Human Pathology (2023) 132, 20e30

www.elsevier.com/locate/humpath

Review

Histologic evaluation in the diagnosis and

management of celiac disease: practical
challenges, current best practice
recommendations and beyond*
Zongming Eric Chen M.D, Ph.D, Hee Eun Lee M.D, Ph.D,
Tsung-Teh Wu M.D, Ph.D*
Division of Anatomic Pathology, Department of Laboratory Medicine and Pathology, Mayo Clinic,
Rochester, MN, 55905, USA

Received 22 June 2022; revised 13 July 2022; accepted 14 July 2022

Available online 4 August 2022

Keywords: Summary Celiac disease (CD) is an immunoallergic enteropathy affecting genetically susceptible in-
Celiac disease; dividuals upon dietary exposure to gluten. In current clinical practice, the diagnosis of CD is based
Ultrashort celiac disease; on a combination of clinical, serologic, and histologic factors with the possible exception of pediatric
Refractory celiac disease; patients. Histopathologic evaluation of small intestinal tissue plays a critical role in the disease diag-
Collagenous sprue; nosis and management, despite many practical challenges. Recently published best practice guidelines
Q-MARSH help to standardize biopsy sample procurement, tissue preparation, histology interpretation, and report-
ing, to optimize patient care. In addition, an increasing demand for monitoring the disease course,
particularly demonstrating the efficacy of dietary and nondietary interventions for disease manage-
ment, calls for the use of quantitative histology. With the advent of a gradual transition
toward digital pathology in routine diagnostic practice, quantitative histopathologic evaluation in
CD shows a promising future.
© 2022 Elsevier Inc. All rights reserved.

1. Introduction

Celiac disease (CD) is an immune-mediated enteropathy

occurring in genetically susceptible individuals after expo-
sure to gluten in their diets. Its prevalence is increasing [1]. A
*
Competing interests: None. recent meta-analysis study of epidemiologic surveys showed
* Corresponding author. Mayo Clinic, 200 First Street SW, Rochester, an estimated seroprevalence of 1.4% and biopsy-proven
MN, 55905, USA. prevalence of 0.7% [2]. Increased incidences occurred
E-mail address: wu.tsungteh@mayo.edu (T.-T. Wu).

