losartan-potassium and Polycythemia-Vera

Polycythemia vera (PV) is classically thought to be associated with low erythropoietin (EPO) levels. Here, we present a review of the utility of using EPO levels in diagnosing polycythemia.. We conducted a systematic literature review of the Medline data through Pubmed and Google Scholar. We included the articles which described confirmed PV associated with elevated EPO level. Our search strategy included the following terms in Pubmed (((polycythemia vera[MeSH Terms]) OR (jak2 protein tyrosine kinase[MeSH Terms])) OR (Myeloproliferative Disorders[MeSH Terms])) AND (Erythropoietin[MeSH Terms]), and 'polycythemia vera with erythropoietin' in Google Scholar.. Our research yielded four cases of PV with elevated EPO levels. The most common symptom was a headache. Thrombotic phenomena happened in a single case in the form of Budd-Chiari syndrome. The mean Hb level was 20.2 gm/dl, and the EPO level was 213 mlU/mL.. Although PV is usually associated with low EPO levels, high levels do not exclude this diagnosis. Workup should include testing for JAK2 mutation and bone marrow biopsy in the presence of suggestive signs and symptoms. Novel biomarkers are also being proposed to aid in the diagnosis.. Although elevated EPO levels suggest secondary causes of polycythemia, cases where elevated EPO levels were associated with an underlying PV are reported in the literature, and we have summarized a review of them. Workup for polycythemia should include JAK2 mutation testing if signs and symptoms suggest PV even if EPO is elevated.

Topics: Bone Marrow; Erythropoietin; Humans; Janus Kinase 2; Polycythemia; Polycythemia Vera

An absolute erythrocytosis is present when the red cell mass is greater than 125 % of the predicted value for sex and body mass. It can have a primary or secondary and congenital or acquired cause. New causes particularly congenital continue to be discovered and investigated. Investigation for the cause starts with repeat and confirmation of the raised hemoglobin and measurement of an erythropoietin level to indicate whether to pursue primary or secondary causes and then further investigations as appropriate. Management options include low dose aspirin and venesection. Specific management advice is available for certain specific clinical situations.

Topics: Adaptor Proteins, Signal Transducing; Aspirin; Erythropoietin; Humans; Intracellular Signaling Peptides and Proteins; Mutation; Phlebotomy; Polycythemia; Polycythemia Vera; Proteins; Receptors, Erythropoietin; Von Hippel-Lindau Tumor Suppressor Protein

Topics: Anemia, Sickle Cell; Biopsy; Complementary Therapies; Diagnosis, Differential; Erythropoietin; Hemochromatosis; Hepatitis C, Chronic; Humans; Iron; Kidney Transplantation; Liver; Metabolic Syndrome; Phlebotomy; Polycythemia; Polycythemia Vera; Porphyria Cutanea Tarda; Postoperative Complications

The concept of prodromal chronic myeloproliferative neoplasms has been endorsed by the WHO classification implicating a stepwise evolution of disease. Histology of the bone marrow (BM) and borderline to mildly expressed clinical features play a pivotal role for diagnosing prefibrotic-early primary myelofibrosis. By lowering the platelet count for essential thrombocythemia and regarding BM morphology, early manifestations are tackled. Pre-polycythemic stages of polycythemia vera with a low hemoglobin level at onset are diagnosed by positive JAK2V617F mutation status, a low erythropoietin value, and characteristic BM features. The revised WHO classification incorporates hematological, morphological, and molecular-genetic parameters to generate a consensus-based working diagnosis.

Topics: Bone Marrow Examination; Disease Progression; Erythrocyte Volume; Erythropoietin; Female; Humans; Janus Kinase 2; Male; Mutation; Platelet Count; Polycythemia Vera; Primary Myelofibrosis; Risk; Thrombocythemia, Essential; World Health Organization

Topics: Anemia, Hypochromic; Antineoplastic Agents; Breast Neoplasms; Carcinoma, Small Cell; Clinical Protocols; Darbepoetin alfa; Data Interpretation, Statistical; Erythropoietin; Female; Filgrastim; Follow-Up Studies; Graft vs Host Disease; Granulocyte Colony-Stimulating Factor; Granulocytes; Hematinics; Hematologic Neoplasms; Hematopoietic Cell Growth Factors; Hematopoietic Stem Cell Transplantation; Humans; Immunoglobulins, Intravenous; Lung Neoplasms; Lymphoma; Lymphoma, Non-Hodgkin; Male; Multicenter Studies as Topic; Neoplasms; Neutropenia; Platelet Aggregation Inhibitors; Polycythemia Vera; Polyethylene Glycols; Randomized Controlled Trials as Topic; Recombinant Proteins; Research Design; Survival Analysis; Thrombocythemia, Essential; Treatment Outcome

Erythrocytosis results when there is an increased red cell mass and thus an increased hemoglobin. The causes can be divided into primary intrinsic defects of the erythroid progenitor cell and secondary defects, where factors external to the erythroid compartment are responsible. Both can then be further divided into congenital and acquired categories. Congenital causes include mutations of the erythropoietin receptor and defects of the oxygen-sensing pathway including VHL, PHD2 and HIF2A mutations. When fully investigated there remain a number of patients in whom no cause can be elucidated who are currently described as having idiopathic erythrocytosis. Investigation should start with a full history and examination. Having eliminated the common entity polycythemia vera, further direction for investigation is guided by the erythropoietin level. Clinical consequences of the various erythrocytoses are not clear, but in some groups thromboembolic events have been described in young patients. Evidence is lacking to define best management, but aspirin and venesection to a target hematocrit should be considered.

Topics: Adult; Aspirin; Blood Viscosity; Diagnosis, Differential; Erythropoiesis; Erythropoietin; Female; Hematocrit; Humans; Hypoxia; Male; Mutation; Phlebotomy; Platelet Aggregation Inhibitors; Polycythemia; Polycythemia Vera; Receptors, Erythropoietin; Signal Transduction; Thrombophilia

An absolute erythrocytosis is present when the red cell mass is raised and the haematocrit is elevated above prescribed limits. Causes of an absolute erythrocytosis can be primary where there is an intrinsic problem in the bone marrow and secondary where there an event outside the bone marrow driving erythropoiesis. This can further be divided into congenital and acquired causes. There remain an unexplained group idiopathic erythrocytosis. Investigation commencing with thorough history taking and examination and then investigation depending on initial features is required. Clear simple criteria for polycythaemia vera are now defined. Those who do not fulfil these criteria require further investigation depending on the clinical scenario and initial results. The erythropoietin level provides some guidance as to the direction in which to proceed and the order and extent of investigation necessary in an individual patient. It should thus be possible to make an accurate diagnosis in the majority of patients.

Topics: Erythrocyte Volume; Erythropoietin; Hematocrit; Hemoglobins; Humans; Hypoxia; Mutation; Polycythemia; Polycythemia Vera

Optimal clinical management of patients with primary myelofibrosis and post-essential thrombocythemia/polycythemia vera myelofibrosis is a challenge, given the typically advanced age of presentation and variability of the disease course and prognosis. Current medical therapeutic options have not demonstrated an impact on the disease course, which exceeds the palliation of disease-related extramedullary hematopoiesis and alleviation of cytopenias. In contrast, allogeneic stem cell transplantation (SCT) can lead to 'cure' but is limited due to patient's age or comorbidities. Currently, in patients, who are reasonable candidates, SCT (frequently with a reduced intensity conditioning regimen) is employed for intermediate- to high-risk disease. Current pharmaco-medical therapy is used as a bridge to transplant, or instead of transplant in poor transplant candidates. Pathogenetic insights, especially the discovery of the Janus kinase (JAK)2(V617F) mutation, have ushered in a host of new potential therapeutic agents that may augment the role of medical therapy. Similarly, the boundaries of transplantation continue to alter with strategies that decrease conditioning-related toxicity, improved antimicrobial prophylaxis and decreased graft-versus-host disease. The potential for continued improvements in both medical and transplant therapy suggests that for the immediate future the optimal choices for an individual patient will remain potentially volatile and present complex decisions.

Topics: Adult; Androgens; Clinical Trials as Topic; Cytostatic Agents; Disease Management; Drugs, Investigational; Erythropoietin; Etanercept; Hematopoietic Stem Cell Transplantation; Humans; Immunoglobulin G; Janus Kinase 2; Middle Aged; Myeloablative Agonists; Palliative Care; Polycythemia Vera; Primary Myelofibrosis; Prognosis; Receptors, Tumor Necrosis Factor; Risk; Thrombocythemia, Essential; Transplantation Conditioning; Treatment Outcome

The clinical criteria for the diagnosis of essential thrombocythemia (ET) according to the polycythemia vera study group (PVSG) do not distinguish between ET and thrombocythemia associated with early stage PV and prefibrotic chronic idiopathic myelofibrosis (CIMF). The clinical criteria of the PVSG for the diagnosis of polycythemia vera (PV) only detects advanced stage of PV with increased red cell mass. The bone marrow criteria of the World Health Organization (WHO) are defined by pathologists to explicitly define the pathological criteria for the diagnostic differentiation of ET, PV, and prefibrotic and fibrotic CIMF. As the clinical PVSG and the pathological WHO criteria show significant shortcomings, an updated set of European Clinical and Pathological (ECP) criteria combined with currently available biological and molecular markers are proposed to much better distinct true ET from early PV mimicking ET, to distinguish ET from thrombocythemia associated with prefibrotic CIMF, and to define the various clinical and pathological stages of PV and CIMF that has important therapeutic and prognostic implications. Comparing the finding of clustered giant abnormal megakaryocytes in a representative bone marrow as a diagnostic clue to MPD, the sensitivity for the diagnosis of MPD associated with splanchnic vein thrombosis was 63% for increased red cell mass, 52% for low serum EPO level, 72% for EEC, and 74% for splenomegaly indicating the superiority of bone marrow histopathology to detect masked early and overt MPD in this setting. The majority of PV and about half of the ET patients have spontaneous EEC, low serum EPO levels and PRV-1 over-expression and are JAK2 V617F positive. The positive predictive value for the diagnosis of PV of spontaneous growth of endogenous erythroid colonies (EEC) of peripheral blood (PB) and bone marrow (BM) cells is about 80-85% when either PB or BM EEC assays, and up to 94% when BM and PB EEC assays were performed. The diagnostic impact of low serum EPO levels (ELISA assay) in a large study of 186 patients below the normal range (<3.3 IU/l) had a sensitivity specificity and positive predictive value of 87%, 97% and 97.8%, respectively, for the diagnosis of PV. There is a significant overlap of serum EPO levels in PV versus control and controls versus SE. The specificity of a JAK2 V617F PCR test for the diagnosis of MPD is high (near 100%), but only half of ET and MF (50%) and the majority of PV (up to 97%) are JAK2 V617F positi

Topics: Amino Acid Substitution; Biomarkers; Blood Cell Count; Bone Marrow; Cell Lineage; Colony-Forming Units Assay; Disease Progression; Enzyme-Linked Immunosorbent Assay; Erythrocyte Volume; Erythroid Precursor Cells; Erythropoietin; GPI-Linked Proteins; Humans; Isoantigens; Janus Kinase 2; Membrane Glycoproteins; Mutation, Missense; Point Mutation; Polycythemia Vera; Polymerase Chain Reaction; Predictive Value of Tests; Primary Myelofibrosis; Receptors, Cell Surface; Sensitivity and Specificity; Thrombocythemia, Essential; World Health Organization

Topics: Alleles; Amino Acid Substitution; Bone Marrow; Cohort Studies; Erythroid Precursor Cells; Erythropoietin; Exons; Humans; Ireland; Janus Kinase 2; Polycythemia; Polycythemia Vera; Prevalence; United Kingdom

Historically, red cell mass (RCM) measurement has been promoted as an accurate indicator of the body's red cell content. Both the Polycythemia Vera Study Group and a committee sponsored by the World Health Organization have endorsed its inclusion in their diagnostic criteria without supporting evidence of diagnostic accuracy from a systematic study. Over the years, it has become evident that RCM measurement is a cumbersome and costly test and, as a result, it has been abandoned by many hematologists in practice. The recent discovery that a somatic JAK2-activating mutation, JAK2(V617F), is almost invariably associated with polycythemia vera further supports the use of diagnostic algorithms that are based on biologic parameters in place of traditional diagnostic criteria that are based on RCM measurement. Therefore, a contemporary approach to the diagnosis of polycythemia vera starts with peripheral blood mutation screening for JAK2(V617F) as well as measurement of serum erythropoietin. The results of these tests, along with the clinical scenario, determine the need for further investigation, including bone marrow examination.

Topics: Algorithms; Amino Acid Substitution; Bone Marrow Examination; Diagnosis, Differential; Erythrocyte Volume; Erythropoietin; Hematocrit; Humans; Janus Kinase 2; Mutation, Missense; Point Mutation; Polycythemia Vera; Predictive Value of Tests; Reproducibility of Results; Sensitivity and Specificity

Recent insights into the molecular mechanisms of polycythemia vera (PV) and essential thrombocythemia (ET) are challenging the traditional diagnostic classification of these myeloproliferative disorders (MPDs). Clonality analysis using X-chromosome inactivation patterns has revealed apparent heterogeneity among the MPDs. The recently discovered single somatic activating point mutation in the JAK2 gene (JAK2-V617F) is found in the great majority of patients with PV, but also in many patients with phenotypically classified ET and other MPDs. In contrast to the acquired MPDs, mutations of the erythropoietin receptor and thrombopoietin receptor have been identified in familial forms of nonclonal erythrocytosis and thrombocytosis, respectively. The mechanisms of major clinical complications of PV and ET remain poorly understood. Quantitative or qualitative abnormalities of red cells and platelets do not provide clear explanations for the thrombotic and bleeding tendency in these MPDs, suggesting the need for entirely new lines of research in this area. Recently reported randomized clinical trials have demonstrated the efficacy and safety of low-dose aspirin in PV, and an excess rate of arterial thrombosis, major bleeding, and myelofibrotic transformation, but decreased venous thrombosis, in patients with ET treated with anagrelide plus aspirin compared to hydroxyurea plus aspirin.

Topics: Erythropoietin; Humans; Janus Kinase 2; Polycythemia Vera; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Thrombocythemia, Essential; Thrombopoietin

Formation of endogenous erythroid colonies (EECs) or endogenous megakaryocytic colonies (EMCs) is a hallmark of myeloproliferative disorders (MPDs). The diagnostic value of EEC for polycythemia vera (PV) using standardized media has been demonstrated, and has led clinicians to consider EEC as a major diagnostic criterion in the WHO classification. The interest of EEC currently needs to be considered taking into account recent data about V617F JAK2 mutation in MPD. In particular, EECs and EMCs should be helpful for the diagnostic and the vascular risk evaluation of essential thrombocythemia (ET) and for mutation negative patients. A low serum erythropoietin (EPO) level is a consistent finding in PV. Recent studies have shown that commercial serum EPO assays provided a reliable, accurate, and low-cost criterion for the diagnosis of a significant proportion of PV. It suggests that diagnostic algorithms integrating serum EPO level could be elaborated. The diagnostic value of EPO assay for thrombocytosis has not been proved, but some data suggest a predictive value of low EPO levels for thrombosis in ET.

Topics: Colony-Forming Units Assay; Erythropoietin; Humans; Myeloproliferative Disorders; Polycythemia Vera; Thrombocythemia, Essential

Pathogenetically fundamental observations have identified polycythemia vera (PV) as a clonal stem cell disease with bone marrow histological and other biological features that distinctly differentiate it from other causes of 'increased' hematocrit. However, relatively little attention has been given to the effective utilization of pathology and laboratory markers of clonal myeloproliferation as diagnostic tools in PV. In contrast, the diagnostic use of red cell mass (RCM) measurement in PV stemmed from the accidental endorsement, as 'diagnostic criteria', of 'study eligibility criteria' that were formulated for clinical trials. It has since become evident that RCM measurement is a tedious procedure that is fraught with multiple-level imprecision, as well as suboptimal diagnostic accuracy. Therefore, it is reasonable to consider dispensing with RCM measurement as a diagnostic test for PV and instead utilize a diagnostic algorithm that combines clinical information with easily accessible laboratory data, including serum erythropoietin level and bone marrow histology. Recent discoveries of myeloproliferative-disease-specific molecular markers, including the JAK2 V617F tyrosine kinase mutation that is found in the majority of patients with PV, provide further support for such a measure.

Topics: Algorithms; Bone Marrow; Diagnosis, Differential; Erythrocyte Volume; Erythropoietin; Humans; Polycythemia Vera

The total blood volume (BV) consists of the part occupied by red blood cells (RBC), which is referred to as red cell mass (RCM), and that occupied by plasma (ie, plasma volume). Quantitative laboratory measurements that are pertinent to RBC, including RBC count, hematocrit (Hct), and hemoglobin (Hgb) are expressed in reference to a given volume of whole blood and are therefore influenced by plasma volume. Consequently, a "direct" RCM measurement has been promoted as a more accurate indicator of the body's red cell content. In accordance with this view, an international group of investigators, then identified as the Polycythemia Vera Study Group (PVSG), recommended that RCM be measured and only if elevated should a patient be considered for participation in a series of clinical trials in PV that were conducted more than 30 years ago. By default, the 'study eligibility' criteria used in these studies became 'diagnostic' criteria without any systematic evaluation for diagnostic accuracy. Furthermore, over the years, it has become evident that RCM measurement is a cumbersome and costly test that is also suboptimal in its diagnostic accuracy. As a result, the specific procedure has been abandoned by the majority of hematologists in certain countries and instead physicians are increasingly using bone marrow histology, serum erythropoietin level, and other disease-characteristic biologic markers as diagnostic tools.

Topics: Algorithms; Bone Marrow Examination; Diagnosis, Differential; Erythrocyte Volume; Erythropoietin; Female; Humans; Male; Polycythemia Vera

This review will focus on the molecular basis of certain polycythemic disorders. Primary polycythemias are characterized by acquired somatic or inherited germ-line mutations expressed within hematopoietic progenitors that cause increased accumulation of red blood cells. Polycythemia vera (PV), an acquired condition, is the most common primary polycythemia; although some progress has been made in the understanding of PV, its molecular basis remains unknown. In contrast, recent advances in delineating the molecular defects of some inherited polycythemias have greatly furthered our knowledge of the regulation of erythropoiesis and hypoxia sensing; however, more work needs to be done.

Topics: Adult; Animals; Child; Cohort Studies; Disease Models, Animal; DNA-Binding Proteins; Erythropoiesis; Erythropoietin; Ethnicity; Female; Founder Effect; Germ-Line Mutation; GPI-Linked Proteins; Humans; Hypoxia; Hypoxia-Inducible Factor 1; Hypoxia-Inducible Factor 1, alpha Subunit; Isoantigens; Janus Kinase 2; Male; Membrane Glycoproteins; Mice; Nuclear Proteins; Polycythemia; Polycythemia Vera; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Cell Surface; Receptors, Erythropoietin; Russia; Transcription Factors; Tumor Suppressor Proteins; Ubiquitin; Ubiquitin-Protein Ligases; Von Hippel-Lindau Tumor Suppressor Protein

By definition, myeloproliferative disorders (MPDs) are caused by an acquired somatic mutation of a hematopoietic progenitor/stem cell and have sporadic occurrence. However, well-documented families exist with first-degree relatives acquiring one or several MPDs. It is reasonable to assume that the germ-line mutation(s) or genetic background must facilitate or predispose for one or several somatic mutation(s) that lead to the MPD that is indistinguishable from the sporadic form. This is best documented in familial polycythemia vera (PV), which appears to be inherited as an autosomal dominant disorder with incomplete penetrance. However, there are also families wherein members develop any combination of MPDs, including PV, essential thrombocythemia (ET), chronic myelocytic leukemia (CML), and idiopathic myelofibrosis (IMF). A separate group of familial diseases is the familial thrombocythemias, wherein germ-line mutations in the genes for thrombopoietin or its receptor, MPL, cause polyclonal hereditary thrombocythemia, which may be clinically indistinguishable from ET. Patients with the congenital polycythemic condition "primary familial and congenital polycythemia" (PFCP) have characteristically decreased erythropoietin (Epo) levels similar to PV, hypersensitive erythroid progenitors, and low Epo levels; as such, this condition is often confused with PV. Therefore, PFCP will also be discussed here, while other congenital polycythemic states such as the Chuvash polycythemia that have elevated or inappropriately normal Epo levels will be omitted from this review in view of their distinct phenotype and unique laboratory features.

Topics: Chromosomes, Human, X; Erythroid Precursor Cells; Erythropoietin; Genetic Markers; Humans; Loss of Heterozygosity; Phenotype; Polycythemia Vera

Polycythemia vera (PV) is a chronic myeloproliferative disorder due to a haematopoietic stem cell's clonal proliferation. PV is also characterized by independency or hyper sensibility of haematopoietic progenitors to several cytokine as erythropoietin. This acquired disorder is often associated with thrombocytosis, leukocytosis and splenomegaly. Generally, diagnosis remains easy, based on basic clinical and biological abnormalities. Sometimes, positive diagnosis required more sophisticated tests as assay of endogenous erythroid colony, erythropoietin blood level and bone marrow biopsy. Usually natural history of disease remains long with a good quality of life. In some cases complications occur: mainly thrombosis and late myeloid metaplasia with myelofibrosis and acute leukemia. Therapeutic approachs remain complex and difficult to optimize based up on age and disease severity. Treatment searchs for reducing hyper viscosity complications and for avoiding therapeutic induced leukemia.

Topics: Biopsy; Bone Marrow; Diagnosis, Differential; Erythroid Cells; Erythropoietin; Humans; Leukemia; Polycythemia Vera; Quality of Life; Severity of Illness Index

Topics: 2,3-Diphosphoglycerate; Bisphosphoglycerate Mutase; Erythropoiesis; Erythropoietin; Gene Expression Regulation; Hemoglobinopathies; Homeostasis; Humans; Hypoxia; Kidney Diseases; Liver Diseases; Neoplasms; Oxygen; Polycythemia; Polycythemia Vera

Topics: Anemia; Apoptosis; Clone Cells; Disease Progression; Erythropoiesis; Erythropoietin; Female; Growth Substances; Hematologic Tests; Hematopoietic Stem Cells; Humans; Iron; Leukemia, Myeloid; Male; Myeloproliferative Disorders; Phlebotomy; Phosphorus Radioisotopes; Polycythemia Vera; Pregnancy; Pregnancy Complications, Hematologic; Receptors, Erythropoietin; Receptors, Growth Factor; Thrombocytosis; Thrombosis

By definition, idiopathic erythrocytosis (IE) applies to a group of patients characterised by having a measured RCM above their predicted normal range (an absolute erythrocytosis) and following investigation do not have a form of primary or secondary erythrocytosis. Patients with IE are heterogenous. The possibilities include physiological variation, 'early' polycythaemia vera (10-15% develop clear features of PV over a few years), unrecognized congenital erythrocytosis, unrecognized or unrecognizable secondary acquired erythrocytosis or a currently undescribed form of primary or secondary erythrocytosis. Patients are more commonly male with a median age at presentation of 55-60 years. Approximately half of the patients present with vascular occlusive complications. Retrospective evidence indicates that vascular occlusion occurs less frequently when the PCV is controlled at normal levels. Venesection is the treatment of choice to lower the PCV. As a general approach to management, all patients with a PCV above 0.54 should be venesected to a PCV less than 0.45. This target PCV should also apply to patients with lesser degrees of raised PCV who have additional other risk factors for vascular occlusion.

Topics: Aged; Arterial Occlusive Diseases; Bone Marrow; Chlorambucil; Diagnosis, Differential; Endocrine System Diseases; Erythrocyte Volume; Erythroid Precursor Cells; Erythropoietin; Genetic Predisposition to Disease; Humans; Hypoxia; Kidney Diseases; Leukemia; Leukemia, Radiation-Induced; Middle Aged; Phosphorus Radioisotopes; Polycythemia; Polycythemia Vera; Receptors, Erythropoietin; Sequence Deletion; Smoking; Stroke

Topics: Age Factors; Chromosomes, Human, Pair 8; Chromosomes, Human, Pair 9; Erythrocyte Volume; Erythropoietin; Female; Hematocrit; Humans; Male; Polycythemia Vera; Prognosis; Reference Standards; Sex Factors; Survival Rate; Time Factors; Trisomy

Differentiation of an elevated hemoglobin/hematocrit level warrants not only the determination of relevant laboratory values, including erythropoietin, but also an elaborate evaluation of bone marrow histopathology. Particularly in the initial stages of polycythemia vera (PV), when not all stringent clinical criteria are completely fulfilled, a more refined analysis of morphological features helps to distinguish autonomous (PV) from spurious or secondary erythrocytosis - polyglobuly (PG). PV is characterized by a panmyelosis, i.e., a trilinear proliferation of all cell lineages, whereas in PG erythropoiesis predominates. Megakaryopoiesis exerts a significant impact on differentiation, because in PV there is a conspicuous grouping associated with a strikingly expressed pleomorphous appearance. This peculiar feature implicates assemblies of small- to medium-sized megakaryocytes lying adjacent to giant cells with extensively lobulated, staghorn-like nuclei. A further discriminating parameter is presented by the interstitial lesions frequently occurring in PG. These, according to the usually underlying inflammatory cause (chronic bronchitis-recurrent bronchopneumonias), include iron-laden macrophages, a prominent perivascular plasmacytosis, phagocytosis of cell debris by histiocytic reticular cells and often an in- crease in the number of eosinophils. Comparable findings are not, or only to a minor ex-tent, detectable in PV. In conclusion, by regarding hematopoiesis and the myeloid stroma initial stages of PV may be definitively distinguished from PG.

Topics: Bone Marrow; Diagnosis, Differential; Erythropoietin; Hematopoiesis; Hematopoietic Stem Cells; Humans; Polycythemia; Polycythemia Vera

Polycythemia rubra vera (PV) is one of four diseases collectively called the myeloproliferative disorders (MPDs). Each disorder leads to an increased production of one or several hematopoietic cell lineages. MPDs arise from acquired mutations in a pluripotent hematopoietic stem cell. However, the molecular mechanisms leading to the development of these diseases are poorly understood. This review will summarize and evaluate recent advances in our understanding of one particular MPD, PV.

Topics: bcl-X Protein; Erythropoietin; GPI-Linked Proteins; Growth Substances; Humans; Isoantigens; Membrane Glycoproteins; Myeloproliferative Disorders; Neoplasm Proteins; Phosphoprotein Phosphatases; Polycythemia Vera; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Receptors, Cell Surface; Receptors, Cytokine; Receptors, Erythropoietin; Receptors, Thrombopoietin; Signal Transduction; Stem Cells

Topics: Erythropoietin; Humans; Hypertension; Polycythemia; Polycythemia Vera

The primary objective during the evaluation of erythrocytosis is to ascertain the presence or absence of polycythemia vera (PV). Because of prognostic and treatment differences, PV must be distinguished from relative polycythemia and secondary erythrocytosis. This distinction is currently accomplished through the laboratory measurement of red blood cell mass, plasma volume, and arterial oxygen saturation and determination of oxygen pressure at 50% hemoglobin saturation (P50). Furthermore, according to the Polycythemia Vera Study Group guidelines, the demonstration of an increased red blood cell mass is an absolute criterion for the diagnosis of PV. This article discusses the use of the serum erythropoietin level and endogenous erythroid colony assay as a potential alternative in the diagnosis of PV.

Topics: Decision Trees; Diagnosis, Differential; Erythroid Precursor Cells; Erythropoietin; Humans; Polycythemia Vera

The production of erythroid cells is a dynamic and exquisitely regulated process. The mature red cell is only the final phase of a complex but orderly series of genetic events that are initiated at the time a multipotent stem cell becomes committed to expressing the erythroid programme. Aberrations either in the intrinsic generation and/or amplification of functional erythroid cells or in the regulatory influences of microenvironment or cytokines form the basis for a number of blood diseases. In this review we focus upon abnormalities in red blood cell production and discuss how alterations in cytokine regulation of red blood cell production may contribute to these disease processes. We discuss clinical states in which blood red cell numbers are altered, including primary familial and congenital polycythaemia, the myeloproliferative disorder polycythaemia vera, erythroleukaemia, and Diamond-Blackfan anaemia. These disorders are briefly described and evidence supporting a potential role of specific cytokine receptor signalling defects as contributing to these phenotypes is discussed.

Topics: Erythropoietin; Fanconi Anemia; Hematologic Diseases; Hematopoiesis; Humans; Leukemia, Erythroblastic, Acute; Mutation; Polycythemia; Polycythemia Vera; Receptors, Cytokine; Receptors, Erythropoietin; Signal Transduction

We investigated the in vitro erythroid progenitor growth and the effects of sera on normal-marrow CFU-E (colony-forming units - erythroid) growth in 2 patients with renal failure on regular hemodialysis following a prior history of polycythemia vera (PV). PV was diagnosed 3 and 11 years, respectively, before the development of terminal renal failure. One of the patients had entered a spent phase of PV as characterized by diffuse extensive myelofibrosis and anemia; the other also had mild myelofibrosis. The serum erythropoietin (EPO) levels were low or normal on serial measurements by radioimmunoassay. There was no correlation between the hematocrit values and serum EPO levels. EPO-independent erythroid colonies were present in the cultures of bone marrow and peripheral blood cells from both patients after renal failure in the anemic state. With the addition of various concentrations of EPO, the number of erythroid colonies increased as the concentrations of EPO increased which was in accordance with the clinical observation that 1 patient with postpolycythemic myeloid metaplasia partially responded to recombinant human EPO therapy. In the EPO-dependent CFU-E assay, normal-marrow CFU-E numbers supported by 10% of the patient sera were less than those by normal sera. In the absence of EPO in cultures, no erythropoietic activity was found in the patients' sera. Our study on uremic patients with underlying PV showed that the biologic characteristics of autonomous erythroid progenitor growth for PV persisted during the spent phase and after the development of terminal renal failure with anemia. The erythroid progenitors responded to EPO both in vitro and in vivo. Their sera exhibited an inhibiting effect on the growth of normal-marrow erythroid progenitors.

Topics: Adult; Anemia; Blood Proteins; Cell Division; Cells, Cultured; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Kidney Failure, Chronic; Middle Aged; Polycythemia Vera; Recombinant Proteins; Renal Dialysis

Current methodology for the immunoassay of erythropoietin (EPO) in human plasma or serum is reviewed, with an emphasis on measurement of EPO concentrations in the low and normal ranges, analytical interference and blood sampling requirements. In only 2 out of 8 research or in-house immunoassays reported since 1987 was there evidence that patients with polycythaemia vera (PV) could be identified, PV being an EPO-independent form of polycythaemia in which EPO concentrations are low in untreated cases. The same was true for only 1 out of 13 currently available kit methods. Remarkable differences in sample stability have been observed with different methods. Measurement of EPO in serum is recommended in most published articles. However, only EDTA plasma seems to be acceptable for the one generally available method with proven high diagnostic sensitivity for PV. It is concluded that most EPO assay methods have not been shown to be adequate for the measurement of the low EPO concentrations, and thus have poor diagnostic sensitivity for PV. It is inferred that they might not be appropriate to assess states of EPO deficiency. Only when a sufficiently sensitive diagnostic method becomes generally available will it be possible to define the various causes of low EPO concentrations. As in other fields of polypeptide hormone measurement, further developments in the field of EPO assay may be expected to be important in diagnostic medicine.

Topics: Drug Stability; Erythropoietin; Humans; Immunoassay; Polycythemia Vera; Quality Control; Reagent Kits, Diagnostic; Sensitivity and Specificity

Polycythemia vera shares basic features of pathogenesis with other subtypes of the group of chronic myeloproliferative disorders. All myelopoietic cells are derived from one transformed hemopoietic stem cell. Genetic instability of mitotic clonal cells explains the risk of leukemic transformation, which may be enhanced by cytoreductive treatment. Recent data show that erythroid hyperplasia is not due to erythropoietin hypersensitivity, but rather to abnormal stimulation by other cytokine growth factors. Treatment as established by clinical trials has almost normalized life expectancy in older patients, but the optimal strategy for subgroups of patients is still unknown. For younger patients, new and potentially curative approaches should be investigated.

Topics: Animals; Chronic Disease; Clinical Trials as Topic; Cytokines; Disease Progression; Erythropoietin; Growth Substances; Humans; Life Expectancy; Polycythemia Vera; Practice Guidelines as Topic; Primary Myelofibrosis; Rabbits

There is no single diagnostic marker for polycythaemia vera (PV). The Polycythemia Vera Study Group established diagnostic criteria more than twenty years ago. Since some new laboratory and clinical investigations have developed, these criteria can now be updated. It is proposed that the overall pattern of major and minor criteria should remain. A raised red cell mass (greater than 25% above the patient's mean normal predicted value based on surface area) and absence of a cause of secondary polycythaemia are essential criteria. Then either palpable splenomegaly or the presence of a marker of clonal haemopoiesis would support the diagnosis of PV. In the absence of both of these latter major criteria, at least two minor criteria must be present to secure the diagnosis. These are: platelet count > 400 x 10(9)/l; neutrophil count > 10 x 10(9)/l; splenomegaly demonstrated by a scanning technique; characteristic BFU-E growth or reduced serum erythropoietin value.

Topics: Alkaline Phosphatase; Biomarkers; Blood Cell Count; Bone Marrow; Clone Cells; Diagnosis, Differential; Dosage Compensation, Genetic; Erythrocyte Volume; Erythroid Precursor Cells; Erythropoietin; Female; Humans; Male; Oxygen; Polycythemia Vera; Splenomegaly

PV represents a clonal disorder characterized by excessive erythropoiesis accompanied by low serum EPO levels. Two populations of erythroid progenitors have been identified in the BM of patients with PV. One population responds normally to EPO and the other, the malignant clone, is exquisitely sensitive to EPO. The latter phenomenon is regarded as the most readily accepted explanation for the pathophysiology of this disease, although other mechanisms have been proposed. Thrombohemorrhagiccomplications, which usually correlate with the hematocrit level, are the most important because of their frequency and severity. Preventing such complications represents one of the most important therapeutic goals. Diagnostic criteria have been established enabling an accurate diagnosis of PV, in which the majority of patients live an excess of 10 years. Some will develop a falling hematocrit secondary to PPMM, also termed the spent phase. Patients may also develop acute leukemia, which is increased in incidence if myelosuppressive therapy is used, compared with phlebotomies alone. The initial treatment is phlebotomy to an hematocrit of 45% or less. The studies of the PVSG have shown that chlorambucil produces excessive mortality from malignancy, and 32P and phlebotomy are associated with superior survivals which are equivalent. Because of the substantial risk of thrombotic complications, elderly patients, ie, those older than 70 years, should be treated initially with phlebotomy and myelosuppressive therapy, usually 32P. Hydroxyurea appears less leukemogenic than 32P or alkylating agents; however, recent reports have questioned this. IFN is a promising new agent which may become a standard form of therapy in the future.

Topics: Adult; Aged; Antineoplastic Agents; Bloodletting; Erythropoiesis; Erythropoietin; Female; Hematocrit; Humans; Male; Middle Aged; Polycythemia Vera

Erythropoietin (EPO) is a prime stimulating factor for red cell production. EPO is a glycoprotein which has a molecular weight of 34,000, and is mainly produced by the kidney. EPO stimulates the differentiation and proliferation of erythroid progenitor cells in the bone marrow. The rate of production of EPO is regulated primarily by renal oxygen availability. Because anemia reduces renal oxygen availability, anemic stress accelerates EPO production in the kidney. Recently, EPO has mainly been determined by radioimmunoassay. Serum EPO titer is usually inversely correlated with hemoglobin concentration, as typically shown in iron deficiency anemia. Serum EPO titers in aplastic anemia are much higher than those in iron deficiency anemia relative to the hemoglobin concentration. Serum EPO titers in anemia caused by malignancies sometimes differ considerably among patients. Serum EPO in renal anemia usually show low titers irrespective of the degree of anemia. Serum EPO titers in untreated polycythemia vera are lower than those in treated polycythemia vera or secondary polycythemia. Determination of serum EPO is useful in differential diagnosis of polycythemia vera. Recombinant human EPO has been used to treat various anemias including renal anemia, refractory anemia, anemia in malignancies and secondary anemia. Determination of serum EPO titers is also valuable in many other situations of clinical medicine.

Topics: Anemia; Circadian Rhythm; Diagnosis, Differential; Erythropoietin; Humans; Polycythemia Vera; Radioimmunoassay

The red blood cell (RBC) has a major contribution to biological research. For example, the basic knowledge on the structure and function of the plasma membrane and the structure, function and genetics of proteins came from studies of RBC and hemoglobin (Hb). The reasons why the RBC has become such a useful experimental tool is due to several facts: (a) peripheral blood (PB) is readily available; (b) pure populations of RBC can be easily isolated from other blood cells; (c) RBC can be maintained in vitro for weeks under simple conditions (physiological salt solution at 4 degrees C) and finally, (d) the mature RBC is a rather simplified cell that lost many cellular structures and functions as a result of its extreme specialization. When studying the developmental aspects of erythroid cell differentiation and maturation, one faces a more complicated situation. Like other hemopoietic cells, the origin of RBC is in precursors in the bone marrow (BM). However, unlike PB, BM is not readily available and it consists of a mixture of cells of various types and stages of differentiation, which makes the purification of a particular cell population quite difficult.

Topics: Biomarkers; Cell Differentiation; Cell Division; Culture Media; Culture Techniques; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Humans; Polycythemia Vera

The remarkable capacity of the bone marrow to compensate for blood loss and for reduced atmospheric oxygen tension has been found to be mediated by a renal hormone, named erythropoietin. It is produced by peritubular interstitial cells in response to renal hypoxia, but molecular engineering has permitted large scale production of an identical recombinant erythropoietin in vitro. When used as a replacement hormone in patients with impaired endogenous production it has been found to be capable of improving or eliminating the anemia of chronic kidney disease and the anemia of prematurity. In the future it may also be used as a pharmacologic agent and possibly be able to control the anemia of patients with bone marrow failure and make them transfusion-independent.

Topics: Anemia; Bone Marrow; Erythropoiesis; Erythropoietin; Humans; Kidney; Kidney Failure, Chronic; Polycythemia Vera; Recombinant Proteins

Topics: Cell Differentiation; Cell Division; Cells, Cultured; Colony-Forming Units Assay; Colony-Stimulating Factors; Erythroblasts; Erythropoiesis; Erythropoietin; Growth Substances; Hematopoietic Stem Cells; Hormones; Humans; Interleukin-3; Leukemia, Myeloid; Leukemia, Myeloid, Acute; Lymphokines; Neoplastic Stem Cells; Polycythemia Vera; Primary Myelofibrosis; Thrombocytosis

The principal function of erythrocytes is the transport of oxygen. Erythropoiesis proceeds at a rate consistent with the demand for oxygen-carrying capacity, and the major regulator of erythrocyte production is erythropoietin. Erythropoietin is produced primarily by the kidney under control of a tissue oxygenation sensor. The recently developed erythropoietin radioimmunoassay should provide a clinically useful tool. Erythrocytosis is a pathologic state characterized by an elevated erythrocyte mass, which may result from increased proliferation of erythroid progenitors due to an intrinsic cellular defect or in response to extrinsic signals. Secondary erythrocytosis results from either physiologically appropriate compensation for inadequate tissue oxygenation or from inappropriate stimulation of erythropoiesis. Erythrocytosis increases oxygen-carrying capacity of the blood, but at high hematocrit levels increased blood viscosity may result in decreased tissue oxygen delivery. Polycythemia vera is a hematopoietic stem cell disease of clonal origin. Initial results from the Polycythemia Rubra Study Group suggest that therapy with chlorambucil is associated with an unacceptably high risk for development of acute leukemia, and 32P is preferred for situations in which phlebotomy alone is insufficient.

