19 - Thymus
Editors: Mills, Stacey E.
Title: Histology for Pathologists, 3rd Edition
Copyright 2007 Lippincott Williams & Wilkins
> Table of Contents > VII - Alimentary Tract > 25 - Colon
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25
Colon
Julia Dahl
Joel K. Greenson
Embryology
The gastrointestinal tract is a remarkably complex, three-dimensional, specialized, and vital organ system derived from a simple tubal structure composed of all three germ layers (endoderm, mesoderm, and ectoderm). Early in development, the gut is patterned into four asymmetrical axes anterior-posterior (AP), dorsoventral (DV), left-right (LR), and radial (RAD), utilizing critical developmental pathways that are directed by reciprocal mesodermal (mesenchymal) to endodermal (epithelial) cell-cell interactions and endodermal to endodermal cell-cell interactions (1,2,3,4,5,6,7,8,9). Because gut epithelium is a constitutively developing tissue, constantly differentiating from a stem cell in a progenitor pool throughout adult life, these developmental pathways, axes of development, and cell-cell cross talk continue to be important in cell differentiation, homeostasis, and apoptosis of the adult intestinal epithelium (8,9,10,11,12).
The adult colon displays a morphologic and functional pattern clearly identifiable in three of the four embryonic axes, and interacts with the gut derivatives (thyroid, lung, liver, and pancreas) produced in the fourth embryonic axis (13,14). Development and differentiation along the AP axis gives rise to the foregut, the midgut, and hindgut, resulting in regionally specific differentiation from mouth to anus. The significant variation in patterns of gene expression, physiologic function, disease distribution, and variations in histologic appearance between the right and left colon reflects the combined midgut and hindgut derivation (8,15,16,17,18,19,20,21,22,23,24,25,26,27,28,29,30). The cecum, appendix, ascending, and proximal portion of the transverse colon (right colon) are derived from the midgut while the distal transverse, descending, sigmoid colon, and rectum (left colon) are derived from the hindgut (13,14).
The LR axis is manifested in the colon by characteristic turning and looping of the gut, resulting in portions of colon with varying mesentery and fixation within the abdominal cavity (13).
The fundamental axis maintained in the adult is the radial (crypt to surface) axis. Homeostasis of intestinal epithelium occurs throughout life along this radial axis, with the epithelial and mesenchymal progenitor/proliferative cells being deeper in the radial axis than the differentiated functional cells and the apoptotic cells being luminal (11,31,32,33).
Abnormalities of any of the developmental pathways or along any axis during organogenesis may result in gross morphologic malformations, including diverticula, rotational malformations, stenoses, atresias, duplications, aganglionic segments, imperforate anus, and others (13,14,34,35,36,37). Perturbations of developmental pathways used for organ homeostasis may result in metaplasias, polyposis syndromes, and malignant transformation (10,38,39,40,41,42).
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Anatomic Considerations
The colon is the terminal 1.0 to 1.5 m segment of the gastrointestinal tract, following the periphery of the peritoneal cavity, with the rectum extending into the pelvis and concluding at the anal canal (Figure 25.1) (43,44). There is considerable anatomic variation in the position of the colon segments, mesenteric coverings, and attachments to the posterior abdominal wall (44,45). Nonetheless, consistency of vascular supply, venous drainage, and innervation distinguishing the two primary (right and left) colon segments along the embryologic AP (midgut/hindgut) axis is maintained. The right colon receives its blood supply from the superior mesenteric artery, parasympathetic nervous innervation from the vagus nerve, and sympathetic innervation from the superior mesenteric ganglia. The left colon receives its blood supply from the inferior mesenteric artery, parasympathetic innervation from sacral nerves S2, S3, and S4 through the nervi erigentes; and sympathetic innervation from the inferior mesenteric ganglia. Venous drainage is predominantly portal. The rectum receives blood from the middle and inferior rectal arteries, parasympathetic innervation from the nervi erigentes, and sympathetic innervation through the hypogastric plexus through lumbar spinal segments L1, L2, and L3 (19,43,44,45).
Unique external features of the colon include the teniae coli and haustra, visible through the investing serosa and subserosal tissue. The muscular layers of the large intestine are composed of both longitudinally and circularly arranged fibers. The longitudinal fibers are present circumferentially through the length of the colon but are primarily concentrated into three flat bands called the teniae coli (43,44,45). Convexities of the circular muscular layer, the haustra, are transient and probably the result of structural and functional properties of the colon.
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Figure 25.1 Major regions of the colon. |
From the luminal perspective, various landmarks are recognized by the endoscopist. The cecum is readily identified by the ileocecal valve, appendiceal orifice, and blind-ended, saclike appearance. The subjacent portal vasculature imparts a blue hue to the mucosa at the hepatic flexure. Orientation of the teniae coli within the transverse colon results in a T-shaped lumen, ending in a slitlike orifice and acute angle of the splenic flexure. Although the descending and sigmoid colon may have thickened mucosal folds and diverticular orifices, calibration marks on the colonoscope are more reliable means of approximating the location within this region.
Function
The basic function of the large bowel is conservation of salt and water and facilitation of orderly disposal of waste materials (30). Considerable advancements have been made recently in characterizing the vast and interrelated colon functions; these further support the concept of two primary, unique colon segments, the right and left colon (15,19,46). In addition to their distinct embryologic derivation, the right and left colon display segment-specific arrays of physiologic functions, patterns of motility, commensal bacterial populations, and metabolic intermediate and end products; as well as local and systemic immune functions (15,21,23,24,26,29,30,47,48,49,50,51). These varied functions are reflected in differing patterns of gene, lectin and surface marker expression, varying disease distributions, and rates of (and sequences involved in) neoplastic transformation (23,24,28,29,46). Subtle regional variations of the colonic mucosa, reflecting the segmental nature of the right and left colon, have been well recognized by gastrointestinal pathologists (15,20,22) and are described in the following sections.
