Chronic Thrombocytosis Research Articles

Acquired Von Willebrand Disease Associated with Hematologic Malignancies: Case Series and Literature Review

Objective: To describe a single institution experience of AvWD of the spectrum of presentation in hematologic malignancies. Introduction: Acquired von Willebrand disease (AvWD) is a rare acquired bleeding disorder characterized by mucocutaneous bleeding and functional alterations in von Willebrand factor (vWF). AvWD can be caused by numerous conditions but we are going to focus on those associated with hematologic malignancies. The pathogenic mechanisms responsible for the vWF abnormalities can include abnormal clearance due to binding of paraproteins, inhibition of vWF, adsorption to the surface of platelets, proteolysis from increased fluid shear stress, and decreased synthesis. Methods: Clinical review of 4 cases of AvWD at a tertiary academic medical center from January 2016 to January 2022. Results: Case 1: 57-year-old female with history of chronic thrombocytosis of 1.6 million and was diagnosed with a JAK2 V617F mutation positive essential thrombocythemia and concurrent iron deficiency anemia. She was placed on low dose aspirin for thromboprophylaxis and presented with hematochezia with a drop in hemoglobin from 9.7g/dL to 4.3g/dL, platelets were 857k/uL. At that time her aPTT was mildly elevated at 37.0 (normal 36.5) This prompted testing for von Willebrand disease which showed low vWF activity 50% and vWF GP I binding activity 47% with normal vWF antigen activity 82% and factor VIII clotting activity of 100 IU/ml. Aspirin was held and hydroxyurea initiated at 1000mg daily with clinical response. Coagulation testing including aPTT and vWF antigen, Activity and multimers normalized after cytoreduction of her platelets with hydrea. Case 2: 75-year-old male with history of stroke on aspirin. We saw the patient for erythrocytosis and made the diagnosis of JAK 2 V617F mutation positive polycythemia rubra vera. Phlebotomy was initiated however, he presented with large ecchymoses at the venipuncture sites following phlebotomy. Testing showed quantitative vWF and factor VIII were normal, abnormal GPIbM: vWF ratio and loss of highest molecular weight multimers. He was maintained on hydroxyurea 1500mg daily for target hematocrit of 40 g/dL and low dose aspirin thromboprophylaxis without recurrent bleeding. Case 3: A 68-year-old female who we were following with a history of an IgM monoclonal gammopathy of undetermined significance (MGUS) reported mild ecchymosis on the extremities. Testing showed low factor VIII level of 34% and low vWF antigen and activity at 29% and 22%, and normal vWF multimers. She has been observed without any therapy given lack of clinically significant bleeding. Three years later, she has now developed of cytopenias and bone marrow biopsy showed 30 - 40% lymphoplasmacytic infiltrate consistent with progression to Waldenström Macroglobulinemia. Case 4 60 year-old-male with history of an IgG lambda MGUS who presented with new onset recurrent melena without a source of bleeding found on endoscopy. Testing showed decreased vWF antigen 30% and vWF activity 21%, normal factor VIII activity 69% and all multimers decreased consistent with AvWD from his known MGUS. His recurrent mucosal bleeding was managed with weekly Humate P. Revlimid 10mg per day was initiated for MGUS. Two months later, his indices improved with normalized VWF multimers and VWF antigen 70% and factor VIII activity 142%, and with now only mildly decreased VWF activity at 48%. Recurrent bleeding ceased. Conclusions: Patients with hematologic malignancies may present with mucocutaneous bleeding which may mimic congenital vWD however, AvWD should be considered in this population of patients. In addition, patients with AvWD can present with varying degrees of bleeding which might be mistaken for platelet dysfunction due to the underlying malignancies or the addition of aspirin or anticoagulation. One must maintain high clinical suspicion for AvWD with new onset bleeding in setting of known myeloproliferative or lymphoproliferative disorder. vWF antigen, activity, factor VIII activity and multimer testing should be obtained to secure the diagnosis. Treatment of the underlying disorder will result in clinical and laboratory improvement.