Topics: Blood Gas Analysis; Bone Marrow; Erythropoietin; Hematopoietic Stem Cells; Hemoglobins; Humans; Polycythemia; Polycythemia Vera

Topics: Animals; Antibody Specificity; Erythropoietin; Female; Humans; Male; Mice; Polycythemia; Polycythemia Vera; Radioimmunoassay; Rats; Sheep

Topics: Anemia, Aplastic; Animals; Cell Division; Colony-Stimulating Factors; Cyclic AMP; Erythrocytes; Erythrocytes, Abnormal; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Humans; Polycythemia Vera; Prostaglandins E

Topics: Anemia; Animals; Biological Assay; Chemical Phenomena; Chemistry; Culture Techniques; Cyclic AMP; Cyclic GMP; DNA; Erythroblasts; Erythropoiesis; Erythropoietin; Humans; Kidney; Liver; Mice; Polycythemia; Polycythemia Vera; Prostaglandins E; Radioimmunoassay; Rats; RNA; RNA, Messenger; Sheep

Topics: Alkylating Agents; Androgens; Blood; Bloodletting; Carbon Dioxide; Cobalt; Erythropoiesis; Erythropoietin; Humans; Hydrogen-Ion Concentration; Oxygen; Phosphorus Radioisotopes; Polycythemia; Polycythemia Vera

Data presently available suggest that polycythemia vera is an acquired clonal disorder of the pluripotent stem cell resulting in expansion of committed stem cell pools, most prominently in the erythroid line. In vitro and in vivo studies suggest that erythropoiesis in polycythemia vera is at least partially modulated by erythropoietin.

Topics: Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Humans; Polycythemia Vera

Topics: Arteries; Basophils; Blood Platelets; Blood Volume Determination; Bone Marrow; Erythrocytes; Erythropoiesis; Erythropoietin; Granulocytes; Hematocrit; Hemoglobins; Hepatomegaly; Humans; Megakaryocytes; Myeloproliferative Disorders; Osteosclerosis; Oxygen Consumption; Polycythemia; Polycythemia Vera; Primary Myelofibrosis; Splenomegaly

Topics: Anemia; Animals; Diagnosis, Differential; Erythropoietin; Humans; Mice; Neoplasms; Polycythemia; Polycythemia Vera

Topics: Alkylating Agents; Blood Cell Count; Blood Volume; Erythrocyte Count; Erythropoiesis; Erythropoietin; Hematocrit; Hemoglobinopathies; Hemoglobins; Humans; Hypoxia; Oxygen Consumption; Polycythemia; Polycythemia Vera; Protein Binding

Topics: Aminolevulinic Acid; Anemia; Animals; Bone Marrow; Bone Marrow Cells; Depression, Chemical; Erythrocytes; Erythropoiesis; Erythropoietin; Estrogens; Feedback; Female; Heme; Hemoglobins; Humans; Hypoxia; Immunoglobulin G; In Vitro Techniques; Infant, Newborn; Iron; Male; Mice; Models, Biological; Nephritis; Polycythemia; Polycythemia Vera; Rats

Topics: Adrenal Cortex Hormones; Alpha-Globulins; Androgens; Anemia, Aplastic; Animals; Base Sequence; Blood Proteins; Cell Division; Chemical Phenomena; Chemistry; Dogs; Drug Synergism; Endocrine Glands; Erythropoiesis; Erythropoietin; Glycoproteins; Hematopoiesis; Humans; Kidney; Leukocytes; Leukocytosis; Leukopenia; Mice; Polycythemia; Polycythemia Vera; Radiation Effects; Rats; Thrombopoietin; Thyroid Hormones

Topics: Anemia; Animals; Chemical Phenomena; Chemistry; Erythrocyte Aging; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Kidney Failure, Chronic; Neoplasms; Oxygen Consumption; Polycythemia Vera

Topics: Anemia; Anemia, Aplastic; Animals; Blood Transfusion; Bone Marrow; Bone Marrow Cells; Cell Differentiation; Chronic Disease; Erythropoiesis; Erythropoietin; Humans; Immune Sera; Kidney; Kidney Diseases; Lipids; Mercaptopurine; Polycythemia Vera

Topics: Adult; Aged; Blood Cell Count; Blood Coagulation Tests; Blood Gas Analysis; Bone Marrow Examination; Bromine; Erythrocytes; Erythropoietin; Female; Hepatomegaly; Humans; Male; Mannitol; Megakaryocytes; Middle Aged; Phosphorus Isotopes; Polycythemia Vera; Respiratory Function Tests; Retinal Vessels; Skin; Splenomegaly

Topics: Animals; Chemical Phenomena; Chemistry, Physical; Dogs; Erythropoiesis; Erythropoietin; Humans; Hypoxia; Kidney; Mice; Neurons, Efferent; Neurosecretory Systems; Neurotransmitter Agents; Polycythemia; Polycythemia Vera; Rabbits; Rats; Reticulocytes

Topics: Animals; Central Nervous System Diseases; Erythropoiesis; Erythropoietin; Female; Genes, Regulator; Genital Diseases, Female; Hemodynamics; Humans; Hypertension; Hypoxia; Kidney; Kidney Diseases; Liver Diseases; Male; Polycythemia; Polycythemia Vera; Rats

Topics: Anemia; Animals; Bone Marrow; Dogs; Erythrocytes; Erythropoiesis; Erythropoietin; Hematopoiesis; Hemorrhage; Humans; Hypoxia; Iron Isotopes; Kidney; Kidney Diseases; Mice; Placenta; Polycythemia Vera; Rabbits; Rats; Stimulation, Chemical

Topics: Anemia; Anemia, Macrocytic; Blood Platelets; Eosinophilia; Epoetin Alfa; Erythropoietin; Humans; Iron; Leukemia; Polycythemia Vera; Vitamin B 12

The diagnosis of polycythemia vera (PV) is based on clinical and biological criteria defined by either the Polycythemia Vera Study Group (PVSG) or the World Health Organization (WHO). Both the PVSG and WHO PV criteria have proved helpful and are extensively used, yet diagnostic strategies and scheduling of biological investigations vary. We assessed the value of measuring serum erythropoietin (Epo) as a first intention diagnostic test in patients with absolute erythrocytosis (AE).. Serum and bone marrow (BM) samples of 241 patients with a suspicion of erythrocytosis were collected in 8 hospital centers. One hundred and ninety had an absolute erythrocytosis (116 had PV, 66 had secondary erythrocytosis and 4 had idiopathic erythrocytosis). Serum Epo was assayed (ELISA) in 186. Statistical analysis (ROC curves) was used to define serum Epo thresholds that were specific for PV and secondary erythrocytosis and to analyze the diagnostic value of a low or high serum Epo level.. A large majority of PV patients (87% or 101/116) had a serum Epo level below the normal range in healthy patients (3.3 IU/L), giving this value a specificity of 97% with a 97.8% positive predictive value for the diagnosis of PV. Statistical analysis (ROC curves) defined two thresholds allowing a specific and direct diagnosis of 65.6% (65/99) of untreated PV (Epo < 1.4 IU/L) and 19.7% (13/66) of those with secondary erythrocytosis (Epo > 13.7 IU/L).. Based on these data, we propose that measurement of serum Epo level, a simple, reliable and inexpensive test, should be considered as a first intention diagnostic test for patients with absolute erythrocytosis.

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Bone Marrow; Collagen; Colony-Forming Units Assay; Culture Media; Cytokines; Diagnosis, Differential; Enzyme-Linked Immunosorbent Assay; Erythroid Precursor Cells; Erythropoietin; Female; Humans; Male; Middle Aged; Polycythemia; Polycythemia Vera; Predictive Value of Tests; ROC Curve; Sensitivity and Specificity

The aim of the present work was to investigate the effect of hydroxyurea (HU) treatment on haematopoietic progenitors and CD34 positive (CD34+) cells in patients with polycythaemia vera (PV) and essential thrombocythaemia (ET). Of the PV patients were 10 treated with phlebotomy only and 10 were on HU therapy. Seven ET patients were untreated and 10 received HU. In each subject peripheral blood was obtained for in vitro colony growth, determination of CD34+ cells and plasma erythropoietin (EPO) concentration. The mean number of EPO independent erythroid colonies (EEC) was higher in the group of PV patients on phlebotomy therapy compared to the PV patients treated with HU (74.4 and 8.0 colonies/10(5) cells, respectively) but the difference did not reach statistical significance. The corresponding means for the untreated ET patients and ET patients treated with HU were 13.0 and 1.3 colonies/10(5) cells, respectively, this difference being statistically significant (p = 0.012). The mean EEC for combined groups of PV and ET without myelosuppressive treatment were compared with the results for PV and ET patients on HU therapy; this difference was statistically significant (p = 0.014). The same pattern was observed for total erythroid growth with EPO. The relationship between the concentration of CD34+ cells and total EEC in peripheral blood was statistically significant for both PV (p<0.005) and ET (p<0.01). This finding supports the hypothesis that the level of CD34+ cells in peripheral blood could be used as a proliferation marker in these two myeloproliferative entities. No relationship between plasma EPO and EEC was present. It therefore appears that the reported differences in plasma/serum EPO concentrations between PV patients on phlebotomy treatment compared to patients on myelosuppressive treatment are not likely to be found at the production site for erythrocytes.

Topics: Adult; Aged; Aged, 80 and over; Antigens, CD34; Cell Division; Cells, Cultured; Colony-Forming Units Assay; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Hydroxyurea; Male; Middle Aged; Phlebotomy; Polycythemia Vera; Thrombocythemia, Essential

Topics: Cross-Over Studies; Double-Blind Method; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Humans; Polycythemia Vera; Reticulocytes

Acquired erythrocytosis can be classified into polycythemia vera (PV) and non-neoplastic erythrocytosis (NNE). The vast majority of PV patients harbor JAK2 mutations, but differentiating JAK2 mutation-negative PV from NNE is challenging due to a lack of definitive molecular markers.. We studied the clinical features of 121 patients with erythrocytosis of which 47 (38.8%) were JAK2 mutation-positive and also fulfilled the diagnostic criteria for PV, and 67 (55.4%) JAK2 mutation-negative erythrocytosis patients who were diagnosed as NNE. Diagnosis was strictly based on driver mutation analysis and central pathology review.. No JAK2 mutation-negative PV patients were found in our cohort. The NNE group showed significantly younger (p < 0.01) age with higher frequency of smoking (p < 0.001), alcohol consumption (p < 0.001), and diabetes mellitus (p < 0.05), whereas the PV group (n = 47) showed significantly higher white blood cell count, platelet count, and lactate dehydrogenase (p < 0.001). Although serum erythropoietin (EPO) levels were significantly higher in NNE compared to PV (p < 0.001), approximately 40% of the NNE patients had EPO levels below the lower range of normal, fulfilling a minor diagnostic criterion of PV and raising the possibility of PV misdiagnosis.. Low EPO levels in JAK2 mutation-negative erythrocytosis may not be a reliable diagnostic criterion for distinguishing PV from NNE.

Topics: Biomarkers; Erythropoietin; Humans; Mutation; Polycythemia; Polycythemia Vera

We assessed the diagnostic performances of erythropoietin and JAK2 mutations in 1,090 patients with suspected polycythemia who were referred for red cell mass (RCM) measurement. In patients with a high haematocrit and/or haemoglobin level, a low erythropoietin level (<=3·3 mUI/ml) and JAK2 mutation showed comparable positive predictive value (PPV) for true polycythemia (RCM>=125%), 92·1% and 90% respectively. A very-low erythropoietin level (<=1·99 mUI/ml) had a PPV of 100% for polycythemia vera (PV) diagnosis. We confirmed the correlations between RCM, erythropoietin and JAK2 variant allelic frequency in PV patients. This study prompts the need to revisit the role of EPO in PV diagnostic criteria.

Topics: Alleles; Amino Acid Substitution; Clinical Decision-Making; Disease Management; Erythrocyte Indices; Erythrocyte Volume; Erythropoietin; Gene Frequency; Genetic Association Studies; Genetic Predisposition to Disease; Humans; Janus Kinase 2; Mutation; Plasma Volume; Polycythemia Vera; Sensitivity and Specificity

Discriminating polycythemia vera (PV) from secondary polycythemia (SP) is crucial due to the inherent risk of thrombosis in PV and different treatment approaches. The majority of PV patients have subnormal serum erythropoietin levels and harbor Janus kinase 2 (JAK2) mutations; however, serum erythropoietin levels may be normal in approximately one third of PV patients and mutational analysis is costly and requires access to specialized laboratories. Recently, neutrophil-to-lymphocyte (NLR) and platelet-to-lymphocyte ratios (PLR) emerged as rapidly available biomarkers to identify PV patients under an increased risk of thrombosis and death. This multicenter retrospective study investigated whether these two biomarkers may also be used to differentiate PV from SP. A total of 207 subjects were included (103 PV and 104 SP) with both baseline NLR (median 4.33 vs. 1.89) and PLR (median 259.12 vs. 81.11) being significantly higher in PV than in SP (p < 0.001 for both analyses). According to the receiver operating curve analysis, PLR (area under the curve, AUC 0.936, the optimal cut-off value of > 138.1 had 82.5% sensitivity and 91.67% specificity for the detection of PV) outperformed other tested variables (NLR, total leukocytes, neutrophils, lymphocytes and platelets) and its cut-off values with 100% specificity and sensitivity were able to confirm (PLR > 224.56; 31% patients) and to exclude (PLR < 68.8; 13% patients) the highest proportions of PV patients. Therefore, PLR may represent a cheap and a rapidly available biomarker with valuable diagnostic and prognostic properties. This information may be particularly useful in resource-limited settings; however, our results need validation on larger datasets.

Topics: Biomarkers; Blood Platelets; Erythropoietin; Humans; Lymphocytes; Neutrophils; Platelet Count; Polycythemia; Polycythemia Vera; Prognosis; Retrospective Studies; Thrombosis

Considering the evolving diagnostic criteria of polycythemia vera (PV), we analyzed the utility of serum erythropoietin (EPO) as a predictive marker for differentiating polycythemia vera (PV) from other etiologies of erythrocytosis.. We conducted a retrospective study after a review of electronical medical records from January 2005 to December 2016 with diagnosis of erythrocytosis using International Classification of Disease-specific codes. To evaluate the diagnostic performance of EPO levels and JAK2-V617F mutation, we constructed a receiver-operated characteristic curve of sensitivity versus 1-specificity for serum EPO levels and JAK2-V617F mutation as predictive markers for differentiating PV from other causes of erythrocytosis.. We surveyed 577 patients with erythrocytosis. Median patient age was 59.2 years, 57.72% (n = 329) were male, 86.3% (n = 491) were white, and only 3.3% (n = 19) were African American. A total of 80.88% (n = 351) of those diagnosed with PV had a JAK2-V617F mutation compared to only 1.47% (n = 2) whose primary diagnosis was secondary polycythemia. When comparing JAK2-V617 mutation to the EPO level, the area under the curve of JAK2-V617 (0.8970) was statistically larger than that of EPO test (0.6765). Therefore, the PV diagnostic methodology using JAK2-V617 is better than the EPO test. An EPO level of < 2 mIU/mL was > 99% specific to predict PV but was only 12% sensitive.. In the appropriate clinical setting, cytogenetic and molecular studies such as JAK2 mutation status prevail as the most useful tools for PV case identification. The use of isolated EPO to screen patients with erythrocytosis is not a good diagnostic approach.

Topics: Biomarkers, Tumor; Cancer Care Facilities; Erythropoietin; Female; Genetic Testing; Humans; Janus Kinase 2; Male; Middle Aged; Mutation; Polycythemia; Polycythemia Vera; Predictive Value of Tests; Retrospective Studies; ROC Curve

Myeloproliferative neoplasms (MPNs) such as polycythemia Vera (PV) and Essential Thrombocythemia (ET) can be associated with a high risk of both venous and arterial thrombosis. However, the co-existence between these two complications is very rare and has never been described before, especially in young adults with no known history of MPNs.. We report the case of a 39 year-old Caucasian Moroccan male patient without cardiovascular risk factors (CVRF), who presented with acute chest pain. He also suffered from a severe headache since 2 weeks. Electrocardiogram (ECG) showed ST segment elevation myocardial infarction in the posterolateral leads. Cerebral Computed Tomography (CT) scan revealed subarachnoid hemorrhage (SAH), and cerebral Magnetic Resonance Angiography (MRA) found a Superior Sagittal Sinus Thrombosis (SSST). Routine blood tests showed raised hemoglobin and hematocrit in addition to leukocytosis and thrombocythemia. His coronary angiography revealed a thrombus in the ostial left circumflex artery (LCX). Further testing revealed positive Janus kinase 2 (JAK2) V617F mutation and low erythropoietin level, confirming the diagnosis of PV according to the 2008 World Health Organization (WHO) criteria. Antithrombotic and anti-ischemic treatments, in addition to myelosuppressive therapy with hydroxyurea, were initiated with a good clinical and biological evolution.. This case shows that MPNs are an important cause of thrombosis, especially in young patients with no other risk factors. Early diagnosis and appropriate management are fundamental before the occurrence of life-threatening complications that can sometimes present in unusual forms associating arterial and venous thrombotic events.

Topics: Adult; Chest Pain; Coronary Vessels; Erythropoietin; Headache; Humans; Janus Kinase 2; Male; Mutation; Polycythemia Vera; Superior Sagittal Sinus; Venous Thrombosis

In the 2016 WHO classification, hemoglobin and hematocrit thresholds for diagnosing polycythemia vera (PV) have been lowered, increasing the number of consultations for polycythemia investigations. In PV, beta-2 microglobulin (B2m) levels are reportedly increased, whereas erythropoietin (EPO) levels are usually low. Most secondary polycythemia cases (SP) are caused by tobacco use. We decided to analyze the relevance of these three parameters in all patients seen for polycythemia investigations to help differentiate PV from SP cases. A cohort of 257 patients (123 PV; 134 SP) was identified. The median B2m level was higher for PV patients (3.16 vs 1.98 mg/l, p < 0.0001). Increased B2m levels were observed in 83.7% of PV patients (11.9% in SP). The median EPO level was lower in PV patients (4.4 vs 12.3 UI/l, p < 0.0001). Tobacco was used by 42.8% of SP patients (8% in PV, p < 0.0001). Increased B2m, low EPO and no tobacco exposure was predictive of PV (specificity and positive predictive value = 100%). Normal B2m, normal EPO and tobacco exposure was predictive of SP (positive predictive value = 100%). These simple and inexpensive parameters could be used to rapidly differentiate PV from SP cases, before prescribing time-consuming JAK2 V617F mutation analysis by specialists.

Topics: beta 2-Microglobulin; Biomarkers; Diagnosis, Differential; Disease Susceptibility; Erythropoietin; Humans; Janus Kinase 2; Mutation; Polycythemia; Polycythemia Vera; Prognosis; Tobacco Use

A low erythropoietin (EPO) level is a minor diagnostic criterion for polycythemia vera (PV). Controversies exist regarding the diagnostic value of a low EPO level when considering increasing availability of advanced molecular testing. We assessed the role of low EPO level for PV diagnosis in the context of positive JAK2 mutation status as well as other diagnostic parameters. Of 138 patients, 75 patients had PV and 63 had secondary erythrocytosis. Of the 75 patients with PV, 32% had EPO levels within the normal range. EPO level positively correlated with obesity and smoking status, making it an unreliable diagnostic marker in those patients. Although EPO level below normal as a standalone diagnostic modality was significantly associated with PV (odds ratio [OR] 0.857; p < 0.001), when JAK2V617F mutation status was included in the prediction model, the association of low EPO was not statistically significant (OR 0.962, p = 0.269). Positive JAK2V617F demonstrated a strong predictive value for PV (OR 670.5, p = 0.006) either alone or in combination with other variables. Results show that EPO level is not a reliable diagnostic marker due to physiologic variation in association with obesity and smoking.

Topics: Biomarkers; Biopsy; Bone Marrow; Diagnosis, Differential; Erythropoietin; Female; Humans; Janus Kinase 2; Male; Mutation; Polycythemia Vera; Prognosis; Reproducibility of Results; Retrospective Studies; ROC Curve

Topics: Alleles; Biomarkers; Erythropoietin; Exons; Humans; Janus Kinase 2; Mutation; Polycythemia Vera; Prognosis

Topics: Diagnostic Screening Programs; Erythropoietin; Female; Humans; Janus Kinase 2; Male; Middle Aged; Mutation; Myeloproliferative Disorders; Polycythemia Vera; Pruritus; Retrospective Studies; Water

Topics: Biomarkers; Diagnosis, Differential; Erythropoietin; Hematopoiesis; Humans; Janus Kinase 2; Phenotype; Polycythemia; Polycythemia Vera; Practice Patterns, Physicians'; Severity of Illness Index; Symptom Assessment

Topics: Calreticulin; Disease Progression; DNA Mutational Analysis; Erythropoietin; Hematopoietic Stem Cell Transplantation; Humans; Immunologic Factors; Janus Kinase 2; Male; Middle Aged; Mutation; Polycythemia Vera; Primary Myelofibrosis; Transplantation, Homologous; Treatment Outcome

Topics: Adult; Aged; Aged, 80 and over; Biomarkers; Disease Progression; Erythropoietin; Female; Humans; Male; Middle Aged; Phenotype; Polycythemia Vera; Primary Myelofibrosis; Prognosis; Thrombocythemia, Essential; Young Adult

Topics: Adult; Aged; Biopsy; Bone Marrow; Erythropoietin; Female; Humans; Janus Kinase 2; Male; Middle Aged; Polycythemia Vera

We describe a novel ERBB1/EGFR somatic mutation (p. C329R; c.985 T > C) identified in a patient with JAK2

Topics: Amino Acid Sequence; Cell Differentiation; Cell Line, Tumor; Clone Cells; ErbB Receptors; Erythroblasts; Erythropoietin; Gene Expression; Humans; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Mutation; Polycythemia Vera; Primary Myelofibrosis; Protein Multimerization; Sequence Alignment; Sequence Homology, Amino Acid; Signal Transduction

Polycythemia Vera (PV) and essential thrombocythemia (ET) are myeloproliferative neoplasms respectively characterized by erythrocytosis and thrombocytosis; other disease features include leukocytosis, splenomegaly, thrombosis, bleeding, microcirculatory symptoms, pruritus, and risk of leukemic or fibrotic transformation.. PV is defined by a JAK2 mutation, whose absence, combined with normal or increased serum erythropoietin level, makes the diagnosis unlikely. JAK2, CALR, and MPL mutations are the mutually exclusive "driver" mutations in ET with respective incidences of 55%, 25%, and 3%; approximately 17% are triple-negative. However, the same molecular markers might also be present in prefibrotic myelofibrosis, whose morphological distinction from ET is prognostically relevant.. Median survivals are approximately 14 years for PV and 20 years for ET; the corresponding values for younger patients (age <60 years) are 24 and 33 years. Life-expectancy in ET is inferior to the control population. Driver mutational status has not been shown to affect survival in ET whereas the presence of JAK2/MPL mutations has been associated with higher risk of arterial thrombosis and that of MPL with higher risk of fibrotic progression. Risk factors for overall survival in both ET and PV include advanced age, leukocytosis and thrombosis. Leukemic transformation rates at 20 years are estimated at <10% for PV and 5% for ET; fibrotic transformation rates are slightly higher. Most recently, ASXL1, SRSF2, and IDH2 mutations have been associated with inferior overall, leukemia-free or fibrosis-free survival in PV; similarly adverse mutations in ET included SH2B3, SF3B1, U2AF1, TP53, IDH2, and EZH2.. Current risk stratification in PV and ET is designed to estimate the likelihood of recurrent thrombosis. Accordingly, PV includes two risk categories: high-risk (age >60 years or thrombosis history) and low-risk (absence of both risk factors). In ET, risk stratification includes four categories: very low risk (age ≤60 years, no thrombosis history, JAK2/MPL un-mutated), low risk (age ≤60 years, no thrombosis history, JAK2/MPL mutated), intermediate risk (age >60 years, no thrombosis history, JAK2/MPL un-mutated), and high risk (thrombosis history or age >60 years with JAK2/MPL mutation). In addition, presence of extreme thrombocytosis (platelets >1000 × 10(9)/L) might be associated with acquired von Willebrand syndrome (AvWS) and, therefore, risk of bleeding.. The main goal of therapy in PV and ET is to prevent thrombohemorrhagic complications. All patients with PV require phlebotomy to keep hematocrit below 45% and once-daily aspirin (81 mg). In addition, high-risk patients with PV require cytoreductive therapy. Very low risk ET patients might not require any form of therapy while low-risk patients require at least once-daily aspirin therapy. Cytoreductive therapy is also recommended for high-risk ET patients but it is not mandatory for intermediate-risk patients. First-line drug of choice for cytoreductive therapy, in both ET and PV, is hydroxyurea and second-line drugs of choice are interferon-α and busulfan. We currently do not recommend treatment with ruxolutinib or other JAK2 inhibitors in PV or ET, unless in the presence of severe and protracted pruritus or marked splenomegaly that is not responding to the aforementioned drugs. Screening for AvWS is recommended before administrating aspirin, in the presence of extreme thrombocytosis. Am. J. Hematol. 92:95-108, 2017. © 2016 Wiley Periodicals, Inc.

Topics: Aspirin; Calreticulin; Diagnosis, Differential; Erythropoietin; Humans; Hydroxyurea; Interferon-alpha; Janus Kinase 2; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mutation; Polycythemia Vera; Primary Myelofibrosis; Receptors, Thrombopoietin; Risk Assessment; Thrombocythemia, Essential; Thrombocytosis

The study mainly aimed at investigating possible correlations between peripheral blood counts, erythropoietin (EPO), JAK2 V617F mutation, and vascular complications prior to diagnosis of a population-based cohort of newly diagnosed patients with myeloproliferative neoplasms (MPN).. The study comprises 1105 patients with polycythemia vera (PV) and 1284 patients with essential thrombocythemia (ET) registered in the Swedish MPN Registry.. We conclude that vascular complications among newly diagnosed patients had affected more than one-third of our study population. Risk factors for vascular complications prior to diagnosis were lower hemoglobin in PV, and the presence of JAK2 V617F mutation, higher age, and leukocytosis in ET.

Topics: Age Factors; Aged; Aged, 80 and over; Biomarkers, Tumor; Erythropoietin; Female; Hemoglobins; Humans; Janus Kinase 2; Leukocytosis; Male; Middle Aged; Mutation; Polycythemia Vera; Prospective Studies; Registries; Risk Factors; Sweden; Thrombocythemia, Essential; Venous Thromboembolism

Increased fragility has been described in humans with polycythemia vera (PV). Herein, we describe an osteoporotic phenotype associated with decreased osteoblast activity in a mouse model of PV and another mouse of polycythemia and elevated circulating erythropoietin (EPO). Our results are important for patients with PV or those treated with recombinant EPO (rEPO).. PV and other myeloproliferative syndromes have been recently associated with an increased risk for fractures. However, the presence of osteoporosis in these patients has not been well documented. EPO, a hormone primarily known to stimulate erythropoiesis, has been shown recently to regulate bone homeostasis in mice. The aim of this study was to examine the bone phenotype of a mouse model of PV and compare it to that of animals with polycythemia caused by elevated circulating EPO.. Bone mass and remodeling were evaluated by micro-computed tomography and histomorphometry. The JAK2(V617F) knock-in mouse, a model of human PV, manifests polycythemia and low circulating EPO levels. Results from this mouse were compared to wild type (wt) controls and the tg6 transgenic mouse that shows polycythemia caused by increased constitutive expression of EPO.. Compared to wt, both JAK2(V617F) and tg6 mice had a decrease in trabecular bone mass. Tg6 mice showed an additional modest decrease in cortical thickness and cortical bone volume per tissue volume (P < 0.01) suggesting a more severe bone phenotype than JAK2(V617F). Decreased osteoblast numbers and bone formation along with normal osteoclast numbers and activity were found in both mice.. This study indicates that PV is associated with low bone mass and decreased osteoblast activity in mice. Our results support future studies of osteoporosis in affected humans. Polycythemia caused by chronically elevated circulating EPO also results in bone loss, and implications on patients treated with rEPO should be evaluated.

Topics: Animals; Bone Density; Bone Diseases, Metabolic; Bone Remodeling; Disease Models, Animal; Erythropoietin; Male; Mice, Knockout; Mice, Transgenic; Osteoblasts; Osteogenesis; Polycythemia; Polycythemia Vera; X-Ray Microtomography

A 5-y-old female ferret (Mustela putorius furo) was evaluated for diarrhea, anorexia, and lethargy for 1 wk. Only mild dehydration was detected on physical examination. CBC analysis revealed marked erythrocytosis with an unremarkable plasma biochemistry panel; follow-up CBC analyses revealed a consistent primary erythrocytosis. Whole-body radiographs and abdominal ultrasonography were unremarkable except for a small nephrolith in the right kidney and a small cyst in the left kidney. The plasma erythropoietin level was 17.0 mIU/mL and considered normal. In light of the diagnostic work-up and consistent erythrocytosis, a diagnosis of polycythemia vera (primary erythrocytosis) was made. The initial presentation of diarrhea resolved after treatment with oral metronidazole (20 mg/kg PO BID for 7 d). Treatment for the polycythemia consisted of a phlebotomy initially followed by chemotherapy with hydroxyurea (10 mg/kg PO BID). During the subsequent 12 mo, the hydroxyurea dose adjusted according to follow-up CBC results, and finding an optimal dosage regimen proved to be challenging. One year after the initial diagnosis, the ferret presented to an emergency clinic for acute and severe hemorrhagic diarrhea and died shortly thereafter. The postmortem diagnosis was acute venous infarction of the small and large intestine. To our knowledge, this report is the first to describe the diagnosis and long-term management of polycythemia vera in a ferret and the use of hydroxyurea for this purpose.

Topics: Animals; Antineoplastic Agents; Diarrhea; Erythropoietin; Fatal Outcome; Female; Ferrets; Hydroxyurea; Phlebotomy; Polycythemia Vera; Radiography; Ultrasonography

Topics: Adult; Biomarkers, Tumor; Bone Marrow; Calreticulin; Erythropoietin; Female; Fusion Proteins, bcr-abl; Hematocrit; Hemoglobins; Humans; Janus Kinase 2; Male; Middle Aged; Mutation; Myeloproliferative Disorders; Polycythemia Vera; Receptors, Thrombopoietin; Thrombocythemia, Essential; World Health Organization

Low serum erythropoietin (EPO) is a minor criterion of Polycythaemia Vera (PV) but its diagnostic usefulness relies on studies performed before the discovery of JAK2 V617F mutation. The objective of the present study was to evaluate the diagnostic accuracy of serum EPO and JAK2 V617F allele burden as markers of PV as well as the combination of different diagnostic criteria in 287 patients (99 with PV, 137 with Essential Thrombocythaemia and 51 with non-clonal erythrocytosis). Low EPO showed good diagnostic accuracy as a marker for PV, with the area under the curve (AUC) of the chemiluminescent-enhanced enzyme immunoassay (CEIA) being better than that of radioimmunoassay (RIA) (0·87 and 0·76 for CEIA and RIA, respectively). JAK2 V617F quantification displayed an excellent diagnostic accuracy, with an AUC of 0·95. A haematocrit >52% (males) or >48% (females) plus the presence of the JAK2 V617F mutation had a sensitivity and specificity of 79% and 97%, respectively. Adding low EPO or the JAK2 V617F allele burden did not improve the diagnostic accuracy for PV whereas the inclusion of both improved the sensitivity up to 83% and maintaining 96% specificity. Haematocrit and qualitative JAK2 V617F mutation allow a reliable diagnosis of PV. Incorporation of EPO and/or JAK2 V617F mutant load does not improve the diagnostic accuracy.

Topics: Alleles; Amino Acid Substitution; Area Under Curve; Biomarkers; Diagnosis, Differential; Erythropoietin; Female; Hematocrit; Hemoglobins; Humans; Janus Kinase 2; Leukocyte Count; Male; Mutation, Missense; Platelet Count; Point Mutation; Polycythemia; Polycythemia Vera; Reproducibility of Results; ROC Curve; Sensitivity and Specificity; Thrombocythemia, Essential

Polycythemia vera (PV) is a chronic myeloproliferative neoplasm associated with JAK2 mutations (V617F or exon 12) in almost all cases. The World Health Organization has defined the criteria for diagnosis, but it is still unclear which parameter (hemoglobin or hematocrit) is the most reliable for demonstrating increased red cell volume and for monitoring response to therapy; also, the role of bone marrow biopsy is being revisited. PV is associated with reduced survival because of cardiovascular complications and progression to post-PV myelofibrosis or leukemia. Criteria for risk-adapted treatment rely on the likelihood of thrombosis. Controlled trials have demonstrated that incidence of cardiovascular events is reduced by sustained control of hematocrit with phlebotomies (low-risk patients) and/or cytotoxic agents (high-risk patients) and antiplatelet therapy with aspirin. Hydroxyurea and interferon may be used as first-line treatments, whereas busulfan is reserved for patients that are refractory or resistant to first-line agents. However, there is no evidence that therapy improves survival, and the significance of reduction of JAK2 mutated allele burden produced by interferon is unknown. PV is also associated with a plethora of symptoms that are poorly controlled by conventional therapy. This article summarizes my approach to the management of PV in daily clinical practice.

Topics: Aged; DNA Mutational Analysis; Erythropoietin; Humans; Janus Kinase 2; Male; Middle Aged; Physician-Patient Relations; Polycythemia Vera; Precision Medicine

Polycythaemia vera (PV) is a clonal disorder of bone marrow stem cells characterised by erythrocytosis. Diagnosis of PV requires exclusion of secondary causes of polycythaemia. It has been held that an elevated erythropoietin (Epo) level strongly indicates secondary erythrocytosis and excludes PV diagnosis, to the extent that the reduced serum Epo level is currently listed as a minor criterion in the WHO classification scheme for PV. However, patients with PV who co-present with Budd-Chiari syndrome have been documented with elevated serum Epo levels. For these patients, identification of the Janus kinase 2 (JAK2) V617F point mutation along with the transient nature of the Epo elevation provides certainty of PV diagnosis, as illustrated by the proband. In this case report, the patient's positive response to cytoreductive therapy (hydroxyurea 500 mg daily) and phlebotomy (750 mL over three phlebotomies) further supports validity of PV diagnosis with elevated Epo. The patient remains on rivaroxaban (Xarelto) for treatment of her portal vein thrombosis.

Topics: Ascites; Biomarkers; Budd-Chiari Syndrome; Diagnosis, Differential; Erythropoietin; Female; Humans; Middle Aged; Polycythemia Vera

Erythropoiesis is a tightly regulated process which becomes decoupled from its normal differentiation program in patients with polycythemia vera (PV). Somatic mutations in JAK2 are commonly associated with this myeloid proliferative disorder. To gain insight into the molecular events that are required for abnormally developing erythroid cells to escape dependence on normal growth signals, we performed in vitro expansion of mature erythroblasts (ERY) from seven normal healthy donors and from seven polycythemic patients in the presence of IL3, EPO, SCF for 10, 11, or 13 days. Normal ERYs required exposure to the glucocorticoid dexamethasone (Dex) for expansion, while PV-derived ERYs expanded in the absence of dexamethasone. RNA expression profiling revealed enrichment of two known oncogenes, GPR56 and RAB4a, in PV-derived ERYs along with reduced expression levels of transcription factor TAL1 (ANOVA FDR < 0.05). While both normal and polycythemic-derived ERYs integrated signaling cascades for growth, they did so via different signaling pathways which are represented by their differential phospho-profiles. Our results show that normal ERYs displayed greater levels of phosphorylation of EGFR, PDGFRβ, TGFβ, and cKit, while PV-derived ERYs were characterized by increased phosphorylation of cytoplasmic kinases in the JAK/STAT, PI3K, and GATA1 pathways. Together these data suggest that PV erythroblast expansion and maturation may be maintained and enriched in the absence of dexamethasone through reduced TAL1 expression and by accessing additional signaling cascades. Members of this acquired repertoire may provide important insight into the pathogenesis of aberrant erythropoiesis in myeloproliferative neoplasms such as polycythemia vera.

Topics: Adult; Aged; Basic Helix-Loop-Helix Transcription Factors; Case-Control Studies; Cells, Cultured; Dexamethasone; Erythroblasts; Erythropoiesis; Erythropoietin; Female; Gene Expression Profiling; Gene Expression Regulation; Humans; Interleukin-3; Male; Middle Aged; Phosphoproteins; Polycythemia Vera; Proteomics; Proto-Oncogene Proteins; rab4 GTP-Binding Proteins; Receptors, G-Protein-Coupled; Signal Transduction; Stem Cell Factor; T-Cell Acute Lymphocytic Leukemia Protein 1

We prospectively evaluated the accuracy of the 2007 World Health Organization (WHO) criteria for diagnosing polycythemia vera (PV), especially in "early-stage" patients. A total of 28 of 30 patients were diagnosed as PV owing to an elevated Cr-51 red cell mass (RCM), JAK2 positivity, and at least 1 minor criterion. A total of 18 PV patients did not meet the WHO criterion for an increased hemoglobin value and 8 did not meet the WHO criterion for an increased hematocrit value. Bone marrow morphology was very valuable for diagnosis. Low serum erythropoietin (EPO) values were specific for PV, but normal EPO values were found at presentation (20%). We recommend revision of the WHO criteria, especially to distinguish early-stage PV from essential thrombocythemia. Major criteria remain JAK2 positivity and increased red cell volume, but Cr-51 RCM is mandatory for patients who do not meet the defined elevated hemoglobin or hematocrit value (>18.5 g/dL and 60% in men and >16.5 g/dL and 56% in women, respectively). Minor criteria remain bone marrow histology or a low serum EPO value. For patients with a normal EPO value, marrow examination is mandatory for diagnostic confirmation. Because the therapies for myeloproliferative disorders differ, our data have major clinical implications.

Topics: Bone Marrow; Erythrocyte Volume; Erythropoietin; Hematocrit; Hemoglobins; Humans; Janus Kinase 2; Polycythemia Vera; Practice Guidelines as Topic; Prospective Studies; Sensitivity and Specificity; World Health Organization

The molecular etiology of polycythemia vera (PV) remains incompletely understood. Patients harbor increased numbers of hematopoietic stem cells and display Epo-independent erythroid maturation. However, the molecular mechanism underlying Epo hypersensitivity and stem cell expansion is unclear. We have previously shown that the transcription factor nuclear factor erythroid-2 (NF-E2) is overexpressed in the majority of PV patients. Here we demonstrated that elevation of NF-E2 expression in healthy CD34(+) cells to levels observed in PV caused Epo-independent erythroid maturation and expansion of hematopoietic stem cell (HSC) and common myeloid progenitor (CMP) cell numbers. Silencing NF-E2 in PV patients reverted both aberrancies, demonstrating for the first time that NF-E2 overexpression is both required and sufficient for Epo independence and HSC/CMP expansion in PV.

