Port Wine Stain Skin Research Articles

The Relationship Between the Effectiveness of HMME-PDT and the Dermoscopic Features of Port-wine Stains in Chinese Pediatric Patients: A Retrospective Study.

IntroductionAlthough pulsed dye laser (PDL) remains the gold standard for the treatment of port-wine stains (PWS), hematoporphyrin monomethyl ether photodynamic therapy (HMME-PDT) is another treatment modality that has been shown to be effective in the treatment of PWS. This study aimed to observe the clinical efficacy and therapeutic response of HMME-PDT in the treatment of pediatric Chinese patients with PWS and to analyze the association between the efficacy of therapy and the dermoscopic features of PWS.MethodsPediatric patients with PWS and negative HMME skin test were enrolled between December 2017 and May 2021. Patients received an intravenous injection of 5 mg/kg HMME, and lesions were irradiated with 532-nm LED green light with a power density of 70–80 mW/cm2 for 20–25 min. Digital photographs and dermoscopic images were taken before and after two treatment sessions, and the clinical response was observed. The relationship between the efficacy of HMME-PDT and the dermoscopic features of PWS was retrospectively analyzed.ResultsA total of 216 pediatric patients (1–14 years) were recruited. Sixty-six patients had the pink type, while 150 had the purple type. After two HMME-PDT sessions, 55 patients showed excellent efficacy (25.46%), 77 patients showed good efficacy (35.65%), 69 patients showed fair efficacy (31.94%), and 15 patients showed no improvement (6.95%). Dotted and globular vessels were highly associated with excellent efficacy (41.82%); linear vessels were mainly associated with good efficacy (54.55%); reticular vessels were mainly associated with fair (55.07%) and mixed vessels were mainly associated with no improvement (26.66%).ConclusionHMME-PDT is an effective and safe treatment for pediatric patients with PWS. Dotted and globular vessels as well as linear vessels showed better efficacy compared to the other dermoscopic patterns in patients with PWS. Dermoscopy can provide useful clinical information about treatment outcomes.

Quantitative assessment of vascular features in port wine stains through optical coherence tomography angiography

BackgroundVascular lesions such as port wine stains (PWS) lead to facial and psychological problems, which require careful and precise treatments. The key point of treating PWS is to selectively destroy the abnormal blood vessels. Hence, the in vivo monitoring of targeted vessels is crucial. Optical coherence tomography angiography (OCTA), an emerging label-free imaging tool, facilitates the evaluation of skin structure and vasculature at a high resolution. In this study, we utilised OCTA to capture the structural and vascular morphology in patients with PWS. Moreover, we quantitatively characterised the morphological features of different types of PWS. MethodsThis observational clinical study was conducted on 3 patients with flat PWS and 3 patients with thickened PWS. The age range was 4–27 years, and all of them had not received any treatment before this study. The OCTA images of the PWS lesions and contralateral skin were compared. Vascular morphology was characterized, and ectatic vessel depth was quantified according to the OCTA images. ResultsThe blood vessels of the PWS lesions tend to had larger diameters and higher densities than those in the contralateral normal skin. The vessel diameters of PWS lesions were 73 ± 14 μm, with high heterogeneity ranging from 10 to >150 μm, however, the vessel diameters of normal skin were 28 ± 2 μm, ranging from 10 μm to 60 μm. In terms of different PWS lesions, the thickened type showed a trend of larger vessel diameter and higher density than those of the purplish red type. The ectatic vessels were located at the depth of 216 ± 13 μm in the PWS skin. ConclusionsOCTA can facilitate the in vivo three-dimensional visualization of structure and vasculature for PWS lesions. Various quantitative analysis parameters, such as vessel diameter, density, and depth, are typically measured using OCTA. This fact demonstrates the superior capability of OCTA for the precise and comprehensive assessment of PWS lesions.

Bifocal 532/1064 nm alternately illuminated photoacoustic microscopy for capturing deep vascular morphology in human skin.

