Criteria for the diagnosis of coeliac disease vary. A report from the United European Gastroenterology Week40
emphasised the importance of small bowel biopsy in the diagnosis of coeliac disease, stating “The finding of circulating antibodies . . . supports the diagnosis but is not essential, and should not be used for diagnosis without histologic confirmation.” In people suspected of having coeliac disease, the American Gastroenterological Association mandates a biopsy to confirm the diagnosis.41
Although many still regard the small bowel biopsy as a “gold standard” in the diagnosis of coeliac disease,41,42
the US National Institutes of Health43
recently released a consensus statement recommending biopsies only after a positive serology or when faced with incongruous or indeterminate serological results.
Box 1 Summary of major features of mucosa suggestive of coeliac disease
- Proximal small bowel involvement, decreasing distally
- Patchy distribution, in some cases
- Mucosal architectural changes, including
- Mucosal inflammation
- Enterocyte changes
Box 2 Differential diagnosis of small bowel biopsy specimens sharing features of coeliac disease
- Increased intraepithelial lymphocytes
Allergies to proteins other than gluten (eg, chicken, cow's milk, eggs, fish, rice and soy; entities cause both raised intraepithelial counts and villous architectural changes)
Autoimmune conditions, various (eg, systemic lupus erythematosus)
Blind loop syndrome
Inflammatory bowel disease
Irritable bowel syndrome
Non‐steroidal anti‐inflammatory drugs
Tropical sprue (entities cause both raised intraepithelial counts and villous architectural changes)
Crypt hyperplasia or villous flattening
Allergies to proteins other than gluten (eg, chicken, cow's milk, eggs, fish and soy; entities cause both raised intraepithelial counts and villous architectural changes)
Common variable immunodeficiency
T cell lymphoma, associated enteropathy
Histopathologically, the protean manifestations of coeliac disease (box 1) display a range in severity. An ever‐expanding array of entities is known to produce similar histopathological findings (box 2), thereby potentially complicating the histopathological diagnosis of coeliac disease. For this reason, some prudently suggest that the pathologist's role should remain more indirect, affording the clinician a descriptive report indicating that the lesion is “. . . consistent with CS [celiac sprue]”.44
Depending on the level of serological antibody titres, others advocate abandoning biopsies altogether.45
Presently, we consider the small bowel biopsy essential for diagnosing coeliac disease; likewise, we also acknowledge the multitude of entities that may present with similar histological findings. For this reason it is imperative that there be candid and regular communication between the clinician and the pathologist. Although not always feasible, the clinical–pathological conference can be an ideal venue for such an exchange.
A modest literature proposes a diagnostic role for nasal or rectal biopsies following gluten challenge.46,47
It has been acknowledged that such a technique may afford limited diagnostic value in the context of existing clinical tools, without further investigation.48
For the time being, pathologists are unlikely to encounter such specimens outside an experimental setting.
Normal variations in small bowel histology
The small bowel exhibits a range of morphological variation that should be considered to be normal both among individuals and across populations. A brief overview of normal histological variants follows (for a comprehensive review, see Segal and Petras49
). Natural differences in villous architecture across populations can be dramatic, and it is important that pathologists appreciate these differences. Variants of villous morphology include
- finger‐like, with a cylindrical core and rounded apex;
- leaf‐like, with a broad flattened base with a tapering apex;
- tongue‐like, with a broad flattened base and rounded apex; and
- ridge‐like, with a flat linear base that is less in width than its height.
In the duodenum it is not unusual to see branched villi or villi containing fused tips.50
Mixed populations of villi are common.49
In adults, the proximal villi of the duodenum are broad, and a leaf‐like morphology is common. In the upper jejunum, tongue‐like villi are often seen. More distally, the villi gradually become elongated, assuming a finger‐like morphology.51
Although villi overlying Brunner's glands in the duodenum may exhibit a finger‐like appearance, these are typically shorter in length.50
Similar villous changes may be seen in those overlying lymphoid aggregates; in fact, villi may be absent in these areas altogether.50
A larger concentration of IELs may be seen over lymphoid aggregates.
The pathologist should also be beware that gastric metaplasia, gastric heterotopia and heterotopic pancreas can be observed within the small bowel.49
Differences among populations
The developing fetus first develops finger‐like villi, followed by crypts.52,53
Exposure of the neonate mucosa to ingested material and intestinal flora results in blunting of the villi; this is relatively transient among infants in temperate regions and more persistent among those in the tropics.54
It may not be until well into childhood that the finger‐like morphology replaces the leaf‐like variant, assuming the environment is conducive to this change. Yet, the broader villi may represent a normal observation in the proximal small bowel.54
In adults, villous morphology does not seem to change in elderly people.55
The finger‐like morphology may predominate in people residing in temperate areas, whereas those indigenous to more tropical climates often have the leaf‐like and ridge‐like variants proximally and the finger‐like variant distally. In its mildest form, this may represent a normal histological variant.56
Interestingly, tropical migrants to temperate regions exhibit some reversal of villous blunting,57
whereas those moving to tropical regions incur villous blunting.58
Although differences in intestinal flora have been proposed as an explanation for loss of the finger‐like variant, these differences may also relate in part to dietary differences.56
Differences attributable to sampling
The volume of case material derived from small bowel biopsies is ever increasing, owing in large part to the relative ease of performing upper gastrointestinal endoscopy and procuring tissue. We have found conflicting results on the benefit of suction capsule versus endoscopic pinch biopsies.59,60
Regardless, endoscopic pinch biopsies of the duodenum have largely replaced suction capsule biopsies of the jejunum.
