Efficacy of Miniature Punch Grafting Followed by Puvasol Therapy in Refractory Stable Vitiligo

Introduction: Vitiligo affects approximately 2% of the worldwide population regardless of race, ethnic background or gender. When the medical methods are not successful, surgical procedures may be considered with the aim of placing a new source of pigment cells to reinitiate melanogenesis within the affected areas. This study was conducted to evaluate the efficacy of combining punch grafting followed by PUVASOL therapy in vitiligo patients. After punch grafting patients are given PUVA/PUVASOL exposure. Objective: To study the efficacy of miniature punch grafting followed by PUVASOL therapy in refractory stable vitiligo. Material and Methods: The present study was conducted in the Department of Dermatology and Venereology of Dayanand Medical College & Hospital, Ludhiana from May 2005 to March 2007. Fifteen patients of chronic stable vitiligo not responding to medical treatment and attending the outpatient department of dermatology were included in study. Appropriate statistical methods were used to analyse the data. Conclusion: Patients subjected to Miniature Punch Grafting showed more than 80% acceptance in all selected age groups and among both the sex. There was an inverse relationship between number of grafts inserted and rejection rate. Most common side effect seen was cobblestoning (60%) with few patients showing graft displacement and hematoma formation too.


Introduction
V i ligo aff ects approximately 2% of the worldwide popula on regardless of race, ethnic background or gender.About 10% of cases are localized and 90% are generalized.There are a number of medical therapies that may restore, improve or at least decrease the depigmenta on.These are topical cor costeroids, calcipotriol, topical or systemic methoxypsoralen, and oral psoralen plus ultraviolet A radia on (PUVA), ultraviolet B (UVB) radia on, pseudocatalase plus calcium plus UVB, vitamin supplementa on, human placental extract, systemic cor costeroids, other immunomodulators, and topical L-Phenylalanine in combina on with UVA (PAUVA) 1,2 .However, such treatments usually induce incomplete repigmenta on and occasionally the outcome is poor [2][3][4] .
When the medical methods are not successful, surgical procedures may be considered with the aim of placing a new source of pigment cells to reini ate melanogenesis within the aff ected areas.Several surgical procedures for the treatment of intractable lesions have been reported to be eff ec ve, including thin Thiersch gra s, suc on-blistered epidermis 1,3 , minigra ing 4,5 and injec on of non-cultured and various cultured cell gra ing techniques.Only pa ents in whom the vi ligo is stable (i.e.no progression within 4 to 6 months) are considered good candidates for gra ing.
On the other hand PUVA, either systemic or topical, increases the number of melanocytes and synthesis of melanin 2 .Topical outdoor PUVA is a popular and effi cacious therapeu c op on for pa ents with limited vi ligo 6 .The mechanism of PUVA rests on the synergis c interac on of the two components (8methoxypsoralen and UVA light) in the skin 7 .
This study was conducted to evaluate the effi cacy of combining punch gra ing followed by PUVASOL therapy in vi ligo pa ents.This procedure is adapted only when the disease is stable for a suffi cient period of me.In punch gra ing small (1-2 mm) round, full thickness gra s are harvested with a trephine from a pigmented site and transplanted into an area of vi ligo.It causes restora on of melanocytes in the depigmented skin.Such repigmented skin then regains its normal immune / infl ammatory func on 8 .
A er punch gra ing pa ents are given PUVA/ PUVASOL exposure.It s mulates tyrosinase ac vity in melanocytes and may s mulate other components of skin, such as kera nocytes to release infl ammatory mediators.Some of those may act as basic fi broblast growth factor (bFGF) and may enhance further prolifera on of melanocytes.Giving PUVASOL therapy a er punch gra ing helps in perifollicular repigmenta on.It has been found that during adequate systemic methoxypsoralen therapy, melanocytes are s mulated, migrate from the hair follicle reservoir, and spread centrifugally from the infundibulum into the basal cell layer, recolonizing the epidermis with func onal pigmentary cells 9,10 .

Objective
To study the effi cacy of miniature punch gra ing followed by PUVASOL therapy in refractory stable vi ligo.

