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Early View Article: Online published version of an accepted article before publication in the final form.

Journal Name: International Journal of Hepatobiliary and Pancreatic Diseases (IJHPD)

Type of Article: Original Article

Title: A histopathological and histochemical study of cholecystitis

Authors: Ponniah Anupama, Menon Nirmala V, Ramakrishnan Janaky, llias Laila

Mohammed, Mohammed Babitha Alingal, Nalakath Asiq Sideeque

doi: To be assigned

Received: 26th September 2014

Accepted: 29th October 2014

How to cite the article: Anupama P, Menon NV, Janaky R, Mohammed lL, Alingal MB, Sideeque NA. A histopathological and histochemical study of cholecystitis. International Journal of Hepatobiliary and Pancreatic Diseases (IJHPD). Forthcoming 2014.

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TYPE OF ARTICLE: Original Article 1

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TITLE: A histopathological and histochemical study of cholecystitis 3

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AUTHORS: 5

Ponniah Anupama1, Menon Nirmala V2, Ramakrishnan Janaky3, llias Laila 6

Mohammed4, Mohammed Babitha Alingal5, Nalakath Asiq Sideeque6 7

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AFFILIATIONS: 9

1MBBS, MD, Assistant Professor, Department of Pathology, MES Medical College, 10

Perinthalmanna, Kerala, India. Email ID: [email protected] 11

2MBBS, MD, Professor, PSG Institute of Medical Sciences and Research, 12

Coimbatore, Tamil Nadu, India. Email ID: [email protected] 13

3MBBS, Junior Resident, Department of Pathology, MES Medical College, 14






6MBBS, MD, Professor and Head of Department, Department of Pathology, MES 20

Medical College, Perinthalmanna, Kerala, India. Email ID: [email protected] 21

22

CORRESPONDING AUTHOR DETAILS 23

Dr. Ponniah Anupama, 24

Department of Pathology, MES Medical College and Hospital, Malaparamba, 25

Palachode P. O, Kolathur via, Perinthalmanna, Malapuram District, Kerala, India. 26

Postal Code -679338. 27

Contact Phone Number: +919447973557 28

Email ID: [email protected] 29

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Short Running Title: A histopathological and histochemical study of cholecystitis 33

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ABSTRACT 67

Aims: 68

The present study mainly aimed at to assess the qualitative and quantitative 69

assessment of gallbladder mucins in chronic calculous cholecystitis, correlating the 70

mucin histochemistry and morphology of the gallbladder in chronic calculus 71

cholecystitis with each other and with chemical composition of gallstones. 72

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Methods: 74

Haematoxylin & Eosin stained sections of the selected specimens were screened 75

and one to two sections with adequate amount of well preserved mucosa with lining 76

epithelium were chosen. The corresponding paraffin blocks were selected and 77

isolated. Four sections were cut on each of the selected blocks for special stains. 78

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Results: 80

Comparison of inflammation and mucin histochemistry, inflammation and metaplasia, 81

composition of calculi and grade of inflammation, composition of stones and mucin 82

histochemistry, composition of calculi and metaplasia, fibrosis and mucin 83

histochemistry, fibrosis and stone composition indicated a decrease in intraepithelial 84

total acid mucin content in chronic calculous cholycystitis. Cases with severe 85

inflammation showed the maximum decrease in sulphomucin, concomitant increase 86

in sialomucin scores and a high incidence of gastric metaplasia. Intestinal metaplasia 87

on other hand did not correlate with the degree of inflammation or sialomucin 88

content. 89

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Conclusion 97

This study concludes that normally gallbladder epithelium contains sulphated acid 98

mucins with traces of neutral and sialomucins. The sulphomucin content decreases 99

in chronic calculous cholecystitis and with severe inflammation, total acid mucin 100

content decreases, neutral mucin increases, and there is a higher incidence of 101

gastric metaplasia and pigment stones and correlating with pigment stones. This 102

tends to have an association with severe inflammation, higher degree of fibrosis, 103

