combination of follicular and epidermal cell suspension as

1

PLAN OF THESIS 1

2

TRANSPLANTATION OF AUTOLOGOUS NONCULTURED EXTRACTED HAIR 3

FOLLICLE OUTER ROOT SHEATH CELL AND AUTOLOGUS NONCULTURED 4

EPIDERMAL CELL SUSPENSION IN COMBINATION AS A NOVEL METHOD IN 5

VITILIGO SURGERY 6

7

8 SUBMITTED IN PARTIAL FULFILLMENT OF THE DEGREE 9

10

OF 11

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MD (DERMATOLOGY, VENEREOLOGY AND LEPROLOGY) OF THE 13 POST GRADUATE INSTITUTE OF MEDICAL EDUCATION AND RESEARCH 14

CHANDIGARH 15

16

BY 17

18

19

DR.MUHAMMED RAZMI T 20

21 JUNIOR RESIDENT 22

DEPARTMENT OF DERMATOLOGY, VENEREOLOGY AND LEPROLOGY 23

PGIMER, CHANDIGARH 24

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GUIDE: 29

30

DR. DAVINDER PARSAD 31 32

PROFESSOR 33

DEPARTMENT OF DERMATOLOGY, VENEROLOGY AND LEPROLOGY 34


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CO-GUIDE: 39

40

DR SENDHIL KUMARAN M 41 42

ASSISTANT PROFESSOR 43

DEPARTMENT OF DERMATOLOGY, VENEROLOGY AND LEPROLOGY 44


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Downloaded From: https://jamanetwork.com/ on 11/19/2021

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SUMMARY OF PROPOSED RESEARCH 49

50

Vitiligo is a complex disease that causes a selective, often progressive, loss of functioning 51

melanocytes from basal layer of epidermis, leaving white patches on the skin and occasionally 52

mucosae.1 Worldwide prevalence of vitiligo is around 1% whereas in India it is around 3-4%, 53

ranging from 0.46% to 8.8%.2 54

Neural crest derived melanocytes are located mainly in the basal layer of the epidermis 55

and in the matrix of hair follicles.3 Melanocytes synthesize melanin pigment, transfer mature 56

melanosomes to basal keratinocytes and are responsible for skin color.4 Etiopathogenesis of 57

vitiligo is multifactorial and polygenic consisting of genetic, immunological and 58

environmental factors. Environmental and genetic factors act in concert to destroy 59

melanocytes. 60

The key clinical finding in vitiligo is the acquired onset of an increasing number of 61

initially hypopigmented and then depigmented macules, patches and later even wide spread 62

involvement of skin. It is often associated with leucotrichia. Vitiligo is divided into three 63

types: localized, generalized, and universal. Localized vitiligo is further sub typed into focal, 64

segmental and mucosal. Generalized vitiligo may be acrofacial, vulgaris or mixed. Universal 65

vitiligo involves more than 80% of the skin. Generalized vitiligo is the most common type, 66

and vulgaris is the most common subtype. The sites of predilection for vitiligo vulgaris are the 67

fingers and wrists, axillae and groin, and body orifices, such as the mouth, eyes, and genitals.5 68

Though the condition is non-contagious and asymptomatic, it has significant psychosocial 69

implications and can lead to an exaggerated sense of humiliation, loss of self-esteem and job 70

discrimination among patients.6 71

Various modalities of treatment (both medical and surgical) have been described for 72

vitiligo. Although medical treatment is the mainstay of treatment, it is not effective in all and 73

residual lesions require surgical treatment. Amongst the surgical options, replenishing 74

melanocytes selectively within vitiliginous macules by autologous melanocyte 75

transplantation, in the form of either tissue graft or cellular graft, is a promising treatment. 76

Surgical method of vitiligo treatment doesn’t alter the natural course of the disease and 77

treatment is mainly symptomatic. Transplantation of autologous noncultured extracted hair 78

follicle outer root sheath cell and autologous noncultured epidermal cell suspension in 79

combination, hereafter denoted as FCS + NCES (a mode of cellular grafting technique) is a 80

novel surgical method for the treatment of vitiligo. 81


3

This pilot study is planned to introduce FCS + NCES as a novel method in vitiligo surgery 82

and compare its outcome with NCES in the same stable vitiligo patient with regard to extent 83

of repigmentation, color matching of repigmented area, patient satisfaction and any adverse 84

events if any. There are studies comparing effectiveness of FCS or NCES separately in 85

vitiligo surgery giving comparable results. But, this is the first study using FCS + NCES as a 86

new modality in vitiligo surgery. 87

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REVIEW OF LITERATURE 117

118

Vitiligo, the most common depigmenting disorder7 is an ‘idiopathic’, acquired pigmentary 119

disorder caused by the loss of functional melanocytes from the basal layer of epidermis. The 120

term is coined from the Latin word ‘vitelius’ that means calf,8 where the characteristic lesions 121

of skin resemble the milky white macules of spotted calf. The disease runs an unpredictable 122

course but is often progressive with phases of stabilized depigmentation.9 It usually begins 123

during childhood or young adulthood. In approximately 50% of all cases, vitiligo appears 124

before the age of 20 years, and 70–80% of patients develop the disease by the age of 30 125

years.10

A large series on childhood vitiligo conducted in India reported a prevalence of 126

2.4%.11

The presence of vitiligo on exposed areas of body leads to social embarrassment, 127

psychological turmoil, and cosmetic disfigurement in those affected.12

128

Vitiligo is known since times immemorial. The oldest information on vitiligo comes from 129

the period of Aushooryan (2200 BC), in the classical Tarikh-e-Tib-e-Iran.9 The disease is 130

mentioned as ‘Shweta kushtha’9 in the ancient Indian sacred book ‘Atharva Veda’(1400 BC). 131

Its prevalence is 1%, ranging from 0.1 to > 8.8% in different countries of the globe. The 132

highest incidence of the condition has been recorded in inhabitants of the Indian subcontinent. 133

The reported difference in incidence between different communities may be due to a higher 134

reporting of vitiligo in certain populations, where an apparent color contrast and stigma 135

attached to the condition may force the patients to seek early medical advice. Both sexes are 136

equally affected although the greater number of reports among females is probably due to the 137

greater cosmetic concern and social consequences to women affected by this condition. 138

CLASSIFICATION

139

Classification of vitiligo according to the distribution of lesions1 140

1. Localized 141

a) Focal 142

b) Segmental 143

c) Mucosal 144

2. Generalized 145

a) Vulgaris 146

b) Acrofacial 147

c) Mixed 148

3. Universal 149


6

Classification of vitiligo with emphasis on segmental vitiligo 150

1. Segmental (unilateral) 151

2. Non-segmental (bilateral) 152

Localized 153

a) Focal 154

b) Mucosal 155

Generalized 156

a) Vulgaris 157

b) Acrofacial 158

c) Universal 159

3. Mixed: segmental and nonsegmental 160

161

There are two main clinical presentations of vitiligo: unilateral (segmental, asymmetric) 162

and bilateral (nonsegmental, symmetric). 163

Clinical features of segmental vitiligo 164

Segmental vitiligo, a subtype of vitiligo, is characterized by its early onset, rapid 165

stabilization and unilateral distribution. The reported prevalence of segmental vitiligo ranges 166

from 3.5% to 20.5% of all patients with vitiligo. Associated autoimmune diseases in patients 167

with segmental vitiligo and their family members are reported less frequently than in 168

generalized vitiligo. Face is the most common site of segmental vitiligo regardless of the 169

gender of the patient. The trunk, neck, extremities, and scalp are involved in descending 170

order. So far, several hypotheses for segmental vitiligo have been put forward, including (i) 171

neuronal mechanisms, (ii) somatic mosaicism and (iii) microvascular skin homing, whether or 172

not leading to an autoimmune destruction of melanocytes.13

Most of the patterns of segmental 173

vitiligo did not follow a dermatomal distribution. High rates of repigmentation with surgical 174

techniques are frequently achieved. 175

Clinical characteristics of non-segmental (bilateral) vitiligo 176

Bilateral vitiligo is a slowly developing condition, with a tendency to progress throughout 177

life. Arrest of the condition may occur14

in a small percentage of these individuals. Focal 178

vitiligo exhibits one or few macules in one area, most commonly in the distribution of 179

trigerminal nerve, although neck and trunk are also commonly involved. Focal vitiligo is a 180

starting point leading to other types of vitiligo. Mucosal vitiligo affects mucosae of the mouth 181


7

and genitalia. Acrofacial vitiligo encompasses depigmentation of the distal parts of the 182

extremities (hands rather than feet) and facial orifices, the latter in a circumferential pattern. 183

Lip-tip vitiligo is a variety in which tips of fingers, toes, nipples, penis and lips become 184

depigmented. Vitiligo vulgaris is composed of several scattered macules and is the most 185

common form of the disease. Depigmented patches are widely and usually symmetrically 186

distributed. Universal vitiligo implies loss of pigment over the entire body surface area and 187

complete or nearly complete depigmentation can be noted. 188

More important is to differentiate between active and stable vitiligo for the initiation of 189

appropriate therapy. Active vitiligo usually requires medical therapy. Surgical therapy is 190

indicated when medical therapy fails and could actually be considered as the first therapeutic 191

option for the treatment of stable vitiligo.15

Active vitiligo is characterized by increase in size 192

of old lesions, development of newer lesions, and appearance of white macules after trauma 193

(Koebnerisation). 194

In patients affected by segmental vitiligo, the causative factor(s) usually disappears, 195

leaving well-defined depigmented lesions. Even generalized vitiligo can enter long phases of 196

clinical remission in which the size and number of lesions are stationary for several years and 197

the Koebner phenomenon is absent. This stage of the disease is referred to as stable vitiligo.16

198

Sometimes vitiligo is a slowly spreading disease or is limited to a specific anatomic region, 199

and on other occasions it becomes aggressive dermatosis developing in a relatively short 200

period of time. Fortunately, most patients have a slow and prolonged course over several 201

years, but progression is the rule, especially with vitiligo vulgaris.17

202

Various hypotheses, not mutually exclusive, have been proposed for pathogenesis of 203

vitiligo. Of these, the most accepted theories include genetic, autoimmune, neurogenic, and 204

the melanocyte self-destruction hypothesis.18

So far, no convincing model describing the 205

interplay of these contributing factors has been formulated. A multi-factorial etiology has 206

been proposed based on existing research. 207

ETIOLOGY 208

Though vitiligo is an ancient disease, the exact etiology still eludes us. There appears to be 209

a combination of genetic predisposition and a number of potentially precipitating factors. 210

Heritability 211

Vitiligo is a heritable condition, upto 30% of the patients have a positive family history and 212

