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Test Code LABPVJAK Polycythemia Vera, JAK2 V617F with Reflex to JAK2 Exon 12-15, Sequencing for Erythrocytosis, Varies

Additional Codes

Test Name in EPIC EPIC Test Code Mnemonic Mayo Test ID
POLYCYTHEMIA VERA, JAK2 V617F WITH REFLEX TO JAK2 EXON 12-15, SEQUENCING FOR ERYTHROCYTOSIS LABPVJAK PVJAK PVJAK

 


Shipping Instructions


Specimen must arrive within 5 days of collection.



Necessary Information


 



Specimen Required


Submit only 1 of the following specimens:

 

Specimen Type: Blood

Container/Tube: Lavender top (EDTA) or yellow top (ACD-B)

Specimen Volume: 10 mL

Collection Instructions:

1. Invert several times to mix blood.

2. Send whole blood specimen in original tube. Do not aliquot.

3. Label specimen as blood.

 

Specimen Type: Bone marrow aspirate

Container/Tube: Lavender top (EDTA) or yellow top (ACD-B)

Specimen Volume: 4 mL

Collection Instructions:

1. Invert several times to mix blood.

2. Send bone marrow specimen in original tube. Do not aliquot.

3. Label specimen as bone marrow.


Forms

If not ordering electronically, complete, print, and send a Hematopathology/Cytogenetics Test Request (T726) with the specimen.

Useful For

Aiding in the distinction between the myeloproliferative neoplasm polycythemia vera and other secondary erythrocytosis

Reflex Tests

Test ID Reporting Name Available Separately Always Performed
JAKXR JAK2 Exon 12-15 Sequencing, Reflex Yes, (order JAKXB-blood or JAKXM-bone marrow), Bill Only No

Testing Algorithm

The test starts with a highly sensitive DNA-based JAK2 V617F test by allele-specific polymerase chain reaction. If the JAK2 V617F result is negative or very low positive (0.06%-2%), JAK2 exon 12-15 Sanger sequencing will be performed on the stored RNA sample. If a JAK2 V617F mutation (>2%) is detected, no further testing will be performed.

 

The Sanger sequencing covers JAK2 exons 12 through the first 90% of exon 15, which spans the region containing essentially all mutations reported in myeloproliferative neoplasms. For more information see:

-Erythrocytosis Evaluation Testing Algorithm

-Myeloproliferative Neoplasm: A Diagnostic Approach to Bone Marrow Evaluation

-Myeloproliferative Neoplasm: A Diagnostic Approach to Peripheral Blood Evaluation

Method Name

Allele-Specific Polymerase Chain Reaction (PCR) and Sanger Sequencing

Reporting Name

PV (JAK2 V617F, Exon 12-15) Reflex

Specimen Type

Varies

Specimen Minimum Volume

Blood: 8 mL; Bone marrow: 2 mL

Specimen Stability Information

Specimen Type Temperature Time Special Container
Varies Refrigerated (preferred) 5 days
  Ambient  5 days

Reject Due To

Gross hemolysis Reject
Paraffin-embedded bone marrow aspirate clot or biopsy blocks
Slides
Paraffin shavings
Moderately to severely clotted		 Reject

Clinical Information

The Janus kinase 2 (JAK2) gene codes for a tyrosine kinase (JAK2) that is associated with the cytoplasmic portion of a variety of transmembrane cytokine and growth factor receptors important for signal transduction in hematopoietic cells. Signaling via JAK2 activation causes phosphorylation of downstream signal transducers and activators of transcription (STAT) proteins (eg, STAT5) ultimately leading to cell growth and differentiation. The JAK2 V617F mutation is located in exon 14 and present in 50% to 60% of primary myelofibrosis and essential thrombocythemia and in 95% to 98% of polycythemia vera (PV). In the rest of the PV cases, over 50 different mutations have been reported within exons 12 through 15 of JAK2, and essentially all non-V617F JAK2 mutations have been identified in PV. These mutations include point alterations and small insertions or deletions. Several of the exon 12 mutations have been shown to have biologic effects similar to those caused by the V617F mutation such that it is currently assumed other nonpolymorphic mutations have similar clinical effects. However, some mutations may not be well characterized and require further clinical and research evaluation.

