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
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).
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.
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.
|Type||Examples||Related ARUP Consult Topics and Test Fact Sheets|
|RBC enzyme defects||
Glucose phosphate isomerase deficiency
Glutathione reductase deficiency
Phosphoglycerate kinase deficiency
Triosephosphate isomerase deficiency
Adenylate kinase 1 deficiency
Hexokinase 1 deficiency
|G6PD Deficiency Test Fact Sheet|
|RBC membrane defects||
|Hemoglobin synthesis abnormalities||
Qualitative hemoglobinopathies (eg, sickle cell disease)
|Immune, infectious causes||
Hepatitis C virus
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||
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|
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.
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.
|Spherocytes, elliptocytes, poikilocytes||
Immune-mediated hemolytic anemias (eg, autoimmune hemolytic anemia, cold agglutinin disease, paroxysmal cold hemoglobinuria)
Physical disruption (eg, of mechanical cardiac valve)
|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
|Positive Heinz body stain||
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.
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.
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.
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.
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.
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.
|Cause of RBC Fragmentation||Detailed Laboratory Testing Information|
|DIC||Disseminated Intravascular Coagulation|
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.
|Hereditary RBC Enzyme Defects|
|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|
|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
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
Preferred initial screening test for G6PD deficiency
Preferred initial screening test for PK deficiency
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
Qualitative Flow Cytometry
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
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)
Polymerase Chain Reaction/Fluorescence Monitoring
Preferred test to detect glucose-6-phosphate dehydrogenase (G6PD) variants in females or any individual with reduced G6PD enzyme activity
Massively Parallel Sequencing
Use to test for a known familial sequence variant previously identified in a family member
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