Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder of undefined aetiology. The disease presents with severe thrombocytopenia and absence of megakaryocytes in the bone marrow. Furthermore, CAMT patients may develop bone marrow aplasia. To obtain more insight into the mechanism underlying CAMT, five children were analysed. All patients had increased plasma thrombopoietin (Tpo) levels, indicating a platelet production defect. Bone marrow‐derived CD34+ stem cells from three patients were cultured in an in vitro liquid culture system to study megakaryocytopoiesis. CD34+ cells from two of the three patients failed to differentiate into megakaryocytes. The lack of megakaryocyte formation could imply that a defect in the c‐mpl gene, encoding the Tpo receptor, exists. Sequencing of c‐mpl revealed mutations in four of five patients. Three patients had point mutations and/or a deletion in the coding regions of c‐mpl. All point mutations led to an amino acid substitution or to a premature stop codon. In one patient, a homozygous mutation in the last base of intron 10 was found that resulted in loss of a splice site. This study showed that mutations in c‐mpl could be the cause of thrombocytopenia in CAMT in the majority of patients. Furthermore, Tpo has been shown to have an anti‐apoptotic effect on stem cells. Therefore, mutations in c‐mpl might not only affect megakaryocyte formation but may also impair stem cell survival, which could explain the occurrence of bone marrow failure as final outcome in patients with CAMT.

SummaryIt has been reported that blood trombopoietin (TPO) levels can discriminate between thrombocytopenia due to increased platelet destruction and decreased platelet production. With our TPO ELISA and a glycocalicin ELISA we analysed a large group of patients in detail and could confirm and amplify the above notion in detail.TPO levels were determined in plasma from 178 clinically and serologically well-defined thrombocytopenic patients: 72 patients with idiopathic autoimmune thrombocytopenia (AITP), 29 patients with secondary AITP, 5 patients with amegakaryocytic thrombocytopenia and 72 patients who suffered from various diseases (46 in whom megakaryocyte deficiency was not and 26 in whom it was expected). In addition, we measured the level of glycocalicin as a marker of total body mass of platelets.In all patients with primary AITP and secondary AITP, TPO levels were within the normal range or in some (n = 7) cases only slightly increased. The level of glycocalicin was not significantly different from that of the controls (n = 95). The patients with amegakaryocytic thrombocytopenia had strongly elevated TPO levels and significantly decreased glycocalicin levels. Similarly, among the 72 thrombocytopenic patients with various disorders, elevated TPO levels were only found in patients in whom platelet production was depressed. The mean level of glycocalicin in these patients was decreased compared to that in controls and patients with AITP, but was not as low as in patients with amegakaryocytic thrombocytopenia.In conclusion, all patients with depressed platelet production had elevated levels of circulating TPO, whereas the TPO levels in patients with an immune-mediated thrombocytopenia were mostly within the normal range. Therefore, measurement of plasma TPO levels provides valuable diagnostic information for the analysis of thrombocytopenia in general.Moreover, treatment with TPO may be an option in AITP.

SummaryThrombopoietin is produced at a constant rate by the liver and kidney and is removed from the circulation upon binding and subsequent uptake via the Tpo receptor, c-Mpl, expressed by platelets and megakaryocytes. Apart from uptake, this study shows that platelets can also function as a storage pool for Tpo.Upon stimulation with various platelet agonists, full-length biologically active Tpo was released by platelets. Platelet fractionation experiments indicated that this Tpo most likely is contained in the granules. When platelets were preincubated with Tpo-peptide mimetic or truncated Tpo prior to maximal activation, a three- to fivefold increment in Tpo release was seen, whereas, the release of other granule proteins such as vWF-propeptide or serotonin remained unchanged. Therefore, the Mpl agonists might compete with Mpl-bound Tpo, thereby releasing Tpo into the platelet supernatant.Intravascular release of Tpo by platelets might occur in patients with massive platelet activation, as occurs in patients with disseminated intravascular coagulation. The Tpo concentration in these patients is elevated (p <0.01) and correlates with markers for thrombin generation, TAT complexes and Fl+2 (rp =0.8 and 0.9; p <0.01). This suggests that the increment in Tpo concentration was attributed to Tpo release by activated platelets in vivo, which might be instrumental in subsequent stimulation of thrombocytopoiesis.

