Gallbladder, Extrahepatic Biliary Tract, and Pancreas Tissue Processing Techniques and Normal Histology
James M. Crawford
Introduction
Disorders of the biliary tract affect a significant portion of the world’s population. More than 95% of biliary tract disease is attributable to cholelithiasis (i.e., gallstones). It is estimated that more than 20 million persons in the United States have gallstones, with a total weight of 25 to 50 tons.1 The annual cost of treatment for cholelithiasis and its complications was estimated in 1998 to exceed $6 billion, representing approximately 0.2% of the national health care budget.2 More than 900,000 cholecystectomies are performed annually in the United States, making it one of the most common abdominal operations performed.3,4
Carcinoma of the pancreas is the fifth most frequent cause of death from cancer in the United States. Its incidence has remained unchanged during the past 50 years. The 5-year survival rate remains a dismal 5%, and there are almost 30,000 deaths per year in the United States from pancreatic cancer.5
This chapter presents an approach to the processing of surgical specimens of the gallbladder, extrahepatic biliary tract, and pancreas. Guidelines for handling intraoperative frozen section specimens are also discussed.
Normal Anatomy and Histology
Gallbladder and Extrahepatic Biliary Tract
The liver secretes as much as 1 L of bile per day. Between meals, bile is stored in the gallbladder, which in adults has a capacity of approximately 50 mL. Bile is concentrated 5- to 10-fold in the gallbladder through active absorption of electrolytes combined with passive movement of water. After ingestion of food that stimulates cholecystokinin and neural activation, the gallbladder contracts and propels the concentrated bile into the tubal gut through the extrahepatic biliary system. The gallbladder is not considered essential for adequate biliary function because humans do not suffer from maldigestion or malabsorption of fat after cholecystectomy.
The gallbladder is an elongated sac that lies in a fossa on the inferior surface of the right hepatic lobe. In adults, it is 7 to 10 cm long and 3 cm at its widest part. It is covered by peritoneum over its inferior aspect, but it is directly apposed to the surface of the liver along its superior aspect. The domelike fundus projects beyond the inferior border of the liver and slightly downward, and it is covered entirely with peritoneum. The body projects upward, backward, and to the left, approaching the porta hepatis. The neck corresponds to the tapered portion of the gallbladder. It curves backward and abruptly downward to enter the delicate connective tissue investment of the porta hepatis.
At the most proximal end of the cystic duct, there is a discrete area of narrowing that helps to regulate the flow of bile into and out of the gallbladder. The cystic duct is approximately 3 to 4 cm long and joins the common hepatic duct to form the common bile duct. This junction lies at the lowest region of the porta hepatis. The common bile duct then courses downward about 7 cm, passes behind the superior part of the duodenum, and enters the pancreas. In 80% to 90% of individuals, the common bile duct and main pancreatic duct unite and emerge as a common channel through the ampulla of Vater. However, in a minority of individuals, the common bile duct and pancreatic duct remain separate when they pass through the pancreas and ampulla.6
The common bile duct is the most anterior structure of the three major structures of the porta hepatis: common bile duct, portal vein, and hepatic artery. The common bile duct lies at the edge of the peritoneal reflection, which is the anterior edge of the epiploic foramen, the orifice between the greater and lesser peritoneal cavity located behind the stomach. The portal vein lies posterior to and to the left of the common bile duct. The hepatic artery and peripheral nerves lie most posteriorly. There may be marked variation in the relationship of the biliary tree to the portal vein and hepatic artery where these structures approach the porta hepatis and enter the liver.7
Unlike the remainder of the gastrointestinal tract, the gallbladder lacks a discrete muscularis mucosae and submucosa. It consists only of a mucosal lining with a single layer of simple columnar cells; a fibromuscular layer; a layer of subserosal fat with arteries, veins, lymphatics, nerves, and paraganglia; and a peritoneal covering, except where the gallbladder lies adjacent to or is embedded in the liver (Fig. 33.1). The epithelium is arranged in numerous interlacing folds, which imparts a honeycombed pattern to the surface. In the neck of the gallbladder, these folds coalesce to form the spiral valves of Heister, which extend into the cystic duct. In combination with cystic duct muscle action, the valves of Heister assist in retaining bile between meals. The rapid tapering of the gallbladder neck just proximal to the cystic duct is the primary site of gallstone impaction.
