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Progrès dans les méthodes diagnostiques du déficit en cellules souches limbiques. Apport de la microscopie confocale et de la tomographie en cohérence optique
摘要： The limbus is the anatomical and functional barrier between corneal and conjunctival epithelia. It is characterized by presence of the limbal stem cell niche which allows corneal homeostasis to be maintained. Limbal stem cell deficiency is characterized by a dual process: insufficient regeneration of corneal epithelium, which cannot therefore assure its function of physiological support, associated with corneal invasion by conjunctival proliferation. Diagnosis is currently made via routine clinical examination, corneal impression cytology and in vivo confocal microscopy (IVCM). Slit lamp examination shows abnormal limbal anatomy, thin and irregular epithelium with late fluorescein staining, and superficial vascularization. With its high resolution, IVCM allows identification of limbal and corneal epithelial changes at a cellular level in en face views, parallel to the corneal surface, but with a restricted viewing field of the corneal surface. It shows a poor transition between the corneal and conjunctival epithelia, associated with a loss of the normal corneal epithelial stratification, low basal cell and sub-basal nerve plexus densities, even with sub-epithelial fibrosis. Optical coherence tomography in central cornea and at the limbus, with scans in different orientations, allows a quick, global and non-invasive analysis of normal eyes and those with limbal stem cell deficiency. It shows a thin limbal epithelium, lacking normal thickening, featuring absence of stromal undulations and limbal crypts in cross-sections and sections parallel to the limbus, lack of visible limbal crypts in en face sections, loss of clear transition between the hyporeflective corneal epithelium and the hyperreflective conjunctival epithelium, and hyperreflective sub-epithelial fibrosis.
关键词： Limbal stem cell deficiency,Limbus,Optical coherence tomography,In vivo confocal microscopy
更新于2025-09-23 15:23:52
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In vivo confocal microscopy morphometric analysis of corneal subbasal nerve plexus in dry eye disease using newly developed fully automated system
摘要： Purpose To evaluate in vivo confocal microscopy (IVCM) features of corneal subbasal nerve plexus (SNP) in the setting of dry eye disease (DED) using fully automated software BACCMetrics,^ and to further investigate its diagnostic performance in discriminating DED patients. Methods IVCM exams of SNP in DED patients and matched control subjects were performed using Heidelberg Retina Tomograph with the Rostock Cornea Module. The following parameters were obtained with ACCMetrics: corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), corneal nerve fiber length (CNFL), corneal nerve total branch density (CTBD), corneal nerve fiber area (CNFA), corneal nerve fiber width (CNFW), and corneal nerve fractal dimension (CNFrD). The Mann–Whitney U test was used to compare variables. Receiver operating characteristic curves with calculations of the area under the curve (AUC) were used to describe the accuracy of IVCM parameters for discriminating DED patients from controls. Results Thirty-nine DED patients and 30 control subjects were included. Significantly, lower values of CNFD, CNBD, and CNFL and higher value of CNFW were found in DED patients compared to controls (respectively, 20.5 ± 8.7 vs 25.4 ± 6.7 n/ mm2; 25.6 ± 20.1 vs 37.6 ± 21.5 n/mm2; 12.6 ± 4.4 vs 14.5 ± 2.9 mm/mm2; 0.021 ± 0.001 vs 0.019 ± 0.001 mm/mm2; always p < 0.024). CNFW value had the highest diagnostic power in discriminating DED patients (AUC = 0.828). When the diagnosis of DED was made based on either CNFW or CNBD, the sensitivity was 97.4% and the specificity 46.7%. Conclusions The software ACCMetrics was able to rapidly detect SNP alterations occurring in the setting of DED and showed good diagnostic performance in discriminating DED patients.
关键词： Dry eye,Sub-basal nerve plexus,Automated analysis,ACCMetrics,In vivo confocal microscopy
更新于2025-09-23 15:22:29
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In vivo confocal microscopy classification in the diagnosis of meibomian gland dysfunction
摘要： Aim Meibomian gland dysfunction (MGD) is one of the most common disorders in ophthalmology. The aim of this study was to evaluate the use of this in vivo confocal microscopy (IVCM)-MGD description to classify patients affected by clinical MGD and measure the correlation with standard clinical criteria and subjective symptoms. Methods One hundred eyes of 100 patients suffering from MGD and 15 eyes of normal subjects were included. A comprehensive evaluation with the ocular surface disease index (OSDI), Schirmer test, tear break-up time (TBUT), tear osmolarity, Oxford score, Meibomian gland expression, palpebral IVCM, and meibography was performed. Then each patient was classi?ed using a new IVCM classi?cation: type 0 for normality, type 1 for meibum obstruction, type 2 for in?ammation, and type 3 for ?brosis. Results The mean age of patients was 52 ± 20 years old, the OSDI was 38 ± 23, the BUT 5 ± 2.6 s, the Schirmer test 13 ± 7 mm, tear osmolarity 300 ± 11 osmol/L, the Oxford score 0.5 ± 0.6, the meibum expression score 1.7 ± 1.02, and the meibography score 1.3 ± 0.9. The IVCM MG classi?cation of the 15 normal subjects was 0. For MGD patients, 29% were in type 1, 40% were type 2, and 31% were type 3. The patients in IVCM MG type 2 had a higher OSDI (p = 0.001) compared with the other types. There was a strong correlation between the IVCM score and the meibography score (r = 0.71 p < 0.0001). Conclusion This new IVCM classi?cation provided a practical pathophysiological system for MGD. By giving objective criteria, this IVCM classi?cation may help advance the understanding of patients’ symptoms and enhance treatment effectiveness in MGD.
