IgA class anti-endomysial and anti-tissue transglutaminase antibodies in relation to duodenal mucosa changes in coeliac disease

Transcription

1 Pathology (2003) 35, pp IMMUNOLOGY IgA class anti-endomysial and anti-tissue transglutaminase antibodies in relation to duodenal mucosa changes in coeliac disease LORETE MARIA DA SILVA KOTZE *, SHIRLEY RAMOS DA ROSA UTIYAMA {, RENATO MITSUNORI NISIHARA {, VANESSA FERREIRA DE CAMARGO { AND SERGIO OSSAMU IOSHII * *Service of Gastroenterology and Digestive Endoscopy, Cajuru University Hospital, Pontifical Catholic University of Paraná, and ÀLaboratory of Immunopathology, Clinical Hospital, Federal University of Paraná, Curitiba, Paraná, Brazil Summary Aim: The aim of the present study was to correlate the serological methods of coeliac disease diagnostic tests (IgA EmA and IgA anti-ttg) with the histological findings of the duodenal mucosa. Methods and Results: Forty-seven patients were studied and the data were analysed by the Pearson correlation. Seven patients (15%) with normal mucosa were negative for both assays. Forty untreated patients showed 89% agreement between the two serological methods, with all samples (40/40) positive to EmA and 80% (32/40) positive to antittg. Eight positive samples to EmA, that were negative to anti-ttg, presented an increased number of intra-epithelial lymphocytes in the duodenal biopsy and clinical improvement with a gluten-free diet. Partial or total villous atrophy was detected with EmA titres equal to or higher than 1/10. The correlation coefficient between the two serological methods was R~ Conclusions: Both serological tests correlated very well with histological findings in negative patients and in those with high levels of antibodies. For patients with clinical evidence of CD but with low levels of antibodies, the combination of serological tests and intestinal biopsy is recommended. Key words: Coeliac disease, anti-endomysial antibodies, tissue transglutaminase antibody, duodenal mucosa. Received 26 March, revised 4 July, accepted 9 August 2002 INTRODUCTION Coeliac disease (CD) can be defined as a permanent intolerance to dietary gluten from wheat, barley, rye and oats, with high cellular and humoral immunological responses. According to Marsh, 1 the intestinal mucosa can be normal or present changes ranging from mild alterations in mucosal architecture to severe atrophy. Intra-epithelial lymphocytes (IEL) are increased in number 1 3 independently of the histological changes. Although the gold standard for the diagnosis of CD continues to be a small bowel biopsy, tests searching for circulating antibodies to gliadin (AGA), reticulin (ARA), endomysium (EmA) and, more recently, for tissue transglutaminase (ttg) are useful for screening, diagnosis and monitoring of compliance with a gluten-free diet. 4 6 IgA class anti-endomysial antibodies (IgA EmA) detected by indirect immunofluorescence techniques present high specificity and sensitivity. 7 EmA has been considered to be an autoantibody to ttg, an enzyme that cross-links and stabilises extracellular matrix proteins. 8 Tissue transglutaminase is the predominant antigen of endomysial antibodies, but is not the only one, at least in some patients. 9 The tissue transglutaminase antibody assay (IgA anti-ttg) is currently considered by some authors to be as good as IgA EmA testing and of more practical use because it is an immunoabsorbent test (ELISA), mainly for CD screening in large populations. 5,10 12 Many papers have reported a close correlation between the two tests in adults and children. 8,13 15 Several studies have demonstrated a correlation between IgA EmA titres and alterations in small bowel mucosa, 5,16 but few studies comparing the simultaneous results of EmA, anti-ttg and histological findings of the duodenal mucosa are available in the literature. 17 The aim of this study was to correlate simultaneously the titres of IgA EmA, the determination of anti-ttg IgA and the histological aspects of the duodenal mucosa in coeliac patients. PATIENTS AND METHODS A total of 47 patients from southern Brazil was studied: 40 with clinical evidence of CD (dyspepsia, chronic anaemia, diarrhoea, intestinal malabsorption, failure to thrive, delayed menarche, recurrent abortion) and seven on a gluten-free diet for more than 2 years, diagnosed by intestinal biopsy. Thirty-six were females and 11 were males, with a mean age of 35.7 years (range, 3 73 years) (Table 1). After the patients gave informed consent to participate in the study, upper gastrointestinal endoscopy was performed and three to five biopsy specimens were obtained with a standard forceps from the second part of duodenum of each patient, mounted on filter paper and stored in formalin. The material was stained with H&E. The histopathologist detected excess IEL, crypt hyperplasia and villous atrophy, classified as partial (PVA) or total (TVA). 18 IEL were counted by the method of Ferguson and Murray. 3 The normal number estimated by Kotze for the healthy population from the same geographic area was 24% (24 IEL per 100 epithelial cells). 19 Total serum levels of immunoglobulin A (IgA) were determined by turbidimetry 20 in order to avoid a false-negative result due to IgA deficiency. ISSN printed/issn online/03/ # 2003 Royal College of Pathologists of Australasia DOI: /

