is poorly investigated in oral pathology due to the peculiar anatomical and topographical oral mucosa features. A dedicated handheld confocal microscope with an intra-oral probe was developed for oral mucosa imaging. The main objective was to describe the healthy oral mucosa and the cytoarchitectural findings detectable in different oral disorders by means of the newly designed handheld confocal microscope. Secondary aim was to identify the main RCM criteria that differentiate oral lesions in order to provide algorithm for a rapid non-invasive evaluation. This observational retrospective study included all consecutive patients with oral disorders and volunteers with healthy oral mucosa who underwent RCM examination in our outpatient clinic from September 2018 to December 2021. Three different investigators examined together the RCM images to detect the key features and secondary criteria for each type of oral lesion collected. The study population included 110 patients affected by oral lesions and seven volunteers with healthy oral mucosae. A total of 15 oral disorders were imaged and divided in three main groups: white, red and pigmented lesions. Key features and secondary criteria were identified for every single type of oral disease. RCM permits a cytoarchitectural evaluation of the oral mucosae affected by inflammatory, dysplastic and neoplastic diseases, thus orienting the clinicians towards non-invasive diagnosis and enhancing the diagnostic management. The "tree diagrams" proposed allow a schematic and simplified view of confocal features for each type of oral disease, thus drastically reducing the diagnostic timing.

The role of reflectance confocal microscopy in the diagnosis of eccrine poroma: A retrospective case-control study.

Eccrine poroma (EP) is a rare benign adnexal tumor that may mimic benign or malignant tumors and differential diagnosis may be difficult under clinical and dermoscopic examination. Reflectance confocal microscopy (RCM) examination may add important information to diagnosis and subsequent management of solitary lesions for which dermoscopy can be challenging. The aim of the present study was to investigate features of EP at RCM in order to detect the characteristics that might aid in the differential diagnosis of EP versus other solitary lesions (benign or malignant). Secondary objective was to correlate the resulting features with histopathological findings. This monocentric retrospective observational case-control study included all EPs registered with RCM between January 2007 and May 2018. Control cases were benign or malignant lesions similar in clinical appearance, morphology, and dermoscopic features to EPs. RCM evaluators were blinded to clinical-dermoscopic images and to final histopathological diagnoses. Finally, RCM-histopathological correlation was performed. A total of 11 EPs and 33 controls were included in the present study. Among RCM parameters, "cords without palisading," "dark holes," "prominent vascularization" and "abundant stroma" resulted positively associated with EP in univariate analysis. RCM features correspond to the histopathological diagnosis of EP in 97% of cases, as illustrated by the cluster analysis. An excellent correlation between diagnostic features of conventional histopathology and RCM was observed. RCM assists in the differential diagnosis of solitary lesions, allowing to reach a correct diagnosis of EP through the identification of its four characteristics.

Correlation of Probe-Based Confocal Laser Endomicroscopy (pCLE) and Mucosal Integrity Testing (MIT) with Epithelial Barrier Function and Presence of Gastroesophageal Reflux Disease (GERD).

INTRODUCTION: Epithelial barrier function (EBF) disruption is a key mechanism underlying gastroesophageal reflux disease (GERD). Our aim was to assess whether two novel technologies, probe-based confocal laser endomicroscopy (pCLE) and mucosal integrity testing (MIT), could assess EBF. METHODS: We prospectively enrolled patients undergoing upper endoscopy for refractory GERD or non-GERD conditions. Patients underwent esophagogastroduodenoscopy, pCLE, MIT, esophageal biopsy at 2 cm and 6 cm above the esophagogastric junction, and wireless pH testing. To assess EBF in vitro, biopsies were mounted in a mini-Ussing chamber, 1 ml of fluorescein was instilled on the mucosal side, and concentration of fluorescein on the serosal side was measured at 3 h. RESULTS: We enrolled 54 subjects (28 GERD, 26 non-GERD based on Lyon consensus criteria). In vivo permeability assessed by pCLE did not differ significantly between GERD vs. non-GERD patients and did not correlate with in vitro permeability. Mean MIT at 2 cm was lower in GERD compared to non-GERD (1914 vs. 3727 ohms). MIT correlated inversely with in vitro permeability at 2 cm and at 6 cm. Using a predictive model that used slope and intercept of MIT at 2 cm and 6 cm, sensitivity and specificity of MIT at identifying GERD was 76% and 72%, respectively. CONCLUSION: pCLE did not differentiate GERD vs non-GERD and did not correlate with EBF measured in vitro. MIT, on the other hand, may be more promising as it differentiated GERD vs non-GERD and correlated with EBF measured in vitro.