关键词： classification,Meibomian gland dysfunction,diagnosis,in vivo confocal microscopy
更新于2025-09-23 15:19:57
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Apport de la microscopie confocale par réflectance et de l’OCT dans le diagnostic d’un carcinome basocellulaire nodulaire kystique
摘要： The role of reflectance confocal microscopy and OCT in the diagnosis of nodular kystic BCC
关键词： Basal cell carcinoma,Noninvasive imaging,Optical coherence tomography,In vivo confocal microscopy,Dermoscopy,In vivo reflectance confocal microscopy,Cystic basal cell carcinoma
更新于2025-09-19 17:15:36
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Mapping of dendritic lesions in patients with herpes simplex keratitis using in vivo confocal microscopy
摘要： Purpose: To produce a two-dimensional reconstruction map of dendritic lesions in patients with herpes simplex keratitis (HSK) using in vivo confocal microscopy. Methods: Four eyes of four patients (mean 65.8 years) with HSK presenting with a dendritic lesion were enrolled. Slit-lamp biomicroscopy and in vivo laser confocal microscopy were performed. Acquired confocal images at the level of the epithelium were arranged and mapped into subconfluent montages. Changes in the shape and degree of light reflection of abnormal cells and deposits around dendritic lesions as well as other corneal layers were qualitatively evaluated. Results: Mapping of dendritic lesion was successful in all cases, and the subconfluent montages clearly showed the larger image of dendritic lesion. In all cases, the dendritic lesion consisted of hyperreflective irregular epithelial cells, and was surrounded by distorted and elongated epithelial cells. In three cases, hyperreflective deposits were noted at the midline of the lesion. The corneal stroma showed a hyperreflective honeycomb pattern. In two cases, inflammatory cells were observed at the level of endothelial cell layer. Conclusion: Mapping of dendritic lesions in patients with HSK was successful in all patients using in vivo confocal microscopy. Cellular level observation of dendritic lesion at a relatively larger magnification may help understand the in vivo morphological change of HSK. Further study in more patients with HSK and nonherpetic dendritic lesion is needed to utilize confocal microscopy images in differential diagnosis and follow-up of the epithelial lesions with dendrite.
关键词： hyperreflective irregular epithelial cells,herpetic epithelial keratitis,in vivo confocal microscopy,fluorescein slit-lamp photograph,dendritic lesion
更新于2025-09-10 09:29:36
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Diagnostic accuracy of ex vivo fluorescence confocal microscopy for Mohs surgery of basal cell carcinomas: a prospective study on 753 margins
摘要： Background: Frozen histological sections are currently used for intra-operative margin assessment during Mohs surgery. Fluorescence confocal microscopy (FCM) is a new tool, which offers a promising and faster alternative to frozen histology. Objective: The aim of the present study is to prospectively evaluate in a clinical setting, the accuracy of FCM as compared to frozen sections in BCC’s margin assessment. Methods: Patients with BCC, scheduled for Mohs surgery were prospectively enrolled. Freshly-excised surgical specimens were first examined through FCM and then frozen sections were evaluated. Permanent sections were finally obtained, in order to validate our sample technique. A blind re-evaluation was also performed for discordant cases between FCM and frozen sections. Sensitivity and specificity levels, as well as positive and negative predicting values were calculated and ROC curves were generated. Results: We enrolled 127 BCCs in as many patients (40.2% females). A total number of 753 sections were examined. All BCCs were located on the head and neck area. When evaluating the performance of FCM as compared to frozen sections 79.8% sensitivity, 95.8% specificity, 80.5% positive predicting and 95.7% negative predicting values were found (Area Under the Curve: .88, 95%CI .84-.92; P<.001). A total of 49 discordant cases between FCM and frozen sections evaluations were blindly re-evaluated, of which 24 were false positive and 25 false negative. The performance of FCM and frozen sections was also evaluated according to the final histopathological assessment. Conclusions: We found high levels of accuracy for FCM as compared to frozen sections evaluation, in intra-operative BCC’s margin assessment during Mohs surgery. Some technical issues still prevent a wide use of this technique, but new upcoming devices promise to overcome these limitations.
