Introduction
Endoscopy provides a unique opportunity to visualize the mucosal surface of the gastrointestinal (GI) tract as well as a variety of extraluminal and extraintestinal organs and structures. When considered within the context of a specific clinical picture, endoscopic images may be all that is needed to establish a specific diagnosis or provide sound clinical management. However, endoscopists often need to sample tissue. Examination by a qualified pathologist of specimens obtained at endoscopy is a routine and critical part of managing disorders of the alimentary tract. The purpose of this chapter is to orient the pathologist to the clinical and technical considerations unique to specimens obtained endoscopically from the alimentary tract. This is followed by a discussion of the normal anatomy of the tubal gut.
Bowel Preparation
The effectiveness of endoscopy often depends on the quality of the bowel preparation. Preparation of the upper GI tract for endoscopy typically involves, at minimum, a 6-hour fast. Preparation for colonoscopy is achieved by use of oral purging agents, either with or without enemas. Most colonoscopy preparation regimens include the use of a clear liquid diet for 1 to 2 days, followed by cleansing with oral polyethylene glycol (PEG)-electrolyte solution or other oral laxatives (e.g., magnesium citrate, senna) and rectal enemas ( Table 1.1 ). In general, vomiting is reported more frequently with oral PEG-based high-volume lavage regimens than with other agents. PEG lavage regimens reportedly provide more consistent cleansing.
|
Purgative- and laxative-based regimens are more likely to cause flattening of surface epithelial cells, goblet cell depletion, lamina propria edema, mucosal inflammation, and increased crypt cell proliferation, although these effects occur infrequently. Osmotic electrolyte solutions, such as PEG-based solutions, are better agents for preserving mucosal histology. In the most severe form of mucosal damage due to purgatives such as sodium phosphates, sloughing of the surface epithelium, neutrophilic infiltration of the lamina propria, and hemorrhage may be encountered, and the changes may even resemble pseudomembranous colitis, nonsteroidal antiinflammatory drug-induced injury, or inflammatory bowel disease. Oral sodium phosphate bowel preparations were removed from the market in 2008 after their use was associated with renal injury. Chemical-induced colitis, caused by inadequate cleaning of endoscopic instruments, also has been reported but is very rare. Sodium phosphate–based preparations may also cause endoscopically visible aphthoid-like erosions similar in appearance to Crohn’s disease. Mucosal changes in this situation may also resemble pseudomembranous colitis, both endoscopically and microscopically.
Methods for Obtaining Tissue Specimens
There are a limited number of methods available for obtaining tissue via GI endoscopy. This section describes several of these methods and the common situations in which they are used.
Endoscopic Pinch Biopsy
Pinch biopsy, performed with the use of a biopsy forceps during endoscopy, is the most common form of tissue sampling; the biopsy site is usually fully visualized at the time of sampling. Suction capsule biopsy requires fluoroscopic guidance to position a long tube with the biopsy apparatus and is done separately from endoscopy without visualization. Suction capsule biopsy without bowel visualization is still performed in some centers, but it is less successful than endoscopy-guided biopsy in obtaining tissue and therefore has fallen out of favor. Pinch biopsies may be small or large (the latter are referred to as “jumbo” biopsies) and can be obtained with or without the use of electrocautery. Electrocautery has value for hemostasis and destruction of residual tissue but introduces burn artifact into the harvested tissue.
All standard biopsy forceps have a similar design ( Fig. 1.1 ). The sampling portion consists of a pair of small cups that are in apposition when closed. In this manner, they can be passed through the channel of a gastroscope or colonoscope. Some biopsy forceps have a spike at the base of the cup or teeth to help seat the forceps against the mucosa. The spike also helps to impale multiple biopsy specimens before the forceps is removed from the endoscope.
After insertion into the endoscope and emergence from the distal end, routine biopsy forceps can be opened to a 4- to 8-mm width. The opened forceps is pressed against the mucosal surface for tissue sampling. Large-cup (jumbo) biopsy forceps have jaws that open to a width of 7 to 9 mm. The biopsy forceps is closed against the mucosal surface, and the endoscopist pulls the forceps away from the mucosa to remove the fragment of tissue. This method often yields samples that include muscularis mucosae, except in regions such as the gastric body, where the mucosal folds are quite thick. The submucosa is sampled occasionally with either standard or jumbo forceps.