https://doi.org/10.1016/j.humpath.2022.07.017
0046-8177/© 2022 Elsevier Inc. All rights reserved.
Diagnosis and management of celiac disease
globally, even in traditionally low incidence geographic re-
gions such as Asia [3]. Many contributing factors have been
identified including the common use of gluten in food,
increased disease awareness in the population, and improved
detection methodology. There are also emerging data to
suggest other environmental factors may play a role, such as a
microbial impact on disease development [4]. Nevertheless,
because of the variable clinical presentation of the disease,
which includes many asymptomatic patients, and limited
accessibility to endoscopic biopsy and sensitive and specific
serologic tests in many regions of the world, underdiagnosis
and misdiagnosis of CD remains an important worldwide
challenge [5]. Currently, the only effective treatment for CD
is life-long gluten-free diet (GFD), which is costly and
difficult to strictly follow. There is still an unmet clinical need
for highly effective novel therapies [6].
There have been great advances in understanding the
major immunopathogenetic mechanisms of CD [7,8]. The
antigenic stimulators are a group of proteins collectively
called gluten, including gliadins in wheat, hordeins in
barley, and secalins in barley. They are rich in proline,
which makes them resistant to gastric acid digestion. When
these proteins pass down into duodenum and permeate
through the mucosa, they can become immunogenic,
particularly after their binding to tissue transglutaminase
(tTG) in the duodenal mucosa. This process releases
deaminated peptides that can be presented by human
leukocyte antigen (HLA) to activate specific CD4þ T cells,
a critical step in initiating a cascade of innate and adaptive
immune responses. So far, HLA haplotypes of D2 (2.5),
D8, and rarely D7 are responsible for the genetic suscep-
tibility of CD [5]. The cytokines released from the initial
immune reaction, particularly interleukin (IL)-15 and
interferon, are important in recruiting cytotoxic CD8þ T
cells (more gd subtype than ab subtype) into the epithe-
lium, causing enterocyte death and a breakdown of the
mucosal barrier, leading to escalating mucosal inflamma-
tion and injury [9,10].
With recent advances in bioengineering capacity, it is
now possible to synthesize specific HLA-DQ molecules to
form tetramer complexes with known peptides that cause
CD. These artificial molecular complexes are extremely
sensitive and specific to identify gluten-responsive CD4þ T
cells in the blood or small intestinal mucosa of susceptible
individuals. As such, they have the potential to be a novel,
noninvasive biomarker for CD and revolutionize the current
clinical diagnostic algorithm [11]. On the therapeutic front,
several emerging therapies targeting specific immunopa-
thogenic mechanisms of CD have shown promising results.
The number of clinical trials for CD novel therapies has
also grown significantly in the last few years [6]. However,
a common challenge for all of these trials is how to reliably
demonstrate therapeutic efficacy. Currently, quantitative
histologic evaluation to demonstrate mucosal changes
before and after therapeutic intervention appears the only
21
sensitive and reproducible method necessary to address this
issue [12e14].
In current clinical practice, the diagnosis of CD is based
on a combination of clinical, serologic, and histologic
factors, with one exception [15e17]. European pediatric
guidelines suggest that, in a subset of children, duodenal
biopsies can be avoided in the presence of strict symp-
tomatic and serological criteria (serum IgA anti-tTG anti-
body 10 times or greater than the upper limit of normal,
positive anti-EMA, and the presence of HLA-DQ2/8) [17].
The no-biopsy approach, however, remains controversial.
There are recent reports concerning the reliability of serum
biomarker performance in the real world [18,19]. In addi-
tion, the lack of direct assessment of mucosal injury makes
histologic comparisons of before and after GFD treatment
impossible and may miss or delay the diagnosis of sero-
negative or refractory CD (RCD) [20].
On the other hand, to make the best use of histologic
evaluation in CD diagnosis and management, there are
many challenging issues to consider, beyond the scope of
the histology itself. A holistic approach is often necessary
to deal with perplexing clinical scenarios. Recently pub-
lished best practice guidelines help to standardize tissue
procurement, specimen preparation, and histologic inter-
pretation, as well as reporting to optimize patient care [21].
These best practice principles are also mentioned in
recently published excellent review articles [22,23].
The focus of the current review is to highlight these best
practice recommendations, considering the most frequently
encountered practical challenges, particularly focusing on
histologic variants of CD and their specific differential di-
agnoses. The use of quantitative histology is not recom-
mended in the current best practice guidelines for several
reasons, including a lack of definitive clinical value and the
process being too time-consuming for routine diagnostic
practice. However, its value for disease management,
including monitoring disease course and particularly in
demonstrating the efficacy of dietary and nondietary in-
terventions, has been clearly demonstrated [12]. With
advent adoption of whole slide imaging technology and
digital imaging analysis applications into routine surgical
pathology practice [24], quantitative histopathologic eval-
uation in CD has a promising future. Therefore, also
included is the Quantitative-Mucosal Algorithmic Rules for
Scoring Histology (Q-MARSH) [12] and potential chal-
lenges for its clinical adoption.
2. Practical challenges of tissue histologic
evaluation for CD diagnosis
The histologic changes of CD occur primarily in the
small intestine. However, systemic presentations, such as
dermatitis herpetiformis [25], lymphocytic and collagenous
gastritis [26], and colitis [27], also occur and are beyond the
scope of this review. The most characteristic histologic
22 Z.E. Chen et al.

