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
||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
|Hereditary Spherocytosis Test Fact Sheet
Paroxysmal Nocturnal Hemoglobinuria
|Hemoglobin synthesis abnormalities
||Qualitative hemoglobinopathies (eg, sickle cell disease)
|Immune, infectious causes
|Cytomegalovirus Test Fact Sheet
Hepatitis C Virus
Human Immunodeficiency Virus
Leptospira Species - Leptospirosis
Plasmodium Species - Malaria
Mycoplasma pneumoniae Infection
Babesia microti - Babesiosis
||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
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; 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 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.
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
|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
||HSt (including dehydrated HSt, or xerocytosis; and overhydrated HSt, or hydrocytosis)
|Positive Heinz body stain
Chemical or toxin exposure
||Cold agglutinin disease
Warm autoimmune hemolytic anemia
|Unusual RBC inclusions
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 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.
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 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.
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
|Hemolytic uremic syndrome
|Atypical hemolytic uremic syndrome
|Disseminated intravascular coagulation
||Disseminated Intravascular Coagulation
|Hemolysis, elevated liver function tests, and low platelet count constellation (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
|Glucose phosphate isomerase deficiency
|Glutathione reductase deficiency
|Phosphoglycerate kinase deficiency
|Triosephosphate isomerase deficiency
|Adenylate kinase 1
|Phosphofructokinase deficiency (glycogen storage disease VII, Tauri disease)
||ANK1, SLC4A1, SPTB, EPB42
||SPTA1, SPTB, EPB41
|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.