Morphometric Analysis of Histoarchitectural Changes in Cholecystitis

MORPHOMETRIC ANALYSIS OF HISTOARCHITECTURAL CHANGES IN CHOLECYSTITIS

DISSERTATION

SUBMITTED TO THE TAMILNADU DR.M.G.R. MEDICAL UNIVERSITY

CHENNAI

In partial fulfillment of the requirements for the degree of

M.D. (PATHOLOGY) BRANCH – III

DEPARTMENT OF PATHOLOGY

TIRUNELVELI MEDICAL COLLEGE HOSPITAL TIRUNELVELI – 627011

CERTIFICATE

This is to certify that the dissertation titled “MORPHOMETRIC

ANALYSIS OF HISTOARCHITECTURAL CHANGES IN

CHOLECYSTITIS” is a bonafide work done by Dr.R.PRIYADARSHINI,

Department of Pathology, Tirunelveli Medical College, Tirunelveli – 627011,

during her post graduate degree course from 2014 to 2017. This work has not

formed the basis for previous award of any degree.

Prof. Dr. K. SITHY ATHIYA MUNAVARAH, MD.,

Dean,

CERTIFICATE

I hereby certify that this dissertation entitled “MORPHOMETRIC

ANALYSIS OF HISTOARCHITECTURAL CHANGES IN

CHOLECYSTITIS” is arecord of work done by Dr.R.PRIYADARSHINI,

Department of Pathology, Tirunelveli Medical College & Hospital, Tirunelveli, in

partial fulfilment of university rules and regulations for the award of M.D.

DEGREE in PATHOLOGY BRANCH III,under my guidance and

supervisionduring the academic period from 2014 to 2017.

Prof. Dr.K. SHANTARAMAN,MD.,

Professor and Head,

Tirunelveli Medical College Tirunelveli-627011.

Prof. Dr. S. VASUKI,MD.,

DECLARATION

I solemnly declare that the dissertation titled “MORPHOMETRIC

ANALYSIS OF HISTOARCHITECTURAL CHANGES IN

CHOLECYSTITIS” was done by me at Tirunelveli Medical College, Tirunelveli

– 627011, during the period of 2014 to2017 under the guidance and supervision of

Prof. S. VASUKI.MD, to be submitted toThe Tamil Nadu Dr. M.G.R. Medical

University towards thepartial fulfilment of requirements for the award of MD

DEGREE in PATHOLOGY BRANCH-III.

Place : Tirunelveli Date :

Dr.R.PRIYADARSHINI,

ACKNOWLEDGEMENT

This thesis owes its existence to the help, support and inspiration of several

people. First, I thank Professor Dr. K. SITHY ATHIA MUNAVARAH, M.D.

Dean, Tirunelveli Medical College, for having permitted me to conduct the study

and use the hospital resources in the study.

I would like to express my sincere and heartfelt gratitude to Professor Dr.

SHANTARAMAN. K. MD, Professor and Head, Department of Pathology, for

his guidance, inspiration and suggestions for the development of this thesis

context.

I would like to express my special appreciation and thanks to my mentor

Professor Dr. S. VASUKI.M.D., and Professors DR. SWAMINATHAN.K.

M.D., DR.SURESH DURAI. J. M.D., DR. ARASI RAJESH.M.D., Department

of Pathology, for guiding me during the period of study.

I am extremely thankful to Professor DR. A.SELVAMURUGAN,

Professor and Head,Department of Forensic Medicinefor supporting me

academically and professionally during the period of study.

I also thank all the lab technicians especially Mrs. Veeralakshmi for their

valued assistance and my fellow postgraduates for their cooperation which

enormously helped me in the study.

Finally, but by no means least, my deepest gratitude goes to my husband

Dr. N. KARTHIKEYAN, mom and dad for their unflagging love and

ABBREVIATIONS

GB – Gall bladder

EHBD – Extra hepatic bile duct

IHBD – Intra hepatic bile duct

CBD – Common bile duct

LMW – Low Molecular weight keratin

EMA – Epithelial Membrane Antigen

CEA – Carcino Embryonic Antigen

R.A sinus – Rokitansky Aschoff sinus

DM – Diabetes Mellitus

Ca(HUCB)2 – Calcium monohydrogen bilirubinate

HPE – Histopathological Examination

MALT – Mucosa Associated Lymphoid Tissue

CLL – Chronic Lymphocytic Leukemia

SLL – Small Lymphocytic Lymphoma

IgG 4 – Immunoglobulin G 4

HMG- COA - 3-Hydroxy 3- Methyl Glutaryl Coenzyme A

CONTENTS

1. INTRODUCTION 1

2. AIMS OF THE STUDY 3

3. REVIEW OF LITERATURE 4

4. MATERIALS AND METHODS 45

5. OBSERVATIONS AND RESULTS 49

6. DISCUSSION 69

7. SUMMARY 75

8. BIBILIOGRAPHY

1

INTRODUCTION

Cholecystitis is often thought to be a major ailment in modern society.

Gallstones are more likely associated with humans from antique era, since they

have been found in the gallbladders of Egyptian mummies dating back to 1000

BC. Historical evidence suggests that Alexander The Great had suffered from

acute gall bladder infection[1]. Nowadays, it is a worldwide medical problem, with

wide areas of geographical distribution.

Recently, gallstones are becoming increasingly common due to sedentary

lifestyle and socio economic status. Its frequency has been strongly integrated

with the epidemics of obesity and the metabolic syndrome[2].

Risk factors associated with cholecystitis are multifactorial. Many risk

factors for gallstone formation are not modifiable such as ethnicity, older age,

female sex and family history or genetics. On contrary, modifiable factors like

diet, drugs, physical activity also contributes for stone formation. Other

attributable risk factors related to biliary sludge include pregnancy, drugs and total

parenteral nutrition or starvation.

Certain diseases like cirrhotic liver, chronic hemolytic diseases and ileal

Crohn’s disease are more prone for black pigment stones. Gallstone disease in

childhood, was once considered rare but its occurrence has been increased these

days because of lifestyle modification particularly childhood obesity[3].

Population-based surveys have uncovered an overall increase in mortality

2

incidence of gallstone disease, there is a concomitant increase in certain

complications like gallstone-related pancreatitis[4].

Gall bladder is one of the commonest specimens received in the

histopathology laboratory. Cholecystitis is classified based on the presence of

stones as calculous (cholelithiasis) and acalculous cholecystitis. Both are

associated with significant macroscopic and microscopic changes.

Histopathological alterations such as epithelial hyperplasia, dysplasia with

erosion of mucosa and inflammatory cellular infiltrates were encountered in both

cholelithiasis and acalculous cholecystitis. Therefore it will automatically warrant

a study to distinguish and estimate the changes evident in both types. In the recent

decades,morphometrictechniques have been developed rapidly which permits

quantification of structural data facilitating correlation with functional studies.

Morphometry is a field concerned with studying variations and alterations

in forms such as size and shape. It can be used to assess the malignant nature of

cells and its classification by quantitative measurements of nuclei such as density,

area, etc. For example, in uveal melanoma, the nuclear perimeter shows good

correlation with prognosis[5]. In addition, it can also be used to estimate the

predominant alteration with statistical evidence. With the recent advent of

personal computers, morphometric measurement could be easily merged with the

computer for rapid and better analysis.

