In 1994, three groups independently cloned TPO, as the ligand for orphan cytokine receptor, c-Mpl [15–17]. TPO is a single 95 kDa glycoprotein consisting of 332-amino acid protein that is the primary regulator of normal platelet production. TPO is synthesized primarily in the liver and is secreted into circulation. Although it has long been thought that hepatic transcription and translation of the TPO gene appear constant, recent study reveals that TPO production is stimulated, at least in part, by desialylated, senescent platelets clearance via hepatocyte Ashwell-Morrell receptor. Hepatocyte Ashwell-Morrell receptor regulates TPO production via JAK2-STAT3 signaling [18, 19]. Secreted TPO is removed from circulation by binding of the TPO receptor (c-Mpl) on platelets and bone marrow megakaryocytes. Thus, plasma TPO levels usually increase in response to the decreased in platelet/megakaryocyte mass. Actually, plasma TPO levels are markedly increased in patients with congenital amegakaryocytic thrombocytopenia, aplastic anemia, or chemotherapy-induced thrombocytopenia (CIT). In sharp contrast, in patients with ITP (or thrombocytopenia due to increased platelet destruction or consumption), plasma TPO levels are within normal range or slightly increased [20–23]. Thus, plasma TPO levels may have diagnostic utility in discriminating between patients with ITP and hypoplastic thrombocytopenia [13, 14, 24].

Reticulated platelets (RPs) are reported to be younger platelets (i.e., immature platelets) that have been released recently into the circulation and are probably analogous to reticulocytes reflecting erythropoiesis. RPs can be distinguished from mature platelets by their RNA contents using flow cytometry with an RNA-binding fluorochrome, such as thiazole orange, and RP% and absolute number of RPs are reflecting platelet production and hence platelet turnover [25–27]. In patients with ITP, RP% was markedly increased compared with healthy controls, whereas RP% in patients with AA or CIT was within normal range [13, 25, 26]. Thus, RP% could also be a good marker to differentiate ITP from hypoplastic thrombocytopenia. However, the method for the measurement of RP% is a time-consuming, laboratory-based assay and has not been standardized yet, because RP% is measured by flow cytometry. To measure RP% in daily practice, percentage of immature platelet fraction (IPF%) could alternatively be measured by automated hematology analyzer such as Sysmex^® XN-1000. As compared to XE-2100 (Sysmex Corp., Kobe, Japan), XN-1000 is a newer generation that uses oxazine to detect RNA and PLT-F channel to more accurately detect platelets (Fig. 1). We have compared RP%, IPF% measured by XE-2100, and IPF% measure by XN-1000 (newer generation) in parallel to differentiate patients with ITP from aplastic anemia or CIT who were already well diagnosed. The sensitivity and specificity for the diagnosis of ITP were 83.0 and 75.0% for IPF% (XE), 85.1 and 89.3% for IPF% (XN), and 93.6 and 89.3% for RP%, respectively. Moreover, examination of patients with paroxysmal nocturnal hemoglobinuria (PNH) revealed that hemolysis and/or red blood cell fragments interfered with IPF% (XE) values, but not with RP% or IFP% (XN) values [28]. Thus, IPF% measured by XN-1000 appears comparable to RP% measured by flow cytometry and may have diagnostic utility in discriminating between patients with ITP and hypoplastic thrombocytopenia in daily practice.