The sample size varies according to the amount of pressure the endoscopist applies to the forceps. In addition, application of a fully opened biopsy forceps flush against the mucosa before closure usually yields larger pieces of tissue, compared with tissue obtained by tangential sampling or incomplete opening of the forceps. In general, biopsy specimens are 4 to 8 mm in length. The forceps shape does not impart a significant difference in either size or adequacy of biopsy specimens. Single-use disposable biopsy forceps also have been shown to provide excellent samples. In essence, there are no differences in the quality of tissue samples obtained among the dozen or more biopsy forceps currently available, so the primary considerations in the selection of an endoscopic biopsy forceps are usually related to cost.
After the biopsy specimens have been obtained and the forceps have been removed from the endoscope, an assistant dislodges the tissue fragments from the forceps with a toothpick or a similar small, sharp instrument. The tissue is then placed into a container containing appropriate fixative and labeled according to instructions provided by the endoscopist.
Specimens obtained with a jumbo forceps often exceed 6 mm in maximum diameter, but these are not necessarily deeper than standard biopsies. Rather, a jumbo forceps typically provides more mucosa for analysis. This is particularly useful during surveillance tissue sampling, such as in patients with Barrett’s esophagus or ulcerative colitis. Jumbo biopsy forceps are as safe as standard biopsy forceps. However, use of jumbo forceps is limited by their diameter because the instrument cannot fit through a standard endoscope accessory channel. Jumbo forceps require a 3.2-mm-diameter channel, characteristic of therapeutic endoscopes, which may be less comfortable for patients. In addition, although jumbo biopsy specimens are larger than standard biopsy specimens, this does not necessarily mean that they are of greater diagnostic value.
Upper endoscopy or colonoscopy may be performed for clinical indications driven by symptomatology. Colonoscopy, in particular, may also be performed for screening purposes in clinically asymptomatic individuals. Selection of a biopsy site at the time of endoscopy is driven by the need to assess visible mucosal abnormalities. In addition, it is advantageous to establish the inflammatory status of the “background” mucosa by sampling normal-appearing mucosa during the evaluation of conditions such as gastroesophageal reflux disease (GERD), nonulcer dyspepsia, diarrhea, polyps, and nodules and for surveillance of premalignant conditions, including Barrett’s esophagus and inflammatory bowel disease. For example, in patients with inflammatory or dysplastic polyps of the stomach, it is essential to sample adjacent nonpolypoid mucosa to help determine the background disorder in the stomach in which the polyp has developed. As a second example, the ampulla of Vater may be biopsied to exclude adenomatous change in patients with familial adenomatous polyposis, because the lifetime incidence of ampullary adenomas in these patients exceeds 50%.
Biopsy of biliary or pancreatic strictures may be carried out under fluoroscopic guidance during endoscopic retrograde cholangiopancreatography (ERCP) with the use of either standard or specially designed biopsy forceps. Even gallbladder lesions observed on ERCP may be amenable to endoscopic biopsy, although this is rarely performed clinically. Endoscopy-directed diagnostic biopsies are extremely safe. In one study of 50,833 consecutive patients who underwent upper endoscopy, none had any biopsy-associated complications. The risk of perforation after diagnostic or therapeutic colonoscopy (with polypectomy) is extremely low.
Occasionally, an endoscopist uses a specialized insulated biopsy forceps to sample a small polyp (“hot biopsy”), after which remaining tissue is ablated in situ using electrocautery. Unfortunately, cautery artifact in such small tissue samples often makes histologic interpretation difficult (or impossible). In addition, the electrocautery technique carries an excessive risk of perforation resulting from deep tissue burn, particularly in the cecum and ascending colon. Finally, destruction of residual dysplastic tissue by electrocautery may be incomplete in as many as 17% of cases. For these reasons, hot biopsies have been largely abandoned by most endoscopists.
There is a limited literature available regarding the relatively new and controversial “resect and discard” concept for diminutive colorectal polyps found during screening colonoscopy. This concept suggests that one can safely perform endoscopic removal of diminutive colon polyps (usually by simple pinch biopsy removal using cold forceps or cold snare polypectomy) without the need for pathologic analysis of the polyp. The reasoning is that diminutive polyps have a very low likelihood of harboring either malignancy or advanced adenomatous features such as high-grade dysplasia or villiform change. Therefore, discarding these lesions without histologic evaluation should be cost-effective and clinically efficacious, with an endoscopic sensitivity for correctly classifying adenomas of 94% and specificity of 89%. Furthermore, these polyps may be assessed by in situ optical scanning techniques that can help predict polyp histology. For instance, narrow band imaging has a reported negative predictive value of 95%, which further increases physician confidence that the polyps can be discarded without pathologic examination.
