—  SHORT COURSE #22  —

Diagnostic Problems in GI Pathology

Case 1 - Significance of Intraepithelial Lymphocytes in the Small Bowel

Lisa Yerian, John Hart and Amy Noffsinger


Introduction:
Small bowel biopsy is an indispensable component of the diagnostic work-up of patients with malabsorption and/or diarrhea secondary to mucosal damage. A systematic approach to the light microscopic examination of duodenal biopsy specimens is important in order to make a specific histologic diagnosis whenever possible. Many authors [1, 2, 3] have adopted an approach in which diagnostic categories are constructed based on mucosal morphology, generally in the following fashion:
  1. Complete villous flattening with crypt hyperplasia

  2. Villous and crypt shortening (mucosal atrophy)

  3. Variable villous flattening with crypt hyperplasia (focal or diffuse)

  4. Normal mucosal architecture
There are disease entities in each of these morphologic categories in which intraepithelial lymphocytosis can occur. Some of these disorders exhibit no additional diagnostic abnormalities, and the pathologist can offer only a differential diagnosis. Other disorders manifest additional histologic features that allow a specific diagnosis to be made.

Intraepithelial Lymphocytosis
The number of intraepithelial lymphocytes that separates normal individuals from those with small bowel disease has, surprisingly, not been studied extensively. Forty IELs per 100 enterocytes was adopted as the cutoff for the diagnosis of celiac disease by Furguson and Murray because the highest level in their control group of normal individuals was 40, and the mean value plus two standard deviations was 36.1 [4]. However, a recent larger study of a Swedish population concluded that a value of 20 IELs per 100 enterocytes was more appropriate (their mean plus 3 SD was 18.5). The authors calculated that using that cutoff no more that 1 in 1000 healthy persons would be falsely diagnosed with intraepithelial lymphocytosis [5]. They also studied the use of CD3 immunostains to highlight IELs and calculated a cutoff of 30 per 100 enterocytes, with 25-29 IELs reported as "borderline" [5]. The authors stress the importance of not performing counts of IELs in the epithelium anywhere in the vicinity of even small lamina propria lymphoid aggregates, since increased numbers of IELs are normal there. This is particularly critical in the evaluation of biopsies of the terminal ileum, since lymphoid aggregates and follicles often occupy large portions of such samples. Immunohistologic studies to highlight IELs utilizing a CD3 antibody may aid in the recognition of intraepithelial lymphocytosis in biopsies without villous blunting [6], but the use of this strategy is generally not required in daily practice.

Recently it has been proposed that an abnormal distribution of IELs along the length of the villi, even if the overall number is not significantly increased, is suggestive of celiac disease [7]. This proposal is based on the observation that in healthy individuals there is a progressive decrease in the density of IELs from the base of a villus to its tip. In contrast, in a subset of patients with celiac disease this normal "decrescendo pattern" of IEL distribution is lost, and instead, the number of IELs is similar along the entire length of the villus, or is actually higher at the tip than at the base. However, the author emphasizes that this pattern of IEL distribution merely suggests the need for serologic testing to rule out celiac disease, as there are other causes of this histologic finding, and some patients may have no disease state at all [8].

Complete Villous Flattening Pattern

Celiac Disease
Celiac disease occurs because of the ingestion of alpha-gliadin within gluten containing foods by sensitive individuals, and can be diagnosed in patients of any age after institution of gluten to the diet. Gluten is present at high levels in wheat, rye and barley but is absence in corn and rice. Oats (in moderate amounts) has been shown to be tolerated by some celiac patients without adverse effects [9]. Gliadin injures the enterocytes in celiac disease patients, causing them to aberrantly express HLA antigens and secrete IL-15. This, in turn may lead to the intraepithelial infiltration of CD8+ T-cells that is so characteristic of celiac disease. The gliadin is deaminated by tissue transglutaminase in the interstitium, resulting in a peptide that is recognized by an expanded population of CD4+ T-cells (DQ2 and DQ8 restricted) in the lamina propria [10].