Small tubular channels are occasionally found buried in the gallbladder wall subjacent to the liver. These “subvesical bile ducts” occur in approximately 4% of gallbladders. Although their anatomy is quite variable, approximately half originate in the biliary tree of the right lobe of the liver and drain directly into the gallbladder; the remainder traverse the mesenchyme but do not directly communicate with the gallbladder lumen.
Small outpouchings of the gallbladder mucosa (i.e., Rochitansky-Aschoff sinuses) may penetrate into and occasionally through the muscle layer. Their prominence in the setting of inflammation and gallstone formation suggests that they are a type of acquired herniation.
Scattered along the length of the intrahepatic and extrahepatic biliary tree are mucin-secreting submucosal glands. They become prominent near the terminus of the common bile duct, appearing as microscopic outpouchings that interdigitate with the spiraling smooth muscle of the ampullary sphincter. To the unwary, buried glands may be mistaken for invasive cancer.
Pancreas
In adults, the average length of the pancreas is approximately 15 cm, and the average weight is 60 to 140 g. Embryologically, the pancreas arises from the duodenum as a dorsal bud and a smaller ventral bud. Fusion of the two creates the composite head; the dorsal bud is the primary source of the tapering body and tail. Fusion of the ventral duct and distal portion of the dorsal duct creates the main pancreatic duct (i.e., duct of Wirsung). Occasionally, the proximal portion of the dorsal duct persists as the accessory duct of Santorini.
The head represents the residuum of the embryologic ventral bud of the pancreas, having fused with the dorsal bud that constitutes the body and tail. From the ampulla of Vater, the main pancreatic duct traverses diagonally upward through the pancreatic head to the angle of the pancreatic duct of the body and tail, representing the duct of the embryologic dorsal bud. The bulbous head of the pancreas is situated in the curve of the duodenum and is the widest portion of the pancreas. The uncinate process is the portion of the pancreatic head that is most caudad and imparts a comma shape to the pancreas.
The body of the pancreas normally has a uniform diameter and a prismoid cross section with three surfaces; the posterior surface is the most vertical. The tail of the pancreas consists of the terminal few centimeters. It tapers gently to a point in the splenorenal ligament that contains the splenic artery and vein.
The pancreas is a pinkish-tan organ with grossly distinct, coarse lobulations, which result from delicate collagen septa that subdivide the parenchyma into macroscopic lobules. Histologically, the pancreas has two separate glandular components, the exocrine and endocrine glands (Fig. 33.2). The exocrine portion, which constitutes 80% to 85% of the organ’s volume, is composed of numerous small glands (i.e., acini) that contain columnar to pyramidal epithelial cells oriented in a radial fashion in the individual glands. The fine duct channels that drain each secretory acinus progressively converge to form the main pancreatic ductal system. The epithelium lining the smallest ducts is cuboidal but gradually evolves into a tall, columnar epithelium in the main duct. The main duct’s epithelium secretes electrolytes and mucin. Surrounding the larger pancreatic ducts are numerous accessory mucous glands.
The endocrine pancreas consists of approximately 1 million microscopic clusters of cells, the islets of Langerhans. Most islets are 100 to 200 µm in diameter and consist of four major and two minor cell types. The four main types are beta, alpha, delta, and pancreatic polypeptide (PP) cells. They make up approximately 68%, 20%, 10%, and 2%, respectively, of the adult islet cell population. Beta cells produce insulin, alpha cells secrete glucagon, delta cells produce somatostatin, and PP cells contain a unique type of pancreatic polypeptide. There are rare cell types referred to as D1 cells and enterochromaffin cells. D1 cells elaborate vasoactive intestinal polypeptide. Enterochromaffin cells synthesize serotonin and are the progenitors of pancreatic endocrine tumors that cause the carcinoid syndrome.