The most well-recognized function of the colon is absorption of water, desiccation, and transient storage of the feces. The cecum receives 1.3 to 1.8 liters of electrolyte-rich ileal effluent daily and is a high-capacity absorptive surface, effectively absorbing 80% of the chylous water (and sodium ions, Na+) during the prolonged mucosal exposure created by the retrograde peristalsis unique to the cecum (21,26,27,30). Bulk absorption (of water and sodium) occurs via electroneutral sodium chloride (NaCl) transport at the surface and in the superficial portions of the crypts (21,30,52). Within the left colon, as fecal contents are propagated distally, low-capacity electrogenic absorption via luminal sodium channels, regulated by aldosterone and angiotensin, serve in further absorption of water, as well as preservation of sodium (21,30,53,54), particularly during periods of salt deprivation and/or mucosal injury. An additional mechanism for water absorption unique to the left colon, involving formation of
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a hyperosmolar (Na+) compartment between the colonocytes and pericrypt myofibroblasts, has been observed and may be vital in extracting water from the osmotically dense feces, allowing final compaction and storage with a stool output of 200 to 500 g daily (27,55,56).
The colon participates in several diverse and integral metabolic processes through absorption, secretion, fermentation, oxidation, and other processes unique to the colonic epithelium or in concert with the colonic commensal bacteria. Rivaling London's East End or New York City in cultural diversity, there are an estimated 400 to 500 microbial species (bacteria, fungi, and a few protozoa) forming complex ecosystems from the terminal ileum to the rectum (49,58,59,60). Bacterial cells outnumber human cells roughly 1000:1, with highest concentration in the cecum and decreasing gradient and varying composition proceeding distally. In the colon of healthy humans, commensal bacteria are required for numerous vital processes: formation of short-chain fatty acids, metabolic intermediates and vitamins; metabolism of proteins and amino acids; detoxification or biotransformation of bile acids, and natural sterols and phytoestrogens, as well as phosphate and oxalate excretion (29,49,50,57,58,60,61). Fermentation of carbohydrates to form short-chain fatty acids, particularly butyrate, serves as a major source of energy for colonocytes, and butyrate plays a crucial role in colonocyte growth and differentiation (51,57,60,62,63). Nearly equal to the activity observed in the liver, colonocytes have the capacity to mediate biotransformation of bile salts, drugs, and xenobiotics (49,50). Many of these processes are segment-specific to either the right or the left colon.
Colonic commensal bacteria are integral to the health and function not only of the large intestine, but to the host at large through activity in local and systemic immune function and regulation. Locally, the colonic epithelium and commensal flora serve as important barriers to infection via tight junctions and secretion of antimicrobial substances, as well as competition for nutrient substrates (64,65,66,67,68).
The intestinal mucosa (terminal ileum through rectum) is the largest immune organ of the body (49,58,59,68,69). Lamina propria and lymphoid aggregate plasma cells represent 80% of the antibody-producing cells within the entire body and produce more antibodies than any other part of the body (49,58,59,69). The process of antigen sampling across specialized regions of the colon results in gut priming and routinely confers protection from subsequent infection locally and in other mucosal tissues (systemic immunization) (70,71,72,73,74,75,76,77). At baseline, the gut immune system is highly activated in response to normal flora so called physiologic inflammation in which the intestinal microflora and the intestinal immunologic mechanisms influence each other locally and systemically, forming an interdependent mutualistic ecosystem, the balance of which is required for maintenance of health and prevention of disease (71,73,74,78,79).
Light Microscopy
Although regional histologic differences exist within the right and left colon, the overall microscopic structure is similar throughout its length. The colon contains four histologically distinct compartments: (a) mucosa, (b) submucosa, (c) muscularis externa (or muscularis propria), and (d) serosa. The enteric nervous system spans all four compartments with cell bodies (ganglia and plexi) in both the submucosa and muscularis propria extending processes throughout the lamina propria, submucosa, and muscular layers, while receiving and transmitting information through synapses with the central nervous system via parasympathetic and sympathetic autonomic nervous systems.
Mucosa
The luminal colonic mucosa is the most metabolically and immunologically active compartment of the colon, as reflected in its organization and relative complexity. The luminal surface is covered by glycocalyx (glycans, enzymes, lectins, and mucin), facilitating formation of the commensal microbial ecosystem and serving as an integral barrier function (80,81,82). Beneath the glycocalyx is polarized columnar epithelium lining millions of regularly spaced crypts that span the depth of the lamina propria. The crypts are aligned perpendicular to and extend to the muscularis mucosae, imparting the well-known rack of test tubes appearance (Figure 25.2). Some variation in space between crypts is expected in biopsies of normal individuals; however, irregularly oriented or bifurcated crypts are considered
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abnormal (see Regional Variation in Histologic Features, below). Variation in the usual pattern of the mucosa is seen at innominate grooves (regularly occurring folds in the mucosa), adjacent to lymphoid follicles, with lymphoglandular complexes and with the normal mucosal folds and ridges created by contraction of the muscularis mucosae (Figures 25.3,25.4,25.5). Each of these variations must be distinguished from the histologic changes of chronic mucosal injury (inflammatory bowel disease, or IBD) (Table 25.1).
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Figure 25.2 Normal colonic mucosa. The histologic section of this endoscopic mucosal biopsy specimen is oriented so the simple columnar surface epithelium facing the lumen is at the top of the figure and the cut surface of the specimen is at the bottom. The mucosal crypts are lined up in parallel, and their mouths are open to the lumen. The lamina propria consists of the stromal elements investing the crypts and extending from the surface epithelium to the smooth muscle cells of the muscularis mucosae at the bottom. |
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Figure 25.3 Innominate grooves of colonic mucosa. Multiple crypts open in a mirror image across a common crypt lumen at the groove, with the common crypt lumen opening to the colonic lumen. This normal finding is not a true branching of the crypts and should not be misinterpreted as architectural distortion indicative of chronic mucosal injury (i.e., inflammatory bowel disease). The innominate groove common lumen is generally within the superficial one-third of the mucosa. |
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Figure 25.4 Colonic lymphoid aggregate. This lymphoid aggregate splays the adjacent crypts, which appear in a diagonal or near horizontal axis (rather than vertical), and mimics basal lymphoplasmacytosis at the crypt/lymphoid aggregate margin, resembling minor architectural disarray. Goblet cells are absent in the crypt epithelium adjacent to the follicle, while retained on the opposite side of the crypt. These normal features of colonic lymphoid aggregates should not be misinterpreted as architectural distortion indicative of chronic mucosal injury (i.e., inflammatory bowel disease) (see Table 25.2). These aggregates may contain well-formed germinal centers and appear as small polyps endoscopically. |
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Figure 25.5 Lymphoglandular complex. In this tangential section, crypt epithelium is present within a lymphoid follicle that extends from the mucosa through the muscularis mucosae into the submucosa. |
Advances in immunohistochemistry and development of new antibodies has allowed further classification of cell types, with utility in assessing both normal and pathologic histologic patterns and providing useful adjuncts to standard histochemical stains (Table 25.2) (52,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123).
