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Endoscopic and Histologic Diagnosis of Barrett Esophagus
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Endoscopic and Histologic Diagnosis of Barrett Esophagus ELIZABETH RAJAN, MD; LAWRENCE J. BURGART, MD; AND CHRISTOPHER J. GOSTOUT, MD resolution or magnified endoscopy is simple, safe, and desirable for surveillance but requires additional procedural time. The use of light-induced fluorescence endoscopy and light-scattering spectroscopy (ie, optical biopsy) is appealing for the diagnosis and characterization of suspicious lesions. Adjunctive endoscopic techniques and adherence to a protocol for performing biopsies facilitate the early detection and subsequent surveillance of Barrett esophagus. Mayo Clin Proc. 2001;76:217-225
Endoscopy plays an important role in the identification, diagnosis, and treatment of Barrett esophagus. Short-segment (<2-3 cm) and traditional long-segment (>2-3 cm) Barrett esophagus are distinguished solely on the length of metaplastic tissue above the esophagogastric junction. The histologic hallmark of intestinal metaplasia is required to confirm diagnosis. Biopsy specimens obtained from tissue of presumed Barrett esophagus or an irregular Z line confirm metaplastic glandular mucosa and permit evaluation of dysplastic or neoplastic changes. In the appropriate clinical setting, the use of adjunctive diagnostic techniques may facilitate the diagnosis of Barrett esophagus and sequelae such as dysplasia. Chromoendoscopy with high-
EGJ = esophagogastric muscular junction; SCJ = squamocolumnar junction
I
esophagogastric junction (Figure 1, left). The line of demarcation between the 2 types of mucosa is readily identifiable in the absence of pathologic changes. The esophagogastric muscular junction (EGJ), with only partial insufflation of the lumen, is located at the point where the mucosal vascular pattern disappears cephalad to the most proximal extent of the gastric rugal folds. This more subtle characterization of the EGJ is useful when distinguishing between a small hiatal hernia and a Barrett segment. The distal mucosal columns or folds converge directly into the EGJ. These columns, when visible with only partial insufflation of the esophageal lumen, denote the relative location of the lower esophageal sphincter. Once the endoscope is passed into the proximal stomach and retroflexed to examine the proximal stomach, the insertion tube of the endoscope can be seen coming through a tightly fitting junction. In some patients, a to-and-fro movement of the instrument can reveal the Z line. A horseshoe-shaped ring of tissue can be seen surrounding the endoscope, with the open ends of the horseshoe marking the left and right lateral boundary of the lesser curvature. This prominent ring of tissue is referred to as the angle of His (Figure 1, right).
n Barrett esophagus, the stratified squamous epithelial lining of the esophagus is replaced by metaplastic specialized columnar epithelium. Suggestive endoscopic findings and subsequent histologic confirmation of intestinal metaplasia anywhere within the tubular esophagus is considered diagnostic of Barrett esophagus. Endoscopy plays an important role in the identification, diagnosis, and treatment of Barrett esophagus. The endoscopist must be able to discriminate Barrett mucosa from an irregular squamocolumnar junction (SCJ), from esophagitis, and from a small diaphragmatic hernia. ENDOSCOPIC LANDMARKS Normal Anatomy The location of the SCJ usually varies with the patient’s height; it is generally situated at or just distal to the diaphragmatic hiatus between 35 and 45 cm from the incisors. It is recognized grossly as an abrupt color change from the pale esophageal squamous epithelium to the salmon-colored columnar epithelium of the cardia. This typically appears as a slightly undulating circumferential border called the Z (zigzag) line. In addition to gross color, the distal esophageal squamous epithelium can be identified by the presence of superficial, thin, capillary-type vessels that disappear at the
Diaphragmatic Hernia The importance of endoscopy lies less in demonstrating the presence of a hernia than in establishing the existence of associated reflux disease and distinguishing a Barrett esophagus. A small (2-3 cm) diaphragmatic hernia appears tubular, with the typical gastric epithelium and rugal folds inferior to the Z line and above the diaphragmatic hiatus (Figure 2, left). The distal end of the diaphragmatic hernia
From the Division of Gastroenterology and Hepatology and Internal Medicine (E.R., C.J.G.) and Department of Laboratory Medicine and Pathology (L.J.B.), Mayo Clinic, Rochester, Minn. Individual reprints of this article are not available. The entire Alan J. Cameron Symposium on Barrett Esophagus and Gastroesophageal Reflux Disease will be available for purchase as a bound booklet from the Proceedings Editorial Office at a later date. Mayo Clin Proc. 2001;76:217-225
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Figure 1. Left, Squamocolumnar junction depicted by both a Z line (arrows) and the distinctive mucosal microvasculature of the distal esophageal mucosa interfacing with the proximal ends of the gastric rugal folds. Right, Retroflexed view of the prominent ring of tissue referred to as the angle of His (arrows).
is identified by the “rosette” of rugal folds. It is confirmed when the patient is asked to take a quick breath in, and the contracture of the diaphragm is seen at the level of the rosette. Other endoscopic landmarks include the proximal displacement of the Z line and the radiographic ring structures described by Wolf.1 The retroverted endoscope can be pulled back to the level of the diaphragmatic hiatus or even a short distance into the hernia pouch, distinctively permitting recognition of the SCJ from below (Figure 2, right). ENDOSCOPIC DIAGNOSIS Barrett Esophagus In Barrett esophagus, the SCJ is displaced upward and has an asymmetric, irregular appearance with salmon-pink,
tonguelike mucosal projections extending up into the squamous epithelium. Barrett epithelium may take on several configurations, including a circumferential segment or isolated islands of gastric-appearing mucosa contiguous or close to the Z line. Short-segment (<2-3 cm) and traditional long-segment (>2-3 cm) Barrett esophagus are arbitrarily distinguished solely on the length of metaplastic tissue above the EGJ (Figure 3). Furthermore, short-segment Barrett esophagus may manifest as an irregular Z line and consequently may be missed easily during endoscopy unless there is suspicion and biopsy specimens are taken (Figure 4). The histologic hallmark of intestinal metaplasia is needed to confirm the diagnosis of Barrett esophagus. Whether shortsegment and long-segment Barrett metaplasia are equiva-
Figure 2. Left, Small diaphragmatic hernia with clearly seen squamocolumnar junction. Right, Distinctive retroflexed appearance of the squamocolumnar junction only seen in patients with a diaphragmatic (hiatal) hernia.
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Figure 3. Left, Barrett esophagus. A long segment of columnar-appearing tissue is present within the tubular esophagus. Right, Short-segment Barrett esophagus with columnar-appearing mucosa extending 2 cm into the esophagus.
lent diseases with regard to risks and the need for surveillance has yet to be determined. Intestinal metaplasia may also occur at a normal-appearing EGJ, posing a further management dilemma. The need for surveillance in these patients has not been established. It is sometimes difficult to ascertain whether specialized columnar epithelium arises from the esophagus or from the gastric cardia. Barrett epithelium in this region is clearly metaplastic and abnormal, irrespective of origin and extent of involvement. In general, biopsy of a normal-appearing EGJ is unnecessary when screening for Barrett esophagus. The accuracy of diagnosing Barrett esophagus by visual inspection at endoscopy has been reported to have a sensitivity as low as 60% and a false positivity of 31%.2 Spechler et al3 studied 142 patients not known to have Barrett esophagus and found endoscopically apparent Barrett esophagus in 1% of patients, whereas 18% of patients without endoscopically apparent Barrett esophagus had specialized columnar epithelium on histology, suggesting that adults frequently have unrecognized segments of specialized columnar epithelium at the EGJ; the importance of this finding remains unclear. Kim et al4 studied the reproducibility of endoscopic techniques used in diagnosing Barrett esophagus and showed substantial variability, with 18% of patients with Barrett esophagus meeting the diagnostic criteria on only 1 of 2 closely spaced examinations. The study concluded that Barrett epithelium involving less than 3 cm of the distal esophagus is particularly susceptible to diagnostic errors, with apparent regression or progression caused by errors in measurement and tissue sampling rather than true epithelial changes. A further prospective study of patients with symptoms of gastroesophageal reflux showed endoscopy to be 92% sensitive in detecting Barrett esophagus.5 These studies
highlight the potential for underdiagnosis when mucosal abnormalities are not recognized and for overdiagnosis when biopsies of normal-appearing EGJs are performed. Intestinal Metaplasia of the Cardia Methylene blue has been used for improved diagnosis of intestinal metaplasia at the gastric cardia. Morales et al6 obtained 4 random biopsy specimens followed by 4 methylene blue–stained biopsy specimens. The sensitivity for
Figure 4. Highly irregular Z line or squamocolumnar junction. Arrows indicate cephalad irregular extension of the Z line.
