Macroanatomy

The foregut-derived gallbladder is a pear-shaped hollow viscus attached to the posterior visceral surface of the right lobe of the liver near the caudate lobe. The inferior aspect of the gallbladder is near the pylorus, duodenum and colonic hepatic flexure. The adult gallbladder measures on average 10 cm in length and 3–4 cm in width with a wall thickness of 1–2 mm. Its volume is usually 40–70 mL, sometimes increasing to 100 mL. The superior (hepatic) surface is adherent to the liver while the inferior (free) surface is peritonealized in continuity with the visceral peritoneum of the liver. The gallbladder has traditionally been divided into three parts: fundus, body and neck. The dilated blind end, known as the fundus, bulges out from the edge of the liver. The infundibulum is the tapered portion proximal to the neck. The neck connects into the cystic duct. Hartmann pouch, a small bulge where the neck joins the cystic duct, is thought to be a consequence of chronic inflammation. Smooth muscle bundles in the cystic duct region form the spiral valves of Heister, which help control release of bile from the gallbladder. The cystic duct usually measures 3 cm in length before its confluence with the common hepatic duct to form the common bile duct.

Arterial blood is provided by the cystic artery, which usually branches from the right hepatic artery. The gallbladder’s veins drain into the liver through the hepatic surface. Most of the gallbladder’s lymphatics drain to lymph nodes in the region of the neck and cystic duct; the remaining lymphatics drain to hepatic hilar or inferior hepatoduodenal ligament and pancreatoduodenal lymph nodes and ultimately to the celiac axis.

Microanatomy

The gallbladder wall is composed of mucosa (surface epithelium and lamina propria), a layer of loose smooth muscle bundles (tunica muscularis), perimuscular connective tissue (subserosa or adventitia) and (on the free surface) the serosa. The gallbladder does not have a muscularis mucosae or submucosa ( Fig. 10.1 ).

Normal gallbladder histology. The degree of distension of the gallbladder helps determine the prominence of the mucosal folds. The lamina propria contains very few mononuclear inflammatory cells; stromal cells account for most of its cellularity. Note the porosity between the loosely-arranged smooth muscle bundles of the tunica muscularis. The fibrous connective tissue directly beneath the tunica muscularis is relatively more condensed than the looser fibroadipose tissue found deeper in the perimuscular connective tissue. (Haematoxylin and eosin. [H&E] stain.)

Mucosa

The mucosa is variably folded, more so when the gallbladder is contracted. The mucosa is lined by columnar epithelial cells with pale eosinophilic cytoplasm and basally located nuclei containing inconspicuous nucleoli ( Fig. 10.2 ). The epithelial cells mainly contain acidic sulfomucins (predominantly MUC5B), contrasting with the mucin of metaplastic glands. These cells do not have the characteristic acidophilic granularity (abundance of mitochondria) characteristic of the absorptive cells of the small intestine, and compared to gastric foveolar cells, they are less mucinous. Scattered among these columnar cells are thin, slender, darker cells designated ‘pencillate’ (or ‘peg’ or ‘dark’) cells, the nature of which is unclear, differing in only a few ways enzymatically and ultrastructurally from the columnar cells ( Fig. 10.2 ). Just above and oriented parallel to the basement membrane are a few randomly dispersed, inconspicuous, oval-shaped cells sometimes referred to as ‘basal’ cells, although there is no continuous basal layer in the gallbladder mucosa. Scattered T lymphocytes may be present in the epithelium. There are no melanocytes or goblet cells in the normal gallbladder. Tubuloalveolar mucous glands (which histologically resemble pyloric metaplasia that occurs in chronic cholecystitis and cholelithiasis but differ ultrastructurally and histochemically) are present in the neck region only ( Fig. 10.3 ). Gastric and intestinal metaplasia may occur in chronically irritated gallbladders.

Normal gallbladder, high magnification. Tall, thin, dark pencillate cells are scattered among the columnar epithelial cells. Note the scant cellularity of the lamina propria. (H&E stain.)

Peribiliary mucous glands. Peribiliary mucous glands are small, round, and composed of cuboidal cells containing abundant apical pale to basophilic mucin and basal nuclei, closely resembling pyloric glands. The glands are arranged in a lobular architecture and are separated from one another by fibrous stroma. Such glands are found mainly in the neck of the gallbladder, as well as throughout the extrahepatic bile ducts. (H&E stain.)

Perimuscular connective tissue and serosa

Nerves, vessels, ganglia and paraganglia reside within the variable amounts of fibroelastic and adipose tissue that compose the perimuscular connective tissue of the gallbladder. On careful inspection the perimuscular tissue has two subtle layers: a thin, denser layer adjacent to the muscularis and a looser, adipocyte-rich zone. The inner fiber-rich layer also often contains thick-walled medium-sized vessels. The interlobular connective tissue of the liver is continuous with the perimuscular connective tissue of the gallbladder along the hepatic surface. Paraganglia are usually concentrated in subserosal or perimuscular connective tissue. Ganglion cells are most concentrated in the gallbladder neck and may be in the perimuscular connective tissue as well as within the lamina propria and tunica muscularis. The perimuscular connective tissue (particularly adjacent to the liver) may contain bile ductules, known as Luschka ducts, thought to represent remnants of the embryonic hepatic primordium ( Fig. 10.4 ). They are lined by cuboidal biliary epithelium and are cuffed by fibrous tissue. They may measure up to 1–2 mm and have been reported in 10% of cholecystectomy specimens. In the setting of cholecystitis, the ducts may have a pseudoinfiltrative appearance. Luschka ducts do not communicate with the lumen or Rokitansky–Aschoff sinuses. Pericholecystic lymph nodes may contain lipogranulomas, as does any intra-abdominal lymph node above the ligamentum teres.

Luschka ducts. Variably sized biliary-type glands are present in the deep perimuscular connective tissue on the hepatic aspect of the gallbladder. These are thought to be derived from the liver tissue and do not communicate with the gallbladder lumen or with Rokitansky–Aschoff sinuses. (H&E stain.)

Immunohistochemistry

The columnar epithelial cells of the gallbladder stain for keratin 7 (K7) but usually not K20. These cells also express cell adhesion molecule (CAM) 5.2 and anion exchanger (AE) 1 and 3. Normal columnar epithelial cells show apical cytoplasmic staining for carcinoembryonic antigen (CEA), especially with polyclonal antibody. In contrast, carcinoma cells stain diffusely for CEA. If present, neuroendocrine cells are sparse.

