Improving the Diagnosis of Dermatitis Herpetiformis Using the Intraepithelial Lymphogram.

Dermatitis herpetiformis is a cutaneous manifestation of celiac disease. Phenotyping of intraepithelial lymphocytes in the small bowel mucosa can strengthen the diagnosis of celiac disease when it is not clear-cut. We aim to evaluate the usefulness of the intraepithelial lymphogram to confirm dermatitis herpetiformis in equivocal cases. We performed a retrospective multicenter study on patients diagnosed with dermatitis herpetiformis and collected data from the intraepithelial lymphogram assessed by flow cytometry. A total of 36 patients were analyzed in relation to the severity of intestinal damage (18 had non-atrophic mucosa) at baseline (N = 28) and/or after the adoption of a gluten-free diet (median follow-up of three years, N = 16). We observed that patients with atrophy more often had positive celiac serology (p = 0.019), celiac clinical symptoms (p = 0.018), and iron-deficiency anemia (p = 0.018), but the severity of skin damage was similar in both groups (p = 0.79). At baseline, increased TCRγδ+ cells were present in 94% of patients with atrophy and 67% with non-atrophic lesions (p = 0.13). After a gluten-free diet, increased TCRγδ+ cells persisted in 100% and 63% of cases, respectively (p = 0.21). We concluded that increased TCRγδ+ cells may be helpful in confirming the diagnosis of dermatitis herpetiformis in equivocal cases, even in patients who were started on a gluten-free diet.

Assessment of activated gut-homing CD8+ T cells in blood by flow cytometry during a 3-day gluten challenge.

Accurate celiac disease (CD) diagnosis must be performed in individuals following a gluten containing diet. Diagnostic procedures for individuals already on a gluten-free diet (GFD) avoiding long gluten reintroductions are still challenging. To deal with this issue, we developed an accurate but simple method that requires only a 3-day gluten challenge and circumvents the main limitations of previously suggested proposals such as requirement of specific peptides and unusual specialized lab facilities or high cost. In an attempt to standardize this methodology to be used in daily clinical practice, we describe here an optimized protocol for assessing activated gut-homing CD8+ T cells in blood combined with a short gluten challenge. Details about the amount and type of gluten antigen and the starting material are included, as well as the strategy to easily characterize and identify the cells of interest using flow cytometry. This methodology constitutes a diagnostic tool for CD diagnosis of high specificity and sensitivity for seropositive disease (>95%) as an alternative to long-term gluten challenge and open new possibilities to test the response to gluten in research and clinical trials.

Isolation and cryopreservation of peripheral blood mononuclear cells.

The study of peripheral blood mononuclear cells (PBMCs) in immune-mediated diseases, such as celiac disease (CD), is important to uncover pathogenesis, find new biomarkers and discover and evaluate new treatments. Many studies have been published about the use and value of PBMCs in CD such as those including enzyme-linked immunospot (ELISPOT) assays, flow cytometry, peptide-MHC tetramers, genetic and proteomic analyses, and in vitro and proliferation assays. We present here and easy and efficient method for isolation of PBMCs using density gradient centrifugation. We also describe a simple way to freeze PBMCs in order to preserve their number and viability and a thawing procedure leading to high rates of viability of the cryopreserved cells to be used in subsequent applications.

Flow cytometric analysis of duodenal intraepithelial lymphocytes (celiac lymphogram): A diagnostic test for celiac disease.

Celiac disease (CD) diagnosis in adults and certain cases of children mainly relies on the assessment of histopathological features in duodenal biopsies. However, none of the histological findings that characterize CD are pathognomonic. This, in addition to the clinical heterogeneity of the disease and the presence of seronegative forms, makes the diagnosis of CD still a challenge. A hallmark of the celiac mucosa is the elevated number of TCRγδ intraepithelial lymphocytes (IEL) in the epithelium, which may remain increased even long after gluten withdrawal. Active disease is also characterized by the decreased CD3- IEL subset. The use of flow cytometry enables a precise cell counting and phenotyping, allowing the ascertainment of both TCRγδ+ and CD3- IEL subsets, what is known as the "IEL lymphogram." Although determination of this lymphogram has become a routine evaluation tool in numerous hospitals, standardization of the technical method will guarantee an accurate performance in order to become a pivotal technique for CD diagnosis. Here we describe the protocol to process duodenal biopsies in order to obtain the IELs from the mucosa and to characterize lymphocyte populations by flow cytometry to obtain the IEL lymphogram.