Presentation with the classic symptoms of malabsorption (diarrhea, steatorrhea, abdominal bloating and pain, weight loss, poor weight gain, failure to thrive, fatigue, metabolic bone disease) is becoming less and less frequent. Instead, a large (and ever increasing) number of "atypical" presenting symptoms are being reported, including low serum folate, calcium, magnesium or phosphorus levels, intra-cranial calcifications causing seizures, and growth retardation in children [11]. Unexplained iron deficiency anemia is now one of the leading presenting signs of celiac disease, particularly among adolescent patients [12, 13]. There has also been a significant increase in diagnosis through screening of patients with Down's syndrome, and juvenile onset diabetes mellitus and other autoimmune disorders (e.g. PBC). Virtually every patient with dermatitis herpetiformis has or will develop celiac disease. About 90% of patients with celiac disease carry a HLA-DQ2 of HLA-DQ8 allele.

In the past elevation of serum anti-gliadin and anti-endomysial antibody titers were required to establish a diagnosis of celiac disease. In patients with a high clinical suspicion of celiac disease (i.e. those with classic symptomatology) the sensitivity and specificity of these assays were in the range of 90%. However, as screening tests in asymptomatic adult blood donors, for example, the positive predictive value for a positive anti-gliadin antibody test was only 20% [14]. Moreover, the value of these serologic tests varied by geographic area and ethnicity, even among high-risk patient populations [15]. Most studies have shown that the anti-endomysial antibody test is more sensitive and specific than the anti-gliadin antibody test [16]. It is important to remember, though, that the anti-endomysial antibody test only detects IgA antibodies, while both IgA and IgG anti-gliadin antibody tests are available. This is important because the frequency of selective IgA deficiency is more than 10 times higher in patients with celiac disease than in the general population [17].

Recently it was demonstrated that the primary antigen detected by the anti-endomysial indirect immunofluorescent test is a peptide portion of tissue transglutaminase (tTG) [18]. Automated ELISA assays utilizing either human or guinea pig tTG have become the primary test for celiac disease, and have supplanted the more time consuming and subjective anti-endomysial antibody test in some laboratories [19, 20]. However, no single test is 100% sensitive and specific in all testing situations, and currently a panel including anti-tTG, anti-endomysial and anti-gliadin antibody tests is usually performed [21]. One recent screening study of unselected healthy infants reported that nine of 484 had a positive anti-tissue transglutaminase test at age 2 ½ years. In seven of these children duodenal biopsies confirmed the diagnosis of celiac disease [22].

Classic histologic features of untreated celiac disease include: 1) total villous blunting (flat mucosa) and crypt hyperplasia, with an increased number of mitotic figures in the crypts; 2) a dense inflammatory cell infiltration in the lamina propria, including numerous plasma cells and lymphocytes, admixed with eosinophils and neutrophils, and 3) an increased number of intraepithelial lymphocytes (IELs). If the duodenal mucosa is completely flat the diagnosis of celiac disease is almost assured, although rarely autoimmune enteropathy, viral enteritis, common variable immunodeficiency syndrome and tropical sprue can result in a flat mucosa. Disorganization, flattening, and/or vacuolization of the surface epithelium are also evident in some cases of celiac disease. After withdrawal of gluten from the diet there is slow resolution towards normal villous architecture. The mucosa of the most distal portion of the small bowel recovers most quickly, while the duodenum is the last to normalize. It may take several months or longer of a strict gluten free diet before the biopsy appearance returns to normal.

As more patients with "atypical" symptoms underwent duodenal biopsy to rule out celiac disease it became clear that a range of pathologic abnormalities could expected. Marsh has proposed a classification for the morphologic appearance of duodenal biopsies in celiac disease patients [23, 24], which can be briefly summarized as follows:
  • Type 0 – normal crypt & villous architecture; no increase in IELs

  • Type 1 – normal crypt & villous architecture; > 40 IELs/100 enterocytes

  • Type 2 – crypt hyperplasia but normal villous length; > 40 IELs/100 enterocytes

