Polycythemia Vera Workup

The Polycythemia Vera Study Group (PVSG) was the first to set rigorous criteria for the diagnosis of polycythemia vera (PV) in the 1970s. With the establishment of polymerase chain reaction (PCR)–based methods for detecting the JAK2 V617F mutation, this may become the first molecular diagnostic marker for PV, similar to BCR/ABL for chronic myelogenous leukemia (CML). However, because of a paucity of centers doing red blood cell mass measurements, demonstrating an elevated red blood cell mass continues to become more difficult.

The diagnostic criteria set by the PVSG are organized into two categories, A and B. The diagnosis of PV is established if all three category A criteria are present, or if criteria A1 plus A2 plus any two criteria from category B are present.

Category A criteria are as follows:

1. Total red blood cell mass ≥36 mL/kg in males or ≥32 mL/kg in females
2. Arterial oxygen saturation ≥92%
3. Splenomegaly

Category B criteria are as follows:

• Thrombocytosis, with platelet count > 400,000/μL
• Leukocytosis, with a white blood cell count > 12,000/μL
• Increased leukocyte alkaline phosphatase (ALP) > 100 U/L
• Serum vitamin B12 concentration > 900 pg/mL or binding capacity > 2200 pg/mL

Total red blood cell mass is measured by labeling the cells with chromium 51 (⁵¹Cr). Documentation of an elevated total red blood cell mass with ⁵¹Cr-labeled red blood cells and, ideally, an iodine-131 (¹³¹I) plasma volume dual technique differentiates true erythrocytosis from pseudoerythrocytosis (decreased plasma volume).

However, the red blood cell mass is becoming difficult to obtain because the ⁵¹Cr isotope needed to perform the test is no longer readily available, and institutions willing to perform the test are few as a result of small demand and lack of profit in performing the test.

Diagnostic criteria for PV as per the 2016 revised World Health Organization (WHO) guidelines include three major criteria and a minor criterion. Diagnosis requires the presence of either all three major criteria or the first two major criteria and the minor criterion. ^[5]

Major WHO criteria are as follows:

1. Hemoglobin >16.5 g/dL in men and >16 g/dL in women, or hematocrit >49% in men and >48% in women, or red cell mass >25% above mean normal predicted value

2. Bone marrow biopsy showing hypercellularity for age with trilineage growth (panmyelosis) including prominent erythroid, granulocytic, and megakaryocytic proliferation with pleomorphic, mature megakaryocytes (differences in size)

3. Presence of JAK2V617F or JAK2 exon 12 mutation

The minor WHO criterion is as follows:

• Serum erythropoietin level below the reference range for normal

Criterion 2 (bone marrow biopsy) may not be required in patients who have sustained absolute erythrocytosis (in men, hemoglobin/hematocrit of >18.5 g/dL/55.5% or in women, >16.5 g/dL/49.5%) if major criterion 3 and the minor criterion are present. However, bone marrow biopsy is the only way to detect initial myelofibrosis, which is present in up to 20% of patients and may predict a more rapid progression to overt myelofibrosis. ^[5]

JAK2 mutations also occur in about 60% of patients with essential thrombocythemia. PV is mainly related to JAK2 mutations, whereas a wider mutational spectrum is detected in essential thrombocythemia (ET) with mutations in JAK2, the thrombopoietin (TPO) receptor (MPL), and the calreticulin (CALR) genes. ^[15]

In patients who are positive for JAK2 and whose hemoglobin/hematocrit level is diagnostically equivocal (ie, as in "masked" PV), bone marrow examination is necessary to distinguish the two conditions. ^[16]  Masked PV includes both early forms of PV as well as a distinct form marked by male predominance, a more frequent history of arterial thrombosis and thrombocytosis, and significantly higher rates of progression to myelofibrosis and acute leukemia and inferior survival. ^[17]

If the JAK2 V617F mutation is absent but the Epo level is low, then testing for JAK2 exon 12 and 13 mutations would be helpful for making a diagnosis of PV in the 2-3% of PV patients who are negative for JAK2 V617F mutation. Patients who are negative for JAK2 mutations and have a normal or high Epo level have secondary erythrocytosis.

Automated red blood cell counts and hematocrit values (including hemoglobin levels) may be deceptive with regard to the total red blood cell mass in patients with polycythemia vera (PV). Direct measurement of the red blood cell mass should show an increase with a normal or slightly decreased plasma volume. This is a nuclear medicine test that uses radiochromium-labeled red blood cells to measure actual red blood cell and plasma volume. However, patients with hemoglobin concentrations of at least 20 g/dL or hematocrit values of at least 60% in males and 56% in females always have an elevated red blood cell mass.

The red blood cells in patients with PV are usually normochromic and normocytic, unless the patient has been bleeding from underlying peptic ulcer disease or phlebotomy treatment (in which case the cells may be hypochromic and microcytic, reflecting low iron stores). See the image below.

This blood film at 10,000X magnification shows a giant platelet and an eosinophil. Erythrocytes show signs of hypochromia as a result of repeated phlebotomies. Courtesy of U. Woermann, MD, Division of Instructional Media, Institute for Medical Education, University of Bern, Switzerland.