20% have an affected first degree relative. The pattern of inheritance points to a polygenic 213


8

trait with the involvement of 3 or more diallelic alleles.19

HLA studies have shown an 214

aggregation of HLA-DR4 in blacks and HLA-B13 in Moroccan Jews.9 HLA-B12 has also 215

been shown to be associated with vitiligo.18

Recently HLA-A2 has been correlated with 216

vitiligo.20

It has been suggested that the genetic background of these patients may render them 217

more susceptible to melanocyte damage and hence to vitiligo. 218

Precipitating factors 219

Patients frequently attribute the onset of vitiligo to a specific life event such as an accident, 220

crisis, physical or emotional stress. It may follow a cut or abrasion due to Koebner 221

phenomenon. 222

PATHOGENESIS 223

The importance of the disease makes the understanding of the pathophysiological, 224

biological and molecular events leading to melanocyte death or dysfunction, crucial to the 225

outcome. Multiple theories have been postulated to explain the appearance of vitiligo patches. 226

The proposed causative factors are not mutually exclusive. 227

Autoimmune hypothesis 228

It is the most popular hypothesis. Vitiligo is considered as an autoimmune disease due to 229

the following features: presence of auto-antibodies directed against melanocytes and related 230

structures in patient’s sera,21

the associations of vitiligo with other autoimmune conditions, 231

the detection of organ specific antibodies in patient’s sera, the detection of auto-antibodies in 232

first degree relatives of subjects with vitiligo and the association of the disease with HLA –233

DR4, HLA-DR1. 234

In vitiligo, there is production of auto antibodies against melanocyte antigens. These anti 235

melanocyte antibodies have different target antigens on the surface of melanocytes. The titer 236

of these antibodies correlates with the activity and extent of the disease.22

Recently anti-237

tyrosinase antibodies were also detected. Baharav et al22

demonstrated these antibodies to be 238

more in extensive widespread vitiligo than in localized vitiligo. Whether these antibodies 239

represent a primary event or are secondary to the release of antigens from previously damaged 240

melanocytes is not yet known. In a recent study conducted in Mumbai, antibodies were 241

detected against tyrosinase, tyrosine hydroxylase, thyroid peroxidase, thyroglobulin and 242

keratinocytes at frequencies of 11%, 22%, 18%, 24% and 27% respectively. Overall, 243

antibodies were more common in patients with nonsegmental vitiligo (50–67%) than in those 244

with segmental disease (0–17%), and were detected more frequently in patients with shorter 245


9

disease durations (<10 years).23

Patients with vitiligo have been reported to have a reduced 246

number of lymphocytes and helper T-cells and an increased number of natural killer cells in 247

the serum whereas in inflammatory vitiligo there is an increase in T – cell infiltration, 248

predominantly CD-8 + T-cells at the periphery of the lesions. A statistically significant 249

decrease in Helper T –cells/Suppressor T-cells ratio was obtained in the study as compared to 250

controls. 251

Neural hypothesis 252

Proposed by Lerner24

about 40 years ago, this theory states that there is liberation of a 253

neurochemical mediator that is toxic to the melanocytes from nearby nerve endings. Support 254

for this hypothesis comes from a number of observations namely: vitiligo in neurologically 255

compromised skin, vitiligo sparing paralyzed limbs, onset of vitiligo following peripheral 256

nerve injury and vitiligo limited to a single dermatome, though strictly not following a 257

particular nerve course.25

The neural hypothesis is based on the presence of segmental vitiligo. 258

An ultrastructural study of normal dermal nerves was performed recently. Subtle 259

ultrastructural differences were observed between biopsies taken from marginal and central 260

parts of vitiliginous skin and non vitiliginous skin. The most consistent feature seen in all the 261

biopsies from vitiliginous skin was an increase in thickness of basement membrane of 262

Schwann cells. This change was seen in approximately ¾ of dermal nerves in vitiligo biopsies 263

and ¼ of dermal nerves of normal control biopsies. About half of the dermal nerves showed 264

minor axonal damage, whereas indicators of regeneration predominated in others. In addition, 265

relation between the nervous stem cell and epidermal melanocyte has recently been 266

provided.26

Abnormalities reflecting possible nerve mediated aberrations in beta-endorphins 267

and met-encephalin secretion in vitiligo patients and increased immunoreactivity to 268

neuropeptide Y and vasoactive intestinal polypeptide in vitiligo skin have been reported. 269

Although little is known about the effects of neuropeptides on human melanocytes, the 270

nervous system may exert a tonic effect on melanocytes in normal or diseased human skin, 271

especially through calcitonin related peptide secretion26

, yet the role of the nervous system in 272

the pathogenesis is yet to be elucidated. 273

Biochemical support for this hypothesis arises from the observation that acetylcholine has 274

been shown to have an inhibitory effect on DOPA oxidase activity in marginal melanocytes in 275

vitiligo and acetylcholinesterase activity has been shown to be absent in depigmenting skin 276

and present in repigmenting skin.14

277


10

Self destruct hypothesis27

or autocytotoxic hypothesis 278

This postulates that an intermediate metabolite of melanin synthesis, particularly quinine is 279

toxic to melanocytes. Melanin repigmentation is produced in the melanocytes through the 280

tyrosinase activity. Tyrosinase gene family consists of tyrosinase enzyme, TRP-1&2, 281

Calnexin and LAMP-1. It has been postulated that mutation in TRP-1 protein is involved in 282

cell degeneration or death, associated with faulty scavenging of intermediates of melanin 283

pigmentation leading to apoptosis of melanocytes. 284

Compartmentalization of melanosomes normally protects melanocytes from destruction by 285

such substances. However, it is thought that leaky melanosomes or high quinine : melanin 286

ratio could damage the pigment cell. The free radical scavenging function of melanin may be 287

insufficient to prevent damage by highly toxic quinine.19

288

Biochemical theory 289

It has also been shown that both lesional and non-lesional epidermis has decreased catalase 290

activity, that leads to an increase in peroxidase concentration in it. Hydrogen peroxide 291

functions as a reversible inhibitor of human tyrosinase. 292

Role of Liver X receptor (LXR) expression in vitiligo 293

LXR regulate a variety of cellular functions, they have robust anti-inflammatory activity in 294

skin, but they also modulate epidermal proliferation, carcinogenesis, differentiation and 295

permeability barrier function. Kumar et al28

demonstrated in their study that expression of 296

LXR-α/ b at both mRNA and protein level was significantly higher in perilesional skin as 297

compared to the normal skin of vitiligo patient. 298

The new hypotheses: 299

The melanocyte growth factor deficient theory – Defective growth and passage capacity of 300

melanocytes derived from uninvolved and perilesional skin could be due to decreased 301

concentration of melanocyte growth factors in vitro.29

302

Decreased melanocyte lifespan hypothesis -- Several cytokines, such as interleukin-1 and 303

interferon-gamma, mainly produced and released by keratinocytes, may induce apoptosis of 304

melanocytes due to deficiency in survival signals by interfering with the melanocyte 305

membrane tyrosine kinase receptor, C-KIT.30

Reduced levels of C-KIT receptors in vitiligo 306

melanocytes or of growth factors could induce premature apoptosis and decreased melanocyte 307

survival. 308

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11

New Integrated Theory of Non Segmental Vitiligo (NSV):- A Melanocytorrhagic disorder31

-310

The new integrated theory takes into account melanocyte detachment and transepidermal 311

elimination, neural-biochemical and autoimmune hypotheses. This new theory proposes that 312

NSV is a primary melanocytorrhagic disorder with altered melanocyte responses to friction 313

and possibly other types of stress, inducing their indolent detachment and subsequent 314

transepidermal loss. Further it was shown that melanocytorrhagy and apoptosis is seen only in 315

patches of unstable vitiligo.32

316

1. Melanocyte defective adhesion: - The melanocyte adhesion system is less well 317

organized and far weaker than the system which firmly holds epidermal keratinocytes 318

bound to each other and to the basement membrane. No melanocyte – keratinocyte 319

adhesion structures can be detected by electron microscopy. 320

2. Loss of dendricity: Dendrites are critically important for melanosome transfer, 321

because one melanocyte contacts several keratinocytes in the epidermis through 322

dendritic cell processes. Moreover, ultrastructural observations suggest that dendrites, 323

independently of structural junctions, may dramatically increase the adhesion and 324

anchoring of melanocytes within the basal layer of the epidermis. It has been 325

suggested that the loss of dendricity induced either by oxyradicals (impaired redox 326

status hypothesis) or by increased release of catecholamines (neural biochemical 327

hypothesis) exaggerates transepidermal loss induced by minor mechanical trauma. 328

This loss of dendricity could also affect melanosome transfer and contribute to 329

depigmentation. 330

3. Weakening of melanocyte attachment and melanocyte detachment after friction: 331

Ultrastructural abnormalities of the basement membrane have been observed 332

frequently in vitiligo, namely multiple replication or layering of the basement 333

membrane directly beneath melanocytes and focal gaps in the basement membrane. As 334

a result of the weakening of their basal anchoring, melanocytes could be detached by 335

mechanical or chemical injury. Human skin is repeatedly exposed to mechanical 336

stimuli usually grouped under the term friction. During friction, an alternation of 337

stretching and relaxation sometimes results in epidermal disruption, degeneration of 338

keratinocytes and widening of intercellular spaces. Extracellular granular material 339

deposits are found ultrastructurally in NSV skin after severe frictional injury of normal 340

skin. Altered synthesis of extracellular matrix components (such as tenascin) may be 341

produced by damaged keratinocytes. 342


12

4. Transepidermal elimination of melanocytes: After their detachment, melanocytes 343

are seen in a mid-spinous location as early as 8 hours after friction. Twenty-four hours 344

later, some melanocytes reach the stratum corneum. 345

The ultimate consequence of all the pathogenic mechanisms described above is 346

melanocyte destruction, and therefore the final outcome is absence of pigmentation. Initially 347

only epidermal melanocytes are affected, but as the condition progresses, the most important 348

pigment cell reservoir, the hair follicle, may also become involved and leucotrichia develops, 349

thus making repigmentation difficult. How and to what extent this phenomenon occurs is 350

dependent on the individual response of the affected patient and the aggressiveness of the 351

pathogenic process. 352

HISTOPATHOLOGY 353

There is a marked absence of melanocytes and melanin in the epidermis. Histochemical 354

studies show a lack of dopapositive melanocytes in the basal layer of the epidermis . Recent 355

immunohistochemical studies with a large panel of antibodies show only an occasional 356

melanocyte in lesional skin.33

Electron microscopy studies confirm the loss of melanocytes, 357

which appear to be replaced by Langerhans’ cells. Areas around the margins of vitiligo show 358

abnormalities of keratinocytes as well as degenerating melanocytes. In inflammatory vitiligo, 359

where there is a raised erythematous border, there is an infiltrate of lymphocytes and 360

histiocytes. This infiltrate is also found in the marginal areas of some biopsies.34