Embryology

The gallbladder originates from an evagination of primitive endodermal cells from the embryonic foregut, as do the liver and bile ducts. The underlying mesodermal cells appear to participate by inducing the endodermal cells. An outgrowth that buds from the ventromedial endoderm opposite the dorsal pancreatic bud near the yolk sac-foregut junction by the fourth week of fetal development eventually becomes the hepatic diverticulum as it pushes into the splanchnic mesenchyme. The liver and intrahepatic bile ducts derive from the cranial portion of the hepatic diverticulum, whereas the gallbladder and cystic duct derive from the caudal portion. The ventral pancreatic bud, which gives rise to the pancreatic head and uncinate process, develops from the hepatic diverticulum near its junction with the foregut. The common embryologic origin of the gallbladder with the pancreas and other elements of the biliary tract is reflected in many pathologies involving this organ, including the different types of neoplasms. The common bile duct (CBD) is formed by elongation and canalization of the original hepatic diverticulum. The gallbladder is recognizable as a small tubular structure by 5 weeks of gestation, when the embryo measures 5 mm. At this time, it is connected to the CBD by a short stem that will later become the cystic duct. Most of the biliary tract has canalized by the eighth week of gestation. By 15–18 weeks, the layers of the gallbladder wall are completely differentiated, and by about the third month of gestation, the fetal liver begins to secrete bile.

Physiology

The gallbladder has a relatively small volume (40–70 mL) relative to the 1000 mL of bile secreted by the liver on a daily basis. To compensate for this volume disparity, gallbladder epithelial cells absorb water from bile through an osmotic gradient generated by a Na ⁺ /K ⁺ -ATPase-mediated sodium-coupled transport of chloride, allowing a larger quantity of bile constituents to be effectively stored through concentration. Bile flow into the duodenum is facilitated by contraction of smooth muscle in the gallbladder and reciprocal relaxation of the sphincter of Oddi, both stimulated by ingestion of food. Gallbladder contractions are induced by the action of cholecystokinin (CCK), a peptide hormone released by the neuro­endocrine cells of the small intestine, on the CCK-A receptors of the interstitial cells of Cajal. Counterintuitively, ingestion of a fatty meal also stimulates the release of somatostatin, a hormone that inhibits gallbladder contraction, from the intestine and pancreas. In light of their simultaneous release yet opposing actions, CCK and somatostatin are thought to act as physiological ‘balances’ for each other. Bile accumulates in the CBD during fasting while the sphincter of Oddi is contracted. Once the pressure in the CBD exceeds the resting pressure of the gallbladder (~10 mm Hg), bile flows into the gallbladder.

The peribiliary mucous glands in the neck and the surface epithelial cells produce mucin, most of which is neutral, heavily sulphated, and contains a few sialic residues. The most abundant mucin in the human gallbladder is MUC5B. These mucosubstances are receiving more attention recently as their contribution to the formation of gallstones is elucidated. Gallbladder bile is sterile in most individuals, likely attributable to a combination of the antibacterial action of bile acids and cholangiocyte secretion of immunoglobulin A (IgA) and defensins.

Epidemiology

In developed countries, cholelithiasis is present in 10–20% of adults ; it is very uncommon in the paediatric population ( Table 10.2 ). It has a female predilection and is more common with advancing age, perhaps related to increased biliary cholesterol secretion. Amerindian populations of both North and South America have a very high incidence (75%) of cholesterol gallstones. These populations may harbor dominant lithogenic genes.

Cholesterol gallstones

Cholesterol stones are formed when the bile is supersaturated with cholesterol, which may result from decreased bile acid production, increased cholesterol output in bile or both. Stone formation by cholesterol monohydrate crystals is enhanced by mucin hypersecretion and gallbladder hypomotility. Cholesterol stone formation is promoted by conditions that increase hepatic cholesterol excretion, such as oral contraceptives, pregnancy, anticholesterol medications, rapid weight loss, obesity, total parenteral nutrition (TPN) and hypertriglyceridaemia, or by conditions that induce gallbladder stasis (e.g. mechanical obstruction, neurogenic/hormonal factors). Conditions that deplete bile salts (e.g. Crohn disease) may also promote cholesterol stone formation. It is not yet clear whether there is increased risk of stone formation in diabetic patients. Alcohol consumption seems to be protective against cholelithiasis.

The degree of cholesterol content influences the gross appearance of cholesterol stones ( Fig. 10.6 A ). Ten percent of gallstones are pure cholesterol stones, which measure up to 4 cm. These are smooth, round to ovoid and yellow-white and have a laminated or crystalline cut surface. Mixed stones have lower cholesterol content and thus may be layered grey-white to black depending on the bilirubin, calcium carbonate and phosphate content. Mixed stones tend to be multiple, smaller and faceted and may have a dark core and layered cut surface. At least 80% of cholesterol stones are radiolucent because they do not contain calcium carbonate.

Gallstones. A, Cholesterol gallstones. These are usually numerous; the high concentration of calcium bilirubinate at the centre of the stone imparts a dark-brown appearance surrounded by concentric layers of yellow concretions. B, Black-pigmented gallstones. Compared to cholesterol stones, these are usually smaller and on section exhibit a homogeneous black appearance. C, Brown-pigmented gallstones have a soft consistency and frequently form in situ within the extrahepatic bile ducts.

Pigmented gallstones

Worldwide (particularly in Asia), pigmented stones are the most common, resulting from parasitic infections. In the United States, less than 25% of gallstones are pigmented. Pigmented stones are composed of insoluble calcium salts which form as a result of increased unconjugated bilirubin in bile and, by definition, contain no more than 30% cholesterol. Haemolytic syndromes and severe ileal dysfunction or bypass (which increase the concentration of unconjugated bilirubin in bile) predispose to pigmented stone formation, as do parasitic and bacterial infections of the biliary tract.

