An erythrophage

Extravascular hemolysis occurs when RBCs are phagocytized by macrophages in the spleen, liver and bone marrow (see image of an erythrophage to the right). Extravascular hemolysis is the most common form of hemolytic anemia in animals. It usually occurs alone (without intravascular hemolysis), but will always (to some extent) accompany intravascular hemolysis. Note that during the normal aging of red cells in the circulation, effete red cells are destroyed by macrophages, i.e. extravascular hemolysis is always occurring to some degree. However, this is a physiologic process and does not result in anemia or excessive unconjugated bilirubin production.

With extravascular hemolysis, the erythrocytes are degraded within macrophages, so hemoglobin is not released free into the cytoplasm. Thus, we do not see hemoglobinemia or hemoglobinuria with extravascular hemolysis alone, unless it is accompanying intravascular hemolysis.

Within macrophages, the hemoglobin is broken down into its constituents, i.e. the heme ring (a porphyrin ring with iron in its center) and globin chains.

The globins are broken down to amino acids, which are then used for protein synthesis. The porphyrin ring of heme is oxidized by microsomal heme oxygenase, producing biliverdin and releasing the iron (Fe3+). The iron can then be exported into plasma through iron channels, where it binds to apotransferrin (the iron transport protein) forming transferrin or can be stored within cells as ferritin (i.e. the iron is bound to the storage protein, apoferritin). With time, ferritin becomes oxidized and degrades to form hemosiderin. Hemosiderin can be visualized within macrophages as a dusky blue-gray pigment and can be definitively stained with Prussian blue (which turns hemosiderin blue). Biliverdin is reduced by biliverdin reductase to unconjugated bilirubin (water insoluble). The unconjugated bilirubin is released into the plasma, where it binds to albumin (to render it water-soluble) and is taken up by hepatocytes.

There are many causes of extravascular hemolysis. Hemolytic anemias (intravascular or extravascular) are usually regenerative (if sufficient time is given for the marrow to regenerate and if there are no additional factors suppressing erythropoiesis).

• Immune-mediated hemolytic anemia (IMHA) :

  Immune-mediated hemolytic anemia in a dog. Many spherocytes (smaller RBCs which lack central pallor) can be seen in the smear.

  Attachment of IgG or IgM causes fixation of complement (to C3b) on red cell membranes. Macrophages possess receptors for the Fc portion of IgG and IgM as well as for C3b, thus causing red cells with attached immunoglobulin or C3b to be phagocytized (see image below). Partial phagocytosis of erythrocytes forms spherocytes which, in large numbers, are pathognomonic for IMHA. Note, that spherocytes are most readily seen in the dog, because central pallor is usually present in canine erythrocytes. The immunoglobulin- and complement-coated red cells can be detected in a direct Coombs test using a Coombs reagent, which consists of species-specific anti-Ig and/or anti-C3. Thus, a positive Coombs test is further supportive evidence of IMHA, but false positives and negatives do occur. IMHA can be primary or secondary to drugs (e.g. penicillin in horses) or erythroparasites.

Auto-reactive B cells secrete immunoglobulins (IgG and/or IgM) that recognize a "self" epitope on red blood cells. Strong complement-fixing antibodies result in formation of the membrane attack complex, which punches holes in the red cell membrane causing them to rupture within the circulation (intravascular hemolysis). Weaker complement-fixing antibodies only generate the opsonin C3b, which also attaches to RBC membranes. Ig- or C3b-bound red blood cells are destroyed by macrophages (which contain receptors for C3b and the Fc portion of immunoglobulins) as they traverse through organs such as the spleen (extravascular hemolysis). Multivalent antibodies, such as IgM, can crosslink adjacent red blood cells, resulting in agglutination.

• Erythroparasites: Many erythroparasites cause a hemolytic anemia due to extravascular hemolysis, e.g. Mycoplasma hemofelis (feline infectious anemia), Anaplasma bovis. With many of these organisms, there is a concurrent immune-mediated component to the anemia (the organisms make the red cells antigenic).
  
  
• Other organisms: Bacteria, such as Clostridium sp and Leptospira, can cause an extravascular hemolytic anemia, as can rickettsial and viral (e.g. equine infectious anemia) agents.
  

  Oxidant-induced hemolytic anemia due to acetaminophen toxicity in a cat. Large heinz bodies (arrowheads) can be seen on the erythrocytes.

  
  
• Oxidant injury: Oxidant injury (e.g. acetaminophen toxicity in cats) can result in extravascular hemolysis. Heinz bodies, eccentrocytes and pyknocytes are seen with oxidant injury (although this is species dependent). The Heinz body-containing red blood cells are removed prematurely from the circulation by macrophages (principally in the spleen). Inherited defects in red cell enzymes that help the red blood cell combat oxidant injury (e.g. glucose-6-phosphate dehydrogenase deficiency in horses) can result in an oxidant-induced hemolytic anemia.
  
  
• Fragmentation injury: This usually occurs secondary to vascular disease (e.g. hemangiosarcoma) or disseminated intravascular coagulation (DIC). Keratocytes, schistocytes, and acanthocytes are observed in peripheral blood in fragmentation anemias. A few spherocytes may be observed in fragmentation anemias and do not indicate immune-mediated disease in this setting. Fragmentation anemias may be non-regenerative as cytokines associated with the primary disease often suppress the bone marrow. Note that some degree of intravascular hemolysis does also occur with fragmentation injury (particularly when fibrin strands shear red cells in DIC), however the amount of hemoglobin released into the circulation is insufficient to cause visible hemoglobinemia or hemoglobinuria.
  
  
• Histiocytic disorders: In these disorders, instead of red cell destruction occuring due to the red cells being abnormal, they are destroyed because the macrophages are stimulated by cytokines (usually liberated from T cells, i.e. the macrophages are reactive) or are neoplastic (e.g. histiocytic sarcoma). Macrophage variants of histiocytic sarcoma have been identified in dogs (particularly large breeds, like Golden Retrievers and Labradors) and can produce an extravascular hemolytic anemia, that can mimic IMHA.
  
  
• Inherited red cell defects: Inherited defects of red cell enzymes (e.g. pyruvate kinase deficiency) and membranes (e.g. hereditary stomatocytosis) can result in extravascular hemolytic anemias. These have primarily been identified in dogs, but can also occur in cats.