Establishment of New Immunostatistical Equations for Diagnosis and Follow-Up of Gluten-Free Diet in Patients with Celiac Disease

MATERIALS AND METHODS

Samples collection: Seventy blood samples were taken from people who were diagnosed with or suspected of having CD, with ages ranging from 5 to 40 years. They were separated into two groups, the first of which contained 40 people of both sexes who had previously been diagnosed with CD, while the second group included 30 individuals of both sexes, with ages ranging from 5 to 35 years, suspected of having CD due to the appearance of some clinical symptoms, such as diarrhoea, general pallor and fatigue. Samples were collected from patients at Al-Salam Teaching Hospital in Mosul for one year from 9th April, 2022 to 25th December, 2022 and the study was carried out at Mosul University College of Sciences/Iraq. The control group also included 30 blood samples from healthy individuals of both sexes, with ages ranging from 5 to 40 years. A survey, which included name, age, gender, weight and height, was collected from all members of the study sample.

Samples: A 5 mL blood sample was withdrawn for each patient using 5 mL medical syringes and 2 mL of the blood samples were distributed into tubes containing EDTA anticoagulant and gently shaken to prevent blood clotting. Until being used for molecular tests, the remaining 3 mL blood samples were placed in gel tubes to separate the serum by centrifugation at 3,000 rpm for 15 min. After separation, the blood serum was distributed into small plastic Eppendorf tubes and frozen at a temperature of -20°C until the serological tests were conducted.

Measurement of the concentration of tTG/IgA antibodies and antigliadin antibodies (DGP/IgA): Serological analysis of serum samples was performed to measure the concentration of tTG/IgA antibodies. Proprietary test kits by the manufacturer (AESKULISA) for determination of tTG/IgA antibodies and by (DiaMetra) for DGP/IgA antibodies were used, following the company’s instructions and utilizing indirect Enzyme-Linked Immunosorbent Assay (ELISA) technology. Serum samples were added to the pits of the ELISA plate and then a plate-washing process was performed to remove the unbound parts and enzyme-linked antibodies that interact with the antibody-antigen complex were added followed by a plate-washing process to remove the unbound paired antibodies. Then, the base material was added, followed by a solution to stop the reaction, which leads to a colour change-the intensity of which depends on the concentration of the target antibodies. The optical absorbance was measured at the wavelength of 450 nm¹⁰.

Estimation of total IgA level in serum: The level of total IgA in the body was estimated using the sandwich ELISA technique, where the principle of the assay is based on the use of identical pairs of antibodies, in which each antibody recognizes a distinct, non-overlapping area of the antigen molecule. The first antibody, known as capture, is coated on the pits. Then the samples are added to the etching. A second antibody known as detection, which is bound to the enzyme, is added. Then the base material is added, which allows the formation of a colour reaction. Then, a solution to stop the reaction is added and the absorbance is measured¹¹.

Statistical analysis: To statistically compare the means, which included comparisons between the levels of tTG/IgA and DGP/IgA gliadin in patients with CD and those suspected of having CD in addition to the control sample, a test was used with a probability of p<0.05. To investigate the association between the variables, a regression coefficient analysis was carried out. The SPSS 24.0 software was used for statistical analysis.

RESULTS AND DISCUSSION

Several statistical indicators, such as sample size, arithmetic mean and standard deviation,for assessing tTG/IgA concentrations for the study groups, which were comprised of CD patients, those suspected of having CD and controls as shown in Table 1.

The results of the Duncan’s Test appeared in Table 2. The results of the test measure the concentration of tTG/IgA among the three groups as follows. There was a significant increase in the concentration of tTG/IgA in CD patients compared with both those suspected of having CD and the controls. There were no significant differences in the concentration of tTG/IgA between those suspected of having CD and the controls in terms of the probability value associated with Duncan’s Test, which amounted to 0.874, which is greater than 0.05.

Some statistical variables for evaluating the DGP/IgA concentration of the study groups, which included CD patients, those suspected of having CD and observers compared with the control sample, as indicated by sample size, mean and standard deviation as shown in Table 3.

The results of Duncan’s Test, which measured the concentration of DGP/IgG among the three groups, appeared in Table 4. There was a significant increase in the concentration of DGP/IgA in CD patients compared with both those suspected of having CD and the controls. There were no significant differences in the concentrations of DGP/IgA between those suspected of having CD and the controls in terms of the probability value associated with Duncan’s Test, which amounted to 0.945, the given number is more than 0.05.

