Hemolytic anemia

Hemolytic anemias are a group of conditions characterized by the breakdown of red blood cells. Hemolysis is caused by either abnormalities in the RBCs themselves (in hemoglobin, the RBC membrane, or intracellular enzymes), known as corpuscular anemia, or by external causes (immune-mediated or mechanical damage), known as as extracorpuscular anemia. All hemolytic anemias result in varying degrees of fatigue, pallor, and weakness (from asymptomatic disease to life-threatening hemolytic crisis), although some forms of hemolytic anemia have more specific findings (e.g., venous thrombosis in paroxysmal nocturnal hemoglobinuria). They also have common laboratory findings, including elevated indirect bilirubin and lactate dehydrogenase, reticulocytosis, and decreased haptoglobin levels. The Coombs test helps to distinguish between autoimmune (positive Coombs test) and nonautoimmune anemias (negative Coombs test). Treatment involves RBC transfusions as required but otherwise depends on the specific type of hemolytic anemia and its causes.

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).

Type	Definition	Causes
By RBC pathology
Intrinsic hemolytic anemia	Increased destruction of RBCs due to a defect within the RBC	Erythrocytic membrane defects: hereditary spherocytosis, paroxysmal nocturnal hemoglobinuria (PNH) Enzyme defects: glucose-6-phosphate dehydrogenase deficiency (G6PD deficiency), pyruvate kinase deficiency (PKD) Hemoglobinopathies: sickle cell disease, thalassemia, hemoglobin C disease
Extrinsic hemolytic anemia	Abnormal breakdown of normal RBCs	Mechanical destruction in large vessels: e.g., prosthetic heart valves Shearing of RBCs due to occlusion of small vessels by microthrombi: e.g., hemolytic uremic syndrome (HUS), thrombotic thrombocytopenic purpura (TTP) Immune reactions due to infections (e.g., Mycoplasma) or tumors (e.g., chronic lymphocytic leukemia) Infections causing increased destruction of RBCs (e.g., Babesia, malaria, Bartonella bacilliformis) Isoimmune reactions: e.g., ABO/Rh incompatibility Autoimmune reactions: e.g., autoimmune hemolytic anemia (AIHA) Increased degradation by the spleen (hypersplenism)
By location of RBC breakdown
Intravascular hemolytic anemia	Increased destruction of RBCs within the blood vessels	Toxins (e.g., snake bites) and oxidizing agents (e.g., copper poisoning) [1][2] G6PD deficiency Immune-mediated anemia (PNH, transfusion ABO incompatibility, hemolytic anemia of the newborn, cold agglutinin disease) Macroangiopathic anemia (mechanical destruction by, e.g., prosthetic cardiac valves) Microangiopathic anemia (e.g., TTP, HUS, disseminated intravascular coagulation, HELLP syndrome, systemic lupus erythematosus)
Extravascular hemolytic anemia	Increased destruction of RBCs by the reticuloendothelial system (primarily the spleen)	RBC defects (e.g. sickle cell disease, spherocytosis, pyruvate kinase deficiency) Immune-mediated anemia (warm and cold agglutinin disease, PNH)

• 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]

Serum studies ^[5][6][7]

General findings

• ↓ Hemoglobin, hematocrit, and RBC count
• WBC count: can be increased in inflammation/malignancy
• Platelets: increased in microangiopathic hemolytic anemia
• ↑ Reticulocytes: loss of erythrocytes → reactive increase in erythropoiesis → increase in reticulocytes in the peripheral blood
• MCV: depends on the underlying condition and acuity of hemolysis
  • Decreased in thalassemia and normal or decreased in anemia of chronic disease (infection, malignancy, or inflammation)
  • Normal in most hemolytic anemias
  • Increased in severe reticulocytosis (when the bone marrow becomes highly active to compensate for the RBC loss) ^[7]
• Iron studies are usually normal.

