Key Points• The platelet defect associated with Paris-Trousseau thrombocytopenia and Jacobsen syndrome is caused by an abnormal transcription factor FLI1.• FLI1 DNA-binding ETS domain mutations cause bleeding disorders with both autosomal dominant and recessive patterns of inheritance.Hemizygous deletion of a variable region on chromosome 11q containing FLI1 causes an inherited platelet-related bleeding disorder in Paris-Trousseau thrombocytopenia and Jacobsen syndrome. These multisystem disorders are also characterized by heart anomalies, changes in facial structure, and intellectual disability. We have identified a consanguineous family with autosomal recessive inheritance of a bleeding disorder that mimics Paris-Trousseau thrombocytopenia but has no other features of the 11q23 deletion syndrome. Affected individuals in this family have moderate thrombocytopenia; absent collagen-induced platelet aggregation; and large, fused a-granules in 1% to 5% of circulating platelets. This phenotype was caused by a FLI1 homozygous c.970C>T-point mutation that predicts an arginine-to-tryptophan substitution in the conserved ETS DNA-binding domain of FLI1. This mutation caused a transcription defect at the promoter of known FLI1 target genes GP6, GP9, and ITGA2B, as measured by luciferase assay in HEK293 cells, and decreased the expression of these target proteins in affected members of the family as measured by Western blotting of platelet lysates. This kindred suggests abnormalities in FLI1 as causative of Paris-Trousseau thrombocytopenia and confirms the important role of FLI1 in normal platelet development. (Blood. 2015;126(17):2027-2030

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We sought to determine the effect of time and temperature of blood sample storage before preparation of human PBMCs by Ficoll-hypaque density gradient centrifugation. Blood samples from healthy donors were stored at room temperature (RT) or refrigerated at 4oC before preparation of PBMCs. Cell yield and viability, and proportions of major cell populations within PBMCs, as determined by fluorescence flow cytometry, were assessed for both fresh and cryopreserved samples. Highly multiparametric mass cytometry was performed on cryopreserved PBMCs. We found that refrigeration had marked negative effects on subsequent PBMC yield. Storage at RT led to co-purification of low density neutrophils with PBMCs, but had no detectable effects on the proportions of multiple cell subsets including, but not limited to, monocytes, NK cells, B cells, Treg cells, and naïve, central memory and effector memory CD4+ and CD8+ T cells and CD45RA-positive terminal effector CD8+ T cells. Expression of a number of cell surface receptors, including CXCR5, CCR6, CXCR3 and TIGIT, but not CD247 was reduced after RT storage before PBMC preparation, and this effect correlated with the degree of low density neutrophil contamination. As such, when PBMC preparation cannot be undertaken immediately after blood draw, storage at RT is far superior to refrigeration. RT storage leads to neutrophil activation, but does not compromise measurement of PBMC subset distribution. However caution must be applied to interpretation of cytometric measurements of surface molecules such as chemokine receptors.

Two new murine monoclonal antibodies, AK 1 and SZ 1, reactive with the human platelet glycoprotein (GP) Ib-IX complex have been produced by the hybridoma technique. Both AK 1 and SZ 1 immunoprecipitated the GP Ib-IX complex from Triton X-100-solubilized, periodate-labeled platelets. With trypsinized, labeled platelets, AK 1, SZ 1, and FMC 25 (epitope on GP IX) immunoprecipitated a membrane-bound proteolytic fragment of the GP Ib-IX complex consisting of GP IX and an congruent to 25,000 mol wt remnant of the alpha-chain of GP lb disulfide-linked to the beta-subunit. Unexpectedly, although AK 1 and SZ 1 immunoprecipitated purified GP Ib-IX complex, neither antibody immunoprecipitated the individual components of this complex, GP Ib or GP IX. When GP Ib and GP IX were recombined, however, AK 1 and SZ 1 again immunoprecipitated the reformed complex, strongly suggesting that both antibodies were recognizing an epitope present only on the intact complex. Cross-blocking studies indicated that AK 1 and SZ 1 recognized a very similar or identical epitope that was proximal to the epitope for FMC 25. Both AK 1 and SZ 1 bound to a similar number of binding sites (congruent to 25,000) on intact platelets as monoclonal antibodies directed against either GP lb or GP IX. The combined data suggests that GP lb and GP IX are fully complexed in the intact platelet membrane.