gastric metaplasia and presence of sialomucin. 104

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Keywords: Gall bladder stones, chronic calculous cholecystitis, mucin 106

histochemistry, metaplasia. 107

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INTRODUCTION 131

Million people’s are affected by gallstones and are common major factor of morbidity 132

throughout the world, necessitating hospitalization and cholecystectomy. Cholesterol 133

saturated as ‘lithogenic’ bile originating from the liver and considered an important 134

factor in gallstones formation has been found in healthy individuals [1]. Lithogenic 135

bile therefore cannot be the only factor involved in the process. Other factors such as 136

super saturation of bile with calcium [2], gallbladder mucus, prostaglandins and 137

functional failure of electrolyte absorption by gallbladder mucosa also may influence 138

gallstone formation. Biliary calcium can reduce the solubility of cholesterol [3] 139

rendering the bile lithogenic. Apart from being a critical initiating factor, calcium is 140

physically incorporated into gallstones as well. Gallbladder mucus has long been 141

recognized as an important 142

factor contributing to gall stone development [4, 5]. The implication of mucin in 143

gallstone formation has been widely studied. Hypersecretion of mucus occurs during 144

gallstone formation in humans [6-9]. and experimental animals [10]. Apart from 145

forming the nucleus for calculus, the mucins form a structural component of 146

gallstones as shown by histochemical studies on calculi [11, 12]. Calcium and 147

prostaglandins can stimulate mucus secretion by gallbladder mucosa [13-15]. Normal 148

human gallbladder contains predominantly sulphated acid mucin [16]. It is this 149

sulphated mucin content that is increased in gallstone disease. Metaplastic and 150

neoplastic gallbladder epithelium on the other hand shows an increase in 151

sialomucins and decrease in sulphomucins. Several studies have suggested 152

progression from metaplasia, through dysplasia, to adenocarcinoma of gallbladder 153

[17]. The existence of such a pathway has not been definitely proven. 154

Mucins 155

Mucins are the chemical components of the secretion delivered by certain types of 156

epithelial and connective tissue cells. The original term mucin was coined by 157

Carpenter as early as in the year 1846 [18]. Reid and Clamp in 1978 suggested 158

glycoconjugates as general term, which could be subdivided into ‘proteoglycans’ and 159

‘glycoproteins’ [19]. 160


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Free hexose groups are often available, together with certain acidic moieties, the 161

presence of which will markedly influence histochemical reactivity. The different 162

mucins may be present as a single type within a given tissue unit, or more usually as 163

a mixture of different types. The synthesis of mucin is initiated in the rough 164

endoplasmic reticulum of the producing cells and is completed in the golgi 165

apparatus. Sulphation of the hexosamine molecule occurs in the golgi region [8, 20]. 166

Scheme of an easy method to classify the mucin like a proteins the only two classical 167

techniques [8]. 168

The different types of mucins which can be distinguished histochemically are as 169

follows [21]. 170

ACID MUCINS 171

1. Strongly sulphated 172

a. connective tissue 173

b. epithelial 174

2. Weakly sulphated 175

Sulphated; histochemically atypical 176

3. Carboxylated; sialomucin 177

a. enzyme – labile (N-acetyl form) 178

b. enzyme – Resistant (N-acetyl O-acetyl form) 179

4. Sulfated sialomucin 180

5. Carboxylated; nonsulphated uronic acid (Hyaluronic acid) 181

NEUTRAL MUCINS 182

There are no subdivisions to this group 183

184

MATERIALS AND METHODS 185

A total number of 40 specimens were selected from gallbladders with clinical and 186

histopathological diagnosis of chronic calculous cholecystitis received in the 187

department of pathology PSG INSTITUTE OF MEDICAL SCIENCES AND 188

RESEARCH during the period of 2005 to 2007. Criteria for selection were 189

1) Histopathological confirmation of chronic calculous cholecystitis. 2) Presence of 190

calculous accompanying the specimen. 3) Availability of sufficient mucosa and well 191

preserved lining epithelium in sections. 4) Availability of corresponding paraffin 192


