JAK2 V617F MUTATION ANALYSIS
Label Name: JAK2 V617F M
Lab Discipline: Molecular Diagnostics
Subdiscipline: Molecular Diagnostics
Institution:  Duke University Health System 
EAP ID:  LAB6605 
Last Review:  3/17/2017 10:03:13 AM
Container & Volume
  Age Group   Container   Volume  
  0  - 18 Years LAVENDER TOP TUBE 3  ML
Label Reminders
  Be sure to include patient's name, history #, date and time of collection, and collector's initials.
Collection Notes
  All:
  • Bone Marrow: One lavender-top EDTA tube (minimum of 1 ml) is required for testing. Forward unprocessed bone marrow promptly at ambient temperatures. THE SPECIMEN CANNOT BE FROZEN. GREEN-TOP (HEPARIN) TUBES ARE NOT ACCEPTABLE FOR TESTING.

    Peripheral Blood: One lavender-top EDTA tube (minimum of 3 mls) is required for testing. Forward unprocessed peripheral blood promptly to the laboratory at ambient temperatures. THE SPECIMEN CANNOT BE FROZEN. GREEN-TOP (HEPARIN) TUBES ARE NOT ACCEPTABLE FOR TESTING.
 
Transport
  Deliver peripheral blood and bone marrow specimens to lab at ambient temperature. If there is a delay of more than 24 hours in delivery, refrigerate the sample. DO NOT FREEZE.
Turn Around Time -  Routine: 10 days   Stat: N/A
Reference Values
JAK2 V617F M
NEGATIVE
Methodology
  This assay uses a Taqman real time PCR based technique to detect the presence of the V617F mutation (c.1849G>T) in the JAK2 gene. The immunomagnetically isolated granulocyte fraction of the patient peripheral blood or bone marrow is used for genomic DNA extraction. A single real time PCR reaction is performed using an oligonucleotide primer pair that amplifies both the mutant and wild-type JAK2 alleles and allele specific Taqman probes that are fluorescently labeled with VIC-TAMRA (wild-type JAK2) or FAM-TAMRA (V617F mutant JAK2). A threshold cycle value (Ct) is measured for each Taqman probe. This represents the PCR cycle at which amplified product is first detected. For each sample, a delta Ct value is calculated as the difference in Ct value between the JAK2 wild-type and JAK2 V617F mutant probes. A 1% sensitivity control, a 50% quantitative control, and 100% mutant and wild type controls are included in each assay. Results are interpreted based on the patient delta Ct value relative to the controls. A delta Ct between the 1% sensitivity control and 50% quantitative control is interpreted as positive for the V617Fmutation. A delta Ct between the 50% quantitative control and 100% mutant control is interpreted as containing a homozygous population for the V617F mutation. If the patient delta Ct falls between the 1% sensitivity control and negative control for the assay, it is interpreted as indeterminate, and retesting is recommended. This test is performed using an ABI Prism 7500 Sequence Detection System.

This test was developed and its performance characteristics determined by the DUHS Clinical Molecular Diagnostics Laboratory. It has not been cleared or approved by the U.S. Food and Drug Administration. This test is used for clinical purposes. It should not be regarded as investigational or for research. This laboratory is certified under the Clinical Laboratory Improvement Amendments of 1988 ("CLIA") as qualified to perform high complexity clinical testing.
   
   
Clinical Significance and Interpretive Data
    BACKGROUND AND CLINICAL UTILITY:

The V617F mutation is an acquired activating mutation in JAK2 which is found in the granulocytes of a majority (greater than 90%) of polycythemia vera (PCV) patients. JAK2 V617F is also seen in other myeloproliferative disorders including essential thrombocythemia (ET) and primary myelofibrosis (PMF). Approximately 50-60% of ET and PMF patients have an acquired JAK2 V617F mutation. The presence of a clonal JAK2 V617F mutation is highly suggestive of a myeloproliferative disorder such as PCV, ET or PMF, but not diagnostic of any one of these diseases. To make a specific diagnosis, correlation with other clinical and laboratory findings is required. Similarly, the absence of the JAK2 V617 mutations does not exclude the diagnosis of ET, PMF or PCV.