Material & Methods
The present study was conducted in the Department of Dermatology and Venereology of Dayanand Medical College & Hospital, Ludhiana from May 2005 to March 2007.Fi een pa ents of chronic stable vi ligo not responding to medical treatment and a ending the outpa ent department of dermatology were included in the study.In stable vi ligo there were no new lesions and no progression seen in the previous lesions for the past one year.A er doing punch gra ing PUVASOL therapy was given and pa ents were followed for a period of 12 weeks i.e. 3 months.
Pa ents with unstable vi ligo, children < 12 years of age, pregnant and lacta ng women, pa ents with history of intolerance to PUVA/PUVASOL, pa ents with past history or family history of skin cancers, pa ents with renal disease / liver disease, pa ents having any chronic debilita ng diseases and tendency to develop hypertrophic scars or keloids were excluded from the study.
Preliminary inves ga ons which were carried out are :-1.Complete blood counts 2. Urine rou ne 3. Liver func on tests 4. Renal func on tests 5. Ophthalmoscopic examina on.
Informed consent of the pa ent was taken before miniature punch gra ing.Both donor and recipient areas were prepared asep cally by cleaning them thoroughly with savlon, followed by betadine and spirit.At the recipient area 2.5 mm punches were taken out at a distance of 5-10 mm.The punched out ssue was discarded.Donor area was bu ock or front of thigh.From donor area 3 mm punches were taken and kept in a petridish containing normal saline.The donor area was dressed with an sep c dressing.The 3 mm punches from the donor site were placed in 2.5 mm crators created at the recipient site.The edges were ironed out and gra ed area was covered with nons cking framyce n dressing and then pressure dressing was given using dynaplast.The pa ents were also advised a course of an bio c therapy and analgesics for a week and allowed to go home immediately a er dressing with the instruc ons to avoid any vigorous ac vity which could displace the dressing.Pa ents were then called a er 48 hrs to change the dressing and further follow up was a er one week when pa ents were examined and complica ons if any were noted.
A er 15 days of punch gra ing, pa ents were given PUVASOL therapy, where we gave tab 8-methoxypsoralen in the dosage of 0.6 mg/kg/day twice a week and then the pa ents were asked to expose the aff ected area to natural sunlight a er 2 hours.While on PUVASOL pa ents were instructed to protect their eyes from direct sunlight with the help of UV opaque goggles on the day of PUVASOL.Ini ally sun exposure was for 5 minutes and 5 minute increment was given a er every three si ngs, subject to a maximum of 30 minutes.Pa ents were followed at 2 weekly intervals for total of 12 weeks.On every follow up visit, the gra ed area was examined for evidence of new pigment development (i.e.repigmenta on).Close up clinical photographs were taken.The results obtained were recorded in performa.
In the end of the study, the results obtained were compiled and subjected to appropriate sta s cal procedure to arrive at valid conclusions.

Result
Fi een pa ents of chronic stable vi ligo who were not having any problem men oned in the exclusion criteria and reported in the study period were taken for the study.The mean age group of the study popula on was 23.93 years.Majority of them were females and from urban areas (Table 1).Most of the pa ents had surface area involvement in the range of 0.3-0.5% with a mean of 0.49 of total body surface area.The dura on of the disease varied from 1 to 16 years.Majority of the pa ents (53.33%) had disease dura on ` 6-10 years.Most of the pa ents had segmental form of the disease (67%) with (13%) having generalized and (20%) focal form of the disease (Table 2).
The number of gra s inserted per pa ent varied from 12 to 62 (Table 3).Percentage acceptance in diff erent age groups varied from 81.36 to 87.26%.The diff erence in the gra acceptance rate in diff erent age groups was not sta s cally signifi cant (Table 4).Percentage acceptance in males was 92.10 while in females was 83.53.However the diff erence in gra acceptance between males and females was not sta s cally signifi cant.The mean of gra s accepted was 30.67 with SD of 13.66 and mean of gra s rejected was 5.00 with SD of 2.36 (Table 5).
Most common side eff ect observed was cobblestoning which was observed in 60% of the pa ents.Other side eff ects which were observed were hematoma forma on and displacement of the gra (Table 7).Mean pigmenta on a ained a er 4 weeks of PUVASOL was 78.67%, which progressively increased and maximum repigmenta on was seen at 12 weeks i.e. 90.13%.So there was signifi cant repigmenta on a er 12 weeks a er PUVASOL.Most common side