S1711 Cerebral Infarction as a Rare Downstream Complication of Pancreatitis

Introduction: Pancreatitis is characterized by progressive and irreversible loss of function. Long term complications of pancreatitis include refractory abdominal pain, malabsorption, and diabetes. The downstream complications may also be systemic. In the case below we suggest that the acute release of pancreatic enzymes led to a hypercoagulable state and severe inflammatory reaction contributing to an acute cerebrovascular stroke presentation. Case Description/Methods: 55-year-old right-handed woman with a past medical history significant for chronic idiopathic pancreatitis, HTN, chronic thrombocytosis, and HLD presented to the hospital with complaints of nausea and vomiting. Initial work-up was significant for a lipase of 1,178 U/L and subsequent imaging showed peritoneal thickening and nodularity extended from the pancreatic head / neck junction most likely due to ongoing pancreatitis. Patient was admitted to hospital medicine but unfortunately that night the patient became acutely aphasic and confused. She developed right-sided neglect, profound aphasia, and left gaze preference. She was immediately evaluated by neurology and a stat CT revealed acute ischemic changes but no large vessel occlusion. She received intravenous tPA and was transferred to the critical care unit. CT angiogram revealed thrombus within the aortic arch at the origins of the right brachiocephalic arteries and follow-up MRI brain revealed scattered infracts within the left MCA territory. It was suspected that her stroke was likely precipitated by artery-to-artery embolus worsened in the setting of her acute on chronic pancreatitis attack. Patient worked extensively with rehabilitation services for weeks before eventually being discharged home with residual weaknesses. Discussion: Acute on chronic pancreatitis is an inflammatory process that can produce complications through both loss of endocrine and exocrine function. Additionally, the thrombotic tendency in pancreatitis may be related to systemic hypercoagulability resulting from inflammatory cytokines, endothelial dysfunction due to release of proteases and lytic enzymes, and compression of vessels from surrounding inflammation. These factors could contribute to the development of acute cerebrovascular stroke as seen in our patient. Furthermore, this case serves to highlight the need for further study into the systemic effects of acute on chronic pancreatitis and to serve as a reminder that though rare cerebral infarction can be a rare complication of pancreatitis.

Genetic alterations in patients with chronic leucocytosis and persistent thrombocytosis

To elucidate the relevance of genetic alterations, we analysed 17 genes known to be involved in haematological neoplasms in patients with chronic leucocytosis and patients with persistent thrombocytosis. Mutations of the JAK2, SETBP1 and ASXL1 genes were found in 1/13, 1/13, and 2/13 patients with leucocytosis, respectively. Mutations of the JAK2, CALR, SETBP1 and ASXL1 genes were found in 1/5, 1/5, 1/5 and 2/5 patients with thrombocytosis, respectively. One leucocytosis patient with a JAK2 V617F mutation developed polycythaemia vera. Another leucocytosis patient developed Philadelphia chromosome-negative chronic myeloid leukaemia (Ph(-) CML) accompanied by t(9;12)(q34.1;p13.?3) (Mori et al. 2016). Another leucocytosis patient with mutations of the SETBP1 and ASXL1 genes progressed to blast crisis of Ph(-) CML accompanied by i(17)(q10). Chronic leucocytosis patients who had genetic alterations tended to develop haematological neoplasms, while thrombocytosis unexpectedly resolved in two persistent thrombocytosis patients with genetic alterations.

In Vitro Functional Rescue of a Double MPL K39N/W272R Mutant Associated with Congenital Amegakaryocytic Thrombocytopenia (CAMT) Using Crispr/Cas9