As a promising technology, photoacoustic microscopy (PAM) plays a critical role in diagnosis and assessment of dermatological conditions by providing subtle vascular networks non-invasively. However, the established PAMs are insufficient for clinical dermatology when faced with complex structures of human skin instead of animal models owing to high melanin content and superimposed vasculature for Asians, which cannot balance the spatial resolution and the imaging depth. To evaluate the ability of bifocal 532/1064-nm alternately illuminated photoacoustic microscopy (BF-PAM) to non-invasively reveal the morphological structure of human skin for improving the diagnosis and therapeutic efficacy of skin diseases. A BF-PAM was developed to capture biopsy-like information of human skin from epidermis to hypodermis. The optical foci of the two excitation beams are staggered in the axial direction to form an extended depth-of-field, which can maintain the lateral resolution and the contrast of PA image. The imaging capability of the BF-PAM was demonstrated by depicting the vascular morphology of multilayered skin with imaging depth of ˜3 mm. Furtherly, vascular malformations in port-wine stains skin were quantitatively assessed without the need for any contrast agent, and the distribution, depth and diameter of the ectatic vessels can determine an optimal treatment protocol for port-wine stains lesions. The quantitative vascular morphology in the dermis can be used to accurately assess vascular characteristics, in which case it enables clinicians to determine optimum treatment parameters in individual patients. As a non-invasive imaging technique, BF-PAM holds great potential to provide objective assessment to enhance the therapeutic efficacy. The study was conducted in accordance with the Declaration of Helsinki (as revised in 2013). The study was approved by the Chinese Ethics Committee of Registering Clinical Trials (ChiECRCT20200184) and registered with Chinese Clinical Trial Registry (ChiCTR2000034400). Before skin imaging, written informed consent was taken from all individual participants.

Quantification of skin lesions using high-frequency ultrasound and shear wave elastography in port-wine stain patients: a clinical study

This study aimed to assess the different types of port-wine stain (PWS) skin lesions quantitatively using high-frequency ultrasound (US) and shear wave elastography (SWE) before and after treatment, and investigate the feasibility and application value of high-frequency US and SWE in PWSs. A total of 195 PWS patients with 238 skin lesions before treatment and 72 follow-up PWS patients with 90 skin lesions were assessed using high-frequency US and SWE. The skin lesions were divided into four groups: pink-type, purple-type, thickened-type, and nodular-type PWSs. Gray-scale US was used to observe normal skin, observe the skin changes of lesions, and assess the skin thickness. The thickened skin was calculated. Power Doppler (PD) signal grades were used to assess the skin blood signals. SW velocity (in m/s) and Young's elastic modulus (in kPa) were used to assess the stiffness of normal skin and skin lesions. The heightened SWE was also calculated. The dermis hypoechogenicity, thickness of thickened skin, and skin PD signal grades were significantly higher in all PWS-type groups compared with the normal-skin group (all P<0.05). The thickened skin and skin PD signal grades in the nodular-type and thickened-type group were significantly thicker and higher than those in the pink-type and purple-type group (all P<0.05). The PD signal grades in the purple-type was significantly higher than that in the pink-type group (P<0.05). All SWE values of PWS lesions were significantly higher in the transverse section than those in the longitudinal section (all P<0.05). The differences in heightened Emean, Emin, Cmean, and Cmin between each PWS group and the normal-skin group were not significant. The heightened Emax and Cmax in the nodular-type PWS group was significantly higher than those in the normal-skin group and pink-type, and purple-type PWS groups (all P<0.05). The heightened Emax and Cmax were significantly higher in the thickened-type PWS group than those in the normal-skin group (all P<0.05). In the evaluation of therapeutic effects, the ratio of dermis hypoechogenicity in pink-type lesions significantly decreased, and thickened skins in all groups were significantly thinned (all P<0.05). The differences of PD signal grades, heightened Emax, and Cmax in all groups between pre-treatment and post-treatment showed no significance. High-frequency US and SWE show feasibility and application values assessing PWS skin lesions. Their features include dermis hypoechogenicity, thicker skin, higher PD signal grades, higher Emax, and higher Cmax. Thicker skin is thus the best feature for assessing therapeutic effect.