Pigmented stones come in black and brown varieties. Black stones may be black or deep brown with an irregular, shiny outer surface, are difficult to crush, are small (2–5 mm) and resemble glass on fracturing ( Fig. 10.6 B ). They form most often in older individuals in sterile bile but are also the most common gallstone type in patients with chronic haemolytic anaemia, cirrhosis or sclerosing cholangitis. Many black stones (50–75%) are radiopaque secondary to calcium carbonate and calcium phosphate. Brown stones are rough, flaky and soft and may appear greasy; they are usually radiolucent ( Fig. 10.6 C ). Brown stones usually form in the context of biliary stasis and bacterial (e.g. Escherichia coli ) or parasitic (e.g. Ascaris lumbricoides, Opisthorchis sinensis ) infection. Stones are produced when free fatty acids released by bacterial phospholipases complex with calcium, and unconjugated bilirubin is formed from hydrolysis of conjugated bilirubin by β-glucuronidases.

Rarely, previously documented gallstones may disappear, presumably either secondary to spontaneous dissolution or passage through the CBD or a cholecystoenteric fistula. Some gallstones become embedded within the gallbladder wall, through Rokitansky–Aschoff sinuses or mucosal ulceration. Some gallstones are observed radiographically to float in bile when they are less dense than the bile itself (sometimes induced by fasting); these gallstones may appear to contain gas radiographically, possibly produced by bacteria.

Clinical features and treatment

Gallstones grow most rapidly during the first 2 to 3 years of their production, at a rate of about 1–2 mm per year, after which time growth subsides. Most gallstones are asymptomatic. Symptomatic patients most frequently complain of right upper quadrant pain (that may be colicky) and flatulence occurring after ingestion of fatty foods. Ultrasonography can detect not only gallstones measuring over 2 mm, but also biliary ‘sludge’. Plain abdominal radiographs detect 10–25% of gallstones.

The treatment of choice for symptomatic patients is cholecystectomy. Cholecystectomy for gallstones is also indicated in children and in patients with sickle cell disease, at high risk of developing gallbladder cancer (Native Americans), with porcelain gallbladder (hyalinizing cholecystitis) or with stones >3 cm. Rarely, less invasive therapy, such as extracorporeal shock-wave lithotripsy and oral or contact dissolution therapy, may be administered, but with limited success.

Complications

There are many possible complications of cholelithiasis. Some are covered in this section; others, including cholecystitis, hydrops/mucocele and cancer, are described in other sections.

Erosion of stones through mucosa may lead to inflammatory adhesions between organs (e.g. between gallbladder and colon or duodenum) through which gallstones may ultimately pass via fistulous tracts. Stones may pass through these fistulous tracts or through the CBD without sequelae, but they may also lead to obstruction. Various obstruction-related complications include choledocholithiasis; obstructive jaundice; pancreatitis secondary to ampullary obstruction; small intestinal obstruction, usually in the ileum resulting in ‘gallstone ileus’ ; and rarely, appendiceal or colonic obstruction. Stones that are small enough to pass through the CBD and ampulla of Vater typically do not cause obstruction at any downstream site. Stones that are large enough to cause obstruction of the intestine or colon are therefore assumed to have passed through a cholecystoenteric fistula, which may or may not remain patent until surgery. Gallbladders tend to shrink and become fibrotic and embedded in adhesions when there is a healed cholecystoenteric fistula. Rarely, obstructive jaundice may result from extrinsic compression of the CBD by a gallstone in the cystic duct or gallbladder neck (Mirizzi syndrome).

Hydrops and mucocele

Hydrops refers to gallbladder distension by clear, watery liquid, and mucocele refers to distension by mucoid material. The literature cites a 3% incidence of hydrops and mucocele (combined) among pathologically examined gallbladders, but in our experience, they occur less frequently. In adults the cause is usually cystic duct obstruction secondary to an impacted stone or less often, tumours, fibrosis, kinking of the cystic duct, cystic fibrosis or extrinsic compression from a mass, as from the liver. These conditions usually cause vomiting and a right upper quadrant abdominal mass, with or without pain and tenderness. Hydrops and mucocele may require cholecystectomy in adults because they tend not to resolve spontaneously. Hydrops in children is frequently reversible and may be caused by infections (e.g. Epstein-Barr virus [EBV], streptococcal infections, typhoid, leptospirosis, viral hepatitis) or other causes of inflammation, such as Kawasaki syndrome (mucocutaneous lymph node syndrome) and Henoch–Schönlein purpura. Congenital cystic duct abnormality should also be excluded.

Hydrops and mucocele may enlarge the gallbladder significantly; up to 2 kg in some patients. Histological findings are typically unimpressive and nonspecific. Epithelial attenuation and variable Rokitansky–Aschoff sinus formation are typically observed with sparse inflammation. In some examples of mucocele, the precipitated mucin can be seen clinging to the mucosa. The gallbladder wall is usually thin in paediatric cases because of the acute nature of the obstruction in most children. In adults, however, the gallbladder shows mural thickening, sometimes with fibrosis of the tunica muscularis and stromal muciphages, reflecting the chronic nature of the process in adults.

Acute cholecystitis

Rapid-onset injury to the gallbladder causes acute cholecystitis, which is defined clinically by leucocytosis combined with the persistence beyond 24 hours of the constellation of acute biliary pain, right upper quadrant tenderness, guarding and fever. Acute cholecystitis is not necessarily characterized by neutrophilic inflammation, but rather is related to ischaemia with resultant congestion, vascular leakage, oedema, fibrin deposition and epithelial denudation.

Pathological findings

Acute calculous and acalculous cholecystitis share similar pathological findings and are discussed together. Gross findings indicating acute inflammation may include oedema and distension of the gallbladder, fibrinous serosal exudate, friability or frank gangrene ( Fig. 10.7 ). Additional findings that may be appreciated at surgery may include adhesions between the omentum and gallbladder and pericholecystic fluid collections. There may be areas of serosal haemorrhage and congested subserosal vessels. The wall may be oedematous and haemorrhagic, thickened up to 2 cm. The mucosa may be oedematous and congested, ulcerated or necrotic. In the histological preparations, acute cholecystitis is often readily recognizable on visual examination of the slides, revealing a pink thickened wall caused by oedema and congestion. In calculous cholecystitis a stone may be observed obstructing the cystic duct or gallbladder neck. Sanguineous biliary material and turbid liquid fill the lumen; although this may grossly resemble pus, the cloudy nature is actually imparted by calcium carbonate and cholesterol.

Acute cholecystitis, gross appearance. The mucosal surface exhibits haemorrhagic necrosis.

