Hemolytic Anemias

Last Literature Review: February 2021 Last Update:

Hemolytic anemias, which result from premature destruction of red blood cells (RBCs),  may be hereditary or acquired. Hemolytic anemias can result from numerous causes, including RBC membrane disorders, RBC enzyme defects, immune conditions, hemoglobinopathies, and thrombotic microangiopathies, among others (see Classification).  Common hemolytic anemias include glucose-6-phosphate dehydrogenase (G6PD) deficiency, pyruvate kinase (PK) deficiency, and hereditary spherocytosis. Laboratory testing includes an initial evaluation for hemolysis, secondary testing to determine the etiology of hemolytic anemia, and, in some cases, molecular testing to confirm the diagnosis or determine recurrence risk.

Quick Answers for Clinicians

How does hemolytic anemia present clinically?

Hemolytic anemia is a clinically heterogeneous disorder with equally heterogeneous presentations. Patients may be asymptomatic or may present with anemia and related symptoms (eg, fatigue, dyspnea, tachycardia) or hemolysis-related symptoms (jaundice, hematuria).   Patients with chronic hemolysis may exhibit hepatosplenomegaly, lymphadenopathy, cholestasis, and choledocholithiasis.  Additional disease-specific symptoms may occur (eg, Raynaud phenomenon in patients with cold agglutinin syndrome). 

Which initial tests should be ordered in cases of suspected hemolytic anemia?

The first step in the evaluation of suspected hemolytic anemia is confirmation of hemolysis. A standard workup for hemolysis includes lactate dehydrogenase (LDH), unconjugated bilirubin, and haptoglobin tests, as well as a reticulocyte count. Hemolysis is confirmed by increases in the reticulocyte count, LDH, and unconjugated bilirubin, along with decreased haptoglobin.   A peripheral smear should be ordered after hemolysis is confirmed to provide clues to the etiology of hemolytic anemia.

What can and cannot be deduced from a peripheral smear?

Although findings on a peripheral smear may suggest a particular etiology for disease (refer to Peripheral Smear), the peripheral smear is not a standalone diagnostic test. Most findings are not specific for hemolytic anemia or particular etiologies thereof. A workup for hemolysis is recommended to confirm hemolytic anemia and inform secondary testing. In the absence of hemolysis, the use of flow cytometry tests may be considered to begin investigation of abnormal cells on the peripheral smear.

Indications for Testing

Patients with unexplained anemia, particularly with reticulocytosis and/or evidence of hemolysis, should be evaluated for hemolytic anemias. Suggestive symptoms that may prompt investigation include acute jaundice and hematuria, as well as nonspecific symptoms of anemia such as dyspnea and fatigue. 


The hemolytic anemias are a clinically heterogeneous group of disorders that can be classified by class or type, mechanism, and whether hemolysis is intravascular or extravascular.

Types of Hemolytic Anemia
Type Examples Related ARUP Consult Topics and Test Fact Sheets
RBC enzyme defects

G6PD deficiency

PK deficiency

Glucose phosphate isomerase deficiency

Glutathione reductase deficiency

Phosphoglycerate kinase deficiency

Triosephosphate isomerase deficiency

Adenylate kinase 1 deficiency

P5N deficiency

Hexokinase 1 deficiency

G6PD Deficiency Test Fact Sheet
RBC membrane defects

Hereditary spherocytosis

Hereditary elliptocytosis

Hereditary pyropoikilocytosis



Paroxysmal Nocturnal Hemoglobinuria

Hemoglobin synthesis abnormalities

Qualitative hemoglobinopathies (eg, sickle cell disease)



Unstable Hemoglobinopathies


Immune, infectious causes


Epstein-Barr virus

Hepatitis C virus




Mycoplasma pneumoniae


Babesia microti


Epstein-Barr Virus

Hepatitis C Virus

Human Immunodeficiency Virus

Plasmodium Species - Malaria

Babesia microti - Babesiosis

Immune, autoimmune

Warm autoimmune hemolytic anemia (primary or secondary)

Cold agglutinin syndrome (including cold hemagglutinin disease and paroxysmal cold hemoglobinuria)

Mixed autoimmune hemolytic anemia (primary or secondary)

Immune, microangiopathic RBC destruction





HELLP syndrome

Thrombotic Microangiopathies

Disseminated Intravascular Coagulation


Physical disruption (eg, due to mechanical valves)

Envenomation (eg, from brown recluse spider venom)

Systemic disease (eg, malignant hypertension)

