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HelpTest ID: HAEV1 Hemolytic Anemia Evaluation, Blood
Ordering Guidance
Preliminary screening tests, such as complete blood cell count with peripheral smear and direct Coombs test with a negative result, should be run before ordering this evaluation.
Cold agglutinin disorders and autoimmune disorders should be excluded prior to testing. This evaluation is not suitable for acquired causes of hemolysis.
Shipping Instructions
Specimens must arrive within 72 hours of collection.
Necessary Information
At minimum, include recent transfusion information and 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 specimen 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.
Specimen Required
The following specimens are required for testing:
2 Whole blood EDTA specimens
2 Whole blood ACD specimens
1 EDTA control specimen
2 Well-made peripheral blood smears (Wright stained or fixed in absolute methanol)
Patient:
Specimen Type: Whole blood
Container/Tube: Lavender top (EDTA) and yellow top (ACD)
Specimen Volume:
EDTA: Two 4 mL tubes
ACD: Two 6 mL tubes
Collection Instructions:
1. Immediately refrigerate specimens after collection.
2. Send whole blood specimens in original tubes. Do not aliquot.
3. Rubber band patient specimen and control vial together.
Specimen Type: Slides
Container/Tube: Blood smears
Specimen Volume: 2 Peripheral blood smears
1. Prepare 2 peripheral blood smears from 1 of the EDTA tubes collected from the patient
2. Either stain the smear with Wright stain or fix the smear with absolute methanol prior to shipping.
Normal Shipping Control:
Specimen Type: Whole blood
Container/Tube: Lavender top (EDTA)
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. Clearly hand write normal control on the outermost label.
3. Immediately refrigerate specimen after collection.
4. Send specimen in original tube. Do not aliquot.
5. Rubber band patient specimen and control vial together.
Forms
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
Evaluation of lifelong or inherited hemolytic anemias, including red blood cell membrane disorders, unstable or abnormal hemoglobin variants, and red blood cell enzyme disorders
This evaluation is not suitable for acquired causes of hemolysis.
Profile Information
| Test ID | Reporting Name | Available Separately | Always Performed |
|---|---|---|---|
| HAEVI | Hemolytic Anemia Interpretation | No | Yes |
| HGBCE | Hb Variant, A2 and F Quantitation,B | Yes | Yes |
| HPLC | HPLC Hb Variant, B | No | Yes |
| UNHB | Hb Stability, B | No | Yes |
| FRAGO | Osmotic Fragility | Yes, (Order FRAG) | Yes |
| SCTRL | Shipping Control Vial | No | Yes |
| BND3 | Band 3 Fluorescence Staining, RBC | No | Yes |
| G6PDC | G6PD Enzyme Activity, B | Yes, (Order G6PD1) | Yes |
| PKC | PK Enzyme Activity, B | Yes, (Order PK1) | Yes |
| GPIC | Glucose Phosphate Isomerase, B | Yes, (Order GPI1) | Yes |
| HKC | Hexokinase, B | Yes, (Order HK1) | Yes |
| AKC | Adenylate Kinase, B | Yes, (Order AK1) | Yes |
| PFKC | Phosphofructokinase, B | Yes, (Order PFK1) | Yes |
| PGKC | Phosphoglycerate Kinase, B | Yes, (Order PGK1) | Yes |
| TPIC | Triosephosphate Isomerase, B | Yes, (Order TPI1) | Yes |
| GSH | Glutathione, B | Yes | Yes |
| P5NT | Pyrimidine 5' Nucleotidase, B | Yes | Yes |
| PBSM | Morphology Review | No | Yes |
Reflex Tests
| 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 |
| HPFH | Hb F Distribution, B | No | No |
| WASQR | Alpha Globin Gene Sequencing, B | Yes, (Order WASEQ) | No |
| WBSQR | Beta Globin Gene Sequencing, B | Yes, (Order WBSEQ) | No |
| WGSQR | Gamma Globin Full Gene Sequencing | Yes, (Order WGSEQ) | No |
| HAEV0 | Hemolytic Anemia Summary Interp | No | No |
| WAGDR | Alpha Globin Clustr Locus Del/Dup,B | Yes, (Order AGDD) | No |
| WBGDR | Beta Globin Gene Cluster, Del/Dup,B | Yes, (Order WBGDD) | No |
Testing Algorithm
This is a consultative evaluation in which the case will be evaluated, the appropriate tests performed at an additional charge, and the results interpreted. If a peripheral blood smear is provided, the morphologic features will be incorporated into the interpretation.