Histologically, early acute cholecystitis is manifested by mural oedema, vascular congestion, haemorrhage and fibrin deposition with mucosal erosion. Eosinophils are usually scattered throughout the oedematous wall, perhaps a response to bile contents. Neutrophils, on the other hand, may not play a role in acute cholecystitis unless there is secondary infection. When choledocholithiasis exists, intraepithelial neutrophil aggregates may be observed and typically also accompany mucosal ulcers, creating a picture that some refer to as ‘active cholecystitis’. Between the 5th and 10th days of the process, a tissue culture-like myofibroblastic proliferation accompanied by lymphocytes, eosinophils, plasma cells and pigment-laden macrophages contributes to the mural thickening ( Fig. 10.8 ). This myofibroblastic proliferation with ‘tissue culture’ appearance may be misinterpreted as a sign of chronicity, but counterintuitively, it is characteristic of acute cholecystitis. However, acute cholecystitis may also be superimposed on changes of true chronic cholecystitis. Small vessels may contain fibrin thrombi. Marked reactive epithelial atypia in acute cholecystitis may mimic dysplasia (see later). Muscular arteries may demonstrate fibrinoid necrosis; in this case, the differential diagnosis of a collagen vascular disease may be noted. Surgery becomes more difficult and risky after about 72 hours into the process, secondary to organizing fibrous adhesions.

Acute cholecystitis, microscopic appearance. The perimuscular connective tissue is oedematous and cellular secondary to a combination of myofibroblastic proliferation (which counterintuitively is a feature of acute cholecystitis) and inflammation. The mucosa in this case is necrotic. On high magnification (inset) an oedematous tissue culture-like myofibroblastic proliferation with a smattering of inflammatory cells, including eosinophils, is observed in the perimuscular connective tissue. (H&E stain.)

Focal or diffuse transmural infarction may lead to perforation, which is uncommon since cholecystectomy is usually performed early. Perforations may be walled off by the omentum, resulting in abscess formation. Fistulae to other organs may form in the setting of perforation. Bile peritonitis, which is frequently fatal, may result from bile leakage through a distended gallbladder even in the absence of an identifiable perforation. True mural necrosis (not to be confused with severe mucosal ulceration) characterizes acute gangrenous cholecystitis. Secondary bacterial infection may occur. Diabetic patients are particularly at risk of acute gaseous cholecystitis or emphysematous cholecystitis (mural gas gangrene), presenting radiographically as pneumobilia, which is caused by gas-forming bacterial such as Clostridium perfringens . Gas gangrene may histologically be recognized by neutrophils, eosinophils and giant cells surrounding empty spaces.

Severe acute cholecystitis with a pus-filled lumen is classified as ‘empyema’. These cases may be missed clinically secondary to atypical presentation, and mortality is unfortunately high (15–25%). Histologically, empyema is characterized by extensive mucosal ulceration with abundant suppurative inflammation.

Chronic cholecystitis and variants

The vast majority of cholecystectomies are performed for chronic cholecystitis, which is the most common disease of the gallbladder. More than 90% of cases are associated with stones, and it accordingly has a female predominance, as with cholelithiasis (see Fig. 10.6 ). Chronic cholecystitis may or may not be preceded by prior attacks of acute cholecystitis, and many cases are discovered incidentally during workup for other conditions when gallstones are noted radiographically.

The number and size of stones do not correlate well with the severity of injury in chronic cholecystitis, and it is not clear whether or how much stones actually contribute to the inflammation and pain. Intermittent obstruction and changes in bile composition are thought to play a role in mucosal injury. It has been postulated that in some populations with a high incidence of chronic cholecystitis, the exuberant inflammatory responses that gallbladders mount to injury may have been beneficial and protective against parasitic biliary infections in their ancestors. Bile cultures are positive in one-third of patients (frequently growing E. coli or enterococci). Helicobacter species have been identified in some studies, although their contribution to the injury is debated. Some patients may be chronic carriers of Salmonella typhi in the gallbladder. Patients with IgA deficiency, achlorhydria or malabsorption are more likely to harbor the otherwise uncommon Giardia lamblia .

The duration and severity of chronic cholecystitis impact the pathological findings. More than 90% of chronic cholecystitis patients have gallstones. The gallbladder wall may be normal or thickened. The gallbladder may be enlarged or fibrotic and small ( Fig. 10.9 ). Prior episodes of acute cholecystitis may be suggested by serosal adhesions. The mucosa may be preserved, flattened, ulcerated or granular. Regenerative areas may appear polypoid, corresponding microscopically to clusters of glands exhibiting pyloric metaplasia admixed with fibrosis and myoid cells. Hyalinizing cholecystitis, characterized by replacement of the gallbladder wall by a homogeneous thin band of hyalinized fibrous tissue with or without calcification, is found in about 1% of cholecystectomies. Cancer is often found in cases with little or no calcification.

Chronic cholecystitis, gross appearance. A, Numerous cholesterol stones were present. Areas of mucosal prominence alternate with areas showing an atrophic appearance. B, This small, scarred, nonfunctioning gallbladder contained a single large gallstone.

Histological findings in chronic cholecystitis include mural thickening, variable fibrosis and variable chronic inflammation mainly by T lymphocytes, accompanied by fewer plasma cells, eosinophils and histiocytes ( Fig. 10.10 ). Patients may experience symptoms from cholelithiasis even if the gallbladder appears microscopically unremarkable, and on the lower end of the spectrum, interpretation of subtle microscopic findings as ‘mild’ chronic cholecystitis becomes subjective. Even normal gallbladders accompanying livers used for transplantation from healthy individuals dying from trauma contain sparse lymphoid cells. The term chronic active cholecystitis is applied when neutrophils are present within the epithelium; this may be a sign of obstruction and is not considered a manifestation of acute cholecystitis, and it is often not a sign of infection either ( Fig. 10.10 , inset).

Chronic cholecystitis, microscopic appearance. The tunica muscularis is thickened, and the perimuscular connective tissue is densely fibrotic secondary to chronic injury. Rokitansky–Aschoff sinuses invaginate into the perimuscular connective tissue more prominently than in normal gallbladders. Scattered, loose lymphoid aggregates are present. When neutrophils infiltrate the surface epithelium (inset), the term chronic active cholecystitis may be applied. (H&E stain.)

