Diagnosis of methemoglobinemia and sulfhemoglobinemia and possible hereditary (congenital) causes
Differentiation of methemoglobinemia and sulfhemoglobinemia from other causes of cyanosis (eg, congenital heart disease)
| Test Id | Reporting Name | Available Separately | Always Performed |
|---|---|---|---|
| MEVI | Methemoglobinemia Interpretation | No | Yes |
| HGBCE | Hb Variant, A2 and F Quantitation,B | Yes | Yes |
| HPLC | HPLC Hb Variant, B | No | Yes |
| METH | Methemoglobin, B | Yes, (Order MET) | Yes |
| SULF | Sulfhemoglobin, B | Yes, (Order MET) | Yes |
| METR1 | Cytochrome b5 Reductase, B | Yes | Yes |
| Test Id | Reporting Name | Available Separately | Always Performed |
|---|---|---|---|
| SDEX | Sickle Solubility, B | Yes | No |
| IEF | Isoelectric Focusing, B | No | No |
| MASS | Hb Variant by Mass Spec, 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, (Order WBDD) | No |
| WGSQR | Gamma Globin Full Gene Sequencing | Yes, (Order WGSEQ) | No |
| MEV0 | Methemoglobin Summary Interp | No | No |
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 is an evaluation for methemoglobin and sulfhemoglobin levels and possible hereditary causes. Methemoglobin, sulfhemoglobin levels, cytochrome-b5 reductase (methemoglobin reductase) activity, and protein analysis screening for hemoglobin variants (capillary electrophoresis, cation exchange high performance liquid chromatography and capillary electrophoresis) will always be performed. If additional hemoglobin variant confirmatory testing is required, appropriate reflex testing will be performed. This will vary from additional protein analysis methods to molecular testing, as needed.
One or more of the following molecular tests may be 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
After all test results are finalized, an additional consultative interpretation that summarizes all testing and incorporates subsequent genetic results will be provided.
See Benign Hematology Evaluation Comparison in Special Instructions.
MEVI, MEV0: Medical Interpretation
HGBCE: Capillary Electrophoresis
HPLC: Cation Exchange/High-Performance Liquid Chromatography (HPLC)
METH, SULF: Spectrophotometry
METR1: Kinetic Spectrophotometry
IEF: Isoelectric Focusing
HPFH: Flow Cytometry
UNHB: Isopropanol and Heat Stability
MASS: Mass Spectrometry (MS)
Cb5r deficiency
Congenital methaemoglobinemia
Congenital methemoglobinemia
cytochrome b5 reductase deficiency
Dominant congenital methemoglobinemia
Hemoglobin M
M Hemoglobins
Methaemoglobinemia
Methemoglobin Evaluation
NADH cytochrome b5 reductase 3
NADH methemoglobin reductase deficiency
RCM
Recessive congenital methemoglobinemia
Sulfhemoglobinemia
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 is an evaluation for methemoglobin and sulfhemoglobin levels and possible hereditary causes. Methemoglobin, sulfhemoglobin levels, cytochrome-b5 reductase (methemoglobin reductase) activity, and protein analysis screening for hemoglobin variants (capillary electrophoresis, cation exchange high performance liquid chromatography and capillary electrophoresis) will always be performed. If additional hemoglobin variant confirmatory testing is required, appropriate reflex testing will be performed. This will vary from additional protein analysis methods to molecular testing, as needed.
One or more of the following molecular tests may be 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
After all test results are finalized, an additional consultative interpretation that summarizes all testing and incorporates subsequent genetic results will be provided.
See Benign Hematology Evaluation Comparison in Special Instructions.
Whole Blood ACD-B
Whole Blood EDTA
Specimen must arrive within 3 days (72 hours) of collection.
Include recent transfusion information.
Include most recent complete blood cell count results.
Metabolic Hematology Patient Information (T810) is strongly recommended. Testing may proceed without this information, however if the information requested is received, any pertinent reported clinical features and data will drive the focus of the evaluation and be considered in the interpretation.
The laboratory has extensive experience in hemoglobin variant identification and many cases can be confidently classified without molecular testing. However, molecular confirmation is always available, subject to sufficient sample quantity (eg, multiplex ligation-dependent probe amplification testing requires at least 2 mL of sample in addition to protein testing requirements). If no molecular testing or specific molecular tests are desired, utilize the appropriate check boxes on the form. If the form or other communication is not received, the reviewing hematopathologist will select appropriate tests to sufficiently explain the protein findings, which may or may not include molecular testing.