361

Mechanisms of repigmentation in vitiligo 362

Some vitiligo patients show spontaneous repigmentation even though all have a permanent 363

melanocyte loss. Spontaneous repigmentation of the vitiligo patches is a regular feature when 364

exposed to sun. After therapy, repigmentation can occur in four ways: follicular, marginal, 365

diffuse and combined. Repigmentation usually occurs in the follicular pattern, suggesting that 366

follicular melanocytes colonize vitiliginous skin. In most patients of repigmenting vitiligo, 367

studies also argue for a proliferation of melanocytes, followed by their migration; however 368

less commonly, repigmentation might occur from residual intraepidermal melanocytes. Based 369

on follicular repigmentation, the existence of a melanocyte reservoir has been postulated. The 370

existence of a population of intraepithelial cells that have immunopathological characteristics 371

of mature melanocytes within the upper epidermal region has been shown.35

These KIT (+), 372

BCL-2(+), TRP-1(-) cells may contribute to the precursor melanocyte reservoir of human 373

skin. 374


13

During repigmentation, melanocytes migrate from the outer root sheath of the hair follicle 375

to the basal layer of the epidermis just above the basement membrane. Because keratinocytes 376

are attached to each other by desmosomes and to the basement membrane by 377

hemidesmosomes, migration of melanocytes involves several complex processes that are not 378

yet understood. 379

Treatment options 380

A number of therapeutic options for vitiligo are available but there is still a need for a 381

treatment that is promptly effective. There is no curative treatment for this condition. 382

Management of vitiligo is a real challenge for a dermatologist. 383

Medical therapies: 384

Corticosteroids (Topical, intralesional and systemic)36

, Oral mini pulse37

, PUVA (topical 385

and systemic)38

, NBUVB39

, calcipotriol40

and tacrolimus41

are used most widely. Some of the 386

less commonly used medical modalities include phenylalanine41

, khellin41

, topical 387

minoxidil42

, levamisole43

and melagenina.44

Recently oral minocycline was shown to be 388

effective in treating vitiligo.45

389

Most of these therapies aim to restore melanocyte function by their anti-inflammatory or 390

immunomodulatory action and by preventing melanocyte auto destruction so that normal skin 391

appearance and function is restored. 392

Surgical therapies: 393

All patients with vitiligo should be initially treated with medical methods. Surgical 394

methods are important solutions for stable vitiligo refractory to medical treatment. High 395

repigmentation rates are obtained with all procedures so far described in most anatomic 396

locations, but they are of little help for acral areas and bony prominences. Unilateral vitiligo is 397

the clinical form with the best response to grafting and transplant methods, and a good 398

proportion of patients with stable bilateral disease also respond adequately. Nevertheless, 399

appropriate patient selection is important to achieve maximal results.14

However none of the 400

surgical modalities developed so far is uniformly effective in all patients and body sites and 401

there is need for constant research and innovations for better surgical therapeutic options for 402

vitiligo. 403

404

Aims of various surgical procedures:46

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A) Camouflage Tattooing: Introduction of artificial pigments into the lesions for 406

permanent camouflage. 407

B) Excision: Removal of the depigmented areas, e.g. excision with primary closure and 408

covering with thin Thiersch's graft. 409

C) Melanocyte transplantation:29, 47

Commonly used methods of autologous transplant of 410

melanocytes are 411

Tissue grafts: 412

1. Thin and ultra-thin split thickness grafts (STSG) 413

2. Suction blister epidermal grafts(SBEG) 414

3. Mini punch grafts (MPG) 415

4. Hair follicular grafts (HFG) 416

Cellular grafts: 417

5. Noncultured epidermal cell suspension (NCES) 418

6. Cultured “pure” melanocytes (CM) 419

7. Cultured epithelial grafts (CE) 420

8. Autologous noncultured extracted hair follicle outer root sheath cell suspension also 421

called follicular cell suspension (FCS) 422

D) Therapeutically wounding the lesion to stimulate the melanocytes from the periphery 423

and the black hair follicles to proliferate, migrate and re-pigment the lesion, e.g. 424

therapeutic dermabrasion, laser ablation, cryosurgery (liquid nitrogen spraying), 425

needling and local application of phenol or trichloroacetic acid.47

426

Every method has its own advantages and disadvantages. As there are no specific data 427

available from the prospective studies in this field, it is not easy to recommend which surgical 428

approach to vitiligo offers the best result. 429

430

Several points need to be assessed in patients when surgical treatment is planned.14

431

Stable Disease:48

432

The most important factors indicating stability are 433

(1) No progression of lesions for at least 1 year. 434

(2) Spontaneous repigmentation. 435

(3) A positive minigrafting test disclosing repigmentation around four to five minigrafts 436

(1.0 or 1.2 mm), implanted 3 to 4 mm apart within an achromic lesion, is an indication 437


15

of future recovery by surgery. So far, this test is the most accurate evidence of vitiligo 438

stability. 439

(4) Absence of new koebner phenomenon (KP), including the donor site for the 440

minigrafting test.14

441

(5) Unilateral vitiligo is almost a synonym of stable disease with an excellent 442

repigmentation response. 443

However, these criteria may be challenged by clinical observations in which KP and 444

minigraft testing are discordant. Data obtained from minigraft testing in case series suggest 445

that the minigraft test provides a reflection of the stability of defined individual lesions, which 446

does not necessarily reflect global stability of the disease.49

447

‘Vitiligo global issues consensus conference, 2011’ convened by Vitiligo European Task 448

Force (VETF), concluded that assessment of ‘overall’ stability is inaccurate and unreliable, 449

whereas individual lesion stability is more reliable, especially when used in the context of 450

surgical intervention.50

451

Methods and Size of Lesions: 452

Depending on the size of the treated area, the method may vary. Simple methods such as 453

minigrafting and suction epidermal grafting are useful for small or medium sized lesions. On 454

the contrary, for extensive depigmented defects, cellular transplants may be required. 455

Age: 456

Because of the invasive nature of surgical procedures, they are not recommended in 457

children; nevertheless, highly motivated preadolescents can be treated under sedation or 458

general anesthesia. Also, it is not surprising to see patients beyond the age of 50 years who 459

may be interested in surgical repigmentation. 460

Psychological Aspects: 461

Some patients with high emotional trauma because of depigmentation may seek advice for 462

invasive procedures. However, a psychological evaluation may be needed to ascertain the real 463

need for surgical treatment. 464

Photographic Record: 465

Illustrations are recommended to help in determining the percentage of improvement, 466

quality of repigmentation and possible side effects. 467

468

469


16

Patient's Expectations: 470

Repigmentation is not always comparable with normally pigmented skin and the final 471

results vary considerably from patient to patient. However, most individuals are pleased with 472

the achieved results; minor imperfections are far less important than the noticeable 473

repigmentation of vitiliginous skin, mainly in ethnic skin patients with a dark complexion; 474

sometimes surgical repigmentation may look even better than what is observed in many 475

patients after medical therapy. 476

Method and Donor Site: 477

Appropriate training with a specific method is an important prerequisite for surgical 478

therapy. Donor site should be as hidden as possible and the gluteal region may be suitable for 479

this purpose in most patients. 480

Serial Procedures: 481

Most procedures require more than one intervention and several sessions may be needed 482

to accomplish full recovery or to complete repigmentation of minor depigmented defects. 483

Combination methods may be of value for this purpose. 484

Difficult Areas for Surgical Treatment: 485

With surgical procedures much improvement is achieved, particularly in unilateral vitiligo, 486

but certain areas are difficult to repigment, such as joints, lips, eyelids, genitalia, cutaneous 487

folds, the dorsum of hands and feet, especially fingers and toes. In some of these areas, 488

inadequate immobilization prevents a good take of grafts and repigmentation is difficult to 489

achieve; some of these areas may need regrafting, and recovery is possible in some patients. 490

Nevertheless, other factors not known at present may prevent a good repigmentation response. 491

Success rates of different surgical options: 29

492

Among all procedures, suction blister epidermal grafts and thin and ultra-thin split-493

thickness grafts seem to be the most effective procedures, with overall success rates of 80.3% 494

(CI 76.4–84.2%) and 77.9% (CI 72.2–83.6%) respectively. But, a recent randomized study 495

directly comparing NCES and SBEG showed NCES is significantly better than SBEG.51

496

Among cellular grafts, all techniques seem to be equally effective with success rates of 61.1% 497

(CI 56.1–66.1%), 63.6% (CI 57.2–70%), and 63.6% (CI 55.8–70.6%) for noncultured 498

epidermal cell suspension, cultured melanocytes and cultured epidermis respectively. The 499

mean repigmentation with noncultured extracted outer root sheath cell suspension is about 500

65.7%. 501


17

Cases with more extensive vitiligo vulgaris, involving greater than 30% body surface area, 502

are generally considered unsuitable for transplantation procedures as chances of retention of 503

the pigment are less. Extensive areas may be best treated with cellular grafts. Theoretically, 504

culture methods would provide an unlimited number of cells/tissue for transplantation, while 505

NCES would provide up to 8–10 times donor-to-recipient expansion. Therefore, it seems that 506

larger areas may be treated with cellular grafts and thin and ultra-thin split-thickness grafts 507

and moderate areas may be treated with cellular grafts and minigrafts. Smaller areas may be 508

easily treated with suction blister epidermal grafts which gives good aesthetic results and is 509

technically less challenging. Overall, better results are reported in focal and segmental vitiligo 510

(75%-95%) than in generalized vitiligo. Young, dark complexioned patients have better 511

results. Comparatively, acral areas, malleoli, knees, and elbows are less responsive to surgery. 512

Smaller patches respond better. Addition of PUVA/PUVASOL therapy enhances 513

repigmentation and increases the repigmentation rate (90-95%). 514

Adverse events:29

515

No serious adverse events have been reported with any of the transplantation methods. 516

Cellular grafts appear to have the least frequency of adverse events. Adverse events reported 517

at recipient sites are infection, milia, scarring, rejection of the graft. Complication at donor 518

sites reported are infection, milia, scarring and pigmentary changes. Commonly recipient site 519

is prone to secondary bacterial infections if asepsis is not followed. This can be minimized by 520

following strict asepsis in procedure. If dermabrasion or skin harvesting is too deep it will 521

lead to milia ( keratin filled cysts ), scarring and pigmentary changes. Rejection of graft 522