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Findings that are often present in patients with PV, but are not required for diagnosis, include the following ^[1] :

• Thrombocytosis (>400,000 platelets/µL)
• Leukocytosis (>12,000/µL)
• Leukocyte alkaline phosphatase score >100 units/L in the absence of fever or infection

The platelet count is elevated to 400,000-800,000/µL in approximately 50% of patients. The release of potassium into the serum caused by the increased number of platelets during in vitro coagulation may cause a pseudohyperkalemia in the serum, whereas the true plasma potassium level in vivo is actually within the reference range, as shown by measuring plasma levels and by the lack of electrocardiography (ECG) changes. Morphologic abnormalities in platelets include macrothrombocytes and granule-deficient platelets.

An elevated white blood cell count (>12,000/µL) occurs in approximately 60% of patients. It is mainly composed of neutrophils with a left shift and a few immature cells. Mild basophilia occurs in 60% of patients.

The leukocyte alkaline phosphatase (LAP) score is elevated (>100 U/L) in 70% of patients. This technique is only semiquantitative and is susceptible to interobserver and laboratory errors unless it can be performed by flow cytometry, which is not routinely available.

Abnormal platelet function (as measured by platelet aggregation tests with epinephrine, adenosine diphosphate [ADP], or collagen) may be demonstrated, but bleeding time may be normal. Some patients' platelet-rich plasma spontaneously aggregates without the addition of any of the above substances. This indicates a propensity for thromboses.

Routine coagulation test results are normal, with a high turnover rate for fibrinogen. The prothrombin time (PT) and activated partial thromboplastin (aPTT) time may be artifactually prolonged, however, because the erythrocytosis results in the collection of a low amount of plasma in relation to the anticoagulant in the test tube. Thus, the volume of the ratio of anticoagulant to blood must be modified when drawing blood for coagulation tests in patients who are polycythemic.

Vitamin B-12 levels are elevated to more than 900 pg/mL in approximately 30% of patients, and 75% of patients show an elevation in the unbound vitamin B-12 binding capacity greater than 2200 pg/mL. This is because of increased transcobalamin-III, a binding protein found in white blood cells, and it reflects the total white blood cell counts in the peripheral blood and bone marrow.

Hyperuricemia occurs in 40% of patients and reflects the high turnover rate of bone marrow cells releasing DNA metabolites.

The most important diagnostic tests are JAK2 mutation analysis and the serum erythropoietin (Epo) level. A positive JAK2 V617F mutation and a low Epo level confirms the diagnosis of PV.

A low serum Epo level, which is decreased in nearly all patients with PV who have experienced no recent hemorrhage, distinguishes polycythemia from secondary causes of polycythemia in which the serum Epo level is generally within the reference range or is elevated. Each laboratory has its own reference range for serum Epo levels.

Endogenous erythroid colony (EEC) formation, a minor diagnostic according to 2008 WHO diagnostic criteria for PV, has been eliminated from the 2016 criteria. Insulin-like growth factor 1 receptor (IGF-1R) has been found to be responsible for the EEC formation in PV, and Wang et al found significantly elevated IGF-1R levels in the peripheral blood of 14 of 16 (87%) PV patients. ^[18]

In comparison, none of 33 patients with secondary polycythemia and 29 normal controls had elevated IGR-1R levels. In addition, IGF-1R levels were significantly higher in patients with PV who were treated with phlebotomy only, compared with those treated with hydroxyurea or ruxolinitinib. ^[18]

An enlarged spleen is often palpable and in such cases, imaging studies are not required. In some patients with posteriorly enlarged spleens or in those who are obese, ultrasonography or computed tomography scans may be able to detect splenic enlargement that was not evident on physical examination.

Measuring arterial oxygen saturation (SaO₂) and carboxyhemoglobin (COHb) levels is important to rule out hypoxia as a secondary cause for erythrocytosis. Pulse oximetry is the most convenient method for measuring SaO₂; however, in people who smoke cigarettes, the COHb must be determined directly and subtracted to give an accurate SaO₂ value. A value below 92% indicates a causal relationship with erythrocytosis. If the fall is due to increased COHb, this is less likely to cause erythrocytosis.

Nocturnal oxygen desaturation due to sleep apnea is observed in 20% of patients.

Bone marrow studies are not necessary to establish the diagnosis of polycythemia vera. If such studies are performed, however, the finding of hypercellularity and hyperplasia of the erythroid, granulocytic, and megakaryocytic cell lines or myelofibrosis supports the diagnosis of a myeloproliferative process. See the image below.

Bone marrow film at 400X magnification demonstrating dominance of erythropoiesis. Courtesy of U. Woermann, MD, Division of Instructional Media, Institute for Medical Education, University of Bern, Switzerland.

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Iron stores are decreased or absent because of the increased red blood cell mass, and macrophages may be masked in the myeloid hyperplasia that is present. Fibrosis is increased and detected early by silver stains for reticulin.

Cytogenetics of the bone marrow cells show a clonal abnormality in 30% of patients who are not treated and in 50% of patients who are treated with alkylating or myelosuppressive agents. These chromosomal abnormalities include deletions of the long arm of chromosome 5 or 20 (5q-, 20q-) and trisomy 8 (+8) or 9 (+9). Leukemic transformation is usually associated with multiple or complex abnormalities.

Measuring spontaneous growth of erythroid progenitors in cultures (burst-forming unit, erythroid [BFU-E]) in the absence of Epo is a very sensitive test for polycythemia vera (PV) or familial erythrocytosis. However, it is not routinely available for clinical use.

The hemoglobin-oxygen dissociation curve may be useful in rare cases to detect a congenital hemoglobinopathy with increased oxygen affinity. This condition can occur in families.