Antineutrophil Cytoplasmic Antibodies Testing in a Large Cohort of Unselected Greek Patients

Objective. To retrospectively evaluate ANCA testing in a cohort of unselected Greek in- and outpatients. Methods. In 10803 consecutive serum samples, ANCA were tested by indirect immunofluorescence (IIF) and ELISA. ELISA in inpatients was performed only on IIF positive sera. Results. Low prevalence (6.0%) of IIF positive samples was observed. Among these samples, 63.5% presented perinuclear (p-ANCA), 9.3% cytoplasmic (c-ANCA) and 27.2% atypical (x-ANCA) pattern. 16.1% of p-ANCA were antimyeloperoxidase (anti-MPO) positive, whereas 68.3% of c-ANCA were antiproteinase-3 (anti-PR3) positive. Only 17 IIF negative outpatients' samples were ELISA positive. ANCA-associated vasculitides (AAV), connective tissue disorders and gastrointestinal disorders represented 20.5%, 23.9%, and 21.2% of positive results, respectively. AAV patients exhibited higher rates of MPO/PR3 specificity compared to non-AAV (93.8% versus 8%). Conclusions. This first paper on Greek patients supports that screening for ANCA by IIF and confirming positive results by ELISA minimize laboratory charges without sacrificing diagnostic accuracy

Short Tandem Repeats Loci in Parentage Testing

The need for confirmation or exclusion of biological father and / or biological mother is a social phenomenon, which is imposed by socio-economic and, sometimes, by moral-psychological factors. Modern science has significantly contributed to solving this problem, as many medical methods have been applied for this purpose. Biological markers that have been conducted for distinguishing between individuals were the human ABO blood groups, the Rh, MNS, Duffy, Kidd, and Kell systems, as well as the human leukocyte antigens (HLA) system. For a long time theHLA testing represented the standard testing in forensic genetics, but, due to the linkage disequilibrium and the predominance of certain HLA alleles and as the demand for parentage investigations is rapidly increasing during the recent years, this serological era has been replaced by molecular markers through the introduction of “DNA profiling”, which is based on polymorphisms of short tandem repeats (STRs) loci. Nowadays, “DNA profiling” by analysis of STR loci is the method of choice for human identification and parentage investigations. This technique is the most informative, accurate, robust, rapid, cost-effective method of genotyping and has worldwide acceptance in the courts, as the probability of parentage will typically be greater than 99.99999%

Discrimination Power Assessment of STR Genotyping in Parentage Investigation

OBJECTIVE Nowadays, the application of DNA-typing in laboratory medicine is increasing rapidly for paternity/maternity disputes. The goal of this study was to evaluate the use of polymorphic microsatellite marker DNA analysis and to establish this analysis as the method of choice for parentage investigations.SUBJECTS AND METHODS Among 708 civil parentage tests addressed to our Laboratory previously examined for HLA class I (-A*,-B*,-Cw*), and class II (-DRB1*,-DQB1*,-DPB1*) alleles using PCR-SSOP and/or PCR-SSP methodologies, a cohort of 50 cases (137 individuals) of disputed parentage was selected. In these cases DNA-typing was generated from co-amplification of 15 autosomal STR DNA markers (D3S1358, HUMTH01, D21S11, D18S51, Penta E, D5S818, D13S317, D7S820, D16S539, HUMCSF1PO, Penta D, HUMvWA, D8S1179, HUMTPOX, HUMFGA and the sex determining Amelogenin marker HUMAMEL), using fragment analysis methodology.RESULTS The evaluation of the results showed that 15 out of 50 cases, were sufficient for exclusion of fatherhood by both approaches (HLA and STRs). In all remaining 35 non-excluded cases, the PI value using HLA genotyping ranged from 76 to 6,452,794, whereas using aSTR genotyping ranged from 15,173 to 9.2 x 1010. In one non-excluded motherless case the alleged father showed one genetic discrepancy with the child at D21S11 locus, due to a mutation event.CONCLUSION The use of DNA-typing with 15 aSTR loci for parentage testing provides an accurate and high-sensitivity method which is simpler to perform and more rapid than an accepted standard technology, such as HLA genotyping. The analysis of aSTR loci offers a highly discriminating test suitable for trio paternity testing, increasing the W rate in comparison to HLA genotyping. Nevertheless, when a mutation event occurs in motherless cases, combination of HLA and STR polymorphisms offers high level of information, and also diminishes the possibility of false exclusion due to aSTRs mutations

