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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.

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.

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.

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.

To evaluate the effect of illumination, camera orientation, and camera distance on color consistency of different skin tones, several swatches from a Pantone SkinTone Guide deck were selected and photographed at varying distances, illuminations, and angles. The RGB values from each selected swatch from each image were converted to LAB units and compared with the converted “true” values provided by Pantone. The ability of various color references to correct different skin tones was tested by comparing color values from a subject’s forearm skin to “true” color values of the closest Pantone SkinTone swatch found by visual comparison.

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.

Author(s): Lin, Jessica; Saknite, Inga; Valdebran, Manuel; Balu, Mihaela; Lentsch, Griffin; Williams, Joshua N; Koenig, Karsten; Tromberg, Bruce J; Atanaskova Mesinkovska, Natasha | Abstract: ObjectivesNon-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.MethodsMPM 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.ResultsWe 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 l 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).ConclusionsThis 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.

Background Through noninvasive imaging of live, individual immune cells of patients post allogeneic hematopoietic cell transplantation (HCT), we characterized differences in microvasculature among patients with cutaneous acute graft-versus-host disease (aGVHD) compared to post-HCT patients without any organ aGVHD (controls). Traditional histopathology is often non-specific for cutaneous aGVHD. We present parameters characteristic of cell motion within upper dermal capillaries, which could enable noninvasive tracking of disease development. Objective Test the feasibility of eight quantitative parameters extracted from reflectance confocal videos to distinguish between patients with and without cutaneous aGVHD following HCT. Methods We used a clinical reflectance confocal microscope to noninvasively image left volar forearm dermal capillaries of 5 cutaneous aGVHD patients and 5 post-HCT controls prior to topical and systemic therapies. All diagnoses were retrospectively confirmed on day 100 by a transplant physician (MB) based on initiation and response to therapy. Two raters (JRP, IS) independently estimated capillary density and size, and the number and size of paused (temporarily stopping), adherent (stationary for 30+ sec), and rolling leukocytes. The mean value per two raters was calculated for further analysis. Due to the small sample size, descriptive statistics were used. Results Patients in both study groups were similarly distributed in terms of gender (40% female), age (median 63 and 63, respectively), days post-HCT (median 44 and 41), and underlying disease (60% and 40% acute myeloid leukemia; the rest: myelodysplastic syndrome). 4 patients had only skin involvement: grade 1 (N=1 patient), grade 2 (N=3), whereas one patient had grade 2 cutaneous with symptoms of chronic ocular GVHD. One patient required therapy with systemic steroids. We found an increased number of rolling (median: 2 vs 1), adherent (3 vs 1) and paused (2 vs 1) leukocytes in aGVHD patients, compared to post-HCT controls (Figure 1). The average time of a leukocyte being paused was higher in aGVHD patients (6±7 seconds) than post-HCT controls (2±3 seconds). The size of paused leukocytes was greater in aGVHD patients (9±2¼m) compared to controls (6±4¼m) (Figure 2). Capillary size (8±2 vs. 8±2¼m) and density (4±1 vs. 3±2 capillaries) were similar among both groups. Conclusion In similar-sized capillaries, we observed increased leukocyte interactions with the endothelium in aGVHD patients compared to post-HCT controls. Despite similar sized rolling and adherent leukocytes, the size of paused leukocytes was larger in aGVHD patients. Future studies could evaluate the change of these parameters over time.

We explore the potential of reflectance confocal video microscopy of human skin microvasculature to noninvasively track immune cell activity following hematopoietic cell transplantation. In three patients, we tracked leukocyte-endothelial interactions over time.

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.