Introduction: Thrombopoietin (Tpo) and its receptor, Mpl, are the principal regulators of early and late thrombopoiesis. Mutations in MPL can drastically impair its function and be a contributing factor in chronic thrombocytosis and in congenital amegakaryocytic thrombocytopenia (CAMT). CAMT is a rare inherited syndrome characterized by thrombocytopenia at birth, progressing to bone marrow failure and pancytopenia. The functional impact of CAMT mutations on Mpl signaling/trafficking is yet to be determined and could be relevant to multiple hematologic malignancies. Here we report unique familial cases of CAMT presenting with a previously unreported MPL mutation: T814C (W272R) in the background of the MPL K39N (Baltimore mutation), known to cause hereditary thrombocytosis (HT).Patients: Consanguineous parents and their eldest daughter, all heterozygous for Mpl K39N/W272R, do not present any signs of disease. Their monozygotic twin daughters presented at birth with severe thrombocytopenia (platelet counts: 12,000/L and 14,000/L), low hemoglobin levels (10.4 mg/dL and 7.8 mg/dL) and very high Tpo levels (3,650 pg/mL and 3115 pg/mL). Bone marrow smears performed 19 days after birth showed severe megakaryocytopenia (only 1 megakaryocyte (MK) seen). Bone marrow colony formation assays yielded 3 and 9 MK colonies vs 84 MK colonies/105 mononuclear cells for the control, leading to a diagnosis of CAMT type I. Whole blood sequencing revealed the presence of a homozygous double MPL K39N/W272R mutation. One of the twins died after bone marrow transplant. A younger male sibling, homozygous for MPL K39N/W272R mutation, has also been diagnosed with CAMT type I.Objectives: This study focuses on the functional characterization of the novel MPL W272R and K39N/W272R mutations and in vitro genetic engineering as a potential therapeutic option for CAMT.Methods: Human megakaryoblastic UT-7 and murine Ba/F3 cells stably expressing human wild-type (WT) Mpl or K39N, W272R or doubly mutated K39N/W272R Mpl fused to mNeonGreen were used as models. Confocal microscopy, proliferation and surface biotinylation assays, as well as co-immunoprecipitation (co-IP) and western blotting analysis, were used to elucidate the function and trafficking of Mpl mutants. CRISPR/Cas9 genetic engineering was used to repair mutant MPL and rescue its function.Results: Confocal microscopy shows that a significant fraction of chimeric WT Mpl protein reaches the cell surface. Significant surface expression is also noted for Mpl K39N. In contrast, the chimeric Mpl protein bearing the W272R mutation, alone or together with the K39N mutation, showed no detectable surface expression of the Tpo receptor. These results were confirmed by surface biotinylation assay. Co-expression of WT CALR fused to RFP, used as an ER marker, showed significantly higher co-localization (Pearson's R value) with mutant Mpl than with WT Mpl, evidence that the large majority of receptors were retained within the ER. We also evaluated Tpo signaling through the JAK/STAT, MAPK and PI3K pathways and Tpo-induced proliferation. Both WT and K39N-mutated Mpl were competent for signaling, while single or double mutants bearing W272R were unresponsive to Tpo. Tpo-induced signaling was partially restored via GRASP55 over-expression (forcing ER-trapped Mpl to traffic to the cell surface). Genetic engineering performed on cells carrying the W272R mutation restored the WT sequence and the response to Tpo, with similar cell proliferation as WT Mpl cells. In addition, co-IP studies indicate that Jak2 associates strongly with WT Mpl and Mpl K39N but not with Mpl W272R.Conclusion: We report a new mutation of Mpl (W272R) present in cis with HT-causing K39N mutation in the context of CAMT. The absence of symptoms in the Mpl K39N/W272R-mutated parents (found to be heterozygous) can be explained by the opposite (apparently neutralizing) effects of the two mutations on the trafficking and signaling of Mpl, as shown by confocal microscopy, western blotting and proliferation assays. In children homozygous for Mpl K39N/W272R, the W272R mutation prevented Mpl binding of Jak2 and expression at cell surface, rendering Tpo signaling impossible despite the presence of the K39N mutation. Function of the deficient Mpl receptor could be rescued using two separate approaches: CRISPR/Cas9 genetic engineering and GRASP55 over-expression. Cell-permeable Tpo analogs will also be tested. DisclosuresNo relevant conflicts of interest to declare.

Fever of unknown origin (FUO) and a renal mass: Renal cell carcinoma, renal tuberculosis, renal malakoplakia, or xanthogranulomatous pyelonephritis?

Often patients with fevers of unknown origin (FUOs) present with loss of appetite, weight loss, and night sweats, without localizing signs. Some are found to have a renal mass during diagnostic evaluation. In patients with FUOs and a renal mass, the differential diagnosis includes renal tuberculosis, renal cell carcinoma (hypernephroma), renal malakoplakia, and xanthogranulomatous pyelonephritis. A 68-year-old woman presented with an FUO during her diagnostic workup. She manifested an irregularly enlarged kidney on abdominal computed tomography (CT) scan, as well as a highly elevated erythrocyte sedimentation rate of more than 100 mm/hour, an elevated serum ferritin level, and chronic thrombocytosis, which favored a diagnosis of renal cell carcinoma. Renal malakoplakia and renal tuberculosis comprised further differential diagnostic considerations. Microscopic hematuria may be present with any of the disorders in the differential diagnosis, but was absent in this case. An abdominal CT scan was suggestive of xanthogranulomatous pyelonephritis. Because of concerns regarding renal cell carcinoma, the patient received a nephrectomy. The pathologic diagnosis was of xanthogranulomatous pyelonephritis, without renal cell carcinoma.

Is Obesity Alone Associated with Increased Blood Cell Counts in Otherwise Healthy Children?

