Reliable markers of early developing coeliac diseases are needed. Coeliac autoantibodies in the serum or Marsh I inflammation may be indicators of subsequent coeliac disease.
To investigate whether determination of intestinal transglutaminase 2-targeted autoantibody deposits would detect early developing coeliac disease better than previous methods.
The study investigated patients previously excluded for coeliac disease: 25 had positive serum coeliac autoantibodies (endomysial), 25 antibody-negative had Marsh I, and 25 antibody-negative had Marsh 0 finding. Seven (median) years after baseline investigation, new coeliac cases were recorded, and small bowel biopsy was offered to the rest of the patients. Serum and intestinal coeliac autoantibodies and intraepithelial lymphocytes were assessed as indicators of developing coeliac disease.
Seventeen patients had developed coeliac disease: 13 in the autoantibody-positive group, three in the Marsh I group and one in the Marsh 0 group. At baseline, intestinal coeliac autoantibody deposits had a sensitivity and specificity of 93% and 93% in detecting subsequent coeliac disease, CD3+ 59% and 57%, γδ+ 76% and 60%, and villous tip intraepithelial lymphocytes 88% and 71%, respectively.
Endomysial antibodies with normal histology indicates early developing coeliac disease. Transglutaminase 2-targeted intestinal autoantibody deposits proved the best predictor of subsequent coeliac disease.
The diagnosis of coeliac disease requires the presence of small intestinal mucosal villous atrophy and crypt hyperplasia (Marsh III).1, 2 However, mucosal intraepithelial lymphocytosis evincing normal villous architecture (Marsh I) precedes this lesion. Marsh I lesion may thus indicate early developing coeliac disease, but this finding is also associated with other disorders. Only 2–10% of patients are eventually shown to suffer from genetic gluten intolerance.3, 4 Increased densities of γδ+5 or villous tip intraepithelial lymphocytes (IELs)6 have been of better positive predictive value than IELs in general in the diagnosis of early developing coeliac disease, but so far no single marker is considered a reliable indicator of this condition.
Serum immunoglobulin (Ig)A-class endomysial (EmA)7 and transglutaminase 2 (TG2)8, 9 antibodies are powerful tools in disclosing coeliac disease with overt villous atrophy. Some studies have suggested, however, that these autoantibodies might be less useful in the case of early developing coeliac disease,10–12 as the antibodies are thought to enter the circulation only after severe villous atrophy has developed. In contrast, there are also cases where coeliac autoantibodies have appeared in the serum before the development of villous atrophy, implying that they might be valuable in identifying patients with early developing coeliac disease.5, 13, 14
Previous studies have demonstrated that TG2 is the antigen for in vitro binding of coeliac IgA-class antibodies to intestinal and extraintestinal tissues.15, 16 Coeliac autoantibodies are produced in the small bowel mucosa,17–19 and it has been recognized that the small intestinal epithelial basement membrane region contains deposited IgA in untreated coeliac disease, though the target of this deposition remained unknown for decades.20, 21 We recently demonstrated in vivo that deposited extracellular IgA in the small intestinal mucosa targets TG2 in untreated coeliac disease patients.22 Further, our previous findings have suggested that TG2-targeted intestinal autoantibody deposits might be detectable before the development of overt villous atrophy and precede the appearance of serum coeliac autoantibodies.22, 23 These preliminary results raised the intriguing hypothesis that intestinal autoantibody deposits targeted against TG2 would precede manifest coeliac mucosal lesion, and further that this might furnish a method applicable in the diagnosis of early developing coeliac disease.
Recent evidence shows that coeliac disease is no longer restricted to severe enteropathy,24 and that patients can suffer from gluten-dependent clinical symptoms and complications of coeliac disease even before small bowel mucosal villous atrophy has developed.25, 26 Such patients benefit of gluten-free diet, and therefore, reliable markers of early developing coeliac disease are needed. However, previously published evidence of this condition has mainly consisted of case reports. This long-term follow-up study focused for the first time on early developing coeliac disease in a large, well-defined patient material. The aim was first to establish whether patients with normal villous architecture but positive serum coeliac autoantibodies, or Marsh I lesion in the small bowel mucosa, suffer from early developing coeliac disease. Secondly, we investigated whether intestinal TG2-targeted autoantibody deposits are superior in detecting early developing coeliac disease than the CD3+, γδ+ and villous tip IELs currently employed.
