Digestive and Liver Disease 43S (2011) S385–S395

Coeliac disease: The histology report

Vincenzo Villanacci a,*, Paola Ceppa b , Enrico Tavani c , Carla Vindigni d, Umberto Volta e
On behalf of the “Gruppo Italiano Patologi Apparato Digerente (GIPAD)” and of the “Società Italiana
di Anatomia Patologica e Citopatologia Diagnostica”/International Academy of Pathology,
Italian division (SIAPEC/IAP)
a Department of Pathology, Spedali Civili, Brescia, Italy
b Surgical Department, Integrated Morphological and Methods Section of Pathological Anatomy, University of Genova, Genova, Italy
c Department of Pathology, G. Salvini Hospital Rho, Rho, Italy
d Department of Pathology and Human Oncology, University of Siena, Siena, Italy
e Department of Diseases of the Digestive System and Internal Medicine, Policlinico S. Orsola – Malpighi, Bologna, Bologna, Italy

Abstract

To this day intestinal biopsy is justly considered the “gold standard” for the diagnosis of coeliac disease (CD). The aim of the authors
in setting up these guidelines was to assist pathologists in formulating a more precise morphological evaluation of a duodenal biopsy in the
light of clinical and laboratory data, to prepare histological samples with correctly oriented biopsies and in the differential diagnosis with other
pathological entities and complications of the disease. A further intention was to promote the conviction for the need of a close collaborative
relationship between different specialists namely the concept of a “multidisciplinary team”.
© 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.

Keywords: Coeliac disease; GIPAD report; T lymphocytes; Malabsorption

1. Introduction based on the most recent acquisitions regarding the diagnosis

and pathogenesis of coeliac disease, is the only specialist that
These guidelines are intended as an aid for pathologists, can make the final diagnosis of coeliac disease. A number
in order to allow a more precise morphological evaluation of specific points in the diagnostic process will thus be dealt
of duodenal biopsies in the light of clinical and laboratory with, considering issues and concerns of differential diagnosis
data. The aim is to arrive at the conviction of the need for with other similar diseases before the final diagnosis can be
a close collaborative relationship between different specialists reached.
such as adult or pediatric gastroenterologists, endoscopists, This document is an update of the “guidelines” published
laboratory staff, endoscopy room nurses, pathology laboratory by the Italian Group of Digestive Disorders (GIPAD) in 1998
technicians and pathologists. This implies the creation of [1].
a “multidisciplinary team” led by a gastroenterologist who, The document, after a brief historical and epidemiological
address, considers the following points:
• Clinical and laboratory aspects
List of abbreviations: CD, coeliac disease; tTGA, antitransglutaminase anti- • The methodological approach to duodenal biopsies
bodies; EMA, antiendomysial antibodies; AGA, antigliadin antibodies; IEL, • Aspects of normal and pathological duodenal mucosa
intra epithelial lymphocytes.
• Diagnosis
* Correspondence to: Vincenzo Villanacci, MD, Department of Pathology,
Spedali Civili, Piazzale Spedali Civili 1, 25100 Brescia Italy. • Differential diagnosis
E-mail address: villanac@alice.it (V. Villanacci). • Complications that can be confirmed histologically.

1590-8658/$ – see front matter © 2011 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.
S386 V. Villanacci et al. / Digestive and Liver Disease 43S (2011) S385–S395