SummaryIn this report a sensitive enzyme-linked immunosorbent assay (ELISA) for the measurement of plasma thrombopoietin (Tpo) is described that is solely based on monoclonal antibodies (MoAbs).The assay has an intra and inter-assay variance of 5-7% and 7-13%, respectively. Native and recombinant human Tpo (rhTpo) were recognized equally well, no cross reactivity with other cytokines was found and rhTpo added to plasma and serum was completely recovered. With the ELISA, Tpo concentrations in EDTA-anticoagulated plasma of all controls (n = 193) could be determined, since the limit of detection (2 ± 0.8 A.U./ml, mean ± sd) was lower than the concentration found in controls (11 ± 8 A.U./ml, mean ± sd; 2.5th-97.5th percentile: 4-32 A.U./ml). Tpo levels in serum were on average 3.4 times higher than in plasma.We showed in vivo that Tpo is bound by platelets, as in thrombocytopenic patients (n = 5) a platelet transfusion immediately led to a drop in plasma Tpo level, whereas in patients receiving chemotherapy the induced thrombocytopenia was followed by a rise in plasma Tpo levels.In summary, these results indicate that this ELISA is a reliable tool for Tpo measurements and is applicable for large scale studies.

Summary Background. Febrile conditions are often associated with increased platelet turnover and refractoriness to platelet transfusions, although several pyrogenic cytokines enhance thrombopoiesis. This study aimed to characterize the effects of experimental human endotoxemia on platelet turnover and thrombopoiesis. Methods. Endotoxin (4 ng/kg) was infused into 30 healthy men to study the regulation of thrombopoiesis in systemic human inflammation. Platelet counts, plasma thrombopoietin (TPO) and glycocalicin levels, and reticulated platelets (RP) were measured to evaluate the effect of acute endotoxemia on thrombopoiesis. Ten subjects received pretreatment with 1000 mg aspirin po. to evaluate possible effects of aspirin on platelet turnover, and ten subjects received paracetamol to control for effects of anti-pyresis. Results. Platelet counts dropped by about 15% (p <0.001) one hour after LPS infusion, began to recover at 24 h, and exceeded baseline values by 8% (CI: 5-12; p <0.001) at 7 days after LPS iv. Reticulated platelet counts increased from 1.62% (CI: 1.24-2.0) to a maximum of 2.39% (CI: 1.81-2.98; p = 0.003) at 6 h. TPO levels increased from baseline values of 10 A.U/ml (CI: 8.8-11.2) to 15.5 A.U/ml (CI: 13.6-17.3) at 24 h (p <0.001), whereas plasma glycocalicin was not changed (p >0.05). The number of circulating platelet-neutrophil aggregates increased more than 100% at 6 h (p <0.001). Neither aspirin nor paracetamol affected changes in any of the parameters measured. Conclusion. Low grade endotoxemia induces a rapid fall of platelet counts, which is followed by an early increase in reticulated platelets and TPO levels but not of glycocalicin levels. Finally peripheral platelet counts increase several days after LPS infusion.

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Thrombopoietin (Tpo), the main regulator of thrombocytopoiesis, is a probable candidate to play a role in the increase in platelet counts that is frequently seen after surgery. In the current study, serial blood samples of patients that underwent major surgery were analysed with respect to Tpo kinetics, platelet turnover and inflammatory cytokines. Platelet Tpo content and plasma Tpo levels rose before platelet counts increased, suggesting that Tpo was indeed responsible for the elevation in platelet counts. In addition, an increase in interleukin 6 (IL‐6) levels, but not in IL‐11 and tumour necrosis factor alpha levels, was seen before the rise in Tpo concentration. In vitro, IL‐6 was shown to enhance Tpo production by the HepG2 liver cell line. Thus, increased Tpo levels after surgery, possibly resulting from enhanced Tpo production under the influence of IL‐6 or other inflammatory cytokines, are involved in an enhanced thrombocytopoiesis.

BACKGROUND: The Transfusion Register of IrregularAntibodies and Cross-match Problems (TRIX) is a unique national database in the Netherlands that was launched in 2007. Transfusion laboratories register the presence of irregular RBC alloantibodies for their patients and can consult the database for information that is relevant for pretransfusion testing, unknown in their own laboratory information system. STUDY DESIGN AND METHODS:Data from the TRIX database 10 years after implementation have been analyzed to demonstrate the added value of TRIX for transfusion practice. TRIX antibody registration, antibody disappearance likelihood, and differences between men and women have been analyzed and evaluated.ABBREVIATIONS: ADL = antibody disappearance likelihood; ADRs = antibody disappearance registrations; LISs = laboratory information systems; TRIX = Transfusion Register of Irregular Antibodies and Cross-match Problems.From the 1 Amphia Hospital, Breda, the