Advanced Imaging of the Biliary System and Pancreas.

Despite advances in noninvasive techniques for imaging the pancreaticobiliary system, several disease processes including indeterminate biliary strictures as well as neuroendocrine tumors, inflammatory pseudotumors, and complex cysts of the pancreas remain difficult to characterize. New endoscopic imaging technologies have emerged to address these challenges. Cholangioscopy and intraductal ultrasound (IDUS) are powerful tools to characterize subtle biliary concretions and strictures. Confocal Laser Endomicroscopy (CLE) and Optical Coherence Tomography (OCT) are emerging approaches for the most difficult biliary lesions. Contrast harmonic endoscopic ultrasound (CH-EUS), elastography, and 3D-EUS are improving the approach to subtle pancreatic lesions, particularly in the context of indeterminate tissue sampling. Pancreatoscopy, pancreatic IDUS, and intracystic CLE hold promise to further improve the assessment of pancreatic cysts. We aim to comprehensively review the emerging clinical evidence for these innovative endoscopic imaging techniques.

Sorting of Arabidopsis NRAMP3 and NRAMP4 depends on adaptor protein complex AP4 and a dileucine-based motif.

Adaptor protein complexes mediate cargo selection and vesicle trafficking to different cellular membranes in all eukaryotic cells. Information on the role of AP4 in plants is still limited. Here, we present the analyses of Arabidopsis thaliana mutants lacking different subunits of AP4. These mutants show abnormalities in their development and in protein sorting. We found that growth of roots and etiolated hypocotyls, as well as male fertility and trichome morphology are disturbed in ap4. Analyses of GFP-fusions transiently expressed in mesophyll protoplasts demonstrated that the tonoplast (TP) proteins MOT2, NRAMP3 and NRAMP4, but not INT1, are partially sorted to the plasma membrane (PM) in the absence of a functional AP4 complex. Moreover, alanine mutagenesis revealed that in wild-type plants, sorting of NRAMP3 and NRAMP4 to the TP requires an N-terminal dileucine-based motif. The NRAMP3 or NRAMP4 N-terminal domain containing the dileucine motif was sufficient to redirect the PM localized INT4 protein to the TP and to confer AP4-dependency on sorting of INT1. Our data show that correct sorting of NRAMP3 and NRAMP4 depends on both, an N-terminal dileucine-based motif as well as AP4.

[Imaging features of Fuchs endothelial corneal dystrophy].

Objective: To investigate the imaging features of Fuchs endothelial corneal dystrophy (FECD) and to provide imaging evidence for the diagnosis of this disease. Methods: Retrospective case series study. A total of 128 eyes (64 patients, including 19 males and 45 females) diagnosed with FECD at the Beijing Tongren Eye Center of Capital Medical University from January 2014 to December 2016 were enrolled. The average age was 57.8±12.9 years. There were 25 eyes of stage Ⅰ (19.5%), 81 eyes of stage Ⅱ (63.3%), 16 eyes of stage Ⅲ (12.5%) and 6 eyes of stage Ⅳ (4.7%).All patients underwent specular microscopy, and 41 patients (82 eyes) had in vivo confocal microscopy (IVCM). The patients' general data, clinical stage, and image features of specular microscopy and IVCM were analyzed. The enumeration data was compared by chi-square test. Differences of measurement data were compared by ANOVA. Data which cannot be accurately measured was compared by rank sum test. Results: As the disease progressed, the number, incidence rate, and fusing rate of dark"holes"on specular microscopy increased. The number of guttata on IVCM increased, and the fusing pattern of guttata developed from pair-like, chain-like to group-like. On specular microscopy, the mean rank of stage Ⅰ (78.2), stage Ⅱ (228.4), stage Ⅲ (284.5) and stage Ⅳ (288.5) was statistically different (χ²=84.183, P=0.000). All positions of all eyes of stage I had no fusion of the dark "holes". The incidence of fusion on the peripheral cornea gradually increased significantly (χ²=27.167, P=0.000) from stage Ⅱ (45.1%, 146/324), stage Ⅲ (76.3%, 45/59) to stage Ⅳ (83.3%, 15/18). Conclusions: The imaging features of specular microscopy and IVCM can be applied as an important basis for early diagnosis of FECD. Specular microscopy is a practical method for rapid screening of FECD. IVCM is an important imaging basis for clarifying the appearance of guttata and analyzing fusion features, so as to guide the differentiation of stages. (Chin J Ophthalmol, 2020, 56:938-943).

[In vivo confocal microscopic characteristics of limbal stem cell deficiency].

