Journal of Cosmetic and Laser Therapy, 2014; 16: 21–25
ORIGINAL RESEARCH REPORT
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The assessment of pulsed dye laser treatment of port-wine stains with reflectance confocal microscopy JIE REN∗1, HUI QIAN∗1, LEIHONG XIANG1, ZHANYAN PAN1, LU ZHONG1, SHUXIAN YAN1 & MICHAEL H. GOLD1,2 1Department of Dermatology, Huashan Hospital, Fudan University, Shanghai, P. R. China, and 2Gold Skin Care Center, Nashville, TN, USA
Abstract Background: Reflectance confocal microscopy (RCM) is a noninvasive technique for evaluating cutaneous lesions with cellular level resolution close to conventional histopathology. The aim of this study is to observe the vascular changes in Port-wine (PWS) lesions and assess the clinical efficacy of Pulsed Dye Laser (PDL) treatment by examining vessel diameter and density with RCM. Materials and methods: Eleven adult patients with PWS, each had four test patches carried out with different pulse durations (1.5, 3, 6, and 10 ms), respectively; fluences of 9–12 J/cm2; and a spot size of 7 mm. The PDL treatment was repeated 3–5 times at a 2-month interval. Photographs and measurements with RCM were taken before each treatment and 2 months after the last treatment. Results: The PDL treatment exhibited increasing clearance with reducing pulse durations. Vessel diameters and densities were significantly decreased in the same pulse-duration groups after treatment. There was significant difference between 1.5 ms pulse-duration group and other pulse-duration groups in reducing blood vessel diameter at the depth of 150 μm. Conclusions: RCM can be used to assess the clinical efficacy of PDL treatment. Key Words: reflectance confocal microscopy, port-wine stains, pulsed dye laser
Introduction Port-wine stains (PWS) are benign vascular malformations consisting of ectatic blood vessels situated predominantly in the superficial dermis. A reduction of neural innervations around the ectatic blood vessels appears to be the pathogenesis of PWS (1). Pulsed dye laser (PDL) is the modality of choice for the treatment of PWS. PDL treatment achieves observable lightening of PWS by reducing the number, size, and therefore, erythrocyte content of these vessels following the principles of selective photothermolysis proposed by Anderson and Parrish (2). PDL incorporates the use of larger spot sizes, higher influences, varying pulse durations, and dynamic cooling for more effective treatment of PWS via greater vessel heating and deeper vascular injury (3). However, despite technological advances,
a considerable number of patients with PWS still do not achieve complete lesion removal with PDL treatment. Relationships between laser parameters and their effect on PWS treatment outcome are complex and not fully understood. It would be very useful to further understand the vascular responses of PWS to lasers through noninvasive imaging. However, we currently do not have an established noninvasive method of accurately assessment of treatment efficacy. In vivo reflectance confocal microscopy (RCM) is a noninvasive technique for evaluation of cutaneous lesions with cellular level resolution close to conventional histopathology (4). RCM has previously been used for the noninvasive visualization of benign and malignant inflammatory and proliferative skin disorders (5). It has already reported that RCM may aid in the evaluation of vascular lesions. RCM
∗Co-first author. Correspondence: LeiHong Xiang, MD, PhD, Department of Dermatology, Huashan Hospital, Fudan University, Shanghai 200040, P. R. China. Tel: ⫹ 86-021-52887765. E-mail: [email protected] (Received 14 August 2013 ; accepted 17 October 2013) ISSN 1476-4172 print/ISSN 1476-4180 online © 2014 Informa UK, Ltd. DOI: 10.3109/14764172.2013.862552
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allows the in vivo examination of the diameter of vessels and degree of vascular tortuosity or dilation (6). The aim of this study is to use RCM to observe the vascular changes in PWS lesions, including the diameter and density of vessels, before and after PDL treatment, and to assess the clinical efficacy of PDL with different pulse durations. Materials and methods
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Patient selection and treatment We recruited 11 patients (6 males and 5 females, aged 24.82 ⫾ 9.82 years, Fitzpatrick skin phototypes III–IV) with PWS who had no previous treatments after the project was approved by the Ethics Committee of Huashan Hospital, Fudan University, Shanghai, China. For ten patients, lesions were distributed only on the face while one patient had lesions on the neck as well. The lesions appeared pink, red, and purple in color. The 595-nm pulsed dye laser (V-Beam, Candela, USA) with a dynamic cooling device was used for the study. Each patient had four test patches carried out with different pulse durations (1.5, 3, 6, and 10 ms), respectively, fluences of 9–12 J/cm2, and a spot size of 7 mm. On each patient the four test patches were performed with four different pulse durations, while the same fluence was selected for each session. The treatment endpoint occurred when the color of the treated cutaneous vessel showed purpura. The PDL treatment was repeated 3–5 times at a 2-month interval. Once a patient’s lesion was completely clear in any of the four pulse-duration groups, the treatment would be ended. Post-treatment discomfort was relieved by immediate application of cold spray or an ice pack to the treated areas. Clinical evaluation Efficacy was determined by comparison of digital photographs (Cannon EOS 400D) obtained before each treatment and 2 months after the last treatment. One independent blinded dermatologist graded lesion clearance (%) on a four-point scale: poor (⬍ 25%), fair (26–50%), good (51–75%), and excellent (76–100%).