Effect relationship analysis in patients with CD
Analysis of the effect of total IgA on tTG/IgA in patients with CD: The best model for the effect of total IgA on tTG/IgA is the Exponential model, as it represents the best relationship between these two variables in terms of the probability value accompanying the F test, which amounted to 0.001, which is less than 0.05. The percentage of the effect of total IgA on tTG/IgA amounted to 11%, as indicated by the value of the clarifications coefficient (R²), meaning that 11% of the changes that occurred in tTG/IgA were caused by total IgA, or the remaining percentage of 89% is for other variables that were not included in the model. There was a significant effect of total IgA on tTG/IgA in terms of the value of the regression coefficient, which amounted to -3.131 and this effect was significant in terms of the probability value associated with the t test, which amounted to 0.038, which is less than 0.05. The mathematical relationship between these two variables, according to the Exponential model, was shown in Table 5 and Fig. 1.

Effect analysis of total IgA on DGP/IgA in patients with CD: The best model for the effect of total IgA on DGP/IgA is the Power model, as it represents the best relationship between these two variables in terms of the probability value accompanying the F test, which amounted to 0.001, which is less than 0.05.

The percentage of the effect of total IgA on DGP/IgA amounted to 25%, as indicated by the value of the clarification coefficient (R²), meaning that 25% of the changes that occurred in DGP/IgA were caused by total IgA, or the remaining percentage of 75% is for other variables that were not included in the model. There was a significant effect of total IgA on DGP/IgA in terms of the value of the regression coefficient, which amounted to -1.818 and this effect was significant in terms of the probability value associated with the t test, which amounted to 0.001, which is less than 0.05.

Fig. 1:

Effect of total IgA on tTG/IgA in patients with CD

Fig. 2:

Effect of total IgA on DGP/IgA in patients with CD

The mathematical relationship between the two variables according to the Power model was shown in Table 6 and Fig. 2.

Effect relationship analysis for patients with suspected CD
Effect analysis of total IgA on tTG/IgA: The best model for the effect of total IgA on tTG/IgA is the Compound model, as it represents the best relationship between these two variables in terms of the probability value accompanying the F test, which amounted to 0.049, which is less than 0.05.

The percentage of the effect of total IgA on tTG/IgA amounted to 11%, as indicated by the value of the clarifications coefficient (R²), meaning that 11% of the changes that occurred in tTG/IgA were caused by total IgA, or the remaining percentage of 89% is for other variables that were not included in the model. There was a significant effect of total IgA on tTG/IgA in terms of the value of the regression coefficient, which amounted to 0.540 and this effect was significant in terms of the probability value accompanying the t test, which amounted to 0.044, which is less than 0.05. The mathematical relationship between the two variables according to the combined model was shown in Table 7 and Fig. 3.

Analysis of the effect of total IgA on DGP/IgA in patients with suspected CD: The best model for the effect of total IgA on DGP/IgA is the combined model compound, as it represents the best relationship between these two variables in terms of the probability value accompanying the F test, which amounted to 0.017, which is less than 0.05. The percentage of the effect of total IgA on DGP/IgA amounted to 14%, as indicated by the value of the clarification coefficient (R²), meaning that 14% of the changes that occurred in tTG/IgA were caused by total IgA, or the remaining percentage of 86% was for other variables that were not included in the model. There was a significant effect of total IgA on DGP/IgA in terms of the value of the regression coefficient, which amounted to 0.597 and this effect was significant in terms of the probability value associated with the t test, which amounted to 0.019, which is less than 0.05. The mathematical relationship between the two variables was shown in Table 8 and Fig. 4.

Total IgA, anti-tTG and gliadin DGP (D-linked antibodies) are the most significant serological tests for CD diagnosis nowadays. They are all members of the IgA and IgG classes. Only members of the extremely sensitive IgA class, however, may be termed CD-specific. Except for DGP, the use of IgG markers should be limited to people with IgA deficiency due to the high occurrence of false positives⁹. When patients maintain a gluten-free diet (GFD), these types of antibodies can be utilized alone or in combination to provide a trustworthy diagnosis in virtually all cases of CD.

Fig. 3:

Effect of total IgA on tTG/IgA in patients with suspected CD

Fig. 4:

Effect of total IgA on DGP/IgA in patients suspected of CD

They can also be used to track a patient’s reaction to a GFD, as levels of these antibodies drop when gluten is removed from the diet. Therefore, these serologic selections have a dual role in monitoring GFD efficacy and patient adherence after diagnosis, but first, they precisely and reliably identify patients with CD.

