The role of flow cytometry in celiac disease screening using human leukocyte antigen in adult patients with type 1 diabetes mellitus

Study population

This study was performed at a tertiary hospital with an area of influence of approximately 352,000 adult individuals. We initially evaluated 1056 patients with a diagnosis of DM1. Patients previously diagnosed with CD were excluded from our study.

It was calculated that a sample of 142 individuals would be needed to estimate the proportion of patients with CD among those diagnosed with DM1 (1056 DM1 patients, estimated CD prevalence of 4% in this group) with an accuracy of ±3%. Based on a previous study performed by our group [14], we considered a 70% participation acceptance rate; thus, we aimed to include 200 patients. These subjects were selected consecutively according to their order of attendance at the endocrinology consulting room. There were no first-degree relatives, twins or patients with latent autoimmune diabetes of adults.

Study design

Epidemiologic data, as well as antigliadin (AGA), anti-tissue transglutaminase (AtTG) and antiendomysium (EMA) antibody titers, total IgA levels and HLA typing results, were collected from the 200 DM1 patients. In those patients with IgA deficiency, AtTG IgG class levels were ordered.

HLA-DQ2 or DQ8 positive patients were assessed at an outpatient visit and were invited to have an upper gastrointestinal endoscopy (UGIE). In those patients who consented, 5 distal duodenum biopsies were taken, 2 from the gastric antrum and 2 from the gastric corpus. Of the 5 duodenal biopsies, 1 was collected in saline solution for the study of the lymphocytic population with flow cytometry and the other 4 remaining samples were collected in formaldehyde solution for histological examination. Patients with histological findings that suggested CD were contacted and seen again in the gastroenterology consulting room.

Antibody determination

EMA were quantified by indirect immunofluorescence on sections from the distal portion of monkey esophagus as an antigenic substrate (MeDiCa Diagnostics, Encinitas, CA, USA). Sera were diluted at a 1:5 concentration in a 50 mM and pH 7.8 phosphate buffer. Concentrations above these dilution values were considered as positive.

AGA were quantified by an immunoenzymatic method, using a commercial kit with gliadin extracted from wheat as a substrate (Orgentec Diagnostika, GMBH, Mainz-Germany). Values over 10 arbitrary units were considered as positive.

AtTG were assessed, following the manufacturer’s instructions, using an immunoenzymatic method that uses human recombinant transglutaminase as a substrate (Orgentec Diagnostika, GMBH, Mainz-Germany). Values above 15 U/mL were considered as positive.

HLA determination

Blood samples that had been anticoagulated with ethylenediaminetetraacetic acid (EDTA) were obtained from each patient. DNA extraction was achieved using paramagnetic particles (Maxwell® 16 System). DNA concentrations for each sample were measured by spectrophotometry (NanoDrop®) and further concentration adjustments were performed in order to study DRB1 and DQB1 loci with a low resolution PCR-SSO technique (sequence-specific oligonucleotides; Luminex®). Samples identified as DRB1*03/DQB1*02 or DRB1*04/DQB1*03 were subsequently studied for the allelic assignation, including the DQA1 locus, using a high resolution polymerase chain reaction technique with sequence specific primers (PCR-SSP). Results were presented as a serological annotation in which patients were DQ2 and/or DQ8 either positive or negative.

Histological examination

Four distal duodenum biopsies and four gastric biopsies were collected for the histological examination. Biopsy material was fixed in a 4% formaldehyde dilution and embedded in paraffin. Three 4-μm thick slices were later obtained for each sample and hematoxylin-eosin stain was applied. Gastric antral biopsies were additionally stained with Giemsa. All of the samples were studied by the same expert pathologist. Duodenal lesions, if any, were classified following Oberhuber’s [15] modification of Marsh’s criteria [16].

Lymphocytic population study

One of the duodenal biopsies was collected in a saline solution and was incubated in culture medium, with the addition of 50 µL EDTA + 50 µL dithiothreitol (0.1 M), for 60-90 min at room temperature under moderate stirring using a vertical rotor. Once the cell suspensions were obtained, the mixture was centrifuged for 5 min at 1500 rpm (no brake). The supernatant was then decanted and the sample was resuspended in 1 mL of culture medium. Culture medium was prepared with 10 mL of fetal serum (hemotherapy), 88 mL of culture medium, 1 mL of antibiotic and 1 mL of glutamine.

