Comparative Study of Efficacy Of

SYNOPSIS

COMPARATIVE STUDY OF EFFICACY OF

EPIDERMAL MELANOCYTE TRANSFER

VERSUS

HAIR FOLLICULAR MELANOCYTE TRANSFER IN

STABLE VITILIGO

FOR REGISTRATION OF SUBJECT FOR DISSERTATION TO

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BENGALURU

IN PARTIAL FULFILMENT OF REGULATIONS

IN REGARD FOR THE AWARD OF

MASTERS DEGREE IN DERMATOLOGY, VENEREOLOGY

AND LEPROSY

IN RESPECT OF

DrD NAVYA

DEPARTMENT OF DERMATOLOGY

COMMAND HOSPITAL, AIR FORCE, BENGALURU 560007

RAJIV GANDHI UNIVERSITY OF HEALTH SCIENCES

KARNATAKA, BENGALURU

ANNEXURE II

PROFORMA FOR REGISTRATION OF SUBJECTS

FOR DISSERTATION

1 / Name of candidate and address ( in block letters) / DR D NAVYA
DEPARTMENT OF DERMATOLOGY COMMAND HOSPITAL (AIR FORCE) BENGALURU 560007
2 / Name of institution / COMMAND HOSPITAL, AIR FORCE
3 / Course of study and subject / MD (DERMATOLOGY, VENEREOLOGY AND LEPROSY)
4 / Date of admission to the course / JULY 2013
5 / Title of the topic / COMPARATIVE STUDY OF EFFICACY OF
EPIDERMAL MELANOCYTE TRANSFER VERSUS HAIR FOLLICULAR
MELANOCYTE TRANSFER IN
STABLE VITILIGO
6 / Brief resume of intended work
6.1 Need for study
6.2 Review of literature
6.3 Objectives of study / AS PER APPENDIX I
AS PER APPENDIX II
AS PER APPENDIX III
7 / Material and methods
7.1 Source of data
7.2 Method of collection of data
7.3 Does the study require any
investigation to be
conducted on patient if so,
please describe briefly
7.4 Has ethical clearance been
obtained from your
Institution in case of 7.3 / AS PER APPENDIX IV
AS PER APPENDIX IV
AS PER APPENDIX IV
YES, ATTACHED
8 / List of references / AS PER APPENDIX V
9 / Signature of the candidate
10 / Remarks of the guide / AS PER APPENDIX VI
11
/ 11.1 Name and designation of
guide ( in block letters)
11.2 Signature of guide
11.3 Head of the Department
11.4 Signature of HOD / LT COL (DR)AJAY CHOPRA
ASSOCIATEPROFESSOR
DEPARTMENT OF DERMATOLOGY, VENEREOLOGY AND LEPROSY
COL(Dr)K RADHAKRISHNAN ,MD
HEAD OF THE DEPARTMENT
DEPARTMENT OF DERMATOLOGY, VENEREOLOGY AND LEPROSY
12 / 12.1 Remarks of the Principal
12.2 Signature

APPENDIX I

6. Brief Resume of the intended work

6.1 Need for study:

Vitiligo is a specific,common, often heritable acquired disorder characterized by well circumscribed milky white cutaneous macules or patches devoid of identifiable melanocytes on otherwise healthy looking skin.

Vitiligo vulgaris is a condition that has immense socio-psychological impact in addition to its cosmetic disability.Surgical treatment is indicated in stable disease not responding to medical treatment. There are various surgical modalities available for vitiligo, whichare based on the idea of restoring melanocytes on the recipientsite. These can be tissue grafting such as suction blister epidermal grafting, thin and ultrathin split-thickness skin grafting, minigrafting (punch grafting) and follicular grafting,or cellular transplantation such as cultured pure melanocyte transplantation, cocultured melanocyte–keratinocyte suspension cell transplantation, cultured epidermis, and noncultured basal cell layer enriched epidermal cell suspension transplantation. Each of these techniques has some advantages and some disadvantages and attempts are ongoing todevelop a better technique.