Reference Values

An interpretive report will be provided.

Interpretation

The results will be reported as 1 of the 3 following states:

-Positive for JAK2 V617F mutation

-Positive for JAK2 mutation (other than V617F)

-Negative for JAK2 mutations

 

If the result is positive, a description of the mutation at the nucleotide level and the altered protein sequence are reported.

 

A positive mutation status is highly suggestive of a myeloid neoplasm and may support a diagnosis of polycythemia vera in the appropriate clinical setting. Correlation with clinicopathologic findings and other laboratory results is necessary in all cases.

 

A negative mutation status makes a diagnosis of polycythemia vera highly unlikely, although it does not completely exclude this possibility, other myeloproliferative neoplasms, or other neoplasms.

Cautions

A positive result is not specific for a particular diagnosis. Correlation with clinicopathologic findings and other laboratory results is necessary in all cases.

 

If this test is ordered in the setting of erythrocytosis and suspicion of polycythemia vera, interpretation requires correlation with a concurrent or recent prior bone marrow evaluation.

Method Description

Genomic DNA is extracted, and 2 polymerase chain reaction (PCR) reactions are used for each sample. In each reaction, a short fragment of genomic DNA, including the JAK2 V617 site, is amplified using quantitative PCR in a real-time PCR instrument. In the first reaction, the 5' terminal base of the reverse primer matches the JAK2 V617F mutated sequence and the PCR conditions are such that it will only bind the mutated DNA. In the second reaction, the 5' terminal base of the reverse primer matches the wild-type sequence, and the PCR conditions are such that it will only bind the wild-type sequence. In both reactions, the PCR is monitored using TaqMan probe chemistry. The amount of mutated DNA and the amount of wild-type DNA is measured for each sample. In each run, the amount of mutated and wild-type DNA in a calibrator DNA sample is also measured. The calibrator is a mixture of DNA from a positive cell line and a negative cell line and used to mitigate the run-to-run variation. Following each reaction, Relative Quantification Software is used to calculate the normalized mutated:wild type ratio, which is expressed as a unitless ratio following correction with the calibrator data.

 

The formula for the normalized ratio is as follows:

 

Normalized ratio =

mutated/wild type (sample)

mutated/wild type (calibrator)

 

The final result is reported as % JAK2 V617F of total JAK2 (ie, [mutated/mutated + wild type] x 100%).(Unpublished Mayo method)

 

For the Sanger sequencing, total RNA is extracted from whole blood or bone marrow and complementary DNA synthesized from JAK2 messenger RNA. A fragment spanning exons 12 through 15 is then amplified using standard PCR and the sequence is obtained using Sanger sequencing with analysis on an automated genetic analyzer.(Unpublished Mayo method)

Day(s) Performed

Monday through Saturday

Report Available

7 to 10 days

Specimen Retention Time

Blood/Bone marrow: 2 weeks; Extracted DNA and RNA: 3 months

Performing Laboratory

Mayo Clinic Laboratories in Rochester

Test Classification

This test was developed and its performance characteristics determined by Mayo Clinic in a manner consistent with CLIA requirements. It has not been cleared or approved by the US Food and Drug Administration.

CPT Code Information

81270-JAK2 V617

0027U (if appropriate)

LOINC Code Information

Test ID Test Order Name Order LOINC Value
PVJAK PV (JAK2 V617F, Exon 12-15) Reflex In Process

 

Result ID Test Result Name Result LOINC Value
42395 PV Reflex Result 43399-5
MP037 Specimen Type 31208-2
42394 Final Diagnosis 50398-7