Materials and methods
Patients and study design
Patients who initially had normal villous architecture but were subsequently shown to develop manifest coeliac disease with villous atrophy and crypt hyperplasia are referred as early developing coeliac disease patients in this study. This condition has also been regarded as latent coeliac disease in the literature.
From 1995 to 1999 altogether 707 adult patients underwent small intestinal biopsy at the Department of Gastroenterology and Alimentary Tract Surgery in Tampere University Hospital due to suspicion of coeliac disease. Small bowel mucosal villous morphology and the densities of CD3+ and γδ+ IELs were determined in all. A coeliac disease diagnosis was established when villous atrophy and crypt hyperplasia (Marsh III lesion) were present in the biopsy specimens.2 In 545 patients the villous architecture was normal and coeliac disease was hence excluded. However, 29 of them had positive coeliac autoantibodies in the serum and either Marsh 0 or Marsh I findings in the mucosa; in addition, 69 antibody-negative patients had Marsh I lesions in the small bowel mucosa.
According to power calculations the number of patients in each study group was set at 25. The study groups consisted of age- and sex-matched patients with normal villous architecture: a random sample of 25 of 29 patients with positive serum coeliac autoantibodies, 25 antibody-negative with Marsh I and 25 antibody-negative with Marsh 0 finding in the small bowel mucosa. The demographic data and primary reasons for coeliac disease suspicion in the 75 study patients are shown in Table 1.
|Patients with positive coeliac autoantibodies in the serum (n = 25)||Patients with Marsh I finding in the small bowel mucosa* (n = 25)||Patients with Marsh 0 finding in the small bowel mucosa* (n = 25)|
|Female, n (%)||19 (76)||19 (76)||19 (76)|
|Age, median (range)||39 (18–67)||39 (17–73)||39 (18–68)|
|Primary reason for coeliac disease suspicion, n (%)|
|Abdominal symptoms†||8 (32)||18 (72)||14 (56)|
|Anaemia or malabsorption||2 (8)||2 (8)||6 (24)|
|Extraintestinal symptoms‡||7 (28)||5 (20)||5 (20)|
|Coeliac disease at-risk groups§||8 (32)||–||–|
The follow-up investigation took place in 2004. Altogether 69 of the original 75 subjects could be traced. The emergence of coeliac disease during the follow-up was recorded in patient files, and all patients without prior development of coeliac disease were invited for a follow-up visit a median of 7.3 years after baseline investigation. At the follow-up, clinical symptoms were recorded. The daily consumption of gluten was analysed by a dietician according to a 4-day record of food intake to ascertain that patients had not reduced their gluten intake. Patients were asked to undergo upper gastrointestinal endoscopy and small bowel biopsy.
Finally, baseline (Figure 1) histological and serological findings in patients who were shown to have developed mucosal lesion compatible with coeliac disease during the whole follow-up were compared with patients again excluded for the disease by small bowel biopsy.
Serum coeliac autoantibodies
EmA (primate-type reticulin) and reticulin antibodies (ARA, rodent-type reticulin) have been shown to be directed exclusively to TG2 in both rodent and primate tissues.15, 16 During the study period, EmA replaced ARA in clinical practice. These two coeliac autoantibody tests have proved to be virtually identical in our laboratory,27 and are thus termed here coeliac autoantibodies. At the follow-up visit EmA was used together with the TG2 antibody test.
