|Year : 2021 | Volume
| Issue : 1 | Page : 35-41
Simplification of noncultured epidermal cell suspension: A need of the hour
Sidharth Tandon, Kezhaleto Zutso, Konchok Dorjay, Ajeet Singh, Surabhi Sinha
Department of Dermatology, Dr Ram Manohar Lohia Hospital and PGIMER, New Delhi, India
|Date of Submission||01-Nov-2018|
|Date of Decision||26-Feb-2019|
|Date of Acceptance||19-Mar-2019|
|Date of Web Publication||07-Apr-2021|
Department of Dermatology, Dr Ram Manohar Lohia Hospital and PGIMER, New Delhi, 110001
Source of Support: None, Conflict of Interest: None
Context: Vitiligo is the most common acquired disorder of pigmentation. The treatment options for vitiligo are either medical or surgical modalities. Noncultured epidermal cellular grafting is an innovative surgical technique that can be used for the treatment of stable vitiligo. Aim: To evaluate the effect of noncultured epidermal cellular grafting in stable vitiligo utilizing normal saline as a trypsin inhibitor. Materials and Methods: Thirty-three patients with stable vitiligo for the past 2 years were recruited in the study. Noncultured epidermal cell suspension grafting was done with normal saline as trypsin inhibitor. Result: Out of 33 patients, maximum patients had focal vitiligo (21), followed by segmental (7) and generalized (5). Response was excellent in 7 patients (21%), good in 16 (48%), regular in 7 (21%), and poor in 3 patients. Thus, 23 (69%) patients had a satisfactory (excellent to good) response. Satisfactory pigmentation over trunk was seen in 83%, face and neck in 90%, and limbs in 57%. Three patients had lesions involving the fingertips and toes and only one of them had a good response with the other two patients showing a poor response. Conclusion: The noncultured epidermal cell suspension grafting is an effective surgical procedure for stable vitiligo patients. In this study, we have tried to simplify the procedure by utilizing only normal saline as a trypsin inhibitor.
Keywords: Vitiligo, noncultured epidermal cell suspension, normal saline
|How to cite this article:|
Tandon S, Zutso K, Dorjay K, Singh A, Sinha S. Simplification of noncultured epidermal cell suspension: A need of the hour. Pigment Int 2021;8:35-41
| Introduction|| |
Vitiligo is the most commonly encountered depigmenting dermatosis affecting 0.5 to 1% of the general population and can be caused by both genetic and acquired factors.,
The treatment options for vitiligo are either medical or surgical. The medical line of treatment includes topical corticosteroids, topical tacrolimus, systemic steroids, and phototherapy using psoralens. The repigmentation derived with these methods can sometimes be less than satisfactory, leading to the usage of surgical modalities for treatment.
The surgical methods available for treatment are punch grafting, split skin grafting, suction blister grafting, hair follicle graft, cultured and noncultured melanocyte suspension, laser, and micropigmentation.
Billingham and Medawar performed the first noncultured epidermal cellular suspension on piebald guinea pig skin. Since then the procedure has undergone lots of modifications. In this study, we used normal saline as a trypsin inhibitor to make the procedure simpler without affecting the response.
| Materials and Methods|| |
Thirty-three patients with stable vitiligo for the past 2 years with no increase or decrease in size and number of the lesions were included in the study. The inclusion criteria included stable vitiligo for the past 2 years, not on any immunosuppressive therapy, absence of keloidal tendency, and absence of any bleeding diathesis. The exclusion criteria used in the study were more than 30% body surface area involvement, presence of any autoimmune disorder, past history of any malignancy, age less than 10 years, and pregnant females.
Patients were classified according to the site of lesions and the type of vitiligo into generalized, focal, and segmental vitiligo.
The technique described by Mulekar et al. was used with a few modifications (procedure described below) to reduce the cost and complexity of the procedure. Photographs were taken preoperatively and at 3 and 6 months postoperatively ([Figure 1],[Figure 2],[Figure 3],[Figure 4]).
|Figure 1 (a) Vitiligo patch at the dorsum of left foot. (b) 3 months post op. (c) 6 months post op.|
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|Figure 2 (a) Vitiligo patch at the anterior aspect of left leg. (b) 1 month post op. (c) 3 months post op. (d) 6 months post op.|
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|Figure 3 (a) Vitiligo patch at the anterior aspect of left leg. (b) 3 months post op. (c) 6 months post op.|
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|Figure 4 (a) Halo phenomenon post suction blister and punch grafting. (b) Failed punch grafting at forehead in the same patient. (c) Complete resolution of the halo at 6 months post op. (d) Complete resolution of the forehead patch at 6 months post op.|
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The anterolateral aspect of left thigh was used as the donor area for the graft. The size of the donor area was 1/10th of the recipient area. The area was made sterile by cleaning with Savalon followed by betadine and lubricating the area with paraffin gauze. Anesthesia in the region was achieved by infiltrating the area with 2% plain lignocaine injection. A thin epidermal graft was taken using a shaving blade held with a needle holding forceps. The donor site was dressed with a betadine-soaked sterile gauze using a micropore tape.
The harvested graft was washed with normal saline and transferred to a petri dish containing ∼5 ml of 0.2% trypsin EDTA solution and was incubated at 37 °C for a period of 50 minutes. Thereafter, the trypsinized graft was again washed with normal saline to stop the process of trypsinization and was transferred to a petri dish containing another 10 ml of normal saline. The epidermis was separated from the dermis using jewelers forceps. The dermis was discarded and then cells were teased off from the epidermis using the same forceps, leading to the appearance of turbidity in normal saline. The remaining stratum corneum was discarded. The suspension prepared was transferred to a 15 ml falcon tube, which was then centrifuged at 1800 rpm for 10 minutes to obtain a cell pellet. Nine milliliters of the supernatant fluid was discarded and the pellet obtained was resuspended in the remaining 1 ml normal saline.
