Investigations
1st investigations to order
haemoglobin
Test
An elevated haemoglobin, detected either at initial presentation or incidentally, warrants further investigation.
Frequently, patients are asymptomatic and elevated haemoglobin is discovered on a blood count performed for other reasons.
Elevated haemoglobin has good diagnostic sensitivity (with a few exceptions) but poor specificity.
Elevated haemoglobin or haematocrit are required for PV diagnosis; both should be examined.[7][8]
If possible, it is important to determine if the patient ever had normal blood counts.
In the setting of hypoxia, a repeat test, several weeks after the hypoxia has been corrected, is recommended.
Adjust appropriately for those living at high altitude.
Some patients may have pathologically and molecularly proven PV, without overt elevations in haemoglobin or haematocrit.[16] This is known as 'masked PV' as the diagnosis is not obvious from the patient’s haemoglobin/haematocrit. Masked PV patients are more frequently male and tend to present with higher platelet counts and increased reticulin fibrosis in the bone marrow.[16]
Masked PV requires careful pathological distinction from essential thrombocythaemia when accompanied by thrombocytosis.[18] High clinical vigilance for a possible PV diagnosis is essential in the presence of suggestive symptoms or complications (e.g., otherwise unexplained thrombosis).[18]
Result
>165 g/L (>16.5 g/dL) in men; >160 g/L (>16.0 g/dL) in women
haematocrit
Test
Elevated haemoglobin or haematocrit is required for PV diagnosis; both should be examined.[7][8]
Some patients may have pathologically and molecularly proven PV, without overt elevations in haemoglobin or haematocrit.[16] This is known as 'masked PV' as the diagnosis is not obvious from the patient’s haemoglobin/haematocrit. Masked PV patients are more frequently male and tend to present with higher platelet counts and increased reticulin fibrosis in the bone marrow.[16]
Masked PV requires careful pathological distinction from essential thrombocythaemia when accompanied by thrombocytosis.[18]
High clinical vigilance for a possible PV diagnosis is essential in the presence of suggestive symptoms or complications (e.g., otherwise unexplained thrombosis).[18]
Result
World Health Organization: >49% in men, >48% in women; British Committee for Standards in Haematology: >52% in men, >48% in women
white blood cell (WBC) count
Test
Polycythaemia vera (PV) is often associated with at least a modest leukocytosis.
Leukocytosis is not a particularly sensitive finding and is non-specific.
However, WBC count ≥15 × 10⁹/L (15 × 10³/microlitre or 15,000/microlitre) has been shown to independently predict both inferior leukaemia-free and overall survival in patients with PV.[14][84][94]
Result
elevated
platelet count
Test
Polycythaemia vera is often associated with at least a modest thrombocytosis.
Thrombocytosis is not a particularly sensitive finding and is non-specific.
There is no evidence that thrombocytosis is a risk factor for thrombosis.[61]
Result
elevated
mean corpuscular volume (MCV)
Test
Usually low in patients with polycythaemia vera (PV).
Low values usually indicate iron deficiency.
Some cases of PV can present with iron deficiency and a normal haemoglobin level. Therefore, iron deficiency can mask erythrocytosis in PV.
A low MCV in the setting of iron deficiency and a normal haemoglobin suggests PV as a possible diagnosis.
Result
usually low and co-existent with iron deficiency and normal haemoglobin
peripheral blood smear
Test
In patients with polycythaemia vera, red blood cells (RBCs) are abundant and usually normochromic and normocytic, although iron stores may be depleted leading to hypochromic and microcytic RBCs. A leukoerythroblastic blood smear suggests disease progression.
Result
abundant normochromic/normocytic or hypochromic/microcytic RBCs
liver function tests (LFTs)
Test
Liver function is generally normal in patients with polycythaemia vera.
Abnormality of bilirubin, aminotransferases, or alkaline phosphatase should prompt screening for hepatic or portal vein thrombosis.
It is important to note that normal LFTs do not rule out the presence of thromboses.
Result
usually normal
serum ferritin
Test
Performed to screen for iron deficiency if a low mean corpuscular volume (MCV) is found.
Some cases of polycythaemia vera (PV) can present with iron deficiency and a normal haemoglobin level. Therefore, iron deficiency can mask erythrocytosis in PV.