2 COELIAC DISEASE DIAGNOSTIC TESTS 57 Tests for EmA IgA were performed by the indirect immunofluorescence assay as previously described, 21 using human umbilical cord as substrate. 22 Sera were considered positive if fluorescence was observed at a dilution of 1/2.5 or more. All positive sera were titred up to the end point. The highest dilution yielding a positive reaction was reported as the result. Positive and negative controls were used for each batch. Serum IgA anti-tissue transglutaminase antibody (ttg) titres were measured by an enzyme-linked immunosorbent assay (ELISA) with guinea pig liver ttg, using a commercial kit (Inova Diagnostics Inc., USA), based on the method described by Dieterich et al. 23 Results higher than 20 units were considered positive. 24 The results were ordered according to the IgA EmA titres for correlation with IgA anti-ttg and with the histological findings. Data were analysed with Statistica (Microsoft, USA) software, using the method of Pearson correlation for the serological tests. The x 2 -test was used to compare the different male/female frequency. Values of Pv0.05 were considered significant. RESULTS There was a preponderance of female patients (Pv0.0001). Table 1 shows the age of the patients and the results of the determination of IgA EmA and IgA anti-ttg and the findings in the duodenal biopsies. In seven samples (15%) from patients on a gluten-free diet, the results were negative by both serological methods, characterising 100% concordance between the two techniques and total compatibility with the normal results observed in the biopsies. In the other samples (n~40) obtained from patients at diagnosis, the concordance between serological methods was 89%. We obtained 100% (40/40) positivity to IgA EmA with titres of 1/2.5 to 1/80, and 80% positivity (32/40) for IgA anti-ttg characterised as units. Of the eight IgA EmA-positive samples with titres of 1/2.5, six were negative for IgA anti-ttg, as were two of four IgA EmA-positive samples with titres 1/5 (Table 1). In these eight samples with discordant serological results, the intestinal biopsies, although normal in architecture, presented an increased number of IEL. These patients improved clinically with a gluten-free diet. The samples with IgA EmA titres equal to or higher than 1/10 were also positive for IgA anti-ttg and the duodenal mucosa presented partial or total atrophy with crypt hyperplasia, except for a 3-year-old child with a normal biopsy (Table 1). Statistical analysis by Pearson correlation yielded a correlation coefficient of R~0.797 between the two serological methods (Fig. 1). DISCUSSION The southern region of Brazil was settled mainly by Europeans and presents a high degree of ethnic admixture. Even considering variations determined by environmental factors, CD described in this geographic area is very similar to that described in developed countries. 19 As CD can be found at any age, we studied patients aged 3 73 years, with a mean age of 35.7 years. We included children older than 2 years because IgA EmA can appear after the second year of life, suggesting that the sensitivity of these antibodies may be age-dependent. 25 In this study there was a preponderance of female patients, in agreement with data reported by authors from different countries. 12,26 Routine checking for IgA deficiency is recommended in studies for CD serology because the sera can be falsely negative for IgA EmA and for IgA anti-ttg in IgAdeficient individuals. 4,6 In the present study, none of the patients presented IgA deficiency. IgA EmA have been considered to be a specific and sensitive marker of CD. These antibodies are detected by immunofluorescence techniques using monkey s oesophagus or human umbilical cord as substrate. 22 Recently, they were identified as antibodies to ttg, an enzyme that crosslinks and stabilises extracellular matrix protein. 8 Tissue transglutaminase has been found to be the major, if not the only, autoantigen of gluten-sensitive enteropathy. 23,27 The ELISA for IgA anti-ttg antibodies is quantitative and easy to perform and is a valid alternative to indirect immunofluorescence for IgA EmA in screening for suspected CD. 28 Many papers have reported high sensitivity and specificity of both IgA EmA 7 and IgA anti-ttg. 5,6,28 The concordance of the two tests varied from author to author, as shown in Table 2. In our study, the concordance between the tests was 100% only in the negative samples (n~7), in accordance with normal biopsies. These samples corresponded to coeliac patients on a gluten-free diet. Similar data were reported by different authors 6,13,23 confirming the tendency of EmA and anti-ttg to disappear with exclusion of gluten from the diet, although other studies showed positivity at low titres for prolonged periods of time in patients on a gluten-free diet. 14,28 Cataldo et al. 29 reported that anti-ttg assays are less satisfactory for monitoring dietary compliance, but our data showed that both tests yielded similar results. EmA IgA were positive in 100% (40/40) of the samples TABLE 1 Patient age and test data based on IgA EmA titres Number (n~47) Patient age mean (range) IgA EmA (titres) IgA anti-ttg mean (range) Duodenal biopsy (11 73) negative 8.14 (3 15) 7N (32 71) 1/ (2 21) 8N (21 45) 1/ (6 100) 2N, 1PVA, 1TVA (25 65) 1/ (65 390) 3TVA (3 53) 1/ (36 640) 1N*, 1PVA, 7TVA (5 60) 1/ (91 640) 8TVA* (18 63) 1/ ( ) 8TVA *Child. Abbreviations: N, normal; PVA, partial villous atrophy; TVA, total villous atrophy.