Topics: Antigens, CD34; Cell Proliferation; Cells, Cultured; Erythropoiesis; Erythropoietin; Flow Cytometry; Gene Expression; Gene Silencing; Hematopoietic Stem Cells; Humans; Myeloid Progenitor Cells; NF-E2 Transcription Factor, p45 Subunit; Polycythemia Vera

My diagnostic approach in case of isolated erythrocytosis is based on the visit and the interview of patients, and on checking the causes of secondary erythrocytosis. If causes of secondary erythrocytosis are not evident and serum erythropoietin level is low-normal, I study JAK2 mutations. In the case of a patient with erythrocytosis and other signs of myeloproliferation, such as leukocytosis, thrombocytosis or splenomegaly, the diagnosis of polycythemia vera (PV) is likely, and I test serum erythropoietin and JAK2 mutations first. I stratify patients at diagnosis of PV according to age and history of thrombosis. I start hydroxyurea for patients who are at a high risk of thrombosis (that is, with one or two risk factors), while I continue only phlebotomy in other cases. All PV patients, if not contraindicated, receive aspirin. I follow up patients monthly until normalization of their blood cell counts or splenomegaly, and afterwards every 2 months with visit, cell blood count and blood smear evaluation. After diagnosis, I perform bone marrow biopsy only in the case of clinical signs of disease evolution.

Topics: Adult; Combined Modality Therapy; Erythropoietin; Female; Humans; Hydroxyurea; Janus Kinase 2; Male; Middle Aged; Mutation; Phlebotomy; Polycythemia Vera

Topics: Aged, 80 and over; Comorbidity; Drug Resistance; Erythropoiesis; Erythropoietin; Humans; Hydroxyurea; Janus Kinase 2; Male; Mutation, Missense; Phlebotomy; Point Mutation; Polycystic Kidney, Autosomal Dominant; Polycythemia; Polycythemia Vera; Ultrasonography

Topics: Aged; Algorithms; Alleles; Bone Marrow Examination; Diagnosis, Differential; Erythropoietin; Female; Hemoglobinometry; Humans; Hydroxyurea; Janus Kinase 2; Male; Middle Aged; Phlebotomy; Point Mutation; Polycythemia; Polycythemia Vera; Prognosis; Thromboembolism

A high percentage of patients with the myeloproliferative disorder polycythemia vera (PV) harbor a Val617→Phe activating mutation in the Janus kinase 2 (JAK2) gene, and both cell culture and mouse models have established a functional role for this mutation in the development of this disease. We describe the properties of MRLB-11055, a highly potent inhibitor of both the WT and V617F forms of JAK2, that has therapeutic efficacy in erythropoietin (EPO)-driven and JAK2V617F-driven mouse models of PV. In cultured cells, MRLB-11055 blocked proliferation and induced apoptosis in a manner consistent with JAK2 pathway inhibition. MRLB-11055 effectively prevented EPO-induced STAT5 activation in the peripheral blood of acutely dosed mice, and could prevent EPO-induced splenomegaly and erythrocytosis in chronically dosed mice. In a bone marrow reconstituted JAK2V617F-luciferase murine PV model, MRLB-11055 rapidly reduced the burden of JAK2V617F-expressing cells from both the spleen and the bone marrow. Using real-time in vivo imaging, we examined the kinetics of disease regression and resurgence, enabling the development of an intermittent dosing schedule that achieved significant reductions in both erythroid and myeloid populations with minimal impact on lymphoid cells. Our studies provide a rationale for the use of non-continuous treatment to provide optimal therapy for PV patients.

Topics: Animals; Blotting, Western; Cell Proliferation; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Enzyme Inhibitors; Erythropoietin; Flow Cytometry; Humans; Janus Kinase 2; Mice; Mice, Inbred C57BL; Polycythemia Vera; STAT5 Transcription Factor

TET2 mutations are found in polycythemia vera and it was initially reported that there is a greater TET2 mutational burden than JAK2(V617F) in polycythemia vera stem cells and that TET2 mutations precede JAK2(V617F). We quantified the proportion of TET2, JAK2(V617F) mutations and X-chromosome allelic usage in polycythemia vera cells, BFU-Es and in vitro expanded erythroid progenitors and found clonal reticulocytes, granulocytes, platelets and CD34(+) cells. We found that TET2 mutations may also follow rather than precede JAK2(V617F) as recently reported by others. Only a fraction of clonal early hematopoietic precursors and largely polyclonal T cells carry the TET2 mutation. We showed that in vitro the concomitant presence of JAK2(V617F) and TET2 mutations favors clonal polycythemia vera erythroid progenitors in contrast with non-TET2 mutated progenitors. We conclude that loss-of-function TET2 mutations are not the polycythemia vera initiating events and that the acquisition of TET2 somatic mutations may increase the aggressivity of the polycythemia vera clone.

Topics: Amino Acid Substitution; Blood Platelets; Cell Proliferation; Cells, Cultured; Chromosomes, Human, X; Clone Cells; Dioxygenases; DNA-Binding Proteins; Erythroid Precursor Cells; Erythropoietin; Female; Flow Cytometry; Granulocytes; Hematopoietic System; Humans; Janus Kinase 2; Mutation; Polycythemia Vera; Proto-Oncogene Proteins; Reticulocytes; T-Lymphocytes; Time Factors

Myeloproliferative neoplasms (MPNs) are associated with recurrent activating mutations of signaling proteins such as Janus kinase 2 (JAK2). However, the actual downstream signaling events and how these alter myeloid homeostasis are poorly understood. We developed an assay to measure basal levels of phosphorylated signaling intermediates by flow cytometry during myeloid differentiation in MPN patients. Our study provides the first systematic demonstration of specific signaling events and their comparison with disease phenotype and JAK2 mutation status. We demonstrate increased basal signaling in MPN patients, which occurs in both early and later stages of myeloid differentiation. In addition, the pattern of signaling is not correlated with JAK2 mutation status and signaling intensity is poorly correlated with mutant JAK2 allele burden. In contrast, signaling differences are detected between different MPN disease phenotypes. Finally, we demonstrate that signaling can be inhibited by a JAK2-selective small molecule, but that this inhibition is not JAK2 V617F specific, because MPN patients with mutant JAK2, wild-type JAK2, and control patients were inhibited to a similar degree. Our data suggest that, in addition to JAK2 mutations, other factors contribute significantly to the MPN phenotype, results that are relevant to both the pathogenesis and therapy of MPN.

Topics: Aged; Blotting, Western; Cell Line, Tumor; Erythropoietin; Female; Flow Cytometry; Genotype; Hematopoietic Stem Cells; Humans; Intracellular Space; Janus Kinase 2; Male; Middle Aged; Mutation; Myeloproliferative Disorders; Phenotype; Phosphoproteins; Phosphorylation; Polycythemia Vera; Primary Myelofibrosis; Pyrimidines; Signal Transduction; Sulfonamides; Thrombocythemia, Essential

A somatic point mutation (V617F) in the JAK2 tyrosine kinase was found in a majority of patients with polycythemia vera (PV), essential thrombocythemia, and primary myelofibrosis. However, contribution of the JAK2V617F mutation in these 3 clinically distinct myeloproliferative neoplasms (MPNs) remained unclear. To investigate the role of JAK2V617F in the pathogenesis of these MPNs, we generated an inducible Jak2V617F knock-in mouse, in which the expression of Jak2V617F is under control of the endogenous Jak2 promoter. Expression of heterozygous mouse Jak2V617F evoked all major features of human polycythemia vera (PV), which included marked increase in hemoglobin and hematocrit, increased red blood cells, leukocytosis, thrombocytosis, splenomegaly, reduced serum erythropoietin (Epo) levels and Epo-independent erythroid colonies. Homozygous Jak2V617F expression also resulted in a PV-like disease associated with significantly greater reticulocytosis, leukocytosis, neutrophilia and thrombocytosis, marked expansion of erythroid progenitors and Epo-independent erythroid colonies, larger spleen size, and accelerated bone marrow fibrosis compared with heterozygous Jak2V617F expression. Biochemical analyses revealed Jak2V617F gene dosage-dependent activation of Stat5, Akt, and Erk signaling pathways. Our conditional Jak2V617F knock-in mice provide an excellent model that can be used to further understand the molecular pathogenesis of MPNs and to identify additional genetic events that cooperate with Jak2V617F in different MPNs.

Topics: Amino Acid Substitution; Animals; Disease Models, Animal; Erythropoietin; Extracellular Signal-Regulated MAP Kinases; Gene Dosage; Gene Expression Regulation; Homozygote; Humans; Janus Kinase 2; Mice; Mice, Transgenic; Mutation, Missense; Polycythemia Vera; Promoter Regions, Genetic; Proto-Oncogene Proteins c-akt; Signal Transduction; STAT5 Transcription Factor

We report a Jak2V617F knockin mouse myeloproliferative neoplasm (MPN) model resembling human polycythemia vera (PV). The MPN is serially transplantable and we demonstrate that the hematopoietic stem cell (HSC) compartment has the unique capacity for disease initiation but does not have a significant selective competitive advantage over wild-type HSCs. In contrast, myeloid progenitor populations are expanded and skewed toward the erythroid lineage, but cannot transplant the disease. Treatment with a JAK2 kinase inhibitor ameliorated the MPN phenotype, but did not eliminate the disease-initiating population. These findings provide insights into the consequences of JAK2 activation on HSC differentiation and function and have the potential to inform therapeutic approaches to JAK2V617F-positive MPN.

Topics: Amino Acid Substitution; Animals; Antigens, CD; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Cell Count; Cell Differentiation; Disease Models, Animal; Erythroid Precursor Cells; Erythropoietin; Gene Expression; Gene Expression Profiling; Hematocrit; Hematopoietic Stem Cell Transplantation; Hematopoietic Stem Cells; Heterozygote; Humans; Janus Kinase 2; Megakaryocyte Progenitor Cells; Megakaryocyte-Erythroid Progenitor Cells; Mice; Mice, Inbred C57BL; Mice, Transgenic; Myeloid Progenitor Cells; Myeloproliferative Disorders; Polycythemia Vera; Protein Kinase Inhibitors; Pyrrolidines; Spleen; Sulfonamides; Survival Analysis

To understand the changes in gene expression in polycythemia vera (PV) progenitor cells and their relationship to JAK2V617F.. Messenger RNA isolated from CD34(+) cells from nine PV patients and normal controls was profiled using Affymetrix arrays. Gene expression change mediated by JAK2V617F was determined by profiling CD34(+) cells transduced with the kinase and by analysis of leukemia cell lines harboring JAK2V617F, treated with an inhibitor.. A PV expression signature was enriched for genes involved in hematopoietic development, inflammatory responses, and cell proliferation. By quantitative reverse transcription-PCR, 23 genes were consistently deregulated in all patient samples. Several of these genes such as WT1 and KLF4 were regulated by JAK2, whereas others such as NFIB and EVI1 seemed to be deregulated in PV by a JAK2-independent mechanism. Using cell line models and comparing gene expression profiles of cell lines and PV CD34(+) PV specimens, we have identified panels of 14 JAK2-dependent genes and 12 JAK2-independent genes. These two 14- and 12-gene sets could separate not only PV from normal CD34(+) specimens, but also other MPN such as essential thrombocytosis and primary myelofibrosis from their normal counterparts.. A subset of the aberrant gene expression in PV progenitor cells can be attributed to the action of the mutant kinase, but there remain a significant number of genes characteristic of the disease but deregulated by as yet unknown mechanisms. Genes deregulated in PV as a result of the action of JAK2V617F or independent of the kinase may represent other targets for therapy.

Topics: Amino Acid Substitution; Antigens, CD34; Bone Marrow Cells; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Cells, Cultured; Cluster Analysis; Erythroid Cells; Erythropoietin; Gene Expression Profiling; Humans; Janus Kinase 2; Kruppel-Like Factor 4; Mutation; Oligonucleotide Array Sequence Analysis; Polycythemia Vera; Reverse Transcriptase Polymerase Chain Reaction; Transfection

Somatic mutations in Janus kinase 2 (JAK2), including JAK2V617F, result in dysregulated JAK-signal transducer and activator transcription (STAT) signaling, which is implicated in myeloproliferative neoplasm (MPN) pathogenesis. CYT387 is an ATP-competitive small molecule that potently inhibits JAK1/JAK2 kinases (IC(50)=11 and 18 nM, respectively), with significantly less activity against other kinases, including JAK3 (IC(50)=155 nM). CYT387 inhibits growth of Ba/F3-JAK2V617F and human erythroleukemia (HEL) cells (IC(50) approximately 1500 nM) or Ba/F3-MPLW515L cells (IC(50)=200 nM), but has considerably less activity against BCR-ABL harboring K562 cells (IC=58 000 nM). Cell lines harboring mutated JAK2 alleles (CHRF-288-11 or Ba/F3-TEL-JAK2) were inhibited more potently than the corresponding pair harboring mutated JAK3 alleles (CMK or Ba/F3-TEL-JAK3), and STAT-5 phosphorylation was inhibited in HEL cells with an IC(50)=400 nM. Furthermore, CYT387 selectively suppressed the in vitro growth of erythroid colonies harboring JAK2V617F from polycythemia vera (PV) patients, an effect that was attenuated by exogenous erythropoietin. Overall, our data indicate that the JAK1/JAK2 selective inhibitor CYT387 has potential for efficacious treatment of MPN harboring mutated JAK2 and MPL alleles.

Topics: Alleles; Animals; Cell Line, Tumor; Cells, Cultured; Colony-Forming Units Assay; Drug Evaluation, Preclinical; Erythropoietin; Humans; Inhibitory Concentration 50; Janus Kinase 1; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Mice; Mutation; Oncogene Proteins, Fusion; Polycythemia Vera; Protein Kinase Inhibitors; Protein-Tyrosine Kinases; Receptors, Thrombopoietin; STAT Transcription Factors; Substrate Specificity

Recently several different JAK2 exon12 mutations have been identified in V617F negative polycythaemia vera (PV) or idiopathic erythrocytosis (IE) patients. The patients present with erythrocytosis, ligand-independent cell growth and low serum erythropoietin (EPO) levels. Within this group, a deletion of amino acids 542-543 (N542-E543del) of JAK2 is most prevalent. We have previously shown that in the presence of JAK2(V617F), suppressor of cytokine signalling 3 (SOCS3) is unable to negatively regulate EPO signalling and proliferation of V617F-expressing cells. Here we report a PV patient heterozygous for the somatic JAK2(N542-E543del) mutation and a previously unreported germline mutation within the SH2 domain of SOCS3 (F136L). Interestingly, the SOCS3(F136L) mutation was detected in a Japanese myeloproliferative disorder patient cohort at double the frequency of healthy controls. Cells expressing SOCS3(F136L) had markedly elevated EPO-induced proliferation and extended EPO-induced JAK2 phosphorylation. Additionally, compared to wild-type SOCS3, mutant SOCS3 had an extended half-life in the presence of JAK2 and JAK2(N542-E543del). Our findings suggest that this loss-of-function SOCS3 mutation may have contributed to disease onset by causing deregulated JAK2 signalling in the presence of a constitutively active JAK2(N542-E543del) mutant.

Topics: Adult; Base Sequence; Cell Proliferation; Cells, Cultured; Erythropoietin; Female; Germ-Line Mutation; Humans; Janus Kinase 2; Mutation; Phosphorylation; Polycythemia Vera; Suppressor of Cytokine Signaling 3 Protein; Suppressor of Cytokine Signaling Proteins

The Janus kinase 2 (JAK2) is important for embryonic primitive hematopoiesis. A gain-of-function JAK2 (JAK2(V617F)) mutation in human is pathogenetically linked to polycythemia vera (PV). In this study, we generated a zebrafish ortholog of human JAK2(V617F) (referred herewith jak2a(V581F)) by site-directed mutagenesis and examined its relevance as a model of human PV.. Zebrafish embryos at one-cell stage were injected with jak2a(V581F) mRNA (200pg/embryo). In some experiments, the embryos were treated with a specific JAK2 inhibitor, TG101209. The effects of jak2a stimulation on hematopoiesis, jak/stat signaling, and erythropoietin signaling were evaluated at 18-somites.. Injection with jak2a(V581F) mRNA significantly increased erythropoiesis, as enumerated by flow cytometry based on gfp(+) population in dissociated Tg(gata1:gfp) embryos. The response was reduced by stat5.1 morpholino coinjection (control: 4.37% +/- 0.08%; jak2a(V581F) injected: 5.71% +/- 0.07%, coinjecting jak2a(V581F) mRNA and stat5.1 morpholino: 4.66% +/- 0.13%; p<0.01). jak2a(V581F) mRNA also upregulated gata1 (1.83 +/- 0.08 fold; p=0.005), embryonic alpha-hemoglobin (1.61 +/- 0.12 fold; p=0.049), and beta-hemoglobin gene expression (1.65 +/- 0.13-fold; p=0.026) and increased stat5 phosphorylation. These responses were also ameliorated by stat5.1 morpholino coinjection or treatment with a specific JAK2 inhibitor, TG101209. jak2a(V581F) mRNA significantly reduced erythropoietin gene (0.24 +/- 0.03 fold; p=0.006) and protein expression (control: 0.633+/-0.11; jak2a(V581F) mRNA: 0.222+/-0.07 mIU/mL; p=0.019).. The zebrafish jak2a(V581F) model shared many features with human PV and might provide us with mechanistic insights of this disease.

Topics: Amino Acid Sequence; Amino Acid Substitution; Animals; Base Sequence; Blotting, Western; Disease Models, Animal; Embryo, Nonmammalian; Erythropoiesis; Erythropoietin; Gene Expression Regulation, Developmental; Humans; Janus Kinase 2; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Polycythemia Vera; Protein-Tyrosine Kinases; Pyrimidines; Reverse Transcriptase Polymerase Chain Reaction; Sequence Homology, Amino Acid; STAT5 Transcription Factor; Sulfonamides; Zebrafish; Zebrafish Proteins

Splenic enlargement (splenomegaly) develops in numerous disease states, although a specific pathogenic role for the spleen has rarely been described. In polycythemia vera (PV), an activating mutation in Janus kinase 2 (JAK2(V617)) induces splenomegaly and an increase in hematocrit. Splenectomy is sparingly performed in patients with PV, however, due to surgical complications. Thus, the role of the spleen in the pathogenesis of human PV remains unknown. We specifically tested the role of the spleen in the pathogenesis of PV by performing either sham (SH) or splenectomy (SPL) surgeries in a murine model of JAK2(V617F)-driven PV. Compared to SH-operated mice, which rapidly develop high hematocrits after JAK2(V617F) transplantation, SPL mice completely fail to develop this phenotype. Disease burden (JAK2(V617)) is equivalent in the bone marrow of SH and SPL mice, however, and both groups develop fibrosis and osteosclerosis. If SPL is performed after PV is established, hematocrit rapidly declines to normal even though myelofibrosis and osteosclerosis again develop independently in the bone marrow. In contrast, SPL only blunts hematocrit elevation in secondary, erythropoietin-induced polycythemia. We conclude that the spleen is required for an elevated hematocrit in murine, JAK2(V617F)-driven PV, and propose that this phenotype of PV may require a specific interaction between mutant cells and the spleen.

Topics: Alleles; Animals; Bone Marrow; Bone Marrow Transplantation; Erythropoietin; Hematocrit; Janus Kinase 2; Mice; Mice, Inbred BALB C; Mice, Inbred C57BL; Mutation; Phenotype; Polycythemia Vera; Spleen; Splenectomy

Polycythemia vera (PV) is characterized by erythrocytosis associated with the presence of the activating JAK2(V617F) mutation in a variable proportion of hematopoietic cells. JAK2(V617F) is detected in other myeloproliferative neoplasms, does not appear to be the PV-initiating event, and its specific role in deregulated erythropoiesis in PV is incompletely understood. We investigated the pathogenesis of PV to characterize abnormal proliferation and apoptosis responses and aberrant oncogenic pathway activation in primary PV erythroid precursors.. Peripheral blood CD34(+) cells isolated from PV patients and healthy controls were grown in liquid culture to expand a population of primary erythroblasts for experiments designed to analyze cellular proliferation, apoptosis, JAK2(V617F) mutation status, cytokine-dependent protein phosphorylation and gene expression profiling using Affymetrix microarrays.. The survival and proliferation of PV erythroblasts were growth factor-dependent under strict serum-free conditions requiring both erythropoietin (EPO) and stem cell factor. PV erythroblasts exhibited EPO hypersensitivity and enhanced cellular proliferation associated with increased EPO-mediated extracellular signal-regulated kinases 1 and 2 phosphorylation. EPO-induced AKT phosphorylation was observed in PV but not normal erythroblasts, an effect associated with apoptosis resistance in PV erythroblasts. Analysis of gene expression and oncogenic pathway activation signatures revealed increased RAS (p<0.01) and phosphoinositide-3 kinase (p<0.05) pathway activation in PV erythroblasts.. Deregulated erythropoiesis in PV involves EPO hypersensitivity and apoptosis resistance of erythroid precursor cells associated with abnormally increased activation of RAS-ERK and phosphoinositide-3 kinase-AKT pathways. These data suggest that investigation of the mechanisms of abnormal RAS and phosphoinositide-3 kinase pathway activation in erythroblasts may contribute to our understanding of the molecular pathogenesis of PV.

Topics: Antigens, CD34; Apoptosis; Cell Proliferation; Cells, Cultured; Culture Media, Serum-Free; Dose-Response Relationship, Drug; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Flavonoids; Gene Expression; Humans; Immunoblotting; Insulin-Like Growth Factor I; Janus Kinase 2; MAP Kinase Kinase Kinase 1; Mutation; Phosphatidylinositol 3-Kinases; Phosphorylation; Polycythemia Vera; Proto-Oncogene Proteins c-akt; ras Proteins; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Stem Cell Factor

Elevated haemoglobin and erythrocytosis are frequent causes for investigation.. A 50-year-old woman with elevated haemoglobin and erythrocytosis was referred to our hospital for investigation.. She had elevated serum erythropoietin and iron deficiency. Mutational status of the JAK2 gene was negative with respect to polycythemia vera, and no secondary reasons for polycythemia - including tumours - were revealed. A bone marrow biopsy and bone marrow smear showed a moderately increased number of lambda- positive monoclonal plasma cells, and small amounts of lambda light chains were detected in the urine. Serum electrophoreses showed no gammopathy. The reason for her elevated haemoglobin could be an erythropoietin-producing tumour or an idiopathic erythrocytosis based on a mutation in exon 12 of the JAK2 gene. Investigation of her plasma cells revealed a 10-fold increase in erythropoietin mRNA expression, indicating the reason for her elevated serum erythropoietin and haemoglobin values.

Topics: Bone Marrow Examination; Diagnosis, Differential; Erythropoietin; Female; Gene Expression; Hemoglobins; Humans; Iron Deficiencies; Middle Aged; Multiple Myeloma; Myeloproliferative Disorders; Polycythemia; Polycythemia Vera; RNA, Messenger

Topics: Diagnosis, Differential; Erythropoietin; Hemoglobins; Humans; Myeloproliferative Disorders; Plasma Cells; Polycythemia Vera; Risk Factors; RNA, Messenger

Topics: Diagnosis, Differential; Erythropoietin; Female; Hematocrit; Hemoglobins; Humans; Janus Kinase 2; Male; Mutation; Polycythemia Vera; Prognosis

Regulation of normal hematopoiesis by neuropeptide substance P (SP) and its amino terminal fragment, SP(1-4), has been reported. Endogenous erythroid colony (EEC) formation without erythropoietin is characteristic of polycythemia vera (PV), a chronic myeloproliferative disorder. We investigated the effect(s) of SP and SP(1-4) on EEC formation from PV BM mononuclear cells (BMMCs) and purified CD36+ erythroid progenitors. We found a potent in vitro inhibitory effect of SP and SP(1-4) on PV EEC formation for both BMMCs and CD36+ erythroid progenitors. The influence of SP and SP(1-4) on PV progenitor erythroid differentiation and cell viability was also investigated, and the impact of neurokinin receptors and G proteins in the inhibition were analyzed by quantitative PCR and with specific inhibitors. This progenitor inhibition was: (1) not mediated by accessory cells; (2) characterized by an increase in cell death and inhibition of the EPOindependent terminal erythroid differentiation; and (3) not mediated by the same neurokinin receptor. NK-1R and NK-2R antagonists completely abrogated the SP inhibitory effect but not SP(1-4)-induced inhibition. Furthermore, the truncated form of the NK-1R was predominant over the full-length mRNA and could mediated the SP inhibitory effect on PV CD36+ erythroid progenitors. Different G proteins were also implicated according to the erythroid differentiation stage of the PV cells. The observation of an inhibitory effect of SP and its related peptide, SP(1-4), on PV EEC formation at physiological concentrations (10-8M) suggests that neuropeptides represent a way to downregulate pathologic expansion of PV progenitors.

Topics: Cell Differentiation; Cell Proliferation; Cell Survival; Erythroid Precursor Cells; Erythropoietin; GTP-Binding Proteins; Humans; Peptide Fragments; Polycythemia Vera; Receptors, Tachykinin; RNA, Messenger; Substance P

HLA-G5 is secreted by erythroblasts in all hematopoietic organs, suggesting a role for this protein in erythropoiesis. To examine this, we analyzed whether HLA-G5 affects the proliferation of UT7/EPO and HEL erythroleukemia cells and characterized the mechanism by which HLA-G5 influences erythropoietin receptor (EPOR) signaling. We show that HLA-G5 inhibits the proliferation of UT7/EPO cells, the EPOR signaling of which is similar to that of normal erythroid progenitors. HLA-G5-mediated inhibition was associated with reduced phosphorylation of JAK2 kinase and that of the downstream signaling proteins STAT-5 and STAT-3. Involvement of JAK2 in erythroid cell proliferation has been highlighted by the role of JAK2 V617F mutation in polycythemia vera (PV), a myeloproliferative disorder characterized by erythroid lineage overproduction. We demonstrate that HLA-G5 downregulates EPOR constitutive signaling of JAK2 V617F-expressing HEL cells, leading to inhibition of cell proliferation through G1 cell cycle arrest. Combination of HLA-G5 with JAK inhibitor I further decreases HEL cell growth. Clinical relevance is provided by analysis of PV patients who carry JAK2 V617F mutation, showing that HLA-G5 inhibits the formation of erythropoietin-independent erythroid colonies. Such HLA-G5-mediated inhibition constitutes a new parameter to be considered in the design of future approaches aimed at treating JAK2 V617F-positive myeloproliferative disorders.

Topics: Cell Division; Cell Line, Tumor; Colony-Forming Units Assay; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; G1 Phase; Histocompatibility Antigens Class I; HLA Antigens; HLA-G Antigens; Humans; Janus Kinase 2; Leukemia, Erythroblastic, Acute; Microspheres; Mutation, Missense; Phosphorylation; Point Mutation; Polycythemia Vera; Protein Processing, Post-Translational; Receptors, Erythropoietin; Recombinant Fusion Proteins; STAT3 Transcription Factor; STAT5 Transcription Factor

CD177 (PRV1) expression is strongly related to polycythaemia vera (PV). Whilst studying CD177 expression in PV patients and controls, individuals with beta-thalassaemia minor were found to display an elevated expression of CD177. The study was expanded to include patients with thalassaemia intermedia, sickle cell/beta-thalassaemia and thalassaemia major. CD177 expression was increased in these thalassaemic groups and correlated with their erythropoietic activity, as assessed by the measurement of serum erythropoietin and soluble transferrin receptor levels. Within this context, elevated CD177 expression is not only a specific feature of PV but may be an indicator of increased erythropoietic activity in thalassaemia syndromes.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; beta-Thalassemia; Erythropoiesis; Erythropoietin; Gene Expression; Genotype; GPI-Linked Proteins; Humans; Isoantigens; Janus Kinase 2; Membrane Glycoproteins; Middle Aged; Mutation; Polycythemia Vera; Receptors, Cell Surface; Receptors, Transferrin; RNA, Messenger

The somatic activating janus kinase 2 mutation (JAK2)(V617F) is detectable in most patients with polycythemia vera (PV). Here we report that CP-690,550 exerts greater antiproliferative and pro-apoptotic activity against cells harboring JAK2(V617F) compared with JAK2(WT). CP-690,550 treatment of murine factor-dependent cell Patersen-erythropoietin receptor (FDCP-EpoR) cells harboring human wild-type or V617F JAK2 resulted in inhibition of cell proliferation with a 50% inhibitory concentration (IC(50)) of 2.1 microM and 0.25 microM, respectively. Moreover, CP-690,550 induced a significant pro-apoptotic effect on murine FDCP-EpoR cells carrying JAK2(V617F), whereas a lesser effect was observed for cells carrying wild-type JAK2. This activity was coupled with inhibition of phosphorylation of the key JAK2(V617F)-dependent downstream signaling effectors signal transducer and activator of transcription (STAT)3, STAT5, and v-akt murine thymoma viral oncogene homolog (AKT). Furthermore, CP-690,550 treatment of ex-vivo-expanded erythroid progenitors from JAK2(V617F)-positive PV patients resulted in specific, antiproliferative (IC(50) = 0.2 microM) and pro-apoptotic activity. In contrast, expanded progenitors from healthy controls were less sensitive to CP-690,550 in proliferation (IC(50) > 1.0 microM), and apoptosis assays. The antiproliferative effect on expanded patient progenitors was paralleled by a decrease in JAK2(V617F) mutant allele frequency, particularly in a patient homozygous for JAK2(V617F). Flow cytometric analysis of expanded PV progenitor cells treated with CP-690,550 suggests a possible transition towards a pattern of erythroid differentiation resembling expanded cells from normal healthy controls.

Topics: Animals; Apoptosis; Blotting, Western; Bone Marrow; Cell Cycle; Cell Nucleus; Cell Proliferation; Cells, Cultured; Erythroid Precursor Cells; Erythropoietin; Flow Cytometry; Humans; Immunoprecipitation; Janus Kinase 2; Membrane Potential, Mitochondrial; Mice; Mutation; Phosphorylation; Piperidines; Polycythemia Vera; Protein Kinase Inhibitors; Protein Transport; Pyrimidines; Pyrroles; Receptors, Erythropoietin; Signal Transduction; STAT3 Transcription Factor

The somatic JAK2(V617F) mutation is seen in most polycythemia vera (PV) patients; however, it is not clear if JAK2(V617F) is the PV-initiating mutation.. In order to examine this issue, we developed a novel real-time quantitative allele-specific PCR, in which allelic discrimination is enhanced by the synergistic effect of a mismatch in the -1 position, and a locked nucleic acid (LNA) nucleoside at the -2 position.. Determination of allelic frequencies was reproducible (SD = 1.5%) and sensitive--0.1% mutant allele detected in 40 ng of DNA. The JAK2(V617F) frequency in clonal granulocytes from 3 PV females was less than 50% (27.5 +/- 11) and in 7 females greater than 50% (75 +/- 10.5). We also found that wild-type JAK2 BFU-E colonies from PV patients can grow without erythropoietin. The identification of the primary genetic lesion resulting in PV is essential for the development of novel therapeutic strategies.. Our studies correlating the frequency of JAK2(V617F) mutant allele and clonality, as well as the presence of homozygous wild-type JAK2 erythropoietin-independent erythroid colonies, provide compelling evidence that the JAK2(V617F) is not the PV-initiating mutation. This supports a model wherein the JAK2(V617F) mutation arises as a secondary genetic event. Furthermore, our results indicate that an undefined molecular lesion, preceding JAK2(V617F), is responsible for clonal hematopoiesis in PV. We conclude that development of therapeutic strategies that target the JAK2(V617F) clonal cells may not be sufficient for eradication of PV.

Topics: Clone Cells; DNA Probes; Erythropoietin; Female; Gene Frequency; Humans; Janus Kinase 2; Mutation, Missense; Polycythemia Vera; Polymerase Chain Reaction

Three consecutive polycythemia vera (PV) patients were analyzed before and during pegylated-interferon (rIFNalpha) treatment for the following markers: (1) granulocyte and CD34(+) cell clonality, (2) Jak2V617F expression, (3) PRV-1 mRNA overexpression, and (4) Epo-independent colony (EEC) growth. Before rIFNalpha therapy, all patients displayed clonal hematopoiesis, 100% Jak2V617F expression as well as PRV-1 overexpression, and EEC growth. After rIFNalpha treatment, all three patients demonstrated polyclonal hematopoiesis. Nonetheless, Jak2V617F expression, PRV-1 overexpression, and EEC-growth remained detectable, albeit at lower levels. We conclude that reemergence of polyclonal hematopoiesis after rIFNalpha treatment may be achieved in a substantial proportion of patients. However, this does not constitute elimination of the PV clone. These data demonstrate the usefulness of novel markers in monitoring minimal residual disease and caution against discontinuation of rIFNalpha treatment after hematologic remission. Long-term follow-up of large patient cohorts is required to determine whether rIFNalpha treatment can cause complete molecular remissions in PV.

Topics: Adult; Amino Acid Substitution; Antigens, CD34; Biomarkers; Cell Proliferation; Clone Cells; Erythropoietin; Female; Gene Expression; GPI-Linked Proteins; Granulocytes; Hematopoiesis; Humans; Immunologic Factors; Interferon-alpha; Isoantigens; Janus Kinase 2; Membrane Glycoproteins; Middle Aged; Mutant Proteins; Polycythemia Vera; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; T-Lymphocytes

The predictive values of common biological criteria for the diagnosis of polycythemia vera were studied in a cohort of patients with high hematocrit. We found JAK2V617F and erythropoietin assays were the most relevant first tests. Classification of patients according to their JAK2V617F status and erythropoietin levels facilitated the choice of further diagnostic investigations.

Topics: Amino Acid Substitution; Cohort Studies; Diagnosis, Differential; Erythropoietin; Female; Gene Frequency; Hematocrit; Humans; Janus Kinase 2; Male; Mutant Proteins; Mutation, Missense; Point Mutation; Polycythemia; Polycythemia Vera; Predictive Value of Tests

A G>T transversion in a tyrosine kinase JAK2 (V617F) was reported in over 80% of patients with polycythemia vera (PV). Current evidence suggests that JAK2(V617F) somatic mutation is involved in the pathogenesis of PV, as it confers erythropoietin-independent proliferation to erythroid progenitor cells. However, several unanswered questions regarding the essential role of JAK2(V617F) arose as 1) it is not a dominant mutation, 2) it is not PV-specific as it is found in several myeloproliferative disorders, and 3) some ( approximately 20%) PV patients lack the JAK2(V617F) mutation. We investigated the relative frequency of JAK2(V617F) in in vitro-expanded PV progenitors.. In vitro expansion of erythroid progenitors from mononuclear cells was optimized. Frequency of JAK2(V617F) allele was measured by using allele-specific real-time polymerase chain reaction. Clonality was performed using established procedure.. In vitro expansion of PV erythroid progenitors and differentiated dendritic cells resulted in a decrease of the frequency of JAK2(V617F) allele compared with granulocytes or CD235(+) erythroid progenitors. Clonality analysis demonstrated that although granulocytes of these PV patients were clonal, expanded erythroid cells were polyclonal. However, in vitro-expanded PV erythroid progenitors still had approximately a twofold increased proliferative capacity in comparison with erythroid progenitors from healthy individuals. Erythropoietin favors the cells without JAK2(V617F) allele. Dendritic cells in one out of three patients remained clonal.. JAK2(V617F) mutation does not provide a proliferative/survival advantage to the PV clone during in vitro expansion. These data suggest that the JAK2(V617F) mutation plays an important role in the biology of PV, yet it may not be the PV-initiating event.

Topics: Alleles; Base Sequence; Cell Proliferation; Cells, Cultured; Child; Culture Media; DNA Primers; Erythroid Precursor Cells; Erythropoietin; Female; Flow Cytometry; Humans; Janus Kinase 2; Polycythemia Vera; Polymerase Chain Reaction

The JAK2 617V>F mutation is frequent in polycythemia vera (PV) and essential thrombocythemia (ET). Using quantitative polymerase chain reaction (PCR), we found that high levels of JAK2 617V>F in PV correlate with increased granulocytes and high levels of hemoglobin and endogenous erythroid colony formation. We detected normal progenitors and those that were heterozygous or homozygous for the mutation by genotyping ET and PV clonal immature and committed progenitors. In PV patients, we distinguished homozygous profiles with normal, heterozygous, and homozygous progenitors from heterozygous profiles with only heterozygous and normal progenitors. PV patients with a heterozygous profile had more mutated, committed progenitors than did other PV and ET patients, suggesting a selective amplification of mutated cells in the early phases of hematopoiesis. We demonstrated that mutated erythroid progenitors were more sensitive to erythropoietin than normal progenitors, and that most homozygous erythroid progenitors were erythropoietin independent. Moreover, we observed a greater in vitro erythroid amplification and a selective advantage in vivo for mutated cells in late stages of hematopoiesis. These results suggest that, for PV, erythrocytosis can occur through two mechanisms: terminal erythroid amplification triggered by JAK2 617V>F homozygosity, and a 2-step process including the upstream amplification of heterozygous cells that may involve additional molecular events.

Topics: Aged; Amino Acid Substitution; Cells, Cultured; Erythroid Precursor Cells; Erythropoietin; Female; Granulocytes; Hematopoiesis; Heterozygote; Homozygote; Humans; Janus Kinase 2; Male; Mutation, Missense; Polycythemia Vera; Thrombocythemia, Essential

Adaptive mechanisms to hematocrit levels of 0.9 in our erythropoietin-overexpressing mice (tg6) include increased plasma nitric oxide levels and erythrocyte flexibility. Doubled reticulocyte counts in tg6 suggest an increased erythrocyte turnover. Here we show that compared with wild-type (wt) animals, erythrocyte lifespan in tg6 is 70% lower in tg6 mice. Transgenic mice have a younger erythrocyte population as indicated by higher intercellular water and potassium content, higher flexibility, decreased density, increased surface to volume ratio, and decreased osmotic fragility. Interestingly, despite being younger, the tg6 erythrocyte population also harbors characteristics of accelerated aging such as an increased band 4.1a to 4.1b ratio, signs of oxidative stress, or decreased surface CD47 and sialic acids. In tg6, in vivo tracking of PKH26-labeled erythrocytes revealed dramatically increased erythrocyte incorporation by their liver macrophages. In vitro experiments showed that tg6 macrophages are more active than wt macrophages and that tg6 erythrocytes are more attractive for macrophages than wt ones. In conclusion, in tg6 mice erythrocyte aging is accelerated, which results, together with an increased number and activity of their macrophages, in enhanced erythrocyte clearance. Our data points toward a new mechanism down-regulating red cell mass in excessive erythrocytosis in mice.

