State-of-the-art technologies provide new insights linking skin and blood vessel abnormalities in SSc-related disorders.

OBJECTIVE: A key unanswered question in systemic sclerosis (SSc) is how microvascular abnormality and fibrosis inter-relate. Our aim was to use state-of-the-art non-invasive imaging methods to gain new insights into pathophysiology, comparing patients with different subtypes of SSc, including early dcSSc, not only to healthy controls but also to patients with causes of Raynaud's phenomenon not progressing to fibrosis. METHODS: Laser Doppler imaging, nailfold capillaroscopy, spectroscopy, and ultrasound measured (respectively) perfusion, microvascular structure, oxygenation/oxidative stress, and skin thickening in the hands of 265 subjects: 31 patients with primary Raynaud's phenomenon (PRP), 35 with undifferentiated connective tissue disease (UCTD), 93 with limited cutaneous SSc (lcSSc), 46 with diffuse cutaneous SSc (dcSSc, including 27 'early') and 60 healthy controls. RESULTS: Mean perfusion was reduced in SSc groups compared to controls (lcSSc 172 perfusion units [standard deviation 157], late-dcSSc 90 [145], early-dcSSc 68 [137] vs. controls 211 [146]; p = 0.0002) as was finger-oxygenation (lcSSc 12.1 [13.6] arbitrary units [AU], late-dcSSc 12.2 [8.4], early-dcSSc 11.1 [11.3] vs controls 14.9 [10.5]; p = 0.0049). Oxidative stress was increased at the hand-dorsum in SSc groups (p = 0.0007). Perfusion positively correlated with oxygenation (r = 0.23, p < 0.001), and capillary density negatively with skin thickness (r = -0.26, p < 0.001). CONCLUSION: Our findings lend support to the hypothesis that in SSc, particularly early dcSSc, (but not in PRP or UCTD), reduced perfusion (together with structural microvascular abnormality) associates with reduced oxygenation, with oxidative stress and with skin thickening/fibrosis, most likely driving a vicious cycle which ultimately results in irreversible tissue injury. Findings in skin may mirror alterations in internal organs.

Non-invasive Imaging of Localised Scleroderma for Assessment of Skin Blood Flow and Structure.

Extensive morphoea causes major morbidity, disability and disfigurement; pathophysiology is poorly understood. The aim of this study was to investigate, with non-invasive imaging, the relationship between localised abnormalities of skin structure and perfusion, which characterise morphoea. Thirty-two patients with morphoea underwent imaging at affected and unaffected sites. Skin thickness was imaged with optical coherence tomography (OCT) and high-frequency ultrasound (HFUS). Perfusion was imaged with dual-wavelength laser Doppler imaging (LDI) and thermography. Epidermal thickness showed a small increase from affected to unaffected site (OCT, active and inactive plaques [p = 0.005 and p = 0.004], HFUS active plaques only [p = 0.03]). Deeper perfusion was higher within affected than unaffected sites (LDI p < 0.001, thermography p < 0.0001, active and inactive plaques). Epidermal thickness was inversely related to superficial (but not deeper) perfusion. This novel study of OCT, HFUS, LDI and thermography confirms loss of epidermal thickness and increased deeper perfusion in morphea plaques.

The influence of measurement location on reliability of quantitative nailfold videocapillaroscopy in patients with SSc.

