20: Hemolysis and Hemolytic Anemia

• Hemolysis is an increased rate of red blood cell (RBC) destruction, shortening their lifespan.

Hemolytic Disorder vs. Hemolytic Anemia

• Hemolytic disorder: increased RBC destruction, potentially without anemia if bone marrow compensates.
• Hemolytic anemia: RBC destruction exceeds bone marrow's production capacity, resulting in lower hemoglobin and hematocrit levels.

Laboratory Findings in Hemolytic Disorder

• Hemoglobin levels can be normal if the bone marrow compensates effectively.
• Elevated reticulocyte count indicates increased RBC production by the bone marrow.

Laboratory Findings in Hemolytic Anemia

• Decreased hemoglobin levels indicate anemia.
• Elevated reticulocyte count, but not enough to compensate for RBC destruction.
• Elevated lactate dehydrogenase (LDH) released from lysed cells.
• Decreased haptoglobin levels due to binding with free hemoglobin.
• Increased indirect (unconjugated) bilirubin due to heme catabolism.
• Presence of schistocytes (fragmented RBCs) in peripheral blood smears.

Classifying Hemolytic Anemias

• Acute vs. chronic
• Inherited vs. acquired
• Intrinsic vs. extrinsic
• Intravascular vs. extravascular
• Fragmentation vs. macrophage-mediated

Mechanism of Hemolysis

• Fragmentation: physical fragmentation of RBCs due to shear stress, as in microangiopathic hemolytic anemia (MAHA).
• Macrophage-Mediated: RBCs destroyed by macrophages, often due to immunologic causes, as in autoimmune hemolytic anemia (AIHA).

Acute vs. Chronic Hemolysis

• Acute Hemolysis: rapid RBC destruction with sudden triggers.
• Examples: paroxysmal cold hemoglobinuria (PCH) and hemolytic transfusion reactions.
• Chronic Hemolysis: persistent low-level hemolysis with periodic exacerbations.
• Examples: glucose-6-phosphate dehydrogenase (G6PD) deficiency and sickle cell disease.

Inherited vs. Acquired Hemolytic Anemias

• Inherited: genetic defects.
• Examples: thalassemia and hereditary spherocytosis.
• Acquired: external factors.
• Examples: autoimmune hemolytic anemia (AIHA) and malaria.

Intrinsic vs. Extrinsic RBC Defects

• Intrinsic Defects: abnormalities within RBCs.
• Examples: membrane defects (hereditary spherocytosis), enzyme deficiencies (G6PD deficiency), and hemoglobinopathies (sickle cell disease).
• Extrinsic Defects: external factors.
• Examples: immune-mediated (AIHA), mechanical damage (prosthetic heart valves), infectious agents (malaria), and chemical agents.

Site of Hemolysis

• Intravascular Hemolysis: occurs within blood vessels.
• Results in hemoglobinemia and hemoglobinuria.
• Extravascular Hemolysis: occurs primarily in the spleen and liver.
• Characterized by splenomegaly.

Fragmentation (Intravascular) Hemolysis

• Trauma to RBC membrane causes contents to spill directly into plasma.
• Schistocytes form, and hemoglobin is released into the blood as α/β dimers.
• Haptoglobin binds hemoglobin dimers.
• Hemoglobin-haptoglobin complex binds to CD163 on macrophages.
• The complex is internalized, hemoglobin is degraded to heme, iron is released, and protoporphyrin is converted to unconjugated bilirubin.
• Haptoglobin is degraded.
• Free hemoglobin is oxidized to methemoglobin; heme dissociates from globin.
• Hemopexin binds free metheme.
• Hemopexin-metheme complex binds to CD91 on hepatocytes.
• Metheme is converted to unconjugated bilirubin.
• Hemopexin is recycled.

Macrophage-Mediated (Extravascular) Hemolysis

• RBCs are engulfed by macrophages and lysed inside via digestive enzymes.
• Occurs primarily in the spleen and liver.
• Hemoglobin is degraded into heme, releasing iron, and protoporphyrin is converted to unconjugated bilirubin.
• Macrophages release unconjugated bilirubin into the blood, binding to albumin for transport to the liver.
• Unconjugated bilirubin enters the hepatocyte and is converted to conjugated bilirubin.
• Conjugated bilirubin exits via bile to the small intestine.
• Bacteria convert conjugated bilirubin to urobilinogen, excreted in stool.
• Some urobilinogen is reabsorbed, recycled, and excreted.

