ORIGINAL ARTICLEUsefulness of the organ culture system when villousheight/crypt depth ratio, intraepithelial lymphocytecount, or serum antibody tests are not diagnosticfor celiac disease

ANTONIO PICARELLI, MARCO DI TOLA, MARIACATIA MARINO, VALERIO LIBANORI,RAFFAELE BORGHINI, ELISA SALVI, GIUSEPPE DONATO, DOMENICOVITOLO, ANTONIO TIBERTI,ADRIANA MARCHEGGIANO, GABRIO BASSOTTI, and ENRICO CORAZZIARI

ROME AND PERUGIA, ITALY

From the Department of Internal M

Sapienza University, Rome, Ita

Adolescent Neuropsychiatry, Sap

Department of Clinical Medicine,

Department of Experimental Medi

Italy; Department of Clinical

University of Perugia, Perugia, Ital

Conflict of interest: All authors hav

closure of potential conflicts of inte

nancial or other relationship that m

The existence of mild forms of celiac disease (CD) canmake the histology-based di-agnosis difficult to reach. Since anti-endomysium (EMA) and anti-tissue transgluta-minase (anti-tTG) are detectable in culture supernatants of duodenal biopsies fromCD patients, our aim was to assess if this system can support the histology in the di-agnostic work-up. A total of 559 suspected CD patients underwent serum EMA/anti-tTG detection, upper endoscopy with duodenal biopsy sampling, histologicanalysis, and organ culture to detect EMA/anti-tTG in supernatants. A subgroup of30 patients with organ culture positive results were put on a gluten-free diet (GFD).Their gluten-dependency was evaluated by the psychological general well-beingandbeck depression inventory indexes. Statistical analysis was performedbyCohenk inter-test, Friedman test, and Dunnmultiple comparison. Two hundred forty-one outof 559 (43.1%) patients showed intestinal villous atrophy, whereas serum and organculture EMA/anti-tTG were positive in 293/559 (52.4%) and 334/559 (59.7%) patients,respectively. The strength of agreement resulted good for serology vs histology (k 50.730), good for organ culture vs histology (k5 0.662), and very good for serology vsorgan culture (k5 0.852). After 12 months of GFD, psychological general well-beingindex significantly increased, and beck depression inventory index significantly de-creased (P, 0.001 for each one). Data highlight the organ culture system as a usefultool to assist the histology in diagnosing CD,mainly in cases without villous atrophy orin seronegative patients. Themarked improvement in quality of life after aGFD furthersupports the reliability of this system in diagnosing CD. (Translational Research2012;-:1–9)

edicine and Medical Specialties,

ly; Department of Child and

ienza University, Rome, Italy;

Sapienza University, Rome, Italy;

cine, Sapienza University, Rome,

and Experimental Medicine,

y.

e read the journal’s policy on dis-

rest and state that there are not fi-

ight lead to a conflict of interest.

Submitted for publication September 4, 2012; revision submitted

October 24, 2012; accepted for publication October 25, 2012.

Reprint requests: Antonio Picarelli, MD, Department of Internal Med-

icine and Medical Specialties, Sapienza University, Polyclinic Um-

berto, Viale del Policlinico, 155, 00161, Rome, Italy; e-mail:

antonio.picarelli@uniroma1.it.

1931-5244/$ - see front matter

� 2012 Mosby, Inc. All rights reserved.

http://dx.doi.org/10.1016/j.trsl.2012.10.010

1

AT A GLANCE COMM

Picarelli A, et al.

Background

The mild forms of celia

histology-based diagnosis

endomysium and anti-tiss

detectable in culture supe

opsies from celiac patient

Translational Significanc

Our data highlight the or

useful tool to assist the his

liac disease, mainly in cas

or in seronegative patien

quality of life and clinica

a gluten-free diet in patie

mild enteropathy celiac d

culture alone, further su

this system in diagnosing

Translational Research2 Picarelli et al - 2012

Abbreviations: anti-tTG ¼ anti-tissue transglutaminase; BDI ¼ beck depression inventory; CD¼ celiac disease; ELISA ¼ enzyme-linked immunosorbent assay; EMA ¼ anti-endomysium;GFD ¼ gluten-free diet; IEL ¼ intraepithelial lymphocyte; PGWB ¼ psychological generalwell-being

ENTARY

c disease can make the

difficult to reach. Anti-

ue transglutaminase are

rnatants of duodenal bi-

s.

e

gan culture system as an

tology in diagnosing ce-

es without villus atrophy

ts. The improvement in

l picture observed after

nts identified as having

isease through the organ

pports the reliability of

celiac disease.