Ordered-domain unfolding of thermophilic isolated ß subunit ATP synthase.

The flexibility of the ATP synthase's ß subunit promotes its role in the ATP synthase rotational mechanism, but its domains stability remains unknown. A reversible thermal unfolding of the isolated ß subunit (Tß) of the ATP synthase from Bacillus thermophilus PS3, tracked through circular dichroism and molecular dynamics, indicated that Tß shape transits from an ellipsoid to a molten globule through an ordered unfolding of its domains, preserving the ß-sheet residual structure at high temperature. We determined that part of the stability origin of Tß is due to a transversal hydrophobic array that crosses the ß-barrel formed at the N-terminal domain and the Rossman fold of the nucleotide-binding domain (NBD), while the helix bundle of the C-terminal domain is the less stable due to the lack of hydrophobic residues, and thus the more flexible to trigger the rotational mechanism of the ATP synthase.

Intestinal and blood lymphograms as new diagnostic tests for celiac disease.

Accurate celiac disease (CD) diagnosis is still challenging for some specific patients or circumstances. Thus, much effort has been expended last decades focused on seronegative or low grade enteropathy CD and, especially, on enable early diagnosis of individuals on a gluten-free diet (GFD). We discuss here two diagnostic approaches based on immunophenotyping by flow cytometry that we expect to reduce the persistent low diagnostic rates and the common diagnostic delay. The intraepithelial lymphogram is based on determining the percentage of TCRγδ+ and surface CD3- lymphocytes in the intestinal epithelium. The concomitant increase in TCRγδ+ and decrease in surface CD3- intraepithelial lymphocytes has been termed the celiac lymphogram and has been proved to be discriminative in seronegative, low grade enteropathy and potential CD, as well as in most CD patients on a GFD. A blood lymphogram based on the analysis of activated gut-homing CD8+ T cells combined with a 3-day gluten challenge is also considered, which has shown high sensitivity and specificity to diagnose seropositive Marsh 1 and Marsh 3 CD in individuals following a GFD. In addition, flow cytometry can be extremely useful in cases of refractory CD type II to identify aberrant cells. Those approaches represent highly accurate methods for CD diagnosis, being simple, fast, highly reproducible and of easy implementation in clinical practice.

Activated gut-homing CD8+ T cells for coeliac disease diagnosis on a gluten-free diet.

BACKGROUND: The diagnosis of coeliac disease (CD) in individuals that have started a gluten-free diet (GFD) without an adequate previous diagnostic work-out is a challenge. Several immunological assays such as IFN-γ ELISPOT have been developed to avoid the need of prolonged gluten challenge to induce the intestinal damage. We aimed to evaluate the diagnostic accuracy of activated gut-homing CD8+ and TCRγδ+ T cells in blood after a 3-day gluten challenge and to compare it with the performance of IFN-γ ELISPOT in a HLA-DQ2.5 subsample. METHODS: A total of 22 CD patients and 48 non-CD subjects, all of them following a GFD, underwent a 3-day 10-g gluten challenge. The percentage of two T cell subsets (CD8+ CD103+ ß7hi CD38+/total CD8+ and TCRγδ+ CD103+ ß7hi CD38+/total TCRγδ+) in fresh peripheral blood drawn baseline and 6 days after the challenge was determined by flow cytometry. IFN-γ ELISPOT assays were also performed in HLA-DQ2.5 participants. ROC curve analysis was used to assess the diagnostic performance of the CD8+ T cell response and IFN-γ ELISPOT. RESULTS: Significant differences between the percentage of the two studied subsets of CD8+ and TCRγδ+ cells at days 0 and 6 were found only when considering CD patients (p < 10-3 vs. non-CD subjects). Measuring activated CD8+ T cells provided accurate CD diagnosis with 95% specificity and 97% sensitivity, offering similar results than IFN-γ ELISPOT. CONCLUSIONS: The results provide a highly accurate blood test for CD diagnosis in patients on a GFD of easy implementation in daily clinical practice.

Coeliac Disease in Elderly Patients: Value of Coeliac Lymphogram for Diagnosis.