The only surfaces of the pancreas that are covered by peritoneum are the most anteroinferior aspect of the head, the anterosuperior surface of the body and tail, and the portion of the anteroinferior surface of the body and tail that is not apposed to the transverse mesocolon. The remainder of the pancreas is devoid of peritoneum and is apposed to some of the most vital structures of the mesenteric root and retroperitoneum.
Depending on the body mass of the host, the pancreas may lie in a bed of adipose tissue. This has relevance in determining where the corpus of the pancreas ends. The mesenchyme of the pancreas corpus consists of delicate connective tissue septa that separate the pancreatic parenchyma into lobules and a delicate sheath of connective tissue, vasculature, and nerves that travels along the pancreatic ductal system. Scattered adipocytes are located within the connective tissue septa, even in the normal pancreas of a lean individual. When the pancreas becomes damaged, inflamed, scarred, or atrophied, the boundaries of residual pancreatic exocrine glandular tissue with the peripancreatic mesenchymal adipose tissue may be difficult to establish with certainty. Pancreatic exocrine atrophy may leave residual islets of Langerhans, giving the impression that they are floating in adipose tissue.
Gallbladder
Gallstones afflict 10% to 20% of the adult population in developed countries. More than 80% of gallstones are clinically silent. Most individuals remain free of biliary pain or gallstone complications for decades, but biliary colic develops in approximately 25% during 10 years.8,9 Each year in the United States, approximately 1,000,000 symptomatic patients are found to have gallstones, ensuring a steady future population for the 900,000 cholecystectomies performed annually.
Gallbladders removed during open laparotomy or laparoscopic cholecystectomy are common specimens. Gallbladder cancer arises most frequently in the setting of long-standing cholecystitis with gallstones. The symptoms associated with gallbladder cancer usually mimic those of chronic cholecystitis, and the cancer may be advanced at the time of diagnosis. Occasionally, previously undetected gallbladder cancer is found only on examination of a resected specimen. Gallbladder lesions identified by endoscopic retrograde cholangiopancreatography (ERCP) may be amenable to endoscopic biopsy,10 and percutaneous fine-needle aspiration of the gallbladder may be performed.11–13 However, most often, gallbladder cancer is encountered in cholecystectomy specimens.
Gallstones
The two main types of gallstones are cholesterol stones and pigment stones. In the West, approximately 80% are cholesterol stones, which contain more than 50% crystalline cholesterol monohydrate. The remainder are pigment stones, which are composed predominantly of bilirubin calcium salts. There is a greater preponderance of pigmented gallstones in Asia because of the high prevalence of fluke infections of the biliary tract.
Cholesterol Stones
Cholesterol stones develop exclusively in the gallbladder and are composed of various amounts of cholesterol, ranging from 100% (which is rare) to approximately 50%. Pure cholesterol stones are pale yellow and round to ovoid, and they have a finely granular, hard external surface (Fig. 33.3), which on transection reveals a glistening, radiating crystalline palisade. With increasing proportions of calcium carbonate, phosphates, and bilirubin, the stones exhibit discoloration and may be lamellated and grayish-white to black on transection.
Most often, multiple stones may range as large as several centimeters in diameter. Rarely, there is a single, large stone that may fill the entire fundus or obstruct the gallbladder neck. The surfaces of stones may be rounded or faceted; the latter results from tight apposition when they occur in multiples.
Cholesterol stones are gross-only specimens. Regardless of their color, they are hard and cannot be crushed easily between the thumb and forefinger, unlike pigmented gallstones. Larger stones may be crushed intraoperatively during laparoscopic cholecystectomy to minimize the size of the abdominal wall incision required to remove the gallbladder. In this situation, the lamellated interior of the stones is usually evident. Chemical analysis of stones is not required for clinical care and is of value for research purposes only.
Cholesterolosis is an incidental finding pertinent to cholesterol biology but not directly related to gallstone formation. Cholesterol normally enters the gallbladder mucosa by free exchange with the lumen and is esterified by acyl-coenzyme A : cholesterol acyltransferase. Cholesterol hypersecretion by the liver promotes excessive accumulation of cholesterol esters in the lamina propria of the gallbladder. The mucosal surface may be studded with minute, yellow flecks, giving a strawberry appearance to the gallbladder mucosa (Fig. 33.4).
