Blood Smear Evaluation Research Articles

Analytic errors in Sysmex-generated hematology results in blood from a dog with chronic lymphocytic leukemia.

A blood sample from a 14-year-old dog was submitted to the veterinary diagnostic laboratory of the University of Milan for marked leukocytosis with atypical cells. A diagnosis of chronic T-cell lymphocytic leukemia (CLL) was made based on blood smear evaluation and flow cytometric phenotyping. A CBC by Sysmex XT-2000iV revealed a moderate normocytic normochromic anemia. Red blood cells counted by optic flow cytometry (RBC-O) resulted in a higher value than using electrical impedance (RBC-I). The relative reticulocyte count based on RNA content and size was 35.3%, while the manual reticulocyte count was <1%. The WBC count of 1,562,680cells/μL was accompanied by a flag. Manual counts for RBC and WBC using the Bürker chamber confirmed the Sysmex impedance results. Finally the manual PCV was lower than HCT by Sysmex. While Sysmex XT can differentiate between RBC and WBC by impedance, even in the face of extreme lymphocytosis due to CLL, RBC-O can be affected by bias, resulting in falsely increased RBC and reticulocyte numbers. Overestimation of RBC-O may be due to incorrect Sysmex classification of leukemic cells or their fragments as reticulocytes. This phenomenon is known as pseudoreticulocytosis and can lead to misinterpretation of regenerative anemia. On the other side PCV can be affected by bias in CLL due to the trapping of RBC in the buffy coat, resulting in a pink hue in the separation area. As HGB concentration is not affected by flow cytometric or other cell-related artifacts it may represent the most reliable variable to assess the degree of anemia in cases of CLL.

Rhinocerebral mucormycosis in a patient with acute promyelocytic leukemia.

A 63-year-old male presented with headache and weakness lasting for 2 months. Laboratory studies revealed the following: hemoglobin of 7.1 g/dL, white blood cells of 105x109/L, and platelets of 34x109/L. A diagnosis of acute promyelocytic leukemia was made by peripheral blood smear and bone marrow evaluations including morphological and genetic studies. The patient received one cycle of a chemotherapy regimen including cytarabine and idarubicin. During the aplastic phase, on day +6 from the end of chemotherapy, the patient developed fever, swelling on the left side of the face, infraorbital edema, and yellow-black discoloration of the upper palate (Figure 1). A paranasal sinus CT scan revealed a defect of the nasal septum and skin, and subcutaneous edema was seen at the maxillary and nasal level (Figure 2). The patient underwent surgical debridement. Histopathological assessment of the debridement specimen showed mucormycosis-associated hyphae. Culture of the nasal discharge was positive for Mucor spp. Liposomal amphotericin B was initiated at 5 mg/kg/day, but the patient died on the 30th hospital day. Informed consent was obtained. Figure 1 Yellow-black discoloration of upper palate. Figure 2 Paranasal sinus CT scan revealed a defect of the nasal septum and skin and subcutaneous edema. Mucormycosis is rapidly progressive and mortality for this infection is high [1,2,3,4]. Hematologic malignancies, long-term corticosteroid use, and immunosuppressive therapies are predisposing factors for mucormycosis. For management, mucormycosis should be considered early in high-risk patients, and surgical debridement together with effective antifungal therapy should be applied as soon as possible.

Lipid peroxidation and decrease on the activities of antioxidant enzymes in experimental infection by Babesia bovis in cattle

Babesia bovis is one of the causative agents of bovine babesiosis, a disease with high morbidity and mortality in susceptible populations. The aim of this study was to evaluate the occurrence of oxidative stress in cattle experimentally infected with attenuated B. bovis. For that, eight healthy cattle were used and divided into two groups: animals infected with B. bovis (n = 4; group A) and non-infected animals (n = 4; group B). Blood samples of all animals were collected at 0, 7, 11, and 15 days post-infection (DPI) for red blood cells (RBCs) count, and measurement of thiobarbituric acid reactive substance (TBARS) levels in serum (lipid peroxidation) and activity of antioxidant enzymes in whole blood (catalase (CAT) and superoxide dismutase (SOD)). The parasitemia was determined by blood smear evaluation and conventional PCR for B. bovis. Blood smears were negative throughout the experiment; however, infection was confirmed by PCR positive for B. bovis at 15 DPI. A slight reduction on RBC count was observed in cattle of group A at 11 and 15 DPI (P < 0.05). The same animals showed an increased level of TBARS (P < 0.05) at 11 DPI, suggesting lipid peroxidation, while the activities of CAT and SOD decreased (P < 0.05) at 7 and 15 DPI, respectively. Our data support the occurrence of an oxidative/antioxidant imbalance in cattle infected with B. bovis.