Regional Variation in Histologic Features
Although the overall mucosal pattern as described above is maintained, the right and left colon display subtle histologic differences in the ratio of surface epithelial colonocytes to goblet cells, thickness of mucus layer, normal presence of Paneth's cells, crypt length, quantity of surface intraepithelial lymphocytes, lamina propria mononuclear cell distribution, and density, as well as lymphoid aggregate density (Figure 25.6). These subtle, but significant, differences make it essential to dissuade our clinical colleagues from the practice of utilizing pooled biopsies simply labeled colon. We encourage gastroenterologists and surgeons to uniformly adopt the practice of separately submitting and appropriately labeling biopsy material from the right and left colon, whether for evaluation of diarrhea or classification of polyps, because the range of normal histologic appearance differs significantly (15,20,22) and neoplastic sequence and progression varies within the right and left colon (19,28,99).
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Table 25.1 Common Artifacts and Variants: A Guide to Evaluation and Interpretation | ||||||||||||||||||||||||||||||||||||||||||
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Table 25.2 Predominant Immunohistochemical and Histochemical Staining Patterns of many of the Normal Cell Types Present in the Human Colon (51,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105,106,107,108,109,110,111,112,113,114,115,116,117,118,119,120,121,122,123) | ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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Figure 25.6 Normal mucosa from the cecum (A) and rectum (B). A. The mucosa in the cecum has more absorptive cells and fewer goblet cells compared to the rectum. The lamina propria is more cellular, with greater density of plasma cells, eosinophils, and lymphoid aggregates in the cecum as compared to the rectum. Paneth's cells are normally present, residing at the base of the crypts. B. The rectal mucosa has a higher ratio of goblet cells to absorptive cells, with a less dense lamina propria and more easily identified muciphages. Paneth's cells are not normally seen within the rectum. |
Reflecting a dominant function in absorption and antigen processing, the right colon displays a higher colonocyte (absorptive enterocyte + M cell) to goblet cell (roughly 5:1) ratio as compared to the left colon (Figure 25.6). Proceeding distally, an increase in goblet cells are apparent, with a ratio of 3 or 4:1 (colonocyte:goblet cell), facilitating increased formation of gel-type mucin in the descending and sigmoid colon necessary for consolidation and transit of the increasingly formed fecal matter (15,26,27,124). Paneth cells are normally present at the base of crypts within the midgut-derived right colon but are indicative of metaplasia secondary to chronic mucosal injury after the distal one-third of the transverse colon (Figure 25.6).
Surface intraepithelial lymphocytes are seen in greater concentrations in the right colon than the left and can be marked overlying lymphoid aggregates (20,22,125,126,127). Similarly, lamina propria mononuclear cell density is also greater in the right colon than the left (descending and sigmoid colon), as are organized lymphoid aggregates, possibly related to the higher concentration of commensal microorganisms and resultant antigen sampling activities (20,22). As goblet cell concentration and mucin increases, lamina propria macrophages, specifically scavenging mucin (muciphages) are observed with an increasing gradient proceeding distally. In the sigmoid colon and rectum, most gastrointestinal pathologists will accept a few bifurcated crypts as being within the range of normal (20), although this has not been systematically studied nor reported.
Epithelium
The colonic mucosa is composed of continuous, polarized surface and crypt epithelium forming millions of crypts (26,128) invested in basement membrane, surrounded by a variably cellular and vascular lamina propria (20,125,127), and with a deep boundary formed by the muscularis mucosae.
The colonic mucosal microarchitecture shows remarkable stability along both the AP and radial axes, despite its high turnover rate and variety of highly specialized cell types (129,130). Albeit based upon circumstantial evidence, it is generally accepted that mucosal renewal is attributed to colonic epithelial stem cells located and maintained within a mesenchymal niche, situated near the base of the intestinal crypts (13,31,32,128,129,131,132,133,134). Multipotent stem cells slowly divide, under influence of reciprocal signaling by mesenchymal and other cells, giving rise to a transient population of progenitor cells that rapidly divide and undergo a well-controlled maturation process while migrating toward the lumen (31,128,134). The granule-containing epithelial cell types appear to ignore the rule of luminal progression during maturation, with Paneth's cells (of the right colon) migrating to the crypt base (13,64,135,136) and enteroendocrine cells (throughout the colon) homing to the mid- and deeper regions of the crypts (Figure 25.7) (13,15,137).
During migration, dividing transit cells commit and differentiate to one of five distinct epithelial cell types: absorptive colonocyte, mucus-secreting goblet cell, enteroendocrine cell, Paneth's cell, or M cell. At any given time, 75 to 80% of all colonocytes are associated with the crypts, and only 10 to 15% of colonocytes form the surface epithelium (intercrypt table) (126,130).
Absorptive Colonocytes
Absorptive colonocytes compose the majority of the surface epithelium (80,82,138). The luminal surface is characterized by rigid, tightly packed apical microvilli, (13,130), the tips of which contain integral membrane mucinlike glycoproteins that form a continuous, filamentous brush border glycocalyx (139) that is visible as a striate luminal border. Absorptive
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colonocyte cytoplasm is lightly eosinophilic, with small apical vesicles containing mucin (of different composition than goblet cell mucin) positioned for luminal release (21,63,89,99). The apical poles of columnar cells fan out over goblet cells such that only the small apexes of goblet cells contact the lumen (130). Basally aligned colonocyte nuclei are oval, uniformly sized, and aligned with the long axis parallel to the long axes of the cells, with smooth nuclear contours and frequently observed nucleoli.
