Plasma TPO Research Articles

Plasma thrombopoietin level after liver transplantation: relationship to cold ischemia time and coagulation parameters.

To determine the relation between thrombopoietin (Tpo) levels following orthotopic liver transplantation (OLT), cold ischemia time and postoperative peripheral blood platelet count and prothrombin activity. Prospective clinical study. Intensive care unit. Fourteen patients with uncomplicated postoperative course after OLT. Plasma Tpo, as quantified by enzyme immunoassay, rose significantly from 194.9 +/- 45.7 pg/ml on day 1 after OLT to a peak value of 500.7 +/- 94.1 pg/ml on day 5 while platelet count was below normal values. Then the platelet count increased and reached normal values while Tpo decreased to normal. The rise of Tpo levels was associated with normalization of prothrombin time but peak Tpo concentrations were in inverse correlation with cold ischemia times. The extent of production of Tpo in the liver graft following OLT is affected by cold ischemia time. This observation may be applicable in the prevention of bleeding complications associated with postoperative thrombocytopenia.

Plasma thrombopoietin levels in liver cirrhosis and kidney failure.

Recently, c-Mpl ligand (thrombopoietin, TPO) has been cloned by several groups and found to be a primary regulator of thrombopoiesis. Its mRNA expression has been detected in several organs including kidneys, bone marrow stroma cells, muscles, and is very strongly expressed in the liver. To clarify thrombopoiesis and the regulation of TPO in severe liver and renal failure. We analysed plasma TPO levels in patients with biopsy verified liver cirrhosis (n = 18; mean platelet count 115 +/- 54 x 109 L-1), in patients on chronic haemodialysis as a result of end-stage renal failure (n = 20; mean platelet count 295 +/- 94 x 109 L-1), and in healthy individuals (n = 20; mean platelet count 250 +/- 40 x 109 L-1). Plasma was prepared from EDTA-anticoagulated whole blood and a commercially available ELISA kit was used for the analysis. The mean plasma TPO concentration amongst the normal individuals was 50 +/- 14 pg mL-1. In the patients with liver cirrhosis and in patients on haemodialysis the mean TPO levels were 62 +/- 19 pg mL-1 and 46 +/- 17 pg mL-1, respectively. The mean plasma TPO concentration for the cirrhotic patients was significantly higher than the mean recorded for the healthy volunteers (P = 0.031), whereas no statistically significant differences in plasma TPO were seen between the group of end-stage renal failure and normals. Our results suggest that TPO production is maintained in liver cirrhosis and in renal failure, and that the thrombocytopenia in liver cirrhosis is not due to an impaired TPO production.

Age-related changes in thrombopoietin in children: reference interval for serum thrombopoietin levels.

We studied thrombopoietin (TPO, Mpl ligand) values using a sensitive ELISA in 254 serum samples obtained from disease-free children and adult volunteers. TPO was detected in all samples, and its values ranged widely from 0.25 to 9.18 fmol/ml. When analysed by dividing the subjects into 11 age groups, the mean TPO levels from birth to 1 month of age were increased (3.73-5.92 fmol/ml). The highest values were found 2 d after birth; TPO levels then gradually decreased to adult levels (0.83 fmol/ml). The relationship between TPO values and platelet counts was not significant in all subjects (r = 0.27) or in children alone (r = 0.12). In children > 1 month of age a 95% reference interval for serum TPO values was determined from 0.58 to 3.27 fmol/ml. A significant correlation was found between TPO values in serum and plasma; serum TPO values = -0.257 + 4.039 x plasma TPO values (r = 0.951, P < 0.001, n = 22). This study is the first to report age-dependent changes in blood TPO levels throughout child development. Serum TPO values were significantly high up to 1 month of age and were correlated with plasma TPO levels.

Plasma thrombopoietin concentrations in thrombocytopenic and non-thrombocytopenic patients in a neonatal intensive care unit.