3 58 KOTZE et al. Pathology (2003), 35, February Fig. 1 Anti-endomysial (IgA EmA) 6 anti-transglutaminase (IgA anti-ttg) antibodies. Correlation: R~ TABLE 2 Correlation between IgA EmA and IgA anti-ttg in sera from coeliac patients Author (country) Sensitivity (%) Specificity (%) EmA ttg EmA ttg Palacios Sarrasqueta et al. 13 (Spain) Dickey et al. 5 (Northern Ireland) Levine et al. 8 (Israel) Gillett and Freeman 6 (Canada) Lock et al. 32 (United Kingdon) from patients at diagnosis, with titres ranging from 1/2.5 to 1/80, and IgA anti-ttg were positive in 80% (32/40) of the samples, ranging from 20 to 640 units. The concordance between the methods in 40 positive samples was 89%. Similar results have been observed by Palacios Sarrasqueta et al. 13 and Sugai et al. 24 with concordance of 84 and 87%, respectively, when testing sera of coeliac patients on a normal diet. Of eight samples with low EmA titres (1/2.5), six were negative for IgA anti-ttg, and of four with 1/5 IgA EmA, two were also negative for IgA anti-ttg. High titres of IgA anti-ttg were only seen in CD. Koop et al. 30 suggested the specificity of 76% for IgA anti-ttg arose from false positivity in low titres in patients with inflammatory bowel disease, chronic liver disease and diabetes mellitus. These results were not observed with IgA EmA. Similarly, Reeves et al. 31 obtained false-positive results for CD using IgA anti-ttg in patients with systemic autoimmune disease and Down syndrome. Thus, low titres of IgA anti-ttg may not be disease specific. Our results agree with those reported by Palacios Sarrasqueta et al. 13 and Levine et al. 8 who showed that the sensitivity and specificity of IgA EmA are greater than IgA anti-ttg. Lock et al., 32 in a pilot study, also reported that IgA EmA assay was a better test. Dickey et al. 5 stated that, although the ELISA IgA anti-ttg assay could be more convenient than IgA EmA testing, it offered no advantages in sensitivity or specificity if used in isolation. However, incomplete concordance between the two tests in terms of positivity means that combination screening with both assays offers higher sensitivity, since almost one-third of patients have only one antibody. Since some coeliac patients with normal serum IgA could be negative for both antibodies, biopsies should be performed in seronegative individuals deemed at high risk for CD. 5,33 It is not easy to compare different methods because of different patterns; titres of IgA EmA may not correspond exactly to units of IgA anti-ttg. In this study, 100% concordance was observed in the negative samples and in six positive samples with 1/2.5 IgA EmA titres, and two with 1/5 titres negative for IgA anti-ttg. We draw attention to the fact that there was wide variation in units of IgA anti-ttg for the same titre of IgA EmA (Table 1), mainly in titres of 1/10 or more. Table 1 reports the mean value of IgA anti-ttg corresponding to each titre of IgA EmA, which shows that there was no linear correspondence of values. These aspects justify the correlation coefficient obtained in the analysis of the results (R~ ) which was similar to those reported by Dieterich et al. 23 (R~0.862) and Sugai et al. 24 (R~0.9020). The conclusion is that a superposition of the two methods exists but is incomplete (Fig. 1). Another subject to consider when we compare data from different laboratories is the discrepancy in the techniques and kits employed. 34 Antiendomysium and anti-ttg antibodies are both highly efficient for routine laboratory screening. The choice of one or the other will depend on the available facilities. 11 The correlation between EmA and histological findings was 100% for the negative samples (patients on a glutenfree diet). For low titres (1/2.5) the mucosal architecture was preserved in all cases (100% concordance) and for titres of 1/5, two were normal, one presented partial villous atrophy, and one total villous atrophy. Kaukinen et al. 35 reported 10 cases of CD without villous atrophy; eight were EmA-positive and nine were anti-ttg-positive. Although all patients had clinical complaints compatible