Membrane trafficking and exocytosis are upregulated in port wine stain blood vessels.

Port wine stain (PWS) is characterized as a progressive dilatation of immature venule-like vasculatures which result from differentiation-impaired endothelial cells. In this study, we aimed to identify the major biological pathways accounting for the pathogenesis of PWS. Sequential windowed acquisition of all theoretical fragment ion mass spectra (SWATH-MS) was used to identify differentially expressed proteins in PWS lesions, followed by confirmative studies with immunohistochemistry, immunoblot and transmission electron microscopy (TEM). 107 out of 299 identified proteins showed differential expressions in PWS lesions as compared to normal skin, mainly involving the functions of biosynthesis, membrane trafficking, cytoskeleton and cell adhesion/migration. The confirmative studies showed that expressions of membrane trafficking/exocytosis related proteins such as VAT1, IQGAP1, HSC70, clathrin, perlecan, spectrin α1 and GDIR1 were significantly increased in PWS blood vessels as compared to normal ones; while collagen subtypes 6A1 and 6A3 were decreased in PWS skin. Furthermore, TEM studies showed there is a significant upregulation of extracellular vesicle exocytosis from PWS blood vessels as compared to control. The biological process of membrane trafficking and exocytosis is enhanced in PWS blood vessels. Our results imply that the extracellular vesicles released by lesional endothelial cells may act as potential intercellular signaling mediators to contribute to the pathogenesis of PWS.

Coexistence of Eph receptor B1 and ephrin B2 in port-wine stain endothelial progenitor cells contributes to clinicopathological vasculature dilatation.

Port-wine stain (PWS) is a vascular malformation characterized by progressive dilatation of postcapillary venules, but the molecular pathogenesis remains obscure. To illustrate that PWS endothelial cells (ECs) present a unique molecular phenotype that leads to pathoanatomical PWS vasculatures. Immunohistochemistry and transmission electron microscopy were used to characterize the ultrastructure and molecular phenotypes of PWS blood vessels. Primary culture of human dermal microvascular endothelial cells and in vitro tube formation assay were used for confirmative functional studies. Multiple clinicopathological features of PWS blood vessels during the development and progression of the disease were shown. There were no normal arterioles and venules observed phenotypically and morphologically in PWS skin; arterioles and venules both showed differentiation impairments, resulting in a reduction of arteriole-like vasculatures and defects in capillary loop formation in PWS lesions. PWS ECs showed stemness properties with expression of endothelial progenitor cell markers CD133 and CD166 in non-nodular lesions. They also expressed dual venous/arterial identities, Eph receptor B1 (EphB1) and ephrin B2 (EfnB2). Co-expression of EphB1 and EfnB2 in normal human dermal microvascular ECs led to the formation of PWS-like vasculatures in vitro, for example larger-diameter and thick-walled capillaries. PWS ECs are differentiation-impaired, late-stage endothelial progenitor cells with a specific phenotype of CD133+ /CD166+ /EphB1+ /EfnB2+ , which form immature venule-like pathoanatomical vasculatures. The disruption of normal EC-EC interactions by coexistence of EphB1 and EfnB2 contributes to progressive dilatation of PWS vasculatures.

Relationship between the blood perfusion values determined by laser speckle imaging and laser Doppler imaging in normal skin and port wine stains

ObjectiveLaser Doppler imaging (LDI) and laser speckle imaging (LSI) are two major optical techniques aiming at non-invasively imaging the skin blood perfusion. However, the relationship between perfusion values determined by LDI and LSI has not been fully explored. Methods8 healthy volunteers and 13 PWS patients were recruited. The perfusions in normal skin on the forearm of 8 healthy volunteers were simultaneously measured by both LDI and LSI during post-occlusive reactive hyperemia (PORH). Furthermore, the perfusions of port wine stains (PWS) lesions and contralateral normal skin of 10 PWS patients were also determined. In addition, the perfusions for PWS lesions from 3 PWS patients were successively monitored at 0, 10 and 20min during vascular-targeted photodynamic therapy (V-PDT). The average perfusion values determined by LSI were compared with those of LDI for each subject. ResultsIn the normal skin during PORH, power function provided better fits of perfusion values than linear function: powers for individual subjects go from 1.312 to 1.942 (R2=0.8967–0.9951). There was a linear relationship between perfusion values determined by LDI and LSI in PWS and contralateral normal skin (R2=0.7308–0.9623), and in PWS during V-PDT (R2=0.8037–0.9968). ConclusionThe perfusion values determined by LDI and LSI correlate closely in normal skin and PWS over a broad range of skin perfusion. However, it still suggests that perfusion range and characteristics of the measured skin should be carefully considered if LDI and LSI measures are compared.

