Aiding in the distinction between the myeloproliferative neoplasm polycythemia vera and other secondary erythrocytosis
| 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 |
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
Allele-Specific Polymerase Chain Reaction (PCR) and Sanger Sequencing
JAK2 V617F
JAK2 exon 12
Polycythemia Vera
Myeloproliferative Neoplasm (MPN)
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
Varies
Specimen must arrive within 5 days of collection.
| Question ID | Description | Answers |
|---|---|---|
| MP037 | Specimen Type |
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.
If not ordering electronically, complete, print, and send a Hematopathology/Cytogenetics Test Request (T726) with the specimen.
Blood: 8 mL; Bone marrow: 2 mL
| Gross hemolysis | Reject |
| Paraffin-embedded bone marrow aspirate clot or biopsy blocks Slides Paraffin shavings Moderately to severely clotted | Reject |
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| Varies | Refrigerated (preferred) | 5 days | |
| Ambient | 5 days |
Aiding in the distinction between the myeloproliferative neoplasm polycythemia vera and other secondary erythrocytosis
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
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.
An interpretive report will be provided
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.
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.
1. Baxter EJ, Scott LM, Campbell PJ, et al. Acquired mutation of the tyrosine kinase JAK2 in human myeloproliferative disorders. Lancet. 2005;365(9464):1054-1061. doi:10.1016/S0140-6736(05)71142-9
2. James C, Ugo V, Le Couedic JP, et al. A unique clonal JAK2 mutation leading to constitutive signaling causes polycythaemia vera. Nature. 2005;434(7037):1144-1148. doi:10.1038/nature03546
3. Kralovics R, Passamonti F, Buser AS, et al. A gain-of-function mutation of JAK2 in myeloproliferative disorders. N Engl J Med. 2005;352(17):1779-1790. doi:10.1056/NEJMoa051113
4. Steensma DP, Dewald GW, Lasho TL, et al. The JAK2 V617F activating tyrosine kinase mutation is an infrequent event in both "atypical" myeloproliferative disorders and the myelodysplastic syndrome. Blood. 2005;106(4):1207-1209. doi:10.1182/blood-2005-03-1183
5. Ma W, Kantarjian H, Zhang X, et al. Mutation profile of JAK2 transcripts in patients with chronic myeloid neoplasias. J Mol Diagn. 2009;11(1):49-53
6. Kilpivaara O, Levine RL. JAK2 and MPL mutations in myeloproliferative neoplasms: discovery and science. Leukemia. 2008;22(10):1813-1817. doi:10.1038/leu.2008.229
7. Kravolics R: Genetic complexity of myeloproliferative neoplasms. Leukemia. 2008;22(10):1841-1848. doi:10.1038/leu.2008.233
8. Defour JP, Chachoua I, Pecquet C, Constantinescu SN. Oncogenic activation of MPL/thrombopoietin receptor by 17 mutations at W515: implications for myeloproliferative neoplasms. Leukemia. 2016;30(5):1214-1216. doi:10.1038/leu.2015.271
9. Tefferi A. The classic myeloproliferative neoplasms: Chronic myelogenous leukemia, polycythemia vera, essential thrombocythemia, and primary myelofibrosis. In: Valle DL, Antonarakis S, Ballabio A, Beaudet AL, Mitchell GA, eds. The Online Metabolic and Molecular Bases of Inherited Disease. McGraw-Hill; 2019, Accessed January 5, 2024. Available at https://ommbid.mhmedical.com/content.aspx?sectionid=225078035&bookid=2709
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)
Monday through Saturday
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.
81270-JAK2 V617
0027U (if appropriate)
| 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
Applies only to results expressed in units of measure originally reported by the performing laboratory. These values do not apply to results that are converted to other units of measure.
|
|---|---|---|
| MP037 | Specimen Type | 31208-2 |
| 42394 | Final Diagnosis | 50398-7 |
| 42395 | PV Reflex Result | 43399-5 |