Polycythemia Vera (PV) represents one of the classic myeloproliferative neoplasms (MPNs), a group of clonal hematopoietic stem cell disorders characterized by excessive production of one or more blood cell lineages. First described by William Osler in 1903, PV has evolved from a clinical diagnosis based primarily on physical examination findings to a molecularly defined entity with precise diagnostic criteria established by the World Health Organization (WHO).

The 2016 revision of the WHO classification of myeloid neoplasms and acute leukemia introduced updated diagnostic criteria for PV that incorporate both morphological and molecular features. These criteria represent a significant advancement in the diagnostic approach to PV, allowing for earlier and more accurate diagnosis while distinguishing PV from secondary causes of erythrocytosis and other MPNs.

Pathophysiology and Disease Mechanism

Polycythemia Vera arises from acquired mutations in hematopoietic stem cells that lead to autonomous, cytokine-independent proliferation of erythroid, myeloid, and megakaryocytic lineages. The vast majority of PV cases (approximately 95%) harbor mutations in the Janus kinase 2 (JAK2) gene, most commonly the JAK2 V617F point mutation located in exon 14. This mutation results in constitutive activation of the JAK-STAT signaling pathway, leading to enhanced cell proliferation and survival.

The JAK2 V617F mutation occurs in a valine-to-phenylalanine substitution at position 617, which disrupts the autoinhibitory function of the JAK2 pseudokinase domain. This results in ligand-independent activation of the receptor, even in the absence of erythropoietin binding. A smaller subset of PV patients (approximately 3-4%) harbor mutations in JAK2 exon 12, which also lead to constitutive JAK2 activation through different mechanisms.

The autonomous proliferation of hematopoietic cells in PV leads to several characteristic features: increased red blood cell mass (absolute erythrocytosis), leukocytosis, thrombocytosis, and bone marrow hypercellularity. These changes result in the clinical manifestations of the disease, including hyperviscosity symptoms, thrombotic complications, and splenomegaly.

Major Criterion 1: Elevated Hemoglobin or Hematocrit

The first major criterion requires documentation of elevated hemoglobin or hematocrit levels that exceed gender-specific thresholds. For men, the threshold is hemoglobin greater than 16.5 g/dL or hematocrit greater than 49%. For women, the threshold is hemoglobin greater than 16.0 g/dL or hematocrit greater than 48%. Importantly, only one of these parameters needs to be elevated to meet this criterion.

These thresholds are intentionally set higher than normal reference ranges to increase specificity for PV. Normal hemoglobin levels typically range from 13.5-17.5 g/dL in men and 12.0-15.5 g/dL in women, while normal hematocrit ranges from 40-50% in men and 36-46% in women. The WHO criteria use more stringent cutoffs to distinguish PV from other causes of mild erythrocytosis.

Several important considerations apply when evaluating this criterion. Hemoglobin and hematocrit measurements should be obtained when the patient is in a stable state, not actively bleeding, and has not recently undergone therapeutic phlebotomy. Recent phlebotomy can temporarily lower these values, potentially masking the diagnosis. Additionally, measurements should be obtained using standard laboratory techniques, and results should be interpreted in the context of the patient's overall clinical presentation.

It is crucial to recognize that elevated hemoglobin or hematocrit alone is not specific for PV. Secondary causes of erythrocytosis must be considered, including chronic hypoxia (from conditions such as chronic obstructive pulmonary disease, sleep apnea, or high-altitude residence), renal disease (including renal artery stenosis, polycystic kidney disease, or post-renal transplant erythrocytosis), and tumors producing erythropoietin (such as renal cell carcinoma, hepatocellular carcinoma, or uterine fibroids).

Major Criterion 2: Bone Marrow Biopsy Findings

The second major criterion requires histopathologic examination of bone marrow biopsy demonstrating hypercellularity for age with trilineage growth, also known as panmyelosis. This finding reflects the clonal nature of PV, where the mutated hematopoietic stem cell gives rise to increased production of all three major blood cell lineages: erythroid, granulocytic, and megakaryocytic.

Hypercellularity refers to increased cellularity compared to age-matched normal bone marrow. Normal bone marrow cellularity decreases with age, from approximately 80-100% in children to 30-70% in adults, with the lower range more common in elderly individuals. In PV, the bone marrow typically shows 80-100% cellularity regardless of age, reflecting the autonomous proliferation of hematopoietic cells.

The trilineage growth pattern is a hallmark of PV and helps distinguish it from other MPNs. The bone marrow should demonstrate:

• Prominent erythroid proliferation: Increased numbers of erythroid precursors at various stages of maturation, reflecting the primary abnormality in red blood cell production.
• Granulocytic proliferation: Increased numbers of granulocytic precursors, typically with a left-shifted maturation pattern, accounting for the leukocytosis commonly seen in PV.
• Megakaryocytic proliferation: Increased numbers of megakaryocytes that are characteristically pleomorphic (varying in size and shape) and mature, explaining the thrombocytosis observed in many PV patients.