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Figure 25.7 Normal right colon with Paneth's cells and an endocrine cell. Within the crypt on the left are three Paneth's cells at the base of the crypt. Note the basal nucleus and coarse, luminal-facing granules that empty into the crypt lumen. Within the crypt on the right is an endocrine cell at the base of the crypt. The endocrine cell is smaller, has a luminal nucleus and fine, basally facing granules, that empty into the pericrypt myofibroblast sheath and adjacent vasculature. |
Goblet Cells
Goblet cells are dispersed throughout the surface epithelium and crypts and are distinguished by their wine goblet shape, which results from a large number of mucous granules in the apical pole. Mucin composition varies regionally along the length of the colon due to differential synthesis of the several known secreted and membrane-bound mucins (15). This variation is reflected in the differential histochemical staining patterns commonly observed (99,140,141). Goblet cell cytoplasm is relatively clear with standard hematoxylin and eosin (H&E) stains; however, mucin granules become distinct with mucicarmine, Alcian blue pH 2.5, and periodic acid-Schiff stains (15). Goblet cell nuclei, when compared to adjacent absorptive colonocytes, appear hyperchromatic, dense, and irregular (130).
Endocrine Cells
Endocrine cells within the gut epithelium represent the largest population of hormone-producing cells in the body (128,137,142,143), comprising approximately 1% of the individual cells lining the intestinal lumen, predominantly located in the crypts and, rarely, scattered within the lamina propria (13,15,28,122,130,142,146,147). More than 30 peptide hormone genes are known to be expressed throughout the digestive tract, in a regionally and spatially distinct pattern (142,148). Enteroendocrine cells contain basally oriented, small, but distinct, deeply eosinophilic granules (137,142). The round, smoothly contoured nuclei of enteroendocrine cells are pushed lumenally, with opposite polarity to the other epithelial cell types (Figure 25.7).
Enteroendocrine cells may be further identified by their histochemical silver staining properties and may also be identified immunohistochemically with varying immunoreactivity to chromogranin A, synaptophysin, neuron-specific enolase, and specific antibodies to the putative peptide hormone of the cell or cell proliferation (i.e., carcinoid tumor) (Table 25.1).
Paneth's Cells
Paneth's cells disregard the rule of luminal migration and are normally encountered at the base of the crypts, within the midgut-derived right colon (13,65,114,128,135). These pyramidal-shaped cells have basally aligned oval nuclei and apical coarse, densely eosinophilic cytoplasmic granules (Figure 25.7) (13,65). Granule and cellular contents include: -defensins, -defensins, NOD2, lysozyme, phospholipase A2, secretory leukocyte inhibitor, monomer IgA, TNF- , heavy metal ions, zinc binding protein, trypsin and trypsinogen, EGF, osteopontin, FAS ligand (CD95L), CD44v6, CD15, REG protein, and numerous others (65,128,135,149,150). The diverse Paneth's cell contents reflect their significant role in innate immunity. Additionally putative roles in regulation of cell matrix interactions, apoptosis, and cellular immunity, as well as stem-cell niche maintenance, have been proposed (65,128,135,149,151).
In addition to characteristic granule staining with H&E stains, granules are conspicuously stained by periodic acid-Schiff, and phloxine-tartrazine (65); and, interestingly, Paneth's cell autofluorescence is elicited by eosin stain (Table 25.2) (114).
M Cells and Follicle-Associated Epithelium
Membranous (M) cells are distinctive epithelial cells that occur in the dome region of organized lymphoid follicles and associated with both the immunologic cells and variants of absorptive colonocytes (the follicle associated epithelium) unique to the dome region (59,126,128,152,154). Estimates of M cells in human colon vary widely, reported from rare to approximately 10% of surface epithelial cells (126,153,154,155,156). Light microscopy has insufficient magnification to distinguish the unique features of M cells (reduced numbers of an irregular microvilli, apical microfolds, absence of thick filamentous brush border glycocalyx, and the presence of an unusual subdomain of the basolateral membrane that amplifies the cell surface and forms an intraepithelial pocket) visible with electron microscopy. The M cell intraepithelial pocket provides a
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docking site for special populations of intraepithelial B and T lymphocytes, along with a small number of macrophages; and immediately overlies the dome region of lymphoid follicles (126,152,153,154). These unique morphologic features provide local, functional openings in the epithelial barrier through which M cells sample the contents of the lumen and transfer antigents to antigen-presenting cells via a specialized method of transcytosis (67,71,152,155). The follicle associated crypts contains few or no goblet cells, enteroendocrine cells or Paneth cells (153,155). These closely apposed columnar enterocytes may mimic features of low grade cytologic dysplasia (adenoma), particularly with distortion of the crypt architecture generally produced by the adjacent lymphoid aggregate.
Intraepithelial Inflammatory Cells
Intraepithelial lymphocytes (IELs) occur in two compartments: within the paracellular spaces of the absorptive epithelium and in highest density associated with lymphoid aggregates within M-cell pockets (Figure 25.8) (125,127,157,158,159). The former are predominantly CD3+, CD8+, TCR- + suppressor T cells, with between 15 and 40% TCR- + T cells, while the latter are mixture of CD3+/CD45RO+ activated memory, some CD45RA+ naive T cells, and IgM-secreting B cells (86,127,160,161,162). Intraepithelial lymphocytes are the first members of the immune system to encounter dietary antigens and commensal and pathologic microorganisms, and they likely play an integral role in oral tolerance (58,158,161,163,164,165). The IELs home toward their intraepithelial destination, migrating along various chemokine gradients produced by adjacent epithelial, inflammatory, and mesenchymal cells (59,166).
Nuclear molding and indistinct cytoplasmic contours are characteristic of IELs as they extend through the basement membrane to occupy paracellular spaces (Figure 25.8). Retention of the classic lymphocyte round nucleus and thin rim of cytoplasm is more common in IELs overlying aggregates. Normal IEL density ranges from 1 to 5 lymphocytes per 100 colonocytes, except in follicle-associated epithelium, where M-cell associated IELs are abundant (162). The number of IELs decreases from the ascending colon to rectum, with highest concentration in the lymphoid aggregate and commensal bacteria-rich cecum (125,126). It is imperative to ascertain the site of each colon biopsy to avoid misinterpretation of normal IEL density in right colon biopsies as lymphocytic colitis (Table 25.2). Generally, 20 or greater lymphocytes per 100 colonocytes are considered pathologic (59,125,127,168).