Pigment Stones
Pigment gallstones are a complex mixture of insoluble calcium salts of unconjugated bilirubin combined with inorganic calcium salts. Pigment gallstones are classified as black or brown. Black pigment stones usually are found in sterile gallbladder bile, and brown stones are found in infected intrahepatic or extrahepatic ducts. Black pigment stones contain oxidized polymers of calcium salts of unconjugated bilirubin; lesser amounts of calcium carbonate, calcium phosphate, and mucin glycoprotein; and a modicum of cholesterol monohydrate crystals. Brown pigment stones contain pure calcium salts of unconjugated bilirubin, mucin glycoprotein, a substantial cholesterol fraction, and calcium salts of palmitate and stearate.
Black stones are rarely greater than 1.5 cm in diameter and are almost invariably occur in large numbers (with an inverse relationship between size and number) (Fig. 33.5). Their contours are usually spiculated and molded. Brown stones tend to be laminated and soft, and they may have a soaplike or greasy consistency, resembling fecal material.
Pigment stones are gross-only specimens. Black and brown stones crumble or crush easily between the thumb and forefinger, which constitutes the primary confirmatory finding for distinguishing true pigment stones from darkly pigmented cholesterol stones, which do not crush.
Processing Gallbladder Specimens
Cholecystectomy is performed for gallstone disease, cholecystitis, cancer or presumed neoplasia, or incidental findings. Gallbladders removed at open laparotomy usually are intact. Because those removed during laparoscopic cholecystectomy may be torn, spillage of bile and gallstones into the abdominal cavity is a complication of this procedure.
Cholecystitis
Acute cholecystitis may be acalculous (without stones) or calculous (with stones). Chronic cholecystitis almost always occurs in the setting of gallstones. In acute cholecystitis, the gallbladder is usually enlarged and tense, and it may assume a bright red or blotchy, violaceous to green-black discoloration because of subserosal hemorrhages. The serosal covering is frequently layered by fibrin and, in severe cases, by a suppurative, coagulated exudate. There are no specific morphologic differences between acute acalculous and calculous cholecystitis, except for the absence of macroscopic stones in the former. In the latter, an obstructing stone is usually found in the neck of the gallbladder or the cystic duct.
In addition to gallstones, the gallbladder lumen is usually filled with cloudy or turbid bile that may contain large amounts of fibrin, pus, and blood. When the exudate is composed of pure pus, the condition is referred to as empyema of the gallbladder. In mild cases of cholecystitis, the gallbladder wall is thickened, edematous, and hyperemic. In more severe cases, the organ is green-black and necrotic, with or without perforation; this condition is called gangrenous cholecystitis. The inflammatory reactions are not histologically distinctive and consist of the usual patterns of acute inflammation (i.e., edema, leukocytic infiltration, vascular congestion, frank abscess formation, or gangrenous necrosis).
The morphologic changes in chronic cholecystitis are extremely varied and sometimes minimal. The serosa is usually smooth and glistening, but it may be dulled by subserosal fibrosis. Dense fibrous adhesions may remain as a sequela of preexistent acute inflammation. On tissue sectioning, the wall is thickened to some degree but rarely to more than three times normal. The wall of the gallbladder has an opaque gray-white appearance and may be less flexible than normal. In uncomplicated cases, the lumen contains fairly clear, greenish-yellow, mucoid bile and usually contains stones. The mucosa typically is well preserved.
In rare instances, extensive dystrophic calcification in a chronically inflamed gallbladder wall may produce a porcelain gallbladder, which is associated with a markedly increased incidence of cancer. Xanthogranulomatous cholecystitis is a rare condition in which the gallbladder is shrunken, nodular, and chronically inflamed with foci of necrosis and hemorrhage. Gallstones are usually found. This rare condition can be confused macroscopically with a malignant neoplasm. An atrophic, chronically obstructed gallbladder may contain only clear secretions, a condition known as hydrops of the gallbladder.