Celiac disease (CD), the most common systemic au-toimmune disorder with a local inflammatory compo-nent, is induced in HLA-DQ2/8 positive individualsby the ingestion of wheat gluten and related prolaminesin rye and barley.1 Gliadins, the alcohol-soluble glutenprotein fractions, are the external trigger of CD, whichin turn represents an unique and privileged model ofboth adaptive and innate immune response,2 where theenzyme tissue transglutaminase (tTG) is known to bethe specific autoantigen.3

The gold standard for CD diagnosis is still now a histo-logic finding of total, subtotal, or partial intestinal villousatrophy (type III of Marsh-Oberhuber classification) re-coverable by a correct gluten-free diet (GFD),4 exceptfor the symptomatic patients showing high serum anti-body levels.5 However, the existence of mild forms ofCD, including patchy atrophy or latent CD, can makea correct diagnosis difficult to reach. Some patients canindeed present serum antibody positive results but normalsmall intestine mucosal architecture.6

On the other hand, several pathologists consider theintraepithelial lymphocytosis a pathognomonic feature

of CD, even in the absence of villous atrophy. Sincethe intraepithelial lymphocytes (IELs) are a functionalcomponent of the gut-associated lymphoid tissue, it isnot unexpected that an immunologic stimulation perpe-trated by endogenous or ingested antigens, includingwheat gluten, can lead to increased IELs number. Possi-ble causes of increased IELs number not related to CDinclude food allergies (eg, cow’s milk, cereals, fish), in-fections (eg, enteric viruses, Helicobacter pylori, Giar-dia intestinalis), bacterial overgrowth, adverse effects todrugs (eg, nonsteroidal anti-inflammatory drugs), andchronic idiopathic inflammatory colitis or colitis witha different etiology.7 Therefore, for patients with in-creased IELs number but normal small intestine muco-sal architecture (types I–II of Marsh-Oberhuberclassification), the major problem is to discern in whichcases the intraepithelial lymphocytosis is gluten-dependent through a proper differential diagnosis.8

Previous studies demonstrated that gluten-dependentmorphologic and immunologic features can be repro-duced in vitro by using the organ culture system.9,10 Inaddition, it has been shown that anti-endomysium(EMA) and anti-tTG antibodies are detectable in culturesupernatants of duodenal biopsy specimens from un-treated CD patients.11,12 On these bases, our aim wasto evaluate if the organ culture system can be a supportto IEL count and serum antibody tests in diagnosing themild forms of CD. For this purpose, we assessed the or-gan culture system with respect to villous height/cryptdepth ratio, IEL count, and serum antibody tests per-formed in a large number of patients waiting for a diag-nosis of CD.

MATERIALS AND METHODS

Patients. A total of 559 consecutive outpatients (206male/353 female, mean age 34.21, range 9–73 years),referred to our Gastrointestinal Unit from February2008 to October 2010 with symptoms suggestive ofCD (eg, abdominal pain and swelling, diarrhea, dyspep-sia, osteopenia or osteoporosis, weight loss, and ane-mia) on a gluten-containing diet, were enrolled in thestudy (Table I). The presence of gastrointestinalcancers, cow’s milk allergy, or other enteropathiesknown to be associated with CD-like duodenalalterations7,8 was considered as exclusion criterion.All patients underwent serum IgA EMA and/or anti-

tTG antibody detection, upper endoscopy with multiple

Table I. Demographic characteristics and clinical

spectrum of the 559 patients enrolled in the study

Age [mean (range)] 34.21 (9–73) ySex [M/F] 206/353Relatives with CD [No. of pts (%)]

First-degree 163/559 (29.2%)Second-degree or higher 129/559 (23.1%)

Intestinal symptoms [No. of pts (%)]Abdominal pain/swelling 405/559 (72.5%)Diarrhea 317/559 (56.7%)Dyspepsia 152/559 (27.2%)

Extraintestinal symptoms [No. of pts (%)]Osteopenia/osteoporosis 189/559 (33.8%)Dental enamel hypoplasia 171/559 (30.6%)

Malabsorption/anemia [No. of pts (%)]Weight loss 233/559 (41.7%)Iron deficiency anemia 241/559 (43.1%)

Abbreviation: CD, celiac disease; pts, patients.