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Figure 25.8 Intraepithelial lymphocytes (IELs) overlying a lymphoid follicle. Large numbers of IELs are typically seen overlying lymphoid aggregates. This should not be misinterpreted as lymphocytic colitis. |
Intraepithelial eosinophils may occasionally be seen in the normal colon, although at much lower numbers than lymphocytes (168,169,170).
Stem and Dividing Transit Cells
It is estimated that between four and six stem cells are present per crypt, with some dividing transit cells apparently able to be recruited to serve as stem cells following injury with stem cell loss (31). These proliferative and undifferentiated cells are morphologically indistinct; however, they appear to have a large nucleus with diffuse chromatin and scant cytoplasm with few small organelles (13,128). Mitotic activity is frequently encountered in the basal one-fifth of the crypt, and apoptosis may also be seen (129,131,134).
Apoptosis
The epithelial cells of the colon have remarkably short life spans (Table 25.3), during which they mature, migrate, and function (13,21,33,47,131,134,171). Programmed cell
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death (apoptosis), is the conclusion of the normal process of colonocyte turnover, recognizable histologically by identification of apoptotic bodies and debris predominantly in the surface epithelium (Figure 25.9), and less frequently within the colonic crypts. Apoptotic bodies consist of vacuoles containing pyknotic nuclear debris, are surrounded by free space, and generally are at the basal portion of the epithelium or immediately subjacent to the basement membrane (23,132,134,172,173,174,175,176,177,178,179,180). Lamina propria inflammatory cells similarly undergo apoptosis; however, this is frequently overlooked histologically (174,181,182). Sodium phosphate bowel preparations transiently increase the rate of apoptosis (see Bowel Preparation, below); similar effects are seen with other physical and chemical agents. Increased apoptosis (both surface and/or crypt) may also be seen in several disease states, including graft-versus-host disease, autoimmune enteropathies, systemic autoimmune disorders, and with certain medications (70,183,184). Altered apoptosis (increased, decreased, and abnormal localization) is seen in neoplastic progression (185).
Table 25.3 The Life Span of the Various Colon Epithelial Cells and Number of Replacements Per Average Human Life Span Vary Between Cell Types. Despite the High Rate of Turnover, Preservation of Genetic Information is the Rule Rather than the Exception | |||||||||||||||||||||
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Basement Membrane
The basement membrane complex anchors the various epithelial cells to the underlying myofibroblast network and lamina propria. This fenestrated extracellular support matrix is produced collaboratively by epithelial and mesenchymal cells (98,126,129,186). Basement membrane composition varies, regulated by several factors produced by the epithelium, myofibroblasts, and lamina propria cells (126,129,186,187). In addition to allowing intraepithelial lymphocytes to traverse the basement membrane, the fenestrations allow epithelial, mesenchymal, and dendritic cell processes to sample and/or present antigens and have functional implications in water and ion transport (21,32,129).
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Figure 25.9 Apoptosis. Two apoptotic bodies are seen within the surface epithelium (arrows). |
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Figure 25.10 Normal basement membrane. The normal basement membrane is 3 to 5 m thick and has a crisp, delicate, and regular lower border; it blends into the crypt sheath imperceptibly (trichrome stain). |
In well-oriented sections, the normal basement membrane is between 3 m and 5 m thick, regular, and stains with connective tissue stains (Masson's trichrome, saffron, eosin von Gieson elastin) (Figure 25.10) (187,188). Similar to Paneth's cells, autofluorescence is elicited with eosin stain (115). Basement membrane thickness greater than 10 mm is considered pathologic, as is irregularity of the basement membrane, particularly entrapping superficial lamina propria capillaries (115,187).
Lamina Propria
The lamina propria invests the colonic crypts, extending from the fenestrated basement membrane complex to the muscularis mucosae. The various lamina propria inflammatory and mesenchymal cells are organized within extracellular matrix, each performing integral immunologic, metabolic, proliferative, and motility functions.
Lamina Propria Inflammatory Cells
As dictated by the wide array of immunologic functions performed by the colonic mucosa, the lamina propria houses localized antigen-sampling and processing factories, with over 30,000 discrete lymphoid aggregates, concentrated within the blind-ended cecum and distributed along the length of the colon (189,190). In addition to lymphoid aggregates, the normal colon contains mature B lymphocytes, plasma cells, T lymphocytes [helper, suppressor, and lymphokine-activated killer (LAK) cells, but unlikely natural killer (NK) cells], eosinophils, mast cells, and macrophages, in combination filling between 30 and 50% of the free lamina propria space (22). Normally, there is a decreasing inflammatory cell gradient from lumen to
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muscularis mucosae, in which the lamina propria loose connective tissue is obscured within the superficial aspect of the lamina propria but becomes visible approaching the muscularis mucosa (191). The predominant cell type of the lamina propria is the IgA-secreting plasma cell, with much smaller proportions of IgM-, IgE-, and IgG-secreting plasma cells also present (181,192). Secreted IgA and IgM are transported luminally, providing humoral immune protection (138,181,192). The distinct cartwheel nucleus, perinuclear Golgi zone, and amphophilic cytoplasm characteristic of plasma cells observed in other tissues are retained in colonic plasma cells. Of the remaining lamina propria lymphocytes, more than 90% of the lymphocytes were CD3+ T cells, with fewer than 50% also CD8+ (126,127,159). There are also CD 20-positive B lymphocytes present within and adjacent to lymphoid follicles (71,76,78,138,193).
