Diagnostic and prognostic value of bone marrow angiogenesis and megakaryocyte c-Mpl expression in essential thrombocythemia

The lack of diagnostic certainty in some patients makes it difficult to distinguish between primary and secondary forms of thrombocytosis. To augment current diagnostic studies for thrombocytosis, we retrospectively evaluated clinical records and bone marrow trephine specimens of 183 patients with thrombocytosis-164 with essential thrombocythemia (ET), 19 with reactive thrombocytosis (RT)-for bone marrow angiogenesis, bone marrow megakaryocyte c-Mpl staining, and morphologic evidence of megakaryocyte proliferation. Angiogenesis was increased in patients with ET compared with healthy controls (P <.0001) and patients with RT (P =.006). In addition, an increase in angiogenesis was associated with certain disease features such as splenomegaly (P =.004) and reticulin fibrosis (P =.005). Decreased megakaryocyte c-Mpl staining was observed in a heterogeneous pattern in ET compared with healthy controls (P <.0001) and RT (P <.0001). Histologic stratifying criteria incorporating increased angiogenesis, decreased megakaryocyte c-Mpl expression, and marked megakaryocyte proliferation in the bone marrow was highly sensitive (97%) and specific (95%) for distinguishing ET from RT (P <.0001). However, with the current duration of follow-up available on the patients, none of the histologic features evaluated have yet demonstrated prognostic value for subsequent clinical course, vascular events, or survival.     

Platelet c-mpl expression is dysregulated in patients with essential thrombocythaemia but this is not of diagnostic value

Essential thrombocythaemia (ET) can be difficult to discriminate from an occult case of reactive thrombocytosis (RT). Since thrombopoietin (TPO) is the primary regulator of thrombopoiesis, we have investigated whether levels of TPO and/or its receptor, c-mpl, are of value in the differential diagnosis of ET. Plasma TPO levels in patients with ET, RT and other myeloproliferative disorders (MPDs) did not differ significantly from normal controls. However, surface c-mpl expression was significantly reduced in platelets from 18 ET patients, 0-65.5% of controls (P < 0.001). Immunoblots on five of these and five additional patients were consistent with absent or reduced c-mpl protein levels. The surface c-mpl expression results were significantly different from those in eight RT patients (21. 3-95.5%, P = 0.0015), but there was considerable overlap between the two groups and a reduced level was not restricted to ET. Furthermore, c-mpl expression in ET patients was not different from eight patients with other MPDs (0-87.6%, P = 0.06), nor could it differentiate between ET patients with monoclonal and polyclonal haemopoiesis. Although a low or absent c-mpl level is suggestive of a primary rather than a secondary thrombocytosis, it is insufficiently discriminatory to be used as a diagnostic marker for ET.     

Thrombopoietin levels in patients with primary and reactive thrombocytosis

Human c-mpl ligand or thrombopoietin (TPO) has been proved to be a critical cytokine in the physiological regulation of thrombopoiesis. Previous evidence suggested that TPO production is constitutive and TPO plasma levels are regulated by the platelet–megakaryocyte mass through c-mpl receptor-mediated uptake and metabolism. To evaluate whether this mechanism of TPO level regulation is also operative in subjects with an elevated platelet count, we evaluated serum TPO in 32 patients with thrombocytosis due to essential thrombocythaemia (ET) or polycythaemia vera (PV) and in 70 subjects with reactive thrombocytosis; 32 healthy subjects were also studied. TPO levels were significantly higher in the ET and PV groups (median 246.2 pg/ml, range 93.5–4596) and reactive thrombocytosis patients (median 287 pg/ml, range 82.7–1960.0) than in normal subjects (median 156.7 pg/ml, range 62.2–352.7). No significant difference was found between the two groups of patients, indicating that serum TPO levels cannot differentiate between primary and reactive thrombocytosis. No significant correlation was found between platelet count and TPO levels in either ET-PV or reactive thrombocytosis, whereas a trend toward a correlation between acute-phase reactants and TPO was observed in patients with reactive thrombocytosis. These results indicate that TPO clearance by platelets–megakaryocytes is not fully operative or is not the only mechanism of TPO level regulation in primitive and reactive thrombocytosis.     

