Hemolytic anemia is characterized by the breakdown of red blood cells (RBCs). Hemolysis can either be caused by abnormalities in RBCs (hemoglobin, the RBC membrane, or intracellular enzymes), which is called intrinsic hemolytic anemia, or by external causes (immune-mediated or mechanical damage), which is called extrinsic hemolytic anemia. Hemolysis can be further categorized depending on whether it occurs inside the blood vessels (intravascular hemolysis), in the reticuloendothelial system (extravascular hemolysis), or both. Hemolytic anemias cause varying degrees of fatigue, pallor, and weakness, ranging from asymptomatic disease to life-threatening hemolytic crisis; although, some hemolytic anemias have more specific findings (e.g., thrombosis in paroxysmal nocturnal hemoglobinuria). Hemolytic anemia should be suspected in patients with anemia and laboratory findings of hemolysis (e.g., elevated indirect bilirubin and lactate dehydrogenase, reticulocytosis, and decreased haptoglobin levels). The Coombs test helps to distinguish between antibody-mediated (positive direct Coombs test) and nonantibody-mediated (negative direct Coombs test) anemias. Further tests should be performed to investigate the underlying etiology. Treatment involves RBC transfusions as required. Additional treatment is based on the type of hemolytic anemia and its cause.
Types and etiologies of hemolytic anemia
Hemolytic anemias are characterized by an excessive breakdown of red blood cells (RBCs). They can be classified according to the cause of hemolysis (intrinsic or extrinsic) and by the location of hemolysis (intravascular or extravascular).
|By RBC pathology|
|Intrinsic hemolytic anemia|
|Extrinsic hemolytic anemia|| || |
|By location of RBC breakdown|
|Intravascular hemolytic anemia|| || |
|Extravascular hemolytic anemia|
- Signs of anemia
- Signs of hemolysis
- Signs of increased hematopoiesis (mostly in severe chronic anemias, e.g., thalassemia)
- Perform initial laboratory studies to confirm anemia and hemolysis and .
- Obtain a DAT) to narrow the differential: (i.e.,
- Consider further investigations of the underlying etiology based on clinical suspicion and DAT results.
If the patient has severe symptoms of anemia or a life-threatening cause is suspected (i.e., , , , ), proceed directly to treatment in parallel with diagnostic evaluation.
Routine laboratory studies 
- ↓ Hb, Hct, and RBC count 
- WBC count: can be elevated due to inflammation or malignancy
- Platelets: decreased in MAHA and in
- : Use the for patients with anemia.
While no single test can be used to confirm hemolysis, the finding of anemia in the presence of accelerated erythropoiesis (i.e., reticulocytosis) in addition to evidence of RBC destruction in serum and/or urine studies is highly suggestive of hemolytic anemia.
Typical biochemical findings in hemolysis include ↓ haptoglobin, ↑ LDH concentration, ↑ indirect bilirubin concentration, peripheral blood smear abnormalities (e.g., ↑ reticulocytes, schistocytes, spherocytes, polychromasia), and urinalysis abnormalities (e.g., hemoglobinuria, hemosiderinuria, and urobilinogen).
|Evidence of hemolysis in serum studies |
|Parameter||Description||Features of intravascular hemolysis||Features of extravascular hemolysis|
|Haptoglobin || || |
|Lactate dehydrogenase|| || |
|Indirect (unconjugated) bilirubin|| || |
|Evidence of hemolysis in urine studies |
|Intravascular hemolysis||Extravascular hemolysis |
|Hemoglobinuria || || |
|Hemosiderinuria || || || |
|Urobilinogen || || |
Description: A special reagent is added to patients' blood samples: Coombs serum, which contains antihuman globulins (AHGs) that detect and adhere (with 2 binding sites) to immune proteins that mediate hemolysis, i.e., antibodies (IgG) and/or complement
- If these proteins are coating the RBC surface when the serum is added, AHGs will cause multiple RBCs to adhere to each other in a process called agglutination.