change in small intestinal mucosa is increased intra-

Table 1 A list of common histologic differential diagnoses
epithelial lymphocytosis (IEL), which occurs in essentially of CD.
all affected individuals. However, it is also the most
Common and rare entities Common and rare entities
nonspecific histologic finding, one that may occur in many
show duodenal IEL and show duodenal mucosal
conditions unrelated to CD [28,29]. Mucosal architectural preserved villous atrophy with or without IEL
changes, including villous blunting and crypt hyperplasia, architecture
are also characteristic findings, representing a dynamic
 Infectious etiologies  Tropical sprue
balance between mucosal injury and regeneration. These
including H. pylori  Collagenous sprue
changes are almost always associated with increased lam- gastritis, bacterial over-  Refractory CD
ina propria lymphoplasmacytic infiltration. In fact, the growth syndrome, giardia,  Whipple’s disease
absence of plasma cells can indicate common variable cryptosporidiosis, and viral  Mycobacterial infection
immunodeficiency (CVID) [30]. Cryptal architectural infection including HIV  Drugs including angio-
destruction, with or without missing normal cell compo-  Drugs including NSAIDs tensin 2 receptor blockers
nents such as goblet cells and endocrine cells, almost never  Food hypersensitivity such as Olmesartan and
occurs in CD and should lead to evaluation for autoimmune including Cow’s milk, soy, immune checkpoint inhibi-
diseases (including autoimmune enteropathy and inflam- etc. tor such as Nivolumab
matory bowel disease) or drug-induced injury [31e34].  Peptic duodenitis  Autoimmune enteropathy
 Immunodeficiency  GVHD
Frequently, neutrophils and eosinophils are also present in
including CVID, IgA  Crohn’s disease
the inflammatory milieu of the lamina propria with CD, and
deficiency  Eosinophilic enteritis
rarely neutrophilic or eosinophilic cryptitis may also exist,  Systemic autoimmune dis-  Microvillous inclusion
particularly in cases with significant mucosal flattening. ease including rheumatoid disease
However, the presence of significant neutrophilic or arthritis, SLE, Hashimoto  Tufting enteropathy
eosinophilic cryptitis with crypt destruction is rare, and thyroiditis  Nutritional deficiency
when present should be considered as a potential red flag  Chemotherapy
for nonceliac injury [23]. Other common but nonspecific  Lymphoma
cytologic features in CD include enterocyte vacuolar Abbreviations: CD, celiac disease; IEL, intraepithelial lymphocytosis;
changes, focal gastric foveolar metaplasia, and slightly HIV, human immunodeficiency virus; NSAID, nonsteroidal anti-
increased apoptosis in crypt cells [35]. Markedly increased inflammatory drug; CVID, common variable immunodeficiency;
crypt cell apoptosis is rare for CD, however, and should SLE, systemic lupus erythema; GVHD, graft-versus-host disease.
lead to exclusion of drug-induced injury or graft-versus-
host disease [36].
As already noted, the characteristic histologic alterations ctly taking care of the patients are necessary. For example, a
seen in CD are not pathognomonic for CD. Other etiologies CD patient may also suffer from Crohn’s disease and/or H.
can cause similar mucosal histologic changes, including pylori gastritis. These challenging clinical situations require
infection, autoimmune enteropathy, drug effect, and immu- close clinical follow-up and monitoring and may require
nodeficiency. For example, Helicobacter pylori gastritis may further tissue sampling to assess histologic changes during
induce significant IEL in the duodenum mucosa, mimicking the disease course, such as after GFD or other relevant
CD. Some drugs, particularly angiotensin 2 receptor blockers treatments.
such as Olmesartan, are known to induce IEL with villous The histological changes of CD are often unevenly
atrophy, mimicking severe active CD [31]. More recently, distributed and of variable severity [38]. Limited tissue
immune checkpoint inhibitors (nivolumab, ipilimumab, and sampling, along with suboptimal orientation, is an important
pembrolizumab) have been reported to cause duodenal injury practical challenge for histologic evaluation. The misinter-
that can share similarities with severe active CD [37]. To pretation of mucosal injury patterns may occur when tissue
differentiate CD from these morphologic mimickers can be a sampling is limited, resulting in underdiagnosis or a missed
major challenge in some cases. Table 1 summarizes common diagnosis [39,40]. The recently described CD patterns of
differential diagnoses that need to be considered when duodenal bulb-only or bulb-predominant CD injury are seen
evaluating tissue histology for CD. These entities can be in a subset of patients and clearly illustrate the clinical
roughly divided into 2 categories based on the predominant importance of obtaining bulb tissue for evaluation (see
histologic patterns, ie, (1) IEL with preserved villous archi- detailed discussion below). Tissue fragmentation and crush
tecture, and (2) villous atrophy with or without IEL. In most artifact secondary to endoscopic biopsy handling techniques
cases, with proper clinical information including medication can also severely impair the pathologists’ ability to discern
use and CD serologic testing results, pathologists can histologic details and make an accurate diagnosis. These