2. Brief history risk groups in which, on the basis of intestinal and extrain-
testinal symptoms, and of the presence of any associated
The first descriptions of coeliac disease can be found in the diseases and familiarity, the possibility of coeliac disease
first century A.D. when the physician Celsus introduced the must be investigated [4,5].
Latin term “coeliac” to indicate a diarrhea-like disease. Later, These risk groups are made up of:
in 250 A.D., Areteo Cappadocia described the clinical signs 1. Subjects in whom coeliac disease is strongly suspected
of a prolonged intestinal disease that was very difficult to (cases with severe malabsorption and with highly predic-
treat, using the Greek word koiliakos to identify “those who tive associated diseases):
suffer in their intestines”. In 1856, Francis Adams translated • malabsorption syndrome with repeated diarrhea-like
this Greek word into English, coining the term “coeliac”. A bowel movements, abdominal pain and marked weight
few years later, in 1888, Samuel Gee described the detailed loss;
symptoms of this condition both in adults and in children, pre- • dermatitis herpetiformis, also called coeliac disease of
dicting that the only treatment consisted of an appropriate diet, the skin, since in practically all cases there is more or
with few items derived from flour. Only halfway through the less severe gluten-dependent intestinal damage.
twentieth century, however, did it become clear that coeliac 2. Subjects in whom coeliac disease is moderately suspected
disease occurs in some individuals following the ingestion (cases with atypical or extraintestinal symptoms and asso-
of wheat proteins, which damage the intestinal mucosa. The ciated diseases):
systematic description of the histopathological alterations of • atypical gastro-intestinal symptoms (dyspepsia, consti-
coeliac disease (CD) is mainly due to the work of Marsh pation, vomiting and intestinal subocclusion);
[2,3]. Today we know that CD is a chronic, immune-mediated • extraintestinal symptoms (anemia – most often due to a
disease occurring in genetically predisposed individuals due lack of iron but also to a lack of folic acid and vitamin
to an intolerance to gluten-containing foods and, in particular, B12), hyposomia, oral ulcers, hypertransaminasemia,
to some of its proteins, called gliadins. This intolerance leads osteopenia or osteoporosis, tooth enamel abnormalities,
to abnormal immune response, which is followed by a chronic hemorrhagic syndrome due to vitamin K malabsorption,
inflammation of the small intestinal mucosa with progressive changes in the female reproductive system (late menar-
disappearance of intestinal villi. che, early menopause, recurrent miscarriage, premature
labour);
• associated diseases (diabetes mellitus type 1, Hashimoto
3. Epidemiology thyroiditis, Graves’ disease, selective IgA deficiency,
alopecia areata, piebald skin, psoriasis, Addison’s dis-
The disease has a variable incidence, which in Europe ease, Systemic Lupus Erythematosus, polymyositis,
is estimated between 0.3 and 1.2%, similar percentages are rheumatoid arthritis, cerebellar ataxia, epilepsy with
reported in North America and Australia. Recently, a high or without cerebral calcifications, peripheral neuropa-
prevalence has been reported in people of Northern Africa thy, autoimmune hepatitis, primary biliary cirrhosis,
(5–6% in populations of Western Sahara). In Italy the most idiopathic dilated cardiomyopathy, Berger’s disease,
recent statistics estimate a prevalence of 1/100, and each year Down’s syndrome, Turner’s syndrome, Williams’ syn-
about 5000 new cases are diagnosed. CD, once considered a drome).
disease of childhood, can affect individuals of all ages, with a 3. 1st degree relatives of coeliac patients (high familiarity
preference for females (male/female ratio 1 :2). of coeliac disease that is present in 4–17% of 1st degree
relatives of coeliac patients, but may also be found in high
proportions in 2nd degree relatives).
4. Clinical and laboratory aspects A major role in the diagnostic process of coeliac disease
is played by serology, which allows identification within the
4.1. Clinical aspects of CD at-risk groups of the subjects who should undergo intestinal
biopsy. While making it clear that no positive antibody test
The variety of clinical manifestations which coeliac disease allows a diagnosis of coeliac disease without the necessary
may present complicates its recognition. A correct diagnosis confirmation provided by an intestinal biopsy, some of the
can not rely on a single test, but requires a precise recon- antibody markers show such a high diagnostic accuracy (with
struction of a puzzle, whose pieces are represented by the levels of sensitivity and specificity >95%) that they are highly
clinical, serological, genetic and histological aspects. The predictive of coeliac disease.
evaluation of all these factors, apart from genetics, must take
place while the patient is still on a diet containing gluten, 4.2. Antibody markers
since a gluten-free diet changes the clinical, serological and
histological pattern, making it impossible to recognize the • IgA class antitransglutaminase antibodies (tTGA) are the
characteristic aspects of disease. tests with the highest sensitivity for coeliac disease (98%)
The significant improvement in our knowledge of intoler- with specificity estimated at around 90%. High titres of
ance to gluten has made it possible to identify the so-called IgA class tTGA (>5 times the cut-off) are almost always
 V. Villanacci et al. / Digestive and Liver Disease 43S (2011) S385–S395 S387