Megakaryocytic colony formation (CFU-Meg) in essential thrombocythemia: quantitative and qualitative abnormalities of bone marrow CFU-Meg

For the purpose of examining the mechanisms for the overproduction of megakaryocytes and platelets in essential thrombocythemia (ET), marrow megakaryocytic colony-forming units (CFU-Meg) were characterized using a methylcellulose culture system in nine patients with ET, and they were compared with those of 16 control subjects and four patients with secondary thrombocytosis (ST). The number of CFU-Meg per 10(5) cells in ET was significantly higher (p less than .001) than that in the controls (5.8 times of the controls) or that in ST. ET showed endogenous CFU-Meg (58 +/- 16% of the total CFU-Meg) that could form megakaryocytic colonies without phytohemagglutinin-stimulated leukocyte-conditioned medium (PHA-LCM) as a source of Meg-CSA. The controls and the patients with St revealed no endogenous CFU-Meg. The dose-response studies with PHA-LCM revealed that CFU-Meg from ET are highly sensitive to low concentrations of PHA-LCM compared to CFU-Meg from the control and ST. Erythropoietic burst-forming units (BFU-E) were monitored as a control progenitor, because the patients with ET studied did not have erythrocytosis. Although five out of nine patients with ET had endogenous BFU-E, the percentages of endogenous BFU-E were small (4-10%). The number of BFU-E in ET was not different from the control value. Thus, ET is associated with an enlarged CFU-Meg compartment that shows abnormal growth patterns when cultured in vitro, while the growth abnormality of BFU-E is small, which may underlie the hematological features of ET. The culture of CFU-Meg should be clinically useful for differentiating ET from ST.     

Marked heterogeneity in protein levels and functional integrity of the thrombopoietin receptor c-mpl in polycythaemia vera

Polycythaemia vera (PV) is a myeloproliferative disorder (MPD) characterized by an increased production of mature blood cells. The underlying pathogenic mechanisms behind PV are largely unknown. Thrombopoietin (TPO) is the most important cytokine for stimulation of megakaryocyte growth and formation of functional platelets. Recently, it has been shown that the receptor for TPO, c-mpl, is expressed on haematopoietic stem cells, and that TPO promotes the growth of these stem cells via binding to c-mpl. Quantitative or qualitative abnormalities of c-mpl function could thus theoretically play a role in the pathogenesis of different MPDs. Previous studies of the integrity of the c-mpl system in PV have produced conflicting results. We therefore studied c-mpl protein expression using immunoblot analysis in 15 PV patients and 10 healthy controls. Seven out of 15 PV patients (47%) exhibited similar c-mpl protein levels to the controls, whereas eight out of 15 patients (53%) showed either markedly reduced or absent levels of c-mpl. Five of the seven c-mpl-positive patients had only been treated by phlebotomy, whereas six out of eight c-mpl-negative patients were receiving treatment with hydroxyurea, anagrelide or alpha-interferon. Disease duration tended to be slightly longer in c-mpl-negative patients compared with c-mpl-positive patients (mean = 55 vs. 43 months). Tyrosine phosphorylation of JAK-2 in immunoprecipitates of platelets obtained after stimulation with TPO (100 and 1000 ng/ml) was normal in c-mpl-positive patients, whereas it could not be detected in c-mpl-negative patients. We therefore conclude that there exists a marked heterogeneity in c-mpl protein levels and functional integrity in PV. However, it seems less likely that c-mpl abnormalities per se are directly involved in the pathogenesis leading to the occurrence of PV, as c-mpl levels were similar to those seen in healthy individuals in about half of the patients under study.     

Predictive values of X-chromosome inactivation patterns and clinicohematologic parameters for vascular complications in female patients with essential thrombocythemia