(failure to repigment after surgery ) is seen if surgery is done on unstable vitiligo patches or 523

resistant sites. Cultured melanocytes and NCES have a mean of 0.0 and 0.08 adverse events at 524

recipient site respectively, and 0.01 and 0.009 at the donor site respectively. Tissue grafts are 525

reported to be associated with more adverse effects and the maximum number of adverse 526

events on the recipient site is seen with MPG (0.7) and STSG (0.5). 527

Response:29

528

The treated area appears bright pink immediately after removal of the dressing. The earliest 529

pigmentation was noticed 3 weeks post surgery. Many patients showed hyperpigmentation 530

which gradually blended with the surrounding skin over 6–8 months. The donor area healed 531

rapidly and soon became indistinguishable from the surrounding skin. Occasionally, the donor 532

area healed with hyperpigmentation. 533


18

NONCULTURED EPIDERMAL CELL SUSPENSION 534

The technique of noncultured epidermal suspension was pioneered by Gauthier et al52

. The 535

suspension was prepared by incubating the donor skin obtained from the scalp in trypsin 536

0.25% for 18 hrs. The suspension was injected into blisters raised by cryotherapy. Eight out of 537

the 12 patients treated had > 70% repigmentation at the vitiligo site. It was proposed that the 538

presence of keratinocytes in the suspension supplies essential growth factors for melanocyte 539

growth. They stated that this technique could emerge as simple and effective alternative to the 540

costly cultured melanocyte transplantation technique.52

541

Olsson and Juhlin53

first used the M2 medium for suspension of the noncultured epidermal 542

cells. A total of 20 vitiligo patients were included and results showed 100% repigmentation in 543

all 3 patients with segmental vitiligo and 80 % repigmentation in 12 patients of stable 544

generalized vitiligo. The research group found the transplantation as effective as 545

transplantation of cultured melanocytes. However, several practical problems surfaced with 546

the procedure. Usage of cryotherapy to raise blisters damaged the melanocytes resulting in 547

hypopigmentation. Significant run-off of suspension from recipient site was associated with 548

the high fluidity of suspension. Blisters were difficult to raise at the bony prominences by the 549

use of cryotherapy.

550

Van Geel et al54

added hyaluronic acid to the cellular suspension to improve the viscosity 551

and fixation, CO2 laser was used to obtain a depth-controlled and precise dermabrasion at the 552

recipient site and adjuvant PUVA or UVB therapy was added 3 weeks after grafting to 553

stimulate and homogenize the repigmentation. First, a pilot study was conducted in 4 patients 554

all of whom achieved 80 % pigmentation. The therapeutic value of this procedure was 555

increased with the further evaluation in 28 patients in a double blind placebo controlled study. 556

70% or more repigmentation was observed in 55% of the patients. 557

It is concluded that the transplantation of noncultured epidermal cell suspension is an 558

efficacious and safe procedure. The technique requires special laboratory equipment. 559

However, in comparison to cultured melanocytes, this is an inexpensive and simple OPD 560

procedure requiring 4-6 hrs. Large areas, 8-10 fold the size of donor skin, can be treated with 561

this procedure. A temporary color mismatch is observed in all patients, which improved over 562

5-6 months. No scarring is observed at the donor or recipient site. They stressed on proper 563

aseptic precautions and the use of prophylactic antibiotics to prevent postoperative infections. 564

The use of post-operative UVB therapy helped to achieve uniform pigmentation. In one 565


19

patient it was observed that leucotrichia adjacent to depigmented eyebrow was also 566

repigmented supporting retrograde migration of pigment cells.54, 55

567

Transplantation of noncultured melanocytes is the method that results in least 568

hyperpigmentation. This is because the cells are diluted and are transplanted in smaller 569

numbers than in other methods. Halo phenomenon that noted in transplantation of cultured 570

melanocytes is noted even in grafting of noncultured epidermal cell suspension. The 571

technique shows excellent results in segmental vitiligo, focal stable vitiligo and piebaldism. 572

Age and gender seem to have no significant effect on repigmentation. Acral and periorificial 573

vitiligo has the poorest response.56

574

Mulekar et al57

conducted an extensive study recruiting 49 patients with segmental vitiligo 575

and 15 with focal vitiligo. They used Hams F-12 medium for suspension. 95-100% 576

repigmentation was observed in 41 patients of segmental vitiligo and 11 patients of focal 577

vitiligo. The percentage of patients with segmental vitiligo showing an excellent response was 578

84%, while 6% of the patients had a good response to treatment. 10%

of patients failed to 579

produce any pigmentation. In focal vitiligo 73% showed an excellent response, while 20% had 580

poor repigmentation at the end of the respective follow-up period. Response to the treatment 581

on the lips was not encouraging. Patients with both focal and segmental vitiligo had retained 582

the pigment at the end of respective follow-up periods (5 yrs). Repigmentation failed

to be 583

produced in 10% of patients in the segmental vitiligo group and 20% in the focal vitiligo 584

group. 585

Pandya et al58

abraded the recipient area with a high speed motor dermabrader and the 586

denuded area was covered with saline moistened gauze piece. The suspension was poured 587

evenly from the pipette and covered with a collagen dressing. This is covered with a small 588

gauze piece moistened with MK medium. The dressing was kept in place by a Tegaderm 589

dressing. 590

In Swedish procedure of melanocyte transplantation pioneered by Olsson and Juhlin,59

they 591

used phosphate buffered saline (PBS) to wash the denuded area and they put PBS moistened 592

gauze over denuded area. 593

NONCULTURED EXTRACTED HAIR FOLLICLE OUTER ROOT SHEATH CELL 594

SUSPENSION (FCS) 595

Hair follicle is an important reservoir of melanocytes and their precursor cells. 596

Melanocyte-lineage antigens plus c-Kit (the receptor for stem cell factor) stained cells are 597

localized in the outer layer of the outer root sheath of the infundibulum and mid-follicle and 598


20

the matrix of the hair bulb.60

This reservoir of melanocytes and melanocyte stem cells ARE 599

important in the treatment of vitiligo as the initial repigmentation in vitiligo patches often 600

occurs around the hair follicles and vitiligo patches on skin lacking hair follicles such as 601

palms and eyelids are often resistant to medical therapies. 602

There are other populations of cells which might constitute the hair follicular cell 603

suspension. These include basal cells high in α6-integrin/keratin 14 (K14) expression, 604

suprabasal cells low in α6-integrin/K14 expression, hair germ cells expressing Lgr5, P-605

cadherin and S100A4, bulge cells expressing CD34 and CD200 and a more distal population 606

expressing MTS24.61

The perifollicular connective tissue sheath and the papilla is a potential 607

source for mesenchymal stem cells in the cell suspension obtained from extracted hair 608

follicles.62

609

Vanscheidt et al,63

in a small case series used single cell suspension of ‘plucked’ hair 610

follicles in the treatment of vitiligo. They found almost complete (>90%) repigmentation in 3 611

of 5 patients with vitiligo, around 50% repigmentation in one patient and less than 10% 612

repigmentation in one patient. Their technique is simple, non-invasive and allows immediate 613

and repeated application. However the cell yield is less in case of plucked hair follicles and 614

optimization of cell harvest form the hair follicular unit needs to be standardized for optimum 615

yield. Cell suspension prepared from hair follicles obtained by FUE method contains more 616

CD200+ cells (a marker for hair follicle bulge stem cells) as compared to plucked hair.64

This 617

is further supported by the observation that transplantation of plucked hair doesn’t result in 618

hair growth, however transplantation of extracted follicular unit promptly accepted by the 619

recipient site with resulting hair growth.65, 66

620

The dye exclusion test is used to determine the number of viable cells present in a cell 621

suspension. It is based on the principle that live cells possess intact cell membranes that 622

exclude certain dyes such as trypan blue, eosin or propidium whereas dead cells do not. 623

Mohanty et al successfully used trypan blue dye exclusion method to show viability of 624

melanocytes in their study.67

625

Hair follicle is a rich source of three different types of stem cells and it appears that all of 626

them are important in hair growth. These stem cells include melanocyte stem cells, 627

keratinocyte stem cells and mesenchymal stem cells.68

Melanocyte stem cells in a melanocyte 628

reservoir, located in the upper "permanent" outer root sheath, have the capacity to migrate and 629

enter vacant niches in epidermis. This phenomenon might be responsible for perifollicular 630

pigmentation seen in vitiligo in response to phototherapy. It can be used to cover large 631

depigmented areas. 632


21

In an open labeled pilot study Mohanty et al performed FCS in fourteen patients of vitiligo 633

and achieved >75% pigmentation in 9 patients. The procedure involved removal of only 15-634

25 follicular units, which provide 25000 to 50000 cells sufficient to treat up to 25 cm2. From 635

the experience gained from NCES, the authors recommended the desired number of cells for 636

repigmentation as 2000 cells/cm2.67

There are no control studies that have determined the 637

precise number of melanocyte concentration required for achieving pigmentation. In follicular 638

melanin unit, there is one melanocyte for every five keratinocytes in the hair bulb,69

which is 639

much higher than epidermal melanin unit which has one melanocyte for every thirty-six 640

keratinocytes. In comparison to epidermal melanocytes, anagen hair bulb melanocytes are 641

larger, more dendritic, produce larger melanosomes and with more extensive golgi and rough 642

endoplasmic reticulum. Hair melanocytes have remarkable synthetic capacity so that a 643

relatively small number of melanocytes can potentially produce sufficient melanin to pigment 644

up to 1.5 m of hair shaft.61

Melanocyte stem cell has been recognized in the hair follicle but 645

not in the epidermis. Melanocyte stem cells are less in number in epidermis in comparison to 646

hair follicle. All these properties make hair a more attractive source of melanocytes than 647

epidermis for cell based therapies in vitiligo.65

Because of the above mentioned factors FCS 648

might require lesser melanocyte concentration compared to epidermal cell suspension. 649

650

651


22

Justification for the proposed study 652

FCS + NCES – added benefit of two successful methods 653

Same patient – no confounding factor 654

Similar anatomical area – no question of koebnerisation or other local factors 655

Both FCS and NCES are proved to be efficacious for vitiligo surgery in terms of extent of 656

pigmentation and colour matching. Recent randomized study comparing effects of FCS and 657

NCES in two different groups of patients showed result more favouring to NCES.70

Here, we 658

are doing NCES in one group of vitilgo patches and FCS + NCES in other group of patches in 659

the same patient. So we are treating the vitiligo patches with the modality with proven 660

efficacy in one area, and in other area we are combining two modalities which proved their 661

efficacy while used exclusively. 662

Theoretically one will expect more repigmentation with FCS, due to the presence of 663

various melanocyte stem cells, better melanocyte – keratinocyte ratio and morphological 664

properties of melanocytes in hair follicle, compared to NCES. But, in various studies there is 665

no statistically significant superior result with FCS, and there are even studies showing 666

inferior results with FCS than NCES.70

It was proposed that the presence of keratinocytes in 667

the suspension supplies essential growth factors for melanocyte growth.52

Melanocytic 668

homeostasis is modulated via a complex network of autocrine and paracrine factors. 669

