Every hemolytic condition can be classified according to each of these descriptors. Table 22-1 shows this and provides a noncomprehensive list of hemolytic anemias. This chapter focuses on the distinction between intravascular and extravascular hemolytic conditions. The other classifying schemes are summarized here briefly and are used to organize the chapters that follow.

TABLE 22-1

Classification of Selected Hemolytic Anemias by Primary Cause and Type of Hemolysis

Acute versus chronic hemolysis delineates the clinical presentation. Acute hemolysis has a rapid onset and is isolated (sudden), episodic, or paroxysmic, as in paroxysmal cold hemoglobinuria or paroxysmal nocturnal hemoglobinuria. Patients with paroxysmal cold hemoglobinuria experience hemolysis only after exposure to cold, and patients with paroxysmal nocturnal hemoglobinuria experience hemolysis while sleeping. Whatever the cause, acute hemolysis either disappears or subsides between episodes, during which time the patient’s condition returns to normal. A hemolytic transfusion reaction is an example of a single acute incident.

Chronic hemolysis may not be evident if the bone marrow is able to compensate, but may be punctuated over time with hemolytic crises that cause anemia. Glucose-6-phosphate dehydrogenase deficiency is such a condition. RBC life span is chronically shortened, but bone marrow compensation prevents anemia. When the cells are challenged with oxidizing agents such as antimalarial drugs, a dramatic acute hemolytic event occurs. When the drug is withdrawn, compensation returns.

Other chronic conditions result in anemia that is so severe that the bone marrow cannot generate cells fast enough to compensate for the anemia. Thalassemia is an example of such a condition. Although cell production is brisk, each cell possesses an inadequate complement of one type of globin, and hemoglobin (Hb) production is decreased. As a result the oxygen-carrying capacity of the blood is chronically low. Cells lyse in thalassemia because excess normal globin chains precipitate inside the cells, which leads to hemolysis and chronic anemia.

Inherited hemolytic conditions, such as thalassemia, are passed to offspring by mutant genes from the parents. Acquired hemolytic disorders develop in individuals who were previously hematologically normal but acquire an agent or condition that lyses RBCs. Infectious diseases such as malaria are an example.

The hemolytic conditions are also classified as involving intrinsic or extrinsic RBC defects, with the latter caused by the action of external agents. This is the classification scheme used for subsequent chapters in this book. Examples of intrinsic hemolytic disorders are abnormalities of RBC membranes, enzymatic pathways, or the hemoglobin molecule. With intrinsic defects, if the RBCs of the affected patient were to be transfused into a healthy individual, they would still have a shortened life span, because the defect is in the RBC. If normal RBCs are transfused into a patient who has an intrinsic defect, the transfused cells have a normal life span, because the transfused cells are normal.

Extrinsic hemolytic conditions are those that arise from outside the RBC, typically substances in the plasma or conditions affecting the anatomy of the circulatory system. Even though malaria protozoa and other infectious agents are within the RBC, they are classified as extrinsic because the RBC was normal until it was invaded by an outside agent. An antibody against RBC antigens and a prosthetic heart valve are examples of noninfectious extrinsic agents. In extrinsic hemolysis, cross-transfusion studies have shown that the patient’s RBCs have a normal life span in the bloodstream of a normal individual, but normal cells are lysed more rapidly in the patient’s circulation. These studies confirm that something outside the RBCs is causing the hemolysis. (Of course, in the case of intracellular parasites, the cross-transfusion study is not applicable.) Most intrinsic defects are inherited; most extrinsic ones are acquired (see Table 22-1). A few exceptions exist, such as paroxysmal nocturnal hemoglobinuria, an acquired disorder involving an intrinsic defect (see Chapter 23).

Intrinsic disorders are subclassified as membrane defects, enzyme defects, and hemoglobinopathies. Extrinsic hemolysis may be immunohemolytic, traumatic, or microangiopathic, or may be caused by infectious agents, chemical agents (drugs and venoms), or physical agents (see Table 22-1).

Another classification scheme is based on the site of hemolysis and the general mechanism of lysis. Intravascular hemolysis occurs by fragmentation. Although this takes place most often within the bloodstream, cells can lyse by fragmentation in the spleen and bone marrow as well. Extravascular hemolysis occurs when RBCs are engulfed by macrophages and lysed by their digestive enzymes. The designation extravascular, meaning outside the vessels, can refer either to lysis within the macrophage and not in the bloodstream, or to the fact that most of the macrophages are in tissues, chiefly the spleen and the liver, and thus are outside the bloodstream. This classification is useful because laboratory testing readily distinguishes the two types. However, the specific cause of the hemolysis must still be determined for appropriate treatment.