Essential thrombocythemia (ET) is a heterogeneous disorder in which the clonality of hematopoiesis varies. The clinical significance of clonality status in ET remains to be determined. We used the human androgen receptor gene (HUMARA)-polymerase chain reaction assay to investigate X-chromosome inactivation patterns (XCIPs) and their value in predicting vascular complications in 89 female patients with ET. Fifty-four (68.4%) patients had a clonal pattern of XCIP, and 15 (19.0%) had a polyclonal pattern. The remaining 20 patients had either an ambiguous or a homozygous pattern of XCIP and were therefore excluded from further analysis. Patients with clonal XCIPs were older (P =.029) and were at greater risk for thrombosis (P =.007) than were those with polyclonal XCIPs. We did not find a correlation between the occurrence of hemorrhage and XCIP (P =.492). Advanced age was predictive of thrombosis and hemorrhage. Platelet count did not influence the risk for vascular complications. Hypertension was significantly correlated with thrombotic events (P =.002), whereas diabetes mellitus and hypercholesterolemia were of no predictive value. In a multivariate analysis, age was the significant predictor of thrombosis (P =.030); however, XCIPs (P =.083) and hypertension (P =.073) tended to predict thrombosis. Our results suggest that older patients who have clonal XCIPs or hypertension are at increased risk for thrombosis and should be monitored closely for this complication.     

Philadelphia (Ph) chromosome-positive thrombocythemia without features of chronic myeloid leukemia in peripheral blood: natural history and diagnostic differentiation from Ph-negative essential thrombocythemia

We have evaluated the clinical symptoms, hematological features, and natural history of 3 cases and 20 reported cases described as Philadelphia chromosome-positive (Ph⁺) essential thrombocythemia (ET). The presence of increased small mononuclear megakaryocytes in bone marrow smears and biopsy material in patients with pronounced thrombocytosis and no evidence of chronic myeloid leukemia (CML) in peripheral blood appeared to be a diagnostic clue to the diagnosis of Ph⁺ (essential) thrombocythemia. As compared to cases of reactive thrombocytosis, the megakaryocytes in Ph⁺ thrombocythemia are smaller than normal ones and typically have hypolobulated round nuclei. This contrasts with the finding of clustered mature and enlarged megakaryocytes in Ph-negative true ET. Patients diagnosed as Ph⁺ ET may progress to CML and show a high tendency to myelofibrosis and blastic transformation. These observations indicate that both Ph⁺ ET and Ph⁺ thrombocythemia associated with CML can be regarded as early manifestations of the chronic stable phase of CML.     

Sex and age as prognostic factors in essential thrombocythemia.

BACKGROUND. The major causes of morbidity and mortality in essential thrombocythemia (ET) are bleeding and thrombotic accidents, but a prognostic pattern for these complications has not yet been discovered. MATERIALS AND METHODS. In this study we report data from a large cohort of patients with thrombocytosis, distinguished for sex and age, in order to define their thrombotic risk. The prevalence of vascular complications recognized in 86 patients with essential thrombocythemia was studied. In addition, 91 patients with polycythemia vera (PV), 20 with myelofibrosis (MF) and 63 with secondary thrombocytosis (ST) were evaluated. RESULTS. 6.3% of ET subjects younger than 40 (4.6% of males and 7.0% of females), 11.8% of patients between 40 and 65 years old (14.9% of males and 9% of females), and 16.8% of subjects over 65 (14.6% of males and 17.8% of females) showed thrombotic accidents. In the PV and MF groups thromboses occurred more frequently than in the ET groups for all ages and for both sexes. On the contrary, ST subjects showed fewer thromboses than ET patients, but their incidence rose with patient age; moreover the prevalence of males in this group was limited. In ET patients, particularly in females, the incidence of thrombosis was low under 40 years of age, but rapidly increased later. CONCLUSIONS. ET females over 40 must be followed with particular attention in order to prevent thrombotic complications.     

Flow cytometric analysis of megakaryocyte ploidy in chronic myeloproliferative disorders and reactive thrombocytosis

Abstract: Megakaryocyte (MK) ploidy patterns were analysed by flow cytometry in 29 newly diagnosed and previously untreated patients with chronic myeloproliferative disorders (MPD) and concomitant thrombocytosis, in 9 patients with reactive thrombocytosis (RT) and in 12 healthy individuals. Unfractionated bone marrow from routine aspirates was used. MKs were identified with a fluorescein labelled monoclonal antibody specific for glycoprotein IIIa (GPIIIa) and DNA was stained with propidium iodide. For the 12 healthy volunteers the mean modal ploidy number was 16 N; the 9 patients with RT displayed an identical MK ploidy pattern. The frequency of MKs with a ploidy ≥32 N was 45% among the patients with essential thrombocythaemia (ET) compared to 32% among the healthy volunteers (p < 0.001). MKs with ploidy number ≥ 64 N, comprising approximately 13% of the total number of MKs, was a characteristic finding in the patients with ET. Similar findings were present in 8 patients with polycythaemia vera (PV). In patients with PV 34% and 6% of the MKs displayed ploidies ≥32 N and ≥64 N, respectively. In contrast, a distinct shift towards lower ploidy number, with 63% of MKs ≤ 8 N, was found among the 4 patients with chronic myeloid leukaemia (CML). The present results indicate that by using flow cytometric analysis of MK ploidy distribution in patients with thrombocytosis, those with a reactive cause are likely to be discriminated from patients with myeloproliferative thrombocytosis, i.e. PV and ET on one hand and CML on the other hand. The distinction between ET and PV, however, has to be made on other grounds.     