skin with an adhesive. This RCM steel ring fixture stabilized the area of interest and helps maintain a constant mechanical contact between the skin and the RCM. A small drop of immersion oil was applied to the skin lesion. On each patient four test patches were selected for RCM imaging. Transparent body charts were used to outline the skin areas at baseline to permit co-localization during follow-up examination. RCM imaging was performed before each treatment and 2 months after the last treatment. RCM images were acquired using a standardized protocol for each patient. The protocol included the acquisition of three RCM datasets per site: (i) four 2 ⫻ 2 mm mosaics at the depth of 100 μm; (ii) four 2 ⫻ 2 mm mosaic at the depth of 150 μm; and (iii) four 0.5 ⫻ 0.5 mm z-stack with 2 μm spacing from the center of the 2 ⫻ 2 mm mosaic. The density and diameter of blood vessels were counted at the 2 ⫻ 2 mm mosaic images. Statistical analysis Data were expressed as mean ⫾ S.D. Paired t-test was used to determine the significance between groups before and after treatment, and a one-way ANOVA test with Bonferroni correction was used to analyze the significance when three or more groups were compared. Statistical significance was defined as P ⬍ 0.05. The SPSS 13.0 software was used for all statistical analyses.
Results Clinical response Clearance rates of patients for PDL treatment with different pulse duration are shown in Table I. Overall, clearance was excellent in 27.28% of patients, good in 20.45%, fair in 36.36%, and poor in 15.91%. The PDL treatment exhibited increasing clearance with reducing pulse duration. The “excellent” clearance increased from 18.18 to 18.18, 27.27, and 45.46% at pulse duration of 10, 6, 3, and 1.5 ms, respectively, while the “poor” clearance decreased
Table I. Number of patients achieving various clearance rates for PDL treatment with different pulse durations.
RCM evaluation RCM was performed with the commercially available near infrared RCM Vivascope 1500 (Lucid Inc., Rochester, NY, USA). This system uses a diode laser at a wavelength of 830 nm, a power less than 30 mW, and a ⫻30 water immersion objective lens with numeric aperture of 0.9. The RCM objective was attached to the skin via a stainless steel ring, which in turn was attached to the corneal layer of the
Clearance rate (%) Pulse duration
76–100 (excellent)
1.5 ms 3 ms 6 ms 10 ms
5 (45.45%) 3 (27.27%) 2 (18.18%) 2 (18.18%) Total ⫽ 12 27.28%
2 4 2 1
51–75 (good)
25–50 (fair)
(18.18%) (36.37%) (18.18%) (9.09%) Total ⫽ 9 20.45%
3 (27.27%) 3 (27.27%) 5 (45.46%) 5 (45.46%) Total ⫽ 16 36.36%
⬍ 25 (poor) 1 1 2 3
(9.09%) (9.09%) (18.18%) (27.27%) Total ⫽ 7 15.91%
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Pulsed dye laser treatment of port-wine stains
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Figure 1. (i) A 50-year-old male before treatment with the 595-nm pulsed dye laser, and (ii) after four treatments with different pulse duration of 1.5 (a), 3 (b), 6 (c), 10 ms (d). The lesion was cleared by more than 75% in (a) after PDL treatments with the pulse duration of 1.5 ms.