Immunophenotyping was performed using one tube per sample and a control tube. The following monoclonal antibodies, tagged with different fluorochromes, were added to 200 μL of cellular suspension: 10 μL of anti-CD103-fluorescein isocyanate, 10 μL of anti-TCRγδ-phycoerythrin (PE), 5 μL of anti-CD3-PE- cyanine 7, and 5 μL of anti-CD45-allophycocyanin (Becton, Dickinson [BD] and Company). After 30 min incubation at 4°C in a dark box, cells were washed with phosphate buffered saline (PBS) for 7 min at 1200 rpm (no brake) and the sample was decanted; 500 μL of PBS were then added to each tube.

Flow cytometry analysis was performed using BD’s FACSCalibur™ cytometer. Forward and side scatter signals (FSC, SSC) were recorded on a linear scale, whereas fluorescence signals were registered on a logarithmic scale. Multiparametric data were collected using a scatter threshold that excluded erythroid cells and cellular debris. Lymphocytes were selected using a lymphocyte selection gate based on a SSC/CD 45 (light dispersion at 90°) histogram. A negative isotopic control was applied in order to adjust the fluorescence intensities. Data were analyzed using BD’s CellQuest™ program.

CD diagnosis

Patients were classified as having possible CD when they had histological lesions suggestive of CD and all other pathological entities with similar histological changes had been ruled out. Thus, taking into account the high specificity of the antibodies, patients who had histological changes suggestive of CD and positive antibodies were considered as possible CD cases.

Special attention was paid to excluding other possible diagnoses in Marsh 1 and 2 type cases with CD negative antibodies. Thus, as well as considering patients’ personal history and clinical records, we asked individuals with Marsh 1 and 2 type lesions and negative antibodies about their use of nonsteroid anti-inflammatory drugs (NSAIDs) in the previous month and tested them for Helicobacter pylori (HP). Those patients with a positive HP test received eradication therapy and had a new UGIE with duodenal biopsies at least 6 months after treatment was completed, to evaluate the presence of histological changes. None of the patients with Marsh 1 type changes had taken NSAIDs in the month before the endoscopy was performed. Those patients who were HP negative, with no NSAID consumption and without any symptoms, were followed at the outpatient clinic and underwent an additional UGIE with duodenal biopsies at 2 years to assess whether histological lesions persisted or had spontaneously reverted. Cases in which histological changes persisted either after HP eradication therapy or after 2-year follow up were classified as possible CD.

Patients with Marsh 2 type lesions and negative antibodies were asked about previous NSAID consumption and their HP status was assessed. Again, none of the patients in this group had taken NSAIDs during the month before the endoscopy was performed. Those cases that were found to be positive for HP were treated in the same way as patients with lymphocytic enteritis. HP negative patients and those without a history of NSAID consumption were classified as possible CD cases.

All of the patients with a “possible CD” diagnosis were advised to follow a gluten-free diet (GFD). Definite CD diagnosis was made in those cases with a symptomatic improvement (when symptoms had been present) and a serological negativization (when antibodies had been positive) or a histological improvement (when antibodies had been negative) after at least 1 year on a GFD.

GFD compliance follow up

GFD compliance was assessed by a detailed anamnesis during the subsequent interviews. Serological response (antibody negativization) was evaluated in those patients with positive antibodies at the beginning of the study. In those cases with negative antibodies, histological response was studied by performing an UGIE with duodenal biopsies after two years.

Statistical analysis

Study variables were recorded in a database that was set up with the IBM SPSS Statistics 15.0 program (Chicago, USA). Comparisons were performed with two-tailed tests. P-values less than 0.05 were considered statistically significant. A normal distribution could not be assumed for the CD patients’ group, because of the small number of individuals with this diagnosis. Therefore, comparisons related to lymphocytic populations between CD and non-CD patients were performed using the Mann–Whitney U test. The predictive power of flow cytometry was calculated with a receiver operating characteristic (ROC) curve, taking the value at which the sum of the sensitivity and specificity was the highest as the optimal cutoff point.