Topics: Animals; Disease Models, Animal; Erythrocytes; Erythropoietin; Flow Cytometry; Gene Expression Regulation; Humans; Macrophages; Mice; Mice, Transgenic; Phagocytosis; Polycythemia Vera; Potassium; Reticulocytes

Erythropoiesis is critically dependent on erythropoietin (EPO), a glycoprotein hormone that is regulated by hypoxia-inducible factor (HIF). Hepatocytes are the primary source of extrarenal EPO in the adult and express HIF-1 and HIF-2, whose roles in the hypoxic induction of EPO remain controversial. In order to define the role of HIF-1 and HIF-2 in the regulation of hepatic EPO expression, we have generated mice with conditional inactivation of Hif-1alpha and/or Hif-2alpha (Epas1) in hepatocytes. We have previously shown that inactivation of the von Hippel-Lindau tumor suppressor pVHL, which targets both HIFs for proteasomal degradation, results in increased hepatic Epo production and polycythemia independent of Hif-1alpha. Here we show that conditional inactivation of Hif-2alpha in pVHL-deficient mice suppressed hepatic Epo and the development of polycythemia. Furthermore, we found that physiological Epo expression in infant livers required Hif-2alpha but not Hif-1alpha and that the hypoxic induction of liver Epo in anemic adults was Hif-2alpha dependent. Since other Hif target genes such phosphoglycerate kinase 1 (Pgk) were Hif-1alpha dependent, we provide genetic evidence that HIF-1 and HIF-2 have distinct roles in the regulation of hypoxia-inducible genes and that EPO is preferentially regulated by HIF-2 in the liver.

Topics: Animals; Basic Helix-Loop-Helix Transcription Factors; Disease Models, Animal; Erythropoietin; Gene Expression Regulation; Hepatocytes; Liver; Mice; Mice, Knockout; Polycythemia Vera; von Hippel-Lindau Disease; Von Hippel-Lindau Tumor Suppressor Protein

Recent discoveries in the molecular pathogenesis of both polycythemia vera (PV) and congenital polycythemia (CP) underline the prospect of a genetic diagnosis in these disorders. At the forefront are the mutually exclusive exon 14 (JAK2V617F) and exon 12 JAK2 mutations that are almost always present in PV but not in polycythemias of other causes. Similarly, the molecular basis of CP is being unraveled, and several cases are now associated with germline mutations involving the von Hippel-Lindau (VHL) or erythropoietin receptor (EPOR) genes. Therefore, current diagnostic work-up for acquired polycythemia should start with peripheral blood JAK2 mutation screening, whereas VHL and/or EPOR mutations should be considered when CP is suspected. In all instances, serum erythropoietin measurement provides complementary information; the serum erythropoietin level is expected to be decreased in PV regardless of JAK2 mutation status, increased in VHL mutation-associated CP, and decreased or normal in the presence of an EPOR mutation.

Topics: Algorithms; Carrier Proteins; Cytoskeletal Proteins; Erythropoietin; Germ-Line Mutation; Humans; Janus Kinase 2; Molecular Chaperones; Mutation; Oxygen; Polycythemia; Polycythemia Vera; Receptors, Erythropoietin; Transcriptional Activation; Translocation, Genetic

In the present study, we report that media conditioned by polycythemia vera (PV) CD3+ cells promote BFU-E and CFU-Mk colony formation by both cord blood and PV peripheral blood CD34+ cells in the absence of exogenous cytokines and promoting megakaryocyte proplatelet formation. CD3+ cells constitutively produce elevated levels of IL-11, while stimulation with the addition of phytohemagglutinin (PHA) increased GM-CSF levels in most of the patients with PV. Anti-IL-11-neutralizing antibody partially inhibited the formation of BFU-E and CFU-Mk colonies promoted by PV CD3+ cell-conditioned media. Although IL-11 is not produced by normal T cells, real-time PCR and flow cytometric analysis showed that IL-11 was upregulated in the CD3+ cells of most PV patients as compared to normal CD3+ cells. In addition, a greater percentage of BFU-E colonies formed by PV CD34+ cells in the presence of PV CD3+ cell-conditioned media alone were JAK2V617F-positive as compared with that induced by EPO. We conclude that dysregulated production of soluble growth factor(s), including IL-11 and GM-CSF by PV T cells, contributes to the in vitro formation of erythroid colonies in the absence of exogenous cytokines by PV CD34+ cells and likely plays a role in sustaining hematopoiesis in PV.

Topics: Cells, Cultured; Colony-Forming Units Assay; Culture Media, Conditioned; Erythroid Precursor Cells; Erythropoietin; Fetal Blood; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoiesis; Hematopoietic Cell Growth Factors; Humans; Interleukin-11; Janus Kinase 2; Megakaryocytes; Mutation, Missense; Phytohemagglutinins; Polycythemia Vera; T-Lymphocytes

Idiopathic erythrocytosis (IE) is characterized by erythrocytosis in the absence of megakaryocytic or granulocytic hyperplasia, and is associated with variable serum erythropoietin (Epo) levels. Most patients with IE lack the JAK2 V617F mutation that occurs in the majority of polycythemia vera patients. Four novel JAK2 mutant alleles have recently been described in patients with V617F-negative myeloproliferative disorders presenting with erythrocytosis. The aims of this study were to assess the prevalence of JAK2 exon 12 mutations in IE patients, and to determine the associated clinicopathological features.. A cohort of 58 IE patients with low to normal serum Epo levels and no known causative mutation were identified from 181 individuals diagnosed with IE. Patients' DNA samples were screened for the presence of a JAK2 exon 12 mutation by allele-specific polymerase chain reaction and sequencing. Bone marrow trephines were examined for morphological abnormalities and the erythroid activity assessed immunohistochemically.. Eight mutation-positive cases were identified, including one with a previously undescribed mutant JAK2 exon 12 allele and another with biallelic involvement. The hematologic features of mutation-positive and mutation-negative patients were similar, although Epo-hypersensitive erythroid progenitors occurred exclusively in patients with an exon 12 mutation (p=0.0002; n=15). Patients' bone marrows were moderately hypercellular, as the result of erythroid hyperplasia, and several had mild megakaryocyte atypia.. JAK2 exon 12 mutations were detected in 27% of patients with low serum Epo levels, all of whom had Epo-independent erythroid progenitors. Consequently, IE patients presenting with either of these features should be tested for the presence of a JAK2 mutation.

Topics: Alleles; Amino Acid Substitution; Bone Marrow; Cohort Studies; Erythroid Precursor Cells; Erythropoietin; Exons; Humans; Ireland; Janus Kinase 2; Polycythemia; Polycythemia Vera; Prevalence; United Kingdom

We describe an unusual presentation of toe ulceration in a 39-year-old otherwise healthy man. The left fifth toe was painful for several months with ulceration at the tip. The other toes appeared cyanotic and discolored. Foot pulses were normal. Laboratory testing revealed the platelet count at 1208 thousand/microliter, with hemoglobin 19.2g/dl and hematocrit 57%. The erythropoietin level was found to be markedly decreased at 1 mU/ml. The patient was given the diagnosis of polycythemia vera with iron deficiency. Phlebotomy was performed and aspirin and cytoreductive therapy with hydroxyurea was prescribed with resolution of the toe ulceration.

Topics: Adult; Aspirin; Erythropoietin; Fibrinolytic Agents; Foot Ulcer; Hematocrit; Humans; Hydroxyurea; Male; Phlebotomy; Platelet Count; Polycythemia Vera; Thrombocytosis; Toes

The biologic hallmark of polycythemia vera (PV) is the formation of endogenous erythroid colonies (EECs) with an erythropoietin-independent differentiation. Recently, it has been shown that an activating mutation of JAK2 (V617F) was at the origin of PV. In this work, we studied whether the STAT5/Bcl-xL pathway could be responsible for EEC formation. A constitutively active form of STAT5 was transduced into human erythroid progenitors and induced an erythropoietin-independent terminal differentiation and EEC formation. Furthermore, Bcl-xL overexpression in erythroid progenitors was also able to induce erythroid colonies despite the absence of erythropoietin. Conversely, siRNA-mediated STAT5 and Bcl-xL knock-down in human erythroid progenitors inhibited colony-forming unit-erythroid (CFU-E) formation in the presence of Epo. Altogether, these results demonstrate that a sustained level of the sole Bcl-xL is capable of giving rise to Epo-independent erythroid colony formation and suggest that, in PV patients, JAK2(V617F) may induce EEC via the STAT5/Bcl-xL pathway.

Topics: bcl-X Protein; Cell Differentiation; Colony-Forming Units Assay; Erythropoiesis; Erythropoietin; Gene Deletion; Humans; Polycythemia Vera; RNA, Small Interfering; STAT5 Transcription Factor; Stem Cells

It is widely accepted that an increased serum endogenous erythropoietin (Epo) level in a patient presenting with an elevated red cell mass makes a diagnosis of clonal polycythemia vera (PV) extremely unlikely. However, until the recent description of the constitutively activating V617F point mutation of the Janus 2 tyrosine kinase (JAK2)--a high-frequency molecular marker that is extremely specific for clonal chronic myeloproliferative disorders--distinction of PV from secondary erythrocytosis or other conditions has often been difficult. The purpose of this study was to use JAK2 V617F analysis to re-evaluate the validity of elevated Epo levels as a PV-exclusion criterion in patients with hepatic vein thrombosis [Budd-Chiari syndrome (BCS)].. We reviewed clinical data from 30 patients at our institution who presented with erythrocytosis and BCS. We isolated myeloid cells from fresh or archival bone marrow samples from four patients with BCS and an elevated serum Epo level, and analyzed them for the presence of the JAK2 V617F mutation.. All four samples were positive for JAK2 V617F, confirming the presence of a clonal hematopoietic disorder consistent with PV.. In the presence of BCS, elevated serum Epo levels do not exclude the diagnosis of PV.

Topics: Adult; Biomarkers; Budd-Chiari Syndrome; DNA Mutational Analysis; Erythropoietin; Female; Humans; Janus Kinase 2; Male; Middle Aged; Mutation, Missense; Polycythemia; Polycythemia Vera; Protein-Tyrosine Kinases; Proto-Oncogene Proteins

The determination of serum/plasma erythropoietin (EPO) concentration has gained widespread use in the diagnosis of polycythaemia vera (PV). A reduced EPO concentration in a newly diagnosed essential thrombocythaemia (ET) seems to be a risk factor for thromboembolic events. In this study plasma EPO concentration was determined before and after initiated hydroxyurea (HU) therapy, 14 patients with PV or ET were included. After 1 month on HU therapy 11 of 14 patients had increased their EPO concentration compared with prior to medication. The plasma EPO was increased in all, except one patient, after 4 months HU therapy. If EPO is incorporated in the diagnostic or prognostic procedures it should be determined before myelosuppressive therapy is initiated.

Topics: Aged; Aged, 80 and over; Blood Platelets; Erythropoietin; Female; Humans; Hydroxyurea; Male; Middle Aged; Platelet Count; Polycythemia Vera; Thrombocythemia, Essential; Time Factors

The JAK2(V617F) mutation is present in almost all patients with polycythemia vera (PV), large proportions of patients with essential thrombocythemia and idiopathic myelofibrosis, and less frequently in atypical myeloproliferative disorders (MPD). We show that transplantation of JAK2(V617F)-transduced bone marrow into BALB/c mice induces MPD reminiscent of human PV, characterized by erythrocytosis, granulocytosis, extramedullary hematopoiesis, and bone marrow fibrosis, but not thrombocytosis. Fluorescence-activated cell sorting of bone marrow and spleen showed proportional expansion of common myeloid progenitors, granulocyte-monocyte and megakaryocyte-erythrocyte progenitors. Megakaryocyte and late erythroid progenitors were dramatically increased, with only modest expansion of early erythroid progenitors. Erythropoietin (Epo) receptor expression was reduced on early, but normal on late erythroblasts. Serum levels of Epo and granulocyte colony-stimulating factor, but not granulocyte macrophage colony-stimulating factor, were reduced, whereas tumor necrosis factor-alpha was increased, possibly exerting a negative effect on JAK2(V617F)-negative hematopoiesis. These data suggest that erythrocytosis and granulocytosis in JAK2(V617F) mice are the net result of a complex interplay between cell intrinsic and extrinsic factors. There were no thromboembolic events and no animals succumbed to their disease, implicating additional factors in the manifestation of human disease. The disease was not transplantable and prolonged observation showed normalization of blood counts in most JAK2(V617F) mice, suggesting that the mutation may not confer self-renewal capacity.

Topics: Animals; Bone Marrow; Bone Marrow Cells; Bone Marrow Transplantation; Cell Line; Clone Cells; Erythropoietin; Fibrosis; Granulocyte Colony-Stimulating Factor; Hematopoiesis, Extramedullary; Hematopoietic Stem Cells; Humans; Janus Kinase 2; Mice; Mice, Inbred BALB C; Mutation, Missense; Myeloproliferative Disorders; Polycythemia; Polycythemia Vera; Receptors, Erythropoietin; Spleen; Time Factors; Transfection; Tumor Necrosis Factor-alpha

Polycythemia vera (PV) is an acquired myeloproliferative disorder with primary expansion of the red cell mass leading to an increased risk of thrombosis and less frequently to myelofibrosis and secondary acute leukemia. Standard therapies include cytoreduction with either phlebotomy or chemotherapeutic agents and antithrombotic drugs. Because long-term exposure to cytotoxic chemotherapy may increase the risk of acute transformation, new therapeutic options are needed. Tipifarnib is a nonpeptidomimetic inhibitor of farnesyl transferase that was developed as a potential inhibitor of RAS signaling. In the present study we report that tipifarnib used at pharmacologically achievable concentrations strongly inhibits the erythroid burst-forming unit (BFU-E) autonomous growth that characterizes patients with PV. Moreover, at low tipifarnib concentrations (0.15 muM), the inhibitory effect was preferentially observed in PV BFU-E progenitors and not in normal BFU-E progenitors and was not rescued by erythropoietin (EPO). Thus tipifarnib may specifically target PV stem cells and may be of clinical interest in the treatment of patients with PV.

Topics: Alkyl and Aryl Transferases; Case-Control Studies; Cell Proliferation; Cells, Cultured; Dose-Response Relationship, Drug; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Farnesyltranstransferase; Humans; Polycythemia Vera; Quinolones

Myeloproliferative disorders are clonal haematopoietic stem cell malignancies characterized by independency or hypersensitivity of haematopoietic progenitors to numerous cytokines. The molecular basis of most myeloproliferative disorders is unknown. On the basis of the model of chronic myeloid leukaemia, it is expected that a constitutive tyrosine kinase activity could be at the origin of these diseases. Polycythaemia vera is an acquired myeloproliferative disorder, characterized by the presence of polycythaemia diversely associated with thrombocytosis, leukocytosis and splenomegaly. Polycythaemia vera progenitors are hypersensitive to erythropoietin and other cytokines. Here, we describe a clonal and recurrent mutation in the JH2 pseudo-kinase domain of the Janus kinase 2 (JAK2) gene in most (> 80%) polycythaemia vera patients. The mutation, a valine-to-phenylalanine substitution at amino acid position 617, leads to constitutive tyrosine phosphorylation activity that promotes cytokine hypersensitivity and induces erythrocytosis in a mouse model. As this mutation is also found in other myeloproliferative disorders, this unique mutation will permit a new molecular classification of these disorders and novel therapeutical approaches.

Topics: Animals; Base Sequence; Bone Marrow Transplantation; Cell Line, Tumor; Cell Proliferation; Erythropoietin; Exons; Humans; Interleukin-3; Janus Kinase 2; Mice; Mutation; Polycythemia; Polycythemia Vera; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; RNA, Small Interfering; Signal Transduction

Controversy continues to persist about the role of histopathology regarding diagnosis of polycythemia vera (PV). For this reason, a clinicopathological study was performed on 334 patients presenting with a sustained borderline to marked erythrocytosis (hemoglobin >17 g/dl in men and >15 g/dl in women). The aim was to elucidate the discriminating impact of bone marrow biopsy examinations in an independent fashion from laboratory parameters. According to morphological findings based on a semiquantitative evaluation of standardized features, cellularity, megakaryocytes (quantity, size, pleomorphous aspect, clustering, nuclear lobulation), eosinophils, cellular debris, perivascular plasmacytosis and iron-laden macrophages exerted a distinctive value. Comparison with clinical data and follow-up revealed that in only 13 patients (4%), histopathology failed to differentiate clearly between PV (208 patients) and secondary polycythemias (113 patients). In conclusion, certain sets of morphological parameters allow a distinction between autonomous and reactive polycythemias and therefore enhance significantly diagnostic validity.

Topics: Adult; Aged; Bone Marrow; Bone Marrow Cells; Bone Marrow Examination; Cell Count; Cell Size; Eosinophils; Erythropoietin; Female; Hematocrit; Humans; Iron; Macrophages; Male; Megakaryocytes; Middle Aged; Neoplasms; Organelles; Plasma Cells; Pneumonia; Polycythemia; Polycythemia Vera; Primary Myelofibrosis; Reticulin; Retrospective Studies; Sensitivity and Specificity; Smoking

Polycythemia vera (PV) is a human clonal hematological disorder. The molecular etiology of the disease has not been identified. PV hematopoietic progenitor cells exhibit hypersensitivity to growth factors and cytokines, suggesting possible abnormalities in protein-tyrosine kinases and phosphatases. By sequencing the entire coding regions of cDNAs of candidate enzymes, we identified a G:C--> T:A point mutation of the JAK2 tyrosine kinase in 20 of 24 PV blood samples but none in 12 normal samples. The mutation has varying degrees of heterozygosity and is apparently acquired. It changes conserved Val(617) to Phe in the pseudokinase domain of JAK2 that is known to have an inhibitory role. The mutant JAK2 has enhanced kinase activity, and when overexpressed together with the erythropoietin receptor in cells, it caused hyperactivation of erythropoietin-induced cell signaling. This gain-of-function mutation of JAK may explain the hypersensitivity of PV progenitor cells to growth factors and cytokines. Our study thus defines a molecular defect of PV.

Topics: Amino Acid Sequence; Animals; Base Sequence; Cytokines; DNA Mutational Analysis; DNA, Complementary; Erythropoietin; HeLa Cells; Heterozygote; Humans; Immunoblotting; Janus Kinase 2; Molecular Sequence Data; Mutation; Phenylalanine; Point Mutation; Polycythemia Vera; Polymerase Chain Reaction; Protein Structure, Tertiary; Protein-Tyrosine Kinases; Proto-Oncogene Proteins; Receptors, Erythropoietin; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Signal Transduction; Transfection; Tyrosine; Valine

The prevailing attitudes regarding diagnostic and therapeutic procedures in patients with polycythaemia vera (PV) among Swedish haematologists were surveyed by way of a mailed questionnaire in August 2002. Among diagnostic procedures frequent use is reported for arterial O(2) saturation, spleen size determination, bone marrow histology, serum erythropoietin, serum cobalamins and leukocyte alkaline phosphatase score, while direct determination of the red blood cell mass is used infrequently (seldom or never by 82%). Among therapeutic modalities hydroxyurea and phlebotomy alone were most frequently used. The (32)P therapy was used at least sometimes by 57% of the physicians, and more widely in the university clinics. Anagrelide and alfa-interferon was used in a minority of patients only. The use of prophylactic acetylsalicylic acid was very variable. The majority of the physicians had an aim for their phlebotomy treatment at a level of 0.45 or less, but 21% used a level of 0.46-0.49 and 8% a level of 0.55-0.60 (in younger patients). The platelet level, at which myelosuppressive therapy was initiated, also varied, from 400 x 10(9)/L to >1500 x 10(9)/L. It can be concluded that in practical clinical work in Sweden the diagnosis of PV is established by frequent use of serum erythropoietin, bone marrow examination and spleen size determination. The use of different therapeutic modalities is very variable. Many physicians carry out their phlebotomy treatment with less intensity compared with national and international recommendations.

Topics: Antineoplastic Agents; Attitude of Health Personnel; Erythrocyte Volume; Erythropoietin; Health Care Surveys; Humans; Interferon-alpha; Phlebotomy; Phosphorus Radioisotopes; Physicians; Polycythemia Vera; Surveys and Questionnaires; Sweden

Polycythaemia vera is a chronic myeloproliferative disease with no single diagnostic marker. The Polycythaemia Vera Study Group used a combination of major and minor diagnostic criteria. In the presence of newer diagnostic tools, low serum erythropoietin level has been proposed as an important diagnostic criterion for polycythaemia vera, whereas an elevated erythropoietin value contradicts the diagnosis. We report a case of polycythaemia vera with extreme haemoconcentration in which the serum level of erythropoietin was above the upper reference limit.. A 53-year-old man with erythrocytosis (packed cell volume 0.68, hemoglobin 23.6 g/dl), thrombocytosis, leukocytosis and no underlying disease had a serum level of erythropoietin of 29.0 U/l (normal range 1.3-21.5 U/l).. The patient was treated with venesection. After one month the haemoglobin level had decreased to 15.7 g/dl and packed cell volume to 0.48. The symptoms of polycythaemia disappeared and the serum level of erythropoietin declined to low normal values (2.2 U/l after five weeks and 5.4 U/l after 1.5 year). We propose that local hypoxia in the kidneys might be responsible for the elevated erythropoietin value at the time of diagnosis. The present case shows that high erythropoietin values may not necessarily exclude the diagnosis of polycythaemia vera.

Topics: Attention; Bone Marrow; Erythrocyte Count; Erythropoietin; Headache; Humans; Male; Middle Aged; Phlebotomy; Polycythemia Vera

Endogenous erythroid colonies (EECs), formed in vitro without erythropoietin (EPo) or other exogenous cytokines, are characteristic of Polycythemia vera (PV). Our aim was to identify specific conditions of culture of bone marrow (BM) progenitors allowing formation of erythroid colonies without EPo.. BM mononuclear cells (BMMCs), purified CD34+ cells, and purified CD36+ erythroid progenitors were cultured in serum-free media without and with cytokines: EPo, stem cell factor (SCF), and interleukin (IL)-11 and IL-8, produced by BM stromal cells and found elevated in PV.. EECs were formed in PV cultures of either BMMCs or CD34+ cells, which include cytokine-secreting cells, but not in cultures of purified CD36+ erythroid progenitors (EP). Despite expression of V617F JAK-2, no constitutive activation of JAK-2, Stat-5, or Erk-1/2 was detected in erythroblasts issued from PV CD36+ progenitors. However, when SCF was provided, PV CD36+ progenitors formed erythroid colonies without EPo. The ability to form erythroid colonies with SCF alone was conferred to BM progenitors of healthy donors and secondary erythrocytosis by exposure to IL-11 and IL-8. Both IL-11 and IL-8 enhanced formation of erythroid colonies in response to EPo and interfered with the activation of Erk-1/2 and Stat-5 induced, respectively, by SCF and EPo in erythroblasts. Anti-IL-11 antibody inhibited formation of erythroid colonies by PV BMMCs and CD34+ cells.. The data indicate that PV erythroid progenitors remain cytokine-dependent and that normal BM progenitors exposed to IL-11 and IL-8 can acquire the ability to form erythroid colonies without EPo.

Topics: Bone Marrow Cells; Cell Culture Techniques; Coculture Techniques; Cytokines; Erythroid Precursor Cells; Erythropoietin; Hematopoietic Stem Cells; Humans; Interleukin-11; Interleukin-8; Polycythemia Vera; Signal Transduction

Topics: Erythrocyte Count; Erythropoietin; Humans; Polycythemia Vera

Myeloproliferative disorders (MPDs) are heterogeneous diseases that occur at the level of a multipotent hematopoietic stem cell. They are characterized by increased blood cell production related to cytokine hypersensitivity and virtually normal cell maturation. The molecular pathogenesis of the MPDs has been poorly understood, except for chronic myeloid leukemia (CML), where the Bcr-Abl fusion protein exhibits constitutive kinase activity. Since some rare MPDs are also related to a dysregulated kinase activity, a similar mechanism was thought to be likely responsible for the more frequent MPDs. We investigated the mechanisms of endogenous erythroid colony formation (EEC) by polycythemia vera (PV) erythroid progenitor cells and found that EEC formation was abolished by a pharmacological inhibitor of JAK2 as well as an siRNA against JAK2. JAK2 sequencing revealed a unique mutation in the JH2 domain leading to a V617F substitution in more than 80% of the PV samples. This mutation in the pseudokinase autoinhibitory domain results in constitutive kinase activity and induces cytokine hypersensitivity or independence of factor-dependent cell lines. Retroviral transduction of the mutant JAK2 into murine HSC leads to the development of an MPD with polycythemia. The same mutation was found in about 50% of patients with idiopathic myelofibrosis (IMF) and 30% of patients with essential thrombocythemia (ET). Using different approaches, four other teams have obtained similar results. The identification of the JAK2 mutation represents a major advance in our understanding of the molecular pathogenesis of MPDs that will likely permit a new classification of these diseases and the development of novel therapeutic approaches.

Topics: Amino Acid Substitution; Cell Division; Cytokines; Erythrocytes; Erythropoietin; Humans; Janus Kinase 2; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Mutation; Myeloproliferative Disorders; Polycythemia Vera; Receptors, Erythropoietin; Signal Transduction

The plasma erythropoietin (EPO) concentration is subnormal in the vast majority of all patients at the time of diagnosis of polycythaemia vera (PV) and in 33-50% of patients with essential thrombocythaemia (ET). At equivalent haemoglobin concentrations patients with PV and a substantial number of ET patients have subnormal EPO concentrations compared with those of healthy subjects. A possible explanation could be altered haemoglobin (Hb) oxygen affinity. Plasma EPO concentration and the oxygen pressure at 50% Hb saturation (p50) were measured in 29 patients with PV, 23 patients with ET and 34 healthy controls. There was no significant correlation between p50 and plasma EPO concentration. However, the mean p50 for PV patients exceeded the mean for healthy controls (p = 0.004). Furthermore, the mean p50 for ET patients significantly (p = 0.012) exceeded the mean for controls but there was no significant difference in p50 between patients with PV and ET. It could be hypothesized that the lower oxygen affinity to Hb in PV and ET patients partly explains the decreased EPO production.

Topics: Adult; Aged; Aged, 80 and over; Erythropoietin; Female; Hemoglobins; Humans; Male; Middle Aged; Oxygen; Partial Pressure; Polycythemia Vera; Thrombocytosis

The in vitro growth of erythroid colonies in the absence of erythropoietin, known as endogenous erythroid colonies (EEC) forms part of the diagnostic criteria for polycythaemia vera (PV). The availability of EEC culture in routine laboratory setting is limited as culture methods are technically demanding, difficult to standardize, expensive and laborious. In this study, we assessed the performance characteristics of a simplified method using ammonium chloride red cell lysis followed by culture on commercially available, batch-tested, methylcellulose media. Seventy-six patients were included; four were secondarily excluded on the basis of culture failure. Of the 14 patients with PV, 13 (93%) were positive for EEC on at least one occasion: 90% (nine of 10) of bone marrow and 67% (six of nine) of peripheral blood specimens were positive. All 30 patients with secondary polycythaemia (n = 12) or apparent polycythaemia (n = 18) were negative for EEC. The incidence of EEC in idiopathic erythrocytosis was 40% (eight of 28); 50% (five of 10) in those who met one of the minor criteria for PV and 17% (three of 18) in those who did not. We conclude that our EEC assay yield results comparable with that of more elaborate methods.

Topics: Ammonium Chloride; Bone Marrow; Cell Culture Techniques; Colony-Forming Units Assay; Erythroid Precursor Cells; Erythropoietin; Female; Hemolysis; Humans; Male; Polycythemia Vera; Predictive Value of Tests; Sensitivity and Specificity

Polycythemia vera (PV) is a myeloproliferative disorder arising in a multipotent hematopoietic stem cell. The pathogenesis of PV remains poorly understood; however, the biologic hallmark of this disease is the presence of erythropoietin (Epo)-independent colony formation (endogenous erythroid colony [EEC]) and cytokine hypersensitivity. We have developed a simple liquid culture from CD34+ cells to study PV erythroid differentiation. PV erythroid differentiation was characterized in this culture system by two types of abnormalities: 1) an increased proliferation of progenitors in response to cytokines, associated with strict cytokine dependency for preventing apoptosis; and 2) Epo-independent terminal erythroid differentiation in the presence of stem cell factor and interleukin-3 as evidenced by the acquisition of glycophorin A. The level of Epo-independent terminal differentiation correlates in PV patients with the number of EEC. Epo-independent terminal differentiation as well as normal Epo-induced differentiation were repressed by inhibitors of JAK2 (AG490), PI3K (LY294002), and the Src family kinases (PP2). In contrast, an inhibitor of the ERK/MAP kinase pathway (PD98059) had no effect on Epo-independent terminal differentiation. These signaling abnormalities were not mediated by a decreased expression or activity of the membrane tyrosine phosphatase CD45, which dephosphorylates JAK2 and Src family kinases. This study demonstrates that early steps of PV erythroid differentiation are strictly cytokine dependent. In contrast, late erythroid differentiation is an Epo-independent phenomenon that is mediated by signaling pathways identical to those in Epo-induced differentiation.

Topics: Antigens, CD34; Cell Differentiation; Cell Division; Erythroid Precursor Cells; Erythropoietin; Humans; Leukocyte Common Antigens; Polycythemia Vera; Signal Transduction

Polycythemia vera (PV) is a malignant disorder of hemaopoietic stem cells which is characterized by clonal hyperproliferation and a low rate of apoptosis. This study was to assess endogenous erythroid colony (EEC) formation in the bone marrow of PV patients and determine its clinical significance.. The bone marrow mononuclear cells of 26 patients with PV, 2 patients with secondary erythrocytosis (SE), and 19 normal controls were cultured by Marsh's method for EEC evaluation, and the clinical significance was evaluated.. EECs appeared in 25 patients with PV but not in 2 patients with SE and 19 normal controls. The number of EECs and the EEC ratio [EEC/erythropoietin (EPO)-dependent colony forming unit-erythroid (CFU-E)] in PV patients positively correlated with hemoglobin (Hb) levels. Their EEC number did not correlate with white blood cell (WBC) counts, platelet (PLT) counts, or leukocyte alkaline phosphatase (LAP) scores. Their EEC did not correlate with serum EPO levels. Fifteen patients with PV were treated with hydroxyurea (Hu) and/or interferon-alpha (IFN-alpha). Their EEC ratio before treatment positively correlated with the treatment time required for complete remission (CR) and negatively correlated with the time before relapse. The EEC numbers of 7 PV patients treated with Hu/IFN-alpha decreased after the blood cell counts dropped to normal levels. There was a positive correlation between the EEC ratio and the incidence of attacks of vascular thrombosis in PV patients. The numbers of apoptosised bone marrow mononuclear cells in PV patients were lower than those in normal controls. The EEC numbers of PV patients negatively correlated with the rate of apoptosis of bone marrow mononuclear cells.. EEC formation is characteristic in PV patients. EEC number in PV patients positively correlates with Hb levels, the time required for CR, and the incidence of attacks of vascular thrombosis. EEC number negatively correlates with the time before relapse. Bone marrow suppressive treatment might decrease EEC number. Thus, EEC number is a sensitive and specific parameter reflecting the abnormal hematopoietic clone burden induced by polycythemia vera. EEC number is an important diagnostic parameter for PV patients.

Topics: Adult; Aged; Apoptosis; Colony-Forming Units Assay; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Female; Humans; Male; Middle Aged; Polycythemia Vera; Thrombosis

Topics: Biomarkers; Diagnosis, Differential; Enzyme-Linked Immunosorbent Assay; Erythrocyte Count; Erythropoietin; Humans; Polycythemia; Polycythemia Vera; World Health Organization

Topics: Erythrocyte Indices; Erythropoietin; Humans; Polycythemia Vera; Sensitivity and Specificity; Thrombocythemia, Essential

Essential thrombocythemia (ET) and polycythemia vera (PV) are clonal myeloproliferative disorders that are often difficult to distinguish from other causes of elevated blood cell counts. Assays that could reliably detect clonal hematopoiesis would therefore be extremely valuable for diagnosis. We previously reported 3 X-chromosome transcription-based clonality assays (TCAs) involving the G6PD, IDS, and MPP1 genes, which together were informative in about 65% of female subjects. To increase our ability to detect clonality, we developed simple TCA for detecting the transcripts of 2 additional X-chromosome genes: Bruton tyrosine kinase (BTK) and 4-and-a-half LIM domain 1 (FHL1). The combination of TCA established the presence or absence of clonal hematopoiesis in about 90% of female subjects. We show that both genes are subject to X-chromosome inactivation and are polymorphic in all major US ethnic groups. The 5 TCAs were used to examine clonality in 46 female patients along with assays for erythropoietin-independent erythroid colonies (EECs) and granulocyte PRV-1 mRNA levels to discriminate polycythemias and thrombocytoses. Of these, all 19 patients with familial polycythemia or thrombocytosis had polyclonal hematopoiesis, whereas 22 of 26 patients with clinical evidence of myeloproliferative disorder and 1 patient with clinically obscure polycythemia were clonal. Interestingly, interferon alpha therapy in 2 patients with PV was associated with reversion of clonal to polyclonal hematopoiesis. EECs were observed in 14 of 14 patients with PV and 4 of 12 with ET, and increased granulocyte PRV-1 mRNA levels were found in 9 of 13 patients with PV and 2 of 12 with ET. Thus, these novel clonality assays are useful in the diagnosis and follow-up of polycythemic conditions and disorders with increased platelet levels.

Topics: Adolescent; Adult; Agammaglobulinaemia Tyrosine Kinase; Aged; Child; Chromosomes, Human, X; Clone Cells; Colony-Forming Units Assay; Diagnosis, Differential; Dosage Compensation, Genetic; Erythroid Precursor Cells; Erythropoietin; Exons; Female; Genetic Markers; GPI-Linked Proteins; Granulocytes; Hematopoiesis; Homeodomain Proteins; Humans; Interferon-alpha; Isoantigens; Membrane Glycoproteins; Middle Aged; Polycythemia; Polycythemia Vera; Polymorphism, Genetic; Polymorphism, Single-Stranded Conformational; Protein-Tyrosine Kinases; Receptors, Cell Surface; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Thrombocytosis

Familial clustering of malignancies provides a unique opportunity to identify molecular causes of cancer. Polycythemia vera (PV) is a myeloproliferative disorder due to an unknown somatic stem cell defect that leads to clonal myeloid hyperproliferation. We studied 6 families with PV. The familial predisposition to PV appears to follow an autosomal dominant inheritance pattern with incomplete penetrance. All examined females informative for a transcriptional clonality assay had clonal hematopoiesis. We excluded linkage between PV and a number of previously proposed candidate disease loci (c-mpl, EPOR, 20q, 13q, 5q, 9p). Therefore, mutations at these loci are unlikely primary causes of familial PV. The finding of erythropoietin-independent erythroid progenitors in healthy family members indicated the presence of the PV stem cell clone in their hematopoiesis. This finding, together with clonal hematopoiesis in the affected individuals, supports the hypothesis of multiple genetic defects involved in the early pathogenesis of PV.

Topics: Adult; Aged; Aged, 80 and over; Child, Preschool; Clone Cells; Erythroid Precursor Cells; Erythropoietin; Family Health; Female; Genetic Linkage; Hematopoiesis; Humans; Inheritance Patterns; Male; Middle Aged; Mutation; Neoplastic Stem Cells; Pedigree; Polycythemia Vera; Time Factors

Topics: Adult; Darbepoetin alfa; Epoetin Alfa; Erythropoiesis; Erythropoietin; Female; Hematinics; Humans; Middle Aged; Osteomyelitis; Polycythemia Vera; Primary Myelofibrosis; Recombinant Proteins; Stem Cell Transplantation; Treatment Failure; Treatment Outcome

To investigate the growth of endogenous erythroid colony (EEC) in polycythemia vera (PV) patients and its clinical significance.. Bone marrow mononuclear cells of 26 PV patients, 2 secondary erythrocytosis (SE) and 19 normal controls were cultured by Marsh's method for EEC.. 1. EEC was present in 25/26 (96.2%) PV patients and was not found in 2 SE patients and 19 normal controls. 2. The number of EEC and the ratio of EEC/Epo-dependent CFU-E (EEC ratio) were positively correlated with the hemoglobin (Hb) levels (r = 0.608, P = 0.01) in PV patients, but did not correlate with white blood cell (WBC) counts, platelet counts and neutrophil alkaline phosphatase scores. 3. EEC did not correlate with PV patients' serum Epo levels (r = 0.518, P = 0.125). 4. Fifteen PV patients were treated with hydroxyurea and/or interferon-alpha. Their EEC ratio before treatment was correlated positively with the time required for complete remission (CR) (r = 0.651, P = 0.009) and negatively with the time before relapsing (r = -0.529, P < 0.02). 5. EECs of 7 PV patients treated with HU/IFN were decreased after their blood cell counts normalization. 6. There was a positive correlation between the EEC ratio and the attacks of vascular thrombosis (r = 0.524, P = 0.01). (7) The apoptosis of bone marrow mononuclear cells of PV patients was less than that of normal controls. PV patients' EEC was negatively correlated with the apoptosis of their bone marrow mononuclear cells (r = -0.192, P < 0.045).. EEC is peculiarly present in PV patients, and is a sensitive parameter in reflecting the abnormal hematopoietic clone burden and in diagnosing and monitoring the disease.

Topics: Adult; Aged; Apoptosis; Bone Marrow Cells; Erythroid Precursor Cells; Erythropoietin; Female; Humans; Male; Middle Aged; Polycythemia Vera

Most frequently, an elevated packed cell volume (PCV) value arouses the suspicion of a polycythaemic state. The aim of the present work was to assess a few readily available variables which could help the clinician to differentiate between polycythaemia vera (PV) and apparent polycythaemia (AP). During a 5-year period, 31 consecutive newly diagnosed patients with PV were identified, and during a 4-year period 38 consecutive subjects were considered to be afflicted with AP. In each subject: (i) the red cell mass (RCM) and plasma volume were measured, (ii) the spleen size was assessed using gamma-camera imaging, and (iii) the plasma erythropoietin (EPO) concentration was determined. The diagnosis of PV was based upon recently proposed criteria. By definition, all PV patients had absolute erythrocytosis, i.e. the RCM was greater than 25% above the mean normal predicted value for the individual. There was no statistical difference between the plasma volumes for PV and AP patients. However, the mean measured/predicted plasma volume for subjects with AP was significantly lower than the mean for PV. The means for spleen scan areas (posterior and left lateral projections) for AP patients were identical to the mean reference values for our laboratory. As compared to AP, in PV the spleen scan areas were significantly increased, and the lateral spleen scan area was significantly larger than the posterior area. It was also shown that, in contrast to AP, both spleen scan areas were significantly larger in male than in female PV patients. All PV patients had plasma EPO concentrations below the lower reference limit, and in 68% of the patients undetectable EPO concentrations were present. Most AP patients (84%) had EPO values within the reference range; 8% had slightly subnormal, but not undetectable, plasma EPO levels.

