False positive reactions for IgA and IgG anti-tissue transglutaminase antibodies in liver cirrhosis are common and method-dependent

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Abstract

Background

Conflicting results were obtained in the assay of anti-transglutaminase (anti-tTG) autoantibodies in patients with chronic liver disease. In order to establish whether this was attributable to methodological differences, anti-tTG antibodies were assayed in a large number of patients suffering from liver cirrhosis (LC).

Methods

54 patients with LC and 29 patients suffering from celiac disease (CD), used as controls, were tested for IgA and IgG anti-tTG with 11 different commercial methods.

Results

In the patients with LC, positivity ranged from 0% to 33.3% for IgA anti-tTG and from 0% to 11.1% for anti-tTG of the IgG class. The largest number of false positives was found with methods that used tTG in association with gliadin peptides as antigen substrate. A significant association was found between IgA anti-tTG antibodies and serum immunoglobulin concentration.

Conclusions

The results of the various methods of assaying anti-tTG antibodies in patients with LC are highly variable, and the positives found are generally false positives, partly due to the high immunoglobulin concentration.

Introduction

Celiac disease (CD) is an immunomediated disorder characterised by multiform clinical expressions and the presence of circulating antibodies [1]. Anti-endomysial autoantibodies (EMA) appear at the early stages of the disease, and the EMA test is considered an excellent, non-invasive diagnostic test due to its high sensitivity and specificity [1], [2]. However, this test, based on the indirect immunofluorescence method using primate esophagus as substrate, is not easily applied for large-scale screening. Since Dieterich et al. [3] identified tissue transglutaminase (tTG) as the main autoantigen responsible for EMA positivity in CD patients, it has been possible to prepare immunoenzymatic assays (ELISA) to test for anti-tTG autoantibodies. The first ELISA tests, using guinea pig liver (gp-tTG) as tTG antigen source, demonstrated good diagnostic sensitivity [4], [5], [6], [7], but also low specificity due to the presence of a large proportion of false positive results, especially in patients with chronic liver disease [8], [9]. Later studies demonstrated that these false positives were due to impurities in the antigen source [9], [10], and that the use of the ELISA gp-tTG test is consequently not very practicable for screening patients with persistent, otherwise unexplained hypertransaminasemia.

Subsequently, in a trial conducted on a large number of patients with chronic liver disease, Carroccio et al. [9] demonstrated that the use of recombinant human tTG (rh-tTG) considerably improved the specificity of the test. Recently, however, Vecchi et al. [11] found a high rate (31.6%) of positivity for anti-tTG in patients with liver cirrhosis (LC) not associated with CD, although they also used a method with rh-tTG. In order to establish whether the conflicting results of the above studies were attributable to differences in the methods used, we determined the anti-tTG autoantibodies in a large number of LC patients with 11 different commercial methods, all of which used rh-tTG or human native tTG (nh-tTG) as antigen source.

Section snippets

Patients

54 patients suffering from LC were studied (34 men, mean age 66.7±11.7 years, and 20 women, mean age 68.6±10.2 years), 32 of whom were HCV+ and 10 HBV+, and 12 of whom suffered from alcoholic or cryptogenetic cirrhosis, all treated at the Liver Unit of the Department of Medicine, Pordenone Hospital, Italy. Patients suffering from cirrhosis secondary to autoimmune hepatitis or primary biliary cirrhosis were excluded from the trial. The first step was to conduct the EMA test on all patients;

IgA anti-tTG

None of the healthy controls or those affected by autoimmune diseases tested positive with the various methods used.

All the CD patients tested positive for IgA anti-tTG with 8 out of 11 methods; the remaining three methods failed to detect positivity in only one case (Table 1 and Fig. 1a) with a low antibody content, and Cronbach's alpha test showed a high degree of agreement between the methods (α=0.979).

However, the methods performed differently in the LC patients, with positivity ranging

Discussion

CD often accompanies liver damage, and hypertransaminasemia, sometimes isolated, can be present in 10–54% of patients [12], [13], [14], [15], [16]. Serological screening for CD is therefore mandatory in patients with otherwise unexplained hypertransaminasemia. Although the EMA test has proved to be highly sensitive and specific, identification of tTG as the main, if not the only antigen responsible for EMA positivity [3] means that the ELISA tests for anti-tTG have rapidly become the most

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