Red blood cell enzymes will always be performed. Capillary electrophoresis, cation exchange high-performance liquid chromatography, and hemoglobin stability studies will always be performed. Reflex testing required to positively identify a hemoglobin abnormality may be added as the case requires. Osmotic fragility (OF) and eosin-5-maleimide binding band 3 flow cytometry will be performed on all cases. A normal shipping control for OF is necessary to exclude false-positive results due to preanalytic artifact.
The protein and molecular test results will be reported separately, which may result in incomplete data until all testing has been finalized.
One or more of the following molecular tests may be reflexed on this test:
-WAGDR / Alpha Globin Cluster Locus Deletion/Duplication, Blood
-WASQR / Alpha Globin Gene Sequencing, Blood
-WBSQR / Beta-Globin Gene Sequencing, Blood
-WBGDR / Beta-Globin Gene Cluster Deletion/Duplication, Blood
-WGSQR / Gamma-Globin Full Gene Sequencing, Varies
An additional comprehensive consultative interpretation that summarizes all results will be provided after all tests are completed to incorporate results into an overall evaluation.
For more information see Hereditary Hemolytic Anemia Evaluation Testing Algorithm
Special Instructions
Method Name
HAEVI: Medical Interpretation
HGBCE: Capillary Electrophoresis
HPLC: Cation Exchange/High-Performance Liquid Chromatography (HPLC)
UNHB: Isopropanol and Heat Stability
FRAGO, SCTRL: Osmotic Lysis
BND3: Flow Cytometry
G6PDC, PKC, GPIC, HKC, AKC, PFKC, PGKC, TPIC, GSH, P5NT: Kinetic Spectrophotometry (KS)
PBSM: Consultant Review
MASS: Mass Spectrometry (MS)
HPFH: Flow Cytometry
IEF: Isoelectric Focusing
Reporting Name
Hemolytic Anemia EvaluationSpecimen Type
ControlWhole Blood ACD-B
Whole Blood EDTA
Whole Blood Slide
Specimen Minimum Volume
EDTA Whole blood: 3 mL; ACD Whole blood: 5 mL
Specimen Stability Information
| Specimen Type | Temperature | Time | Special Container |
|---|---|---|---|
| Control | Refrigerated | 72 hours | PURPLE OR PINK TOP/EDTA |
| Whole Blood ACD-B | Refrigerated | 72 hours | |
| Whole Blood EDTA | Refrigerated | 72 hours | |
| Whole Blood Slide | Refrigerated | CARTRIDGE | |
Clinical Information
Hemolytic anemia (HA) is characterized by increased red blood cell (RBC) destruction and a decreased RBC life span. Patients usually have decreased hemoglobin concentration, hematocrit, and RBC count, but some can have compensated disorders, and symptoms such as reticulocytosis, pigmented gallstones, and decreased haptoglobin are factors that raise clinical suspicion. Blood smear abnormalities may include variable amounts of poikilocytosis including spherocytes, elliptocytes, schistocytes, stomatocytes, echinocytes, polychromasia, basophilic stippling, and target cells. Osmotic fragility can be increased due to the presence of spherocytes. These are all nonspecific features that can be present in both hereditary and acquired hemolytic disorders.
Inherited hemolytic disorders may include RBC membrane disorders, RBC enzyme defects, or abnormalities in the hemoglobin molecule in the RBC. This panel assesses for possible causes of congenital/hereditary causes of HA and does not evaluate for acquired causes. Therefore, the anemia should be lifelong or familial in nature. Examples of acquired HA include autoimmune HA (Coombs-positive HA, Coombs-negative autoimmune HA), cold agglutinin disease, paroxysmal nocturnal hemoglobinuria, paroxysmal cold hemoglobinuria, mechanical hemolysis (aortic stenosis or prosthetic heart valves), disseminated intravascular coagulation/thrombotic microangiopathy, and drug-induced HA.
This consultation evaluates for a hereditary cause of increased RBC destruction and includes testing for RBC membrane disorders, such as hereditary spherocytosis and hereditary pyropoikilocytosis, hemoglobinopathies, and red blood cell enzyme abnormalities.
This panel is of limited use in patients with a history of recent transfusion and should be ordered as remote a date from transfusion as possible in those patients who are chronically transfused.