OBJECTIVES: Nailfold videocapillaroscopy is being increasingly used as a marker of SSc-related microvascular disease, including in response to treatment. However, it requires further validation. Our aim was to assess the inter-observer, intra-observer and test-retest variability of semi-automated measurement of capillary features as well as of a manual density measurement. METHODS: All capillary apexes in images from 58 patients with SSc were marked up independently by two trained observers (inter-observer variability). The first observer then re-marked the images (intra-observer variability), and finally, the first observer marked up a second image of the same nailfold (test-retest). Mark-up of capillaries was carried out on cropped mosaic images (cropped independently by the observers to a fixed width, to allow the same length of nail bed to be studied for each patient) and on whole mosaic images (examining the whole nail bed). RESULTS: Reproducibility of independently cropped mosaic images was poor and was due to the variation in the positioning of the cropped area. However, quantification of whole mosaic images was highly reproducible, e.g. for inter-capillary distance, the intra-class correlation coefficient for inter-observer, intra-observer and test-retest reliability was 0.95, 0.98 and 0.90 (compared with 0.88, 0.79 and 0.89 for cropped mosaic images), respectively. Intra-observer limits of agreement for whole mosaic images were better than inter-observer reproducibility. CONCLUSION: Quantitative assessment of SSc-related change in nailfold capillaries is unreliable if examination of the same set of capillaries cannot be guaranteed. Conversely a wide-field, high-magnification system that allows visualization of the whole nail bed offers a highly reproducible approach for quantitative assessment and therefore has potential as an outcome measure.

A multicentre study on the reliability of qualitative and quantitative nail-fold videocapillaroscopy assessment.

OBJECTIVE: To investigate the inter- and intra-observer reliability of both qualitative and quantitative parameters used in the assessment of nail-fold capillaroscopy images. METHODS: Fifty mosaic nail-fold images of healthy controls (n = 10), patients with primary RP (n = 10) and SSc (n = 30) were assessed in random order by two blinded observers on two occasions at centres in Sweden, UK and The Netherlands. Each image was therefore scored by six observers twice. RESULTS: Inter- and intra-observer reliability of quantitative parameters showed substantial to almost perfect agreement [inter- and intra-observer weighted κ's for the number of widened capillaries was 0.75 and 0.87 and giant capillaries was 0.84 and 0.92, intra-class correlation coefficients (ICCs) for capillary density was 0.87 and 0.92 and total loop width was 0.94 and 0.98, respectively]. Qualitative parameters including architecture, avascularity, haemorrhage, crossed, ramified and bushy capillaries showed moderate to substantial inter-observer reproducibility (weighted κ ranging from 0.47 to 0.73), and substantial intra-observer repeatability (weighted κ ranging from 0.71 to 0.80), whereas the scoring of tortuous and bizarre capillaries showed poor inter-observer and substantial intra-observer agreement (inter-observer weighted κ's was 0.39 and 0.21 and intra-observer weighted κ's was 0.68 and 0.76, respectively). CONCLUSION: All quantitative and certain qualitative parameters are highly reliable in terms of inter- and intra-observer agreement. A combination of parameters with the highest reliability should be incorporated into future capillaroscopic scoring systems in studies of prediction and monitoring of SSc spectrum disorders.

Nail-fold capillary abnormalities are associated with anti-centromere antibody and severity of digital ischaemia.

OBJECTIVES: Advances in nail-fold capillaroscopy allow capillary abnormalities to be quantified. Our aim was to investigate, in patients with SSc, the relationships between the degree of nail-fold capillary abnormality and disease subtypes (lcSSc and dcSSc), duration of RP and the presence of (i) severe digital ischaemia (as defined by previous i.v. vasodilators, debridements or amputations), (ii) a positive ACA, (iii) clinically evident calcinosis, (iv) pulmonary arterial hypertension and (v) telangiectases. METHODS: This was a retrospective study of 176 patients. Six capillary measurements (four semi-automated and two manual) were calculated (automated width, distance between capillaries, tortuosity and derangement, and manual distance and density). Relationships between these measurements and the different clinical features of SSc were examined using multiple linear regressions (adjusted for age, sex and smoking status). RESULTS: One hundred and forty-two patients had lcSSc and 34 had dcSSc. Sixty-eight (39%) had a history of severe digital ischaemia, 66 (38%) were anti-centromere positive, 53 (30%) had clinically evident calcinosis and 26 (15%) had an estimated pulmonary artery pressure of >30 mmHg. Positive associations were found between both automated and manually measured distance between capillaries and (i) presence of severe digital ischaemia and (ii) positive ACA, and reduced density was also associated with positive anti-centromere. Patients with moderate/severe telangiectases had wider capillaries compared with those with 'mild' lesions. CONCLUSIONS: Both severe digital ischaemia and positive ACA are associated with measurable nail-fold capillaroscopic change, which has the potential of being an outcome measure for the structural microvascular disease associated with SSc-spectrum disorders.