This article gives a valuable audit on calculous and acalculous cholecystitis

and spotlights current information about the pathogenesis, risk factors,

histopathological changes and morphometric analysis of the predominant

3

AIMS OF THE STUDY

The aim of this study is

1) To morphometrically analyse the histomorphological pattern in the wall

of human gall bladder in cholecystitis.

2) To compare them statistically with normal human gall bladder through

4

REVIEW OF LITERATURE

HISTORY :

Cholecystectomy is the commonest operative procedure of biliary tract and

second most common surgery performed today[6]. History of surgery is weighed

down with names of well known German specialists like Kocher, Czerny,

Courvoisier and Mikulicz.In 1687,Stal Pert Von Der Weil while operating a

patient with purulent peritonitis inadvertently discovered gall stones[1]. But Carl

Langenbuch spearheaded the idea and executed first gall bladder (GB) extraction

in 1882 [7].

In 1987, Philippe Mouret in France did the first laparoscopic

cholecystectomy which immediately reformed the treatment of gall stones[8].

Today laparoscopic cholecystectomy is the preferred treatment for symptomatic

gall stones.

EMBRYOLOGY :

Embryologically, GB and biliary tree anlage begins around fourth week of

gestation. They are endodermal in origin and develop as a ventral bud known as

hepatic diverticulum[9]. It stretches out into septum transversum as rapidly

multiplying cell strands and partitioned into two parts(FIG. 1) - a larger cranial

part pars hepatica which is the liver primordium and smaller caudal part pars

5

At 29 days of gestation, pars cystica will start protruding as anterolateral

dilatation which later develops into GB along with a cystic duct visible at 34

days[11]. Initially GB is an empty organ however as a consequence of multiplying

strands of epithelial lining, it turns out to be briefly solid. Further recanalization

happens by vacuolation of epithelium .

FIG.1 – Diaphragmatic representation of hepatic diverticulum.

The external layers of GB wall and cystic duct develop from consolidating

mesenchyme around the epithelial mass. Myoblasts develop around 30mm stage

resulting in establishment of each of the three layers of wall of future gall bladder

6

For up to 56 days of gestation, the extra hepatic biliary tree (EHBD)

develops from extension of the caudal part of the hepatic diverticulum. From the

beginning, it remains patent and in coherence with the developing liver at all

stages.By 12 weeks of gestation, tubular structures are formed from the distal part

of right and left hepatic ducts.

The intra hepatic bile ducts (IHBD) require finely tuned epithelial –

mesenchymal interactions for its developement, which continue from the hilum of

the liver toward its fringe along the branches of the developing portal vein[12].

ANATOMY :

Gall bladder is a pear shaped organ that lies in the shallow fossa on visceral

surface of liver[13].It usually lies at junction of right lobe and medial lobe of left

lobe[14] (FIG. 2). In adult, it measures 7 to 10 cm in length, 3 to 4 cm in width and

thickness of 1 to 2 mm[15].

Its capacity is usually 40 to 70 ml [15]. But when obstructed, it can distend

markedly and contain upto 300 ml[16]. It is divided into four anatomic areas

7

FIG. 2 – showing anatomy of gall bladder and its relations.

FUNDUS :

It is the rounded blind end that usually extends 1 to 2 cm beyond hepatic margin.

8

FIG. 3 – Parts of gall bladder – fundus, body and neck.

CORPUS :

The body extends from fundus and tapers into neck connecting with the

cystic duct. It contains most of the elastic tissue and therefore serves as the main

storage area.

NECK :

The neck lies in the most profound part of GB fossa and extends into free

portion of hepatoduodenal ligament. True glands are present only in the neck.

INFUNDIBULUM :

The infundibulum or Hartmann’s pouch is an out pouching of wall in the

region of neck. It is the result of some pathological conditions such as dilatation

9

BLOOD SUPPLY

ARTERIAL SUPPLY :

GB is supplied by cystic artery, usually given off in Calot’s triangle. It is a

branch of right hepatic artery. The course of cystic artery may vary and enters GB

in the body or neck[17]. It is divided into anterior and posterior branches.

VENOUS DRAINAGE:

It is carried through small veins that enter directly into liver. Rarely, a large

cystic vein carries blood back to portal vein [19].

LYMPHATIC DRAINAGE:

Lymphatics drain into nodes present at the neck of GB. From here it flows

to nodes near hepatic hilum and in inferior portion of hepato– duodenal ligament

reaching the nodes on celiac axis[20] .

NERVE SUPPLY :

Nerves of GB arises from vagus and from sympathetic branches T8 &

T9 that pass through the celiac plexus[21] .

BILE DUCT:

Bile duct comprises intra hepatic and extra hepatic bile duct (EHBD).

The EHBD consists of right hepatic and left hepatic duct, common hepatic duct,

cystic duct, common bile duct (CBD) or choledochus. The CBD enters second

10

HISTOLOGY :

GB is a muscular sac. The wall of GB is composed of mucosa comprising

surface epithelium & lamina propria (LP), smooth muscle – tunica muscularis,

perimuscular subserosal connective tissue&serosa. There is no submucosa or

muscularis mucosa (FIG. 4). True glands are present only in the neck and are of

tubulo-alveolar type[23].

MUCOSA :

The mucosa is thrown into variably sized branching folds lined by single

layer of columnar cells(FIG. 4) having pale cytoplasm with occasional small

apical vacuoles & basally located nuclei . Ultra structurally, these cells have

abundant micro villi with filamentous glycocalyx on apical surfaces and core

rootlets. Small darker staining columnar cells called pencillate cells are scattered

among columnar cells.Tubulo alveolar mucous glands are seen only in the neck of

GB.

Histochemically, the epithelial cells contain mainly sulfomucin in contrast

11

FIG 4 – showing normal histology of GB, mucosa thrown into folds, lamina propria and tunica muscularis.

IMMUNO HISTOCHEMISTRY :

By IHC, the epithelial cells are reactive to both Low molecular weight

keratin (LMW) and Epithelial membrane antigen (EMA). Carcino embryonic

antigen (CEA) positivity is confined to apical cells[23].

LAMINA PROPRIA :

It lies between surface epithelium and muscularis and fills the cores of

mucosal folds. It is composed of loose connective tissue, lymphocytes, plasma

cells & macrophages. Nerve fibers, small blood vessels and lymphocytes are also

12

MUSCULAR LAYER :

It is made up of haphazardly arranged bundles of smooth muscle

fibers.Unlike in GI tract, the GB has only one muscle layer, tunica muscularis.

They are highly porous, allowing numerous invaginations from the mucosa.

Mucosal invaginations known as RokitanskyAschoff (R.A) sinuses (FIG. 13) may

be seen in normal GB but more prone in inflammation[25].

PERIMUSCULAR LAYER :

This sub serosal layer contains variable amount of fibro elastic & adipose

tissueas well as nerve cells. Bile ductules also known as Luschka ductules (FIG.

5) which are thought to be remnants of primordium liver may likewise be seen [26].

They are lined by cuboidal epithelium alike intra hepatic bile duct surrounded by

collar of fibrous tissue. They have no relation with R.A sinuses. They have been

seen in 10 % of cholecystectomy specimens[23].