- If no such proteins are present, AHGs will not bind to anything.
- RBC agglutination is considered a positive result, while the absence of RBC agglutination is considered a negative result.
Direct Coombs test (DAT) 
This is a key test in the workup of hemolytic anemia.
- Indications: All patients with hemolysis
- Positive result
- Negative result: Suggests nonantibody-mediated hemolysis
Indirect Coombs test 
- Goal: is the detection of anti-RBC antibodies in the serum of, for example:
- The patient's blood sample is purified so that only the serum remains.
- The patient's serum is incubated with test RBCs.
- If there are anti-RBC antibodies in the patient's serum, they will bind to the test RBCs.
- Coombs serum is added and AHGs bind to the patient's antibodies, which are bound to the test RBCs, leading to RBC agglutination.
- Positive result
- Negative result: No detectable anti-RBC antibodies
Differences between the Coombs tests
|Key differences between direct and indirect Coombs testing|
|Direct Coombs test ||Indirect Coombs test |
|Clinical applications|| |
|Location of antibodies detected|| || |
|Coombs serum added to:|| || |
|AHGs in Coombs serum bind to:|
Investigation of underlying causes
Perform further diagnostic workup according to the suspected etiology; specialist consultation is advised. 
- Hb electrophoresis: abnormal Hb patterns, e.g., in thalassemia
- Flow cytometry
- Genetic analysis: mutations in congenital hemolytic anemia
- Bone marrow biopsy
Antibody-mediated hemolysis (DAT positive)
|Laboratory investigations for antibody-mediated hemolysis |
|Concerning initial features||Suggested additional investigations|
|AIHA ||Warm AIHA|| |
|Cold AIHA|| |
Nonantibody-mediated hemolysis (DAT negative)
|Laboratory investigations for nonantibody-mediated hemolysis |
|Concerning initial features||Suggested additional investigations|
|Intrinsic hemolytic anemia||Hereditary spherocytosis|| |
|Hereditary elliptocytosis|| |
|Paroxysmal nocturnal hemoglobinuria || |
|Pyruvate kinase deficiency|
|G6PD deficiency|| |
|Extrinsic hemolytic anemia|
|Intracellular pathogens|| || |
- Physiologically, a membrane-bound glycosylphosphatidylinositol (GPI) anchor protects RBCs against complement-mediated hemolysis.
- Acquired mutation on the PIGA gene located on the X chromosome → GPI anchor loses its protective effect → RBC destruction by complement and reticuloendothelial system → intravascular and extravascular hemolysis 
- The GPI anchor proteins involved in PNH are:
- PNH can also occur in patients with aplastic anemia 
- Pallor, excessive fatigue; , weakness
- Intermittent jaundice
- Episodes of hemoglobinuria causing pink/red/dark urine which usually occurs in the morning due to the concentration of urine overnight. 
- Vasoconstriction 
- Venous thrombosis in unusual locations (e.g., hepatic, cerebral, and/or abdominal veins) 
- Increased risk of infections (in case of pancytopenia)
- CBC: anemia, thrombocytopenia, and/or pancytopenia ; usually ↑ reticulocytes 
- : ↓ haptoglobin, hemosiderinuria, hemoglobinuria 
- Direct Coombs test: negative 
- Flow cytometry of peripheral blood (confirmatory test for PNH): can show deficiency of GPI-linked proteins on the surface of RBCs and WBCs (e.g., CD55, CD59) 
- Bone marrow biopsy: : not required for the diagnosis of PNH, but indicated in patients with significant pancytopenia
- Additional studies: to assess for complications, for example:
- Patients with mild or no clinical manifestations: with close surveillance
- Patients with significant clinical manifestations: Start treatment.
- Provide supportive care for all patients.