Key points in common between Intravascular and Extravascular Hemolysis

• Processes of Hemolysis
  • Intravascular hemolysis: Occurs due to mechanical trauma to RBCs within circulation, leading to the release of hemoglobin into the plasma. Hemoglobin binds haptoglobin and is processed via macrophages and hepatocytes.
  • Extravascular hemolysis: Occurs when RBCs are engulfed by macrophages, primarily in the spleen and liver, where hemoglobin is broken down into its components.
• Sites of Hemolysis
  • Intravascular: Within blood vessels.
  • Extravascular: Within macrophages of the spleen and liver.
• Catabolic Products
  • Both pathways produce:
    • Unconjugated bilirubin (from protoporphyrin breakdown)
    • Iron (which is recycled)
    • Methemoglobin & hemopexin complexes (specific to intravascular hemolysis)
    • Urobilinogen & stercobilin (from bilirubin metabolism, particularly in extravascular hemolysis)
• Time Frame for the Appearance of Products
  • Hemoglobin and haptoglobin complexes appear immediately after intravascular hemolysis.
  • Unconjugated bilirubin is released into plasma soon after hemolysis, peaks within hours, and is conjugated in the liver.
  • Urobilinogen and bilirubin excretion occur over the next several hours to days as part of normal metabolic processing.

Protoporphyrin Ring Catabolism

• Heme Oxygenase: removes iron from heme, forming biliverdin, and also opens the protoporphyrin ring.
• Biliverdin Reductase: converts biliverdin to unconjugated bilirubin.

Metabolites and Sites of Production

• Unconjugated Bilirubin: secreted into the blood, binds to albumin, and is transported to the liver.
• Hepatocyte (Liver Cell): converts unconjugated bilirubin to conjugated bilirubin via UGT1A1.
• Conjugated Bilirubin: referred to as bisglucuronosyl bilirubin, is further processed and excreted.

Mechanisms That Salvage Hemoglobin and Heme During Fragmentation Hemolysis

• Fragmentation of RBCs:
• Trauma causes membrane breach, releasing hemoglobin into plasma, accounting for 10-20% of normal RBC breakdown.
• Haptoglobin-Hemopexin-Methemalbumin System:
  • Haptoglobin Mechanism:
    • Haptoglobin binds hemoglobin dimers, preventing filtration and saving iron.
    • The complex sequesters hemes, protecting cells from oxidative properties.
    • Macrophages internalize the complex via CD163, salvaging iron and converting protoporphyrin to unconjugated bilirubin.
  • Hemopexin Mechanism:
    • Free hemoglobin oxidizes to methemoglobin.
    • Metheme binds to hemopexin, saving iron and preventing oxidant injury.
    • The complex is internalized by hepatocytes via CD91, where iron is salvaged and protoporphyrin is converted to unconjugated bilirubin. Preventing Oxidative Damage
• Nitric Oxide Scavenging:
  • Hemoglobin and heme scavenge nitric oxide, causing oxidative damage.
  • Binding to haptoglobin and hemopexin prevents oxidative damage. Additional Mechanisms
• Iron Salvage and Oxidation Prevention:
  • Normal versus Accelerated Fragmentation Hemolysis:
    • Haptoglobin levels are adequate in normal fragmentation, but can be depleted in diseases like sickle cell, hemolytic transfusion reactions, and sepsis.

Changes to Bilirubin Metabolism During Excessive Fragmentation Hemolysis

• Increased RBC Removal - During increased macrophage-mediated hemolysis, more than the usual number of red blood cells (RBCs) are removed from circulation daily. This occurs due to the expression of surface markers on senescent or defective RBCs, leading to their premature removal by macrophages.
• Rise in Total Plasma Bilirubin
  • As RBCs are lysed prematurely, the total plasma bilirubin level rises, primarily due to an increase in the unconjugated bilirubin fraction. Unconjugated bilirubin is produced from the degradation of hemoglobin within macrophages.
• Liver Processing:
  • A healthy liver processes the increased load of unconjugated bilirubin by converting it to conjugated bilirubin. Conjugated bilirubin is then excreted into the intestine.
• Increased Urobilinogen Formation: - In the intestines, increased conjugated bilirubin is converted to urobilinogen by bacteria. The urobilinogen is subsequently absorbed by the portal circulation and excreted by the kidneys.
• Urine Detection: - Increased urobilinogen is detectable in the urine because it is absorbed into the portal circulation and incompletely reprocessed by the liver. Although unconjugated bilirubin levels rise in the plasma, it does not appear in the urine because it is bound to albumin and cannot pass through the glomerulus.