Serum IgA-class ARA and EmA were determined by an indirect immunofluorescence (IF) method; a serum dilution of 1:≥5 was considered positive in both. In ARA, a typical R1 pattern was required in rat kidney and liver sections;28 in EmA human umbilical cord was used as a substrate.7 Assessment of serum IgA-class TG2 was carried out by enzyme-linked immunosorbent assay (ELISA; Celikey, Pharmacia Diagnostics, GmbH, Freiburg, Germany) using human recombinant TG2 as antigen, and the lower limit of positivity was a unit value of 5.29
Small bowel mucosal morphology and IELs
Upon endoscopy, seven forceps biopsy specimens were taken from the distal part of the duodenum. Five of these were processed, stained with haematoxylin and eosin (H & E) and studied under light microscopy. The villous height–crypt depth ratios (Vh/CrD) were determined from several well-oriented biopsy samples from multiple sites in order to detect patchy forms of villous atrophy, as described previously.30 Vh/CrD < 2 was considered compatible with coeliac disease (Marsh III).
The villous tip IEL score/20 enterocytes was calculated from the mean value of five random villi. The reference value was set at 4.2 IELs/20 enterocytes.31 The correlation coefficients for intraobserver variation for villous tip IELs were 0.89 and for interobserver variation 0.87 in our laboratory.
Two small bowel biopsy specimens were freshly embedded in optimal cutting temperature compound (OCT; Tissue-Tec, Miles Inc, Elkhart, IN, USA), snap-frozen in liquid nitrogen and stored at −70 °C. Immunohistochemical stainings were carried out on 5-μm-thick frozen sections. CD3+ IELs were stained with monoclonal antibody Leu-4 (Becton Dickinson, San Jose, CA, USA) and γδ+ IELs with T-cell receptor (TCR)-γ antibody (Endogen, Woburn, MA, USA). Positive IELs were counted with a ×100 flat field light microscope objective throughout the surface epithelium; at least 30 fields measuring 1.6 mm in epithelial length were counted and IEL density expressed as cells/mm of epithelium.32 For comparison, in a case report the values of IELs are also given as IELs/100 enterocytes. CD3+ IELs correlated well with the total IEL density in the small bowel mucosa32 and thus a normal density of these cells indicated a Marsh 0 and increased density of a Marsh I finding.2 The reference values were set at 37 cells/mm for CD3+ IELs and 4.3 for γδ+ IELs.33 In our laboratory the correlation coefficients for intraobserver variation for CD3+ and γδ+ IELs were 0.95 and 0.98, and those for interobserver variation were 0.92 and 0.98 respectively. Evaluations of the specimens and calculations of the IELs were carried out by one investigator and without prior knowledge of disease history or laboratory findings.
Small bowel mucosal TG2-specific IgA deposits
In earlier studies we have shown that untreated coeliac disease patients have in vivo in situ IgA deposits on TG2 in their small bowel mucosa. When this IgA was eluted from the tissues, it targeted purified TG2 both in ELISA and Western blot. In addition, when TG2 binding to fibronectin was disrupted by chloroacetic acid, disappearance of extracellular IgA deposits was demonstrated in coeliac small bowel samples.22 The method used here was based on our previous experiments to detect TG2-specific antibodies in situ in tissue sections by their co-localization with TG2 when double-labelled by IF.
In the present study, small bowel mucosal TG2-specific IgA deposits were investigated in each patient with an available frozen small bowel specimen at baseline. From each of these cases altogether six unfixed, 5-μm-thick sections from frozen small bowel specimens were processed, three for investigating IgA deposits and three for double-colour labelling for both IgA and TG2 by direct IF. IgA was detected using fluorescein isothiocyanate-labelled rabbit antibody against human IgA (Dako AS, Glostrup, Denmark) at a dilution of 1:40 in phosphate-buffered saline (PBS, pH 7.4). In coeliac disease a clear subepithelial IgA deposition can be found below the basement membrane along the villous and crypt epithelium and around mucosal vessels; in contrast in normal small bowel samples IgA is detected inside the plasma and epithelial cells only.22, 23 The evaluation was carried out blindly without knowledge of the disease history or laboratory findings. For the double labelling, sections were stained for human IgA (green, as above) and for TG2 (red) using monoclonal mouse antibodies against TG2 (CUB7402, NeoMarkers, Fremont, CA, USA) followed by rhodamine-conjugated antimouse immunoglobulin antibodies (Dako), both diluted 1:200 in PBS. More than 500 small bowel specimens have been investigated for IgA deposits in our laboratory so far, and intraobserver and interobserver variations have both been 98% in the detection of the presence or the absence of TG2-targeted IgA deposits between five investigators.