Translational ResearchVolume -, Number - Picarelli et al 3

duodenal biopsy sampling, histologic analysis, and or-gan culture to detect IgA EMA and/or anti-tTG anti-bodies in supernatants. All procedures followed in thisstudy were made for diagnostic purposes and, therefore,were in accordance with the ethical standards of the in-stitutional committee responsible for human experi-mentation. Furthermore, a written informed consentwas obtained from each patient being studied.

Upper endoscopy. All patients underwent upper en-doscopy after at least 8 hours of complete fasting.Five biopsy samples of the second portion of duodenumwere collected from each patient. Three biopsies weresubmitted to histologic analysis, whereas the other 2samples were used for the organ culture system.

Histologic analysis. Duodenal biopsy samples werefixed overnight in 4% formalin. After orientation, inclu-sion in paraffin, and cutting, morphologic analysis wasperformed by hematoxylin-eosin staining. To betterevaluate the small intestine mucosal architecture,villous height/crypt depth ratio was measured andvalues ,3:1 were considered indicative of morphol-ogic alteration. The number of IELs per 100 intestinalepithelial cells (IECs) was evaluated by the immuno-histochemical staining for CD3 lymphocyte surfacemarker. The upper limit of the reference interval was40 IELs/100 IECs. The histologic pattern wasevaluated blindly by 3 gastrointestinal patho-logists(agreement rate 97.5%) according to Marsh-Oberhuber classification, in which type 0 indicatesa normal histology sufficient to exclude CD, type I(infiltrative) is characterized by an increased numberof IELs, type II (hyperplastic) also indicates crypthyperplasia, and type III (destructive) is alsocharacterized by a partial (IIIa), subtotal (IIIb), ortotal (IIIc) villous atrophy.4 All type III lesions were

considered diagnostic for CD, whereas types I and IIwere considered indicative of CD-related earlyintestinal alterations. The occurrence of patchyabnormalities, evaluated on 3 duodenal biopsysamples collected from each patient, was interpretedas positive histologic finding.

Organ culture system. The culture medium, preparedwith Roswell Park Memorial Institute 1640 medium17 mL, fetal bovine serum 3 mL, l-glutamine (200mM) 0.2 mL, penicillin (10,000 UI/mL)-streptomycin(10,000 mg/mL) 2 mL, and gentamicin (10 mg/mL)0.04 mL (Gibco/Invitrogen, Carlsbad, Calif), waspreventatively stabilized at pH 7.4 and sterilized byfiltration with a 0.22 mm pore size filter (SigmaChemical, St. Louis, Mo). For each patient, 2duodenal biopsy samples (minimal weight required: 5mg/sample) were washed in physiological solution(NaCl, 9 g/L), placed in sterile tubes containing 500mL of medium, and then cultured for 48 h at 37�C,one in the presence and one in the absence of peptic-tryptic digest of gliadin (1 mg/mL) according to the‘‘in batch’’ method.13 Thereafter, culture supernatantswere collected and stored at –20�C until used. Alloperations were performed in a sterile environment.

Anti-endomysium antibody detection. IgA EMAweresearched in sera diluted 1:5 and in undiluted culture su-pernatants by indirect immunofluorescence analysis oncryostat sections of monkey esophagus (Eurospital,Trieste, Italy). After serum or supernatant incubation,the sections were stained with a fluoresceinisothiocyanate-conjugated anti-human IgA. Theresults, expressed as ‘‘positive/negative,’’ were eval-uated blindly by 2 trained observers, whose agreementrate was 99.6%. Positive EMA results were identifiedby the typical honeycomb-like staining pattern alongmuscularis mucosae (endomysium) that marks thecollagenous matrix of type 3 connective tissuesurrounding the smooth muscle fibers of the primateesophagus.

