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Test ID REVE2 Erythrocytosis Evaluation, Blood

Ordering Guidance

Polycythemia vera and acquired causes of erythrocytosis should be excluded before ordering this evaluation.

Necessary Information

Send the following information with the specimen:

-Recent transfusion information

-Most recent complete blood cell count (CBC) results and serum erythropoietin (EPO) levels, if known


Metabolic Hematology Patient Information (T810) is strongly recommended and should include clinical and family history, CBC results, EPO levels, and JAK2 testing results, if known. Testing may proceed without this information; however, it allows for a more complete interpretation.

Specimen Required


Preferred: Lavender top (EDTA)

Acceptable: Yellow top (ACD solution B), green top (sodium heparin)

Specimen Volume: 5 mL

Collection Instructions: Send whole blood specimen in original tube. Do not aliquot.


1. New York Clients-Informed consent is required. Document on the request form or electronic order that a copy is on file. The following documents are available:

-Informed Consent for Genetic Testing (T576)

-Informed Consent for Genetic Testing-Spanish (T826)

2. Metabolic Hematology Patient Information (T810)

3. If not ordering electronically, complete, print, and send a Benign Hematology Test Request (T755) with the specimen.

Useful For

Definitive, comprehensive, and economic evaluation of an individual with JAK2-negative erythrocytosis associated with lifelong sustained increased hemoglobin or hematocrit

Profile Information

Test ID Reporting Name Available Separately Always Performed
REVEI Erythrocytosis Interpretation No Yes
HGBCE Hb Variant, A2 and F Quantitation,B Yes Yes
HPLC HPLC Hb Variant, B No Yes
MASS Hb Variant by Mass Spec, B No Yes

Reflex Tests

Test ID Reporting Name Available Separately Always Performed
SDEX Sickle Solubility, B Yes No
HEMP Hereditary Erythrocytosis Mut, B Yes No
IEF Isoelectric Focusing, B No No
UNHB Hb Stability, B No No
HPFH Hb F Distribution, B No No
ATHAL Alpha-Globin Gene Analysis Yes No
WASQR Alpha Globin Gene Sequencing, B Yes, (Order WASEQ) No
WBSQR Beta Globin Gene Sequencing, B Yes, (Order WBSEQ) No
WBDDR Beta Globin Cluster Locus Del/Dup,B Yes, (WBDD) No
WGSQR Gamma Globin Full Gene Sequencing Yes, (Order WGSEQ) No
BPGMM BPGM Full Gene Sequencing Yes No
REVE0 Erythrocytosis Summary Interp No No
VHLE VHL Gene Erythrocytosis Mutations Yes, (Order VHLZZ) No

Testing Algorithm

This is a consultative evaluation in which the case will be evaluated at Mayo Clinic Laboratories, the appropriate tests will be performed at an additional charge, and the results interpreted.


This profile evaluates for hereditary (congenital) causes of erythrocytosis. Symptoms should be long-standing or familial in nature. All cases will be tested for hemoglobin variants (cation exchange high performance liquid chromatography, capillary electrophoresis, and mass spectrometry) with an interpretative report. Additional testing is guided in a reflexive manner and may include molecular testing of the HBA1/HBA2, HBB, EPOR, VHL, EGLN1(PHD2), EPAS1(HIF2a), and BPGM genes, among others, as appropriate. For more information see Erythrocytosis Evaluation Testing Algorithm.

Note: p50 evaluation is no longer available due to assay issues.


If any of the following molecular tests are performed, an additional consultative interpretation that summarizes all testing will be provided to incorporate subsequent results into an overall evaluation:

-ATHAL / Alpha-Globin Gene Analysis, Varies

-WASQR / Alpha -Globin Gene Sequencing, Blood

-WBSQR / Beta-Globin Gene Sequencing, Blood

-WBDDR / Beta-Globin Cluster Locus Deletion/Duplication, Blood

-WGSQR / Gamma-Globin Full Gene Sequencing, Varies


Additional reflex tests are performed if the hemoglobin testing does not explain the patient's phenotype/hereditary erythrocytosis. Each of the following reflex tests contains an individual interpretative report.

