Definitions of Histological Abnormalities in Inflammatory Bowel Disease: an ECCO Position Paper

Abstract Histological assessment of endoscopic biopsies in inflammatory bowel disease [IBD] plays an important role in clinical management, investigative studies, and clinical trials. Scoring schemes consisting of multiple histological items and offering considerable precision are widely available. However, definitions of histological abnormalities are often inconsistent. Furthermore, interobserver variability for their recognition and assessment may be high. The European Crohn’s and Colitis Organisation [ECCO] formed an expert panel to explore definitions of histological abnormalities in IBD, with the aim of improving the quality of diagnosis and facilitating development of scoring schemes. The process confirmed that the current definitions often have no evidence base and vary between sources. Using available evidence and expert knowledge, the panel produced a series of ECCO consensus position statements on histological features in IBD.


Introduction
Microscopic examination and interpretation of ileocolonic biopsies help to optimise the management of inflammatory bowel disease [IBD].Histology can confirm the diagnosis, aid subclassification, determine severity, estimate the efficacy of drug therapy, and detect complications. 1,2The pathologist's focus depends on the setting.][8][9][10][11] However, there is often no consensus on basic definitions of histological abnormalities, 9 and many definitions are inevitably arbitrary.Table 1 outlines broad categories of histological abnormality.

Materials and Methods
The project leaders assembled a panel of 15 expert histopathologists and gastroenterologists to develop consensus position statements on histological definitions in IBD.Table 2 summarises the aims.The ECCO Positions appear below with accompanying supporting text.Details of the methodology and additional text are in the Supplementary Materials.
Normal crypts are parallel to one another and extend from the mucosal surface to the underlying muscularis mucosae ['test tube' arrangement; Figure 1]. 1,12Assessment of architecture and of features that rely on spatial distribution requires biopsies of adequate size with optimal orientation that also ideally include muscularis mucosae [Figure 1].
Suggestions for thresholds to define crypt distortion include the following: a minimum of 10% crypts showing any of the features in ECCO Position 1.1.4][15] However, there is no consensus on these suggestions.
Crypt atrophy encompasses crypt shortening and wider crypt spacing [Figures 2 and 3], but assessment of these features is subjective. 13,16,17][17][18][19][20][21] Several exceptions are worth noting.Sampling from innominate grooves can demonstrate apparent branching. 22The mucosa up to a distance of three crypts from a lymphoid aggregate is probably unreliable for assessment of architecture because the aggregates can distort adjacent glands.Normal rectal or caecal mucosa may show a minor degree of crypt distortion or shortening [Figure 1]. 1 There is no consistent definition of surface irregularity [or the analogous terms villous surface, villiform surface, villous mucosa].The impression of a villiform surface is partly the result of separation of crypts [Figure 4]. 21,23,24CO Position 1.7.
Villous atrophy in the ileal mucosa is defined as the presence of shortened villi with a villus:crypt ratio lower than 3:1 Agreement: 93%

Produce ECCO Positions on histological features in inflammatory bowel disease [IBD]
Reduce interobserver and intra-observer variability  Definitions of mucin depletion may specify a reduction in the number of goblet cells or a reduction in mucin droplets [Figure 2]. 5,13Definitions describe 'significant' or 'unequivocal' reduction but do not usually offer quantification.One suggestion is one or less goblet cell in eight to ten enterocytes [anonymous personal communication].][30] Pyloric metaplasia [PM; broadly synonymous with pseudopyloric metaplasia / ulcer-associated cell lineage / spasmolytic polypeptide-expressing metaplasia] comprises glands lined by columnar cells that have clear or pale PASpositive cytoplasm with neutral mucin granules and oval or round basal nuclei, thus resembling normal gastric antral and Brunner glands [Figure 6]. 31,32PM is associated with chronicity, particularly in the small bowel in CD.[35][36]

In inflammatory bowel disease biopsies, chronic inflammation is defined as an increase in the number of lymphocytes and plasma cells in the lamina propria
Agreement: 100%