Endoscopic Snare Polypectomy
During endoscopy, a loop of wire may be placed around a polypoid lesion that protrudes into the lumen of the gut for the purpose of removing the polyp ( Fig. 1.2 ). This technique is used primarily for colonic polyps, but polyps throughout the alimentary tract may be excised in this manner. Depending on their size, excised polyps are either retrieved through the suction channel of the endoscope or held by the snare after resection while the colonoscope is removed from the patient.
Many endoscopists have reported successful removal of diminutive polyps (<0.5 cm in diameter) during both “hot” (with electrocautery) and “cold” (without electrocautery) snare polypectomy. These endoscopists use small metal snares, termed mini-snares , that open to a size of either 1 to 2 cm or 2 to 3 cm. Polyps larger than 0.5 cm are amenable to snare polypectomy, although the size of the polyp that can be excised may be limited by the size of the loop placed around it (and the endoscopist’s estimation of perforation risk). Alternatively, large polyps can be removed in a piecemeal fashion and submitted to pathology in several parts. This technique usually requires multiple transections of the lesion until the entire polyp has been removed. One caveat with this technique is that identifiable tissue margins may be lost, so that the pathologist is often unable to determine the status of the resection margins.
A hot snare allows the endoscopist to apply modulated electrosurgical current to a metal wire that cuts through pedunculated polyps at the base. This assists tissue cutting and coagulation. Electrocautery also minimizes bleeding from larger blood vessels located in the stalk of the polyp. Cold polypectomy, without electrical current, avoids use of cautery, thereby limiting the amount of burn artifact in the specimen and minimizing the risk of perforation. In general, the risk of perforation from either mechanical or electrical injury is minimal but is greater in portions of the colon that are covered by a free serosal surface, such as the transverse colon. Information on the relative risk of clinically significant hemorrhage after hot polypectomy is limited, but the risk is generally considered to be low (0.4%). A large cross-sectional study from South Korea established that loop polypectomy is only rarely performed without electrical current (i.e., cold polypectomy), but this is usually inadvertent, resulting from failure of application of the current. Absence of electrical current is associated with an increased risk of clinically significant postpolypectomy hemorrhage. A higher risk of postpolypectomy hemorrhage also occurs in patients with pedunculated polyps larger than 1.7 cm or a stalk diameter larger than 0.5 cm, sessile polyps, or malignant lesions.
For polyps excised in one piece by either hot or cold polypectomy, the polyp base constitutes the surgical margin of resection. This is true for both pedunculated and sessile polyps. For polyps removed by hot snare polypectomy, the cauterized portion of the specimen constitutes the surgical margin. An artificial stalk can be created when large sessile lesions are loop-excised. A true pedunculated polyp, with a stalk, has a narrow base that persists after removal; the base of a sessile polyp is typically as wide as the mucosal surface that is sampled.
Snares are available in a variety of shapes and sizes. Some snares can be rotated, which provides the endoscopist with greater control of snare placement. The choice of snare size is usually based on the size of the lesion being removed. The selection of a particular snare shape is a matter of personal choice.
Snare polypectomy is performed in a similar fashion regardless of whether colonic, esophageal, gastric, or small bowel lesions are being removed. The ampulla of Vater may be resected by standard snare techniques if an ampullary lesion is discovered. The risk of perforation during snare polypectomy is less than 0.1%, and perforation usually results from transmural burn secondary to cautery. One commonly used technique aimed at decreasing the risk of perforation is to pull the snared polyp away from the mucosa so that less cautery is applied to the underlying tissue.
Another commonly used method is saline-assisted polypectomy. A small needle is passed through the endoscope and inserted into the gut wall adjacent to the polyp. A bolus of normal saline is then injected. Fluid collects within the submucosal plane, lifting the mucosal-based polyp away from the muscularis propria. A standard snare polypectomy is then performed, but the cushion of saline insulates the deeper tissue layers from the electrical current. Saline-assisted polypectomy is usually reserved for large sessile polyps and ampullary polyps and, theoretically, results in a decreased rate of polypectomy-associated perforation.