The megakaryocytes in PV are particularly distinctive. They are typically large, mature cells with abundant cytoplasm and well-lobulated nuclei. Unlike the small, hypolobulated megakaryocytes seen in essential thrombocythemia or the bizarre, dysplastic forms seen in primary myelofibrosis, PV megakaryocytes are pleomorphic but generally appear mature and functional.

Bone marrow biopsy is essential not only for establishing the diagnosis of PV but also for excluding other conditions that might mimic PV, such as secondary erythrocytosis (which typically shows normal or only mildly hypercellular bone marrow), other MPNs, or myelodysplastic syndromes. The biopsy also provides important prognostic information, as the presence of significant fibrosis may indicate progression toward post-PV myelofibrosis.

Major Criterion 3: JAK2 Mutation

The third major criterion requires detection of a JAK2 mutation, specifically JAK2 V617F or JAK2 exon 12 mutation. This criterion reflects the central role of JAK2 mutations in the pathogenesis of PV and represents one of the most significant advances in MPN diagnosis.

The JAK2 V617F mutation is the most common genetic abnormality in PV, present in approximately 95% of cases. This point mutation results from a G-to-T transversion at nucleotide 1849 in exon 14 of the JAK2 gene, leading to substitution of valine by phenylalanine at position 617. This mutation disrupts the autoinhibitory function of the JAK2 pseudokinase domain, resulting in constitutive activation of the kinase even in the absence of cytokine binding.

JAK2 exon 12 mutations are found in approximately 3-4% of PV patients who are negative for JAK2 V617F. These mutations include various insertions, deletions, and point mutations within exon 12, all of which lead to constitutive JAK2 activation. Patients with JAK2 exon 12 mutations often present with isolated erythrocytosis without significant leukocytosis or thrombocytosis, making them clinically distinct from JAK2 V617F-positive patients.

JAK2 mutation testing can be performed on peripheral blood or bone marrow samples using various molecular techniques, including allele-specific polymerase chain reaction (PCR), quantitative PCR, next-generation sequencing, or digital PCR. The sensitivity of these assays varies, with allele-specific PCR and quantitative PCR being highly sensitive and capable of detecting mutations present in as few as 1-5% of cells.

The presence of a JAK2 mutation is highly specific for MPNs, particularly PV, essential thrombocythemia, and primary myelofibrosis. However, it is important to note that JAK2 mutations are not exclusive to PV and can be found in other MPNs. The clinical context, bone marrow findings, and other laboratory parameters must be considered together to establish the correct diagnosis.

Rare cases of PV may be negative for both JAK2 V617F and JAK2 exon 12 mutations. These JAK2-negative PV cases are extremely uncommon and may require alternative diagnostic approaches, including evaluation for other MPN-associated mutations (such as CALR or MPL mutations, though these are more commonly associated with essential thrombocythemia and primary myelofibrosis) or reliance on the first two major criteria plus the minor criterion.

Minor Criterion: Subnormal Serum Erythropoietin Level

The minor criterion requires documentation of a subnormal serum erythropoietin level, defined as a level below the reference range for normal. This criterion reflects the autonomous nature of erythropoiesis in PV, where red blood cell production occurs independently of erythropoietin stimulation.

Erythropoietin is a glycoprotein hormone produced primarily by the kidneys in response to tissue hypoxia. Under normal circumstances, decreased oxygen delivery to the kidneys stimulates erythropoietin production, which then promotes red blood cell production in the bone marrow. In PV, however, the JAK2 mutation leads to autonomous erythropoiesis that is independent of erythropoietin signaling, resulting in suppression of erythropoietin production through negative feedback mechanisms.

Normal serum erythropoietin levels vary by laboratory but typically range from approximately 3.7 to 31.5 mU/mL. In PV, erythropoietin levels are characteristically low or undetectable, often measuring less than 3.7 mU/mL. This finding helps distinguish PV from secondary erythrocytosis, where erythropoietin levels are typically normal or elevated in response to tissue hypoxia or autonomous erythropoietin production by tumors.

Several important considerations apply when interpreting erythropoietin levels. The measurement should be obtained when the patient is in a stable state, not actively bleeding or undergoing phlebotomy, as these conditions can affect erythropoietin levels. Additionally, erythropoietin levels should be measured before initiating cytoreductive therapy, as some treatments may affect erythropoietin production.