Specific workup for hemolytic anemia ^[5][6]

• ↑ Lactate dehydrogenase (LDH): nonspecific parameter; indicates increased cellular breakdown
• ↓ Haptoglobin
  • Only free (unbound) haptoglobin is measured.
  • Hemoglobin released from broken down erythrocytes binds to haptoglobin, resulting in decreased free circulating haptoglobin.
  • Occurs mainly in intravascular hemolysis
  • Acute phase protein can be increased or normal as a response to inflammation and thus possibly mask hemolysis.
• ↑ Indirect (unconjugated) bilirubin:
  • Hemoglobin is released from RBCs
  • Heme is catabolized to unconjugated bilirubin.
  • Unconjugated bilirubin accumulates, resulting in jaundice.
• ↑ Urobilinogen in urine
  • Common in extravascular hemolysis
  • Unconjugated bilirubin binds to albumin.
  • Complexes of unconjugated bilirubin and albumin are transferred to the liver and metabolized into conjugated bilirubin.
  • Conjugated bilirubin is excreted via bile and turned into urobilinogen by colonic bacteria
  • Urobilinogen is reabsorbed into enterohepatic circulation and excreted in urine
• Hemosiderinuria ^[8]
  • Mostly occurs in intravascular hemolysis
  • Hemoglobin is taken up by proximal convoluted tubule.
  • Hemic iron is trapped within the epithelial cells of the tubule, forming hemosiderin.
  • Proximal tubule cells containing the hemosiderin are sloughed off and excreted into urine, lending it a brown color.
• In extreme hemolysis (e.g., hemolytic crisis): increased free hemoglobin →; hemoglobin binds to transport proteins when RBC are degraded in the bloodstream → if maximum capacity of these proteins is reached, free hemoglobin circulates in the bloodstream → hemoglobinuria → brown-colored urine

Coombs test ^[9]

This test detects antibodies and/or complement proteins on the surface of RBC; (direct test) or in serum (indirect test). The test uses Coombs serum, which contains anti-human globulins. A positive result in a patient with hemolysis supports the diagnosis of antibody-mediated, extracorpuscular anemia.

Direct Coombs test

• The blood sample is purified so that only erythrocytes remain.
• Coombs serum is added.
• The sample is analyzed for erythrocyte agglutination.
  • Erythrocyte agglutination indicates the test is positive meaning that the patient's erythrocytes are coated with autoantibodies.
  • If there is no erythrocyte agglutination, the test is negative.
• Indicated to confirm:
  • Autoimmune reactions (e.g., cold autoimmune hemolytic anemia)
  • Isoimmune reactions (e.g., hemolytic disease of the newborn)
  • Drug-induced immune reactions (e.g., methyldopa, penicillin)

Indirect Coombs test

• The patient's blood sample is purified so that only serum remains.
• A donor's blood (RBC) sample is added.
• Coombs serum is added as well.
• The sample is analyzed for erythrocyte agglutination.
  • Erythrocyte agglutination indicates the test is positive: Preexisting, circulating, freeantibodies within patient's serum bind to the surface of the donor's RBCs, causing agglutination.
  • Absence of erythrocyte agglutination indicates the test is negative.
• Indicated for detecting antibodies in:
  • Antenatal care (e.g., screening of pregnant women for antibodies that may cause hemolytic disease of the newborn)
  • Blood transfusion preparation (RBC phenotyping )

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

Peripheral blood smear ^[5][10]

• Used to detect pathologic RBC forms
• Common findings
  • Intravascular hemolytic anemia
    • Schistocytes
    • Heinz bodies and bite cells in G6PD deficiency
    • ↑ Reticulocytes
  • Extravascular hemolytic anemia
    • Sickle cells in sickle cell disease
    • Target cells in sickle cells disease, thalassemia, and hemoglobin C disease
    • Teardrop cells in thalassemia
    • Hemoglobin crystals within RBCs in hemoglobin C disease
    • Spherocytes in spherocytosis and possibly in cold and warm agglutinin disease
    • Smudge cells (Gumprecht shadows) in CLL
    • ↑ Reticulocytes

Additional findings ^[11][12]

• Hemoglobin electrophoresis: abnormal hemoglobin 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).

