HPLC and flow cytometry combined approach for HbF analysis in fetomaternal haemorrhage evaluation.

Introduction: Recently, a flow cytometric (FC) based test has been developed for detection of circulating fetal cells to replace the less accurate and reproducible Kleihauer-Betke test.FC test is easier to perform, it can distinguish the origin of fetal cells, but it is expensive and available in highly specialized laboratories. We evaluated the introduction of high-performance liquid chromatography (HPLC) approach as initial screening to identify patients who need an additional FC test to better discriminate the nature of haemoglobin-F (HbF) positive cells. Methods: Blood samples from 130 pregnant women suspected to have fetomaternal haemorrhage were analysed with HPLC and FC methods. The cut-off for HbF HPLC concentration was calculated. Statistical analyses for the evaluation of HPLC as a screening method were performed. The positivity cut-off of HbF to be used as decision-making value to continue the investigation was calculated. Results: An excellent agreement (R2 > 0.90) was observed between the percentage of HbF obtained by HPLC and the percentage of fetal cells detected by FC. Results obtained from each assay were compared to define the HPLC threshold below which it is not necessary to continue the investigations, confirming the maternal nature of the HbF positive cells detected. Our study demonstrated that a cut-off of 1.0 % HbF obtained by HPLC was associated with the lowest rate of false negative results in our patient cohort. Conclusions: This study provides a new FMH investigation approach that possibly leads to a reduction in times and costs of the analysis.

Rapid evaluation of T cell clonality in the diagnostic work-up of mature T cell neoplasms: TRBC1-based flow cytometric assay experience.

The identification of mature T cell neoplasms by flow cytometry is often challenging, due to overlapping features with reactive T cells and limitations of currently available T cell clonality assays. The description of an antibody specific for one of two mutually exclusive T cell receptor (TCR) ß-chain constant regions (TRBC1) provides an opportunity to facilitate the detection of clonal TCRαß+ T cells based on TRBC-restriction. Here we prospectively analyzed 14 healthy controls and 63 patients with the flow cytometry protocol currently used for suspected T cell neoplasm implemented with immunostaining targeting TRBC1. Specimens were firstly classified in 3 groups based on clinical records data, laboratory findings and immunophenotypic features. T cell clonality was assessed by TCR Vß repertoire analysis and the new rapid TRBC1 assay. Results showed that TRBC1 unimodal expression was unequivocally associated with samples presenting with immunophenotypic aberrancies. Moreover, we demonstrated that the use of TRBC1 is useful in solving uncertain cases and confirmed the high sensitivity of the method in identifying small T cell clones of uncertain significance (T-CUS). Finally, we found a high degree of concordance (97%) comparing the currently available clonality assessment methods with the proposed new method. In conclusion, our results provided real-life evidence of the utility of TRBC1 introduction in the flow cytometric clonality evaluation for the routine diagnostic work-up of T cell neoplasms.

[Current incidence of congenital heart disease diagnosed in the first year of life: results of a 20-year registry with one-year follow-up and comparison with the literature]. / Incidenza attuale delle cardiopatie congenite diagnosticate nel primo anno di vita: risultati di un registro di 20 anni con follow-up ad un anno e confronto con la letteratura.

BACKGROUND: The epidemiological data on the incidence of congenital heart defects derive from retrospective registries based on birth discharge codes with methodological limits and different selection criteria. Our aim was to determine the actual incidence of congenital heart defects in the first year of life in a population of residents in a province of Tuscany, Italy. METHODS: This prospective study was conducted in 31 185 newborn residents in the province, enrolling a consecutive population throughout the first year of life and followed up at least for one year. The population cohort was controlled and merged with a retrospective research of the diagnostic codes derived from hospital discharge records of the region of Tuscany. RESULTS: A congenital heart disease was suspected in 10 167 newborns, 32.6% of all live births. Overall, 524 defects were diagnosed (5.2% of the exams), with an incidence at birth of 16.8/1000/year (M/F ratio 0.84). Isolated ventricular septal defects were 343 and 198 spontaneously closed within one year, therefore, the total number dropped to 326 with a one-year incidence of 10.35/1000/year. Besides ventricular septal defects, the most common defects were atrial septal defects (7.3%), followed by ductus arteriosus (4.2%), aortic coarctation (4%), pulmonary stenosis (3.3%) and tetralogy of Fallot (3.1%). CONCLUSIONS: The one-year inclusion period and follow-up allowed us to exclude those defects whose hemodynamic significance is not clear at birth, or are spontaneously reversible within the first year of life. Nevertheless, with the inclusion of defects not evident at birth, congenital heart defects are still common.