There is evidence supporting a strong correlation in the histological observations in coeliac disease, between the duodenum and jejunum.61
Larger biopsy specimens are generally considered to be more amenable to histological evaluation,41,47,62
although “jumbo” forceps have been reported to confer no marked advantage over a standard bite size.63
Endoscopic biopsies have the advantage of targeting grossly abnormal regions of the bowel to efficiently procure multiple biopsy specimens. Newer endoscopic methods, such as push enteroscopy and double‐balloon enteroscopy, allow access to the entire length of small bowel to biopsy forceps. When using pinch biopsy forceps, the endoscopist must be careful not to leave the specimen within the bowel or lose it to the suction chamber. Biopsy forceps crush and destroy tissue and thus evaluation of specimen margins should be limited; in addition to damaging cells, this mode of tissue procurement may introduce artefactual haemorrhage in the sample. Superficial biopsy specimens lacking a muscularis mucosa can cause artefactual separation of the villous bases, resulting in the appearance of shorter and thicker villi.49
Tissue derived from a suction capsule may incur partial or even complete separation of the epithelium from the lamina propria. The absence of a host response to ulceration, such as an acute inflammatory response or granulation tissue, will alert the pathologist to potential sampling difficulties. Due care must be exercised in removing samples from the capsule.50
Histopathology of coeliac disease
Symptomatology in coeliac disease seems to be related to the length of affected bowel, and not to the severity of the mucosal lesion.64,65
Thus, insults compromising the inherent compensatory ability of the small bowel—such as worsening extent of disease, infection, ischaemia and short bowel, among other things—may suffice to unmask previously compensated coeliac disease. Conflicting reports exist on the distribution of lesions in coeliac disease along the small bowel mucosa. It has been suggested that villous lesions rarely coexist with histologically normal mucosa.66
Others describe coeliac disease as exhibiting a patchy distribution,32,50,67,68
thus implying a need for multiple biopsy specimens to secure a diagnosis. Traditionally, the severity of intestinal pathology is regarded as greatest in the proximal small bowel with distal lessening. Lesions affect the mucosa and the submucosa.4
Coeliac disease has also been reported to affect other mucosal sites, such as the oesophagus, stomach and large bowel.
Healing of the small bowel mucosa proceeds in a caudal to cephalad direction.47
This may take anywhere from 6 to 24 months after induction of treatment and in some cases the extent of recovery may remain incomplete.69
The migratory rate of epithelial cells from the crypts to the villous surface is reduced from 3–5 to 1–2 days in coeliac disease.41
Lesions can be described in terms of a range of architectural, cytological and ultrastructural features (table 1) that, combined, create a blurring array of histopathological permutations. Alone, these features are non‐specific and should be regarded as shared among a range of ailments afflicting the small bowel (box 2). When screening small bowel biopsy specimens several features may be suggestive of coeliac disease, such as increased IELs, crypt hyperplasia and villous atrophy.67,70
Table 1The modified Marsh–Oberhuber classification Intraepithelial lymphocytes
Because of the nature of the immunopathological basis of coeliac disease, the pathologist encounters a complex and heterogeneous population of lymphocytes and granulocytes in the mucosal biopsy specimens. The lamina propria is the seat of a brisk immunological response, and grading this reaction is difficult and impractical. Fortunately, early work in a murine model showed IELs to be a surrogate marker of immune activity in the lamina propria.71
Researchers in the 1970s extended these results to humans72
and it is from this work that current standards for IEL counts were derived.
Traditionally, counts >40 IELs per 100 epithelial cells were considered to be abnormal.72
This benchmark was subsequently challenged, with 12 IELs per 100 epithelial cells offered as the actual value.73
More recent estimates include 25 IELs,74,75
or 20 IELs per 100 epithelial cells.77
The trend towards a lower “normal” number of IELs is reflected by the proposal that 30 IELs per 100 epithelial cells were incorporated into revised classification schemes.42
It remains to be determined whether lowering the upper limit for IELs will adversely decrease the specificity of the small bowel biopsy in the diagnosis of coeliac disease; nevertheless, it is conceivable that this number may be further reduced in the future.
