Hyperspectral imaging to accurately segment skin erythema and hyperpigmentation in cutaneous chronic graft-versus-host disease.

In 51 lesions from 15 patients with the inflammatory skin condition chronic graft-versus-host-disease, hyperspectral imaging accurately delineated active erythema and post-inflammatory hyperpigmentation. The method was validated by dermatologist-approved confident delineations of only definitely affected and definitely unaffected areas in photographs. A prototype hyperspectral imaging system acquired a 2.5 × 3.5 cm2 area of skin at 120 wavelengths in the 450-850 nm range. Unsupervised extraction of unknown absorbers by endmember analysis achieved a comparable accuracy to that of supervised extraction of known absorbers (melanin, hemoglobin) by chromophore mapping: 0.78 (IQR: 0.39-0.85) vs. 0.83 (0.53-0.91) to delineate erythema and 0.74 (0.57-0.87) vs. 0.73 (0.52-0.84) to delineate hyperpigmentation. Both algorithms achieved higher specificity than sensitivity. Whereas a trained human confidently marked a median of 7% of image pixels, unsupervised and supervised algorithms delineated a median of 14% and 27% pixels. Hyperspectral imaging could overcome a fundamental practice gap of distinguishing active from inactive manifestations of inflammatory skin disease.

Effect of camera distance and angle on color of diverse skin tone-based standards in smartphone photos.

Accurate and reproducible color capture is vital in medical photography. Camera distance and angle are particularly important as they are highly variable in a clinical setting. To account for variability in illumination, camera technology, and geometric effects, color standards are often used for color correction. To explore how geometry affects color, we quantified the change in CIELAB color value of a color standard for diverse skin tones at varying smartphone camera distances and angles. Whereas both chromaticity (a* and b*) and lightness (L*) were affected by angle, distance only affected L* (standard error of measurement, SEM > 1 CIELAB unit). Flash usage did not generally reduce distance and angle associated variability. Compared to compressed (JPG) format, raw (DNG) images had decreased median variability across different distances and angles. These findings suggest that in medical photography, inconsistent camera distance and angle can increase variability in photographed skin appearance over time.

Non-Expert Markings of Active Chronic Graft-Versus-Host Disease Photographs: Optimal Metrics of Training Effects.

Lack of reliable measures of cutaneous chronic graft-versus-host disease (cGVHD) remains a significant challenge. Non-expert assistance in marking photographs of active disease could aid the development of automated segmentation algorithms, but validated metrics to evaluate training effects are lacking. We studied absolute and relative error of marked body surface area (BSA), redness, and the Dice index as potential metrics of non-expert improvement. Three non-experts underwent an extensive training program led by a board-certified dermatologist to mark cGVHD in photographs. At the end of the 4-month training, the dermatologist confirmed that each trainee had learned to accurately mark cGVHD. The trainees' inter- and intra-rater intraclass correlation coefficient estimates were "substantial" to "almost perfect" for both BSA and total redness. For fifteen 3D photos of patients with cGVHD, the trainees' median absolute (relative) BSA error compared to expert marking dropped from 20 cm2 (29%) pre-training to 14 cm2 (24%) post-training. Total redness error decreased from 122 a*·cm2 (26%) to 95 a*·cm2 (21%). By contrast, median Dice index did not reflect improvement (0.76 to 0.75). Both absolute and relative BSA and redness errors similarly and stably reflected improvements from this training program, which the Dice index failed to capture.

Association of Leukocyte Adhesion and Rolling in Skin With Patient Outcomes After Hematopoietic Cell Transplantation Using Noninvasive Reflectance Confocal Videomicroscopy.