Melanocyte proliferation, melanogenesis, migration, dendricity, and differentiation are 670

influenced by keratinocytes and fibroblasts, as well as the melanocyte-derived growth factors 671

and cytokines.71

Keratinocyte derived factors that help in melanogenesis include Endothelin-1 672

(ET-1)72

, Stem cell factor (SCF),73

also known as steel factor, Basic fibroblast growth factor 673

(bFGF)74

, Nerve growth factor (NGF)75

etc. So there may be keratinocyte growth factors 674

lacking if we use FCS alone, that is contributing to inferior outcome, which can be overcome 675

by combining NCES to FCS. 676

Also, there are studies showing keratinocyte damage in vitiligo. Bhawan J et al76

677

performed light and electron-microscopic studies on the amelanotic and adjacent normal-678

appearing skin in patients with vitiligo. The amelanotic skin revealed complete loss of 679

pigment and absence of melanocytes. In addition to severe degenerative changes of 680

melanocytes, varying degree of damage was also seen in the keratinocytes of normal-681

appearing skin adjacent to amelanotic skin. Kumar R et al77

proposed that oxidative stress 682

may also manifest as the ultrastructural and functional changes observed in keratinocytes 683


23

extracted from the perilesional and normal skin of NSV patients. There are notable 684

morphological alterations like swollen mitochondria with disrupted cristae, a pathognomonic 685

feature of apoptosis in keratinocytes. So, by combining NCES to FCS, we are replenishing 686

healthy keratinocytes to the vitiligo patches. 687

The drawback of many efficacy studies comparing various modalities in vitilgo surgery is, 688

they used different patient population for comparing. Every individual is different. Especially 689

in the case of vitiligo, there may be alteration in outcome based on patient characteristics such 690

as type of vitiligo, immunological profile, disease activity, family history, adherence to the 691

medical advice etc. Moreover, there may be difference in results due to the quality and 692

concentration of suspension used, care taken during surgery; if different modalities of vitiligo 693

surgery are done on different time. Here, we are performing both methods of surgery on the 694

same patient and at the same sitting, so that avoiding above mentioned confounding factors. 695

The success of vitiligo surgery also depends on the anatomical area of vitiligo patch. Acral 696

areas, joints, lips show poor response to any therapeutic method. In the literature, various 697

anatomical areas are selected randomly for comparing different surgical methods. We selected 698

vitiligo patches which are bilaterally symmetrical or different patches on the similar 699

anatomical sites for the comparison of outcome. This overcomes the issue of koebnerisation, 700

lack of quiescence after surgery at areas like joints and the innate resistance of acral areas78

to 701

vitiligo surgery. 702

703

704

705

706

707

708

709

710

711

712


24

713

714

715


25

AIMS AND OBJECTIVES 716

AIM: To compare the effect of transplantation of autologous noncultured extracted hair 717

follicle outer root sheath cell suspension and epidermal cell suspension in combination v/s 718

transplantation of autologous noncultured epidermal cell suspension alone in stable vitiligo in 719

the same patient using primary and secondary outcome parameters. 720

721

722

723


26

MATERIALS AND METHODS 724

Subjects will be recruited from the patients attending Pigmentary and Dermatosurgery 725

Clinic of Department of Dermatology, Venereology and Leprology; Postgraduate Institute of 726

Medical Education and Research, Chandigarh, India. A total of 30 subjects of stable vitiligo 727

(lesional stability defined as individual lesions not increasing in size for the last 1 year), who 728

are satisfying inclusion/exclusion criteria would be recruited. In one group of vitiligo patches, 729

(say, right side lesions if bilaterally symmetrical vitiligo or proximal/medial lesions if more 730

than one vitiligo patch in same anatomical region), FCS + NCES will be done and in other 731

group of vitiligo patches (say, left side lesions if bilaterally symmetrical vitiligo or 732

distal/lateral lesions if more than one vitiligo patch in same anatomical region) NCES alone 733

will be done. This will be exercised using random number table. Follow ups will be done at 734

day 8, week 4, week 8 and week 16. Patient assessment will be done by digital photographs in 735

the same settings with respect to patient positioning, background, lighting and camera 736

settings; and questionnaire to know extent of repigmentation and compare the efficacy of both 737

methods using primary and secondary outcome parameters. 738

739

Primary outcome : 740

Extent of repigmentation. 741

Secondary outcomes: 742

Pattern of repigmentation. 743

Extent of repigmentation and type of vitiligo 744

Extent of repigmentation and site of lesions 745

Color matching of repigmented area. 746

Patient satisfaction (patient global assessment). 747

Adverse events if any. 748

749

750

INCLUSION CRITERIA: 751

1. Subjects with clinical diagnosis of focal, segmental or generalized vitiligo which has been 752

stable for more than 1year. 753

2. Vitiligo patches should be in same anatomical region bilaterally or two or more patches in 754

the same anatomical region separated by a stretch of normal skin (at least 1cm) 755


27

756

757

EXCLUSION CRITERIA: 758

1) Age less than 10 years 759

2) Pregnancy 760

3) Patient with actively spreading vitiligo 761

4) Appearance of new lesions 762

5) History of Koebnerization 763

6) History of hypertrophic scars or keloidal tendency 764

7) Bleeding disorders 765

8) Patients with unrealistic expectation 766

767

At the first visit, a pro forma is filled noting the baseline characteristics, history and 768

examination findings. Informed consent is taken for the procedure. 769

770

Difficult and Simple types of vitiligo 771

Repigmentation in generalised vitiligo (VV) and acrofacial vitiligo (AFV) is variable and 772

often more disappointing while results in segmental (SV) and focal vitiligo (FV) is in general 773

consistently high. So we planned to assess the extent of repigmentation separately for difficult 774

type of vitiligo defined as VV and AFV and simple type of vitiligo defined as SV and FV. 775

776

Subcategorisation based on sites of surgery 777

Acral lesions as well as bony areas, joints and eyelids are inherently resistant to any method 778

of surgery while face, trunk and proximal limbs show good response. A separate analysis of 779

extent of repigmentation will be carried out for the lesions on resistant sites defined as fingers, 780

hands, feet, joints, bony areas and eye lids; also for those on easy sites defined as face, trunk, 781

arms and legs. 782

783

TECHNIQUE OF TRANSPLANTATION: 784

785

Noncultured Epidermal Cell Suspension Method 70

786

Harvesting the graft: 787


28

1. About one-tenth the size of recipient area will be selected as the donor site, usually on 788

non-cosmetically important site like the thighs. 789

2. Donor area will be shaved, cleaned with betadine and surgical spirit and anaesthetized 790

with mixture of 2% lignocaine and normal saline, NS (1:1). 791

3. Split thickness skin graft will then be taken with the help of a shaving blade held firmly by 792

a straight artery forceps. 793

4. Haemostasis is established and the area will be dressed with Bactigras gauze. 794

5. Suitable antibiotic and analgesic will be prescribed. 795

796

Preparing noncultured epidermal cell suspension: 797

1. Split thickness skin specimen will be transferred under aseptic conditions to a container 798

with NS and transferred to laboratory. There, the skin graft will be transferred to Trypsin-799

EDTA solution (0.25% trypsin and 0.02% EDTA) in a Petri dish and incubated at 37°C in 800

5% CO2 for one hour to separate the epidermis from the dermis. 801

2. Afterwards, the Trypsin-EDTA solution will be removed and PBS will be added and 802

pipetted well so as to separate the cells from the tissue. 803

3. The solid waste of tissue will be removed and the suspension will be centrifuged at 1000 804

rpm for 5 minutes. 805

4. The supernatant will then be discarded and the pellet, containing cells from the stratum 806

basale and lower half of the stratum spinosum that are rich in melanocytes will be taken. 807

5. The melanocytes will be stained with trypan blue and counted simultaneously with 808

Neubauer's chamber under the light microscope. This will help to identify whether the 809

melanocytes are viable as the dead cells would pick up the blue stain. 810

6. Phosphate buffer saline is added to make suspension of noncultured epidermal cells. 811

812

Transplantation procedure: 813

1. The recipient site will be shaved, cleaned with betadine and surgical spirit and 814

anaesthetized with mixture of 2% lignocaine and NS (1:1). 815

2. Dermabrasion will be done until tiny pinpoint bleeding spots are seen which imply that 816

the dermo-epidermal junction has been reached. Dermabrasion will be extended 5mm 817

beyond margins to prevent halo phenomenon. 818

3. The denuded area will be washed with PBS and covered with a PBS moistened gauze 819

piece. 820


29

4. The noncultured epidermal cell suspension will be carefully transferred to a tuberculin 821

syringe. 822

5. With 18g needle attached to this syringe, few small drops of suspension will be placed 823

over the denuded surface which will be then spread evenly with the help of needle. 824

6. This will be covered with sterile Vaseline gauze or Bactigras after washing with NS. Once 825

again small drops of suspension will be placed over this gauze and spread evenly. 826

7. After washing with NS a meshed collagen sheet (Kollagen M) will be put over the gauze 827

with suspension. 828

8. This will be then covered by a small gauze piece moistened with PBS. 829

9. Tegaderm will be placed over this so that an artificial blister will be formed which holds 830

melanocytes with PBS over the recipient site. At difficult areas like lips surgical glue will 831

be used to put Tegaderm in place. 832

10. Over this, surgical pad is put and the dressing will be stabilized by placing the elastic 833

plaster (Dynaplast). 834

The patient will be observed for 1 hour after procedure and then allowed to go home. The 835

dressing will be removed at the first follow-up visit after 5-7 days in the hospital. 836

Noncultured Extracted Hair Follicle Outer Root Sheath Cell Suspension and 837

Noncultured Epidermal Cell Suspension Combination67

838

Anagen hairs are extracted from occipital area of scalp. In pigmented populations, it is not 839

very difficult to recognize anagen hair clinically. An anagen hair is an active, growing hair. 840

From the surface, anagen hairs tend to be stronger in hair shaft tensile strength and more 841

pigmented, that is, these hairs have more melanin. Follicular unit extraction method (FUE) is 842

used for hair follicle tissue harvest. Samples are sent to the laboratory for processing within 843