Canine vector-borne co-infections: Ehrlichia canis and Hepatozoon canis in the same host monocytes

The protozoon Hepatozoon canis and the rickettsia Ehrlichia canis are tick-borne pathogens, transmitted by Rhipicephalus sanguineus, which cause canine hepatozoonosis and canine monocytic ehrlichiosis, respectively. Co-infection of the same host monocytes with H. canis and E. canis confirmed by molecular characterization of the infecting agents and quantitative assessment of co-infected cells is described for the first time in three naturally-infected dogs. Blood smear evaluation indicated that at least 50% of the leukocytes infected with H. canis gamonts contained E. canis morulae. Co-infection of the same host cell demonstrated in this report suggests that infection with one pathogen may permit or enhance invasion or prolonged cellular survival of the other.

Evaluation of oxidative stress in the anemia of dogs with chronic kidney disease.

Anemia and systemic oxidative stress may occur in dogs with chronic kidney disease (CKD). Only scarce information regarding the intraerythrocytic redox status under these conditions is available at this time. The aim of this study was to evaluate the indicators of oxidative stress and intraerythrocytic antioxidant defense in dogs with anemia of CKD. Thirty dogs with CKD in stages 3 or 4 with nonregenerative anemia (HCT ≤37%) were compared to 20 healthy dogs. Complete blood count, reticulocyte %, blood smear evaluation, intraerythrocytic concentrations of total (GSHt), reduced (GSH), and oxidized glutathione (GSSH), and activities of glutathione peroxidase, glutathione reductase and superoxide dismutase (SOD), as well as plasma concentrations of thiobarbituric acid-reactive substances (TBAR) were determined. Anemia of CKD dogs was nonregenerative (reticulocytes ≤0.2% with scarce anisocytosis and poikilocytosis). Intraerythrocytic GSSH and SOD, and plasma TBAR were higher in dogs with CKD. There was a positive correlation between the creatinine concentration and TBAR, and negative correlations between creatinine concentration and HCT, as well as between HCT and TBAR. In CKD dogs with a higher degree of anemia, SOD levels were higher and GSSH concentrations were lower. Despite the evidence of increased systemic oxidative stress, the compensatory response of SOD and the sustained intraerythrocytic concentrations of GSSH in CKD dogs with anemia indicated that the erythrocytes maintained the antioxidant defense. There was no strong evidence that oxidative stress was associated with higher degrees of anemia in dogs with CKD.

Hematologic findings predictive of bone marrow disease in dogs with multicentric large-cell lymphoma.

Evaluation of the bone marrow is needed for complete staging in dogs with multicentric large-cell lymphoma, but is often omitted in clinical practice. The objective was to determine if routine peripheral blood findings, including microscopic evaluation of blood smears, can predict the presence of bone marrow involvement in dogs with lymphoma. Hematologic data including evaluation of blood smears and bone marrow aspirates from 107 dogs newly diagnosed with large-cell lymphoma were retrospectively evaluated. Neoplastic lymphocytes were identified based on cell size, nuclear size, chromatin pattern, and the presence of nucleoli. Positive specimens were defined as having ≥10% neoplastic lymphocytes. Two groups were established based on the presence or absence of lymphoma in the bone marrow. Variables (positive blood smear, HCT, platelet count, and total and differential WBC counts) were evaluated to determine if they were predictive of bone marrow involvement using univariate and multivariate logistic models. Thrombocytopenia and the presence of >10% neoplastic lymphocytes on blood smears were identified as significant variables for predicting the presence of bone marrow involvement. When considered independently, either a positive blood smear or thrombocytopenia had low sensitivity (60%) and moderate specificity (89% and 87%, respectively). Sensitivity increased when these variables were evaluated together (80%). In dogs with multicentric large-cell lymphoma, thrombocytopenia or the presence of neoplastic lymphocytes in circulation is suggestive of bone marrow involvement, but not definitive. Normal peripheral blood findings do not exclude the possibility of lymphoma in the bone marrow.