The study patients were genotyped for human leucocyte antigen (HLA)-DQB1*02, DQB1*0302 and DQA1*05 alleles using the DELFIA Coeliac Disease Hybridization Assay (Perkin-Elmer Life and Analytic Sciences, Wallac Oy, Turku, Finland). DQB1*02 and DQA1*05 are associate alleles for HLA DQ2 and DQB1*0302 for HLA DQ8, and these haplotypes are found in 96–100% of coeliac disease patients.34, 35
The size of the study groups was determined by power calculations: the assumption was that at least 50% of patients with positive coeliac autoantibodies in the serum and at most 10% of patients with negative autoantibodies having a Marsh I or a Marsh 0 finding at baseline would develop overt coeliac disease during the study. α was given the value of 0.05 and the power was 90%. In order to elicit significant differences between the study groups the number of patients in each group had to be at least 23, and was finally set at 25.
Quantitative data were expressed as medians and ranges. Statistical differences between study groups were evaluated using the Pearson chi-square test, Fisher's exact test or Mann–Whitney U-test, as appropriate. P < 0.05 was considered statistically significant. Sensitivities and specificities of different markers in detecting early developing coeliac disease were calculated. All calculations were performed with spss (version 12.0.1).
The study protocol was approved by the Ethics Committee of Tampere University Hospital. Informed consent was obtained from all study subjects.
Coeliac disease diagnosed on clinical grounds during the follow-up
In 2004, altogether 15 with initially normal villous architecture had developed small bowel mucosal villous atrophy and crypt hyperplasia, and hence were diagnosed with coeliac disease by routine clinical follow-up before study enrolment. Twelve were in the autoantibody-positive group, two in Marsh I and one in Marsh 0 group (Figure 1, Table 2). In addition, two patients from the original study group had adopted a complete gluten-free diet during the follow-up without proper histological verification of the disease. They were excluded from further evaluations and considered not to have coeliac disease.
|Number and study group||Gender||Age (years)||Primary reason for CD suspicion||HLA DQ2/ DQ8||Serum IgA-class coeliac autoantibodies||Intestinal TG2- specific IgA deposits||CD3+ IELs (Marsh I)||γδ+ IELs||Villous tip IELs||Follow-up time before the development of villous atrophy (years)||Clinical manifestations at the time of the CD diagnosis|
|1. Ab+||F||64||Mouth ulcers||ND||+||+||+||+||+||0.3||Mouth ulcers|
|2. Ab+||F||36||Family history||ND||+||+||+||+||+||1.0||None|
|3. Ab+||F||27||Family history||+/−||+||+||−||+||+||1.0||Abdominal pain|
|4. Ab+||M||44||Neurological symptoms||+/−||+||+||+||+||ND||1.4||Neurological symptoms|
|5. Ab+||F||62||Abdominal distension||ND||+||+||+||−||+||1.5||Abdominal distension|
|6. Ab+||F||47||Abdominal distension||+/−||+||+||−||−||+||2.0||Abdominal distension|
|7. Ab+||F||42||Autoimmune thyroid disease||+/−||+||+||+||+||+||2.2||Fatigue|
|8. Ab+||F||50||Abdominal pain||ND||+||ND||−||+||+||2.6||Abdominal pain|
|9. Ab+||M||32||Diarrhoea||+/−||+||+||−||+||+||4.0||Abdominal pain|
|10. Ab+||F||39||Loose stools||+/−||+||+||+||+||+||4.3||Loose stools|
|11. Ab+||F||67||Mouth ulcers||ND||+||+||−||+||+||4.3||Loose stools|
|12. Ab+||F||26||Elevated liver enzymes||+/−||+||+||+||+||−||4.7||Abdominal pain|
|13. Ab+||F||66||Type 1 diabetes||+/−||+||+||−||−||+||5.2*||Mouth ulcers|
|14. Marsh I||F||67||Loose stools||+/−||−||+||+||+||−||1.7||Loose stools|
|15. Marsh I||M||32||Arthritis||+/−||−||ND||+||+||+||3.4||Arthritis|
|16. Marsh I||M||34||Diarrhoea||+/+||−||−||+||−||+||7.4*||Diarrhoea|
|17. Marsh 0||F||68||Abdominal pain||+/−||−||+||−||+||+||1.3||Abdominal pain|
At the follow-up visit two new coeliac disease patients with typical small bowel histological findings were detected: one in the autoantibody-positive group and one in the Marsh I group (Table 2, Figures 1 and 2). At follow-up, both new coeliac disease patients had positive serum EmA and TG2 antibodies. During the follow-up, three patients had developed severe heart disease and endoscopy was considered unethical (Figure 1).
