Hemolytic Anemias

Hemolytic anemias, which result from premature destruction of red blood cells (RBCs),  may be hereditary or acquired. Hemolytic anemias can be due to numerous causes, including RBC membrane disorders, RBC enzyme defects, immune conditions, hemoglobinopathies, and thrombotic microangiopathies, among other causes (see Classification section).  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

What is the clinical presentation of hemolytic anemia?

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 section), 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.

Which testing algorithms are related to this topic?

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



Hereditary Spherocytosis Test Fact Sheet
Paroxysmal Nocturnal Hemoglobinuria
Hemoglobin synthesis abnormalities Qualitative hemoglobinopathies (eg, sickle cell disease)



Unstable Hemoglobinopathies


Immune, infectious causes Cytomegalovirus

Epstein-Barr virus

Hepatitis C




Mycoplasma pneumoniae


Babesia microti

Cytomegalovirus Test Fact Sheet

Epstein-Barr Virus

Hepatitis C Virus

Human Immunodeficiency Virus

Leptospira Species - Leptospirosis

Plasmodium Species - Malaria

Mycoplasma pneumoniae Infection

Parvovirus B19

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)

Cold Agglutinin Disease
Immune, microangiopathic RBC destruction TTP




HELLP syndrome

Thrombotic Microangiopathies

Disseminated Intravascular Coagulation

HELLP Syndrome

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


HELLP syndrome



Physical disruption (eg, of mechanical cardiac valve)


Malignant hypertension

Polychromasia without other morphologic abnormality, with or without platelet decrease PNH

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 Hemoglobinopathy

G6PD deficiency

Chemical or toxin exposure

Agglutination 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 will be negative for antibodies, and the results of an osmotic fragility or cryohemolysis test 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.  Screening tests exist for the most common RBC enzymopathies, including G6PD and PK deficiency.  For other RBC enzymopathies, activity is generally determined using a spectrophotometric assay for enzyme activity,  and 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 make a definitive diagnosis of 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 for different subtypes of the disease.  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 support 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 may also point to additional testing (eg, a positive C3 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
Thrombotic thrombocytic purpura Thrombotic Microangiopathies
Hemolytic uremic syndrome Thrombotic Microangiopathies
Atypical hemolytic uremic syndrome Thrombotic Microangiopathies
Disseminated intravascular coagulation Disseminated Intravascular Coagulation
Hemolysis, elevated liver function tests, and low platelet count constellation (HELLP syndrome) HELLP Syndrome

Hemoglobinopathies and Thalassemias​

For more information on secondary testing for hemoglobinopathies, including unstable hemoglobinopathies and thalassemias, see the Hemoglobinopathies, Unstable Hemoglobinopathies, and Thalassemias ARUP Consult 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 result during pregnancy, or if autoimmune hemolytic anemia is diagnosed, cord blood should be tested with a direct Coombs test following delivery.  If jaundice is observed, or if the direct Coombs tests is positive, a CBC, reticulocyte count, and bilirubin test are recommended.  Monitoring for anemia and hyperbilirubinemia is recommended. 

ARUP Laboratory Tests

Initial Evaluation

Evaluate for hemolysis, thrombocytopenia, and leukopenia

Assess bone marrow response to anemia

Assess hemolysis

Evaluate cellular morphology

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

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

May be detected a week after onset in cases of intravascular hemolysis

Secondary Evaluation

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

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

Confirm diagnosis of hereditary spherocytosis when hemolytic anemia and spherocytes are present

Autoimmune Hemolytic Anemias

Determine if Donath-Landsteiner antibodies are present

Diagnose paroxysmal cold hemoglobinuria

Identify antibodies as cause of hemolysis

Molecular Genetic Testing

Determine etiology, elicit inheritance pattern, and assess recurrence risk in individuals with unexplained hemolytic anemia, unexplained hyperbilirubinemia, family history of unexplained hemolytic anemia, or pregnancy with hydrops fetalis of unknown etiology

Preferred genetic test for individuals of African descent

Preferred test for individuals of high-risk ethnic backgrounds other than those of African descent

Appropriate test for symptomatic individuals of African descent who do not carry the A- allele

For initial screening for GP6D deficiency, refer to glucose-6-phosphate dehydrogenase

Confirm PK deficiency in individuals with abnormal PK enzyme activity and/or clinical findings

Assess carrier status for PK deficiency 

Test for a known familial sequence variant previously identified in a family member

Medical Experts



Archana Mishra Agarwal, MD
Associate Professor of Clinical Pathology, University of Utah
Medical Director, Hematopathology and Special Genetics, ARUP Laboratories


Additional Resources
Resources from the ARUP Institute for Clinical and Experimental Pathology®