4 COELIAC DISEASE DIAGNOSTIC TESTS 59 with CD, if only duodenal biopsies were performed in these 10 patients the diagnosis of CD could have been missed. With the exclusion of gluten from the diet, all the patients improved. Thus, IgA EmA can be positive before histological changes in the mucosa can be demonstrated, even with an increased number of IEL. This fact emphasises the need for both serum determinations and a duodenal biopsy for the correct diagnosis of the nontypical forms of clinical presentation of CD, as suggested by other authors. 5,36 Some IgA EmA-positive patients may also have milder degrees of enteropathy with intact villi. 37 However, clinicians need to be aware of the fact that villous atrophy is common in seronegative patients and a duodenal biopsy should be considered if the clinical picture is suggestive. A biopsy is also more likely to identify milder degrees of enteropathy without villous atrophy. 1 Also, we should keep in mind that, even though IgA EmA is currently the preferred serological test for selecting suspected coeliac patients for duodenal biopsies, false-negative cases have been reported and may be more common than suggested by previous studies. 38 Rostami et al. 16 reported disappointing sensitivity of EmA, particularly for PVA, in contrast to the present results. With 1/10 to 1/80 IgA EmA titres, the correlation with histological changes was evident. An exception was a 3-year-old child with normal mucosa but with 1/20 IgA EmA. Probably the site of biopsy did not demonstrate lesions because in CD the lesions can be patchy. 39 Picarelli et al. 40 reported CD with mild enteropathy and recommended multiple biopsies. We should also consider that susceptibility to gluten damage may vary in severity. The correlation between the determinations of IgA antittg and histological findings was similar to those described for IgA EmA: 100% concordance in the negative samples; discordance in one positive case with 21 units and normal histology, and in two positive cases with 52 and 100 units but also with preserved mucosa. In the presence of high levels, a maximum of 640 units, partial or total villous atrophy was detected. Fabiani and Catassi 17 reported that in active CD, anti-ttg antibodies were significantly higher in patients with grade III enteropathy than in those with grade II enteropathy. Koop et al. 30 reported that detection of mild to high levels of IgA antittg is highly specific for CD; however, in the presence of low levels, there is an overlap with liver disease, inflammatory bowel disease and diabetes. It has been suggested that susceptibility to gluten damage may vary in severity among individuals and in the same individual with time, as also does the recovery with an adequate diet. 41 Seven patients on a gluten-free diet for at least 24 months had negative serological tests and showed complete recovery of the mucosal changes. Thus, the determination of IgA EmA or IgA anti-ttg could be indicated for monitoring the diet in CD patients. The earlier appearance of IgA EmA instead of IgA antittg in the serum of coeliac patients may be related to the amount of gluten in the diet or may occur on an individual basis. Troncone et al. 42 reported that in five of 10 patients undergoing a gluten challenge, IgA EmA were detected earlier than IgA anti-ttg antibodies. For the authors, ttg-based ELISA is an effective diagnostic test, although immunofluorescent-based assays are more sensitive, particularly during a gluten challenge. Human ttg-based ELISA represents a cost-effective strategy for identifying both symptomatic and atypical forms of CD. Both children and adults presented the same results for serum antibodies. 15 Identification and treatment of CD in an outpatient setting could have significant implications for reducing long-term morbidity and can produce major savings in future health care costs. 43 According to Feighery et al., 37 small bowel histology and or serology show similar predictive values in the diagnosis of CD. A combination of clinical data, serology and histology is required for an effective diagnosis of this disorder. Exclusive reliance on histology or serology will result in failure to make a diagnosis in a significant proportion of patients. 37 CONCLUSIONS The present results revealed that in patients with high antibody levels there was an excellent correlation between IgA EmA and IgA anti-ttg, with no risk of false-negative results. However, at low levels, the results obtained with IgA EmA were more reliable. Both serological tests correlated very well with histological findings in negative patients and in those with high antibody levels. For patients with clinical evidence of CD but with low antibody levels, a combination of serological tests and intestinal biopsy is recommended. Address for correspondence: Professor L. M. S. Kotze, Rua Bruno Filgueira, 369 Conjunto 1205, CEP , Curitiba, Paraná, Brazil. loretekotze@hotmail.com References 1. Marsh MN. Coeliac Disease. Oxford: Blackwell, 1992; Chapter 6, Mucosal pathology in gluten sensitivity. 2. Ferguson A. Intraepithelial lymphocytes of the small intestine. Gut 1977; 18: Ferguson A, Murray D. Quantitation of intraepithelial lymphocytes in human jejunum. Gut 1971; 12: Chan AW, Butzner JD, McKenna R, Fritzler MJ. Tissue transglutaminase enzyme-liked immunosorbent assay as a screening test for coeliac disease in pediatric patients. Pediatrics 2001; 107: E8. 5. 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