Serological and Genetic Testing in Coeliac Disease

Serological testing is often an initial step in the assessment of a patient for possible CD.¹² Recent developments have led to a selection of highly sensitive and specific antibody tests, which are more reliable than the older anti-gliadin antibody tests used previously in the diagnosis of CD.¹² These newer antibodies, which include IgA-tTG antibody, IgA-endomysial antibody (EMA), and IgA/IgG de-amidated gliadin peptide antibody (DPG), have a reported specificity and sensitivity in excess of 90%.¹² Currently, the American College of Gastroenterology clinical guidelines recommend tTG for detecting CD in patients over 2 years of age.¹⁵

In patients with an IgA deficiency, IgG-DPG testing is appropriate.¹⁵ IgA-EMA testing remains the most sensitive serological test but requires immunofluorescence. Therefore, it is subject to interobserver variation.^12,16

More than 98% of CD patients express the HLA-DQ2 or HLA-DQ8.¹⁷ Therefore, HLA typing can be of help to exclude the diagnosis, particularly in those patients who are already on a GFD or have had equivocal serological/histological results.^11,17 However, it cannot confirm the diagnosis.

Histopathology

Despite the advances in serological testing, the duodenal biopsy remains essential for the diagnosis of CD.^10,11 It is worthwhile considering several points before undertaking a thorough morphological assessment of the duodenal biopsy.

Ideally, the patient is on a gluten-containing diet at the time of biopsy. Unfortunately, patients commonly present after a variable amount of time on a GFD.¹⁰ In this circumstance, there may be a degree of mucosal healing, and the histology report should consider this possibility. Mucosal injury in CD tends to be more severe in the proximal small intestine and may be patchy. Therefore, guidelines usually recommend multiple biopsies (four to six).^11,18

While biopsies from the duodenal bulb may show features related to peptic injury, and while prominent Brunner’s glands can affect the interpretation of the villous architecture, biopsies from this site are nevertheless important because the duodenal bulb may be particularly sensitive to gluten-induced injury.¹⁸ Good orientation of the biopsies is crucial in permitting accurate assessment of the villous architecture. As a minimum, the pathologist should look for at least three or four adjacent, well-visualised crypt-villous units.¹⁸

The main histological features of CD are villous atrophy, crypt hyperplasia, increased chronic inflammatory cells IELs and mononuclear cells in the lamina propria) and morphological changes in the enterocytes (Fact Sheet 15.1).¹⁰

Increased IELs are a non-specific finding but may be the only histological manifestation of CD^19,20 (Figure 15.1). In recent years, several groups have addressed the question of exactly what constitutes raised IELs, partly because of the replacement of capsule biopsies of the jejunum with the duodenal biopsies that are now standard in CD diagnosis.^20–22 These studies suggested a cut-off of 20–25 lymphocytes per 100 enterocytes as the upper limit for normal duodenal mucosa.^20–22 Immunohistochemistry for CD3 may be helpful in giving a more accurate IEL count^20,22 (Figure 15.2). However, this is a subject of controversy and indeed some experts advise strongly against the use of CD3 staining. CD3 immunostaining is not necessary routinely but may be useful when the IEL count is close to the cut-off for abnormality. IEL counts between 25 and 29 on CD3-immunostained sections are usually regarded as ‘borderline’, with counts above 30 considered as ‘pathological intraepithelial lymphocytosis’.^20,22

Figure 15.1 Intraepithelial lymphocytosis with normal villous architecture in a duodenal biopsy. Note that the intraepithelial lymphocytes (IELs) are present along the entire villus and over its tip. The normal villous IEL decrescendo pattern is lost. Marsh-Oberhuber type 1 change.

Figure 15.2 Intraepithelial lymphocytes (IELs) are positive for CD3. These cells are easier to identify with CD3 immunostaining than with H&E staining. CD3 immunostaining can help to give a more accurate IEL count in difficult or borderline cases.