All patients except those with histologically proven coeliac disease were consuming a normal, gluten-containing diet throughout the study period. The remaining 30 patients who underwent endoscopy were again excluded for coeliac disease; all showed normal villous architecture in the follow-up biopsy specimens and were negative for serum EmA and TG2 antibodies. This means that five patients who were autoantibody-positive at baseline had undergone negative seroconversion; at baseline, one of three with a frozen sample available had detectable intestinal IgA deposits, while at the end of the follow-up it was one of four. All five patients undergoing negative seroconversion had clinical symptoms suggesting coeliac disease at baseline, and all had Marsh 0 finding in the small bowel mucosa.
Characteristics of early developing coeliac disease patients
Baseline symptoms, serological and histological findings in the 17 patients with established early developing coeliac disease (at the time coeliac disease was excluded) are shown in Table 2. Fifty-two percentage (13 of 25) of patients with baseline-positive autoantibodies in the serum, 12% (three of 25) of patients with Marsh I and 4% (one of 25) of patients with Marsh 0 at baseline were later shown to have developed villous atrophy compatible with coeliac disease (P < 0.001). Of the autoantibody-positive patients with early developing coeliac disease, seven had Marsh I and six had Marsh 0 findings in the small bowel mucosa at baseline. The presence of intestinal IgA deposits in patients with early developing coeliac disease and in patients again excluded for coeliac disease at baseline and at the end of the follow-up is shown in Table 3. Baseline serum TG2 antibody results were available in five patients developing coeliac disease during the follow-up. Two of these were positive for these antibodies, and also for EmA; three were negative for TG2 antibodies (one of whom had positive EmA).
|Baseline, n (%)||End of the follow-up, n (%)|
|Early developing CD patients, all||14/15 (93)*||15/15 (100)*†|
|CD autoantibody-positive group||12/12 (100)||12/12 (100)|
|Marsh I group||1/2 (50)||2/2 (100)|
|Marsh 0 group||1/1 (100)||1/1 (100)|
|Patients excluded for CD by biopsy, all||1/14 (7)*||1/28 (4)*‡|
|CD autoantibody-positive group||1/3 (33)||1/4 (25)|
|Marsh I group||0/8 (0)||0/12 (0)|
|Marsh 0 group||0/3 (0)||0/12 (0)|
There were no differences in median age or gender distribution between patients with early developing coeliac disease and patients excluded for coeliac disease. Forty-four percentage of patients with early developing coeliac disease and 10% of those excluded for coeliac disease had first-degree relatives affected by coeliac disease (P = 0.021). HLA DQ2 or DQ8 was detected in every early developing coeliac disease patient with an available sample (n = 12), and in 10 of 29 (35%) patients excluded for coeliac disease (P < 0.001).