the expression of coeliac disease, while false positives find that they are serologically negative for coeliac disease.
(about 10%) almost always present medium-low titres (<2 • According to NIH guidelines [7], a duodenal biopsy is not
times the cut-off) essential in patients with dermatitis herpetiformis if the
• IgA class antiendomysial antibodies (EMA), while having diagnosis is supported by immunofluorescence detection
a lower sensitivity compared to IgA class tTGA (90% of granular deposits of IgA in the dermis. In these cases,
vs. 98%), show an almost absolute specificity for coeliac in fact, the gluten-dependent intestinal damage is always
disease. Antibody titres from >1 :40 correlate with a present in a more or less severe form and gluten-free diet
greater severity of intestinal lesions, and low titre positives will lead to resolution of the skin lesions.
(1 :5) are often an expression of infiltrative lesions of the When the results of the intestinal biopsy and serological
intestinal mucosa (type 1), suggestive of coeliac disease. tests are consistent, the clinician is able to make the diagnosis
• IgA class antigliadin antibodies (AGA) are now an obsolete of coeliac disease. The diagnosis is confirmed with the
test with levels of sensitivity and specificity significantly resolution of the clinical symptoms and negative serology
lower than tTGA and EMA, and the search for their tests after a reasonable period of strict gluten-free diet
presence is useful only in early childhood (children aged (usually 12 months). Therefore, provided that the clinical
<2 years); because they are the first antibodies to appear, situation has improved and serological tests have become
they show a higher sensitivity than other tests in this age negative as a result of following this diet, an intestinal biopsy
group. Positivity for IgA AGA associated with negativity after gluten withdrawal is no longer considered essential for
for EMA and tTGA is almost never an expression of the definitive diagnosis of coeliac disease, not only in children
coeliac disease in adults and in children aged >2 years. [6], but also in adults [7].
• With regard to the IgG class of antibodies, their use should
be restricted to patients with selective IgA deficiency, 4.5. Genetic testing
because only in this subgroup of patients is the response
indicative of coeliac disease. The recommended test is the Coeliac disease is closely associated with histocompatibil-
detection of tTGA (+ AGA in children aged <2years). The ity antigens (HLA) DQ2 and DQ8. Practically all patients
negativity of IgA tTGA with very low values (<0.1 AU) with coeliac disease are positive for one or both of these
always suggests the presence of a selective IgA deficiency HLAs or for a fraction of the heterodimer, but genetic testing
and indicates a search for IgG class tTGA. is never diagnostically significant since at least 30% of the
general population present the same HLAs as coeliac patients.
4.3. Indications for biopsy in suspected coeliac disease 1. When the genetic test should be performed:
• In cases where there is a discrepancy between serology
Intestinal biopsies taken from the first and second duodenal and histology.
portion remain an essential means of confirming the diagnosis • In 1st degree relatives to assess the genetic predisposi-
of coeliac disease. To retain its diagnostic validity, it is tion to coeliac disease.
fundamental for the patient to be on a normal diet containing 2. Significance of genetic testing:
gluten at the time of the biopsy (often due to incorrect • The main clinical significance of genetic testing is to
information, patients may have already been on a gluten-free exclude a diagnosis of coeliac disease in the absence
diet for some time when they undergo the biopsy). of HLA-DQ2 (and its fractions) and -DQ8 in cases of
diagnostic doubt.
4.4. Who should undergo an intestinal biopsy? • Exclusion of predisposition to coeliac disease in family
members of coeliac patients in the absence of HLA-
• Subjects with positive serology characterized by the pres- DQ2 (and fractions) and -DQ8.
ence of IgA class antitransglutaminase and antiendomysial
antibodies, and children younger than 2 years with isolated 4.6. Clinical notes
IgA AGA positivity. In some cases, the detection of very
low antibody titres, particularly for IgA tTGA (test with A close collaboration between pathologist and clinician
10% false positives), in the absence of EMA, suggests is essential in order to address the concerns relating to
monitoring the patient for some time and re-testing before the diagnosis of coeliac disease especially in cases that are
proceeding with an endoscopy investigation. difficult to evaluate.
• Subjects with deficiency of IgA positive for IgG tTGA (and The information that the clinician should provide the
even children aged <2 years with positivity for AGA IgG pathologist may be summarized as follows:
with or without IgG tTGA) should also undergo intestinal • Details of the patient’s diet (normal or gluten-free, in the
biopsy. second case specifying how long the patient has been on a
• Subjects in whom coeliac disease is strongly suspected, gluten-free diet).
in whom a severe malabsorption syndrome is present, • Level of clinical suspicion: high or moderate based on the
irrespective of antibody test results (in practice it should be symptoms.
performed even if all the antibodies are negative), precisely • Whether the patient has a family history of coeliac disease
because in highly symptomatic subjects it is possible to (defining the degree of relationship).
S388 V. Villanacci et al. / Digestive and Liver Disease 43S (2011) S385–S395

• Serology with absolute (EMA), high (tTGA) or low

(AGA) predictability. Always specify the antibody class,
the presence of selective IgA deficiency, and the antibody
titre if available (with its cut-off).
• Genetic test results (if performed in accordance with the
indications), especially with regard to DQA1 05, DQB1 02
and DQB1 0302.