Inflammatory tissue response is one of the first and most common manifestations of acute graft-versus-host disease (aGVHD), a potentially deadly immune-mediated disease that occurs in 30-60% of patients after stem cell transplantation. A fundamental challenge in developing effective treatment strategies for aGVHD is the lack of tools to study disease biology in real-time in post-transplant patients. The inflammatory tissue response causes increased expression of specialized endothelial proteins on vessel walls making leukocytes to roll, adhere and eventually extravasate into the tissue at a higher rate than in normal conditions. Although the importance of leukocyte-endothelial interactions to detect and track inflammation has been well shown in murine models, there are no published clinical studies in humans. In this study, we explore the feasibility to detect presence of aGVHD in post-transplant patients through the imaging of in vivo leukocyte motion. We used a clinical confocal microscope (Vivascope 1500) to acquire videos of 5 aGVHD patients and 5 controls (no aGVHD) within 50±30 days post-transplant. The microscope is capable of real-time imaging of individual cells in the postcapillary vessels at 9 frames per second. Through video analysis, we extracted five quantitative parameters: number and velocity of rolling leukocytes, number of adherent leukocytes (stationary >30 s), blood flow velocity, and number of vessels. In a limited number of subjects, we show that parameters characteristic of the dynamic motion in skin capillaries can be observed noninvasively in post-transplant patients. Further studies are needed to test the diagnostic potential of these parameters.

Leukocyte-endothelial interactions have been well-characterized by intravital microscopy in mice. Quantitative parameters descriptive of these dynamic processes, e.g. the level of leukocyte rolling, adhesion and extravasation, can detect and track inflammation. Despite technology available to study individual cell motion noninvasively in human skin, we are not aware of any published exploratory or clinical studies. In this preliminary study, we explore the feasibility to extract parameters characteristic of individual cell motion in the postcapillary vessels of healthy human skin from videos taken by a noninvasive clinical confocal microscope (Vivascope 1500). The microscope is capable of real-time imaging of individual cells at 9 frames per second. We took videos of ten cutaneous vessels per each of two body sites (volar forearm and upper anterior chest) of ten healthy subjects. We then characterized the dynamic motion of cells via subsequent video analysis by extracting the following parameters: blood flow velocity, number of adherent leukocytes (stationary

A commercially available point measurement device, the Mexameter(®), and an experimental RGB imaging prototype device were used for erythema index estimation of 50 rosacea patients by analysing the level of skin redness on the forehead, both cheeks and both sides of a nose. Results are compared with Clinician's Erythema Assessment (CEA) values given by two dermatologists. The Mexameter uses 568 nm and 660 nm LEDs and a photodetector for estimation of erythema index, while the used prototype device acquired RGB images at 460 nm, 530 nm and 665 nm LED illumination. Several erythema index estimation algorithms were compared to determine which one gives the best contrast between increased erythema and normal skin. The erythema index estimations and CEA values correlated much better for the RGB imaging data than for those obtained by the conventional Mexameter technique that is widely used by dermatologists and in clinical trials. In result, we propose an erythema index estimation approach that represents increased erythema with higher accuracy than other available methods.

We explore challenges and opportunities for clinical implementation of optical technologies in dermatology. Examples include a confocal diagnostic service and cross-polarized 3D photography to train neural networks for assessments following stem cell transplantation.

RGB images of rosacea were analyzed using segmentation maps of principal component analysis (PCA) and erythema index (EI). Areas of segmented clusters were compared to Clinician’s Erythema Assessment (CEA) values given by two dermatologists. The results show that visible blood vessels are segmented more precisely on maps of the erythema index and the third principal component (PC3). In many cases, a distribution of clusters on EI and PC3 maps are very similar. Mean values of clusters’ areas on these maps show a decrease of the area of blood vessels and erythema and an increase of lighter skin area after the therapy for the patients with diagnosis CEA = 2 on the first visit and CEA=1 on the second visit. This study shows that EI and PC3 maps are more useful than the maps of the first (PC1) and second (PC2) principal components for indicating vascular structures and erythema on the skin of rosacea patients and therapy monitoring.

Diffuse reflectance spectroscopy system was developed for estimation of skin hydration in the near-infrared spectral range of 900-1700 nm. Experimental setup consisted of a near-infrared spectrometer, Y-type fiber optics probe with 1 detection and 6 illumination fibers, halogen-tungsten light source and a PC. By analyzing diffuse reflectance spectrum, a parameter representing skin hydration by performing baseline correction and calculating the area under the 1450 nm water absorption maximum is proposed. A clinical study was performed acquiring data of skin hydration of 39 patients' forearm skin. Results of the developed system are compared to results obtained by a commercial device based on skin conductance measurements.