reasonably confidently make an accurate diagnosis. How- preanalytical variables have a direct impact on proper his-
ever, for complicated clinical scenarios, such as when mul- tologic evaluation and its outcome.
tiple disorders may coexist in individual patients, close How to properly apply CD histologic classification
communications between pathologists and clinicians dire- grading schemes is another practical challenge. Since the
Diagnosis and management of celiac disease
original Marsh criteria proposed in 1990s [41], there have
been several classification grading schemes developed
through years, with the basic purpose of capturing the dy-
namic mucosal changes of CD and to semiquantitatively
document disease status and activity. Table 2 summarizes
commonly used classification grading schemes. Currently,
the most popular grading scheme is the so-called Marsh-
Oberhuber system [42]. It is a semiquantitative descrip-
tion of IEL and villous atrophy, with or without crypt hy-
perplasia. The classification relies on subjective judgment
based on global assessment. The interobserver agreement is
generally low and the reproducibility poor. To simplify the
grading method, Corazza-Villanacci developed a 3-grade
system: grade A shows normal crypts and villus architec-
ture but increase IEL (>25 IELs per 100 enterocytes);
grade B1 shows villous atrophy but clearly detectable villi
are still present, whereas grade B2 shows villous atrophy to
the extent of complete flattening, with no detectable villi
[43]. This simplified system showed better agreement be-
tween pathologists and is commonly used in clinical
practice. However, the subjectivity of individual patholo-
gists at the time of tissue evaluation still exists.
3. Current best practice recommendations for
histopathology evaluation in CD
To address and overcome these practical challenges,
experts from the Rodger C. Haggitt Gastrointestinal Pa-
thology society and the North American Association for the
Study of Celiac Disease recently published a set of best
practice recommendations [21]. These help to standardize
preanalytical and analytical factors impacting tissue histo-
logic evaluation for CD. Table 3 summarizes the key rec-
ommendations. It is formally recommended to obtain
adequate biopsy specimens when CD is considered to be
clinically possible, regardless of the endoscopic mucosal
appearance. The biopsy series should contain at least 2
biopsies from the duodenal bulb and 4 biopsies from the
distal (postbulbar) duodenum. To ensure tissue integrity, it
Table 2 Comparing different histologic classification grading schemes used for celiac disease.
IEL Mucosal Architectural
Abnormality
Normal Normal
Increased Normal
Increased Normal villous height
but increased crypt depth
Increased Partial villous atrophy
Increased Subtotal villous atrophy
Increased Total villous atrophy
Increased Hypoplastic
Abbreviations: IEL, intraepithelial lymphocytosis; Vh, villous height; Cd, crypt depth.
23
is also recommended to have one biopsy per pass as the
standard endoscopic tissue sampling technique. Impor-
tantly, it is formally recommended to have clinicians to
supply relevant clinical information when submitting tissue
for histologic evaluation. This is extremely helpful in set-
tings where a pathologist’s access to electronic medical
records is limited.
Tissue orientation is a major preanalytical factor impacting
proper histologic evaluation and applying CD classification
grading schemes. It was recognized that, although there are
insufficient data to recommend special orientation efforts dur-
ing tissue preparation and embedding, using serial sectioning
to obtain well-oriented villus-crypt units is still recommended.
Identifying reasonable numbers of well-oriented villus-crypt
units is also a key determinant for successful quantitative
tissue evaluation (see more discussion below).
For histologic analysis, the recommendations also pro-
vide helpful standardization. For counting IELs, the recom-
mendations recognize several practical scenarios: (1) A
normal or “non-CD” pattern with occasional IEL, up to 25/
100 enterocytes, with a distribution skewed toward the lateral
aspects of the villi and decrease lymphocytes toward the
villous tips, the so-called “decrescendo pattern”; (2) IEL in
CD patients with normal villi, showing >25/100 enterocytes
with an even distribution over the entire villous or more
numerous lymphocytes in the villous tips (>6/20 enter-
ocytes); (3) The number of IEL in CD patients with abnormal
villi almost always exceeds 40/100 enterocytes. It is also
formally recognized that routinely applying CD3 immuno-
staining for IEL counting is not necessary [44], and therefore
discouraged. The recommendations also call for increased
awareness of unusual histologic changes that may indicate
non-CD etiology during histologic evaluation. In addition,
care must be taken to avoid overinterpreting villous abnor-
malities in areas of Brunner gland hyperplasia or lympho-
glandular complexes.
These recommendations also clearly recognize the clin-
ical necessity of comparing mucosal histologic changes with
previous biopsies for certain indications, and the utility of
Marsh Marsh Corazza QMARSH
-Oberhuber -Villanacci
Vh:Cd ratio
0 0 Normal 3.0
1 1 Grade A 3.0
2 2.0 to <3.0
3 3a Grade B1 1.0 to <2.0
3b 0.5 to <1.0
3c Grade B2 <0.5
4
24 Z.E. Chen et al.