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intestinal metaplasia at the cardia increased from 38% (for random biopsy alone) to 67% with a targeted methylene blue–stained biopsy specimen. Positive staining was defined as blue-stained mucosa that persisted despite vigorous rinsing. Methylene blue staining was graded by Fennerty et al7 as negative, subtle focal, subtle diffuse, prominent focal, prominent diffuse, and equivocal when it was unclear if blue discoloration was secondary to the stain or an artifact. The most common pattern of mucosal staining was subtle focal staining, which was associated with focal glands of intestinal metaplasia, whereas generalized intestinal metaplasia was associated with prominent staining. Difficulties in interpretation during chromoendoscopy are equivocal results and false positives that may occur from inadequate mucolysis, staining or rinsing, or mucosal damage from either instrumentation or erosive disease. Biopsy Protocol The suggested protocol for surveillance for Barrett esophagus recommends 4-quadrant biopsies taken at 2-cm intervals along the entire length of specialized columnar epithelial lining.8 With the exception of short-segment Barrett esophagus, this format should be adhered to as well during an index endoscopy both to establish the diagnosis of a suspected Barrett eosophagus and to provide baseline surveillance for the patient. In patients with short-segment Barrett esophagus, especially those with 2 cm or less of suspected mucosa, a sufficient number of biopsies should be taken to represent the mucosa in question. A jumbo spiked biopsy forceps has been advocated to obtain a larger specimen and improve histologic interpretation during surveillance. These biopsy devices require the use of larger endoscopes with sufficiently sized channels to accommodate the larger forceps. They are infrequently used during established surveillance and are impractical to use during an index diagnostic or a screening endoscopy. Most important, biopsies of any mucosal abnormalities seen during surveillance or at an initial index examination, no matter how trivial, should be assessed to exclude high-grade dysplasia and early carcinoma. Active inflammation with erythema, erosions, ulceration, and exudate impair the ability to detect Barrett esophagus. If there is concern about the coexistence of a Barrett segment under these circumstances, the patient should undergo a course of proton pump inhibitor therapy with plans for follow-up endoscopy with assessment of biopsy specimens, as indicated, in 4 to 6 weeks. In a prospective controlled trial, patients with biopsyproven Barrett esophagus underwent both 4-quadrant jumbo random biopsy and methylene blue–directed jumbo biopsy in a randomized order.9 Methylene blue–directed biopsy led to the identification of a much larger proportion of specialized columnar epithelium compared with random
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biopsy (limited segment, 54% vs 94%; long-segment Barrett esophagus, 72% vs 92%). Methylene blue–directed biopsy diagnosed dysplasia or cancer in significantly more biopsy samples (12% vs 6%) than did random biopsies. The use of vital staining and optical biopsy should improve the diagnosis of and enhance the endoscopic surveillance for Barrett esophagus. HISTOLOGIC CONFIRMATION OF BARRETT ESOPHAGUS Biopsy specimens obtained in presumed Barrett esophagus or irregular Z line serve 2 primary functions: confirmation of metaplastic glandular mucosa and evaluation of dysplastic or neoplastic changes. Both functions require only basic tissueprocessing technology and light microscopy. Most centers fix biopsy specimens in formalin, a staple in histopathology laboratories, which allows for standard handling, avoidance of heavy metals, maximum flexibility for special stains, and potential utility in polymerase chain reaction–based molecular studies.10 Heavy metal–containing fixatives have been advocated by some subspecialists because they facilitate nuclear fixation, preserving some subtle nuclear morphologic features not uniformly present in formalin-fixed material. However, these cytologic variations are out of the mainstream of diagnostic pathology and may actually result in increased diagnostic variability among medical centers. Routine hematoxylin and eosin–stained sections allow for confirmation of glandular metaplasia, subclassification into metaplastic subtypes (eg, intestinal, cardiac, and fundic), and evaluation of dysplasia and neoplasia. Some authors originally considered any variant of columnar metaplasia in the esophagus as equivalently diagnostic of Barrett esophagus.11 Studies in the 1980s offered 2 key observations underscoring the primary importance of intestinal metaplasia as the critical subtype of columnar mucosa in Barrett esophagus12-15 (Figure 5, top). First, introduction of standard biopsy protocols resulting in improved sampling demonstrated that 96% or more of true Barrett metaplasia contained a considerable component of intestinal metaplasia, usually a vast majority. In other words, it is rare to have Barrett esophagus without intestinal metaplasia, whereas hiatal hernia or irregular Z line most often are not accompanied by intestinal metaplasia. Second, dysplasia and its inherent risk of malignancy were intimately associated with intestinal metaplasia and not gastric metaplasia. Therefore, the rare patients with Barrett metaplasia without intestinal metaplasia appear to have minimal risk of adverse outcome. The histologic evaluation of dysplasia is based on cytologic and architectural criteria.16,17 Low-grade dysplasia is dependent on the presence of epithelial hyperchromasia (ie, an increased nucleus-cytoplasmic ratio), resulting in the appearance of clonal expansion of atypical cells involving
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contiguous crypts, typically with minimal crowding or intact architecture (Figure 5, middle). These cytologic changes do overlap somewhat with those observed in reactive or regenerative epithelium. High-grade dysplasia is based on the presence of more notable nuclear irregularity, including loss of basal polarity, irregular chromatin distribution, nuclear contour irregularities, and presence of nucleoli (Figure 5, bottom). Architectural criteria such as atypical branching, crowding, and cribriforming of glandular acini also come into consideration. Interpretation of the above morphologic parameters comprising criteria for dysplasia grading are inherently subjective. Furthermore, the delimitation of low-grade from high-grade dysplasia is somewhat arbitrary. It is not surprising, therefore, that intra- and interobserver variation in the evaluation of dysplasia has been shown to be significant. A group of expert gastrointestinal pathologists with an interest in this topic and preset criteria—differentiation of high-grade dysplasia and intramucosal carcinoma from low grade or no dysplasia (a critical management breakpoint)— could not achieve 90% agreement.17 Results have been even more variable with general community pathologists.18 Nonetheless, histologic evaluation of dysplasia remains the cornerstone of stratifying treatment options in patients with Barrett metaplasia. It seems most reasonable for several pathologists to review borderline, difficult, and critical individual cases with the goal of achieving consensus within pathology groups. Periodic consultation from expert pathologists outside an individual group minimizes diagnostic drift in the important but subjective evaluation of dysplasia in Barrett esophagus.
Figure 5. Esophagectomy specimen for Barrett metaplasia with high-grade dysplasia. Top, The low-power overview shows nondysplastic intestinal metaplasia to the left and dysplastic metaplasia to the right. There is a sharp, “clonal” morphologic breakpoint (arrows) between the nondysplastic and dysplastic areas. The epithelial hyperchromasia indicative of dysplasia involves the entire mucosal thickness including the luminal surface (hematoxylineosin, original magnification ×100). Middle, High-power view demonstrating an area of low-grade dysplasia. The epithelium displays hyperchromasia (increased nuclear-cytoplasmic ratio) but retains a high degree of intercellular uniformity and nuclear basal polarity (hematoxylin-eosin, original magnification ×400). Bottom, High-power view demonstrating an area of high-grade dysplasia. The epithelium demonstrates hyperchromasia with nuclear pleomorphism and loss of basal polarity. A modest degree of architectural complexity typical of high-grade dysplasia can be seen (hematoxylin-eosin, original magnification ×400).