A low MCV in the setting of iron deficiency and a normal haemoglobin suggests PV as a possible diagnosis
Result
normal; or low with co-existent low MCV and normal haemoglobin
JAK2 gene mutation testing
Test
Molecular testing of blood or bone marrow for Janus kinase 2 (JAK2) V617F mutations is recommended for all patients with suspected myeloproliferative neoplasm.[72]
The JAK2 V617F mutation is present in approximately 95% of patients with polycythaemia vera (PV).[3][4][5][6] However, this mutation is not specific for PV, as it is found in other myeloproliferative neoplasms (essential thrombocythaemia, primary myelofibrosis), chronic myelomonocytic leukaemia, and acute myeloid leukaemia.[32][77] Furthermore, it is among the most common mutations detected in those with 'clonal haematopoiesis of indeterminate potential' who have no particular haematological phenotype.[78]
Presence of both a JAK2 mutation and low serum erythropoietin in a patient with sustained absolute erythrocytosis confirms diagnosis of PV.[8][7]
Quantitative testing for JAK2 V617F mutant allele burden may add important prognostic information. Patients with a higher JAK2 V617F allele burden appear to be at greater risk for thrombosis and myelofibrotic progression.[49][50][51]
If JAK2 V617F mutation is not detected, a diagnosis of PV is less likely. However, some patients have JAK2 exon 12 mutations (approximately 3% of patients with PV).[29]
Testing for JAK2 exon 12 mutation should be carried out in patients with suspected PV who are negative for the JAK2 V617F mutation.[72]
Result
JAK2 V617F mutation usually present
bone marrow biopsy
Test
Mandated in the diagnostic criteria for polycythaemia vera (PV), unless there is sustained absolute erythrocytosis in the presence of both a JAK2 mutation and low serum erythropoietin level.[7][8][80][81]
Bone marrow biopsy allows for karyotype analysis and assessment of reticulin fibrosis. Abnormal karyotype is a marker of inferior survival in PV.[14] Reticulin fibrosis in the marrow at presentation (initial myelofibrosis) correlates with an increased risk of post-PV myelofibrosis.[8][80][82]
Several histological features (e.g., increased bone marrow cellularity and increases in all three primary cell lines) may distinguish secondary polycythaemia from PV, and discriminate among the various myeloproliferative neoplasms.[83] For example, bone marrow examination is critical for making the distinction between 'masked PV' and essential thrombocythaemia.[18] It can also help to distinguish early PV with concomitant thrombocytosis from essential thrombocythaemia.[18]
Result
hypercellularity for age with trilineage growth (panmyelosis), including prominent erythroid, granulocytic, and megakaryocytic proliferation with pleomorphic, mature megakaryocytes
Investigations to consider
oxygen saturation
Test
A low oxygen saturation may suggest a secondary cause for elevated haemoglobin such as smoking, sleep apnoea, or other lung disease.
This requires further investigation and correction of hypoxia.
If haemoglobin does not normalise after secondary causes of erythrocytosis have been corrected, then further investigation is necessary and a primary haematological disorder should be considered.
Result
normal or low
serum erythropoietin
Test
Low erythropoietin strongly suggests polycythaemia vera (PV), although alone it is not diagnostic. Low erythropoietin is also reported in those with primary familial and congenital polycythaemia associated with gain-of-function erythropoietin receptor mutations.[79]
A normal value makes the diagnosis of PV less likely but does not rule it out. The erythropoietin level returns to normal in some patients with PV after phlebotomy.
High erythropoietin is consistent with secondary erythrocytosis.[67] In the absence of an obvious secondary cause of erythrocytosis, patients are screened for erythropoietin-secreting tumours (chest x-ray, abdominal imaging, and brain MRI or CT scan). If the diagnosis is still unclear, referral to a haematologist is appropriate; a primary haematological disorder is unlikely.
Result
usually low; occasionally normal
red blood cell (RBC) mass
Test
RBC mass measurement may be considered if haemoglobin/haematocrit is equivocal, or affected by certain clinical conditions (e.g., volume overload).[53]
This test is not commonly performed in practice, and is no longer included in the WHO diagnostic criteria. However, it may have a role if diagnosis cannot be definitively established by other tests.[8]
RBC mass is always elevated in PV. RBC mass test is only standardised at sea level and may not be applicable at other elevations.
Result
elevated (>25% above mean normal predicted value)
further mutational testing
Test
Performed to exclude differential diagnoses and for prognostication.[37][84]
Testing for calreticulin (CALR) and thrombopoietin receptor (MPL) mutations can be performed if thrombocytosis is the predominant clinical manifestation, and JAK2 V617F and JAK2 exon 12 testing is negative.[72] A positive CALR or MPL mutation generally excludes PV, and suggests a diagnosis of essential thrombocythaemia or primary myelofibrosis.[85][86][87][88] However, there have been reports of CALR mutations in patients with polycythaemia vera (PV).[89]
Next-generation sequencing assays can test for multiple gene mutations simultaneously (including JAK2, CALR, MPL).[72][77]
Mutations in SRSF2, ASXL1, and IDH2 are associated with inferior overall survival and inferior myelofibrosis-free survival.[37]
Result
CALR and MPL mutations typically absent in PV
cytogenetic and molecular testing: BCR::ABL1
Test
Fluorescence in situ hybridisation (FISH) or polymerase chain reaction (PCR) for BCR::ABL1 (Philadelphia chromosome) should be carried out to exclude chronic myeloid leukaemia (CML).[72]
Prognosis and management of CML differs considerably to that of polycythaemia vera.
Result
negative for BCR::ABL1 (Philadelphia chromosome)
serum uric acid
Test
Commonly elevated owing to rapid cell turnover from expanded haematopoiesis.
Elevated levels predispose to gout and urate kidney stones.
Result
usually elevated; may be normal
abdominal imaging
Test
Generally, clinically significant splenomegaly is appreciated on physical examination.
Abdominal imaging may be performed if splenomegaly is suspected but physical examination is equivocal. Ultrasound may be preferred to avoid radiation exposure.
Symptoms suggestive of splanchnic vein thrombosis should also prompt imaging studies.
Result
splenomegaly
vascular imaging
Test
At initial presentation, 12% to 39% of patients have major thrombosis (e.g., stroke, myocardial infarction, pulmonary embolism, superficial thrombophlebitis, deep vein thrombosis).[2][12]
Thrombosis may occur in both usual and unusual locations.
Vascular imaging (CT scan or MRI) is performed if there are any symptoms or physical examination findings suggestive of thrombosis.
Abdominal vessel thromboses should be ruled out in people with abnormal liver function tests, recurrent abdominal pain, or evidence of the Budd-Chiari syndrome.
Routine screening in asymptomatic patients is not indicated.
Result
may demonstrate thrombosis
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