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Test 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 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.



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 vials

ACD: Two 6-mL vials

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
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
HAEV0 Hemolytic Anemia Summary Interp No 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:

-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

 

An additional comprehensive consultative interpretation that summarizes all results will be provided after all tests are completed to incorporate results into an overall evaluation.

Method Name

HAEVI, HAEV0: Medical Interpretation

HGBCE: Capillary Electrophoresis

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

UNHB: Isopropanol and Heat Stability

FRAGO: 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 Evaluation

Specimen Type

Control
Whole Blood ACD-B
Whole Blood EDTA
Whole Blood Slide

Specimen Minimum Volume

EDTA Blood: 3 mL
ACD 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 red blood cell 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

 

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 Aug;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 Jun;37(3):304-325

5. Lux SE: Anatomy of the red cell membrane skeleton: unanswered questions. Blood. 2016 Jan 14;127(2):187-199. doi: 10.1182/blood-2014-12-512772

6. Gallagher PG: Abnormalities of the erythrocyte membrane. Pediatr Clin North Am. 2013 Dec;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. 2012 Apr;97(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 Jun;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. This test 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 4576-5
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 4551-8
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 days