Investigation of photodynamic therapy optimization for port wine stain using modulation of photosensitizer administration methods

To raise photosensitizer concentration level during the photodynamic therapy process, two new methods of photosensitizer administration were investigated. The first method involves the slow intravenous injection of photosensitizer throughout the first 15 min of irradiation, and the second method involves 30 min fomentation before photosensitizer injection and irradiation. The fluorescence spectra of port wine stain skin were monitored and the therapeutic effect correlated index was calculated with a previously published spectral algorithm. Thirty cases were divided into group A (slow injection of photosensitizer during the first 15 min), group B (fomentation), and group C (control group, traditional injection method), with 10 cases in each group. To analyze the effect of these two new methods, the change of therapeutic effect correlated index values of two photodynamic therapy sessions for each patient were calculated, and the photodynamic therapy outcome was compared. The results showed that the change of therapeutic effect correlated index in group A was slightly more remarkable than that in the control group. The change of therapeutic effect correlated index in group B was similar to that in the control group. Slow injection of photosensitizer during photodynamic therapy has a potential to increase photosensitizer concentration level during photodynamic therapy. However, fomentation before photodynamic therapy has no such potential. There is a need for new methods to be attempted.

A new Monte Carlo program for simulating light transport through Port Wine Stain skin

A new Monte Carlo program is presented for simulating light transport through clinically normal skin and skin containing Port Wine Stain (PWS) vessels. The program consists of an eight-layer mathematical skin model constructed from optical coefficients described previously. A simulation including diffuse illumination at the surface and subsequent light transport through the model is carried out using a radiative transfer theory ray-tracing technique. Total reflectance values over 39 wavelengths are scored by the addition of simulated light returning to the surface within a specified region and surface reflections (calculated using Fresnel's equations). These reflectance values are compared to measurements from individual participants, and characteristics of the model are adjusted until adequate agreement is produced between simulated and measured skin reflectance curves. The absorption and scattering coefficients of the epidermis are adjusted through changes in the simulated concentrations and mean diameters of epidermal melanosomes to reproduce non-lesional skin colour. Pseudo-cylindrical horizontal vessels are added to the skin model, and their simulated mean depths, diameters and number densities are adjusted to reproduce measured PWS skin colour. Accurate reproductions of colour measurement data are produced by the program, resulting in realistic predictions of melanin and PWS blood vessel parameters. Using a modest personal computer, the simulation currently requires an average of five and a half days to complete.

Microarray analysis of port wine stains before and after pulsed dye laser treatment

Neither the pathogenesis of port wine stain (PWS) birthmarks nor tissue effects of pulsed dye laser (PDL) treatment of these lesions is fully understood. There are few published reports utilizing gene expression analysis in human PWS skin. We aim to compare gene expression in PWS before and after PDL, using DNA microarrays that represent most, if not all, human genes to obtain comprehensive molecular profiles of PWS lesions and PDL-associated tissue effects. Five human subjects had PDL treatment of their PWS. One week later, three biopsies were taken from each subject: normal skin (N); untreated PWS (PWS); PWS post-PDL (PWS + PDL). Samples included two lower extremity lesions, two facial lesions, and one facial nodule. High-quality total RNA isolated from skin biopsies was processed and applied to Affymetrix Human gene 1.0ST microarrays for gene expression analysis. We performed a 16 pair-wise comparison identifying either up- or down-regulated genes between N versus PWS and PWS versus PWS + PDL for four of the donor samples. The PWS nodule (nPWS) was analyzed separately. There was significant variation in gene expression profiles between individuals. By doing pair-wise comparisons between samples taken from the same donor, we were able to identify genes that may participate in the formation of PWS lesions and PDL tissue effects. Genes associated with immune, epidermal, and lipid metabolism were up-regulated in PWS skin. The nPWS exhibited more profound differences in gene expression than the rest of the samples, with significant differential expression of genes associated with angiogenesis, tumorigenesis, and inflammation. In summary, gene expression profiles from N, PWS, and PWS + PDL demonstrated significant variation within samples from the same donor and between donors. By doing pair-wise comparisons between samples taken from the same donor and comparing these results between donors, we were able to identify genes that may participate in formation of PWS and PDL effects. Our preliminary results indicate changes in gene expression of angiogenesis-related genes, suggesting that dysregulation of angiogenic signals and/or components may contribute to PWS pathology.