Sepsis is a potentially fatal disease with mortality rate as high as 50% in patients with septic shock; mortality rate can increase by 7.6% per hour if appropriate treatment is not started. Internationally accepted guidelines for diagnosis of sepsis rely on vital sign monitoring and laboratory tests in order to recognize organ failure. This pilot study aims to explore the potential of hyperspectral and thermal imaging techniques to identify and quantify early alterations in skin oxygenation and perfusion induced by sepsis. The study comprises both physiological model experiments on healthy volunteers in a laboratory environment, as well as screening case series of patients with septic shock in the intensive care department. Hyperspectral imaging is used to determine one of the main characteristic visual signs of skin oxygenation abnormalities - skin mottling, whereas changes in peripheral perfusion have been visualized by thermal imaging as heterogeneous skin temperature areas. In order to mimic septic skin mottling in a reproducible way in laboratory environment, arterial occlusion provocation test was utilized on healthy volunteers. Visualization of oxygen saturation by hyperspectral imaging allows diagnosing microcirculatory alterations induced by sepsis earlier than visual assessment of mottling. Thermal images of sepsis patients in the clinic clearly reveal hotspots produced by perforating arteries, as well as cold regions of low blood supply. The results of this pilot study show that thermal imaging in combination with hyperspectral imaging allows the determination of oxygen supply and utilization in critically ill septic patients.

Mūsdienās multispektrālās attēlošanas iekārtas izmanto ādas parametru un fizioloģisko procesu aprakstīšanai gan pētniecības, gan diagnostikas nolūkiem. Iekārtu darbības uzlabošanai ir nepieciešams labāk saprast gaismas mijiedarbību ar audiem, kā arī veikt šo iekārtu kalibrēšanu ar ādas maketu. Redzamā un tuvā infrasarkanā optiskā diapazona spektroskopijā ir svarīgi ādas maketi, kas simulē audu slāņaino struktūru un ķīmiskās īpašības, kā arī maketi, kas ir bioloģiski līdzvērtīgi. Šajā pētījumā tika izveidots ādas makets no bioloģiskām un ķīmiski sintezētām struktūrām. Ādas maketa izveidei tika izmantota fibrīna matrica ar dermālo un epidermālo šūnu piejaukumu, lai imitētu ādas slāņaino struktūru. Fibrīna matrica tiek veidota no 0,47 ml asins plazmas, 0,4 ml fizioloģiskā šķīduma, 0,8 μl treneksāmskābes un 89,4 μl kalcija glukanāta. Izveidoto matricu ievieto šūnu inkubatorā, lai tā polimerizētos. Nākošais slānis tiek veidots ar dermālo šūnu piejaukumu (180-270 šūnas), un pēdējais fibrīna matriksa slānis tiek veidots ar epidermālo šūnu piejaukumu (270 šūnas) un šūnu augšanu veicinošu serumu (FBS). Šūnu kultivēšanai nepieciešamas vismaz divas nedēļas. Šajā slāņainajā struktūrā ir iespējams pievienot ādas hromoforu simulējošus iekļāvumus. Optiskajā diapazonā no 450-900 nm ādas hromoforas, kurām ir visizteiktākais spektrs, ir bilirubīns, melanīns un hemoglobīns. Lai simulētu ādas hromoforu spektrālās īpašības, tika izmantots sintezēts bilirubīns, eritrocītu masa un nigrozīns. Lai izpētītu šī maketa iekārtu kalibrēšanas potenciālu, tika izveidoti 76 paraugi, kur katros 24 paraugos bija pievienots viens no absorbentiem ar dažādām koncentrācijām. Pilna ādas maketa audzēšanai nepieciešamas divas nedēļas, lai ātrāk tiktu iegūti pirmie rezultāti tika veidoti maketi bez dermālo un epidermālo šūnu piejaukuma. Fibrīna matricas un ādas imitējošā maketa absorbcijas spējas ir mazas salīdzinājumā ar hromoforu absorbcijas spējām. Lai novērtētu maketu, kas paredzēti konkrētu hromoforu spektrālo īpašību imitēšanai, iespējams veikt eksperimentus ar fibrīna matricu, kuras izveidošanai ir nepieciešama viena diena. Sintezētā bilirubīna koncentrācijas tika mainītas robežās no 0,01-2,00 mg/ml, melanīna optisko īpašību simulējošās vielas nigrozīna koncentrācija tika mainīta no 1,5 - 312,8 μg/ml, eritrocītu masas koncentrācija mainījās no 0,2 - 42,4 mg/ml.Mērījumi tika veikti, izmantojot multispektrālās attēlošanas iekārtu Cri Nuance 2.4. (Cambridge Research & Instrumentation, Inc., Amerikas Savienotās Valstis). Absorbcijas spektrs tika apstrādāts, izmantojot Microsoft Office Excel 2007. Iegūtajos rezultātos ir iespējams redzēt, ka piedāvātais ādas makets spēj simulēt ādas optiskās īpašības. Izmantotie absorbenti - sintezētais bilirubīns, nigrozīns un eritrocītu masa - spēj simulēt ādas hromoforu spektrālās īpašības. Palielinot absorbentu koncentrāciju paraugā, palielinās absorbcijas spektra maksimālā intensitāte. Izveidotais ādas makets varētu būt izmantojams iekārtu kalibrēšanai, taču šis makets nav piemērots starplaboratoriju iekārtu salīdzināšanai, jo tas nav stabils laikā