Drug induced (>150 associated drugs)

aHUS, atypical hemolytic uremic syndrome; DIC, disseminated intravascular coagulation; HELLP, hemolysis, elevated liver function tests, and low platelet count; HSt, hereditary stomatocytosis; HUS, hemolytic uremic syndrome; n/a, not applicable; PNH, paroxysmal nocturnal hemoglobinuria; P5N, pyrimidine 5’-nucleotidase; TTP, thrombotic thrombocytopenic purpura

Laboratory Testing

Initial Evaluation

CBC and Workup for Hemolysis

The first step in the evaluation of hemolytic anemia is a CBC, along with a complete clinical examination and family history.  If the CBC reveals normocytic or macrocytic anemia, a reticulocyte count should be performed. A standard workup for hemolysis, including lactate dehydrogenase (LDH), haptoglobin, and unconjugated bilirubin tests, is also recommended.   Urinalysis may be useful and may reveal hemoglobinuria even in the absence of visible RBCs.   Urinary hemosiderin may be detected a week after onset in cases of intravascular hemolysis. 

The reticulocyte count, which indicates the bone marrow response to hemolysis, is generally increased in hemolytic anemia unless there is simultaneous iron deficiency or bone marrow suppression.  LDH and unconjugated bilirubin are usually elevated.   Haptoglobin is most often decreased.   Together, reticulocytosis, increased LDH, increased unconjugated bilirubin, and decreased haptoglobin confirm hemolysis. 

Peripheral Smear

If not performed with the CBC, a peripheral smear with Heinz body stain should be obtained after confirmation of hemolysis.  Findings noted on the peripheral smear may suggest possible diagnoses that can be determined with the appropriate secondary tests (see Secondary Testing). For a complete testing strategy based on peripheral smear morphology, see the Hemolytic Anemias Testing Algorithm.

Possible Etiologies of Hemolysis Based on Peripheral Smear Findings
Finding Possible Etiologies
Spherocytes, elliptocytes, poikilocytes

Hereditary spherocytosis

Hereditary elliptocytosis

Hereditary pyropoikilocytosis

Immune-mediated hemolytic anemias (eg, autoimmune hemolytic anemia, cold agglutinin disease, paroxysmal cold hemoglobinuria)

Schistocytes/fragmented cells



HELLP syndrome



Physical disruption (eg, of mechanical cardiac valve)


Malignant hypertension

Polychromasia without other morphologic abnormality, with or without platelet decrease


RBC enzyme defects

Sickle cells, target cells Hemoglobinopathy
Stomatocytes HSt (including dehydrated HSt, or xerocytosis; and overhydrated HSt, or hydrocytosis)
Basophilic stippling Lead poisoning

P5N deficiency

Positive Heinz body stain


G6PD deficiency

Chemical or toxin exposure


Cold agglutinin disease

Warm autoimmune hemolytic anemia

Unusual RBC inclusions Infection

Direct Coombs Test

A direct Coombs test (also known as a direct antiglobulin test, or DAT) is useful to distinguish between immune and nonimmune hemolysis.  The detection of immunoglobulins (IgG, IgM, or IgA) or complement (C3) in the context of hemolysis suggests immune hemolytic anemia, although the direct Coombs test is not specific.  In some cases of immune hemolytic anemia, a direct Coombs test will not result in the detection of antibodies; if hemolysis cannot be otherwise explained after a negative direct Coombs test result, consider retesting with a column agglutination method, followed by secondary tests. 

Secondary Testing

RBC Enzyme Defects

RBC enzymopathies are diagnosed primarily by exclusion of other potential causes of hemolysis.  In patients with RBC enzymopathies, the direct Coombs test result will be negative for antibodies, and an osmotic fragility or cryohemolysis test result will be normal.   Specific abnormalities will not be observed on the peripheral smear, although nonspecific features (eg, basophilic stippling in P5N deficiency, Heinz bodies in G6PD deficiency, and poikilocytosis in PK deficiency) may be present with certain RBC enzymopathies.  

To identify the specific RBC enzymopathy responsible for hemolysis, tests for reduced enzyme activity are recommended.  The most common RBC enzymopathies, including G6PD and PK deficiency, may be identified by specific screening tests.  For other RBC enzymopathies, activity is generally determined using a spectrophotometric assay for enzyme activity ; such assays are recommended even for the more common RBC enzymopathies.  All enzyme activity assays require careful interpretation because results may be influenced by erythrocyte age, recent transfusion, the presence of leukocytes, and other factors.  Molecular genetic testing is considered complementary to enzyme testing and is often required to definitively diagnose a suspected RBC enzymopathy.  

