A 4-year follow-up study of 20 cases of stable localized childhood vitiligo treated by autologous, noncultured melanocyte–keratinocyte cell transplantation : Pigment International

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Original Article

A 4-year follow-up study of 20 cases of stable localized childhood vitiligo treated by autologous, noncultured melanocyte–keratinocyte cell transplantation

Abdul Latheef, Ettappurath N.; Rahima, S.; Muhammed, K.; Riyaz, Najeeba

Author Information

Department of Dermatology and Venereology, Government Medical College, Kozhikode, Kerala, India

Address for correspondence: Ettappurath N. Abdul Latheef, Department of Dermatology and Venereology, Government Medical College, Kozhikode, Kerala, Postal Code: 673008, India. E-mail: [email protected]

Received July 19, 2021

Received in revised form December 08, 2021

Accepted December 18, 2021

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Pigment International 10(1):p 46-51, Jan–Apr 2023. | DOI: 10.4103/pigmentinternational.pigmentinternational_
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Abstract

Background: 

Vitiligo is a common skin disease affecting 1% to 2% of general population. Even though different modalities of treatment available, it remains as a difficult disease to treat. Many cases remain stable after a period of medical therapy without any further response. For treating pediatric vitiligo, currently available and result providing therapies are limited. Surgical techniques are the only hope in such patients. Only limited studies are available regarding surgical interventions in childhood vitiligo.

Aim: 

To evaluate the response of autologous, noncultured melanocyte–keratinocyte cell transplantation in childhood stable vitiligo.

Methods: 

A retrospective analysis was conducted in 20 cases of stable localized childhood vitiligo (10 segmental and 10 focal) treated by autologous, noncultured melanocyte–keratinocyte cell transplantation. Serial photographs were taken from the first visit onwards. The patients were followed for a period of 4 years. The results were analyzed on a visual analog scale.

Results: 

Out of total 20 cases, 11 had excellent (90–100%) response, 4 had good (60–89%), 2 had fair (25–59%), and 3 had poor response (0–24%). Age and sex of the patients and size and duration of lesions did not show significant influence on results of transplantation.

Conclusion: 

This is a simple, safe, and effective surgical therapy for replenishing the missing melanocytes even in resistant cases of vitiligo. Only limited studies are there in childhood vitiligo. In spite of its invasive nature, it can be used to treat localized stable vitiligo in children.

Introduction

Vitiligo is an idiopathic acquired melanocytopenic condition affecting the skin characterized by well demarcated white patches. The condition affects 1% to 2% of the general population. The common age group is 10 to 30 years, and as often in males as in females.[1] Multifactorial causation is postulated. Various therapeutic options are available to treat vitiligo and many articles are being published about the efficacy and safety of various repigmenting therapies. Majority of the studies are of adult vitiligo, and there is a dearth of literature on childhood vitiligo.[2] Many authors express that there are differences between childhood and adult vitiligo that have importance in the management of this disease. The gender predilection is almost same in adults but childhood vitiligo seems to occur more frequently in girls.[2,3] The important epidemiologic difference between adult and childhood vitiligo is the increased prevalence of segmental vitiligo in children.[4] For treating pediatric vitiligo, currently available and result providing therapies are limited. They include topical PUVA therapy or narrow band ultraviolet B, class 3 topical steroids,[5,6] and topical tacrolimus ointment.[7,8] These medical therapies have poor response in lesions with leukotrikia and in glabrous skin. Even though this is the current situation, most doctors do not consider surgical therapies in children due to lack of evidence and published data.[2,9] Localized vitiligo may be either segmental, which is characterized by unilateral macules in quasidermatomal distribution or focal vitiligo, which is described as depigmented macules in a localized, nondermatomal distribution. Focal vitiligo could be considered an initial stage of generalized or immunologic vitiligo or may be an abortive form of the segmental type.[2]

Objective

To assess the repigmentation and sustainability of pigmentation after autologous, noncultured melanocyte–keratinocyte (MK) cell transplantation in localized stable childhood vitiligo over a period of 4 years follow-up.

Methods

After obtaining institutional ethics committee clearance, we conducted a retrospective study on all stable childhood vitiligo patients (patients aged 16 years and below) who attended our tertiary care center from January 2013 to December 2015 and who were treated with autologous, noncultured MK cell transplantation. A diagnosis of stable vitiligo was made when there were no new lesions or no increase in size of existing lesion for a period of 1 year. Case records with insufficient data were excluded. Patients on immunosuppressive therapy, history of keloid, or bleeding tendency were not considered for surgery. Written consent was taken from the parents of the children for surgery and follow-up photographs. With a preset proforma, we collected information on age and gender profile, clinical details, previous treatment received and response to treatment during the 4 years follow-up period. Topical anesthesia (lignocaine plus prilocaine cream) was used for anesthesia over donor and recipient areas. The surgical procedure adopted was as followed.

Equipments

The equipments used are skin grafting knife (Silvers knife), marking pen, spatula, test tubes, petri dishes, iris scissors, jeweller’s forceps electrical dermabraders, incubator, and centrifuge.