  • Type 3 – crypt hyperplasia &villous blunting (mild to flat); >40 IEL/100 enterocyte
Type 1 morphology (also known as the "infiltrative lesion"), which represents the earliest recognizable light microscopic change, was first documented in biopsies from first-degree relatives of celiac disease patients and in patients with dermatitis herpetiformis. These patients had no gastrointestinal complaints and were considered to suffer from a form fruste of celiac disease [23]. It has been shown that the infiltrative lesion can be induced in full fledged celiac disease patients who have been on a gluten free diet (with a documented entirely normal duodenal mucosa) by administering a low dose of dietary gluten. Increasing the load of dietary gluten can produce evolution to a flat mucosa. [25, 26]. It has recently been estimated that only 30% of "gluten sensitive" patients exhibit a flat mucosa [24]. The number of duodenal biopsies that are obtained has been shown to influence the likelihood of identifying flat mucosa, indicating that the morphologic changes in celiac disease can be patchy [27, 28]. Since a flat mucosa with increased IELs is much more specific for celiac disease than an increase in IELs alone, the procurement of multiple biopsies (4 to 6) is clearly desirable [29]. It is currently unknown what percentage of patients with the "infiltrative lesion" will go on to develop flat mucosa and the full blown clinical syndrome of celiac disease. It is important to recognize this morphologic expression of gluten sensitivity because the atypical symptoms of these patients will respond to dietary gluten withdrawal. For that reason, and because of the greater risk of lymphoma in untreated patients, a gluten-free diet is recommended for all celiac patients, regardless of the presence or absence of villous blunting.

Complications of Celiac Disease
Some celiac patients who are asymptomatic on a gluten free diet sudden redevelop symptoms of malabsorption. Most of these patients are ultimately discovered to have discontinued the gluten free diet, inadvertently or not. Some patients, however, relapse despite strict adherence to the proper diet, and are said to suffer from refractory sprue.

Collagenous sprue is a very rare condition, with less than 50 cases reported in the literature [30, 31]. As the name suggests it refers to the development of a thickened band of collagen beneath the flat surface mucosa of the small bowel (analogous to collagenous colitis). This is a serious complication because it is often impossible to re-establish normal villous architecture, despite the use of high dose immunosuppression. The patients die either of malnutrition or due to the development of a lymphoma.

The other feared complication of celiac disease is the development of small bowel lymphoma, which is sometimes heralded by the redevelopment of malabsorption. These lymphomas are unusual in that they are almost always of T-cell phenotype [32], while almost all sporadic gastrointestinal lymphomas are of B-cell origin. In many patients gene rearrangement studies are necessary to confirm the diagnosis of a clonal T-cell proliferation, since significant cytological atypia may not be present. The relative risk of small bowel lymphoma in celiac patients has been variously estimated at 40 to 100 fold greater that the normal population [33], but there is some evidence that strict adherence to a gluten free diet may prevent the development of lymphoma [34]. The mucosa in celiac patients with lymphoma often appears atrophic, with both crypt hypoplasia and total villous blunting (Marsh type 4 morphology).

The evolution to lymphoma was initially overlooked in some celiac patients who developed diffuse ulceration of the small bowel mucosa (so-called ulcerative jejuno-ileitis), making it difficult to discern the underlying clonal lymphoid infiltrate, especially in biopsies [35, 36]. There has been some confusion in the literature, however, in that the term ulcerative jejuno-ileitis is also used to describe large areas of small bowel mucosal ulceration in non-celiac patients. In this population there is no association with lymphoma.

A small number of patients also develop lymphocytic or collagenous colitis, which may manifest simultaneously, before or after the diagnosis of sprue [37]. In one study of 21 patients with "refractory sprue" collagenous colitis was responsible for the development of diarrhea in three patients on a strict gluten free diet [38].