Enhanced port-wine stain lightening achieved with combined treatment of selective photothermolysis and imiquimod

Pulsed dye laser (PDL) is the gold standard for treatment of port-wine stain (PWS) birthmarks but multiple treatments are required and complete resolution is often not achieved. Posttreatment vessel recurrence is thought to be a factor that limits efficacy of PDL treatment of PWS. Imiquimod 5% cream is an immunomodulator with antiangiogenic effects. We sought to determine if application of imiquimod 5% cream after PDL improves treatment outcome. Healthy individuals with PWS (n = 24) were treated with PDL and then randomized to apply posttreatment placebo or imiquimod 5% cream for 8 weeks. Chromameter measurements (Commission Internationale de l'Eclairage L∗a∗b∗ colorspace) for 57 PWS sites (multiple sites per patient) were taken at baseline and compared with measurements taken 8 weeks posttreatment. The Δa∗ (change in erythema) and ΔE (difference in color between normal-appearing skin and PWS skin) were measured to quantify treatment outcome. Two patients developed minor skin irritation. Other adverse effects were not noted. Average ∆a∗ was 0.43 for PDL + placebo sites (n = 25) and 1.27 for PDL + imiquimod sites (n = 32) (P value = .0294) indicating a greater reduction in erythema with imiquimod. Average ∆E was 2.59 for PDL + placebo and 4.08 for PDL + imiquimod (P value = .0363), again indicating a greater color improvement with imiquimod. Effects were evaluated after a single treatment and duration of effect is unknown. Combined selective photothermolysis and antiangiogenic therapy may enhance PWS treatment efficacy.

An Overview of Three Promising Mechanical, Optical, and Biochemical Engineering Approaches to Improve Selective Photothermolysis of Refractory Port Wine Stains

During the last three decades, several laser systems, ancillary technologies, and treatment modalities have been developed for the treatment of port wine stains (PWSs). However, approximately half of the PWS patient population responds suboptimally to laser treatment. Consequently, novel treatment modalities and therapeutic techniques/strategies are required to improve PWS treatment efficacy. This overview therefore focuses on three distinct experimental approaches for the optimization of PWS laser treatment. The approaches are addressed from the perspective of mechanical engineering (the use of local hypobaric pressure to induce vasodilation in the laser-irradiated dermal microcirculation), optical engineering (laser-speckle imaging of post-treatment flow in laser-treated PWS skin), and biochemical engineering (light- and heat-activatable liposomal drug delivery systems to enhance the extent of post-irradiation vascular occlusion).

Choosing optimal wavelength for photodynamic therapy of port wine stains by mathematic simulation

Many laser wavelengths have been used in photodynamic therapy (PDT) for port wine stains (PWS). However, how these wavelengths result in different PDT outcomes has not been clearly illuminated. This study is designed to analyze which wavelengths would be the most advantageous for use in PDT for PWS. The singlet oxygen yield in PDT-treated PWS skin under different wavelengths at the same photosensitizer dosage was simulated and the following three situations were simulated and compared: 1. PDT efficiency of 488, 532, 510, 578, and 630 nm laser irradiation at clinical dosage (100 mW∕cm(2), 40 min); 2. PDT efficiency of different wavelength for PWS with hyperpigmentation after previous PDT; 3. PDT efficiency of different wavelengths for PWS, in which only deeply located ectatic vessels remained. The results showed that singlet oxygen yield is the highest at 510 nm, it is similar at 532 nm and 488 nm, and very low at 578 nm and 630 nm. This result is identical to the state in clinic. According to this theoretical study, the optimal wavelength for PDT in the treatment of PWS should near the absorption peaks of photosensitizer and where absorption from native chromophores (haemoglobin and melanin) is diminished.