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blocks. While 36 specimens fulfilled the above criteria, 4 cases had only biliary sand 193

in the container. 3 gallbladders resected for choledochal cysts, were taken as 194

controls. Haematoxylin & Eosin stained sections of the selected specimens were 195

screened and one to two sections with adequate amount of well preserved mucosa 196

with lining epithelium were chosen. The corresponding paraffin blocks were selected 197

and isolated. Four sections were cut on each of the selected blocks for special 198

stains. A proforma was prepared for assessment. The slides were assessed 199

according to the proforma. 200

Haematoxylin & Eosin Stain 201

Sections stained with Haematoxylin & Eosin were assessed for the intensity of 202

inflammation and degree of fibrosis, which in turn were graded as mild, moderate 203

and severe (1+, 2+, 3+) (Figure 1, 2). The number of Rokitansky Aschoff sinuses 204

was indicated as many, few and nil (Figure 6). Gastric metaplasia and intestinal 205

metaplasia were also noted and indicated as present or absent (Figure 3, 4). 206

Mucin Histochemistry 207

The following special stains for mucin were done with a view to assess the quantity 208

and quality of mucins in the superficial and deep parts of gallbladder mucosa. 209

1. High Iron Diamine – Alcian Blue Stain (HID-AB) 210

2. Alcian Blue – Periodic Acid Schiff (AB-PAS) 211

A scoring system was devised, based on the percentage positivity of cells in each 212

field under low power examination (10X), as shown below: 213

75% -100 % - 5+ 214

50% - 75% - 4+ 215

25% - 50% - 3+ 216

5% - 25% - 2+ 217

0% - 5% - 1+ (Figures 5-8) 218

The values were tabulated and statistically assessed using ANOVA, t-test, x2 (Chi2) 219

test. 220

Biochemical analysis of calculi, wherever available (36 samples) was performed to 221

determine the chemical composition of calculi. Gross parameters such as external 222

appearance, colour, number of stones and weight were noted. The following 223

biochemical procedures were carried out on the 36 samples of calculi. 224


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Biochemical analysis [22] 225

Cholesterol 226

Wash the gallstones with water and dry. Powder the stone and heat some with 227

successive small portions of ether in a test tube by inserting the tube in some warm 228

water and filter. Dissolve a little of residue obtained on evaporation of the ether in 229

chloroform and add a little of mixture of acetic anhydride and sulphuric acid (in the 230

proportion of 10 ml to 0.1 ml). A dark green colour develops rapidly. 231

Phosphate And Calcium Oxalate 232

Treat the remaining residue after ether extraction, with dilute hydrochloric acid 233

(25%). This dissolves the inorganic salts present. Filter and test the filtrate for 234

phosphate with molybdate. Make some of the solution alkaline with ammonia and 235

add acetic acid and ammonium oxalate solution. If calcium is present a precipitate of 236

calcium oxalate is formed. 237

Bile Pigment 238

Test the precipitate remaining after treatment with hydrochloric acid for bile 239

pigments. Wash the material remaining on the filter paper and extract with warm 240

chloroform. Examine the chloroform extract for 241

bilirubin by means of diazo reagent. Change to pink colour indicates presence of bile 242

pigment. 243

The mucin histochemistry scores obtained were tabulated, separately for the three 244

grades of inflammation and fibrosis. An attempt was made to correlate the score with 245

degree of inflammation, fibrosis, presence of metaplasia (intestinal and pyloric) and 246

type of stone. Statistical assessment of the results was also performed. 247

248

RESULTS 249

The three specimens of gall bladder used as a control in the present study showed 250

predominantly alcian blue (AB) positive mucins in the surface and deep mucosal 251

epithelium.Periodic Acid Schiff (PAS) positivity was seen in traces . Comparison of 252

inflammation and mucin histochemistry, inflammation and metaplasia, composition of 253

calculi and grade of inflammation, composition of stones and mucin histochemistry, 254

composition of calculi and metaplasia, fibrosis and mucin histochemistry, fibrosis and 255

stone composition was done. 256


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High Iron diamine and alcian blue (HID-AB)stain was done in the three controls and 257

it showed strong HID-AB positivity indicating the predominance of sulphomucin in 258

superficial and deep mucosa and evaluated by mean score which is normally 259

observed in the normal gall bladder mucosa (Table:1). 260

Comparison of inflammation and mucin histochemistry (Tables 2 & 3) showed that 261

out of the total 36 cases studied, 9 (25%) cases showed mild (Grade I) inflammation, 262

