Hemolytic anemia

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.

See also “Sickle cell disease”, “Paroxysmal nocturnal hemoglobinuria”, and “Autoimmune hemolytic anemia.”

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).

• Signs of anemia
  • Pallor
  • Fatigue
  • Exertional dyspnea
  • In severe cases: tachycardia, angina pectoris, leg ulcers
• Signs of hemolysis
  • Jaundice
  • Pigmented gallstones
  • Splenomegaly
  • Back pain and dark urine in severe hemolysis with hemoglobinuria
• Signs of increased hematopoiesis (mostly in severe chronic anemias, e.g., thalassemia)
  • Bone marrow expansion: widening of the diploic space of the skull, biconcave deformity of the vertebral bodies
  • Cortical thinning and softening of bone → ↑ risk of pathologic fractures
  • Extramedullary hematopoiesis: hepatosplenomegaly

References: ^[3][4]

Consider hemolysis in patients with acute or chronic anemia in whom an obvious cause (e.g., bleeding) is not apparent. See “Diagnosis of anemia” for details on the general approach for a patient with anemia. ^[5]

Approach ^[5][6]

1. Perform initial laboratory studies to confirm anemia and hemolysis and classify anemia by morphology.
   • Anemia workup: CBC with MCV, reticulocyte count
   • Hemolysis workup: Add LDH, haptoglobin, bilirubin, urinalysis, and peripheral blood smear (PBS)
2. Obtain a direct Coombs test (i.e., DAT) to narrow the differential:
   • DAT positive: antibody-mediated hemolytic anemia
   • DAT negative: nonantibody-mediated hemolytic anemia
3. Consider further investigations of the underlying etiology based on clinical suspicion and DAT results.

Rule out hemolysis in any patient with unexplained anemia, even if the urine dipstick test is negative for blood and jaundice is not evident on physical examination.

If the patient has severe symptoms of anemia or a life-threatening cause is suspected (i.e., TTP/HUS, disseminated intravascular coagulation, HELLP syndrome, acute hemolytic transfusion reaction), proceed directly to treatment in parallel with diagnostic evaluation.

Routine laboratory studies ^[5][6]

• CBC
  • ↓ Hb, Hct, and RBC count ^[6]
  • MCV
    • Normocytosis: typical finding for most hemolytic anemias; consider other anemia of chronic disease
    • ↑ MCV: Can be due to severe reticulocytosis ; consider other causes of macrocytosis (e.g., megaloblastic anemia)
    • Microcytosis: consider thalassemia and/or iron deficiency
  • WBC count: can be elevated due to inflammation or malignancy
  • Platelets: decreased in MAHA and in Evan syndrome
• Reticulocyte count: Use the corrected reticulocyte count for patients with anemia.
  • Reticulocytosis: most common ;
  • Can be normal in patients with:
    • A delayed bone marrow response
    • Concomitant bone marrow suppression or disease ^[6]
  • Reticulocyte production index: usually ≥ 2% in hemolytic anemia ^[7][8]
  • See also “Basics of hematology” and “Hematological parameters.”

Iron studies are usually normal in hemolytic anemia, however, iron deficiency can be seen in chronic intravascular hemolysis ^[9]

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).

Serum studies

Haptoglobin levels can be low in both intravascular hemolysis and extravascular hemolysis and, therefore, should not be used to differentiate between the two. ^[10]

Urine studies

• 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) ^[16]

This is a key test in the workup of hemolytic anemia.

• Goal
  • Detection of antibodies and/or complement proteins on the surface of RBCs
  • Narrowing the differential of hemolysis into two groups:
    • Antibody-mediated hemolysis
    • Non-antibody-mediated hemolysis
• Indications: All patients with hemolysis
• Method
  • The patient's blood sample is purified so that only RBCs remain.
  • Coombs serum is added and AHGs bind to the patient's antibodies and/or complement already present on the surface of their RBCs, leading to RBC agglutination.
• Positive result
  • Indicates that the patient's RBCs are coated with autoantibodies and/or complement that are causing hemolysis. ^[17]
  • Etiologies include:
    • Autoimmune hemolytic anemia (warm AIHA or cold AIHA)
    • Alloimmune hemolytic anemia
      • Transfusion reaction
      • Hemolytic disease of the newborn
    • Drug-induced immune-mediated hemolysis (e.g., due to methyldopa, penicillin)
• Negative result: Suggests non-antibody-mediated hemolysis