-BPGMM / 2,3-Bisphosphoglycerate Mutase, Full Gene Sequencing Analysis, Varies

-HEMP / Hereditary Erythrocytosis Mutations, Whole Blood

-VHLE / VHL Gene, Erythrocytosis, Mutation Analysis, Varies


For more information, see:

-Myeloproliferative Neoplasm: A Diagnostic Approach to Bone Marrow Evaluation

-Myeloproliferative Neoplasm: A Diagnostic Approach to Peripheral Blood Evaluation

-Benign Hematology Evaluation Comparison

Method Name

REVEI, REVE0: Medical Interpretation

HGBCE: Capillary Electrophoresis

HPLC: Cation Exchange/High Performance Liquid Chromatography (HPLC)

MASS: Mass Spectrometry (MS)

SDEX: Hemoglobin S Solubility

HEMP: Polymerase Chain Reaction (PCR) Amplification/Sanger Sequence Analysis

IEF: Isoelectric Focusing

HPFH: Flow Cytometry

UNHB: Isopropanol and Heat Stability

ATHAL: Alpha-Globin Gene Analysis, Varies

WASQR, WBSQR, WGSQR, BPGMM: Polymerase Chain Reaction (PCR)/Sanger Sequencing

WBDDR: Polymerase Chain Reaction (PCR) Analysis/Multiplex Ligation-Dependent Probe Amplification (MLPA)

VHLE: Polymerase Chain Reaction (PCR) followed by DNA Sequence Analysis

Reporting Name

Erythrocytosis Evaluation

Specimen Type

Whole Blood EDTA

Specimen Minimum Volume

2.5 mL

Specimen Stability Information

Specimen Type Temperature Time Special Container
Whole Blood EDTA Refrigerated 7 days

Reject Due To

Gross hemolysis Reject

Clinical Information

Erythrocytosis (polycythemia) is identified by a sustained increase in hemoglobin or hematocrit. An isolated increase in red blood cell count (in the absence of chronic phlebotomy or coincident iron deficiency) may occur in thalassemia or other causes and does not indicate erythrocytosis. Erythrocytosis may occur as a primary disorder, due to an intrinsic defect of bone marrow stem cells, or secondary in response to increased serum erythropoietin (EPO) levels. Secondary erythrocytosis is associated with a number of disorders, including chronic lung disease, chronic increase in carbon monoxide, cyanotic heart disease, high-altitude living, kidney cysts and tumors, hepatoma, and other EPO-secreting tumors. Rare plasma cell dyscrasia-associated syndromes such as POEMS (polyneuropathy, organomegaly, endocrinopathy, monoclonal protein, skin changes) and TEMPI (telangiectasias, elevated erythropoietin and erythrocytosis, monoclonal gammopathy, perinephric fluid collections, and intrapulmonary shunting) can be associated with increased hemoglobin levels. When these causes of secondary erythrocytosis are excluded, a heritable cause involving hemoglobin or erythrocyte regulatory mechanisms may be present. It is important to differentiate polycythemia vera (PV) from heritable causes of erythrocytosis, the latter of which can be passed to progeny but does not carry the risks of clonal evolution or marrow fibrosis associated with PV.


The most common cause of hereditary erythrocytosis is the presence of a high-oxygen-affinity (HOA) hemoglobin variant. A subset of hemoglobins with increased oxygen (O2) affinity results in clinically evident erythrocytosis caused by decreased O2 unloading at the tissue level. Many are asymptomatic; however, some patients have recurrent headaches, dizziness, fatigue, and restless legs. A subset of patients experience thrombotic episodes. Affected individuals can be plethoric, and many are misclassified as polycythemia vera, particularly prior to more recent genetic testing availability. The oxygen dissociation curve is left-shifted (p50 values are decreased) in HOA variants. Changes to the amino acid sequence of the hemoglobin molecule may distort the protein structure, affecting O2 transport or unloading and the binding of 2,3-bisphosphoglyceric acid (2,3-BPG). 2,3-BPG stabilizes the deoxygenated state of hemoglobin. Therefore, a decrease in the 2,3-BPG concentration results in greater O2 affinity of the normal hemoglobin molecule. Rare cases of erythrocytosis have been associated with a reduction in 2,3-BPG formation. This is due to variants in the converting enzyme, bisphosphoglycerate mutase (BPGM). Truncating variants in the erythropoietin receptor gene, EPOR, have been shown to be a cause of the autosomal dominant primary familial and congenital polycythemia (OMIM 133100).