5. Approach to duodenal biopsy: the method

Fig. 1. Cellulose acetate filter with three easily-detachable cut filters.
The biopsies that the pathologist receives nowadays are
all performed by endoscopic examination, which, in addition
to the duodenum, makes it possible to explore other districts When properly carried out, this method is of great benefit
of the gastro-intestinal tract. Biopsies performed through the to the pathologist, but also to the technician who during the
use of the Crosby-Watson capsule by perioral route are now embedding phase does not have to search for the individual
considered outdated and are no longer performed. biopsies, which are sometimes fragmented and have no
Here are some points which require a close working guiding landmark.
relationship between the endoscopist, the endoscopy-room The use of cellulose acetate filters allows perfect adhesion
nurse, the pathology laboratory technician and the pathologist. of the biopsies, avoiding their dispersion in the fixation
medium. These filters also do not react chemically with
5.1. Site of the biopsy the fixatives and reagents used during the processing of the
sample; during the cutting phase they do not offer resistance
Biopsy by endoscopy is always performed in the second to the blade and, unlike tissue paper, they do not fray.
and third duodenal portion, as the bulb and the proximal This method, which can be applied on all segments of
duodenum can be a source of incorrect assessments; we rec- the gastro-intestinal tract, has led to considerable diagnostic
ommend at least 4 biopsies, 2 for each of the areas mentioned and economic benefits by reducing the time, the number of
above. Performing a biopsy only in the duodenal bulb may be embeddings, and consequently the number of sections to be
a source of error, or may at least greatly reduce the sensitivity cut and colored.
of the examination, and hence is strongly discouraged [8]. For its obvious advantages, the use of the kit is strongly
recommended.
5.2. Orientation of the biopsy sample
5.3. Stains
This is essential for proper histological assessment.
It is sufficient to stain with Haematoxilin & Eosin,
Positioning of biopsies on cellulose acetate filters is possibly associated with an Alcian Blue-PAS, to assess all the
advisable, with benefits for: necessary morphological elements (one or two sections can be
1. the laboratory staff, since with a simple 90° rotation it is used for immunohistochemical assessment if necessary).
possible to embed the combined biopsy-filter;
2. for subsequent histological evaluation by the pathologist.
The method based on experience acquired at St Mark’s 6. Histopathological aspects of normal and pathological
Hospital in London ensures histological samples in which duodenal mucosa
it is possible to analyze the mucosa and, if necessary, the
submucosa of the removed tissue, thus respecting the normal 6.1. Normal intestinal mucosa
anatomical relationship between the different layers of the
intestinal wall. In particular, after initial experience with Villi: Digitiform appearance with the ratio between the
filters that needed to be cut with obvious waste of time and height of the villi and of the crypts always in favor of the
human resources, a comprehensive kit has been developed, on villus (3 : 1 or more).
which three easily-detachable cut filters with a bevelled end Enterocytes: Normal height with 29–34 μm clear brush-
shaped like a clarinet mouthpiece are already fixed (Fig. 1). border.
After the fixing stage, the filter-biopsy combination is Intra-epithelial lymphocytic infiltrate: The number of intra-
processed and then embedded. During this last phase the epithelial lymphocytes (T lymphocytes) is subject to individ-
technician rotates the filter-biopsy combination 90 degrees in ual variability. The majority of normal subjects have less than
order to place the samples in their natural position. 20 lymphocytes per 100 epithelial cells; based on the experi-
After cutting, the biopsies are placed on a slide and, if ences of Hayat [9] and Veress [10], a count of IEL between
necessary, the position of the bevelled end is marked on the 25 and 29/100 epithelial cells is considered borderline and
label, to indicate the first biopsy. pathological over 30/100 epithelial cells.
 V. Villanacci et al. / Digestive and Liver Disease 43S (2011) S385–S395 S389

Fig. 2. Normal duodenal mucosa: villus/crypt ratio 3 : 1. Intraepithelial lymphocytes within the normal range.