Indirect Coombs test ^[18]

• Goal: is the detection of anti-RBC antibodies in the serum of, for example:
  • Transfusion recipients
  • Patients with hemolytic disease of the fetus and newborn (HDFN)
  • Pregnant women at risk of developing antibodies that cause HDFN
• Indications
  • Any patient requiring pretransfusion testing, e.g., blood typing
  • Patients with suspected transfusion reactions
  • Newborns with signs of hemolysis
  • Screening for Rh-negative mothers to determine if anti-D immunoglobulin is indicated
• Method
  • 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
  • Indicates the presence of freely circulating anti-RBC antibodies present in the patient's serum that may be responsible for HDFN or transfusion reactions.
  • Etiologies include:
    • RBC alloimmunization in transfusion recipients or newborns
    • Maternal alloimmunization
• Negative result: No detectable anti-RBC antibodies

Differences between the Coombs tests

The direct Coombs test detects antibodies that are directly attached to the RBC surface. The indirect (or not direct) Coombs test detects serum antibodies that are not bound to RBCs.

Perform further diagnostic workup according to the suspected etiology; specialist consultation is advised. ^[17][19]

• Hb electrophoresis: abnormal Hb patterns, e.g., in thalassemia
• Flow cytometry
  • Absence of CD55 and CD59 on RBC surface in PNH
  • Detection of CD19, CD20, and CD23, light chain restriction (kappa or lambda) in CLL
• Genetic analysis: mutations in congenital hemolytic anemia
• Bone marrow biopsy
  • Rarely used in the workup of anemia due to its invasiveness
  • Indication: pancytopenia, presence of abnormal cells (e.g., blasts) in CBC/peripheral blood smear
  • Pathologic findings are most common in malignancies that replace bone marrow (e.g., CLL).

Antibody-mediated hemolysis (DAT positive)

Nonantibody-mediated hemolysis (DAT negative)

Hemolytic anemia that is caused by structural or functional RBC abnormalities can be further classified as one of the following based on pathogenesis:

• RBC membrane defects
  • Paroxysmal nocturnal hemoglobinuria
  • Hereditary spherocytosis
  • Hereditary elliptocytosis
• Enzyme defects
  • Pyruvate kinase deficiency
  • Glucose-6-phosphate dehydrogenase deficiency
  • Hexokinase deficiency
  • Adenylate kinase deficiency
• Hemoglobinopathies
  • Hemoglobin C disease
  • Hemoglobin Zurich
  • Sickle cell disease
  • Thalassemia

See respective articles for more detailed information.

• Etiology: autosomal recessive defect of pyruvate kinase
• Pathophysiology
  • 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
  • Seldom asymptomatic
  • Typically newborn jaundice due to hemolysis and history of exchange transfusions
  • Splenomegaly
  • Pallor, fatigue, weakness
  • In rare cases: hydrops fetalis
• Diagnosis
  • ↓ Pyruvate kinase enzyme activity
  • PKLR gene mutation
  • Blood smear: burr cells
• Therapy
  • Phototherapy and/or exchange transfusions
  • In the case of severe anemia or excessively enlarged spleen: splenectomy

References ^[22][23][24]

Definitions

• 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)

Pathophysiology ^[25]

Glutamic acid can also be replaced with a lysine, creating hemoglobin C.

• 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 RBC dehydration (↑ MCHC).
• RBCs have reduced oxygen-binding capacity and a shorter lifespan.

In hemoglobin C disease, lyCine (lysine) replaces the amino acid glutamic acid.

Clinical features ^[25]

• Hemolytic anemia (usually mild)
• Cholelithiasis
• Jaundice
• Splenomegaly
• Patients with HbSC gene mutation (one HbC and one HbS trait) have milder symptoms than HbSS patients.

Diagnostics

Treatment ^[28]

• Most patients do not require treatment.
• Consider folic acid supplements in hemoglobin C disease.
• Consider cholecystectomy in patients with symptomatic gallstones.
• Splenectomy is rarely indicated.