关键词： basal cell carcinoma,fluorescence confocal microscopy,Mohs surgery,skin cancer,ex vivo confocal microscopy
更新于2025-09-09 09:28:46
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Classification of Limbal Stem Cell Deficiency Using Clinical and Confocal Grading
摘要： To grade the severity of limbal stem cell deficiency (LSCD) based on the extent of clinical presentation and central corneal basal epithelial cell density (BCD). This is a retrospective observational comparative study of 48 eyes of 35 patients with LSCD and 9 eyes of 7 normal subjects (controls). Confocal images of the central cornea were acquired. A clinical scoring system was created based on the extent of limbal and corneal surface involvement. LSCD was graded as mild, moderate, and severe stages based on the clinical scores. The degree of BCD reduction was given a score of 0 to 3. Compared with BCD in controls, BCD decreased by 23.0%, 40.4%, and 69.5% in the mild, moderate, and severe stages of LSCD classified by the clinical scoring system, respectively. The degree of BCD reduction was positively correlated with larger limbal and corneal surface involvement and when the central visual axis was affected (all P # 0.0005). Mean corrected distance visual acuity logarithm of the minimum angle of resolution was 0.0 6 0.0 in control eyes, 0.2 6 0.5 in mild LSCD, 0.6 6 0.4 in moderate LSCD, and 1.6 6 1.1 in severe LSCD (P , 0.0001). There was a significant correlation between a higher clinical score and corrected distance visual acuity logarithm of the minimum angle of resolution (rho = 0.82; P , 0.0001) and a greater decrease in BCD (rho = 20.78; P , 0.0001). A clinical scoring system was developed to assess the extent of clinical presentation of LSCD. A classification system to grade the severity of LSCD can be established by combining the BCD score with the clinical score.
关键词： diagnosis,classification system,limbal stem cell,in vivo confocal microscopy,limbal stem cell deficiency
更新于2025-09-09 09:28:46
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Ex Vivo Confocal Microscopy Of Basal Cell Carcinoma On A 3-Color Scale
摘要： Ex vivo confocal microscopy (CM) is capable of visualizing freshly excised tissue in real-time with cellular resolution without routine processing. Depending on the laser wavelength, either reflectance (RCM) or fluorescence (FCM) is utilized. Ex vivo CM is useful for the rapid evaluation of tumor margins during Mohs micrographic surgery (MMS). Initially, ex vivo RCM studies used acetic acid as contrast agent to enhance basal cell carcinoma (BCC) cell nuclei. However, thin strands of BCC were frequently missed. The use of fluorophores improves contrast, so that even small strands of BCC can be spotted in FCM mosaics. Acridine orange (AO) is the dye most widely used. AO binds specifically to DNA and emits fluorescence, so images of living cell nuclei can be enhanced and displayed as bright structures in FCM mosaics. Even though good resolution and morphological correlation are achieved with this standard technique, nowadays confocal mosaics are displayed in a grey scale format. However, dermato-pathologists are often neither familiar with nor comfortable assessing these black-and-white images. We herein report a new technique for obtaining 3-color scale confocal mosaics (3CS-FCM) with the simultaneous use of AO and ethidium bromide (EB) as fluorescent dyes. In this technique, the excised skin sample is first soaked with liquid nitrogen. The sample is then sectioned into 20-30 μm-thick slices using a cryostat and stained with the dye mixture (AO 0.1 mM + EB 0.25 mM) for about one minute. The sample is then placed in the confocal microscope plate for imaging (Nikon A1R+, NIKON CORPORATION?, Japan). The tissue is scanned simultaneously with two different wavelength lasers (405 and 488 nm) and the collected fluorescence displayed on the screen as a 3-color-scale mosaic. Around 10 to 15 minutes are required for completion of the tissue processing and for final mosaics to be developed. Unlike AO, EB binds specifically to the DNA of BCC cells that are damaged due to freezing. As a result, BCC nests are stained by EB and emit red fluorescence after laser stimulation; in contrast, the epidermis and dermis are stained by AO and emit green fluorescence. Blue color corresponds to the background tissue autofluorescence. All fluorescence is collected by the microscope displaying the final images in a 3-color scale format. AO and EB staining do not affect additional fixation or staining of the sample. Figure 1 shows completed BCC mosaics displayed with this new technique. Each color represents a different skin structure, making the mosaics easier to read. In this way, 3CS confocal mosaics are more user-friendly and can be interpreted by healthcare professionals without previous experience with FCM. Moreover, with frozen sample processing, the tissue is completely flattened and the entire sample can be displayed on the screen. These developments represent important advantages over previously described images obtained with CM. In conclusion, 3CS-FCM is an innovative technique that provides colored images, expanding significantly the applicability of FCM. Larger studies are nevertheless required to validate the technique for MMS and other applications.
关键词： fluorescence confocal microscopy,3-color scale mosaics,basal cell carcinoma,ethidium bromide,acridine orange,ex vivo confocal microscopy,Mohs micrographic surgery
更新于2025-09-04 15:30:14