SEROSA :

It covers the entire unattached GB surface. When GB is attached to liver

13

FIG 5– Luschka ductules seen in perimuscular layer at the hepatic surface of gall bladder.

EHBD :

It is lined by single layer of tall epithelium similar to GB.The epithelium

invaginates into the stroma known as Sacculi of Beale surrounded by mucinous

glands[27].

CHOLECYSTITIS :

It is defined as inflamed GB in which inflammation may be acute, acute on

chronic, or chronic. 90% cases involve stones in GB i.e calculous cholecystitis

14

GALL STONES :

EPIDEMIOLOGY :

Gall stone is a vital problem affecting 5 – 20% of adults[29].The prevalence

ranges widely across ethnicities and approximately twice as high in women as in

men[30]. It is considered as one of the leading causes of gastrointestinal

problems[31].They are often asymptomatic but 1- 3% of patients per annum may

present with symptoms[32].

RISK FACTORS :

Risk factors are multifactorial and classified as modifiable and non –

modifiable factors[33]. (Table 1)

1) ETHNICITY :

Geography and particularly ethnicity contribute to prevalence of gallstone

disease and also the type of stones. Cholesterol gallstones are more common in

the developed countries of the Western world while brown pigment stones in the

bile ducts in Asia[29]. White Americans show an overall predominance of 16.6% in

women and 8.6% in men[34]. Intermediate prevalence rates occur in Asian[35]and

Black Americans while Sub-Saharan Black Africans have very low frequencies

15

Table 1 – Risk factors of gall stones.

NON MODIFIABLE MODIFIABLE

GENETICS OBESITY

ETHINICITY REDUCED ACTIVITY

FEMALE TPN

FAMILY HISTOTY DIET

AGE DRUGS

2) GENETICS :

Genetic susceptibility is a key factor in gallstone formation. Familial

studies reveal an increased frequency of nearly five times increased risk in the

relatives of gallstone patients. These rates are even higher in monozygotic twins

than dizygotic twins[37].

3) AGE :

The frequency of gallstones markedly increases with age which are 4 to 10

times more likely in older individuals[38]. It also corresponds with the type of

stone formation initially being composed predominantly of cholesterol but later

16

4) GENDER :

Women show increased frequency for gall stones formation among which

premenopausal are at higher risk[33]. Risk factors include parity, oral

contraceptives and estrogen replacement therapy[40]. Female sex hormones show

strong association with GB function and bile secretion. Estrogen acts by

augmenting the secretion of cholesterol and decreases bile salt formation, whereas

progestins reduces bile salt secretion impeding GB emptying leading to stasis[39].

5) OBESITY :

Obesity, in particular, centripetal obesity, is a common risk factor for gall

stone disease[41]. It affects the rate limiting step in cholesterol synthesis by

enhancing HMG COA reductase activity. It eventually promotes increased

cholesterol synthesis in the liver and its secretion into bile[42].

6) DYSLIPIDEMIA & DIABETES MELLITUS :

Gallstone disease is a metabolic problem, in association with lipid

abnormalities & diabetes mellitus. A low HDL cholesterol[43] and

hypertriglyceridemia[44] carry an increased risk of developing stones. In DM,

insulin resistance alters cholesterol and bile salt metabolism predisposing to gall

stone formation[45].

7) DIET & TOTAL PARENTERAL NUTRITION :

Diets rich in cholesterol[46], fatty acids[47], carbohydrates[48] and legumes[49]

are associated with the risk of development of cholelithiasis whereas unsaturated

17

the other hand, is a risk factor for developing microlithiasis and gallstone disease

besides acute acalculous cholecystitis[55].It may be due to loss of the enteric

stimulation of the GB which occurs as a result of starvation, prompting

gallbladder stasis[56].

8) LIFESTYLE :

The relationship between socioeconomic status and gallstones is still

controversial[33]. Reduced physical activity increases the risk of gallstone disease

and increased physical activity helps in preventing cholelithiasis[55].

9) DRUGS :

Certain medications like estrogen therapy, thiazide diuretics[57],

ceftriaxone[42] and octreotide[58] show predilection for gall stone formation

whereas statins reduces the risk[59].

10) UNDERLYING ILLNESS :

Advanced cirrhosis[60], Crohn’s disease[61], cystic fibrosis[62] and other

injuries such as spinal cord injuries[63] are well known risk factors.

PATHOGENESIS :

Gall stones are formed of abnormal constituents of bile. Biliary sludge

precedes stone formation which contains mucous gel, hydrophobic bile pigments,

cholesterol lecithin liquid crystals and solid cholesterol mono hydrate crystals[64].

First, cholesterol precipitates into crystals when its concentration in bile exceeds

the solubilizing ability of bile salts and phospholipids. Biliary accumulation along

18

and mucous hypersecretion facilitating stone formation[65]. There are two different

types of stones - cholesterol stones and pigment stones.(Table 2)

CHOLESTEROL STONES :

Cholesterol stones comprises of 70% of gall stones in western countries[66].

Its formation is related to super saturation of bile with cholesterol and rapid

nucleation of cholesterol crystals[67]. Other mechanisms include GB hypomotility

resulting in stasis.More than 80% of cholesterol stones lack calcium carbonate and

hence radiolucent[23].

MORPHOLOGY :

Pure cholesterol stones accounts for 10% of gall stones. They are

yellowish to white, round to ovoid, crystalline or laminated cut surface measuring

upto 4 cm[23] (FIG. 6).

Stones with lower cholesterol level are designated as mixed. Depending on

the proportion of calcium carbonate, bilirubin and phosphates, mixed stones may

be lamellated, greyish white to black in colour. They are usually smaller and

19

Table.2 – Classification of stones.

PIGMENT STONES :

It is classified as black stones which are hard in consistency and brown

stones which are soft.

BLACK STONES :

Black stones form in sterile GB bile. It contains enough calcium carbonates

and phosphates to render them radiopaque[68].It is associated with states of

hyperbilirubinemia [69],all hemolytic anemias like spherocytosis[70],sickle cell

disease[71], thalassemia[72], malaria[73]and foot trauma in long distance runners[74]. GALL STONES

PIGMENT STONES

BLACK STONES

BROWN STONES

20

FIG. 6 – showing multiple cholesterol stones each measuring less than 4 cm.

MORPHOLOGY:

Black stones are black or deep brown, (FIG 7) relatively small measuring

about 2-5 mm. They are brittle and sometimes spiculated. On fracturing, these

stones have a glass like appearance[23].

BROWN STONES :

Brown stones are more common in Asia[33]. It forms secondaryto stasis,

anaerobic bacterial infection and parasitic infestation from nematodes and flukes

21

FIG. 7 – showing multiple black pigment stones.

It contains unpolymerized Ca(HUCB)2 and calcium soapscontaining

amorphous calcium salts of palmitate and stearate rendering them as

radiolucent[68].Parasites in the bile duct may induce stone formation by the

calcified overcoat of parasite’s egg which serves as a nidus and enhance

precipitate of calcium bilirubinate[66].