Hemolysis

Normal Bilirubin Metabolism

Detection of hemolysis depends partly on detection of RBC breakdown products. A prominent product is bilirubin. The process of normal bilirubin production is described to clarify the relationship between hemolysis and increased bilirubin levels.

The story of bilirubin production is, in part, a story of iron salvage. The body salvages and recycles iron like a precious metal. If this were not true, there would perhaps be a hemoglobin and iron excretion system, but there is not. Because iron must be salvaged, however, there is a process for saving heme and globin. Bilirubin is the excretory product for the protoporphyrin component of heme.

RBCs live approximately 120 days. During this time, they undergo various metabolic and chemical changes, which result in a loss of deformability. Under normal circumstances, macrophages of the mononuclear phagocyte system (or reticuloendothelial system) recognize these changes and phagocytize the aged erythrocytes (see Chapter 8). The organs of this system include the spleen, bone marrow, liver, lymph nodes, and circulating monocytes, but it is primarily the spleen and liver that process senescent RBCs. The macrophages in the spleen (littoral cells) are especially sensitive to subtle RBC changes, whereas the macrophages of the liver (Kupffer cells) detect and destroy more severely damaged RBCs. This is an extravascular hemolytic process. Hemolysis occurs within macrophages; thus macrophage-mediated hemolysis is a more precise term.

The majority of RBC degradation occurs extravascularly as enzymes of the macrophage lyse the phagocytized erythrocytes (Figure 22-1). Hemoglobin is hydrolyzed into heme and globin; the latter is further degraded into amino acids that return to the amino acid pool. Iron is released from the heme, returned to the plasma via ferroportin, bound to its protein carrier molecule (transferrin), and recycled to developing RBCs. The remaining protoporphyrin is degraded through a series of biochemical reactions in different tissues and organs.

Figure 22-1 Normal catabolism of aged red blood cells.

Figure 22-1 illustrates protoporphyrin catabolism. While protoporphyrin is inside the macrophage, heme oxygenase acts on it, breaking the porphyrin ring to yield a linear molecule, biliverdin. The lungs excrete a by-product of that reaction, carbon monoxide. The green biliverdin is reduced to bilirubin, a nonpolar yellow molecule that is secreted into the plasma (Box 22-1). Because it is hydrophobic, this bilirubin must bind to albumin to be transported in plasma to the liver. The bilirubin-albumin complex enters the liver parenchymal cells, where the bilirubin dissociates from the albumin and is joined (i.e., conjugated) with two molecules of glucuronic acid by glucuronyl transferase to form bilirubin diglucuronide.² The addition of the two sugar acid molecules makes the molecule polar and water soluble. Bilirubin diglucuronide is also called conjugated bilirubin. The bilirubin originally released from macrophages that lacks these sugars is termed unconjugated.

BOX 22-1 Visualizing the Color Changes of Hemoglobin Degradation

The degradation of heme can be seen in bruises in fair-skinned individuals or in the sclera of the eye after a vascular bleed. The same process that macrophages facilitate can occur in tissues. At first, the extravasated but deoxygenated blood gives the injury the purple-red appearance of hemoglobin. As the hemoglobin is degraded, the color changes to a greenish hue due to biliverdin, but ultimately it becomes yellow due to bilirubin.

Conjugated bilirubin is excreted as bile acid into the intestines. There it assists with the emulsification of fats for absorption from the diet. Conjugated bilirubin is oxidized by gut bacteria into various water-soluble compounds, collectively called urobilinogen. Most urobilinogen is oxidized further to stercobilin and similar compounds that give the brown color to stool, which is the ultimate route for excretion of protoporphyrin.

Because they are water soluble, some conjugated bilirubin and urobilinogen molecules are absorbed from the intestines and can be detected in the plasma. The portal circulation, the blood vessels that surround the intestines to absorb nutrients, collect these bile products by osmosis. The portal circulation carries blood directly to the liver, so most of the absorbed conjugated bilirubin and urobilinogen is recycled directly into the bile again. Some remains in the plasma, however, and is filtered by the renal glomerulus and excreted in the urine. Direct bilirubin is virtually undetectable in urine, but a measurable amount of urobilinogen can be expected normally. The yellow color of urine is due to urobilin, a derivative of urobilinogen, that is also water soluble and absorbed into the portal circulation. Urobilinogen is colorless.

Normal Plasma Hemoglobin Salvage During Intravascular Hemolysis

Intravascular hemolysis is trauma to the RBC membrane that causes a breach sufficient for the cell contents, chiefly hemoglobin, to spill directly into plasma (Box 22-2). Approximately 10% to 20% of normal RBC destruction is intravascular,³ secondary to turbulence and anatomic restrictions in the vasculature.

