Anti-Emm in a pregnant patient--case report.

A 23-year-old primigravida of North African origin presented with a positive antibody screen at booking at 15 weeks of gestation. An antibody to a high-frequency antigen (HFA) of unknown identity was detected, which was reactive with the red blood cells of the father. This led to several challenges including antibody identification, clinical monitoring to detect signs of haemolytic disease of the foetus and newborn (HDFN) and compatible blood in case perinatal transfusion was needed. Anti-Emm was identified 2 months post-partum. This is the first published case which describes a pregnant patient with anti-Emm.

Fetal and neonatal thrombopoietin levels in alloimmune thrombocytopenia.

Thrombopoietin (Tpo) is the main hematopoietic growth factor for platelet production. Plasma Tpo levels in autoimmune thrombocytopenic patients are normal or slightly elevated. Although thrombocytopenia exists, Tpo levels are not increased because the produced megakaryocytes and platelets can bind circulating Tpo, thereby normalizing Tpo levels. In this study, plasma samples from fetuses and neonates with neonatal alloimmune thrombocytopenia (NAIT), a different form of immune thrombocytopenia, were measured. Umbilical cord samples from 50 fetuses before treatment because of severe thrombocytopenia and 51 fetuses after treatment, and peripheral blood samples of 21 untreated newborns with NAIT were analyzed. As controls, plasma Tpo levels were determined in 21 umbilical cord samples of 14 nonthrombocytopenic fetuses with hemolytic disease resulting from red blood cell alloimmunization and in umbilical cord samples of 51 healthy newborns. The values were also compared with the plasma Tpo levels in 193 healthy adults. Mean Tpo levels from the groups of fetuses and neonates, including both NAIT and control plasma, were slightly but significantly elevated compared with levels in healthy adults. Tpo levels in NAIT samples were not significantly different from the levels in hemolytic disease samples or in samples from healthy newborns. Thus, as in autoimmune thrombocytopenic patients, normal Tpo levels are present in NAIT patients.

The role of thrombopoietin in post-operative thrombocytosis.

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.

In multiple myeloma increased thrombopoietin (Tpo) production may be involved in the maintenance of platelet production.

In multiple myeloma (MM), suppression of haematopoiesis occurs as a result of expansion of malignant cells in the bone marrow. Thrombopoietin (Tpo) levels in patients with impaired platelet production are generally found to be highly elevated. To examine the circulating Tpo levels in patients with MM, Tpo levels were measured in 50 serum samples from 34 patients. Tpo levels were subsequently related to disease stage, and cell numbers and markers, i.e. platelet count, leukocyte count and haemoglobin (Hb) concentration. Elevated Tpo levels were found in association with decreased platelet counts (n=8), but also in patients with normal platelet counts (n=14). The latter group included patients without and with signs of impaired haematopoiesis, i.e. with decreased Hb concentration and decreased leukocyte count. These results show that neither platelet counts nor Tpo levels are reliable parameters to judge bone-marrow failure in patients with MM. Furthermore, in patients with MM, increased Tpo levels may play a role in the maintenance of thrombocytopoiesis. The origin of the increased Tpo levels remains to be determined.

Analysis of the kinetics of TPO uptake during platelet transfusion.

BACKGROUND: It has been shown in several studies that platelets play a role in the removal of TPO from the circulation. For instance, in vitro studies have shown that platelets can bind and internalize TPO, and transfusion studies have shown that the concentration of circulating TPO decreased after platelet transfusion. In the current study, the in vivo kinetics of plasma TPO levels and TPO uptake by transfused platelets is analyzed in more detail. STUDY DESIGN AND METHODS: Serial blood samples from patients who received a platelet transfusion were analyzed with respect to platelet count, plasma TPO concentration, and TPO content per platelet. In addition, the capacity of transfused platelets to bind TPO in vitro was assessed. RESULTS: Platelet counts increased immediately after transfusion, but subsequently started to decrease. Conversely, TPO levels decreased significantly but then returned to baseline level by 44 hours after transfusion. Platelet count and plasma TPO concentration were inversely correlated (r(p) = -0.9; p<0.05). The decrease in TPO concentration upon transfusion was accompanied by a significant increase in the platelet-associated TPO concentration. After transfusion, platelets isolated from the patient still displayed functional TPO receptors, as indicated by their intact capacity to bind TPO in vitro. CONCLUSION: The decrease in plasma TPO followed by the increase in platelet TPO provides evidence that platelets are responsible for the clearance of TPO in circulation. In vivo, platelets can bind and may degrade TPO upon platelet transfusion.

High levels of reticulated platelets and thrombopoietin characterize fetal thrombopoiesis.