Incidence and natural history of neonatal isolated ventricular septal defects: Do we know everything? A 6-year single-center Italian experience follow-up.

BACKGROUND: Despite ventricular septal defects (VSDs) are the most common congenital heart diseases (CHDs) in the neonatal period, their incidence and natural history are still debated and their follow-up and management strategies remain controversial. Our aim was to evaluate the incidence and natural history of isolated VSDs. METHODS: From January 1996 to December 2015 all neonates with a CHD suspicion were referred to the Cardiological Department of Grosseto Misericordia Hospital. Only newborns with confirmed isolated VSD were enrolled in this study and followed for 6 years. RESULTS: Our 343 newborns with an isolated VSD (incidence of 10.45/1000/births) account for 64% of all detected CHDs. VSDs location were as follows: muscular (73.8%), perimembranous (11.3%), inlet (1%), and outlet (0.8%). Of the located VSDs, 90% were small, 7.5% moderate, and 2.5% large, respectively. Spontaneous closure was observed in 96 (29.2%) of the VSD patients at 6-month, 198 (60.2%) at 1-year, 261 (79.3%) at 2-year, and in 302 (91.8%) at 6-year follow-up. Risk factors for defect persistence were a perimembranous location (P = .001; HR: 0.508, CI: 0.342-0.755), detection of multiple defects (P = .043; HR: 0.728, CI: 0.536-0.990), and male gender (P < .048; HR: 0.783, CI: 0.615-0.998), respectively. CONCLUSIONS: We here provide an incidence and natural history of neonatal isolated VSDs in a neonatal Caucasian population. These data may be useful for the development of expert consensus/standard recommendation guidelines for the follow-up and VSD management, data that are currently lacking.

[Myocardial infarction as the first manifestation of an atrial myxoma: the bomb in the heart. Case report and literature review]. / Infarto miocardico come prima manifestazione di un mixoma atriale: la bomba nel cuore. Caso clinico e revisione della letteratura.

Atrial myxoma is a cardiac tumor often histologically benign but very insidious for its mechanical complications. Among these, myocardial infarction can be an expression of coronary embolism. Imaging techniques are essential for the diagnosis and the therapeutic steps. We describe the clinical case of a 52-year-old woman with acute myocardial infarction and normal coronary arteries with left atrial myxoma. We conducted a review of published case reports over the last 45 years on the rare association between atrial myxoma and acute myocardial infarction, to obtain pathogenic and epidemiological information from the real world.

Antibiotics GE23077, novel inhibitors of bacterial RNA polymerase. II. Structure elucidation.

During the screening program for new antibacterial agents produced by actinomycetes, GE23077 was isolated from fermentation broths of an Actinomadura sp. strain as a complex of factors A1, A2, B1, B2. NMR, MS and GC/MS analysis carried out on the isolated components led to the conclusion that GE23077 is a novel cyclic heptapeptide consisting of common and unusual amino acids. The chemical structures of the complex components were elucidated. Components A and B differ in the structure of the acyl group connected to a 2,3-diaminopropanoic acid moiety. A alpha-amino-malonic acid residue in the peptidic sequence is the origin of an isomerization process between A1 and A2 as well as B1 and B2. The chirality of the alpha-amino-malonic acid residue can be inverted easily via keto-enol tautomerism. Factors A2 and B2 should be considered as epimers of A1 and B1 respectively. By degradation studies the absolute configuration of some amino acids were determined. Chiral GC-MS and Micellar Electrokinetic Capillary Chromatography (MEKC) were used to define the absolute stereochemistries of five out of ten chiral centers.

Antibiotics GE23077, novel inhibitors of bacterial RNA polymerase. I. Taxonomy, isolation and characterization.

GE 23077 factors A1, A2, B1 and B2 are novel antibiotics isolated from fermentation broths of an Actinomadura sp. strain. GE23077 antibiotics are cyclic peptides, which inhibit Escherichia coli RNA polymerase at nM concentrations. Both rifampicin-sensitive and rifampicin-resistant polymerases are inhibited, whereas E. coli DNA polymerase and wheat germ RNA polymerase are substantially not affected. In spite of the potent activity on the enzyme, the antibiotics generally show poor activity against whole cell bacteria. The spectrum of activity is restricted to Moraxella catarrhalis, including clinical isolates, with partial activity against Neisseria gonorrhoeae and Mycobacterium smegmatis.