Three-dimensional optoacoustic imaging of nailfold capillaries in systemic sclerosis and its potential for disease differentiation using deep learning.

The autoimmune disease systemic sclerosis (SSc) causes microvascular changes that can be easily observed cutaneously at the finger nailfold. Optoacoustic imaging (OAI), a combination of optical and ultrasound imaging, specifically raster-scanning optoacoustic mesoscopy (RSOM), offers a non-invasive high-resolution 3D visualization of capillaries allowing for a better view of microvascular changes and an extraction of volumetric measures. In this study, nailfold capillaries of patients with SSc and healthy controls are imaged and compared with each other for the first time using OAI. The nailfolds of 23 patients with SSc and 19 controls were imaged using RSOM. The acquired images were qualitatively compared to images from state-of-the-art imaging tools for SSc, dermoscopy and high magnification capillaroscopy. The vascular volume in the nailfold capillaries were computed from the RSOM images. The vascular volumes differ significantly between both cohorts (0.216 ± 0.085 mm3 and 0.337 ± 0.110 mm3; p < 0.0005). In addition, an artificial neural network was trained to automatically differentiate nailfold images from both cohorts to further assess whether OAI is sensitive enough to visualize anatomical differences in the capillaries between the two cohorts. Using transfer learning, the model classifies images with an area under the ROC curve of 0.897, and a sensitivity of 0.783 and specificity of 0.895. In conclusion, this study demonstrates the capabilities of RSOM as an imaging tool for SSc and establishes it as a modality that facilitates more in-depth studies into the disease mechanisms and progression.

A Multicenter Study of the Validity and Reliability of Responses to Hand Cold Challenge as Measured by Laser Speckle Contrast Imaging and Thermography: Outcome Measures for Systemic Sclerosis-Related Raynaud's Phenomenon.

OBJECTIVE: Reliable and objective outcome measures to facilitate clinical trials of novel treatments for systemic sclerosis (SSc)-related Raynaud's phenomenon (RP) are badly needed. Laser speckle contrast imaging (LSCI) and thermography are noninvasive measures of perfusion that have shown excellent potential. This multicenter study was undertaken to determine the reliability and validity of a hand cold challenge protocol using LSCI, standard thermography, and low-cost cell phone/mobile phone thermography (henceforth referred to as mobile thermography) in patients with SSc-related RP. METHODS: Patients with RP secondary to SSc were recruited from 6 UK tertiary care centers. The patients underwent cold challenge on 2 consecutive days. Changes in cutaneous blood flow/skin temperature at each visit were imaged simultaneously using LSCI, standard thermography, and mobile thermography. Measurements included area under the curve (AUC) for reperfusion/rewarming and maximum blood flow rate/skin temperature after rewarming (MAX). Test-retest reliability was assessed using intraclass correlation coefficients (ICCs). Estimated latent correlations (estimated from multilevel models, taking values between -1 and 1; denoted as rho values) were used to assess the convergent validity of LSCI and thermography. RESULTS: In total, 159 patients (77% with limited cutaneous SSc) were recruited (84% female, median age 63.3 years). LSCI and standard thermography both had substantial reliability, with ICCs for the reperfusion/rewarming AUC of 0.67 (95% confidence interval [95% CI] 0.54, 0.76) and 0.68 (95% CI 0.58, 0.80), respectively, and ICCs for the MAX of 0.64 (95% CI 0.52, 0.75) and 0.72 (95% CI 0.64, 0.81), respectively. Very high latent correlations were present for the AUCs of LSCI and thermography (ρ = 0.94; 95% CI 0.87, 1.00) and for the AUCs of standard and mobile thermography (ρ = 0.98; 95% CI 0.94, 1.00). CONCLUSION: This is the first multicenter study to examine the reliability and validity of cold challenge using LSCI and thermography in patients with SSc-related RP. LSCI and thermography both demonstrated good potential as outcome measures. LSCI, standard thermography, and mobile thermography had very high convergent validity.