from 27.27 to 18.18, 9.09, and 9.09%, respectively. Clinical examples are shown in Figure 1. RCM examination Differences in blood vessel diameter and density using RCM evaluation are shown in Table II. Overall, RCM imaging at the depth of 100 and 150 μm showed greater vessel diameter before treatment and the vessels tended to increase in caliber with increasing depth, as shown in Figure 2. The mean blood vessel diameters of all examined regions at the depth of 100 and 150 μm before treatment were 72.11 ⫾ 27.50 and 98.18 ⫾ 33.02 μm, respectively, and these decreased to 31.82 ⫾ 17.39 and 67.11 ⫾ 31.07 μm, respectively, after treatment
(P ⬍ 0.05). Meanwhile, the mean blood vessel densities at the depth of 100 and 150 μm were 15.09 ⫾ 5.06 and 18.55 ⫾ 3.46, respectively, before treatment; both decreased significantly to 8.89 ⫾ 3.34 and 11.89 ⫾ 2.81, respectively, after PDL treatment (P ⬍ 0.05). After treatment, both diameter and density of blood vessel decreased statistically as compared with those before treatment in the same pulse-duration groups (P ⬍ 0.05). There was significant difference between 1.5 ms pulse-duration group and other pulseduration groups in reducing blood vessel diameter at the depth of 150 μm (P ⬍ 0.05), while no significant difference among each pulse-duration groups at the depth of 100 μm (P ⬎ 0.05). Statistically, there was no difference between different pulse-duration
Table II. Diameter and density of blood vessel examined in 1 mm2 RCM images among different pulseduration groups before and after treatment. 100 (μm) depth Pulse duration 1.5 ms Before Tx After Tx Difference 3 ms Before Tx After Tx Difference 6 ms Before Tx After Tx Difference 10 ms Before Tx After Tx Difference
150 (μm) depth
Blood vessel diameter (μm)
Blood vessel density
Blood vessel diameter (μm)
Blood vessel density
74.64 ⫾ 25.16 31.82 ⫾ 17.39∗ 42.82 ⫾ 19.94
16.45 ⫾ 7.12 7.27 ⫾ 2.00∗ 9.18 ⫾ 7.22
95.64 ⫾ 27.83 56.00 ⫾ 27.38 ∗ 39.64 ⫾ 29.30#
18.91 ⫾ 3.99 10.82 ⫾ 2.27∗ 8.09 ⫾ 3.18
70.27 ⫾ 31.62 36.91 ⫾ 21.81∗ 33.36 ⫾ 28.21
13.91 ⫾ 5.13 7.82 ⫾ 3.28∗ 6.09 ⫾ 5.41
94.55 ⫾ 33.18 66.91 ⫾ 28.97∗ 27.64 ⫾ 23.64
16.91 ⫾ 2.77 10.91 ⫾ 2.55∗ 6.00 ⫾ 3.29
69.73 ⫾ 27.78 41.55 ⫾ 21.44∗ 28.18 ⫾ 20.03
14.82 ⫾ 3.60 9.73 ⫾ 2.69∗ 5.09 ⫾ 3.18
99.09 ⫾ 39.26 69.91 ⫾ 32.42∗ 29.18 ⫾ 25.13
19.36 ⫾ 3.67 13.09 ⫾ 3.21∗ 6.27 ⫾ 3.41
73.82 ⫾ 28.77 45.36 ⫾ 18.95∗ 28.46 ⫾ 23.39
15.18 ⫾ 4.07 10.73 ⫾ 4.22∗ 4.45 ⫾ 6.07
103.45 ⫾ 34.90 75.64 ⫾ 25.89∗ 27.81 ⫾ 25.65
19.00 ⫾ 3.22 12.73 ⫾ 2.72∗ 6.27 ⫾ 3.47
Data correspond to the mean ⫾ S.D. Tx: treatment. Difference ⫽ the diameter (or density) before treatment-the diameter (or density) after treatment. ∗P ⬍ 0.05 vs. Before Tx. #P ⬍ 0.05 1.5 ms pulse-duration group compared with 3, 6, 10 ms groups separately.
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Figure 2. (a, b) RCM images of a 50-year-old male before and (c, d) after PDL treatment. Compared with (a, b), diameter of blood vessel decreased significantly in (c, d). Scale bar 100 μm.
groups in reducing vessel density at the depth of both 100 and 150 μm (P ⬎ 0.05).