15-20 min of harvesting of the tissue. 844

Follicular unit extraction (FUE): 845

1. Hairs are trimmed to a length of approximately 2 mm. 846

2. Field block anaesthesia is given with 2% lignocaine, which is infiltrated in the skin 847

encircling the area chosen for FUE. 848

3. To obtain follicular units, 1-mm punch is rotated till mid-dermis in the direction of hair 849

follicle. Care is taken not to go up to subcutaneous space to avoid transaction of the hair 850

follicle. 851


30

4. Then follicular unit is pulled out gently using hair follicle holding ring forceps by holding 852

the skin surrounding the hair shaft(s). 853

5. Transacted hair follicles are discarded. Depending on the area to be transplanted, 854

approximately 15-25 pigmented follicles are extracted per subject and collected in NS. 855

6. The procedure of FUE takes approximately 25-30 minutes. 856

Preparation of single cell suspension: 857

1. The extracted hair follicles are transported to the laboratory under sterile conditions and 858

washed three times with phosphate buffered saline containing the antibiotics and 859

antimycotics. 860

2. The follicles are then incubated with 0.25% trypsin-0.05% EDTA at 37 ºC for 90 minutes 861

to prepare the single cell suspension. 862

3. Cells started loosening up within 15-20 minutes. After every 30 minutes the hair follicles 863

are placed in a new tube of trypsin EDTA and the reaction in the previous tube is 864

terminated by adding the trypsin inhibitor (Sigma-Aldrich). 865

4. This is done to prevent digestion of separated cells by trypsin. After cell separation only 866

thin keratinous shafts of the hairs are left, which are discarded. 867

5. The cell suspensions of all the three tubes are added in a single tube and then filtered 868

through a 70μm cell strainer to prepare a single cell suspension. 869

Finally, the cell suspension is centrifuged for 5 minutes at 1000rpm to obtain a cell pellet, 870

which is re-suspended in a small amount of PBS and transported to the operation theatre 871

for transplantation, where NCES is added to FCS and gently mixed to prepare FCS + 872

NCES combination. 873

6. The whole procedure of preparation of cell suspension took approximately 2-3 hours. 874

Transplantation procedure: 875

1. The recipient site will be shaved, cleaned with betadine and surgical spirit and 876

anaesthetized with mixture of 2% lignocaine and NS (1:1). 877

2. Dermabrasion will be done until tiny pinpoint bleeding spots are seen, which imply that 878

the dermo-epidermal junction has been reached. Dermabrasion will be extended 5mm 879

beyond margins to prevent halo phenomenon. 880

3. The denuded area will be washed with PBS & covered with a PBS moistened gauze piece. 881

4. The FCS + NCES combination will be carefully transferred to a tuberculin syringe. 882


31

5. With 18g needle attached to this syringe, few small drops of suspension will be placed 883

over the denuded surface which will be then spread evenly with the help of needle. 884

6. This will be covered with sterile Vaseline gauze/Bactigras after washing with NS. Once 885

again small drops of suspension will be placed over this gauze and spread evenly. 886

7. After washing with NS a meshed collagen sheet (Kollagen M) will be put over the gauze 887

with suspension. 888

8. This will be then covered with a small gauze piece moistened with PBS. 889

9. Tegaderm will be placed over this so that an artificial blister will be formed which holds 890

melanocytes with PBS over the recipient site. At difficult areas like lips surgical glue will 891

be used to put Tegaderm in place. 892

10. Over this, surgical pad is put and the dressing will be stabilized by placing the elastic 893

plaster (Dynaplast). 894

The patient will be observed for 1 hour after procedure and then allowed to go home. The 895

dressing will be removed at the first follow-up visit after 5-7 days in the hospital. 896

897

FOLLOW UP: 898

The patients will be asked to follow up at the clinic on day 8, week 4, week 8 and week 16 899

after the transplantation procedure and percentage of repigmentation will be assessed by 900

blinded investigator (Dr. Davinder Parsad) subjectively by serial digital photographs in the 901

same settings with respect to patient positioning, background, lighting and camera settings 902

and objectively by serial paper markings. No intermittent treatment will be given during this 903

post-surgery period. 904

Repigmentation will be assessed as follows: 905

≤25% Minimal repigmentation 906

26-50% Mild repigmentation 907

51-75% Moderate repigmentation 908

76-90% Marked repigmentation 909

>90% Excellent repigmentation 910

Also, the repigmentation pattern will be noted as ‘diffuse’, ‘perifollicular’ or ‘migrating 911

from the borders’. A note will also be made of the colour matching of repigmented skin as 912

‘somewhat lighter than’, ‘same as’ or ‘somewhat darker than’ normal skin. 913


32

At each visit, patient will be assessed for any complications at the donor and recipient 914

sites. Patient will be asked to fill a patient satisfaction questionnaire at week 16. Hence, both 915

objective and subjective evaluation of the results shall be done. 916

917

918

919

920

921

922

923

924

925

926

927

928


33

STATISTICAL METHODS AND DATA ANALYSIS PROCEDURES 929

Sample size was estimated based on the previous study,51

assuming excellent (> 90%) 930

repigmentation in one group as 71% and in the other group as 27%. Our sample size came out 931

to be 31 lesions per group at a power of 95% and confidence interval of 95%. It was decided 932

to include extra ssubjects for the possible lost to follow up cases. Finally, we included 42 933

lesions per group for the proposed study. 934

The statistical analysis will be carried out using Statistical Package for Social Sciences (SPSS 935

Inc., Chicago, IL, version 16.0 for Windows). All quantitative variables will be expressed 936

using measures of central tendency (mean, median) and measures of dispersion (standard 937

deviation). Normality of data will be checked by Kolmogorov Smirnov test. For normally 938

distributed data, means will be compared using student's t-test for outcome. For skewed data 939

or scores Mann–Whitney test will be used. Qualitative or categorical variables will be 940

described as frequencies and proportions. For significance of changes within the group over a 941

period Wilcoxon signed-rank test will be used. Proportions will be compared between 942

groups using Chi square test or Fisher's exact test whichever will be applicable. All 943

statistical tests will be two-sided and performed at a significance level of p < 0.05. 944

945

946

947

948

949


34

ETHICAL JUSTIFICATION 950

This planned study is to be undertaken in stable vitiligo patients not responding to medical 951

treatment. Informed consent will be obtained from all patients and they will be explained that 952

surgical treatment is for the existing lesions of vitiligo and new lesions of vitiligo may still 953

appear in future. Patients will not be denied of medical treatment and only those who failed 954

medical treatment will be chosen for surgery. To detect any adverse effect at the earliest, 955

periodic visits of the patient along with active intervention are planned at regular intervals. All 956

necessary steps would be undertaken to ensure safety and convenience to the patients during 957

entire study period. Moreover, the patients who may deny participating would be excluded 958

from study without asking any reason thereof. 959

Nowadays surgical modalities have become treatment of choice for stable vitiligo not 960

responding to medical treatment. Both autologous noncultured epidermal cell suspension 961

(NCES) and autologous noncultured extracted hair follicle outer root sheath cell suspension 962

(FCS) have been shown to be safe and efficacious in the repigmentation of stable vitiligo 963

patches. So we hypothysise that FCS + NCES will result in better repigmentation in stable 964

patches of vitiligo and can be used in areas that show inferior result to above two methods like 965

acral areas. These surgical modalities are affordable to most of the patients and impart not 966

much financial burden. 967

By this study our aim is to establish FCS + NCES as a novel method in the treatment of 968

stable vitiligo using NCES as a control. This in the long run will be helpful to the patients in 969

terms of cost-effectiveness and ultimately the outcome. Thus, the cost and risk of undergoing 970

an invasive procedure by the patient can be ethically justified. 971

972

973

974

975

976

977

978

979

980

981


35

BIBLIOGRAPHY 982

1. Hercogova J, Schwartz RA, Lotti TM. Classification of vitiligo: a challenging endeavor. 983 Dermatol Ther. 2012;25 Suppl 1:S10-6. 984 2. Behl PN, Bhatia RK. 400 cases of vitiligo. A clinico-therapeutic analysis. Indian J Dermatol. 985 1972;17:51-6. 986 3. Sommer L. Generation of melanocytes from neural crest cells. Pigment Cell Melanoma Res. 987 2011;24:411-21. 988 4. Schallreuter KU. A review of recent advances on the regulation of pigmentation in the 989 human epidermis. Cell Mol Biol (Noisy-le-grand). 1999;45:943-9. 990 5. Alikhan A, Felsten LM, Daly M, Petronic-Rosic V. Vitiligo: a comprehensive overview Part I. 991 Introduction, epidemiology, quality of life, diagnosis, differential diagnosis, associations, 992 histopathology, etiology, and work-up. J Am Acad Dermatol. 2011;65:473-91. 993 6. Parsad D, Dogra S, Kanwar AJ. Quality of life in patients with vitiligo. Health Qual Life 994 Outcomes. 2003;1:58. 995 7. Taieb A, Picardo M. The definition and assessment of vitiligo: a consensus report of the 996 Vitiligo European Task Force. Pigment Cell Res. 2007;20:27-35. 997 8. Mosher DB, Fitzpatrick TB, Ortonne JP. Disorders of pigmentation in: Dermatology in General 998 Medicine. 6th ed. New York: Mc Graw Hill Inc; 2003. Vol 1 p. 839-847. 999 9. Sehgal VN, Srivastava G. Vitiligo: compendium of clinico-epidemiological features. Indian J 1000 Dermatol Venereol Leprol. 2007;73:149-56. 1001 10. Herane MI. Vitiligo and leukoderma in children. Clin Dermatol. 2003;21:283-95. 1002 11. Handa S, Dogra S. Epidemiology of childhood vitiligo: a study of 625 patients from north 1003 India. Pediatr Dermatol. 2003;20:207-10. 1004 12. Parsad D, Pandhi R, Dogra S, Kanwar AJ, Kumar B. Dermatology Life Quality Index score in 1005 vitiligo and its impact on the treatment outcome. Br J Dermatol. 2003;148:373-4. 1006 13. van Geel N, Mollet I, Brochez L, Dutre M, De Schepper S, Verhaeghe E, et al. New insights in 1007 segmental vitiligo: case report and review of theories. Br J Dermatol. 2012;166:240-6. 1008 14. Falabella R. Surgical approaches for stable vitiligo. Dermatol Surg. 2005;31:1277-84. 1009 15. Guerra L, Capurro S, Melchi F, Primavera G, Bondanza S, Cancedda R, et al. Treatment of 1010 "stable" vitiligo by Timedsurgery and transplantation of cultured epidermal autografts. Arch 1011 Dermatol. 2000;136:1380-9. 1012 16. Njoo MD, Das PK, Bos JD, Westerhof W. Association of the Kobner phenomenon with 1013 disease activity and therapeutic responsiveness in vitiligo vulgaris. Arch Dermatol. 1999;135:407-13. 1014 17. Falabella R. Surgical treatment of vitiligo: why, when and how. J Eur Acad Dermatol 1015 Venereol. 2003;17:518-20. 1016 18. Koranne RV, Sachdeva KG. Vitiligo. Int J Dermatol. 1988;27:676-81. 1017 19. Le Poole C, Boissy RE. Vitiligo. Semin Cutan Med Surg. 1997;16:3-14. 1018 20. Liu JB, Li M, Chen H, Zhong SQ, Yang S, Du WD, et al. Association of vitiligo with HLA-A2: a 1019 meta-analysis. J Eur Acad Dermatol Venereol. 2007;21:205-13. 1020 21. Moellman GE, Kross P, Halaban R. On the subject of serum antibodies to melanocytes. J 1021 Invest Dermatol 1985; 84: 333-4 1022 22. Baharav E, Merimski O, Shoenfeld Y, Zigelman R, Gilbrud B, Yecheskel G, et al. Tyrosinase as 1023 an autoantigen in patients with vitiligo. Clin Exp Immunol. 1996;105:84-8. 1024 23. Pradhan V, Patwardhan M, Thakkar V, Kharkar V, Khopkar U, Ghosh K, et al. Vitiligo patients 1025 from India (Mumbai) show differences in clinical, demographic and autoantibody profiles compared 1026 to patients in western countries. J Eur Acad Dermatol Venereol. 2011. 1027 24. Lerner AB. Vitiligo. J Invest Dermatol. 1959;32:285-310. 1028 25. Mosher DB FT, Ortonne JP. Disorders of pigmentation in: Dermatology in General Medicine. 1029 New York: Mc Graw Hill Inc. 2003;Vol 1:p. 839-47. 1030


