INTRALESIONAL INOCULATION OF AUTOLOGOUS NON-CULTURED EPIDERMAL MELANOCYTE SUSPENSION IN VITILIGO- A NOVEL METHOD.
Vitiligo is a cosmetic problem often associated with psycho-social problems. The factors responsible for unsatisfactory outcome of medical therapeutic regimens include long duration, acrofacial and segmental vitiligo, poor compliance and the occurrence of adverse effects.
Vitiligo can be treated with a number of surgical interventions as punch graft, thin Thiersch's graft, melanocytes transplantation, tattooing and epidermal grafting. In autologous melanocytes transplantation both cultured  and non-cultured  either in pure form  or along with other epidermal cells  is used. Transplantation of cultured autologous melanocytes is costly, time consuming with high chances of contamination. There are reports that the presence of keratinocytes is beneficial as they produce growth factors and melanogenic peptides such as bFGF, mast cell growth factor and endothelin-1. 
It has been possible to make economical transplantation of the autologous non-cultured epidermal suspension containing melanocytes (ANEM) by inoculating it in blisters.  Delayed healing of transplanted sites and increased chances of infection in blister inoculative technique can be minimized by spreading the melanocytes over dermabraded skin but this is associated with a risk of drying up and dislodgement of ANEM. These disadvantages of dermabraded skin can be reduced by putting proper occlusive bandage at the transplanted sites. The present study (TANEM) for repigmenting depigmented skin lesions was based on the method described by Gauther and Surleve-Bazielle. 
The aim of present study was to experiment a novel method of transplantation of ANEM by direct inoculation without production of blister. Furthermore, an attempt was also made to compare the effect with that of inoculation of ANEM in blister.
MATERIALS AND METHODS
This study was experimental study. The participants were selected by convenient sampling. A total of 50 patients of Vitiligo attending the outpatient department of Skin and V.D, M.L.B. Medical College, Jhansi, over a period of six months, were included in the study. Inclusion criteria were patient with stable vitiligo not responding to PUVA/PUVASOL therapy given for at least one year. Exclusion criteria were, age below 8 years, women with pregnancy or lactation and any concurrent illness. Informed consent from the patients and routine investigations were done before performing the procedure. Since the study duration was short, we had to limit the sample size for convenience.
After preparation of the part (the medial side of thigh) injection of xylocaine with 2% adrenaline (1/2, 00, 000) was given. The superficial punch grafts were, obtained with the help of disposable punches (3 mm or 4 mm) and collected in solution containing, 0. 25% trypsin, versene (EDTA), glucose in phosphate buffer saline (TVPG solution) and transported to the department of Microbiology where the tissue was incubated for 18-24 hrs at 40 C.
Three identical Vitiligo sites were selected test site (T), blister site B and control site (C). Equal number of ten sites on face, neck, trunk, upper limb and lower limb were selected for transplantation. At site (B) blister was produced by applying liquid nitrogen with cotton tipped applicator for 20-30 second. The patients were advised to avoid rupture of blister which usually appeared after 8-10 hours of application.
After 18-24 hours in incubation, the graft was taken out of TVPG solution and put in Petri dish containing EDTA (1/5, 000 in saline) for 15 min at 37[degrees]C in incubatory. The epidermis was separated from dermis with the help of tweezers and transferred in petri dish containing phosphate buffer solution (pH 7.4-7.8) the basal layer of epidermis was rubbed gently with the help of blunt side of surgical blade and solution was vigorously pipetted out to obtain epidermal suspension. After that the cellular debris was removed and suspension was collected in sterile capped bottle.
The recipient site was prepared. At the test site (T) ANEM suspension was given direct intralesionally with the help of insulin syringe. The injection was carefully given at a depth of 1 mm approximately at the dermal epidermal junction 0.1 ml of solution (ANEM) was inoculated to produce a wheal. Several such injections were given to cover the whole of the test side. At the selected control site (C) direct intralesional inoculation of PBS solution alone was given.