Collections of bile- and lipofuscin-laden macrophages (usually accompanied by a mild lymphocytic infiltrate) characterize cholegranulomatous cholecystitis , occurring as a response to bile penetration through the epithelium. Small vessels may show luminal obliteration. Mucosal neural proliferations may occur either in a traumatic neuroma-like pattern or in a ganglioneuroma-like pattern. The impact and indentations caused by gallstones may be seen on the mucosa. The perimuscular tissue may show elastotic changes. The epithelium may be atrophic, normal or hyperplastic. Focal or patchy ulcerations are usually signs of superimposed acute/subacute injury. Epithelial regeneration or metaplasia may be observed. Pyloric gland metaplasia, which may be the only finding in some cases of chronic cholecystitis, is frequently present. Other forms of metaplasia, which may also represent general mucosal responses to chronic injury, are discussed later.

Rokitansky-Aschoff sinuses are irregular, tubular mucosal invaginations through gaps in the tunica muscularis, analogous to diverticula of the colon ( Fig. 10.10 ). They are common in chronic cholecystitis and increase in prominence with its severity. These sinuses may contain bile or stones. When they are lined by atypical reactive epithelium (a common occurrence in chronic cholecystitis), they may mimic invasive carcinoma. Conversely, many invasive gallbladder carcinomas appear deceptively bland.

As discussed previously, a septum of adenomyomatous hyperplasia or inflamed fibrous tissue may result in the formation of hourglass gallbladder. It is as yet unclear whether adenomyomatous nodules/hyperplasia represent prominent Rokitansky–Aschoff sinuses (manifesting chronic injury) or whether they are a developmental phenomenon; we suspect the latter.

Hyalinizing cholecystitis (‘porcelain gallbladder’)

From 1% to 2% of surgically removed gallbladders show replacement of the wall by a dense homogeneous band of hyalinized fibrous tissue ( Fig. 10.11 ). This may be a manifestation of longstanding injury to the gallbladder, since patients with this finding are about a decade older than those with the usual form of chronic cholecystitis. The dense, paucicellular hyalinized fibrous tissue completely or nearly completely replaces all elements of the gallbladder wall, including the muscular elements and epithelium. It often shows artefactual clefting ( Fig. 10.11 , inset) and calcifications (frequently present but not always easy to identify). Such gallbladders with entirely calcific walls were termed ‘porcelain gallbladder’ in past literature, but it has recently been demonstrated that hyalinized gallbladders with no or minimal calcification (‘incomplete porcelain’) are the entities associated with an increased cancer risk. Because the epithelium usually is almost absent in cases of hyalinizing cholecystitis, and because carcinomas that arise in this setting can be quite subtle, any epithelium observed in completely hyalinized zones of gallbladder is concerning for carcinoma ( Fig. 10.12 ). Features that help distinguish carcinoma from reactive epithelium include glands oriented parallel to the mucosal surface, nuclear enlargement and clear cytoplasm. Carcinomas arising in hyalinizing cholecystitis are difficult to stage since the normal layers of the wall are usually not recognizable; consequently, they should all be regarded as advanced lesions (at least pT2).

Hyalinizing cholecystitis. All layers of the gallbladder wall, including the mucosa, tunica muscularis and perimuscular connective tissue, are replaced by densely hyalinized collagen. Staging of carcinomas in hyalinizing cholecystitis may be problematic because the mural layers are no longer recognizable. High-power inset, Artefactual clefts are formed within the densely hyalinized collagen. Note the virtual absence of epithelium. (H&E stain.)

Cancer in hyalinizing cholecystitis. In completely hyalinized portions of gallbladder, carcinomatous glands may be quite attenuated and inconspicuous, as in this case. The infiltrative pattern, nuclear atypia with prominent nucleoli, and luminal necrosis are helpful clues to the diagnosis. Any epithelium within such hyalinized gallbladders with minimal to no calcifications (‘incomplete porcelain’) should raise suspicion for carcinoma. (H&E stain.)

Mucosal-predominant lymphoplasmacytic cholecystitis

Some cases of chronic cholecystitis show diffuse mucosal involvement by dense lymphoplasmacytic inflammation within the lamina propria that does not extend into other layers of the gallbladder wall ( Fig. 10.13 ). It has recently been demonstrated that this form of chronic cholecystitis is likely a manifestation of downstream biliary obstruction from any of a variety of causes, such as biliary, ampullary or pancreatic tumours; stones impacted in the cystic duct or CBD; or sclerosing inflammatory immune-mediated conditions. Since biliary obstruction occurs in a variety of conditions, such as ulcerative colitis, primary sclerosing cholangitis and autoimmune pancreatitis, at one point this form of cholecystitis was thought to be directly associated with these conditions, although downstream obstruction instead seems to be the common denominator for this pattern of inflammation. Some studies applied the term ‘sclerosing cholecystitis’ to cases thought to be on the spectrum of IgG4-related sclerosing disorders of the pancreatobiliary tract, as discussed later. The epithelium in primary sclerosing cholangitis-related cases may be attenuated, and peribiliary mucous gland hyperplasia may be present. Intraepithelial neutrophils and storiform fibroplasia may be observed in IgG4-related cases. Table 10.3 outlines features helpful in the differential diagnosis between mucosal-predominant lymphoplasmacytic cholecystitis and IgG4-related sclerosing cholecystitis. Mucosal hyperplasia with ectatic vessels in the superficial lamina propria tend to be observed in cases related to obstruction. Nevertheless, none of these histologic findings has proved specific for the underlying cause of the cholecystitis.

Mucosal-predominant lymphoplasmacytic cholecystitis. A dense lymphoplasmacytic infiltrate expands the lamina propria while largely sparing the rest of the gallbladder wall. This pattern of inflammation is typically observed in the setting of downstream biliary obstruction, which may be secondary to virtually any cause. (H&E stain.)