Anti-Ro60 Seropositivity Determines Anti-Ro52 Epitope Mapping in Patients With Systemic Sclerosis

Epitope mapping of anti-Ro52 antibodies (Abs) has been extensively studied in patients with Sjögren's syndrome (SjS) and systemic lupus erythematosus (SLE). Comprehensive epitope mapping in systemic sclerosis (SSc), where anti-Ro52 antibodies are also frequently detected, has not been performed. The aim of the present study was to fully characterize Ro52 epitopes in anti-Ro52-positive SSc using Ro52 fragments spanning the full antigen. Further analysis was made according to anti-Ro60 status. Epitope mapping was performed in 43 anti-Ro52-positive SSc patients. Seventy eight anti-Ro52-positive pathological controls, including 20 patients with SjS, 28 patients with SLE, 15 patients with dermatomyositis (DM), and 15 patients with primary biliary cholangitis (PBC), and 20 anti-Ro52-negative healthy individuals as normal controls were also tested. Five recombinant Ro52 fragments [Ro52-1 (aa 1-127), Ro52-2 (aa 125-268), Ro52-3 (aa 268-475), Ro52-4 (aa 57-180), and Ro52-5 (aa 181-320) were used to test reactivity by line-immunoassay and in house ELISA. Anti-Ro60 reactivity was tested by ELISA. All anti-Ro52 positive sera reacted with Ro52-2; none recognized Ro52-3. Antibodies against Ro52-1 were less frequently found in SSc than in SjS/SLE (11.6 vs. 41.7%, p = 0.001); and antibodies against Ro52-4 were less frequently found in SSc than in SjS/SLE (27.9 vs. 50%, p = 0.03). In SSc patients, reactivity against Ro52-1 was more frequent in anti-Ro52+/anti-Ro60+ than in anti-Ro52+/anti-Ro60-patients (33.3 vs. 0%, p = 0.003). In this comprehensive analysis of Ro52 epitope mapping in SSc, the coiled coil domain remains the predominant epitope on Ro52. Contrary to SjS and SLE, patients with SSc fail to identify epitopic regions within the N-terminus of the protein, especially if they lack con-current anti-Ro60 reactivity

Could Immunophenotype Guide Molecular Analysis in Patients with Myeloid Malignancies?

Objective: Immunophenotype has been correlated with molecular aberrations in several studies. The aim of this study was the discovery of immunophenotypic features related to mutations in AML and MDS patients connected to prognostic factors. Moreover, an effort to evaluate a method for the detection of the most common NPM1 mutations of exon12 and Internal Tandem Duplications (ITD) mutations of FLT3 gene by flow cytometry was performed. Method: Patients with de novo myeloid neoplasms [ AML and MDS (AML-M3 patients were excluded)] were included. FLT3/ITD/TKD and NPM1 mutations were detected by PCR and fragment analysis. The immunophenotypic analysis was performed by multi-dimensional flow cytometry (FC) with a standardized panel of monoclonal antibodies on peripheral blood or bone marrow samples. Nucleophosmin Antibody and CD135 were used for the mutations immunophenotypic detection. Results: NPM1 and/or FLT3 mutations correlated with low or no expression of more immature cells markers such as CD34, CD117, HLADR, as well as higher expression of more mature markers such as CD11b. The higher expression of CD33 should be mentioned as well. The presence of NPM1mut and FLT3/ITD does not seem to be detectable by FC at least using these two monoclonal antibodies. The presence of CD7 aberrant lymphoid marker’s expression was associated with FLT3mut, NPM1wt genotype. CD56 or CD2 positivity was found only in patients’ samples negative for NPM1 and/or FLT3 mutations. Conclusions: Certain immunophenotype findings including the presence of aberrant lymphoid markers may be indicative of the presence of mutations in NPM1 and FLT3 linked to prognosis

Detection of specific autoantibodies in rheumatoid arthritis using immunoblotting technique ( new techniques)