ENDOSCOPIC TECHNIQUES TO ENHANCE DETECTION OF BARRETT ESOPHAGUS A variety of adjunctive endoscopic techniques are available for the detection of Barrett esophagus. Chromoendoscopy is a simple technique that has yet to be used widely in clinical practice. Methylene blue is readily available and preferentially used, as reflected in the majority of published data available on Barrett esophagus and chromoendoscopy. Lugol iodine solution and toluidine blue have also been used, primarily in screening for esophageal squamous cell cancers. Magnification and high-resolution endoscopy and light analysis techniques may enhance the diagnosis and characterization of suspicious lesions, but their use in clinical practice is limited by the cost of additional equipment and adequate personnel training. Chromoendoscopy Chromoendoscopy, or vital staining, refers to the topical application of chemical stains or pigments to improve characterization and localization of mucosal abnormalities dur-
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Figure 6. Left, Unstained, irregular Z line. Right, Intensive methylene blue staining of a focal patch of Barrett epithelium.
ing endoscopy. Vital stains that have been used in Barrett esophagus include methylene blue, indigo carmine, Lugol iodine solution, and toluidine blue. Absorptive stains identify specific epithelial cells or cellular constituents by preferential diffusion or absorption across the cell membrane. Contrast staining highlights tissue topography and accentuates irregularities in surface contour by outlining mucosal elevations, depressions, or crevices. The technique and yield of chromoendoscopy is improved when either magnification or high-resolution video endoscopes are used. Methylene Blue.—The use of methylene blue may be of particular benefit in detecting short-segment Barrett esophagus, identifying potential foci of dysplasia within a segment of Barrett epithelium, or recognizing residual foci of metaplastic tissue after endoscopic treatment. Methylene blue (methylthionine chloride) is an absorptive stain that is available as a 1% sterile solution. It is taken up by actively absorbing tissues such as small intestinal and colonic epithelial tissue, but importantly, it will not stain nonabsorptive squamous mucosa or gastric mucosa. The exact mechanism of entry across the cell membrane into the cytoplasm remains unclear. Surface mucus impairs the uptake of an absorptive dye into epithelial cells. The segment of esophagus to be studied is first washed and bathed for several minutes with a mucolytic agent such as 10% acetylcysteine. The mucolytic action is related to the sulfhydryl group in the molecule that disrupts disulfide linkages in mucus, thereby reducing or destroying mucus viscosity over a span of several minutes.19 Methylene blue reversibly stains. The staining methods add no more than 10 minutes to the procedure time. The intestinal metaplasia of Barrett esophagus stains blue, which is useful to confirm a suspected short-segment Barrett esophagus and residual foci of metaplastic tissue
after endoscopic treatment (Figure 6). Absent or inhomogeneous staining targets possible dysplasia or cancer (Figure 7). An explanation for the differential staining of dysplastic and nondysplastic Barrett esophagus is the decrease in cytoplasm (increase in the nuclear-cytoplasmic ratio) and the decrease in the number of goblet cells that are characteristic of dysplastic epithelium.2 The sensitivity and specificity of vital staining is known to be significantly affected by the presence of ulcers and esophagitis. Dye can bind nonspecifically to exudate or can fail to stain areas of denuded epithelium. False negatives due to staining technique occur if insufficient time is spent on mucolysis and staining.
Figure 7. Chromoendoscopy of Barrett esophagus showing a focal patch of unstained mucosa within densely stained (methylene blue) Barrett tissue.
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Canto et al20 classified patterns of mucosal staining as either diffuse (>75% of the pink columnar epithelium) or nondiffuse (<75% of pink columnar epithelium). The majority (72%) of patients with positive staining had a nondiffuse staining pattern. Furthermore, a reproducible pattern of staining was demonstrated during repeat endoscopy within 2 to 4 weeks of a previously positive stain. The accuracy of methylene blue staining for detecting specialized columnar epithelium was reported at 95%.20 Methylene blue staining characteristics for dysplastic and malignant Barrett esophagus were further studied prospectively by Canto et al.21 The stain intensity was characterized as dark blue, moderate blue, light blue, and unstained, whereas stain heterogeneity (the degree of variation in stain intensity) was classified as absent, mild, moderate, or marked. A total of 92% of biopsy specimens with high-grade dysplasia or cancer were unstained or stained light blue, compared with 82% with low-grade dysplasia and 38% with no dysplasia. Furthermore, the presence of moderate to marked stain heterogeneity was present in all patients with severe dysplasia or adenocarcinoma, compared with 21% of patients with low-grade dysplasia and 3% without dysplasia. This study concluded that increased heterogeneity and decreased stain intensity are strong independent predictors of high-grade dysplasia or cancer and may help direct tissue biopsies. However, the reproducibility of staining patterns needs to be further studied. Indigo Carmine.—Indigo carmine is a blue contrast stain that can highlight the villiform appearance of intestinal metaplasia in Barrett esophagus and accentuate subtle mucosal abnormalities. Because it is a contrast agent, endoscopy with higher-resolution instruments is advantageous. It is simple to use and does not require a mucolytic agent. A 0.5% to 0.8% solution is sprayed onto the mucosa and provides a dark layer that rapidly disperses because of the effects of secretions and gut motility. Lugol Iodine Solution.—Lugol iodine solution (named after the 19th-century French physician Jean Guillaume Lugol) is an iodine-based absorptive stain with an affinity for glycogen in nonkeratinized squamous epithelium. After the solution is sprayed onto the mucosa, the normal esophageal mucosa turns a prominent green-brown color within moments of application, gradually fading over minutes to hours. Absence of staining indicates diminished or absent glycogen content, as seen in squamous cell cancers, dysplasia, Barrett epithelium, gastric metaplasia, and some degrees of inflammatory esophagitis.22,23 It is used primarily in screening for early esophageal squamous cell carcinoma, particularly in areas where it is endemic, such as Japan and China.24 The sensitivity and specificity of Lugol iodine solution–enhanced endoscopy for diagnosing Barrett esophagus have been reported at 89% and 93%, respectively.25