BACKGROUND. The nature and kinetics of leukocyte migration are intimately connected to the pathophysiology of acute graft-versus-host disease (aGVHD). Real-time visualization of leukocyte-endothelial interactions in patients post hematopoietic transplantation (HCT) may augment clinical diagnosis and patient management. In this cross-sectional pilot study, we aimed to test the feasibility of parameters characteristic of leukocyte motion in skin capillaries as potential imaging biomarkers to detect aGVHD. STUDY POPULATION. We enrolled 16 post-HCT patients: 8 patients with any organ aGVHD and 8 patients with no organ aGVHD on the day of the imaging. Diagnoses were retrospectively confirmed on day 100 by a transplant physician (MB), who was blinded to the results of the confocal imaging. 7 out of 8 aGVHD patients had only skin involvement: stage 1 (N=2 patients), stage 2 (N=2), and stage 3 (N=3) cutaneous aGVHD, whereas one patient had stage 1 gut aGVHD with no skin involvement. 6 out of 8 aGVHD group patients required systemic therapy with steroids. METHODS. To noninvasively visualize leukocyte motion in the topmost capillaries of post-HCT patients' skin, we used a clinical reflectance confocal microscope (Vivascope 1500, Caliber I.D.). It enables real-time en face view of individual cells at 9 frames per second. We took twenty 30-second videos of different capillaries in the left volar forearm and left upper chest. We counted the number of adherent and rolling leukocytes per 10 minutes of videos per patient. RESULTS. We found increased leukocyte rolling in the aGVHD group (average of 3 rolling leukocytes per patient), compared to post-HCT controls (average of 1 rolling leukocyte). Highest leukocyte rolling (>8 rolling leukocytes) was associated with increased all-cause mortality, but not predictive of transplant-related mortality (TRM). Similarly, we found increased leukocyte adhesion in the aGVHD group (average of 3 adherent leukocytes per patient), compared to post-HCT controls (average of 1 adherent leukocyte). Interestingly, we observed 7 adherent leukocytes in one patient who had isolated gut aGVHD and no skin involvement. DISCUSSION. Our preliminary results show altered leukocyte-endothelial interactions in the skin capillaries of aGVHD patients, suggesting that confocal microscopy may be an important tool in augmenting the clinical diagnosis and managing post-HCT patients. Future studies should test whether there is a difference in the pattern of change in parameter values between patients who did and who did not develop aGVHD. ACKNOWLEDGEMENT. This work was supported in part by Career Development Award Number IK2 CX001785 from the United Sates Department of Veterans Affairs Clinical Science R&D (CSRD) Service. Disclosures Byrne: Karyopharm: Research Funding. Jagasia:Janssen: Research Funding; Kadmon: Consultancy; Incyte: Consultancy. Tkaczyk:Incyte: Consultancy.