Endoscopic Mucosal Resection
The use of a liquid cushion to expand the submucosa and minimize transmural cautery damage is a principal feature of endoscopic mucosal resection (EMR). This technique is commonly used to resect premalignant and malignant lesions confined to the mucosa. In general, EMR requires some measure of confidence that a lesion is, in fact, confined to the mucosa or submucosa. Many endoscopists now rely on endoscopic ultrasonography (EUS) to determine the depth of a particular lesion before EMR. The accuracy of high-frequency EUS (15 or 20 MHz) may be as great as 95% for determining whether a lesion is limited to the mucosa.
Several variations of the EMR technique are currently used. Many rely on submucosal injection of liquid, but there is no agreement regarding the type or quantity of liquid that should be injected. Most endoscopists advocate the use of saline alone. Others add diluted epinephrine to saline in an attempt to constrict small blood vessels at the base of the lesion. Submucosal fluid collections are absorbed in a relatively short time period, which can limit their value. To lengthen the amount of time that the submucosal cushion may last, and thus maximize the time available for performing a safe resection, investigators have used hypertonic solutions of 3.5% saline or 50% dextrose. Others advocate use of sodium hyaluronate instead of saline. None of these agents is used more often than simple saline. The quantity of liquid injected also varies. There is general agreement that the selected lesion should appear, endoscopically, to be raised by the cushion of liquid before the EMR is performed. Failure to lift the lesion despite generous use of submucosal saline (the so-called nonlifting sign) may be a sensitive indicator that a lesion has spread deeper into the bowel wall.
Two major types of resection techniques are used—those that do not use suction and those that do. When suction is not used, the endoscopist uses a dual-channel endoscope. A snare, passed through one instrument channel, is opened and placed around the lesion. A biopsy forceps is passed through the second channel and used to grab the lesion and pull the mucosa through the snare farther away from the muscularis propria. The snare is then closed around the base of the tented lesion, and electrocautery is applied ( Fig. 1.3 ). This method is referred to as the lift-and-cut technique or a strip biopsy .
Suction methods of EMR incorporate the use of a cap fitted onto the tip of an endoscope. The cap presents an open surface to the mucosa and creates a short chamber into which the selected lesion may be aspirated and held by suction, which is applied through a single-channel endoscope. A specialized snare is opened in the cap before aspiration of the lesion. Once the mucosa has been drawn into the cap, the snare may be closed around the lesion and cautery applied in the usual fashion. This technique, also called aspiration mucosectomy , has been widely successful for removing lesions throughout the GI tract.
A newer EMR technique is similar to aspiration mucosectomy, but after the lesion has been suctioned into the cap, a tiny rubber ring is released around the base of the lesion, similar to the method used during endoscopic variceal ligation. Once suction is released, the lesion appears contained within a “pseudopolyp” that may be removed by snare cautery. This is known as band-ligation EMR ( Fig. 1.4 ).
EMR allows the endoscopist to attempt an en bloc resection and thus, potentially, to completely resect an early malignant lesion. En bloc resection is limited, however, to small lesions (1.5 to 2 cm in largest diameter). If deep margins are positive for neoplasia, surgical resection of the affected region is advocated. Current indications for EMR include superficial carcinoma of the esophagus or stomach in patients who are not candidates for surgery; unifocal high-grade (or low-grade) dysplasia in Barrett’s esophagus; and large, flat colorectal adenomas (which might otherwise require piecemeal resection) regardless of the degree of dysplasia. EMR as a form of primary therapy for small, superficial cancer has gained increasing popularity in the United States but is even more widely used in Japan, where early gastric cancer is more common. EMR is also used as a form of primary therapy for small submucosal lesions such as rectal carcinoid tumors or leiomyomas. In many cases, the submucosal lesion can be completely resected ( Fig. 1.5 ).
Major complications of EMR include bleeding and perforation. Bleeding occurs in fewer than 1% to as many as 20% of cases, depending on the size of the lesion and its location. Clinically significant bleeding is rare and usually is amenable to endoscopic hemostatic cauterization. Perforation rates are lower than 2% in the hands of experienced operators. A recent recommendation is that the endoscopist should immediately inspect the underside of the resected specimen for the presence of a “target sign”: a pale ring of muscularis mucosa resected with the mucosal lesion. Applying endoscopic clips to the corresponding defect in the resection site is purported to represent an effective method to mitigate against the risk of postprocedure bleeding. In the hands of experienced operators, EMR results in large specimens for pathologic analysis, even in the absence of complete resection. Success rates for en bloc resection of early gastric cancers by EMR range from 36% to 74%, greatly reducing the need for full-thickness surgical resection.