Well-orientated villi are optimal for the IEL count, which should include at least 300 enterocytes. The final IEL count should be a mean number per 100 enterocytes.^20,22 The count should avoid the villi close to lymphocytic aggregates, as IEL counts are always higher in these areas. Only those lymphocytes residing above the basement membrane should be included in the IEL count.²²

In addition to the IEL count, the distribution of the IELs is worth examining when considering a diagnosis of CD.²³ In normal small intestinal mucosal biopsies obtained from the distal duodenum (Figure 15.3), the density of IELs decreases from the middle to the distal (upper) third of the villi, resulting in a ‘decrescendo pattern’.²³ In a study of 78 patients, Goldstein et al. found that a loss of this ‘decrescendo pattern’, leading to an even distribution of IELs along the sides and tips of the villi, was more common in patients with gluten sensitivity (Figure 15.4).²³

Figure 15.3 Normal duodenal mucosa. Occasional intraepithelial lymphocytes (IELs) are present and their density is greatest along the lower and middle thirds and sharply decreases along the upper third of the villus.

Figure 15.4 Immunohistochemistry for CD3 highlights increased intraepithelial lymphocytes along the sides and tips of the villi and loss of the normal ‘decrescendo’ pattern.

The majority of IELs in CD are positive for CD3 and most IELs express CD8 (70%), with a smaller number (5%–10%) expressing CD4, in keeping with the helper/inducer phenotype.¹ A fifth of IELs are negative for both CD4 and CD8.¹ The relative proportions are probably similar in CD and in normal mucosa.

Together with IELs, villous atrophy and crypt hyperplasia are the key parameters in the classification schemes (see later) that some pathologists use to stage CD (Figure 15.5).^24–26 Villous atrophy / crypt hyperplasia is detectable in well-orientated sections, when there is a reduction in the normal villous height to crypt depth ratio of between 5:1 and 3:1.¹ The presence of increased mitotic figures (normally seen only in the lower third of the crypt) is another feature of CD, and may indicate the presence of crypt hyperplasia, even in suboptimally orientated biopsies.^1,19

Figure 15.5 Together with increased intraepithelial lymphocytes, villous atrophy and crypt hyperplasia are the key parameters used to assess the duodenal damage caused by gluten sensitivity. Biopsy from a non-responsive coeliac disease patient showing subtotal villous atrophy with intraepithelial lymphocytosis and a chronic inflammatory infiltrate in the lamina propria (Marsh-Oberhuber type 3b change).

The lamina propria may be expanded by a mixed inflammatory cell infiltrate.¹⁹ Plasma cells and T lymphocytes are the predominant cell types, but smaller numbers of neutrophils, eosinophils and mast cells can also be present.¹⁹ In a study of 150 newly diagnosed CD patients, Brown et al. observed at least focal neutrophilic infiltration of the lamina propria in more than 56% of the cohort.² Furthermore, eosinophils, with a density of 20 per high-power field or more, were present in almost a quarter of cases.² Raised eosinophils and an increased neutrophil count were both associated with a more advanced stage of disease.²

Several morphological alterations in the surface enterocytes occur in CD.^1,2,19 These changes, which tend to be more prominent when associated with villous atrophy, include cuboidalisation and a loss of enterocyte height (resulting in an increased nuclear/cytoplasmic ratio), cytoplasmic basophilia, and cytoplasmic vacuolation (Figure 15.6).^1,2,19 Another mucosal change that can occur is mild thickening of the basement membrane. Up to 45% of CD biopsies may have at least focal thickening (i.e.>1.5 µm) of the subepithelial collagen band (see later).²

Figure 15.6 Duodenal biopsy from a patient with untreated coeliac disease showing surface epithelial cytoplasmic vacuolation.

In practice, identification of otherwise unexplained intraepithelial lymphocytosis, even if villous architecture is normal or minimally disturbed, raises the possibility of CD. A count of more than 30 per 100 epithelial cells can be regarded as abnormal, and a count of 25 to 29 as borderline and probably abnormal. The pathology report should advise the clinical team to exclude CD by reviewing the clinical findings and, in particular, by performing serological tests. The report should also state that there are other causes of intraepithelial lymphocytosis, particularly drugs and infection (Fact Sheet 15.2). Identification of unexplained villous atrophy without intraepithelial lymphocytosis may also be difficult to interpret. The pathologist should initially consider the possibility of artefactual changes, e.g. biopsy malorientation, trauma, etc., and of proximal (D1) origin, as they can mimic atrophy. In addition, there are many causes other than CD of genuine villous atrophy, including infection, drugs, and non-specific (peptic) duodenitis. As is the case for intraepithelial lymphocytosis, clinical and serological exclusion of CD may be appropriate if there is villous atrophy, while also bearing in mind the differential diagnosis. If both intraepithelial lymphocytosis and villous atrophy are present and are otherwise unexplained, the recommendation to exclude CD should be stronger because there are few other causes of this histological picture.