Abstract 3135 Introduction:Childhood obesity has been linked to continued obesity and cardiovascular disease in adulthood. Studies in adults have established the relationship between morbid obesity and an increase in inflammatory markers. However, data in children is limited, with few studies examining white blood cell counts, and no studies that address platelet count. The objective of this study is to examine the relationship of obesity with hemoglobin, leukocyte and platelet counts among generally healthy children. Methods:Using the 2007–08 NHANES dataset, we analyzed data for children 2 to 17 year of age, including demographic characteristics, body mass index (BMI) and complete blood count (CBC) results. We excluded children with chronic disease and those reported as being in “fair” or “poor” health. We also excluded children whose caregiver reported them having an acute illness in the previous 30 days. Our main exposure variable was weight status as defined by BMI percentile for age. Weighted multiple regression modeling was used to examine relationships between obesity and white blood cells (WBC), platelets, hemoglobin concentration and CRP, controlling for age, gender and race. Results:There were 1482 eligible children aged 2 – 17 years in the dataset, of which 18.6% (276) were found to be obese and 16.2 % (240) were overweight. Multiple linear regression modeling revealed that being obese or overweight was significantly associated with increased cell counts when controlling for age, gender and race, but not hemoglobin concentration (see Table 1). Specifically, obesity was positively associated with WBC, neutrophil, and platelet counts (all p-values < 0.01.) Discussion:The present study found that obesity is an independent risk factor for having higher cell counts in children, specifically total WBC, neutrophils and platelets. Excess adipose tissue is characterized by inflammation and progressive infiltration by macrophages as obesity develops. The association of increased BMI with inflammatory cells likely exists through multiple pathways. For example, it has been suggested that the increase in leptin produced by adipose tissue may be responsible for the increase in inflammatory cells, possibly through and interaction with multi-lineage CD34+ cells. It has also been shown that the increase in inflammatory markers can lead to endothelial damage and structural arterial changes in children, placing them at increased risk for cardiovascular disease in adulthood. It is unclear as to whether our finding of increased platelets contributes to this risk. However, chronic thrombocytosis has been associated with a higher risk for cardiovascular disease in adults with essential thrombocythemia.If obesity is an independent risk factor for having higher cell counts at baseline, then this should be taken into account during hematologic evaluation. It should also be considered when assessing the potential impact of the current childhood obesity epidemic, and the possible long-term consequences. Further study is needed to confirm our findings.TableAssociations of Obesity or Overweight with Blood Cell Counts in Children Ages 2–17 years (n=1482)Outcome VariableExposure GroupRegression CoefficientSE95% CIs of Coefficientsp-valueWBC (1000 cells /μL)Obese0.980.210.53, 1.430.0003Overweight0.360.1540.03, 0.690.0330Neutrophils (1000 cells /μL)Obese0.680.1810.30, 1.060.0017Overweight0.280.1180.03, 0.530.0317Platelets (1000 cells /μL)Obese28.767.23413.42, 44.090.0011Overweight8.883.4281.61, 16.140.0198Hemoglobin (g/dL)Obese−0.040.113−0.27, 0.200.7559Overweight0.050.080−0.12, 0.220.5220CRP (mg/dL)Obese0.170.0480.07, 0.270.0027Overweight0.090.046−0.01, 0.180.0806 Disclosures:No relevant conflicts of interest to declare.

Fever of unknown origin (FUO) due to a solitary cavitary lung lesion: The deadly ferritin-laced doughnut

Fever of unknown origin (FUO) is the clinical designation for patients who have fevers >101F that have persisted for >3 weeks that remain undiagnosed, after an intensive ambulatory/in-hospital workup. Fevers of unknown origin may be due to wide variety of infectious, neoplastic, or rheumatic/inflammatory disorders. The most common causes of FUOs in elderly patients are infectious and neoplastic diseases. With FUOs, the clinical presentation and routine laboratory tests are usually sufficient to narrow differential diagnostic possibilities. We present a case of an elderly Italian woman who presented with an FUO and a solitary, thick-walled cavitary lesion on chest x-ray (CXR). The infectious disease differential diagnosis of her FUO included lung abscess, M. tuberculosis (TB), systemic mycoses, and echinococcal-cyst (or hydatid-cyst) disease. The malignancy and neoplastic differential diagnosis included bronchogenic carcinoma, lymphoma, and metastatic carcinoma. Her nonspecific laboratory tests indicated a highly elevated erythrocyte sedimentation rate (ESR) >100 mm/hour, chronic thrombocytosis, relative lymphopenia, and highly elevated serum ferritin levels. Excluding highly elevated serum ferritin levels, the differential diagnosis of her FUO with a solitary, thick-walled cavitary lesion was lung abscess vs tuberculosis. However, her highly elevated serum ferritin levels proved to be the critical diagnostic clue in predicting the diagnosis of squamous-cell carcinoma. We conclude that serum ferritin levels are an important part of the laboratory workup. As with other nonspecific laboratory tests, the diagnostic significance of highly elevated ferritin levels depends associated clinical features in the clinical presentation.