Rheumatoid Arthritis (RA), the most commonly occurring autoimmune disorder, if untreated leads progressively to permanent joint damage and disability. Early therapeutic intervention may prevent progression of joint destruction, however for the initiation of an aggressive treatment more sensitive and specific diagnostic tests are required. In 1987 the American College of Rheumatology (ACR) in addition to clinical criteria, proposed the Rheumatoid Factor (RF) as the only serological diagnostic marker. This marker lacks specificity and has very low sensitivity in the early stages of the disease. In order to meet the need for improved diagnostic and prognostic tests various biomarkers and autoantibodies have being assessed. Among them, only the anti-Cyclic Citrullinated Peptide (anti-CCP) has gained wide acceptance and was included in 2010, in the ACR/EULAR classification criteria for RA. Aim of the study The aim of this study is the detection and measurement of several biomarkers and autoantibodies in Greek patients with RA, the evaluation of their role in the diagnosis of RA and the prediction of the evolution of an inflammatory arthritis to RA. In addition we have investigated their importance in the prognosis of the course of the disease, the response to therapeutic protocols and their use in routine clinical practice, with the formation of an algorithm. Patients and Methods A total of 426 patients were included in the study. Of those 278 (64.4%) were diagnosed as having RA (group A) and 148 (34.6%) suffered from other autoimmune or inflammatory diseases (group B) such as Systemic Lupus Erythematosus (N=31), Mixed Connective Tissue Disease (N=4), Polyarthritis Syndrome (N=47), Seronegative Spondylarthritis (N=36) and Idiopathic Inflammatory Bowel Disease (N=30). Moreover, 32 blood donors were included as healthy controls. As a special category 131 the 47 patients with Polyarthritis Syndrome, 17 (36.2%) of whom developed RA during a six months’ follow up, were studied. The laboratory tests applied in the serum samples of the patients and controls included: 1) the measurement of RF, of C-Reactive Protein (CRP) and of complement factors C3, C4 by nephelometry, 2) the titration of antinuclear (ANA) and anti-double stranded DNA (anti-dsDNA) using indirect immunofluorescence, 3) the detection of autoantibodies to extractable nuclear antigens (anti-ENA) using the method of ELISA and Immunoblotting, and 4) the anti-RA33, anti-CCP2, anti-CCP3.1, anti-MCV antibodies as well as and the RF IgG, IgM and IgA classes, using ELISA. For the statistical analysis of the results the program SPSS 18.0 and STATA 8.0, were used. Results 1. The comparison of group A patients with RA versus group B showed the following results : a) The presence of positive ANA and anti-RA33 antibodies or of abnormal CRP values does not differ significantly between the two groups of patients. b) Anti-ENA antibodies were detected in 47 (16.8%) of 279 samples studied. There was no significant difference in the percentages of positive anti-Ro (SSA) antibodies between the two groups. However the investigation of the specificity of the anti-Ro antibodies for the antigenic epitopes (Ro52, Ro60) showed a higher percentage of anti-Ro52 antibodies in the group A samples in comparison with group B (57.14% vs 21.4%, p= 0.025). c) The percentage of RA patients with positive anti-CCP2, anti-CCP3.1, anti-MCV antibodies and abnormal RF values was significantly higher in group A in comparison to group B patients (p2 years), showed no significant difference regarding any of those parameters. 5. The index for disease activity (DAS28) was not shown to be significantly correlated with CRP, RF or anti-CCP2 levels whereas a positive correlation was found with the levels of anti-MCV antibodies (r=0.69, p 2 έτη δεν ανέδειξε στατιστικά σημαντική διαφορά για καμία παράμετρο. 5. Η μεταβολή του δείκτη ενεργότητας της νόσου (DAS28) δεν βρέθηκε να συσχετίζεται σημαντικά με τα επίπεδα των αντι-CCP2, του ΡΠ και της CRP ενώ βρέθηκε σημαντική θετική συσχέτιση με τα επίπεδα των αντι-MCV αντισωμάτων (r=0,69, p<0,001). Επίσης, ο βαθμός μεταβολής των επιπέδων όλων των παραμέτρων δεν φαίνεται να επηρεάζεται από το θεραπευτικό σχήμα που ακολουθούσαν οι ασθενείς για το χρονικό διάστημα 2 ετών που μελετήθηκε. Συμπεράσματα Τα αντι-CCP2 αντισώματα εμφανίζουν την υψηλότερη προγνωστική ικανότητα για τη ΡΑ τόσο σε σχέση με άλλα μη ΡΑ νοσήματα, όσο και σε σχέση με τους υγιείς. Σημαντική καταγράφεται η προγνωστική ικανότητα του ΡΠ τόσο για τη ΡΑ σε σύγκριση με άλλα μη ΡΑ νοσήματα, όσο και για την εξέλιξη του Πολυαρθρικού Συνδρόμου σε ΡΑ μέσα στους πρώτους 6 μήνες από την αρχική διάγνωση, υπολείπεται όμως της προγνωστικής ικανότητας των αντι-CCP2. Έτσι και οι δύο παράμετροι φαίνεται να αποτελούν αξιόλογους δείκτες για την πρόγνωση, τη διάγνωση αλλά και την πρόβλεψη της εξέλιξης της νόσου. Επιπλέον, η υψηλότερη ειδικότητα (83,3%) των αντι-MCV αντισωμάτων για τη ΡΑ σε σχέση με τα μη ΡΑ νοσήματα καθώς και η θετική συσχέτιση της μεταβολής του DAS28 με τη μεταβολή του επιπέδου των αντι-MCV αντισωμάτων, τα αναδεικνύει ως μοναδικό αξιόπιστο δείκτη για την παρακολούθηση της δραστηριότητας της νόσου, απαραίτητο εργαλείο για τον κλινικό γιατρό στη λήψη αποφάσεων αναφορικά με τη διατήρηση ή τροποποίηση του θεραπευτικού σχήματος για τον ασθενή με ΡΑ. 13