Significance of teardrop cells in peripheral blood smears

Abstract Teardrop erythrocytes (syn. dakryocytes) play a key role in the evaluation of peripheral blood smears in patients with anemia, especially as part of the “leukoerythroblastic picture”. Teardrop-shaped red blood cells can be seen in a wide range of diseases that lead to bone marrow fibrosis, which is often accompanied by extramedullary hematopoesis. The differential diagnoses encompass primary myelofibrosis (PMF), myelodysplastisc syndromes during the late course of the disease, rare forms of acute leucemias and myelophtisis caused by metastatic carcinoma. Rarely, MF associated with post-irradiation, toxins, autoimmune diseases, metabolic conditions, inborn hemolytic anemias, iron-deficient anemia or β-thalassaemia can lead to the formation of teardrops as visualized on peripheral blood smears.

Molecular identification of Theileria parasites of northwestern Chinese Cervidae.

BackgroundTheileria and Babesia protozoan parasites are transmitted mainly by tick vectors. These parasites cause heavy economic losses to the live-stock industry, as well as affecting the health of wild animals in parasite-endemic areas. Identification of infectious agents in wild animals is not only crucial for species preservation, but also provides valuable information on parasite epidemiology. Here, we conducted a molecular surveillance study in Northwestern China to assess the prevalence of blood pathogens in cervids.MethodsPCR analysis and microscopic evaluation of blood smears to detect Theileria- and Babesia-related diseases in Cervidae were conducted, in which 22 blood samples from red deer (n = 22) in Qilian Mountain and 20 from sika deer (n = 20) in Long Mountain were collected and tested for the presence of Theileria and Babesia. The 18S rRNA gene was amplified, and selected polymerase chain reaction (PCR)-positive samples were sequenced for species identification.ResultsPCR revealed that 9.1% of the Qilian Mountain samples and 20% of the Long Mountain samples were positive for Theileria uilenbergi; 90.09% of the Qilian Mountain samples (n = 22) were positive for T. capreoli, but all of the Long Mountain samples (n = 20) were negative for T. capreoli; no other Theileria or Babesia species were found. PCR showed that T. uilenbergi and T. capreoli were present in red deer in Qilian Mountain, while only T. uilenbergi was found in sika Deer in Long Mountain. The 18S rRNA gene sequences were aligned against the corresponding GenBank sequences of known isolates of Theileria and Babesia and subjected to phylogenetic analysis. The phylogenetic tree showed that the newly isolated Theileria spp. could be classified as belonging to two clades: one group belonged to the same clade as T. uilenbergi, the other to a clade containing T. capreoli.ConclusionsOur results provide important data to increase understanding of the epidemiology of Cervidae theileriosis, and will assist with the implementation of measures to control theileriosis transmission to Cervidae and small ruminants in central China.

Engraftment of canine peripheral blood lymphocytes into nonobese diabetic-severe combined immune deficient IL-2R common gamma chain null mice

To study the canine immune system we generated a mouse model engrafted with canine lymphocytes using NOD SCID IL2R common gamma chain −/− (NSG) mice as recipients (Ca-PBL-SCID). Engraftment of canine peripheral blood lymphocytes (PBLs) was determined post-injection with 107 peripheral blood mononuclear cells (PBMCs) into irradiated NSG mice using flow cytometry and fluorescently labeled antibodies specific to canine helper T cells (CD45+ CD4+), cytotoxic lymphocytes (CD45+ CD8+), regulatory T cells (CD45+ CD4+ Foxp3+), and B cells (CD45+ Ig+ CD21lo). Canine CD45+ lymphocytes were detectable as early as day 1 in the peritoneal cavity, and beginning at 9 days in the blood, bone marrow, and spleen. CD4+ T cells, of which Foxp-3+ CD25hi cells constituted a minor percentage, were the predominant lymphocyte population at 9 days post engraftment contrasting with increasing proportions of CD8+ CTL's and Ig+ B cells beginning at 16 days. Canine immunoglobulin was initially detected in the serum of Ca-PBL-SCID mice at 9 days post-engraftment and peaked in concentration at day 36. From day 28 to 52 post-engraftment 30% of the Ca-PBL-SCID mice became markedly anemic and thrombocytopenic, yet gross and histopathologic examination of bone marrow, kidneys, spleen, liver, and intestine revealed no obvious lesions. Blood smear evaluation revealed agglutination of mature red blood cells, reticulocytes and a regenerative anemia. These findings demonstrate that NSG mice are capable of engraftment of canine PBLs yet develop graft versus host disease similar to Hu-PBL-SCID mice.