Topics: Adult; Aged; Aged, 80 and over; Bone Marrow; Comorbidity; Diagnosis, Differential; Erythrocyte Volume; Erythropoietin; Female; Humans; Leukocyte Count; Male; Middle Aged; Platelet Count; Polycythemia; Polycythemia Vera; Predictive Value of Tests; Prospective Studies; Radionuclide Imaging; Spleen; Splenomegaly

Angiogenesis has been implicated in the growth, dissemination and metastasis of solid tumours. Several recent studies have shown increased bone marrow vasculature in acute and chronic leukaemia as well as in myelofibrosis and myelodysplastic syndromes. Increased serum and/or cellular levels of vascular endothelial growth factor (VEGF) as the most potent and specific angiogenic factor were reported in acute and chronic leukaemia and multiple myeloma and also predict poor prognosis in these haematological malignancies. Little is known about angiogenesis and VEGF levels in polycythemia. Thus, the serum levels of VEGF (pg/ml) and erythropoietin (U/l) were determined using ultra-sensitive ELISA assays in 16 polycythemia patients (10 with polycythemia vera (PV) and 6 with secondary polycythemia) and correlated with their clinical and laboratory features. The serum levels of VEGF were significantly higher in 90% of PV cases and 60% of secondary polycythemia compared to healthy controls. The median VEGF levels (pg/ml) were 622 (range, 272-4760), 306 (range, 111-408) and 143 (range, 91-282) in PV, secondary polycythemia and healthy controls, respectively. Since serum VEGF reflects both plasma VEGF and platelet released VEGF, the concentration of VEGF per platelet (VEGF/PLT) as pg per 10(6) platelet was used as a more standardised measure. Splenomegaly emerged as the main factor associated with a marked increase in serum VEGF/PLT levels. On the other hand, the serum erythropoietin levels (U/l) were significantly reduced in PV (range, 1.2-14.3) raised in secondary polycythemia (range, 26-104) compared with normal controls (range, 9.7-31.1), P<0.01. In conclusion, the present study shows increased VEGF levels in most polycythemia patients, and splenomegaly is associated with a profound increase in VEGF serum levels in these patients. Also, patients with PV have significantly reduced serum levels of erythropoietin compared with secondary polycythemia. Also, serum VEGF/PLT was higher in PV patients treated with phlebotomy alone rather than oral chemotherapy, suggesting a possible advantage for chemotherapy over phlebotomy alone in suppressing the disease progression. Further studies with large number of polycythemia cases are warranted to explore the role of these cytokines in the pathogenesis, diagnosis and/or therapy of polycythemia.

Topics: Adult; Aged; Aged, 80 and over; Bone Marrow; Case-Control Studies; Endothelial Growth Factors; Enzyme-Linked Immunosorbent Assay; Erythropoietin; Female; Humans; Intercellular Signaling Peptides and Proteins; Lymphokines; Male; Middle Aged; Phlebotomy; Platelet Count; Polycythemia Vera; Spleen; Splenomegaly; Vascular Endothelial Growth Factor A; Vascular Endothelial Growth Factors

The aim was to assess how the diagnosis of polycythaemia vera (PV) was established and to study to which extent the classic Polycythemia Vera Study Group (PVSG) criteria and the revised criteria for the diagnosis of PV as proposed by Pearson and Messinezy in 1996 (PM criteria) were fulfilled when the diagnosis of PV was made.. A questionnaire was sent to physicians in charge of haematological patients at the departments of medicine in 12 hospitals in the Health and Medical Care in the Västra Götaland Region (VGR), Sweden, with a population of 1.5 million inhabitants; they were asked to provide reports as regards all patients with an unequivocal diagnosis of PV during a 5-year period from January 1994 to December 1998.. Full reports were obtained from six hospitals, which serve about 1.1 million inhabitants. The results from a total of 129 PV patients, 62 from two university hospitals (UHs) and 67 from four county hospitals (CHs), were the subject of the present analysis. Results. It was shown that measurement of the red cell mass (RCM) had been carried out in 61 of 62 (98%) patients in UHs compared with 24 of 67 (36%) patients at CHs (P < 0.01). By using ultrasound imaging and/or scintigraphy the spleen size had been determined in 55 of 62 (89%) patients at UHs and in 24 of 67 (36%) patients at CHs (P < 0.01). At the UHs, arterial oxygen saturation had been measured in 32 of 62 (52%) patients; the corresponding figure for the CH patients was 22 of 67 (33%). Plasma or serum erythropoietin (EPO) concentrations had been measured in 44 of 62 (71%) of PV patients at UHs and in 31 of 67 (46%) of patients at CHs; in all these cases the EPO concentrations were subnormal or not measurable.. At the UHs only 37% of the PV patients fulfilled the PVSG criteria for the diagnosis of PV whereas the corresponding figure for CHs was 4% (P < 0.01). The adherence to PM criteria was, however, 71% at UHs compared with 16% at CHs (P < 0.01).

Topics: Adult; Erythrocyte Volume; Erythropoietin; Female; Humans; Male; Polycythemia Vera; Surveys and Questionnaires; Sweden

Plasma thrombopoietin (TPO) was measured, by immunoenzymometric assay, in 39 patients with polycythaemia vera (PV), 33 patients with essential thrombocythaemia (ET) and 10 healthy volunteers. The mean TPO concentration was significantly higher in ET patients than in PV patients (p=0.04) and normals (p<0.001). The 6 untreated ET patients had a significantly lower mean TPO concentration compared to the 27 ET patients who were on myelosuppressive regimens (p=0.01). The mean plasma TPO for the 5 PV patients treated with phlebotomy only did not differ significantly from the corresponding mean for the 34 PV patients treated with myelosuppressive agents. Concomitantly, plasma EPO was measured in 25 of the PV patients and in 30 of the ET patients by an immunoradiometric assay with normal reference interval in adults 3.7-16 IU/L. In the 14 PV patients with EPO <3.7 IU/L mean plasma TPO did not differ significantly from the mean for the 11 PV patients with EPO >or=3.7 IU/L; neither of these two groups had plasma TPO concentrations significantly different from the mean for the control subjects. The 7 ET patients with subnormal plasma EPO had significantly lower mean plasma TPO compared to the ET patients with normal and high plasma EPO concentrations (p=0.03 and p=0.02, respectively). Also, the 16 ET patients with normal plasma EPO had significantly lower plasma TPO compared to the 8 patients with high plasma EPO (p=0.04). The mean plasma TPO for each of these three groups of ET patients was significantly higher than the corresponding mean for the controls (p<0.001 for each group). The results of the present study indicate that a relationship between plasma EPO and TPO concentrations may exist and that myelosuppressive treatment affects the TPO concentration in ET but not in PV patients.

Topics: Adult; Aged; Aged, 80 and over; Case-Control Studies; Erythropoietin; Female; Humans; Male; Middle Aged; Platelet Count; Polycythemia Vera; Thrombocythemia, Essential; Thrombopoietin

In 80 patients with polycythaemia vera (PV) a total of 108 venous blood samples were obtained and analysed for EDTA-plasma erythropoietin (EPO) concentration. At the time of study 21 of the PV patients were newly diagnosed and had prior to blood sampling neither received phlebotomy treatment nor therapy with myelosuppressive agents; these subjects had a mean plasma EPO concentration of 0.5+/-0.9 IU/L. Thirty-seven patients treated with phlebotomy only had a mean plasma EPO concentration of 2.5+/-2.9 IU/L. The mean plasma EPO concentrations for 26 patients treated with hydroxyurea, 13 patients treated with radiophosphorous and 11 patients given a combination of myelosuppressive agents were 8.9+/-8.0, 10.9+/-12.6 and 7.2+/-7.4 IU/L, respectively. Untreated patients and patients on phlebotomy only had significantly lower values for plasma EPO than patients on therapy with myelosuppressive drugs. This finding persisted also after a correction for differences in haemoglobin levels had been introduced. Thereby, the present results would suggest a difference in the EPO feedback system in untreated and phlebotomised PV patients compared to PV patients treated with myelosuppressive agents.

Topics: Adult; Aged; Busulfan; Erythropoietin; Female; Humans; Hydroxyurea; Immunosuppressive Agents; Interferon-alpha; Male; Middle Aged; Phlebotomy; Phosphorus Radioisotopes; Polycythemia Vera

A 58 year old male heavy smoker presented with intracranial haemorrhage and erythrocytosis. Four aetiologies of polycythaemia--polycythaemia rubra vera (PRV), renal cell carcinoma, sleep apnoea syndrome, and relative polycythaemia--were found to be associated with the underlying causes of erythrocytosis. He did not fulfill the diagnostic criteria for PRV at initial presentation, but an erythropoietin independent erythroid progenitor assay identified the masked PRV, and the low post-phlebotomy erythropoietin concentration also suggested the likelihood of PRV evolution. This case demonstrates that a search for all the possible causes of erythrocytosis is warranted in patients who already have one aetiology of polycythaemia.

Topics: Carcinoma, Renal Cell; Erythroid Precursor Cells; Erythropoietin; Humans; Intracranial Hemorrhages; Kidney Neoplasms; Male; Middle Aged; Polycythemia; Polycythemia Vera; Reverse Transcriptase Polymerase Chain Reaction; RNA, Messenger; Sleep Apnea Syndromes; Smoking

Topics: Aged; Epoetin Alfa; Erythropoietin; Humans; Injections, Subcutaneous; Male; Mouth Diseases; Polycythemia Vera; Primary Myelofibrosis; Recombinant Proteins; Skin Diseases

The SHP-1 phosphatase associates with the receptors for erythropoietin, stem cell factor, and interleukin-3, and negatively regulates the mitogenic signals generated during engagement by their respective ligands. The erythroid progenitors of patients with polycythemia vera are hypersensitive to the mitogenic effects of these growth factors despite the fact that the numbers and binding affinities for their receptors are not increased. To determine whether post-receptor signaling defects may account for growth factor-hypersensitivity in polycythemia vera, we determined the expression of SHP-1 in highly purified erythroid progenitors from polycythemia vera patients. Our data demonstrate that in approximately 60% of the patients, expression of SHP-1 in the colony forming unit-erythroid population is diminished. The decreased expression of the protein may result from a transcriptional defect, as suggested by the diminished SHP-1 mRNA expression in the erythroid progenitors of these patients. Studies to determine the level of maturation of polycythemia vera and normal cells indicated that there was no difference between the two at early colony forming unit-erythroid stage of differentiation although polycythemia vera cells showed retarded differentiation kinetics at late colony forming unit-erythroid stage of differentiation. Furthermore, SHP-1 expression in normal colony forming unit-erythroid demonstrated downregulation of mRNA and protein levels during terminal differentiation, suggesting that its function is required for growth control during the early stages of erythropoiesis. These results indicate an important role for SHP-1 in the regulation of normal human erythroid progenitors and suggest that defective expression of the protein may contribute to the pathogenesis of polycythemia vera.

Topics: Cell Differentiation; Colony-Forming Units Assay; Enzyme Induction; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Heme; Humans; Intracellular Signaling Peptides and Proteins; Phosphorylation; Polycythemia Vera; Protein Processing, Post-Translational; Protein Tyrosine Phosphatase, Non-Receptor Type 11; Protein Tyrosine Phosphatase, Non-Receptor Type 6; Protein Tyrosine Phosphatases; Proto-Oncogene Proteins c-kit; Receptors, Erythropoietin; Receptors, Interleukin-3; RNA, Messenger; Signal Transduction; Transcription, Genetic

To report a case of erythrocytosis in a patient with a hydronephrotic horseshoe kidney and normal erythropoietin values.. A hydronephrotic horseshoe kidney was discovered during evaluation to determine the etiology of the erythrocytosis in a 23-year-old male with normal erythropietin values. Blood parameters returned to normal following heminephrectomy. The hydronephrosis had been caused by stenois of the pyeloureteric junction.. Although erythropoietin values may be normal, hydronephrosis can cause secondary erythrocytosis.

Topics: Adult; Diagnosis, Differential; Erythropoietin; Humans; Hydronephrosis; Kidney; Kidney Diseases, Cystic; Male; Polycythemia; Polycythemia Vera; Tomography, X-Ray Computed

We studied the suitability of collagen-based semisolid medium for assay of endogenous erythroid colony formation performed in myeloproliferative disorders. Bone marrow (BM) mononuclear cells (MNC) from 103 patients suspected of having polycythemia vera (PV, 76 patients) or essential thrombocythemia (ET, 27 patients) were grown in collagen-based, serum-free, cytokine-free semisolid medium. Colony analysis at day 8 or 10 showed that this collagen assay is specific, as endogenous growth of erythroid colonies was never observed in cultures of 16 healthy donors and 6 chronic myelogenous leukemia (CML) patients. Endogenous erythroid colony formation was observed in 53.3% of patients suspected of PV, with only 15.4% of positive cultures for patients with 1 minor PV criterion and 72% (p = 0.009) of positive cultures for patients with > or =2 minor or 1 major PV criterion. Similarly, endogenous growth of erythroid colonies was found in 44.4% of patients suspected of ET, with 31.6% of positive cultures for patients with 1 ET criterion versus 75% for patients with > or =2 ET criteria. In addition, we found that in collagen gels, tests of erythropoietin (EPO) hypersensitivity in the presence of 0.01 or 0.05 U/ml of EPO and tests of endogenous colony-forming units-megakaryocyte (CFU-MK) formation cannot be used to detect PV or ET, as these tests were positive for, respectively, 21.4% and 50% of healthy donors and 83% and 50% of CML patients. A retrospective analysis suggests that collagen assays are more sensitive than methylcellulose assays to assess endogenous growth of erythroid colonies. In summary, serum-free collagen-based colony assays are simple and reliable assays of endogenous growth of erythroid colonies in myeloproliferative diseases. They also appear to be more sensitive than methylcellulose-based assays.

Topics: Bone Marrow; Cells, Cultured; Collagen; Colony-Forming Units Assay; Culture Media, Serum-Free; Erythrocytes; Erythroid Precursor Cells; Erythropoietin; Female; Gels; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Megakaryocytes; Methylcellulose; Polycythemia Vera; Thrombocytosis

Deregulating the expression of Bcl-xL, an inhibitor of apoptosis, in an erythropoietin-dependent erythroblast cell line averts apoptosis induced by the withdrawal of erythropoietin. Since in polycythemia vera an abnormal clone of erythroid progenitors is independent of erythropoietin, we investigated whether the endogenous expression of Bcl-xL was deregulated in these cells.. Erythroid colonies from patients with polycythemia vera and normal subjects were cultured in the presence and absence of erythropoietin and assessed by immunocytochemical and flow-cytometric analysis with anti-Bcl-x antibodies that recognize the two species of Bcl-x (Bcl-xL and Bcl-xS). Reverse-transcriptase-polymerase-chain-reaction analysis was used to determine which one of the two species was responsible for anti-Bcl-x staining. Bone marrow mononuclear cells from 8 healthy bone marrow donors, 14 patients with polycythemia vera, 19 patients with other myeloproliferative syndromes, and 12 patients with secondary erythrocytosis were analyzed by flow cytometry with antibodies against Bcl-x and glycophorin A, an erythroid marker.. Erythroid cells from patients with polycythemia vera survived in vitro without erythropoietin, and this finding correlated with the expression of Bcl-x protein (Bcl-xL messenger RNA was the main species of Bcl-x found), even in mature erythroblasts that normally do not express Bcl-x. The mean (+/-SD) percentage of cells positive for both glycophorin A and Bcl-x in the 14 patients with polycythemia vera (21.8+/-3.6 percent) was significantly higher than that in 8 normal donors (6.62+/-1.58 percent), 12 patients with secondary erythrocytosis (6.87+/-1.95 percent), 9 patients with essential thrombocythemia (3.81+/-0.97 percent), and 10 patients with chronic myeloid leukemia (2.7+/-0.41 percent).. Deregulated expression of Bcl-x may contribute to the erythropoietin-independent survival of erythroid-lineage cells in polycythemia vera and thereby contribute to the pathogenesis of this disease.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; bcl-X Protein; Case-Control Studies; Cell Survival; Child; Erythroid Precursor Cells; Erythropoietin; Female; Flow Cytometry; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Male; Middle Aged; Polycythemia Vera; Proto-Oncogene Proteins c-bcl-2; Reference Values; Thrombocytosis

By using an immunoradiometric method with a stated detection limit of < or =1 IU/l (stated normal reference limit in adults 3.7-16 IU/l) we determined EDTA-plasma erythropoietin (EPO) in 58 patients with polycythaemia vera (PV) and 49 patients with essential thrombocythaemia (ET). At the time of blood sampling, 20 of the PV patients were newly diagnosed and untreated, 23 were treated by phlebotomy only, and 30 also received myelosuppressive treatment (with 32P, hydroxyurea or alpha-interferon). Of the ET patients 24 were untreated and 28 received myelosuppressive therapy. For comparison plasma EPO was also determined in 10 patients with pseudopolycythaemia (PP). In this latter group the results for plasma EPO agreed well with the cited normal reference limits. The majority of untreated PV patients (12/20) had undetectable plasma EPO concentration, and the remainder all had values below the lower normal reference limit. Plasma EPO in PV was not significantly influenced by phlebotomy therapy. Twelve of the 24 untreated ET patients (50%) had plasma EPO values below the reference interval (undetectable in 2 patients). The mean EPO concentration was significantly lower in PV patients receiving phlebotomy therapy than in patients with untreated ET. In the total material of PV and ET treated with myelosuppressive agents the PV patients showed significantly lower values for EPO concentration than did patients with ET. The present results support the view that EPO measurements by high-detectability methods are diagnostically useful and should be included in the panel of new criteria for the diagnosis of PV.

Topics: Adult; Aged; Aged, 80 and over; Erythropoietin; Female; Hemoglobins; Humans; Hydroxyurea; Immunoradiometric Assay; Interferon-alpha; Male; Middle Aged; Phlebotomy; Platelet Count; Polycythemia Vera; Reference Values; Thrombocythemia, Essential

We assessed the in vitro culture growth of erythroid progenitors [burst forming unit-erythroid (BFU-E)] and serum erythropoietin (EPO) levels in different groups of polycythaemia to determine the discriminative power in differential diagnosis of polycythaemia.. We used the methylcellulose culture technique to study the growth of endogenous erythroid colonies (EECs) and EPO-dependent BFU-E from bone marrow (BM) and/or peripheral blood (PB) cells from 40 patients with polycythaemia vera (PV), 13 with secondary polycythaemia (SP), 19 with pure erythrocytosis (PE), five with PE and PV evolution later (PE-PV), and 12 with relative polycythaemia (RP). The serum EPO levels were measured by radioimmunoassay before treatment in 47 patients, 23 SP patients, 19 PE patients, five PE-PV patients and 16 RP patients, as well as after treatment in 38 PV patients, five PE-PV patients and 12 PE patients.. The results of the erythroid progenitor culture assay showed that the numbers of EPO-dependent BFU-E in BM did not differ significantly among groups. The PB BFU-E were significantly higher in PV than in SP or PE, and no statistical difference were found among patients with SP, PE and RP. There was a correlation between BM BFU-E and PB BFU-E in the individual PV and PE patients. EECs were present in all BM and PB cultures of untreated and phlebotomy-treated PV and PE-PV patients, but were absent in 6 of 17 PV patients who had received cytotoxic therapy. EECs were not found in SP, PE and RP. PB could substitute for BM in the EEC or the BFU-E assay. Both pretreatment and post-treatment serum EPO levels of PV and PE-PV were similar, which were significantly lower than SP, PE or RP. The serum EPO levels in treated PV or PE-PV patients who had normal haematocrit values were not significantly different from those in untreated patients. In contrast, the phlebotomy-treated PE patients had significantly higher serum EPO values than untreated PE patients. In the differentiation between PV and PE, the sensitivity, specificity, positive predictive value and negative predictive value of post-treatment serum EPO levels at a cut-off level of < or = 9 UL-1 were 74%, 92% and 52% respectively. The discriminative power of post-phlebotomy serum EPO levels was even higher with a positive predictive value of 80% and negative predictive value of 92% for the prediction of PV evolution in patients with pure erythrocytosis of unknown origin.. The present study showed that apart from EEC assay, the post-phlebotomy serum EPO level was a sensitive and specific parameter in the differential diagnosis of polycythaemia, in particular for the identification of PV among patients with unclassifiable polycythaemia.

Topics: Adult; Biomarkers; Bone Marrow; Cell Culture Techniques; Cell Division; Cells, Cultured; Colony-Forming Units Assay; Diagnosis, Differential; Erythropoietin; Hematopoietic Stem Cells; Humans; Phlebotomy; Polycythemia; Polycythemia Vera; Radioimmunoassay

In polycythemia vera (PV) erythroid colonies that grow in vitro in the absence of exogenous erythropoietin (EPO) arise from the abnormal clone that is responsible for overproduction of red blood cells. Although the mechanism of autonomous formation of burst-forming units-erythroid (BFU-E) is not fully understood, a spontaneous release of growth regulatory molecules by PV cells and/or by accessory cells is likely to be involved. Because of its cytokine synthesis inhibiting action, interleukin-10 (IL-10) could be a potentially useful molecule to modulate abnormal erythropoiesis in PV. We studied the effect of recombinant human IL-10 on the EPO-independent growth of erythroid bursts derived from peripheral blood mononuclear cells (PBMNCs) of patients with PV. IL-10 showed a profound, dose-dependent, and specific inhibitory effect on autonomous BFU-E formation. Ten nanograms per milliliter of IL-10 significantly suppressed spontaneous growth of erythroid colonies in methylcellulose in five of five PV patients tested with a mean inhibition by 81% (range, 72-94). To elucidate the possible mechanism of the inhibitory action of IL-10 we further studied the effect of anticytokine antibodies on autonomous BFU-E growth and the ability of exogenous cytokines to restore IL-10-induced suppression of erythroid colony growth. Among a panel of growth regulatory factors tested (granulocyte-macrophage colony-stimulating factor [GM-CSF], IL-3, granulocyte colony-stimulating factor, stem cell factor, and insulin-like growth factor-1) GM-CSF was the only molecule for which both an inhibition of spontaneous BFU-E formation by its respective antibody as well as a significant restimulation of erythroid colonies in IL-10-treated cultures by exogenous addition was found. Moreover, inhibition of GM-CSF production by IL-10 was shown in PV PBMNCs at the mRNA level. Our data indicate that autonomous BFU-E growth in PV can be profoundly inhibited by IL-10 and that this inhibitory effect seems to be at least in part secondary to suppression of endogenous GM-CSF production.

Topics: Adult; Aged; Aged, 80 and over; Cell Division; Erythroid Precursor Cells; Erythropoietin; Female; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Inhibitors; Growth Substances; Humans; Immune Sera; Interleukin-10; Leukocytes, Mononuclear; Male; Middle Aged; Polycythemia Vera

Erythropoietin (EP) and stem cell factor (SCF) are essential growth factors for erythroid progenitor cell proliferation and differentiation in serum-free culture. It has been previously shown that burst-forming units-erythroid and colony-forming units-erythroid from patients with polycythemia vera (PV) have enhanced sensitivity to EP and SCF compared with normal erythroid progenitors, but little is known about the mechanism for this difference. In the present investigation, the effect of EP and SCF on protein tyrosine phosphorylation in day-8 normal and PV erythroid colony-forming cells, which give rise to colonies of 2-49 hemoglobinized cells, was studied. EP rapidly induced tyrosine phosphorylation of the EP receptor, whereas the most prominent phosphorylated protein induced by SCF was identified as the SCF receptor. No additional phosphorylated proteins were evident when PV cells were compared with normal cells. Culture of normal erythroid progenitors with orthovanadate, an inhibitor of protein tyrosine phosphatases, resulted in an increased number of erythroid colonies and enhanced protein tyrosine phosphorylation. However, in contrast, little enhancement was evident with PV cells. These results indicate that, although vanadate may be acting in normal erythroid progenitors as a phosphatase inhibitor that potentiates the kinase activity induced by SCF and EP, this function is diminished in PV cells. Because erythropoiesis is regulated by a balance between protein tyrosine kinase activity and protein tyrosine phosphatase activity, PV patients may have an abnormal phosphatase activity allowing increased cell proliferation.

Topics: Cell Division; Cells, Cultured; Colony-Forming Units Assay; Culture Media, Serum-Free; Erythroid Precursor Cells; Erythropoietin; Humans; Phosphorylation; Polycythemia Vera; Protein Processing, Post-Translational; Protein Tyrosine Phosphatases; Protein-Tyrosine Kinases; Proto-Oncogene Proteins c-kit; Recombinant Proteins; Stem Cell Factor; Vanadates

The present study revises the criteria of the Polycythemia Vera Group (PVSG) for the diagnoses of essential thrombocythemia (ET) and polycythemia vera (PV) in view of accumulating data on in vitro cultures of hematopoietic progenitors and by adding histopathology from bone marrow biopsies. The majority of ET patients show spontaneous megakaryocyte or erythroid growth or both, but in about 35% the growth pattern is normal. So far none of the patients with reactive thrombocytosis have shown either spontaneous megakaryocyte or erythroid colony growth. Virtually all PV patients show spontaneous or endogenous erythroid colony (EEC) formation, whereas patients with secondary erythrocytosis and healthy controls do not show any erythroid colony growth in the absence of erythropoietin (EPO). Some rare patients with a disorder other than a myeloproliferative disease (MPD) may show spontaneous growth of erythroid colonies caused by a mutation in the EPO receptor. Megakaryocytes in bone marrow smears and biopsy material from ET and PV patients are typically increased in number and size. Enlarged megakaryocytes with mature cytoplasm and multilobulated nuclei and the tendency of these megakaryocytes to cluster in a normal or slightly increased cellular bone marrow represent the diagnostic hallmark of ET. Increase and clustering of enlarged, mature, and pleiomorphic megakaryocytes in a moderate to marked hypercellular bone marrow with hyperplasia of dilated sinuses is the diagnostic feature of untreated PV. In reactive thrombocytosis and secondary erythrocytosis the size and morphology of megakaryocytes remain normal and there is no tendency of the megakaryocytes to cluster. Both spontaneous EEC and histopathology of bone marrow biopsies appear to offer specific clues to the diagnosis of overt and latent ET or PV and have the potential to differentiate ET from reactive thrombocytosis and PV from secondary erythrocytosis. Moreover, bone marrow histopathology has the diagnostic power to distinguish and to stage the various MPDs without regard to clinical and laboratory data.

Topics: Biopsy; Bone Marrow; Bone Marrow Examination; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Female; Humans; Male; Megakaryocytes; Myeloproliferative Disorders; Polycythemia Vera; Primary Myelofibrosis; Thrombocythemia, Essential

The purpose of the present work was to evaluate the proliferative character of polycythaemia vera (PV). Therefore, in 15 patients with different stages of PV we assessed the level of CD34 positive (CD34+) cells in peripheral blood and bone marrow, erythroid colony growth of bone marrow cells and plasma erythropoietin (EPO). The mean concentration of CD34+ cells in blood was significantly increased in PV patients (9.0 +/- 11.2 x 10(3)/mL) compared to healthy controls (2.0 +/- 1.7 x 10(3)/mL). In aspirated bone marrow no such difference between PV and control subjects was present. Six patients with splenomegaly and/or requirement for chemotherapy had significantly higher mean blood levels of CD34+ cells compared to the remaining PV patients. All PV patients presented EPO independent erythroid colonies. Three PV patients with anaemia and long disease duration had high EPO levels.

Topics: Aged; Aged, 80 and over; Antigens, CD34; Bone Marrow Cells; Cell Division; Cells, Cultured; Colony-Forming Units Assay; Erythroid Precursor Cells; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Male; Middle Aged; Polycythemia Vera

The evaluation of the RNA content in reticulocytes by means of flow cytometry allows us to differentiate three reticulocyte populations by their degree of maturity: LFR, MFR and HFR. We have studied the value of those reticulocyte subpopulations for the differential diagnosis in polycythemia.. We have studied 25 polycythemia patients, 12 of these were diagnosed of polycythemia vera (PV) and 13 of secondary polycythemia (PS). Reticulocytes and their different reticulocyte populations were automatically analyzed by flow cytometry using the Sysmex R-2000 analyzer (TOA Corp. Japan). We calculated the reticulocyte maturity index (RMI) which is the percentage of the equation (MFR + HFR) x 100/LFR (9). We have also studied hemogram parameters (Hct, Hb, VCM, HCM, RDW, leukocytes and platelets), iron metabolism (serum iron, ferritin, transferrin and transferrin saturation index), and erythropoietin levels. The findings were statistically analyzed using the Pearson correlation test and the comparison tests of averages of independent samples (Student's t).. Patients with PV present high RMI and MFR + HFR (medium and high fluorescence reticulocytes) as compared with secondary polycythemia . We did not find any correlation between RMI and other analytical parameters (erythropoietin, ferritin, serum iron, transferrin), except for the transferrin saturation index (TSI), with a correlation factor r: 0.5486 for p < 0.05.. This higher proportion of younger reticulocytes in PV might be explained because they are a parameter that would reflect the expansion of erythropoietic bone progenitor cells, which would be more increased in PV than in PS. However, another possible explanation would consist in the existence of associated ferropenia in PVs, which lead to an increase in cytoplasmic levels of transferrin receptor mRNA.

Topics: Adult; Aged; Blood Cell Count; Erythrocyte Indices; Erythropoiesis; Erythropoietin; Evaluation Studies as Topic; Female; Ferritins; Flow Cytometry; Humans; Iron; Male; Middle Aged; Polycythemia; Polycythemia Vera; Receptors, Transferrin; Reticulocyte Count; Reticulocytes; Transferrin

The case of an 11 year old girl with three line type of polycythaemia vera, with 4 cm splenomegaly and a plethoric complexion is presented. Peripheral blood values were as follows: RBC: 7.75 x 10(12)/l, Hb: 18.8 g/l, WBC: 15.2 x 10(9)/l, platelets: 920 x 10(9)/l. Serum erythropoietin level: < 1 mU/ml. In vitro, erythroid colonies developed from the bone marrow in the absence of added erythropoetin. For three years the haematocrit value has been controlled by regular venesections. Since extreme thrombocytosis developed, the treatment was continued with interferon alpha. Different treatment protocols are discussed.

Topics: Age Factors; Child; Erythropoietin; Female; Hematocrit; Humans; Interferon-alpha; Phlebotomy; Polycythemia Vera; Splenomegaly

Correction of anaemia with recombinant human erythropoietin (rHu-EPO) improves the responsiveness of thyroidal and gonadal axes to exogenous TRH and GnRH in chronic haemodialysis patients, but the mechanisms remain to be fully elucidated. In order to assess the influences of endogenous erythropoietin on the hypothalamo-hypophyseal thyroidal and gonadal axes, we studied the response of polycythaemic and anaemic patients, in comparison to normal controls, after the administration of exogenous TRH and GnRH.. Exogenous hypothalamic factors, 500 micrograms TRH and 100 micrograms GnRH, were administered as a bolus and blood samples were obtained over a 3-hour period at 30, 60, 90, 120 and 180 minutes.. Five male polycythaemic patients (low EPO), three male anaemic patients (high EPO) and six normal age and sex matched controls were studied.. Blood samples were centrifuged immediately and the serum was stored at -20 degrees C until assayed for total T4, free T4, free T3, TSH, prolactin, growth hormone (TRH test), and FSH, LH, testosterone (GnRH test). Haematological parameters and biochemical profiles were also measured.. After TRH administration, both patient groups showed a normal TSH response; however, their free T4 and free T3 secretion was blunted compared to controls. Normal basal PRL levels increased in an exaggerated fashion, whereas, when compared to chronic renal failure patients on chronic haemodialysis, we did not see a paradoxical GH response or a basal GH increase in these 5 patients. GnRH administration in the study groups elicited a normalization in the LH response without an increase in testosterone levels; however, an exaggerated FSH response was found in the polycythaemic patients (low EPO).. Thus by investigating the role of low endogenous EPO levels in non-anaemic and anaemic patients with high EPO levels, our study suggests that the underlying chronic disease state may be the major contributing factor in the regulation of the hypothalamo-hypophyseal thyroid and gonadal axes, rather than the EPO levels.

Topics: Aged; Anemia, Aplastic; Erythropoietin; Follicle Stimulating Hormone; Gonadotropin-Releasing Hormone; Growth Hormone; Humans; Luteinizing Hormone; Male; Middle Aged; Pituitary Hormones, Anterior; Polycythemia Vera; Prolactin; Testosterone; Thyroid Hormones; Thyrotropin; Thyrotropin-Releasing Hormone

Human recombinant interleukin-1 (IL-1) was studied for its effects on the erythroid progenitors from normal subjects and from patients with polycythaemia vera (PV). No supportive effect of IL-1 was noticed on the normal, erythropoietin (Epo) dependent, erythroid burst-forming unit (BFU-E) using peripheral blood or bone marrow. In contrast, the Epo-independent BFU-E from peripheral blood of PV patients could be stimulated significantly. This enhancing effect of IL-1 was not only observed with unsorted but also with sorted CD34+ cells. In addition, it was shown that IL-1 indirectly stimulated the Epo-independent BFU-E because anti-GM-CSF could abrogate the supportive effects of IL-1. In contrast to the Epo-independent BFU-E, the Epo-dependent erythroid colony formation from PV patients could not be augmented by IL-1. Finally, we studied the effects of IL-4 on the Epo-independent BFU-E, because IL-4 can affect the erythroid colony formation and modulate the effects of IL-1. IL-4 suppressed the Epo-independent BFU-E. This effect could be counteracted by the addition of IL-1 to the culture medium. However, the suppressive effect of IL-4 was not related to a decline in spontaneous release of IL-1, because an anti-IL-1 antibody did not modify the spontaneous erythroid colony formation. These data indicate that IL-1 and IL-4 exert separate influences on the Epo-independent erythroid colony formation in PV.

Topics: Bone Marrow; Cell Division; Cells, Cultured; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Erythroid Precursor Cells; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Humans; Interleukin-1; Interleukin-4; Polycythemia Vera; Recombinant Proteins

Erythropoietin (EPO) is a potent regulator of the viability, proliferation and differentiation of erythroid progenitor cells. Its effect is mediated by binding to the erythropoietin receptor (EPO-R), a member of a new cytokine receptor family. Alterations of the EPO/EPO-R system have recently been shown to be involved in the pathogenesis of familial erythrocytosis and polycythaemia vera (PV). In order to define whether genetic changes in the EPO-R gene and its ligand play a role in the development of PV, the structure and expression levels of the EPO-R and EPO genes were examined in samples from bone marrow and/or peripheral blood mononuclear cells of 24 patients with PV. As expected, EPO serum levels were low and no detectable level of EPO mRNA was found by reverse polymerase chain reaction (RT-PCR) in the peripheral blood mononuclear cells of our PV patients. To search for structural alterations of the EPO-R, cDNA samples were subjected to PCR and SSCP analysis as well as sequencing. Heterogenous expression of EPO-R mRNA was observed without any structural changes, as revealed by RT-SSCP analysis using overlapping primers spanning the whole coding region of the EPO-R gene. Structural integrity of the EPO-R was further confirmed by sequencing of cloned PCR products. These data suggest that the mechanisms for the development of PV do not involve structural changes of the EPO-R gene.

Topics: Adult; Aged; Base Sequence; Bone Marrow; Erythropoietin; Female; Humans; Male; Middle Aged; Molecular Sequence Data; Oligonucleotide Probes; Polycythemia Vera; Polymerase Chain Reaction; Polymorphism, Single-Stranded Conformational; Receptors, Erythropoietin; RNA, Messenger; Transcription, Genetic

Erythroid progenitor cells isolated from patients with polycythemia vera (PV) proliferate and differentiate in methylcellulose in the absence of exogenous erythropoietin (EPO). To investigate the potential role of the erythropoietin receptor (EPO-R) in the pathogenesis of PV, we cultured bone marrow-derived or peripheral blood-derived erythroid progenitors in the presence of neutralizing monoclonal antibodies (MoAbs) specific for EPO or EPO-R. Mononuclear cells were obtained from 9 healthy adults and 9 PV patients by Ficoll-Hypaque gradients and cultured with or without EPO in methylcellulose for 12 days under standard or serum-free conditions. Neutralizing anti-EPO and anti-EPO-R MoAbs, added to cultures at day 0, caused dose-dependent growth inhibition of all normal burst-forming units-erythroid (BFU-E) derived from health adult controls. The MoAbs had no effect on the growth of nonerythroid progenitor cells under the same culture conditions. In contrast, neutralizing antibodies distinguished two classes of BFU-E derived from PV patients. Class I BFU-E from PV patients were EPO-dependent. These progenitors, like those derived from healthy adults, had normal EPO dose-dependent growth characteristics and showed a normal period of EPO requirement in vitro that extended 6 days after the initiation of culture. These results indicate that EPO exerts its critical effect early during erythroid differentiation; the addition of neutralizing antibodies to normal progenitors after 6 days had no effect on the subsequent size or maturation of the colonies. Class II BFU-E from PV patients were EPO-independent. They proliferated and differentiated even in the presence of high concentrations of neutralizing anti-EPO or anti-EPO-R MoAbs. We conclude that the class II BFU-E from PV patients are independent of free EPO.

Topics: Antibodies, Monoclonal; Bone Marrow; Cell Differentiation; Cell Division; Cells, Cultured; Erythroid Precursor Cells; Erythropoietin; Female; Humans; Male; Polycythemia Vera; Receptors, Erythropoietin

A 20-year-old woman presented with polycythemia vera and was treated with phlebotomy alone for eleven years, following which all clinical manifestations of the disease disappeared. The clinical remission with normal physical findings and normal peripheral blood counts has persisted for a further 11 years. Erythroid colony culture results have paralleled the clinical state. Initial bone marrow cultures revealed spontaneous growth of erythroid burst-forming units (BFU-E). Subsequent cultures of peripheral blood cells throughout most of the period of spontaneous clinical remission have revealed little or no spontaneous growth of BFU-E. This suggests a suppression of the abnormal stem cell clone during the period of remission.

Topics: Adult; Bloodletting; Cells, Cultured; Erythroid Precursor Cells; Erythropoietin; Female; Humans; Polycythemia Vera; Remission, Spontaneous

We have investigated the question of erythropoietin (Epo) hypersensitivity versus Epo independence as the basis for the endogenous erythroid bursts (EEBs) that develop in cultures without added Epo from hematopoietic cells of polycythemia vera (PV) patients. Using an improved serum-free (SF) medium containing interleukin (IL)-3, but no insulin-like growth factor-1 (IGF-1), and devoid of contaminants that influence erythropoiesis, we compared circulating normal and PV early erythroid progenitors (BFU-E) with respect to their responses in vitro to recombinant human (rHu) Epo. Cultures were seeded with Ficoll-Hypaque density-separated peripheral blood (PB) mononuclear cells (MNCs), and erythroid bursts, together with their component colonies of > or = 50 cells, were scored in situ at 13 to 16 days of culture. The Epo dose-response curve of BFU-E from PV patients was found to be statistically indistinguishable from that of normal subjects. This observation provides compelling evidence against the Epo-hypersensitivity hypothesis. In the complete SF medium minus Epo, the sensitivity of BFU-E to IGF-1 was much greater in PV than in normals, the dose-response curve being shifted to the left by at least 2 orders of magnitude. These data show that the erythroid progenitor cell response in PV is hypersensitive to IGF-1, and independent of Epo. The data also emphasize the importance of truly SF medium conditions for assessment of progenitor cell sensitivities to recombinant growth factors. Depletion of adherent cells totally prevented erythroid burst formation by normal circulating progenitors, but did not prevent the hypersensitive response to IGF-1 of such cells from PV patients. Hence, again unlike its normal counterpart, the progenitor cell response in PV appears to be independent of adherent cell control.