Classical and Non-Classical HLA Alleles as Supplementary Markers in Indirect Kinship Parentage Testing

A civil paternity investigation involving the parents of the deceased alleged father in order to establish a family relationship is presented. On the basis of the 23 autosomal short tandem repeat (aSTR) genotyping results, conclusive proof of paternity was not achieved, as the probability of paternity (W) was calculated to 0.99988. Additional genetic data of 17 classical and non-classical human leukocyte alleles (HLA) typing by next-generation sequencing (NGS) at a high-resolution level supported the hypothesis of grandpaternity over the hypothesis of coincidental paternal obligate allele (POA) sharing (total WaSTR&HLA = 0.9999998). The present study demonstrates the utility of 17 HLA genetic markers-typing in the solution of deficiency cases of disputed parentage

Celiac Disease in Adult Patients: Specific Autoantibodies in the Diagnosis, Monitoring, and Screening

The increasing prevalence of celiac disease (CD), especially in adults, its atypical clinical presentation, and the strict, lifelong adherence to gluten-free diet (GFD) as the only option for healthy state create an imperative need for noninvasive methods that can effectively diagnose CD and monitor GFD. Aim. Evaluation of anti-endomysium (EmA) and anti-tissue transglutaminase IgA (tTG-A) antibodies in CD diagnosis, GFD monitoring, and first degree relatives screening in CD adult patients. Methods. 70 newly diagnosed Greek adult patients, 70 controls, and 47 first degree relatives were tested for the presence of EmA and tTG-A. The CD patients were monitored during a 3-year period. Results. EmA predictive ability for CD diagnosis was slightly better compared to tTG-A (P=0.043). EmA could assess compliance with GFD already from the beginning of the diet, while both EmA and tTG-A had an equal ability to discriminate between strictly and partially compliant patients after the first semester and so on. Screening of first degree relatives resulted in the identification of 2 undiagnosed CD cases. Conclusions. Both EmA and tTG-A are suitable markers in the CD diagnosis, in the screening of CD among first degree relatives, having also an equal performance in the long term monitoring