(1) Background: Although a meta-analysis reported that the sensitivity of CD3+ TCRγδ+ cells for coeliac disease diagnosis was >93%, a recent study has suggested that sensitivity decreased to 65% in elderly patients. (2) Aim: To evaluate whether the sensitivity of intraepithelial lymphocyte cytometric patterns for coeliac disease diagnosis changes with advanced age. (3) Methods: We performed a multicentre study including 127 coeliac disease patients ≥ 50 years: 87 with baseline cytometry (45 aged 50-59 years; 23 aged 60-69 years; 19 aged ≥ 70 years), 16 also with a follow-up cytometry (on a gluten-free diet); and 40 with only follow-up cytometry. (4) Results: In Marsh 3 patients, a sensitivity of 94.7%, 88.9% and 86.7% was observed for each age group using a cut-off value of TCRγδ+ >10% (p = 0.27); and a sensitivity of 84.2%, 83.4% and 53.3% for a cut-off value >14% (p = 0.02; 50-69 vs. ≥70 years), with difference between applying a cut-off of 10% or 14% (p = 0.008). The TCRγδ+ count in the ≥70 years group was lower than in the other groups (p = 0.014). (5) Conclusion: In coeliac patients ≥ 70 years, the TCRγδ+ count decreases and the cut-off point of >10% is more accurate than >14%.

The HLA complex and coeliac disease.

The Human Leukocyte Antigen (HLA) has a crucial role in the development and pathogenesis of coeliac disease (CD). The genes HLA-DQA1 and HLA-DQB1, both lying in this region and encoding the HLA-DQ heterodimer, are the main genetic predisposing factors to CD. Approximately 90% of CD patients carry the heterodimer HLA-DQ2.5, leaving only a small proportion of patients with lower risk heterodimers (HLA-DQ8, HLA-DQ2.2 or HLA-DQ7.5). These HLA-DQ molecules act as receptors present in the surface of antigen presenting cells and show high affinity for deamidated gluten peptides, which bind and present to CD4+ T cells. This triggers the immunological reaction that evolves into CD. Since specific HLA genetics is present in almost the totality of CD patients, HLA typing has a very high negative predictive value, and it can be used to support diagnosis in specific scenarios. HLA risk has been associated to different CD-related features, such as age at onset, clinical outcomes, antibody levels and grade of histological lesion; but further research is needed. HLA-DQ genotypes have been also suggested to modulate the composition of the gut microbiota.

Hallazgos ultrasonográficos en articulaciones de muñecas y manos de pacientes con sospecha clínica de artritis reumatoide en fase temprana / Ultrasonographic findings in wrists and hands of patients with clinical suspicion of early phase rheumatoid arthritis

La Artritis Reumatoide (AR) es una enfermedad crónica y autoinmune cuyo primer año de evolución es considerado por el Colegio Americano de Reumatología como su fase temprana. Con el objetivo de describir los hallazgos ultrasonográficos en las articulaciones de muñecas y manos de pacientes con sospecha clínica de AR en fase temprana referidos de la consulta de Reumatología del Hospital Central Universitario Dr. Antonio María Pineda durante el lapso junio-agosto de 2018, se realizó un estudio descriptivo transversal evaluando 126 articulaciones de 21 pacientes según la escala modificada del OMERACT. Los pacientes se caracterizaron por un promedio de edad de 51,4 ± 11,1 años, siendo el grupo etario más afectado el de 41-50 años y 51-60 años. Hubo un predominio del sexo femenino (85,7%) y una media de inicio de síntomas de 5,2 ± 2,8 meses. Las principales alteraciones encontradas fueron derrame sinovial (54,7%), engrosamiento sinovial (28,5%), tenosinovitis en el grupo extensor (28,5%), erosiones óseas (11,1%) y tenosinovitis en flexores (9,5%). Los hallazgos mostraron mayor afectación de las articulaciones radiocarpianas; 12,7% y 7,9% mostraron hipertrofia sinovial y sinovitis grado I, 15% derrame sinovial grado 1 y 2 y 7,9% erosiones óseas pequeñas. El 8,7% de las II metacarpofalángicas mostraron hipertrofia sinovial grado I, 6,3% sinovitis, 13,4% derrame sinovial y 1,5% erosiones óseas medianas; el 0,79% de las II interfalángicas proximales presentaron derrame sinovial grado 1. Se observó tenosinovitis grado 1 en 25,4% de extensores y 7,9% de flexores. En conclusión, la ultrasonografía es una herramienta complementaria en el diagnóstico y seguimiento de la enfermedad reumatoide en fase temprana por lo que se sugiere fomentar su uso evitando gastos innecesarios y retrasos en el inicio del tratamiento(AU)

