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HelpTest Code LAB8865 Polycythemia Vera, JAK2 V617F with Reflex to JAK2 Exon 12-15, Sequencing for Erythrocytosis, Varies
Additional Codes
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.
Secondary ID
65116Useful 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
Special Instructions
Method Name
Allele-Specific Polymerase Chain Reaction (PCR) and Sanger Sequencing
Specimen Type
VariesSpecimen 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 |
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.
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 daysSpecimen Retention Time
Blood/Bone marrow: 2 weeks; Extracted DNA and RNA: 3 monthsPerforming Laboratory
Mayo Clinic Laboratories in Rochester
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 |