Thrombocytopenia is a frequent occurrence in the neonatal intensive care unit (NICU), but the role of thrombopoietin (Tpo) in the pathophysiology is unknown. We obtained serial plasma Tpo concentrations in 20 thrombocytopenic neonates in our NICU, and performed bone marrow studies in 15. The initial Tpo levels ranged from undetectable (<41 pg/ml) to 1112 pg/ml and did not correlate with gestational age or platelet count. Neonates with decreased marrow megakaryocytes did not have plasma Tpo levels as high as those reported in adults, particularly in small for gestational age infants (Tpo < 300 pg/ml). In 14/15 neonates followed until resolution, the Tpo concentration decreased as the platelet count increased.

Plasma thrombopoietin levels in thrombocytopenic states: implication for a regulatory role of bone marrow megakaryocytes.

To evaluate the diagnostic value of thrombopoietin (TPO, c-mpl ligand) measurements, and clarify the regulatory mechanisms of TPO in normal and in thrombocytopenic conditions, the plasma TPO concentration was determined in normal individuals (n = 20), umbilical cord blood (n = 40), chronic idiopathic thrombocytopenic purpura (ITP; n = 16), in severe aplastic anaemia (SAA; n = 3), chemotherapy-induced bone marrow hypoplasia (n = 10), myelodysplastic syndrome (MDS; n = 11), and sequentially during peripheral blood progenitor cell transplantation (n = 7). A commercially available ELISA and EDTA-plasma samples were used for the analysis. The plasma TPO concentration in the normals and umbilical cord blood were 52 +/- 12 pg/ml and 66 +/- 12 pg/ml, respectively. The corresponding values in patients with SAA and chemotherapy-induced bone marrow hypoplasia were 1514 +/- 336 pg/ml and 1950 +/- 1684 pg/ml, respectively, and the TPO concentration, measured sequentially after myeloablative chemotherapy and peripheral blood progenitor cell transplantation, was inversely related to the platelet count. In contrast, the plasma TPO recorded in patients with ITP (64 +/- 20 pg/ml) and MDS (68 +/- 23 pg/ml) were only slightly higher than normal levels. In conclusion, TPO levels were significantly elevated in patients in which bone marrow megakaryocytes and platelets in circulation were markedly reduced, whereas TPO levels were normal in ITP patients, and only slightly increased in the MDS patients. These latter patients displayed a preserved number of megakaryocytes in bone marrow biopsies. Our data support the suggestion that megakaryocyte mass affects the plasma TPO concentration. In thrombocytopenic patients a substantially increased plasma TPO implies deficient megakaryocyte numbers. However, TPO measurements do not distinguish between ITP and thrombocytopenia due to dysmegakaryopoiesis, as seen in MDS patients.

Adenovector-Mediated Expression of Human Thrombopoietin cDNA in Immune-Compromised Mice: Insights into the Pathophysiology of Osteomyelofibrosis

Thrombopoietin (TPO) cDNA can be effectively delivered in vivo by adenovectors. Immune normal mice (BALB/c) and syngeneic mice with variable degrees of immune dysfunction nu, SCID, and NOD-SCID) were treated with an adenovirus vector expressing the human TPO cDNA (AdTPO). Platelet peaks were significantly higher in SCID and NOD-SCID mice compared with BALB/c and nu mice. Human plasma TPO concentration correlated with the platelet counts. SCID and NOD-SCID mice exhibited also granulocytosis and increased numbers of hemopoietic progenitors in bone marrow. Following platelet peak, BALB/c mice developed autoantibodies against murine TPO leading to thrombocytopenia and depletion of megakaryocytes and hemopoietic progenitors in bone marrow. AdTPO-treated SCID mice developed osteomyelofibrosis and extramedullary/extrasplenal hemopoiesis. In contrast, NOD-SCID mice with a similar magnitude of TPO overexpression did not show fibrotic changes in bone marrow. We conclude, first, that a chronic high level of TPO overexpression stimulates megakaryocytopoiesis and myelopoiesis leading to thrombocytosis and granulocytosis. Second, increased megakaryocytopoiesis is not sufficient for development of secondary osteomyelofibrosis. The functionally deficient monocytes and macrophages of NOD-SCID mice probably prevented fibrotic marrow changes. Third, immune deficiency enhances expression of adenovirally mediated transgenes, and fourth, xenogeneic transgene delivered by adenovector to a host with normal immune functions may induce loss of immune tolerance and autoimmune phenomenon.