Objective: To investigate the cellular changes and quantitative analysis of basal cell density (BCD) and corneal epithelial thickness (CET) in limbal stem cell deficiency (LSCD) by in vivo confocal microscopy (IVCM). Methods: Prospective case-control study. A total of 35 eyes of 23 patients diagnosed with LSCD and 25 eyes from normal subjects were included in this study. Based on slit-lamp presentation and the global consensus on classification, the LSCD patients were classified into LSCD Ⅰ, LSCD Ⅱ and LSCD Ⅲ. Confocal images of the central cornea, and the superior, inferior, nasal and temporal limbus were inspected by IVCM. Morphologic characteristics of LSCD were summarized. The BCD and CET in all locations were measured. ANVOA or Kruskal-Wallis test was used for analysis when appropriate. A receiver operating characteristic was used to detect the diagnosis efficiency of BCD and CET. Results: The characteristics in the corneal epithelium of LSCD on IVCM included nested corneal epithelial cells, goblet cells with hyper-reflective spots, irregular basal cells and decreasing subbasal nerve density. The mean BCD of the LSCD group was (8 976±1 096) cells/mm2 in the central cornea. Compared to the control group, the BCD in the central cornea, superior, inferior, nasal and temporal limbus decreased by 30.2%, 26.0%, 28.7%, 29.3% and 30.2%, respectively (all P<0.007). The CET in the central cornea was (47.3±8.1) µm. The CET in the central cornea, superior, inferior, nasal and temporal limbus decreased by 27.9%, 23.7%, 20.6%, 26.9% and 23.1%, respectively, compared to the control group (all P<0.007). There was a decline of BCD and CET in more serious LSCD. Additionally, the decline of BCD and CET was shown in the unaffected region. The receiver operating characteristic showed the diagnosis efficiency of BCD in the corneal center and limbus (0.931 and 0.916) was superior to CET (0.853 and 0.817). Conclusions: There was a series of characteristic cellular changes in LSCD on IVCM. Both BCD and CET decreased significantly in LSCD. The BCD had higher diagnostic efficiency for LSCD.(Chin J Ophthalmol, 2020, 56:447-455).

[Observation and quantification of diabetic keratopathy in type 2 diabetes patients using in vivo laser confocal microscopy].

Objective: To study the diabetic keratopathy in type 2 diabetes patients with retinopathy by in vivo laser confocal microscopy. Methods: This was a case-control study. Ninety type 2 diabetes patients were involved in this study from May 2015 to December 2019 in Qingdao Eye Hospital. According to the diabetic retinopathy clinical stage, these patients were divided into the non-proliferative diabetic retinopathy (NPDR) group (30 cases), early stage proliferative diabetic retinopathy (PDR) group (30 cases) and intermediate to late stage PDR group (30 cases). Thirty non-diabetic healthy volunteers were included in the control group. The central cornea was observed with an in vivo laser confocal microscope. The corneal nerve fiber density, nerve fiber length, nerve branch density, and nerve fiber tortuosity were compared between groups. The corneal Langerhans cells, epithelial cells, stromal cells and endothelial cells were also compared. Results: There were more nerve fibers and branches in the control group than the other three diabetic groups. The nerve fiber length in the control group, NPDR group, early stage PDR group and intermediate to late stage PDR group was (21.55±2.57), (14.73±1.56), (11.23±1.40) and (8.02±1.33) mm/mm2, respectively, and there were statistically significant differences between the groups (F=316.17, P=0.00). In the nerve fiber density, nerve branch density and curvature, there were statistically significant differences between the groups (F=345.72, 479.46, 167.00, all P=0.00). The basal cell density in the control group, NPDR group, and two PDR groups was (5 761±303), (5 336±367), (4 146±379) and (3 658±365) cells/mm2, respectively, and there were statistically significant differences between the groups (F=234.94, P=0.00). The anterior stromal cell density in the four groups was (836±30), (727±57), (544±59) and (360±47) cells/mm2, respectively, and there were statistically significant differences between the groups (F=535.08, P=0.00). The hexagonal endothelium cell rate in the four groups was 62.0%±5.5%, 51.1%±3.7%, 40.2%±4.0% and 27.8%±3.9%, respectively, and the Langerhans cell density was (1.5±0.6), (4.2±1.3), (6.8±2.1) and (10.9±2.1) cells/mm2, respectively; there were statistically significant differences between the groups (F=342.28, 179.78, all P=0.00). There was no statistically significant difference between the groups in the corneal endothelial cell density (F=1.58, P=0.20). Conclusions: In type 2 diabetes patients with diabetic retinopathy, the corneal nerve fiber and branch density can be significantly reduced, and the density of the hexagonal corneal endothelial cells, epithelial basal cells and anterior stromal cells can also decrease. Langerhans cells may be involved in the development diabetic keratopathy. (Chin J Ophthalmol, 2020, 56: 754-760).