Myeloid cells that normally reside in the lamina propria include eosinophils and mast cells. In the normal colon, the number of eosinophils is highly variable, dependent upon both the region of colon sampled (168,169,170,171,172,194) and the geographic residence of the patient (195). A range of normal eosinophil counts in the lamina propria has been reported as 0 to 8 per high-power field (hpf); however, the eosinophil concentration should be interpreted on the basis of the company it keeps (i.e., other features of colitis versus otherwise normal) (Rodger C. Haggitt, MD, personal communication). Higher mean eosinophil concentration is seen in biopsies of patients from the southern United States, compared to the northern United States, with a rather extreme degree of variability (195). Although eosinophils are increased in parasitic and allergic disease, collagenous colitis, ulcerative colitis, Crohn's disease, and other pathologic conditions, consideration of the geographic residence and site of biopsy are integral prior to considering increased eosinophils (as an isolated histologic finding) to be pathologic (170,195). Mast cells, or tissue based basophils, are less numerous than eosinophils, and their density appears to be increased in the ileocecal region compared with other sites of the colon (196,197). Mast cells are difficult to distinguish with routine H&E, but stain well with Giemsa, toluidine blue, tryptase, and CD117 (c-kit) (Figure 25.11, Table 25.2) (194). Neutrophils are not normally seen in any significant number within the lamina propria, although they may be seen in areas of hemorrhage and within blood vessels.
Macrophages are commonly seen scattered throughout the lamina propria and are occasionally concentrated at the basal aspect of the crypts (70,198,199,200,201) (Figure 25.12). While macrophages are generally difficult to see with H&E stains, visualization may be enhanced by specific histochemical stains that detect the variety of materials they scavenge and store: apoptotic debris, microbes, lipofuscin, cholesterol esters, gangliosides, mucolipids, glycogen, mucopolysaccharides, and others (Figure 25.12, Table 25.2) (199,200). Muciphages are the most commonly recognized macrophages, ingesting mucin exuded from adjacent goblet cells (and to a lesser extent enterocytes) that crosses the basement membrane. These normal constituents of the lamina propria have increased concentration within the left colon, in keeping with the increased number of goblet cells present in this location. Distention of the lamina propria with the appearance of replacement of other lamina propria inflammatory cells is rarely normal and may indicate bacterial or fungal ingestion/infection (e.g., Tropheryma whippelii, Mycobacterium avium-intracellulare complex, Histoplasma capsulatum, and others) or various metabolic storage disorders, requiring use of histochemical, PCR, or electron microscopic methodologies as well as further laboratory evaluation (97,105,202,203,204,205).
 |
Figure 25.11 Mucosal mast cells. Although difficult to discern on H&E-stained sections, mucosal mast cells are easily identified with CD117. Mucosal mast cells serve well as an internal control when evaluating CD117 stains of gastrointestinal tract mesenchymal tumors. (Anti-CD117 stain.) |
Plasmacytoid dendritic cells are scattered throughout the lamina propria, while stellate dendritic cells are concentrated in the subepithelial dome space associated with lymphoid follicles (78,138,206,207). These are histologically indistinct and frequently require immunohistochemistry for definitive identification (Table 25.2) (70,71,76,138). The former have recently been implicated in allergic and autoimmune disorders (88), while the latter are integral in antigen presentation (70,73,208).
 |
Figure 25.12 Lamina propria muciphages. A. These pale macrophages at the base of the mucosa are stuffed with mucins. This finding is not infrequent and does not generally correlate with disease. B. The same area stained with Alcian blue pH2.5; the muciphages show strong cytoplasmic staining. Similar findings are seen with PAS with diastase digestion. Of note, bacteria-laden macrophages in Whipple's disease are generally negative when stained with Alcian blue pH 2.5 but densely stain with PAS with diastase. |
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Myofibroblasts (Pericrypt Myofibroblast Sheath and Lamina Propria Myofibroblasts)
The lamina propria contains two distinct populations of myofibroblasts: the pericrypt myofibroblast sheath and the subepithelial myofibroblast (SEM) syncytia. Interacting closely with the epithelium, lamina propria inflammatory cells, and the muscularis mucosa, myofibroblasts function in absorption, ion and mucin secretion, immune regulation, and differentiation (maintenance of stem cell niche) (521,124,209). The rim of fusiform cells organized in close apposition to each colonic crypt was originally designated the pericryptal fibroblastic sheath (Figure 25.13) (186,209). This specialized population of mesenchymal cells is now known to be a syncytium (both anatomically and functionally) of cells that surrounds the crypts and extends into the lamina propria, forming a reticular network within the extracellular matrix, attaching to one another with both gap and adherens junctions (83,92,186,209,210), and displaying distinct immunophenotypes (Table 25.2).
In the region of the crypts, the myofibroblasts are oval and scaphoid in appearance and appear to overlap like shingles on a roof. The SEMs exist in two distinct morphological states: (a) the activated myofibroblast and (b) the stellate transformed myofibroblast, similar in appearance to macrophage dendritic cells (186,209). Myofibroblasts often are surrounded by an incomplete basal lamina and embedded in a subepithelial sheet of reticular fibers that also contains fenestrae or foramina through which lymphocytes and macrophages traverse. Gap junctions couple some myofibroblasts to the tissue smooth muscle, and the cells are commonly in close apposition to varicosities of nerve fibers; however, it is has not been determined whether the interstitial cell of Cajal network is physically connected to the SEM network (186,209,211).
Vasculature and Lymphatics
Vasculature is limited to capillaries and high endothelial venules scattered throughout lamina propria and to
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lymphatic channels immediately superficial to the muscularis mucosa (212,213). Capillaries are composed of a circumferential endothelial lining and may contain red blood cells as well as inflammatory cells. Irregularly shaped, distorted, and engorged capillaries frequently indicate prolapse of the mucosa. In addition to provision of oxygen and nutrients to mucosal cells, vascular adhesion molecules participate in appropriate homing of circulating lymphocytes to their appropriate colonic microenvironment. Lymphatic tributaries rarely initiate within the lamina propria; however, when present, they appear to have thinner walls and cross the muscularis mucosae to join the readily observed submucosal lymphatics (213,214,215). Definitive differentiation between capillaries and lymphatics requires immunohistochemical analysis (Table 25.2) (84,94,100,216).
