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.

Characterization of lymphocyte subsets in ascitic fluid and peripheral blood of decompensated cirrhotic patients with chronic hepatitis C and alcoholic liver disease: A pivotal study.

Hepatitis C virus and alcoholic liver disease are major causes of chronic liver diseases worldwide. Little is known about differences between chronic hepatitis C and alcoholic liver disease in terms of lymphocytes' sub-population. Aim of the present study was to compare the sub-populations of lymphocytes in both ascitic compartment and peripheral blood in patients with decompensated liver cirrhosis due to chronic hepatitis C and alcoholic liver disease. Patients with decompensated liver cirrhosis due to hepatitis C virus or alcoholic liver disease evaluated from April 2014 to October 2016 were enrolled. Whole blood and ascitic fluid samples were stained with monoclonal antibodies specific for human TCRɑß, TCRɣδ, CD3, CD4, CD8, CD19, CCR6, CD16, CD56, CD25, HLA-DR, Vɑ24. Sixteen patients with decompensated liver cirrhosis were recruited (9 with hepatitis C virus and 7 with alcoholic liver disease). In ascitic fluid, the percentage of both CD3+CD56- and CD3+CD56+iNKT cells resulted higher in hepatitis C virus patients than in alcoholic liver disease patients (1.82 ± 0.35% vs 0.70 ± 0.42% (p < 0.001) and 1.42 ± 0.35% vs 0.50 ± 0.30% (p < 0.001), respectively). Conversely, in peripheral blood samples, both CD3+CD56- and CD3+CD56+iNKT cells resulted significantly higher in alcoholic liver disease than in hepatitis C virus patients (4.70 ± 2.69% vs 1.50 ± 1.21% (p < 0.01) and 3.10 ± 1.76% vs 1.00 ± 0.70% (p < 0.01), respectively). Both elevation of iNKT cells in ascitic fluid and reduction in peripheral blood registered in hepatitis C virus but not in alcoholic liver disease patients might be considered indirect signals of tissutal translocation. In conclusion, we described relevant differences between the two groups. Alcoholic liver disease patients displayed lower number of CD3+CD4+ cells and a higher percentage of CD3-CD16+, Vα24+CD3+CD56- and Vα24+CD3+CD56+iNKT cells in ascitic fluid than hepatitis C virus positive subjects. Further studies might analyze the role of immune cells in the vulnerability toward infections and detect potential targets for new treatments especially for alcoholic liver disease patients.

Bench to bedside review of myositis autoantibodies.

Idiopathic inflammatory myopathies represent a heterogeneous group of autoimmune diseases with systemic involvement. Even though numerous specific autoantibodies have been recognized, they have not been included, with the only exception of anti-Jo-1, into the 2017 Classification Criteria, thus perpetuating a clinical-serologic gap. The lack of homogeneous grouping based on the antibody profile deeply impacts the diagnostic approach, therapeutic choices and prognostic stratification of these patients. This review is intended to highlight the comprehensive scenario regarding myositis-related autoantibodies, from the molecular characterization and biological significance to target antigens, from the detection tools, with a special focus on immunofluorescence patterns on HEp-2 cells, to their relative prevalence and ethnic diversity, from the clinical presentation to prognosis. If, on the one hand, a notable body of literature is present, on the other data are fragmented, retrospectively based and collected from small case series, so that they do not sufficiently support the decision-making process (i.e. therapeutic approach) into the clinics.