Methods of Processing Tissue for Pathologic Evaluation
A general framework for processing biopsy specimens is provided in Table 1.2 .
| Technique | Comment |
|---|---|
| Formalin fixation | Routine processing of all alimentary tract biopsies; immediate immersion in fixative. Permits immunohistochemistry, molecular analysis. |
| Flow cytometry | Suspected hematologic malignancy; fresh tissue in sterile culture medium |
| Electron microscopy | Suspected poorly differentiated malignancy, infection (e.g., Whipple disease, microsporidiosis); immediate immersion in electron microscopy fixative |
| Electron microscopy fixative only | Suspected systemic mastocytosis, for which plastic-embedded thick sections with toluidine blue staining may be used for identification of mast cells |
| Microbial culture | Suspected viral, fungal, or parasitic infection; sterile tissue |
| Biochemical analysis | Suspected metabolic deficiency (e.g., disaccharidase deficiency); frozen tissue |
| Cytogenetics * | Suspected neoplasm (benign or malignant); fresh tissue in sterile culture medium |
| Cell culture * | Suspected neoplasm (benign or malignant); fresh tissue in sterile culture medium |
Formalin
Of the many types of fixatives used for human tissue, 10% buffered formalin remains the standard and is well suited for mucosal biopsies of the GI tract. It is inexpensive, is harmless to the tissue even after long periods, and is compatible with most of the stains commonly used for morphologic assessment. Hollende solution, B5, and Bouin fixative have been used for mucosal biopsies because of better preservation of nuclear morphology compared with formalin. However, the heavy metal content of these fixatives creates biohazard disposal problems that are greater than those of formaldehyde-based fixatives. These fixatives also interfere with isolation of nucleic acid from tissue; the search for substitute fixatives and new tissue processing techniques is an active area of scientific investigation.
On occasion, the formaldehyde in formalin may be irritating to the eyes and upper respiratory tract of personnel. The level at which formalin is considered carcinogenic is well above the level that causes sensory irritation, which has a threshold of 1.0 ppm. Accordingly, in pathology suites, proper ventilation should be used to maintain exposure below 1.0 ppm, the lowest concentration that may exert a cytotoxic effect in humans. A workplace surveillance program for formalin exposure is recommended. Typical occupational exposure in endoscopy suites is exceedingly brief, so special ventilation is not usually required in that hospital area.
Alimentary tract biopsy specimens should be placed in a volume of formalin fixative that is at least 10 times greater than that of the tissue, and the fixative should surround the specimen completely. These parameters are usually easily met with small endoscopic tissue samples and even with those obtained by EMR, using small tubes of fixative solution. For routine processing, it is a common mistake to place specimens on saline-soaked gauze for delivery to the pathology suite: Severe drying may occur. Complete immersion of these biopsies in formalin should always occur at the bedside. Formaldehyde diffuses into tissue at a rate of approximately 1.0 mm per hour at room temperature. Therefore, up to 1 hour is often needed to adequately fix a specimen with a diameter greater than 1.0 mm, and more time is needed for larger specimens. Controlled microwave fixation at 63° to 65° C can greatly speed the process and is useful for rapid processing of specimens.
Orientation of Formalin-Fixed Tissue Obtained at Endoscopy
Esophageal, gastric, and colonic mucosal biopsies do not require precise orientation before tissue processing and embedding. Until the mid-1980s, most peroral small intestinal biopsies were obtained by either a Crosby suction capsule or a Quinton hydraulic assembly. These two methods were performed fluoroscopically and therefore did not permit direct visualization of the alimentary tract. Biopsies obtained by these methods were carefully oriented under a dissecting microscope before fixation and embedding. By the late 1980s, the fluoroscopy had been replaced by a suction capsule biopsy procedure in direct endoscopic biopsy of the small intestine. Biopsies obtained by this technique are not usually oriented before immersion in fixative, processing, and embedding. Rather, microscopic examination of multiple tissue sections usually permits identification of portions of the small intestinal mucosa that are well oriented and therefore can be assessed satisfactorily for tissue architecture.
In contrast, processing of an endoscopic polypectomy specimen in the pathology suite requires diligent effort. The size and surface configuration (bosselated or villiform) of the polyp should be noted, and the base of the polyp should be identified and described as to whether it is sessile or contains a cylindrical stalk. Regardless of the configuration of the stalk, the base of the polyp should always be inked. Ink and cautery artifact on a microscopic slide are valuable landmarks for locating the relevant resection margins. Small polyps (<1 cm in diameter) should be bisected along the vertical plane of the stalk so that the surgical margin is included. Both halves of the specimen can then be submitted in one cassette.