On the blister site (B) the blister fluid was partially aspirated with the help of insulin syringe and 0.5 ml of solution (ANEM) was inoculated in the blister and the patients were asked to lie supine for 15 minutes so that the solution could settle down. The transplanted site B was then dressed with Sofratulle.
Monthly follow up of the patient was carried out up to 3 months. During the follow up, the patients were given PUVA SOL therapy thrice weekly using 8-methoxy psoralen at the dose of 0.6 mg/kg weight/treatment day. The observations obtained were tabulated and statistical analysis was done.
To compare the results between test sites and blister sites Z test was applied using SPSS software. Chi square test was used for the distribution of positive pigmentation produced on various part of body according to sites.
Equal number of ten sites on face, neck, trunk, upper limb and lower limb were selected for transplantation of ANEM suspension with above mentioned procedures. Localized Vitiligo was the most common variety.
After one week, 30 tests sites (T) out of 50 (60%) showed only erythema. While on the blister site (B) only erythema was observed in 14 (28%), erythema plus crusting was observed in 20 (40%) and erythema crusting, and discharge was observed in 7 (14%) (Table-1).
After one month, 25 (50%) test sites and 35 (70%) blister sites showed pigmentation whiles no pigmentation was seen at control sites (Table-2). It was also revealed that the chances of positive responses at various sites of inoculation at both test and blister sites by this method was similar in case of face and neck region while response was better on blister site in case of trunk and limbs (~ 60-80%) as compared to test sites (~40-50%). After applying Chi-square test, [chi square] value obtained was 1.905 and p value was 0.7532 which was found to be insignificant. This shows that pigmentation produced after inoculation was distributed uniformly on various part of body and is associated with the procedures.
To compare the results between test sites and blister sites Z test was applied.
After applying Z test for comparison between Test site (T) and Blister site (B), [absolute value of (Z)] = 5.57773 and p value was <0.0001 which is highly significant i.e. the difference in the responses between the test and blister sites are significant and the response of the above method was more consistent on blister sites as compared to Test site (T) (Table 3).
Pigmentation produced on Test site (25) was excellent at 3 sites (6%), good at 7 (14%), satisfactory at 8 (16%) and poor at 7 sites (14%). The response of transplantation varied according to type of Vitiligo with better response in localized type (Table-4).
Direct intralesional inoculation of ANEM was planned to overcome shortcomings of blister technique such as it can be performed only at limited sites, may cause hypo pigmentation/ hyper pigmentation and hypertrophic scar.
On applying the z-test to compare the pigment production at the test and blister sites, the p-value observed was less than 0.0001, implicating that the difference in the responses between the test and blister sites are significant and the response of the procedure was more consistent on blister sites than test site. Absence of pigmentation on all control sites showed that the result was not due to injection only.
The aim was to inoculate the ANEM solution at dermoepidermal junction, less response on the test sites could be due to inappropriate depth of the injection. There was significant lower response on the lower limb (30%) by the intralesional inoculation (Test site) that could be due to difficulty in giving injection on the shin. In one patient with leucotrichia, the depigmented eyebrow was also repigmented supporting the hypothesis of retrograde migration of the melanocytes.
Outcome of transplant depended upon the type of Vitiligo and there was better response in cases of localized Vitiligo (73%) in comparison to generalized Vitiligo (27.78%). Thus, TANEM seems to be ideally suited for stable localized Vitiligo. Production of pigment by inoculation of ANEM does not seem to be influenced by the age, sex, site of the lesion, duration of disease and duration of PUVA therapy.
Follow up of most of the patients showed that the pigmentation produced was stable and the pigmentation was of similar colour as of adjoining normal skin. Repigmentation is likely to remain for a prolonged period, as was observed in this study.
Since the calculated sample size was too high and thereby not feasible to include in this limited period of study, we had to limit the sample size for convenience.