Anti-tissue transglutaminase antibody detection. IgAanti-tTG antibodies were measured in sera diluted1:101 and in culture supernatants diluted 1:5 byenzyme-linked immunosorbent assay (ELISA) onmicrotiter-plate wells coated with recombinant humantTG (INOVA Diagnostics, San Diego, Calif;distributed by Instrumentation Laboratory, Milan,Italy). Serum results, firstly quantified by an ELISAreader at 450 nm (A450nm), were compared witha standard reference curve and then expressed in U/mL. According to the manufacturer’s instructions, theantibody level 4 U/mL was used as a cutoff value toidentify anti-tTG positive results. As previouslyreported,12 culture supernatant results were expressed

Translational Research4 Picarelli et al - 2012

in A450nm, and the antibody level 0.300 was used ascutoff value to identify anti-tTG positive results.To investigate the reproducibility of the ELISA test,

we calculated the intra- and inter-assay coefficient ofvariations (CV). For the intra-assay variation, 20 refer-ence samples (10 sera and 10 culture supernatants) weretested 4 times in the same run; CV (range) was 0.8%–3.1%. For the inter-assay variation, the same 20 refer-ence samples were tested 1 time in 3 different runs;CV (range) was 1.9%–8.2%.

Follow-up protocol. To emphasize the diagnostic util-ity of the organ culture system, 30 patients from a totalof 95 cases identified as having mild enteropathy CDthrough the organ culture alone were put on a GFDand regularly monitored for 12 months to evaluate theirgluten-dependency. Because the above mentioned 30patients agreed to undergo the follow-up protocolwhereas the remaining 65 refused this procedure, it isreasonable to consider them as randomly selected,excluding any clinical or laboratory bias. Consistently,these patients appeared to be sex- and age-reflective ofthe overall group.Since the suitability of oat as part of a GFD is still

controversial,14 all the GFDs administered in this studyexcluded any oat-containing food. The compliance toGFD was assessed every month by means of a patientfood diary (control level 1) and a specific interviewwith a dietician (control level 2). At the same timepoints, serum EMA/anti-tTG antibodies were detectedas further index of adherence to GFD (control level 3)except for the seronegative patients, who were moni-tored up to the control level 2. All the patients showedan excellent compliance to GFD and completed thefollow-up protocol.The gluten-dependency was assessed by means of 2

quality of life standard indexes determined at baselineand after 6 and 12 months of GFD, as well as by evalu-ation of the clinical spectrum before and after dietarytreatment.

Quality of life assessment. As previously reported,15,16

the psychological general well-being (PGWB) and beckdepression inventory (BDI) questionnaires wereadministered to each one of the above mentionedpatients to assess, respectively, the well-being anddepression state.The PGWB questionnaire, a reliable and validated

tool to appraise the distress and the inner personal state,consists of 22 items each using a 6-graded Likert scale(1–6 rating scale). These 22 items explore 6 psycholog-ical subdimensions (anxiety, depressed mood, positivewell-being, self-control, general health, and vitality).The PGWB index is a sum score of the six sub-dimension measures ranging from 22 to 132, with

higher scores representing better the psychologicalwell-being.The BDI questionnaire, a self-reported instrument in-

tended to assess the presence and severity of depression,consists of 21 items each using a 0 to 3 rating scale. TheBDI index is a sum score of the individual item mea-sures ranging from 0 to 63, with higher scores indicativeof depression (,10: none or minimal depression;11–17: mild depression; 18–29: moderate depression;30–63: severe depression).Both PGWB and BDI questionnaires were adminis-

tered and evaluated blindly by an expert psychologist.