Pulsed Dye Laser Treatment of Portwine Stain (PWS) and the Other Vascular Lesions, and the Evaluation of the PWS Skin Color Using a Colorimeter

Pulsed Dye Laser Treatment of Portwine Stain (PWS) and the Other Vascular Lesions, and the Evaluation of the PWS Skin Color Using a Colorimeter

Spectrophotometers for the clinical assessment of port-wine stain skin lesions: a review

Reflectance spectrophotometry is the most established and widely used objective technique for the assessment of port-wine stain (PWS) skin, and has been applied extensively in other dermatological applications. To date, no review has been published regarding the different spectrophotometric devices used on PWS skin. This paper comprises such a review, introducing the reader to the relevant background material and then discussing scanning, narrow-band and tristimulus spectrophotometers in turn. Scanning spectrophotometry is the most versatile of the three methods but it is noted that considerable expertise is required to interpret the acquired data. Narrow-band and tristimulus devices are available at a much lower price and can be considerably simpler to use. They do, however, provide limited information that does not account for the complex effects of melanin and other chromophores within the skin. Although scanning spectrophotometers would be the preferred choice for most investigations, cheaper, simpler and equally reliable options are available and may better suit the needs of some research projects.

Imaging port wine stains by fiber optical coherence tomography

We develop a fiber optical coherence tomography (OCT) system in the clinical utility of imaging port wine stains (PWS). We use our OCT system on 41 patients with PWS to document the difference between PWS skin and contralateral normal skin. The system, which operates at 4 frames with axial and transverse resolutions of 10 and 9 mum, respectively, in the skin tissue, can clearly distinguish the dilated dermal blood vessels from normal tissue. We present OCT images of patients with PWS and normal human skin. We obtain the structural parameters, including epidermal thickness and diameter and depth of dilated blood vessels. We demonstrate that OCT may be a useful tool for the noninvasive imaging of PWS. It may help determine the photosensitizer dose and laser parameters in photodynamic therapy for treating port wine stains.

Blood flow dynamics after laser therapy of port wine stain birthmarks

During laser therapy of port wine stain (PWS) birthmarks, regions of perfusion may persist. We hypothesize that such regions are not readily observable even when laser surgery is performed by highly experienced clinicians. The objective of this study was to use objective feedback to assess the acute vascular response to laser therapy. A clinic-friendly laser speckle imaging (LSI) instrument was developed to provide the clinician with real-time images of blood flow during laser therapy. Images were acquired from patients undergoing laser therapy of PWS birthmarks at Scripps Clinic and the Beckman Laser Institute and Medical Clinic. Blood flow maps were extracted from the acquired imaging data. Histogram-based analysis was applied in grading the degree of heterogeneity present in the blood flow maps after laser therapy. Collectively, two types of patient responses were observed in response to laser exposure: (1) an immediate increase in perfusion within minutes after laser therapy; and (2) an overall decrease in blood perfusion approximately 1 hour after laser therapy, with distinct regions of persistent perfusion apparent in the majority of post-treatment blood-flow images. A comparison of blood flow in PWS and adjacent normal skin demonstrated that PWS blood flow can be greater than, or sometimes equivalent to, that of normal skin. In general, a decrease in skin perfusion is observed during pulsed laser therapy of PWS birthmarks. However, a heterogeneous perfusion map was frequently observed. These regions of persistent perfusion may be due to incomplete photocoagulation of the targeted vessels. We hypothesize that immediate retreatment of these regions identified with LSI, will result in enhanced removal of the PWS vasculature. Lasers Surg. Med. 41:563-571, 2009. (c) 2009 Wiley-Liss, Inc.