The intra-epithelial lymphocyte count is very important mitosis per crypt. Alongside the epithelial cells are endocrine
and should always be done, especially in the initial lesions, cells, goblet cells and Paneth cells, but these have no value as
following the indications given below: regards the diagnosis of coeliac disease.
• always count the T lymphocytes with the help of immuno- Lamina propria: Plasma cells, eosinophils, histiocytes,
histochemical investigations using anti-CD3 antibodies; mast cells and lymphocytes are normally found in the lamina
• evaluate the biopsies perfectly oriented with a precise propria. Neutrophils are generally absent, except in cases
alignment of the surface-coating epithelial cells; of active duodenitis with possible gastric metaplasia closely
• do the count both in the apical portions and along related to Helicobacter pylori infection.
the edges of the villi; it is important to have accurate The cellular component mainly consists of plasma cells and
and reproducible fields. Counts done only on the apical lymphocytes, the latter sometimes in the form of lymphoid
portions have proved unreliable (Fig. 2A–D). aggregates and eosinophilic granulocytes whose value must
Glandular crypts: The crypts basically have the task of never be greater than 60 for 10 fields of vision examined at
performing a regenerative function, which means it is possible 40×.
to find evidence of mitosis; the normal range is usually one
S390 V. Villanacci et al. / Digestive and Liver Disease 43S (2011) S385–S395

7. Pathological intestinal mucosa Marsh classification

Type 1 or infiltrative lesion
7.1. Basic lesions
1. Villi architecturally within normal morphological limits
(normal villa/crypt ratio 3 :1);
The histological diagnosis of CD consists of an integrated
2. Increased number of intraepithelial lymphocytes (greater
assessment of the following elementary lesions:
than 25–30 per 100 epithelial cells) (Fig. 3A–D).
• Increased intraepithelial T lymphocytes: a value between
25 and 29 IEL/100 enterocytes is considered border-line Type 2 or hyperplastic lesion
value; >30 IEL/100 enterocytes represents a pathological 1. Villi architecturally within normal morphological limits
“lymphocytosis”. (like type 1);
• Decreased enterocyte height, flattening of enterocytes, in- 2. Increased number of intraepithelial lymphocytes (greater
tracytoplasmic vacuolation as well as reduction or absence than 25–30 per 100 epithelial cells) (like type 1);
of brush-border are possible but not specific. 3. Hyperplasia of the glandular elements (regenerative aspect
• Crypt hyperplasia: extension of the regenerative epithelial of the glandular elements highlighted by the reduced
crypts associated with changes in the presence of more muciferous activity and increased number of mitoses).
than 1 mitosis per crypt.
Type 3 or destructive lesion
• Villous atrophy: decrease in villous height, alteration of
1. Varying degrees of villous atrophy associated with hyper-
normal crypt/villous ratio (3 :1) until total disappearance
plasia of glandular crypts;
of villi. This assessment requires proper orientation of the
2. Reduced surface enterocyte height, with irregular brush-
biopsies.
border and sometimes cytoplasmic vacuoles;
None of these elementary lesions of CD is exclusive; the
3. Increased number of intraepithelial lymphocytes (like type
diagnosis of CD is based on the identification of histological
1 and 2 lesions).
lesions accompanied by clinical and serological consistent
data. On the basis of the presence of one or more of these The combination of the three factors described above is
elementary lesions the histopathology of CD is subdivided consistent with a diagnosis of coeliac disease or gluten-
into different diagnostic categories according to the Marsh sensitive enteropathy, which should be considered as syn-
classification [2]. onyms. These three patterns, albeit schematic, represent the
histological lesions seen in coeliac disease and it is important

Fig. 3. Type 1/2 infiltrative lesion according to Marsh-Oberhuber. Grade A new classification.
 V. Villanacci et al. / Digestive and Liver Disease 43S (2011) S385–S395 S391

Fig. 4. From Marsh [2], amended.

to consider them as dynamic and progressive both in one di- An amendment to this classification has been proposed by
rection and the other and not static, since they depend on how Oberhuber et al. [11], who divided the Marsh type lesion 3
much gluten the patient has been exposed to and for how long. into three subgroups.
Figure 4 summarizes the above description. 3a mild villous atrophy and pathological increase of intraep-
This classification is universally recognized for the diag- ithelial lymphocytes.
nosis of coeliac disease, and extensively validated; the only 3b moderate villous atrophy and pathological increase of
point worthy of observation and critical analysis is that the intraepithelial lymphocytes (Fig. 5A, B).
cases with mild, moderate or severe atrophy (total villous 3c total villous atrophy and pathological increase of intraep-
flattening) are all grouped together in a single category: the ithelial lymphocytes (Fig. 6A, B).
type 3 lesion.

Fig. 5. Type 3a–3b lesion according to Marsh-Oberhuber. Grade B1 new classification.

Fig. 6. Type 3c lesion according to Marsh-Oberhuber. Grade B2 new classification.