16 (44.4%) cases showed moderate (Grade II) inflammation and 11(30.5%) cases 263

showed severe (Grade III) inflammation. The mean scores for AB positive (acid 264

mucins) in the superficial and deep mucosa, in the three grades of inflammation 265

showed a progressive decrease for Alcian Blue positive (acid) mucins in the 266

superficial and deep mucosal epithelium, with increasing grades of inflammation 267

(Table:2). The mean scores for PAS positive mucins in the superficial and deep 268

mucosa were higher in Grade III inflammation, than in Grade 1 inflammation. Grade 269

II inflammation however shows random scores, not conforming to any pattern (Table 270

2). The mean scores for HID (sulfomucin) positive are the lowest for Grade III 271

inflammation. A progressive decrease in HID scores was observed in the superficial 272

mucosal epithelium (Table 3). Alcian Blue (sialomucin) scores were seen to be 273

highest in Grade III inflammation (Table 3). 274

Comparison of grades of inflammation and metaplasia (Table 4) showed that out of 275

the 36 cases, 13 (36.1%) showed intestinal metaplasia, 12 (33.3%) showed gastric 276

metaplasia and 11(30.5%) did not show any metaplasia. Metaplastic epithelium is 277

characterized by presence of PAS positive mucins and / or goblets cells containing 278

AB positive mucins in AB – PAS stains sections (Figures 9-11). Intestinal metaplasia 279

has the highest incidence in Grade I inflammation (56%), and is lowest in Grade II 280

inflammation (19%).The reverse is seen with gastric metaplasia, which has the 281

highest incidence in Grade III Inflammation (46%), and a considerably lower 282

incidence in Grade I inflammation (11%). 283

Comparison of composition of calculi and grade of inflammation (Table 5), 3 cases 284

out of 36 cases did not show calculi. 13 (39.3%) showed cholesterol calculi and 20 285

(60.6%) showed pigment stone calculi. The presence of pigment stones appears to 286

correlate with severity of inflammation [(3/8(37.5%) in Grade I inflammation, 287


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9/11(81.8%) in Grade III inflammation)] as against cholesterol stones [(5/8(62.5%) in 288

Grade I inflammation, 2/11(18.1%) in Grade III inflammation]. 289

Comparison of stones and mucin histochemistry (Table 6) showed that the mean 290

scores of sialomucins in the superficial and deep mucosal epithelium are higher in 291

cases with pigment stones (0.85,0.72),when compared with those having cholesterol 292

stones (0.24,0.58) the number of cases that show presence of sialomucins is low 293

with both types of calculi (5/20 cases with pigment stones and 6/13 cases with 294

cholesterol stones). 295

Comparison of composition of calculi and metaplasia (Table 7) showed that intestinal 296

metaplasia is more or less equally associated with pigment and cholesterol calculi 297

(35% and 30% incidence). Gastric metaplasia shows a considerably higher 298

incidence (40%) in association with pigment stones, when compared with cholesterol 299

stones (23%). 300

Comparison of fibrosis and mucin histochemistry (Table 8), out of 40 cases, 33 301

(82.5%) cases showed fibrosis. Alcian Blue scores (acid mucins) are slightly lower in 302