[The fundus autofluorescence of retinal astrocytic hamartomas in tuberous sclerosis complex].

Objective: To investigate the autofluorescence findings of retinal astrocytic hamartoma (RAH) in patients with tuberous sclerosis complex (TSC). Methods: It was a retrospective case series study. Twenty-three patients (35 eyes) who were referred to Department of Internal Medicine and Department of Ophthalmology, Peking Union Medical College Hospital between November 2012 and June 2018 with established TSC-associated RAH diagnosis were included. The findings of fundus autofluorescence, fundus photos and spectral-domain optical coherence tomography (SD-OCT) were retrospectively reviewed. RAH lesions were classified into three types based on the morphology shown in fundus photos. The fundus autofluorescence features of TSC-associated RAH were described. The Welch's test and Fisher's exact test were used for statistical analysis. Results: The patients were 8 males and 15 females aged (28±9) years old (range, 15-55 years). Seventy-two RAH lesions were examined, including 59 type 1 RAHs, 7 type 2 RAHs and 6 type 3 RAHs. According to fundus autofluorescence, type 1 RAHs could be further divided into reduced, speckled and background autofluorescence patterns, among which the hypoautofluorescence pattern accounted for the majority (69.5%, 41/59), while the speckled pattern was usually accompanied by outer retinal disorganization and discontinuation of photoreceptor outer segment as revealed by SD-OCT. No significant difference was revealed in tumor thickness for reduced, speckled and background autofluorescence patterns of type 1 RAHs [(490.2±97.9) vs. (589.2±221.6) vs. (463.0±76.2) µm respectively, F=1.426, P=0.283]. Among type 1 RAHs, the number of reduced autofluorescence pattern lesions found in perifoveal, peripapillary, inferonasal, inferotemporal, superonasal, superotemporal quadrants were 9, 4, 4, 7, 4, 13 respectively, while that of speckled autofluorescence pattern lesions were 3, 0, 3, 2, 3, 2 and background autoflurorescence pattern lesions 3, 0, 1, 1, 0, 0. No significant difference was revealed in location distribution (P=0.452) either. Type 2 RAHs featured numerous hyperautofluorescent spots or plaques, and calcification in type 2 RAHs varied in autofluorescence intensity. Type 3 RAHs, combining the features of type 1 and 2 RAHs, were characterized by central hyperautofluorescent spots and hypoautoflurescent rim, but the area of hyperautofluorescence was smaller than that of calcification as shown in fundus photos. Conclusions: In TSC, the fundus autofluorescence of RAHs varies from hypoautofluorescence to hyperautofluorescence patterns according to RAH types. The retinal involvement and calcification degree of TSC-associated RAHs could be reflected on the autofluorescence, which was beneficial to the full assessment. (Chin J Ophthalmol, 2020, 56: 211-216).

[Histopathology manifestation and imaging characteristics of in vivo confocal microscopy for diagnosis of ocular surface squamous neoplasia].