Statistical analysis. Qualitative variables includingEMA/anti-tTG antibody results, histologic findings,and clinical parameters were expressed as frequencies(both absolute and relative), whereas quantitative datasuch as PGWB and BDI indexes were expressed asmedian, quartile cutpoints (first and third quartile),and range (minimum and maximum value).Both inter-analysis and inter-patient differences

among EMA/anti-tTG antibody results determined inserum and organ culture of the study participantsdivided according to Marsh-Oberhuber histologic clas-sification were assessed by c2 test for qualitative and in-dependent data. The significance level was set at P #0.05.The strength of agreement among serum, organ cul-

ture, and histologic findings was estimated by Cohen’sk pairwise inter-test for nominal scales. In this test,the coefficient of agreement (Cohen’s kappa statistic,k) is 1 when there is perfect agreement between the clas-sification systems, 0 when there is no agreement betterthan chance, and ,0 when agreement is worse thanchance.17 According to Altman’s classification,18 thestrength of agreement was considered poor fork ,0.20, fair for 0.21 # k # 0.40, moderate for 0.41# k # 0.60, good for 0.61 # k # 0.80, and very goodfor 0.81 # k # 1.00.Comparisons among PGWB or BDI indexes deter-

mined at baseline, after 6 and 12 months of GFD in30 patients identified as having mild enteropathy CDthrough the organ culture alone, were performed by us-ing the analysis of variance for the Friedman ranks asfirst test (Friedman test). If the overall P value was#0.05, Dunn’s multiple comparison was used as posttest. The P values of#0.05 were considered significant.Differences between each clinical parameter evalu-

ated before and after 12 months of GFD in the same pa-tients described above were evaluated by McNemar testfor qualitative and paired data. The significance levelwas set at P # 0.05.The statistical evaluations were carried out by using

MedCalc Software v. 11.2.1 (MedCalc Software bvba,

Table II. EMA/anti-tTG antibody results in serum and organ culture of the study participants divided according

to Marsh-Oberhuber histologic classification

Study participants (n 5 559)

EMA/anti-tTG positive results (%) Inter-analysis differences

Serum Organ culture c2 P

Marsh-Oberhuber 0 type (n 5 244) 7 (2.9%) 27 (11.1%) 11.413 0.0007Marsh-Oberhuber I type (n 5 68) 51 (75%)* 62 (91.2%)* 5.233 0.0222Marsh-Oberhuber II type (n 5 6) 6 (100%)* 6 (100%)* na naMarsh-Oberhuber III type (n 5 241) 229 (95%)*,† 239 (99.2%)*,‡ 5.959 0.0146

Abbreviations: anti-tTG, anti-tissue transglutaminase; EMA, anti-endomysium; na, not applicable.The c2 and P values reported in the table refer to c2 test applied between EMA/anti-tTGantibody results determined in serumandorgan culture(inter-analysis differences). The symbols reported in the table refer to c2 test applied among EMA/anti-tTG antibody results highlighted in thestudy participants divided according to Marsh-Oberhuber histologic classification (inter-patient differences: * P , 0.0001 vs type 0 patients,† P , 0.0001 vs type I patients, ‡ P 5 0.0012 vs type I patients).

Table III. Strength of agreement among serum,

organ culture, and histologic findings

k SE 95% CIStrength ofagreement

Serology and histology 0.730 0.029 0.673–0.786 GoodOrgan culture and

histology0.662 0.031 0.601–0.723 Good

Serology and organculture

0.852 0.022 0.808–0.896 Very good

k 5 Cohen’s kappa statistic.

Translational ResearchVolume -, Number - Picarelli et al 5

Mariakerke, Belgium) for qualitative data and Graph-Pad Prism package v. 3.0 (GraphPad Software Inc.,San Diego, Calif) for quantitative data. For statisticalsamples below 8 patients, any inferential test was con-sidered not applicable.

RESULTS

Serum EMA/anti-tTG antibodies were positive in293/559 (52.4%) patients enrolled in the study. At thehistologic examination, 241/559 (43.1%) patients hadevidence of villous atrophy (78 with IIIa, 43 with IIIb,and 120 with IIIc type of Marsh-Oberhuber classifica-tion), 74/559 (13.2%) patients presented only intraepi-thelial lymphocytosis (68 with I and 6 with II type ofMarsh-Oberhuber classification), and 244/559 (43.7%)patients showed no histologic alterations suggestive ofCD (0 type of Marsh-Oberhuber classification). Organculture EMA/anti-tTG antibodies were positive in334/559 (59.7%) patients, without any difference be-tween the biopsies cultured in medium alone and thosesubjected to the in vitro gliadin-challenge.All patients being studied were divided in 4 groups

according to Marsh-Oberhuber histologic classification.Serum and organ culture antibody results of thesegroups were evaluated for inter-patient and inter-analysis differences (Table II). In patients with Marsh-