Table 3 A summary of GISP-NAASCD best practice recommendations.

Tissue procurement and
clinical information
submission
Recommendations  Taking biopsy regardless
of endoscopic appearance
 At least 4 specimens from
the distal (postbulbar)
duodenum and 2 speci-
mens from the duodenal
bulb
 Obtaining one specimen
per pass of the biopsy
forceps
 Supply relevant clinical
information including
signs and symptoms,
endoscopic findings,
medications, patient and
family history, current
adherence to GFD, and
serological or genetic test
results
Abbreviations: CD, celiac disease; IEL, intraepithelial lymphocytosis; GFD, gluten-free diet.
Tissue preparation and
orientation
 Special effort at tissue
orientation in the labo-
ratory are not required
for the diagnosis of ce-
liac disease
 Adequate numbers of
biopsies, along with the
appropriate use of serial
sectioning typically
result in a sufficient
number of well-oriented
villus crypt units to
accurately determine
architecture in the
majority of cases with
randomly embedded
tissue
Histologic evaluation Pathology reporting
 Occasional IEL (up to  Pathology reports should
25/100 enterocytes) are mention semi-
present in non-CD quantitatively the degree
samples with a of villous blunting and
“decrescendo pattern” should compare the
(more numerous along villous architecture with
the lateral aspects of villi existing previous bi-
and decrease toward the opsies if clinically indi-
villous tips) cated in patients with
 IEL in CD samples are suspected or proven CD
evenly distributed over  A named classification
the entire villous (>25/ system score may be
100 enterocytes) or are included in pathology
more numerous in the reports if it is understood
villous tips (>6/20 by, and enhances
enterocytes); may communication with
prompt additional sero- clinicians
logic studies if they have  No specific system is
not already been carried endorsed as superior
out.
 Immunohistochemical
stains for T-lymphocyte
markers do not improve
the detection of CD; “up
front” ordering of
immunohistochemical
stains not recommended
 Histologic changes are
sensitive but not specific
for CD; differential
diagnosis should be
clinically and histologi-
cally excluded
 Avoid overinterpreting
villous abnormalities in
areas of Brunner gland
hyperplasia or lympho-
glandular complexes

including semiquantitative descriptions of villous architec- 4. Histologic variants of CD