Basal plasmacytosis is defined as an increase in plasma cells at the basal aspect of the colorectal mucosa, typically between the base of the crypts and the muscularis mucosae
Agreement: 100% The criteria and thresholds for grading chronic inflammation as absent, mild, moderate, or severe are defined vaguely or are not defined at all Agreement: 100% Histological manifestations of mucosal chronic inflammation include basal plasmacytosis, lymphoid aggregates, and a diffuse or variable increase in lamina propria chronic inflammatory cells.8][39][40] When basal plasmacytosis is present, the density of plasma cells at the base of the mucosa is high and may be similar to or higher than the density of plasma cells in the upper mucosa [Figures 2, 7, and 9]. 1,13,20Basal plasmacytosis can be diffuse and band-like, focal, or patchy. 1,41,42][45] There is no agreement on how to measure the number of mucosal chronic inflammatory cells or on grading of basal plasmacytosis. 40,43,46Although IBD is a chronic inflammatory process, 47,48 there is less emphasis in clinical practice and in scoring systems on chronic inflammation than on acute or active inflammation. 6ome authors refer to basal lymphoplasmacytosis, for which definitions are even less precise than for basal plasmacytosis.Determining colorectal lamina propria lymphocyte numbers or density or defining an increase in lymphocytes is more difficult and subjective than assessing plasma cells.According to some sources, up to two basal lymphoid aggregates may be present in a normal colorectal mucosal biopsy [Figure 8].However, distinction between normal and abnormal numbers is difficult.Scoring schemes that include lymphoid aggregates do not quantify these aggregates. 49,50Architectural distortion and epithelial metaplasia are the result of crypt destruction and injury associated with chronic and active inflammation, but they do not define 'chronic inflammation' in IBD. 51

Eosinophils do not define chronic inflammation or acute inflammation reliably in inflammatory bowel disease
Agreement: 93%

ECCO Position 4.2.
There is no widely accepted definition of a significant increase in colorectal mucosal eosinophils in inflammatory bowel disease Agreement: 93%

ECCO Position 4.3.
Eosinophilic cryptitis is defined as the presence of at least one eosinophil in the crypt epithelium Agreement: 93% ECCO Position 4.4.

An eosinophilic crypt abscess is defined as eosinophils in a crypt lumen without the presence of luminal neutrophils Agreement: 100%
There is limited information about the normal number of intestinal mucosal eosinophils, the definition of an eosinophil crypt abscess, 50,49 and the criteria for a mild, moderate, or severe increase in intestinal mucosal eosinophils [Figures 7, 9].The significance of focal eosinophil cryptitis in the absence of other changes is also uncertain.The panel agreed that an eosinophilic crypt abscess comprises at least two eosinophils.If neutrophils are also present, the term for the lesion is a crypt abscess [ie, neutrophil crypt abscess; Figure 10].

ECCO Position 5.1.
In inflammatory bowel disease, a granuloma is a distinct collection of at least five histiocytes/macrophages that can also contain multinucleate giant cells.It may have a welldefined or poorly defined outline, lacks necrosis, and may have a lymphoid cuff Agreement: 93% ECCO Position 5.2.

A cryptolytic granuloma [mucin granuloma] is a collection of histiocytes, neutrophils, and mucin associated with an injured crypt and can be present in any form of inflammatory bowel disease
Agreement: 100% The number of histiocytes necessary to define a granuloma in IBD is at least five. 13,44,52The threshold may vary with the setting. 15Histiocyte counts may depend partly on section thickness, number of serial sections examined, and the individual observer.In CD, a granuloma is almost always nonnecrotising, never has caseous necrosis, and typically shows no confluence with other granulomas [Figure 11]. 44,45,51,53,54ant cells are acceptable.Conventional practice classifies granulomas as either present or absent, with no quantification.
A cryptolytic granuloma characteristically lies adjacent to a crypt that exhibits rupture [Figure 12]. 55,56In addition to mucin, neutrophils, and histiocytes, the lesion may contain lymphocytes, multinucleate foreign body-type giant cells, and parts of epithelial cells. 44,57It may consist largely of histiocytes.Close approximation of a granuloma to a crypt suggests that it is cryptolytic, but identification of crypt injury may require serial tissue sections and is sometimes not possible. 51lthough granulomas help distinguish CD from UC, most biopsy samples from CD do not contain granulomas. 58bsence of a demonstrable association with crypt injury or with ulceration strengthens the discriminatory value of a granuloma towards CD.Cryptolytic granulomas can occur in both CD and UC, 1 but appear to be more common in CD.
The definition of microgranuloma varies in the literature and there is no widely accepted definition.