Erythrocytic membrane defects

Paroxysmal nocturnal hemoglobinuria (PNH)

• Definition: : an acquired genetic defect of the hematopoietic stem cell characterized by a triad of hemolytic anemia, pancytopenia, and thrombosis ^[13]
• Pathophysiology ^[14]
  • 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:
    • CD55/DAF (Decay accelerating factor)
    • CD59/MIRL (Membrane inhibitor of reactive lysis)
  • PNH can also occur in patients with aplastic anemia ^[15][16]
• Clinical symptoms
  • Pallor, excessive fatigue; , weakness
  • Intermittent jaundice
  • Episodes of hemoglobinuria causing pink/red/dark urine which usually occurs in the morning due to concentration of urine overnight. ^[17][13]
  • Venous thrombosis in unusual locations (e.g., hepatic, cerebral, and/or abdominal veins) ^[14]
  • Vasoconstriction ^[17]
    • Headache, pulmonary hypertension
    • Abdominal pain, dysphagia, erectile dysfunction
  • Increased risk of infections (in case of pancytopenia)
• Diagnostics ^[17]
  • CBC: anemia or pancytopenia ^[15][16]
  • Flow cytometry: absence of CD55 and CD59 on RBC surface
  • Coombs test: negative
  • Serum studies: decreased levels of haptoglobin
• Therapy
  • Prednisone for initial therapy
  • Eculizumab: anti-C5 antibody (complement inhibitor)
  • Bone marrow transplantation: in case of bone marrow hypoplasia ^[18]
  • Allogeneic stem cell transplantation
  • Iron and folate supplementation
• Complications
  • Vasoconstriction and thrombotic emboli leading to thrombotic complications, e.g., infarctions, Budd-Chiari syndrome^[19]
  • Increased risk of acute leukemias

Suspect PNH if a patient presents with hemolytic anemia, venous thrombosis, and pancytopenia!

Other examples

• Hereditary spherocytosis
• Hereditary elliptocytosis

Enzyme defects

Pyruvate kinase deficiency ^[20][21][22]

• 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-bisphosphoglcyerate → ↑ 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
• Therapy
  • Phototherapy and/or exchange transfusions
  • In the case of severe anemia or excessively enlarged spleen: splenectomy

Other examples

• Glucose-6-phosphate dehydrogenase deficiency
• Hexokinase deficiency
• Adenylate kinase deficiency

Hemoglobinopathies

Hemoglobin C disease ^[23]

• Pathophysiology: 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.
• Clinical features
  • Hemolytic anemia (usually mild)
  • Jaundice
  • Splenomegaly
  • Patients with HbSC gene mutation (one HbC and one HbS trait) have milder symptoms than HbSS patients.
• Diagnostics:
  • Peripheral smear in homozygous HbC disease is characterized by ^[24]
    • Anisopoikilocytosis
    • Target cells and spherocytes
    • Rod-shaped RBCs containing precipitated hemoglobin C crystals
  • ↑ MCHC
• Therapy: folic acid supplements

Hemoglobin Zurich ^[25]

• Pathophysiology: Replacement of the 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%)
• Clinical symptoms:
  • Often asymptomatic
  • Hemolysis upon exposure to certain chemicals and diet (e.g., sulfonamides, fava beans), stress, and infection
  • Binding of carbon monoxide stabilizes the hemoglobin chain; hence, smokers have less prominent hemolysis.
• Diagnosis: newborn screening
• Treatment
  • Avoid triggering agents
  • Severe hemolytic anemia: blood transfusion, plasmapharesis

Other examples

• Sickle cell disease
• Thalassemia

In hemoglobin C disease, there is an excess of lyCine (lysine) which substitutes the amino acid glutamic acid.

Isoimmune hemolytic anemia

• ABO incompatibility
• Rhesus incompatibility
• See hemolytic disease of the fetus and newborn.