Table 10.3

Differential diagnosis between mucosal-predominant lymphoplasmacytic cholecystitis (MPLC) and IgG4-related sclerosing cholecystitis (IgSC)

	Associated diseases	Histology	Immunology
MPLC	Downstream biliary obstruction (e.g. from tumour, stone or inflammation)	Diffuse plasma cell infiltrate in lamina propria	Less frequent IgG4-producing plasma cells
IgSC	Autoimmune pancreatitis type 1, other IgG4-related diseases	Plasma cells lying singly in lacunae within a band of wavy basophilic sclerosis that surrounds the epithelium	Abundant IgG4-producing plasma cells (>50/high-power field)

 

Infections

Gallbladder infections are usually secondary and superimposed on another process such as cholelithiasis or neoplasia and thus typically present as chronic cholecystitis. These secondary infections are most often caused by enteric bacteria, especially E. coli . The gallbladder serves as a primary site of involvement for other bacterial infections such as cholera and Salmonella . Although Salmonella does not injure the gallbladder itself, it uses the gallbladder as a reservoir during active typhoid fever and in the ‘carrier’ state. Patients with gallbladder cancer may have higher rates of Salmonella positivity. The renewed interest in the association between Salmonella and gallbladder cancer is based on epidemiological studies, leading to the speculation that the salmonellae that linger on the surface of gallstones may be an instigator for carcinogenesis. Opportunistic microorganisms (see AIDS-related cholecystitis earlier), viruses (e.g. CMV, EBV, hepatitis A) and parasites (e.g. Schistosoma , Paragonimus westermani , Ascaris lumbricoides , Giardia , amoebae) may also involve the gallbladder.

Vasculitis

Transmural inflammation of vessels (with or without fibrinoid necrosis) without inflammation in the surrounding tissue may present clinically as acalculous cholecystitis or may be an incidental finding in a gallbladder with stones. True vasculitis is not to be confused with secondary inflammation in the setting of a diffuse inflammatory process (‘innocent bystander’ phenomenon). Vasculitis of the type characteristic of polyarteritis nodosa may be isolated to the gallbladder, or it may be a manifestation of a systemic disorder such as systemic lupus erythematosus or scleroderma. Vasculitis in the setting of systemic diseases such as rheumatoid arthritis, mixed connective tissue disease, Henoch–Schönlein purpura, Churg–Strauss syndrome, Behçet syndrome or Wegener granulomatosis may also be observed in the gallbladder. Intrahepatic arterial chemotherapy has also been associated with vasculitis.

Cholesterolosis

Lipid-laden macrophages containing cholesterol esters and triglycerides accumulate within the lamina propria and epithelium to form cholesterolosis. This is most common in adults and multiparous women and is thought to result from abnormally increased transmucosal lipid transport and supersaturated esterified cholesterol within bile. Cholesterol stones coexist with cholesterolosis in 50% of cholecystectomies and 10% of autopsy cases; formation of cholesterol stones is also promoted by supersaturated bile. Cholesterolosis is not associated with hypercholesterolaemia or coronary artery disease. It typically spares the cystic duct and may involve the gallbladder in a diffuse or patchy fashion. Cholesterol polyps may form with or without background cholesterolosis. Diffuse cholesterolosis is appreciated grossly as golden-yellow flecks in the mucosal prominences, which imparts the appearance of a ‘strawberry gallbladder’ when the intervening mucosa is congested. The luminal bile is often dark and viscid, rich in cholesterol and sometimes contains yellow particles of floating lipid termed ‘lipoidic corpuscles’.

Microscopically, cholesterolosis appears as lipid-laden macrophages filling (and often confined to) the lamina propria ( Fig. 10.14 ). Expansion of the lamina propria may impart a microvillous architecture to the mucosa. Special stains may reveal lipid accumulation within the epithelial cells as well. On occasion, deeper mural aggregates of foamy macrophages may be present. Gallbladders with cholesterolosis usually are not inflamed or involved by neoplasia, suggesting that it may induce or may be a marker of a protective environment. Also, lipid droplets might be resorbed in the setting of inflammation, preventing the development of cholesterolosis.

Cholesterolosis. Lipid-laden macrophages fill the lamina propria. (H&E stain.)

Epithelial hyperplasia

Exaggerated folding of otherwise normal-appearing epithelium constitutes hyperplasia in the gallbladder ( Table 10.4 ). Focal or patchy microscopic hyperplasia is frequently seen in response to injury, such as by cholesterolosis or stones. The cores of the mucosal folds may be oedematous, especially when there is downstream obstruction. The extremely unusual phenomenon in which noninflamed mucosa lined by normal epithelium with no metaplasia is thickened by tall, closely apposed, villous-like structures without nodule formation is called primary papillary hyperplasia ; it may be diffuse, segmental or focal and does not develop dysplasia. The rarity of this entity and lack of a clear definition have led some to question its existence. A complex appearance of the mucosa likely resulting from tangential sectioning superimposed on hyperplasia of otherwise normal epithelium has been called spongioid hyperplasia . Gallbladders in patients with anomalous junction of the CBD and pancreatic duct outside the duodenal wall may form atypical papillary epithelial proliferations that are probably best classified as ‘dysplasia’ or ‘intracholecystic papillary tubular neoplasms’.

Papillary hyperplasia secondary to metachromatic leukodystrophy

Abnormal accumulation of sulphatide within tissues characterizes the disorder metachromatic leukodystrophy ( sulfatide cholecystosis ), which results from mutation in the gene encoding arylsulphatase, responsible for degrading sulphatide (a sphingolipid, also known as 3-0-sulphogalactosylceramide). Sulphatide accumulation in the gallbladder is manifested by an increase of variably basophilic, periodic acid-Schiff (PAS)- and cresyl violet-positive macrophages within the lamina propria. The gallbladder mucosa in children with metachromatic leukodystrophy is reported to form distinctive papillary proliferations that may or may not be accompanied by intestinal metaplasia; it is unclear whether these represent dysplasia or primary or secondary hyperplasia.

Adenomyomatous ‘hyperplasia’

The so-called adenomyomatous hyperplasia, which we prefer to call ‘adenomyomatous nodule’, may either represent a localized, distinctive type of diverticulum (probably congenital) or a malformation. Despite the name, there is no epithelial hyperplasia ( Fig. 10.15 ) (see later Non-neoplastic tumour-like lesions ).

Adenomyomatous nodule. Cystically dilated glands are present within a relatively well-circumscribed area of enhanced fibrous stroma in the perimuscular connective tissue. Note that despite the name, there is minimal to no appreciable myoid component in the lesional stroma. (H&E stain.)

Metaplasia

The gallbladder epithelium may exhibit metaplasia, most frequently of the gastric type, followed by intestinal type and rarely pancreatic acinar or squamous types ( Table 10.5 ). Metaplasia is often associated with cholelithiasis, is more frequent with increasing age and may be seen anywhere within the gallbladder in a focal or diffuse fashion.