Changes to Iron Salvage Systems During Excessive Fragmentation Hemolysis

• Excessive Fragmentation Hemolysis: - Excessive fragmentation hemolysis can be caused by traumatic physical lysis of RBCs, such as from prosthetic heart valves or intracellular parasites like malaria protozoa. This results in a significant release of RBC contents, including hemoglobin, into the plasma.
• Hemoglobinemia: - The development of (met)hemoglobinemia occurs as free hemoglobin and its oxidized form, methemoglobin, appear in the plasma. Iron salvage proteins form complexes with their respective ligands, such as hemoglobin-haptoglobin, metheme-hemopexin, and metheme-albumin.
• The haptoglobin-hemopexin-methemalbumin system plays a crucial role in salvaging hemoglobin and heme during hemolysis.
  • هHaptoglobin: Haptoglobin binds to free hemoglobin dimers, forming a complex that prevents filtration through the glomerulus and saves iron from urinary loss.
    • The hemoglobin-haptoglobin complex is internalized by macrophages, where iron is salvaged, globin is catabolized, and protoporphyrin is converted to unconjugated bilirubin.
  • Hemopexin:
    • Hemopexin binds free metheme (oxidized heme), preventing oxidative damage and saving iron from urinary loss.
    • The metheme-hemopexin complex is internalized by hepatocytes, where iron is salvaged, and protoporphyrin is converted to unconjugated bilirubin.
  • Macrophage Role:
    • Macrophages express receptors such as CD163 for haptoglobin-hemoglobin complexes and CD91 for hemopexin-metheme complexes. These receptors facilitate the internalization and processing of these complexes.
• Prevention of Heme Toxicity: - Hemopexin plays a critical role in preventing heme toxicity to other cells. Macrophages possess a heme exporter, FLVCR, to rid themselves of excess heme, which is then accepted by hemopexin.

Tests Indicating Increased Hemolysis

• Bilirubin Levels:
  • Increased plasma unconjugated bilirubin and carbon monoxide are indicators of increased hemoglobin catabolism.
  • Elevated bilirubin in serum or plasma causes icteric serum. Assays showing increased indirect bilirubin result in higher total bilirubin levels.
  • Increased urinary urobilinogen can also indicate increased hemolysis.
• Plasma and Urine Hemoglobin: - Visual examination of plasma and urine can suggest fragmentation hemolysis, with color changes indicating the presence of hemoglobin degradation products. - The presence of hemoglobin/heme in urine suggests that plasma salvage systems are exceeded. - Urine Hemosiderin: Detection of hemosiderin (iron) in urine indicates past hemoglobinuria.
• Glycated Hemoglobin:
  • Reduced glycated hemoglobin levels indicate shortened RBC survival in chronic hemolytic disease.
  • Lower glycated hemoglobin values reflect decreased RBC lifespan due to early lysis. Variability in RBC life span among individuals affects interpretation, necessitating baseline values for accuracy.
• Lactate Dehydrogenase (LDH):
• Increased serum LDH activity points to fragmentation hemolysis due to enzyme release from ruptured RBCs. Elevated LDH levels suggest fragmentation hemolysis rather than damage to other organs.

