Test Id : REVE1

This is a consultative evaluation in which the case will be evaluated at Mayo Clinic Laboratories, the appropriate tests 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 p50 (if shipping control is received) and 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. See Erythrocytosis Evaluation Testing Algorithm in Special Instructions.

An additional consultative interpretation that summarizes all testing, will be provided after test completion to incorporate subsequent results into an overall evaluation if any of the following molecular tests are reflexed:

-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

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

-HEMP / Hereditary Erythrocytosis Mutations, Whole Blood

-VHLE / VHL Gene, Erythrocytosis Mutation Analysis, Varies

The following are available in Special Instructions:

-Myeloproliferative Neoplasm: A Diagnostic Approach to Bone Marrow Evaluation

-Myeloproliferative Neoplasm: A Diagnostic Approach to Peripheral Blood Evaluation

-Benign Hematology Evaluation Comparison

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

All 3 specimens must arrive within 72 hours of collection.

Include recent transfusion information.

A total of 3 specimens are required to perform this profile. The following specimens are required for testing:

-Whole blood EDTA

-Whole blood sodium heparin for P50*

-Normal shipping control: Whole blood sodium heparin for P50*

*Note: If no sodium heparin patient or control specimens are received, the P50 test cannot be performed.

Patient:

Container/Tube: Lavender top (EDTA) and green top (heparin)

Specimen Volume:

EDTA: 5 mL

Heparin: 4 mL

Collection Instructions:

1. Immediately refrigerate specimens after collection.

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

3. Rubber band patient specimen and control vial together.

Normal Shipping Control:

Container/Tube: Green top (heparin)

Specimen Volume: 4 mL

Collection Instructions:

1. Collect a control specimen from a normal (healthy), unrelated, nonsmoking person at the same time as the patient.

2. Label clearly on outermost label normal control.

3. Immediately refrigerate specimen after collection.

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

5. Rubber band patient specimen and control vial together.

• Informed Consent for Genetic Testing
• Myeloproliferative Neoplasm: A Diagnostic Approach to Peripheral Blood Evaluation
• Myeloproliferative Neoplasm: A Diagnostic Approach to Bone Marrow Evaluation
• Erythrocytosis Evaluation Testing Algorithm
• Metabolic Hematology Patient Information
• Benign Hematology Evaluation Comparison
• Informed Consent for Genetic Testing (Spanish)

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 in Special Instructions:

-Informed Consent for Genetic Testing (T576)

-Informed Consent for Genetic Testing-Spanish (T826)

2. Metabolic Hematology Patient Information (T810) is available in Special Instructions.

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

EDTA blood: 2.5 mL

Heparin blood: 1 mL

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

This is a consultative evaluation in which the case will be evaluated at Mayo Clinic Laboratories, the appropriate tests 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 p50 (if shipping control is received) and 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. See Erythrocytosis Evaluation Testing Algorithm in Special Instructions.

An additional consultative interpretation that summarizes all testing, will be provided after test completion to incorporate subsequent results into an overall evaluation if any of the following molecular tests are reflexed:

-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

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

-HEMP / Hereditary Erythrocytosis Mutations, Whole Blood

-VHLE / VHL Gene, Erythrocytosis Mutation Analysis, Varies

The following are available in Special Instructions:

-Myeloproliferative Neoplasm: A Diagnostic Approach to Bone Marrow Evaluation

-Myeloproliferative Neoplasm: A Diagnostic Approach to Peripheral Blood Evaluation

-Benign Hematology Evaluation Comparison

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, renal cysts and tumors, hepatoma, and other EPO-secreting tumors. When these common 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 do not carry the risks of clonal evolution or marrow fibrosis associated with PV.

The most common cause of hereditary erythrocytosis is the presence of high-oxygen-affinity (HOA) hemoglobin. A subset of hemoglobins with increased oxygen (O2) affinity result 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 minor subset of patients has thrombotic episodes. Affected individuals can be plethoric and many are misclassified as polycythemia vera. The oxygen dissociation curve is left-shifted (p50 values are decreased). 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. A few cases of erythrocytosis have been associated with a reduction in 2,3-BPG formation. This is most commonly due to variants in the converting enzyme, bisphosphoglycerate mutase. 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, but 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 important with p50 results further stratifying JAK2-negative cases. Importantly, a significant subset of HOA hemoglobin variants can be electrophoretically silent on multiple routine screening platforms; however, most, if not all, of HOA hemoglobin variants can be identified with addition of the mass spectrometry method. Our extensive experience with these disorders allows an economical, comprehensive evaluation with high sensitivity.

Definitive results and an interpretive report will be provided.

The evaluation includes testing for a hemoglobinopathy and oxygen (O2) affinity of the hemoglobin molecule. An increase in O2 affinity is demonstrated by a shift to the left in the O2 dissociation curve (decreased p50 result). Reflex testing for EPOR, EGLN1 (PHD2), EPAS1 (HIF2a), VHL, and BPGM will be performed as needed.

A hematopathologist expert in these disorders will evaluate the case, appropriate tests are performed, and an interpretive report is issued.

The shipping control specimen is required to adequately interpret the oxygen dissociation result, as temperature extremes can impact the integrity of the specimen.

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.

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;30:447-459

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;52:280-284

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

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

• Informed Consent for Genetic Testing
• Myeloproliferative Neoplasm: A Diagnostic Approach to Peripheral Blood Evaluation
• Myeloproliferative Neoplasm: A Diagnostic Approach to Bone Marrow Evaluation
• Erythrocytosis Evaluation Testing Algorithm
• Metabolic Hematology Patient Information
• Benign Hematology Evaluation Comparison
• Informed Consent for Genetic Testing (Spanish)

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 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 electrophoregrams 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 (HPLC). 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; instruction manual: Bio-Rad Variant II Beta-thalassemia Short Program Instructions for Use, L70203705. Bio-Rad Laboratories, Inc; 11/2011)

Oxygen Dissociation, P50:

The operating principle of the Hemox Analyzer is based on dual wave-length spectrophotometry for the measurement of the oxygen saturation of hemoglobin (in percent) and a Clark electrode for measuring the oxygen partial pressure in millimeters of mercury. The resulting output signals from both measuring systems are fed into a computer and analyzed.(Guarnone R, Centenara E, Barosi G: Performance characteristics of Hemox-Analyzer for assessment of the hemoglobin dissociation curve. Haematologica. 1995;80:426-430; Vanhille DL, Nussenzveig RH, Glezos C, Perkins S, Agarwal AM: Best practices for use of the HEMOX analyzer in the clinical laboratory: quality control determination and choice of anticoagulant. Lab Hematol. 2012 Sep;18[3]):17-19. doi: 10.1532/LH96.12001)

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 historic 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)

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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.

83020-26-Erythrocytosis Interpretation

83020-Hemoglobin Electrophoresis

83021-HPLC Hb Variant

82820-Hemoglobin O2 affinity (p50)

83789-Hemoglobin Variant by Mass Spectroscopy (MS), Blood

83068 (if appropriate)

82664 (if appropriate)

88184 (if appropriate)

Sample Reports

Normal and Abnormal sample reports are provided as references for report appearance.

Normal Reports | Abnormal Reports

SI Sample Reports

International System (SI) of Unit reports are provided for a limited number of tests. These reports are intended for international account use and are only available through MayoLINK accounts that have been defined to receive them.

SI Normal Reports | SI Abnormal Reports