Carcinoma of the Gallbladder
Carcinomas of the gallbladder exhibit two patterns of growth: infiltrating or exophytic. The infiltrating pattern is more common and usually appears as a poorly defined area of diffuse thickening and induration of the gallbladder wall that may cover several square centimeters or may involve the entire gallbladder. Deep ulceration may cause direct penetration of the gallbladder wall or fistula formation into adjacent viscera. These tumors are scirrhous and have a very firm consistency.
The exophytic pattern grows into the lumen as an irregular, cauliflower-shaped mass, but it may also invade the underlying gallbladder wall. The luminal portion may be necrotic, hemorrhagic, and ulcerated. The most common sites of involvement are the fundus and neck; approximately 20% of cases involve the lateral walls. By the time these neoplasms are discovered, most have invaded the liver centrifugally, and many have extended to the cystic duct, adjacent bile ducts, and portohepatic lymph nodes.
Specimen Appearance
Perforations or tears in the gallbladder specimen should be documented. The serosa should be evaluated for its color and consistency. Inflammation usually imparts a dull and irregular appearance to the serosa. Adhesions may be evident, particularly in the region adjacent to the liver bed. In many cases, adherent cauterized liver tissue may be included with the gallbladder specimen. The finding of a fibrinous or purulent exudate points to a severe form of acute cholecystitis. Gangrenous gallbladders are blue-black, typically with a green bile discoloration; gross necrosis and perforation also are seen. The wall of a gallbladder with chronic cholecystitis may be markedly thickened. With extensive calcification, the gallbladder may take on a shiny, white, and hard porcelain appearance (Fig. 33.6).
Carcinoma in a gallbladder specimen may be evident on external examination as a dense mass bulging out of or penetrating into the gallbladder wall. Given the frequent advanced nature of gallbladder cancer at the time of diagnosis or resection, the tumor may be grossly transected at the specimen margin. Lymph nodes in the vicinity of the cystic duct should be identified and evaluated for metastasis. Serosal tumor implants from elsewhere in the abdominal cavity may also be found by the surgeon.
Specimen Dissection
The length of the gallbladder should be measured, and it should be opened longitudinally. The luminal contents are documented, including their volume and color, mucin content, and the presence or absence of sludge or stones. Biliary sludge consists of microcrystalline aggregates suspended in the mucinous fluid. The gallbladder wall thickness and circumference should be measured. The cystic duct is often tortuous and difficult to open, but an attempt should be made to examine the cystic duct margin at minimum. Gallstones in the gallbladder lumen, gallbladder neck, or cystic duct should be reported. Their gross features should be specifically described.
Occasionally, gallstones are returned to the patient. This should be done in a sealed container without formalin because formalin is a chemical biohazard. If the gallstones have been immersed in formalin, they should be rinsed in water before release to the patient. If chemical analysis is requested, the gallstones should be placed in a dry, sterile container and forwarded to the appropriate laboratory.
The mucosa of the gallbladder should be inspected and described. Normal mucosa has a honeycomb appearance and is tan and velvety. In cases of acute or chronic cholecystitis, the mucosa becomes effaced and loses its velvety appearance; it may be ulcerated and friable or flattened and atrophic, respectively. Cholesterolosis produces diffuse, small, yellow flecks on the mucosal surface.
Mucosal polyps are usually delicate, protuberant, papillary structures that are easily disrupted. Alternatively, flat areas of mucosal dysplasia or nascent adenocarcinoma may appear as firm, plaquelike elevations of the mucosa or exophytic tumors (Fig. 33.7) with effacement of the layers of the gallbladder wall in cases of invasive carcinoma. Invasive carcinoma should not be confused with adenomyosis, which is displacement of benign glandular epithelium into the gallbladder wall and which is usually associated with marked muscular hypertrophy. Adenomyosis may appear as a dense, focal, tumor-like thickening of the gallbladder wall (Fig. 33.8). Massively dilated Rokitansky-Aschoff sinuses in chronic cholecystitis may impart tumor-like thickening of the gallbladder wall.