Discussion PDL treatment is based on the theory of selective photothermolysis, which induces selective photocoagulation of the target blood vessels. The appropriate setting of laser parameters such as spot diameter, fluence and pulse duration can improve PWS clearance rates. Current studies show that less than 20% of PWS patients can be lightened completely with PDL treatment, 50% patients achieve 70% improvement while around 20–30% patients barely showed minimal response to the treatment (7). However, pulse durations selected in these studies vary greatly. There is experimental evidence that the ideal pulse duration shall be from 1 to 10 ms as PWS blood vessels range from 20 to 150 μm in diameter (8). To achieve better comparability, we minimized the variables for the PDL treatment. On each patient four test patches were performed with four different pulse durations (1.5, 3, 6, and 10 ms), while the same fluence and spot diameter were selected for each session. In our study, more than 75% lightening was achieved by 45.45% of the patients in 1.5 ms pulse-duration
group, which surpassed other pulse-duration groups in terms of efficacy. Histopathological examinations of PWS showed a normal epidermis overlying an abnormal plexus of dilated blood vessels located as a layer in the upper dermis (9). Recently several noninvasive imaging techniques have been used to identify morphology, physiology, and dynamics of PWS, which may provide further objective measurement on treatment response. RCM, a promising, noninvasive, high-resolution new imaging tool, can perform histologic evaluation of the skin in vivo. The correlation between RCM features and histopathologic findings has been demonstrated in pigmented (10), neoplastic (11), and benign vascular lesions (12). RCM evaluation revealed a great number of large dilated vessels in mid- to superficial dermis in PWS (6). We used this new technology to study the features of blood vessels in the upper dermal layer. Images of PWS before and after treatment were obtained to compare the changes of mean blood vessel diameter and density, thus evaluating the response to PDL treatment. The abnormal vessels in PWS lesions are composed of dilated blood vessels of various diameters and depths. We found the vessels morphology of RCM features varies at different scanning depths. As scanning depths increased, both the mean diameter
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Pulsed dye laser treatment of port-wine stains and maximum diameter of vessels become larger. Therefore, it is necessary to mark the scanning depth when measuring vessel diameter with RCM, in order to have an objective and reliable evaluation. As our data showed, both the mean blood vessel diameter and the density decreased after treatment as compared with those before treatment in the same pulse-duration groups. It is interesting that there was a significant difference between the 1.5 ms pulseduration group and the other pulse-duration groups in vessel diameter reduction at the depth of 150 μm, while there was no significant difference among all pulse-duration groups at the depth of 100 μm. This seems to indicate that the ectatic vessels in upper dermis have better response to PDL treatment regardless of pulse duration variation. It is important to note that laser energy must be sufficient to coagulate PWS blood vessels. A previous study showed that higher fluences were required for longer pulse durations (13). Our study clearly showed that PDL with a pulse duration of 1.5 ms was easier to destroy the target dilated vessels, and that significant improvement of PWS could be achieved with the PDL with longer pulse durations. In our study, patients in the 1.5 ms pulseduration group achieved more lightening and clinical efficacy, while they surpassed others in reducing vessel diameters at 150 μm depth for RCM imaging. It is confirmed that the correlation between the clinical therapeutic outcome and the vessel diameter after treatment is consistent. Furthermore, there was no contrast among different pulse-duration groups in reducing the vessel density both at the depth of 100 and 150 μm, which proves once again that blanching efficacy is more related to the diameter of blood vessel. PWS initially appear to be flat, pink/red patches that may gradually develop with age into hypertrophic, red/purple lesions, leading to further cosmetic disfigurement and psychologic distress (14). A few studies have indicated that treatment at younger ages require fewer sessions and might improve the PWS clearance (15). Our data also revealed such a trend that both the mean diameter and maximum diameter of vessels decreased after PDL treatment. It seems to be another explanation for lesional lightening outcome. We propose that patients should receive several more sessions at regular intervals to constrain the development of lesion color and thickness, even after their lesions stop to further lighten in subsequent treatments. Further observations and studies are needed. In conclusion, RCM enhances our understanding of the interactions between PDL treatment and PWS so that the treatment parameter settings can be optimized accordingly. The therapeutic outcome of PDL treatment is related to blood vessel diameter of PWS. RCM can also help with the pre-treatment prediction of efficacy for individuals with different PWS
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characteristics. The application of RCM in assessment of laser efficacy for other vascular lesions, such as spider angioma and cherry angioma, should also be further studied. Declaration of interest: The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