The following specimens are required for testing:
Whole blood ACD-B specimen
2 Whole blood EDTA specimens
Container/Tube: Lavender top (EDTA) and yellow top (ACD solution B)
Specimen Volume:
EDTA: Two 4-mL tubes
ACD: One 6-mL tube
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 in Special Instructions:
-Informed Consent for Genetic Testing (T576)
-Informed Consent for Genetic Testing-Spanish (T826)
2. Metabolic Hematology Patient Information (T810) in Special Instructions
3. If not ordering electronically, complete, print, and send a Benign Hematology Test Request (T755) with the specimen
| Gross hemolysis | Reject |
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| Whole Blood ACD-B | Refrigerated | 72 hours | |
| Whole Blood EDTA | Refrigerated | 72 hours |
Diagnosis of methemoglobinemia and sulfhemoglobinemia and possible hereditary (congenital) causes
Differentiation of methemoglobinemia and sulfhemoglobinemia from other causes of cyanosis (eg, congenital heart disease)
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 is an evaluation for methemoglobin and sulfhemoglobin levels and possible hereditary causes. Methemoglobin, sulfhemoglobin levels, cytochrome-b5 reductase (methemoglobin reductase) activity, and protein analysis screening for hemoglobin variants (capillary electrophoresis, cation exchange high performance liquid chromatography and capillary electrophoresis) will always be performed. If additional hemoglobin variant confirmatory testing is required, appropriate reflex testing will be performed. This will vary from additional protein analysis methods to molecular testing, as needed.
One or more of the following molecular tests may be 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
After all test results are finalized, an additional consultative interpretation that summarizes all testing and incorporates subsequent genetic results will be provided.
See Benign Hematology Evaluation Comparison in Special Instructions.
Methemoglobin:
Methemoglobin forms when the hemoglobin (Hb) molecule iron is in the ferric (Fe3[+]) form instead of the functional ferrous (Fe2[+]) form. Methemoglobinemia can be hereditary or acquired and is present by definition when methemoglobin levels are greater than the normal range. Acquired methemoglobinemia results after toxic exposure to nitrates and nitrites/nitrates (fertilizer, nitric oxide), topical anesthetics (“caines"), dapsone, naphthalene (moth balls/toilet deodorant cakes), and industrial use of aromatic compounds (aniline dyes).
Congenital methemoglobinemias are rare. They are due either to:
-A deficiency of cytochrome b5 reductase (methemoglobin reductase) in erythrocytes, an autosomal recessive disorder resulting from genetic variants in either CYB5R3 or CYB5A.(1,2) Type IV is thought to be extraordinarily rare. Type III is no longer a category.
-One of several intrinsic structural disorders of Hb, called M-Hbs; all of which are inherited in an autosomal dominant manner.(3,4) Classically, M-Hbs result from histidine-to tyrosine substitutions at the proximal or distal histidine important in coordinating the oxygen molecule. These include alpha-, beta- and gamma-chain variants. Rarely, other substitutions outside the proximal and distal histidine location can cause Hb variants that increase methemoglobin or sulfhemoglobin levels. Most M-Hb variants are readily identified by high performance liquid chromatography (HPLC) or mass spectrometry methods with characteristic electrophoresis patterns; however, some require more specialized techniques. Most are associated with increased methemoglobin with or without an increase in sulfhemoglobin. Alpha chain M-Hb variants can be associated with increased sulfhemoglobin without an increase in methemoglobin.
Sulfhemoglobin:
Sulfhemoglobin cannot combine with oxygen. When acquired, sulfhemoglobinemia can be associated with cyanosis and often accompanies methemoglobinemia. Sulfhemoglobinemia has been associated with exposure to sumatriptan, sulfonamides, metoclopramide, paint or varnish vapors, dimethyl sulfoxide, acetanilide, phenacetin, trinitroluene, zinc ethylene bisdithiocarbamate (a fungicide), and flutamide. It is important to note that some Hb variants are known to interfere with this test (especially M-Hbs) and sulfhemoglobin absorbance can be increased due to the Hb variant. Hb evaluation that includes the HPLC method is recommended to exclude this possibility.
In contrast to methemoglobinemia, sulfhemoglobinemia persists until the erythrocytes containing it are destroyed. Therefore, blood level of sulfhemoglobin declines gradually over a period of weeks.
Definitive results and an interpretive report will be provided.
This is a consultative evaluation in which the history and previous laboratory values are reviewed by a hematologist who is an expert on these disorders. Appropriate tests are performed, and an interpretive report is issued.
Sulfhemoglobin is exceedingly stable and does not change in stored or shipped specimens.
Methemoglobin is unstable and can degrade at a rate of about 40% per 24 hours.
A normal methemoglobin value obtained with stored or shipped specimens does not exclude prior methemoglobinemia of minimal degree. However, significant methemoglobinemia will still be demonstrable.
