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Manuscript Accepted Early View Article
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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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Manuscript Accepted Early View Article
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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
Perinthalmanna, Kerala, India. Email ID: [email protected] 15
4MBBS, MD, Assistant Professor, Department of Pathology, MES Medical College, 16
Perinthalmanna, Kerala, India. Email ID: [email protected] 17
5MBBS, MD, Assistant Professor, Department of Pathology, MES Medical College, 18
Perinthalmanna, Kerala, India. Email ID: [email protected] 19
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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TITLE: A histopathological and histochemical study of cholecystitis 65
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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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TITLE: A histopathological and histochemical study of cholecystitis 129
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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
415
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CONFLICT OF INTEREST 416
The authors declare no conflict of interests. 417
418
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23. Laitio M. Morphology and histochemistry of non tumorous gallbladder 480
epithelium. A series of 103 cases. Path.Res.Pract.1980 ; 167(2-4):335-345. 481
24. Hakkinen, I .,Laitio M, Epithelial glycoproteins of human gallbladder. Arch 482
Pathol.1970;90:137-142 483
25. Lee SP, Hypersecretion of mucus glycoproteins by the gallbladder 484
epithelium in experimental cholelithiasis. J Pathol 1981; 134:199-207. 485
26. Harvey PRC, Rupar C, Gallinger S, Petrunka CN and Strasberg SM. 486
Quantitive and qualitive comparison of gallbladder mucus glycoprotein from 487
patients with and without gallstones. GUT 1986; 27:374-81 488
27. Albores - Saavedra J, Nadji M, Henson DE, Ziegels – Weissman J, Mones 489
JM. Intestinal metaplasia of the gallbladder: A morphologic and 490
immunocytochemical study. Human Path 1986;17:614-20 491
28. Gupta SC, Misrav, Singh PA, Roy A, Misra SP , Gupta AR. Gallstones and 492
carcinoma gallbladder.Dig Dis Sci.2000;45:1061-1071 493
29. Basu R, Baig SJ,Biswas ,Das S, Baju R,Chattopadhya,YG Histopathological 494
changes in gall bladder mucus in cholelithiasis correlation with chemical 495
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
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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
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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
Manuscript Accepted Early View Article
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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
Manuscript Accepted Early View Article
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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
Manuscript Accepted Early View Article
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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
Manuscript Accepted Early View Article
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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
Manuscript Accepted Early View Article
Page 24 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%
Manuscript Accepted Early View Article
Page 25 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
Manuscript Accepted Early View Article
Page 26 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
Manuscript Accepted Early View Article
Page 27 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 7: Score 4 - About 75% of cells contain Mucin AB-PAS (X 100). 633
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
Figure 12: Intestinal Metaplasia - Gall Bladder Goblet cells contain Alcian Blue 642
Manuscript Accepted Early View Article
Page 28 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
Manuscript Accepted Early View Article
Page 29 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
Manuscript Accepted Early View Article
Page 30 of 34
662
Figure 5: Score I - About 5% of cells contain Mucin AB-PAS (X 100). 663
664
665
Figure 6: Score 3 - About 50% of cells contain Mucin AB-PAS (X 100). 666
667
Manuscript Accepted Early View Article
Page 31 of 34
668
Figure 7: Score 4 - About 75% of cells contain Mucin AB-PAS (X 100). 669
670
671
Figure 8: Score 5 - About 75-100% of cells contain Mucin HID-AB (X 100). 672
673
Manuscript Accepted Early View Article
Page 32 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
Figure 10: Intestinal Metaplasia - Gall Bladder Goblet cells contain Alcian Blue 680
positive mucin AB-PAS (X 100). 681
682
Manuscript Accepted Early View Article
Page 33 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
Figure 12: Intestinal Metaplasia - Gall Bladder Goblet cells contain Alcian Blue 688
positive mucin. The columnar cells are HID positive (Brown) HID-AB (X 400). 689
690