What are the steps to order JAK2 (Janus Kinase 2) mutation testing?

How to Order JAK2 Mutation Testing

JAK2 V617F should be the first test ordered in any patient suspected of having a Philadelphia chromosome-negative myeloproliferative neoplasm (MPN), using peripheral blood or bone marrow samples sent for molecular genetic analysis. 1

Clinical Indications for Testing

Order JAK2 mutation testing when patients present with:

• Erythrocytosis: Hemoglobin >16.5 g/dL in men or >16.0 g/dL in women, or hematocrit >49% in men or >48% in women 1
• Thrombocytosis: Sustained platelet count ≥450 × 10⁹/L 1
• Suspected myelofibrosis: Presence of megakaryocyte proliferation and atypia with bone marrow fibrosis 1
• Unexplained generalized pruritus with elevated hemoglobin or hematocrit 1

Sequential Testing Algorithm

Step 1: Initial JAK2 V617F Testing

• Order JAK2 V617F mutation analysis first on peripheral blood or bone marrow samples 1
• This mutation is present in >90% of polycythemia vera cases and approximately 50% of essential thrombocythemia and primary myelofibrosis cases 1
• The assay should have sensitivity to detect mutant allele burden as low as 1-3% 2

Step 2: If JAK2 V617F is Negative

For suspected polycythemia vera:

• Order JAK2 exon 12 mutation testing 1
• JAK2 exon 12 mutations are found in JAK2 V617F-negative PV patients 1

For suspected essential thrombocythemia or myelofibrosis:

• Order CALR mutation testing first, then MPL mutation testing if CALR is negative 1
• This sequential approach follows the diagnostic algorithm for driver mutations 1

Step 3: Additional Molecular Testing (if triple-negative)

If JAK2, CALR, and MPL are all negative in patients with bone marrow features consistent with myelofibrosis:

• Search for complementary clonal markers: ASXL1, EZH2, IDH1/IDH2, and SRSF2 1
• These help establish clonal nature of disease when driver mutations are absent 1

Sample Requirements and Testing Methods

Specimen type:

• Peripheral blood (preferred for initial screening) or bone marrow 1
• RNA-based testing is highly sensitive and can replace DNA-based testing due to relative abundance of target transcripts 3

Testing methodology:

• Quantitative real-time PCR (QPCR) is commonly used for JAK2 V617F detection 4
• High-resolution melt-curve analysis (HRM) is useful for detecting multiple mutations within JAK2 exon 12 4
• Reverse transcription-PCR with direct sequencing can detect mutations throughout the pseudokinase domain 3

Clinical Decision Support

JAK2-tree screening tool can optimize test utilization by applying basic CBC parameters before ordering 5:

• This decision rule uses hemoglobin, platelet, and white blood cell counts to identify patients most appropriate for testing 5
• Sensitivity for JAK2 V617F detection is 91-94% while potentially reducing unnecessary testing volume by 15% 5

Common Pitfalls to Avoid

• Do not skip JAK2 V617F testing and proceed directly to other mutations - this is the most common mutation and should always be tested first 1
• Do not order all mutation panels simultaneously - follow the sequential algorithm to optimize cost-effectiveness 1
• Do not rely solely on JAK2 V617F for diagnosis - the mutation is not specific for any single MPN subtype and additional WHO criteria must be met 1
• Do not use insensitive assays - ensure the laboratory method can detect low mutant allele burdens (1-3% sensitivity) 2

Integration with Other Diagnostic Tests

JAK2 mutation testing should be ordered alongside:

• Bone marrow biopsy (required for diagnosis except in PV with hemoglobin >18.5 g/dL in males or >16.5 g/dL in females) 1
• Serum erythropoietin level (should be below reference range in PV) 1
• Complete blood count with differential 1
• Conventional cytogenetics and FISH if indicated 1