Oberhuber III, II, and I type, both serum and organculture antibody positive results were higher than inthose with Marsh-Oberhuber 0 type (P , 0.0001 foreach one). In patients with Marsh-Oberhuber III type,serum and organ culture antibody positive results werealso higher than in those with Marsh-Oberhuber I type(P , 0.0001 and P 5 0.0012, respectively). Antibodypositive results determined in organ culture were higherthan those highlighted in serum of patients with Marsh-Oberhuber III, I, and 0 type (P 5 0.0146, P 5 0.0222,and P 5 0.0007, respectively). As reported inTable III, the strength of agreement resulted good for se-rology vs histology (k5 0.730), good for organ culturevs histology (k5 0.662), and very good for serology vsorgan culture (k 5 0.852).In Table IV was reported the clinical spectrum of the

95 patients with organ culture positive results, negativehistology (27 with 0, 62 with I, and 6 with II type ofMarsh-Oberhuber classification), and serum positive re-sults in 64 of them (67.4%). As scheduled, 30 of thesepatients were treated for CD, and their quality of lifewas monitored over time. After 12 months of GFD,PGWB index significantly increased and BDI index sig-nificantly decreased (P, 0.001 for each one), testifyinga marked improvement in the quality of life (Figs 1 and2). Both PGWB and BDI indexes showed a similar im-proving trend among patients with Marsh-Oberhuber 0,I, and II type, as well as between seropositive and sero-negative patients (data not shown). In the same condi-tions, an improving trend of the clinical spectrum wasobserved without apparent differences among patientswith Marsh-Oberhuber 0, I, and II type, as well as be-tween seropositive and seronegative patients (Table V).

DISCUSSION

According to the latest European Society for PediatricGastroenterology, Hepatology, and Nutrition guide-lines, if the clinical spectrum and serum antibody tests

Table IV. Clinical spectrum of the 95 patients with organ culture positive results but negative histology

Relatives with CD (patients) HLA (patients) Clinical presentation (patients) Hemato-chemical data (patients)

Father/mother (n 5 41) DQ2 (n 5 89) Typical (n 5 68) Y Hemoglobin (n 5 54)Brother/sister (n 5 26) DQ8 (n 5 6) Atypical (n 5 27) Y Iron (n 5 54)Cousin (n 5 14) Y Ferritin (n 5 28)

Y Cholesterol (n 5 53)Y Triglycerides (n 5 15)[ Transaminase (n 5 27)

Abbreviation: CD, celiac disease.The Marsh-Oberhuber histologic classification and serum results for these patients are deductible in Table I.

Fig 1. Box and whiskers plots (median, first-third quartile, minimum-

maximum value) showing PGWB index changes from baseline to 6

and 12 months of GFD. Number of patients identified as having

mild enteropathy CD through the organ culture alone and subjected

to follow-up protocol, as well as median and quartile cutpoints (first

and third quartile) of PGWB index are reported in the graph bottom.

The symbol reported in graph refers to Friedman first test (Friedman

statistic: 29.19328, overall P, 0.0001) and Dunn’s multiple compar-

ison post test (*P, 0.001 vs tCD pts 2) applied among the aforemen-

tioned parameters.

uCD pts5 untreated CD patients; tCD pts 15 treated CD patients on

a GFD from 6months; tCD pts 25 treated CD patients on a GFD from

12 months.