S392 V. Villanacci et al. / Digestive and Liver Disease 43S (2011) S385–S395

Without prejudice to all the other morphological criteria The final diagnosis in the case of an atrophic lesion,
described above, this classification provides a better descrip- culminating in a “consistent” with a CD with atrophic
tion of the spectrum of lesions that may occur both in coeliac lesions (type 3a, 3b or 3c); in the case of non-atrophic
disease patients on a normal diet and in those on a gluten-free lesions culminating in finding attributable to intraepithelial
diet. lymphocytosis, stressing that these injuries are “suggestive
Along the same lines, and in an attempt to simplify and for” but not exclusive of CD and should therefore necessarily
standardize the work of pathologists and facilitate the rela- be placed in the right clinical setting and supported by a
tionship between pathologists and clinicians, a new version serological confirmation.
of the histological classification has recently been proposed As a brief addition to the above we propose that the
by Corazza and Villanacci [12,13]; in particular, the lesions term sub-atrophy, in itself unclear and misleading, should no
that characterize coeliac disease have been divided into two longer be used. Instead, it is better to specify whether the
categories: Non-atrophic (grade A) and atrophic (grade B). villi are normal or atrophic, and in the latter case, the degree
Grade A lesions are characterized by a pathological of atrophy, from mild to moderate to severe. In the event of
increase in intraepithelial lymphocytes, best recognized by the severe atrophy it is possible to use the term total or severe
use of immunohistochemical techniques. atrophy. Scores should not be attributed to the individual
Grade B lesions are further subdivided into: morphological elements as they are too subjective and of little
Grade B1 in which the villus/crypt ratio is less than 3 :1, or no use for the final diagnosis.
with villi still identifiable, and
Grade B2 in which the villi are no longer identifiable: 8.2. The histology report: checklist

Name Sex M/F Date of Birth / /

1st Biopsy No. Control No.
No. of biopsies Oriented Non-Oriented
Villi: normal atrophy mild moderate severe
Villus/crypt ratio: normal [3 : 1] altered
Intraepithelial lymphocytes: normal increased
(normal: less than 25–30 lymphocytes/100 epithelia;
increased: more than 25–30 lymphocytes/100 epithelia)
Evaluation with CD3
Glands: normal hyperplastic
Lamina propria
DIAGNOSIS
Oberhuber-Marsh: New grading system:
Type 1 Grade A
Type 2
Type 3a Grade B1
Type 3b
Type 3c Grade B2
8. Diagnostic criteria Comments:

The diagnosis represents the culmination of what is

described above and must comprise all the morphological 9. Immunohistochemistry
requirements so as to allow a direct, clear and simple
understanding of the morphological situation of the duodenal One of the key points in the diagnosis of coeliac disease
mucosa by the clinician. is the number of intraepithelial lymphocytes, which are CD3
A two-step proposal is presented below: and CD8 positive T lymphocytes; in pathological conditions,
their number should be more than 25 lymphocytes per 100
8.1. Assessment of the morphological pattern divided epithelial cells (border-line value 25–30).
according to description and diagnosis The definition of a precise cut-off between normal, ab-
normal and border-line pathological lesions is of particular
The description should report, in sequence, the same mor- importance given the increase in coeliac disease diagnosed in
phological elements listed above, namely: villous trophism, the early/subclinical stage.
number of intraepithelial lymphocytes, features and glandular The counts can be performed reasonably well on the
structures of the lamina propria, concluding with compatibil- normal and irreplaceable hematoxylin-eosin but we suggest,
ity or non-compatibility with the pattern of a coeliac patient especially in the initial forms, that an immunohistochemical
based on complete clinical and laboratory data. assessment should always be carried out with monoclonal
 V. Villanacci et al. / Digestive and Liver Disease 43S (2011) S385–S395 S393