References 1. Selim MM, Kelly KM, Nelson JS, Wendelschafer-Crabb G, Kennedy WR, Zelickson BD. Confocal microscopy study of nerves and blood vessels in untreated and treated port wine stains: preliminary observations. Dermatol Surg. 2004; 30:892–897. 2. Anderson RR, Parrish JA. Selective photothermolysis: precise microsurgery by selective absorption of pulsed radiation. Science. 1983;220:524–527. 3. Ortiz AE, Nelson JS. Port-wine stain laser treatments and novel approaches. Facial Plast Surg. 2012;28:611–620. 4. Calzavara-Pinton P, Longo C, Venturini M, Sala R, Pellacani G. Reflectance confocal microscopy for in vivo skin imaging. Photochem Photobiol. 2008;84:1421–1430. 5. Pan ZY, Liang J, Zhang QA, Lin JR, Zheng ZZ. In vivo reflectance confocal microscopy of extramammary Paget disease: diagnostic evaluation and surgical management. J Am Acad Dermatol. 2012;66:e47–53. 6. Astner S, González S, Cuevas J, Röwert-Huber J, Sterry W, Stockfleth E, Ulrich M. Preliminary evaluation of benign vascular lesions using in vivo reflectance confocal microscopy. Dermatol Surg. 2010;36:1099–1110. 7. Maguiness SM, Liang MG. Management of capillary malformations. Clin Plast Surg. 2011;38:65–73. 8. Jasim ZF, Handley JM. Treatment of pulsed dye laser-resistant port wine stain birthmarks. J Am Acad Dermatol. 2007;57: 677–682. 9. Kelly KM, Choi B, McFarlane S, Motosue A, Jung B, Khan MH, Ramirez-San-Juan JC, Nelson JS. Description and analysis of treatments for port-wine stain birthmarks. Arch Facial Plast Surg. 2005;7:287–294. 10. Busam KJ, Charles C, Lee G, Halpern AC. Morphologic features of melanocytes, pigmented keratinocytes, and melanophages by in vivo confocal scanning laser microscopy. Mod Pathol. 2001;14:862–868. 11. Braga JC, Scope A, Klaz I, Mecca P, González S, Rabinovitz H, Marghoob AA. The significance of reflectance confocal microscopy in the assessment of solitary pink skin lesions. J Am Acad Dermatol. 2009;61:230–241. 12. Aghassi D, Anderson RR, González S. Time-sequence histologic imaging of laser-treated cherry angiomas with in vivo confocal microscopy. J Am Acad Dermatol. 2000;43: 37–41. 13. Kono T, Groff WF, Sakurai H, Takeuchi M, Yamaki T, Soejima K, Nozaki M. Evaluation of fluence and pulseduration on purpuric threshold using an extended pulse pulsed-dye laser in the treatment of port wine stains. J Dermatol. 2006;33:473–476. 14. Chen JK, Ghasri P, Aguilar G, van Drooge AM, Wolkerstorfer A, Kelly KM, Heger M. An overview of clinical and experimental treatment modalities for port wine stains. J Am Acad Dermatol. 2012;67:289–304. 15. Geronemus RG, Quintana AT, Lou WW, Kauvar AN. High-fluence modified pulsed dye laser photocoagulation with dynamic cooling of port-wine stains in infancy. Arch Dermatol. 2000;136:942–943.
ORIGINAL RESEARCH REPORT
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The assessment of pulsed dye laser treatment of port-wine stains with reflectance confocal microscopy JIE REN∗1, HUI QIAN∗1, LEIHONG XIANG1, ZHANYAN PAN1, LU ZHONG1, SHUXIAN YAN1 & MICHAEL H. GOLD1,2 1Department of Dermatology, Huashan Hospital, Fudan University, Shanghai, P. R. China, and 2Gold Skin Care Center, Nashville, TN, USA
Abstract Background: Reflectance confocal microscopy (RCM) is a noninvasive technique for evaluating cutaneous lesions with cellular level resolution close to conventional histopathology. The aim of this study is to observe the vascular changes in Port-wine (PWS) lesions and assess the clinical efficacy of Pulsed Dye Laser (PDL) treatment by examining vessel diameter and density with RCM. Materials and methods: Eleven adult patients with PWS, each had four test patches carried out with different pulse durations (1.5, 3, 6, and 10 ms), respectively; fluences of 9–12 J/cm2; and a spot size of 7 mm. The PDL treatment was repeated 3–5 times at a 2-month interval. Photographs and measurements with RCM were taken before each treatment and 2 months after the last treatment. Results: The PDL treatment exhibited increasing clearance with reducing pulse durations. Vessel diameters and densities were significantly decreased in the same pulse-duration groups after treatment. There was significant difference between 1.5 ms pulse-duration group and other pulse-duration groups in reducing blood vessel diameter at the depth of 150 μm. Conclusions: RCM can be used to assess the clinical efficacy of PDL treatment. Key Words: reflectance confocal microscopy, port-wine stains, pulsed dye laser
Introduction Port-wine stains (PWS) are benign vascular malformations consisting of ectatic blood vessels situated predominantly in the superficial dermis. A reduction of neural innervations around the ectatic blood vessels appears to be the pathogenesis of PWS (1). Pulsed dye laser (PDL) is the modality of choice for the treatment of PWS. PDL treatment achieves observable lightening of PWS by reducing the number, size, and therefore, erythrocyte content of these vessels following the principles of selective photothermolysis proposed by Anderson and Parrish (2). PDL incorporates the use of larger spot sizes, higher influences, varying pulse durations, and dynamic cooling for more effective treatment of PWS via greater vessel heating and deeper vascular injury (3). However, despite technological advances,
a considerable number of patients with PWS still do not achieve complete lesion removal with PDL treatment. Relationships between laser parameters and their effect on PWS treatment outcome are complex and not fully understood. It would be very useful to further understand the vascular responses of PWS to lasers through noninvasive imaging. However, we currently do not have an established noninvasive method of accurately assessment of treatment efficacy. In vivo reflectance confocal microscopy (RCM) is a noninvasive technique for evaluation of cutaneous lesions with cellular level resolution close to conventional histopathology (4). RCM has previously been used for the noninvasive visualization of benign and malignant inflammatory and proliferative skin disorders (5). It has already reported that RCM may aid in the evaluation of vascular lesions. RCM
∗Co-first author. Correspondence: LeiHong Xiang, MD, PhD, Department of Dermatology, Huashan Hospital, Fudan University, Shanghai 200040, P. R. China. Tel: ⫹ 86-021-52887765. E-mail: [email protected] (Received 14 August 2013 ; accepted 17 October 2013) ISSN 1476-4172 print/ISSN 1476-4180 online © 2014 Informa UK, Ltd. DOI: 10.3109/14764172.2013.862552
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J. Ren et al.