Bioclimatic consideration of structural materials of tram stops in Athens

207 σ.Ο στόχος της παρούσας διπλωματικής εργασίας είναι η βιοκλιματική προσέγγιση των δομικών υλικών που απαρτίζουν τις στάσεις του τραμ στην Αθήνα, στο πλαίσιο του βιοκλιματικού σχεδιασμού των υπαίθριων αστικών χώρων. Επιχειρείται η κατανόηση του τρόπου με τον οποίο τα υλικά που χρησιμοποιούνται στις εξωτερικές επιφάνειες των πόλεων συμμετέχουν στις διαδικασίες μετάδοσης θερμότητας, και επηρεάζου το αστικό μικροκλίμα και τις συνθήκες θερμικής άνεσης. Ειδικότερα, διερευνάται η θερμική συμπεριφορά των κυριότερων υλικών που συναντώνται στις στάσεις του τραμ. Η τεκμηρίωση της θερμικής συμπεριφοράς βασίζεται σε μετρήσεις που έλαβαν χώρα τόσο τη χειμερινή όσο και τη καλοκαιρινή περίοδο, κατά τη διάρκεια των οποίων μετρήθηκαν με θερμοκάμερα οι επιφανειακές θερμοκρασίες που αναπτύσσουν τα υλικά ενώ παράλληλα καταγράφηκαν με κατάλληλα όργανα τα μετεωρολογικά δεδομένα που επικρατούσαν στο περιβάλλον κάθε στάσης. Όπως προέκυψε από την επεξεργασία των μετρήσεων τα δομικά υλικά των στάσεων του τραμ διαμορφώνουν ικανοποιητικές συνθήκες θερμικής άνεσης στο περιβάλλον των στάσεων, καθιστώντας την όλη κατασκευή βιοκλιματική και υποδειγματική για το σχεδιασμό υπαίθριων αστικών χώρω που εξυπηρετούν τις μεταφορικές ανάγκες των χρηστών.The aim of this thesis is to present a bioclimatic approach to the structural materials used for the construction of tram stations in Athens, in the context of the bioclimatic design of urban outdoor spaces. There is an attempt to comprehend the way that these materials, used at the external surfaces of the cities, are involved in heat transfer processes affecting the urban microclimate and the thermal comfort conditions. More specifically, this thesis investigates the thermal behavior of the most common materials used in the construction of tram stops. The documentation of the thermal behavior is built on measurements that took place both in winter and summer season. during which were measured the surface temperatures developed by the materials with the use of a thermal imaging camera, while at the same time, with the appropriate equipment was noted down the meteorological data prevailing in the tram stations' environment, Based on the elaboration of measurements it is deduced that the structural materials of the tram stops create satisfying conditions of thermal comfort in the stations' environment, making, thus, the whole construction bioclimatic , as well as exemplary for the design of urban open spaces that serve the passengers' needs.Αλεξάνδρα Τ. ΤσαγδήΑθηνά Ν. Τσιρογιάνν

Elevated circulating ghrelin, but not peptide YY(3-36) levels, in term neonates with infection

Background: Early diagnosis and treatment of neonatal infection is important to prevent morbidity and mortality. The gastrointestinal tract-derived hormones ghrelin and peptide YY (PYY), which participate in the regulation of food intake and energy balance, may also play roles in the inflammatory response. Their involvement in neonatal infection is not known. Methods: Plasma ghrelin and PYY(3-36) levels were serially measured (by ELISA) on Days 0, 1, 2, 3 and 7 following admission in 36-term neonates with febrile infection (22 of them were septic) and once in 20 healthy term neonates of similar postnatal age and gender distribution, as controls. Associations of ghrelin and PYY(3-36) levels with clinical and laboratory parameters, including anthropometrics, fever, leukocyte and platelet counts, serum glucose, C-reactive protein (CRP) and serum amyloid A levels, were assessed. Results: Plasma ghrelin levels were significantly higher in infected neonates than in controls at each study day (p=0.009), whereas PYY(3-36) levels did not differ significantly between patients and controls at any day. In infected neonates, ghrelin levels on admission correlated negatively with serum glucose levels (p=0.003), whereas fever change during the course of infection was significantly associated with change of ghrelin levels (p=0.01). Receiver operating characteristic analysis of ghrelin levels resulted in significant areas under the curve (AUC) for detecting infected neonates on admission (AUC=0.728, p=0.005). Conclusions: Circulating ghrelin, but not PYY(3-36), levels are increased in neonates with infection, possibly reflecting and/or participating in the inflammatory process