Reference Values
Hemoglobin Variant, A2 and F Quantitation
HEMOGLOBIN A
0-30 days: 5.9-77.2%
1-2 months: 7.9-92.4%
3-5 months: 54.7-97.1%
6-8 months: 80.0-98.0%
9-12 months: 86.2-98.0%
13-17 months: 88.8-98.0%
18-23 months: 90.4-98.0%
≥24 months: 95.8-98.0%
HEMOGLOBIN A2
0-30 days: 0.0-2.1%
1-2 months: 0.0-2.6%
3-5 months: 1.3-3.1%
≥6 months: 2.0-3.3%
HEMOGLOBIN F
0-30 days: 22.8-92.0%
1-2 months: 7.6-89.8%
3-5 months: 1.6-42.2%
6-8 months: 0.0-16.7%
9-12 months: 0.0-10.5%
13-17 months: 0.0-7.9%
18-23 months: 0.0-6.3%
≥24 months: 0.0-0.9%
VARIANT 1
0.0
VARIANT 2
0.0
VARIANT 3
0.0
Hemoglobin Stability
Normal (reported as normal [stable] or abnormal [unstable])
OSMOTIC FRAGILITY
≥12 months:
0.50 g/dL NaCl (unincubated): 3-53% hemolysis
0.60 g/dL NaCl (incubated): 14-74% hemolysis
0.65 g/dL NaCl (incubated): 4-40% hemolysis
0.75 g/dL NaCl (incubated): 1-11% hemolysis
NaCl = sodium chloride
Reference values have not been established for patients who are younger than 12 months of age.
BAND 3 FLUORESCENCE STAINING RED BLOOD CELLS(RBC)
≥12 months: Normal (reported as normal, decreased, or equivocal)
Reference values have not been established for patients who are younger than 12 months of age.
Glucose 6 Phosphate Dehydrogenase Enzyme Activity
≥12 months of age: 8.0-11.9 U/g Hb
Reference values have not been established for patients who are younger than 12 months of age.
Pyruvate Kinase Enzyme Activity
≥12 months of age: 5.5-12.4 U/g Hb
Reference values have not been established for patients who are younger than 12 months of age.
Glucose Phosphate Isomerase Enzyme Activity
≥12 months of age: 40.0-58.0 U/g Hb
Reference values have not been established for patients who are younger than 12 months of age.
Hexokinase Enzyme Activity
≥12 months: 0.7-1.7 U/g Hb
Reference values have not been established for patients who are younger than 12 months of age.
Adenylate Kinase Enzyme Activity
≥12 months: 195-276 U/g Hb
Reference values have not been established for patients who are younger than 12 months of age.
Phosphofructokinase Enzyme Activity
≥12 months of age: 5.8-10.9 U/g Hb
Reference values have not been established for patients who are younger than 12 months of age.
Phosphoglycerate Kinase Enzyme Activity
≥12 months: 142-232 U/g Hb
Reference values have not been established for patients who are younger than 12 months of age.
Triosephosphate Isomerase Enzyme Activity
≥12 months of age: 1033-1363 U/g Hb
Reference values have not been established for patients who are younger than 12 months of age.
Glutathione
≥12 months: 46.9-90.1 mg/dL RBC
Reference values have not been established for patients who are younger than 12 months of age.
Pyrimidine 5' Nucleotidase
Normal
Interpretation
A hematopathologist expert in these disorders evaluates the case, appropriate tests are performed, and an interpretive report is issued.