Autoimmune Enteropathy
This ill-defined disorder shares many clinical and pathologic features with celiac disease, but there is no relationship with the ingestion of gluten and a gluten free diet has no effect on the severe, protracted watery diarrhea [39]. Symptoms usually begin in the first year of life, but an adult onset form is also described [40]. Serum autoantibodies directed against enterocytes or goblet cells are characteristic of this disorder [41]. However, this assay is not routinely available, it is not well standardized, and the epitopes have not been completely defined. The lack of a clear clinical, morphologic or genetic marker for this disease indicates that there are probably a number of unrelated disorders that share common clinical and pathologic features. These patients often have (or develop) other autoimmune diseases. In most patients both the small and large intestine are severely affected, with biopsies showing dense lamina propria infiltrates and intraepithelial lymphocytosis. Apoptosis of crypt epithelium is usually also a prominent feature. The duodenal mucosa is usually flat; compensatory crypt hyperplasia may or may not be evident.

Crypt and Villous Atrophy Pattern
This pattern is probably the most uncommonly encountered. Crypt atrophy is usually a result of the patient's inability to increase cell proliferation because of severe malnutrition. Infants with cow's milk protein intolerance [42, 43] or microvillous inclusion disease [44, 45] may develop this pattern for this reason (given the remarkably high caloric need at this age). Some chemotherapeutic agents (e.g., vincristine, vinblastine, VP-16) may also produce this pattern due to interference with the process of cell division (mitotic arrest). However, intraepithelial lymphocytosis is not a feature of any of the foregoing conditions. There are rare reports of autoimmune enteritis with both crypt and villous atrophy, but in most cases while the mucosa is flat, there is some degree of crypt hyperplasia (see above).

Variable Villous Flattening with Crypt Hyperplasia Pattern
Many malabsorptive disorders produce mild to moderate villous blunting and crypt hyperplasia but exhibit no other diagnostic feature, which means that the pathologist is not able to make a specific diagnosis. Therefore, correlation with the clinical history is of paramount importance. Of course patients with celiac disease may also exhibit this pattern during the sometimes lengthy process of mucosal healing following institution of a gluten free diet.

The recognition of mild villous blunting can itself be a difficult task, since poor biopsy orientation is common. Some degree of villous shortening is common over Brunner's glands, and is not indicative of a disease state. Therefore, small bowel biopsies performed to evaluate diarrhea and/or malabsorption should always be obtained as distally as possible to avoid this possible confounding factor. Also, shallow biopsies that don't include fibers of the muscularis mucosae can tend to stretch out during processing, producing an artifactual appearance of mild villous blunting. In most cases villous blunting does not occur in isolation, and the absence of an inflammatory component (either an intraepithelial lymphocytosis or significant lamina propria infiltrate including neutrophils) should raise the possibility that the apparent villous shortening is in fact artifactual.

Viral Enteritis
In most cases an etiologic agent is never cultured from the stool and thus only a presumptive diagnosis is possible [46]. Norwalk agent, rotavirus, and other viruses have been implicated in various studies. These viruses do not produce inclusions visible by light microscopy. Villous blunting and an increase in intraepithelial lymphocytes are often evident. In some patients (primarily children, but also adults) the mucosa may become completely flat, and an erroneous diagnosis of celiac disease may be made. Since these infections are self-limited the patient will appear to respond to a gluten free diet, thus "confirming" the misdiagnosis. This highlights the importance of serologic tests for anti-tTG, anti-endomysial and anti-gliadin antibodies in making a diagnosis of celiac disease [46].

Tropical Sprue
In the U.S. cases are primarily seen among patients who have spent prolonged periods of time in the Caribbean islands. This disorder is presumed to be a result of overgrowth of toxic strains of non-anaerobic bacteria and will respond to long term therapy with antibiotics, B12 and folate [47]. The villous flattening is said to be more severe distally in the small bowel, and occasionally completely flat mucosa is present. An intraepithelial lymphocytosis is also evident, which can lead to confusion with celiac disease.

Bacterial Overgrowth
Bacterial overgrowth occurs in the setting of reduced bowel motility, due to a surgical procedure (eg Roux-en-Y limb), fistula, or organic disease (eg diverticulum, scleroderma). Malabsorption is caused by villous blunting (? due to the toxic products of the bacteria themselves) and the bacterial deconjugation of bile salts, interfering with luminal digestion). A mild intraepithelial lymphocytosis may also be present.

