Extensive Vitiligo Treated with Melanocyte Cell TransplantVitiligo Before SurgeryVitiligo Surgery : Mega Sessions Vitiligo is an acquired pigmentary disorder that is characterizedby white patches, corresponding to substantial loss of functioning epidermal orsometimes hair follicle melanocytes. The worldwide average incidence isestimated to be 1%. Clinically it can be classified as localized, segmental, generalizedor universal. The courseof the disease id unpredictable it can stay stable for years and then suddenlyturn active. The etio- pathogenesis ofvitiligo is not yet fully understood, but the autoimmune hypothesis is the mostcommonly accepted one, based on which, many treatment modalities have beendescribed.1.Theface, neck, trunk respond favourably to medical treatments including topicalcorticosteroids2, topical immunomodulators like calcineurininhibitors3, Vitamin D derivatives4, Phototherapy ( PUVA,NB UVB, PUVA sol)5.Forthe patients having extensive vitiligo which is spreading actively thetreatments reported to be effective are systemic steroids6 which areusually given in a pulsed manner i.e. Oral mini pulse therapy, steroid sparingagents like Azathioprine7, methotrexate are being used morefrequently to outweigh the long term side effects of steroidsByand large vitiligo patches bearing pigmented hair respond well to medicines andphototherapy, the repigmentation induced being marginal and perifolliclular. However,vitiligo patches associated with leukotrichia or vitiligo patches on the acralareas i.e. lip tip variety including dorsum of hand, foot, finger tips, toes, knees,elbows tend to respond poorly to medical treatment and the only treatmentoption is surgical. Surgery can be undertaken once the disease is stable for atleast one year.Thebasic principle of surgical therapy is to replenish the depleted melanocytereservoir by transplanting autologous melanocytes from a pigmented area to the depigmented skin.The various surgicaloptions available include various tissue graft techniques and cellular graftstechniques.Tissue grafts techniquesinclude minipunch grafting, thin split thickness grafting, suction blistergrafting and hair follicle grafts.Cellulargrafting techniques include the cultured and the non cultured cell suspensiontechnique.Non culturedepidermal cell suspension techniques have revolutionized the surgicalmanagement of vitiligo. The technique is relatively simple and the results interms of colour and texture matching are excellent. NCECS can be done onvirtually any site including the traditionally considered difficult to operate sites e.g. eyelids, lips, neck, mobile areas as the elbows, knees, acral areas including fingers , toes,dorsum of hand, foot etc. All sites of the body except the palms and soles canbe easily managed with this technique. Donor recipient ratioThe biggestadvantage of NCES lies in the fact that with a small skin skin graft from thedonor area, a large recipient vitiligo area can be treated. The commonest donorrecipient ratio being used is 1: 10 i.e. if we harvest a skin graft of 3cm X 3cm i.e. 9 cm2 we can almost treat 90 cm2 of vitiligo area.Now this ratio isobviously so important because a smaller graft implies minimum scar over the donorarea which will fade way over a period of time Punch grafting (tissuegraft) is the easiest and least expensive method, but it is not suitable forlarge lesions and seldom produces even repigmentation.8 in minipunch grafting the donor recipient ratio is 1: 3 i.e. a punch canonly spread the pigment three times its size. Mini punch grafting can create cobblestoning both at the donor and recipient ratio, depigmented junctional zone andscarring.9 In suction blistergrafting the donor recipient ratio is 1:1 and although the cosmetic results aregood there is limitation in raising blisters hence most of the times blistergrafting is suitable only in small areas10. Ultrathin epidermal sheetgrafting can treat larger areas (up to 200 cm2) but requires skilland experience.8 Ultrathin split thicknessgrafting involves harvesting of large sheets of skin; the donor recipient ratiois 1:1 or at times even 2: 1 because ifthe graft harvested is relatively thick it has to be discarded as only thethinnest graft would give a better cosmetic result. Hence if we were to graft90 cm2 we would need a skin graft measuring anywhere from 90 to 180cm2 which is a large area that will have a risk of scarring.11 anddisfigurement at the donor site. The graft on the recipient site can have astuck appearance and in may cases hyperpigmentation, perigraft halo , miliaformation is encountered. The overall cosmetic result leaves a lot to bedesired. Hence the NCECS isthe clear choice for vitiligo surgery Operating large areas A patient of stablevitiligo may have a large area to be operated and currently there are no definiteguidelines as to how much maximum area can be operated in a single sessionMost melanocyte transplantation techniques areperformed under local anaesthesia in an outpatient facility. However,transplantation for extensive areas will require general anaesthesia. Allmethods require strict sterile conditions .If a case is being done under local anaesthesia lignocaine (2%)with or without adrenaline is generally