Thrombocytémie essentielle. Apport de la mutation V617F de JAK2 pour la stratégie diagnostique, la physiopathologie et les modalités évolutives

An increased platelet number in blood depends on a limited spectrum of causes, which aren't always simple to identify. Secondary thrombocytosis is a reactive process in relation with acute or chronic inflammatory diseases, or asplenia. The infrequent inherited thrombocytoses disorders are suspected when similar cases are observed in the same family. However, the most frequent causes of chronic thrombocytosis in adults are the so-called chronic myeloproliferative syndromes (chronic myelocytic leukaemia, polycythemia vera, primary myelofibrosis, essential thrombocytemia), and to a lesser extent, myelodysplastic syndromes. In the course of these disorders, thrombocytosis is often the first recognized abnormality. Chronic myelocytic leukaemia is easily diagnosed owing to the presence of either the Philadelphia chromosome or the BCR-ABL fusion gene product. The next step still relies upon a distinction according to the PVSG or the WHO criteria of Polycythemia Vera (PV) and Idiopathic myelo fibrosis (IMF) to finally confirm genuine Essential Thrombocythemia (ET). The recent description of the V617F mutation of JAK2 in 90% of PV patients, 43 to 67% with IMF and 50% of ET diagnosed according to either the PVSG or the WHO criteria is a definite characteristic of clonality now accessible in haematology practice. However, this mutation is neither specific nor constant in any of the Philadelphia negative myeloproliferative disorders, which outlines the importance of the WHO criteria of megakaryocytic abnormalities on bone marrow biopsy as the hallmark of Ph negative MPDs. The exclusion of PV and of IMF, including pre fibrotic and early fibrotic forms is still required for the diagnosis of "true" ET. Disease stratification and treatment strategy are targeted on the evaluation and prevention of vascular complications. Acute leukaemia or myelodysplasia, and other clonal progressions like myelofibrotic transformation, are infrequent and delayed events. However, according to the present data, the risk of fibrotic progression or of leukaemic transformation is not related to the mutation status of ET patients.

The Mevalonate Pathway as a Therapeutic Target in the Ph-Negative Chronic Myeloproliferative Disorders

The Ph-negative chronic myeloproliferative disorders (CMPDs) polycythaemia vera, essential thrombocytosis and idiopathic myelofibrosis are acquired stem cell disorders, which pathophysiologically are featured by clonal myeloproliferation and accumulation of myeloid cells, the latter being consequent to decreased apoptosis. Myelofibrosis and neoangiogenesis in the bone marrow and spleen are the histopathological hallmarks of idiopathic myelofibrosis but may develop in the other diseases as well. In patients with myelofibrosis elevated levels of circulating CD34+ cells are highly characteristic being partly explained by a proteolytic bone marrow mileu owing to excessive release of various proteases with ensuing extracellular matrix degradation and constitutive mobilisation of CD34+ cells into the peripheral blood. Thrombohaemorrhagic complications are major clinical problems contributing significantly to morbidity and mortality. Based upon in vitro and in vivo studies of the effects of statins (antithrombotic, antiproliferative, antiangiogenic, antiproteolytic) and zoledronic acid (antiproliferative antiproliferative, antiangiogenic, antiproteolytic) this review focusses on the translation of these effects into potential clinical benefits of combinational therapy with these agents in patients with CMPDs.

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-α 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.