Rheumatoid Arthritis (RA) is a chronic and autoimmune disease whose first year of clinical manifestations is considered the early phase of the disease according to the American College of Rheumatology. With the aim of describing the ultrasonographic findings in the wrists and hands of patients with clinical suspicion of early phase RA referred to the Rheumatology Service of the Hospital Central Universitario Dr. Antonio Maria Pineda during the period June-August 2018, a cross-sectional descriptive study was conducted evaluating 126 joints of 21 patients according to the modified scale of the OMERACT. Patients had an average age of 51.4 ± 11.1 years and the most affected age groups was the 41-50 years and 51-60 years. Predominance of female sex (85.71%) as well as an average of 5.2 ± 2.8 months of time of symptoms onset was observed. The main alterations observed were synovial effusion (54.7%), synovial thickening (28.5%), tenosynovitis in extensor tendons (28.5%), bone erosions (11.1%) and tenosynovitis in flexor tendons (9.52%). The radiocarpal joints were the most affected showing grade 1 synovial hypertrophy and synovitis in 12.7% and 7.9% of joints, respectively; grade 1 and 2 synovial effusion was observed in 15% of joints and small bone erosions in 7.9%. For the second metacarpophalangeal joint, grade I synovial hypertrophy was found in 8.7% of joints, synovitis in 6.3%, synovial effusion in 13.4% and medium-sized bone erosions in 1.5%; in 0.79% of the proximal interphalangeal joints grade I synovial effusion was observed. Tenosynovitis grade 1 was observed in 25.4% of extensor tendons and 7.9% flexors. The use of ultrasonography should be encouraged as a complementary tool for the diagnosis of RA, avoiding unnecessary expenses and delay in treatment(AU)

Gamma delta+ intraepithelial lymphocytes and coeliac lymphogram in a diagnostic approach to coeliac disease in patients with seronegative villous atrophy.

BACKGROUND: The causes of seronegative villous atrophy can be grouped as coeliac or noncoeliac related. There is no consensus on how to approach subjects with seronegative coeliac disease. AIM: To evaluate the accuracy of both an increase in CD3+ T-cell receptor gamma delta+ (TCRγδ+ ) intraepithelial lymphocytes and coeliac lymphogram for the diagnosis of coeliac disease in patients with seronegative villous atrophy. METHODS: Sixty-seven consecutive patients with seronegative villous atrophy were included. Duodenal biopsies to assess TCRγδ+ and CD3- by flow cytometry were performed at the index endoscopy. Coeliac lymphogram was defined as an increase in TCRγδ+ plus a decrease in CD3- intraepithelial lymphocytes. Sensitivity, specificity and Fagan's nomogram were calculated. RESULTS: Coeliac disease was diagnosed in 37 patients and noncoeliac villous atrophy in 30. Coeliac patients were younger (39 ± 3 vs 55 ± 3 years; P = 0.001), more often showed HLA-DQ2/8 (97.6% vs 61%; P = 0.002) and had a more severe histology (61% vs 32% Marsh 3b-c; P = 0.055), as compared to noncoeliac ones. Coeliac lymphogram was associated with a sensitivity of 87% (CI, 73.7-95) and specificity of 96.7% (82.7-99.9), whereas evaluating only TCRγδ+ yielded a sensitivity of 91.3% (79.2-97.6) and specificity of 83.3% (65.3-94.3). Among patients with a pre-test coeliac disease probability of 30%, post-test probabilities were 92% and 5% for positive and negative coeliac lymphogram, and 70% and 4% for positive and negative TCRγδ+ . CONCLUSIONS: Coeliac lymphogram was associated with a high level of diagnostic evidence either against or in favour of coeliac disease in patients with seronegative villous atrophy.

Exploring undiagnosed celiac disease in women with recurrent reproductive failure: The gluten-free diet could improve reproductive outcomes.