Autoimmune hemolytic anemia (AIHA)

Cold agglutinin disease ^[26][27][28]

• Definition: an autoimmune disease characterized by cold-sensitive antibodies binding to RBC, which causes agglutination and lysis of these RBCs
• Etiology ^[27]
  • Cold-sensitive antibodies: have an optimal temperature for antigen-antibody reaction at 0–4°C ^[27]
    • Mostly IgM antibodies
    • Cause acute intravascular and extravascular hemolysis
    • Intravascular hemolysis is mediated through the complement system, which is activated by IgM antibodies bound to erythrocytes.
    • Antigen-antibody reaction is triggered by low body temperature and/or a cold ambient temperatures
  • Usually idiopathic
  • Secondary causes
    • Mycoplasma pneumoniae or EBV infection
    • Malignancy (e.g. non-Hodgkin lymphoma, chronic lymphocytic leukemia)
    • Waldenstrom macroglobulinemia
• Epidemiology: Median age at diagnosis is approximately 70 years. ^[29][30]
• Clinical features
  • 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
• Diagnosis ^[28]
  • Peripheral blood smear: spherocytes, polychromasia; , agglutination of RBCs
  • Coombs test: positive
  • ↑ Cold agglutinin titer
  • ↓ C3 and C4
• Therapy
  • Mild disease: Avoid cold exposure.
  • Moderate/severe disease: immunosuppressive therapy with rituximab (anti-CD20 antibody)
• Complications
  • Venous thromboembolism ^[31]
  • Associated lymphoproliferative disorders
• Prognosis: Spontaneous remission within a few weeks is common.

Cold weather is MMMMiserable: Cold (IgM) AIHA is seen in Malignancy (CLL), Mycoplasma pneumoniae, and Mononucleosis.

Warm agglutinin disease ^{[32][33][34][28][29]}

• Definition: an autoimmune disease characterized by heat-sensitive antibodies binding to RBC, which triggers inappropriate phagocytosis of these RBCs ^[34]
• Etiology:
  • Heat-sensitive antibodies
    • Mostly IgG antibodies that bind Fc receptor of phagocytes
    • Binding of the antibodies to RBCs leads to agglutination followed by ↑ extravascular hemolysis, which is mediated by the reticuloendothelial system of the spleen and liver. ^[35]
  • Triggered by increased body temperature (> 37°C/98.6° F)
  • Mostly idiopathic
  • Secondary causes
    • Malignancy (lymphoma, chronic lymphocytic leukemia)
    • Autoimmune diseases (e.g., SLE)
    • Certain drugs (e.g., rifampin, phenytoin, penicillins, α-methyldopa) ^[36]
• Clinical features
  • Onset of symptoms is typically gradual.
  • Signs of anemia (pallor, fatigue, weakness)
  • Mild splenomegaly
• Diagnosis
  • Peripheral blood smear: spherocytes, agglutinated RBCs,polychromasia
  • Coombs test: positive
• Therapy
  • Initial treatment of choice: glucocorticoids
  • If glucocorticoids are unsuccessful
    • IV immunoglobulin
    • Rituximab, especially in children
    • Splenectomy

“Warm weather is Great”: Warm AIHA is IgG mediated.

Microangiopathic hemolytic anemia ^[37][10]

• Etiology
  • Hemolytic uremic syndrome (HUS)
  • Thrombotic thrombocytopenic purpura (TTP)
  • Malignant hypertension
  • Systemic lupus erythematosus (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 passing through these small vessels → intravascular hemolysis, schistocytes, ↑ free hemoglobin
  • Characteristically accompanied by thrombocytopenia (otherwise normal coagulation)
• Clinical features
  • Features of anemia (e.g., pallor, fatigue)
  • Jaundice
  • Organ dysfunction due to microthrombi formation (e.g., renal dysfunction, mental status changes)
  • Petechiae due to thrombocytopenia
• Management: cause-specific treatment

Macroangiopathic hemolytic anemia

• Etiology
  • Congenital cardiovascular anomalies (e.g., bicuspid aortic valve, coarctation of the aorta) ^[38]
  • 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 hemoglobin
• Clinical features
  • Features of anemia (e.g., pallor, fatigue)
  • Jaundice