- Indications include severe anemia, thrombosis, severe fatigue, pain crises, and end-organ damage
- First-line treatment: complement inhibition with an anti-C5 antibody (e.g., eculizumab , ravulozumab)
- Second-line treatment:
Supportive care 
- Neisseria meningitidis infection
- Etiology: autosomal recessive defect of pyruvate kinase
- Glucose is the only energy source in RBCs
- Pyruvate kinase catalyzes the last step of glycolysis (i.e., irreversibly converts phosphoenolpyruvate into pyruvate)
- Absence of pyruvate kinase → ATP deficiency in RBC
- ATP deficiency disrupts the cation gradient along the RBC membrane → rigid RBCs → ↑ hemolysis (extravascular)
- Accumulation of 2,3-bisphosphoglycerate → ↑ release of O2 from Hb → masks symptoms of anemia
- Clinical symptoms
- Hemoglobin C disease: occurs in individuals who are homozygous for the hemoglobin C mutation (HbCC)
- Hemoglobin C trait: occurs in individuals who are heterozygous carriers of the hemoglobin C mutation (HbAC)
- HbC precipitates as crystals → ↑ RBC rigidity and ↓ deformability → extravascular hemolysis
- β-globin mutation (glutamate replaced by lysine)
- HbC is less soluble than HbA and tends to form hexagonal crystals, which lead to RBehydration (↑ MCHC).
- RBCs have reduced oxygen-binding capacity and a shorter lifespan.
Clinical features 
- Hemolytic anemia (usually mild)
- Patients with HbSC gene mutation (one HbC and one HbS trait) have milder symptoms than HbSS patients.
|Characteristic findings in hemoglobin C disease compared with hemoglobin C trait |
|Hemoglobin C disease (HbCC)||Hemoglobin C trait (HbAC)|
| || |
- Clinical features
- Antibody-mediated destruction of RBCs
- Mechanical destruction of RBCs
Cold agglutinin hemolytic anemia 
- Cold agglutinin hemolytic anemia (cold AIHA): a group of autoimmune disorders characterized by hemolysis that is caused by the binding of cold-sensitive autoantibodies to RBCs 
- Cold agglutinin disease (CAD): a primary (idiopathic) form of cold AIHA in which hemolysis is mediated by monoclonal cold-sensitive IgM autoantibodies produced by a low-grade clonal B-cell lymphoproliferative disorder of the bone marrow
- Cold agglutinin syndrome (CAS): a rare form of cold AIHA in which cold-sensitive autoantibody-mediated hemolysis occurs secondary to another condition (e.g., infection, lymphoma)
- Epidemiology: Median age at diagnosis is approximately 70 years. 
Cold-sensitive antibodies (cold agglutinins):
- Mostly IgM antibodies
- Cause acute intravascular hemolysis and extravascular hemolysis
- Intravascular hemolysis is mediated through the complement system, which is activated by IgM antibodies bound to RBCs.
- The antigen-antibody reaction is triggered by low body temperature and/or cold ambient temperatures. 
- CAD: idiopathic
- CAS occurs secondary to other diseases, including:
- Cold-sensitive antibodies (cold agglutinins):
- Classically associated with cold exposure
- Symptoms are usually more acute and severe than in warm AIHA.
- Pallor, fatigue, weakness
- Painful cyanosis of the extremities (acrocyanosis)
- Livedo reticularis
- Raynaud phenomenon
- Skin ulcerations
- Indications for testing: typical cold-induced symptoms (e.g., acrocyanosis, livedo reticularis) or unexplained anemia
- Initial investigations: CBC, reticulocyte count, , DAT, and cold agglutinin titer
- Additional investigations are indicated to identify secondary causes (i.e., to confirm CAS), for example:
- Diagnosis of cold AIHA can be established in patients with all of the following:
- Other potential diagnostic findings (nonspecific)
Hematology consult is advised.
- Goal: Reduce cold-induced symptoms, control hemolysis, and improve anemia.
- Compensated hemolysis and mild or absent circulatory symptoms : close surveillance
- Significant clinical manifestations: Start acute therapy.
