and C
and C.R.-K. one EMA positive. Both antibodies, EMA and anti-TG2, reached very similar sensitivities, 98% and 99% respectively, while EMA acquired an increased specificity (99%) than anti-TG2 (93%). Through the use of both markers mixed, in comparison to using anti-TG2 by itself, 5.7% of sufferers are better diagnosed. Nevertheless, whenever we evaluate the efficiency of EMA and anti-TG2 in symptomatic and asymptomatic sufferers, the sensitivity of EMA is usually 98% irrespective of symptoms, thus higher than for anti-TG2 10 upper limit of normal (ULN) (respectively 77% and 84%). Our results support the use of EMA to increase CD diagnostic accuracy in a non-biopsy approach, especially in asymptomatic children. (ESPGHAN) guidelines published in 2012, allow for a diagnosis of CD without biopsies in children and adolescents with symptoms and levels of immunoglobulin A against anti-tissue transglutaminase antibodies (anti-TG2) 10 occasions the upper limit of normal (ULN), confirmed by anti-endomysium antibodies (EMA) and positivity for HLA DQ2 and/or DQ8 [1]. In these cases, the enteropathy, detected by a small intestinal biopsy (SIB), is an additional diagnostic element but is not an essential criterion. Thus, CD antibodies are considered highly specific, especially in children. Moreover, EMA testing reaches a higher specificity (98%C100%) when it is carried out by experienced professionals, therefore EMA is considered the reference standard for CD-specific antibodies. A recent multinational prospective study (ProCeDE) [2] validates this non biopsy approach in children presenting clinical symptoms whenever anti-TG2 levels are 10 ULN and with positive EMA in a second blood sample, thus supporting the use of EMA as a confirmatory test when CD diagnosis is performed without biopsy. The authors also conclude HLA does not improve the diagnostic accuracy if the abovementioned criteria are met. Similarly, Wolf and colleagues [3] observed in a prospective study that testing for EMA and HLA did not increase the positive predictive value (PPV) in cases with anti-TG2 10 ULN. However, the majority of patients were included based on prior positive anti-TG2 assessments, and because of the patients preselection the specificity of EMA is lower (94%) than commonly described. Based mainly on these two studies, the 2019 ESPGHAN guidelines state that the non-biopsy approach is safe in children with anti-TG2 10 ULN and Carboxypeptidase G2 (CPG2) Inhibitor positive EMA without the need for HLA assessment [4]. An evidence-based review of the accuracy of serological markers for CD diagnosis reports an overall slightly better sensitivity for anti-TG2 compared to EMA, and conversely a higher specificity for EMA (98%) compared to anti-TG2 (90%C95%) [5]. However, the specific role of EMA in combination with anti-TG2 has been addressed by a limited number of studies and is still a matter of debate. The aim of our study is to assess the contribution of EMA to the accuracy of serology-based CD diagnosis in the non-biopsy approach, not only in symptomatic, but Carboxypeptidase G2 (CPG2) Inhibitor also in asymptomatic patients. 2. Patients and Methods 2.1. Study Design and Participants We have retrospectively evaluated pediatric patients, aged 0.8 to HSF 15 years, who were referred to the Pediatric Gastroenterology and Hepatology Unit of La Fe University Hospital between 2009 and 2017, for serological evaluation because of clinical symptoms suggesting CD or as screening in at risk groups. Only those in whom serological CD markers and total serum IgA levels were available were considered for statistical analysis. Additional inclusion criteria were: Determination of EMA and anti-TG2 antibodies from the same serum sample, serum samples must be collected no earlier than 3 weeks before the biopsy, if performed, and patients were on a gluten-containing diet at the time of biopsy and blood sampling. Patients Carboxypeptidase G2 (CPG2) Inhibitor who did not have a final diagnosis and/or their histopathological study was not valid Carboxypeptidase G2 (CPG2) Inhibitor for interpretation and/or had an IgA deficiency, were excluded from the study. CD diagnosis was based on ESPGHAN 1990 and 2012 criteria [1,6] Data on clinical symptoms, final diagnosis, degree of histological lesion, and HLA genotyping (DQ2 and/or DQ8) were obtained from the clinical files. The present study was approved by the Ethics Committee of La Fe University Hospital. The number of ethical approval: 2017/0002. 2.2. Methodology 2.2.1. Serology EMA antibodies were routinely tested by an indirect immunofluorescence method (IFI) using monkey esophagus sections (Biosystems?, Barcelona, Spain). The test serum samples were diluted 1:5 and incubated for 30 minutes with anti-human immunoglobulin A conjugated with fluorescein isothiocyanate (FITC) (Dako?, Glostrup, Denmark) 1:20 dilution. Vectashield mounting medium for fluorescence (Vector Laboratories, Burlingame, CA, USA) was added before carefully covering with a coverslip. Slides were blindly examined using a Motic Trinocular BA400 with filter for FITC microscope (20) by two experienced professionals with more than ten years of experience reading EMA results. A positive result was recorded if the connective tissue surrounding the muscle cells was brightly fluorescent,.