Cerebral babesiosis and acute respiratory distress syndrome in a dog

To describe a case of cerebral babesiosis and acute respiratory distress syndrome (ARDS) in a dog. A 5-year-old male neutered Scottish Terrier was referred to the emergency department of the Ecole Nationale Vétérinaire de Toulouse for evaluation of progressive dyspnea and clinical signs suggestive of central neurological disease. Thoracic radiographs showed a diffuse and heavy interstitial/alveolar lung pattern. Babesiosis was diagnosed based on blood smear evaluation. The dog died of cardiopulmonary arrest 6 hours after presentation. Cerebral babesiosis and ARDS were confirmed at necropsy. Major pathological findings included erythrocyte aggregation in the lungs, liver, and brain. This case report describes an unusual clinical presentation of Babesia canis canis infection, the most common species associated with babesiosis in Europe. In addition, this is to our knowledge the first case of Babesia-associated ARDS confirmed by histopathology in a dog.

Plasmodium knowlesi and HIV co-infection in a German traveller to Thailand

A case of Plasmodium knowlesi and HIV co-infection is reported in a German traveller returning from Thailand. The 54 year-old patient presented to the Institute of Tropical Medicine in Tübingen with a 11-day history of daily fever and chills. Initial microscopic evaluation of Giemsa-stained thin blood smears was suggestive of a mixed infection with Plasmodium falciparum and Plasmodium malariae. However, PCR amplification of small subunit ribosomal RNA gene revealed a P. knowlesi infection. Parasitaemia was 473 parasites/μl and the platelet count was within the normal range. Oral treatment with Malarone® was initiated and resulted in a fast recovery without any complications.As part of routine screening the patient also underwent HIV testing and was found to be HIV positive with a CD4 cell count of 115/μl and a viral load of 34,799 copies/ml. A follow-up measurement of the viral load seven days after the first quantification revealed an increase to 102,000 copies/ml. Three months after the first quantification the viral load had dropped to 10,000 copies/ml without the initiation of antiretroviral treatment. This suggests the possibility of a P. knowlesi malaria-induced temporary elevation of viral load similar to that reported for P. falciparum and HIV co-infection.

Babesia odocoilei infection in a Saskatchewan elk (Cervus elaphus canadensis) herd

An 8-year-old female elk (Cervus elaphus canadensis) cow, presented for chronic severe weight loss and unthriftiness, was diagnosed with Babesia odocoilei infection based on blood smear evaluation, polymerase chain reaction (PCR), and DNA sequence analysis. Subsequently, velvet antler from a male that died acutely on the same farm was also PCR positive for Babesia spp. Both animals originated from a game ranch of Saskatchewan-bred and -raised animals with no known history of tick exposure, but with a history of numerous sudden deaths of unknown etiology. The presence of B. odocoilei in Canada might be a result of a recent introduction that could have deleterious effects on local wild ungulates or may represent discovery of a previously unrecognized endemic disease in local wildlife.

Comparison of automated versus manual neutrophil counts for the detection of cellular abnormalities in dogs receiving chemotherapy: 50 cases (May to June 2008)