MORPHOLOGY :

These stones are much softer, laminated and may have soap like or greasy

consistency[75] (FIG. 8)

CLINICAL PRESENTATION :

Majority of patients have silent stones without any symptoms and may not

22

repeated episodes of right upper quadrant or epigastric pain accompanied by

nausea or vomiting [77]. Often attacks occur after fatty meals and almost at

night[78].

FIG. 8 – shows multiple soft brown pigment stones.

Sometimes, patients may experience referred pain between the shoulder

blades or below right shoulder region (Boas sign)[79]. On examination, there will

be tenderness in right quadrant (Murphy’s sign)[80]. Occasionally, gall stone ileus

may occur by a large stone eroding the adjacent loop of small bowel which in turn

results in intestinal obstruction (Bouverets syndrome) [81].

ACALCULOUS CHOLECYSTITIS :

Cholecystitis can occur without the evidence of gall stones. It is

23

hyperalimentation. Extensive burns, sepsis, major operations and multiple trauma

are at higher risk. Decreased GB perfusion and ischemia caused by shock,

congestive cardiac failure and arteriosclerosis are the contributing mechanisms[82].

It usually presents with nonspecific clinical features similar to gall stone disease.

Microscopically, it shows leukocyte margination of blood vessels,

lymphatic dilatation and bile infiltrating deeper into the muscular layer[83]. There

will be associated edema of serosa and muscular layers with patchy thrombi of

arterioles and venules[14]. Due to nonspecific presentations, diagnosis will be

delayed and tends to have fulminant complications such as gangrene, perforation

and empyema. Majority of the patients will improve by percutaneous

cholecystostomy.

ACUTE CHOLECYSTITIS :

It is an acute inflammation of GB.It is more often a clinical entity and does

not necessarily imply acute inflammation of GB i.e. neutrophils are often

lacking[23].

It is precipitated by obstruction of stones in neck or cystic duct (90%) and

rest includes acute acalculous cholecystitis[84]. Its etiology may not be

demonstrable but it is typically seen in debilitating patients[85].

PATHOLOGIC FINDINGS :

The gross and histopathological findings of GB wall in both acute

24

GROSS FINDINGS :

GB is usually enlarged, tense and external surface shows bright red or

green black discolouration[75].

HISTOPATHOLOGY :

In acute cholecystitis, the mucosa is often eroded and shows prominent

edema, congestion, hemorrhage and fibrin deposition in all layers(FIG. 9). In

extreme cases, necrosis and neutrophils are seen[23]. Sometimes fibrin thrombi

may be seen in small veins[20].On addition, the adventitia shows activated myo

fibroblastic cells in a loosely edematous stroma which is considered as one of the

hallmarks of acute cholecystitis[23]. (FIG. 10)

25

VARIANTS :

The variants of acute cholecystitis are :

1) Empyema of GB

2) Acute emphysematous cholecystitis.

EMPYEMA OF GB :

It is a severe form of acute calculous cholecystitis with superadded

suppuration[86]. In other words, it is an inflammation of GB with contained pus.

The presentation is often atypical[87]. Microscopically, the mucosa is often

extensively ulcerated and contains numerous neutrophils[23]. Laparoscopic

cholecystectomy is contra indicated because of fear of life threatening

complications[88]. It carries high mortality rate[87].

26

ACUTE EMPHYSEMATOUS CALCULOUS CHOLECYSTITIS :

It is a life threatening form of acute calculous cholecystitis caused by

presence of gas on GB lumen or wall[89]. Clostridium species, Escherchia coli,

streptococcus and Klebsiella are the most common gas forming organism[90]. It is

a consequence of ischemic injury associated with predisposing conditions like

atherosclerosis, arterial embolism, vasculitis[91]. It is more common in older

patients with DM[89]. On HPE, the inflammatory reactions are histologically

indistinct and consists of usual pattern of acute inflammatory changes[75] and

ischemic changes formed by progressive vascular incompetence leading to

necrosis and perforationof GB wall. It is associated withcomplications like

perforation and abscess formation leading to peritonitis and carries high mortality

rate[92].

CHRONIC CHOLECYSTITIS :

ETIOLOGY :

It is often associated with cholelithiasis. It occurs as a result of chronic

irritation of GB wall by recurrent attacks of cholecystitis secondary to transient

obstruction by gallstones[93].

EPIDEMOLOGY :

Women are more affected than men. It usually occurs in 5th to 6th

decade. It is strongly associated with GB cancer with an incidence ranging from

27

CLINICAL FEATURES:

Intermittent or constant pain in right upper quadrant is the most specific

symptom. Many patients have nausea with colic pain. Other non-specific

symptoms are belching, bloating, flatulence or upper abdomen fullness. A

palpable GB mass in upper abdomen can sometimes be seen on clinical

examination suggesting chronic hydrops[94].

PATHOPHYSIOLOGY :

Characteristic finding of chronic calculous cholecystitis is fibrosis which leads

to shrunken or contracted GB[93].

GROSS :

Calculi are present in greater than 90% of cases.GB is itself normal or

show thickening of wall. In more severe cases the GB may be shrunken with

marked fibrosis, thickening and serosal scarring [23].

PORCELAIN GB :

Long standing chronic calculous cholecystitis may develop mural

calcification and referred as porcelain GB. Its prevalence is 1 – 2% in chronic

choloecystitis specimens. It is strongly associated with GB cancer with an

incidence ranging from 12-61%. Grossly, it appears bluish and brittle in

28

FIG. 11– porcelain GB with brittle in nature.

HISTOPATHOLOGY :

On HPE, the epithelium may be relatively normal, atrophic and show

hyperplastic or metaplastic changes[95]. Metaplasia can be of intestinal with goblet

cell (FIG. 12) or pyloric (antral), the former being accompanied by paneth cells

and endocrine cells. Of these, pyloric metaplasia is more common[96].R.A sinuses

(FIG.13) results from increased intra luminal pressure. It is a pseudo diverticula

formed by deeply invaginating mucosa without a complete muscular coat. It is

common finding and more prominent in severe cases[23]. Luschka’s ducts are

29

Fig.12– shows intestinal metaplasia of GB with paneth cells.

The degree of chronic inflammation varies with predominance of

lymphocytes and some plasma cells, macrophages and occasionally eosinophils.

When neutrophils are present, a diagnosis of chronic active cholecystitis may be

30

FIG. 13– showing R.A sinuses associated with thickened wall and inflammatory cells

In some cases, there will be collection of macrophages containing bile and

lipofuschin which is termed as cholegranulomatous cholecystitis[23].The different

31

VARIANTS :

1) Xanthogranulomatouscholecystitis.

2) Hyalinisingcholecystitis.

3) Follicular cholecystitis.

4) Diffuse lymphoplasmacyticcholecystitis.

5) Eosinophiliccholecystitis.

6) IgG4 related sclerosingcholecystitis

1) XANTHOGRANULOMATOUS CHOLECYSTITIS :

It is considered as an uncommon variant and clinically similar to acute or

chronic cholecystitis. Grossly there is large or small contracted GB usually

associated with cholelithiasis and greyish yellow nodules in outer layers.

Recurrent inflammation and calculi incite degeneration and necrosis of GB wall

which subsequently leads to intra mural abscess and eventual replacement by

32

FIG.14 – shows foamy macrophages containing bile and cholesterol clefts.