Fever of unknown origin caused by late-onset rheumatoid arthritis

Fever of unknown origin (FUO) is always a diagnostic challenge. The causes of FUO are legion and may be due to malignancy, infection, collagen vascular disease, and a variety of other unusual disorders. Currently, malignancies-followed by infectious etiologies-are the most common cause of FUO. We present an elderly female patient with an FUO who was thought to have subacute bacterial endocarditis because of an antecedent history of recent dental work. Subacute bacterial endocarditis was ruled out on the basis of negative cultures and negative transesophageal echocardiography. No evidence for an infectious disease or neoplastic etiology could be demonstrated in this patient. The diagnosis of FUO is most difficult when there is a paucity of clues from the history and physical examination, as was the case in this patient. Nonspecific laboratory tests included highly increased erythrocyte sedimentation rate (>or=100 mm/h), highly increased C-reactive protein, relative lymphocytopenia, and chronic thrombocytosis. These findings are compatible with a variety of infectious and inflammatory disorders. No evidence could be found for vasculitis. The only laboratory diagnostic findings present in her case were a highly increased rheumatoid factor titer and perinuclear antineutrophilic cytoplasmic antibody level. Polymyalgia rheumatica/temporal arteritis, systemic lupus erythematosus, and adult Still's disease were ruled out. The patient's FUO was best explained by the finding of late-onset rheumatoid arthritis (LORA), which is characterized by acute onset in elderly patients without the usual musculoskeletal manifestations of rheumatoid arthritis. Both the highly increased rheumatoid factor titer and perinuclear antineutrophilic cytoplasmic antibody level in the absence of an alternate explanation indicate that the FUO in this patient was caused by LORA.

A Genetic Mutation on Murine Chromosome 7 Causes Thrombocytosis Associated with Thrombopoietin Excess - A Model of Jak2V617F Negative Idiopathic Thrombocytosis.

A substantial proportion of patients with Essential Thrombocythemia (ET) have evidence of an acquired Jak2V617F mutation that causes the myeloproliferative phenotype. Jak2V617F negative ET patients presumably have mutations in other related pathways or alternatively may have chronic thrombocytosis secondary to factors extrinsic to the hemopoietic system. This second hypothesis is supported by clonality studies that demonstrate at least 30% of women with ET have evidence of polyclonal granulocytes. As part of an ENU mutation screen to identify novel genes associated with thrombocytosis, we have studied a mutant mouse strain, called plt2, with autosomal recessive thrombocytosis associated with increased thrombopoietin production. This mutation is linked to mouse chromosome 7, and therefore is unrelated to mutations in Jak2, thrombopoietin or the thrombopoietin receptor, c-Mpl. plt2/plt2 mice have platelet counts of 2038±347x109/L (n=107) increased by 47% compared to wild-type mice (1386±223; n=107, p<0.001). This thrombocytosis was associated with increased numbers of megakaryocyte progenitor cells (plt2/plt2 28.8±4.6; n=4, Meg-CFC/5x105 cells to maximal cytokine stimulation compared to wild-type 18.3±5.5; n=4, p<0.05) and megakaryocytes (108±15; n=7 megakaryocytes per hpf compared to 70±13; n=7, p<0.01) that demonstrated a normal maturation profile based on ploidy measurement. plt2/plt2 mutant mice exhibited mild hepatomegaly (liver weight 1.6±0.2g; n=34 compared to 1.3±0.2g; n=23, p<0.001) and serum thrombopoietin levels were elevated by 76% (4930±1309pg/mL; n=18 compared to 2802±1031; n=15, p<0.001). There was no difference in relative TPO transcription between plt2/plt2 mice and wild-type mice in the liver (p=0.23), however, the TPO content of whole liver lysates was increased by 50% in the mutant mice (150%±50; n=8 per 1g liver weight compared to 100±32; n=8, p=0.03). To examine how the plt2 mutation interacts with other components of the TPO pathway, plt2/plt2 mice were intercrossed with mice lacking the TPO receptor (Mpl−/−). Mpl−/−plt2/plt2 mice have thrombocytopenia with platelet counts that are indistinguishable from Mpl−/−/ mice confirming that the plt2 mutation causes thrombocytosis by acting through the Mpl receptor. Surprisingly, Mpl+/−plt2/plt2 mice, with only one copy of the Mpl gene, had marked thrombocytosis with platelet counts of 2656±459x109/L (n=25) increased by 92% over wild-type mice suggesting an additive effect between excess thrombopoietin and Mpl heterozygosity on platelet count. We have utilized ENU mutagenesis to identify a novel mouse pedigree with heritable thrombocytosis linked to chromosome 7 that demonstrates altered cytokine production can mimic the chronic thrombocytosis that is the cardinal diagnostic feature of ET.