Section levels should be numbered consecutively; the first level is the one usually located closest to the middle of the polyp stalk. Large polyps (≥1 cm in diameter) may be sectioned differently if the polyp head is too wide to fit into a single cassette. First, the polyp should be bisected along its long axis and fixed overnight in formalin. Once fixed, the sides of the polyp may be trimmed away from the stalk on a vertical axis and submitted in separate cassettes that are labeled accordingly. The middle of the polyp, including the base, should be sectioned vertically and submitted in an appropriate number of cassettes. If a stalk is identified histologically, the status of the margins should always be noted in the surgical pathology report.
If the polyp has been excised in a piecemeal fashion, the size, color, surface configuration (bosselated or villiform), and aggregate dimensions of the tissue fragments should be noted. It is important to record the number of tissue fragments received in the pathology suite.
Flow Cytometry
GI lesions suspected of representing a lymphoproliferative process are usually submitted for histology but should also be processed for flow cytometry. Biopsy specimens intended for flow cytometric analysis, such as gastric biopsies of a mass lesion or EUS-guided fine-needle aspiration (FNA) samples from concerning lymph nodes, should be placed in sterile culture medium and delivered as rapidly as possible to the flow cytometry laboratory. Ideally, this should occur within several hours, but storage of specimens at 4° C overnight is an acceptable alternative.
On receipt in the laboratory, the tissue specimen is disaggregated, and a cell suspension is prepared. Cocktails of fluorescently labeled antibodies appropriate to the diagnostic question are applied to the cell suspension. Current flow cytometry machines can analyze 5000 to 10,000 cells per second, measuring multiple wavelengths of laser-induced fluorescence simultaneously and thus permitting rapid and highly efficient analysis of cell populations. This technique cannot be performed on fixed tissue. It is therefore incumbent on the endoscopist to consider the possibility of a lymphoproliferative disorder at the time of endoscopy in order to ensure that tissue is preserved in a fresh state. Communication between the endoscopist and the pathologist before or immediately after the procedure increases the likelihood that the flow cytometry sample will be received and processed in a timely fashion.
Electron Microscopy
For the rare instances in which electron microscopy of an alimentary tract biopsy is contemplated, tissue samples should be placed directly into the appropriate fixative, which usually consists of a mixture of paraformaldehyde and glutaraldehyde. Unlike formaldehyde-based fixatives, bifunctional glutaraldehyde fixatives penetrate only approximately 0.5 mm into the tissue. Therefore, tissue fragments to be placed in fixative for subsequent electron microscopy should, ideally, measure less than 1.0 mm in maximal dimension. Indications for electron microscopy of endoscopic biopsy specimens are now largely limited to examination of unusual tumors. However, this technique is also helpful in cases of unknown diarrhea in children and in patients with the acquired immunodeficiency syndrome (AIDS) for detection of parasitic organisms.
Endoscopy-Induced Artifacts
Many types of tissue artifacts may be introduced into tissues as a result of bowel preparation, endoscopic trauma, or tissue handling. Some of these are listed in Table 1.3 . Histologic features of artifacts are provided in Table 1.4 . The most common type of artifact (or effect) is lamina propria edema and intramucosal hemorrhage, known as scope trauma ( Fig. 1.6 ). Other effects include aggregation and clumping of inflammatory cells in the lamina propria, surface flattening, mucin depletion, and even erosion and influx of air into the tissue (pseudolipomatosis). The most common histologic artifacts include cautery and crush artifacts occurring as a complication of tissue resection techniques. These may be difficult to avoid in clinical practice given the nature of current endoscopic resection technologies ( Fig. 1.7 ). Cautery artifact as a result of hot biopsy is a normal and expected component of endoscopic polypectomy with electrocautery. Specifically, the region of cauterization may provide a useful landmark of the surgical margin.