Objective: To study the histopathology manifestation and imaging characteristics of in vivo confocal microscopy (IVCM) for diagnosis of ocular surface squamous neoplasia (OSSN) and analyze the reliability of IVCM in differential diagnosis from OSSN cases. Methods: A prospective study. Twenty-three patients (23 eyes) with OSSN were collected from September 2015 to November 2017 in Beijing Tongren Eye center. They were underwent the examinations sequentially as follows: visual examination; slit-lamp microscope examination and ocular surface photography; color fundus image; anterior segment optical coherence tomography examination; IVCM examination and histopathology examination after surgery. With histopathology diagnosis, all subjects were divided into two groups: conjunctival intraepithelial neoplasm (CIN) and squamous cell carcinoma (SCC). Compared with histopathological findings, the IVCM results (cell morphology, cytoplasm and nucleus) of OSSN were analyzed. The difference analysis between the count data of two groups was carried out by the chi square test or Fisher's exact test. Receiver operating characteristic curve and area under the curve (AUC) were used to describe the accuracy of each parameter to differentiate SCC from OSSN. Results: There were 12 males and 11 females among the 23 patients aged (62±15) years. With the histopathological diagnosis, there were 15 cases (15 eyes) in the CIN group and 8 cases (8 eyes) in the SCC group. Eye redness, pain, foreign body sensation and tears were the symptoms of OSSN. There was no significant difference in clinical symptoms between the CIN and SCC groups. The size of tumors and the incidence of trophoblastic vessels in the SCC group were greater than those in the CIN group [8.5(6.5-15.5) mm vs. 5.8(4.0-8.5)mm, Z=4.702,P=0.029; 7/8 vs. 5/15, χ2=6.135, P=0.013). In histopathology, multilayered epithelia with cellular polymorphism, varied cytoplasm staining, and slightly thicker nuclei were observed in the CIN group. The corneal epithelial cells of SCC patients were markedly heterogeneous, and the intercellular bridge structure can be detected. Proliferative cells appeared with spindle shaped cells, nuclear mitotic figures and syncytial cells in the SCC group. The IVCM examination in the CIN group and SCC group showed that the cytoplasm was high and the ratio of nuclear plasma increased. There was no significant difference between the two groups in the size of epithelial cells, cytoplasm reflectivity, and nuclear cytoplasm ratio (P=0.053, 0.108, 0.067). The proportions of nuclear mitotic figures, spindle or chimeric cells, nests of whirlpool cells and the abnormal cells of the superficial stroma layer (8/8, 8/8, 8/8 and 8/8) in the SCC group was higher than those (2/15, 1/15, 4/15 and 0/15) in the CIN group, and the difference was statistically significant (P=0.001, 0.001, 0.003; P<0.001). Among these IVCM parameters, the abnormal cells of the superficial stroma layer had the highest diagnostic ability with the AUC value of 1.000, and the following AUC values were 0.933 for mitotic or double nuclei, 0.901 for spindle or chimeric cells, and 0.867 for the nests of whirlpool cells. Conclusions: The IVCM characteristics of OSSN are the dysplastic cells, hyper-reflective cytoplasm and increased nuclear plasma ratio in the corneal epithelium. Dysplastic cells appearing in the superficial stroma layer, nuclear mitotic figures and nests of vortex cells are the main IVCM parameters to support the malignant change of OSSN lesions. (Chin J Ophthalmol, 2018, 54: 652-660).

[In vivo confocal microscopic observation of corneal changes in patients with chronic Stevens-Johnson syndrome or toxic epidermal necrolysis].

Objective: To describe corneal alterations visible on in vivo confocal microscopy in patients with debilitating ocular sequelae caused by Stevens-Johnson syndrome (SJS) or toxic epidermal necrolysis (TEN). Methods: Twenty-two eyes of 11 consecutive patients suffering from chronic SJS or TEN were studied using slit lamp and in vivo confocal microscopy in the Ophthalmology Outpatient Department of Peking Union Medical College Hospital from March 2014 to April 2015. Results: Dry eye with meibomian gland dysfunction (19 eyes of 10 patients, 86.4%) was the most frequent clinical sequelae. Two patients (3 eyes, 13.6%) had severe vison loss with corneal neovascularization and conjunctivization. One patient had asymmetric binocular disease. Corneal epithelial cells were observed at an active state in 10 patients (19 eyes, 86.4%) and corneal stromal cells at an active state in all patients (22 eyes, 100%). Twenty eyes (10 patients, 91%) were noted with abnormal nerve layout and 10 eyes (5 patients, 45.5%) with dendritic cell infiltration around pathological nerve damages. Numerous inflammory cells were observed in 6 eyes (3 patients, 27.3%). Conclusions: The corneas of patients with chronic ocular sequelae associated with SJS or TEN present a number of abnormalities. In vivo confocal microscopy is a potetial useful tool for therapeutic indications and for follow-up of the debilitating chronic ocular problems linked to SJS and TEN. (Chin J Ophthalmol, 2017, 53:177-181).

Fiber-Optic Confocal Laser Endomicroscopy of Small Renal Masses: Toward Real-Time Optical Diagnostic Biopsy.

PURPOSE: The incidental detection of small renal masses is increasing. However, not all require aggressive treatments as up to 20% are benign and the majority of malignant tumors harbor indolent features. Improved preoperative diagnostics are needed to differentiate tumors requiring aggressive treatment from those more suitable for surveillance. We evaluated and compared confocal laser endomicroscopy with standard histopathology in ex vivo human kidney tumors as proof of principle towards diagnostic optical biopsy. MATERIALS AND METHODS: Patients with a solitary small renal mass scheduled for partial or radical nephrectomy were enrolled in study. Two kidneys were infused with fluorescein via intraoperative intravenous injection and 18 tumors were bathed ex vivo in dilute fluorescein prior to confocal imaging. A 2.6 mm confocal laser endomicroscopy probe was used to image tumors and surrounding parenchyma from external and en face surfaces after specimen bisection. Confocal laser endomicroscopy images were compared to standard hematoxylin and eosin analysis of corresponding areas. RESULTS: Ex vivo confocal laser endomicroscopy imaging revealed normal renal structures that correlated well with histology findings. Tumor tissue was readily distinguishable from normal parenchyma, demonstrating features unique to benign and malignant tumor subtypes. Topical fluorescein administration provided more consistent confocal laser endomicroscopy imaging than the intravenous route. Additionally, en face tumor imaging was superior to external imaging. CONCLUSIONS: We report what is to our knowledge the first feasibility study using confocal laser endomicroscopy to evaluate small renal masses ex vivo and provide a preliminary atlas of images from various renal neoplasms with corresponding histology. These findings serve as an initial and promising step toward real-time diagnostic optical biopsy of small renal masses.