Post-splenectomy pseudohyperkalemia in a patient with chronic idiopathic myelofibrosis and thrombocytosis

Post-splenectomy pseudohyperkalemia in a patient with chronic idiopathic myelofibrosis and thrombocytosis

Inducible expression of BCR/ABL using human CD34 regulatory elements results in a megakaryocytic myeloproliferative syndrome

AbstractThe BCR/ABL fusion protein is found in more than 90% of patients with chronic myeloid leukemia (CML) as well as in a subset of patients with acute B-cell leukemia. We have previously described a transgenic model for an inducible and reversible acute B-cell leukemia caused by p210 BCR/ABL. Here, we describe a new model of an inducible BCR/ABL disease by directing the expression of the oncogene to megakaryocytic progenitor cells within the murine bone marrow using the tetracycline-responsive expression system under the control of human CD34 regulatory elements. The predominant feature was the development of a chronic thrombocytosis. The condition progressed with the development of splenomegaly accompanied by lymphadenopathy in some mice. Affected animals demonstrated a dramatic increase in the number of megakaryocytes in the bone marrow and the spleen. Immunohistochemistry demonstrated that the reporter gene was expressed in hematopoietic stem cells (HSCs), common myeloid progenitor (CMP) cells, as well as in megakaryocytic/erythroid progenitor cells (MEPs). Although these mice did not display the increase in granulopoiesis commonly found in chronic myeloid leukemia (CML), the phenotype closely resembles a myeloproliferative disorder affecting the megakaryocytic lineage observed in some patients with the BCR/ABL P210 translocation.

Development of thrombocytosis in HIV+ drug users: impact of antiretroviral therapy

With the exception of hemolytic anemia, the potential hematological toxicity of antiretrovirals (ARV) and combination treatments in HIV treated individuals has not been well established. We report, for the first time, hematological toxicity defined as thrombocytosis in 9% of the HIV+ patients receiving highly active antiretroviral treatment (HAART) being followed in a nutritional clinical trial. Participants were evaluated every 6 months during a 2-year period (1998-2000) and blood drawn for biochemical, hematological and immunological parameters. NK cells were negatively correlated with platelet counts in the total cohort ( P = 0.018) and persistently elevated with ARVT. Chronic thrombocytosis was associated with significantly lower NK percentages ( P = 0.005). Twenty-five percent of the patients with thrombocytosis developed a cardiovascular disease. Together, these results support the proposal that HAART may increase the risk of hematological dysfunction and impact the risk of cardiovascular disease.

Correlation of Thrombosis With Increased Platelet Turnover in Thrombocytosis

AbstractThere are no readily applicable methods to routinely assess thrombosis risk and treatment response in thrombocytosis. Reticulated platelets (RP) define the most recently released platelets in the circulation, and the RP% has been shown to estimate platelet turnover in thrombocytopenic states. We examined whether increased RP values were associated with thrombotic complications in thrombocytosis. Platelet count, RP%, and absolute RP count were measured at presentation in 83 patients with chronic or transient thrombocytosis, 46 patients with deep vein (DVT) or arterial (ART) thrombosis and normal platelet counts, and 83 healthy controls with normal platelet counts. Chronic thrombocytosis patients presenting with thrombosis (n = 14) had significantly higher RP% (14.7% ± 10.1%, mean ± SD) than asymptomatic chronic thrombocytosis patients (n = 23, RP% = 3.4% ± 1.8%), healthy controls (3.4% ± 1.3%), DVT patients (n = 21, 3.8% ± 2.1%), or ART patients (n = 25, 4.5% ± 4.1%, P < .05 for all comparisons). Chronic thrombocytosis patients with thrombosis also had significantly higher absolute RP counts than asymptomatic chronic thrombocytosis patients (98 ± 64 × 109/L [range, 54 to 249 × 109/L] v 30 ± 13 × 109/L [range, 11 to 51 × 109/L]; P = .0004), whereas healthy controls, DVT, and ART patients had similarly low absolute RP counts (6 ± 6 × 109/L, 9 ± 7 × 109/L, and 11 ± 7 × 109/L, respectively; P > .49). The RP% and absolute RP counts remained significantly higher in chronic thrombocytosis patients with thrombosis when patients were further subdivided into primary myeloproliferative disorders versus secondary thrombocytosis. Similarly elevated RP percentages and absolute counts were also noted in transient thrombocytosis patients with thrombosis (n = 6, 11.5% ± 4.4% and 90 ± 46 × 109/L, respectively) when compared with asymptomatic transient thrombocytosis patients (n = 40, 4.5% ± 2.7% and 35 ± 16 × 109/L, respectively) and to all control groups (P < .05 for all comparisons). In addition, 7 of 8 thrombocytosis patients who were studied before developing symptoms of thrombosis had elevated absolute RP counts compared with only 1 of 63 thrombocytosis patients who remained asymptomatic. Follow-up studies in seven chronic thrombocytosis patients showed that successful aspirin treatment of symptomatic recurrent thrombosis significantly reduced the RP% from 17.1% ± 10.9% before therapy to 4.8% ± 2.0% after therapy; absolute RP counts decreased from 102 ± 67 × 109/L to 26 ± 10 × 109/L (P < .01 for both). We conclude that thrombosis in the setting of an elevated platelet count is associated with increased platelet turnover, which is reversed by aspirin therapy. Measurement of reticulated platelets to assess platelet turnover may be useful in evaluating both treatment response and thrombotic risk in thrombocytosis.