allows the in vivo examination of the diameter of vessels and degree of vascular tortuosity or dilation (6). The aim of this study is to use RCM to observe the vascular changes in PWS lesions, including the diameter and density of vessels, before and after PDL treatment, and to assess the clinical efficacy of PDL with different pulse durations. Materials and methods
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Patient selection and treatment We recruited 11 patients (6 males and 5 females, aged 24.82 ⫾ 9.82 years, Fitzpatrick skin phototypes III–IV) with PWS who had no previous treatments after the project was approved by the Ethics Committee of Huashan Hospital, Fudan University, Shanghai, China. For ten patients, lesions were distributed only on the face while one patient had lesions on the neck as well. The lesions appeared pink, red, and purple in color. The 595-nm pulsed dye laser (V-Beam, Candela, USA) with a dynamic cooling device was used for the study. Each patient had four test patches carried out with different pulse durations (1.5, 3, 6, and 10 ms), respectively, fluences of 9–12 J/cm2, and a spot size of 7 mm. On each patient the four test patches were performed with four different pulse durations, while the same fluence was selected for each session. The treatment endpoint occurred when the color of the treated cutaneous vessel showed purpura. The PDL treatment was repeated 3–5 times at a 2-month interval. Once a patient’s lesion was completely clear in any of the four pulse-duration groups, the treatment would be ended. Post-treatment discomfort was relieved by immediate application of cold spray or an ice pack to the treated areas. Clinical evaluation Efficacy was determined by comparison of digital photographs (Cannon EOS 400D) obtained before each treatment and 2 months after the last treatment. One independent blinded dermatologist graded lesion clearance (%) on a four-point scale: poor (⬍ 25%), fair (26–50%), good (51–75%), and excellent (76–100%).
skin with an adhesive. This RCM steel ring fixture stabilized the area of interest and helps maintain a constant mechanical contact between the skin and the RCM. A small drop of immersion oil was applied to the skin lesion. On each patient four test patches were selected for RCM imaging. Transparent body charts were used to outline the skin areas at baseline to permit co-localization during follow-up examination. RCM imaging was performed before each treatment and 2 months after the last treatment. RCM images were acquired using a standardized protocol for each patient. The protocol included the acquisition of three RCM datasets per site: (i) four 2 ⫻ 2 mm mosaics at the depth of 100 μm; (ii) four 2 ⫻ 2 mm mosaic at the depth of 150 μm; and (iii) four 0.5 ⫻ 0.5 mm z-stack with 2 μm spacing from the center of the 2 ⫻ 2 mm mosaic. The density and diameter of blood vessels were counted at the 2 ⫻ 2 mm mosaic images. Statistical analysis Data were expressed as mean ⫾ S.D. Paired t-test was used to determine the significance between groups before and after treatment, and a one-way ANOVA test with Bonferroni correction was used to analyze the significance when three or more groups were compared. Statistical significance was defined as P ⬍ 0.05. The SPSS 13.0 software was used for all statistical analyses.
Results Clinical response Clearance rates of patients for PDL treatment with different pulse duration are shown in Table I. Overall, clearance was excellent in 27.28% of patients, good in 20.45%, fair in 36.36%, and poor in 15.91%. The PDL treatment exhibited increasing clearance with reducing pulse duration. The “excellent” clearance increased from 18.18 to 18.18, 27.27, and 45.46% at pulse duration of 10, 6, 3, and 1.5 ms, respectively, while the “poor” clearance decreased
Table I. Number of patients achieving various clearance rates for PDL treatment with different pulse durations.
RCM evaluation RCM was performed with the commercially available near infrared RCM Vivascope 1500 (Lucid Inc., Rochester, NY, USA). This system uses a diode laser at a wavelength of 830 nm, a power less than 30 mW, and a ⫻30 water immersion objective lens with numeric aperture of 0.9. The RCM objective was attached to the skin via a stainless steel ring, which in turn was attached to the corneal layer of the
Clearance rate (%) Pulse duration
76–100 (excellent)
1.5 ms 3 ms 6 ms 10 ms
5 (45.45%) 3 (27.27%) 2 (18.18%) 2 (18.18%) Total ⫽ 12 27.28%
2 4 2 1
51–75 (good)
25–50 (fair)
(18.18%) (36.37%) (18.18%) (9.09%) Total ⫽ 9 20.45%
3 (27.27%) 3 (27.27%) 5 (45.46%) 5 (45.46%) Total ⫽ 16 36.36%
⬍ 25 (poor) 1 1 2 3
(9.09%) (9.09%) (18.18%) (27.27%) Total ⫽ 7 15.91%
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Pulsed dye laser treatment of port-wine stains
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Figure 1. (i) A 50-year-old male before treatment with the 595-nm pulsed dye laser, and (ii) after four treatments with different pulse duration of 1.5 (a), 3 (b), 6 (c), 10 ms (d). The lesion was cleared by more than 75% in (a) after PDL treatments with the pulse duration of 1.5 ms.