To determine the frequency of clinically relevant abnormalities missed by failure to perform a blood smear evaluation in a specific subset of dogs receiving chemotherapy and to compare automated and manual neutrophil counts in the same population. Retrospective case series. 50 dogs receiving chemotherapy with a total nucleated cell count > 4,000 nucleated cells/μL. 50 blood smears were evaluated for abnormalities that have strong potential to change the medical plan for a patient: presence of blast cells, band neutrophils, nucleated RBCs, toxic change, hemoparasites, schistocytes, and spherocytes. Automated and manual neutrophil counts were compared. Blood smears from 10 (20%) patients had ≥ 1 abnormalities. Blast cells were identified on 4 (8%) blood smears, increased nucleated RBCs were identified on 5 (10%), and very mild toxic change was identified on 2 (4%). Correlation coefficient of the neutrophil counts was 0.96. Analysis revealed a slight bias between the automated and manual neutrophil counts (mean ± SD difference, -0.43 × 10(3)/μL ± 1.10 × 10(3)/μL). In this series of patients, neutrophil count correlation was very good. Clinically relevant abnormalities were found on 20% of the blood smears. An automated CBC appears to be accurate for neutrophil counts, but a microscopic examination of the corresponding blood smear is still recommended; further studies are needed to determine whether the detection or frequency of these abnormalities would differ dependent on chemotherapy protocol, neoplastic disease, and decision thresholds used by the oncologist in the ordering of a CBC without a blood smear evaluation.

Botryoid nuclei in the peripheral blood of a dog with heatstroke

An EDTA-anticoagulated blood sample collected from a 1.5-year-old, intact male, English Bulldog was submitted for a CBC. The CBC data and blood smear evaluation revealed borderline high hematocrit (54%, reference interval 37-55%), inappropriate rubricytosis, moderate leukopenia due to both mature neutropenia and lymphopenia, and mild thrombocytopenia. Numerous leukocytes showed evidence of karyolysis, pyknosis, and karyorhexis, and apoptotic bodies were frequent in the background. Many neutrophils had botryoid nuclei characterized by increased numbers of nuclear segments radially arranged with spoke-like, delicate chromatin filaments connecting the segments centrally. The finding of botryoid nuclei and inappropriate rubricytosis was indicative of severe hyperthermia, such as heatstroke. The dog had been exercised a long time during conditions of high temperature and humidity until he collapsed. The dog was diagnosed with severe heatstroke, hypovolemic shock, disseminated intravascular coagulation, and multiorgan dysfunction syndrome. Despite aggressive treatment, the patient died of cardiopulmonary arrest. Botryoid nuclei are frequent in people with heatstroke. In the authors' experience, botryoid nuclei are seen commonly in dogs with heatstroke, but they have never been reported in veterinary medicine. The presence of petechiation with only mild thrombocytopenia and inappropriate rubricytosis also is suggestive of heatstroke and manifests ongoing life-threatening vascular derangement.

Preclinical Potency and Safety Studies of an AAV2-Mediated Gene Therapy Vector for the Treatment of MERTK Associated Retinitis Pigmentosa

Abstract Proof of concept for MERTK gene replacement therapy has been demonstrated using different viral vectors in the Royal College of Surgeon (RCS) rat, a well characterized model of recessive retinitis pigmentosa that contains a mutation in the Mertk gene. MERTK plays a key role in renewal of photoreceptor outer segments (OS) by phagocytosis of shed OS tips. Mutations in MERTK cause impaired phagocytic activity and accumulation of OS debris in the interphotoreceptor space that ultimately leads to photoreceptor cell death. In the present study, we conducted a series of preclinical potency and GLP-compliant safety evaluations of an adeno-associated virus type 2 (AAV2) vector expressing human MERTK cDNA driven by the retinal pigment epithelium-specific, VMD2 promoter. We demonstrate the potency of the vector in RCS rats by improved electroretinogram (ERG) responses in treated eyes compared with contralateral untreated controls. Toxicology and biodistribution studies were performed in Sprague-Dawley (SD) rats injected with two different doses of AAV vectors and buffer control. Delivery of vector in SD rats did not result in a change in ERG amplitudes of rod and cone responses relative to balanced salt solution control-injected eyes, indicating that administration of AAV vector did not adversely affect normal retinal function. In vivo fundoscopic analysis and postmortem retinal morphology of the vector-injected eyes were normal compared with controls. Evaluation of blood smears showed the lack of transformed cells in the treated eyes. All injected eyes and day 1 blood samples were positive for vector genomes, and all peripheral tissues were negative. Our results demonstrate the potency and safety of the AAV2-VMD2-hMERTK vector in animal models tested. A GMP vector has been manufactured and is presently in clinical trial.