2) FOLLICULAR CHOLECYSTITIS :

It is an extremely rare entity seen in older age group usually in mid

60s[99]. Originally described in typhoid fever, it is now known to occur in gram

negative infections[100]. In normal GB mucosa, lymphoid tissue is very sparse.

Some intra epithelial lymphocytes may be evident among surface columnar cells

but lymphoid follicles are never seen[101]. In this variant, there is widespread

formation of lymphoid follicles(FIG.15) in all layers of GB[102]. It is

33

masquerading as cholecystitis are MALT, follicular lymphoma, mantle cell

lymphoma, CLL / SLL[104].

FIG.15 – shows prominent lymphoid follicles in lamima propria.

3) DIFFUSE LYMPHOPLASMACYTIC CHOLECYSTITIS :

It occurs sporadically in almost 7% of GB resected for cholecystitis[105].It

was initially related with chronic inflammation in patients with primary sclerosing

cholangitis[106]. On HPE, it shows triad of diffuse chronic inflammatory infiltrate

with predominant plasma cells accompanied by nodules of mucosal lymphocytes

34

It is a unique form of cholecystitis charcterised by diffuse inflammatory infiltrates

confined to lamina propria not extending to deeper layers[23].

FIG.16 - shows mixture of lymphocytes and plasma cells forming a band in mucosa sparing the deeper layers.

4) EOSINOPHILIC CHOLECYSTITIS :

It is an uncommon condition first described in 1949[108]. Histologically,

eosinophilic cholecystitis is distinguished by a dense transmural infiltration of

eosinophils without evidence of any infestation[109]. The eosinophilic infiltrates

35

It may be associated with parasitic infestations of liver and biliary tract.

It should be distinguished from Churg Strauss syndrome in which gall bladder is

involved in addition to granulomatous angitis and eosinophilia[23].

FIG.17 - shows predominantly esosinophilic infiltrates.

5) HYALINISING CHOLECYSTITIS :

It is a recently described rare subtype clinically referred as porcelain GB. It

is characterised by dense pauci cellular collagenous transmural fibrosis(FIG.18)

replacing the mucosa and muscularis propria[111]. It is often associated with

36

cell cytology, nuclear enlargement and nuclear axis parallel to surface helps in

distinguishing from their malignant counterpart[23].

FIG.18 – shows hyalinization entirely replacing the gall bladder wall.

6) IgG4 RELATED SCLEROSING CHOLECYSTITIS :

In IgG4 related sclerosing pancreatitis, patients may also show changes in

gall bladder in addition to duodenum and pancreas[113]. On HPE, it shows a band

of characteristic basophilic wavy sclerosis with plasma cells lying individually in

37

MORPHOMETRIC ANALYSIS :

HISTORY :

Since ancient era, the human body has been measured for several purposes.

In the older days, human body was measured for figurative arts. Eventually, the

methodology was trailed by the naturalist field and then by anthropologists to

identify basic morphological characteristics of human. The term anthropometria

dates back to the 17th century in the naturalist field, and was initially mentioned

in the short manual of Anthropometria by Johann Sigismund Elsholtz. It is the

first recorded material that researched the human body for scientific and medical

purposes. Elsholtz proposed that anthropometry could be applied in various fields

such as medical practices, physiognomy, the arts, and ethics[114]. By the

application of mathematics, geometry, and statistics, anthropologists proposed

human investigation methodologies.

MORPHOMETRY :

Morphometrics is a branch of anthropometry, which deals with the study of

size and shape of the components of various biological forms. It can also be

characterised as the quantitative analysis of organic structures. It focuses on

studying the variations and changes in forms (i.e., size and shape) of organisms.

The field has emerged rapidly over the last two decades. It is classified into

38

TRADITIONAL MORPHOMETRICS :

It comprises multivariate statistical tools to characterise shape by analysing

conventional estimations between points with biological and anatomical

implications to characterize shapes called landmarks. These measurements usually

depictsthe lengths and widths of various parameters and the distances between

specific landmarks, within and between various populations.[116]

METHOD :

It was done by measuring linear distances such as length, width, and

height. Multiple statistical tools were used to describe patterns of variation in

shape within and among groups. Occasionally,counts, ratios, areas and angles can

be measured.

MERIT :

The advantage of this technique was that it was extremelyfundamental and

simple, however it had certain challenges.

DEMERITS:

1. It is impossible to recoup the shape of the original form using the data

matrices of distance measurements. The overall form is neither archived

nor used in the analysis[117].

2. Linear distance measurements are usually analogous with size and this

39

3. In this technique, the data did not include the area from which the

measurements were taken. Therefore possibility of same results could be

generated from the measurements taken from two different shapes.

4. It was likewise impractical to restore graphical representation of shape

from the taken measurements[118].

MODERN MORPHOMETRICS :

Later, in 1960s, biometricians started applying multivariate statistical tools

to sets of traditional measurements. They used a set of landmarks to describe

shape. Landmark is described as a two or three-dimensional point by a tightly set

of rule. It is considered as more substantial than traditional morphometric methods

in ensuring morphological information and permitting for further analysis of this

information.These information might be two dimensional (2D) or

three-dimensional (3D) organizing with morphological point of interest[117].

It includes the development of powerful statistical methods based on

models that are used to examine the shape variation of all configurations that

correspond to morphologic landmark locations. Indeed, in many biological or

biomedical studies, the most efficient way to analyze the forms of biological

organs or organisms is by registering landmarks[115].

MERIT :

It records the geometry of surveyed objects, and preserves the information

40

DEMERIT :

Disadvantage is that a number of landmarks available can sometimes be

insufficient to capture the shape of an object.

PRINCIPLE :

The fundamental principles of morphometric measurements are :

1. Linear measurements determine the distance between two landmarks by

using an eyepiece graticule with an engraved scale or alternatively with a

ruler on a projected microphotograph.

2. Stereology based on geometric probabilityhighlights the features of

twodimensional images in three dimensions.

3. Computerized image processing and analysis system allows image capture,

stockpiling and analysis using specialized software and hardware.

APPLICATIONS :

Morphometric methods can be applied in creating methods for

identification of tumor in diagnostic context and in characterizing diagnostic

histopathology. In conventional method of subjective diagnostic histopathology,

reproducibility can be defined adequately but the definition of precision is

ambiguous. The pathologists may be interested in morphometry for checking an

intuitive and subjective evaluation of a diagnostic feature on microscopic section

41

A) STUDIES ON CLASSIFICATION OF TUMOR :

It is used to create a classification basis for diagnostic decisions through

two approaches :

1) Initially, it correlates the obtained morphometric information with

traditional tumor classification taking into account of subjective

histopathology.

2) Secondly, by attempting a classification by reviewing the morphometric

data.

It may include correlation of morphometric data with prognostic

parameters like life expectancy, response to therapy and metastasis prediction.

B) STUDIES ON DIAGNOSTIC HISTOPATHOLOGY :

In this technique, the morphometric descriptors are defined to identify

certain type of tumor. It also includes the search for relevant morphometric

parameters.