| Event | Comment |
|---|---|
| Trauma (tissue hemorrhage) | “Scope trauma” (caused by mechanical damage from the endoscope) or excessive mechanical manipulation for access before biopsy |
| Cautery artifact | Excessive use of electrical current during “hot” biopsy |
| Crush artifact | Excessive use of mechanical force during pinch biopsy |
| Inadequate sampling depth | Absence of submucosa (e.g., evaluate submucosal lesion, rule out amyloid) |
| Inadequate sampling location | Absence of muscularis mucosa (for evaluation of Hirschsprung disease) |
| Insufficient regional sampling (e.g., of “normal-appearing” mucosa) | |
| Chemical colitis | Inadequate rinsing of cleaning solution from the endoscope |
| Laxative-induced changes | Edema, damage to surface epithelium from exposure to oral and rectal laxatives |
| Air-drying | Failure to immerse specimen promptly in fixative |
| Postbiopsy healing | Sampling of a previous biopsy site during subsequent endoscopy |
| Wrong fixative | Formalin rather than fixative for electron microscopy (suboptimal but not irretrievable) |
| No fresh tissue | Failure to preserve fresh tissue; precludes flow cytometry, cytogenetics |
| Event | Feature |
|---|---|
| “Scope trauma” | Mucosal lamina propria hemorrhage or edema |
| Changes related to bowel preparation | Clumping of inflammatory cells, mucin depletion, epithelial degenerative changes, focal neutrophilic infiltration, hemorrhage, edema, air in mucosa (pseudolipomatosis) |
| Insufflation of air at endoscopy | Air spaces within mucosa or submucosa (pseudolipomatosis) |
| Cautery artifact | Coagulated, eosinophilic tissue without cellular or nuclear detail |
| Crush artifact | Compressed tissue with markedly elongated, wavy nuclear remnants and no identifiable architecture |
| Chemical colitis from inadequate cleaning of the endoscope | |
| Degenerative damage to or sloughing of surface epithelium, intraepithelial neutrophils and congestion, focal intramucosal hemorrhage | |
| Laxative-induced changes | Lamina propria edema and neutrophilic infiltration, flattening or sloughing of mucosal surface epithelium, decreased goblet cell numbers |
| Air-drying | Eosinophilic and compressed tissue and loss of nuclear detail at edge of tissue fragment |
| Postbiopsy healing | See Table 1.5 |
Pathologic Features of a Healing Biopsy Site
After endoscopic biopsy, the tissue healing process begins quickly ( Table 1.5 ). After endoscopic polypectomy involving removal of both mucosa and a portion of submucosa, granulation tissue forms during the first days after biopsy ( Fig. 1.8, A and B ). Routine superficial biopsies that involve only mucosa reepithelialize within 48 hours (see Fig. 1.8, C ) and heal completely within a few weeks with only mild residual architectural distortion (see Fig. 1.8, D ). Ulcers that penetrate into the muscularis propria, such as those that form after aggressive endoscopic mucosal resection, often take 3 to 6 days to reepithelialize (see Fig. 1.8, F and G ) and as long as a month to heal completely.
| Time | Feature |
|---|---|
| Immediate | Blood clot with coagulum |
| Hours | Acute inflammation; granulation tissue reaction |
| 2 days * | Reepithelialization of inflamed biopsy site by ingrowth of epithelial cells from adjacent preserved epithelium; early formation of submucosal scar |
| 1-4 wk | Restoration of mucosa with rudimentary glandular architecture, maturation of submucosal scar |
| Months | Residual minimal mucosal architectural distortion, submucosal scar |
* Longer with deep biopsies that involve the muscularis propria.
After routine endoscopic mucosal biopsy during upper or lower endoscopy, there is no increased risk of perforation because of subsequent insufflation (as from repeat endoscopy or from barium enema), even immediately after the biopsy. The risk of perforation after a deep biopsy or endoscopic musocal resection that involves the muscularis propria returns to baseline within 3 to 6 days.
Pathologists should be aware of the changes associated with colonic biopsy site repair and not misinterpret focal architectural distortion of the mucosa or focal submucosal scarring as evidence of inflammatory bowel disease. In addition, the finding of muscularis propria in a biopsy specimen in which this depth of sampling is not expected (e.g., in an esophageal biopsy specimen) should be reported to alert the treating physician of the potential risk of perforation.
Methods for Obtaining Cytology Specimens
See Chapter 3 , Chapter 35 , Chapter 45 .