RBC Membrane Defects

Suggestive findings on the peripheral smear may indicate an RBC membrane defect such as hereditary spherocytosis (indicated by spherocytes), hereditary elliptocytosis (suggested by elliptocytes), hereditary pyropoikilocytosis (suggested by poikilocytes), or HSt (indicated by stomatocytes or target cells).  Additional testing may confirm or lead to a diagnosis. 

Hereditary Spherocytosis

Patients with spherocytes on the peripheral smear who have a family history of hereditary spherocytosis, clinical features consistent with this disorder, an increased mean corpuscular hemoglobin concentration (MCHC), and reticulocytosis do not require any additional testing.  Screening tests for hereditary spherocytosis in uncertain cases include the osmotic fragility test, acid glycerol lysis time test, osmotic gradient ektacytometry, and the eosin-5’-maleimide (EMA) binding test.  Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) is recommended if the clinical phenotype is more severe than would be predicted by RBC morphology or when the morphology is more severe than would be expected from studies in an affected family member.  If there is ambiguity in the diagnosis, SDS-PAGE is recommended before proceeding with splenectomy.  Although not generally required in cases of hereditary spherocytosis, molecular genetic testing may be useful if clinical suspicion persists despite negative test results.  

Hereditary Elliptocytosis

Secondary testing for hereditary elliptocytosis is recommended if there is no family history and if there are only a few elliptocytes visible on the peripheral smear.  A diagnosis of hereditary elliptocytosis can be confirmed via SDS-PAGE for protein 4.1 and spectrin analysis, ektacytometry, or laser-assisted optical rotational cell analyzer (LORCA).  Molecular genetic testing may also be useful. 

Hereditary Stomatocytosis

There are several different subtypes of HSt, including dehydrated HSt (also known as xerocytosis), overhydrated HSt (also known as hydrocytosis), cryohydrocytosis (CHC), and familial pseudohyperkalemia (FP).   Obtaining an accurate diagnosis is particularly important in HSt because treatment differs depending on the subtype.  Ion flux measurement, ektacytometry, LORCA, or molecular genetic testing can be used to diagnose HSt and identify the subtype. 

Hereditary Pyropoikilocytosis

A mean corpuscular volume (MCV) of 50-60 fL or a greatly reduced mean cell fluorescence (MCF) as detected by EMA supports a diagnosis of hereditary pyropoikilocytosis.  The diagnosis can be confirmed via spectrin analysis, ektacytometry, LORCA, or molecular genetic testing. 

Autoimmune Hemolytic Anemias​

Because autoimmune hemolytic anemia may arise from a number of causes, including recent transfusion, infections, other autoimmune conditions, drugs, and lymphoproliferative malignancies, careful consideration of the clinical picture is required.  

Autoimmune hemolytic anemia is suggested by a positive result (in the absence of another explanation) on the direct Coombs test.   However, a negative result does not rule out an autoimmune cause of hemolysis. The specific antibodies detected by the direct Coombs test may point to a particular etiology of hemolytic anemia (eg, a positive IgG without positive C3 suggests warm autoimmune hemolytic anemia ). A positive direct Coombs test result may also point to additional testing (eg, a positive C3 result may warrant a cold agglutinins test for cold agglutinin disease or, in the applicable clinical circumstances, a Donath Landsteiner test for paroxysmal cold hemoglobinuria). 

Microangiopathic RBC Destruction

Microangiopathic RBC destruction occurs due to RBC fragmentation and is suggested by the presence of schistocytes on the peripheral smear.  A number of conditions may lead to microangiopathic RBC destruction.

Conditions with Microangiopathic RBC Destruction
Cause of RBC Fragmentation Detailed Laboratory Testing Information
TTP Thrombotic Microangiopathies
HUS Thrombotic Microangiopathies
aHUS Thrombotic Microangiopathies
DIC Disseminated Intravascular Coagulation
HELLP syndrome n/a

Hemoglobinopathies and Thalassemias​

For more information on secondary testing for hemoglobinopathies, including unstable hemoglobinopathies and thalassemias, see the ARUP Consult Hemoglobinopathies, Unstable Hemoglobinopathies, and Thalassemias topics and the Hemoglobinopathies Testing Algorithm.

Other Causes of Hemolytic Anemia

The clinical history, initial evaluation, and peripheral smear may suggest another cause for hemolytic anemia (eg, abnormal inclusions on the peripheral smear in cases of infection).  Selecting the appropriate laboratory tests requires careful clinical judgment. See the Hemolytic Anemias Testing Algorithm for a suggested testing strategy based on peripheral smear and clinical findings.

