Intravascular hemolysis

 

Intravascular hemolysis results from the rupture or lysis of red blood cells within the circulation, i.e. the red cells are lysing in vivo. When the membrane of erythrocytes rupture, they release their hemoglobin into the plasma. The hemoglobin (which is a tetramer) breaks down into hemoglobin dimers in plasma. Haptoglobin (an α-2 globulin produced in the liver) binds the liberated free hemoglobin dimers. However, haptoglobin is readily saturated (this occurs at around a hemoglobin concentration of 150 mg/dL). If intravascular hemolysis continues, the hemoglobin dimers are in excess in plasma and are filtered readily through the glomerulus (because they are < 20 kd in size). This will cause a hemoglobinuria (see image below on right) and a positive reaction for heme protein on the dipstick (with no erythrocytes evident in the urine sediment). Because hemoglobin concentrations >20 mg/dL will cause visible discoloration of plasma (light pink to dark red, depending on how much hemoglobin is present), hemoglobinemia is often visible with intravascular hemolysis. The hemolytic index provided on Cornell University's chemistry panel is often quite high in patients with intravascular hemolysis (i.e. > 200 units). The image on the left shows severe hemolysis (red discolored supernatant plasma of blood centrifuged in a microhematocrit tube) in a dog with an immune-mediated hemolytic anemia (with intravascular hemolysis). The hemolytic index in such a patient would be > 500 units.
hemoglobinuria
Hemoglobinuria

The hemoglobin dimers that remain in circulation are oxidized to methemoglobin, which disassociates into a free heme and globin chains. The oxidized free heme (met-heme) binds to hemopexin (a β-globulin, Hpx) and the met-heme and hemopexin complex (met-heme/Hpx) is taken up by a receptor on hepatocytes and macrophages within the spleen, liver and bone marrow (only hepatocyte uptake is illustrated in the image above). Similarly, the hemoglobin/haptoglobin complex is taken up by hepatocytes and macrophages (to a lesser extent). Within these cells, the hemoglobin disassociates into heme and globin chains. The globins are broken down to amino acids, which are then used for protein synthesis. The heme is oxidized by heme oxygenase forming biliverdin and releasing iron. The iron can be transferred to apotransferrin (the iron transport protein) in plasma or can be stored within cells as ferritin (i.e. the iron is bound to the storage protein, apoferritin). The remaining porphyrin ring (biliverdin) is degraded to unconjugated bilirubin by biliverdin reductase. If the hemoglobin/haptoglobin complex is internalized by macrophages, the unconjugated bilirubin is released into the plasma, where it binds to albumin (to render it water-soluble) and is taken up by hepatocytes through the haptoglobin receptor. Thus, with intravascular hemolysis, increases in bilirubin are usually due to unconjugated bilirubin (indirect) and are likely of macrophage (rather than hepatocyte) origin. Note that it is unusual for intravascular hemolysis to occur alone, i.e. it is usually accompanied by extravascular hemolysis. This extravascular hemolysis is the likely source of most of the unconjugated bilirubin that is produced by macrophages in a hemolytic anemia. Because haptoglobin is consumed during intravascular hemolysis, serum values of this protein usually decline with intravascular hemolytic anemias or when hemoglobin is liberated into plasma by artifactual lysis of red cells in vitro (e.g. freezing of red cells, old samples - see below). Haptoglobin is a positive acute phase reactant and values will increase as part of the acute phase response (an evolutionary conserved innate response to inflammation, injury or infection). In fact, an increase in haptoglobin is one of the main reasons for the high α-2 peak seen in acute phase responses in serum electrophoresis. Corticosteroids will also increase serum values of haptoglobin in dogs.

The process of intravascular hemolysis with resulting hemoglobinemia, hemoglobinuria and bilirubinemia is illustrated in the image below.

Since heme oxygenase is also present in renal tubular cells, the renal epithelium is capable of converting hemoglobin to bilirubin. However, this only occurs when there is intravascular hemolysis with hemoglobinuria (i.e. the renal epithelium does not take up unconjugated bilirubin or hemoglobin from blood!). The renal epithelium absorbs the filtered hemoglobin from the urine, converting it to unconjugated bilirubin and then conjugating it for excretion into the urine (see image below). This may be responsible for some of the bilirubinuria seen in animals with intravascular hemolysis, however in most of these animals, there is concurrent cholestasis that is responsible for the bilirubinuria (which is conjugated).

renal conjugation

Note that red cells can also lyse or rupture in vitro (either in the blood collection tube or during collection). When this occurs, the hemolysis is considered an artifact and does not indicate the animal has a hemolytic anemia.

  • Artifactual hemolysis: Poor venipuncture technique, prolonged blood storage, exposure to temperature extremes (hot or cold enough to freeze the cells), and certain anticoagulants (fluoride-oxalate) will cause artifactual red cell lysis. Red cells are also more fragile in lipemic samples and tend to lyse more readily in these samples, even if the blood is stored or handled correctly. This artifactual red cell lysis can mimic intravascular hemolysis and it can be very difficult to tell them apart (particularly in the laboratory where all we see is the sample and not the patient). However, if the animal is anemic and has hemoglobinuria, true intravascular hemolysis, i.e. a pathological hemolytic anemia, is likely.

Intravascular hemolysis ("true" in vivo hemolysis) results in a hemolytic anemia in affected animals, but is far far less common than hemolytic anemias due to extravascular hemolysis in animals.

There are several causes of intravascular hemolysis:

  • Immune-mediated hemolytic anemia: Complement fixation by IgG or IgM causes assembly of the membrane attack complex (MAC, C6-C9) on red cell membranes in vivo which lyses the cells. A variant of an immune-mediated hemolytic anemia is an acute hemolytic transfusion reaction where transfusion of incompatible blood into an animal will cause acute intravascular hemolysis when antibodies bind to the transfused "foreign" red blood cells and activate the complement cascade.
    Babesia odicoilei infection in a reindeer with intravascular hemolytic anemia. Single (arrowhead) and paired (arrow) pyriform organisms are seen in several RBCs.

  • Erythroparasites: Babesia species replicate inside erythrocytes and rupture the cells when they exit to continue their life cycle. This results in an intravascular hemolysis. Indeed, Babesia bovis infections are often called "red-water" disease due to the accompanying hemoglobinuria.

  • Other organisms: Bacteria, such as Clostridium hemolyticum and Leptospira, can cause lysis of red cells in vivo. There have been reports of bee stings, spider bites and snake venoms causing a hemolytic anemia due to intravascular hemolysis (due to phospholipases in the venom).

  • Oxidant injury: Oxidant injury (e.g. copper poisoning in sheep or dogs, red maple toxicity in horses, zinc toxicity in dogs) can result in an intravascular hemolysis.
    Oxidant-induced hemolytic anemia due to ingestion of wilted red maple leaves in a horse. Eccentrocytes (black arrow) and pyknocytes (black arrowheads) indicate oxidant injury, whereas ghost cells (red arrow) indicate intravascular hemolysis.

  • Metabolic conditions: Acute liver disease in horses can result in intravascular hemolysis. Because phosphate is essential for ATP production and maintenance of the integrity of red cell membranes, intravascular hemolysis can occur with severe hypophosphatemia (e.g. phosphate -depleted dogs and cats with diabetes mellitus that are treated with insulin, post-partum hypophosphatemia in dairy cows).

  • Inherited red cell defects: Dogs with phosphofructokinase deficiency can suffer from bouts of intravascular hemolysis with exercise, due to alkaline fragility of their red blood cells.
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