Acute inflammation is defined histologically by the presence of acute inflammatory cells, namely, neutrophils, in the lamina propria and/or within the surface epithelium, the crypt epithelium, or the lumens of crypts. Acute inflammation can be graded using a validated scoring scheme
Agreement: 93% ECCO Position 6.2.

A crypt abscess is defined as the presence of neutrophils in the crypt lumen Agreement 93%
Most definitions state that the lamina propria and epithelium of normal colorectal mucosa do not contain neutrophils.However, some authors assert that one, two, or even three neutrophils are acceptable, particularly in the lamina propria.There is lack of agreement. 22,24Bowel preparation probably accounts for some instances of small numbers of neutrophils.Neutrophils in the capillary lumen in isolation do not indicate acute inflammation.
The panel agreed that one neutrophil is sufficient to define cryptitis, although some publications specify more than one.At least one neutrophil must be demonstrably intraepithelial [Figure 7].Similarly, there is no universal agreement on the minimum number of neutrophils in a crypt abscess. 15efinitions of a crypt abscess may refer to a cluster or chain of neutrophils.General definitions of 'abscess' usually require more than one neutrophil [Figures 10, 12].The panel agreed that the presence of at least two neutrophils is necessary.By convention, the terms 'cryptitis' and 'crypt abscess' without further qualification refer to neutrophil cryptitis and neutrophil crypt abscesses, respectively.Cryptitis or a crypt abscess can include other types of inflammatory cells in addition to the neutrophil[s] [Figures 10, 12].
Grading of acute inflammation may refer to none, mild, moderate, and severe, 59 although this is often subjective.Many histology scoring schemes include grading of acute inflammation despite the lack of concordance on methodology.[62] Figure 11 A granuloma [circle] in Crohn's disease [CD] includes at least five histiocytes and does not show necrosis.Here, the number of histiocytes is considerably greater than five.Multinucleate giant cells and a lymphoid cuff are present in some granulomas in CD, but not in this example.

An erosion is characterised by injury to the surface epithelium and the underlying mucosa with no extension beyond the muscularis mucosae. An ulcer is characterised by injury that extends beyond the muscularis mucosae. Erosions and ulcers may be difficult to distinguish from one another in a mucosal biopsy Agreement 87%
Distinction between an erosion and an ulcer depends on the depth of penetration into the wall. 63Erosions and ulcers may be associated with granulation tissue formation, fibrin, a surface exudate that includes neutrophils, vascular proliferation in the lamina propria, and fibroblast proliferation in the submucosa. 63Ulcers and erosions may show evidence of re-epithelialisation of the adjacent mucosa.Distinction between erosions and ulcers may be difficult or impossible in practice, especially if biopsies are superficial and lack muscularis mucosae and submucosa [Figures 4, 13

Chronic ileitis is characterised by a chronic inflammatory cell infiltrate and architectural mucosal distortion, but is often difficult to define and recognise. The presence of pyloric metaplasia in the ileum strongly supports a diagnosis of chronic ileitis
Agreement 100% ECCO Position 7.4.

Active chronic ileitis is defined as acute inflammation superimposed upon chronic ileitis Agreement 100%
The normal distal ileal mucosa contains confluent dense lymphoid tissue, lymphoid aggregates, and lymphoid follicles.Accordingly, an increase in chronic inflammatory cells alone is usually insufficient to confirm chronic ileitis. 64Other features, such as villous distortion, villous atrophy and, in particular, pyloric metaplasia support chronicity in this location.
Clinicians may equate 'chronic colitis' with 'ulcerative colitis'.This term requires qualification, particularly in the conclusion of a pathology report.
Biopsies that show chronicity are categorised as inactive chronic or active chronic, the latter requiring at least one feature of activity.The term 'activity' has different meanings depending on the setting [clinical, endoscopy, histology].