Brush Cytology
Brush cytology is a method used for broad sampling of the mucosal surface. Cytology brushes all have a common design: Bristles, usually composed of nylon or metal fibers, branch off a thin metal shaft that runs lengthwise within a protective plastic sheath. The various brushes that are currently available do not seem to vary in terms of performance characteristics. The cytology brush is passed through an accessory channel of an endoscope. The end of the sheath is passed out of the tip of the endoscope, and the bristle portion of the brush is then extended from the sheath. The brush is rubbed back and forth several times along the surface of the lesion or stricture and is then pulled back into the sheath. The sheath is withdrawn from the endoscope, and the brush is pushed out of the sheath, thus exposing the bristles. The bristle portion of the brush may be cut off, placed into fixative, and sent in its entirety to the cytopathology laboratory. Alternatively, the bristles may be rolled against a glass slide in the endoscopy suite. The slides should be immediately sprayed with fixative or submerged within it and subsequently delivered to the cytopathologist. If smears are made in the endoscopy suite, little additional benefit is derived from inclusion of the bristles for cytopathologic analysis. Brush cytology is most often used in the pancreaticobiliary tree to sample strictures in the pancreaticobiliary tract. Another common use is sampling of esophageal plaques or lesions suspected to represent candidiasis.
Fluorescence in situ hybridization (FISH) is an increasingly used technique that can be applied to biliary brush cytology tissue specimens in patients with suspected pancreaticobiliary cancer and in those with primary sclerosing cholangitis (who are by definition at increased risk for cholangiocarcinoma). FISH relies on the fact that a very high percentage of pancreaticobiliary malignancies show chromosomal aneuloploidy, typically with chromosomal gains or additions. The presence of these polysomies is strongly associated with malignancy. Commonly used and commercially available probes are used to target the pericentromeric regions of chromosomes 3 (CEP 3), 7 (CEP 7), and 17 (CEP 17) and the chromosomal band 9p21 (LSI 9p21). FISH can be performed on biliary brush cytology specimens. A dedicated biliary brushing is often obtained for FISH testing. When FISH results are combined with those of routine biliary cytology, the diagnostic yield is much higher.
Fine-Needle Aspiration
FNA is another widely used method for obtaining tissue for cytology. FNA needles may be used during standard endoscopy or EUS. EUS provides endoscopists with the ability to sample tissue from parenchymal lesions and lymph nodes and fluid from cystic lesions. EUS provides real-time imaging to ensure that the intended target is localized and sampled. The needles used for FNA during endoscopy are hollow 19- to 25-gauge needles and are often fitted with a central stylet to avoid gathering of intervening tissue. Some needles can also obtain a “core” of tissue (which may be analyzed histologically) in addition to samples for cytologic evaluation.
Once the lesion of interest has been identified, the sheath is pushed out of the endoscope, and the needle is advanced into the target tissue under ultrasonographic guidance (during EUS). If a stylet is present, it is removed, and suction is applied to a syringe at the proximal end of the needle. The endoscopist moves the needle forward and backward within the lesion, filling the distal needle lumen with tissue. Some endoscopists use suction during EUS FNA of solid lesions, whereas others do not. Suction is used to aspirate cysts for obvious reasons; there is some disagreement regarding the use of stylets in routine practice. The needle is then withdrawn into the sheath, and the entire apparatus is removed from the endoscope. Complications from FNA biopsy occur in fewer than 2% of cases; they include bleeding and, in the setting of pancreatic mass FNA, acute pancreatitis.
Optical Techniques
In recent years, there has been an increase in the use of optical techniques to assess in real time the pathologic status of patients with various disease states. Narrow band imaging (NBI) is a technique in which a high-definition videoendoscope is used to allow evaluation of the GI mucosal surface without the use of dyes. NBI is commercially available, and a high percentage of endoscopists have access to this technology. NBI uses different types of optical filters to apply specific wavelengths of light that can achieve deep penetration of the tissue. Specifically, use of red light results in visualization of deeper tissue layers, because red light penetrates more deeply into tissue than blue light does. NBI allows enhanced mucosal and vascular resolution, compared with white light endoscopy. NBI also allows the endoscopist to evaluate large areas of the GI mucosa without the use of vital dyes.
NBI has several clinical uses. In Barrett’s esophagus, evaluation with NBI in which the vascular pattern and the mucosal regularity are assessed can be used to identify patients with dysplastic mucosa ( Fig. 1.9, A and B ). NBI has also been used to evaluate gastric and colonic lesions, in attempts to increase the polyp detection rate during screening colonoscopy and to assess for dysplasia or malignancy in endoscopically visible lesions. NBI does not appear to increase the adenoma or polyp detection rate during screening colonoscopy, but it does allow real-time detection of adenomas during colonoscopy. This technology raises the possibility of performing an “optical biopsy” to identify adenomas during endoscopy. As stated earlier, whether diminutive, resected lesions optically identified as adenomas should still be sent for formal pathologic evaluation or simply discarded remains a highly controversial issue.