Counting of total IELs (CD3+) at baseline failed to detect early developing coeliac disease (Table 4). Intestinal TG2-specific IgA deposits had the best sensitivity and specificity for the condition, 93% in both. The percentages of γδ+ and villous tip IELs were lower: the sensitivity of γδ+ IELs was 76% and specificity was 60%, and the corresponding values for villous tip IELs were 88% and 71% respectively (Table 5). Nonetheless, even γδ+ and villous tip IELs were statistically significantly more often elevated in patients with early developing coeliac disease than in patients excluded for the disease (Table 4). We also tested different combinations of the tests shown in Table 5. The combination of HLA DQ2/DQ8 and intestinal TG2-specific IgA deposits achieved the sensitivity of 91% and the specificity of 100% and HLA and serum autoantibody testing gave a 67% sensitivity and a 97% specificity. In other combinations, the maximal sensitivity was with villous tip IELs and HLA DQ2/DQ8, 82%, with the specificity of 89%. Some other combinations yielded the 100% specificity, but the sensitivity was at its best only 79% (villous tip IELs and intestinal TG2-specific IgA deposits). Thus, the combination did not provide any additional benefit.
|Early developing coeliac disease (n = 17)||Coeliac disease excluded by biopsy (n = 30)||P-value|
|Vh/CrD, median (range)||2.7 (2.0–5.0)||3.1 (2.4–4.3)||0.063|
|Increased density of CD3+ IELs (Marsh I), n (%)||10/17 (59)||13/30 (43)||0.371|
|Increased density of γδ+ IELs, n (%)||13/17 (76)||12/30 (40)||0.032|
|Increased density of villous tip IELs, n (%)||14/16 (88)||8/28 (29)||<0.001|
|Intestinal TG2-specific IgA deposits present||14/15 (93)||1/14 (7)||<0.001|
|Sensitivity (95% CI)||Specificity (95% CI)|
|Intestinal TG2-specific IgA deposits present||0.93 (0.70–0.99)||0.93 (0.69–0.99)|
|Serum IgA-class coeliac autoantibodies present||0.76 (0.53–0.90)||0.83 (0.66–0.93)|
|Increased density of villous tip IELs||0.88 (0.64–0.97)||0.71 (0.53–0.85)|
|Increased density of γδ+ IELs||0.76 (0.53–0.90)||0.60 (0.42–0.75)|
|Increased density of CD3+ IELs (Marsh I)||0.59 (0.36–0.78)||0.57 (0.39–0.73)|
|HLA DQ2/DQ8 present||1.0 (0.76–1.0)||0.66 (0.47–0.80)|
This study elucidated the natural history of coeliac disease. Altogether 17 patients with normal villous architecture at baseline, while consuming a gluten-containing diet, subsequently developed mucosal damage diagnostic for coeliac disease. Majority of cases had initially positive coeliac autoantibodies in the serum. When these TG2-targeted autoantibodies were examined where they are produced, in the small bowel mucosa, 93% of all patients with early developing coeliac disease were identified in the absence of villous atrophy.
Occasionally coeliac disease diagnosis can be problematic; small bowel biopsy is considered the gold standard in coeliac disease diagnosis despite involving some obvious difficulties in interpretation. Even in the diagnostics of overt coeliac disease, a false diagnosis can be made, because at least 10% of biopsy specimens are of poor quality36 and further, villous atrophy may occasionally be patchy.37 In this study multiple small bowel biopsies were taken from several sites in order to detect also patients with patchy mucosal lesion. There is still always a possibility that a patchy form of villous atrophy is missed, and hence new methods are needed.
The diagnosis of early developing coeliac disease is even more challenging; minor histological abnormalities are unspecific, subjective and difficult to interpret. According to the recent studies focusing on early developing coeliac disease it has become evident that the recognition of coeliac disease without villous atrophy is important,24–26 especially because the patients benefit of dietary treatment. In agreement with this also our patients with early developing coeliac disease suffered from various symptoms typical of coeliac disease before the development of overt villous atrophy (Table 2). However, we demonstrated that Marsh classification could not be utilized reliably in the detection of early developing coeliac disease. Therefore, patients with Marsh I lesion in the small bowel mucosa should not be advised to adhere to a gluten-free diet in the absence of additional evidence.