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Toluidine Blue.—Toluidine blue is a basic absorptive dye that preferentially stains nuclei, thus staining tissue that has increased mitotic activity. It is most commonly used for identifying esophageal squamous cell carcinoma. Following a 1% mucolytic acetic acid rinse, a 1% or 2% aqueous solution of toluidine blue is applied, followed by another 1% acetic acid wash. Positive staining identifies intestinal and gastric metaplastic tissue equally. A study by Chobanian et al26 reported a sensitivity of 98% and specificity of 80% for diagnosing Barrett esophagus. In this study, Barrett epithelium was histologically characterized as specialized columnar epithelium, junctional type, or gastric fundic type. Safety of Chromoendoscopy.—All the endoscopic stains are accepted as nontoxic, although maximal safe doses are not known. Both methylene blue and acetylcysteine are minimally absorbed. Lugol iodine solution must be avoided in patients with a history of iodine allergy. Concentrated Lugol iodine solution (50%) caused heartburn in 3 patients and bronchospasm in 1 patient with iodine sensitivity.27 Methylene blue and indigo carmine have caused serious systemic reactions during parenteral administration.28,29 Stains should be limited to the described concentrations, using the smallest volumes, generally 10 to 20 mL. Pooled stain can be reused to minimize volumes. The patient must be instructed about possible discoloration of urine and feces. The dyes used for chromoendoscopy stain anything they contact, especially clothing fabric, so care must be used to avoid any accidental leakage or spraying outside the patient. They pose no risks to clinical personnel and do not damage the endoscopy equipment, but with frequent use, they may discolor the outer markings on endoscope insertion tubes. Magnification and High-Resolution Endoscopy Endoscopic detection of gastrointestinal pathology depends on the recognition of visible mucosal lesions. Magnification and high-resolution endoscopy enable detection of subtle changes of the gastrointestinal mucosa that may be unrecognized or of indeterminate importance when conventional fiberoptic and video endoscopes are used. Magnification instruments were built in Japan in the 1960s and can magnify the mucosa from ×3 to ×170, with most magnification performed within a more practical range of ×10 to ×35.30 High-magnification endoscopy is best used with absorptive staining because of time-consuming technical aspects of magnification (focusing in and out of magnification) and the need for long-lasting staining. Magnification instruments allow the examination of cellular mucosal patterns. There are limited data on the use of magnification endoscopy in Barrett esophagus. Stevens et al27 described the use of magnification endoscopy in con-
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junction with combined dye spraying. Lugol iodine solution was initially sprayed to define the SCJ, and then areas of unstained mucosa, representing possible Barrett esophagus, were stained with indigo carmine, which was followed by magnification endoscopy to image the resultant fine mucosal pattern. Barrett epithelium was visualized as raised with a villiform surface pattern, an appearance similar to that of small bowel mucosa obtained using the same procedure. This pattern correlated with histologic confirmation of Barrett esophagus. The villous surface appearance seen on magnified examination corresponds to mucosal ridges with depressions (openings of intestinal glands) as seen by electron microscopy and are not true villi. High-resolution instruments are video based and provide greater pixel density along with minor magnification. In our own experience, the perceived advantage of these instruments is their ease of use and the flexibility to use either contrast or absorptive chromoendoscopy stains. Sufficient mucosal surface detail is continually provided without the manipulations needed to focus to a desired magnification and simultaneously readjust the instrument position to accommodate for movement and fluids altering the viewing field. Most of the latest-model commercially available video endoscopes offer higher resolution, which often exceeds 60% of the resolution capabilities of previous video endoscope models. Resolution beyond these instruments is available only in prototype instruments undergoing evaluation. Light Analysis Techniques Spectroscopy, the analysis of wavelength and intensity of light, can also be used to analyze the mucosa. The ability to characterize tissue using light by rapid, contact-only spectroscopic techniques is referred to as optical biopsy and has far-reaching potential. When tissue is illuminated with light (monochromatic laser or nonlaser), photons penetrate into the tissue at varying depths, with some photons absorbed and others reemitted in another wavelength of light (this property is called fluorescence—eg, light-induced fluorescence endoscopy), or the photons can be scattered within the tissue (eg, light-scattering spectroscopy).31 Light-induced fluorescence endoscopy is based on the use of endogenous (naturally occurring) or exogenous (administered) fluorescent agents. Autofluorescence arises from endogenous molecules called fluorophores within tissues such as aromatic amino acids, connective tissue, lipopigments, and by-products of heme synthesis.32 Mucosa, submucosa, and muscularis propria have distinct fluorophore compositions, so that the fluorescence measured at the luminal surface comprises contributions from the various layers. Tissue autofluorescence is sensitive to alterations in tissue morphology and biochemistry and theoretically may be used to detect early malignant transformation. An alternative to optical
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biopsy by autofluorescence is the use of exogenous fluorescence-inducing drugs such as hematoporphyrin derivative, phthalocyanines, and 5-aminolevulinic acid. Drug-induced fluorescence is dependent on the degree of selective localization of the light-sensitizing drug within tissue and drug pharmacokinetics. Both methods are performed with a contact optical fiber probe, typically 0.5 to 1 mm in diameter, passed through the endoscope biopsy channel and placed in contact with the targeted surface of suspicious mucosa. Although this procedure is technically simple, the sampling of only a small volume of tissue (1-3 mm) immediately beneath the probe tip is a disadvantage. Endoscopic systems capable of producing real-time high-resolution fluorescence images and evaluating large areas of mucosal surface in parallel with conventional white light endoscopic examination have recently become available.33 The use of light-induced fluorescence endoscopy to target biopsies may offer more effective screening and surveillance for high-risk patients, such as those with Barrett esophagus. The role of exogenous fluorescenceinducing agents in identifying Barrett mucosa is uncertain. There are 2 contradictory experiences that reported using 5-aminolevulinic acid–induced fluorescence to distinguish between normal squamous and Barrett mucosa.34,35 In light-scattering spectroscopy, the determinants of light scattering within tissue are the size and number of the scatterers (eg, mitochondria or nuclei) and the wavelength of light used to illuminate the tissue.31,36 This property of light has been used during endoscopic procedures to determine the number and size of nuclei and the degree of nuclei crowding in patients with Barrett esophagus. A prospective study that assessed the potential of light-scattering spectroscopy to measure epithelial nuclear enlargement and crowding in patients with Barrett esophagus showed that both the sensitivity and specificity of this technique for detecting dysplasia (either high-grade or low-grade dysplasia) were 90%.37 Dysplasia was diagnosed if 30% or more of the nuclei exceeded 10 µm in diameter. This suggests that light-scattering spectroscopy may readily detect areas of dysplasia during endoscopy. CONCLUSION Endoscopy plays an integral part in the diagnosis and management of Barrett esophagus and its associated complications. In the appropriate clinical setting, the use of adjunctive diagnostic techniques may facilitate the diagnosis of Barrett esophagus. Chromoendoscopy is simple and safe, but this technique requires additional procedural time as well as late-model video endoscopes with improved resolution. To our knowledge, the relative performance, accuracy, ease of use, and cost of the various vital stains for diagnosing Barrett esophagus and the yield in detecting
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dysplasia or cancer have not been formally compared. There are insufficient data to recommend the use of highmagnification endoscopes for screening or surveillance procedures. The use of light-induced fluorescence endoscopy and light-scattering spectroscopy (ie, optical biopsy) is appealing for the diagnosis and characterization of suspicious lesions to target biopsies. Chromoendoscopy and optical biopsy methods are advocated for both the diagnosis and surveillance of Barrett esophagus but require further study. A high level of suspicion and adherence to a protocol for performing biopsies are mandatory.