from 27.27 to 18.18, 9.09, and 9.09%, respectively. Clinical examples are shown in Figure 1. RCM examination Differences in blood vessel diameter and density using RCM evaluation are shown in Table II. Overall, RCM imaging at the depth of 100 and 150 μm showed greater vessel diameter before treatment and the vessels tended to increase in caliber with increasing depth, as shown in Figure 2. The mean blood vessel diameters of all examined regions at the depth of 100 and 150 μm before treatment were 72.11 ⫾ 27.50 and 98.18 ⫾ 33.02 μm, respectively, and these decreased to 31.82 ⫾ 17.39 and 67.11 ⫾ 31.07 μm, respectively, after treatment
(P ⬍ 0.05). Meanwhile, the mean blood vessel densities at the depth of 100 and 150 μm were 15.09 ⫾ 5.06 and 18.55 ⫾ 3.46, respectively, before treatment; both decreased significantly to 8.89 ⫾ 3.34 and 11.89 ⫾ 2.81, respectively, after PDL treatment (P ⬍ 0.05). After treatment, both diameter and density of blood vessel decreased statistically as compared with those before treatment in the same pulse-duration groups (P ⬍ 0.05). There was significant difference between 1.5 ms pulse-duration group and other pulseduration groups in reducing blood vessel diameter at the depth of 150 μm (P ⬍ 0.05), while no significant difference among each pulse-duration groups at the depth of 100 μm (P ⬎ 0.05). Statistically, there was no difference between different pulse-duration
Table II. Diameter and density of blood vessel examined in 1 mm2 RCM images among different pulseduration groups before and after treatment. 100 (μm) depth Pulse duration 1.5 ms Before Tx After Tx Difference 3 ms Before Tx After Tx Difference 6 ms Before Tx After Tx Difference 10 ms Before Tx After Tx Difference
150 (μm) depth
Blood vessel diameter (μm)
Blood vessel density
Blood vessel diameter (μm)
Blood vessel density
74.64 ⫾ 25.16 31.82 ⫾ 17.39∗ 42.82 ⫾ 19.94
16.45 ⫾ 7.12 7.27 ⫾ 2.00∗ 9.18 ⫾ 7.22
95.64 ⫾ 27.83 56.00 ⫾ 27.38 ∗ 39.64 ⫾ 29.30#
18.91 ⫾ 3.99 10.82 ⫾ 2.27∗ 8.09 ⫾ 3.18
70.27 ⫾ 31.62 36.91 ⫾ 21.81∗ 33.36 ⫾ 28.21
13.91 ⫾ 5.13 7.82 ⫾ 3.28∗ 6.09 ⫾ 5.41
94.55 ⫾ 33.18 66.91 ⫾ 28.97∗ 27.64 ⫾ 23.64
16.91 ⫾ 2.77 10.91 ⫾ 2.55∗ 6.00 ⫾ 3.29
69.73 ⫾ 27.78 41.55 ⫾ 21.44∗ 28.18 ⫾ 20.03
14.82 ⫾ 3.60 9.73 ⫾ 2.69∗ 5.09 ⫾ 3.18
99.09 ⫾ 39.26 69.91 ⫾ 32.42∗ 29.18 ⫾ 25.13
19.36 ⫾ 3.67 13.09 ⫾ 3.21∗ 6.27 ⫾ 3.41
73.82 ⫾ 28.77 45.36 ⫾ 18.95∗ 28.46 ⫾ 23.39
15.18 ⫾ 4.07 10.73 ⫾ 4.22∗ 4.45 ⫾ 6.07
103.45 ⫾ 34.90 75.64 ⫾ 25.89∗ 27.81 ⫾ 25.65
19.00 ⫾ 3.22 12.73 ⫾ 2.72∗ 6.27 ⫾ 3.47
Data correspond to the mean ⫾ S.D. Tx: treatment. Difference ⫽ the diameter (or density) before treatment-the diameter (or density) after treatment. ∗P ⬍ 0.05 vs. Before Tx. #P ⬍ 0.05 1.5 ms pulse-duration group compared with 3, 6, 10 ms groups separately.
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J. Ren et al.
