Identification of a potent small molecule capable of regulating polyploidization, megakaryocyte maturation, and platelet production.

BACKGROUND: Megakaryocytic cell maturation involves polyploidization, and megakaryocyte (MK) ploidy correlates with their maturation and platelet production. Retardation of MK maturation is closely associated with poor MK engraftment after cord blood transplantation and neonatal thrombocytopenia. Despite the high prevalence of thrombocytopenia in a range of setting that affect infants to adults, there are still very limited modalities of treatment. METHODS: Human CD34+ cells were isolated from cord blood or bone marrow samples acquired from consenting patients. Cells were cultured and induced using 616452 and compared to current drugs on the market such as rominplostim or TPO. Ploidy analysis was completed using propidium iodide staining and flow cytometry analysis. Animal studies consisted of transplanting human CD34+ cells into NOD.Cg-PrkdcscidIl2rgtm1Wjl/SzJ mice followed by daily injections of 15 mg/kg of 616452. RESULTS: Within one week of culture, the chemical was able to induce polyploidization, the process required for megakaryocyte maturation with the accumulation of DNA content, to 64 N or greater to achieve a relative adult size. We observed fold increases as high as 200-fold in cells of 16 N or greater compared to un-induced cells with a dose-dependent manner. In addition, MK differentiated in the presence of 616452 demonstrated a more robust capacity of MK differentiation than that of MKs cultured with rominplostim used for adult idiopathic thrombocytopenic purpura (ITP) patients. In mice transplanted with human cord blood, 616452 strikingly enhanced MK reconstitution in the marrow and human peripheral platelet production. The molecular therapeutic actions for this chemical may be through TPO-independent pathways. CONCLUSION: Our studies may have an important impact on our fundamental understanding of fetal MK biology, the clinical management of thrombocytopenic neonates and leukemic differentiation therapy.

Contamination of patient blood samples by avian RBCs from control material during automated hematology analysis.

OBJECTIVES: Atypical nucleated RBCs (NRBCs) found on several patient blood smears between 2010 and 2012 were noted to resemble avian RBCs. NRBCs are not normally found in the circulation beyond the neonatal period and may indicate hematologic disease, malignancy in the bone marrow, or other severe conditions. Our blood smears with unusual NRBCs did not contain other abnormalities that typically accompany NRBCs, such as immature cells or dysplastic granulocytes. To investigate this anomaly, we considered possibilities such as contaminated collection tubes and instrument problems. The Retic-C Cell Control used with the LH 750 Hematology Analyzer contains a mixture of human and avian RBCs. METHODS: CBC count with differential tests were performed on blanks and routine laboratory samples run immediately after the Retic-C Cell Control on the LH 750 and LH 780 analyzers to recreate the conditions that might cause spillage into the next tube. RESULTS: We experimentally reproduced the phenomenon of contamination of a subsequent tube with avian cells from a multiply punctured reticulocyte control tube. CONCLUSIONS: We concluded that the NRBCs likely represented avian RBCs from the Retic-C Cell Control that had been introduced into the patient tubes.