Importance: Hematopoietic cell transplantation (HCT) is a potential cure for hematologic cancer but is associated with a risk of relapse and death. Dynamic biomarkers to predict relapse and inform treatment decisions after HCT are a major unmet clinical need. Objective: To identify a quantitative characteristic of leukocyte-endothelial interactions after HCT and test its associations with patient outcomes. Design, Setting, and Participants: In this prospective single-center cohort study from June 2017 to January 2020, patients of any age, sex, race, and ethnicity who had HCT for hematologic cancer were referred by health care professionals as either suspected of having symptoms or not having symptoms of acute graft-vs-host disease between 25 and 161 days after HCT. Patients underwent noninvasive skin videomicroscopy. Videos of dermal microvascular flow were recorded with a reflectance confocal microscope. Two blinded observers (J.R.P. and Z.Z.) counted leukocytes adherent to and rolling along the vessel wall per hour (A&R). Of 57 enrolled patients, 1 relapsed before imaging and was excluded, resulting in 56 patients included in analyses. Main Outcomes and Measures: Relapse of cancer, relapse-free survival, and overall survival. Results: Among the 56 patients (median age, 59 years; 38 [68%] male) who underwent imaging a median of 40 days after HCT, 21 had high A&R and 35 had low A&R. After correcting for the revised Disease Risk Index, patients with high A&R had higher rates of relapse (hazard ratio [HR], 4.24; 95% CI, 1.32-13.58; P = .02), reduced relapse-free survival (HR, 3.29; 95% CI, 1.26-8.55; P = .02), and reduced overall survival (HR, 3.06, 95% CI, 1.02-9.19; P = .05). These associations were preserved after correcting for possible confounders, steroid treatment, and acute graft-vs-host disease status. In the prognostic adequacy calculation by using Cox models, the new imaging biomarker (A&R) accounted for 82% to 95% of the prognostic information to predict each outcome. By contrast, the best existing clinical predictor routinely available, the revised Disease Risk Index, accounted for 10% to 28% of the prognostic information in the same model. Conclusions and Relevance: In this cohort study, leukocyte-endothelial interactions, visualized directly in skin after HCT, were associated with the patient outcomes of relapse, relapse-free survival, and overall survival. Assessing this dynamic marker could help patients at high risk for relapse who may benefit from interventions, such as early withdrawal of immunosuppression.

Novel therapies and innovation for systemic sclerosis skin ulceration.

Skin ulceration is an important cause of morbidity in systemic sclerosis and can occur at anytime during disease progression. Incident disease cohorts are important for understanding whether skin ulceration represents active vasculopathy versus resultant damage. Biomarkers for skin ulcer pathogenesis, both serum and imaging, are under investigation to elucidate the functional consequences of the structural abnormalities. Novel therapeutics for the treatment of vasculopathy benefit from reliable biomarkers able to predict the disease evolution remains an important unmet need. Nonetheless, a diagnostic approach that captures early skin ulceration and treatments that restore vascular and immune homeostasis is critical for effective systemic sclerosis (SSc) vasculopathy management.

Baseline Photos and Confident Annotation Improve Automated Detection of Cutaneous Graft-Versus-Host Disease.

Cutaneous erythema is used in diagnosis and response assessment of cutaneous chronic graft-versus-host disease (cGVHD). The development of objective erythema evaluation methods remains a challenge. We used a pre-trained neural network to segment cGVHD erythema by detecting changes relative to a patient's registered baseline photo. We fixed this change detection algorithm on human annotations from a single photo pair, by using either a traditional approach or by marking definitely affected ("Do Not Miss", DNM) and definitely unaffected skin ("Do Not Include", DNI). The fixed algorithm was applied to each of the remaining 47 test photo pairs from six follow-up sessions of one patient. We used both the Dice index and the opinion of two board-certified dermatologists to evaluate the algorithm performance. The change detection algorithm correctly assigned 80% of the pixels, regardless of whether it was fixed on traditional (median accuracy: 0.77, interquartile range 0.62-0.87) or DNM/DNI segmentations (0.81, 0.65-0.89). When the algorithm was fixed on markings by different annotators, the DNM/DNI achieved more consistent outputs (median Dice indices: 0.94-0.96) than the traditional method (0.73-0.81). Compared to viewing only rash photos, the addition of baseline photos improved the reliability of dermatologists' scoring. The inter-rater intraclass correlation coefficient increased from 0.19 (95% confidence interval lower bound: 0.06) to 0.51 (lower bound: 0.35). In conclusion, a change detection algorithm accurately assigned erythema in longitudinal photos of cGVHD. The reliability was significantly improved by exclusively using confident human segmentations to fix the algorithm. Baseline photos improved the agreement among two dermatologists in assessing algorithm performance.