Grade III fibrosis, compared with Grade I. Neutral mucins (PAS positive) on the other 303

hand shows higher scores in Grade III as against Grade I fibrosis. No correlation is 304

seen between HID scores (for sulphomucins) and degree for fibrosis. Alcian Blue 305

scores (for sialomucins) are the lowest with Grade III fibrosis (Table 8). 306

Comparison of fibrosis and stone composition(Table 9) showed that out of the 33 307

cases pigment stones have a higher incidence in Grade III fibrosis 80%(4/5) as 308

against Grade I fibrosis 52.6%(10/19). 309

310

DISCUSSION 311

Attempts have been made in the past to correlate gallbladder morphology, mucin 312

histochemistry, and composition of calculi in gallstone disease [16, 23, 24]. Most of 313

the previous studies however have combined any two of the three aspects, that is, 314

either morphology with mucin histochemistry, mucin histochemistry with composition 315

of calculi or composition of calculi with morphology. Few studies correlating all the 316

three with one another have been recorded in literature .Purpose of the present 317

study was to determine whether qualitative and / or quantitative variations in 318

gallbladder mucins occurs in chronic calculous cholecystitis and whether the 319


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alterations ,if any, correlate with morphological changes in the gallbladder and / or 320

with the type of calculous present .In short, an attempt has been made through this 321

study, to correlate gallbladder morphology, mucin histochemistry and composition of 322

calculi to one another , in chronic calculous cholecystitis specimens. 323

Gallbladders removed for choledochal cysts were the control in the present study. 324

The mucosal histology was normal and there was no inflammation, fibrosis or 325

metaplasia. AB-PAS and HID-AB staining of sections showed predominantly HID 326

positive sulphated acid mucins throughout the mucosa, with traces of sialomucins 327

and neutral mucins in foci. Normal gallbladder mucosa is known to contain 328

predominantly sulphomucins. Traces of sialomucins and neutral mucins also may be 329

present [24]. Our observations on control samples conform to this well–established 330

normal pattern of mucin histochemistry of gallbladder mucosa. Our results indicate a 331

decrease in intraepithelial total acid mucin content in chronic calculous cholecystitis 332

.Mucin depletion in mucosal epithelial cells is well known in inflammatory conditions 333

of the gastrointestinal tract. In various forms of colitis presenting with mucus 334

diarrhoea or dysentery and showing active inflammation of the mucosa, mucin 335

depletion is a constant finding . Increased mucin secretion by gallbladder mucosa 336

during gallstone formation has been described in literature [6-9, 24]. Earlier 337

investigators have shown that mucins, in addition to being a structural component of 338

gallstones [12], also play an acceleratory role in lithogenesis [25]. Our observation of 339

decreased intraepithelial mucins in inflamed gallbladder mucosa is likely to be a 340

reflection of increased secretion of mucin into bile which is known to occur in 341

calculous disease. The decrease in the intraepithelial mucins in chronic cholecystitis, 342

we found, was due to decrease in sulphomucin which is the predominant type of 343

mucin in gallbladder mucosa. Further, it was observed that cases with severe 344

inflammation showed the maximum decrease in sulphomucin. This was associated 345

with a concomitant increase in sialomucin scores and a high incidence of gastric 346

metaplasia. Intestinal metaplasia on the other hand, did not correlate with the degree 347

of inflammation or sialomucin content .No qualitative changes in gallbladder 348

epithelial mucins have been observed in the earlier studies on chronic cholecystitis 349

[26]. Sialomucins are known to occur in traces in normal gallbladder mucosa and in 350

considerable quantities in metaplastic mucosa. In the present study, an increase in 351


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sialomucins was observed in gallbladders showing severe inflammation 352

.Interestingly, it was in this group that gastric metaplasia had the highest incidence 353

