Use of Sysmex XN-10 red blood cell parameters for screening of hereditary red blood cell diseases and iron deficiency anaemia.

INTRODUCTION: In daily practice in haematology laboratories, red blood cell (RBC) abnormalities are frequent and their management is a real challenge. The aim of this study is to establish a "decision tree" using RBC and reticulocyte parameters from the SYSMEX XN-10 analyser to distinguish between patients with a hereditary RBC disease from iron deficiency anaemia and other patients. METHODS: We analysed results of complete RBC counts in a cohort composed of 8217 adults divided into 5 different groups: iron deficiency anaemia (n = 120), heterozygous haemoglobinopathy (n = 92), sickle cell disease syndrome (n = 56), hereditary spherocytosis (n = 18) and other patients (n = 7931). A Classification And Regression Tree (CART) analysis was used to obtain a two-step decision tree in order to predict these previous groups. RESULTS: Five parameters and the calculated RBC score were selected by the CART method: mean corpuscular haemoglobin concentration, percentage of microcytes, distribution width of the RBC histogram, percentage of nucleated red blood cells, immature reticulocytes fraction and finally RBC Score. When applying the tree and recommended flowchart, 158/166 of the RBC hereditary disease patients and 114/120 iron deficiency anaemia patients are detected. Overall, the correct classification rate reached 99.4%. Sensitivity and specificity for RBC disease detection were 95.2% and 99.9%, respectively. These results were confirmed in an independent validation cohort. CONCLUSION: Based on the XN-10 RBC and reticulocyte parameters, we propose a two-step decision tree delivering a good prediction and classification of hereditary RBC diseases. These results can be used to optimize additional reticulocyte analysis and microscopy review.

Usefulness of thresholds for smear review of neutropenic samples analyzed with a Sysmex XN-10 analyzer.

Neutropenia is one of the main criteria for a blood smear review. The objective of this study was to compare the thresholds proposed by the international consensus group for hematology review (1.0 109/L) and the French speaking Group for Cellular Haematology (1.5 109/L) in terms of the number of useless smears. We collected 112,097 analyzed samples from four laboratories equipped with XN instruments (Sysmex, Kobe, Japan) during early 2016. The only exclusion criterion was a leucocyte count below 0.5 109/L. In the absence of abnormal cells and/or morphology suggesting haematological disease, samples were classified as 'negative for morphology' and the differential from the XN-10 was reported. These smear procedures were considered as uninformative. Some 2202 samples met the criterion for neutropenia (<1.5 109/L) for slide review representing 1.96% of the total. These included 1031 with neutropenia alone and 1171 neutropenia plus other abnormalities. Of the 1031 with neutropenia alone, 886 had a neutrophil count between 1.0 109/L and 1.5 109/L. The smear was uninformative for all of these samples. In conclusion, microscopic examination of a blood smear provided very limited information in cases of neutropenia without other abnormalities.

Regulation of HLA class I surface expression requires CD99 and p230/golgin-245 interaction.

By presenting antigenic peptides on the cell surface, human leukocyte antigen (HLA) class I molecules are critical for immune defense. Their surface density determines, to a large extent, the level of CD8(+) T cell-dependent immune reactions; their loss is a major mechanism of immune escape. Therefore, powerful processes should regulate their surface expression. Here we document the mechanisms used by CD99 to mediate HLA class I modulation. Up-regulation of HLA class I by IFN-gamma requires CD99. In the trans Golgi network (TGN), and up to the cell surface, CD99 and HLA class I are physically associated via their transmembrane domain. CD99 also binds p230/golgin-245, a coiled-coil protein that recycles between the cytosol and buds/vesicles of the TGN and which plays a fundamental role in trafficking transport vesicles. p230/golgin-245 is anchored within TGN membranes via its Golgin-97, RanBP1, IMh1p, P230 (GRIP) domain and the overexpression of which leads to surface and intracellular down-modulation of HLA class I molecules.

Retrovirus-mediated gene transfer in human primary T lymphocytes induces an activation- and transduction/selection-dependent TCR-B variable chain repertoire skewing of gene-modified cells.

In a clinical trial that we recently reported, a suicide gene transfer in human primary T cells required 12 days of ex vivo culture, including activation of peripheral blood mononuclear cells (PBMC) with CD3 monoclonal antibody (CD3 mAb), retrovirus-mediated transduction, and selection of gene-modified cells (GMC) by G418. The aim of the present study was to determine the impact of the initial T cell activation and of the transduction/selection on T cell receptor beta variable chain (TCRBV) repertoire of GMC by using the spectratyping method. The TCRBV repertoires of nontransduced, nonselected control (Co) cells and of GMC generated after an initial stimulation with CD3 mAb, CD3/CD28 beads, or allogeneic PBMC or Epstein-Barr virus-transformed B (B-EBV) cells were compared to the ones of their corresponding PBMC. The TCRBV repertoires were skewed in Co cells generated after CD3 mAb or after allogeneic stimulation, and even more so in their corresponding GMC, demonstrating that both culture-dependent and transduction/selection-dependent events led to TCRBV repertoire alterations. However, TCRBV repertoires were not altered, or to a lesser extent, in Co cells or GMC produced after CD3/CD28 bead activation, demonstrating a protective effect on both culture-dependent and transduction/selection-dependent repertoire alterations. Thus, we suggest to replace the initial CD3 mAb stimulation by CD3/CD28 beads for the production of clinical-grade GMC in the setting of future gene therapy trials.