Endogenous thrombopoietin levels and effect of recombinant human thrombopoietin on megakaryocyte precursors in term and preterm babies.

Thrombocytopenia (platelets <150 x 10[9]/L) is common in the early newborn period. Recent evidence suggests this is due mainly to impaired fetal megakaryocytopoiesis/platelet production. The cause remains unknown. Thrombopoietin (Tpo) is now recognized as the major regulator of platelet production in adults. However, nothing is known about Tpo levels, or the role of Tpo in megakaryocytopoiesis/platelet production, in the fetus/newborn. To answer these questions we: 1) measured plasma Tpo levels by ELISA at birth in healthy term (n = 17) and preterm (gestational age, 24-34 wk) babies (n = 16), and in thrombocytopenic preterm babies (n = 13); and 2) assessed the in vitro Tpo dose response characteristics of circulating megakaryocyte precursor cells (MKp) from all three groups. The median Tpo levels were similar in term babies 145 pg/mL (range 52-237 pg/mL) compared with preterm babies 132 pg/mL (32-318 pg/mL). In the thrombocytopenic preterm babies the median Tpo level of 185 (46-264) was not significantly higher than in healthy babies, despite the fact that their median platelet counts were significantly lower--82 x 10(9)/L (range 21-135), compared with the healthy preterm babies 252 x 109/L (152-320)--p < 0.0001. Tpo levels in the thrombocytopenic preterm babies were also much lower than levels measured in three thrombocytopenic children (905, 2138, and 2700 pg/mL). MKp from all three groups showed dose-dependent proliferation in response to Tpo (p < 0.01 at 100 ng/mL Tpo). Increases in MKp were greater in healthy and thrombocytopenic preterm babies when compared with term babies: 48.2-, 24.6-, and 9.8-fold, respectively (p < 0.05 for both comparisons). These results strongly suggest that: 1) Tpo is a major regulator of megakaryocytopoiesis/platelet production in the fetus/newborn, 2) impaired fetal Tpo production may be a factor in early thrombocytopenia in preterm babies, and 3) recombinant human Tpo is likely to be effective in the treatment of early neonatal thrombocytopenia.

Proliferative effect of postapheresis platelet donor plasma on a megakaryoblastic leukemia cell line.

Platelet donors sometimes show an increased platelet count following apheresis. We analyzed the effect of plasma obtained from such donors, along with thrombopoietin (Tpo), for growth and differentiation-inducing activity on a megakaryoblastic leukemia cell line, Meg-J. Colony formation was stimulated by donor plasma for Meg-J cells in a dose-dependent manner, and the time course of the sampling peaked 1 day after apheresis. Neither the donor plasma nor Tpo induced morphological differentiation. Donor plasma from any sampling time decreased CD41 and CD42b expression by more than 10%, and more than a 10% decrease of CD41 was induced by Tpo. The measurement of plasma Tpo showed no statistically significant increase after platelet donation. Our data suggest that, following apheresis, donor plasma contains factor(s) that: (a) stimulate growth rather than induce differentiation of a megakaryoblastic cell line; (b) are present in increased levels after apheresis; and (c) are different from Tpo.

Sensitive Measurement of Thrombopoietin by a Monoclonal Antibody Based Sandwich Enzyme-linked Immunosorbent Assay

In 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.