• Pathophysiology
  • Replacement of distal histidine in the beta-globin chain with arginine → enlargement of the ligand-binding space around iron → increased affinity for carbon monoxide → increased carboxyhemoglobin levels (≥ 3%)
  • Oxidative stress → Formation of Heinz body and hemolysis
• Clinical features
  • Often asymptomatic
  • Hemolysis upon exposure to certain drugs (e.g., sulfonamides), stress, and infection
• Diagnostics
  • Laboratory findings: normocytic anemia, reticulocytosis
  • Genetic testing
• Treatment
  • Avoid triggering agents.
  • Severe hemolytic anemia: blood transfusion, plasmapheresis

References: ^[29]

Hemolytic anemia that is caused by the destruction of functionally and structurally normal RBCs can be further classified as one of the following based on the mechanism of RBC destruction:

• Antibody-mediated destruction of RBCs
  • Alloimmune hemolytic anemia
    • Hemolytic transfusion reaction
    • Hemolytic disease of the fetus and newborn
  • Autoimmune hemolytic anemia
    • Cold agglutinin hemolytic anemia
    • Warm agglutinin hemolytic anemia
• Mechanical destruction of RBCs
  • MAHA
  • Macroangiopathic hemolytic anemia
• Other
  • Toxin-mediated RBC destruction, e.g., from a snake bite, oxidizing agents (e.g., dapsone, nitrofurantoin, phenazopyridine, primaquine) ^[5]
  • RBC destruction by intracellular pathogens, e.g., in malaria, babesiosis, bartonellosis, rickettsiosis
  • RBC destruction due to hypersplenism

See respective articles for more detailed information.

Microangiopathic hemolytic anemia ^[5][30]

Background

• Etiology ^[31]
  • Primary MAHA
    • Thrombotic thrombocytopenic purpura (TTP)
    • Hemolytic uremic syndrome (HUS)
  • Secondary MAHA ; causes include:
    • Autoimmune disease (e.g., SLE)
    • HELLP syndrome
    • Hypertensive emergency
    • Disseminated intravascular coagulation (DIC)
    • Drug induced (e.g., quinine, trimethoprim/sulfamethoxazole, cyclosporine)
• Pathophysiology
  • Systemic microthrombi plug small vessels → physical intravascular shearing of RBCs that pass through the small vessels → intravascular hemolysis, schistocytes, and ↑ free Hb
  • Characteristically accompanied by thrombocytopenia

Clinical features

• Features of anemia (e.g., pallor, fatigue)
• Jaundice
• Organ dysfunction due to microthrombi formation (e.g., renal dysfunction, altered mental status)
• Petechiae due to thrombocytopenia

Diagnostics

• Consider MAHA in patients with the following:
  • CBC showing anemia and thrombocytopenia
  • Laboratory evidence of hemolysis with a negative DAT
  • PBS showing abundant schistocytes (typically ≥ 2 per high-power field)
• Determine if end-organ damage and/or a secondary cause of MAHA are present.
  • Order routine studies: e.g., coagulation studies, renal function tests, liver chemistries
  • Order further studies based on the patient's clinical presentation: e.g., ECG, cardiac biomarkers, CT head (see “Workup for underlying causes of MAHA”).
• Measure ADAMTS13 activity if there is no obvious secondary cause of MAHA.
  • ADAMTS13 activity ≥ 10%: TTP unlikely
  • ADAMTS13 activity < 10%: TTP likely

Management ^[31][34]

• If an evident secondary cause is identified (e.g., HELLP syndrome, hypertensive emergency, DIC), treat accordingly.
• If suspicion for TTP is high:
  • Do not await ADAMTS13 activity results.
  • Refer for urgent plasma exchange.
  • Consider corticosteroid therapy (e.g., prednisone ).
• Refractory or relapsing TTP : requires treatment with intensified plasma exchange and systemic immunomodulators (e.g., caplacizumab or rituximab)

MAHA is a medical emergency. Consult hematology urgently, especially if there is no immediately evident cause (e.g., hypertensive emergency, preeclampsia).

Consider urgent initiation of plasma exchange for suspected TTP while waiting for ADAMTS13 test results.

Macroangiopathic hemolytic anemia

• Etiology
  • Congenital cardiovascular anomalies (e.g., bicuspid aortic valve, coarctation of the aorta) ^[37]
  • Moderate and severe aortic stenosis; : Heart valve replacement 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
  • Features of anemia (e.g., pallor, fatigue)
  • Jaundice
• Diagnostics
  • Evidence of hemolysis (e.g., ↑ Indirect bilirubin, ↑ LDH, ↓ haptoglobin, reticulocytosis) ^[38]
  • DAT: Negative
  • Blood smear: Schistocytes ^[38]
  • Consider echocardiography ^[39]
• Treatment
  • Address the underlying cause. ^[39]