ture in pathology reports. Although there is no endorsement
for a specific CD classification grading scheme, the appro-
priate use is encouraged and contingent to the clinical setting
where the scheme enhances communication with clinicians.
The recommendations also provide standardized pathology
report templates for CD to illustrate the optimal delivery of
the most valuable histologic information. Pathology reports
that incorporate clinical and pathologic data personalized to
individual patients provide the best value for treating clini-
cians, rather than pathology reports that blindly include every
possible cause of mucosal histologic changes a pathologist
can think of [22].
4.1. CD restrict to duodenal bulb only or ultrashort
CD
As noted earlier, it is not unusual to see patchy mucosal
injury with variable severity in CD patients. Patchy disease is
more frequent in children than in adults [38,45,46]. At the
extreme end of the spectrum, some CD patients only show
mucosal inflammation and injury, particularly villous atro-
phy, within the duodenal bulb (D1 region), without more
distal involvement (Fig. 1). Several prospective studies
confirmed the existence of such patients and illustrate the
Diagnosis and management of celiac disease
Fig. 1 An example of ultrashort celiac disease (USCD). A, Localized typical histologic changes of celiac disease (CD) in duodenal bulb
with increased intraepithelial lymphocytosis (IEL), partial villous atrophy, and crypt hyperplasia with increased lamina propria inflam-
mation. B, Normal histology in the second portion of duodenum biopsied during the same procedure.
importance of including duodenal bulb tissue sampling to
ensure their detection [39,40]. According to a recent meta-
analysis, in 2.5e30% of CD cases, inflammation is restricted
to the duodenal bulb [47] and the study found that duodenal
bulb biopsies improved the rate of detection of CD by 4% in
children and by 8% in adults. Therefore, all published society
guidelines currently recommend examination of bulb bi-
opsies. However, the effectiveness of screening bulb tissue to
improve the detection of CD in some settings is still
controversial. A study by Stoven et al., for example, analyzed
biopsies from duodenal bulbs in a cohort of patients with a
low pretest probability for CD and found only 1 of 679 in-
dividuals had CD disease restricted to bulb [48]. According
to this study, there was only 0.1% improvement in CD
detection in the setting of low pretest probability. Neverthe-
less, the practical importance for pathologists is to pay close
attention to CD-related mucosal histologic changes that are
limited to duodenal bulb, because they may be obscured by
histologic changes unrelated to CD, such as peptic duode-
nitis, Brunner gland hyperplasia, and prominent lymphoid
follicles. In general, marked IEL in bulb biopsies with or with
villous atrophy should always be considered as potentially
representing CD, regardless of the other background fea-
tures.
This D1-only presentation of CD is also referred to as
ultrashort CD (USCD) [49e51]. There are only limited
studies focusing on this topic. From these studies, the
prevalence of USCD ranges from 5 to 10% in both chil-
dren and adults. The patients often show milder symp-
toms, less endoscopic findings, milder histologic abno-
rmalities, and lower serum IgA-tTG titers, when compared
to cases of conventional CD. They also tend to respond
faster to GFD with a complete normalization of mucosal
injury. USCD is generally regarded as a milder phenotype
of CD. Interestingly, a recent study showed a lower fre-
quency of HLA DQ2 and less NK cell suppression in
USCD patients [49], elucidating potential distinct genetic
and immunologic features. Currently, few data exist
regarding the long-term disease course and prognosis of
USCD.
25
4.2. Refractory CD
RCD is a rare complication of CD and an important
clinicopathologic diagnosis [52,53]. The current definition is
that of persistent or recurrent malabsorption and continued
villous atrophy on duodenal biopsies, despite strict avoid-
ance of gluten for a minimum of 12 months, in the absence of
other causes of nonresponsive treated CD and overt lym-
phoma. RCD is further divided into 2 subtypes: RCD type 1
shows IELs without an abnormal T cell phenotype, and RCD
type 2 shows IELs with an abnormal T cell phenotype, or a
monoclonal population of T cells. The distinction between
the 2 types is based on clinical, histologic, and molecular
factors [52,53]. The diagnosis is of paramount importance
because the treatment options and prognosis differ signifi-
cantly, as RCD type 2 is a severe complication that is
considered to be a precursor of T cells lymphoma, with
32e52% of patients transforming to enteropathy-associated
T cells lymphoma in 5 years [53]. It is critical for pathologists
to perform appropriate tests and molecular workup to help
identify these patients at an early stage.
According to a recent systematic review [54], among CD
patients, RCD has a cumulative incidence of 1e4% over a
10-year period and a prevalence of 0.31e0.38%. It usually
presents in 2 clinical scenarios: primary RCD patients form a
minority group who never respond to GFD therapy after the
initial CD diagnosis, while secondary RCD patients make up
the majority of cases and are patients who responded to GFD
for years but then subsequently developed new symptoms or
recurrence of diarrhea. For a primary RCD diagnosis, it
should be noted that the 12-month should not be a rigid time
frame because there are slow-responders who might need a
longer period on a GFD to achieve mucosal healing. In one
series [55], persistent villous atrophy was found in 42% of
patients at 1 year but that reduced to 5% over 2e5 years of
follow-up. Continued “gluten” exposure is one of the main
causes for the slow responsiveness to GFD [52]. CD sero-
logic tests are helpful to investigate this possibility. Other
nonceliac etiologies of mucosal injury should also be clini-
cally excluded.
26 Z.E. Chen et al.