Over the past few decades, IgG and IgA gliadin antibody tests have been replaced by the more sensitive and specific IgA anti-tTG test¹². The use of a tissue biopsy is mandated by the guidelines of gastrointestinal facilities in North America and Europe for the diagnosis of CD¹³. Furthermore, due to the huge variety of preparations for intestinal tissue biopsy, the high financial expense of performing the biopsy and the high frequency of CD in the general population¹⁴, the search for autoantibodies is currently an important initial serological test for individuals who may need a duodenal biopsy. During the past 10 years, very significant advances have taken place in the field of serological testing and the wide availability of these tests has allowed any clinician to rapidly diagnose the pathogenesis of patients with CD¹⁵.

Since CD symptoms can affect people of any age, it is important to note that the first diagnostic step in diagnosing CD is a serological diagnosis. The detection of tTG/IgA and total IgA antibodies is advised by the American Gastroenterology Association. The antibody evaluation of total IgA type, especially in adults, along with the tTG/IgA test and the DGP/IgA test, are some of the most crucial serological tests that attending physicians must utilize throughout the process of diagnosing CD. Additionally, once a discrepancy in the aforementioned antibody readings has been confirmed, oesophageal endoscopy and bowel biopsy cannot be ignored. However, there has already been some discussion about cancelling the biopsy process in children, especially when there is a high concentration of tTG/IgA antibodies as well as a variation in total IgA levels¹⁶.

Because of their limited sensitivity and specificity, the use of natural Antigliadin Antibodies (anti-AGA) antibodies in the diagnosis of CD is no longer recommended¹⁷. The tTG Test, particularly the IgA form, is 99% more sensitive than the other serological assays. In addition to diagnosis, serological testing can be used to track therapy progress¹⁸. While anti-DGP antibodies are a good marker to monitor patients when they are put on a GFD, anti-tTG2 antibodies persist in patients’ sera for a long time, up to several years. Negative test results attest to patients’ adherence to doctors’ dietary suggestions¹⁹.

The most prevalent primary immunodeficiency is IgA antibody deficiency, which is characterized by a lack of IgA antibodies (0.07g L^–1) and normal IgG and antibody levels in the blood and IgM in individuals who are at least four years old. To prevent IgA deficiency in children under the age of four, which is a temporary disease due to the delay in the development of the IgA system after birth, screening for total IgA antibodies should be done after the age of four. Epidemiological studies have already demonstrated that CD is one of the conditions directly linked to IgA deficiency^19-21. In addition to accuracy in acquiring the results, medical laboratories are always searching for the most efficient approaches in terms of diagnostic and economic considerations²². Among the most important disorders that require serological detection of antibodies are primary biliary cirrhosis, or Miller Fisher syndrome, autoimmune gastritis, pernicious anaemia and CD-especially CD²³. Simultaneous detection of serum CD markers using tTG/IgA may reduce the cost of serological CD diagnosis compared with the detection of CD-specific antibodies alone, as well as shorten the time needed to reach the correct result. The determination of tTG/IgA and DGP/IgA concentrations in a patient’s serum celiac IgA plus total IgA takes less than two hours, so this procedure may take a long time in addition to the effort of performing the three tests. It should also be highlighted that the test requires just 250 μL of serum, which is especially essential in young patients^24,25.

Given the importance of tTG/IgA and DGP/IgA in early diagnosis of CD, in addition to the role of serum globulin IgA in shaping the immune response within the small intestine to inflammatory conditions, a regression coefficient test was performed to determine the relationship between tTG/IgA, DGP/IgA and serum globulin IgA. Moreover, statistical immunological equations were extracted that allow treating physicians and laboratories, which may only be able to perform one test, to estimate the level of IgA in the serum and extract the values of both tTG/IgA and DGP/IgA in each patient undertaking a GFD as well as those suspected of having CD due to clinical symptoms without the need for laboratory tests for either tTG/IgA or DGP/IgA.

CONCLUSION

There is a significant increase in the levels of both tTG/IgA and DGP/IgA in CD patients compared to patients suspected of having CD and control. As part of the effect coefficient test, a significant relationship was observed for the effect of total IgA antibodies on each of the tTG/IgA and DGP/IgA antibodies in patients with CD by 11 and 25%, respectively and a significant relationship appeared for the effect of total IgA antibodies in each of antibodies type tTg/IgA and DGP/IgA in suspected cases of CD by 11 and 14%, respectively.

SIGNIFICANCE STATEMENT

The importance of the current study lies in following up on some immunological indicators and molecular manifestations of patients with celiac disease in the city of Mosul, in addition to finding a new diagnostic method that helps researchers, doctors and laboratories to diagnose celiac disease in patients suspected of having the disease, in addition to following up the extent of patients’ commitment to an empty diet of gluten in an easy, fast and inexpensive way and it requires a small amount of blood to conduct the examination, especially for children under the age of four years.