Topics: Aged; Antibodies, Monoclonal; Cells, Cultured; Culture Media, Serum-Free; Erythroid Precursor Cells; Erythropoietin; Female; Humans; Insulin-Like Growth Factor I; Interleukin-3; Male; Middle Aged; Polycythemia Vera; Recombinant Proteins

We used the methylcellulose-culture technique to determine the utility of the erythroid progenitor growth in vitro from nonadherent T-depleted bone marrow and peripheral blood cells in distinguishing polycythemia vera (PV) from essential thrombocythemia. Thirty patients with PV (group A) and 30 patients who presented with idiopathic marked thrombocytosis with platelet count greater than 1,000 x 10(9)/L and a normal or reduced hemoglobin (Hb) level (group B) were studied at initial presentation. Endogenous (erythropoietin-independent) erythroid colonies (EEC) were found in all patients in group A and 13 in group B. The numbers of EEC were comparable between patients in group A and the 13 patients with EEC in group B, 11 of whom with initial Hb levels ranging between 6.4 g/dL and 12.6 g/dL were found to have PV 2 to 45 months after initial evaluation. The number of EEC did not correlate with the time to the progression of polycythemia, whereas myelosuppression delayed the subsequent development of PV. Of the two patients with EEC in group B who did not develop PV, both received chemotherapy soon after presentation, which might preclude the evidence of polycythemia evolution. None of the other patients in group B who did not form EEC developed PV with a median follow-up of 24 months. This study indicates that the assessment of EEC in bone marrow or blood is helpful in early identification of PV or prediction of polycythemia evolution in patients with marked thrombocytosis in whom polycythemia has been initially masked or anemia is present.

Topics: Aged; Erythroid Precursor Cells; Erythropoietin; Female; Humans; Male; Middle Aged; Polycythemia Vera; Thrombocytosis

The effect of mast cell growth factor (MGF) was studied on erythropoietin (Epo)-dependent and Epo-independent ("spontaneous") erythroid colony formation in patients with polycythemia vera (PV). MGF stimulated both Epo-dependent and Epo-independent erythroid colony formation from PV peripheral blood progenitor cells in vitro at a dose similar to normal erythroid progenitor. In addition, evidence was obtained that the stimulating effect of MGF was a direct effect on the erythroid progenitor and independent of serum. Antibodies against interleukin-1 (IL-1), IL-3, granulocyte-macrophage colony-stimulating factor (GM-CSF), and Epo could not abolish the enhancing effect of MGF. This was also supported by the finding that sorted CD34+ cells could be stimulated by MGF in the presence and absence of Epo. Finally, it was demonstrated that the spontaneous erythroid colony formation could not be ascribed to spontaneous release of MGF in the culture medium since anti-MGF did not affect the colony numbers. In conclusion, MGF has a direct stimulatory effect, independent of serum, on both Epo-dependent and Epo-independent erythroid colony formation in PV.

Topics: Antibodies; Antigens, CD; Antigens, CD34; Blood; Cells, Cultured; Erythroid Precursor Cells; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Hematopoietic Cell Growth Factors; Humans; Interleukin-1; Interleukin-3; Polycythemia Vera; Recombinant Proteins; Stem Cell Factor

Patients with polycythaemia and normal controls have been studied to establish and subsequently test non-conventional criteria for the diagnosis of primary polycythaemia (primary proliferative polycythaemia, polycythaemia vera) as compared with conventional Polycythaemia Vera Study Group (PVSG) assessment. One criterion was erythroid colony formation from peripheral blood in a serum-free system, assayed alone and with the addition of recombinant human erythropoietin (Epo), interleukin 3 (IL3), or alpha interferon (alpha-IFN) (Dudley et al. 1990). The remaining criteria were non-culture associated and comprised platelet distribution width (PDW), platelet nucleotide ratio (ATP:ADP), serum erythropoietin and clinical evidence of ischaemic vascular disease. The combination of culture associated and non-culture associated variables, by use of a simple additive scoring system, gave no false positive and only 6% false negative results in distinguishing primary polycythaemia from other polycythaemias and normal controls in those (34 patients Group A) used in its derivation. Testing the scoring system in a newly presenting group (25 patients Group B) was highly satisfactory with no false positives and only a few false negative results (14%). Use of these non-conventional criteria should allow more confident diagnosis of primary polycythaemia, where conventional clinical and laboratory assessment is inconclusive.

Topics: Adenosine Diphosphate; Adenosine Triphosphate; Adult; Biomarkers; Blood Platelets; Cells, Cultured; Colony-Forming Units Assay; Diagnosis, Differential; Erythrocytes; Erythropoietin; Female; Humans; Interferon Type I; Interleukin-3; Ischemia; Male; Polycythemia; Polycythemia Vera; Recombinant Proteins; Reference Values; Thrombosis

During the last three years 77 patients with myelofibrosis were studied by scintigraphy, using 99m Tc colloids and 111 In transferrin as tracers. Low axial uptake of the colloids, extension of the indium uptake beyond the axis towards the knees and sometimes the ankles and elbows, and splenic indium uptake are valuable diagnostic criteria, particularly useful to exclude myelofibrosis associated with a malignant disorder. The clinical severity of the disease, and in particular the disappearance of a physiologically active bone marrow (indium uptake) can be predicted from isotopic studies. Bone marrow scintigraphy could contribute to the difficult decision of splenectomy.

Topics: Adult; Bone Marrow; Erythropoietin; Female; Humans; Leukemia; Male; Middle Aged; Polycythemia Vera; Primary Myelofibrosis; Prognosis; Radionuclide Imaging; Splenomegaly; Thrombocythemia, Essential

We evaluated a newly developed enzymeimmunoassay for serum erythropoietin (Epo) and investigated relationship between Epo levels and hematological disorders. This method has several advantages including simplicity, high sensitivity, good precision. Moreover, the procedure requires only about 2.5 hours. Samples from 134 healthy subjects showed a normal logarithmic distribution, and its normal range was 4.5 approximately 21.3 mU/ml. The levels of Epo in normal subjects and various hematological disorders were as follows: 10.5 +/- 4.1 (mean +/- SD mU/ml) in normal subjects, 2.2 +/- 1.7 in polycythemia vera (PV), 6.1 +/- 3.1 in essential thrombocythemia, 17.8 +/- 27.3 in chronic myelogeneous leukemia, 3.6 +/- 1.8 in stress erythrocytosis, 39.4 and 14.1 in two cases of primary myelofibrosis, 1289 +/- 4798 in iron deficiency anemia and 6564 +/- 10870 in aplastic anemia. In patients with PV, serum Epo were low and did not correlate with hemoglobin concentration. However, inverse correlation was found between changes of Epo levels and hemoglobin levels in most patients. In cases in which PV progressed into myelofibrosis, anemia developed and Epo levels increased accordingly. These results suggest that the method is thought to be useful and reliable for the diagnosis and monitoring of PV and related hematological disorders.

Topics: Erythropoietin; Hematologic Diseases; Hemoglobins; Humans; Immunoenzyme Techniques; Polycythemia Vera; Primary Myelofibrosis; Sensitivity and Specificity

Blood erythropoietin (EPO) concentration was measured by radioimmunoassay in 513 patients with various diseases. Untreated polycythemia vera showed lower EPO concentration than normal. Aplastic anemia (AA) revealed the highest EPO level among all anemic diseases in relation to hematocrit value. EPO level of AA patients who underwent bone marrow transplantation was as low as normal subjects even when the anemia has not fully recovered. Paroxysmal nocturnal hemoglobinuria (PNH) showed unusually high EPO concentration among hemolytic anemias. In normal subjects, blood EPO concentration showed a diurnal rhythm that was higher at night than during the daytime. These findings suggest the diagnostic usefulness of measurement of EPO in blood diseases.

Topics: Adult; Aged; Aged, 80 and over; Anemia, Aplastic; Bone Marrow Transplantation; Circadian Rhythm; Erythropoietin; Female; Hemoglobinuria, Paroxysmal; Humans; Male; Middle Aged; Polycythemia Vera; Radioimmunoassay; Reference Values

Cultures of hematopoietic precursor cells can be helpful in differentiating between primary polycythemia (polycythaemia vera, PV) and reactive secondary polycythemia: in PV erythroid precursors form hemoglobinized colonies in the absence of added erythropoietin (epo) (= endogenous erythroid colonies), whereas in normals and in patients with secondary polycythemia, formation of erythroid colonies is dependent on added epo. We have performed cultures of peripheral blood precursors from 132 patients with elevated hemoglobin in the presence/absence of added epo. In 48/132 patients we assumed that PV was the cause of polycythemia. In 80/132 patients no endogenous colonies appeared and the polycythemia was judged secondary. 23 PV patients were examined repeatedly. In 18 of them the first diagnosis was confirmed by subsequent cultures; in 5 cases endogenous colonies, which had been present in the first cultures, were no longer detectable. A questionnaire on the subsequent clinical course was sent to 108 treating physicians. 77 questionnaires were answered correctly and returned. In 86% of these patients, our culture diagnosis of PV was either confirmed or another myeloproliferative disorder had been found as a cause of endogenous colonies. In 14% our diagnosis of PV had been false positive. On the other hand, our diagnosis of secondary polycythemia was confirmed in 85% of the patients; its most frequent cause was cigarette smoking and chronic bronchitis and only rarely was it associated with heart or kidney disease. 5/77 patients had persistently elevated Hb without an evident cause, and in 3/77 the Hb normalized spontaneously.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Colony-Forming Units Assay; Diagnosis, Differential; Erythroid Precursor Cells; Erythropoietin; False Positive Reactions; Hematopoietic Stem Cells; Humans; Myeloproliferative Disorders; Polycythemia; Polycythemia Vera

Serum erythropoietin (EP) concentration was measured by the recombinant EP-based radioimmunoassay and was examined to standardize the hemoglobin (Hb) related level of 144 normal control and 56 patients with iron deficiency anemia and hemolytic anemia excluding paroxysmal nocturnal hemoglobinuria. The standardization was achieved by logarithmic regression of the EP titier on Hb either by the two-phase linear form or by the third degree sigmoid form at a 95% confidence limit for each regression. The third degree regression was found to be preferable from the view point of both statistics and the negative feedback mechanism. The average and scattering of the deviation from the standard level thus determined of the disease groups indicated that the EP level is: (1) 12 fold higher than the standard level in 42 aplastic anemias (the most in excess and a few in standard). (2) three fold higher than that in 27 myelodysplastic syndromes (relatively higher dispersed state). (3) 29% of the standard level in 33 anemias associated with chronic renal failure (deficient state). (4) 105% of the extrapolated standard level in 22 polycythemia veras (standard state). The standardization of Hb-related Ep titer may provide new pathophysiological approaches in a variety of hematopoietic disorders.

Topics: Anemia, Hemolytic; Anemia, Hypochromic; Erythropoietin; Hemoglobins; Humans; Myelodysplastic Syndromes; Polycythemia Vera; Radioimmunoassay; Reference Standards

An attempt was made to discover the aetiology of acquired portal vein thrombosis in 12 polycythaemic patients who did not show any obvious local or regional cause. In addition to the diagnostic criteria of polycythaemia vera, erythropoietin was determined and cultures of erythroblast precursors were examined. The patients could be divided into 3 groups, in the first of which the definite diagnosis of polycythaemia vera was made on the basis of the PVSG (Polycythaemia Vera Study Group) criteria and bone marrow biopsy (5 patients). In the second group (5 patients), there was a diagnosis of possible polycythaemia vera based essentially on the finding of a spontaneous growth of medullary CFU-E. Finally, diagnostic criteria for polycythaemia vera were absent in two patients. On the basis of these findings, the physiopathology of the association of portal thrombosis and polycythaemia is discussed, in particular polycythaemia secondary to hepatic ischaemia.

Topics: Adult; Blood Volume; Bone Marrow; Erythropoietin; Female; Hematocrit; Humans; Iron Deficiencies; Male; Middle Aged; Polycythemia Vera; Portal Vein; Splenomegaly; Thrombosis

Erythropoietin (Epo) is the major regulator of erythroid viability, proliferation, and differentiation. These functions are transduced following binding of Epo to a specific cell surface receptor, the erythropoietin receptor (EpoR), a member of a new cytokine receptor superfamily of receptors. An activating mutation in the murine EpoR has been described (cEpoR) and confers growth factor-independent growth upon an IL-3-dependent pro-B cell. To determine the effect of an activating mutation in the EpoR upon erythropoiesis specifically and hematopoiesis generally, we infected hematopoietic progenitors and mice with a recombinant erythroleukemic spleen focus-forming virus (SFFV), lacking its pathogenic env gene but expressing cEpoR (SFFVcEpoR). In vitro, infection with SFFVcEpoR resulted in factor-independent growth and development of CFU-Es yet had no effect on BFU-E growth, mixed colony growth, or myeloid colony growth. Mice infected with SFFVcEpoR, but not a virus expressing wild type EpoR (SFFVEpoR), developed erythrocytosis and splenomegaly.

Topics: 3T3 Cells; Animals; Erythrocytes; Erythroid Precursor Cells; Erythropoietin; Mice; Mice, Inbred BALB C; Mutation; Polycythemia Vera; Receptors, Cell Surface; Receptors, Erythropoietin; Splenomegaly

Serum erythropoietin (S-Epo) was measured with a radiommunoassay method in 36 patients with polycythaemia vera, 17 patients with secondary polycythaemia and 14 patients with relative polycythaemia. The diagnoses were made without the aid of the S-Epo values. It was found that S-Epo was below the reference range in 34/36 patients with polycythaemia vera (mean 2.1 +/- 1.0 U/l), elevated in all cases of secondary polycythaemia (mean 121.7 +/- 242 U/l) and normal in all but one of the cases with relative polycythaemia (mean 7.0 +/- 2.5 U/l). Previous studies of S-Epo levels in the differential diagnosis of polycythaemia have shown significant differences between the means of the groups but a considerable overlap. After phlebotomy treatment to normal haematocrit levels, the S-Epo levels remained subnormal in most of the polycythaemia vera patients even after 18 months at a haematocrit around 45%. Two patients who had been kept at normal haematocrit for 6 and 7 years both had subnormal S-Epo. We conclude that with an Epo assay method of high sensitivity and specificity it is possible to differentiate between different forms of polycythaemia with a high degree of certainty, even between patients with relative polycythaemia and polycythaemia vera patients. The reason why S-Epo remains low in spite of a normal haematocrit in treated polycythaemia vera patients is not known.

Topics: Bloodletting; Diagnosis, Differential; Erythropoietin; Female; Hematocrit; Humans; Male; Polycythemia; Polycythemia Vera; Time Factors

Serum erythropoietin (Epo) titers in patients with various hematological malignancies and related diseases were determined by radioimmunoassay. Serum Epo titer was inversely correlated with hemoglobin concentration in iron deficiency anemia, aplastic anemia, myelodysplastic syndromes (MDS), acute leukemia, malignant lymphoma, multiple myeloma and myelofibrosis, but there was no correlation between serum Epo titer and hemoglobin concentration in chronic myelogenous leukemia or polycythemias. Serum Epo titers in aplastic anemia were much higher than those in iron deficiency anemia. Serum Epo titers in MDS, malignant lymphoma and multiple myeloma differed considerably among patients. Serum Epo titers in untreated polycythemia vera were significantly lower than in treated polycythemia vera or secondary polycythemia.

Topics: Anemia; Erythropoietin; Hemoglobins; Humans; Myeloproliferative Disorders; Polycythemia Vera

Topics: Aged; Cells, Cultured; Chromosomes, Human, Pair 8; Erythropoietin; Hematopoietic Stem Cells; Humans; Male; Polycythemia Vera; Trisomy

Polycythemia vera (PV) is a clonal disease of the hematopoietic stem cell characterized by a hyperplasia of marrow erythropoiesis, granulocytopoiesis, and megakaryocytopoiesis. We previously reported that highly purified PV blood burst-forming units-erythroid (BFU-E) are hypersensitive to recombinant human interleukin-3 (rIL-3). Because these cells may be only a subset, and not representative of marrow progenitors, we have now studied partially purified marrow hematopoietic progenitor cells. Dose-response experiments with PV marrow BFU-E showed a 38-fold increase in sensitivity to rIL-3 and a 4.3-fold increase in sensitivity to recombinant human erythropoietin (rEpo) compared with normal marrow BFU-E. In addition, PV marrow colony-forming units-granulocyte-macrophage (CFU-GM) and CFU-megakaryocyte (CFU-MK) also showed a marked hypersensitivity to rIL-3 and to human recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Dose-response curves with rGM-CSF and blood BFU-E showed a 48-fold increase in sensitivity. No effect of rIL-4, rIL-6, human recombinant granulocyte-CSF (rG-CSF), or macrophage-CSF (rM-CSF) was evident, nor was there any effect of PV cell-conditioned medium on normal BFU-E, when compared with normal cell-conditioned medium. Autoradiography with 125I-rEpo showed an increase in Epo receptors after maturation of PV BFU-E to CFU-E similar to that shown with normal BFU-E, but no increase of specific binding of 125I-rIL-3 by PV CD34+ cells was seen compared with normal CD34+ cells. These studies show that PV marrow hematopoietic progenitor cells are hypersensitive to rIL-3 and rGM-CSF, similar to PV blood BFU-E. While the mechanism does not appear to be due to enhanced binding of rIL-3, the hypersensitivity of PV progenitor cells to IL-3 and GM-CSF may be a key factor in the pathogenesis of PV.

Topics: Bone Marrow; Colony-Forming Units Assay; Erythroid Precursor Cells; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Granulocytes; Hematopoietic Stem Cells; Humans; Interleukin-3; Macrophages; Megakaryocytes; Polycythemia Vera; Recombinant Proteins

We examined the conditions necessary for performing a reliable erythropoietin (EPO) assay based on CFU-E colony formation in fetal mouse liver cell (FMLC) microcultures using 96-well microtiter plates. Both linearity of colony numbers with the number of cells plated and comparison among the colony ratios at various densities of seeding cells indicated that the colonies originated from a single progenitor cell when 7500 or fewer cells were plated into individual microtiter wells. About a twofold CFU-E enrichment in 12- to 13-day FMLC was achieved by Ficoll-Paque centrifugation. Plasma treated with acid-boiling stimulated the colony formation most and contained no colony inhibitor. Dose-response curve for the plasma was parallel to the EPO standard curve. The "erythroid colony-stimulating activity" in the plasma was additive to that in the standard EPO, and was completely neutralized by a monoclonal antibody against recombinant human EPO. Using the assay procedure thus established, plasma EPO titer was determined in normal subjects, in patients with nonuremic anemia and polycythemia vera, and in dialysis patients with chronic renal failure. The use of different preparations of standard EPO resulted in a significant difference in the titers because their dose-response curves differed from one another. An inverse relationship was found between EPO titers and hemoglobin concentrations in the nonuremic anemic patients, but not in the dialysis patients with about one half the normal EPO level.

Topics: Anemia; Animals; Biological Assay; Cell Separation; Cells, Cultured; Centrifugation, Density Gradient; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Erythropoietin; Fetus; Hemolysis; Humans; Kidney Failure, Chronic; Liver; Mice; Mice, Inbred ICR; Polycythemia Vera; Reference Values

A link between hyperparathyroidism and the growth of hematopoietic stem cells is suggested by this report of a parathyroid carcinoma with polycythemia vera. A 56-year-old white woman presented with splenomegaly, a palpable neck mass and hypercalcemia, recurrent six years after resection of a parathyroid tumor. She had pancytosis with a subnormal serum concentration of erythropoietin. Radiographs showed subperiosteal erosions an dosteopenia. Nephrocalcinosis was absent. Bone biopsy showed a decreased cortical width with many intracortical osteoclasts. The cancellous bone area remained normal, but the osteoid area/bone area, osteoblast perimeter and osteoclast perimeter were increased. At surgery, a parathyroid carcinoma was found in the same location operated on previously. As in two other reported cases, postoperative improvement in the hypercalcemia was associated with remission of the blood dyscrasia. A novel finding in this case is that when the hypercalcemia eventually recurred, it was again accompanied by pancytosis. With bisphosphonate therapy, the serum intact parathyroid hormone level increased in response to a decrease in the ionized calcium level, indicating that the cancer was not autonomous. This case suggests that in the presence of the ionized hypercalcemia, the parathyroid tumor may have produced or induced production of a growth factor that can stimulate pancytosis. The differential diagnosis of polycythemia and hypercalcemia should be expanded to include parathyroid tumors in addition to hepatic, adrenal, renal, and ovarian neoplasms.

Topics: Bone and Bones; Calcium; Erythropoietin; Female; Follow-Up Studies; Humans; Middle Aged; Parathyroid Hormone; Parathyroid Neoplasms; Polycythemia Vera; Radiography; Radioimmunoassay

Human recombinant interleukin 4 (IL-4) was studied for its effects on erythroid burst-forming units (BFU-E) from normal peripheral blood and from patients with polycythemia vera (PV). IL-4 enhanced the proliferation of normal peripheral blood BFU-E (183% +/- 20% enhancement), whereas in the presence of interleukin 3 (IL-3) no further augmentation was noticed. The IL-4-mediated effects were independent of the absence or presence of adherent cells, B cells, or T cells. These data are in contrast with results obtained from normal human bone marrow cells, in which IL-4 antagonized the enhancing effects of IL-3. In PV a different response pattern was observed. The effects of IL-4 on the erythropoietin (Epo)-independent BFU-E were variable. In five PV patients no suppressive or enhancing effects of IL-4 were observed, whereas in two additional patients a significant decline in the Epo-independent BFU-E was noted. In the presence of IL-3, IL-4 significantly antagonized the IL-3-supported Epo-independent BFU-E in all patients (272% +/- 57% vs 187% +/- 49% enhancement, p less than 0.05). In contrast, IL-4 did not modify the IL-3-supported Epo-dependent BFU-E. In summary, these data suggest a difference between the normal and PV peripheral blood BFU-E. The Epo-dependent erythroid progenitors in PV patients showed a response pattern with IL-3 and IL-4 comparable to that of normal peripheral blood BFU-E, whereas the Epo-independent erythroid progenitors behaved like normal human bone marrow BFU-E, suggesting a shift in the stem cell compartment in PV. This is further supported by the finding that erythroid colony-forming units (CFU-E), normally only present in the bone marrow, could be cultured from the peripheral blood of PV patients in the presence or absence of Epo.

Topics: B-Lymphocytes; Drug Interactions; Erythroid Precursor Cells; Erythropoietin; Humans; Interleukin-3; Interleukin-4; Polycythemia Vera; T-Lymphocytes

Because polycythemia vera (PV) is a clonal hematopoietic stem cell disease with a trilineage hyperplasia, and interleukin-3 (IL-3) stimulates trilineage hematopoiesis, we have studied the response of highly purified PV blood burst-forming units-erythroid (BFU-E) to recombinant human IL-3 (rIL-3). Whereas the growth of normal blood BFU-E in vitro rapidly declined by 40 and 60% after 24 and 48 h of incubation without 50 U/ml of rIL-3, the growth of PV BFU-E declined by only 10 and 30% under the same conditions, demonstrating a reduced dependence on rIL-3. A reduced dependence of PV BFU-E on recombinant human erythropoietin (rEP) was also present. Dose-response experiments showed a 117-fold increase in PV BFU-E sensitivity to rIL-3, and a 6.5-fold increase in sensitivity to rEP, compared to normal BFU-E, whereas blood BFU-E from patients with secondary polycythemia responded like normal BFU-E. Endogenous erythroid colony (EEC) formation, which is independent of the addition of rEP, was reduced by 50% after erythroid colony-forming cells were generated from PV BFU-E in vitro without rIL-3 for 3 d, whereas rEP-stimulated erythroid colonies were unaffected. These studies demonstrate a striking hypersensitivity of PV blood BFU-E to rIL-3, which may be the major factor in the pathogenesis of increased erythropoiesis without increased EP concentrations.

Topics: Dose-Response Relationship, Drug; Erythroid Precursor Cells; Erythropoietin; Humans; In Vitro Techniques; Interleukin-3; Polycythemia Vera; Recombinant Proteins

The results of estimating blood plasma erythropoietic activity in patients with polycythemia of unclear genesis, in 95% of cases coincided with the clinical diagnosis proved during further follow up. These data have permitted recommendation of estimating blood plasma erythropoietic activity in patients to verify the diagnosis of polycythemic states.

Topics: Adult; Child; Diagnosis, Differential; Erythropoietin; Female; Humans; Kidney Diseases; Male; Middle Aged; Polycythemia; Polycythemia Vera

Topics: Adult; Anemia; Erythropoietin; Humans; Kidney Failure, Chronic; Male; Polycythemia Vera; Recombinant Proteins; Renal Dialysis

Topics: Colony-Forming Units Assay; Dose-Response Relationship, Drug; Erythroid Precursor Cells; Erythropoietin; Humans; In Vitro Techniques; Interleukin-3; Polycythemia Vera

We have recently described a new two-phase liquid culture that supports the development of human erythroid progenitors (Fibach et al., Blood 73:100, 1989). The procedure separates the erythroid burst-forming units (BFUe) from the erythroid colony-forming units (CFUe) stage and enables quantitation of the proliferation and differentiation of BFUe into CFUe. In the present study we have utilized this system to study erythroid progenitors in polycythemia vera (PV). The abnormality of the erythroid series in PV has been shown to be associated with an increased responsiveness of the progenitors to the hormone erythropoietin (Epo). A basic question in this clonal stem cell disorder is at what developmental stage this abnormality of the PV clone is phenotypically expressed. We have studied this question by comparing the development of Epo-dependent and Epo-independent CFUe from peripheral blood BFUe of the PV patient during the BFUe to CFUe transition in the liquid culture. The results indicated that both types of CFUe are generated and that in all cases tested the ratio of Epo-independent progenitors at both the BFUe and CFUe stage was similar indicating no preferential development of Epo-independent CFUe. These results suggest that the abnormality of the PV erythroid progenitors is expressed only at the CFUe level. Moreover, since the liquid culture did not contain Epo, the results also support the conclusion that BFUe do not require Epo for proliferation or differentiation into CFUe.

Topics: Adult; Aged; Cell Division; Cells, Cultured; Clone Cells; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Humans; Middle Aged; Polycythemia Vera

Topics: Diagnosis, Differential; Erythropoietin; Female; Humans; Male; Polycythemia Vera

Erythroid progenitors (CFU-E and BFU-E) growth in vitro from bone marrow and peripheral blood of patients with polycythemia vera (PV) was studied using a methylcellulose culture technique. The aim of the study was to find out whether the in vitro colony formation of peripheral blood could be used in the differential diagnosis of PV. In all 25 patients studied, endogenous colonies were found in the bone marrow and peripheral blood. The parallel study of both bone marrow and peripheral blood erythroid progenitors indicates that the presence of endogenous BFU-E in peripheral blood is a dependable test for PV. The results presented here showed that the abnormalities in PV erythroid progenitors are expressed at the level of both CFU-E and BFU-E, suggesting multiple changes in the erythroid progenitors. Our finding indicate that peripheral blood BFU-E differ from bone marrow BFU-E with regard to their dependence for further differentiation on BPA, the activity present in PHA-LCM.

Topics: Adult; Bone Marrow; Cells, Cultured; Dose-Response Relationship, Drug; Erythroid Precursor Cells; Erythropoietin; Female; Humans; Leukocytes; Male; Methylcellulose; Middle Aged; Phytohemagglutinins; Polycythemia Vera

Radioimmunoassay of erythropoietin (EPO) has been used for evaluating its clinical usefulness in distinguishing polycythaemia vera (PV) from pure erythrocytosis (PE). A normal log distribution (13.40 mU/ml +/- 2.45) was observed in the 66 reference samples. Similar results were observed in 29 pure thrombocytaemias (13.23 +/- 5.19). In PV patients, whether in clinical remission or in active phase, the EPO titer was lower than normal values (respectively 7.32 +/- 3.63 and 6.59 +/- 2.75), without any correlation with the haematocrit (44 to 51% and 52 to 71%). In the anaemic cases (excessive therapy, spent phase, myelofibrosis), a slight excess of EPO titer was observed, but less than expected when taking the hematocrit into account. In contrast, pure erythrocytosis is a very heterogeneous group, with the highest EPO values in the well-defined secondary cases, and normal or slightly excessive values in most of the cases, but with some low values similar to those observed in PV cases. In term of PV diagnosis, a 90% predictive value is observed when a "cut-off" value of 11 mU/ml is chosen. When the "cut-off" is 16 mU/ml, no PV case was observed beyond this value. We conclude that EPO assay is useful in myeloproliferative diseases from a practical point of view.

Topics: Diagnosis, Differential; Erythropoietin; Humans; Polycythemia; Polycythemia Vera; Radioimmunoassay; Thrombocytosis

The dependence of the serum erythropoietin (Epo) level on the blood hemoglobin concentration was compared in patients suffering from leukemia and ulcerative colitis. In leukemia, the level of immunoreactive and bioactive Epo was generally much higher than in ulcerative colitis at comparable degrees of anemia. The highest Epo values were found in patients with severe bone marrow insufficiency of erythropoiesis. These findings support the hypothesis that the plasma level of Epo depends not only on the hemoglobin concentration of the blood but is also influenced by the proliferative activity of the erythron.

Topics: Anemia; Animals; Colitis, Ulcerative; Erythropoiesis; Erythropoietin; Gastrointestinal Hemorrhage; Hemoglobins; Humans; Leukemia; Mice; Polycythemia Vera

Forty adult subjects were studied with the aim of establishing positive diagnostic criteria in primary proliferative polycythaemia (polycythaemia vera, PPP). These comprised 14 patients with PPP, eight secondary polycythaemia (SP), five idiopathic erythrocytosis, and 13 normal subjects, classified under standard criteria following comprehensive investigation for causes of SP. Erythroid colony formation from peripheral blood in a serum-free system was assayed with the addition of recombinant human erythropoietin (Epo), interleukin 3 (IL3), or alpha-interferon (alpha-IFN). The differential sensitivity of primitive and mature progenitors (BFU-E) was assessed by counting the number of clusters ('sub-colonies') comprising each erythroid burst. 'Endogenous' erythroid colonies were found in both PPP (56%) and controls (17%). In Epo containing cultures, the mean number of clusters per burst was lower in PPP than controls, and the percentage of small (less than or equal to 8 clusters) bursts was higher. In PPP primitive BFU-E demonstrated greater dependence on IL3 than controls, and mature BFU-E greater inhibition by alpha-IFN. These findings suggest an abnormal response to several growth factors, rather than dysfunction of a single growth factor receptor. Regression analysis of these data defined a discriminant of high diagnostic sensitivity and specificity. This discriminant accurately predicted diagnosis in a further nine polycythaemic patients.

Topics: Adolescent; Adult; Aged; Aged, 80 and over; Cells, Cultured; Diagnosis, Differential; Erythroid Precursor Cells; Erythropoietin; Female; Humans; Interferon Type I; Interleukin-3; Male; Middle Aged; Polycythemia; Polycythemia Vera; Recombinant Proteins

Serum erythropoietin (Ep) was measured by radioimmunoassay before and 24 hours after therapeutic phlebotomies in patients with polycythemia vera (PV) and in normal subjects before and after phlebotomies of comparable volumes. In addition the in vivo oxygen affinity of hemoglobin (P50) was calculated, and red cell indices and 2,3 DPG values were measured. Paired t tests determined whether the differences between pre- and post--phlebotomy values were statistically significant. Blood hemoglobin (Hb) levels declined after phlebotomy, and generally continued to be at or above normal levels. Serum Ep increased after phlebotomy in both groups of subjects. The in vivo P50 value for patients with PV (29.4 +/- 0.4 mmHg) was significantly (p less than 0.005) greater than the normal value (27.2 +/- 0.5). Seven of the PV patients (5 males, 2 females) were restudied. Their Hb and hematocrit values were either normal or slightly higher than normal. The MCV for 4 males and 1 female was below normal. The MCHC was slightly lower than normal and reticulocytosis was not present. The male PV patients had greater than normal 2,3 DPG values and most had right-shifted P50 values. There was no correlation between 2,3 DPG values and P50 values. The female patients did not have 2,3 DPG values consistently greater than normal and their P50 values were not right shifted. These results showed that serum EP increased in response to small reductions in Hb even when subnormal Hb values were not produced. The reduced affinity of Hb in PV patients may explain earlier observations that patients with PV have lower levels of urinary or plasma Ep than normals with the same hematocrits.

Topics: 2,3-Diphosphoglycerate; Adult; Bloodletting; Diphosphoglyceric Acids; Erythrocyte Indices; Erythropoietin; Female; Hemoglobins; Humans; Male; Oxygen; Polycythemia Vera

The role of erythropoietin (EP) in polycythemia vera (PV) is controversial, with some experiments suggesting that erythroid progenitors in PV are exquisitely sensitive to EP and EP dependent, and others suggesting that PV progenitors are EP independent. We have examined the characteristics of the EP receptor (EP-R) on erythroid colony-forming cells (ECFC) from patients with PV. In contrast to normal ECFC, which have two classes of EP-R, with 20% showing high affinity (Kd = 0.13 nM; range, 0.04-0.20 nM) and the remainder lower affinity (Kd = 0.37 nM; range, 0.28-0.57 nM), PV ECFC show a single class of 851 low affinity EP-R with Kd = 0.72 nM (range, 0.36-0.85 nM). ECFC from patients with secondary (EP driven) polycythemia or anemia show two classes of EP-R (Kd = 0.18 and 1.10 nM, respectively). Attempts to remove tightly bound EP from putative high affinity EP-R in PV did not reveal any higher affinity receptors. Determination of molecular size by crosslinking showed two proteins of 90 and 100 kD similar to those seen with normal EP-R. These studies indicate the PV ECFC have EP-R that are structurally similar to normal EP-R but lack the higher binding affinity for EP.

Topics: Cross-Linking Reagents; Electrophoresis, Polyacrylamide Gel; Erythroid Precursor Cells; Erythropoiesis; Erythropoietin; Humans; Polycythemia Vera; Receptors, Cell Surface; Receptors, Erythropoietin

Megakaryocyte colony formation, as identified by conventional techniques, was observed in precursor cell cultures from peripheral blood in 8 of 20 consecutive patients with diagnosis of myeloproliferative disease (4/11 patients with polycythemia vera, 3/5 with essential thrombocythemia, 1/2 with primary osteomyelofibrosis and 2 with a myeloproliferative syndrome not further assessable), but not in 50 healthy controls (p less than 0.0001). 7 cultures showed spontaneous erythroid colonies, but were negative for megakaryocyte colonies. Megakaryocyte colony formation was independent of added erythropoietin, plasma or human leukocyte-conditioned medium, but was dependent on the presence of accessory cells. The cells in megakaryocyte colonies had the characteristic morphology of megakaryocytes and stained positively with the IIIa/IIb monoclonal anti-platelet antibody. Thus, megakaryocyte colony formation by precursor cells from peripheral blood in the absence of exogenous stimulating factors seems to be a phenomenon specific for myeloproliferative disease. Differential diagnosis of thrombocythemia may be facilitated by demonstration of endogenous megakaryocyte colony formation, which does not occur in secondary disease.

Topics: Cell Nucleus; Cells, Cultured; Colony-Forming Units Assay; Culture Media; Cytoplasm; Diagnosis, Differential; Erythropoietin; Fluorescent Antibody Technique; Hematopoietic Stem Cells; Humans; Leukocytes; Megakaryocytes; Myeloproliferative Disorders; Polycythemia Vera; Primary Myelofibrosis; Thrombocythemia, Essential

To further define the growth factors required for the in vitro proliferation of erythroid progenitors in polycythemia vera (PV), we have compared the ability of interleukin 3 (IL-3) and granulocyte-macrophage colony-stimulating factor (GM-CSF) to support the growth of erythropoietin (Epo)-dependent and -independent erythroid colony formation. By using nonadherent mononuclear cells from peripheral blood, Epo-dependent colony formation was enhanced by IL-3 and GM-CSF in PV patients. Comparable results were obtained with normal erythroid progenitors. Augmenting effects of IL-3 and GM-CSF were observed on spontaneous erythroid colony formation, i.e., erythroid colony formation in the absence of exogenous supplied Epo. This was not due to a small amount of Epo in the culture media because an anti-Epo antibody did not prevent endogenous colony formation, nor did it prevent the enhancing effects of IL-3. Finally it was observed that in contrast to IL-3, monocyte depletion was required for the enhancing effects of GM-CSF on erythroid colony formation. These results provide evidence that endogenous colony formation in PV is independent of Epo but can be augmented by IL-3 or GM-CSF.

Topics: Cells, Cultured; Colony-Stimulating Factors; Erythropoiesis; Erythropoietin; Granulocyte-Macrophage Colony-Stimulating Factor; Growth Substances; Humans; Immunologic Techniques; In Vitro Techniques; Interleukin-3; Polycythemia Vera

The ability of erythroid cultures to distinguish among myeloproliferative disorders was examined. We studied 14 patients with polycythemia vera (PV), 11 with chronic myelogenous leukemia (CML), four with non-PV erythrocytosis, two with agnogenic myeloid metaplasia, as well as three normal fetuses and greater than 25 normal adults. Endogenous, i.e. grew without added erythropoietin, bone marrow CFU-E-derived colonies were observed in all but one PV patient. However, endogenous blood BFU-E-derived bursts were observed in only eight of 14 PV patients. Endogenous erythroid colonies were not seen in cultures from any normal adults or fetuses, or patients with CML, erythrocytosis, or myeloid metaplasia. In PV, relative HbF synthesis was always greater in cultures without erythropoietin, while in cultures from all other patients relative HbF synthesis was similar to that observed in cultures from normal individuals. We conclude that PV and CML are distinguishable in culture since CML patients do not have endogenous growth. Most important, endogenous bone marrow CFU-E-derived colonies are the only consistently unique observation in patients with PV, and endogenous CFU-E- and BFU-E-derived colonies and bursts are not uniformly observed in PV blood cultures. In-vitro studies of erythropoiesis to confirm the diagnosis of PV, therefore, require marrow when endogenous colonies and bursts are absent from blood cultures.

Topics: Adolescent; Adult; Aged; Bone Marrow; Child; Colony-Forming Units Assay; Erythroblasts; Erythropoietin; Globins; Hematopoietic Stem Cells; Humans; Leukemia, Myelogenous, Chronic, BCR-ABL Positive; Middle Aged; Polycythemia; Polycythemia Vera

The development of highly specific antibodies against recombinant human erythropoietin (EPO) has recently made the accurate radioimmunological measurement of serum levels of this hormone possible. In this study we determined the serum-EPO levels in 100 healthy volunteers, in 54 patients suffering from polycythemia vera and in 51 patients with secondary polyglobulia. The mean levels for the healthy group were found to be 11.3 +/- 3.4 mU/ml in females and 8.0 +/- 3.2 mU/ml in males. Patients with polycythemia vera had serum-EPO levels of 4.3 mU/ml, while those with secondary polyglobulia had significantly higher levels averaging 30.3 mU/ml (p less than 0.0001). However, an overlapping of serum-EPO values in the range between 10 and 20 mU/ml occasionally occurs. Our results show that measurement of the serum-EPO level can be useful in the differential diagnosis of polyglobulias. Additionally, sequential EPO level measurements after phlebotomy and after hemorrhage show a pronounced increase in serum-EPO in secondary polyglobulia, while in polycythemia vera the level only increases slightly.

Topics: Diagnosis, Differential; Erythropoietin; Female; Male; Polycythemia; Polycythemia Vera; Reference Values

The accurate radioimmunological measurement of serum erythropoietin (EPO) levels has only been possible since the development of highly specific antibodies directed against recombinant human EPO. In the present study, we determined the serum EPO levels in 100 healthy volunteers and in over 300 patients with anemias and hyperglobulinemia of various causes. In the healthy group, the females had levels of 11.3 +/- 3.4 mU/ml, while the males had levels of 8 +/- 3.2 mU/ml. The serum EPO concentrations were inversely related to the degree of anemia in patients with nonrenal anemias, while predialysis patients with renal anemias showed only partially such a tendency. Hemodialysis patients exhibited EPO-levels that were inadequately low relative to the degree of anemia. Patients with hyperglobulinemia had significantly higher serum EPO-levels than healthy individuals and polycythemia vera patients, the latter having particularly low serum EPO levels. Our results show that the determination of serum EPO levels can be of value in the differential diagnosis of hyperglobulinemia. Finally, sequential measurements document fluctuating serum EPO-levels after gastrointestinal hemorrhages and in patients with iron deficiency anemias receiving iron substitution. The probable reason for this phenomenon seems to be the intermittent utilisation of the hormone by EPO-sensitive erythropoietic precursor cells.

Topics: Acquired Immunodeficiency Syndrome; Anemia; Anemia, Hypochromic; Erythropoietin; Female; Gastrointestinal Diseases; Hemoglobins; Humans; Hypergammaglobulinemia; Kidney Diseases; Male; Myelodysplastic Syndromes; Polycythemia Vera; Renal Dialysis

A sensitive competitive radioimmunoassay for the determination of circulating human erythropoietin (Epo) is presented. This RIA of human Epo is founded on the extensive application of a recombinant protein approach. Thus, highly purified recombinant human Epo is employed both as a tracer, immunogen and occasionally as standard in the RIA. The lower limit of detection is approximately 8 mU/ml and well below the mean value of Epo in normal individuals (25.6 +/- 6.6 mU/ml). The intra- and interassay variation are 6.3 and 9.2%, respectively. The documentation of consistent alterations in Epo plasma levels of selected categories of anemic patients establishes the clinical relevance of the immunoassay. The radioimmunoassay of human Epo fulfills reasonable requirements of a clinical routine analysis in terms of both specificity, sensitivity and practicability.

Topics: Anemia; Erythropoietin; Humans; Polycythemia Vera; Radioimmunoassay

The antigenic characteristics of erythropoietin (Ep) dependent and independent ("endogenous") erythroid progenitor cells (BFU-E and CFU-E) from patients with polycythaemia vera (PV) and idiopathic myelofibrosis (MF) were studied using 9 selected murine monoclonal antibodies (McAbs) in a complement dependent cytotoxicity assay followed by culture in methyl cellulose. McAbs L343 (HLA-DR antigen) S3-13 and S17-25 reacted with about 50% of BFU-E in MF and PV. In contrast HLA-DR antigens were not presented on CFU-E. McAbs R1.B19, WHGS29.1, PMN29 and PM81 recognized the antigens on less than 25% of BFU-E and CFU-E. The reactivity of Ep-dependent and Ep-independent BFU-E and CFU-E was similar with the majority of McAbs. However, McAbs S4-7 and VIM-2 reacted with a higher proportion of Ep-independent BFU-E than of Ep-dependent BFU-E. These results indicate that Ep independent, neoplastic BFU-E in PV and MF are more mature than their Ep dependent counterparts. The antigens expressed on erythroid progenitor cells from patients with PV and MF were earlier found on BFU-E and CFU-E from normal bone marrow and peripheral blood, usually on a higher proportion of cells, than in myeloproliferative disorders.

Topics: Aged; Antibodies, Monoclonal; Antigens, Surface; Cytotoxicity Tests, Immunologic; Erythropoietin; Humans; Middle Aged; Polycythemia Vera; Primary Myelofibrosis; Stem Cells

Erythropoietin (Epo) titers in various hematological states were determined by a radioimmunoassay. Epo titers in patients with uremic anemia and iron deficiency anemia were inversely correlated with their respective hemoglobin concentrations. Epo titers in patients with uremic anemia were significantly lower than those in patients with iron deficiency anemia with comparable hemoglobin concentrations.

Topics: Anemia; Anemia, Aplastic; Anemia, Hypochromic; Erythropoietin; Humans; Kidney Failure, Chronic; Polycythemia Vera; Renal Dialysis; Uremia

The definite differential diagnosis between spurious polycythemia (SP) and pure erythrocytosis (PE) was tested. Serum erythropoietin (EPO) levels in 6 patients with PE were 12.8 +/- 3.7 mU/ml and were significantly lower than those of both 19 normal controls (28.5 +/- 15.0 mU/ml) and 9 patients with SP (21.3 +/- 10.2 mU/ml). Three of 11 patients with SP and 1 of 3 patients with PE had significantly higher marrow erythroid progenitor cells (CFU-Es) than those of the normal controls. Spontaneous CFU-E growth (CFU-E growth in the absence of added EPO) was found in 4 of 11 patients with SP, 1 of 3 patients with PE, and all patients with polycythemia vera. However, the number of spontaneous CFU-E was low in SP and PE. The measurements of serum EPO levels and CFU-E growth did not provide differentiation between PE and SP. Even if some patients, whose total red cell volumes are either higher than 12.5% above the mean predicted values or their CFU-E growth is great, are diagnosed as SP, consideration should be made that they might, in fact, have PE.

Topics: Adult; Aged; Bone Marrow Cells; Diagnosis, Differential; Erythrocyte Indices; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Male; Middle Aged; Polycythemia; Polycythemia Vera

Topics: Animals; Blood Cells; Blood Platelets; Colony-Stimulating Factors; Erythropoiesis; Erythropoietin; Granulocytes; Hematopoiesis; Humans; Kidney; Lymphocytes; Megakaryocytes; Neoplasms; Polycythemia Vera; Receptors, Cell Surface; Receptors, Erythropoietin

We assessed the diagnostic value of determinations of serum levels of immunoreactive erythropoietin in 90 patients referred for the investigation of a raised packed red-cell volume (hematocrit) and possible erythrocytosis (a red-cell volume greater than that predicted by weight, body-surface area, or both). The mean values for erythropoietin were 16 mIU per milliliter (range, 8 to 22) in patients with polycythemia rubra vera (n = 24), 30 mIU per milliliter (range, 14 to 123) in patients with secondary erythrocytosis (n = 12), 27 mIU per milliliter (range, 13 to greater than 400) in patients with erythrocytosis of unknown origin (n = 19), and 25 mIU per milliliter (range, 18 to 35) in normal controls (n = 25). The values in the patients with polycythemia rubra vera were lower than those in the other three groups (P less than 0.01, P less than 0.05, and P less than 0.0001, respectively). In subjects with an increased packed red-cell volume but without erythrocytosis (n = 35), the levels of serum erythropoietin were the same as those in normal controls. Among patients with erythrocytosis with an unknown cause, abnormally high serum erythropoietin levels were found in 3 of 19 subjects, and in 1 of these 3 the abnormality was intermittent. Thus, measurement of serum erythropoietin in a single sample may be misleading and may not have high discriminatory value in distinguishing between polycythemia rubra vera and secondary erythrocytosis. This assay is useful in identifying patients with secondary erythrocytosis who have inappropriate erythropoietin secretion.

Topics: Adult; Aged; Diagnosis, Differential; Erythropoietin; Female; Hematocrit; Humans; Male; Middle Aged; Polycythemia; Polycythemia Vera

A two-stage cell culture assay specific for human erythroid burst-promoting activity (BPA) is described. Human peripheral blood mononuclear cells were cultured in suspension with or without a BPA test sample for two days, then transferred to methylcellulose medium with added erythropoietin (EPO) and incubated for ten more days, and finally BFU-E-derived colonies were scored. An increase in number of colonies due to the presence of BPA was observed that was proportional to the concentration of BPA in the test sample. This response was linear with respect to number of cells plated between 2 and 5 X 10(5)/ml. The system was standardized with a partially purified human urinary BPA preparation. Dose responses to urinary protein preparations, plasma, and serum were parallel. The assay system was found to be nonresponsive to highly purified EPO and to bacterial endotoxin. It was determined that BPA action was confined to the suspension culture stage of the assay, while EPO presence was an absolute requirement during methylcellulose culture. In the two-stage assay optimal amounts of BPA caused up to 358% increases of BFU-E-derived colonies; the same amounts of BPA added to conventional methylcellulose cultures caused only up to 54% increases over the number of colonies obtained with EPO alone. Plasma and serum BPA levels of hematologically normal and abnormal individuals showed no correlation with EPO levels and hemoglobin (Hb) concentrations. This seems to rule out the possibility that BPA elaboration is regulated by oxygen availability or the amount of EPO circulating in an organism.

Topics: Anemia, Aplastic; Animals; Cell Count; Cells, Cultured; Colony-Forming Units Assay; Colony-Stimulating Factors; Dose-Response Relationship, Drug; Endotoxins; Erythropoiesis; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Mice; Polycythemia Vera

We assessed the prevalence of overt and latent primary myeloproliferative disorders in hepatic vein thrombosis. Cultures of bone marrow or peripheral blood mononuclear cells were done in 20 patients with Budd-Chiari syndrome. Erythroid colony formation in the absence of erythropoietin, which is a reliable indicator for a primary myeloproliferative disorder, was seen in 16 patients in whom Budd-Chiari syndrome was due to hepatic vein thrombosis, including 13 women aged 18 to 45 years. Among these 16 patients, the conventional criteria for the diagnosis of a primary myeloproliferative disorder were met in only 2. Primary myeloproliferative disorder, often without peripheral blood changes, is a major cause of hepatic vein thrombosis in young women.

Topics: Adolescent; Adult; Budd-Chiari Syndrome; Colony-Forming Units Assay; Contraceptives, Oral; Erythrocyte Volume; Erythropoiesis; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Male; Middle Aged; Myeloproliferative Disorders; Polycythemia Vera; Prospective Studies

Topics: Colony-Forming Units Assay; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Male; Polycythemia Vera

Severe erythrocytosis is associated with increased whole blood viscosity and impaired blood flow. Since a reduced blood flow will cause tissue hypoxia and since tissue hypoxia is associated with increased synthesis of erythropoietin, erythrocytosis per se should cause an increase in the rate of red cell production. This, however, does not occur and severe erythrocytosis in patients with polycythemia vera does not lead to increased synthesis of erythropoietin. We propose here that the reason for this discrepancy is that decreased blood flow to the kidneys, the site of erythropoietin synthesis, does not cause renal tissue hypoxia. The oxygen tension in the kidneys is to a great extent determined by the consumption of oxygen used for sodium reabsorption and since sodium reabsorption is roughly proportional to glomerular filtration, a decreased flow of blood should be matched by a decreased oxygen consumption leaving the tissue tension of oxygen unchanged. Consequently, the location of an oxygen sensor in the kidneys controlling erythropoietin production appears to be most fortuitous since it prevents the development of a vicious circle, with erythrocytosis causing more erythrocytosis.

Topics: Blood Viscosity; Erythropoietin; Glomerular Filtration Rate; Hematocrit; Humans; Hypoxia; Kidney; Oxygen Consumption; Polycythemia; Polycythemia Vera; Radioimmunoassay; Regional Blood Flow; Sodium

A commercially available hemagglutination inhibition assay kit for the measurement of erythropoietin (EPO) was evaluated for its usefulness in the differential diagnosis of the polycythemic states. Serum samples were obtained from patients with polycythemia rubra vera (active and controlled), secondary and relative polycythemia and from normal controls. Firstly, we found that the mean EPO level (+/- SD) of our normal controls (81 +/- 69 miu/ml) was higher than the manufacturer's quoted normal range (15-59 miu/ml) and that there was a significant spread of values (7-233 miu/ml). Secondly, within each patient subgroup studied, the spread of data points was so wide that interpretation of individual data points would be impossible. We conclude that this assay kit is of little value for serum EPO estimation and in the differential diagnosis of polycythemia.

Topics: Diagnosis, Differential; Erythropoietin; Hemagglutination Inhibition Tests; Humans; Polycythemia; Polycythemia Vera

Serum erythropoietin (ESF) levels and the numbers of marrow and blood erythroid progenitors (CFU-Es) of patients with chronic myeloproliferative disorders (CMPD) were studied simultaneously. The numbers of marrow and blood CFU-Es per 1 x 10(5) cells were normal or greatly elevated. There was an inverse correlation between the hemoglobin concentration and the serum ESF level in patients with chronic myelogenous leukemia when the hemoglobin concentration ranged from 9.0 to 13.0 g/100 ml. The serum ESF level was closely related to the hemoglobin concentration in CMPD and it was suggested that the negative feedback mechanism might operate in anemic patients with CMPD.

Topics: Adult; Animals; Bone Marrow Cells; Cell Count; Chronic Disease; Erythropoietin; Female; Hematopoietic Stem Cells; Hemoglobins; Humans; Leukemia, Myeloid; Male; Mice; Middle Aged; Myeloproliferative Disorders; Polycythemia Vera; Primary Myelofibrosis; Thrombocytosis

Topics: Adult; Aged; Blood Cell Count; Erythropoiesis; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Male; Middle Aged; Polycythemia Vera

An in vitro method for the determination of erythropoietin (EPO) titres in human serum samples was developed to detect low levels of EPO titres in various haematological states. Fetal mouse liver cells (FMLC) were incubated in methylcellulose-containing media, and radioiron incorporation into haem was estimated in cultured FMLC. We could determine the concentrations of EPO from around 20 to 600 mU per ml of a very small amount of serum samples from patients and normal adults. Using this method, EPO titres of normal adults (54 +/- 31 mU/ml), chronic renal failure patients (29 +/- 23 mU/ml), aplastic anaemia patients (545 +/- 74 mU/ml), and polycythaemia vera patients (16 +/- 7 mU/ml) were determined.

Topics: Anemia, Aplastic; Animals; Cells, Cultured; Culture Media; Erythropoietin; Humans; Kidney Failure, Chronic; Methods; Mice; Mice, Inbred ICR; Polycythemia Vera

In eight patients with polycythaemia vera and 20 normals, mononuclear cells of the peripheral blood were cultured in methylcellulose with and without exogenously added erythropoietin. The cultures were set up with fetal calf serum and with a serum-free medium of unsaturated fatty acids, lecithin and cholesterol. In patients with polycythemia vera, we found a proliferation of haematopoietic colonies without erythropoietin in both culture systems which could not be seen in normals. The proliferation of endogenous colonies in serum-free media suggests that polycythaemia vera is a clonal disorder which can be modulated by serum factors and by erythropoietin.

Topics: Adult; Aged; Cell Division; Culture Media; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Male; Middle Aged; Polycythemia Vera

Topics: Blood Cell Count; Bloodletting; Diagnosis, Differential; Erythropoiesis; Erythropoietin; Hematocrit; Humans; Kidney Diseases; Lung Diseases; Oxygen; Phosphorus Radioisotopes; Polycythemia; Polycythemia Vera

This is a study of in vitro production of erythropoietic colonies from peripheral blood and bone marrow of normal subjects and patients with different polycythaemic conditions. Proliferative stimuli included: 1. Fetal calf serum (FCS) only. 2. FCS plus a source of erythropoietic-stimulating activity (ESA). 3. FCS + ESA + erythropoietin (Ep). It was found that normal subjects and patients with secondary polycythaemia (SP) exhibited full colony growth only in the presence of both ESA and Ep, while patients with primary proliferative polycythaemia (PPP) showed colony production with FCS alone, further enhanced in the presence of ESA and Ep. A group of patients with idiopathic erythrocytosis (IE), namely with an increase of red cell mass not accompanied by other signs of myeloproliferative disorder, and without underlying cause, showed a heterogeneous response to ESA which in some patients was significantly greater than in normal subjects or in SP patients. It appears therefore that sensitivity of erythropoietic colony formation to Ep and ESA may be helpful in differentiating among various forms of polycythaemia; this study also establishes the heterogenicity of the IE group.

Topics: Bloodletting; Bone Marrow Cells; Colony-Forming Units Assay; Colony-Stimulating Factors; Erythropoiesis; Erythropoietin; Female; Hematocrit; Humans; Male; Polycythemia; Polycythemia Vera

Peripheral blood burst forming units-erythroid (BFU-E) were measured by the plasma clot cell culture technique in patients with the various subtypes of polycythaemia and compared to normal. 10 patients with active polycythaemia rubra vera (PRV) were found to have a mean BFU-E level of 48 +/- 15.8 (SEM) per 5 X 10(5) cultured cells which was significantly different from normals (4 +/- 1.1, P less than 0.02), patients with controlled PRV (7 +/- 1.6, P less than 0.025), secondary polycythaemia (1 +/- 0.3, P less than 0.015) and relative polycythaemia (0, P less than 0.015). Burst forming units were found to fall to normal levels in patients with PRV with appropriate disease control and then to rise again in patients untreated for more than 18 months. Clinical aspects. Measurement of BFU-E levels from the peripheral blood could provide a useful adjunct from an accessible source in the differential diagnosis of polycythaemia as well as being of use in serial monitoring of patients with PRV.

Topics: Bloodletting; Colony-Forming Units Assay; Erythrocytes; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Humans; Polycythemia; Polycythemia Vera

Erythropoietic progenitors from peripheral blood of normal individuals or patients with polycythemia vera (PV) were cultured in methylcellulose medium containing 2.5 U/ml of erythropoietin (Ep). After 7-9 days, colonies considered to be early stage large bursts were individually removed, resuspended in a small volume of fresh methylcellulose medium, and then divided between 2 dishes. To one of these secondary cultures, sufficient Ep was added to bring the concentration of Ep up to approximately 3 U/ml. To the other was added an equal volume of medium but no Ep. The final concentration of Ep in these cultures was determined to be less than 0.01 U/ml. Nine days later, both types of secondary cultures were scored for the presence of colonies containing 8 or more hemoglobinized erythroblasts. Of 90 primary colonies from 3 normal individuals assessed in this way, 59 gave secondary erythroid colonies in the high Ep cultures, while none gave secondary erythroid colonies in the low Ep cultures. Additional control experiments in which primary colonies from normal individuals were divided into duplicate high Ep cultures showed that on average, the procedure used divided primary colonies equally. Of 109 primary colonies from 5 PV patients that yielded secondary erythroid colonies in the high Ep cultures, 21 yielded no secondary erythroid colonies in the low Ep cultures. The other 88 yielded erythroid colonies in both, but the secondary colonies in the low Ep cultures were consistently smaller in size and significantly fewer in number. Similar results were obtained when primary colonies were generated in cultures to which no Ep was added. These findings indicate that primitive BFU-E in patients with PV can be subdivided into 2 populations: a minor population restricted to the production of erythroid colony-forming cells (Ep-dependent progenitors) that require Ep for their detection, and a major population that is not restricted in this way. In addition, these experiments show that most of the primitive BFU-E that generate Ep-independent progenitors also produce significant numbers of cells that are Ep-dependent.

Topics: Adult; Aged; Cell Differentiation; Cells, Cultured; Clone Cells; Colony-Forming Units Assay; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Male; Middle Aged; Phenotype; Polycythemia Vera

Peripheral blood mononuclear cells from five patients with polycythemia vera (P. vera) and three with other polycythemias were cultured in a methylcellulose system. Colony-forming unit-erythroid (CFU-E) colonies appeared after seven days in the absence of added erythropoietin (Ep) in all P. vera cultures. A pattern of growth similar to the one seen for P. vera patients occurred in the culture from a patient in whom that disease was suspected. In the cultures from two of the patients with other polycythemias, erythroid colonies did not appear even in the presence of Ep. These findings emphasize the potential value of culturing peripheral blood for CFU-E colonies in diagnosing polycythemia vera.

Topics: Adult; Aged; Cells, Cultured; Colony-Forming Units Assay; Erythropoietin; Female; Humans; Male; Middle Aged; Monocytes; Polycythemia Vera

Topics: Bone Marrow Cells; Cells, Cultured; Colony-Forming Units Assay; Erythrocytes; Erythropoietin; Hematopoietic Stem Cells; Humans; Polycythemia Vera

A radioimmunoassay for erythropoietin (Ep) in human serum is described. Evidence of assay validity includes parallelism of dilution curves of the 2nd International Reference Preparation (IRP) (used as standard) with test sera, a fall in serum immunoreactive Ep following blood transfusion and an increase in serum immunoreactive Ep with increasing severity of anaemia except in anaemias associated with severe renal failure. The geometric mean estimate of serum immunoreactive Ep in 93 normal men and women was 13.3 miu/ml above the low erythropoietin serum pool used as baseline for the assay. Extra-renal erythropoietin was detectable in the assay and gave a dilution curve parallel to the 2nd IRP.

Topics: Adult; Aged; Anemia; Blood Transfusion; Erythropoietin; Female; Humans; Iodine Radioisotopes; Male; Middle Aged; Polycythemia Vera; Radioimmunoassay; Reference Values

Previous in vitro studies on committed hematopoietic progenitors have suggested that polycythemia vera (PV) is a clonal disorder arising in a pluripotential hematopoietic stem cell. In this study, recently developed technics for clonal assay of a human multipotential progenitor cell (CFU-GEMM) were used to assess the functional characteristics of CFU-GEMM in 19 PV patients. These studies showed: (a) increased numbers of detectable CFU-GEMM in blood and bone marrow samples of PV patients as compared with normals (P less than 0.002 and P less than 0.02, respectively); (b) erythropoietic differentiation of PV CFU-GEMM without exogenous erythropoietin (Ep) in culture (in marked contrast to CFU-GEMM of both normals and subjects with secondary erythrocytosis which require exogenous Ep for terminal hemoglobinization of their erythroid component), a property shown by experiments with an anti-Ep antiserum to be related to increased sensitivity of PV CFU-GEMM to Ep; (c) increased megakaryocyte formation by PV CFU-GEMM as compared with normals (P less than 0.025); and (d) a linear relationship, extrapolating to the origin, between CFU-GEMM detected and cells cultured. These studies demonstrate that at least two clinical features of PV, increased erythropoiesis and megakaryocytopoiesis, are reflected in corresponding functional characteristics of PV CFU-GEMM, and provide direct evidence of distinctive pluripotential stem cell activity in this disorder.

Topics: Bone Marrow; Clone Cells; Colony-Forming Units Assay; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Humans; In Vitro Techniques; Megakaryocytes; Polycythemia Vera

Techniques are described in detail for the RIA of human Ep in unextracted plasma or serum. With 100 microliters of sample, the essay is sensitive at an Ep concentration of approximately 4 mU/ml, and when required, the sensitivity can be increased to 0.4 mU/ml, a range considerably less than the concentration observed in normal human beings. This is approximately 100 times more sensitive than existing in vivo bioassays for this hormone. Studies concerned with the validation of the Ep RIA show a high degree of correlation with the polycythemic mouse bioassay. Dilutions of a variety of human serum samples show a parallel relationship with the standard reference preparation for Ep. Validation of the RIA is further confirmed by observations of appropriate increases or decreases in circulating Ep levels in physiological and clinical conditions known to be associated with stimulation of suppression of Ep secretion. Significantly different mean serum concentrations of 17.2 mU/ml for normal male subjects and 18.8 mU/ml for normal female subjects were observed. Mean plasma Ep concentrations in patients with polycythemia vera are significantly decreased, and those of patients with secondary polycythemia are significantly increased as compared to plasma levels in normal subjects. These results demonstrate an initial practical value of the Ep RIA inthe hematology clinic, which will most certainly be expanded with its more extensive use.

Topics: Adolescent; Adult; Animals; Antibodies; Biological Assay; Erythropoietin; Female; Humans; Male; Mice; Mice, Inbred Strains; Middle Aged; Polycythemia; Polycythemia Vera; Radioimmunoassay; Serum Albumin; Serum Albumin, Bovine; Sex Factors

Topics: Adult; Diagnosis, Differential; Erythropoietin; Humans; Polycythemia; Polycythemia Vera; Prognosis

Circulating erythroid progenitors from 10 patients with polycythaemia vera, nine with secondary polycythaemia and 10 normal subjects were studied in culture to determine their frequency, requirements for erythropoietin (ESF) and physical characteristics. Increased numbers of erythroid progenitors were detected in circulation from the patients with polycythaemia vera when compared to patients with secondary polycythaemia or normals, and 19 +/- 3% of these progenitors formed endogenous colonies without added ESF. Velocity sedimentation analysis showed that the majority of ESF-dependent erythroid progenitors in all study groups sedimented at 6.46 mm/h. The circulating endogenous erythroid colony-forming cells in polycythaemia vera patients sedimented more rapidly. Tritiated thymidine suicide experiments failed to detect significant differences between the percentages of endogenous and ESF-dependent progenitors in DNA synthesis in polycythaemia patients and normals. Thus, differences in cell cycle state were not responsible for the more rapid sedimentation of endogenous colony-forming cells. These results further define characteristics of circulating erythroid progenitors in polycythaemia vera and normal subjects and suggest that simple culture of peripheral blood cells may be useful in the differential diagnosis of polycythaemia.

Topics: Blood Sedimentation; DNA; Erythrocyte Count; Erythrocytes; Erythropoietin; Hematopoietic Stem Cells; Humans; Interphase; Polycythemia; Polycythemia Vera

A radioimmunoassay (RIA) method for erythropoietin (Epo) was developed and validated against the polycythaemic mouse assay. The correlation was good, with a r = 0.94. Several other criteria of specificity were also filled by the RIA, which had a lower detection limit of 5 microU/ml. The mean serum-Epo level in 6 patients with secondary polycythaemia, 50.2 +/- 26.2 microU/ml, was significantly higher than in a group of 11 normal subjects, 28.7 +/- 7.2 microU/ml (P less than 0.0002). However, the Epo level in 31 polycythaemia vera (PV) patients, M = 21.9 +/- 6.6 microU/ml, was not significantly different from normal (P = 0.006). Since previous studies with bioassay of heat-treated and concentrated plasma samples have shown a decreased serum-Epo level in PV, Epo levels were measured before and after heat treatment and concentration of samples from normals and polycythaemics. It was found that the levels of immunoreactive material increased after heat treatment and 40 times concentration in samples from normals and patients with secondary polycythaemias, but decreased in PV. We conclude that the Epo levels in serum in the low range measured by our and previous RIA:s probably are not true Epo levels but are partly due to an unspecific serum effect, that was removed by heat treatment.

Topics: Anemia, Aplastic; Animals; Antibody Affinity; Antibody Specificity; Erythropoietin; Humans; Immune Sera; Mice; Polycythemia Vera; Radioimmunoassay

The ability of circulating progenitor cells from patients with polycythaemia vera (PV) and myelofibrosis with myeloid metaplasia (MMM) to develop erythroid colonies was studied in cultures with and without erythropoietin. In all normal controls, patients with secondary polycythaemia and MMM, erythroid colonies developed only after the addition of erythropoietin. Only in patients with PV, both in the active and spent phases of the disease, erythroid colonies developed in the absence of erythropoietin. The results indicate the perpetuation of erythropoietin-dependent, as well as erythropoietin-independent progenitors in both phases of this disease. Although spent PV often clinically resembles MMM, there is a basic difference in the behaviour of the circulating erythroid progenitors in these diseases which may serve as a useful tool in discriminating MMM from spent PV, when there is no history of active PV.

Topics: Adult; Aged; Cells, Cultured; Colony-Forming Units Assay; Erythrocyte Count; Erythropoietin; Hematopoietic Stem Cells; Humans; Middle Aged; Polycythemia Vera; Primary Myelofibrosis

An RIA for Ep has been developed that is highly sensitive and specific. A homogeneous Ep preparation was labeled with 125 I by the chloramine-T method to a specific activity of 90 to 136 micro Ci/microgram and immunoreactivity of 80%. Ep antiserum, which was produced to a human urinary Ep preparation (80 U/mg of protein), was adsorbed with normal human urinary and serum proteins without any loss in sensitivity of the RIA to increase the specificity of the assay. A good correlation was seen between the RIA and the exhypoxic polycythemic mouse assay (corr. coef. 0.967; slope 1.05 and "y" intercept 0.75). Ep titers in sera from 175 hematologically normal human subjects exhibited a normal frequency distribution and ranged between 5.8 and 36.6 mU/ml with a mean of 14.9 +/- 4.7 (S.D.) and median of 14.3 Serum Ep titers were markedly elevated in seven patients with aplastic anemia and one patient with pure red cell aplasia (1350 to 20,640 mU/ml) and were lower than normal in two patients with polycythemia vera (8.1 and 9.4 mU/ml). The serum Ep titers in a prenephrectomy patient with chronic glomerulonephritis (32.1 mU/ml) decreased to below normal levels (9.04 mU/ml) after nephrectomy. The cord serum erythropoietin titers in 10 IDM [90.82 +/- 134.1 (S.D.) mu/ml] returned to values within the normal range (13.86 +/- 5.55) on day 3 after birth, suggesting the utility of the RIA in elucidating the role of hypoxia and/or insulin in increased erythropoiesis in IDM. The serum Ep titers in patients with anemias and polycythemias were compared to those of normal human subjects and agreed well with pathophysiologic mechanisms of these hemopoietic disorders, confirming the validity of the RIA.

Topics: Anemia; Dose-Response Relationship, Drug; Erythropoietin; Evaluation Studies as Topic; Fetal Blood; Hematologic Diseases; Humans; Iodine; Iodine Radioisotopes; Polycythemia Vera; Radioimmunoassay

2 patients with coexistent polycythaemia vera and chronic lymphocytic leukaemia are described. A 61-year-old man presented with an increased packed cell volume (PCV) and a leucoerythroblastic blood picture, which was reversible upon treatment, neutrophil leucoytosis, bone marrow lymphocyte infiltration and splenomegaly, and subsequently developed lymphomas, blood lymphocytosis and thrombocytosis. The second case was a 58-year-old female presenting with increased PCV and leucocyte alkaline phosphatase score. She later had neutrophil leucocytosis, thrombocytosis, lymphocytosis, lymphomas and splenomegaly. These cases, together with 6 cases published by others, suggest that an association exists between the two diseases.

Topics: Erythropoietin; Female; Hematocrit; Hematopoiesis; Hematopoietic Stem Cells; Humans; Leukemia, Lymphoid; Leukocyte Count; Male; Middle Aged; Neutrophils; Polycythemia Vera

The role of erythropoietin (Ep) in the regulation erythroid committed precursor (ECP) population size was studied. Experiments were performed on polycythemic CBA mice treated with cyclophosphamide (Cy) on the 5th day after hypertransfusion. In these animals no erythropoietin-responsive cell (ERC) regeneration could be detected up to day 10 after Cy. The results obtained demonstrate that regeneration of ERC could be prompted if the test dose of Ep(2U) was preceded by daily injections of 1 U Ep. Furthermore, one injection of 5 U Ep given 72 h before Ep test does, at the time when pre-ERC were nearly completely recovered, was sufficient to stimulate ERC regeneration. At the same time hydroxyurea suicide was not changed in polycythemic mice treated with Cy and daily Ep injections as compared to control animals. The results indicate that Ep does not stimulate only the multiplication of ERC bu also progression of pre-ERC to ERC. In this way the population size of regenerating ECP is Ep dependent.

Topics: Animals; Cell Division; Cyclophosphamide; Erythropoiesis; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Hydroxyurea; Iron Radioisotopes; Mice; Polycythemia; Polycythemia Vera

Recent data concerning in vitro studies of erythroid progenitors in polycythemia vera (PV) are reviewed. As shown by different laboratories, at least two populations of CFUE seem to coexist in the bone marrow of such patients: one exhibits normal in vitro behavior; the other, probably of clonal origin, is abnormally sensitive to erythropoietin. This abnormal population of CFUE is highly sensitive to trace amounts of erythropoietin and is probably responsible for the in vivo development of polycythemia despite a depressed level of erythropoietin. At the BFUE level, two populations also seem to coexist. However, the abnormal behavior of erythroid progenitors is probably not expressed at this early level of differentiation but only at the late CFUE level. This is in agreement with some of the data, which suggest that in the differentiation of erythroid progenitors into erythroblasts, erythropoietin is only needed at a level very close to CFUE.

Topics: Animals; Cell Transformation, Neoplastic; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Humans; Polycythemia Vera

Topics: Erythroblasts; Erythropoietin; Humans; Polycythemia; Polycythemia Vera

Topics: Diagnosis, Differential; Erythropoietin; Polycythemia; Polycythemia Vera; Radioimmunoassay

An in vitro bioassay has been used to measure erythropoietin levels in extracts of serum from 74 patients with polycythaemia. Patients with polycythaemia vera in relapse had subnormal or undetectable levels which did not increase after challenge by venesection. In polycythaemia secondary to hypoxaemia serum erythropoietin concentrations were generally elevated with a further elevation occurring after venesection. Where polycythaemia was secondary to renal polycystic disease or tumours increased levels of erythropoietin could not be consistently demonstrated. In these patients the serum erythropoietin was unaffected by venesection. In other patients in whom the cause for polycythaemia was unknown examples of each of the above patterns were seen. Erythropoietin investigations in such patients may help in the elucidation of the underlying pathology.

Topics: Adult; Aged; Biological Assay; Bloodletting; Child; Diagnosis, Differential; Erythropoietin; Female; Heart Defects, Congenital; Humans; Male; Middle Aged; Polycythemia; Polycythemia Vera

In eight patients suffering from polycythaemia vera, the proliferation of erythroid colonies in methylcellulose with and without exogenous erythropoietin was studied. After three and five weeks, respectively, no colonies grew. Due to addition of erythropoietin more erythroid colonies proliferated which could be cultivated over nine weeks. The experiments suggest that the erythropoiesis in polycythaemia vera depends on erythropoietin.

Topics: Adult; Aged; Cell Division; Culture Techniques; Erythropoiesis; Erythropoietin; Female; Humans; Male; Middle Aged; Polycythemia Vera; Time Factors

Bioassays for human erythropoietin are cumbersome, time-consuming, and insensitive. The purification of human erythropoietin (EP) had provided small quantities of highly bioactive EP (approximately 70,000 U/mg) required for the development of an EP radioimmunoassay (RIA). The RIA for EP described in this investigation, can detect 5 mU/ml of EP in the assay tube; the serum concentration of EP in normal individuals ranged from less than 18 to 81 mU/ml with a mean value of 29 mU/ml. In contrast, nine patients with severe aplastic anemia had markedly elevated serum EP concentrations with a mean value of 3,487 mU/ml, range 984--6,434 mU/ml. In this RIA, patients with Polycythemia vera had consistently undetectable EP concentrations, less than 18 mU/ml. Eleven patients with chronic renal failure had a higher mean serum EP concentration (40.5 mU/ml) than normal individuals, but the range (less than 18-115 mU/ml) overlapped that of normals. By immunologic and gel chromatographic criteria, EP measured in serum was similar to standard urinary EP. The EP immunoassay that we have developed has sufficient sensitivity and specificity not only to quantitate the elevated serum EP levels found in aplastic anemia but also to discriminate decreased from normal serum concentrations of EP in most circumstances. This simple, reliable RIA has provided the necessary framework upon which to increase our understanding of the importance of EP in hematopoiesis.

Topics: Adult; Aged; Anemia, Aplastic; Blood Transfusion; Chromatography, Gel; Erythrocyte Transfusion; Erythropoietin; Female; Humans; Kidney Failure, Chronic; Male; Middle Aged; Polycythemia Vera; Radioimmunoassay

The primary polycythemias result from malignant proliferation of myeloid stem-cell. Typically, an increase of red cell mass and a decrease of erythropoietin is found. In polycythemia vera, augmentation of PCV is frequently associated with elevation of WBC and platelets, as well as splenomegaly. The treatment consists of venosection and administration of P32 or cytostatics; all of which methods exhibit a specific risk. In secondary polycythemias, augmentation of red cell mass is consecutive to increased erythropoietin production; these hypererythropoietinemias may be induced by hypoxia or, rarely, may result from an inappropriate tumoral (malignant or benign) secretion. "Spurious" polycythemias are finally defined by the more or less normal red cell mass. They are divided into three groups: micropolycythemias, relative polycythemias and "spurious" chronic polycythemias. The latter are frequent and exhibit relatively important morbidity and mortality, and therefore the recently proposed new therapeutic approaches should be considered. Tobacco addiction appears to be one of the major causes of these "spurious" polycythemias.

Topics: Diagnosis, Differential; Erythrocyte Volume; Erythropoietin; Humans; Polycythemia; Polycythemia Vera; Smoking

Serum ESF titers were measured in 42 polycythemic patients using the fetal mouse liver cell bioassay. ESF titers in patients with secondary polycythemia differed significantly from those in patients with polycythemia vera (p less than 0.0001). Among the 21 patients with secondary polycythemia, 1 patient had an ESF titer less than 10 mU/ml (the lower limit of sensitivity) and 20 had ESF titers that ranged between 11 and 112 mU/ml, with a mean titer of 56 mU/ml. Among the 21 patients with polycythemia vera, 13 patients had ESF titers less than 10 mU/ml and 8 had ESF titers ranging between 12 and 55 mU/ml, with a mean titer of 26 mU/ml. The mean hemoglobin concentration in the 8 patients with ESF titers greater than 10 mU/ml was significantly below that in the 13 polycythemia vera patients with ESF titers less than 10 mU/ml (p less than 0.03). If ESF titers less than 10 mU/ml had been indicative of polycythemia vera and ESF titers greater than 10 mU/ml had been indicative of secondary polycythemia in patients with hemoglobin concentrations greater than 17.7 g/dl, but not indicative of either condition in patients with hemoglobin concentrations less than 17.7 g/dl, 71.5% of the polycythemic patients in this study would have been diagnosed correctly, 9.5% incorrectly, and in the 19% the diagnosis would have remained uncertain. It was concluded that measurement of serum ESF titers using this in vitro bioassay can be of clinical importance in differentiating between polycythemia vera and secondary polycythemia.

Topics: Adult; Aged; Animals; Erythropoietin; Hematocrit; Hemoglobins; Humans; Liver; Mice; Mice, Inbred Strains; Middle Aged; Polycythemia; Polycythemia Vera

Pluripotent hemopoietic progenitors (CFU-GEMM) give rise to multilineage hemopoietic colonies in culture. We have examined the erythropoietin requirements of CFU-GEMM-derived erythroid progeny in patients with polycythemia vera (PV) and studied their proliferative activity by short-term exposure to 3HTdR. Mixed colonies with erythroid components were observed in all bone marrow and peripheral blood samples from patients with PV that were cultured without addition of exogenous erythropoietin. This response is consistent with previously reported growth patterns for CFU-E and BFU-E. The frequency of mixed colonies increased regularly when erythropoietin was added to the cultures. Short-term exposure of peripheral blood specimens to 3HTdR prior to plating yielded a reduction of the plating efficiency by 20%-70% when compared to cells that were not exposed to 3HTdR. The observation of cycling CFU-GEMM in PV contrasts with the usually quiescent behavior of CFU-GEMM in peripheral blood of normal individuals under steady-state conditions. These results support the view that the increased proliferative rate observed for CFU-GEMM may be responsible for the increased formation of blood cells in PV.