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26. Hara M, Toyoda M, Yaar M, Bhawan J, Avila EM, Penner IR, et al. Innervation of melanocytes 1031 in human skin. J Exp Med. 1996;184:1385-95. 1032 27. Lerner AB. On the etiology of vitiligo and gray hair. Am J Med. 1971;51:141-7. 1033 28. Kumar R, Parsad D, Kaul D, Kanwar A. Liver X receptor expression in human melanocytes, 1034 does it have a role in the pathogenesis of vitiligo? Exp Dermatol. 2010;19:62-4. 1035 29. Gupta S OM, Kanwar AJ, Ortonne JP. Surgical management of vitiligo and other 1036 leucodermas: evidence based practice guidelines. Surgical Management of Vitiligo. 1st ed. 1037 Massachusettes: Blackwell Publishing Ltd 2007. p. 69-79. 1038 30. Kitamura R, Tsukamoto K, Harada K, Shimizu A, Shimada S, Kobayashi T, et al. Mechanisms 1039 underlying the dysfunction of melanocytes in vitiligo epidermis: role of SCF/KIT protein interactions 1040 and the downstream effector, MITF-M. J Pathol. 2004;202:463-75. 1041 31. Gauthier Y, Cario Andre M, Taieb A. A critical appraisal of vitiligo etiologic theories. Is 1042 melanocyte loss a melanocytorrhagy? Pigment Cell Res. 2003;16:322-32. 1043 32. Kumar R, Parsad D, Kanwar AJ. Role of apoptosis and melanocytorrhagy: a comparative 1044 study of melanocyte adhesion in stable and unstable vitiligo. Br J Dermatol. 2011;164:187-91. 1045 33. Le Poole IC, van den Wijngaard RM, Westerhof W, Dutrieux RP, Das PK. Presence or absence 1046 of melanocytes in vitiligo lesions: an immunohistochemical investigation. J Invest Dermatol. 1047 1993;100:816-22. 1048 34. Bleehen SS. Histology of vitiligo. In: Klaus SN ePC, Vol. 5. Basel: Karger, 1979: 54–61. 1049 35. Norris DA, Horikawa T, Morelli JG. Melanocyte destruction and repopulation in vitiligo. 1050 Pigment Cell Res. 1994;7:193-203. 1051 36. Banerjee K, Barbhuiya JN, Ghosh AP, Dey SK, Karmakar PR. The efficacy of low-dose oral 1052 corticosteroids in the treatment of vitiligo patient. Indian J Dermatol Venereol Leprol. 2003;69:135-7. 1053 37. Pasricha JS, Khaitan BK. Oral mini-pulse therapy with betamethasone in vitiligo patients 1054 having extensive or fast-spreading disease. Int J Dermatol. 1993;32:753-7. 1055 38. Halder RM, Brooks HL. Medical therapies for vitiligo. Dermatol Ther. 2001; 14:1–6. 1056 39. Kanwar AJ, Dogra S, Parsad D, Kumar B. Narrow-band UVB for the treatment of vitiligo: an 1057 emerging effective and well-tolerated therapy. Int J Dermatol. 2005;44:57-60. 1058 40. Kumaran MS, Kaur I, Kumar B. Effect of topical calcipotriol, betamethasone dipropionate and 1059 their combination in the treatment of localized vitiligo. J Eur Acad Dermatol Venereol. 2006;20:269-1060 73. 1061 41. Forschner T, Buchholtz S, Stockfleth E. Current state of vitiligo therapy--evidence-based 1062 analysis of the literature. J Dtsch Dermatol Ges. 2007;5:467-75. 1063 42. Srinivas CR, Shenoi SD, Balachandran C. Acceleration of repigmentation in vitiligo by topical 1064 minoxidil in patients on photochemotherapy. Int J Dermatol. 1990;29:154-5. 1065 43. Pasricha JS, Khera V. Effect of prolonged treatment with levamisole on vitiligo with limited 1066 and slow-spreading disease. Int J Dermatol. 1994;33:584-7. 1067 44. Suite M, Quamina DB. Treatment of vitiligo with topical melagenine--a human placental 1068 extract. J Am Acad Dermatol. 1991;24:1018-9. 1069 45. Parsad D, Kanwar A. Oral minocycline in the treatment of vitiligo--a preliminary study. 1070 Dermatol Ther. 2010;23:305-7. 1071 46. Savant SS. Surgical therapy of vitiligo: current status. Indian J Dermatol Venereol Leprol. 1072 2005;71:307-10. 1073 47. Mutalik S, Ginzburg A. Surgical management of stable vitiligo: A review with personal 1074 experience. Dermatol Surg. 2000;26:248-54. 1075 48. Parsad D, Gupta S. Standard guidelines of care for vitiligo surgery. Indian J Dermatol 1076 Venereol Leprol. 2008;74 Suppl:S37-45. 1077 49. Malakar S, Lahiri K, Malakar RS. How unstable is the concept of stability in surgical 1078 repigmentation of vitiligo? Dermatology. 2000;201:182-3. 1079


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50. Ezzedine K, Lim HW, Suzuki T, Katayama I, Hamzavi I, Lan CC, et al. Revised 1080 classification/nomenclature of vitiligo and related issues: the Vitiligo Global Issues Consensus 1081 Conference. Pigment Cell Melanoma Res. 2012;25:E1-13. 1082 51. Budania A, Parsad D, Kanwar AJ, Dogra S. Comparison between autologous noncultured 1083 epidermal cell suspension and suction blister epidermal grafting in stable vitiligo: a randomized 1084 study. Br J Dermatol. 2012;167:1295-301. 1085 52. Gauthier Y, Surleve-Bazeille JE. Autologous grafting with noncultured melanocytes: a 1086 simplified method for treatment of depigmented lesions. J Am Acad Dermatol. 1992;26:191-4. 1087 53. Olsson MJ, Juhlin L. Leucoderma treated by transplantation of a basal cell layer enriched 1088 suspension. Br J Dermatol. 1998;138:644-8. 1089 54. van Geel N, Ongenae K, De Mil M, Naeyaert JM. Modified technique of autologous 1090 noncultured epidermal cell transplantation for repigmenting vitiligo: a pilot study. Dermatol Surg. 1091 2001;27:873-6. 1092 55. van Geel N, Ongenae K, De Mil M, Haeghen YV, Vervaet C, Naeyaert JM. Double-blind 1093 placebo-controlled study of autologous transplanted epidermal cell suspensions for repigmenting 1094 vitiligo. Arch Dermatol. 2004;140:1203-8. 1095 56. Olsson MJ, Juhlin L. Long-term follow-up of leucoderma patients treated with transplants of 1096 autologous cultured melanocytes, ultrathin epidermal sheets and basal cell layer suspension. Br J 1097 Dermatol. 2002;147:893-904. 1098 57. Mulekar SV. Long-term follow-up study of segmental and focal vitiligo treated by 1099 autologous, noncultured melanocyte-keratinocyte cell transplantation. Arch Dermatol. 1100 2004;140:1211-5. 1101 58. Pandya V, Parmar KS, Shah BJ, Bilimoria FE. A study of autologous melanocyte transfer in 1102 treatment of stable vitiligo. Indian J Dermatol Venereol Leprol. 2005;71:393-7. 1103 59. Olsson MJ, Juhlin L. Transplantation of melanocytes in vitiligo. Br J Dermatol. 1995;132:587-1104 91. 1105 60. Randall VA, Jenner TJ, Hibberts NA, De Oliveira IO, Vafaee T. Stem cell factor/c-Kit signalling 1106 in normal and androgenetic alopecia hair follicles. J Endocrinol. 2008;197:11-23. 1107 61. Legue E, Sequeira I, Nicolas JF. Hair follicle renewal: authentic morphogenesis that depends 1108 on a complex progression of stem cell lineages. Development. 2010;137:569-77. 1109 62. Sellheyer K, Krahl D. Skin mesenchymal stem cells: prospects for clinical dermatology. J Am 1110 Acad Dermatol. 2010;63:859-65. 1111 63. Vanscheidt W, Hunziker T. Repigmentation by outer-root-sheath-derived melanocytes: proof 1112 of concept in vitiligo and leucoderma. Dermatology. 2009;218:342-3. 1113 64. Kumar A MS, Sahni K, Kumar R, Gupta S. Extracted hair follicle outer root sheath cell 1114 suspension for pigment cell restoration in vitiligo. J Cutan Aesthet Surg 2013;6:121-5. 1115 65. Gho CG, Braun JE, Tilli CM, Neumann HA, Ramaekers FC. Human follicular stem cells: their 1116 presence in plucked hair and follicular cell culture. Br J Dermatol. 2004;150:860-8. 1117 66. Gho CG, Neumann HA. Hair transplantation of plucked hair biopsies. Dermatol Surg. 1118 2001;27:913. 1119 67. Mohanty S, Kumar A, Dhawan J, Sreenivas V, Gupta S. Noncultured extracted hair follicle 1120 outer root sheath cell suspension for transplantation in vitiligo. Br J Dermatol. 2011;164:1241-6. 1121 68. Nishimura EK, Jordan SA, Oshima H, Yoshida H, Osawa M, Moriyama M, et al. Dominant role 1122 of the niche in melanocyte stem-cell fate determination. Nature. 2002;416:854-60. 1123 69. Tobin DJ, Paus R. Graying: gerontobiology of the hair follicle pigmentary unit. Exp Gerontol. 1124 2001;36:29-54. 1125 70. Singh C, Parsad D, Kanwar AJ, Dogra S, Kumar R. Comparison between autologus 1126 noncultured extracted hair follicle outer root sheath cell suspension and autologous non cultured 1127 epidermal cell suspension in the treatment of stable vitiligo: a randomized study. Br J Dermatol. 1128 2013. 1129