The hair follicle is an important reservoir of melanocytes and their precursor cells.In the follicular melanin unit there is one melanocyte for every five keratinocytes in the hair bulb,which is much higher than in the epidermal melanin unit, which has one

melanocyte for every 36 keratinocytes. In comparison with epidermal melanocytes, anagen hair bulb melanogenic melanocytes are larger, more dendritic, with more extensive Golgi and rough endoplasmic reticulum, and produce larger melanosomes. Hair melanocytes have a remarkable syntheticcapacity and a relatively small number of melanocytes can potentially produce sufficient melanin to pigment up to 1.5 m

of hair shaft. There are fewer melanocyte stem cells in the epidermis in comparison with the hair follicle. All these properties make hair a more attractive source of melanocytes than epidermis for cell-based therapies in vitiligo.1

Traditionally, hair follicles for follicle grafting are obtainedby elliptical scalp biopsy, and then single hair follicles arecut and transplanted to the recipient site. Follicular unit extraction ( FUE )is a novel method for harvesting hair follicles to geta cell suspension of the ORS cells.This techniquehas the advantage of invisible or insignificant scarring.1

As there are few studies with a small sample size,this study would thus help in

comparingefficacy, benefits and side effects between hair follicular melanocyte transfer by FUE and epidermal melanocyte transfer by conventional epidermal shave biopsy.

APPENDIX II

6.2 Review of Literature

Vitiligo is a common skin disease with prevalence of 0.1 to 2% . Commonly begins inchildhood or young adulthood, with peak onset of 10 to 30 years, but it may occur atany age. All races are affected and both sexes are equally affected.2

Vitiligo presents as depigmented macules which may slowly enlarge with concurrent development of new lesions. Most common sites are periorificial, face, genitals, mucous membranes, extensor surfaces, hands, and feet. Association has been reported with autoimmune and endocrine diseases and rarely malignant melanoma.2

Stable cases of vitiligo are defined as no new lesions or increase in size of existing lesions in the past one year.3

In patients with stable vitiligo, lack of effective medical therapies has led to the development of surgical treatment options .Stability of the disease process is the most important parameter to achieve a successful outcome .Conventional surgical modalities are tissue grafting techniques such as split-thickness grafts, punch grafts and suction blister grafts,and recent advances which include autologous non-cultured epidermal cell suspensions and cultured melanocytesuspensions.The choice of surgical treatment depends on type of vitiligo, extent and site of lesions, availability of equipment and expertise.

Among the studies regarding autologous grafting with non cultured melanocytes, Gauthier et al concluded that this technique is an effective and simple method for treating patients with achromic areas lacking melanocytes4.

Mulekar S V et al has done a long term follow up study of 142 patients with vitiligo treated by autologous grafting with non cultured melanocytes and concluded that excellent repigmentation was obtained in 56% patients.5

Njoo et al has compared different surgical techniques in 63 patients and concluded that split thickness skin grafting and epidermal blister grafting are the most effective and safest techniques.6

Follicular unit transplant was used to repigment vitiligo patches in 1998.Ortonne et al.,

proposed that repigmentation of vitiligo was derived from melanocyte reservoir in hair follicles.7

Vanscheidt and Hunziker, in a small case series, have used a single cell suspension of ‘plucked’ hair follicles in the treatment of vitiligo.They found almost complete (>90%) repigmentation in three of five patients with vitiligo, around50% repigmentation in one patient and < 10% repigmentation in one patient.8

S Mohanty et al included 14 patients with vitiligo,stable for atleast 3 months in his study and concluded thatpreparation of outer root sheath (ORS) cell suspension is

technically less challenging than preparation of epidermalcell suspension and

extracted hair follicular ORS cell suspension transplantationis effective in repigmentation of vitiligo stable for a year or more.1

APPENDIX III

6.3 Objectives of the Study

To compare the efficacy, benefits and side effects between epidermal melanocyte transfer versus hairfollicle outer root sheath melanocyte transfer in stable vitiligo.