Determination of the effects of blood depth in the dermis on skin colour in a novel skin phantom using digital imaging

A phantom of human port wine stain (PWS) skin, previously described by the authors, that takes into account its light propagation and scattering properties, was used to model varying depths of blood within PWS skin. Digital images of these phantoms were then acquired under controlled conditions, and the colour information was abstracted with a digital image processing suite. These colour data were analysed quantitatively for each depth of blood, and the relationship between depth of blood and colour was defined. A linear relationship was observed between depth of blood within the phantom and hue, hue being an intuitive measure of how colour is perceived by the human eye. As PWS clearance by laser treatment is dependant, to a large degree, on vessel depth within the skin, the ability to abstract colour data from PWS or, in fact, any vascular lesion within the skin, may help predict the degree of clearance before treatment is actually instigated. In the future, the comparison of phantom colour data with data from actual digital images of affected PWS skin, combined with a knowledge of laser light penetration depths, may provide a useful adjunct to clinical judgement in the prediction of PWS treatment outcomes.

Confocal Microscopy Study of Neurovascular Distribution in Facial Port Wine Stains (Capillary Malformation)

Background/Purpose Vascular ectasia observed in port wine stain (PWS) birthmarks might be secondary to localized reduction of neural innervation and associated loss of autonomic stimulation. Our objective was to investigate this theory and evaluate nerve density, blood vessel density, and average blood vessel size in untreated and pulsed dye laser with cryogen spray cooling (PDL-CSC) treated PWS skin. Methods Biopsy skin specimens were taken from 14 adults with a PWS, categorized by: uninvolved skin; untreated PWS skin; PWS skin with a history of good blanching, and PWS skin with a history of poor blanching both in response to PDL treatment. Seven specimens of normal, unaffected skin were used as the experimental control group. Indirect immunohistochemistry was performed on all specimens followed by confocal microscopy imaging with computer analysis to determine nerve density, blood vessel density, and average blood vessel size. Results Nerve density was significantly decreased in all PWS sites as compared to uninvolved skin ( p < 0.01). Average blood vessel diameter was larger in untreated as compared to treated PWS sites and varied between different sites within a single PWS. Conclusion Nerve density was decreased in all evaluated PWS sites, and this may be a factor in lesion pathogenesis. PWS blood vessel size correlated with the PDL blanching response and may prove to be a useful prognostic indicator of therapeutic outcome.

Noninvasive blood flow imaging for real‐time feedback during laser therapy of port wine stain birthmarks

During laser therapy of port wine stain (PWS) birthmarks, regions of persistent perfusion may exist. Immediate retreatment of such regions may improve PWS laser therapeutic outcome. To address this need, we propose use of laser speckle imaging (LSI) to provide real-time, quantitative feedback during laser surgery. Herein, we present in vitro and in vivo data collected with a clinic-based LSI instrument. Prior to clinical implementation, we first investigated three aspects of LSI deemed important for clinical imaging: (1) instrument depth of field (DOF); (2) effects of laser irradiance on speckle flow index (SFI) values; and (3) measurement repeatability. Clinical measurements were acquired from the lesions of PWS patients immediately prior to and after laser therapy at the Beckman Laser Institute. Our preclinical data suggest the following: (1) instrument DOF was approximately 1 cm; (2) quantitative flow characterization with LSI was practically unaffected at normalized irradiance values between 0.06 and 0.5; and (3) our LSI instrument was capable of highly reproducible SFI values. From our clinical measurements, we found that the relative difference between blood perfusion in PWS lesions and adjacent normal skin was highly variable. Based on SFI images, the perfusion of PWS skin is sometimes indistinguishable from that of adjacent normal skin. With laser therapy, we measured a global decrease in blood perfusion, and we frequently observed distinct regions of persistent perfusion. Our results demonstrate the potential role of image-guided laser therapy of PWS birthmarks. LSI is a promising tool for noninvasive blood flow characterization during laser therapy due to its relative simplicity and low cost.