Donor site

Lateral aspect of the gluteal region was selected as donor area because of heavy density of melanocytes.[10] By a marking pen, one-fifth to one-tenth of the recipient area was marked. Topical anesthesia (lignocaine plus prilocaine cream) is applied over that area under occlusion by a polythene sheet. After keeping for 1 hour, the surgery was processed. The marked area and surrounding skin are cleaned with povidone iodine and 70% ethanol. The skin is stretched uniformly and graft is taken with silvers skin grafting knife. A thin graft at the level of upper dermis characterized by multiple bleeding points is better. The wound so created is covered with vaseline gauze.

Preparation of the cell suspension

The skin sample taken from the donor site is transferred to a petri dish containing about 4 mL of 0.2% w/v trypsin solution.[1] The sample is kept in trypsin solution with epidermis upwards and shaken to ensure that it comes in complete contact with the solution. Then, the petri dish

containing sample is incubated for 50 minutes at 37°C in a medical incubator. After incubation, the sample is taken out from the incubator and about 2 mL of trypsin inhibitor is added to petri dish to neutralize the action of trypsin. Then the epidermis is separated from dermis and is transferred to a test tube containing 3 mL of Dulbecco-modified eagle medium/F12 (DMEM/F12) and vortex mixed for 15 seconds. The dermal pieces are then discarded. The epidermis in petri dish is broken down into multiple small pieces using forceps, washed with

DMEM medium and then transferred to test tube containing the same medium. The test tube is then centrifuged (2000 RPM) for 7 minutes. The cell pellet thus settles to the bottom and is made into a suspension of around 0.5 mL in DMEM medium. The suspension is taken in 1 mL syringe with detachable needle. The suspension thus contains a mixture of melanocytes and keratinocytes.

Recipient site

Recipient area is shaved and marked out with a marker pen, cleaned with povidone iodine and 70% ethanol and anaesthetized with topical anesthesia (lignocaine plus prilocaine cream). The recipient area was abraded with a high-speed dermabrader fitted with a diamond fraise wheel. The dermabrasion continued down till multiple pinpoint bleeding spots appear that indicates the dermoepidermal junction was reached.

Transplantation

The denuded area was covered with gauze pieces moistened with isotonic sodium chloride solution until the cell suspension was applied evenly on the denuded area and covered with a dry collagen sheet. Collagen helps transplanted cells to remain in place, providing an optimal environment for cellular growth and vascularization. This was then covered with sterile gauze pieces moistened with DMEM/F12, held in place with adhesive tapes. The patient was allowed to go home after dressing. The patient was warned against any vigorous activities for 7 days which could displace the dressing. Absolute immobilization was not necessary. Antibiotics were prescribed for 5 days. Dressing was removed after 1 week. PUVA had been given to all patients 2 weeks after transplantation and continued till good repigmentation occur. In majority, good repigmentation occurred in 6 to 8 months time. We stopped PUVA therapy after 1 year in all patients irrespective of pigmentation status.

Results were analyzed and evaluated on the basis of serial photographs taken during the follow-up period. Repigmentation was graded on the basis of a visual analog score as excellent with 90% to 100% pigmentation, good with 60% to 89%, fair with 25% to 59%, and poor with 0% to 24% of the treated area. Data were entered in Microsoft excel sheet and analyzed.

Results

In this retrospective study, 20 childhood vitiligo patients (10 segmental and 10 focal vitiligo, 6 males and 14 females with stable lesions) were included. All of them were on some or other form of medical therapy and some on narrow band UVB therapy till the surgery. The duration of vitiligo varied from 1 to 8 years. The age group ranged from 10 to 16 years. The treated area appeared bright pink immediately after removal of dressing after 1 week. The earliest pigmentation is noticed 3 weeks after surgery. At the end of 4 years follow–up, the following results were observed.

Segmental vitiligo

In this study, 10 patients (four boys and six girls) with segmental vitiligo were followed for a period of 4 years. The maximum area treated in one operative session was approximately 110 cm2 and the minimum area, 10 cm2. Six patients had an excellent response to treatment, two had a good response, one patient with fair response, and one failed to produce any pigmentation at all [Table 1]. One patient with excellent response required two sessions of transplantation to achieve the response. The second session was 8 months after the initial procedure. No new lesions were noticed postgrafting. Donor site had good color matching in seven patients. Three patients showed hyperpigmentation [Figure 1].

T1-7
Table 1:
Segmental vitiligo − response to melanocyte–keratinocyte cell transplantation
F1-7
Figure 1:
(a) 12-year-old girl with depigmented patch of segmental vitiligo over neck. (b) Same patient during follow-up after 4 years showing retention of pigment attained at 6 months of surgery.

Focal vitiligo

A total of 10 patients (two boys and eight girls) with focal vitiligo were followed for 4 years. The maximum area treated in one operative session was approximately 120 cm2 and the minimum area 14 cm2. Five patients had excellent response, two had good response, one fair, and two had poor response to treatment [Table 2]. One patient in poor group failed to produce any pigmentation in spite of two grafting sessions. The size of lesion remained unchanged in these patients. However, 2 of 10 patients experienced appearance of new lesions in untreated area during the follow-up period and the treated area remained pigmented. Donor site had good color matching in seven patients. Two patients showed hyperpigmentation and depigmentation noted in one patient [Figure 2].