Specimen Sectioning
Cross sections of the gallbladder fundus and lateral wall should be submitted along with a section through the neck of the gallbladder and cystic duct, including its margin. Gross lesions, whether non-neoplastic or neoplastic, should be sampled thoroughly, with particular attention paid to the surgical margins of resection. Accompanying lymph nodes should be submitted and evaluated histologically.
If a neoplastic lesion is identified on gross examination, that entire portion of the gallbladder should be submitted for histologic evaluation. If neoplasia is identified on histologic review of standard sections obtained from a gallbladder specimen, the specimen should be revisited and the entire area of interest submitted for histologic evaluation, which often involves submission of the entire gallbladder specimen.
Extrahepatic Biliary Tract
The extrahepatic biliary tract consists of the common bile duct, cystic duct, gallbladder, and common hepatic duct. In approximately 60% to 70% of individuals, the common hepatic duct bifurcates into the right and left hepatic ducts before entry into the liver.14 The most common anatomic variation is congenital absence of the right hepatic duct. Instead, the posterior and anterior branches of the bile ducts that supply the right portion of the liver arise from a hilar confluence with the left hepatic bile duct. This occurs in the form of a three-way branch point with the left hepatic bile duct or by a two-way confluence of the anterior or posterior branches with the left hepatic duct.14 Although the common hepatic duct and its branches lie ventral to the portal vein system, the right posterior bile duct may be situated in an inferior-ventral or superior-dorsal fashion around the right portal vein.
In two situations, the extrahepatic biliary tract should be submitted for surgical pathology analysis. The first is for resection of lesions that are primary to the biliary tree, particularly bile duct cancer15 and choledochal cysts. The second is for resection of the biliary tree as part of a pancreas or liver resection specimen. Pancreaticoduodenectomy (Whipple) specimens are discussed later.
Processing Biliary Tract Specimens
Isolated resection of the extrahepatic biliary tree is performed as part of procedures designed to reconstruct the biliary system. Biliary tract reconstruction in situ is not usually possible except in cases of small perforations or strictures, such as those that occur accidentally during laparoscopic cholecystectomy.16
The most common surgical procedure of the extrahepatic biliary tract is resection combined with the performance of a Roux-en-Y choledochojejunostomy. In this procedure, a blind loop of jejunum is anastomosed end to side or end to end with the remaining common bile duct. This type of reconstructive procedure is a routine part of pancreaticoduodenectomy. Because patients with primary sclerosing cholangitis are not treated with surgery, except for those undergoing liver transplantation, the primary indications for biliary tract extirpation and choledochojejunostomy are biliary tract cancer and bypass and resection of a choledochal cyst.
Biliary Tract Cancer
For patients with bile duct cancer, the resection specimen usually consists of the extrahepatic biliary tree, gallbladder, and accompanying soft tissue with lymph nodes. The surgeon should provide anatomic orientation by applying suture tags at the proximal and distal duct margins. Consultation with the surgeon is mandatory if there is any uncertainty with regard to anatomic orientation. En face sections should be obtained of the proximal (toward the liver) and distal (toward the pancreas) bile duct margins. The soft tissue margins should be marked with ink. However, disruption of anatomic planes during operative dissection may make identification of the true soft tissue margins difficult. The biliary system should be opened in the fresh state and examined for the intraluminal extent of tumor. The specimen should then be fixed in formalin overnight.
After fixation, optimal tissue sections for histologic evaluation include cross sections along the length of the biliary tree and all soft tissue margins: anterior, left lateral, posterior, and right lateral. Depending on the amount of soft tissue, lymph nodes may be included with the cut sections through the biliary tree or may be separately dissected from the soft tissue and submitted for histologic evaluation. Lymph node groups should be submitted in separate cassettes as follows: periductal (i.e., along the common bile duct), perihilar, peripancreatic, subpyloric, and celiac and preaortic.
Choledochal Cyst
Congenital cystic dilations of the common bile duct (i.e., choledochal cyst) are nonheritable, extrahepatic lesions (see Chapter 36
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