MORPHOMETRIC HISTOPATHOLOGY :

It deals with the study of certain parameters measured from tissue sections

or derived from the primary measurements estimated through calculations. For

example, in tumor, certain parameters such as nuclear density, nuclear area,

perimeters, cellularity and number of mitosis per area are measured. For rational

application of morphometric methods,it is very important to make distinction

42

GROUP MORPHOMETRY :

It includes the study of multiple samples from different specimens.Then

the pathologist tries to achieve classifications by using obtained morphometric

methods.Finally based on the data from various samples, the results are

interpreted. This method is controlled and the measurements are mostly carried

out by one and the same investigator who is acquainted with the technique

involved.

DIAGNOSTIC MORPHOMETRY :

On the other hand,it is a process that takes place in a number of different

labs. It is carried out by many investigators and various techniques. This results in

variation in tissue processing, section thickness, and staining intensity[119].

MORPHOMETRIC PARAMETERS :

In HPE, the sections studied are usually two dimensional. Indeed there are

certain conditions in which one may need to characterize the tissue in three

dimensional evaluation. It involves volumetric measurements of tissue,

parameters per tissue volume or 3D structure of tissue. For these, stereological

parameters are recommended. But stereological parameters are always not

necessary for diagnostic context.

REPRODUCIBILITY AND ACCURACY :

In morphometric methods, both reproducibility and accuracy can be

estimated. By estimating reproducibility, we can compare the results of subjective

43

reference by standard units of measurements to estimate the accuracy. However

morphometric methods do not give diagnosis directly[5].

COMPUTER ASSISTED MORPHOMETRY :

With the approach of computers, applications for morphometric analysis

became simpler. Compared to conventional methods, computer-assisted

morphometry is exponentially quicker, more accurate, meticulous, and efficient in

providing a broad scope of estimations of morphological parameters. Using this

methodology, subjective, perception based interpretation have been replaced by

objective quantification. It depends on various factors such as image acquisition,

image display,transmission of images, storage and retrieval of images[120].

A) IMAGE ACQUISITION :

In this technique, the microscopic image is connected to the capturing

device. Devices for acquisition images range from electronic still to video

cameras which obtain electronic images directly in real time. It converts

photographs to digital data suitable for display and storage by computer.

B )IMAGE DISPLAY :

Captured images are usually displayed on computer display monitors. It

depends on the screen size, dot pitch and pixel resolution of the video output card.

C) IMAGE ANALYSIS :

It involves two distinct activities – image processing and morphometry.

Image processing may be either hardware based ie. incorporated into cameras,

44

cropping, rotation, labeling, arrows, horizontal and vertical reflection. Further it

allows for quantification studies like morphometric analysis using the processed

digital data. Some microscope manufacturers like Zeiss and Olympus are offering

image analysis packages interpreted with their microscope.

CASE CONTROL STUDY :

To conduct case control study, the control should be free of disease. If the

study involves a large number, then one control should be used for each case. If

the study group is small, then 2 or more controls can be selected for each study

subject. In order toavoidselection bias, some epidemiologists prefer case from one

45

MATERIALS AND METHODS

This study deals with the methodology and comparative study on

morphometric analysis of histoarchitectural changes in cholecystitis. The present

study was carried out at the Department of Pathology at Tirunelveli Medical

College and Hospital during October 2014 to August 2016. The study population

includes case and control groups. Cases include gall bladder specimen operated

for cholecystitis received from Department of Surgery in our hospital. Control

groups include normal human gall bladder obtained from autopsy subjects from

Department of Forensic Medicine in our hospital.

Inclusion criteria

This study includes

A. Cases with clinical diagnosis of cholecystitis and cholelithiasis irrespective

of age and sex.

B. Control group includes autopsy gall bladder specimens with no obvious

gross changes irrespective of age and sex.

Exclusion criteria

1) Autolysed specimens

2) All the cases of malignant gall bladder lesions

46

Age and Sex :

The age of case and control groups varied from 15 to 80 years. Study

populations were grouped into 4 groups as ≤ 20, 21- 40, 41 – 60, and > 60 based

on age and sex. The male patients were 25 and the remaining 50 were female with

male and female ratio of 1: 2.

In our case control study, 75 normal gall bladders (controls) and 75

cholecystitis specimens (cases) were studied. From each specimen, three sections

were taken from neck, body and fundus. After sectioning, tissues were processed

in an automated tissue processor and paraffin blocks were made. Tissue sections

of 3 mm were cut and stained with hematoxylin and eosin. Then the slides were

examined under light microscope and estimation was done in microns using IS

capture software to find out the predominant histopathological alterations in each

layers of gall bladder.

The predominant histopathological alterations observed in this study were

as follows:

HISTOPATHOLOGICAL ALTERATIONS:

EPITHELIUM:

Intact epithelium

Mucosal erosion

Epithelial distortion

47

LAMINA PROPRIA:

Thickness

Irregularity and gap

Lymphocytes

Macrophages

Plasma cells

MUSCULAR LAYER:

Rokitansky Aschoff sinuses

Macrophages

Plasma cells

Lymphocytes

SEROSAL LAYER:

Macrophages

Plasma cells

Lymphocytes

In epithelial layer, intactness and erosion of mucosa were measured in

microns. In addition to these, epithelial distortion like hyperplasia and metaplastic

changes like gastric and intestinal metaplasia were observed and estimated.

Thickness of lamina propria andirregularity and gap between the lamina propria

48

To count the number of inflammatory cells in each layers such as lamina

propria, muscular and serosal layer, 10 high power fields were chosen. In each

areas, the inflammatory cells such as lymphocytes, plasma cells and macrophages

were counted using 40 X magnification and average was taken.

Control specimens were grouped based on their age and gender. An

average value was calculated from the groups and considered as control values for

49

RESULTS AND OBSERVATIONS

In our case control study, 75 normal gall bladders(controls) and 75

cholecystectomy specimens such as calculous and acalculous (cases) were

studied. Out of 75 cholecystectomy specimens, 56 specimens i.e 74.66% were

associated with gall stones. Among these, 2 were reported as chronic follicular

cholecystitis(2.67%), 2 asacute on chronic cholecystitis(2.67%) and 52 as chronic

calculous cholecystitis(69.33%). Remaining 19 specimens(25.34%) were not

associated with stones, in which 2 were reported as chronic follicular

cholecystitis(2.67%) and 3 as acute on chronic cholecystitis(4%) and 14 as

chronic cholecystitis(18.67%).

With regards to gallstones, 36 were black stones(73.33%), 19 were

cholesterol stones(25.33%) and remaining one case comprised of both black and

cholesterol stones(1.33%). The age of case and control groups varied from 15 to

80 years with maximum frequency of 36 falls in age group of 41 – 60 years.

The mean age for calculous cholecystitis was 44.79 with SD of 13.53 and

50

TABLE 2 :AGE GROUP 1 : ≤ 20, GROUP 2 : 21 – 40, GROUP 3 : 41 – 60,

GROUP 4 : ≥60.