(Figure 12). It therefore follows that, sialomucins in significant quantities tend to 354

appear in the areas of gastric metaplasia in the gallbladder mucosa. Their presence 355

is not confined to the goblet cells of intestinal metaplasia which had the lowest 356

percentage of incidence in severe inflammation, in the cases assessed (Figure 13). It 357

has been suggested that antral and intestinal metaplasia in the gallbladder are 358

histogenetically related having the same progenitor cell, and could therefore be parts 359

of a morphological spectrum [27]. Transition from gastric to intestinal metaplasia is a 360

likely possibility [17]. Pre-neoplastic role of intestinal metaplasia in the gallbladder 361

and the metaplasia → dysplasia → neoplasia sequence have received wide attention 362

among workers [11, 17, 28]. The mucin profile changes with progressive 363

transformation to neoplasia, from normal with sulphomucin predominating through 364

metaplastic and dysplastic showing increasing amounts of sialomucin, to full fledged 365

neoplastic with sialomucins predominating .The high incidence of gastric metaplasia 366

in severe inflammation and its association with increased expression of sialomucins 367

with the concomitant reduction in sulphomucins would point, perhaps tentatively, 368

towards a role for gastric metaplasia in the proposed chain of events stated above. 369

Basu et al studied the morphological changes in chronic calculous cholecystitis in 370

relation to the type of stones [29]. They found that inflammation was more severe 371

with pigment calculi while fibrosis and related complications were more frequent with 372

cholesterol calculi. The present study supports the association between pigment 373

stones and severe inflammation. Fibrosis also was more in cases with pigment 374

calculi in our study. Mucins have been shown to be a structural component of 375

gallstones [11, 30]. Histochemical studies carried out on calculi have demonstrated 376

presence of sulphomucins in them, especially in pigment stones [12]. However no 377

correlation between mucin histochemistry of mucosal epithelium and the type of 378

stone has been recorded in literature. In the present study, pigment stones were 379

found more often in association with severe inflammation, gastric metaplasia and 380

increased expression of sialomucins, as against cholesterol stones. We were unable 381

to establish a statistical significance to the above observations, as the number of 382

cases studied was small, especially in the sialomucin expressing group (even though 383


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the scores were high.)But the scores and percentage values did show a distinct 384

pattern indicating a correlation between a severe inflammation, gastric metaplasia, 385

sialomucins and pigment calculi (Refer tables 3 to 7). 386

Considering the proposed pathway of Gastric metaplasia → Intestinal metaplasia → 387

Dysplasia → Adenocarcinoma of gallbladder and the proven presence of 388

sialomucins in considerable amounts in dysplastic and neoplastic gallbladder 389

mucosa, it is reasonable to speculate on a pigment stone → severe inflammation → 390

gastric metaplasia → sialomucin link up, with possible transition to dysplasia, with or 391

without the intervention of intestinal metaplasia. Further studies on large series are 392

required to enable us to draw definite conclusions. If such a high risk group emerges, 393

it will be of significance from the preventive, prognostic and therapeutic point of view. 394

395

CONCLUSION 396

I. The normal gallbladder epithelium contains sulphated acid mucins with traces of 397

neutral and sialomucins. 398

The sulphomucin content decreases in chronic calculus cholecystitis 399

II. In chronic calculus cholecystitis with severe (Grade III) inflammation (as against 400

mild inflammation): 401

1) Total acid mucin content is decreased. 402

2) This decrease is due to HID positive (sulpho) mucin. 403

3) Neutral mucin and sialomucin contents are increased. 404

4) There is a higher incidence of Gastric metaplasia and pigment stones. 405

III. Pigment gallstones tend to have an association with: 406

1) Severe inflammation 407

2) Higher degree of fibrosis 408

3) Gastric metaplasia 409

4) Presence of sialomucins 410

More number of cases need to be studied to see whether high risk group consisting 411

of pigment stones → severe inflammation → gastric metaplasia → sialomucin 412

emerges. If it does, will be of therapeutic and prognostic significance. 413

414

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CONFLICT OF INTEREST 416

The authors declare no conflict of interests. 417

418

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composition of gall stones.Tropic gastroenterol 2002; 23: 25-27 496