Clinical Reference
1. Steiner LA, Gallagher PG. Erythrocyte disorders in the perinatal period. Semin Perinatol. 2007;31(4):254-261
2. Beutler E: Glucose-6-phosphate dehydrogenase deficiency and other enzyme abnormalities. In: Beutler E, Lichtman MA, Coller BS, Kipps TJ, eds. Hematology. 5th ed. McGraw-Hill Book Company; 1995:564-581
3. Hoyer JD, Hoffman DR: The thalassemia and hemoglobinopathy syndromes. In: McClatchey KD, Amin HM, Curry JL, eds. Clinical Laboratory Medicine. 2nd ed. Lippincott, Williams and Wilkins; 2002:866-895
4. King MJ, Garcon L, Hoyer JD, et al. International Council for Standardization in Haematology. ICSH guidelines for the laboratory diagnosis of nonimmune hereditary red cell membrane disorders. Int J Lab Hematol. 2015;37(3):304-325
5. Lux SE: Anatomy of the red cell membrane skeleton: unanswered questions. Blood. 2016;127(2):187-199. doi:10.1182/blood-2014-12-512772
6. Gallagher PG. Abnormalities of the erythrocyte membrane. Pediatr Clin North Am. 2013;60(6):1349-1362
7. Bianchi P, Fermo E, Vercellati C, et al. Diagnostic power of laboratory tests for hereditary spherocytosis: a comparison study in 150 patients grouped according to molecular and clinical characteristics. Haematologica. 201297(4):516-523
8. Cappellini MD, Fiorelli G. Glucose-6-phosphate dehydrogenase deficiency. Lancet. 2008;371:64-74
9. Glader B: Hereditary hemolytic anemias due to red blood cell enzyme disorders. In: Greer JP, Arber DA, Glader B, et al, eds. Wintrobe's Clinical Hematology. 13th ed. Wolters Kluwer/Lippincott, Williams and Wilkins; 2014:728
10. Gallagher PG. Diagnosis and management of rare congenital nonimmune hemolytic disease. Hematology Am Soc Hematol Educ Program. 2015;2015:392-399
11. Koralkova P, van Solinge WW, van Wijk R. Rare hereditary red blood cell enzymopathies associated with hemolytic anemia - pathophysiology, clinical aspects, and laboratory diagnosis. Int J Lab Hematol. 2014;36(3):388-397
Test Classification
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.CPT Code Information
83020-26-Hemolytic Anemia Interpretation
82657-Hexokinase, B
82955-G6PD Enzyme Activity, B
83020-Hemoglobin electrophoresis
83021-High-Performance Liquid Chromatography (HPLC)
83068-Hemoglobin Stability
84087-Glucose phosphate isomerase, B
84220-Pyruvate Kinase Enzyme Activity, B
82657-Adenylate Kinase, B
82657-Phosphofructokinase, B
82657-Phosphoglycerate Kinase, B
82657-Trisephosphate Isomerase, B
85060-26 -Morphology review
85557-Osmotic fragility
88184-Band 3 Fluorescence Staining, RBC
83915-Pyrimidine 5' Nucleotidase
82978-Glutathione, B
83789 (if appropriate)
82664 (if appropriate)
88184 (if appropriate)
LOINC Code Information
| Test ID | Test Order Name | Order LOINC Value |
|---|---|---|
| HAEV1 | Hemolytic Anemia Evaluation | In Process |
| Result ID | Test Result Name | Result LOINC Value |
|---|---|---|
| PKCL | PK Enzyme Activity, B | 32552-2 |
| GPICL | Glucose Phosphate Isomerase, B | 44050-3 |
| G6PCL | G6PD Enzyme Activity, B | 32546-4 |
| TPICL | Triosephosphate Isomerase, B | 44054-5 |
| PGKCL | Phosphoglycerate Kinase, B | 44053-7 |
| PFKCL | Phosphofructokinase, B | 72664-6 |
| HKCL | Hexokinase, B | 49216-5 |
| AKCL | Adenylate Kinase, B | 44051-1 |
| 65615 | HPLC Hb Variant, B | No LOINC Needed |
| 608409 | Glutathione, B | 2383-8 |
| 608427 | Hemolytic Anemia Interpretation | 59466-3 |
| 2734 | Pyrimidine 5' Nucleotidase, B | 2902-5 |
| 13082 | Morphology Review | 59466-3 |
| 83141 | Band 3 Fluorescence Staining, RBC | 98906-1 |
| 9095 | Hb Stability, B | 4639-1 |
| 9064 | Osmotic Fragility, RBC | 34964-7 |
| SCTRL | Shipping Control Vial | 40431-9 |
| 41927 | Hb A | 20572-4 |
| 41928 | Hb F | 32682-7 |
| 3306 | Osmotic Fragility, 0.50 g/dL NaCl | 23915-2 |
| 608441 | Reviewed By | 18771-6 |
| 3307 | Osmotic Fragility, 0.60 g/dL NaCl | 23918-6 |
| 41929 | Hb A2 | 4552-6 |
| 41930 | Variant 1 | 24469-9 |
| 3308 | Osmotic Fragility, 0.65 g/dL NaCl | 23920-2 |
| 3309 | Osmotic Fragility, 0.75 g/dL NaCl | 23921-0 |
| 41931 | Variant 2 | 24469-9 |
| 41932 | Variant 3 | 24469-9 |
| 3310 | Osmotic Fragility Comment | 59466-3 |
| 41933 | HGBCE Interpretation | 78748-1 |
Day(s) Performed
Monday through Friday
Report Available
3 to 25 daysmml-benign-hematology-disorders