 |
Figure 25.13 Lamina propria myofibroblasts, antimuscle-specific actin (MSA) stain. This stain for muscle-specific actin highlights the pericryptal myofibroblast sheath, muscularis mucosae, and submucosal blood vessels. |
Muscularis Mucosae
Forming the deep limiting boundary of the lamina propria is a thin layer of smooth muscle, the muscularis mucosae. This muscle layer is physically tethered to the mucosa, with occasional smooth muscle cells extending into the lamina propria or coalescing with the pericryptal myofibroblast sheath. The muscularis mucosae receives innervation via the submucosal plexus (92,217,218). Because the colonic glands are tethered to the muscularis mucosa, this structure is valuable in evaluating crypt architecture in endoscopic biopsies. Biopsies that do not contain muscularis mucosae may resemble architectural distortion, with glands adopting horizontal or curved configurations. Careful examination of other biopsy fragments with muscularis mucosae, as well as assessment for other features of mucosal injury (Table 25.1), may allow an accurate diagnosis. The muscularis mucosa is normally traversed by lymphoglandular complexes (Figure 25.5), vascular channels, and neural twiglets and participates in absorptive, secretory, proliferative, and possibly motility functions. Isolated thickening may occur with prolapse of the overlying mucosa and adjacent to diverticular orifices. Clear duplication of the muscularis mucosae is generally considered a feature of chronic mucosal injury.
Submucosa
The submucosa is composed of loosely arranged bundles of smooth muscle, fibroelastic tissue, and adipose, in which the local enteric nervous system, vasculature, and lymphatics are embedded. Lymphatic channels may be conspicuous and dilated immediately beneath the submucosa and do not contain cellular elements (213,214,216). Sparse inflammatory cells (relative to the dense physiologic inflammation of the mucosa) are scattered throughout, occasionally organized as submucosal lymphoid aggregates. The submucosa provides a flexible matrix allowing the mucosa to glide and move freely over the rigid muscularis mucosae during peristalsis.
Submucosal smooth muscle consists of loosely woven fascicles of individual smooth muscle cells, forming small bundles. These smooth muscle collections are closely apposed to interstitial cells of Cajal, which in turn are immediately adjacent to nerve varicosities forming the neuroeffector junctions that receive, transmit, and integrate central, parasympathetic, and sympathetic nervous system commands (211,219,220). The two submucosal neural plexuses are the submucosal plexus of Meissner (located immediately beneath the muscularis mucosae) and Henle's deep submucosal plexus (lying on the inner aspect of the muscularis propria). Neural plexuses are composed of neurons, glial cells, and stromal elements (221,222,223). Ganglion cells are unique in their histologic appearance with round or oval nuclei, a prominent (often eosinophilic) nucleolus, and ample basophilic cytoplasm stippled with Nissl substance (Figure 25.14). Ganglion cells characteristically cluster together and may mimic giant cells, epithelioid cells, or granulomas. Nerve axons are fibrillar and distinguishing these axons from fibroblasts or their elastofibrotic products may require the use of histochemical or immunohistochemical stains (Figure 25.14B, Table 25.2) (43,112,220,221,223,224).
Interstitial cells of Cajal (ICCs) are modified myofibroblasts. Histologic features evident with routine H&E stain include a fusiform cell body and large oval nucleus; silver stain or immunohistochemical evaluation will reveal two or more dendritic processes, connecting ICCs to one another, to ganglion cells, or to adjacent smooth muscle (210,220,225,226,227). These intriguing cells are thought to play an important role in the control of gut motor activity (220,228,229). The normal ICC density within the submucosa is substantially less than that seen surrounding the myenteric plexus (see below) (220,228,229,230). Arterioles (branches from the superior and inferior mesenteric arteries), venules, and lymphatics are present throughout the submucosa (Figure 25.15). These vessels in histologic sections are frequently distended by red blood cells and appear tortuous, with several cross sections of a single arteriole seen adjacent to one another within one field of section.
The amount of adipose within the submucosa varies substantially between the right and left colon and among patients. The ileocecal valve and cecum submucosa may appear particularly expanded by mature adipocytes, resembling a lipoma. However, in the absence of the submucosal adipose forming a discrete, lobulated mass, this may be considered within the range of normal.
Muscularis Externa, Subserosal Zone, and Serosa
The muscularis propria or external smooth muscle layers of the colon consist of an inner circular layer and an outer
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longitudinal layer (Figure 25.16) (231,232). Structural variations of the muscularis propria have been identified, which may reflect different motility and storage functions of various regions of the colon (225,226). Auerbach's plexus lies between the two muscle layers and resembles Meissner's plexus histologically. The interstitial cells of Cajal, the putative pacemaker cells of the gut that drive peristalsis, can be identified throughout the muscularis propria with immunizations for CD117 and CD34 (Figure 25.17) (111,233,234).
 |
Figure 25.14 Ganglion cells of Meissner's plexus. A. Submucosal nerve twigs and clusters of ganglion cells comprising Meissner's plexus. (H&E stain, x20). B. Same area stained with S-100 (hematoxylin counter stain), highlighting the Schwann cells. The ganglion cells on the left are conspicuously negative with S-100 (also x20). |
 |
Figure 25.15 Colonic submucosal vasculature. Most of the blood vessels in this section contain erythrocytes. |
Patients with motility disorders may have decreased numbers of these cells within their bowel walls (231,232). The muscularis is perforated by blood and lymphatic vessels and is encased in a subserosal zone of fibroadipose tissue. Strictly speaking, the serosa is limited to the mesothelial lining and immediately adjacent fibroelastic tissue.