PROBLEM: Which is the prevalence and seroprevalence of celiac disease (CD) in women with recurrent reproductive failure? METHOD OF STUDY: Retrospective study performed in a single infertility clinic from September 2016 to December 2017. A total of 690 women with unexplained history of recurrent miscarriage and/or recurrent implantation failure were consecutively recruited. IgA anti-transglutaminase 2 (TG2) antibody data were collected, as well as IgG anti-TG2 and IgA/IgG anti-deamidated gluten peptide (DGP) data in most cases, and IgG anti-gliadin antibodies occasionally. In selected women, HLA-DQ genotyping was requested. Biopsy was suggested to all women with positive serological results or belonging to CD risk groups. Reproductive outcomes were recorded from women with high suspicion of CD and a control group comprised of 49 women. RESULTS: Anti-TG2-positive women comprised 1% of the sample. An additional 4% was observed considering less-specific antibodies (31 women). Only 39% of sero-positive women accepted duodenal biopsy. HLA and biopsy data discarded CD in 14 sero-positive cases (37%), only one with anti-TG2 antibodies. CD was suggested in 10 sero-positive and three sero-negative women (1.9%). Compared with controls, the live birthrate of the studied women with probable CD was significantly decreased before gluten removal of the diet (P = .015), but significantly increased after that (P = .020). CONCLUSION: One percent CD prevalence should be expected after anti-TG2 serological screening. However, more sensitive approaches should be explored, especially considering the potential beneficial effect of the gluten-free diet on the reproductive outcomes of women with CD.

CX3CL1-CX3CR1 Axis: A New Player in Coeliac Disease Pathogenesis.

BACKGROUND: The CX3CL1-CX3CR1 axis has been related to numerous diseases. The aim of our study was to investigate its involvement in coeliac disease (CD) pathogenesis, particularly in the early phase of the disease. METHODS: We collected peripheral blood from CD patients and controls, enrolled in a 3-day gluten challenge, to study soluble CX3CL1, I-TAC and MIG by Luminex, CX3CL1 and CX3CR1 gene expression by qPCR, and CX3CR1 protein expression in monocytes and CD8+, CD4+ and γδ+ T cells, by flow cytometry. We also analysed the expression of the CX3CL1 and CX3CR1 mRNA and protein in the duodenal biopsies of CD patients with active and treated disease, and in non-CD control individuals, by qPCR and immunohistochemistry. RESULTS: After the gluten challenge, increased levels of CX3CL1, I-TAC and MIG proteins were observed in the peripheral blood of CD patients, with no changes in CX3CL1 mRNA, or CX3CR1 mRNA and protein. Regarding duodenal tissue, CX3CL1 was absent or barely present in the superficial and basal epithelium of CD patients, contrasting with the moderate to high levels present in controls. CONCLUSIONS: CX3CL1 seems to be involved in the appearance and progression of CD, and it appears to be a potential diagnostic biomarker. Its use as an alternative therapeutic target in CD deserves further research.

Systematic Review and Meta-Analysis of Prevalence of Coeliac Disease in Women with Infertility.

We aimed to estimate the seroprevalence and the prevalence of coeliac disease (CD) in women with reproductive problems. A systematic review of English published articles until June 2019 was performed in PubMed and Scopus using the terms: (infertility and (coeliac disease OR gluten) OR (miscarriage and (coeliac disease OR gluten) OR (abortion and (coeliac disease OR gluten). All articles showing numerical data of anti-transglutaminase type 2 or anti-endomisium antibodies, or intestinal biopsy information were included. The study group comprised women with overall infertility, unexplained infertility, or recurrent spontaneous abortions. Two authors independently performed data extraction using a predefined data sheet. The initial search yielded 310 articles, and 23 were selected for data extraction. After meta-analysis, the pooled seroprevalence was very similar for overall and unexplained infertility, with a pooled proportion of around 1.3%-1.6%. This implies three times higher odds of having CD in infertility when compared to controls. The pooled prevalence could not be accurately calculated due to the small sample sizes. Further studies with increased sample sizes are necessary before giving specific recommendations for CD screening in women with reproductive problems, but current data seem to support a higher risk of CD in these women.

Influence of HLA on clinical and analytical features of pediatric celiac disease.