T2-7
Table 2:
Focal vitiligo − response to melanocyte–keratinocyte cell transplantation
F2-7
Figure 2:
(a) 13-year-old boy with multiple depigmented patches of vitiligo vulgaris over chest. (b) Same patient during follow-up after 4 years showing retention of pigment attained at 8 months of surgery.

Out of 20 patients, 11 patients (55%) had excellent pigmentation, 4 (20%) had good, 2 (10%) had fair, and 3 (15%) had poor pigmentation. The poor response was mainly over the nonhairy areas and over bony prominences. Two patients of focal vitiligo (one excellent and one good) had marked pigmentary dilution on follow-up and had undergone retransplantation at 8 months and 1 year to achieve this result. Two patients experienced appearance of new lesions in untreated area during the follow-up period and the treated area remained pigmented. Donor site had good color matching in 13 out of 20 patients and 6 showed hyperpigmentation and depigmentation in one. The presence of some color mismatch at the recipient area (hyper- and hypopigmentation) was observed in 30% and perilesional hypopigmentation was observed in 40% of the evaluated patients. But this color mismatch was not disturbing according to the majority of patients.

Discussion

We previously reported a follow-up study of localized vitiligo in adult patients treated by autologous, noncultured cellular transplantation.[1] Resistant cases of focal and segmental vitiligo may remain as such for many years.[2] Surgical intervention is the only therapeutic option available for such cases. Noncultured MK cell suspension transplantation is a widely used procedure in such cases,[11,12,13,14] but only very limited studies are available in childhood vitiligo. Recently, skin grafting has been recommended for selective use in the pediatric population.[2,4] However, there are many difficulties such as immobilization, difficult to treat anatomic sites, expense, and many are time consuming procedures. These limitations have been decreased as MK cell transplantation can be completed in a short time and does not require absolute immobility and any special precautions to treat “difficult to treat” sites.[15]

Response to MK cell suspension transplantation varied in different studies. Gauthier and Surleve-Bazeille reported zero repigmentation in four of seven patients with focal vitiligo.[16] Falabella observed two of four patients with focal vitiligo improved with complete repigmentation.[17] Guerra et al. reported that of the six patients with focal vitiligo, three had complete and two had partial repigmentation.[18] Mulekar observed that repigmentation failed to be produced in 20% of focal vitiligo group.[19] In this study, 15% of the patients failed to produce any pigmentation. We observed new lesions in 2 of 10 focal vitiligo patients during the respective follow-up period. The result is good in segmental vitiligo comparing to focal vitiligo which may be explained as focal vitiligo acting as a precursor of generalized vitiligo where the immunologic process may be still very active. Another observation is the poor response in nonhairy areas such as lips and bony prominences where the reserve melanocytes will be absent. Some larger lesions showed excellent repigmentation but smaller lesions failed. This shows size of the lesion does not affect the repigmentation.

Conclusion

This is a simple, safe, and effective surgical therapy for replenishing the missing melanocytes even in resistant cases of vitiligo. Repigmentation lasts long with very good cosmetic acceptability and only very small donor area is needed for transplantation of larger areas. Considering these facts, this method can be used to treat localized vitiligo in children, in spite of its invasive nature.

Limitations

This study has got all the limitations of a retrospective study. A large scale blinded trials are needed for establishing statistically significant results.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.

REFERENCES

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2. Mulekar SV, Al Eisa A, Delvi MB, Al Issa A, Al Saeed AH. Childhood vitiligo: a long-term study of localized vitiligo treated by noncultured cellular grafting Pediatr Dermatol. 2010;27:132–6
3. Handa S, Dogra S. Epidemiology of childhood vitiligo: a study of 625 patients from north India Pediatr Dermatol. 2003;20:207–10
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14. van Geel N, Wallaeys E, Goh BK, De Mil M, Lambert J. Long term results of non-culturedepidermal cellular grafting in vitiligo, halo nevi, piebaldism and nevus depigmentosus Br J Dermatol. 2010;163:1186–93
15. Mulekar SV, Al Issa A, Al Eisa A. Treatment of vitiligo on difficult-to-treat sites using autologous noncultured cellular grafting Dermatol Surg. 2009;35:66–71
16. Gauthier Y, Surleve-Bazeille JE. Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions J Am Acad Dermatol. 1992;26:191–4
17. Falabella R. Treatment of localized vitiligo by autologous minigrafting Arch Dermatol. 1988;124:1649–55
18. Guerra L, Primavera G, Raskovic D. Erbium: YAG laser and cultured epidermis in the surgical therapy of stable vitiligo Arch Dermatol. 2003;139:1303–10
19. Mulekar SV. Long-term follow-up study of segmental and focal vitiligo treated by autologous, noncultured melanocyte-keratinocyte cell transplantation Arch Dermatol. 2004;140:1211–5
Keywords:

Autologous; childhood vitiligo; focal vitiligo; noncultured melanocyte–keratinocyte cell transplantation; segmental vitiligo; stable vitiligo

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