 |
Figure 25.16 Muscularis propria and subserosal tissue. Both layers of the muscularis propria can be seen with the neural tissue of Auerbach's plexus. Below the muscle layers is the fibrovascular adipose tissue of the subserosa. |
 |
Figure 25.17 Interstitial cells of Cajal (ICC). The CD117 strongly positive, dendritic-appearing cells between the muscle layers and surrounding Auerbach's plexus are the ICCs. These cells are considered to be the pacemaker cell of the gut and perform other functions in gut motility. (Anti-CD117 stain.) |
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Effects of Preparation and Artifacts
Bowel Preparation Effects
The most commonly used bowel preparations for colonoscopy and sigmoidoscopy (sodium phosphate enemas, bisacodyl enemas and suppositories, dioctyl sodium sulfosuccinate, soapsuds enemas) can produce abnormalities of the mucosa that may mimic or obscure inflammatory conditions and impart an edematous or hyperemic appearance of the mucosa to the endoscopist (235,236,237). Histologic features suggesting bowel preparation include: flattening of the absorptive colonocytes to a cuboidal shape, reduction in goblet cell mucus (due to increased mucus secretion), detached surface epithelium leaving an exposed basement membrane, minimal or focal surface epithelial and crypt neutrophilic infiltrate, accentuated extravasation of red blood cells within the lamina propria, and increase in crypt or surface epithelial apoptosis (Figure 25.18). Oral sodium phosphate incites exaggeration of the previously described features of bowel preparation. Endoscopically visible aphthous erosions, erosions, and uncommonly frank ulcers have been reported. Histologically, neutrophilic cryptitis and increased basal apoptosis may be seen in addition to other common features of bowel preparation (Figure 25.19) (235,238,239). This basal apoptosis is histologically identical to low-grade graft-versus-host disease. Hence, oral sodium phosphate bowel preparations should not be used in bone marrow transplant patients. Although bowel preparation histologic changes may not interfere with rendering a polyp diagnosis, subtle inflammatory changes may be overlooked in the midst of various bowel preparation changes or alternately misdiagnosed as a pathologic condition. In the evaluation of patients for reasons other than colorectal cancer screening, bowel preparation with polyethylene glycol is suggested, as it appears to incite minimal histologic alterations (240,241). Nonetheless, polyethylene glycol preparation has also been reported in randomized trials to result in superficial mucus loss, epithelial cell loss, lymphocyte and neutrophil infiltration, and rarely aphthous erosions (242).
 |
Figure 25.18 Enema effect. There is edema with extravasation and lysis of red blood cells (hemorrhage) within the lamina propria. The surface epithelium is largely denuded. Mucin depletion due to induced goblet cell secretion and increased apoptosis may also be seen. |
Incorrect Tissue Orientation and Tangential Sectioning
Difficulties with proper tissue orientation are most common to endoscopically procured biopsy specimens, owing
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to their small size (sometimes as small as 2 mm). In both endoscopic biopsies and surgical resections, the most accurate interpretation is possible when the tissue is sectioned perpendicular to the plane of the surface epithelium. Evaluation of crypt architecture, inflammatory cell gradient, and thickness and regularity of the subepithelial collagen band may be hindered significantly by tangential sectioning (Table 25.1) (243). The appearance of acini (doughnuts) rather than test tubes within the lamina propria is a clear indication of tangential sectioning. Features of chronic mucosal injury may not be sampled in tangential sections that contain only the superficial most aspects of the mucosa. Cytoplasm of adjacent colonocytes in tangentially sectioned tissue may mimic a thickened (but regular) collagen band, risking a misinterpretation of collagenous colitis (Figure 25.20). Tangential sections with exaggerated samples of the basal portions of colonic crypts show cross sections of less mature colonocytes with larger nuclei, less cytoplasm, and without adjacent goblet cells, thus mimicking the features of a tubular adenoma.
 |
Figure 25.19 Oral sodium phosphate bowel preparation effect. Colonic crypts with apoptotic bodies and neutrophils are secondary to the effects of bowel preparation. Such changes could easily be interpreted as representing infectious colitis or graft-versus-host disease (GVHD) in the right clinical setting. |
 |
Figure 25.20 Normal colon mimicking collagenous colitis. A. This normal mucosa shows blending of the colonocyte cytoplasm with the basement membrane to give the illusion of a thickened subepithelial collagen table. Note that there is no surface damage or colitis present. B. This trichrome stained section shows focal thickening in an area where the crypt sheath joins the surface tangentially. Care must be taken when evaluating tangential sections. Again note the lack of colitis or surface damage. |
Tissue Trauma
Tissue trauma occurs with avulsion of the mucosa during forceps biopsy or in improper handling in the pathology gross room. The former may produce endoscopically visible edema, petechiae, friability, tears, and hemorrhage (241,243), and there may be histologic features of crush artifact. Biopsy samples may contain increased cell free space in the lamina propria, resembling edema and extravasation of red blood cells primarily into the luminal portion of the lamina propria. In the absence of other features of mucosal inflammation (i.e., neutrophilic inflammation), these features should not be considered pathologic. Polyfoam pads may cause triangular artifacts in biopsy material and are not recommended (244). Despite relative fixation, crush artifact may occur with pressure applied to biopsy material with rigid forceps. Use of a plastic pipette with large bore opening (i.e., cutting the tip off of a disposable pipette) to transfer biopsy material to the cassette avoids crush artifact. Crushing of the tissue results in crowding of glands and epithelial cells and is accompanied by stripping of the surface epithelium that then dislodges into the lumen.
Pseudolipomatosis
Pseudolipomatosis is characterized by vacuolated, unlined spaces in the lamina propria and mucosa that resemble loosely arranged adipocytes (Figure 25.21). These lesions are due to air trapping from insufflation of the colon during endoscopy (245).
Electrocautery
Endoscopic removal of polyps with electrocautery ( hot biopsy, or snare) frequently results in thermodessication of the tissue, compressing crypts together and altering the nuclear features. Characteristically, the crypts are closely apposed, with elongated, pyknotic and distorted nuclei (Figure 25.22). These features may be difficult or impossible to distinguish from a tubular adenoma. Prolonged electrodesiccation may result in loss of both overall architecture and nuclear detail.
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 |
Figure 25.21 Pseudolipomatosis. The clear spaces within this lymphoid aggregate represent air bubbles due to insufflation during endoscopy. This artifact is frequently misinterpreted as adipose tissue/lipoma. |
 |
Figure 25.22 Electrodesiccation and compression artifact in the bases of adjacent crypts in normal colonic mucosa produced by an endoscopic electrocautery snare. Affected nuclei are pyknotic and elongated. Colonic crypts distorted by this artifact may be difficult or impossible to differentiate from the tubules of an adenoma. |
Acknowledgments
Dr. Dahl is grateful for the continuing influence of the late Dr. Rodger C. Haggitt in the preparation of this manuscript. Dr. Charles Bevins and Richard Naftalin are thanked for their insight and dialogue.
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