CD3 which often allows for a more accurate display of IELs (>25–30/100 epithelial cells) (lesion type 1 according to
lymphocytes, following a series of procedures (see the section Marsh, Grade A according to the new proposed classification).
on intraepithelial lymphocyte infiltration). Evaluation with These conditions include hypersensitivity to other foods
CD8 may also help, and is particularly useful in cases of (milk, cereals, soybeans, fish, etc.), infections (Helicobacter
elderly subjects where it is possible to find refractory forms pylori, Giardia, etc.), the use of drugs, immunodeficiencies
which do not respond to diet, regarded by many as pre- and immunodysregulation (Hashimoto thyroiditis, systemic
lymphomatous and in which the expression of CD8 may be lupus erythematosus, rheumatoid arthritis, etc.) and, not least,
negative with respect to the “norm” [14]. chronic idiopathic inflammatory bowel colitis or colitis with
As frozen material is available, immunohistochemical typ- a different etiology, such as lymphocytic and collagenous
ing for the gamma-delta receptor of T lymphocytes can be car- colitis.
ried out; in normal conditions this receptor is not expressed by The question that we must therefore ask is: How can we
more than 2–3% of T lymphocytes while in coeliac disease it discriminate between different pathological conditions where
may reach 20–30% – a particularly useful marker in initial le- the morphology is essentially superimposable? A proper clin-
sions. This assessment is, however, based on the use of frozen ical evaluation based on histological and laboratory data is
material and is not therefore recommended in routine practice. crucial. We must not forget that a diagnosis of coeliac disease
is a “marker” which remains throughout life with obvious
therapeutic and behavioral relapses. The table below helps
10. Differential diagnosis understand how important the need for collaboration between
the pathologist and endoscopist is in the detection of other
The above summarizes the morphological lesions which conditions, such as infection with Giardia lamblia or other
may occur with coeliac disease and where the pathologist parasites, the possibility of presentations of immunodeficien-
clearly has a key role, if only to exclude the possibility of cies morphologically superimposable on coeliac disease and
clinically suspected malabsorption which may also be: not least the localization of Crohn’s disease or particular
• Parasitic (Giardia lamblia, Cryptosporidium, Microsporid- forms of enteritis within the sphere of untreatable diarrhea,
ium) such as autoimmune enteritis, tufting enteropathy, a disease
• Infectious (Whipple’s disease) caused by atrophy of the microvilli, and cases of graft-
• Viral (cytomegalovirus, herpes virus) versus-host disease, all conditions in which the morphological
• Idiopathic (Crohn’s disease) element is fundamental.
• Neoplastic. Three conditions, however, deserve special mention:
The most important problem today in the diagnosis of • Forms of so-called “autoimmune enteritis” possible in
coeliac disease is represented by early lesions, i.e. normal villi children with immunological deficiency (common vari-
with a pathologic increase in intraepithelial T lymphocytes. able immunodeficiency, X linked agammaglobulinemia) in
This issue is appropriately dealt with in the excellent review which the intestinal biopsy may be fully comparable to the
by Brown et al. [15], summarized in Table 1. pattern of coeliac disease [16].
Table 1 shows that in addition to coeliac disease, there • Damage by drugs: there is increasing evidence in the
are a number of pathological conditions that have the same literature showing that the use of drugs, especially non-
morphological aspect as coeliac disease in its early stages, i.e. steroidal anti-inflammatory drugs (NSAID), are capable
normal villous architecture but with a pathological increase of of causing morphological alterations identical to those of
coeliac disease, and it is therefore important to keep this
possibility in mind in cases of elderly patients, especially
Table 1 when the serological markers are all negative [15].
Causes of proximal small intestinal intraepithelial lymphocytosis with nor- • The possibility that concurrent infection with Helicobacter
mal villus architecture* pylori in the stomach can produce a morphological pattern
• Gluten sensitivity† very similar to that of initial lesions of coeliac disease as
• Non-gluten food hypersensitivity (e.g., cereals, cow’s milk, soy products, recently reported [17].
fish, rice, and chicken)
• Infections (e.g., viral enteritis, Giardia, Cryptosporidia, Helicobacter
pylori)†
• Bacterial overgrowth 11. Complications that can be confirmed histologically
• Drugs (e.g., NSAIDs)†
• Immune dysregulation (e.g., Hashimoto thyroiditis, rheumatoid arthritis, Unlike what occurs in children, there is considerable
SLE, autoimmune enteropathy)† evidence that coeliac disease in adults, especially if diagnosed
• Immune deficiency (e.g., IgA deficiency, CVID)
• Infammatory bowel disease late and even more so if not dealt with by a timely and
• Lymphocytic and collagenous colitis rigorous gluten-free diet, is burdened by a higher mortality
rate than in the general population.
*NSAIDs, non-steroidal anti-inflammatory drugs; SLE, systemic lupus ery-
The removal of gluten from the diet therefore determines
thematosus; IgA, immunoglobulin A; CVID, common variable immunodefi-
ciency. not only an improvement of the histological and clinical
† Most common associations. aspects, but also prevents the complications which must
S394 V. Villanacci et al. / Digestive and Liver Disease 43S (2011) S385–S395