Collagenous Sprue

Collagenous sprue (CS) is a malabsorptive disorder that commonly presents with diarrhoea, abdominal pain, weight loss, and/or anaemia.²⁷ CS mainly involves the proximal small intestine, although it can also affect the jejunum.^27,28 The disease may be diffuse or patchy, and show variation in severity of the mucosal lesions.^27,28

The histological changes seen in CS include subtotal and total villous atrophy, increased IELs, cytoplasmic vacuolation, detachment of the epithelium, lamina propria inflammation, and neutrophilic infiltration.²⁹ As the name suggests, the hallmark of CS is thickening of the subepithelial collagen plate (Figure 15.7), associated with entrapped capillaries and other cellular elements of the lamina propria.^27,29 There is no precise definition of what constitutes a thickened subepithelial collagen plate. Based on the observation that 60% of biopsies from CD patients showed minimal subepithelial fibrosis, with an average thickness of 3 µm, Vakiani et al. used a cut-off of>5 µm to identify CS.²⁹

Figure 15.7 Duodenal biopsy showing subepithelial collagen formation (collagenous sprue) from a patient with known coeliac disease. There is subtotal villous atrophy, mild intraepithelial lymphocytosis, and focal gastric metaplasia.

The relationship between CD and CS is also unclear. Most commentators acknowledge a possible association between the two conditions, based in part on the clinical, serological, and pathological overlap, and some authors suggest that CS can evolve from pre-existing CD.^27,29 Others claim that CS is a distinct entity and that positive CD serology does not occur in ‘true’ CS.³⁰ A further complication is the finding that biopsies from CD patients who do not respond to a GFD (refractory CD, see later) may also show a thickened collagen band.³¹

In one report, collagenous enteritis (affecting the duodenum or the ileum or occasionally both) was associated with advanced patient age at presentation, an absence of coeliac antibodies, and possibly with several drugs including olmesartan, non-steroidal anti-inflammatory drugs, proton pump inhibitors, and statins. Despite similar HLA profiles, the authors concluded that collagenous enteritis is not a form of CD.³²

From a practical point of view, any significant thickening of the collagen plate (particularly if>5 µm) should be noted in the text of the pathology report and reference made to the possibility of CS. These patients may require close follow-up, as they may be less responsive than those with CD to a GFD, necessitating alternative treatment such as steroids.^30,33

Involvement of Other Parts of the Gastrointestinal Tract by Coeliac Disease

CD may also be associated with gastrointestinal (GI) inflammation outside the small intestine.^2,34,35 Lynch et al. found that 4 of 18 patients with current lymphocytic gastritis (LG, defined as an IEL count more than 25/100 gastric epithelial cells,³⁵ had duodenal or jejunal villous atrophy and were seropositive for IgA EMA.³⁴ Moreover, two patients started a GFD and showed clinical improvement; the one patient who underwent repeat endoscopy showed a non-significant reduction in the number of antral/body mucosal IELs.³⁴

In a study of 70 CD patients, Feeley et al. found that seven (10%) also had LG, and that the risk of LG was unrelated to the presence or absence of Helicobacter pylori infection.³⁵ A more recent study identified LG in more than 30% of CD patients and found that its presence was associated with the intensity of inflammation in the concurrent duodenal material.² Interestingly, these workers also noted lymphocytic colitis (Figure 15.8) in 31% and lymphocytic ileitis in 17% of CD patients, although only a limited number of biopsies were available from these sites.²

Figure 15.8 Colonic biopsy showing microscopic lymphocytic colitis: diffuse increase in inflammatory cells in the lamina propria with intraepithelial lymphocytosis (left, H&E staining). The IELs are positive for CD3 (right).