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.     

Expression of adhesion antigens of human bone marrow megakaryocytes, circulating megakaryocytes and blood platelets.

There is evidence that mature megakaryocytes migrate into sinusoids, enter the blood and fragment in the vascular bed. We wondered whether differences in expression of adhesion antigens could be associated with the egress of megakaryocytes from bone marrow into the peripheral blood or the fragmentation into platelets. Megakaryocytes from human marrow were purified by counterflow centrifugal elutriation followed by a glycoprotein Ib-dependent agglutination procedure. Megakaryocytes from central venous blood and pulmonary arteries were purified by counterflow centrifugal elutriation alone. Adhesion antigens were labelled in an immunohistochemical assay. Both bone marrow megakaryocytes and platelets from healthy volunteers stained > 75% positive for CD36, CD41, CD42, Cdw49b (alpha subunit VLA2), Cdw49e (alpha subunit VLA5), Cdw49f (alpha subunit VLA6) and CD62. Circulating megakaryocytes, although > 75% positive for CD41, had, unlike platelets and bone marrow megakaryocytes, a reduced and remarkable heterogeneous (5-100% positive) labelling with antibodies against Cdw49b, Cdw49e, Cdw49f. These results could be confirmed by comparing the bone marrow megakaryocytes, circulating megakaryocytes and platelets from 7 patients that were recovered and processed at the same time. Morphologically mature, circulating megakaryocytes have, unlike bone marrow megakaryocytes, a heterogeneous expression of adhesion antigens, especially of Cdw49b, Cdw49e, and Cdw49f.     

Initial (latent) polycythemia vera with thrombocytosis mimicking essential thrombocythemia

Patients have previously been described who showed clinical signs and symptoms suggesting essential thrombocythemia (ET), but later transformed to polycythemia vera (PV). From a series of 344 patients with a sustained borderline to moderate erythrocytosis, 44 failed to conform initially with the diagnostic criteria of the WHO for PV, because of their low hemoglobin level. Twenty-three patients of this group presented with a thrombocytosis exceeding 600 x 10(9)/l and therefore suggested ET, but later developed full-blown PV. For comparison we investigated also 164 patients with manifest PV, 90 patients with ET and 22 patients with reactive thrombocytosis (Th). The histopathology of initial PV was evaluated by stepwise discriminant analysis of 17 standardized features. Quantity and left shifting of erythro- and granulopoiesis, giant forms and naked nuclei of megakaryocytes, cellularity and reticulin fibers proved to exert a significant relevance concerning differentiation from true ET and Th. In conclusion, initial PV with thrombocytosis is characterized by a special pattern of BM histopathology. Therefore, so-called masked PV in patients with ET or simultaneous PVR-1 gene expression and endogeneous erythroid colony growth in ET patients are probably in keeping with initial PV mimicking ET.     

Immunohistochemical staining for megakaryocyte c-mpl may complement morphologic distinction between polycythemia vera and secondary erythrocytosis

Recent studies have shown decreased megakaryocyte expression of the thrombopoietin receptor (c-mpl) in patients with polycythemia vera (PV) but not in those with reactive erythrocytosis. We examined the diagnostic utility of this observation in 22 patients with PV, 7 patients with secondary erythrocytosis (SE), and 10 normal controls. Commercial antibodies against c-mpl were used with standard immunoperoxidase methods. Megakaryocyte c-mpl staining intensity was uniformly moderate-to-strong in the healthy controls and in all the patients with SE. In contrast, staining intensity in 9 patients with PV (41%) was uniformly weak. Furthermore, in 12 of the remaining 13 patients with PV, the c-mpl staining pattern in each case was heterogeneous and was associated with weak staining intensity in more than 20% of the megakaryocyte population. These preliminary data suggest that c-mpl immunostains may complement bone marrow histopathology in distinguishing PV from nonclonal causes of erythrocytosis. (Blood. 2000;96:771-772)     