Figure 2. (a, b) RCM images of a 50-year-old male before and (c, d) after PDL treatment. Compared with (a, b), diameter of blood vessel decreased significantly in (c, d). Scale bar 100 μm.
groups in reducing vessel density at the depth of both 100 and 150 μm (P ⬎ 0.05).
Discussion PDL treatment is based on the theory of selective photothermolysis, which induces selective photocoagulation of the target blood vessels. The appropriate setting of laser parameters such as spot diameter, fluence and pulse duration can improve PWS clearance rates. Current studies show that less than 20% of PWS patients can be lightened completely with PDL treatment, 50% patients achieve 70% improvement while around 20–30% patients barely showed minimal response to the treatment (7). However, pulse durations selected in these studies vary greatly. There is experimental evidence that the ideal pulse duration shall be from 1 to 10 ms as PWS blood vessels range from 20 to 150 μm in diameter (8). To achieve better comparability, we minimized the variables for the PDL treatment. On each patient four test patches were performed with four different pulse durations (1.5, 3, 6, and 10 ms), while the same fluence and spot diameter were selected for each session. In our study, more than 75% lightening was achieved by 45.45% of the patients in 1.5 ms pulse-duration
group, which surpassed other pulse-duration groups in terms of efficacy. Histopathological examinations of PWS showed a normal epidermis overlying an abnormal plexus of dilated blood vessels located as a layer in the upper dermis (9). Recently several noninvasive imaging techniques have been used to identify morphology, physiology, and dynamics of PWS, which may provide further objective measurement on treatment response. RCM, a promising, noninvasive, high-resolution new imaging tool, can perform histologic evaluation of the skin in vivo. The correlation between RCM features and histopathologic findings has been demonstrated in pigmented (10), neoplastic (11), and benign vascular lesions (12). RCM evaluation revealed a great number of large dilated vessels in mid- to superficial dermis in PWS (6). We used this new technology to study the features of blood vessels in the upper dermal layer. Images of PWS before and after treatment were obtained to compare the changes of mean blood vessel diameter and density, thus evaluating the response to PDL treatment. The abnormal vessels in PWS lesions are composed of dilated blood vessels of various diameters and depths. We found the vessels morphology of RCM features varies at different scanning depths. As scanning depths increased, both the mean diameter
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Pulsed dye laser treatment of port-wine stains and maximum diameter of vessels become larger. Therefore, it is necessary to mark the scanning depth when measuring vessel diameter with RCM, in order to have an objective and reliable evaluation. As our data showed, both the mean blood vessel diameter and the density decreased after treatment as compared with those before treatment in the same pulse-duration groups. It is interesting that there was a significant difference between the 1.5 ms pulseduration group and the other pulse-duration groups in vessel diameter reduction at the depth of 150 μm, while there was no significant difference among all pulse-duration groups at the depth of 100 μm. This seems to indicate that the ectatic vessels in upper dermis have better response to PDL treatment regardless of pulse duration variation. It is important to note that laser energy must be sufficient to coagulate PWS blood vessels. A previous study showed that higher fluences were required for longer pulse durations (13). Our study clearly showed that PDL with a pulse duration of 1.5 ms was easier to destroy the target dilated vessels, and that significant improvement of PWS could be achieved with the PDL with longer pulse durations. In our study, patients in the 1.5 ms pulseduration group achieved more lightening and clinical efficacy, while they surpassed others in reducing vessel diameters at 150 μm depth for RCM imaging. It is confirmed that the correlation between the clinical therapeutic outcome and the vessel diameter after treatment is consistent. Furthermore, there was no contrast among different pulse-duration groups in reducing the vessel density both at the depth of 100 and 150 μm, which proves once again that blanching efficacy is more related to the diameter of blood vessel. PWS initially appear to be flat, pink/red patches that may gradually develop with age into hypertrophic, red/purple lesions, leading to further cosmetic disfigurement and psychologic distress (14). A few studies have indicated that treatment at younger ages require fewer sessions and might improve the PWS clearance (15). Our data also revealed such a trend that both the mean diameter and maximum diameter of vessels decreased after PDL treatment. It seems to be another explanation for lesional lightening outcome. We propose that patients should receive several more sessions at regular intervals to constrain the development of lesion color and thickness, even after their lesions stop to further lighten in subsequent treatments. Further observations and studies are needed. In conclusion, RCM enhances our understanding of the interactions between PDL treatment and PWS so that the treatment parameter settings can be optimized accordingly. The therapeutic outcome of PDL treatment is related to blood vessel diameter of PWS. RCM can also help with the pre-treatment prediction of efficacy for individuals with different PWS
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characteristics. The application of RCM in assessment of laser efficacy for other vascular lesions, such as spider angioma and cherry angioma, should also be further studied. Declaration of interest: The authors report no declarations of interest. The authors alone are responsible for the content and writing of the paper.
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