Translational Research6 Picarelli et al - 2012

are suggestive of CD, an evidence of intestinal villousatrophy is necessary to confirm the diagnosis.5 How-ever, total villous atrophy is the extreme condition ofa modifying spectrum that can be revealed only in thesevere forms of CD.19 This implies that in case of patchyatrophy or in absence of intestinal damage, a featureeasily found in latent CD,6 the diagnosis could bemissed. Technical difficulties (eg, sufficient size,

correct orientation, and cutting of the biopsy samples),as well as the subjective evaluation of the intestinaldamage add further limitations to the histology-baseddiagnosis of CD.20 Indeed, even in centers witha good expertise in CD, about 11% of biopsy samplesare not suitable for a correct morphologic interpreta-tion.21 Several pathologists consider the intraepitheliallymphocytosis an important element in the diagnosisof patients without villous atrophy, although the currentliterature shows that an increased IELs number can benot specific for CD.7 Therefore, for patients with typesI–II of Marsh-Oberhuber classification, the real diffi-culty is to establish if the intraepithelial lymphocytosisis gluten-dependent or it is due to other possiblecauses.8,20 These considerations highlight the need toidentify a new tool able to fill the gaps currently presentin the diagnostic procedures of CD.Given that EMA and anti-tTG antibodies are detect-

able in culture supernatants of duodenal biopsies fromuntreated CD patients,11,12 we evaluated if this organculture systemmay support the IEL count and serum an-tibody tests in diagnosing the mild forms of CD. For thispurpose, we assessed the organ culture system with re-spect to villous height/crypt depth ratio, IEL count, andserum antibody tests performed in 559 consecutive pa-tients waiting for a diagnosis of CD. Data highlightedthat 241 (43.1%) of these patients belonged to type IIIof Marsh-Oberhuber histologic classification, suggest-ing a clear diagnosis of CD. However, 293 (52.4%) pa-tients showed serum antibody positive results. Giventhat some of these patients presented positive serologybut normal small intestine mucosal architecture, wesuggest that the histology-based diagnosis of CD needsto be supported by specific serum antibody tests.Surprisingly, 334 (59.7%) patients showed organ cul-

ture antibody positive results. In particular, this wasfound in 239 out of the 241 patients belonging to typeIII of Marsh-Oberhuber classification. The 2 patientswith positive histology (one with IIIa and the otherone with IIIb type of Marsh-Oberhuber classification)and organ culture negative results also showed negativeserology. This discrepancy could be due to technical

Fig 2. Box and whiskers plots (median, first-third quartile, minimum-

maximum value) showing BDI index changes from baseline to 6 and

12 months of GFD. Number of patients identified as having mild en-

teropathy CD through the organ culture alone and subjected to

follow-up protocol, as well as median and quartile cutpoints (first

and third quartile) of BDI index are reported in the graph bottom.

The symbol reported in graph refers to Friedman first test (Friedman

statistic: 42.46667, overall P, 0.0001) and Dunn’s multiple compar-

ison post test (*P, 0.001 vs tCD pts 2) applied among the aforemen-

tioned parameters.

uCD pts5 untreated CD patients; tCD pts 15 treated CD patients on

a GFD from 6months; tCD pts 25 treated CD patients on a GFD from

12 months.

Translational ResearchVolume -, Number - Picarelli et al 7

difficulties, such as incorrect antibody detection or inap-propriate cutting of the biopsy samples. After a carefulreview (data not shown), the antibody re-testing con-firmed negative results whereas the histologic revisionwith biopsy re-cutting showed normal mucosal architec-ture (1 case with 0 and the other one with I type ofMarsh-Oberhuber classification), highlighting the highsensitivity and specificity of the CD-associated antibodydetection. However, contrasting with the 239/241 caseswith organ culture positive results, only 229/241 pa-tients showed positive serology. Even in patients belong-ing to types I–II and 0 of Marsh-Oberhuberclassification, the organ culture positive results werehigher than those highlighted in serum. Although thestrength of agreement for serology vs histology and or-gan culture vs histology was good, and an even betterlinkage was observed between serology and organ cul-ture, the entire data highlight that the organ culture sys-tem seems to be more sensitive than both histologic andserologic analyses. In addition, since the organ culture is

able to evaluate the production/release of pathogno-monic antibodies directly from the intestinal mucosa,only this system may be considered really specific forCD, confirming previous suggestions about its employ-ment to diagnose cases without villous atrophy22,23 orseronegative patients.12,24,25 A limitation of our studycould be the assessment of eventual patchy abnormali-ties, which has been performed only on 3 duodenal bi-opsy samples. Further studies evaluating a greaternumber of biopsy samples (4–6 biopsies) could be usefultominimize the rate of false negative histologic findings.As a sign of appropriateness of the organ culture in di-