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Alikhan M, Rex D, Khan A, Rahmani E, Cummings O, Ulbright TM. Variable pathologic interpretation of columnar lined esophagus by general pathologists in community practice. Gastrointest Endosc. 1999;50:23-26. Acosta MM, Boyce HW Jr. Chromoendoscopy—where is it useful? J Clin Gastroenterol. 1998;27:13-20. Canto MI, Setrakian S, Petras RE, Blades E, Chak A, Sivak MV Jr. Methylene blue selectively stains intestinal metaplasia in Barrett’s esophagus. Gastrointest Endosc. 1996;44:1-7. Canto MI, Setrakian S, Willis J, Chak A, Sivak MV Jr. Methylene blue staining of dysplastic and non-dysplastic Barrett’s esophagus: an in vivo and ex vivo study [abstract]. Gastrointest Endosc. 1996:43. Abstract 164. Mori M, Adachi Y, Matsushima T, Matsuda H, Kuwano H, Sugimachi K. Lugol staining pattern and histology of esophageal lesions. Am J Gastroenterol. 1993;88:701-705. Sugimachi K, Kitamura K, Baba K, Ikebe M, Kuwano H. Endoscopic diagnosis of early carcinoma of the esophagus using Lugol’s solution. Gastrointest Endosc. 1992;38:657-661. Okumura T, Aruga H, Inohara H, et al. Endoscopic examination of the upper gastrointestinal tract for the presence of second primary cancers in head and neck cancer patients. Acta Otolaryngol Suppl. 1993;501:103-106. Woolf GM, Riddell RH, Irvine EJ, Hunt RH. A study to examine agreement between endoscopy and histology for the diagnosis of columnar lined (Barrett’s) esophagus. Gastrointest Endosc. 1989; 35:541-544. Chobanian SJ, Cattau EL Jr, Winters C Jr, et al. In vivo staining with toluidine blue as an adjunct to the endoscopic detection of Barrett’s esophagus. Gastrointest Endosc. 1987;33:99-101. Stevens PD, Lightdale CJ, Green PH, Siegel LM, GarciaCarrasquillo RJ, Rotterdam H. Combined magnification endoscopy with chromoendoscopy for the evaluation of Barrett’s esophagus. Gastrointest Endosc. 1994;40:747-749. Naitoh J, Fox BM. Severe hypotension, bronchospasm, and urticaria from intravenous indigo carmine. Urology. 1994;44:271-272. Sills MR, Zinkham WH. Methylene blue-induced Heinz body hemolytic anemia. Arch Pediatr Adolesc Med. 1994;148:306-310. Sorbi D, Gostout CJ. Polyp identification and marking: chromoendoscopy, high-resolution and high-magnification endoscopy, tattooing and clipping. Tech Gastrointest Endosc. 2000;2:2-8. Wallace MB, Van Dam J. Enhanced gastrointestinal diagnosis. Gastrointest Endosc Clin N Am. 2000;10:71-80. DaCosta RS, Wilson BC, Marcon NE. Light-induced fluorescence endoscopy of the gastrointestinal tract. Gastrointest Endosc Clin N Am. 2000;10:37-69. Namihisa A, Watanabe H, Tanaka H, Miwa H, Ogihara T, Sato N. Detection of gastric lesions by endoscopic autofluorescence real time imaging system (light induced fluorescence endoscopy) [abstract]. Gastrointest Endosc. 1997;45:AB35. Abstract 43. Messmann H, Kullmann F, Wild T, et al. Detection of dysplastic lesions by fluorescence in a model of colitis in rats after previous photosensitization with 5-aminolaevulinic acid. Endoscopy. 1998; 30:333-338. van den Boogert J, Houtsmuller AB, de Rooij FWM, de Bruin RWF, Siersema PD, van Hillegersberg R. Kinetics, localization, and mechanism of 5-aminolevulinic acid-induced porphyrin accumulation in normal and Barrett’s-like rat esophagus. Lasers Surg Med. 1999;24:3-13. Perelman LT, Backman V, Wallace M, et al. Observation of periodic fine structure in reflectance from biological tissue: a new technique for measuring nuclear size distribution. Phys Rev Lett. 1998;80:627-630. Wallace MB, Shields SJ, Perelmen LT, et al. Fiber-optic detection of low-grade dysplasia in patients with Barrett’s esophagus using reflectance spectroscopy [abstract]. Gastroenterology. 1998;114(4, pt 2):A327. Abstract G1337.
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Endoscopic and Histologic Diagnosis of Barrett Esophagus ELIZABETH RAJAN, MD; LAWRENCE J. BURGART, MD; AND CHRISTOPHER J. GOSTOUT, MD resolution or magnified endoscopy is simple, safe, and desirable for surveillance but requires additional procedural time. The use of light-induced fluorescence endoscopy and light-scattering spectroscopy (ie, optical biopsy) is appealing for the diagnosis and characterization of suspicious lesions. Adjunctive endoscopic techniques and adherence to a protocol for performing biopsies facilitate the early detection and subsequent surveillance of Barrett esophagus. Mayo Clin Proc. 2001;76:217-225
Endoscopy plays an important role in the identification, diagnosis, and treatment of Barrett esophagus. Short-segment (<2-3 cm) and traditional long-segment (>2-3 cm) Barrett esophagus are distinguished solely on the length of metaplastic tissue above the esophagogastric junction. The histologic hallmark of intestinal metaplasia is required to confirm diagnosis. Biopsy specimens obtained from tissue of presumed Barrett esophagus or an irregular Z line confirm metaplastic glandular mucosa and permit evaluation of dysplastic or neoplastic changes. In the appropriate clinical setting, the use of adjunctive diagnostic techniques may facilitate the diagnosis of Barrett esophagus and sequelae such as dysplasia. Chromoendoscopy with high-
EGJ = esophagogastric muscular junction; SCJ = squamocolumnar junction
I
esophagogastric junction (Figure 1, left). The line of demarcation between the 2 types of mucosa is readily identifiable in the absence of pathologic changes. The esophagogastric muscular junction (EGJ), with only partial insufflation of the lumen, is located at the point where the mucosal vascular pattern disappears cephalad to the most proximal extent of the gastric rugal folds. This more subtle characterization of the EGJ is useful when distinguishing between a small hiatal hernia and a Barrett segment. The distal mucosal columns or folds converge directly into the EGJ. These columns, when visible with only partial insufflation of the esophageal lumen, denote the relative location of the lower esophageal sphincter. Once the endoscope is passed into the proximal stomach and retroflexed to examine the proximal stomach, the insertion tube of the endoscope can be seen coming through a tightly fitting junction. In some patients, a to-and-fro movement of the instrument can reveal the Z line. A horseshoe-shaped ring of tissue can be seen surrounding the endoscope, with the open ends of the horseshoe marking the left and right lateral boundary of the lesser curvature. This prominent ring of tissue is referred to as the angle of His (Figure 1, right).
n Barrett esophagus, the stratified squamous epithelial lining of the esophagus is replaced by metaplastic specialized columnar epithelium. Suggestive endoscopic findings and subsequent histologic confirmation of intestinal metaplasia anywhere within the tubular esophagus is considered diagnostic of Barrett esophagus. Endoscopy plays an important role in the identification, diagnosis, and treatment of Barrett esophagus. The endoscopist must be able to discriminate Barrett mucosa from an irregular squamocolumnar junction (SCJ), from esophagitis, and from a small diaphragmatic hernia. ENDOSCOPIC LANDMARKS Normal Anatomy The location of the SCJ usually varies with the patient’s height; it is generally situated at or just distal to the diaphragmatic hiatus between 35 and 45 cm from the incisors. It is recognized grossly as an abrupt color change from the pale esophageal squamous epithelium to the salmon-colored columnar epithelium of the cardia. This typically appears as a slightly undulating circumferential border called the Z (zigzag) line. In addition to gross color, the distal esophageal squamous epithelium can be identified by the presence of superficial, thin, capillary-type vessels that disappear at the
Diaphragmatic Hernia The importance of endoscopy lies less in demonstrating the presence of a hernia than in establishing the existence of associated reflux disease and distinguishing a Barrett esophagus. A small (2-3 cm) diaphragmatic hernia appears tubular, with the typical gastric epithelium and rugal folds inferior to the Z line and above the diaphragmatic hiatus (Figure 2, left). The distal end of the diaphragmatic hernia
From the Division of Gastroenterology and Hepatology and Internal Medicine (E.R., C.J.G.) and Department of Laboratory Medicine and Pathology (L.J.B.), Mayo Clinic, Rochester, Minn. Individual reprints of this article are not available. The entire Alan J. Cameron Symposium on Barrett Esophagus and Gastroesophageal Reflux Disease will be available for purchase as a bound booklet from the Proceedings Editorial Office at a later date. Mayo Clin Proc. 2001;76:217-225
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Figure 1. Left, Squamocolumnar junction depicted by both a Z line (arrows) and the distinctive mucosal microvasculature of the distal esophageal mucosa interfacing with the proximal ends of the gastric rugal folds. Right, Retroflexed view of the prominent ring of tissue referred to as the angle of His (arrows).
is identified by the “rosette” of rugal folds. It is confirmed when the patient is asked to take a quick breath in, and the contracture of the diaphragm is seen at the level of the rosette. Other endoscopic landmarks include the proximal displacement of the Z line and the radiographic ring structures described by Wolf.1 The retroverted endoscope can be pulled back to the level of the diaphragmatic hiatus or even a short distance into the hernia pouch, distinctively permitting recognition of the SCJ from below (Figure 2, right). ENDOSCOPIC DIAGNOSIS Barrett Esophagus In Barrett esophagus, the SCJ is displaced upward and has an asymmetric, irregular appearance with salmon-pink,
tonguelike mucosal projections extending up into the squamous epithelium. Barrett epithelium may take on several configurations, including a circumferential segment or isolated islands of gastric-appearing mucosa contiguous or close to the Z line. Short-segment (<2-3 cm) and traditional long-segment (>2-3 cm) Barrett esophagus are arbitrarily distinguished solely on the length of metaplastic tissue above the EGJ (Figure 3). Furthermore, short-segment Barrett esophagus may manifest as an irregular Z line and consequently may be missed easily during endoscopy unless there is suspicion and biopsy specimens are taken (Figure 4). The histologic hallmark of intestinal metaplasia is needed to confirm the diagnosis of Barrett esophagus. Whether shortsegment and long-segment Barrett metaplasia are equiva-
Figure 2. Left, Small diaphragmatic hernia with clearly seen squamocolumnar junction. Right, Distinctive retroflexed appearance of the squamocolumnar junction only seen in patients with a diaphragmatic (hiatal) hernia.