Age

group

1 2 3 4 Total p value

Control 2 23 35 15 75 <0.0001 pearson chi square test

Calculous 2 21 25 8 56

Acalculous 7 0 11 1 19

11 44 71 24

In our study, various histopathological alterations were observed in each

layers of calculous and acalculous cholecystitis and compared with control. In

both calculous and acalculous cholecystitis, epithelial hyperplasia, intactness,

minimum and maximum erosion were noted. Thickness of lamina propria and

mucosal invaginations called Rokitansky aschoff sinuses were found and

estimated. Maximum to minimum inflammatory infiltrates such as lymphocytes,

51

CHART NO. 1: GENDER DISTRIBUTION AMONG CASE GROUP

CHANGES IN EPITHELIUM :

In epithelial layer of calculous cholecystitis, predominant histopathological

alterations were estimated. Maximum epithelial erosion was calculated as 95.94%,

minimum epithelial erosion as 12.66%, maximum epithelial hyperplasia as

16.67% and maximum intactness as 87.33%. Epithelial metaplasia such as goblet

and intestinal type were not observed. Compared to control groups, it showed a

significant p value of < 0.0001.

In case of acalculous cholecystitis, maximum and minimum epithelial

erosion were calculated as 93.79% and 15.45% respectively. Epithelial distortion

like hyperplasia was noted and estimated as 1.44% and maximum intactness as

84.55%. Epithelial metaplasia was not found. It showed a significant p value of <

0.0001 when compared to control groups.

66.67% 33.33%

Gender

Females

52

CHART NO. 2: CHANGES SEEN IN EPITHELIUM OF CALCULOUS CHOLECYSTITIS

47.68

52.32

3.54 4.06

12.66

0.60 87.33

95.94

16.67

0.00 20.00 40.00 60.00 80.00 100.00 120.00

INTACT EROSION HYPERPLASIA

Axis Title

Epithelium

53

CHART NO. 3: CHANGES SEEN IN EPITHELIUM OF ACALCULOUS CHOLECYSTITIS.

CHANGES IN LAMINA PROPRIA :

Predominant histopathological alterations of lamina propria were found

and estimated. In calculous cholecystitis, maximum irregularity and gap between

the lamina propria and mucosal layer were estimated as 21.59% and maximum

lymphocytic infiltration as 94%. Percentage of plasma cells and macrophages 54.07 6.21 84.55 45.94 15.45 93.79

2.26 _1.44 3.60

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

Mean Minimum Maximum

Axis Title

Epithelium

54

were estimated as 24% and 15 % respectively. Compared to control groups, it

showed a significant p value of < 0.0001.

CHART NO.4: CHANGES SEEN IN LAMINA PROPRIA OF CALCULOUS CHOLECYSTITIS.

14.30

71.70

6.11 7.72

7.73

16

1 1

21.59

98

24

15

0.00 20.00 40.00 60.00 80.00 100.00 120.00

IRREGULARITY & GAP LYMPHOCYTES PLASMA MACROPHAGES

Axis Title

Lamina propria

55

CHART NO.5 : CHANGES SEEN IN LAMINA PROPRIA OF ACALCULOUS CHOLECYSTITIS.

In acalculous cholecystitis, maximum irregularity and gap between the

lamina propria and mucosal layer were estimated as 63.63% and maximum

lymphocytic infiltration as 98%. Percentage of plasma cells and macrophages

were estimated as 7% and 14% respectively. On comparison with control

groups,it showed a significant p value of < 0.0001. 38.76 62.42 4.83 5.42 13.34 32 2 ₁ 66.63 94 7 14 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

IRREGULARITY & GAP LYMPHOCYTES PLASMA MACROPHAGES

Axis Title

Lamina propria

56

CHART NO. 6: COMPARISON OF LAMINA PROPRIA THICKNESS IN CALCULOUS AND ACALCULOUS CHOLECYSTITIS.

In addition to the above criteria, thickness of lamina propria was estimated

and found to be an average of 332.16 microns in calculous cholecystitis whereas

367 microns in acalculous cholecystitis. 332.16

367

310.00 320.00 330.00 340.00 350.00 360.00 370.00

Calculus Acalculous

Axis Title

Thickness of lamina propria

57

CHART NO. 7: CHANGES SEEN IN MUSCULAR LAYER OF CALCULOUS CHOLECYSTITIS.

CHANGES IN MUSCULAR LAYER :

Predominant changes in muscular layer were observed and found. In

calculous cholecytitis, R.A sinuses were calculated as a maximum of 27.56%.

Infiltrates such as lymphocytes, plasma cells and macrophages were estimated as

maximum of 98%, 11% and 12% respectively. 7.64 66.02 4.74 5.56 3.06 26 1 1 27.56 98 11 12 0.00 20.00 40.00 60.00 80.00 100.00 120.00

R.A SINUS LYMPHOCYTES PLASMA MACROPHAGES

Axis Title

Muscular layer

58

CHART NO. 8: CHANGES SEEN IN MUSCULAR LAYER OF ACALCULOUS CHOLECYSTITIS.

In acalculous cholecytitis, R.A sinuses were calculated as a maximum of

4.14%. Inflammatory infiltrates such as lymphocytes, plasma cells and

macrophages were estimated in muscular layer as maximum of 86%, 8% and 9%

respectively. 3.76 53.26 4.22 5.17 3.37 16.00 1.00 1.00 4.14 86.00 8.00 9.00 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

R.A SINUS LYMPHOCYTES PLASMA MACROPHAGES

Axis Title

Muscular layer

59

CHART NO. 9: CHANGES SEEN IN SEROSA OF CALCULOUS CHOLECYSTITIS.

CHANGES IN SEROSAL LAYER :

Predominant alterations of serosal layer were found in calculous

cholecystitis and estimated as 88% maximum infiltration of lymphocytes. Others

like plasma cells and macrophage showed maximum infiltration of 12% and 24%

respectively. 47.25 10 88 4.94 1 12 7.42 1 24 0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00 100.00

Mean Minimum Maximum

Serosa

60

CHART NO. 10: CHANGES SEEN IN SEROSA OF ACALCULOUS CHOLECYSTITIS.

In acalculous cholecytitis, inflammatory infiltrates such as lymphocytes,

plasma cells and macrophages were estimated in serosal lasyer as maximum of

79%, 4% and 9% respectively. 43.84

11.00

79.00

2.25 5.00 _1.00 2.00 4.00

9.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00 90.00

Mean Minimum Maximum

Axis Title

Serosa

61

TABLE 3 : SHOWS DESCRIPTIVES

Descriptives

N

Mean S.D Min Max

EPI_INTA Control 75 100.00 0.00 100 100

Calculous 56 47.68 22.19 4.06 87.33

Acalculous 19 54.07 24.45 6.21 84.55

PI_EROS Control 0 . . . .

Calculous 56 52.32 22.19 12.66 95.94

Acalculous 19 45.94 24.45 15.45 93.79

EPI_DIST Control 0 . . . .

Calculous 28 3.54 3.69 0.6 16.67

Acalculous 7 2.26 0.78 1.44 3.6

LAM_THIC Control 75 203.01 62.10 110.8 410.1

Calculus 56 332.16 180.51 63.77 875.2

Acalculus 19 367.00 169.79 58.21 748.7

LAM_IR Control 0 . . . .

Calculous 6 14.30 5.38 7.73 21.59

Acalculous 4 38.76 29.11 13.34 66.63

62

Calculous 56 71.70 18.98 16 98

Acalculous 19 62.42 16.05 32 94

LAM_PL Control 0 . . . .

Calculous 36 6.11 4.70 1 24

Acalculous 12 4.83 1.64 2 7

LAM_MAC Control 0 . . . .