Molecular Genetic Testing

Genetic testing can be performed to confirm a diagnosis, determine a diagnosis, or assess recurrence risk for a hereditary hemolytic anemia. Panel testing may be useful to distinguish between disorders with overlapping clinical presentations. Genes tested, clinical sensitivity, costs, and methodology vary between panels; clinical judgment is required to select the appropriate panel test. Familial variant testing may be useful to confirm or rule out a diagnosis in at-risk family members if a known familial variant exists. More comprehensive testing (eg, whole exome sequencing) may also be useful in certain circumstances.

Genes Involved in Hereditary Hemolytic Anemias
Condition Genes Inheritance
Hereditary RBC Enzyme Defects
G6PD deficiency G6PD XR
PK deficiency PKLR AR
Glucose phosphate isomerase deficiency GP1 AR
Glutathione reductase deficiency GSR AR
Phosphoglycerate kinase deficiency PGK1 XL
Triosephosphate isomerase deficiency TPI1 AR
Adenylate kinase 1 AK1 AR
Hexokinase 1 HK1 AR
Phosphofructokinase deficiency (glycogen storage disease VII, Tauri disease) PFKM AR
Hereditary RBC Membrane Defects
Hereditary spherocytosis ANK1, SLC4A1, SPTB, EPB42 AD/AR
Hereditary elliptocytosis/pyropoikilocytosis SPTA1, SPTB, EPB41 AD/AR
AD, autosomal dominant; AR, autosomal recessive; XL, X-linked; XR, X-linked recessive

Testing in Neonates

Autoimmune Hemolytic Anemias

If a mother has a positive direct Coombs test result during pregnancy or is diagnosed with autoimmune hemolytic anemia, cord blood should be tested with a direct Coombs test after delivery.  If jaundice is observed or if the direct Coombs test result is positive, a CBC, reticulocyte count, and bilirubin test are recommended,  as is monitoring for anemia and hyperbilirubinemia. 

ARUP Laboratory Tests

Initial Evaluation

Use to assess hemolysis

Use to evaluate cellular morphology

Use as a nonspecific screen for hemolysis due to drugs/toxins, enzyme deficiencies, thalassemias, and unstable hemoglobins

Use to detect in vivo coating of RBCs with immunoglobulin and complement degradation products on the patient's RBCs

Useful for antibody identification and evaluation of transfusion reactions, hemolytic disease of the fetus and newborn (HDFN), and autoimmune hemolytic anemia associated with disease and/or drugs

Use to detect hemosiderin, which may be detected a week after onset in cases of intravascular hemolysis

Secondary Evaluation

RBC Enzyme Defects

Preferred initial screening test for G6PD deficiency

Preferred initial screening test for PK deficiency

RBC Membrane Defects

Functional testing of RBC sensitivity to osmotic stress

Do not use to distinguish between spherocytes in hereditary spherocytosis and acquired autoimmune hemolytic anemia

Use to confirm diagnosis of hereditary spherocytosis when hemolytic anemia and spherocytes are present

Autoimmune Hemolytic Anemias

Use to determine if Donath-Landsteiner antibodies are present

Use to diagnose paroxysmal cold hemoglobinuria

The presence of other red blood cell antibodies may interfere with testing, leading to inconclusive results

Use to identify antibodies as cause of hemolysis

Molecular Genetic Testing

Optimal test to evaluate individuals with hereditary hemolytic anemia or unexplained long-standing hemolytic anemia

A faculty hematopathologist personally directs and interprets each stage of testing to completion; a comprehensive report is provided


High Performance Liquid Chromatography (HPLC)/Electrophoresis/RBC Solubility/Polymerase Chain Reaction/Fluorescence Resonance Energy Transfer/Sequencing/Spectrophotometry/Visual Identification/Quantitative Enzymatic Assay/Quantitative Flow Cytometry/Cytochemical Stain/Multiplex Ligation-Dependent Probe Amplification/Massively Parallel Sequencing

Use to determine etiology, elicit inheritance pattern, and assess recurrence risk in individuals with unexplained hemolytic anemia, unexplained hyperbilirubinemia (neonates), family history of unexplained hemolytic anemia, or pregnancy with hydrops fetalis of unknown etiology

Preferred genetic test for individuals of African descent

Use to detect the single most common pathogenic G6PD variant (the A- allele) in individuals of African descent

For initial screening for G6PD deficiency, refer to Glucose-6-Phosphate Dehydrogenase (0080135)

Preferred test to detect glucose-6-phosphate dehydrogenase (G6PD) variants in females or any individual with reduced G6PD enzyme activity

Use to test for a known familial sequence variant previously identified in a family member


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