Focal active colitis is a histological pattern characterised by cryptitis and/or crypt abscesses involving one or a few crypts, with no associated chronic inflammatory changes Agreement 85%
Focal crypt injury by neutrophils and focal active colitis [FAC] are common in endoscopic colorectal biopsies.FAC is associated with epithelial injury.[67][68][69]    Assessment of disease distribution macroscopically and microscopically can assist with the distinction between UC and CD.[ Figures 14, 15]. 70,71The Vienna system and WHO classification propose replacement of the term dysplasia with 'intraepithelial neoplasia'. 72The features of conventional IBD dysplasia are analogous to those characterising gastrointestinal [GI] neoplasia in general [eg, in colorectal adenomas]. 14,70,73][76] The category 'indefinite for dysplasia' includes lesions that are 'probably negative' or 'probably positive' for dysplasia and is particularly useful when there is significant acute inflammation or ulceration or erosion with epithelial changes that are difficult to classify [Figure 16].'Indefinite for dysplasia' is not an intermediate category between negative for dysplasia and low-grade dysplasia.

Discussion
The ECCO Positions in this article represent expert consensus with the support of evidence where available.Table 3 summarises additional considerations.Further development of this process will include attempts to grade histological changes, aiming to improve the consistency of pathological diagnosis and to facilitate development of new scoring schemes for IBD.

Figure 1
Figure 1 A well-orientated colorectal biopsy, including muscularis mucosae [stars], is ideal for assessment of mucosal architecture.In normal mucosa, crypts are mostly parallel to each other and the crypt base usually reaches the underlying muscularis mucosae [circle].An occasional branching crypt [arrow] is acceptable as normal.For all colour figures refer to online version.

Figure 2 A
Figure 2 A colonic biopsy that shows extensive crypt distortion, including crypt branching and crypt irregularity.In addition, all crypts show atrophy, with shortening [failure to reach the muscularis mucosae; ellipse] and wider spacing [hexagon].The two most central crypts show severe epithelial mucin depletion [arrows].Diffuse basal plasmacytosis is present [stars].

Figure 3
Figure 3 Crypt atrophy and crypt distortion are milder here than in Figure 2. Crypt shortening ranges from very mild [circle] to a reduction of almost 50% in length [capsule].Mild loss of the normal parallel 'test tube' arrangement of crypts represents crypt distortion.

Figure 4 A
Figure 4 A villiform surface in colorectal mucosa [ellipse] is the consequence of wide crypt mouths and irregular regeneration.At the top left there is an erosion [star], defined as injury to the surface epithelium and underlying mucosa without extension deeper than the muscularis mucosae.

Figure 5
Figure 5 Paneth cells [arrows] with characteristic bright red cytoplasm are numerous at crypt bases in the small bowel and are normal in the right colon.By convention, their presence distal to the splenic flexure constitutes Paneth cell metaplasia.

Figure 6
Figure 6 Pyloric metaplasia in ileal mucosa, comprising two glands [ellipse and rectangle] lined by columnar cells with small basal nuclei and pale or clear cytoplasm and resembling gastric pyloric or duodenal Brunner's glands.

Figure 7
Figure 7 Basal plasmacytosis, comprising an increase in plasma cell numbers at the mucosa base ['crypts with their feet in pools of plasma cells'] 40 and elsewhere [eg, within large ellipse; small ellipse surrounds three plasma cells].Other inflammatory cells [eg, eosinophils] are also apparent.The base of a crypt [arrow] shows cryptitis [ie, at least one neutrophil in the crypt epithelium].

Figure 8
Figure 8 Lymphoid aggregates [ellipse around an aggregate] are nodular collections of lymphocytes that typically lie at the base of the mucosa.They are a normal finding in intestinal mucosa.Their number and density may increase in inflammatory bowel disease [IBD], but defining a significant increase is difficult.

Figure 10
Figure 10This crypt abscess [arrow] consists mainly of eosinophils but also includes neutrophils.Therefore, it is a crypt abscess rather than an eosinophil crypt abscess.