To characterize fetal thrombopoiesis, we determined plasma thrombopoietin (TPO) and glycocalicin levels, platelet counts and reticulated platelets (RP) of fetuses and compared them with the respective values of their mothers. Percutaneous umbilical vein sampling in abnormal pregnancies revealed twofold higher thrombopoietin levels and 20-fold higher reticulated platelet counts, but lower levels of glycocalicin in fetuses compared with their mothers (P < 0.05). Neither the expression of platelet glycoprotein Ib and IIb on platelets nor the platelet counts were different between mothers and their fetuses. These data indicate enhanced thrombopoiesis and/or increased platelet turnover in fetuses.

Effects of anticoagulation on thrombopoietin release during endotoxemia.

Several lines of evidence suggest that coagulation may induce the release of thrombopoietin (TPO) into plasma and that TPO levels are higher in disseminated intravascular coagulation. Therefore we set out to illuminate the mechanism of TPO release in the setting of experimental endotoxemia, which induces activation of coagulation and platelets. Endotoxin (lipopolysachharide [LPS], 2 ng/kg) was infused into a total of 54 healthy men in two subsequent studies. Volunteers received infusions of unfractionated heparin, low-molecular-weight heparin, lepirudin, or placebo in a randomized, placebo-controlled fashion after bolus injection of LPS. TPO levels increased on average by 27% to 38% in all groups at 6 hours (P <.05 vs baseline), although all active drugs effectively blocked coagulation. Platelet counts dropped by about 15% at 1 hour after LPS infusion, recovered after 2 days, and exceeded baseline values by 8% to 18% after 7 days (P <.001 vs baseline for all groups). Yet lepirudin blunted the LPS-induced increase in circulating P-selectin by one half (P <.005 vs placebo), whereas both heparins did not diminish the increase in this platelet or endothelial activation marker as compared with placebo. Endotoxemia enhances TPO plasma levels independent of the degree of coagulation induction, which eventually results in increased platelet numbers. Of potential clinical interest is the observation that the direct thrombin inhibitor lepirudin, in contrast to heparins, mitigated LPS-induced platelet activation.

Increase in glycocalicin levels in platelet concentrates stored in plasma or synthetic medium for 8 days: comparison with other platelet activation markers.

BACKGROUND AND OBJECTIVES: Glycocalicin (GC) is a proteolytic fragment of GpIb and can conveniently be measured in supernatants of platelet concentrates (PCs) by means of a sandwich ELISA. Because of the convenience of the assay and easy sample storage, we tested its suitability as a sensitive platelet activation parameter during PC storage. MATERIAL AND METHODS: Filtered PCs in plasma or additive solution were made from 5 pooled buffy coats and were subsequently stored during 8 days at 22+/-2 degrees C. Correlation coefficients (r) were calculated after comparison of GC levels with platelet parameters. RESULTS: A significant increase in GC concentration was found on all subsequent sampling days. PC stored in plasma showed GC levels that correlated well with the soluble P-selectin levels (r = 0.7506), P-selectin (CD62P) expression on platelet membranes (r = 0. 8843), morphology scores according to Kunicki (r = -0.7102), lactate concentrations (r = 0.9216), glucose concentrations (r = -0.8913) and beta-thromboglobulin (beta-TG) concentrations (r = 0.8913). In PCs stored in additive solution, the correlation coefficients with these markers were 0.9209 with soluble P-selectin, 0.7161 with CD62P expression, -0.7474 with morphology score, -0.8908 with glucose concentrations, 0.8923 with lactate concentrations and 0.8908 with beta-TG concentrations. CONCLUSIONS: The GC concentration correlates well with sensitive platelet (activation) parameters, rendering it a sensitive and convenient parameter for platelet activation.

Mutations in the thrombopoietin receptor, Mpl, in children with congenital amegakaryocytic thrombocytopenia.

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.

Platelets release thrombopoietin (Tpo) upon activation: another regulatory loop in thrombocytopoiesis?

Thrombopoietin 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 mega-karyocytes. 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 F1+2(r(p)= 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.

Serum levels of thrombopoietin, IL-11, and IL-6 in pediatric thrombocytopenias.

We measured serum levels of thrombopoietin (TPO), interleukin (IL)-11, and IL-6 in 90 different samples from 67 pediatric patients with thrombocytopenia (TP). The cytokine levels were determined by enzyme-linked immunosorbent assays (ELISA), and the biological activity of TPO was measured using a cell line transfected with human c-mpl. In patients with impaired megakaryocytopoiesis, as found in diseases such as aplastic anemia, amegakaryocytic TP, or TP with absent radii, we found TPO levels which were highly elevated compared with normal values (mean=261 AU/ml, n=52, vs. 22 AU/ml in healthy controls). In contrast, patients suffering from idiopathic thrombocytopenic purpura (mean=16 AU/ml, n=31) or platelet function defects (mean=23 AU/ml, n=7) demonstrated normal TPO levels. The biological activity tested in the bioassay correlated well with the ELISA data. However, sera of some patients with amegakaryocytic TP demonstrated a remarkably higher biological activity of TPO than expected from the ELISA data. Within the different groups there was no correlation between platelet counts and TPO levels. Only 27% of all samples had elevated levels of IL-11 (mean=450 pg/ml, n=20). Elevated IL-6 serum levels were detected in only 13% of all samples analyzed (mean=42 pg/ml, n=12). We conclude that megakaryocytopoiesis is regulated mainly by TPO, that it is dependent on the platelet and the megakaryocytic mass, and that IL-11 plays an additional role in supporting the platelet production. IL-6 does not appear to be up-regulated in children with thrombocytopenia.