Noninvasive measurement of skin autofluorescence is increased in patients with systemic sclerosis: an indicator of increased advanced glycation endproducts?

OBJECTIVE: Skin autofluorescence noninvasively assesses expression of advanced glycation endproducts and therefore potentially the presence of oxidative stress that is implicated in the pathogenesis of systemic sclerosis (SSc). We investigated whether autofluorescence was increased in patients with SSc, primary Raynaud's phenomenon (RP), and morphea as compared to healthy controls. METHODS: Measurements of autofluorescence were made at 5 upper limb sites in 16 healthy controls, 16 patients with diffuse cutaneous SSc (dcSSc), 15 with limited cutaneous SSc (lcSSc), 15 with primary RP, and 13 with morphea. For patients with morphea, additional measurements were made at the affected and an adjacent unaffected site. RESULTS: Autofluorescence was significantly increased in patients with dcSSc but not lcSSc as compared to controls at the proximal phalanx [dcSSc median 0.15, interquartile range (IQR) 0.10-0.24, vs control 0.10, IQR 0.07-0.13; p = 0.014], dorsum of the hand (dcSSc 0.17, IQR 0.11-0.36, vs control 0.12, IQR 0.09-0.17; p = 0.031), the wrist (dcSSc 0.22, IQR 0.13-0.33, vs control 0.13, IQR 0.09-0.18; p = 0.005), and forearm (dcSSc 0.19, IQR 0.12-0.47, vs control 0.14, IQR 0.10-0.16; p = 0.022). There was a trend for autofluorescence to be increased in patients with lcSSc and at morphea sites, compared to noninvolved skin. CONCLUSION: Autofluorescence is increased in patients with dcSSc compared to primary RP and to healthy controls. This suggests increased oxidative stress and the potential for autofluorescence as a biomarker.

Noninvasive imaging techniques in the assessment of scleroderma spectrum disorders.

OBJECTIVE: Systemic sclerosis (SSc) affects both microvascular structure and function. Laser Doppler imaging (LDI) and thermal imaging can be used to measure cutaneous blood vessel function. Nailfold capillaroscopy (NC) measures capillary morphology. The aim of this study was to investigate the relationship between capillary morphology and blood flow, and to determine which combination of techniques allows the best discrimination between patients with SSc, primary Raynaud's phenomenon (RP), and healthy controls. METHODS: NC was performed in 16 patients with SSc, 14 patients with primary RP, and 16 healthy controls. In addition, participants underwent cold stimulus with cold water. Hands were imaged to monitor rewarming and reperfusion. Nailfold morphologic features were measured and baseline images and rewarming curves were analyzed. RESULTS: Significant differences were found between groups (analysis of variance) for capillary morphologic features and rewarming curve characteristics. A correlation (P < 0.001) was found between LDI and thermal imaging at baseline (0.667) and maximum (0.729) blood flow and skin temperature, and for the areas under the rewarming curves (0.684). Receiver operating characteristic curves indicated that NC, thermal imaging, and LDI allowed 89%, 74%, and 72%, respectively, of SSc patient data to be correctly classified versus primary RP patients and controls. CONCLUSION: NC, LDI, and thermal imaging each independently provide good discrimination between patients with SSc and those with primary RP and healthy controls (NC being the most suitable technique for classifying patient groups). However, a combination of all 3 techniques improves classification. LDI and thermal imaging give equivalent information on dynamic changes in the cutaneous microcirculation; however, these only weakly correspond to capillary morphology.