BACKGROUND. Cutaneous erythema and histopathology features in patients post allogeneic hematopoietic cell transplantation (HCT) are nonspecific, making it difficult to distinguish acute graft-versus-host disease (aGVHD) from engraftment syndrome, drug reactions, viral infections, and other etiologies. Biopsies from different locations on the same patient frequently differ in aGVHD grade, and serial biopsies of a single lesion are not possible. Proper biopsy site selection, timing, and serial noninvasive microscopic monitoring may improve our understanding and management of aGVHD. In this study, we tested the feasibility of noninvasive reflectance confocal microscopy (RCM) to detect key histopathology features of cutaneous aGVHD. STUDY POPULATION. We enrolled 11 patients with high clinical suspicion of cutaneous aGVHD, as determined by a transplant physician. In total, 16 lesions of cutaneous aGVHD-affected site were imaged by RCM (6x6 mm2en face images at four different depths) and subsequently biopsied (4x4 mm punch biopsy). 9 out of 11 patients had only skin involvement, and 2 patients had skin and gut aGVHD. 5 patients had >50% body surface area (BSA) affected, 1 had 18-50% BSA, and 5 had METHODS. We used a clinical confocal microscope (Vivascope 1500, Caliber I.D.), which enables real-time evaluation of epidermal and superficial dermal tissue at sub-cellular resolution. Four reflectance confocal microscopists blinded to histopathology independently evaluated the presence or absence of 18 RCM features1. Concurrently, four dermatopathologists blinded to clinical and confocal information determined the presence or absence of 19 histopathology features, as well as the Lerner aGVHD grade. A histologic feature was determined as "present" in a lesion when marked by most experts (expert vote) or resolved by a fifth expert in case of a disagreement among four experts. The reflectance confocal microscopist vote was then correlated to the dermatopathologist expert vote for 17 overlapping features. We also evaluated the interrater variability among microscopists and among dermatopathologists. RESULTS. The main aGVHD features by Lerner grade had > 88% correlation between RCM and histopathology: (1) basal vacuolar change, (2) presence of dyskeratotic keratinocytes, and (3) dermal inflammation. By contrast, dyskeratotic cells and inflammation at the adnexal structures had CONCLUSIONS. In this pilot study, we show that noninvasive label-free RCM of skin enables detection of the main Lerner grade features of cutaneous aGVHD. We found a similar interrater variability among reflectance confocal microscopists and among dermatopathologists who each independently evaluated the presence or absence of a list of aGVHD features. Future studies can build on this work to evaluate the feasibility of RCM to determine cutaneous aGVHD grade, as well as distinguish between rash due to aGVHD and drug reactions. ACKNOWLEDGEMENT. This work was supported in part by Career Development Award Number IK2 CX001785 from the United Sates Department of Veterans Affairs Clinical Science R&D (CSRD) Service. Saknite I, Gill M, Alessi-Fox C, Byrne M, Jagasia M, Gonzalez S, Ardigo M, Tkaczyk ER. Features of cutaneous acute graft-versus-host disease by reflectance confocal microscopy. Br J Dermatol (2019). Disclosures Christi: Caliber I.D.: Employment, Equity Ownership. Byrne:Karyopharm: Research Funding. Jagasia:Kadmon: Consultancy; Incyte: Consultancy; Janssen: Research Funding. Tkaczyk:Incyte: Consultancy.

This presentation will discuss multiphoton microscopy as a tool for in-vivo imaging of skin pigmentation disorders, such as vitiligo and melasma, in order to advance the understanding of these skin conditions and the treatment impact.

Distant Determination of Bilirubin Distribution in Skin by Multi-Spectral Imaging For mapping the bilirubin distribution in bruised skin the multi-spectral imaging technique was employed, which made it possible to observe temporal changes of the bilirubin content in skin photo-types II and III. The obtained results confirm the clinical potential of this technique for skin bilirubin diagnostics.

Mobile platform for multimodal skin assessment has been developed. Different illumination sources allow switching between modalities. Diffuse reflectance spectral imaging is provided by five LEDs, fluorescence is excited by 405 nm LEDs, and laser speckle by 650 nm laser diode. Handheld, battery powered device includes all light sources and color camera with USB. The core of the system is Linux OS embedded microcontroller. USB, FTP and HTML with JavaScript combination is used to create standard image transfer and control interface. In combination with built in WiFi access point it allows online skin images storage and processing. It means that data processing algorithms are located and updated online.

Early diagnostics of skin cancer is of interest for dermatologists. Atypical nevi are not considered to be malignant, but are suspects that should be detected and monitored over time. The multispectral imaging system Nuance operating in spectral range 450–950 nm was adapted for clinical in vivo measurements in diffuse reflectance and fluorescence mode. Mean and standard deviation values of optical density and fluorescence intensity were extracted from segmented pigmented lesions (21 benign and 26 atypical nevi) and used for further analysis. It was possible to achieve 62% sensitivity and 67% specificity for discrimination between atypical and benign lesions using averaged fluorescence mean intensity and standard deviation values.

Near-infrared spectroscopy has a potential for noninvasive determination of skin moisture level due to high water absorption. In this study, diffuse reflectance spectra of in vivo skin were acquired in the spectral range of 900 nm to 1700 nm by using near-infrared spectrometer, optical fiber and halogen bulb light source. Absorption changes after applying skin moisturizers were analyzed over time at different body sites. Results show difference in absorption when comparing dry and normal skin. Comparison of absorption changes over time after applying moisturizer at different body sites is analyzed and discussed. Some patterns of how skin reacts to different skin moisturizers are shown, although no clear pattern can be seen due to signal noise.

In this study, skin erythema assessment of 90 rosacea patients was estimated by a simple, low-cost RGB imaging device. A new erythema index assessment algorithm is proposed and clinically validated. Comparison with dermatologist’s visual assessment shows high correlation.

Assessment of skin blood flow is of interest for evaluation of skin viability as well as for reflection of the overall condition of the circulatory system. Laser Doppler perfusion imaging (LDPI) and laser speckle contrast imaging (LASCI) are optical techniques used for assessment of skin perfusion. However, these systems are still too expensive and bulky to be widely available. Implementation of such techniques as connection kits for mobile phones have a potential for primary diagnostics. In this work we demonstrate simple and low cost LASCI connection kit for mobile phone and its comparison to laser Doppler perfusion imager. Post-occlusive hyperemia and local thermal hyperemia tests are used to compare both techniques and to demonstrate the potential of LASCI device.

Laser speckle contrast analysis (LASCA) offers a non-contact, full-field, and real-time mapping of capillary blood flow and can be considered as an alternative method to Laser Doppler perfusion imaging. LASCA technique has been implemented in several commercial instruments. However, these systems are still too expensive and bulky to be widely available. Several optical techniques have found new implementations as connection kits for mobile phones thus offering low cost screening devices. In this work we demonstrate simple implementation of LASCA imaging technique as connection kit for mobile phone for primary low-cost assessment of skin blood flow. Stabilized 650 nm and 532 nm laser diode modules were used for LASCA illumination. Dual wavelength illumination could provide additional information about skin hemoglobin and oxygenation level. The proposed approach was tested for arterial occlusion and heat test. Besides, blood flow maps of injured and provoked skin were demonstrated.

The diffuse reflectance multispectral imaging technique has been used for distant mapping of in vivo skin chromophores (hemoglobin and melanin). The fluorescence multispectral imaging is not so common for skin applications due to complicity of data acquisition and processing, but could provide additional information about skin fluorophores. Both techniques are compatible, and could be combined into a multimodal solution. The multispectral imaging system Nuance based on liquid crystal tunable filters was adapted for diffuse reflectance and fluorescence spectral imaging of in vivo skin. Uniform illumination was achieved by LED ring light. Combination of four LEDs (warm white, 770 nm, 830 nm and 890 nm) was used to support diffuse reflectance mode in spectral range 450-950 nm. 405 nm LEDs were used for excitation of skin autofluorescence. Multispectral imaging system was adapted for spectral working range of 450-950 nm with scanning step of 10 nm and spectral resolution of 15 nm. An average field of view was 50x35 mm in size with spatial resolution 0,05 mm (the pixel size). Due to spectrally different illumination intensity and system sensitivity, various exposure times (from 7…500 ms) were used for each image acquisition. The proposed approach was tested for different skin lesions: benign nevus, hemangioma, basalioma and halo nevus. Spectral image cubes of different skin lesions were acquired and analyzed to test its diagnostic potential.