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71. Lee BW, Schwartz RA, Hercogova J, Valle Y, Lotti TM. Vitiligo road map. Dermatol Ther. 1130 2012;25 Suppl 1:S44-56. 1131 72. Lee KY JS, Hong JW, et al. Endothelin-1 enhances the proliferation of normal human 1132 melanocytes in a paradoxical manner from the TNF-alpha-inhibited condition, but tacrolimus 1133 promotes exclusively the cellular migration without proliferation: a proposed action mechanism for 1134 combination therapy of phototherapy and topical tacrolimus in vitiligo treatment. J Eur Acad 1135 Dermatol Venereol 2012: in press. 1136 73. Hachiya A, Kobayashi A, Ohuchi A, Takema Y, Imokawa G. The paracrine role of stem cell 1137 factor/c-kit signaling in the activation of human melanocytes in ultraviolet-B-induced pigmentation. J 1138 Invest Dermatol. 2001;116:578-86. 1139 74. Halaban R, Tyrrell L, Longley J, Yarden Y, Rubin J. Pigmentation and proliferation of human 1140 melanocytes and the effects of melanocyte-stimulating hormone and ultraviolet B light. Ann N Y 1141 Acad Sci. 1993;680:290-301. 1142 75. Botchkarev VA, Yaar M, Peters EM, Raychaudhuri SP, Botchkareva NV, Marconi A, et al. 1143 Neurotrophins in skin biology and pathology. J Invest Dermatol. 2006;126:1719-27. 1144 76. Bhawan J, Bhutani LK. Keratinocyte damage in vitiligo. J Cutan Pathol. 1983;10:207-12. 1145 77. Kumar R, Parsad D. Melanocytorrhagy and apoptosis in vitiligo: connecting jigsaw pieces. 1146 Indian J Dermatol Venereol Leprol. 2012;78:19-23. 1147 78. Huggins RH, Henderson MD, Mulekar SV, Ozog DM, Kerr HA, Jabobsen G, et al. Melanocyte-1148 keratinocyte transplantation procedure in the treatment of vitiligo: the experience of an academic 1149 medical center in the United States. J Am Acad Dermatol. 2012;66:785-93. 1150

1151

1152

1153

1154

1155

1156

1157


39

APPENDIX I 1158

CONSENT PROFORMA 1159

TRANSPLANTATION OF AUTOLOGOUS NONCULTURED EXTRACTED HAIR 1160

FOLLICLE OUTER ROOT SHEATH CELL AND AUTOLOGUS NONCULTURED 1161

EPIDERMAL CELL SUSPNSION IN COMBINATION AS A NOVEL METHOD IN 1162

VITILIGO SURGERY 1163

1164

Name of the participant: ____________________________________________ 1165

Name of the Principal (Co-) Investigator: ______________________________ 1166

Name of the Institution: ____________________________________________ 1167

Name and address of the sponsoring (funding) agency(ies): ________________ 1168

I, , age CR. No. exercising my free 1169

power of choice, hereby give my consent to be included as a subject in “Transplantation of 1170

autologous noncultured extracted hair follicle outer root sheath cell and autologous 1171

noncultured epidermal cell suspension in combination as a novel method in vitiligo surgery.” 1172

I have been explained in a language understandable to me, the nature of the treatment, its 1173

expected benefits and possible side effects and I am willing to undergo any necessary 1174

investigations. 1175

I have been informed that for academic and scientific purposes, the white patches will be 1176

photographed before and after the study. 1177

I will allow the use of my photographs for presentation and publication purposes with the 1178

understanding that I will never be identified by name. 1179

I hereby give permission to the investigators to release the information obtained from me, 1180

as a result of participation in this study, to the sponsors, regulatory authorities, 1181

government agencies, and ethics committee. I understand that they may inspect my 1182

original records. 1183

I am aware that I will have to come to PGIMER, Chandigarh for follow up at least 4 times 1184

over a period of 16 weeks (weeks 1, 4, 8 and 16) for the proper conduct of study. 1185

I am also aware of my right to opt out of the study any time during the course trial without 1186

having to give the reason for doing so. 1187

My signature on this form indicates that I: 1188

o Have carefully read and understood the information provided in this form 1189

o Have been explained the nature of this study and give my consent for inclusion in the 1190

study. 1191

1192

Name and signature of patient Name and signature of physician 1193

1194

1195

Date Name and signature of witness 1196

1197


40

1198


41

PATIENT INFORMATION SHEET 1199

Follicular cell suspension and epidermal cell suspension are recently described surgical 1200

techniques with promising results for the management of stable vitiligo patches. By 1201

combining these two methods, we expect better repigmentation. This new method involve 1202

extraction of 15-25 hair follicles from the back of the head and also harvesting superficial skin 1203

from thigh under local anaesthesia, then treating them with various reagents to form a 1204

combined cell suspension. This cell suspension is rich in pigment forming cells called 1205

melanocytes. This cell suspension is transplanted into vitiligo area after abrading superficial 1206

areas of the skin under local anaesthesia. Melanocytes in the cell suspension home into the 1207

dermabraded area and causes pigmentation in 2-6months. 1208

If patient is not a case of stable vitiligo, there is chance to get new white patch at skin 1209

haervested site and also failure of repigmentation at treated vitiligo site. Hair will regrow from 1210

surroundings of extracted site, the back of the head, covering hairless patch within a month. 1211

Surgical treatment is for the existing lesions of vitiligo and new lesions of vitiligo may still 1212

appear in future. 1213

The surgery and preparation of cell suspension is undertaken in strict aseptic precautions 1214

to minimize the chances of infection. Patient need to come to minor operation theatre in OPD 1215

on 2 consecutive days for surgery. First day, skin harvesting will be done and it will take only 1216

half an hour, then patient can go home. On the second day, hair follicle extraction will be 1217

carried out in the morning, then sample will be sent to laboratory; after getting cell suspension 1218

after 2 hours, dermabrasion at vitiligo site will be carried out followed by application of cell 1219

suspension over it. The surgery site will be dressed neatly, and patient can go home on the 1220

same day after surgery. Patient has to come after 5 days to remove dressings and to rule out 1221

any surgical site infection. 1222

The result obtained from this study may establish a novel method in vitiligo surgery 1223

helping patients suffering from vitiligo. 1224

1225

1226

1227

1228

1229

1230

1231


42

APPENDIX II 1232

DATA RECORD SHEET 1233

1234

Name: Age/ Sex: CR. No. : 1235

Occupation: Pt. No 1236

Address: 1237

1238

Chief complaints: 1239

1240

Total duration: 1241

1242

Site of onset: 1243

1244

Progression of disease: 1245

1246

1247

Precipitating factors: 1248

1249

Present status: stable/ unstable 1250

1251

Koebnerisation: present/ absent 1252

1253

History of past illness: H/o similar disease/ autoimmune disorder (pernicious anaemia, 1254

hyperthyroidism, hypothyroidism, alopecia areata, autoimmune hemolytic anemia and 1255

myasthenia gravis)/ atopy / diabetes/ hypertension/ tuberculosis / photosensitivity / any other 1256

disease. 1257

Treatment history: 1258

Treatment taken Response

1. Topical

2. Systemic

3. Phototherapy

4. Indigenous


43

Personal history: 1259

Smoking 1260

Alcohol 1261

Addictions 1262

Family history 1263

1264

GENERAL PHYSICAL EXAMINATION: 1265

Pulse: BP: Weight: 1266

Pallor: Edema: Clubbing: 1267

Cyanosis: Icterus: Lymphadenopathy: 1268

1269

SYSTEMIC EXAMINATION: 1270

CVS 1271

1272

RS 1273

1274

P/A 1275

1276

1277

CUTANEOUS EXAMINATION: 1278

% BSA involved: 1279

Areas affected: 1280

Head and neck/ trunk/ upper limb/ lower limb/ hands/ feet/ mucosae 1281

Leukotrichia: Present/Absent 1282

Mucosal involvement: Present/Absent 1283

1284


44

1285

1286

1287

1288

1289

1290

1291

1292

1293

1294


45

INVESTIGATIONS: 1295

Investigations

Hb

Total count

Differential Count N L E M B

Platelets

RBS

Bleeding time

Clotting time

HBsAg

HIV

1296

1297

Donor site: 1298

Size of the split thickness graft: 1299

Number of hair follicles extracted: 1300

Site of treated area: 1301

a) NCES b) FCS + NCES 1302

1303

Size of treated area: 1304


1306

1307

1308

1309

1310

1311

1312

1313

1314

1315

1316


46

CLINICAL EVALUATION: 1317

1 = NCES 2 = FCS + NCES 1318

Evaluation Day 8 4 weeks 8 weeks 16 weeks

1 2 1 2 1 2 1 2

Extent of pigmentation

<25%

26-50%

51-75%

75 -90%

>90%

Color match of grafted area with the

normal skin

- somewhat darker

- somewhat lighter

- same

Pattern of repigmentation

- Diffuse

- Perifollicular

- Migrating from the borders

1319

Complications / side effects

a. Recipient site

- infection

- milia

- scarring

- rejection

b. Donor site

- Infection

- Milia

- Scarring

- Hypopigmentation

- Hyperpigmentation

Evaluation NCES FCS + NCES

Repigmentation at 16 weeks

1320


47

APPENDIX III 1321

PATIENT SATISFACTION QUESTIONNAIRE70

1322

1323

1. Patients’ Global Assessment: 1324

1325

1) Grade the change in pigmentation in the transplanted area. (0 to 10) 1326


2) Are you satisfied with the obtained result? (0 to 10) 1328


3) Do you find the treatment worthwhile? (0 to 10) 1330


4) Would you choose this treatment again? (yes / no) 1332


1334

1335

For question 1, ‘0’ means ‘much worse’ and ‘10’ means ‘much improved’. 1336

For question 2 and 3, ‘0’ means ‘not at all’ and ‘10’ means ‘very much’. 1337

1338

1339

1340

1341

1342

1343

1344

1345

1346


combination of follicular and epidermal cell suspension as

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