Discussion
Vi ligo is a common pigmentary disorder leading to great cosme c embarrassment, psychological distress and a cause of disrup on of social rela onship.The outcome of this disorder is o en unpredictable and uncertain.There are many cases of vi ligo who either fail to respond or only par ally respond to the medical line of treatment indica ng that melanocyte reservoir is no more available for repigmenta on in these areas 11 .These non responding cases of stable vi ligo can be treated with autologous skin gra ing.Autologous skin gra ing can be done in various ways such as epidermal culture gra ing 12 , pure melanocyte culture gra ing 14 , epidermal gra ing by suc on blister technique 13 , thin Thiersch's split skin gra ing 15 , or thin split skin miniature punch gra ing.The later method was fi rst advocated by Falabella to treat small to medium sized localized patches of focal and segmental vi ligo 4,5 .
The age range in our study was between 12-50 years.Majority of the pa ents were in the age group of 21-30 years with a mean age being 23.9 years.In a study done by Malakar and Dhar 22 , majority of the cases were in the age of 10-30 years with a mean age of 20 years.So age group included in our study was almost similar to other studies.Vi ligo aff ects both sexes equally.However many studies have shown female preponderance of vi ligo 16,17 .In present study also majority i.e. 66% of pa ents were females.In fact, this may be a refl ec on that females are more concerned about cosme c looks and are more stressed due to social and matrimonial problems due to the disease.Most of the pa ents in our study (73.35%) were residents of urban area and large majori es (86.7%) were educated.This could be due to the fact that urban educated pa ents are more concerned regarding the cosme c disfi gurement resul ng from vi ligo.
Most of our pa ents had segmental vi ligo (67%), followed by focal vi ligo (20%) and generalized vi ligo (13%).This is in concordance to other studies 7,18 that also included cases with focal and segmental vi ligo.The dura on of vi ligo ranged from 1-16 years with a mean of 7.17 years (53.33%).8 pa ents had disease dura on between 6-10 years, 5 pa ents (33.33%) between 1-5 years and rest 2 pa ents disease dura on between 1-16 years.Also the surface area involvement was in our study in range of 0.3-0.5% of total body surface area with a mean of 0.49.
The number of gra s inserted per person in the study varied between 12-62 with a mean of 35.67 gra s and in majority of pa ents 30-50 gra s were put.Mean percentage of acceptance of gra was 84.29%.There was no sta s cally signifi cant diff erence in the gra acceptance rate in two sexes and in diff erent age groups.Percentage gra rejec on was 15.71% and it was found that as the number of gra s inserted increased, rejec on percentage decreased.Percentage of gra rejec on in another study conducted by Rathi 19 due to various reasons like movement, secondary infec on was around 10%.
The most important side eff ect observed was cobblestoning seen in 60% of the pa ents while displacement of gra with hematoma forma on (33%) and hematoma forma on alone was seen in (7%) of the pa ents.This is in concordance with the study done by Savant 20 who also observed cobblestoning as the commonest side eff ect seen in 50% of the pa ents.In a study done by Bajaj 18 , cobblestoning was observed in 22%.It was seen in 10 pa ents (total 32 cases) in the ini al stages but with the passage of me it was rec fi ed in majority of the pa ents.Rathi et al 19 also observed cobblestoning in 22.5% with sinking pits in 10%.No case of sinking pits was observed in the present study.This could be due to rela vely small study group.The displacement of gra (s) observed in the study was due to improper immobiliza on by the pa ents.Savant 20 observed three cases of contact allergic derma s to framyce n but no case showed framyce n allergy in present study.
Postopera ve PUVASOL was given and the commonest side eff ect observed was erythema (40%), itching (20%) and another 40% didn't experience any side eff ect due to PUVASOL.None of the pa ents however required discon nua on of therapy due to side eff ects.Trends in repigmenta on were observed for up to 12 weeks.Mean repigmenta on a ained a er 4 weeks of PUVASOL was 28.67% which progressively increased and maximum repigmenta on was seen at 12 weeks (90.13%).There was signifi cant improvement in the repigmenta on achieved with the passage of me and postopera ve PUVASOL therapy has been found to expediate repigmenta on.In another study, Falabella 5 developed his own technique using minipunch to treat localized vi ligo.Total 15 pa ents received treatment by mini gra ing and remarkable repigmenta on was obtained in 13 cases with 90-100% improvement, in one case 70% and another one 50% improvement.However no sealing solu on was used in this study to secure the gra s as in Falabella's technique since simple pressure was enough to secure them.In a prospec ve study of 1000 pa ents of stable and recalcitrant vi ligo the results of miniature punch gra ing was evaluated by Malakar and Dhar 23 .In 656 (74.55%) pa ents, 90-100% repigmenta on was achieved, in 93 (10.57%) pa ents there was no spread of pigment, while in 21 (2.39%) depigmenta on of the gra was no ced.
In another study 33pa ents were selected by Jha et al 21 for punch gra ing.They modifi ed the method which was described by Falabella and Behl by using one punch per cm 2 using 3 mm punch device.There was complete repigmenta on in 10 pa ents and in 23 pa ents pigmenta on was spreading slowly.No recurrence or scar was no ced at donor or recipient site in any of the pa ents.Rate of repigmenta on observed in the present study is similar to other study.

Conclusion
The study on pa ents of vi ligo who were subjected to Miniature Punch Gra ing showed more than 80% acceptance in all selected age groups and among both the sex.There was an inverse rela onship between number of gra s inserted and rejec on rate.Most common side eff ect seen was cobblestoning (60%) with few pa ents showing gra displacement and hematoma forma on too.Mean pigmenta on achieved a er 12 weeks of PUVASOL was above 90% and there were some adverse eff ects with PUVASOL however none of the pa ents discon nued the treatment due to the side eff ects.Mini punch gra ing with PUVASOL is very eff ec ve for a aining excellent re-pigmenta on.Post-opera ve PUVASOL therapy has been found to expediate this re-pigmenta on.

Table 1 :
Sociodemographic characteris cs of the study group

Table 2 :
Descrip on of the vi ligo

Table 3 :
Age wise dis bu on of Gra inserted

Table 4 :
Gra s acceptance in rela on to sex

Table 5 :
Distribu on of subjects according to no.Of gra s rejected:

Table 6 :
Gra s inserted, rejected and percent rejec on

Table 8 :
Trends in pigmenta on achieved: