Childhood polycythemias/erythrocytoses: classification, diagnosis, clinical presentation, and treatment

Polycythemias or erythrocytoses in childhood and adolescence are very rare. Systematic data on the clinical presentation and laboratory evaluations as well as on treatment regimens are sparse. The diagnostic program in absolute erythrocytosis includes extensive clinical, hematological, biochemical, and molecular biological examinations which should be applied following a stepwise algorithm. Absolute erythrocytoses are usually subdivided into primary and secondary forms. Primary erythrocytosis is a condition in which the erythropoietic compartment is expanding independently of extrinsic influences or by responding inadequately to them. Primary erythrocytoses include primary familial and congenital polycythemia (PFCP) due to mutations of the erythropoietin (Epo) receptor gene and the myeloproliferative disorder polycythemia vera. Secondary erythrocytoses are driven by hormonal factors (predominantly by Epo) extrinsic to the erythroid compartment. The increased Epo secretion may represent either a physiologic response to tissue hypoxia, an abnormal autonomous Epo production, or a dysregulation of the oxygen-dependent Epo synthesis. Congenital secondary erythrocytoses are caused, e.g., by hemoglobin variants with increased oxygen affinity, by 2,3-bisphosphoglycerate deficiency, or by mutations in the von Hippel–Lindau gene associated with a disturbed oxygen-dependent regulation of Epo synthesis.     

The classification and diagnostic criteria of the erythrocytoses (polycythaemias)

The term 'erythrocytosis' has advantages over 'polycythaemia' to describe patients with a raised haematocrit (PCV) and deserves to be more widely used. Measurement of red cell mass (RCM) and its relation to that expected for an individual's height and weight permits initial subdivision of erythrocytosis into absolute (increased RCM) or apparent normal RCM. Absolute erythrocytosis may be primary (intrinsically abnormal marrow erythropoiesis) or secondary (increased erythropoietin drive in response to pathological events outside the bone marrow). Both primary and secondary erythrocytosis may be either congenital or acquired. Idiopathic erythrocytosis is a third, probably heterogenous, group within the absolute erythrocytoses. Familial abnormalities of the erythropoietin receptor underlie the primary congenital subgroup. Polycythaemia vera (PV), the clonal myeloproliferative disorder, is so far, the only primary acquired disorder. Newer diagnostic investigations such as serum erythropoietin estimation, improved karyotypic analysis, in vitro culture of erythroid colonies and estimation of spleen size before splenomegaly is palpable, have permitted some modification of the traditional diagnostic criteria of polycythaemia vera. This may allow more confident diagnosis and, together with improved testing for causes of secondary erythrocytosis, may reduce the number of patients so far unsatisfactorily consigned to the idiopathic erythrocytosis group.     

REVIEWThe classification and diagnostic criteria of the erythrocytoses (polycythaemias)

The term ‘erythrocytosis’ has advantages over ‘polycythaemia’ to describe patients with a raised haematocrit (PCV) and deserves to be more widely used. Measurement of red cell mass (RCM) and its relation to that expected for an individual’s height and weight permits initial subdivision of erythrocytosis into absolute (increased RCM) or apparent normal RCM. Absolute erythrocytosis may be primary (intrinsically abnormal marrow erythropoiesis) or secondary (increased erythropoietin drive in response to pathological events outside the bone marrow). Both primary and secondary erythrocytosis may be either congenital or acquired. Idiopathic erythrocytosis is a third, probably heterogenous, group within the absolute erythrocytoses. Familial abnormalities of the erythropoietin receptor underlie the primary congenital subgroup. Polycythaemia vera (PV), the clonal myeloproliferative disorder, is so far, the only primary acquired disorder. Newer diagnostic investigations such as serum erythropoietin estimation, improved karyotypic analysis, in vitro culture of erythroid colonies and estimation of spleen size before splenomegaly is palpable, have permitted some modification of the traditional diagnostic criteria of polycythaemia vera. This may allow more confident diagnosis and, together with improved testing for causes of secondary erythrocytosis, may reduce the number of patients so far unsatisfactorily consigned to the idiopathic erythrocytosis group.     

Erythropoietin receptor and hematological disease

This review will discuss evidence for the role of the erythropoietin (Epo) receptor in the development of erythrocytosis and other hematological disorders. The possible causative role of mutations of other genes in the pathogenesis of idiopathic erythrocytosis will be considered. Polycythemia vera (PV) is a myeloproliferative disorder that is caused by an undefined stem cell abnormality, characterized by a significant erythrocytosis, leukocytosis, and thrombocytosis. However, erythrocytosis may arise from apparent (or relative) polycythemia in which the hematocrit is raised due to a low plasma volume. In such cases the red cell mass is normal. A group of disorders with increased red cell mass caused by stimulation of erythrocyte production is known as secondary polycythemia. Investigation of such patients may reveal a congenital abnormality such as high affinity hemoglobin or an acquired abnormality caused, for example, by smoking, renal vascular impairment, or an Epo-producing tumor. Even after thorough examination there remains a cohort of patients for whom no definite cause for the erythrocytosis can be established. A careful clinical history may reveal whether this idiopathic erythrocytosis is likely to be congenital and/or familial, in which case the term “primary familial and congenital polycythemia” is sometimes applied. Access to a range of laboratory investigations may define the molecular pathophysiology. We will now discuss how this process can be investigated. Am. J. Hematol. 60:55–60, 1999. © 1999 Wiley-Liss, Inc.     

Pure erythrocytosis classified according to erythropoietin titers

Erythropoietin titers, measured by bioassay of plasma extracts in hypertransfused mice, were determined in 162 patients with absolute erythrocytosis, and the results were correlated with the clinical diagnosis. Fifty-two patients met the diagnostic criteria for polycythemia vera, and all had low or nonmeasurable erythropoietin titers. Of the remaining 110 patients, 62 were suspected clinically as having secondary polycythemia. However, 15 had low erythropoietin titers, casting doubt on the accuracy of the clinical diagnosis. The pathogenesis of the erythrocytosis in the last 48 patients was unknown, and they were designated clinically as having pure erythrocytosis. However, in 20, the erythropoietin titers were increased, and in 28, the titers were low, suggesting that they belonged to at least two different groups. Using erythropoietin titers in the classification of absolute erythrocytosis, the first group should be added to the category of patients with secondary polycythemia as a subgroup with disease due to idiopathic overproduction of erythropoietin (hypererythropoietinemia or essential erythrocytosis). The second group should be added as a subgroup of patients with primary polycythemia under the term erythremia.     

Bone marrow features of diagnostic impact in erythrocytosis

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.     

Somatic mutations of JAK2 exon 12 in patients with JAK2 (V617F)-negative myeloproliferative disorders

We searched for JAK2 exon 12 mutations in patients with JAK2 (V617F)-negative myeloproliferative disorders. Seventeen patients with polycythemia vera (PV), including 15 sporadic cases and 2 familial cases, carried deletions or duplications of exon 12 in circulating granulocytes but not in T lymphocytes. Two of the 8 mutations detected were novel, and the most frequent ones were N542-E543del and E543-D544del. Most patients with PV carrying an exon 12 mutation had isolated erythrocytosis at clinical onset, unlike patients with JAK2 (V617F)-positive PV, most of whom had also elevations in white blood cell and/or platelet counts. Both patients with familial PV carrying an exon 12 mutation had an affected sibling with JAK2 (V617F)-positive PV. Thus, several somatic mutations of JAK2 exon 12 can be found in a myeloproliferative disorder that is mainly characterized by erythrocytosis. Moreover, a genetic predisposition to acquisition of different JAK2 mutations is inherited in families with myeloproliferative disorders.     

Smoking as a cause of erythrocytosis.

Five smokers had erythrocyte masses sufficiently larger than normal to pose a problem in the differential diagnosis of polycythemia. Evaluation excluded lung disease, shunt physiology, hemoglobin with increased oxygen affinity, erythropoietin-producing tumor, renal disease, or polycythemia rubra vera as the primary cause of erythrocytosis in these patients. All were found to have levels of carboxyhemoglobin sufficient to cause clinically significant hypoxemia and to account for the increased erythrocyte masses. In two patients the erythrocytosis improved when they stopped smoking. Heavy smoking is a reversible cause of polycythemia and should be considered in the differential diagnosis of this problem.     

Diagnosis and classification of erythrocytoses and thrombocytoses

An erythrocytosis describes an increased peripheral blood packed cell volume (PCV) and is deemed to be absolute or apparent depending on whether or not the measured red cell mass (RCM) is above the reference range. This reference range must be related to the individual's height and weight to avoid erroneous interpretations using ml/kg total body weight expressions in obesity. Absolute erythrocytoses are divided into primary, where the erythropoietic compartment is intrinsically abnormal, secondary, where the erythropoietic compartment is normal but is responding to external pathological events leading to an increased erythropoietin drive, and idiopathic, where neither a primary nor a secondary erythrocytosis can be established. Both primary and secondary erythrocytoses have congenital and acquired forms. The only form of primary acquired erythrocytosis that has been defined is the clonal myeloproliferative disorder, polycythaemia vera (PV). Modified diagnostic markers for PV are proposed. Thrombocytoses can be classified into primary, where megakaryopoiesis is intrinsically abnormal, secondary, where megakaryopoiesis is normal but increased platelet production is a reaction to some other unrelated pathology, and finally idiopathic. This latter new group would be used for patients not satisfying the criteria for primary or secondary thrombocytoses, if these were more precise and rigidly used than currently is the case. While theoretically congenital and acquired forms of primary and secondary thrombocytoses might exist, only one cause of secondary congenital thrombocytosis has been established, and primary congenital thrombocytosis has not yet been precisely defined. Primary (essential) thrombocythaemia (PT) is one of the forms of primary acquired thrombocytoses. The diagnostic criteria of PT traditionally involve the exclusion of secondary thrombocytoses and other myeloproliferative disorders but marrow histology could hold a key positive diagnostic role if objective histological features of PT were agreed.     

Polycythemia vera with an inhibitor against factor XII

A case of polycythemia vera with an inhibitor against factor XII was reported. A 60-year-old female was admitted to Hokkaido University School Hospital because of erythrocytosis and hepatosplenomegaly. The hemoglobin was 22.5 g/dl and white cell count was 9,500/microliters without immature cells. The platelet count was 484,000/microliters. Bone marrow specimens showed marked hypercellularity. Philadelphia chromosome was not found on chromosome analysis. She was diagnosed as polycythemia vera according to the criteria of polycythemia Vera Study Group. Activity of factor XII was found to be decreased on the initial examination, but she had no personal and familial history of bleeding. In order to clarify the cause of decreased activity of factor XII, her plasma was mixed with normal plasma, and then examined PTT using factor XII deficient plasma. Her plasma mixed with equivalent normal plasma did not show the correction of prolonged PTT. It was suggested that an inhibitor of her plasma was included in the IgG fraction using gel chromatography. The patient was treated with phlebotomy and administration of N4-palmitoyl (1- -D-arabinofurasyl) cytosine (derivative of cytosine arabinoside; PLAC) 200 mg/day and Busulfan (1 mg/day). Factor XII was not corrected by phlebotomy, but corrected gradually by administration of PLAC and Busulfan.     

Increased thromboxane biosynthesis in patients with polycythemia vera: evidence for aspirin-suppressible platelet activation in vivo.

Increased thromboxane (TX) production and modified aspirin sensitivity has been detected in vitro in platelets isolated from patients with polycythemia vera. To verify the relevance of these capacity-related measurements to the actual rate of TXA2 biosynthesis in vivo and its suppression by oral aspirin, we have investigated the urinary excretion of major enzymatic metabolites of TXB2 in 17 patients with polycythemia vera and 23 gender- and age-matched controls. Urinary 11-dehydro-TXB2 and 2,3-dinor-TXB2 were measured by previously validated radioimmunoassays. In addition, urinary immunoreactive leukotriene (LT) E4 was measured to explore the 5-lipoxygenase pathway of arachidonate metabolism. Polycythemic patients had significantly (P < .001) higher excretion rates of both 11-dehydro-TXB2 (1,033 +/- 1,050 v 117 +/- 45 pmol/mmol creatinine; mean +/- SD) and 2,3-dinor-TXB2 (725 +/- 676 v 82 +/- 43 pmol/mmol creatinine) than controls. In contrast, urinary LTE4 was not significantly different. Enhanced metabolite excretion did not correlate with the platelet count or with the hematocrit value, and was not related to the current treatment or to a clinical history of thrombotic complications. Platelet TX receptor studies did not show any significant changes in the binding characteristics of two different ligands. A platelet-selective regimen of aspirin therapy (50 mg/d for 7 to 14 days) was associated with greater than 80% suppression in metabolite excretion in nine patients. These results are consistent with abnormal stimuli operating in polycythemia vera to induce a selective enhancement in the platelet biosynthesis of TXA2 without changes in receptor binding. This in vivo abnormality in platelet biochemistry can be largely suppressed by low doses of aspirin.     