1. OMIM: 250800 Methemoglobinemia due to deficiency of methemoglobin reductase. Updated May 20, 2019. Accessed October 19, 2020. Available at www.omim.org/entry/250800?search=250800&highlight=250800
2. OMIM: 250790 Methemoglobinemia and ambiguous genitalia. Updated May 18, 2018. Accessed October 19, 2020. Available at www.omim.org/entry/250790?search=250790&highlight=250790
3. OMIM: 141800 Hemoglobin alpha locus 1; HBA1. Updated November 1, 2019. Accessed October 19, 2020. Available at www.omim.org/entry/141800?search=141800&highlight=141800
4. OMIM: 141900 Hemoglobin beta locus; HBB. Updated November 14, 2019. Accessed October 19, 2020. Available at www.omim.org/entry/141900?search=141900&highlight=141900
5. Haymond S, Cariappa R, Eby CS, Scott MG: Laboratory assessment of oxygenation in methemoglobinemia. Clin Chem. 2005;51(2):434-444
6. Noor M, Beutler E: Acquired sulfhemoglobinemia. An underreported diagnosis? West J Med. 1998;169(6):386-389
7. Thom CS, Dickson CF, Gell DA, Weiss MJ: Hemoglobin variants: biochemical properties and clinical correlates. Cold Spring Harb Perspect Med. 2013;3(3):a011858
8. Percy MJ, McFerran NV, Lappin TR: Disorders of oxidized haemoglobin. Blood Rev. 2005;19(2):61-68
9. Agarwal AM, Prchal JT. Methemoglobinemia and Other Dyshemoglobinemias. In: Kaushansky K, Lichtman MA, Prchal JT, Levi MM, Press OW, Burns LJ, Caligiuri M, eds. Williams Hematology. 9th ed. McGraw-Hill; 2016: 789-800
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 (Hb) protein fractions is at 415 nm, which is specific to Hbs. The resulting electrophoregrams peaks are evaluated for pattern abnormalities and are quantified as a percentage of the total Hb present. Examples of position of commonly found Hb 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 Hemoglobin Variant:
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 Hbs 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)
Methemoglobin:
The normal absorption spectrum of oxyhemoglobin has very little optical density above 600 nm. The absorption spectrum of methemoglobin exhibits a small, characteristic peak at 630 nm. This peak is abolished as methemoglobin is converted to cyanmethemoglobin upon addition of potassium cyanide, and the drop in optical density is proportional to methemoglobin concentration.(Evelyn KA, Malloy HT: Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood. J Biol Chem. 1938;126:655-662; Fairbanks VF, Klee GG: Biochemical aspects of hematology. In: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. WB Saunders Company; 1999: 1676-1678; Robertson LD, Roper D: Laboratory methods used in the investigation of haemolytic anaemias. In: Bain BJ, Bates I, Laffan MA, eds. Dacie and Lewis Practical Haematology. 12th ed. Elsevier; 2017:214-227)
Sulfhemoglobin:
The normal absorption spectrum of oxyhemoglobin has very little optical density above 600 nm. However, if certain poorly defined Hb denaturation products are present in a hemolysate, there is a broad elevation of the absorption curve in the range of 600 to 620 nm. This sulfhemoglobin plateau is not affected by treatment with cyanide. Sulfhemoglobin is not available, nor can it be prepared, in a pure form for preparation of a sulfhemoglobin standard. In calculating sulfhemoglobin concentration, the factor for sulfhemoglobin quantitation is based on studies of Carrico et al.(Evelyn KA, Malloy HT: Microdetermination of oxyhemoglobin, methemoglobin, and sulfhemoglobin in a single sample of blood. J Biol Chem. 1938;126:655-662; Carrico RJ, Peisach J, Alben JO: The preparation and some physical properties of sulfhemoglobin. J Analyt Biochem. 1978;253:2386-2391; Fairbanks VF, Klee GG: Biochemical aspects of hematology. In: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. WB Saunders Company; 1999: 1676-1678; Robertson LD, Roper D: Laboratory methods used in the investigation of haemolytic anaemias. In: Bain BJ, Bates I, Laffan MA, eds. Dacie and Lewis Practical Haematology. 12th ed. Elsevier; 2017:214-227)
Cytochrome b5 Reductase:
Cytochrome B5 reductase (methemoglobin reductase) catalyzes the 1,4-dihydronicotinamide adenine dinucleotide (NADH)-linked reduction of several substrates, including ferricyanide. The activity at 30 degrees C is followed spectrophotometrically by measuring the oxidation of NADH at 340 nm.(Fairbank VF, Klee GG: Biochemical aspects of hematology. In: Burtis CA, Ashwood ER, eds. Tietz Textbook of Clinical Chemistry. 3rd ed. WB Saunders Company; 1999: 1647-1648; van Solinge WW, van Wijk: Enzymes of the red blood cell. In: Rifai N, Horvath AR, Wittwer CT: eds. Tietz Textbook of Clinical Chemistry and Molecular Diagnostics. 6th ed. Elsevier; 2018:chap 30)
Monday through Saturday
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-Hemoglobinopathy Interpretation
83020-Hb Variant, A2 and F Quantitation
83021-HPLC Hb Variant
82657-Methemoglobin reductase
83050-Methemoglobin, quantitative
83060-Sulfhemoglobin, quantitative
82664 (if appropriate)
83068 (if appropriate)
83789 (if appropriate)
88184 (if appropriate)
| Test Id | Test Order Name | Order LOINC Value |
|---|---|---|
| MEV1 | Methemoglobinemia Evaluation | 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.
|
|---|---|---|
| 8268 | Methemoglobin, B | 2614-6 |
| 8272 | Sulfhemoglobin, B | 4685-4 |
| 41927 | Hb A | 20572-4 |
| 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 |
| 65615 | HPLC Hb Variant, B | No LOINC Needed |
| METRB | Cytochrome b5 Reductase, B | 32703-1 |
| 608086 | Methemoglobinemia Interpretation | 59465-5 |
| 608108 | Reviewed By | 18771-6 |