In addition, oxygen sensing pathway variants, EPAS1(HIF2A) (OMIM 611783); EGLN1(PHD2) (OMIM 609820), and VHL (OMIM 263400) cause hereditary erythrocytosis and a subset are associated with pheochromocytoma and paragangliomas. All have shown an autosomal dominant pattern of inheritance, except VHL-associated erythrocytosis, which is an autosomal recessive disorder. Homozygous VHL R200W alterations have been shown to be causative of Chuvash polycythemia, an endemic heritable erythrocytic disorder first described in Russia but subsequently found in other ethnic groups. The prevalence of causative variants in EPOR and the oxygen sensing pathway genes is unknown; . however, in our experience, they are less prevalent than genetic variants that cause HOA hemoglobin variants and are much less prevalent than polycythemia vera. Because there are many causes of erythrocytosis, an algorithmic and reflexive testing strategy is useful for evaluating these disorders. Initial JAK2 V617F alteration testing and serum EPO levels are useful. Importantly, a significant subset of HOA hemoglobin variants can be electrophoretically silent on multiple routine screening platforms; however, most, and possibly all, HOA hemoglobin variants can be identified with addition of the intact mass spectrometry method. Our extensive experience with these disorders allows an economical, comprehensive evaluation with high sensitivity.

Reference Values

Definitive results and an interpretive report will be provided.


The evaluation includes testing for a hemoglobinopathy. Reflex testing for EPOR, EGLN1 (PHD2), EPAS1 (HIF2a), VHL, and BPGM will be performed as needed.


A hematopathology expert in these disorders will evaluate the case, have the appropriate tests performed, and issue an interpretive report.


An isolated increase in red blood cell count in the setting of normal hemoglobin levels (in the absence of chronic phlebotomy or coincident iron deficiency) may occur in thalassemia or other causes and is not an indication for a thorough erythrocytosis evaluation.

Clinical Reference

1. Patnaik MM, Tefferi A: The complete evaluation of erythrocytosis: congenital and acquired. Leukemia. 2009 May;23(5):834-844

2. McMullin MF: The classification and diagnosis of erythrocytosis. Int J Lab Hematol. 2008 Dec;30(6):447-459

3. Percy MJ, Lee FS: Familial erythrocytosis: molecular links to red blood cell control. Haematologica. 2008 Jul;93(7):963-967

4. Huang LJ, Shen YM, Bulut GB: Advances in understanding the pathogenesis of primary familial and congenital polycythaemia. Br J Haematol. 2010 Mar;148(6):844-852

5. Maran J, Prchal J: Polycythemia and oxygen sensing. Pathol Biol. 2004 Jun;52(5):280-284

6. Lee F: Genetic causes of erythrocytosis and the oxygen-sensing pathway. Blood Rev. 2008 Nov;22(6):321-332

7. Merchant SH, Oliveira JL, Hoyer JD, Viswanatha DS: Erythrocytosis. In: His ED, ed. Hematopathology. 2nd ed. Elsevier Saunders; 2012:722-723

8. Zhuang Z, Yang C, Lorenzo F, et al: Somatic HIF2A gain-of-function mutations in paraganglioma with polycythemia. N Engl J Med. 2012 Sep 6;367(10):922-930

9. Oliveira JL, Coon LM, Frederick LA, et al: Genotype-phenotype correlation of hereditary erythrocytosis mutations, a single center experience. Am J Hematol. 2018 May 23. doi: 10.1002/ajh.25150

10. Gangat N, Oliveira JL, Hoyer JD, Patnaik MM, Pardanani A, Tefferi A. High-oxygen-affinity hemoglobinopathy-associated erythrocytosis: Clinical outcomes and impact of therapy in 41 cases. Am J Hematol. 2021 Dec 1;96(12):1647-1654. doi: 10.1002/ajh.26375

11. Gangat N, Oliveira JL, Porter TR, et al: Erythrocytosis associated with EPAS1(HIF2A), EGLN1(PHD2), VHL, EPOR or BPGM mutations: the Mayo Clinic experience. Haematologica. 2022 May;107(5):1201-1204. doi: 10.3324/haematol.2021.280516

Method Description

Hemoglobin Electrophoresis:

The CAPILLARYS System is an automated system that uses capillary electrophoresis to separate charged molecules by their electrophoretic mobility in an alkaline buffer. Separation occurs according to the electrolyte pH and electro-osmotic flow. A sample dilution with a hemolyzing solution is injected by aspiration. A high-voltage protein separation occurs, and direct detection of the hemoglobin protein fractions is at 415 nm, which is specific to hemoglobins. The resulting electropherogram peaks are evaluated for pattern abnormalities and are quantified as a percentage of the total hemoglobin present. Examples of position of commonly found hemoglobin fractions are, from cathode to anode: Hb A2', C, A2/O-Arab, E, S, D, G-Philadelphia, F, A, Hope, Bart, J, N-Baltimore, and H.(Louahabi A, Philippe M, Lali S, Wallemacq P, Maisin D: Evaluation of a new Sebia kit for analysis of hemoglobin fractions and variants on the Capillarys system. Clin Chem Lab Med. 2006;44[3]:340-345; instruction manual: CAPILLARYS Hemoglobin(E) using the CAPILLARYS 2 flex-piercing instrument. Sebia; 06/2014)


High Performance Liquid Chromatography:

Hemolysate of whole blood is injected into an analysis stream passing through a cation exchange column using high-performance liquid chromatography. A preprogrammed gradient controls the elution buffer mixture that also passes through the analytical cartridge. The ionic strength of the elution buffer is raised by increasing the percentage of a second buffer. As the ionic strength of the buffer increases the more strongly retained hemoglobins elute from the cartridge. Absorbance changes are detected by a dual-wavelength filter photometer. Changes in absorbance are displayed as a chromatogram of absorbance versus time.(Huismann TH, Scroeder WA, Brodie AN, Mayson SM, Jakway J: Microchromotography of hemoglobins. III. A simplified procedure for the determination of hemoglobin A2. J Lab Clin Med. 1975;86:700-702; Ou CN, Buffone GJ, Reimer GL, Alpert AJ: High-performance liquid chromatography of human hemoglobins on a new cation exchanger. J Chromatogr. 1983;266:197-205; Szuberski J, Oliveira JL, Hoyer JD: A comprehensive analysis of hemoglobin variants by high-performance liquid chromatography (HPLC). Int J Lab Hematol. 2012 Dec; 34(6):594-604; instruction manual: Bio-Rad Variant II Beta-thalassemia Short Program Instructions for Use, L70203705. Bio-Rad Laboratories, Inc; 11/2011)


Hemoglobin Variant by Mass Spectrometry

Mass spectrometry (MS) is performed using a quadrupole time-of-flight MS (QTOF-MS), and results are analyzed with Agilent MassHunter software. Whole blood is diluted 1:50 with purified water, and cell debris removed by centrifugation. The supernatant is then diluted 1:10 with running buffer (1:1 water:acetonitrile, 1% formic acid) and analyzed on a Q-TOF MS in MS mode using flow injection. A calculated mass for each variant has been integrated into a database containing historical data of multiple method measurements, and empiric MS mass peaks were used as a search criterion.(Zanella-Cleon I, Joly P, Becchi M, Francina A: Phenotype determination of hemoglobinopathies by mass spectrometry. Clin Biochem. 2009;42[18]:1807-1817; Helmich F, van Dongen JL, Kuijper PH, Scharnhorst V, Brunsveld L, Broeren MA: Rapid phenotype hemoglobin screening by high-resolution mass spectrometry on intact proteins. Clin Chim Acta. 2016 Sep 1;460:220-226. doi: 10.1016/j.cca.2016.07.006)

Day(s) Performed

Monday through Saturday

Report Available

3 to 25 days

Test Classification

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

CPT Code Information





83068 (if appropriate)

82664 (if appropriate)

88184 (if appropriate)

LOINC Code Information

Test ID Test Order Name Order LOINC Value
REVE2 Erythrocytosis Evaluation 43113-0


Result ID Test Result Name Result LOINC Value
60286 Hb Variant by Mass Spec, B No LOINC Needed
41927 Hb A 20572-4
65615 HPLC Hb Variant, B No LOINC Needed
608426 Erythrocytosis Interpretation 59466-3
608440 Reviewed By 18771-6
41928 Hb F 32682-7
41929 Hb A2 4552-6
41930 Variant 1 24469-9
41931 Variant 2 24469-9
41932 Variant 3 24469-9
41933 HGBCE Interpretation 78748-1