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Figure 3. Left, Barrett esophagus. A long segment of columnar-appearing tissue is present within the tubular esophagus. Right, Short-segment Barrett esophagus with columnar-appearing mucosa extending 2 cm into the esophagus.
lent diseases with regard to risks and the need for surveillance has yet to be determined. Intestinal metaplasia may also occur at a normal-appearing EGJ, posing a further management dilemma. The need for surveillance in these patients has not been established. It is sometimes difficult to ascertain whether specialized columnar epithelium arises from the esophagus or from the gastric cardia. Barrett epithelium in this region is clearly metaplastic and abnormal, irrespective of origin and extent of involvement. In general, biopsy of a normal-appearing EGJ is unnecessary when screening for Barrett esophagus. The accuracy of diagnosing Barrett esophagus by visual inspection at endoscopy has been reported to have a sensitivity as low as 60% and a false positivity of 31%.2 Spechler et al3 studied 142 patients not known to have Barrett esophagus and found endoscopically apparent Barrett esophagus in 1% of patients, whereas 18% of patients without endoscopically apparent Barrett esophagus had specialized columnar epithelium on histology, suggesting that adults frequently have unrecognized segments of specialized columnar epithelium at the EGJ; the importance of this finding remains unclear. Kim et al4 studied the reproducibility of endoscopic techniques used in diagnosing Barrett esophagus and showed substantial variability, with 18% of patients with Barrett esophagus meeting the diagnostic criteria on only 1 of 2 closely spaced examinations. The study concluded that Barrett epithelium involving less than 3 cm of the distal esophagus is particularly susceptible to diagnostic errors, with apparent regression or progression caused by errors in measurement and tissue sampling rather than true epithelial changes. A further prospective study of patients with symptoms of gastroesophageal reflux showed endoscopy to be 92% sensitive in detecting Barrett esophagus.5 These studies
highlight the potential for underdiagnosis when mucosal abnormalities are not recognized and for overdiagnosis when biopsies of normal-appearing EGJs are performed. Intestinal Metaplasia of the Cardia Methylene blue has been used for improved diagnosis of intestinal metaplasia at the gastric cardia. Morales et al6 obtained 4 random biopsy specimens followed by 4 methylene blue–stained biopsy specimens. The sensitivity for
Figure 4. Highly irregular Z line or squamocolumnar junction. Arrows indicate cephalad irregular extension of the Z line.
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intestinal metaplasia at the cardia increased from 38% (for random biopsy alone) to 67% with a targeted methylene blue–stained biopsy specimen. Positive staining was defined as blue-stained mucosa that persisted despite vigorous rinsing. Methylene blue staining was graded by Fennerty et al7 as negative, subtle focal, subtle diffuse, prominent focal, prominent diffuse, and equivocal when it was unclear if blue discoloration was secondary to the stain or an artifact. The most common pattern of mucosal staining was subtle focal staining, which was associated with focal glands of intestinal metaplasia, whereas generalized intestinal metaplasia was associated with prominent staining. Difficulties in interpretation during chromoendoscopy are equivocal results and false positives that may occur from inadequate mucolysis, staining or rinsing, or mucosal damage from either instrumentation or erosive disease. Biopsy Protocol The suggested protocol for surveillance for Barrett esophagus recommends 4-quadrant biopsies taken at 2-cm intervals along the entire length of specialized columnar epithelial lining.8 With the exception of short-segment Barrett esophagus, this format should be adhered to as well during an index endoscopy both to establish the diagnosis of a suspected Barrett eosophagus and to provide baseline surveillance for the patient. In patients with short-segment Barrett esophagus, especially those with 2 cm or less of suspected mucosa, a sufficient number of biopsies should be taken to represent the mucosa in question. A jumbo spiked biopsy forceps has been advocated to obtain a larger specimen and improve histologic interpretation during surveillance. These biopsy devices require the use of larger endoscopes with sufficiently sized channels to accommodate the larger forceps. They are infrequently used during established surveillance and are impractical to use during an index diagnostic or a screening endoscopy. Most important, biopsies of any mucosal abnormalities seen during surveillance or at an initial index examination, no matter how trivial, should be assessed to exclude high-grade dysplasia and early carcinoma. Active inflammation with erythema, erosions, ulceration, and exudate impair the ability to detect Barrett esophagus. If there is concern about the coexistence of a Barrett segment under these circumstances, the patient should undergo a course of proton pump inhibitor therapy with plans for follow-up endoscopy with assessment of biopsy specimens, as indicated, in 4 to 6 weeks. In a prospective controlled trial, patients with biopsyproven Barrett esophagus underwent both 4-quadrant jumbo random biopsy and methylene blue–directed jumbo biopsy in a randomized order.9 Methylene blue–directed biopsy led to the identification of a much larger proportion of specialized columnar epithelium compared with random
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biopsy (limited segment, 54% vs 94%; long-segment Barrett esophagus, 72% vs 92%). Methylene blue–directed biopsy diagnosed dysplasia or cancer in significantly more biopsy samples (12% vs 6%) than did random biopsies. The use of vital staining and optical biopsy should improve the diagnosis of and enhance the endoscopic surveillance for Barrett esophagus. HISTOLOGIC CONFIRMATION OF BARRETT ESOPHAGUS Biopsy specimens obtained in presumed Barrett esophagus or irregular Z line serve 2 primary functions: confirmation of metaplastic glandular mucosa and evaluation of dysplastic or neoplastic changes. Both functions require only basic tissueprocessing technology and light microscopy. Most centers fix biopsy specimens in formalin, a staple in histopathology laboratories, which allows for standard handling, avoidance of heavy metals, maximum flexibility for special stains, and potential utility in polymerase chain reaction–based molecular studies.10 Heavy metal–containing fixatives have been advocated by some subspecialists because they facilitate nuclear fixation, preserving some subtle nuclear morphologic features not uniformly present in formalin-fixed material. However, these cytologic variations are out of the mainstream of diagnostic pathology and may actually result in increased diagnostic variability among medical centers. Routine hematoxylin and eosin–stained sections allow for confirmation of glandular metaplasia, subclassification into metaplastic subtypes (eg, intestinal, cardiac, and fundic), and evaluation of dysplasia and neoplasia. Some authors originally considered any variant of columnar metaplasia in the esophagus as equivalently diagnostic of Barrett esophagus.11 Studies in the 1980s offered 2 key observations underscoring the primary importance of intestinal metaplasia as the critical subtype of columnar mucosa in Barrett esophagus12-15 (Figure 5, top). First, introduction of standard biopsy protocols resulting in improved sampling demonstrated that 96% or more of true Barrett metaplasia contained a considerable component of intestinal metaplasia, usually a vast majority. In other words, it is rare to have Barrett esophagus without intestinal metaplasia, whereas hiatal hernia or irregular Z line most often are not accompanied by intestinal metaplasia. Second, dysplasia and its inherent risk of malignancy were intimately associated with intestinal metaplasia and not gastric metaplasia. Therefore, the rare patients with Barrett metaplasia without intestinal metaplasia appear to have minimal risk of adverse outcome. The histologic evaluation of dysplasia is based on cytologic and architectural criteria.16,17 Low-grade dysplasia is dependent on the presence of epithelial hyperchromasia (ie, an increased nucleus-cytoplasmic ratio), resulting in the appearance of clonal expansion of atypical cells involving
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contiguous crypts, typically with minimal crowding or intact architecture (Figure 5, middle). These cytologic changes do overlap somewhat with those observed in reactive or regenerative epithelium. High-grade dysplasia is based on the presence of more notable nuclear irregularity, including loss of basal polarity, irregular chromatin distribution, nuclear contour irregularities, and presence of nucleoli (Figure 5, bottom). Architectural criteria such as atypical branching, crowding, and cribriforming of glandular acini also come into consideration. Interpretation of the above morphologic parameters comprising criteria for dysplasia grading are inherently subjective. Furthermore, the delimitation of low-grade from high-grade dysplasia is somewhat arbitrary. It is not surprising, therefore, that intra- and interobserver variation in the evaluation of dysplasia has been shown to be significant. A group of expert gastrointestinal pathologists with an interest in this topic and preset criteria—differentiation of high-grade dysplasia and intramucosal carcinoma from low grade or no dysplasia (a critical management breakpoint)— could not achieve 90% agreement.17 Results have been even more variable with general community pathologists.18 Nonetheless, histologic evaluation of dysplasia remains the cornerstone of stratifying treatment options in patients with Barrett metaplasia. It seems most reasonable for several pathologists to review borderline, difficult, and critical individual cases with the goal of achieving consensus within pathology groups. Periodic consultation from expert pathologists outside an individual group minimizes diagnostic drift in the important but subjective evaluation of dysplasia in Barrett esophagus.