The most important practical challenge for pathologists
is to distinguish RCD type 1 from type 2. The histologic
features of RCD type 1 are the same as active CD, whereas
RCD type 2 is characterized by the presence of abnormal T
cells that do not express surface CD3 and CD8 molecules
but retain intracellular CD3 expression. They also express
NKp46 and IL15 receptors and show clonal T cell receptor
(TCR) gene rearrangements. According to a recent study
[56], these cells are believed to derive from a subset of T
cells in the normal mucosa and are activated to expand by
IL15 and gain of function somatic mutations within Jak1
and Stat3. Increased levels of Smad7 protein also contribute
to the expansion of these abnormal T cells by sustaining
and amplifying the inflammatory cytokine effect [57].
Currently, immunohistochemistry (IHC) and TCR gene
rearrangement analysis are 2 of the most used ancillary
tests in clinical practice in the United States. Typically, by
IHC, normal IELs in RCD type 1 show a CD3þ/CD8þ
phenotype, whereas abnormal IELs in RCD type 2 show
CD3epsilonþ/CD8-phenotype (Fig. 2). The abnormal IELs
in RCD type 2 also exhibit clonal TCR gene rearrange-
ments. However, the sensitivity and specificity of these tests
in the diagnosis of RCD type 2 should be critically evalu-
ated. One recent study [58] found that clonal T cells could
be seen in biopsies from several benign conditions with
IELs, and similar clonal populations were identified in both
RCD type 1 and type 2 patients, whereas no clonal T cells
were detected in one RCD type 2 patient. The same study
also found that IHC for CD3/CD8 ratio was not sensitive to
reliably differentiate RCD type 1 from type 2. The potential
pitfalls in relying on TCR clonal analysis alone in the
diagnosis of RCD type 2 have also been highlighted by
others [59]. In comparison with IHC and TCR clonal
analysis methods, flow cytometry appears to show superior