used. Maximum dosage that can be usedis 4.5mg/kg (up to 300 mg) and with epinephrine 7 mg/kg (up to 500mg) can beused that implies that in safe limits only a small area can be operated at onetime. Regional blocks can be used but they again require lot of training andexpertise to be really effective.Theuse of GA makes the process more simpler as the issues of pain relief over thedonor and the recipient areas is taken care of and the surgeon as well as thepatient, both are equally comfortable. The setting is thus conducive for takingup larger areas for NCES in single session.Itis imperative to have a complete OT set up which is equipped with Anaesthesiaequipment, cardiac monitors, life saving equipments , the staff should betrained to handle emergencies. An anaesthetist has to be present throughout theprocedure till the patient is completely out of anaesthesia; admissionfacilities are required for short stay to monitor the patient post operativelyfor 6-8 hrs.Preoperative counselling and Informed consent12 The entire process starts with detailed history of the patientpertaining not only to vitiligo but general health and associated diseases. Thepatient needs to be properly counselled about the procedure and the pre andpost op care A detailed consent formelaborating the procedure and possible complications should be signed by thepatient. The patient is informed of the nature of the disease. The consent formshould specifically state the limitations of the procedure, and the possibilitythat the NCECS is replacement of functional melanocytes into the vitiligo areawhich is devoid of melanocytes and the disease can become unstable again in thefuture and there can be a loss of pigment on other sites as well as operatedsites and if the disease shows future progression additional medical line oftreatment and or procedures will be needed for proper results. The patient isalso counselled that he / she may require additional medical treatments and orphototherapy post op to stimulate pigmentation on the operated patches and thatit can take a few months to a year for significant repigmentation and colourmatchPreoperative laboratory studies include complete haemogram including platelet counts,bleeding and clotting time (or prothrombin and activated partial thromboplastintime),Thyroid profile, LFT , KFT, serum electrolytes , Chest X ray PA view, ECGin adults . Screening for antibodies for hepatitis B, C and HIV is recommended.Anaesthesia The type of anaesthesia used would be decided on a case to casebasis. Most cases can be managed with just IV sedation with drugs combinationlike ketamine, midazolam, propofol can be used. There is no need for intubationin most of the cases and the cocktails of anaesthesia drugs used by anexperienced anaesthetist can make the whole process go smooth.Theduration of anaesthesia required is usually very short as it is required onlyat the time of harvesting of skin graft (2- 5 mins) and during dermabrasiontime that varies from 20-30 mins. The skin grafts are trypsinized immediatelywhile the dermabrasion is carried out. The cell separation process andapplication of the NCES on the derma braded sites , dressings etc requires nofurther anaesthesia and patient need not be under anaesthesia at that point oftime. It’s important to have a good motivated team in which person understandstheir responsibilities so that the entire process can be completed in a shortyet unhurried manner Procedure Preparationof the NCECS13,14A skin graft is obtained by shave biopsywhich is then incubated at 37 degrees for 45 mins. After 45 minutes the graftis washed in DMEM media to neutralize the action of tryspin, the dermis isteased away and discarded and the rest of the epidermis is suspended inapproximately 5ml of DMEM medium and vortex mixed.This mixture is then centrifuged for 6mins at 3000 rpm centrifugation leads to the formation of a cellular pelletwhich is rich in keratinocytes and melanocytes. The pellet is resuspended inDMEM media to make a volume that can range from 0.1 ml to 1.5 ml depending onthe area to be covered. Methyl cellulose is added in addition to make thesuspension more viscous and avoids runaways on application. Recipientsite preparation The recipient areas are marked out, surgicallyprepared with povidine iodine, spirit draped and then epidermis is removed eitherwith conventional dermabrasion using diamond burs or a CO2 laser. The level ofdermabrasion is papillary dermis which is indicated by the pin point bleeding. Afterresurfacing, it’s important to attain haemostasis with the use of saline soakedgauze which should be irrigated at frequent intervals with Normal saline to keep the denuded area moistTransferof NCECS The suspension of keratinocytes andmelanocytes is then transplanted to the scarified skin in the recipient areaand the area is covered with dry collagen dressings to hold the cell suspension.This is further covered with paraffin dressings, gauze, pad micropore, bandages. Patient is kept stillfor approximately 30 mins to 1 hour so that the cell suspension can settle downafter which the patient can be shifted to the post op care / room.Postoperative care: Proper postoperative immobilization and care are very importantto obtain