BOX 22-2 Laboratory Impact of Significant Hemoglobinemia

The results of a routine complete blood count are unreliable for patients with significant hemoglobinemia. Under normal circumstances, the measured hemoglobin represents the hemoglobin present inside the red blood cells. For individuals with hemoglobinemia, the intracellular hemoglobin and the plasma hemoglobin both are measured. The hemoglobin value therefore is falsely elevated. An unrealistically high value for mean cell hemoglobin concentration may provide a clue to this problem, which can be remedied in several ways (see Chapters 14 and 39).

Because hemoglobin is filtered by the kidney, iron could be lost in normal intravascular hemolysis. In addition, free hemoglobin and iron can cause oxidative damage to cells. Several mechanisms exist to salvage hemoglobin iron and prevent oxidation reactions, and are collectively called the haptoglobin-hemopexin-methemalbumin system (Figure 22-2).

Figure 22-2 Iron salvage mechanisms during intravascular hemolysis. CO, Carbon monoxide; *RBC,* red blood cell.

When free in the plasma, hemoglobin exists as α/β dimers bound to a liver-produced plasma protein called haptoglobin. This is the first mechanism of hemoglobin iron salvage. By binding to haptoglobin, hemoglobin avoids filtration at the glomerulus and is saved from urinary loss. The complex is carried to the liver, where the haptoglobin-hemoglobin complex is bound to macrophage receptors and internalized into the macrophage.⁴ Inside the macrophage, iron is salvaged, and the remaining protoporphyrin is converted to bilirubin, just as though the RBC had been ingested by the macrophage. If lysis is accelerated, haptoglobin is depleted, because the liver’s production of haptoglobin does not increase in response to the increased hemolysis and is typically adequate to salvage only a normal amount of plasma hemoglobin.

A secondary mechanism of iron salvage involves hemopexin. In hemoglobin that is not bound by haptoglobin, the iron becomes oxidized, forming methemoglobin. The heme molecule (actually metheme) dissociates from the globin and binds to another liver-produced plasma protein, hemopexin.5,6 This binding also saves the iron from urinary loss and prevents oxidation of cell membranes. Hemopexin-metheme binds to hepatocyte receptors and is internalized. There the iron is salvaged, and the protoporphyrin is converted to bilirubin, as in a macrophage. In contrast to haptoglobin, hemopexin is recycled to the plasma from the hepatocyte, so a single hemopexin molecule collects and salvages more free metheme molecules. Although its production is constant, because it is recycled to the plasma, levels of hemopexin do not fall dramatically during periods when there is an increase in intravascular hemolysis.

A third mechanism of iron salvage is the metheme-albumin system. Albumin acts a carrier for many molecules. Metheme can bind to albumin when hemopexin is saturated, which further reduces urinary loss. This is probably a temporary holding state for the metheme, which transfers to hemopexin because it has a higher binding affinity for metheme than does albumin.⁷ It is then transported to the liver for processing.

If the previous systems are overloaded, the excess hemoglobin or heme iron will be filtered into the urine. Iron still can be retained in the kidney, however.8,9 Some hemoglobin or (met)heme that enters the filtrate is reabsorbed, not by specific carriers but as part of the general filtrate in the proximal tubule. When inside the renal tubular cell, the iron is separated from the protoporphyrin. There it is stored as ferritin or hemosiderin. The role of the kidney in salvaging iron and returning it to the circulation is uncertain at this time. Although carrier proteins like divalent metal transporter 1 and ferroportin have been identified in the kidney of experimental mice, their localization, function, and regulation are still not clear. When the amount of hemoglobin presented to the kidney exceeds what can be reabsorbed, the excess is then detectable in the urine.

Excessive Extravascular Hemolysis

Many hemolytic anemias are a result of increased extravascular hemolysis (Figure 22-3) during which more than the usual number of RBCs are removed from the circulation daily. Under normal circumstances, senescent RBCs display surface markers that identify them to macrophages as aged cells requiring removal (see Chapter 8). Pathologic processes also lead to expression of abnormal markers, so that cells are identified and removed. If the number of affected cells increases beyond the quantity normally removed each day due to senescence, and if the bone marrow cannot compensate, then anemia develops. As an example, Heinz bodies, aggregates of denatured hemoglobin formed in various anemias, bind to the inner surface of the RBC membrane, producing changes to the exterior of the membrane that can be detected by macrophages. When something causes increased formation of Heinz bodies or other intracellular inclusions, the cells are removed from the circulation prematurely by macrophages. A similar process occurs when intracellular parasites are present or when complement or immunoglobulins are on the surface of the RBC.