Figure 9
Figure 9 Eosinophils accompany basal plasma cells in this biopsy [circles identify four eosinophils].A crypt shows eosinophil cryptitis [arrow; ie, at least one eosinophil in the crypt epithelium without accompanying neutrophils].The maximum number of lamina propria eosinophils and of foci of eosinophilic cryptitis in normal mucosa is uncertain. ].

3. 7 .
Patterns of injury ECCO Position 7.1.Chronic colitis is a descriptive clinicopathological term that requires histological evidence of chronic mucosal injury.The most consistent and reliable markers of chronic mucosal injury are crypt architectural distortion, basal plasmacytosis, Paneth cell metaplasia, and lamina pro-

Figure 12 A
Figure 12 A crypt abscess [arrow] comprises at least two neutrophils in the crypt lumen and may include other inflammatory cells.The deep part of this crypt shows rupture with extrusion of contents, generating a cryptolytic granuloma [circle].

Figure 13
Figure13 Unlike an erosion, an ulcer extends deep to the muscularis mucosae.In this resection specimen, there is severe ulceration with complete loss of mucosa and with extension into the submucosa [star] and underlying muscularis propria [rectangle].Distinction from an erosion is easier here than in many mucosal biopsies.

Figure 14
Figure 14 Dysplasia is an unequivocal neoplastic epithelial alteration without invasive growth.The crypt at lower left [ellipse] does not show dysplasia.Most other crypts [eg, within the circle] show low-grade dysplasia, with some resemblance to normal mucosa.

Figure 15
Figure 15 High-grade dysplasia showing severe cytological atypia and severe architectural changes.The latter include a cribriform pattern [ellipse].The appearances are less reminiscent than low-grade dysplasia of normality.

Figure 16
Figure16 The term 'indefinite for dysplasia' is appropriate when distinction between non-neoplastic epithelial changes and dysplasia is not possible, as in this example of epithelial atypia [capsule] adjacent to an ulcer/erosion [circle].
high-grade dysplasia over low-grade dysplasia include nuclei in the upper half of a cell, complete loss of nuclear polarity, prominent nucleoli, and vesicular chromatin.Architectural changes are also more severe, with cribriform areas Agreement 93% ECCO Position 9.7.A dysplastic lesion should have at least a few crypts with high-grade changes to be classified as high-grade dysplasia, but the number or proportion required for a final diagnosis of high-grade dysplasia is poorly defined Agreement 93% ECCO Position 9.8.The category of indefinite for dysplasia is appropriate when a definite distinction between non-neoplastic changes and neoplasia is not possible, regardless of the reasons [eg, poor quality sample, severe inflammatory infiltrate, confusing histological features] Agreement 93%Assessment of dysplasia in IBD relies on both cytological and architectural features in haematoxylin and eosin [H&E]stained sections and adheres to the criteria ofRiddell et al.

Table 1
Broad categories of histological abnormality.

Table 2 .
Aims of the consensus process.

Table 3
Considerations regarding definitions of histological features of inflammatory bowel disease [IBD].

ECCO Position 9.1. Dysplasia is an unequivocal neoplastic epithelial alteration without invasive growth Agreement 100% ECCO Position 9.2. Diagnosis of conventional dysplasia is based on cytological [eg, nuclear hyperchromasia, pseudostratification, enlarge- ment, pleomorphism, and loss of polarity] and architectural [eg, glandular crowding, tubular or villiform architecture, and absence of normal base-to-surface epithelial matur- ation] alterations of the epithelium Agreement 83% ECCO Position 9.3. A diagnosis of 'negative for dysplasia' is reserved for non- dysplastic epithelium, either normal or regenerative Agreement 100% ECCO Position 9.4. A standardised classification, the Vienna classification, div- ides dysplasia into low-grade dysplasia and high-grade dys- plasia Agreement 93% ECCO Position 9.5. Features that favour dysplasia over reactive muco- sal changes include diffuse nuclear hyperchromasia, Abnormality Present in normal mucosa? Objective definitions available?
NA, not available.