Constitutive Expression of Mpl Ligand Transcripts During Thrombocytopenia or Thrombocytosis

Mpl ligand (thrombopoietin [TPO]) is the physiological regulator of platelet production. In mice, mRNA encoding the Mpl ligand (Mpl-L) is predominantly found by Northern blot analysis in the liver and kidney. To investigate the mode of regulation of the Mpl-L gene, we have developed several experimental models of severe thrombocytopenia differing in their kinetics and an opposite model of chronic thrombocytosis. Northern analysis performed at various times after induction of a thrombocytopenic state demonstrates that, whatever the number of circulating platelets, no change in Mpl-L mRNA level occurs in liver and kidney. By ribonuclease protection assays, we analyzed the ratios between mRNAs coding for the wild-type Mpl-L form and various splice variants encoding inactive or nonsecreted Mpl-L proteins. No modification in levels of these various isoforms was detected confirming the data of a previous report. Because the highest level of Mpl-L bioactivity in sera was observed only in mice with drastically reduced numbers of both platelets and megakaryocytes, these results further suggest that not only platelets, but also megakaryocytes, must be involved in the regulation of the level of circulating Mpl-L. In addition, we show that no downregulation of wild-type Mpl-L mRNA and no change in the ratio of Mpl-L mRNA isoforms were detected in mice in which a chronic thrombocytosis was induced. Together, these different models extend and further confirm that the regulation of Mpl-L does not occur at a transcriptional level or by a modulation in the ratios of Mpl-L mRNA isoforms.

Clinical Aspects of Chronic Myeloproliferative Diseases

The nonleukemic chronic myeloproliferative disorders, polycythemia vera, essential thrombocytosis, and myeloid metaplasia with myelofibrosis, are clonal disorders with similar but distinct clinical and laboratory findings. This review will discuss the diagnostic criteria for each disease, the variable clinical picture, and the therapeutic modalities, actual and theoretical.

A MATHEMATICAL MODEL OF THROMBOPOIESIS IN RATS

A mathematical model of thrombopoiesis in rats is presented. This has four compartments; stem cells, megakaryocytes, thrombocytes and thrombopoietin. A high thrombopoietin concentration influences bone marrow proliferation in three ways. Firstly the stem cells are stimulated and a slow increase in megakaryocyte number follows. Secondly there are additional endomitoses in the (early) megakaryocytes resulting in an increase in megakaryocyte volume. Thirdly the megakaryocyte maturation time is shortened. The parameters of the model are determined from experimental values for the normal, maximum and minimum proliferation rates, maturation times and destruction rates. The model is tested by comparing simulated results for acute and chronic thrombocytopenia and thrombocytosis with experimental curves from the literature. The model and data agree within the limits of experimental error. Not all of the thrombopoietic regulatory system is known yet, so some important alternative hypotheses are investigated and compared with the model. Several hypotheses have been excluded in this way.

Platelets and atherogenesis: Part 2. Amelioration of cholesterol atherogenesis in rabbits with reduced platelet counts as the result of 32P administration

Summary Two schedules of radiophosphorus administration have been used to prepare thrombocytopenic rabbits for the purpose of uncovering a possible role for platelets in the encrustation phase of cholesterol atheromatosis. The group that was less vigorously treated with 32 P had no deaths from infection, and achieved chronic halving of the platelet count. The latter apparently had no effect on the degree or distribution of cholesterol deposits on the aorta. However, a platelet count of 300,000/mm 3 certainly is not at a thrombocytopenic level and may, in fact, be at the lower end of a permissive range for cholesterol deposition. With the heavier dose schedule of 32 P the platelets were reduced to one-fourth of normal, and this appeared to have an ameliorating effect on the development of aortic atheromatosis. A heavy mortality from infection, undoubtedly the result of an excessive reduction of granulocytes, could have biased the statistical analysis in favor of the observed results. Nevertheless, the findings are encouraging because they seem to be complementary to previous work in our laboratory, which showed that cholesterol atheromatosis in the rabbit may be enhanced in a setting of chronic thrombocytosis. Development of a pharmacologic agent which selectively reduces the platelet count is necessary in order to test the validity of these observations.