Guideline for in vivo assessment of adherent and rolling leukocytes in human skin microvasculature via reflectance confocal videomicroscopy.

OBJECTIVE: To develop a guideline that reliably identifies cutaneous adherent and rolling leukocytes from mimicking scenarios via in vivo reflectance confocal videomicroscopy. METHODS: We used a clinical reflectance confocal microscope, the VivaScope 1500, to acquire 1522 videos of the upper dermal microcirculation from 12 healthy subjects and 60 patients after allogeneic hematopoietic cell transplantation. Blinded to clinical information, two trained raters independently counted the number of adherent and rolling leukocytes in 88 videos. Based on discrepancies in the initial assessments, we developed a guideline to identify both types of leukocyte-endothelial interactions via a modified Delphi method (without anonymity). To test the guideline's ability to improve the inter-rater reliability, the two raters assessed the remaining 1434 videos by using the guideline. RESULTS: We demonstrate a guideline that consists of definitions, a step-by-step flowchart, and corresponding visuals of adherent and rolling leukocytes and mimicking scenarios. The guideline improved the inter-rater reliability of the manual assessment of both interactions. The intraclass correlation coefficient (ICC) of adherent leukocyte counts increased from 0.056 (95% confidence interval: 0-0.236, n = 88 videos, N = 10 subjects) to 0.791 (0.770-0.809, n = 1434, N = 67). The ICC of rolling leukocyte counts increased from 0.385 (0.191-0.550, n = 88, N = 10) to 0.626 (0.593-0.657, n = 1434, N = 67). Intra-rater ICC post-guideline was 0.953 (0.886-0.981, n = 20, N = 12) and 0.956 (0.894-0.983, n = 20, N = 12) for adherent and rolling, respectively. CONCLUSION: The guideline aids in the manual identification of adherent and rolling leukocytes via in vivo reflectance confocal videomicroscopy.

Non-invasive optical biopsy by multiphoton microscopy identifies the live morphology of common melanocytic nevi.

Multiphoton microscopy (MPM) is a promising non-invasive imaging tool for discriminating benign nevi from melanoma. In this study, we establish a MPM morphologic catalogue of common nevi, information that will be critical in devising strategies to distinguish them from nevi that are evolving to melanoma that may present with more subtle signs of malignancy. Thirty common melanocytic nevi were imaged in vivo using MPM. Quantitative parameters that can distinguish between different types of nevi were developed and confirmed by examining the histology of eleven of the imaged nevi. MPM features of nevi examined included cytologic morphology of melanocytes in the epidermis and dermis, the size and distribution of nevomelanocytes both within and around nests, the size of rete ridges, and the presence of immune cells in the dermis. Distinguishing features include cytological morphology, the size of nevomelanocytes, the size of nevomelanocyte nests, and the distribution of nevomelanocytes. Notably, these distinguishing characteristics were not easily appreciated in fixed tissues, highlighting essential differences in the morphology of live skin. Taken together, this work provides a morphologic compendium of normal nevi, information that will be critical in future studies directed at identifying melanocytic nevi that are evolving to melanoma.

Individual cell motion in healthy human skin microvasculature by reflectance confocal video microscopy.

OBJECTIVE: To describe upper dermal microvasculature of healthy human skin in terms of density and size of cutaneous blood vessels, leukocyte velocity, and leukocyte interactions with the endothelium. METHODS: We used a reflectance confocal microscope, the VivaScope 1500, to acquire videos of individual cell motion. RESULTS: We found no rolling leukocytes in the upper microvasculature of ten healthy subjects. We observed "paused" leukocytes, that is, leukocytes that temporarily stop, coinciding with the simultaneous stopping of the rest of the blood flow. We imaged more paused (median: 1.0 per subject) and adherent (1.5) leukocytes in the forearm than in the chest (median 0 paused and 0 adherent per subject) per 5 minutes of videos per body site. Leukocytes were paused for a median of 7 seconds in the forearm and 3 seconds in the chest, and we found no correlation between this parameter and the blood vessel or leukocyte size. We visualized blood flow change direction. Flowing leukocyte velocities followed a lognormal distribution and were on average higher in the chest (117 µm/s) than in the forearm (66 µm/s). CONCLUSION: The proposed method and reported values in healthy skin provide new insights into intact human skin microcirculation.