APPENDIX IV

7. Material and methods

7.1 Source of data

a)Cases: All cases of stable vitiligo attending the department of Dermatology, Command Hospital (Air Force) Bengaluru.

b) Duration of study: 18 months from Oct 2013 to Mar 2015

c) Sample size: Group A(epidermal melanocyte transfer) : 30 sites

Group B(Hair follicular melanocyte transfer) : 30 sites.Total 60 sites

7.2 Inclusion criteria:

(i) Age :12-60 years

(ii) Both sexes

(iii) Stable cases of vitiligo defined as no new lesions or increase

in size of existing lesions in the past one year

7.3 Exclusion criteria:

( i) Progressive vitiligo

(ii) Cases with vitiligo patches on scalp(for hair follicular melanocyte transfer)

(iii) Any chronic ailment/medication

(iv) On immunosuppressant therapy

(v) Keloidal tendencies,infection

(vi) On anticoagulants / bleeding tendencies

7.4 Method of collection of data

(a) Informed consent of the patients will be obtained before the procedure.

(b) Patients with single patch of stable vitiligowill be randomly allotted to each of the surgical modalities.

(I)EPIDERMAL MELANOCYTE TRANSFER

MATERIALS

Equipments: , Skin grafting knife Silvers, dermabrader (mechanical), incubator, centrifuge, petri dishes,pipette, jeweller’s forceps , spatula, centrifuge tubes, marking pen and dry collagen sheets.

Reagents: 0.25% trypsin-EDTA solution, trypsin inhibitor (soya protein), Dulbecco’s modified Eagle’s Medium (DMEM)/Nutrient mixture F-12. 1:1 v/v 15 mmol/l HEPES buffer system.

METHOD6

The method of non-cultured autologous epidermal cell suspension involves the following steps:

(i) Harvesting of split thickness skin graft

(ii) Separation of epidermis and dermis by trypsinisation followed by incubation

(iii) Neutralisation by addition of trypsin inhibitor

(iv) Transfer of epidermal portion into DMEM.

(v) Centrifugation to collect cell pellets.

(vi) Resuspension of pellets in DMEM.

(vii)Transfer of cell pellets suspended in DMEM on to dermabraded recipient area with a pipette and dressing thereafter with dry collagen dressings.

(II)HAIR FOLLICULAR MELANOCYTE TRANSFER

MATERIALS

Equipments:1mm-2 mm punches, dermabrader (mechanical), incubator, centrifuge, petri dishes, pipette, jeweller’s forceps ,hair follicle holding forceps,70 micron cell strainer, spatula, centrifuge tubes, and dry collagen sheets.

Reagents: 0.25% trypsin-EDTA solution, trypsin inhibitor (soya protein), Dulbecco’s modified Eagle’s Medium (DMEM)/Nutrient mixture F-12. 1:1 v/v 15 mmol/l HEPES buffer system.

METHOD1

Follicular unit extraction(FUE)

(i) Scalp hairs are trimmed to a length of approximately 2 mm

(ii) Field block anaesthesia is given with 2% lignocaine

(iii) 1 mm – 2 mm punches are used according to the requirement, rotated in the direction of the hair follicle until it reached the mid-dermis.

(iv) Then the follicular unit is pulled out gently using hair follicle holding forceps andcollected in DMEM

Preparation of cell suspension

(i)Extracted follicles are incubated with 0.25% trypsin at 37˚C for 90 min to separate outer root sheath cells

(ii) Add trypsin inhibitor to terminate the action of trypsin

(iii)Cell suspension is then filtered through 70 micron cell strainer

(iv)Finally, cell suspension is centrifuged for 5 min at 1000 r.p.m. to obtain

a cell pellet, which is resuspended in a small amount of DMEM

Transplantation of cell suspension

(i) The recipient vitiligo patch is anaesthetized first and then dermabraded superficially

(ii) The prepared hair follicle suspension is spread uniformly over the dermabraded area with the use of a pipette

(iii)The area is then covered with a collagen dressing, DMEM moistened gauze, tegaderm and thereafter immobilised for 24 hours.