BACKGROUND: Celiac disease (CD) is triggered by gluten and related prolamines in genetically susceptible individuals. We aimed to investigate the influence of HLA-DQ genotypes in clinical, serological and histological features related to CD. METHODS: A retrospective observational study was performed including 463 Spanish patients with biopsy-proven CD. Clinical, serological, histological and HLA-DQ genetic data were collected from each participant. The presence of a family history of CD was also considered. Bivariate (chi-square tests or the Fisher's exact test) and multivariate (logistic regression after adjusting for age and sex) analyses were performed to assess the association between clinical and laboratory parameters with HLA-DQ. RESULTS: A predominance of females (62%), classical clinical presentation (86%) and positive anti-transglutaminase 2/endomysium antibodies (99%) was observed in our sample, with a mean age at onset of 2.6 ± 0.1 years. Five percent of our patients were first-degree relatives of subjects with CD, with HLA-DQ genetics showing increased homozygosity of HLA-DQ2.5 (p = 0.03) and HLA-DQ8 (p = 0.09). In the non-CD family history group, an association between delayed disease onset and HLA-DQ8 carriage was observed (p < 0.001), besides an influence of HLA-DQB1*02 gene dosage on clinical presentation and severity of histological damage (after adjusting for age and sex, p = 0.05 and p = 0.02, respectively) and a trend towards presence of specific antibodies (p = 0.09). These associations could not be evaluated properly in the group of patients with affected first-degree relatives due to the small sample size. CONCLUSIONS: HLA-DQ genotypic frequencies differ slightly between CD patients depending on their family history of CD. In patients lacking CD first-degree relatives, carriage of HLA-DQ2.5 with double dose of HLA-DQB1*02 seems to be associated with classical clinical presentation and more severe histological damage.

Expression patterns common and unique to ulcerative colitis and celiac disease.

Autoimmune diseases like celiac disease (CeD) and ulcerative colitis (UC) show a common genetic background defined by the existence of shared susceptibility loci. We aimed to go deeper into this common genetic background through performing a cross-disease study based on gene expression. We measured the expression of 21 genes located in 13 CeD-UC susceptibility regions, and 10 genes in five CeD risk regions. Determinations were carried out in colon/rectum samples from 13 UC patients (inflamed and uninflamed tissue) and four colon samples from controls. Duodenal samples from 19 CeD patients and 12 controls were used for comparisons. Differences were analyzed using the Bayesian method. The shared chromosomal regions containing TNFAIP3, PTPN2, ICOSLG, C1orf106, and IL21 showed similar results in both diseases. FASLG, PLEK, CCR4, and TAGAP, all located in CeD risk loci, were up-regulated in both CeD and UC patients. Finally, ZFP36L1, ZMIZ1, PUS10, UBE2L3, and BACH2 showed opposite results in CeD and UC. A high complexity underlies autoimmune common susceptibility loci, as the expression pattern of the studied genes does not always correlate with the one expected attending to the apparent genetic background. Differentially expressed genes such as ZFP36L1, ZMIZ1, PUS10, and BACH2 deserve further research in autoimmune diseases.

HLA-DQ distribution and risk assessment of celiac disease in a Spanish center

Aims: celiac disease is a multisystem immune-mediated disease triggered by gluten in genetically susceptible individuals. The HLA-DQ2 and/or HLA-DQ8 heterodimers are encoded by the main genetic predisposing factors and their presence is required for the development of the immunological response that leads to the disease. However, the HLA-conferred risk can differ within different countries. The aim of the study was to analyze the risk of Spanish children to develop celiac disease according to their HLA-DQ genotype. Methods: a retrospective observational case-control study was performed using a sample of 475 celiac patients and 628 controls. Results: children carrying the HLA-DQ2.5 had the highest disease risk, especially those with two HLA-DQB1*02 alleles. A similar high risk was observed in HLA-DQ8 homozygous individuals. A risk conferred by HLA-DQ8 in heterozygosity and HLA-DQ2.2 was also found and two patients with celiac disease carried the HLA-DQ7.5 haplotype as the only HLA risk factor. Conclusions: there are four genetic risk categories according to the HLA-DQ genotype. The HLA-DQ7.5 genotype does not confer risk but should not be used to rule out celiac disease when a high suspicion of the disease exists. These findings could be relevant to determine when to perform serological screening in asymptomatic subjects at risk of celiac disease

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Recomendaciones para la elaboración e interpretación de informes genéticos en enfermedad celíaca / Recommendations to report and interpret HLA genetic findings in coeliac disease

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