Multimodal Device for Real-Time Monitoring of Skin Oxygen Saturation and Microcirculation Function.

The present study introduces a recently developed compact hybrid device for real-time monitoring of skin oxygen saturation and temperature distribution. The prototype involves a snapshot hyperspectral camera, multi-wavelength illuminator, thermal camera, and built-in computer with custom-developed software. To validate this device in-vivo we performed upper arm vascular occlusion on eight healthy volunteers. Palm skin oxygen saturation maps were analyzed in real-time using k-means segmentation algorithm and two-layer optical diffuse model. The prototype system demonstrated a satisfying performance of skin hyperspectral measurements in the spectral range of 507-625 nm. The results confirmed the reliability of the proposed system for in-vivo assessment of skin hemoglobin saturation with oxygen and microcirculation.

Feature characterization of scarring and non-scarring types of alopecia by multiphoton microscopy.

OBJECTIVES: Non-invasive visualization of hair follicles is important for proper diagnosis and management of alopecia; however, histological assessment remains the gold standard. Laser imaging technologies have made possible noninvasive in vivo evaluation of skin and hair follicle. The aim of this study was to evaluate the ability of multiphoton microscopy (MPM) to non-invasively identify morphological features that can distinguish scarring from non-scarring alopecia. METHODS: MPM images were obtained from areas on the scalp affected by alopecia. Investigators blinded to the diagnosis analyzed hair follicle and shaft sizes. Patients were recruited and imaged at the UC Irvine Health Medical Center and the University of California, Irvine Beckman Laser Institute. Patients with androgenetic alopecia (AGA) and alopecia areata (AA), and scarring alopecia, in particular frontal fibrosing alopecia (FFA) were recruited and imaged from July 2016 to July 2017. RESULTS: We imaged 5 normal scalp subjects and 12 patients affected by non-scarring (7 subjects) and scarring (5 subjects) alopecia. In normal and non-scarring alopecia patients, MPM identified presence of sebaceous glands associated with hair follicles. MPM images of scarring alopecia were characterized by the presence of inflammatory cells surrounding hair follicles. Measurements of hair follicle diameter sizes were found to be significantly smaller in scarring alopecia patients compared to normal (P < 0.001) and compared to non-scarring alopecia patients (P = 0.046); non-scarring hair follicles were also significantly smaller than normal hair follicles (P = 0.043). CONCLUSIONS: This study shows that MPM imaging can non-invasively identify morphological features that distinguish scarring from non-scarring alopecia. Further studies are needed to validate this technique and evaluate its potential to be used as an aid for guiding treatment. Lasers Surg. Med. 51:95-103, 2019. © 2018 Wiley Periodicals, Inc.

Noninvasive optical diagnostics of enhanced green fluorescent protein expression in skeletal muscle for comparison of electroporation and sonoporation efficiencies.

We highlight the options available for noninvasive optical diagnostics of reporter gene expression in mouse tibialis cranialis muscle. An in vivo multispectral imaging technique combined with fluorescence spectroscopy point measurements has been used for the transcutaneous detection of enhanced green fluorescent protein (EGFP) expression, providing information on location and duration of EGFP expression and allowing quantification of EGFP expression levels. For EGFP coding plasmid (pEGFP-Nuc Vector, 10 µg/50 ml 10 µg/50 ml ) transfection, we used electroporation or ultrasound enhanced microbubble cavitation [sonoporation (SP)]. The transcutaneous EGFP fluorescence in live mice was monitored over a period of one year using the described parameters: area of EGFP positive fibers, integral intensity, and mean intensity of EGFP fluorescence. The most efficient transfection of EGFP coding plasmid was achieved, when one high voltage and four low voltage electric pulses were applied. This protocol resulted in the highest short-term and long-term EGFP expression. Other electric pulse protocols as well as SP resulted in lower fluorescence intensities of EGFP in the transfected area. We conclude that noninvasive multispectral imaging technique combined with fluorescence spectroscopy point measurements is a suitable method to estimate the dynamics and efficiency of reporter gene transfection in vivo.