Formal enumeration of IELs includes selecting suitably oriented villi in the biopsy specimens and then counting the total IELs present per 100–1000 epithelial cells along the luminal margin, excluding the crypt. The total number of IELs is expressed relative to 100 epithelial cells. For pathologists this procedure is obviously labour intensive, prompting some of them to consider more efficient means of enumerating IELs.75,78
Quantifying villous tip IELs
A rapid method of screening for coeliac disease includes counting the number of IELs present at the villous apex.78
Basically, the pathologist selects five villi. At the distal apex of each villous, 20 epithelial cells are counted and the number of IELs in this range is similarly enumerated; if desired, the number of IELs can then be expressed relative to 100 epithelial cells. This technique has recently been corroborated by others as an efficient means of objectively quantifying IELs,77,79
and the normal number of IELs at the apex per 20 epithelial cells have been documented as 2.2 (11 IELs per 100 epithelial ells),78
4.6 (23 IELS per 100 epithelial cells)75
and 2.3–3.3 (11.5–16.5 IELs per 100 epithelial cells).79
Given the number of entities causing an increase in IELs, an increased number of IELs at the villous tip is by no means diagnostic of coeliac disease, but should raise this possibility in the differential diagnosis.
Loss of the “crescendo sign”
The normal distribution of IELs along the villi assumes a characteristic “luminopetal” distribution,72
that has recently been likened to that of a musical “crescendo”, with a tapering in IELs as we progress towards the villous apex.64
Patients with coeliac disease, including those with architecturally normal villi, lack this pattern as a result of saturation of the tip by lymphocytes, resulting in a more uniform distribution of IELs along the villous length.64,78
A Gestalt approach is applied in assessing biopsy specimens by this method of screening and an abnormal distribution of IELs should alert the pathologist to the possibility of coeliac disease. Despite a rate of potential false positives approaching 25%, this may be more sensitive than formal IEL counts in screening for coeliac disease.78
Accurately quantifying IELs can be complicated by issues such as nuclear overlap and heterogeneity in nuclear shapes, occasionally making it difficult to distinguish epithelial cells from enterocytes and granulocytes.80
The routine application of immunohistochemical staining for lymphocyte markers such as CD3 has been proposed as a means to better evaluate the number and distribution of IELs in instances in which there is a normal villous architecture and a perceived increase in IELs.77,80
Presently, the benefits of a more rigorous IEL count do not seem to supersede the additional expense, demand on pathologists' time and delay in issuing of reports to clinicians imposed by immunohistochemistry. When faced with intraepithelial lymphocytosis on haematoxylin and eosin staining, it is perhaps not unreasonable to first seek a second opinion, offer a differential diagnosis or recommend serological testing.
Crypt hyperplasia denotes elongation of the length of the crypts of Lieberkühn, a process that initially precedes villous atrophy.65,81
Elongation may be caused by expansion of the lamina propria as a result of the proliferation of stromal cells,81
an influx of inflammatory cells50
and tissue remodelling. The crypts contain stem cells capable of renewing enterocytes and goblet cells, and it is not uncommon to see appreciable mitotic activity in this location. This, unfortunately, is not a reliable indicator of crypt hyperplasia.82
Initial studies investigating the application of proliferative markers such as Ki‐67 (MIB‐1) in discriminating early stages of coeliac disease have shown some promise.82
The normal ratio of villous height to crypt depth is the subject of some controversy. It is generally assumed that the normal range is 3:1 to 5:149
; others have considered other ratios to be acceptable, including 2:1,83
and even 1:1.69
In children, a ratio of 2:1 is considered to be normal by some.84
In coeliac disease, a loss of villous height and elongation of the length of crypts may change this ratio. It remains to be determined whether subcategorisation based on grades of severity is indicated.
A loss of villous height is considered to be pathognomonic for coeliac disease by many clinicians; thus, it is important to emphasise the non‐specific nature of this finding (box 2). The height of the villous is generally three times its base width. Oberhuber et al67
proposed a grading as mild, marked or total. An absence of atrophy implies that the villi are of normal height. Mild atrophy indicates a minor to moderate amount of villous blunting; marked atrophy indicates the presence of truncated villous remnants; and finally, total atrophy implies the complete absence of villi.67,81
It is important that the most severely diseased areas of the biopsy be reported (ie, biopsy results should not be averaged). Given the potential for an irregular distribution of disease, a comment denoting the patchy nature of the lesion may also be indicated.
The observation of moderate to total villous atrophy, particularly in a patient with longstanding coeliac disease or in a patient who proves unresponsive to diet, obliges the pathologist to attempt to exclude the presence of a more sinister concomitant lesion, such as Crohn's disease, autoimmune enteropathy, lymphoma or adenocarcinoma.
Additional histological observations
Several additional cytological features can be appreciated in coeliac disease. Enterocytes, for example, may lose their columnar configuration, yielding to a cuboidal shape. The cytoplasm may be basophilic and in some cases contain apical vacuoles.50,67,85
Furthermore, the nuclei may be pyknotic and lose their basal orientation.50
Ultrastructurally, there are reductions in microvillous height with the appearance of apical lysosomes85
and changes in intraepithelial tight junctions.22
Functionally, there are thought to be differences in the glycocalyx or mucous layer of the bowel lumen, resulting from altered patterns of glycosylation; this may selectively promote bacterial adhesion.86
In coeliac disease, the lamina propria may undergo marked expansion, particularly with lymphocytes and plasma cells. Macrophages, eosinophils and mast cells are often seen and occasional neutrophils may also be present. The presence of cryptitis or crypt abscesses should reflexively prompt consideration of the possibility of Crohn's disease.