Test Code 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 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.

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 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

Reject Due To

Gross hemolysis

Reject

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