More than three-fourths of surgically resected gallbladders harbor the most common form of metaplasia, called pyloric metaplasia (or pseudopyloric , antral , or mucous gland metaplasia ) ( Fig. 10.16 ). Clusters of pyloric-like glands may be present in the mucosa or deeper in the tunica muscularis. Exaggerated collections of mucosal pyloric glands may give the microscopic impression of polyps or nodules. We believe most subcentimetre lesions in the literature reported as ‘pyloric gland adenoma’ actually represent florid polypoid metaplasia; to classify a polyp as ‘neoplastic’ (or adenoma), it must either display unequivocal dysplasia or form a distinct nodule >1 cm. Metaplastic pyloric glands are microscopically, histochemically and ultrastructurally distinct from peribiliary mucous glands in the gallbladder neck and cystic duct region. Some authors prefer the term ‘pseudopyloric metaplasia’, which is more accurate, because in contrast to true pyloric glands, these may express small amounts of sialo- and sulfomucins. Estrogen receptor expression has been detected immunohistochemically in some cases. Pyloric glands admixed with parietal and chief cells represent the uncommon gastric heterotopia and not metaplasia.

Pyloric gland metaplasia. These small tubular glands have abundant pale apical mucin compressing the nuclei to the base, closely mimicking pyloric glands. This is the most common type of metaplasia in the gallbladder. (H&E stain.)

Sialomucin-containing goblet cells characterize intestinal metaplasia ( Fig. 10.17 ), which is much less common than pyloric gland metaplasia. In the gallbladder this is usually ‘incomplete’ intestinal metaplasia because a brush border is usually not observed on the apical aspect of the intervening columnar cells. Paneth cells may also be present. Intestinal metaplasia is present significantly more often in gallbladders with dysplasia or carcinoma and in gallbladders from regions with a high incidence of carcinoma. Additionally, goblet cells (intestinal metaplasia) are often seen in areas with epithelial atypia, and it is difficult to discern whether this atypia represents early dysplastic changes or mere atypia of the metaplastic process. Regardless, intestinal metaplasia in the gallbladder is thought to be associated with development of carcinoma (a precursor of a precursor). Convincing dysplastic epithelium also often contains goblet-like cells, which are relatively infrequently encountered in non-neoplastic cases. For these reasons, when the initial section of a gallbladder submitted for routine pathological examination reveals goblet-like cells or intestinal metaplasia, submission of an additional four blocks, each containing several tissue portions, is recommended.

Intestinal metaplasia. Goblet cells, as seen here, define intestinal metaplasia in the gallbladder. Since intestinal metaplasia is usually present in gallbladders with dysplasia, sampling of an additional four blocks is advisable when this finding is present in initial routine sections. There are no features (e.g. nuclear stratification or atypia) to suggest dysplasia in this particular focus. (H&E stain.)

About 15% of gallbladders harbor neuroendocrine cells that can be highlighted with immunohistochemical stains. Histologically, these contain fine, dull, amphophilic cytoplasmic granules located basally (in contrast to the chunkier, more eosinophilic granules located apically in Paneth cells). Most contain serotonin, whereas others express CCK, gastrin, somatostatin or pancreatic polypeptide.

The rare squamous metaplasia of the gallbladder has been noted adjacent to squamous cell carcinomas and also in hyalinizing cholecystitis (‘porcelain gallbladder’).

Pancreatic acinar cells are sometimes present in gallbladder mucosa. They most likely represent a metaplastic phenomenon rather than heterotopia, based on the absence of other pancreatic elements, as well as their size, distribution and association with other metaplasia types (especially pyloric gland type). True pancreatic heterotopia is characterized by compact, lobulated nodules that often also show islets and ducts.

Polyps ( Table 10.6 )

Mucosal injury polyps

Fibromyoglandular polyps

In chronic cholecystitis the mucosa may become nodular, and some of these nodules stand out from the background mucosa by transforming into broad-based polyps. Histologically, they comprise an admixture of fibrosis, myoid cells and glandular elements, thus the name ‘fibromyoglandular’ polyps ( Fig. 10.18 ). Similar polyps have previously been called ‘fibroepithelial’ or ‘fibroinflammatory’ polyps, depending on the pattern. When the stroma is more fibrous or oedematous and there is less inflammation, the term fibrous polyp has been used. These polyps are fundamentally the same, sharing glands variably distributed throughout a fibromyoid stroma, similar to prolapse-type polyps of the GI tract. Their occurrence in patients 10 years older than those with usual cholecystitis suggests that they result from longstanding injury. Fibromyoglandular polyps are usually multiple and measure less than 0.5 cm; infrequently they may measure >1 cm (<5% of cases). On one end of the spectrum, they resemble healed granulation tissue polyps, whereas in others there are more abundant pyloric glands bordering on polypoid pyloric gland metaplasia. The surface epithelium may show regenerative hyperplasia but is more frequently denuded. Dysplasia is present in about 15% of cases, usually resulting from colonization by dysplasia and carcinoma involving the background mucosa.

Table 10.6

Non-neoplastic polyps (>3 mm) of the gallbladder

	Gross features	Microscopic features	Frequency
Injury-related polyps (mean size, 0.4 cm; commonly multiple)
Fibromyoglandular polyp	All types of injury-related polyps: Raised with flat round surface; may be pedunculated on a broad base; only 5% are >1 cm.	Variably shaped glands (often metaplastic) haphazardly arranged within fibrous or myoid stroma	40%
Polypoid pyloric gland metaplasia		Nodular clusters of small, round, pyloric-type glands	5%
Inflammatory (granulation tissue) polyp		Granulation tissue, inflammation, oedema	<3%
Fibrous (‘stromal’) polyp		Variably inflamed fibrous stroma without glands	<1%
Lymphoid polyp		Prominent lymphoid aggregates or follicles; may have background follicular cholecystitis	<1%
Cholesterol polyps (mean size, 0.5 cm; usually not multiple)
	Pedunculated cauliflower-like or branching architecture; rarely (5%) >1 cm	Cauliflower-like architecture and stromal lipid-laden macrophages or just stromal edema; stroma mostly devoid of glands; background usually uninjured with infrequent cholesterolosis (<30%)
Other rare polyp types (variable sizes; usually solitary)
Hamartoma, inflammatory fibroid polyp, etc.	Variable	Variable	<0.1%

 

Fibromyoglandular polyp with intestinal metaplasia. A polypoid mucosal protrusion is formed by irregularly shaped and spaced glands entrapped within a fibrotic stroma above the tunica muscularis. Not all such cases have intestinal metaplasia. (H&E stain.)