Reticulated platelets are increased in chronic myeloproliferative disorders, pure erythrocytosis, reactive thrombocytosis and prior to hematopoietic reconstitution after intensive chemotherapy

Reticulated platelets with a high RNA content represent the most recently released platelets and are regarded to reflect thrombopoiesis. In the present study we used flow cytometric analysis to determine the percentage of reticulated platelets in peripheral blood for patients with chronic myeloproliferative disorders (polycythemia vera, essential thrombocytosis) and acute myelogenous leukemia (AML) patients with severe chemotherapy-induced thrombocytopenia. Patients with essential thrombocytosis and polycythemia vera showed increased levels of reticulated platelets compared with healthy controls, and these levels persisted after normalization of the platelet count by hydroxyurea or interferon-alpha treatment. Patients with reactive thrombocytosis or thrombocytopenia with increased platelet turnover often had higher levels of circulating reticulated platelets than patients with myeloproliferative disorders. Furthermore, AML patients with severe chemotherapy-induced cytopenia showed low levels that started to increase 1-9 days prior to hematopoietic reconstitution. To summarize and conclude: (i) circulating reticulated platelets are increased in patients with chronic myeloproliferative disorders, reactive thrombocytosis and thrombocytopenia due to increased platelet turnover; (ii) patients with pure erythrocytosis often have additional abnormalities in the thrombopoiesis; and (iii) the levels of reticulated platelets seem to predict hematopoietic reconstitution for patients receiving intensive AML therapy.     

Intracellular growth factors in polycythemia vera and other myeloproliferative disorders.

In polycythemia vera, idiopathic myelofibrosis, and essential thrombocytosis, hematopoietic cell proliferation is increased in the absence of a recognizable stimulus, suggesting the autonomous production of growth factors in these disorders. Sonicates of peripheral blood mononuclear cells (PBMNC) from patients with polycythemia vera, idiopathic myelofibrosis, and essential thrombocytosis contained soluble factors that stimulated the proliferation of quiescent-confluent 3T3 cells. PBMNC sonicates from normal individuals; from patients with secondary erythrocytosis, chronic myelogenous leukemia, B-cell chronic lymphocytic leukemia, and acute myelogenous leukemia; and from K-562 and HL-60 cells did not stimulate proliferation. Polycythemia vera PBMNC sonicates also induced anchorage-independent colony formation in soft agar by normal rat kidney fibroblasts. Both the mitogenic and transforming activities of the polycythemia vera PBMNC sonicates resided in the T-lymphocyte-depleted mononuclear fraction of the PBMNC and were not secreted. By gel filtration, reversed-phase HPLC and NaDodSO4/PAGE, the mitogenic and transforming activities in the polycythemia vera PBMNC were localized to three proteins with molecular masses of 13-, 17-, and 65-kDa. The 13-kDa protein was only mitogenic, and the 17-kDa protein was only transforming, whereas the 65-kDa protein had both mitogenic and transforming activity. These proteins may be involved in the autonomous hematopoiesis that characterizes polycythemia vera, idiopathic myelofibrosis, and essential thrombocytosis.     

Polycythemia vera

The diagnostic approach to a patient with polycythemia has been greatly simplified by the introduction of new genetic testing in addition to traditional tests, such as measurement of red cell mass and serum erythropoietin (Epo) level. Clonal erythrocytosis, which is the diagnostic feature of polycythemia vera (PV), is almost always associated with a JAK2 mutation (JAK2V617F or exon 12). Therefore, in a patient with acquired erythrocytosis, it is reasonable to begin the diagnostic work-up with JAK2 mutation analysis to distinguish PV from secondary erythrocytosis. The clinical course of PV is marked by a high incidence of thrombotic complications that represent the main cause of morbidity and mortality in these patients. Blood hyperviscosity as well as platelet and leukocyte quantitative, and qualitative abnormalities play a major role in the pathogenesis of thrombophilia. Prevention of vascular events and minimizing the risk of disease transition into acute leukaemia are the main targets of the whole PV treatment strategy. This can rely on the use of low-dose aspirin in most patients, while the choice of the optimal cytoreductive strategy is based on the individual vascular risk. Phlebotomy is still the preferred treatment in subjects at low risk, while hydroxyurea or pipobroman is usually administered to most elderly subjects or subjects with a previous vascular history. The use of pegylated interferon, imatinib, and JAK2 inhibitors is currently being evaluated.     