Multivessel coronary thrombosis treated with abciximab (reopro®) in a patient with essential thrombocythemia

A 50-year-old man was admitted with acute inferior and anterior myocardial infarction. The patient was diagnosed with essential thrombocythemia (ET) based on the findings of marked thrombocytosis of 1,113 × 10³/mm³, splenomegaly, and numerous clumping megakaryocytes on bone marrow biopsy. Emergent coronary angiography revealed extensive multivessel thrombosis involving the left main coronary artery and completely occluding the proximal right coronary artery. In addition to standard therapy with aspirin, heparin, and primary angioplasty of the right coronary artery, the patient received additional antiplatelet therapy with ticlopidine, hydroxyurea, and the platelet glycoprotein IIb/IIIa receptor-inhibiting monoclonal antibody drug abciximab (ReoPro®). Serial coronary angiograms 1 and 5 days following the infarction showed progressive thrombus resolution. The pathophysiologic mechanisms and therapeutic challenges of ET-associated coronary thrombosis are discussed in this report.     

Thrombopoietin-producing hepatocellular carcinoma

A 66-year-old man with hepatocellular carcinoma (HCC) showed marked thrombocytosis (110.7 x 10(4)/microl). Bone marrow (BM) aspirates demonstrated an increase of mature megakaryocytes (MgK). The serum thrombopoietin (TPO) level was increased to about 100-fold that of the normal level in the terminal stage. However, the platelet count gradually decreased to 13.5 x 10(4)/microl. The autopsy specimen revealed normoplastic BM with decreased MgK, mainly consisting of the immature type, and it was negative for tumor cells. Liver specimen showed markedly fatty metamorphosis. Immunohistochemical staining of TPO demonstrated that hepatocytes were weakly stained and HCC cells strongly stained, suggesting TPO-producing HCC.     

Ultrastructural study shows morphologic features of apoptosis and para-apoptosis in megakaryocytes from patients with idiopathic thrombocytopenic purpura

To investigate whether altered megakaryocyte morphology contributes to reduced platelet production in idiopathic thrombocytopenic purpura (ITP), ultrastructural analysis of megakaryocytes was performed in 11 ITP patients. Ultrastructural abnormalities compatible with (para-)apoptosis were present in 78% +/- 14% of ITP megakaryocytes, which could be reversed by in vivo treatment with prednisone and intravenous immunoglobulin. Immunohistochemistry of bone marrow biopsies of ITP patients with extensive apoptosis showed an increased number of megakaryocytes with activated caspase-3 compared with normal (28% +/- 4% versus 0%). No difference, however, was observed in the number of bone marrow megakaryocyte colony-forming units (ITP, 118 +/- 93/105 bone marrow cells; versus controls, 128 +/- 101/105 bone marrow cells; P =.7). To demonstrate that circulating antibodies might affect megakaryocytes, suspension cultures of CD34+ cells were performed with ITP or normal plasma. Morphology compatible with (para-)apoptosis could be induced in cultured megakaryocytes with ITP plasma (2 of 10 samples positive for antiplatelet autoantibodies). Finally, the plasma glycocalicin index, a parameter of platelet and megakaryocyte destruction, was increased in ITP (57 +/- 70 versus 0.7 +/- 0.2; P =.009) and correlated with the proportion of megakaryocytes showing (para-) apoptotic ultrastructure (P =.02; r = 0.7). In conclusion, most ITP megakaryocytes show ultrastructural features of (para-) apoptosis, probably due to action of factors present in ITP plasma.     