30. Smith BF and La Monte JT. Identification of gallbladder mucin – bilirubin 497

complex in human cholesterol gallstone matrix. J Clin Invest 1985;76:439-498

445. 499

500

501

502

503

504

505

506

507

508

509


of 34

TABLES 510

Table 1: HID–AB stain showed strong HID positivity indicating predominance of 511

sulphomucins. 512

513

* HID- High iron diamine AB- Alcian blue PAS- Periodic Acid Schiff 514

515

516

517

518

519

520

521

522

523

Techniques AB-PAS HID-AB

Superficial Deep

AB-PAS

AB 4.7 2.6

PAS 0.06 0.03

HID-AB

HID 4.5 2.8

AB 0 0.03


of 34

Table 2: Comparison of inflammation and mucin histochemistry- AB PAS STAIN. 524

525

526

527

528

529

530

531

532

533

534

535

536

537

538

539

540

Inflammation

Grade

Alcian blue

[Acid Mucin]

Mean Score

Pas [Neutral Mucin]

Mean Score

Superficial Deep Superficial Deep

I (9 cases) 2.7

3.4 0.25 0.14

II (16 cases)

2.2 3.1

0.11 0.31

III (11 cases) 1.95 2.5 0.29 0.4


of 34

Table 3: Comparison of inflammation and mucin histochemistry: HID-AB. 541

542

543

544

545

546

547

548

549

550

551

552

553

Inflammation

Grade

HID – [Sulphomucin]

Mean Score

InflammatioN

Grade

Alcian Blue –[Sialomucin]

Mean Score

Superficial

Deep

Superficial

Deep

I (9 cases) 3.9 3.6 I (9 cases) 0.14 0.19

II (16 cases) 2.5 3.9 II (16 cases) 0.062 0.13

III (11 cases) 2.1 2.8 III (11 cases) 0.3 0.56


of 34

Table 4: Comparison of grades of inflammation and metaplasia. 554

555

556

557

558

559

560

561

Type of Metaplasia

Inflammation Grade

I

(9 cases)

II

(16 cases)

III

(11 cases)

Total

36

IM (Intestinalmetaplasia)

5

(56%)

3

(19%)

5

(22%)

13

GM

(Gastric Metaplasia)

1

(11%)

6

(38%)

5

(46%)

12


of 34

Table 5: Comparison of calculi and grade of inflammation. 562

563

564

565

566

567

568

569

570

571

572

573

Grade of Inflammation

Pigment Stone

Cholesterol Stone

I (8 cases)

3

5

II (14 cases)

8

6

III (11 cases)

9

2

TOTAL - 33

TOTAL - 20

TOTAL - 13


of 34

Table 6: Comparison of stones and mucin histochemistry. 574

575

576

577

578

579

580

581

582

583

584

Pigment Stones

Cholesterol Stones

Total Number 20 13

Sialomucins

Present In

5 6

Sialomucin Score Superficial Deep Superficial Deep

0.85 0.72 0.24 0.58


of 34

Table 7: Comparison of composition of calculi and metaplasia. 585

586

587

588

589

590

591

592

593

594

595

Types of Metaplasia

Pigment Stones

Cholesterol Stones

Intestinal Metaplasia

35%

30%

Gastric Metaplasia

40%

23%


of 34

Table 8: Fibrosis and mucin histochemistry AB-PAS & HID-AB stain. 596

597

598

599

600

601

602

603

604

605

606

Fibrosis

Grade

AB[Acid

Mucin]

Mean Score

PAS

[Neutralmucin]

Mean Score

HID

[Sulphomucin]

Mean Score

AB

[Sialo Mucin]

Mean Score

Sup

erfic

ial

Dee

p

Sup

erfic

ial

Dee

p

Sup

erfic

ial

Dee

p

Sup

erfic

ial

Dee

p

I (19 cases) 2.46 2.81 0.13 0.22 2.64 3.52 0.11 0.17

II (9 cases) 3.0 3.1 0.96 0.45 3.08 3.27 0.26 1.97

III (5 cases) 2.0 1.7 0.85 0.32 1.7 3.7 0 0.08


of 34

Table 9: Comparison of fibrosis and stone composition. 607

608

609

610

611

612

613

614

615

616

617

618

619

Grade of

Fibrosis

Pigment

Stone

Cholesterol Stone

No Stones

Total

I 10 6 3 19

II 5 3 1 9

III 4 1 0 5

Total (33) 23 13 4 33


of 34

AB-PAS HID-AB

Neutral Mucins Red Negative

Sulphomucins Red-blue Brown-black

Sialomucins Red-blue Blue

Sulpho-Sialomucins Red-blue Brown-blue

AB-PAS, sequential staining with AB(2.5) (blue) and PAS (red); HID-AB, sequential 620