Intraoperative Optical Biopsy during Robotic Assisted Radical Prostatectomy Using Confocal Endomicroscopy.

PURPOSE: Intraoperative optical biopsy technologies may aid in the identification of important anatomical landmarks and improve surgical outcomes of robotic assisted radical prostatectomy. We evaluate the feasibility of confocal laser endomicroscopy during robotic assisted radical prostatectomy. MATERIALS AND METHODS: A total of 21 patients with biopsy proven prostate cancer scheduled for robotic assisted radical prostatectomy were recruited. After intravenous administration of fluorescein 15 patients underwent in vivo intraoperative confocal laser endomicroscopy of prostatic and periprostatic structures using a 2.6 or 0.85 mm imaging probe. Standard robotic instruments were used to grasp and maneuver the confocal laser endomicroscopy probes for image acquisition. Confocal laser endomicroscopy imaging was performed ex vivo on fresh prostate specimens from 20 patients. Confocal video sequences acquired in vivo and ex vivo were reviewed and analyzed, with additional image processing using a mosaicing algorithm. Processed confocal images were compared with standard hematoxylin and eosin analysis of imaged regions. RESULTS: Confocal laser endomicroscopy was successfully integrated with robotic surgery, including co-registration of confocal video sequences with white light and probe handling with standard robotic instrumentation. Intraoperative confocal laser endomicroscopy imaging of the neurovascular bundle before and after nerve sparing dissection revealed characteristic features including dynamic vascular flow and intact axon fibers. Ex vivo confocal imaging of the prostatic parenchyma demonstrated normal prostate glands, stroma and prostatic carcinoma. CONCLUSIONS: We report the initial feasibility of optical biopsy of prostatic and periprostatic tissue during robotic assisted radical prostatectomy. Image guidance and tissue interrogation using confocal laser endomicroscopy offer a new intraoperative imaging method that has the potential to improve the functional and oncologic outcomes of prostate cancer surgery.

Reflectance confocal microscopy - Consensus terminology glossary in Brazilian Portuguese for normal skin, melanocytic and non-melanocytic lesions.

BACKGROUND: Currently, there is no uniform and official terminology in Portuguese for reflectance confocal microscopy analysis, despite the increasing number of Brazilian dermatologists using this new tool. OBJECTIVE: To present the terminology in Brazilian Portuguese for the description of reflectance confocal microscopy and establish the first Brazilian consensus on terms related to normal skin and cutaneous tumors. METHODS: 10 Brazilian specialists from different institutions and states of Brazil were selected to evaluate the best corresponding terms in Portuguese for normal skin, melanocytic and non-melanocytic tumors. The terms used were translated from international consensuses in the English language. The modified Delphi method was used to create the consensus in 3 steps. RESULTS: The terms considered the most appropriate in the Portuguese language to describe the findings of normal skin, melanocytic and non-melanocytic lesions in the reflectance confocal microscopy analysis were presented. STUDY LIMITATIONS: The limitations of the present study include the number of participants and limited regional representation (only two of the five Brazilian regions were represented). CONCLUSION: This Brazilian consensus represents an opportunity for dermatologists and physicians specializing in cutaneous oncology to become familiar with reflectance confocal microscopy, propagating the technique in clinical and research environments to stimulate national and international publications on this subject.

Confocal laser endomicroscopy reliably detects sepsis-related and treatment-associated changes in intestinal mucosal microcirculation.