The subnormal erythropoietin level is particularly useful in cases where JAK2 mutation testing is negative or unavailable. When combined with elevated hemoglobin/hematocrit and characteristic bone marrow findings, a subnormal erythropoietin level provides strong supportive evidence for the diagnosis of PV.

Diagnostic Pathways

The WHO criteria provide two distinct pathways for establishing the diagnosis of PV, reflecting the central importance of JAK2 mutations while allowing for diagnosis in rare JAK2-negative cases.

Pathway 1: All Three Major Criteria

The first diagnostic pathway requires fulfillment of all three major criteria: elevated hemoglobin or hematocrit, characteristic bone marrow biopsy findings, and presence of a JAK2 mutation. This pathway is applicable to the vast majority of PV patients, who are JAK2-positive. The presence of all three major criteria provides the highest level of diagnostic certainty and is the most commonly used pathway in clinical practice.

This pathway is particularly straightforward when JAK2 V617F mutation is detected, as this mutation is highly specific for MPNs and, in the context of elevated hemoglobin/hematocrit and characteristic bone marrow findings, is virtually diagnostic of PV. The combination of these three criteria effectively excludes secondary causes of erythrocytosis and other MPNs.

Pathway 2: First Two Major Criteria Plus Minor Criterion

The second diagnostic pathway requires fulfillment of the first two major criteria (elevated hemoglobin/hematocrit and characteristic bone marrow findings) plus the minor criterion (subnormal erythropoietin level). This pathway is particularly useful in rare cases of JAK2-negative PV, where JAK2 mutation testing is negative or unavailable, or in cases where there is uncertainty regarding JAK2 mutation status.

This pathway recognizes that while JAK2 mutations are present in the vast majority of PV cases, the disease can occasionally occur in the absence of detectable JAK2 mutations. In such cases, the combination of elevated hemoglobin/hematocrit, characteristic bone marrow findings, and subnormal erythropoietin level provides sufficient evidence for diagnosis, particularly when secondary causes of erythrocytosis have been excluded.

The second pathway also serves as a backup diagnostic approach when JAK2 mutation testing is not readily available or when there are technical issues with mutation detection. However, it is important to note that JAK2-negative PV is extremely rare, and alternative diagnoses should be carefully considered before applying this pathway.

Clinical Applications and Diagnostic Workup

The WHO diagnostic criteria for PV serve multiple important functions in clinical practice, guiding not only the initial diagnosis but also the differential diagnosis, risk stratification, and treatment planning.

Initial Diagnostic Evaluation

When PV is suspected based on clinical presentation (such as symptoms of hyperviscosity, thrombotic events, or splenomegaly) or incidental laboratory findings (elevated hemoglobin/hematocrit), a systematic diagnostic workup should be initiated. This typically begins with a complete blood count with differential, which may reveal not only elevated hemoglobin and hematocrit but also leukocytosis and thrombocytosis.

If hemoglobin or hematocrit levels meet the threshold for major criterion 1, further evaluation should proceed with JAK2 mutation testing and bone marrow biopsy. JAK2 mutation testing can often be performed on peripheral blood, making it a convenient first step in the diagnostic workup. If JAK2 mutation is detected, bone marrow biopsy can be performed to confirm the diagnosis and evaluate for other features.

Bone marrow biopsy should include both aspirate and core biopsy specimens. The aspirate allows for cytogenetic analysis and flow cytometry, while the core biopsy provides the histopathologic evaluation necessary to assess cellularity and trilineage growth. Both specimens are important for a complete diagnostic evaluation.

Differential Diagnosis

The WHO criteria are essential for distinguishing PV from other conditions that may present with similar features. Secondary erythrocytosis, which can result from chronic hypoxia, renal disease, or erythropoietin-producing tumors, typically shows normal or only mildly elevated erythropoietin levels and lacks the characteristic bone marrow findings of PV. Additionally, secondary erythrocytosis is not associated with JAK2 mutations.

Other MPNs must also be considered in the differential diagnosis. Essential thrombocythemia may present with thrombocytosis and occasionally mild erythrocytosis, but typically lacks the degree of erythrocytosis seen in PV and shows different bone marrow characteristics. Primary myelofibrosis may present with various blood count abnormalities but is characterized by bone marrow fibrosis, which is not a feature of early-stage PV.

The WHO criteria help distinguish PV from these other conditions by requiring specific combinations of findings that are characteristic of PV. The presence of JAK2 mutation, while not exclusive to PV, when combined with the other criteria, provides strong evidence for the diagnosis.

Risk Stratification

Once the diagnosis of PV is established using the WHO criteria, risk stratification becomes important for treatment planning. PV patients are typically stratified into low-risk and high-risk categories based on age and history of thrombosis. Low-risk patients are those under 60 years of age without a history of thrombosis, while high-risk patients are those 60 years or older or with a history of thrombosis.