Tests Indicating Increased Erythropoiesis

• Reticulocyte Count:
  • Increased circulating reticulocytes indicate heightened erythropoiesis in response to hypoxia from hemolysis.
  • A rise in reticulocyte count, along with nucleated RBCs in severe cases, points to active erythropoiesis.
• Complete Blood Count (CBC):
  • CBC provides clues to hemolytic processes, reflecting anemia severity and RBC morphology.
  • Hemoglobin, hematocrit, and RBC counts indicate anemia presence.
  • Spherocytes are associated with macrophage-mediated hemolysis, while fragmented cells (schistocytes) indicate fragmentation hemolysis.
• Haptoglobin and Hemopexin:
  • Declines in serum haptoglobin levels indicate increased hemolysis.
  • Substantial declines in haptoglobin levels point to fragmentation hemolysis, whereas modest declines may occur in macrophage-mediated hemolysis. Hemopexin assays complement haptoglobin measurements for a comprehensive hemolytic profile.
• Carbon Monoxide:
  • Rates of endogenous carbon monoxide production reflect heme breakdown rates.
  • Elevated carbon monoxide production indicates increased hemolysis, though not typically required for clinical diagnosis.
  • Red Blood Cell Survival:
  • RBC survival assays using chromium-51 radioisotope measure RBC lifespan. Shorter chromium half-times (e.g., less than 15 days) indicate severe hemolysis.

Differentiation Between Hemolytic Anemias and Other Causes of Increased Erythropoiesis

• Hemolytic Anemias:
  • Cause: Increased RBC destruction, leading to compensatory erythropoiesis.
  • Laboratory Findings:
    • Elevated reticulocyte count.
    • Evidence of hemolysis: elevated lactate dehydrogenase (LDH), indirect bilirubin, and decreased haptoglobin.
    • Peripheral smear: Schistocytes, spherocytes, or other hemolytic features.
    • Normal or slightly elevated ferritin, B12, and folate levels.
  • Clinical Features:
    • Fatigue, pallor, jaundice, dark urine, and possible splenomegaly.
• Post-Acute Hemorrhage:
• Cause: Blood loss stimulates bone marrow erythropoiesis.
• Laboratory Findings:
  • Elevated reticulocyte count without hemolysis markers.
  • Decreased hemoglobin, hematocrit, and total protein (if early).
  • Ferritin may remain normal unless chronic blood loss occurs.
• Clinical Features:
  • History of trauma, surgery, or overt bleeding.
• Recovery From Iron, B12, or Folate Deficiency:
  • Cause: Correction of deficiency leads to increased erythropoiesis.
  • Laboratory Findings:
    • Elevated reticulocyte count, improving hemoglobin.
    • Correction of low ferritin (iron deficiency), elevated homocysteine/methylmalonic acid (B12 deficiency), or reduced RBC folate levels.
  • Absence of hemolysis markers.
  • Clinical Features:
    • Improvement in symptoms of anemia after treatment initiation.

Differentiation Between Hemolytic Anemias and Other Causes of Bilirubinemia

• Hemolytic Anemias:
  • Cause: RBC breakdown releases heme, increasing unconjugated bilirubin.
  • Laboratory Findings:
    • Elevated indirect (unconjugated) bilirubin, LDH, and reticulocytes.
    • Decreased haptoglobin due to binding of free hemoglobin.
    • Peripheral smear: Evidence of hemolysis.
  • Clinical Features:
    • Jaundice, dark urine (hemoglobinuria or urobilinogen).
• Internal Bleeding: Cause: Blood accumulation in tissues leads to heme degradation and bilirubin production.
  • Laboratory Findings:
    • Elevated unconjugated bilirubin (mild, not as pronounced as hemolysis).
    • Normal LDH, haptoglobin, and reticulocyte count (no hemolysis).
  • Anemia without hemolytic findings. Clinical Features: History of trauma or bleeding into body cavities. Hepatic Dysfunction:
  • Cause: Impaired bilirubin conjugation in the liver.
  • Laboratory Findings:
    • Elevated unconjugated bilirubin but may also have elevated conjugated bilirubin.
  • Normal hemolysis markers unless coexisting conditions exist.
  • Clinical Features:
  • Signs of liver disease. Biliary Obstruction:
  • Cause: Impaired bilirubin excretion.
  • Laboratory Findings:
  • Predominantly elevated conjugated bilirubin.
  • Normal hemolysis markers.
  • Clinical Features:
    • Pale stools, dark urine, pruritus, and abdominal pain.
• Summary
  • Hemolytic anemias are uniquely characterized by markers of hemolysis (elevated LDH, indirect bilirubin, reticulocytosis, and decreased haptoglobin) and RBC morphology changes on smear.
  • Other causes of increased erythropoiesis lack hemolysis findings.
  • Bilirubinemia in hemolytic anemias arises from unconjugated bilirubin due to RBC destruction, whereas other conditions involve different mechanisms and bilirubin profiles.