Although, this new technique of direct transplantation of melanocytes suspension into the Vitiligo skin is an experimental study, it seems to be an effective and simple method for treating recalcitrant Vitiligo patients. Though the response of the new method was less on test sites as compared to blister sites but unlike the blister technique, the patients can be treated on an outpatient basis by direct intralesional inoculation method and so the compliance could be better. The use of this technique requires special laboratory equipment. However, in comparison with cultured cell, tissue graft this is an inexpensive and less time-consuming procedure. However, further studies are required with more number of patients and with melanocytes count.
 Olsson MJ, Juhlin L. Repigmentation of vitiligo by transplantation of cultured autologous melanocytes. Acta Derm Venereol 1993;73(1):49-51.
 Gauthier Y, Surieve-Bazeille JE. Autologous grafting with noncultured melanocytes: a simplified method for treatment of depigmented lesions. J Am Acad Dermatol 1992;26(2 Pt 1):191-4.
 Lontz W, Olsson MJ, Moellmann G, et al. Pigment cell transplantation for treatment of vitiligo: a progress report. J Am Acad Dermatol 1994;30(4):591-7.
 Halaban R, Langdon R, Birchall N, et al. Basic fibroblast growth factor from human keratinocytes is a natural mitogen for melanocytes. J Cell Bioi 1988;107(4):1611-9.
 Kumar S, Mishra RK, Kumar V, et al. Grafting of autologous non-cultured melanocytes for the treatment of Vitiligo: a pilot study. Indian J Dermatol 2003;48(4):206-8.
Neeraj Srivastava (1), Mayank Singh (2), Sudhir Kumar (3)
(1) Associate Professor, Department of Skin and VD, MLB Medical College, Jhansi, U. P.
(2) Associate Professor, Department of Pathology, MLB Medical College, Jhansi, U. P.
(3) Professor, Department of Surgery, MLB Medical College, Jhansi, U. P.
'Financial or Other Competing Interest': None.
Submission 31-10-2017, Peer Review 01-02-2018, Acceptance 07-02-2018, Published 12-02-2018.
Corresponding Author: Dr. Mayank Singh, Type IV/2, MLB Medical College Campus, Jhansi-284128, U. P.
Table 1. Presence of erythema/crusting/discharge at 1 week after inoculation Procedure Total Erythema Erythema + Erythema + Sites Crusting Crusting + Discharge Test (T site) 50 30(60%) 0 0 Blister (B site) 50 14(28%) 20(40%) 7(14%) Control (C site) 50 0 0 0 Table 2. Distribution of positive pigmentation produced on various part of body according to sites Sites of No Positive Pigmentation Chi- P value & Inoculation Square Significance Test Blister Value Site(T) Site (B) Face 10 7 (70%) 6 (60) 1.905 0.7532 Neck 10 6 (60%) 7 (70%) df = 4 Insignificant Trunk 10 5 (50%) 8(80%) Upper 10 4 (40%) 6(60%) limb Lower 10 3 (30%) 8(80%) Limb Total 50 25 (50%) 35 (70%) Table 3. Comparison of test site (T) and blister site (B) Tests Mean [+ or -] Standard Number of Deviation Responses on Site of Inoculation Test 5 [+ or -] 1.58113 25 site (T) Blister 7 [+ or -] 1 35 Site (B) Tests [absolute P value value of (Z)] Test 5.57773 <0.0001, site (T) Highly Blister significant Site (B) Table 4. Extent of pigmentation on the test sites Location No. of Pigmentation Excellent Good Sites Produced Face 10 07 01 03 Neck 10 06 01 02 Trunk 10 05 00 02 Upper limb 10 04 01 00 Lower limb 10 03 00 00 Total 50 25 03 07 Location Satisfactory Poor No. Response Face 01 02 03 Neck 02 01 04 Trunk 01 02 05 Upper limb 02 01 06 Lower limb 02 01 07 Total 08 07 25
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|Title Annotation:||Original Research Article|
|Author:||Srivastava, Neeraj; Singh, Mayank; Kumar, Sudhir|
|Publication:||Journal of Evolution of Medical and Dental Sciences|
|Date:||Feb 12, 2018|
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