The value of villous tip IELs in discovering patients with early developing coeliac disease was superior to Marsh I lesion, as has also previously been shown in smaller series.6, 31, 38 The clear advantage of villous tip IELs is that they can be studied in routine H & E-stained sections, whereas other histological methods applied here required frozen samples. In this study increased densities of γδ+ IELs were not only indicative of early developing coeliac disease, but also 40% of patients excluded for coeliac disease also on re-evaluation had increased densities of these cells at baseline when coeliac disease was first suspected. False-positive γδ+ cells have similarly been reported elsewhere.13, 23
The diagnosis of both overt and early developing coeliac disease should be based on reliable evidence. Histology has its limitations, as stated above. Nor are coeliac autoantibodies 100% specific. Five autoantibody-positive patients in this study did not proceed to villous atrophy during the follow-up, though it is possible that a longer follow-up might reveal progression of the disease in these individuals. Antibody testing was changing during the study period, and TG2 antibody test was not available at the time of baseline investigations in most patients. However, EmA and TG2 antibody tests correlate closely,8, 29 and the sensitivity and specificity values of these tests have been equal,39 even though some occasional patients remain negative for EmA despite positive for TG2 antibodies, and vice versa. DQ is mandatory in the identification of patients with genetic gluten intolerance, and patients with HLA DQ2 or DQ8 having positive coeliac autoantibodies in the serum are highly likely to suffer from coeliac disease. However, HLA DQ2 and DQ8 alone can only be used when patients are excluded for coeliac disease, because they are common in the population in general.40
We consider that intestinal IgA deposits targeted against TG2 are currently the best method in revealing early developing coeliac disease. Although serological tests predicted the development of villous atrophy relatively well, four (24%) of 17 patients with histologically confirmed coeliac disease had negative serology at baseline. Furthermore, there were five patients who underwent spontaneous negative seroconversion. Only one of them had intestinal IgA deposits at baseline, again showing that the method is more specific than serology. Investigation of intestinal IgA deposits is a special method requiring frozen small bowel biopsy specimens, which limits its utility. This method should be available at least in special centres, because it is clearly beneficial in cases where the conventional histology is ambiguous, especially in cases where a second investigation and biopsy is considered to confirm the diagnosis of coeliac disease. By investigating these deposits in small bowel biopsy specimens, when coeliac disease was first suspected, we were able in the majority of cases to diagnose early developing coeliac disease before the development of forthcoming villous atrophy. However, patients with early developing coeliac disease do not yet fulfil the traditional European Society for Pediatric Gastroenterology and Nutrition diagnostic criteria for coeliac disease. We have now shown that the criteria are no longer valid; many patients suffer from gluten-dependent symptoms before the development of villous atrophy, and the criteria should thus be revised.
In conclusion, the detection of intestinal TG2-specific IgA deposits proved a powerful diagnostic tool in coeliac disease without villous atrophy. We recommend that these deposits be invariably investigated when early developing coeliac disease is suspected. The presence of these intestinal TG2-targeted deposits strengthens the diagnosis of coeliac disease and dietary treatment should be considered at least in symptomatic patients. Further, patients with coeliac disease suspicion but normal villous architecture having positive coeliac autoantibodies in the serum or increased density of γδ+ or villous tip IELs in the small bowel mucosa should be followed up, whereas patients with Marsh I alone do not require routine surveillance. The detection of intestinal TG2-specific IgA deposits provides a reliable means to discover patients with early developing coeliac disease, and thus the results of this study should be taken into consideration when the diagnostic criteria for coeliac disease are revised.
This study was supported by the Research Fund of the Finnish Coeliac Society, the Medical Research Fund of Tampere University Hospital, the Finnish Medical Foundation, the Foundation for Pediatric Research in Finland, the National Graduate School of Clinical Investigation, the Finnish Foundation of Gastroenterological Research, the Yrjo Jahnsson foundation and the Finnish Medical Society Duodecim.