Effects of endotoxemia on thrombopoiesis in men.

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 antipyresis. 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 81% (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.

The diagnostic value of thrombopoietin level measurements in thrombocytopenia.

It 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.

Defective c-Mpl signaling in the syndrome of thrombocytopenia with absent radii.

Thrombocytopenia with absent radii (TAR) syndrome is a rare congenital defect with severe hypomegakaryocytic thrombocytopenia and bilateral radial aplasia. To elucidate a possible relationship between thrombocytopenia in TAR and defects in the thrombopoietin (TPO)/c-Mpl system, we examined TPO activity in sera from six patients and in vitro reactivity of the patients' platelets to recombinant human TPO. We found elevated TPO serum levels in all patients, excluding a TPO production defect as a pathomechanism for the thrombocytopenia. In contrast to healthy controls, however, platelets of TAR patients failed to respond to recombinant TPO as measured by testing TPO synergism to suboptimal concentration of platelet activators. Most interestingly, TPO-induced tyrosine phosphorylation of platelet proteins was completely absent (four out of five) or markedly decreased (one out of five). More detailed investigations of the signal cascades of c-Mpl demonstrated the absence of Jak2 phosphorylation after TPO stimulation in a TAR patient's platelets. A defect in the early events of c-Mpl signal transduction might be the reason for impaired megakaryocytopoiesis in TAR syndrome.

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.

Platelet: transporter of vascular endothelial growth factor.

In animal models, growth of tumors and their metastases is dependent on factors that stimulate vessel formation (angiogenesis). Most clinical studies confirm the importance of angiogenesis for cancer growth in patients. Recent studies on circulating angiogenic factors in patients have focused on serum vascular endothelial growth factor (VEGF) levels in a variety of cancer types. We measured serum VEGF concentrations and blood counts in 27 breast cancer patients during each of 6 cycles of chemotherapy with doxorubicin and cyclophosphamide supported by granulocyte macrophage colony-stimulating factor. Serum VEGF concentrations highly correlated with platelet counts during chemotherapy (r = 0.8; P < 0.01). In particular, during the first treatment cycle, after an initial episode of thrombocytopenia, a strong platelet rebound coincided closely with a serum VEGF peak (r = 0.9; P < 0.01). In addition, plasma VEGF concentrations from 15 other cancer patients and 30 healthy volunteers were 5- to 8-fold lower than their corresponding serum VEGF concentrations (P < 0.001). Activation of platelets increased the VEGF content 8-10 times. These findings demonstrate that VEGF is released by platelets during serum preparation. In this study, we found evidence for VEGF transport by platelets, indicating that serum VEGF concentrations reflect mainly platelet counts rather than tumor burden in cancer patients, as reported earlier. Platelets, known to be important for wound healing, have also been reported to contribute to metastasis formation and tumor growth in animal models. Indeed, tumors can be regarded as never-healing wounds. Our data suggest that platelets may have a stimulating role on angiogenesis-dependent tumor growth through their function as transporters of VEGF.

Rapid diagnosis of schistosomiasis by antigen detection in urine with a reagent strip.

For the rapid and simple diagnosis of schistosomiasis, a reagent strip assay for detection of circulating cathodic antigen (CCA) in urine was developed. The test was based on a previously developed sandwich enzyme-linked immunosorbent assay (ELISA) using a combination of two anti-CCA monoclonal antibodies. For the reagent strip assay, as the capture matrix, monoclonal antibody was coated onto a nitrocellulose membrane and mounted on polyvinyl chloride strips. Urine samples were then tested in an assay consisting of a combined incubation step of the urine sample and biotinylated detecting antibody followed by incubation in streptavidin peroxidase and a subsequent staining. The specificity and the sensitivity of the assay, as determined with urine samples of 61 uninfected controls and 67 Schistosoma mansoni-infected individuals, were 96.7 and 95.5%, respectively. The results of the reagent strip assay compared very well with microscopical parasitological diagnosis by the standard Kato-Katz method for the same individuals. The reagent strip test has a lower detection limit of 1 ng of CCA per ml and can be completed in 75 min. By the inclusion of two reference bands on the strips, standardized reading could be achieved. This reagent strip assay is a promising tool for qualitative diagnosis of S. mansoni infections in control programs.