Topics: Bloodletting; Cell Differentiation; Cell Division; Colony-Forming Units Assay; Erythropoietin; Hematopoietic Stem Cells; Humans; Polycythemia Vera

The results from a biological test for erythropoietin (using the rate of iron absorption in polycythemic mice) and a commercially-available immunological test (haemagglutination-inhibition test) were compared. Of 19 batches of the immunological test which were investigated, 7 batches were completely inactive and a further 3 batches reacted only with the test serum supplied with the test. There was a poor correlation between the results from the biological and the immunological measurements, both on patients with high and those with low serum erythropoietin levels. The difficulty of the immunological erythropoietin test is that pure erythropoietin is not sufficiently available. The immunological test investigated here does not use pure erythropoietin. Aside from this, pathophysiological considerations would lead one to expect basic differences between the results from immunological and biological tests.

Topics: Adrenal Gland Neoplasms; Anemia, Aplastic; Erythropoietin; Hemagglutination Inhibition Tests; Humans; Kidney Failure, Chronic; Leukemia; Pheochromocytoma; Polycythemia Vera

Mononuclear cells from the peripheral blood of normals and patients with terminal renal insufficiency on hemodialysis were set up in methylcellulose cultures with and without addition of sera of patients with polycythemia vera (pv). In renal patients we found more intensive stimulation of hematopoietic progenitor cells after addition of pv-sera than in normals. The findings suggest a hematopoietic factor in sera of pv which is not identical with erythropoietin.

Topics: Cells, Cultured; Colony-Forming Units Assay; Erythropoietin; Hematopoiesis; Hematopoietic Stem Cells; Humans; Kidney Failure, Chronic; Polycythemia Vera

Topics: Blood Volume Determination; Cardiac Catheterization; Diagnosis, Differential; Erythropoietin; Fluoxymesterone; Hemodynamics; Humans; Male; Middle Aged; Polycythemia; Polycythemia Vera; Radionuclide Imaging; Respiratory Function Tests; Skin Diseases; Vascular Diseases

We measured serum concentrations of erythropoietin in 59 patients with polycythemia using a sensitive and specific radioimmunoassay. The mean concentration was 17.5 +/- 8.4 mU/mL (+/- SD) in 26 patients with polycythemia vera and 14.9 +/- 4.2 mU/mL in 26 normal persons. In contrast, the average concentration was 94.3 +/- 101.2 mU/mL in 33 patients with secondary polycythemia, representing a highly significant elevation (p < 0.0001) compared to both normal and polycythemia vera groups. The average hematocrit value did not differ between the polycythemia vera and the secondary polycythemia patients, and both groups had higher values (median, 55%) than the normal donors (median, 41%). Erythropoietin concentrations ascertained by radioimmunoassay helped discriminate between polycythemia vera and secondary polycythemia. Ninety-two percent of polycythemia vera patients had concentrations less than 30 mU/mL (the concentration used as a cut off point), and 94% of secondary polycythemia patients had concentrations greater than 30 mU/mL. This represents an overall correct classification of 93% of the patients. Serum erythropoietin levels as ascertained by radioimmunoassay can distinguish between most polycythemia vera and secondary polycythemia patients and should prove useful in the differential diagnosis of polycythemia.

Topics: Adult; Diagnosis, Differential; Erythropoietin; Female; Hematocrit; Humans; Male; Middle Aged; Polycythemia; Polycythemia Vera; Radioimmunoassay

Topics: Adult; Aged; Diagnosis, Differential; Erythrocyte Count; Erythropoietin; Female; Humans; Male; Middle Aged; Oxygen; Polycythemia; Polycythemia Vera; Splenomegaly

Peripheral blood and bone marrow specimens from patients with polycythemia vera (PV) and chronic myelogenous leukemia (CML) were assayed for erythroid and granulopoietic progenitor cells. All compartments were increased in CML patients in relapse although the ratio of BFU-E to CFU-C numbers remained constant in all CML patients where values ranged over several orders of magnitude. By comparison with normal ratios there was only a slight shift towards increased CFU-C numbers. No quantitative changes in any progenitor compartment was found in PV except for a marginal increase in marrow CFU-E. Erythropoietin (epo)-independent colony formation has been documented in all 61 cases of PV studied to date, and the proportion of progenitors classified as abnormal on this basis increases on average 3- to 5-fold as they differentiate in vivo from primitive BFU-E to CFU-E. Preliminary replating studies suggest that when this occurs in vitro individual BFU-E produce both normal and abnormal phenotypes. Epo-independent erythropoiesis has also been commonly observed in assays of CML cells, although its expression is more variable and in the absence of epo progenitors in CML usually make fewer erythroblasts containing even less hemoglobin than do their counterparts in PV. Expression of a common regulatory defect in erythroid cells in PV and CML suggests a possible relationship to the initial transformation event(s).

Topics: Antibody Formation; Colony-Forming Units Assay; Erythropoiesis; Erythropoietin; Hematopoietic Stem Cells; Humans; Immunity, Cellular; Leukemia, Myeloid; Polycythemia Vera

Topics: Cell Differentiation; Cells, Cultured; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Erythrocytes; Erythropoietin; Hematopoietic Stem Cells; Humans; Polycythemia Vera; Time Factors

Peripheral blood mononuclear cells from patients with polycythemia vera or myelofibrosis with myeloid metaplasia were studied for their erythroid colony growth characteristics in plasma clot cultures. In both diseases, erythroid colonies formed early in culture in the absence of added erythropoietin (endogenous colonies). In no instance did early, endogenous colony formation occur with peripheral blood cells from normals or patients with secondary polycythemia. A normal response to erythropoietin was observed with both control and patients' peripheral blood cells. Spleen mononuclear cells obtained from one patient with myelofibrosis also produced endogenous colonies and showed a response to erythropoietin. This study suggests that culture of peripheral blood mononuclear cells might serve as a useful tool in discriminating polycythemia vera from secondary polycythemia.

Topics: Cells, Cultured; Erythrocyte Count; Erythrocytes; Erythropoiesis; Erythropoietin; Humans; Polycythemia; Polycythemia Vera; Primary Myelofibrosis; Spleen

Serum of patients suffering from a chronic myeloproliferative disorder (polycythaemia, era, osteomyelofibrosis, chronic myeloid leukaemia) and serum of lethally irradiated rats injected before application of a single doses of erythropoietin did not enhance the effect of erythropoietin -- measured with the iron incorporation rate of polycythemic mice. The rationale for these experiments is to try to find a "myeloproliferative factor", which augments the number of stem cells as described in sera of patients with polycythaemia vera, osteomyelofibrosis, and lethally irradiated mice.

Topics: Adult; Aged; Drug Synergism; Erythropoietin; Humans; Leukemia, Myeloid; Middle Aged; Myeloproliferative Disorders; Polycythemia Vera; Primary Myelofibrosis

Topics: Anemia; Animals; Cells, Cultured; Colony-Stimulating Factors; Dexamethasone; Dogs; Erythropoiesis; Erythropoietin; Female; Hematopoietic Stem Cells; Humans; Liver; Polycythemia Vera; Pregnancy; Thyroid Hormones

Erythropoietin titers of plasma cannot be used to differentiate polycythemia vera from secondary polycythemia since the limit of sensitivity of our current bioassay technics is 50 mU, considerably higher than levels found in normal subjects and in patients with polycythemia. However, erythropoietin is relatively heat stable, and since abundant plasma is available from therapeutic phlebotomies it is possible to prepare and assay highly concentrated, erythropoietin-containing extracts. In 35 normal subjects, erythropoietin levels ranged from less than 5 mU/ml (the limit of sensitivity) to 18 mU/ml with a mean of 7.8 mU/ml. In 21 patients with proved polycythemia vera, the levels were less than 5 mU/ml in all. In 41 patients with suspected secondary polycythemia or polycythemia of unknown origin, the levels ranged from less than 5 to 3,000 mU/ml. Three of the 11 patients with levels less than 5mU/ml were subsequently shown to have polycythemia vera. These results suggest that this refinement of the routine bioassay for erythropoietin may be of clinical importance in the differential diagnosis of polycythemia.

Topics: Erythropoietin; Female; Hematocrit; Humans; Male; Polycythemia; Polycythemia Vera

Erythrocytosis in a young Sinhalese man is described. The patient was known to have had a raised Hb and PCV for at least 10 years. Subsequent investigations failed to support the diagnosis of polycythaemia vera or to reveal a cause for secondary polycythaemia. Blood erythropoietin values were raised, but no cause for inappropriate secretion could be identified. Although there was no evidence of erythrocytosis in the family, the findings in this patient appear to be those of a condition which has been called familial polycythaemia. The spleen was unusually large and was associated with hypersplenism and thrombocytopenia. Problems of diagnosis and management are described. Phlebotomy appears to be the treatment of choice, with a regimen of regular venesection for the control of symptoms due to hyperviscosity and vascular occlusion.

Topics: Adult; Blood Cell Count; Blood Viscosity; Bloodletting; Diagnosis, Differential; Erythropoietin; Humans; Hypersplenism; Male; Phosphorus Radioisotopes; Polycythemia; Polycythemia Vera; Radionuclide Imaging; Thrombocytopenia

In vitro studies using the plasma clot culture system have been performed in order to compare the red cell pregenitors able to rise to erythrocytic colonies in 7 days (CFUE) in the bone marrow of polycythemia vera (PV), secondary polycythemias and normal subjects. In PV but never in normal individuals or secondary polycythemias, the bone marrow cells producing erythroid colonies without addition of erythropoietin were found. The erythropoietin dode response curves in PV is biphasic with a plateau up to a concentration of erythropoietin of 0.02-0.05 i.U./ml followed by a near normal response to erythropoietin at higher doses. Thus our results demonstrate that two populations of erythroid stem cells coexist in PV, one being abnormally sensitive to (or independant of) erythropoietin, the other normally responding to erythropoietin. After remission induced by P32 treatment, the abnormal population can disappear but the prognostic significance of this disappearance is uncertain. On the whole these results are in agreement with those of others laboratories using the plasma clot culture system. The reasons of the disagreement with the data published using the methylcellulose technic of culture are discussed.

Topics: Animals; Bone Marrow Cells; Cells, Cultured; Dose-Response Relationship, Drug; Erythrocytes; Erythropoietin; Hematopoietic Stem Cells; Humans; Mice; Polycythemia Vera

We investigated the pathogenesis of isolated erythrocytosis of 14 yr duration in a 28-yr-old man. The increase in red cell mass was attributed to increased erythropoietin production. An extensive search for recognized causes of secondary erythrocytosis was unrevealing. Family members were found to be hematologically normal. After reduction of the circulating red cell mass by 20%, erythropoietin activity nearly quadrupled, thus suggesting a normal erythropoietin response to phlebotomy. When bone marrow cells of the patient were cultured in plasma clots in the absence of added erythropoietin, endogenous erythroid colony formation was observed, a pattern previously believed to be specific for polycythemia vera bone marrow cells. Our observations suggest that the erythrocytosis in this individual is best explained by an abnormal "servoregulatory" mechanism of erythropoietin production. In addition, this is the first instance in which the rule that endogenous erythroid colony formation is correlated with the diagnosis of polycythemia vera has not held.

Topics: Adult; Colony-Forming Units Assay; Erythrocytes; Erythropoietin; Humans; Male; Polycythemia; Polycythemia Vera

Topics: Blood Volume; Bone Marrow; Cells, Cultured; Diagnosis, Differential; Erythrocyte Volume; Erythropoiesis; Erythropoietin; Female; Humans; International Cooperation; Male; Polycythemia; Polycythemia Vera; United States

Leucocytes of normal individuals and patients with polycythemia vera were isolated from the peripheral blood by Ficoll-Hipaque density gradient centrifugation and cultured in vitro suing the bovine plasma clot culture technique with a minor modification: the addition of fresh normal serum. After 14 days in the presence of sheep erythropoïetin (3U/ml) erythropoïetic bursts containing between 3 and 10 subcolonies were observed in normal and polycythemia vera cultures. Blood leucocytes of patients with polycythemia vera rise to these erythropoïetic bursts without addition of erythropoïetin to the culture. This behavior was never observed in the blood of normal individuals. These results indicate that in polycythemia vera commited erythroïd stem cells of high proliferative capacity closely resembling the murine erythroïd burst forming unit have an abnormal sensitivity to erythropoïetin as well as the immediate precursors of the proerythroblasts. The culture of these cells from the peripheral blood offers some practical advantages.

Topics: Cell Differentiation; Erythroblasts; Erythrocytes; Erythropoietin; Humans; In Vitro Techniques; Leukocytes; Polycythemia Vera

Topics: Animals; Erythropoiesis; Erythropoietin; Humans; Mice; Polycythemia; Polycythemia Vera; Russia

The Effects of Various Substances in the Culture Microenvironment on the Proliferation of Erythroblasts in vitro were Studied and Evaluated by differential cell and mitosis counting in normal and pathologic bone marrow. The mitotic frequency and maturation correlated directly with the erythropoietin content of the medium and were potentiated on addition of either folic acid, etiocholanolon or cAMP. However, erythroblast proliferation was stimulated independently of erythropoietin concentration when either cobalt, and androgen or an anabolic substance was added to the medium. An iron-deficient medium prevented the maturation of erythroblasts to reticulocytes, thereby rendering erythropoiesis ineffective. The existence of an erythrocyte chalone ineffective. The existence of an erythrocyte chalone or an erythropoietin inhibitor could not be deduced from our experiments since the transformation of pluripotent to erythropoetin-sensitive stem cells is not included. A stimulation of the granulopoietic proliferation pool occurred when serum from patients with either polycythemia vera or after acute blood loss was added to the medium.

Topics: Androgens; Cobalt; Culture Media; Cyclic AMP; Erythropoiesis; Erythropoietin; Etiocholanolone; Folic Acid; Hematopoiesis; Humans; Mitosis; Polycythemia Vera

Topics: Bone Marrow; Cells, Cultured; Erythropoiesis; Erythropoietin; Gamma Rays; Heme; Iron; Polycythemia Vera; Time Factors; X-Rays

Topics: Alkylating Agents; Erythropoietin; Hematocrit; Hemorrhage; Humans; Leukemia; Polycythemia; Polycythemia Vera; Smoking; Thromboembolism

Topics: Adult; Aged; Cells, Cultured; Dexamethasone; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Female; Humans; Male; Middle Aged; Polycythemia Vera; Preleukemia; Regression Analysis

Bone marrow cells from two glucose-6-phosphate dehydrogenase (G-6-PD) heterozygotes with polycythemia vera were cultured to determine whether progenitors which wre not of the polycythemia vera clone were present, and, if present, which cell lines contributed to the increase in erythroid colonies observed in response to added erythropoietin (ESF). To accomplish this, the G-6-PD isoenzyme activity of individual erythroid colonies was determined. All of the erythroid colonies analyzed in cultures without added ESF, contained the G-6-PD isoenzyme type characteristic of the abnormal clone. With higher ESF concentrations in the culture, however, there was an increase in the colonies that were not of the polycythemia vera clone. Analysis of the ratio of the various types of colonies indicated that normal and polycythemia vera cells are capable of responding to ESF in vitro. In selected patients, this technique permits analysis of the ratios of normal to abnormal cells during the course of the disease, in response to therapy and during late complications, such as myelofibrosis or leukemic transformation.

Topics: Clone Cells; Erythrocytes; Erythropoiesis; Erythropoietin; Glucosephosphate Dehydrogenase; Granulocytes; Hematopoietic Stem Cells; Humans; Isoenzymes; Polycythemia Vera

Peripheral blood precursor cells from all of 12 patients with polycythaemia vera (PV) formed hemoglobinized colonies in vitro without addition os was strictly epo-dependent in 30 normals and 8 patients with erythrocytosis of other origin. By addition of 1 U epo/ml to the cultures, colony formation was increased up to 5-fold in PV. Untreated patients with a short history had the highest numbers of such epo-responsive precursor cells. In 3 patients with concomitant myelofibrosis, erythroid in vitro growth was abundant, but scattered, single colonies could hardly be identified and their hemoglobinization was poor. This picture did not change with addition of epo, and hemoglobinization did not improve. In vitro grown colonies in PV had morphological abnormalities as compared to colonies grown from normal precursor cells: all stages of erythroid differentiation and up to 50% necrotic cells were found within single colonies. Scattered colonies contained an excess of large, immature, vacuolated erythroblasts. It is concluded that these functionaland morphological abnormalities of in-vitro erythropoiesis are a reliable indicator of PV. Further, transition to myelofibrosis is recognized by a characteristic growth pattern with decreased epo-responsiveness.

Topics: Cells, Cultured; Diagnosis, Differential; Erythropoietin; Hematopoietic Stem Cells; Humans; Polycythemia; Polycythemia Vera; Primary Myelofibrosis

Topics: Bone Marrow Cells; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Erythropoiesis; Erythropoietin; Humans; Polycythemia Vera

In vitro studies using the plasma clot culture system have been performed in order to compare the red cell pregenitors able to rise to erythrocytic colonies in 7 days (CFUE) in the bone marrow of polycythemia vera (PV), secondary polycythemias and normal subjects. In PV but never in normal individuals or secondary polycythemias, the bone marrow cells producing erythroid colonies without addition of erythropoietin were found. The erythropoietin dose response curves in PV is biphasic with a plateau up to a concentration of erythropoietin of 0.02--0.05 i.U./ml followed by a near normal response to erythropoietin at higher doses. Thus our results demonstrate that two populations of erythroid stem cells coexist in PV, one being abnormally sensitive to (or independent of) erythropoietin, the other normally responding to erythropoietin. After remission induced by P32 treatment, the abnormal population can disappear but the prognostic significance of this disappearance is uncertain. In the whole these results are in agreement with those of others laboratories using the plasma clot culture system. The reasons of the disagreement with the data published using the methylcellulose technic of culture are discussed.

Topics: Cell Differentiation; Colony-Forming Units Assay; Dose-Response Relationship, Drug; Erythroblasts; Erythrocytes; Erythropoietin; Hematopoietic Stem Cells; Humans; Polycythemia Vera

Marrow specimens of patients with polycythemia vera rise to erythrocytic colonies in vitro without addition of erythropoietin to the culture. This behavior was never observed with marrow cells of normal subjects or secondary erythrocytosis. These results indicate that the sensitivity of some erythrocytic progenitor cells to erythropoietin is altered in polycythemia vera.

Topics: Bone Marrow; Cells, Cultured; Erythroblasts; Erythrocytes; Erythropoietin; Humans; Polycythemia Vera

Topics: Cells, Cultured; Erythroblasts; Erythropoietin; Humans; Polycythemia Vera

A patient with classical polycythemia vera (PV) was found to have an inappropriately elevated serum erythropoietin (Ep) level. Investigations did not reveal any lesion or blood abnormality known to be associated with excessive Ep production and erythrocytosis. Sudden withdrawal of blood to reduce the Hb and Hct from 18.5 gm% and 56% to 13.6 gm% and 41.5%, respectively, resulted in an increment of serum Ep to abnormal level. With iron treatment there was a brisk return of Hb and Hct to prebleeding levels which was associated with reduction in the serum Ep. The inverse relationship between the Ep and Hb or Hct is inconsistent with the presence of excessive Ep-producing lesion. These results suggested that the threshold for Ep secretion from normal Ep-secreting tissue to Hb and Hct levels is set at an abnormal level. This patient's marrow cells when cultured in vitro in the absence of Ep, unlike other PV patients' (except one) marrow cells, did not grow erythroid colonies. In the presence of Ep, however, the colonies comparable to those formed from normal marrow cultures were obtained. These results suggested that his marrow erythropoietic cells were neither Ep independent nor Ep-hyperresponsive, as has been suggested by some investigators for erythropoiesis in PV. This patient presents phenomena that hitherto have not been reported.

Topics: Animals; Erythropoietin; Hematocrit; Hemoglobins; Humans; Iron; Male; Mice; Middle Aged; Polycythemia Vera

We performed in vitro culture studies examining the interaction of erythropoietin with red cell progenitors in polycythemia vera. Bone marrow was obtained from five patients with typical disease and from five healthy volunteers, and assayed for erythroid colony formation (CFU-E) by the methylcellulose technique. In cultures without added erythropoietin, a mean eightfold greater cloning efficiency was noted with the polycythemia vera marrows, as compared to normal. There was prominent stimulation of colony formation by erythropoietin, and the shape of the erythropoietin dose-response cruves appeared to be similar in both patients and controls. Anti-erythropoietin antibody reduced the number of CFU-E in cultures not containing added erythropoietin, but did not eliminate them. Dexamethasone (10(-9) M) caused a consistent increase in CFU-E in the patients' cultures. These studies provide evidence for functional erythropoietin and glucocorticosteroid receptor mechanisms on erythroid precursors in polycythemia vera. The observations are consistent with a concept of this disease as a disorder of hematopoietic stem cells in which peripheral erythrocytosis is caused by an expanded erythroid progenitor compartment which maintains responsiveness to hormonal modulation.

Topics: Antibodies; Dexamethasone; Dose-Response Relationship, Drug; Erythrocytes; Erythropoiesis; Erythropoietin; Humans; Neutralization Tests; Polycythemia Vera

We found primary erythrocytosis in two male siblings with hematologically normal parents. To clarify the abnormalities in erythropoiesis, we studied erythropoietin production in the older sibling as well as in vivo and in vitro responses of bone marrow to various stimuli. His erythropoietin excretion after a 1000-ml phlebotomy increased by 0 to 11 units per day. In liquid-suspension culture, erythropoiesis was prominently augmented by erythropoietin and unstimulated erythropoiesis was greater and more prolonged than normal. Numbers of erythroid colonies rose in methylcellulose culture without exogenous erythropoietin, and cloning increased with added erythropoietin. Anti-erythropoietin antibody substantially decreased erythropoiesis in vitro. Increased bone-marrow erythropoiesis was also demonstrated in murine diffusion chambers. The principal abnormality in this familial erythrocytosis appears to be a greatly expanded erythropoietic precursor pool that is responsive to erythropoietin in vitro and in vivo. This abnormality is analogous to the functional erythropoietic defect in typical polycythemia vera.

Topics: Adult; Autoantibodies; Cells, Cultured; Diagnosis, Differential; Erythropoiesis; Erythropoietin; Hematocrit; Humans; Male; Methylcellulose; Oxygen; Polycythemia; Polycythemia Vera

Topics: Bloodletting; Cell Differentiation; Diagnosis, Differential; Erythropoiesis; Erythropoietin; Humans; Polycythemia; Polycythemia Vera

In the plasma clot culture system both normal and polycythemia vera (PV) bone marrow cells respond to erythropoietin (Ep), giving rise to large numbers of colonies of erythroid cells. In PV, but not in normal individuals, the marrow produced endogenous erythroid colonies (EED) in the absence of exogenous Ep. The number of EEC formed varied from patient to patient comprising anywhere from 6 to 29% of the total number of colonies formed in the presence of Ep. Exposure, before use in culture, of fetal calf serum and citrated bovine plasma to the gammaglobulin fraction of rabbit anti-Ep serum followed by treatment with goat anti-rabbit gamma-globulin re sulted in a significant decrease in EEC formation. Addition of anti-Ep directly to the culture medium produced similar results. In addition, the production of EEC in response to added Ep was inhibited in the presence of anti-Ep. Addition of very small doses of highly purified Ep to anti-Ep-treated cultures resulted in the reappearance of a significantnumber of EEC formation in PV may be due to a population of erythroid-committed precursors that are abnormally sensitive to small concentrations of Ep which may be present in fetal calf serum and citrated plasma. Although the mechanism of formation of these cells is not known, it appears that the final steps in the formation of red cells derived from this clone of precursors is subject to the usual Ep control.

Topics: Adult; Aged; Bone Marrow; Bone Marrow Cells; Cells, Cultured; Erythropoiesis; Erythropoietin; Humans; Immunoglobulin G; Middle Aged; Polycythemia Vera

Topics: Animals; Bone Marrow; Bone Marrow Cells; Cells, Cultured; Erythropoietin; Female; Hematocrit; Hematopoiesis; Heme; Humans; Hypoxia; Male; Mice; Polycythemia Vera

Topics: Blood Transfusion; Bloodletting; Bone Marrow; Bone Marrow Cells; Chlorambucil; Chronic Disease; Erythrocytes; Erythropoietin; Female; Humans; Leukemia; Liver; Male; Phosphorus Radioisotopes; Polycythemia; Polycythemia Vera; Spleen; United States

Anti-erythropoietin antibodies (anti-ESF) were determined by the capacity of the immunoglobulins to neutralize the biological activity of erythropoietin (ESF). Affinity chromatographic methods were used to initiate the purification of anti-ESF by use of ESF covalently linked to Sepharose. It was observed that anti-ESF bound the ESF-Sepharose. In order to remove the nonspecific immunoglobulins, the anti-ESF preparation was subjected to further chromatography on affinity colums where either serum or urinary proteins from a polycythemic patient were covalently linked to Sepharose. It was demonstrated the affinity chromatography could be used as a method isolate anti-ESF.

Topics: Animals; Antibodies; Binding Sites, Antibody; Chromatography, Affinity; Cyanogen Bromide; Epitopes; Erythropoietin; Humans; Immunization; Immunologic Techniques; Neutralization Tests; Polycythemia Vera; Rabbits; Sepharose

Topics: Anemia, Hypochromic; Arthritis, Rheumatoid; Erythropoietin; Female; Humans; Male; Polycythemia; Polycythemia Vera; Uremia

The immunochemical assay (HAI) for erythropoietin is accurate enough to detect small quantities and sensitive enough to demonstrate consistently and quantitatively the presence of erythropoietin in serum and urine. It will be the purpose of this report to describe in detail the procedure for the assay, the usefulness and value of erythropoietin testing in the clinical laboratory. The relationship of erythropoietin levels in serum and urine in the classification of diseases of the red blood cells are discussed. There is presently no other method available for the measurement of erythropoietin that permits the detection of extremely small differences, sensitive enough to demonstrate consistently and quantitatively the presence of small amounts of erythropoietin.

Topics: Anemia, Aplastic; Blood Transfusion; Erythropoietin; Hemagglutination Inhibition Tests; Hematocrit; Humans; Polycythemia; Polycythemia Vera; Urologic Diseases

A previous report have shown that both intact red blood cells (RBC) and an equal number of haemolyzed cells were able to inhibit erythropoiesis on ESF-induced erythropoiesis, suggesting a cell compound to be responsible for the inhibitiory effect. Haeme and haeme compounds have been found to stimulate or inhibit both erythropoiesis and haeme synthesis. The present work presents data on the inhibitory effect of haemolyzed RBC and compounds from intact RBC on erythropoiesis. The inhibiting factor was found to be of a small molecular size and of the same range as a urinary erythorpoiesis inhibiting factor (EIF). The inhibitor did not contain haeme. Both Fe+2 and Fe+3 were tested, showing no reduction of the 59-Fe incorporation into RBC in the test animals. The inhibition could not be due to dilution of the 59-Fe with unlabelled iron from the haemolyzed cells. On the contrary, Fe+3-ions rather stimulated erythropoiesis, probably due to increased amounts of available iron. Haemolysates were prepared from RBC with different amounts of immature cells. With increasing amounts of reticulocytes, a reduction of the inhibitory effect occurred. Also foetal cells showed less inhibition than an equal amount of adult cells. After high speed centrifugation, the inhibitory effect of haemolysates was found in the supernatant, while ghost cells exerted no inhibition. No species differences were found using both exhypoxic polycythaemic mice and rats. An inhibiting factor was liberated into the incubation medium when RBC were incubated for 20 h. No haemolysis occurred during the incubation period. Mature, adult RBC therefore contain a substance which is different from haeme, with a negative feedback on erythropoiesis.

Topics: Animals; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Hemolysis; Mice; Mice, Inbred Strains; Polycythemia Vera; Rats; Stimulation, Chemical

Topics: Anemia, Aplastic; Anemia, Hypochromic; Erythropoietin; Humans; Polycythemia Vera

A liquid bone marrow culture technique was used to study erythropoiesis in polycythaemia vera. Three patients with the diagnosis of polycythaemia vera treated only by phlebotomy were compared to three healthy volunteers. Erythropoiesis in vitro was assessed by counts of nucleated red cells and 3H-thymidine incorporation into erythroid precursors. Human urinary erythropoietin was found to stimulate erythropoiesis in cultures from the polycythaemia patients as well as normals. The erythropoietin effect was blocked by a rabbit anti-erythropoietin antibody. These results suggest that some erythroid precursor cells maintain an intact erythropoietin response mechanism in polycythaemia vera. They also support clinical studies of this disorder indicating that some regulation by erythropoietin may occur in vivo.

Topics: Cell Count; Erythropoietin; Humans; In Vitro Techniques; Polycythemia; Polycythemia Vera

Topics: Adult; Aged; Alkaline Phosphatase; Blood Platelets; Blood Volume; Bone Marrow; Chlorambucil; Erythrocyte Count; Erythropoietin; Female; Hemoglobins, Abnormal; Hookworm Infections; Humans; Hydrogen-Ion Concentration; Leukocyte Count; Male; Middle Aged; Oxygen; Phosphorus Radioisotopes; Polycythemia; Polycythemia Vera; Pruritus; Riboflavin Deficiency; Uric Acid; Vitamin B 12

Topics: Adult; Aged; Animals; Blood Cell Count; Blood Platelets; Blood Transfusion; Erythropoietin; Female; Hematocrit; Hematopoietic Stem Cells; Humans; Injections, Subcutaneous; Iron; Iron Radioisotopes; Leukemia, Myeloid; Leukocyte Count; Leukocytosis; Male; Mice; Middle Aged; Polycythemia Vera; Primary Myelofibrosis

Topics: Bone Marrow; Bone Marrow Cells; Clone Cells; Culture Media; Erythrocytes; Erythropoietin; Humans; Polycythemia Vera

Topics: Absorption; Animals; Antibodies; Binding Sites, Antibody; Epitopes; Erythropoietin; Goats; Humans; Immune Sera; Immunodiffusion; Polycythemia Vera; Precipitin Tests; Rabbits

Topics: Animals; Biopsy; Bloodletting; Culture Techniques; Diabetes Complications; Erythropoietin; Female; Humans; Kidney Diseases, Cystic; Male; Mice; Middle Aged; Polycythemia Vera; Radiography

Topics: Diagnosis, Differential; Erythropoietin; Humans; Polycythemia; Polycythemia Vera

Topics: Animals; Erythropoietin; Humans; Iron; Mice; Polycythemia Vera; Time Factors

Topics: Adult; Aged; Bone Marrow; Bone Marrow Cells; Cells, Cultured; Erythropoiesis; Erythropoietin; Female; Hematocrit; Heme; Hemoglobinometry; Humans; Iron; Iron Isotopes; Leukemia, Myeloid; Male; Middle Aged; Polycythemia Vera; Reticulocytes

Topics: Animals; Dexamethasone; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Injections, Intraperitoneal; Iron; Iron Isotopes; Mice; Polycythemia Vera; Stimulation, Chemical

Topics: Animals; Blood Transfusion; Erythrocytes; Erythropoiesis; Erythropoietin; Hematocrit; Hypoxia; Iron; Iron Isotopes; Male; Mice; Polycythemia Vera; Radiometry

Topics: Animals; Atmospheric Pressure; Carbon Monoxide; Citrates; Dactinomycin; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Hypoxia; Iron; Iron Isotopes; Mice; Polycythemia Vera; Radiation Effects; Reticulocytes

Topics: Aged; Anemia, Macrocytic; Calcium; Duodenal Ulcer; Erythropoietin; Hemoglobinometry; Humans; Iron; Leukocyte Count; Male; Phosphorus Isotopes; Polycythemia Vera; Postgastrectomy Syndromes; Uric Acid; Vitamin B 12

The quantitative relationship between red cell volume, erythropoietin level, and erythropoiesis was evaluated in 43 human beings. Results in normal man were compared with studies in patients with anemia from bone marrow failure and with polycythemia vera. The maximum erythropoietin excretion after bleeding normal men was similar to the basal levels found in patients with chronic anemia of similar magnitude. Although erythropoietin values were low in patients with polycythemia vera, bleeding evoked a normal response. In patients anemic from bone marrow failure, basal levels were elevated, and phlebotomy resulted in an increase consistent with the new level of anemia. These observations indicate that erythropoietin level is affected primarily by the degree of anemia and is not influenced by the duration of anemia. In normal subjects, a fivefold increase in urinary erythropoietin was associated with a doubling of erythropoiesis. Despite similar degrees of erythropoietin production, anemic patients with evidence of bone marrow in the lower extremities had greater red cell production. In patients with polycythemia vera, red cell production was inappropriately elevated with regard to the urinary erythropoietin excretion. Bone marrow maturation time was not shortened in patients anemic from bone marrow failure to the same degree as in bled, normal volunteers. In addition to an adequate level of erythropoietin production, normal bone marrow function is necessary for maximal shortening of maturation time.

Topics: Adult; Aged; Anemia, Aplastic; Bloodletting; Bone Marrow; Erythrocyte Aging; Erythrocytes; Erythropoiesis; Erythropoietin; Female; Hematocrit; Humans; Iron; Iron Isotopes; Leg; Male; Middle Aged; Polycythemia Vera

Topics: Blood Platelets; Bone Marrow Examination; Erythropoiesis; Erythropoietin; Hematopoiesis; Humans; Iron Isotopes; Megakaryocytes; Phosphorus Isotopes; Polycythemia Vera

Topics: Adult; Aged; Erythropoietin; Female; Humans; Iron Isotopes; Male; Middle Aged; Oxygen Consumption; Polycythemia; Polycythemia Vera

Topics: Adolescent; Adult; Aged; Blood Platelets; Bone Marrow Diseases; Diagnosis, Differential; Erythrocyte Count; Erythropoietin; Female; Hematocrit; Hemoglobinometry; Humans; Leukemia, Myeloid; Leukocyte Count; Male; Middle Aged; Myeloproliferative Disorders; Polycythemia Vera; Primary Myelofibrosis; Retrospective Studies

Topics: Adenocarcinoma; Adolescent; Adult; Aged; Animals; Brain Neoplasms; Child; Child, Preschool; Dogs; Electric Stimulation; Erythropoiesis; Erythropoietin; Ethacrynic Acid; Haplorhini; Humans; Hydronephrosis; Hypertension; Hypothalamus; Infant; Iron Isotopes; Kidney Diseases; Kidney Neoplasms; Lung Neoplasms; Male; Mice; Middle Aged; Polycythemia Vera; Rats; Testicular Neoplasms; Testosterone; Urinary Calculi; Urologic Diseases; Wilms Tumor; Wounds and Injuries

Topics: Adult; Aged; Animals; Biological Assay; Electric Stimulation; Erythrocytes; Erythropoietin; Female; Haplorhini; Humans; Hypophysectomy; Hypothalamus; Hypoxia; Iron; Iron Isotopes; Kidney; Kidney Diseases; Kidney Diseases, Cystic; Male; Middle Aged; Neoplasms, Experimental; Polycythemia Vera; Rats; Wilms Tumor

Topics: Adult; Aged; Bone Marrow; Bone Marrow Cells; Culture Techniques; Erythropoiesis; Erythropoietin; Female; Heme; Humans; Male; Polycythemia Vera

Topics: Animals; Cerebellar Neoplasms; Erythropoietin; Hemangiosarcoma; Hematocrit; Humans; Hypoxia; Mice; Polycythemia; Polycythemia Vera

Topics: Aged; Anemia, Hypochromic; Bone Marrow; Cell Differentiation; Erythropoiesis; Erythropoietin; Female; Heme; Humans; In Vitro Techniques; Male; Middle Aged; Polycythemia Vera

Topics: Adult; Aged; Alkaline Phosphatase; Blood Platelets; Blood Transfusion; Bone Marrow; Chromium Isotopes; Erythropoietin; Female; Hematocrit; Humans; Hypoxia; Leukocyte Count; Male; Middle Aged; Neoplasms; Polycythemia; Polycythemia Vera; Vascular Surgical Procedures

Topics: Erythropoietin; Humans; Methods; Polycythemia Vera

Topics: Adult; Aged; Animals; Blood Cell Count; Blood Platelets; Blood Volume; Bone Marrow Cells; Dehydration; Duodenal Ulcer; Erythrocyte Count; Erythropoietin; Hematocrit; Hemodynamics; Hemoglobinopathies; Humans; Hypoxia; Iron Isotopes; Kidney Neoplasms; Leukocyte Count; Lung Diseases; Male; Mice; Middle Aged; Obesity Hypoventilation Syndrome; Oxygen; Oxygen Consumption; Phosphorus Isotopes; Plasma Volume; Polycythemia; Polycythemia Vera; Pulmonary Emphysema

Topics: Animals; Blood Cell Count; Erythropoietin; Humans; Iron Isotopes; Polycythemia Vera; Pulmonary Heart Disease; Rats

Topics: Erythropoietin; Humans; Hypoxia; Kidney; Polycythemia Vera; Renin

Topics: Adolescent; Erythropoietin; Female; Humans; Nephrectomy; Polycythemia Vera; Wilms Tumor

Topics: Erythropoietin; Humans; Kidney Diseases, Cystic; Kidney Neoplasms; Kidney Transplantation; Polycythemia; Polycythemia Vera

Topics: Anemia, Hemolytic; Blood Transfusion; Bloodletting; Bone Marrow; Erythropoiesis; Erythropoietin; Hematocrit; Humans; Polycythemia Vera

Topics: Animals; Erythropoiesis; Erythropoietin; Humans; Polycythemia Vera

Topics: Epoetin Alfa; Erythropoietin; Humans; Kidney; Kidney Diseases, Cystic; Polycythemia Vera; Urography

Topics: Anemia; Anemia, Aplastic; Anemia, Pernicious; Epoetin Alfa; Erythropoietin; Humans; Polycythemia Vera; Saliva; Splenectomy

Topics: Epoetin Alfa; Erythropoietin; Gout; Humans; Kidney Diseases; Liver Diseases; Neoplasms; Polycythemia Vera; Thyroid Diseases

Topics: Diagnosis, Differential; Epoetin Alfa; Erythropoietin; Humans; Kidney; Polycystic Kidney Diseases; Polycythemia; Polycythemia Vera

Topics: Aged; Epoetin Alfa; Erythropoietin; Humans; Neoplasms; Polycythemia; Polycythemia Vera

Topics: Blood Cell Count; Bone Marrow Examination; Epoetin Alfa; Erythropoietin; Hepatomegaly; Humans; Polycythemia; Polycythemia Vera; Primary Myelofibrosis; Splenectomy; Splenomegaly

Topics: Animals; Biological Assay; Epoetin Alfa; Erythropoietin; Hypoxia; Mice; Polycythemia Vera