Haptoglobin Metabolism in Polycythemia Vera

1. Turnover studies with ¹²⁵I-labeled haptoglobin (Hp) were performed in7 patients with polycythemia vera, 2 patients with erythrocytosis of unknownetiology, and 2 control subjects.

2. The T of plasma radioactivity was shortened in 6 of the 7 patients withpolycythemia vera; 3 of these had diminished plasma Hp levels but lackedother evidence of hemolysis.

3. The fractional catabolic rate exceeded 40 per cent/day in all subjectswith a shortened half-time of plasma radioactivity.

4. Increases in the fractional catabolic rate were not accompanied by increases in Hp turnover (mg./kg./day), suggesting that accelerated Hp catabolism per se does not provide a stimulus to Hp production.

5. It is concluded that patients with polycythemia vera catabolize Hp morerapidly than nonpolycythemic subjects, possibly because of increased formation and removal of the haptoglobin-hemoglobin complex.

Submitted on November 5, 1968 Accepted on January 21, 1969     

JAK2 exon 12 mutations in polycythemia vera or idiopathic erythrocytosis

JAK2 exon 12 mutations were detected in 4 out of 20 polycythemia vera and idiopathic erythrocytosis V617F-negative patients and were only present in the myeloid lineage. Initial hematologic data of these patients differ from those of V617F-positive patients, but there is no difference in thrombotic development and myelofibrotic transformation.     

Endogenous erythroid colony formation by peripheral blood mononuclear cells from patients with myelofibrosis and polycythemia vera.

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.     

Polycythemia vera and other primary polycythemias

PURPOSE OF REVIEW: Diagnosis and therapy of polycythemia vera are controversial since the molecular basis of polycythemia vera remains unknown. Distinguishing between polycythemia vera and other polycythemic disorders can be very challenging. The purpose of this review is to discuss the recent progress in this area and critically review the published data in context of our knowledge of other polycythemic disorders. RECENT FINDINGS: Erythropoietin is the principal regulator of regulator of erythropoiesis; its production is regulated by the degree of hypoxia. Our knowledge of cellular responses to hypoxia has recently exploded and led to the elucidation of the molecular basis of a polycythemia caused by augmentation of hypoxic sensing, Chuvash polycythemia. Similar progress in understanding the molecular basis of polycythemia vera has been elusive. A simple, readily available laboratory test to establish a diagnosis of polycythemia vera would be highly desirable; however, none exists. The value of quantization of neutrophil PRV-1 mRNA, platelet c-mpl expression, in vitro assays of erythroid progenitor cells, serum erythropoietin levels, establishing clonality in female subjects using assays employing X-chromosome-based polymorphism assays, and the progress in the chromosomal location of the gene is discussed. Integration of this information underlies the complexity of the molecular biology of polycythemia vera and indicates likely interaction of multiple genetic events in the genesis of polycythemia vera. SUMMARY: The existence of family clustering of PV may facilitate the search for PV molecular basis. Only collaborative interaction of clinical researchers and laboratory scientists will lead to meaningful progress in determining the molecular basis of PV.     

Pathogenic markers in essential thrombocythemia

Although long recognized as a clinical entity, the diagnosis of essential thrombocythemia (ET) still relies on the exclusion of reactive conditions and other myeloproliferative disorders. Recent studies have attempted to identify new markers for this disorder and suggest that part of the problem may relate to its heterogeneity. Cytogenetic abnormalities are rare, but analysis using X-chromosome inactivation patterns indicates that nearly 50% of evaluable patients have polyclonal myelopoiesis and this is associated with a lower risk of thrombosis. Reduced and heterogeneous staining of c-Mpl in bone marrow biopsies may help to distinguish ET and a reactive thrombocytosis. Increased expression of PRV-1 (polycythemia ruba vera) in granulocytes may assist in discriminating between polycythemia vera and at least some cases of ET. The contribution of these markers to disease pathogenesis is unknown and prospective studies are needed to evaluate their usefulness for predicting clinical outcome and directing patient therapy.