Sixty stable sites will be operated in total.

FOLLOW UP

Patients will be followed up monthly for 6 months with only sun exposure and assessment will be done by objective and subjective parameters at every visit.

Subjective parameters

Poor/good/very good/excellent repigmentation

Objective parameters9

Percentage of area of repigmentation.

(<25%,25-50%,50-75%,>75%)

Time taken to achieve maximum repigmentation

Statistical analysis of the assessment will be done for comparing the extent and timetaken for maximum repigmentation. Comparison of benefits and complications also done. A sample size of 30 sites in each of the groups is adequate to carry out this study.

7.5 Evaluation

For the study, a proforma is prepared to record the relevant details of the patient.

7.6Does the study require any investigation to be conducted on Patients? If so, please describe briefly

TLC

DLC

PLATELETS

BLOOD SUGAR

SERUM ELECTROLYTES

LFT AND RFT

PT/INR

APPENDIX V

8. REFERENCES

1.Mohanty s,kumar a,dhawan j,sreenivas v,gupta s.Non cultured extracted hair

follicle outer root sheath cell suspension for transplantation in vitiligo.Br J Dermatol 2011;164:1241-6

2. Halder RM, Taliaferro SJ. Vitiligo. In: Wolff K, Goldsmith LA, Katz SI et al. editors. Fitzpatrick’s dermatology in general medicine. 7th ed. New York: McGraw Hill; 2008: 616-22.

3.Sahni and Prasad:predicting stability in vitiligo;J cutan aesthet surg 2013: vol6: 75-82

4.Gauthier Y, Surleve-Bazeille JE. Autologous grafting with non-cultured melanocytes: A simplified method for treatment of depigmented lesions. J Am Acad Dermatol 1992;26:191-4.

5.Mulekar SV et al. Long-term follow-up study of 142 patients with vitiligo vulgaris treated by autologous, non-cultured melanocyte-keratinocyte cell transplantation. Int J Dermatol 2005;44:841-5.

6.Njoo MD, Westerhof W, Bos JD et al. A systemic review of autologous transplantation methods in vitiligo. Arch Dermatol 1998; 134:1543–9

7.Chouhan K, Kumar A, Kanwar AJ.Body hair transplantation in vitiligo.J cutan

Aesthet surg 2013;6: 111-2

8. Vanscheidt W, Hunziker T. Repigmentation by outer-root-sheathderived

melanocytes: proof of concept in vitiligo and leucoderma.

Dermatology 2009; 218:342–3.

9. Olsson MJ,Juhlin L. Transplantation of melanocytes in vitiligo. Br J Dermatol 1995;132:587-9

APPENDIX VI

9.REMARKS OF THE GUIDE

The topic of comparative study of efficacy between epidermal melanocyte transfer versus hair follicular melanocyte transfer in stable vitiligois bound to bring out some interesting facts on the efficacy, side effects, and technical aspects in study group. It also helps students in learning novel surgical modalities of treatment for vitiligo.

Date: (Ajay chopra)

Lt Col

AssociateProfessor

Department of Dermatology

Command Hospital (Air Force)

Bengaluru- 560007

Study Information Sheet for Patients

Title: Comparative study of efficacy of epidermal melanocyte transfer versus hair follicular melanocyte transfer in stable vitiligo

Investigators:

Principal worker –Dr D Navya

Guide -Lt Col Ajay chopra

Purpose Of The Study

The purpose of this study will be to evaluate the efficacy of repigmentation by either of the surgical techniques in management of stable vitiligo.

Procedure

For stable vitiligo ,surgical procedures are the treatmentofchoice.Both tissue grafts

and cellular grafts are well established methods of treatment and do not involve any

undue risk orexperimentation .Both the procedures are done under local anesthesia

using plain 2% lignocaine. The volume of local anaesthetic will not exceed 40

ml.Sensitivity testing will be done prior to administration of local anaesthesia.