always be suspected if an adult patient continues to be unwell, lymphocytes is useful in these cases and “must” always be
despite the diet. pathological (>25–30/100 epithelial cells), best evaluated
These complications are due to: both with H&E staining and with immunohistochemistry
• Collagenous sprue: The patient does not respond to diet staining for CD3.
and histology shows fibrous tissue in the intestinal wall – Attention should be paid to biopsies taken from the
at the level of the superficial subepithelial layer. This duodenal bulb, where the presence of Brunner glands can
morphological pattern is very similar to the condition lead to a false diagnosis of atrophy; biopsies of the bulb
of collagenous colitis described in the colon, where the should always be compared with those taken from the
thickness of the connective band best highlighted with distal portions, especially in the early stages of the disease,
Masson’s trichrome is more than 15 millimicrons, although which has a progression of the pathological process in a
this is a very rare event is described in the literature. cranio-caudal direction.
• Refractory sprue: This condition reproduces the same – If there are varying degrees of atrophy, these should all be
clinical picture as collagenous sprue but can be identified described, not just the most severe degree. An assessment
by immunohistochemical staining, demonstrating that T of compatibility should only be included in the description
lymphocytes, which in normal conditions express CD3 and of the case, while the term “coeliac disease” should be
CD8, in this case present only the expression of CD3 and avoided in the final diagnosis, which should be limited to a
not of CD8 [14]. description, giving the clinician a precise “snapshot” of the
• Ulcerative jejunoileitis: Presence of extensive ulceration of state of the duodenal mucosa. The final diagnosis of CD
the intestinal mucosa, often related to refractory sprue. should be made solely and exclusively by the pediatric or
• Lymphoma: This is the most serious complication and adult clinical gastroenterologist.
should always be suspected when histology shows a preva-
lence of atypical monomorphous lymphocytic elements. In 12.2. What are the “doubts” in the diagnosis of coeliac
these cases it is useful to carry out immunophenotyping of disease?
the lymphoid population, which is almost always type T
[18–20]. The points that cause doubt and require caution on the
part of the pathologist in the diagnosis of CD are clearly
represented by the cases in which there are initial lesions
12. Summary (Marsh 1–2, and Grade A in accordance with the new
proposed classification); in these cases it is necessary to:
12.1. What are the “certainties” in the diagnosis of coeliac
disease? 1. Carefully assess the orientation of the biopsies.
2. Consider whether the villus/crypt ratio of at least 3 :1 is
An obvious prerequisite for certainty in the diagnosis of respected.
coeliac disease (CD) from the anatomo-pathological point of 3. Carefully count the number of lymphocytes in the surface
view is the observation and respect of a number of key points: coating epithelium.
4. Always carry out additional immunohistochemical evalua-
1. Close collaboration between clinicians, laboratory techni- tion with CD3.
cians and endoscopists. 5. Compare the clinical and laboratory data.
2. An adequate number of biopsies (at least 4, 2 in the distal
and 2 in the proximal duodenum). – The two key elements that must be assessed are the absence
3. Correct orientation of the biopsy (the use of pre-cut of atrophy and the increase in the number of intra-epithelial
cellulose acetate filters). lymphocytes; it is therefore crucial to always associate
4. Sufficient clinical information. immunohistochemical evaluation with CD3. The presence
5. Excellent quality of the biopsy samples. or absence of hyperplasia of the glandular elements is
totally irrelevant for practical and therapeutic purposes.
– With these details it is clear that “certainty” in the – Do not forget that the “slide” is proof of the assessment
diagnosis of CD is only possible if the villous atrophy by the pathologist and as such can be compared and
is associated with a pathological increase in the number re-assessed by other colleagues and specialists; it must also
of intraepithelial lymphocytes (value exceeding 25–30/100 be strongly emphasized that the histological assessment
epithelial cells). In this situation, by applying the three must be conducted solely by the pathologist and not by
classifications now known and validated (Marsh, Marsh- other “specialists”.
Oberhuber and Corazza-Villanacci) there is no problem in – As with “certain” cases, it is even more important in
the diagnosis and comparison with clinical and laboratory doubtful cases to merely express an opinion of possible
data. compatibility with CD in the description, describing only
– The degree of atrophy should be certain and not merely the histological aspects in the final diagnosis.
pseudo-atrophy due to incorrect orientation and cutting – Exclude, if possible, a concurrent infection due to Heli-
of the villi. Assessment of the number of intraepithelial cobacter pylori (it is advisable to always take biopsies of
 V. Villanacci et al. / Digestive and Liver Disease 43S (2011) S385–S395 S395

the antral and oxyntic gastric mucosa), immunodeficien- gie associate. A cura del Comitato Scientifico Nazionale (CSN) e
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and use of drugs. Gazzetta Ufficiale 7 febbraio 2008 n° 32, S.O.
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