- CAS: Treat the underlying condition
- CAD: Consider chronic therapy (systemic immunomodulatory).
- Advise all patients to avoid exposure to the cold.
- Consider folic acid supplementation in all patients.
- Manage disease complications: Venous thromboembolism (VTE) 
- Prognosis: Spontaneous remission is rare in CAD but is common in CAS secondary to infection (e.g., Mycoplasma) and typically occurs within a few weeks of onset. 
|Treatment of cold AIHA|
Warm agglutinin hemolytic anemia 
- Definition: an autoimmune disease characterized by the binding of heat-sensitive autoantibodies to RBCs, which leads to the phagocytosis and destruction of RBCs in the reticuloendothelial system 
- Heat-sensitive antibodies
- Triggered by body temperatures of ≥ 37°C/98.6°F
- Mostly idiopathic
- Secondary causes
“Warm weather is Great”: Warm AIHA is IgG mediated.
- Order CBC, reticulocyte count, hemolysis workup, DAT, and PBS.
- Consider warm AIHA in patients with:
- Additional investigations: to rule out secondary causes (see “ .”)
Early specialist consultation is advised (e.g., hematology, intensive care).
- Goal: control hemolysis and improve anemia.
- Provide acute management for severe disease.
- Address potential secondary causes (see “Etiology”). 
- Hold any potentially causative medications.
- Identify and manage underlying conditions.
- Consider the need for chronic .
- Observation without immunosuppressive interventions may be appropriate for patients with mild asymptomatic anemia.
- Manage disease complications: VTE
- Prognosis: The disease course is often chronic with recurrent relapses, which require close monitoring and repeated therapeutic intervention.
|Management of warm agglutinin hemolytic anemia |
|Acute management|| |
|Chronic management|| |
Do not delay a potentially life-saving blood transfusion in unstable patients with severe anemia, even if crossmatched blood is unavailable. Use type-specific uncrossmatched blood in close consultation with the blood bank. 
Mechanical destruction of RBCs
Microangiopathic hemolytic anemia 
- Etiology 
- Features of anemia (e.g., pallor, fatigue)
- Organ dysfunction due to microthrombi formation (e.g., renal dysfunction, altered mental status)
- Petechiae due to thrombocytopenia
- Consider MAHA in patients with the following:
- Determine if MAHA are present. and/or a secondary cause of
- Measure ADAMTS13 activity if there is no obvious secondary cause of MAHA.
|Workup for underlying causes of MAHA|
|Suspected etiology||Supportive clinical features||Diagnostic testing and characteristic findings|
|Primary thrombotic microangiopathy||TTP|| || |
|Atypical HUS|| || |
|Drug-induced thrombotic microangiopathy || |
|Malignant hypertension|| |
|HELLP syndrome |
|Disseminated intravascular coagulation|
|Autoimmune disease (e.g., SLE)|| |
|Infection|| || |
- If an evident secondary cause is identified (e.g., HELLP syndrome, hypertensive emergency, DIC), treat accordingly.
- If suspicion for TTP is high:
- Refractory or relapsing TTP : requires treatment with intensified plasma exchange and systemic immunomodulators (e.g., caplacizumab or rituximab)
Macroangiopathic hemolytic anemia
- Congenital cardiovascular anomalies (e.g., bicuspid aortic valve, coarctation of the aorta) 
- Moderate and severe : usually resolves the anemia.
- In other patient groups: Prosthetic heart valves can cause anemia.
- Extracorporeal circulation, dialysis
- Exertional hemoglobinuria (“march hemoglobinuria”): RBC destruction in the feet during strenuous exercise (e.g., running on hard surfaces)
- Pathophysiology: RBC destruction in the systemic circulation (large vessels) due to mechanical forces applied to RBC membrane → intravascular hemolysis, schistocytes, ↑ free Hb
- Clinical features
- Address the underlying cause.