Fig. 2 Examples of refractory CD type 1 (AeC) and type 2 (DeF). Hematoxylin and eosin (H&E) stains show increased IEL in A and D,
with partial villous atrophy in A but not D. CD3 immunostains show positivity in most IEL and T cells in lamina propria in both B and E.
CD8 immunostains show retained CD8 expression in most IELs in refractory CD type 1 as shown in C, but loss of CD8 expression in
refractory CD type 2 as shown in F.
Diagnosis and management of celiac disease
sensitivity and specificity in detecting RCD patients,
particularly with moderately increased number of abnormal
IELs [60]. This method has been used clinically to identify
abnormal IELs in duodenal mucosa from RCD type 2 pa-
tients and is more commonly performed in Europe than in
the United States. It is prudent for pathologists to under-
stand different methods and their analytical performance
characteristics to put them into best use for the clinical
diagnosis of RCD patients.
4.3. Collagenous sprue
This is an entity with characteristic morphologic features
[61,62]. The subepithelial collagen layer of the duodenum is
prominently thickened, with a thickness often exceeding
10 mm, and shows entrapment and dilatation of capillaries
(Fig. 3). The collagen deposition can be diffuse or patchy.
Typically, there is associated villus blunting, lamina propria
inflammation including increased eosinophils, and a variable
increase in the number of IELs. Surface epithelial detach-
ment is quite common and the presence of acute inflamma-
tion within the surface epithelium and crypts is also
frequently seen. These features are pathognomonic and
essentially the same as seen in collagenous gastritis or colitis
[63]. In fact, these entities often coexist and, when this is the
case, may indicate a systemic gastrointestinal tract collage-
nosis. An increased expression of fibrogenic genes, such as
procollagen1 and tissue inhibitor of MMP-1 (TIMP-1), by
myofibroblastic cells is thought to be responsible for the
abnormal collagen deposition [64].
Collagenous sprue (CS) occurs in women more than men,
with a 2:1 ratio, and typically affects middle-aged or elderly
women who present with severe diarrhea and weight loss
[65,66]. There is a close association with CD, and CS is seen
with 30e50% of all RCD cases. However, it is important to
know that CD is not the only cause of CS. There are multiple
other potential causes, including autoimmune enteropathy,
CVID, hypothyroidism, IgG4-related sclerosing disease, and
idiopathic collagenosis involving gastrointestinal tract [62].
It is worth noting that angiotensin 2 receptor blockers
Fig. 3 An example of collagenous sprue. A, H&E stain shows total villous atrophy and slightly increased IEL. There is thickened
subepithelial collagen deposition (arrowheads) with entrapped capillaries and inflammatory cells. Note the detachment of epithelium from
the collagen table. B, Trichrome stain highlights subepithelial collagen deposition.
27
(ARBs), ie, sartan family medications such as Olmesartan,
can also induce the development of CS [67]. An accurate
diagnosis of CS is important for patient management and
treatment [66,68,69].
5. Quantitative histology for clinical
management of CD
CD is a chronic inflammatory process and clinical disease
management often requires a close monitoring of the disease
course and particularly demonstrating the efficacy of dietary
and nondietary interventions. The current best practice
guidelines recognize this need to compare mucosal histo-
logic changes with previous biopsies in certain indications
and recommend including semiquantitative descriptions of
villous architecture in the pathology reports. However,
subjectivity and reproducibility are major limitations of the
semiquantitative approach. The Quantitative-Mucosal
Algorithmic Rules for Scoring Histology (Q-MARSH) sys-
tem (Table 2) uses an averaged assessment of villous height
(Vh), crypt depth (Cd), and IEL count (per 100 enterocytes)
to provide objective measures of histological changes [12]. It
has been mainly used as a research tool and was successfully
applied in several recent clinical trials of CD, some of which
showed promising results [13,14,70].
The proposed rules in Q-MARSH require measurements
of Vh and Cd from 3 to 5 well-orientated villusecrypt units
(deeper levels may be required to achieve this) from at least
4 postbulbar duodenum biopsies. This helps to average out
the variability in disease severity. A Vh/Cd ratio of <3.0
and an IEL count of 30/100 enterocytes are abnormal. A
comparison of Q-MARSH with the other commonly used
CD semiquantitative classification grading systems is
shown in Table 2. It should be noted that Vh:Cd ratio in Q-
MARSH does not discriminate between crypt elongation
and shortening of the villi. In general, Vh:Cd ratio of 2.5
indicates normalization of mucosal injury.
To apply Q-MARSH well, there are 2 critical steps: to
identify enough well-orientated villus-crypt units and to
determine the boundary between a villus and a crypt. A well-
28
orientated villus-crypt unit is defined as a continuous
epithelial lining that is visible from the tip of the villous
structure to the bottom of the crypt where it touches the
muscularis mucosae (Fig. 4). To find enough numbers of such
units, serial sectioning is the most reliable method and is
almost always required. There is no standard approach to
determine the villus and crypt boundary. Some use the nar-
rowest point where a crypt opens to the surface as the
boundary, whereas others draw a virtual boundary line based
on inspection of the overall mucosal topology of a specific
tissue fragment. Both are subjective assessments and tend to
work well for measuring Vh and Cd of healthy mucosa with
slender villi and shallow crypts but face significant chal-
lenges when there is partial villous atrophy and crypt hy-
perplasia. A more subjective way to determine the boundary
is to carefully examine the brush border on enterocytes lining
the villi and crypts. The brush border associated with villous
Fig. 4 An illustration of villous height (Vh; blue lines) and
crypt depth (Cd; red lines) measurements (in mm depicted in the
rectangular boxes) in 2 well-orientated villus-crypt units using a
standard digital pathology platform deployed for routine diag-
nostic use at Mayo Clinic. Note the annotations and measurements
made directly on the tissue section. The V/C boundary is deter-
mined by subjective assessment according to the pathologist’s
experience. (For interpretation of the references to color in this
figure legend, the reader is referred to the Web version of this
article).
Z.E. Chen et al.
cells shows a crispy texture, whereas it is absent or becomes
fuzzy on crypt cells. Recently, IHC for apolipoprotein A4
(APOA4) has been shown to help define the villus-crypt
border because it highlights the brush border of the villous
cells but not the crypt cells [71]. The study demonstrated an
improved interobserver variability of Vh and Cd measure-
ments by applying APO A4 IHC in challenging cases. Fig. 4
illustrates how the measurement of Vh and Cd are performed
on a well-orientated villus-crypt unit using a digitally scan-
ned whole slide image. The figure also shows that angulation
in the villi can be incorporated by measurement around the
angles. Essentially all digital pathology platforms have
specific annotation or measurement tools which make Vh and
Cd assessment simple and quick. With a little effort, specific
applications can be developed to automatically calculate
Vh:Cd ratio and export data to pathology reports. IELs can
also be counted automatically by running commercially
available or self-brew lymphocyte enumeration algorithms.
These potential technical improvements, accompanied by
implantation of routine digital pathology workflows, would
make CD quantitative histology evaluation a real feasibility
in the future.
6. Conclusion
Tissue histologic evaluation plays a critical role in the
diagnosis and management of CD. There are many prac-
tical challenges, and the recent best practice recommen-
dations help to standardize some important pre-analytical
and analytical variants to optimize patient care. Patholo-
gists should be aware of histologic mimickers and variants
of CD to make an accurate diagnosis. With the advent of
transitioning into the digital pathology era, there is a po-
tential to adopt quantitative histologic evaluation of CD
into routine practice.
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