satisfactory results. Most patients require only day care admissionand can be discharged same evening Post surgery patient are advised to take a 5day course of antibiotics. Analgesicsare usually required for 2 days after which they can be given on need basis FollowupThe patients are asked to review after 7days for change of dressing. Precautions to follow during dressing change arethat the dressings (donor & recipient) should be soaked with normal salinefor approximately 30 mins before there removal as this facilitates a painlessremoval and ensures the cells applied are not sheared off . All layers ofdressing except the collagen should be removed. The collagen normally gets digestedby day 7 and if is persisting it should be left alone it sheds on its own infew days RepigmentationThe onset of repigmentation is usually seenafter 3- 4weeks and it can take anywhere between 1 to 3 months to attain moresignificant pigmentationSpecialprecautions while doing large area (> 200 cm2)Donorarea calculationPreoperativelyall recipient areas are marked and photographed and calculation of the areathat needs to be operated as shown in the image so that the donor area can becalculated accordingly which 1/10 of the recipient area is roughly. Wehave coined the following terminology while doing NCECS over large areasVitiligoareas upto 150 cm2 are considered as regular NCECS sessionVitiligoarea from 150 cm2 to 400 cm2 are labelled as mega sessionsVitiligoareas 400 cm2 and above are labelled as gega sessionsIfit’s a confluent single area, we can use donor recipient ratio 1: 10. However, if a patient has multiple patcheswhich are spread over multiple sites we go for donor recipient ratio 1: 5 or 1:8 Recipient area preparationWhiledoing large areas, dermabrasion with diamond burs and with micro motor ispreferred since its very fast and large areas (200-500 cm2) can be done in just20-30 mins When large areas are being done there can beat times some skip zones, these can be because of inadequate level ofdermabrasion as the blood can cloud the operating field. Hence it’s importantto watch for this closely and it isadvisable that after the entire area has been dermabraded adequate haemostasisis achieved with saline soaked gauzes and after 10 mins a relook is taken andthe areas not showing pinpoint bleed are further dermabraded to create an evensurface. This is important to avoid skip zones in the repigmentation. While doing margins its important to go 5mm into the normal skinthis reduces the incidence of perigraft achromic fissures Pain management Adequate care of thepost op pain since is essential since large amount of skin has been dermabraded,patients would require a stronger analgesic. In most of our patients we havefound an IM or IV Diclofenac 2 ml i.e. 80mg injection is adequate to keep thepatient comfortable. Few patients would need an additional shot of pentazocine,phenargan Fluid and electrolyte management NCECS is a surgerywith little blood loss because the level of dermabrasion is only upto thepapillary dermis. There is not much bleeding and even in extensive areas i.e.400cm2 the amount of blood loss is less than 20 ml. Also, since asmall donor area is required and the grafts are ultra thin there is not much ofa blood loss from the donor site as well. This can be well compared to theblood loss if extensive sheets of skin are harvested as in split thicknessgrafting or in punch grafting where since the punches are upto the mid dermisthere is considerable more bleed at both the donor and recipient areas. Even though thereis no major blood loss it’s important to keep the patient well hydrated with IVfluids since most GA patients need to be Nil per Oral (NPO) for almost 8 hourspreop and 6 hours post OP and also sincea large area is operated there can be oozing post op. Cultured vs. non cultured Culture methodsprovide an unlimited number of cells for transplantation; while NCEC wouldprovide up to 5–10 times donor-to-recipient expansion however culture methodsrequire much more extensive labs, equipments and trained personnel and hencethe cost of procedure goes up make it unaffordable for most patients. Alsothere are concerns regarding contamination, bacterial/ fungal overgrowths andthe use of agents which might be mutagenic in the futureResults Thepercentage and extent of repigmentation of the mega and gega session is similarto that of regular sessions The onset of pigmentation colour and texturematching is no different in these large area surgeries, even the retention ofpigmentation and relapse rates were similar to the surgeries of limited areaWedid not observe any koebnerization being induced i.e. the disease becomingunstable immediately after the procedure . Hence it makes sense to cover largerareas per session so that patient’s patches can be covered up significantly andthey can get over the psychological stress factors associated with this disease.ConclusionTaking upmega or gega sessions of NCECS are safe and effective Doing large areas at a gois the only way a lot a patients having extensive vitiligo can be managed Itrequires planning and dedicated team ofdermatologists, anaesthetist , OT nursing staff technicians to make theprocedure safe and comfortable for the patient .