Vascular complications of essential thrombocythaemia: a link to cardiovascular risk factors

Essential thrombocythaemia (ET) is a myeloproliferative disorder characterized by absolute thrombocytosis and increased incidence of thrombosis and haemorrhage. We report higher morbidity in patients with ET due to arterial ischaemic complications when cardiovascular risk factors are present. In this retrospective analysis of 46 patients, arterial complications occurred in 20/46 patients (43.4%); patients with cardiovascular risk factors, especially cigarette smoking, had more than twice as many arterial complications than patients without risk factors (62.5% v 22.7%, P <0.05). Neither age, gender nor degree of thrombocytosis were linked to the number of complications. In contrast, we observed no association between cardiovascular risk factors and venous thrombosis or haemorrhagic complications of ET.     

Megakaryocytopoiesis in different forms of thrombocytosis and thrombocytopenia: identification of megakaryocyte precursors by immunostaining of intracytoplasmic factor-VIII-related antigen

Megakaryocytopoiesis was investigated with a polyclonal (26 cases) and a monoclonal (20 cases) antibody to factor-VIII-related antigen (factor VIII RAg) with the indirect peroxidase-antiperoxidase (PAP) method on air-dried bone marrow aspirates (BM). Numerous megakaryocyte precursors were identified in 5 patients with essential thrombocythemia (ET) and the 2 patients with acute myelogeneous leukaemia (AML) after recovery from therapeutic aplasia. Small megakaryocyte precursors were rare in controls (C), patients with reactive thrombocytosis (RT) and immune thrombocytopenia (IT). In 2 patients with severe alcoholism the number of mature megakaryocytes increased after 5 and 7 days of abstinence, respectively.     

Megakaryocyte ploidy in thrombocytosis: improved microdensitometric measurements with a new image analysis system

Megakaryocyte (MK) nuclear deoxyribonucleic acid (DNA) content was measured on a new image analysis system. Feulgen-stained bone marrow aspirate smears were analysed from 9 patients with thrombocytosis. Average optical density (OD) of stained nuclei was evaluated by pixel discrimination over 128 grey levels. Projected nuclear area was delineated manually. The product of these two parameters gave an index of nuclear DNA content. Neutrophils were used as 2N reference cells. Reproducibility of OD, nuclear area and their product was excellent for individual cells (CV less than 2.0% for MKs, less than 4.0% for neutrophils). The average CV for DNA content of 18 groups of 10 neutrophils was 5.5% (range 3.0-9.7%). A significant linear regression existed between MK nuclear area and DNA content (p much less than 0.001) for 627 MKs in essential thrombocythaemia (ET) and 346 MKs in reactive thrombocytosis (RT). Compared with RT, more 8N and 64N MKs were seen in ET (p less than 0.05). 128N MKs were unique to ET. Image analysis of MK ploidy may assist the clinical discrimination between primary and secondary thrombocytosis.     

Basic sciences of the myeloproliferative diseases: pathogenic mechanisms of ET and PV

The molecular pathogenesis of ET and PV is unknown, although the relatively indolent clinical course observed in most patients suggests that the defect may be subtle and difficult to establish. Clonality analysis using X-chromosome inactivation patterns in females on purified CD34+ cells have confirmed that a defect is present in the hematopoietic stem cell. However, at least in ET, a significant proportion of patients have polyclonal hemopoiesis, and this presumably reflects the heterogeneous nature of the disorder(s). Attention has focussed on the potential disruption of the physiological regulators EPO and TPO and their respective receptors. In familial disorders, pathological mutations have been identified in some, but by no means all, cases: EPO receptor mutations in PFCP, TPO mutations in FT and, conversely, TPO receptor (c-mpl) mutations in CAMT. Equivalent ligand or receptor mutations have not been detected in ET or PV patients. However, there is evidence to suggest that c-mpl expression may be dysregulated, with low or absent c-mpl mRNA or protein reported in ET and/or PV patients. At present it is not clear whether this is the cause or consequence of the paradoxically normal/increased TPO levels found with both primary and secondary thrombocytosis. In vitro culture analysis has demonstrated both cytokine independence and hyper-sensitivity as a generalised feature of progenitor cells from many patients, but differences exist depending on the assays used and there is little understanding of the mechanism(s) underlying these responses. Two genes have recently been identified with increased mRNA expression in PV granulocytes: PRV-1, a novel cell surface receptor closely related to the uPAR/Ly6/CD59/snake toxin family of proteins, and NFI-B, a member of the nuclear factor I family which may be associated with TGF-beta resistance. Investigation of their regulation and biological effects may assist in determining the pathobiology of these elusive disorders.