Calculous 39 7.72 3.27 1 15

Acalculous 12 5.42 4.36 1 14

MUSC_RA Control 0 . . . .

Calculous 14 7.64 6.32 3.06 27.56

Acalculous 2 3.76 0.54 3.37 4.14

MUSC_LYM Control 0 . . . .

Calculous 56 66.02 18.95 26 98

Acalculous 19 53.26 19.92 16 86

MUSC_PL Control 0 . . . .

Calculous 31 4.74 2.71 1 11

Acalculous 9 4.22 2.28 1 8

MUSC_MAC Control 0 . . . .

63

Acalculous 12 5.17 3.10 1 9

SER_LYM Control 0 . . . .

Calculous 56 47.25 21.65 10 88

Acalculous 19 43.84 19.74 11 79

SER_PL Control 0 . . . .

Calculous 18 4.94 3.04 1 12

Acalculous 4 2.25 1.26 1 4

SER_MAC Control 0 . . . .

Calculous 24 7.42 5.06 1 24

Acalculous 5 5.00 2.92 2 9

EPI – Epithelium, INTA - intact, EROS – erosion, DIST – distortion, LAM

– lamina propria, THIC – thickness, IR – irregularity & gap, LYM – lymphocytes,

PL – plasma cells, MAC – macrophages.

The average values for each statistical criteria in both calculous and

64

CHART NO. 11 : COMPARISION OF EPITHELIAL CHANGES BETWEEN CALCULOUS AND ACALCULOUS CHOLECYSTITIS.

47.68

52.32

3.54 45.94

2.26

0.00 10.00 20.00 30.00 40.00 50.00 60.00

INTACT EROSION HYPERPLASIA

Axis Title

Epithelium

65

CHART NO. 12 : COMPARISION OF CHANGES IN LAMINA PROPRIA BETWEEN CALCULOUS AND ACALCULOUS CHOLECYSTITIS

14.30

71.70

6.11 7.72

38.76

62.42

4.83 5.42

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00

IRREGULARITY & GAP LYMPHOCYTES PLASMA MACROPHAGES

Axis Title

Lamina propria

66

CHART NO. 13 : COMPARISION OF CHANGES IN MUSCULAR LAYER BETWEEN CALCULOUS AND ACALCULOUS CHOLECYSTITIS

7.64

66.02

4.74 5.56

3.76

53.26

4.22 5.17

0.00 10.00 20.00 30.00 40.00 50.00 60.00 70.00 80.00

R.A SINUS LYMPHOCYTES PLASMA MACROPHAGES

Axis Title

Muscular

67

CHART NO. 14 : COMPARISION OF CHANGES IN SEROSAL LAYER BETWEEN CALCULOUS AND ACALCULOUS CHOLECYSTITIS

47.25

4.94 7.42

43.84

2.25

5.00

0.00 10.00 20.00 30.00 40.00 50.00 60.00

LYMPHOCYTES PLASMA MACROPHAGES

Axis Title

SEROSA

68

TABLE 4 : STATISTICAL COMPARISON BETWEEN THE STUDY GROUPS

Multiple Comparisons

Bonferroni

Dependent Variable

(I) GROUP (J) GROUP SIG

EPI_INTA Control Calculous <0.0001

Acalculous <0.0001

Calculous Control <0.0001

Acalculous 0.408

Acalculous Control <0.0001

Calculous 0.408

LAM_THIC Control Calculous <0.0001

Acalculous <0.0001

Calculous Control <0.0001

Acalculous 0.975

Acalculous Control <0.0001

Calculous 0.975

LAM_LYM Control Calculous <0.0001

69

EPI - Epithelium, INTA – Intact, LAM – Lamina propria,THIC – Thickness,

LYM- Lymphocytes

Based on statistical evidence, the present study showed a significant p

value of < 0.0001 between control group and calculous cholecystitis. Similarly,

control group and acalculous cholecystitis also showed a significant p value of <

0.0001.

Calculous Control <0.0001

Acalculous 0.152

Acalculous Control <0.0001

70

FIG.19 :A case of calculous cholecystitis showing mucosal erosions and lamina propria thickness with dense inflammatory cells in lamina propria

and muscular layer (10X).

71

FIG. 21: Photomicrograph showing mucosal hyperplasia and dense inflammatory cells in lamina propria and muscular layer (10X).

FIG. 22: Photomicrograph showing mucosal erosions withR.A sinus ifiltrating into the muscular layer and inflammatory cells in lamina propria

72

FIG. 23: Photomicrograph showing inflammatory infiltrates predominantly lymphocytes, few macrophages and plasma cells in muscular layer(40X).

73

DISCUSSION

The study was conducted in Tirunelveli medical college hospital,

Tirunelveli, southern Tamilnadu on operated specimens of gall bladder for both

calculous and acalculous cholecystitis. A total number of 75 cholecystectomy

specimens and 75 controlspecimens obtained from autopsy subjects were

subjected to the histopathological examination and morphometric analysis of

predominant histoarchitectural changes.

In our study, majority of patients were between age group of 41 and 60

years with mean age being 45.58 with S.D of 12.38. The mean ageof the calculous

and acalculous patientswere 44.79 and 46.37 respectively. Tyagi SP et

al[122]studied morphological changes of GB in 415 cholecystectomy specimens at

Aligarh and showed the mean age was 43.6% with most of the cases were seen in

the 4th and 5th decades of life.

With regards to gender, there was a female preponderance in our study

with male to female ratio ratio of 1:2. Of the 75 patients, 66.67% were females

and 33.33% were male. Santhosh Upadhyaya Kafle[123] showed female

predominance in his study conducted in Nepal with male to female ratio of 1:7.16.

In the present study which includes 75 cholecystectomy specimens, 56 specimens

showed the presence of gall stones accounting for 74.66% whereas acalculous

cholecystitis constituted of remaining 25.34% specimens. Among the gall stones,

74

of mixed stones. Tadashi Terada[124] in his study showed 96% among the 540

cholecystectomy specimens in the population of Japan had gall stones among

which 47% were black stones, 19% were cholesterol stones and 33% were having

mixed stones.

In the present study, routine histopathological examination showed the

following lesions : chronic calculous cholecystitis(69.33%), chronic cholecystitis

without gall stones(18.67%), chronic follicular cholecystitis with gall

stones(2.67%), chronic follicular cholecystitis without gall stones(2.67%), acute

on chronic calculous cholecystitis (2.67%) and acute on chronic acalculous

cholecystitis(4%).

Santhosh Upadhyaya Kafle[123] in his study on the population of Nepal

observed acute on chronic cholecystitis without cholelithiasis (8%), chronic

acalculous cholecystitis with cholesterosis(22%), chronic cholecystitis without

cholelithiasis (50%), chronic cholecystitis with cholelithiasis (8%),

follicularcholecystitis (4%), xanthogranulomatous calculous cholecystitis(2%)

and without stones(6%).

In the present study, all the specimens were then subjected to

morphometric analysis using IS capture software to find out the histopathological

alterations in calculous and acalculous cholecystitis. Finally the results were

compared with age and gender based control specimens.

In our study, we observed that the predominant histopathological

alterations in epithelium were found to be erosion and hyperplasia. Maximum