The association between CD and LG has prompted some to speculate that, when superimposed on a background of CD, LG may be a reflection of a diffuse enteropathy that primarily involves the small intestine rather than a distinct entity.^34,35

Refractory Coeliac Disease

RCD is diagnosed when a CD patient continues to experience symptoms despite adhering to a strict GFD for more than 12 months, and after excluding other causes of villous atrophy.⁴² The reported prevalence of RCD is variable and may be as high as 9%.³⁹ A more recent Dutch study found a cumulative incidence of 0.04 over a 6-year period.⁴²

RCD has two subtypes: type 1 RCD (RCD1) shows an essentially normal IEL population, whereas type 2 RCD (RCD2) is characterised by an aberrant, clonal IEL phenotype.⁴³

RCD1

Symptoms of RCD1 include fatigue, weight loss, diarrhoea (sometimes alternating with constipation), steatorrhoea, nausea, and abdominal pain.³¹ Patients may also develop other autoimmune conditions, and infection and thrombo-embolic events may complicate the picture.⁴⁴

Although the morphological changes that occur in RCD may not necessarily be more severe than those in CD, biopsies from most RCD1 patients show at least partial villous atrophy, in keeping with Marsh–Oberhuber type 3 lesions.^31,43 The IELs in RCD1 have a normal cytological appearance, and the lamina propria shows a moderately dense infiltrate of lymphocytes and plasma cells.⁴⁵ In a study of 10 RCD1 patients, Olaussen et al. also identified subepithelial collagenous bands in four patients, similar to that observed in CS.³¹ Another study of RCD patients (no distinction was made between types 1 and 2) found that relative thinning of the mucosa and subcryptal chronic inflammation (inflammatory infiltrates between the crypt base and muscularis mucosae) was more common in the RCD group than in controls.⁴⁶ Other, non-specific findings in RCD patients were concomitant collagenous/lymphocytic colitis, LG, acute inflammation, and gastric metaplasia.⁴⁶

The immunophenotype of IELs in RCD1 is similar to that seen in uncomplicated CD, with surface expression of CD3, CD8, and TCRβ (Figure 15.12).^31,43 Some authors assert that the proportion of CD8/TCRβ-positive IELs should be greater than 50% in RCD1, and that a lower percentage raises the possibility of RCD2.⁴³ In TCR (T-cell receptor) gene re-arrangement studies, most cases of RCD1 show polyclonal IELs, although clonal expansions are detectable in a minority.^31,47

Figure 15.12

(A) Refractory coeliac disease type 1 (RCD1) immunophenotype. Intraepithelial lymphocyte (IEL) phenotyping on duodenal biopsies. The IELs are CD3 positive

(B) CD8 positive.

RCD2

The majority of RCD2 patients present with diarrhoea, abdominal pain, weight loss, and malabsorption.⁴³ Patients may also develop skin lesions reminiscent of pyoderma gangrenosum, or ulceration of the limbs and face; there may be a history of long-standing chest/sinusoidal infections or an unexplained fever.⁴³ As with RCD1, most small intestinal biopsies show Marsh–Oberhuber type 3 lesions, with a variable degree of villous atrophy.⁴⁷

The key feature that differentiates RCD2 from RDC1 is the emergence of a dominant population of morphologically normal but immunophenotypically aberrant IELs that demonstrate clonal rearrangements of the TCR gene⁴⁸ (Figure 15.13). The abnormal IELs in RCD2 show cytoplasmic expression of CD3 (Figure 15.14), but have lost expression of surface CD3, CD4, CD8 (Figure 15.14), and TCR.⁴⁸ The identification of aberrant IELs in the stomach and large intestine of RCD2 patients suggests that, like CD, RCD2 may diffusely involve the GI tract.⁴⁹

Figure 15.13 Radiolabelled polymerase chain reaction (PCR)-single strand conformational polymorphism (SSCP) analysis of TCR Vc gene rearrangements. DNA was isolated from formalin-fixed duodenal biopsy specimens from two patients (labelled 1 and 2) with refractory coeliac disease type 2 (RCD2) and peripheral blood lymphocyte DNA from a healthy control (labelled 3). Each PCR amplicon is run on the SSCP gel in a native (N) and denatured form (D). Dominant clonal populations (discrete bands) are demonstrated in both RCD2 samples (1 and 2), whereas there is a polyclonal pattern (smear) in the healthy control DNA (3).

(A) CD8 negative

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Chapter 4 – Transplantation, Immunodeficiency, and Immunosuppression Chapter 5 – Drug-Induced Gastrointestinal Disease Chapter 14 – Duodenitis Chapter 25 – Ileal Pouch Anal Anastomosis Chapter 20 – Microscopic Colitis Chapter 21 – Inflammatory Bowel Disease Diagnosis

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