The recipient area was made sterile and anaesthetized by using the same procedure as for the donor area. It was then subjected to dermabrasion by using an electrical dermabrader. The suspension was then uniformly spread over the area and it was covered with a sterile collagen dressing (Neuskin-F dressing). It was then covered with a transparent adhesive (Tegaderm) and fixed with dynaplast. The patient was advised to rest for 30 minutes post procedure. The dressing was opened at 1 week. The patient was then advised alternate day sun exposure after taking tablet 8-methoxy psoralen in a dose of 0.6 mg/kg.
The patient was asked to return for follow-up at 1 month, 3 months, and 6 months. The repigmentation was assessed by using the scale used by Bao et al. The repigmentation was graded as excellent (>90%), good (50–89%), regular (20–49%), and poor (<20%). Satisfactory repigmentation was defined as pigmentation >50% of the lesional area. 
| Results|| |
Thirty-three patients were recruited for the study. There were 15 males and 18 females with the age group ranging from 11 to 67 years. All the patients underwent a single session for the melanocyte transplantation. The results were evaluated at 3 and 6 months. Types of vitiligo included in the study were generalized (5 patients), focal (21 patients), and segmental (7 patients) ([TABLE 1]).
|TABLE 1 Classification According to the Type of Vitiligo, Site, and Size|
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We found an excellent response in seven patients (21%) (five had focal vitiligo and one each had segmental and generalized vitiligo). All the patients with segmental vitiligo had a satisfactory response (>50%) to the procedure, with six patients having a good response (minimum pigmentation >65%) and one patient having an excellent response. Majority of the patients with focal vitiligo had good response (47.6%) followed by excellent response in 23.8%, regular response in 19%, and poor response only in 9.5% ([Figure 5] and [TABLE 2]).
Two patients developed hyperpigmentation at the recipient site with rest of the patients having an adequate color matching. Relapse was seen in only one patient having generalized type of vitiligo.
| Discussion|| |
Surgery is one of the mainstays of treatment of stable vitiligo, but the results are variable and larger skin areas cannot be covered adequately with the use of surgical modalities. Cultured melanocyte suspension, which can be used to cover larger areas, requires an expensive laboratory setup and is time-consuming.
Gauthier and Surleve-Bazeille did the first epidermal cellular suspension for stable vitiligo in 1992. The various modifications of the technique mentioned above are detailed in [Table 3].,,
We have further modified this technique and have used only 0.2% trypsin EDTA solution and normal saline to create the suspension. By doing this, we were able to bring down the cost of the procedure and also do away with the problem of storing so many reagents in a sterile environment. Holla et al. had proposed PBS as an inhibitor of trypsin. Also, plasma itself can act as a trypsin inhibitor, as shown by Gauthier and Surleve-Bazeille, since the recipient site, after dermabrasion, has pinpoint bleeding points oozing plasma, which may neutralize the remnants of trypsin after it has been washed away with normal saline. Gupta et al. have shown that the trypsin does not act on dermal tissue and does not impede wound healing.
We found 72% satisfactory response to the treatment irrespective of the duration of vitiligo, which is similar to Komen et al. observed a satisfactory response in 60% of their patients. In a study by Ramos et al. they found, a satisfactory response in 75% of their patients with melanocyte suspension, which is in agreement with our findings. We found an excellent response in seven patients (21%). Mulekar reported that 84% of their patients of segmental vitiligo had an excellent response. Ramos et al. observed a satisfactory response in 84% of their patients in segmental vitiligo. Both of these studies corroborate our results of a high rate of response in segmental vitiligo patients. A single patient with generalized vitiligo had a relapse of the disease, which has been observed by other authors too.
We found that the repigmentation rates also varied according to the different sites that were involved. The best repigmentation rates were observed in the head and neck region with 9 patients out of 10 patients developing a satisfactory repigmentation and one patient with lesion on the lower lip having a poor response. The worst response rate was in acral lesions involving the fingers and toes with a poor response in two out of the three patients included. The findings are in concordance with the study by Ramos et al. in which they found the best repigmentation rates in the head and neck region and the worst response in extremities. Huggins et al. also observed similar findings in their patients. As regards the repigmentation rates in truncal lesions, the findings observed are variable with five out of the six patients having a satisfactory repigmentation in the present study. Ramos et al. observed a satisfactory response in 38% of the patients with truncal involvement. Huggins et al. in their study observed poor response in 80% of their patients with truncal lesions. The variable response rates can be attributed to the complex pathogenesis of the disease, which has a multifactorial etiology.
Thus, the repigmentation rates in the present study are similar to those published in literature; however, our method could prove to be more cost-effective as we have used only normal saline as a trypsin inhibitor. Larger studies in the future could further substantiate our findings.
There are a few limitations of the present study. We did not take any controls for the cases, and the method of assessing repigmentaion is also subjective.
| Conclusion|| |
The technique of epidermal melanocyte noncultured suspension has already undergone many modifications and still remains the most appropriate surgical procedure for vitiligo patients. We have tried to modify it further to bring down the cost and complexity of the procedure by utilizing only normal saline as a trypsin inhibitor. Yet there are many unanswered questions as to why some patients have an excellent response, whereas others have a poor response under similar test conditions. Further studies with larger sample sizes carried out with the above-mentioned modifications could be beneficial.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[TABLE 1], [TABLE 2], [Table 3]