"Normal" Mucosal Architecture
On occasion the pathologist is faced with a patient with diarrheal of malabsorptive symptoms but a biopsy that appears normal on cursory examination. In that situation there are several disorders that can be diagnosed by very careful examination, including the infiltrative lesion of some celiac patients, as discussed extensively above. In addition, an intraepithelial lymphocytosis may be noted in small bowel biopsies obtained incidentally or to rule out a non-diarrheal condition [48, 49, 50]. Disorders that manifest an intraepithelial lymphocytosis but normal mucosal architecture are discussed below.

Giardiasis
Most patients with Giardia infection exhibit completely normal villous architecture and no intraepithelial lymphocytosis [51]. Therefore, it is incumbent on the pathologist to carefully examine every "normal" small bowel biopsy from patients with diarrhea for Giardia organisms. In immunocompetent individuals the number of organisms can be quite small. In fact, light microscopic examination of small bowel biopsies is not a sensitive test for the diagnosis of Giardiasis. Although a Giemsa stain is classically recommended to highlight the organism, a trichrome stain actually may be preferable. The organism is slightly larger than an enterocyte nucleus when seen in full section. Making touch preps from the biopsy tissue may increase the diagnostic yield. Far better is the examination of a duodenal aspirate specimen. In patients with a heavy infestation or organisms a mild intraepithelial lymphocytosis may occur.

Common Variable Immunodeficiency Disease
Any biopsy that reveals the presence of Giardia organisms should be examined carefully for the possibility of common variable immunodeficiency disease. In this condition (which actually includes a spectrum of immunologic disorders) there is a failure of maturation of B-lymphocytes to fully developed plasma cells. The underlying defect may actually be in T-cell function or signal transduction [52]. This results in an absence of or severe decrease in immunoglobulin production, (including IgA antibodies, which are an important host defense against intestinal parasites). These patients may present as children or adults with multiple episodes of infection (predominantly respiratory and gastrointestinal). Biopsies usually reveal at least mild villous blunting, but a completely flat mucosa may be present. A mild intraepithelial lymphocytosis is also present in many cases. The combination of flat mucosa and intraepithelial lymphocytosis may lead to a mis-diagnosis of celiac disease.

The correct diagnosis can be made by noticing the absence of mature plasma cells, which are normally numerous in the lamina propria of the small bowel. In addition, prominent nodular lymphoid hyperplasia is often present, particularly in children. The presence of numerous large lymphoid aggregates in these patients can lead to an erroneous diagnosis of lymphoma, although there may also be a true increase in the incidence of G.I. lymphomas in these patients [53].

Crohn's Disease
Biopsies of the duodenum in patients with an established diagnosis of Crohn's disease involving the colon and/or ileum sometimes reveal a mild intraepithelial lymphocytosis with preserved mucosal architecture [48, 49, 50]. This feature alone should not be taken as evidence of clinically significant involvement of the upper G.I. tract by Crohn's disease.

Helicobacter pylori Gastritis
Severe H. pylori gastritis can also cause an increase in IELs in biopsies of the duodenal bulb, but usually not more distally [49]. The degree of intraepithelial lymphocytosis is usually quite mild, or even only borderline abnormal, and resolves upon antibiotic treatment of the H. pylori infection [54, 55]. Interestingly, the gastric biopsies from such patients usually do not exhibit intraepithelial lymphocytosis [48, 50].

Miscellaneous conditions
It is possible that NSAIDs can cause intraepithelial lymphocytosis [48, 49, 50], although a causative link has not yet been proven by demonstration of resolution following discontinuation of the offending agent. Likewise, intraepithelial lymphocytosis has been reported in patients with various autoimmune conditions (rheumatoid arthritis, Hashimoto's thyroiditis, SLE, etc.), but no direct relationship has been established. Rarely a mild intraepithelial lymphocytosis has been reported in patients with microsporidia or cryptosporidium infection. In a significant proportion of patients with duodenal intraepithelial lymphocytosis no associated disease state can be identified [48, 49, 50].

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