Steps of epidermal melanocyte transfer

(i) Harvesting of split thickness skin graft from donor area (thigh) under local anaesthesia

(ii) Preparation of cell suspension

(iii)Transplantation of suspension on dermabraded recipient area under local anaesthesia

(iv)Both donor area and recipient area are covered with dressing along with immobilization of recipient area for 24 hrs.

Steps of hair follicular melanocyte transfer

(i)Harvesting of individual follicular units from scalp with 1or 2 mm punch under local anasethesia after which no suturing is required

(ii) Preparation of cell suspension

(iii)Transplantation of suspension on dermabraded recipient area under local anaesthesia

(iv) Both donor area and recipient area are covered with dressing along with

immobilization of recipient area for 24 hrs

Potential risk and discomfort

No undue potential and discomfort. The procedure is done under local anesthesia. Any patients with hypersensitivity to local anesthetic will be excluded. Patients with comorbid conditions and on chronic medications are excluded. Repigmentation is expected to start in 1 month which completes in 6-12 months in 90% cases.

Confidentiality

All information that patients provide during the study will be used for study purpose and will not be communicated to others.

Contacts

If you have any further questions at any time during the course of the study you feel that you need additional information about any procedure, you can contact the following:

DrD Navya Lt Col(Dr) Ajay chopra

Resident AssociateProfessor

Department of Dermatology Department of Dermatology

Command Hospital(AF) Command Hospital(AF)

Bangalore-07 Bangalore-07

Consent Form

Title: Comparative study of efficacy of epidermal melanocyte transfer versus hair follicular melanocyte transfer in stable vitiligo

______(Patient Particulars) has been fully informed of the nature and purpose of this study. Details of the procedures involved have been explained. All queries raised by the patient have been answered to the best of my ability. A signed copy of this form will be made available to the patient.

Investigator’s Signature:

Date:

I have been fully informed of the above noted study with its possible benefits, risks and consequences in the language that I understand. I hereby agree to participate in this investigation. I furthermore recognize the fact that I am free to withdraw this consent and to discontinue my participation in this project at any time without prejudice to my care. I further consent to this data being used for research and /or publication provided confidentiality is maintained.

Signature:-

Name :-

Date :-

PROFORMA

Name: Age: Sex:

Relation: Unit:

Address:

Occupation:

Presenting Complaints:

History of Present Illness:

Age of onset

Site of onset

Skin lesions:depigmented macule duration:

Areas involved

Period of stability

Past History:

Hypertension/Diabetes mellitus/Ischaemic heart disease

Treatment History:

Personal History: Veg / Non-veg Smoking: Yes / No Duration

Alcohol: Yes / No Duration

Bowel habits: Bladder habits:

Any Other:

Family History: Vitiligo: Yes / No

If yes, whether first degree relative is affected: Yes / No

Hypertension: Yes / No

Diabetes: Yes / NoAsthma: Yes / No

Any Other:

General Examination:

HeightWeightBMI

T P/R R/R BP

Pallor Icterus Cyanosis

Clubbing Pedal oedema Lymphadenopathy

Systemic Examination:

CVS

Resp System

Abdomen

CNS

Dermatological Examination

(a)Lesion:

Site
Number
Symmetry
Size
Shape
Margin
Pattern
Colour /

(b) Palms and Soles:

(d) Genitalia:

(e) Hair: Any associated leucotrichia/alopecia

(f) Nails:

Investigations:

TLC

DLC

Platelets

Blood sugar ( Fasting, PP)

LFT

BUN

Serum Creatinine

PT/INR

DIAGNOSIS

VITILIGO:Fresh Case / Known Case

Any associated dermatological conditions:

Type:

Number of lesions:

Sub-types:

ACTIVITY OF THE DISEASE:

Stable

TREATMENT

1.EPIDERMAL MELANOCYTE TRANSFER(Group A)