In patients with a diagnosis of ET, the presence of the JAK2 V617F mutation is usually associated with splenomegaly, higher hemoglobin levels, higher white blood cell counts and lower platelet counts relative to patients without JAK2 mutations. ET patients who are homozygous for the JAK2 V617F mutation have a higher rate of thrombotic events relative to JAK2 wild type ET patients. JAK2 V617F heterozygous ET patients may also have an increased risk of thrombosis. However, this is controversial. In patients with a diagnosis of PCV, homozygosity for the JAK2 V617F mutation is associated with higher hemoglobin levels and an increased incidence of pruritus but not with a higher incidence of thrombotic events. In ET, PCV and PMF patients, homozygosity for JAK2 mutations is associated with a need for cytoreductive therapy. Finally, in ET and PCV patients, homozygosity for JAK2 V617F is associated with a higher rate of fibrotic transforamation.

REFERENCES:

Baxter et al (2005) Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 365; 1054-1061.

James et al (2005) A unique clonal JAK2 mutation leading to constitutive signalling casuses polycythaemia vera. Nature. 434; 1144-1148.

Kralovics et al (2005) A Gain-of-Functin Mutation of JAK2 in Myeloproliferative Disorders. New England Journal of Medicine. 352(17); 1779-1790.

Scott et al (2007) JAK2 Exon 12 Mutations in Polycythemia Vera and Idiopathic Erythrocytosis. New England Journal of Medicine 356(5); 459-468.

Tefferi and Vardiman (2008) Classification and Diagnosis of Myeloproliferative Neoplasms: The 2008 World Health Organization Criteria and Point-of-Care Diagnostic Algorithms. Leukemia. 22; 14-22.

Vannucchi et al (2008) Clinical correlates of JAK2V617F presence or allele burden in myeloproliferative neoplasms. a critical reappraisal. Leukemia 22; 1299-1307.
   
Indications
    A clinical suspicion of Polycythemia Vera (WHO diagnostic criteria include: Elevated red cell count, no cause of secondary erythrocytosis, splenomegally, the presence of a clonal abnormality other than t(9;22), thrombocytosis, leukocytosis, a bone marrow biopsy with panmyelosis with prominent erythroid and megakaryocytic proliferation, or low serum EPO levels)

A clinical suspicion of Essential Thrombocythaemia (Diagnostic Criteria Include: Elevated platelet count, Bone marrow biopsy showing proliferation of megakaryocytes with increased numbers of large mature megakaryocytes)

A clinical suspicion of Idiopathic Myelofibrosis (Spenomegally, hepatomegally, Blood count abnormalities which vary depending on the stage of disease, Characteristic bone marrow biopsy findings which vary depending on the stage of disease.)
   
Limitations
    The presence of a clonal JAK2 V617F mutation is highly suggestive of a myeloproliferative disorder such as PCV, ET or PMF, but not diagnostic of any one of these diseases.

Similarly, the absence of the JAK2 V617 mutations does NOT exclude the diagnosis of ET, PMF or PCV.

This test detects only the JAK2 V617F point mutation. Other mutations that may occur in the JAK2 gene would not be detected by this assay.

Other rare mutations or polymorphisms within the JAK2 gene may interfere PCR primer binding and subsequent detection of JAK2 V617F.

DNA degradation or inhibitors of the PCR polymerase (such as heparin) can also interfere with the detection of the JAK2 V617F mutation.

The sensitivity of this assay is ~1%. If cells harboring the JAK2 V617F mutation make up less than 1% of all granulocytes in this specimen they may not be detected by this assay.
   
Test Synonyms
  Synonym(s): ET
Synonym(s): JAK2
Synonym(s): PCV
Synonym(s): PMF
Synonym(s): Polycythemia Vera
Synonym(s): V617F
Molecular Diagnostics Laboratory
(MDX)

Medical Director:
 Michael Datto, M.D., Ph.D.
 Phone: 919-684-6965
 Email: michael.datto@duke.edu
Lab Director:
 Catherine Rehder Ph.D, FACMG
 Phone: 919-613-8434
 Email: catherine.rehder@duke.edu
Lab Director:
 Siby Sebastian Ph.D., DABMG
 Phone: 919-613-8432
 Email: siby.s@duke.edu

Address: 
 Wadsworth Bldg, Cytogenetics, Rm 0220
 2351 Erwin Rd
 Durham,  NC  27705
 Phone: 919-684-2698
 FAX: 919-668-5424

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