Figure 5. Esophagectomy specimen for Barrett metaplasia with high-grade dysplasia. Top, The low-power overview shows nondysplastic intestinal metaplasia to the left and dysplastic metaplasia to the right. There is a sharp, “clonal” morphologic breakpoint (arrows) between the nondysplastic and dysplastic areas. The epithelial hyperchromasia indicative of dysplasia involves the entire mucosal thickness including the luminal surface (hematoxylineosin, original magnification ×100). Middle, High-power view demonstrating an area of low-grade dysplasia. The epithelium displays hyperchromasia (increased nuclear-cytoplasmic ratio) but retains a high degree of intercellular uniformity and nuclear basal polarity (hematoxylin-eosin, original magnification ×400). Bottom, High-power view demonstrating an area of high-grade dysplasia. The epithelium demonstrates hyperchromasia with nuclear pleomorphism and loss of basal polarity. A modest degree of architectural complexity typical of high-grade dysplasia can be seen (hematoxylin-eosin, original magnification ×400).
ENDOSCOPIC TECHNIQUES TO ENHANCE DETECTION OF BARRETT ESOPHAGUS A variety of adjunctive endoscopic techniques are available for the detection of Barrett esophagus. Chromoendoscopy is a simple technique that has yet to be used widely in clinical practice. Methylene blue is readily available and preferentially used, as reflected in the majority of published data available on Barrett esophagus and chromoendoscopy. Lugol iodine solution and toluidine blue have also been used, primarily in screening for esophageal squamous cell cancers. Magnification and high-resolution endoscopy and light analysis techniques may enhance the diagnosis and characterization of suspicious lesions, but their use in clinical practice is limited by the cost of additional equipment and adequate personnel training. Chromoendoscopy Chromoendoscopy, or vital staining, refers to the topical application of chemical stains or pigments to improve characterization and localization of mucosal abnormalities dur-
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Figure 6. Left, Unstained, irregular Z line. Right, Intensive methylene blue staining of a focal patch of Barrett epithelium.
ing endoscopy. Vital stains that have been used in Barrett esophagus include methylene blue, indigo carmine, Lugol iodine solution, and toluidine blue. Absorptive stains identify specific epithelial cells or cellular constituents by preferential diffusion or absorption across the cell membrane. Contrast staining highlights tissue topography and accentuates irregularities in surface contour by outlining mucosal elevations, depressions, or crevices. The technique and yield of chromoendoscopy is improved when either magnification or high-resolution video endoscopes are used. Methylene Blue.—The use of methylene blue may be of particular benefit in detecting short-segment Barrett esophagus, identifying potential foci of dysplasia within a segment of Barrett epithelium, or recognizing residual foci of metaplastic tissue after endoscopic treatment. Methylene blue (methylthionine chloride) is an absorptive stain that is available as a 1% sterile solution. It is taken up by actively absorbing tissues such as small intestinal and colonic epithelial tissue, but importantly, it will not stain nonabsorptive squamous mucosa or gastric mucosa. The exact mechanism of entry across the cell membrane into the cytoplasm remains unclear. Surface mucus impairs the uptake of an absorptive dye into epithelial cells. The segment of esophagus to be studied is first washed and bathed for several minutes with a mucolytic agent such as 10% acetylcysteine. The mucolytic action is related to the sulfhydryl group in the molecule that disrupts disulfide linkages in mucus, thereby reducing or destroying mucus viscosity over a span of several minutes.19 Methylene blue reversibly stains. The staining methods add no more than 10 minutes to the procedure time. The intestinal metaplasia of Barrett esophagus stains blue, which is useful to confirm a suspected short-segment Barrett esophagus and residual foci of metaplastic tissue
after endoscopic treatment (Figure 6). Absent or inhomogeneous staining targets possible dysplasia or cancer (Figure 7). An explanation for the differential staining of dysplastic and nondysplastic Barrett esophagus is the decrease in cytoplasm (increase in the nuclear-cytoplasmic ratio) and the decrease in the number of goblet cells that are characteristic of dysplastic epithelium.2 The sensitivity and specificity of vital staining is known to be significantly affected by the presence of ulcers and esophagitis. Dye can bind nonspecifically to exudate or can fail to stain areas of denuded epithelium. False negatives due to staining technique occur if insufficient time is spent on mucolysis and staining.
Figure 7. Chromoendoscopy of Barrett esophagus showing a focal patch of unstained mucosa within densely stained (methylene blue) Barrett tissue.