staining with HID(brown to black) and AB(2.5) (blue). 621

622

FIGURE LEGENDS 623

Figure 1: Severe (Grade III) Inflammation - Gall Bladder. Dense Sheets of 624

Lymphocytes extended between smooth muscle bundles. H & E. (X 100). 625

Figure 2: severe Fibrosis - Gall Bladder. H & E. (X 100). 626

Figure 3: Photomicrograph shows Pyloric Metaplasia in the deeper mucosa. H & E. 627

(X 100). 628

Figure 4: Intestinal Metaplasia characterised by Goblet cells in the Gall Bladder 629

mucosa. H & E. (X 100). 630

Figure 5: Score I - About 5% of cells contain Mucin AB-PAS (X 100). 631

Figure 6: Score 3 - About 50% of cells contain Mucin AB-PAS (X 100). 632


Figure 8: Score 5 - About 75-100% of cells contain Mucin HID-AB (X 100). 634

Figure 9: This field shows Gastric and Intestinal Metaplasia. The Gastric Metaplastic 635

epithelium shows PAS and Alciam Blue Positive Mucins. Goblet cells of Intestinal 636

Metaplasia are Alcian Blue positive Mucins. AB-PAS (X 100). 637

Figure 10: Intestinal Metaplasia - Gall Bladder Goblet cells contain Alcian Blue 638

positive mucin AB-PAS (X 100). 639

Figure 11: Gastric Metaplasia - Gall Bladder. The Non - Neoplastic epithelium is HID 640

positive (Brown) Metaplastic epithelium Alcian Blue positive (Blue) HID-AB (X 100). 641



of 34

positive mucin. The columnar cells are HID positive (Brown) HID-AB (X 400). 643

Figure 13: Intestinal Metaplasia - Gall Bladder HID-AB (X 400). 644

645

FIGURES 646

647

Figure 1: Severe (Grade III) Inflammation - Gall Bladder. Dense Sheets of 648

Lymphocytes extended between smooth muscle bundles. H & E. (X 100). 649

650

651

Figure 2: severe Fibrosis - Gall Bladder. H & E. (X 100). 652

653


of 34

654

Figure 3: Photomicrograph shows Pyloric Metaplasia in the deeper mucosa. H & E. 655

(X 100). 656

657

658

Figure 4: Intestinal Metaplasia characterised by Goblet cells in the Gall Bladder 659

mucosa. H & E. (X 100). 660

661


of 34

662

Figure 5: Score I - About 5% of cells contain Mucin AB-PAS (X 100). 663

664

665


667


of 34

668


670

671

Figure 8: Score 5 - About 75-100% of cells contain Mucin HID-AB (X 100). 672

673


of 34

674

Figure 9: This field shows Gastric and Intestinal Metaplasia. The Gastric Metaplastic 675

epithelium shows PAS and Alciam Blue Positive Mucins. Goblet cells of Intestinal 676

Metaplasia are Alcian Blue positive Mucins. AB-PAS (X 100). 677

678

679


positive mucin AB-PAS (X 100). 681

682


of 34

683

Figure 11: Gastric Metaplasia - Gall Bladder. The Non - Neoplastic epithelium is HID 684

positive (Brown) Metaplastic epithelium Alcian Blue positive (Blue) HID-AB (X 100). 685

686

687


positive mucin. The columnar cells are HID positive (Brown) HID-AB (X 400). 689

690


of 34

691

Figure 13: Intestinal Metaplasia - Gall Bladder HID-AB (X 400). 692

journal name: international journal of hepatobiliary and ... accepted early view article page 1 of...

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