agnosing the mild forms of CD, the 95 patients identi-fied only through this system presented a familyhistory of CD, HLA-DQ2 or -DQ8 type, and symptomsand signs suggestive of CD despite the absence of vil-lous atrophy. The marked improvement in the qualityof life, together with the improving trend of the clinicalspectrum obtained in a third of these patients after 12months of GFD further support the reliability of the or-gan culture system in diagnosing CD. As a slight limi-tation, it has been recently reported that some non-CDpatients (eg, patients with irritable bowel syndrome)may have an improvement in symptoms on a GFD.26

However, the presence of circulating and/or localEMA/anti-tTG antibodies in association to HLA-DQ2/8 type should question the absence of CD also in pa-tients with negative histology, as the case of our study.It is not easy, therefore, to establish the boundary be-tween CD and non-CD patients who may benefit froma GFD.On the other hand, it has been recently demonstrated

that anti-tTG are able to inhibit intestinal epithelial celldifferentiation, induce intestinal epithelial cell prolifer-ation, increase epithelial permeability, activate mono-cytes, disturb angiogenesis, increase vascularpermeability, and induce neuronal apoptosis, suggestingthat these antibodies may have a functional role in thepathogenesis of CD.27-29 This lays emphasis on theneed to diagnose CD in all antibody-positive patients,also in the absence of intestinal damage. Moreover, al-beit little is known on the long-term effects of CD underdiagnosis in patients presenting 0–I–II type of Marsh-Oberhuber classification, they could have an increasedrisk to develop other autoimmune diseases or malig-nancy.30,31 Together with the evidence that a delay inthe diagnosis of CD causes an increase in healthcarecosts,32 the growing comorbidity and mortality ratessuggest the need to diagnose CD and start a GFD assoon as possible. Data obtained in the present studyshow that the organ culture system may be useful forthese purposes.In conclusion, our data confirm and extend recent

studies highlighting the in vitro diagnosis as useful

Table V. Clinical spectrum of 30 patients with organ culture positive results but negative histology before and

after 12 months of GFD

Marsh-Oberhuber (patients) Clinical spectrum Before GFD After GFD c2 P

0 type (n 5 7) Positive serology 3 (42.9%) 0 (0%) na naIntestinal symptoms 4 (57.1%) 0 (0%) na naExtraintestinal symptoms 1 (14.3%) 1 (14.3%) na naMalabsorption/anemia 2 (28.6%) 0 (0%) na na

I type (n 5 20) Positive serology 18 (90%) 0 (0%) 16.056 ,0.0001Intestinal symptoms 19 (95%) 0 (0%) 17.053 ,0.0001Extraintestinal symptoms 6 (30%) 1 (5%) 3.200 0.0625*Malabsorption/anemia 12 (60%) 1 (5%) 9.091 0.0010

II type (n 5 3) Positive serology 3 (100%) 0 (0%) na naIntestinal symptoms 3 (100%) 0 (0%) na naExtraintestinal symptoms 0 (0%) 0 (0%) na naMalabsorption/anemia 3 (100%) 0 (0%) na na

Abbreviations: GFD, gluten-free diet; na, not applicable.The c2 and P values reported in table refer to McNemar test applied between each clinical parameter evaluated before and after GFD.*Not significant.

Fig 3. Flow diagram suggesting a role for the organ culture system in the diagnostic protocol of CD.

Translational Research8 Picarelli et al - 2012

tool to identify the difficult cases of CD.33,34 In partic-ular, the organ culture system is a relatively inexpen-sive method, easy to set up in each laboratory,13 anduseful to diagnose CD in cases without villous atro-phy22,23 as well as in seronegative patients.12,24,25

Since the upper endoscopy with biopsy sampling ismandatory to diagnose CD, an intestinal fragmentcould be reserved for the organ culture (performedeither in the presence or absence of peptic-tryptic-gliadin) to assist the histology in identifying the diffi-

cult cases of CD (Fig 3), for whom the risk of missingthe diagnosis is very high.

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