Risk stratification guides treatment decisions, with low-risk patients typically managed with phlebotomy and low-dose aspirin, while high-risk patients often require cytoreductive therapy in addition to these interventions. The accurate diagnosis of PV using WHO criteria is essential for appropriate risk stratification and treatment selection.

Important Clinical Considerations

Several important considerations must be kept in mind when applying the WHO diagnostic criteria for PV in clinical practice.

First, the criteria should be applied in the context of the patient's overall clinical presentation. While the criteria provide objective standards for diagnosis, they must be interpreted alongside clinical symptoms, physical examination findings, and other laboratory parameters. A patient who meets the diagnostic criteria but has an alternative explanation for their findings (such as recent blood transfusion or use of erythropoietin-stimulating agents) requires careful evaluation.

Second, secondary causes of erythrocytosis must be systematically excluded before establishing a diagnosis of PV. This includes evaluation for chronic hypoxia (pulmonary function testing, sleep studies), renal disease (renal imaging, renal function testing), and erythropoietin-producing tumors (appropriate imaging studies). The presence of a JAK2 mutation does not completely exclude secondary causes, as rare cases of secondary erythrocytosis with coincidental JAK2 mutations have been reported.

Third, the timing of diagnostic testing is important. Hemoglobin and hematocrit measurements should be obtained when the patient is in a stable state, not actively bleeding, and has not recently undergone phlebotomy. Similarly, erythropoietin levels should be measured before initiating cytoreductive therapy, as some treatments may affect erythropoietin production.

Fourth, bone marrow biopsy is essential for diagnosis and should not be omitted, even when JAK2 mutation is positive. The bone marrow findings provide important diagnostic information and help exclude other conditions. Additionally, the bone marrow evaluation provides baseline information that may be useful for monitoring disease progression, particularly the development of fibrosis.

Fifth, patients with PV are at increased risk of both thrombotic and hemorrhagic complications. Thrombotic events, including arterial and venous thrombosis, are common in PV and represent a major cause of morbidity and mortality. Hemorrhagic complications, while less common, can also occur, particularly in patients with extreme thrombocytosis. These risks must be considered in treatment planning and patient counseling.

Sixth, PV is a chronic condition that requires long-term management and monitoring. Regular follow-up with complete blood counts, clinical assessment, and periodic bone marrow evaluation (particularly if there are concerns about disease progression) is essential. Patients should be educated about the signs and symptoms of disease complications and the importance of adherence to treatment recommendations.

Seventh, consultation with hematology/oncology specialists with expertise in MPNs is recommended for diagnosis and management of PV. These specialists are familiar with the nuances of the WHO diagnostic criteria and can provide expert guidance on diagnostic workup, risk stratification, treatment selection, and long-term management.

Treatment Implications

The accurate diagnosis of PV using WHO criteria has important implications for treatment. Once the diagnosis is established, treatment goals include reducing the risk of thrombotic complications, managing symptoms, and preventing disease progression to myelofibrosis or acute leukemia.

Phlebotomy remains a cornerstone of PV treatment, with the goal of maintaining hematocrit below 45% to reduce the risk of thrombotic complications. Low-dose aspirin (81-100 mg daily) is recommended for all PV patients without contraindications, as it has been shown to reduce the risk of thrombotic events.

Cytoreductive therapy is indicated for high-risk patients or those with symptoms that are not adequately controlled by phlebotomy alone. Options include hydroxyurea, interferon-alpha, and ruxolitinib (a JAK2 inhibitor). The choice of cytoreductive agent depends on various factors, including patient age, comorbidities, and treatment goals.

The accurate diagnosis of PV using WHO criteria ensures that patients receive appropriate treatment while avoiding unnecessary interventions. It also allows for proper risk stratification and treatment selection based on individual patient characteristics.

Monitoring and Follow-up

Regular monitoring is essential for patients with PV, both to assess treatment response and to detect disease progression. Complete blood counts should be performed regularly, with frequency depending on disease activity and treatment phase. Clinical assessment should include evaluation for symptoms of hyperviscosity, thrombotic events, disease-related symptoms, and treatment-related side effects.

Periodic bone marrow evaluation may be indicated in certain circumstances, such as when there are concerns about disease progression, development of cytopenias, or transformation to myelofibrosis or acute leukemia. The baseline bone marrow findings obtained at diagnosis provide an important reference point for comparison.

Molecular monitoring, including assessment of JAK2 mutation burden, may be useful in some cases, particularly for monitoring response to JAK2 inhibitor therapy. However, the role of molecular monitoring in routine PV management is still evolving and is not currently part of standard practice.