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Objectives: Reliable diagnosis of heparin-induced thrombocytopenia and thrombosis (HIT) is mandatory for patient management, yet prompt determination of pathogenic antibodies remains an unmet clinical challenge. Common immunoassays carry inherent limitations and functional assays which detect antibody-mediated platelet activation are not usually readily available to routine laboratories, especially the serotonin release assay (SRA), being technically demanding, time consuming, and requires high level expertise. To overcome some of these limitations, we have developed a practical functional flow cytometric assay (FCA) for routine clinical use., Methods: A simple FCA is described which avoids platelet manipulation, is highly specific and sensitive compared with SRA, and provides rapid results., Results: Of the 650 consecutive samples, from HIT-suspected patients, 99 (15.3%) were positive by the PaGIA Heparin/PF4 immunoassay and 31 (4.8%) by FCA. Average platelet activation was 11-fold higher in PaGIA+/FCA+ vs PaGIA-/FCA- samples. Of 21 SRA-positive samples, 19 were FCA-positive (relative sensitivity 90.5%), and of 42 SRA-negative samples, 40 were FCA-negative (relative specificity 95.2%). The FCA showed significantly higher correlation with the clinical presentation of HIT (4Ts score) performed on 182 patients, compared with PaGIA Heparin/PF4 (ROC-plot analysis, AUC 0.93 vs 0.63, P < 0.001). At a 92% sensitivity, the assay specificity was 96%., Conclusions: The present FCA is practical for routine testing, providing prompt reliable results for initial diagnosis and confirmation, to effectively assist in HIT patient management., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Severe neutropenia and protracted thrombocytopenia remain serious clinical problems following cord blood transplantation (CBT) due to the paucity of stem and progenitor cells in the grafts. Administration of ex-vivo expanded megakaryocyte progenitor cells may facilitate platelet production. We propose a novel strategy to expand these rare cells ex-vivo, from a small portion of the cord blood (CB) unit, using fibronectin (FN), a major component of hematopoietic niches, combined with cytokines, including thrombopoietin and the hematopoietic stress-associated acetylcholinesterase readthrough peptide (ARP). Application of multiple gates and high definition flow cytometry enabled clear resolution of expanded hematopoietic stem/precursor cells (HSPC) and megakaryocyte progenitors (Mk-p) and their early subsets while eliminating positively stained non-relevant cells. FN increased viability, expansion of all CD34+ HSPC populations and Mk-p. The combination of FN + thrombopoietin + ARP maintained and expanded very early myeloid and thrombopoietic precursors, increased the proliferation of megakaryocyte, granulocyte-macrophage and multilineage colony-forming progenitors and supported Mk maturation as measured by ploidy and glycoprotein IIb/IIIa expression by quantiative reverse transcription polymerase chain reaction. This approach, which involves expanding HSPC and Mk precursors from a small portion of the CB unit, without sacrificing the coveted stem cells, may lead to improved cell therapy modalities to facilitate earlier myelopoiesis and platelet production post-CBT. [ABSTRACT FROM AUTHOR]

The front cover image is based on the Original Article* I Pathogenic heparin-induced thrombocytopenia and thrombosis (HIT) antibodies determined by rapid functional flow cytometry i by Michal Cipok** et al., https://doi.org/10.1111/ejh.13277 GRAPH. [Extracted from the article]