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Canto et al20 classified patterns of mucosal staining as either diffuse (>75% of the pink columnar epithelium) or nondiffuse (<75% of pink columnar epithelium). The majority (72%) of patients with positive staining had a nondiffuse staining pattern. Furthermore, a reproducible pattern of staining was demonstrated during repeat endoscopy within 2 to 4 weeks of a previously positive stain. The accuracy of methylene blue staining for detecting specialized columnar epithelium was reported at 95%.20 Methylene blue staining characteristics for dysplastic and malignant Barrett esophagus were further studied prospectively by Canto et al.21 The stain intensity was characterized as dark blue, moderate blue, light blue, and unstained, whereas stain heterogeneity (the degree of variation in stain intensity) was classified as absent, mild, moderate, or marked. A total of 92% of biopsy specimens with high-grade dysplasia or cancer were unstained or stained light blue, compared with 82% with low-grade dysplasia and 38% with no dysplasia. Furthermore, the presence of moderate to marked stain heterogeneity was present in all patients with severe dysplasia or adenocarcinoma, compared with 21% of patients with low-grade dysplasia and 3% without dysplasia. This study concluded that increased heterogeneity and decreased stain intensity are strong independent predictors of high-grade dysplasia or cancer and may help direct tissue biopsies. However, the reproducibility of staining patterns needs to be further studied. Indigo Carmine.—Indigo carmine is a blue contrast stain that can highlight the villiform appearance of intestinal metaplasia in Barrett esophagus and accentuate subtle mucosal abnormalities. Because it is a contrast agent, endoscopy with higher-resolution instruments is advantageous. It is simple to use and does not require a mucolytic agent. A 0.5% to 0.8% solution is sprayed onto the mucosa and provides a dark layer that rapidly disperses because of the effects of secretions and gut motility. Lugol Iodine Solution.—Lugol iodine solution (named after the 19th-century French physician Jean Guillaume Lugol) is an iodine-based absorptive stain with an affinity for glycogen in nonkeratinized squamous epithelium. After the solution is sprayed onto the mucosa, the normal esophageal mucosa turns a prominent green-brown color within moments of application, gradually fading over minutes to hours. Absence of staining indicates diminished or absent glycogen content, as seen in squamous cell cancers, dysplasia, Barrett epithelium, gastric metaplasia, and some degrees of inflammatory esophagitis.22,23 It is used primarily in screening for early esophageal squamous cell carcinoma, particularly in areas where it is endemic, such as Japan and China.24 The sensitivity and specificity of Lugol iodine solution–enhanced endoscopy for diagnosing Barrett esophagus have been reported at 89% and 93%, respectively.25
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Toluidine Blue.—Toluidine blue is a basic absorptive dye that preferentially stains nuclei, thus staining tissue that has increased mitotic activity. It is most commonly used for identifying esophageal squamous cell carcinoma. Following a 1% mucolytic acetic acid rinse, a 1% or 2% aqueous solution of toluidine blue is applied, followed by another 1% acetic acid wash. Positive staining identifies intestinal and gastric metaplastic tissue equally. A study by Chobanian et al26 reported a sensitivity of 98% and specificity of 80% for diagnosing Barrett esophagus. In this study, Barrett epithelium was histologically characterized as specialized columnar epithelium, junctional type, or gastric fundic type. Safety of Chromoendoscopy.—All the endoscopic stains are accepted as nontoxic, although maximal safe doses are not known. Both methylene blue and acetylcysteine are minimally absorbed. Lugol iodine solution must be avoided in patients with a history of iodine allergy. Concentrated Lugol iodine solution (50%) caused heartburn in 3 patients and bronchospasm in 1 patient with iodine sensitivity.27 Methylene blue and indigo carmine have caused serious systemic reactions during parenteral administration.28,29 Stains should be limited to the described concentrations, using the smallest volumes, generally 10 to 20 mL. Pooled stain can be reused to minimize volumes. The patient must be instructed about possible discoloration of urine and feces. The dyes used for chromoendoscopy stain anything they contact, especially clothing fabric, so care must be used to avoid any accidental leakage or spraying outside the patient. They pose no risks to clinical personnel and do not damage the endoscopy equipment, but with frequent use, they may discolor the outer markings on endoscope insertion tubes. Magnification and High-Resolution Endoscopy Endoscopic detection of gastrointestinal pathology depends on the recognition of visible mucosal lesions. Magnification and high-resolution endoscopy enable detection of subtle changes of the gastrointestinal mucosa that may be unrecognized or of indeterminate importance when conventional fiberoptic and video endoscopes are used. Magnification instruments were built in Japan in the 1960s and can magnify the mucosa from ×3 to ×170, with most magnification performed within a more practical range of ×10 to ×35.30 High-magnification endoscopy is best used with absorptive staining because of time-consuming technical aspects of magnification (focusing in and out of magnification) and the need for long-lasting staining. Magnification instruments allow the examination of cellular mucosal patterns. There are limited data on the use of magnification endoscopy in Barrett esophagus. Stevens et al27 described the use of magnification endoscopy in con-
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junction with combined dye spraying. Lugol iodine solution was initially sprayed to define the SCJ, and then areas of unstained mucosa, representing possible Barrett esophagus, were stained with indigo carmine, which was followed by magnification endoscopy to image the resultant fine mucosal pattern. Barrett epithelium was visualized as raised with a villiform surface pattern, an appearance similar to that of small bowel mucosa obtained using the same procedure. This pattern correlated with histologic confirmation of Barrett esophagus. The villous surface appearance seen on magnified examination corresponds to mucosal ridges with depressions (openings of intestinal glands) as seen by electron microscopy and are not true villi. High-resolution instruments are video based and provide greater pixel density along with minor magnification. In our own experience, the perceived advantage of these instruments is their ease of use and the flexibility to use either contrast or absorptive chromoendoscopy stains. Sufficient mucosal surface detail is continually provided without the manipulations needed to focus to a desired magnification and simultaneously readjust the instrument position to accommodate for movement and fluids altering the viewing field. Most of the latest-model commercially available video endoscopes offer higher resolution, which often exceeds 60% of the resolution capabilities of previous video endoscope models. Resolution beyond these instruments is available only in prototype instruments undergoing evaluation. Light Analysis Techniques Spectroscopy, the analysis of wavelength and intensity of light, can also be used to analyze the mucosa. The ability to characterize tissue using light by rapid, contact-only spectroscopic techniques is referred to as optical biopsy and has far-reaching potential. When tissue is illuminated with light (monochromatic laser or nonlaser), photons penetrate into the tissue at varying depths, with some photons absorbed and others reemitted in another wavelength of light (this property is called fluorescence—eg, light-induced fluorescence endoscopy), or the photons can be scattered within the tissue (eg, light-scattering spectroscopy).31 Light-induced fluorescence endoscopy is based on the use of endogenous (naturally occurring) or exogenous (administered) fluorescent agents. Autofluorescence arises from endogenous molecules called fluorophores within tissues such as aromatic amino acids, connective tissue, lipopigments, and by-products of heme synthesis.32 Mucosa, submucosa, and muscularis propria have distinct fluorophore compositions, so that the fluorescence measured at the luminal surface comprises contributions from the various layers. Tissue autofluorescence is sensitive to alterations in tissue morphology and biochemistry and theoretically may be used to detect early malignant transformation. An alternative to optical
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biopsy by autofluorescence is the use of exogenous fluorescence-inducing drugs such as hematoporphyrin derivative, phthalocyanines, and 5-aminolevulinic acid. Drug-induced fluorescence is dependent on the degree of selective localization of the light-sensitizing drug within tissue and drug pharmacokinetics. Both methods are performed with a contact optical fiber probe, typically 0.5 to 1 mm in diameter, passed through the endoscope biopsy channel and placed in contact with the targeted surface of suspicious mucosa. Although this procedure is technically simple, the sampling of only a small volume of tissue (1-3 mm) immediately beneath the probe tip is a disadvantage. Endoscopic systems capable of producing real-time high-resolution fluorescence images and evaluating large areas of mucosal surface in parallel with conventional white light endoscopic examination have recently become available.33 The use of light-induced fluorescence endoscopy to target biopsies may offer more effective screening and surveillance for high-risk patients, such as those with Barrett esophagus. The role of exogenous fluorescenceinducing agents in identifying Barrett mucosa is uncertain. There are 2 contradictory experiences that reported using 5-aminolevulinic acid–induced fluorescence to distinguish between normal squamous and Barrett mucosa.34,35 In light-scattering spectroscopy, the determinants of light scattering within tissue are the size and number of the scatterers (eg, mitochondria or nuclei) and the wavelength of light used to illuminate the tissue.31,36 This property of light has been used during endoscopic procedures to determine the number and size of nuclei and the degree of nuclei crowding in patients with Barrett esophagus. A prospective study that assessed the potential of light-scattering spectroscopy to measure epithelial nuclear enlargement and crowding in patients with Barrett esophagus showed that both the sensitivity and specificity of this technique for detecting dysplasia (either high-grade or low-grade dysplasia) were 90%.37 Dysplasia was diagnosed if 30% or more of the nuclei exceeded 10 µm in diameter. This suggests that light-scattering spectroscopy may readily detect areas of dysplasia during endoscopy. CONCLUSION Endoscopy plays an integral part in the diagnosis and management of Barrett esophagus and its associated complications. In the appropriate clinical setting, the use of adjunctive diagnostic techniques may facilitate the diagnosis of Barrett esophagus. Chromoendoscopy is simple and safe, but this technique requires additional procedural time as well as late-model video endoscopes with improved resolution. To our knowledge, the relative performance, accuracy, ease of use, and cost of the various vital stains for diagnosing Barrett esophagus and the yield in detecting
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dysplasia or cancer have not been formally compared. There are insufficient data to recommend the use of highmagnification endoscopes for screening or surveillance procedures. The use of light-induced fluorescence endoscopy and light-scattering spectroscopy (ie, optical biopsy) is appealing for the diagnosis and characterization of suspicious lesions to target biopsies. Chromoendoscopy and optical biopsy methods are advocated for both the diagnosis and surveillance of Barrett esophagus but require further study. A high level of suspicion and adherence to a protocol for performing biopsies are mandatory.
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