Polypoid pyloric gland metaplasia

Pyloric gland metaplasia, the most common type of metaplasia in the gallbladder that develops in response to chronic mucosal injury, may form nodular protrusions. It is clear from the literature, which reports the average size of pyloric gland adenoma as 0.7–0.9 cm, that many exuberant forms of pyloric gland metaplasia are being classified as ‘pyloric gland adenoma’. We do not consider these to be neoplastic (adenomatous) unless they show unequivocal cytological dysplasia, or unless they form tight nodules that stand out from the background mucosa. We employ a size cutoff of 1 cm to classify a nodular proliferation of normal-appearing pyloric glands as ‘neoplastic’; this is also the size cutoff applied to separate mass-forming preinvasive neoplasms of the pancreas and biliary tract from earlier lesions (see later Mass-forming [tumoural] intraepithelial neoplasia: intracholecystic papillary tubular neoplasms ).

Inflammatory polyps

Polypoid nodules form as a result of a variety of inflammatory conditions in the gallbladder. Severe acute or subacute cholecystitis may be associated with the most common examples of inflammatory polyps, including granulation tissue polyps and polypoid xanthogranulomatous nodules in xanthogranulomatous cholecystitis. Prominent mucosal lymphoid follicles, most frequently seen in follicular cholecystitis, may protrude to form epithelial-covered lymphoid polyps .

Stromal polyps

Stromal polyps form during the organization phase of cholecystitis. As discussed earlier, ‘fibrous polyps’ are composed almost exclusively of fibrous tissue. The term ‘neurofibroma-like polyp’ has been applied to aggregates of nerve sheath cells resembling Schwann cell hamartomas of the GI tract. The precise aetiology is unclear, but they seem to be related to injury of some type and are more common in gallbladders with cholesterolosis.

Cholesterol polyps

Cholesterol polyps are the most common polyps to be found in otherwise uninjured gallbladders. Histologically, they are characterized by a cauliflower-like polyp pedunculated on a thin stalk with lamina propria filled with lipid-laden macrophages, appearing to be a polypoid form of cholesterolosis ( Fig. 10.19 ). They may be multiple. Surprisingly, however, the background mucosa often does not show cholesterolosis, and in 14% of cholesterol polyps themselves, stromal macrophages are undetectable, but the architecture is characteristic of cholesterol polyps. In cases without detectable macrophages, the stroma is often oedematous, presumably containing the broken-down product of macrophages. Gallbladders with cholesterol polyps usually do not have cholelithiasis or any features to suggest chronic cholecystitis. This may be interesting to note considering cholesterol polyps appear to be almost as common in men as in women, whereas most other gallbladder pathology is more common in women.

Cholesterol polyp. Foamy macrophages expand the lamina propria in a localized fashion to such an extent that a polypoid mucosal protrusion is formed. Note the cauliflower-like architecture and relative paucity of glands within the stroma. This cholesterol polyp appears to be floating because the connecting stalk is not present in this plane of section. (H&E stain.)

Cholesterol polyps are non-neoplastic but dysplasia (mostly low grade) has been reported in 4% of cases. Cholesterol polyp-like architecture and lipid-laden macrophages in 35% of patients suggest that the complex-tubular type of intracholecystic papillary neoplasms may arise from cholesterol polyps. Despite the neoplastic nature of the complex-tubular type of intracholecystic papillary neoplasms, invasive carcinoma almost never develops within them. Thus, cholesterol polyps are non-neoplastic and also non-precancerous for all practical purposes.

Cysts

Perhaps the most common cysts in the gallbladder wall are formed from expansion of Rokitansky–Aschoff sinuses or adenomyomatous nodules. Microscopic cysts may also be formed by dilated Luschka ducts. One must be astute not to underinterpret the bland appearance of the tubulocystic variant of biliary adenocarcinoma, which can appear deceptively benign.

Adenomyomatous nodules

Mural nodules of cystically dilated glands within a fibromuscular stroma that occur most often in the gallbladder fundus are referred to as ‘adenomyoma’ or adenomyomatous hyperplasia (see Fig. 10.15 ). Despite the name, there is nothing really hyperplastic about these lesions, and the stroma contains minimal to no myoid component. Grossly, they form well-circumscribed grey or yellow-white, rubbery mural thickenings with trabeculations or a sieve-like arrangement on sections. They can occasionally form mucosal lumps (‘polyps’). In the rare presentation as a diffuse process, it is referred to as ‘adenomyomatosis’. Some authors suggest this may be an exuberant form of Rokitansky–Aschoff sinuses, but many features also point to a developmental nature. For example, the architecture is distinct from that of Rokitansky–Aschoff sinuses, they usually do not communicate with the lumen, and the background gallbladder often shows neither other Rokitansky–Aschoff sinuses nor signs of injury. Instead, some examples show features of duplication.

Adenomyomas may superficially mimic adenocarcinoma secondary to the deep location of their glands; this may be confounded by the occasional finding of impingement of benign glands on nerves, simulating perineural invasion. That said, in situ and invasive carcinoma may occasionally develop within adenomyomas even without such changes in the background gallbladder. In our experience, high-grade dysplasia or carcinoma is seen in <5% of adenomyomatous nodules. The term mural intraductal papillary mucinous neoplasm refers to adenomyomas containing a papillary proliferation of dysplastic epithelium.

Inflammatory pseudotumours

Masses mimicking carcinoma may sometimes be formed by extensive myofibroblastic proliferation resulting from inflammatory injury. This is particularly evident in cases of xanthogranulomatous cholecystitis. Pseudotumours in the gallbladder and extrahepatic biliary tree may be observed in cases of eosinophilic cholecystitis as well. Such findings may also be present in cases of IgG4-related sclerosing disease.

Amyloidosis

The gallbladder may be involved in cases of systemic amyloidosis. As in other parts of the GI tract, the amyloid may deposit in vascular walls and the muscularis.

Differential diagnosis