Epidemiological characteristics

Of the 200 DM1 patients initially evaluated in our study, 87 (43.5%) were female and 113 (56.5%) were male. The median age of our study population was 41 years (range: 18-83).

One hundred ninety patients (95%) were either HLA DQ2 or DQ8 positive. The HLA distribution is shown in Fig. 1. Nineteen patients (9.5%) were positive for at least one of the three antibodies that were tested in our study. All of the patients with positive antibodies were HLA DQ2 or DQ8 positive. The 190 DM1 patients who were HLA DQ2 or DQ8 positive were invited to complete a gastrointestinal study for CD detection. One hundred thirty-six patients finally accepted.

Figure 1 Distribution of human leukocyte antigen in our initially evaluated population and in patients who consented to participate in the study

Fifty-two (38.2%) of the 136 patients who underwent surveillance were female and 84 (61.8%) were male. The median age of the patients in this group was 41.5 years (range 18-74). Median age at DM1 diagnosis in this group was 24.5 years (range: 1-64); the median age of patients with a two-year DM1 progression was 15.5 years (range 0-48). HLA distribution is shown in Fig. 1.

CD prevalence

Of the 136 patients studied, 16 had histological findings that suggested CD. Ten of them were at initial stages (Marsh 1 and 2 type lesions) and 6 had Marsh 3 type lesions (Fig. 2). Table 1 shows the diagnostic sequence followed. Patient number 9 had a previous history of common variable immunodeficiency. This entity can cause an enteropathy that is similar to CD, and some patients show a histological response after a GFD [17]. Thus, patient number 9 was encouraged to follow a GFD for 2 years, after which a new histological assessment was performed. After 2 years on a strict GFD, the histological changes were similar to those at baseline endoscopy, so a diagnosis of CD could not be made. Patient number 12 had recently been admitted with septic shock, and did not receive the recommendation to follow a GFD initially. A new examination was performed after 1 year, showing a normalization of the duodenal biopsies; CD diagnosis was therefore ruled out. Even though patient number 13 acknowledged frequent dietary transgressions, a control UGIE yielded a normal duodenal biopsy. Because the histological improvement could not be explained by the GFD, this patient was not diagnosed with definite CD.

Figure 2 Distribution of histological findings compatible with celiac disease

Table 1 Patients with histological findings that suggested CD

In total, 6 patients were diagnosed with definite CD. This comprised 3% of the initially selected patients and 4.4% of the cases who underwent a complete study. The patient distribution is represented in Fig. 3. All CD patients were positive for HLA-DQ2.

Figure 3 Patients with a definite diagnosis of celiac disease Ab, antibodies

Lymphocytic population study in duodenal biopsies

Comparison between all of the patients with and without CD diagnosis

Table 2 shows the results of the duodenal biopsy flow cytometry analysis. The optimal cutoff values found in the ROC curves in order to distinguish healthy subjects from CD patients were:

Table 2 Lymphocytic population in duodenal biopsies, comparing CD patients with DM1 non-CD patients

• γ/δ IEL: values under 17 γ/δ IELs per 100 were considered as normal. AUC = 0.96 (95% CI 0.91-1)

• CD3 negative IEL: values over 5 CD3 negative IELs per 100 were considered as normal. AUC = 0.83 (95% CI 0.72-0.94)

Comparison between patients with and without CD presenting histological lesions

When only the patients with histological findings compatible with CD were studied, a comparison of the IEL distribution in definite CD patients with the distribution in subjects with lesions in whom CD was finally ruled out revealed no significant differences in the percentage of total IELs between the 2 groups. However, in the group of CD patients the γ/δ IEL proportion was found to be significantly higher and the CD3 negative IEL proportion significantly lower (Table 3).

Table 3 Lymphocytic population in duodenal biopsies, comparing CD patients with DM1 patients presenting histological impairment in which CD diagnosis was finally ruled out

When assessing the optimal cutoff values for distinguishing among patients with CD compatible histological changes, those with and without definite CD, we found that:

• γ/δ IEL: values under 13. 5 γ/δ IELs per 100 were considered as normal. AUC = 1 (95% CI 1-1).

• CD3 negative IEL: values over 5 CD3 negative IELs per 100 were considered as normal. AUC = 0.88 (95% CI 0.71-1).