In this study, we have assessed the diagnostic values of DGP and TTG antibody results measured by MIA in a large group of biopsy selected CD patients with both mild and severe degrees of mucosal damage. We demonstrated that the MIA performs as well as conventional ELISA method in CD diagnosis for either individual DGP or TTG antibodies or various combinations of these tests.
The MIA method has the advantage of measuring several antibodies simultaneously with reduced costs and turnaround time. MIA also incorporates reagents, which allow the operator to identify specimens that have selective IgA deficiency and can identify errors in pipetting that can lead to false negative results.6
In our study group, four CD patients were presumptively identified as having IgA deficiency. Two of these patients were truly IgA deficient with total IgA levels <1 mg/dL (normal range: 50–400 mg/dL). These patients had negative DGP IgA and TTG IgA test results and high titres of DGP IgG and TTG IgG with both MIA and ELISA methods. However, the other two patients were IgA sufficient and had positive DGP IgA and TTG IgA with the ELISA method. The MIA gave negative results for all IgA and IgG antibodies in these two cases, suggesting that a technologist error occurred in the handling of these specimens.
The specificity of serological markers in our study was similar to that in previous reports for both individual and combination testing; however, the sensitivity of the tests was not as high as previously reported.15, 16, 25
All of our seronegative coeliac patients had either a positive clinical or histopathology response to gluten exclusion or carried a coeliac predisposing HLA (DQ2 or DQ8) that supported their CD diagnosis. Nonetheless, our study population included a large number of CD patients with partial degrees of mucosal damage. When we evaluated the MIA results in CD patients with total and subtotal villous atrophy, the sensitivity increased to the same level as other reports in which a majority of patients had severe histological damage.14, 17
Several studies have demonstrated that the degree of intestinal damage is a major indicator of seropositivity.12, 19, 26-29
The inclusion of a high number of seronegative CD patients with mild histological damage in our sample reflects the fact that the selection of subjects in this study was based on pathology and not a previous positive serology. Typically, serological tests and endoscopy are ordered simultaneously at our centre when there is a suspicion for CD; therefore, we were able to avoid a selection bias, which would occur if the CD patients are biopsied on the basis of their positive serology. Such a bias would result in an overestimation of the performance of serological tests.
Based on our results, TTG IgG does not have any additional diagnostic value over DGP IgG in routine CD diagnosis. DGP IgG had a significantly higher sensitivity and accuracy than and a similar specificity to TTG IgG. The two IgA-deficient patients in our sample were identified with both DGP IgG and TTG IgG. Although the usefulness of both IgG antibodies in detecting IgA-deficient CD patients needs to be assessed in a large sample, our results suggest that DGP IgG performs at least as well as TTG IgG for detecting IgA-deficient CD patients.
We assessed the diagnostic values of various combinations of DGP and TTG antibodies in CD diagnosis. As expected, the sensitivity increased with ‘OR’ combinations at the expense of a decrease in the specificity. Based on our results, the best accuracy for a combination of tests was achieved if we considered the final result to be positive if either TTG IgA or DGP IgG was positive. The combination of DGP IgG or TTG IgA is particularly useful because in addition to detection of IgA-deficient CD patients, DGP IgG was able to detect a few more IgA-sufficient patients who were missed by TTG IgA alone. Nonetheless, panels of tests often leave clinicians with ambiguous possibilities. Therefore, it is important that physicians not only assess the performance of each test, but also be able to interpret the results in different clinical settings with different pretest probabilities of disease. For example, in a referral population with very high pretest prevalence of CD,17
the positive predictive value of a combination test is very high when two or more coeliac-related antibodies are positive. In this setting, the likelihood of CD is very high on the basis of serology. Nevertheless, biopsy affords both confirmation and baseline information about the degree of intestinal damage, which is useful in the follow-up of patients and assessment of response to a gluten-free diet.30
However, in such a high risk population, a substantial number of seronegative patients may still have CD. Hence, a small intestinal biopsy should be performed when there is a high clinical suspicion for CD even in the face of negative test results. Conversely, in a situation with a lower prevalence of CD, for example, in general population or in patients being screened only because of history of another autoimmune disease,31-35
the combination testing has a very high negative predictive value when all the tests in that combination are negative. However, the lower specificity of combination testing may lead to a substantial number of false positives in a low risk population. Therefore, in the setting where the pretest probability of CD is low, a single TTG IgA test may be sufficient to rule out the disease.
Despite several strengths of our study, such as biopsy-based selection of subjects and inclusion of a large number of patients with a wide range of histological damage, there are also a few limitations. To have a similar case and control group, we used frequency matching to select potential controls. We did not include Marsh I patients in the analysis because of diagnostic uncertainty.20-22
Therefore, we were not able to calculate the positive and negative predictive values of the diagnostic tests in this study. Further large prospective cohort studies are essential to validate the diagnostic and predictive values of these serological tests for CD diagnosis and screening.
In conclusion, the MIA method we evaluated is practical and useful for the measurement of multiple CD-related antibodies. Considering the pretest probability of disease and performance of each test, combination testing helps physicians to decide whom to refer for biopsy and may reduce the number of unnecessary intestinal biopsies.