Chanarin-Dorfman Syndrome with Multi-System Involvement in Two Siblings

Chanarin-Dorfman syndrome (CDS) is a very rare autosomal recessive inherited neutral lipid metabolism disorder associated with congenital ichthyosis and multi-system involvement. Observation of lipid vacuoles in neutrophils (Jordan’s anomaly) in peripheral blood smears in patients with ichthyosiform erythroderma is diagnostic. Herein we present 2 siblings with CDS that were referred to Dokuz Eylul University School of Medicine Department of Pediatrics due to ichthyosis. They had hepatomegaly, cataract, growth retardation, and sensorineural hearing loss. Some lipid vacuoles in neutrophils were noted in peripheral blood smear evaluation. Genetic analysis showed homozygous N209X mutation in both patients. They were put on a low-fat high-carbohydrate diet supplemented with medium-chain fatty acids. During 6 months of follow-up, no improvement was observed in both patients. In conclusion, although CDS is a rare lipid storage disease, it should always be a consideration in patients with congenital ichthyosis, especially those with extracutaneous symptoms or signs. The diagnosis of CDS is made based on a very simple test-peripheral blood smear.Conflict of interest:None declared.

Carcinocythaemia (carcinoma cell leukaemia) in a dog: an acute leukaemia‐like picture due to metastatic carcinoma

An eight-year-old entire female boxer was presented with a two-week history of anorexia and lethargy and two-day history of unilateral left epistaxis. Clinical findings and laboratory test results suggested disseminated intravascular coagulation. On blood smear evaluation, occasional large epithelioid-like unclassified cells were detected. Occasionally these cells were organised in small clusters. Bone marrow examination revealed a marked infiltration by a malignant population of the same epithelioid-like cells. The dog was euthanased because of the guarded prognosis. Following histology and immunohistochemistry, a widespread undifferentiated carcinoma of unknown primary origin was diagnosed. To the authors' knowledge, this is the first case of carcinoma cell leukaemia reported in a dog. Carcinoma cell leukaemia is a rare oncological condition previously described in humans, characterised by non-haematopoietic neoplastic cells in peripheral blood.

Evaluation of an Automated Digital Imaging System, Nextslide Digital Review Network, for Examination of Peripheral Blood Smears

Several automated digital imaging systems have been introduced in recent years to improve turnaround time and proficiency in examining peripheral blood smears in hematology laboratories. To evaluate a new automated digital imaging system, Nextslide Digital Review Network (Nextslide), for examination of peripheral blood smears. We evaluated 479 peripheral blood smears, of which 247 (51.6%) were included for comparison of Nextslide and manual white blood cell differential counts and morphology evaluation, 204 (42.6%) were included for comparison of Nextslide and CellaVision (DM96) differential counts, and 28 (5.8%) were neonatal samples examined for enumeration of nucleated red blood cells. Results from both method comparisons showed excellent correlation for all major white blood cell classes with correlation coefficients ranging from 0.70 to 0.99. Evaluation of white blood cell, red blood cell, and platelet morphology also showed good correlation among methods. White blood cell preclassification capability in the system was evaluated for rate and accuracy. Leukopenic samples demonstrated markedly decreased review time with Nextslide. Enumeration of nucleated red blood cells showed good correlation among methods. Our evaluation of Nextslide shows excellent correlation when compared with conventional manual differentials and CellaVision (DM96) differentials for evaluation of peripheral blood smears.

Novel mutation in the glycoprotein Ibβ in a patient with Bernard–Soulier syndrome

Bernard-Soulier syndrome (BSS) is a rare autosomal recessive disorder characterized by a prolonged skin-bleeding time and thrombocytopenia with giant platelets. The hallmark of BSS is an abnormal platelet attachment to the vessel wall due to reduced or abnormal glycoprotein Ib/IX/V complex. We present a case of BSS in a 14-month-old boy caused by a novel genetic mutation. The patient has the typical clinical findings of BSS, but he was misdiagnosed for a long period. Evaluation of the peripheral blood smear revealed giant platelets and genetic testing confirmed the diagnosis of BSS. The child was found to be homozygous for a nonsense mutation (c.423C > A) in the glycoprotein Ibβ (GPIbβ) gene. Knowing that we are dealing with a very rare syndrome, the detected mutation in our patient was homozygous. Although the parents were nonconsanguineous, we believe that they were related in a distant parental connection, which the parents and their family were not aware of.

How to manage polycythemia vera

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