BACKGROUND: Microcirculatory alterations play a central role in the pathophysiology of sepsis. We investigated probe-based confocal laser endomicroscopy (pCLE) to assess alterations in mucosal microcirculatory perfusion in vivo in a porcine model of septic shock and in patients fulfilling consensus criteria for severe sepsis. METHODS: Septic shock was induced using a faecal peritonitis model in anaesthetized, mechanically ventilated pigs. Mucosal microcirculation was assessed using pCLE in the stomach, duodenum, terminal ileum, and rectum. Duodenal microcirculation was further evaluated in 10 patients with severe sepsis and in 8 healthy controls to quantify capillary diameter, capillary length, and functional capillary density (FCD). RESULTS: In the animal model, FCD was markedly decreased in duodenal (P<0.001), ileal (P<0.001), gastric (P<0.001), and rectal mucosa (P<0.005) 4 h after induction of sepsis. After volume therapy, FCD partially recovered to 90.0% (duodenum), 94.4% (ileum), 95.4% (gastric), and 97% (rectum) of baseline values, indicating decoupling of microvascular and macrovascular flow. In septic patients, the mean capillary diameter (P<0.01) and FCD (P<0.05) in duodenal mucosa were decreased compared with healthy controls. CONCLUSIONS: pCLE reliably detected and quantified microcirculatory alterations in the gastrointestinal mucosa in a porcine model of sepsis and in patients with severe sepsis. Our data suggest that pCLE is a promising tool to assess the efficacy of therapeutic interventions on mucosal microcirculation in real-time, even in the clinical context.

The skin through reflectance confocal microscopy - Historical background, technical principles, and its correlation with histopathology.

Since its first introduction into medical practice, reflectance confocal microscopy (RCM) has been a valuable non-invasive diagnostic tool for the assessment of benign and malignant neoplasms of the skin. It has also been used as an adjunct for diagnosing equivocal cutaneous neoplasms that lack characteristic clinical or dermoscopic features. The use of RCM has led to a decreased number of biopsies of benign lesions. Multiple published studies show a strong correlation between RCM and histopathology thereby creating a bridge between clinical aspects, dermoscopy, and histopathology. Dermatopathologists may potentially play an important role in the interpretation of confocal images, by their ability to correlate histopathologic findings. RCM has also been shown to be an important adjunct to delineating tumoral margins during surgery, as well as for monitoring the non-surgical treatment of skin cancers. Advanced technology with smaller probes, such as the VivaScope 3000, has allowed access to lesions in previously inaccessible anatomic locations. This review explains the technical principles of RCM and describes the most common RCM features of normal skin with their corresponding histological correlation.

Universal adhesives applied to deep dentin with different bonding treatments.

Eighth-generation adhesives may be applied with total etch, selective-etch or self-conditioning, and serve as primers for non-dental substrates. AIM: To determine the bonding characteristics of universal adhesives applied to the deep pulp wall with different strategies, by means of shear bond strength and laser microscopy. MATERIALS AND METHOD: Cavities 4 mm deep and maximum width were carved in 36 extracted molars. Nine groups were formed according to dental substrate treatment and adhesives, as follows: Total-etch: group 1-Monobond 7 self-etch, group 2-One coat 7 universal, and group 3-Single bond universal; Adamantine etch: group 4-Monobond 7 self-etch, group 5-One coat 7 universal, and group 6-Single bond universal; Self-conditioning: group 7-Monobond 7 self-etch, group 8-One coat 7 universal, and group 9-Single bond universal. Molars were filled following the manufacturer's instructions. Three specimens per group (27 altogether) were used to determine shear bond strength using a universal testing machine, while layer thicknesses were measured on the remaining specimens using microscope images and Olympus LEXT 3D Software. Analysis of variance was used to compare data. RESULTS: Mean (standard deviation) bond strength in megapascals (MPa) was: group 1: 7.06±3.01; group 2: 10.74±4.36; group 3: 8.20±3.92; group 4: 7.41±2.23; group 5: 6.84±1.50; group 6: 5.86±2.10; group 7: 5.83±1.94; group 8: 7.14±2.37; group 9: 8.06±3.51. Bond strength was higher (p=0.049) for total-etch (8.61±3.96) than for selective etch (6.71±1.98) and self-conditioning (6.91±2.68). No significant difference was found among the three adhesives (p=0.205). Adhesive layer in micrometers (µm) was total-etch 8.71±4.93, selective etch 5.49±1.70 and self-conditioning 6.27±3.01, with no significant difference. CONCLUSIONS: There were significant differences among bonding strategies, with the highest values for total-etch. No significant difference was observed between self-conditioning and selective etch. No significant difference was found among the adhesives, which all behaved similarly. The greatest adhesive layer thicknesses were recorded in the total-etch group, with no significant difference among the various adhesive approaches.

Los adhesivos universales de octava generación pueden ser aplicados con diferentes estrategias de unión: grabado total, grabado selectivo o autoacondicionamiento. Además, imprimen sustratos no dentales. OBJETIVO: Determinar las caracteristicas de unión de adhesivos universales con diferentes estrategias en pared pulpar profunda mediante resistencia adhesiva al corte y microscopía laser. Materiales y Método: En 36 molares se tallaron cavidades de 4 mm de profundidad y ancho máximo. Se dividieron en 9 grupos según tratamientos y adhesivos. Grabado total: grupo 1-Monobond 7 self-etching, grupo 2-One coat 7 universal y grupo 3-Single bond universal; Grabado selectivo: grupo 4-Monobond 7 self-etching; grupo 5-One coat 7 universal y grupo 6-Single bond universal y Autoacondicionamiento: grupo 7-Monobond 7 self-etching; grupo 8-One coat 7 universal y grupo 9-Single bond universal. Las obturaciones se realizaron siguiendo las instrucciones del fabricante. La resistencia adhesiva al corte se determinó utilizando una máquina de ensayo universal sobre 27 especímenes mientras que los restantes fueron empleados para evaluar los espesores de la capa generado sobre imágenes obtenidas con microscopía y con el software Olympus LEXT 3D. Se ultilizó análisis de varianza. RESULTADOS: Resistencia adhesiva en megapascal (MPa) media (desviación estándar): grupo 1: 7,06±3,01; grupo 2: 10,74±4,36; grupo 3: 8,20±3,92; grupo 4: 7,41±2,23; grupo 5: 6,84±1,50; grupo 6: 5,86±2,10; grupo 7: 5,83±1,94; grupo 8: 7,14±2,37; grupo 9: 8,06±3,51. Grabado total (8,61±3,96) registró los valores mayores (p=0,049) en comparación a grabado selectivo (6,71±1,98) y autoacondicionamiento (6,91±2,68). Los adhesivos no tuvieron diferencias significativas (p=0,205). Capa adhesiva en µm: Grabado total (8,71±4,93); grabado selectivo (5,49±1,70) y autoacondicionamiento (6,27±3,01) sin diferencias significativas (p=0,073). CONCLUSIONES: Las estrategias de unión mostraron diferencias significativas; los valores más altos se obtuvieron con grabado total y entre autoacondicionamiento y grabado selectivo no hubo significancia. Los adhesivos evidenciaron comportamientos similares sin registrar diferencias significativas.

Systematic classification of confocal laser endomicroscopy for the diagnosis of oral cavity carcinoma.

INTRODUCTION: Confocal laser endomicroscopy (CLE) is an optical imaging technique that allows in vivo microscope-like images of the upper aerodigestive tract's mucosa in 1000-fold magnification. The assessment of morphological tissue characteristics for the correct differentiation between healthy and malignoma suspected mucosa requires strict evaluation criteria. This study aims to validate a score for oral cavity squamous cell carcinoma (OCSCC) diagnostic. METHODS: We performed CLE and examined a total of twelve patients. All 95 sequences (778 s, 6224 images) originate from the area of the primary tumor 260 s, 2080 images) and unsuspicious mucosa of the oral cavity (518 s, 4144 images). Specimen were taken at corresponding locations and analyzed histologically in H&E staining as a reference standard. A total of eight examiners (four experienced and four inexperienced) evaluated the sequences based on a scoring system. The primary endpoints are sensitivity, specificity, and accuracy. Secondary endpoints are inter-rater reliability and receiver operator characteristics. RESULTS: Healthy mucosa showed epithelium with uniform size and shape with distinct cytoplasmic membranes and regular vessel architecture. CLE of malignant cells demonstrated a disorganized arrangement of variable cellular morphology. We calculated an accuracy, sensitivity, specificity, PPV, and NPV of 88.7 %, 90.1 %, 87.4 %, 87.5 %, and 90.0 %, respectively, with inter-rater reliability and κ-value of 0.775, and an area under the curve of 0.935. CONCLUSIONS: The results confirm that this scoring system is applicable in the oral cavity mucosa to classify benign and malignant tissue.

Bowen's disease of the penile shaft presenting as a pigmented macule: dermoscopy, reflectance confocal microscopy and histopathological correlation.

The penile localization of pigmented Bowen's disease has been rarely reported and has been mostly related to human papillomavirus infection. Early diagnosis and treatment are important to prevent progression to invasive squamous cell carcinoma. However, diagnosis can be challenging because it may be difficult to distinguish from melanoma, even using dermoscopy. Reflectance confocal microscopy may be useful in suggesting the bedside diagnosis before the histopathological confirmation. A case of penile pigmented Bowen's disease is described along with its dermoscopy and reflectance confocal microscopy findings and their correlation with histopathology.