Rodak's Hematology Clinical Principles and Applications

PART IV

Erythrocyte Disorders

CHAPTER 23

Introduction to increased destruction of erythrocytes

Kathryn Doig

OUTLINE

Classification

Hemolysis

Normal Bilirubin Metabolism

Normal Plasma Hemoglobin Salvage During Fragmentation Hemolysis

Excessive Macrophage-Mediated (Extravascular) Hemolysis

Excessive Fragmentation (Intravascular) Hemolysis

Clinical Features

Laboratory Findings

Tests of Accelerated Red Blood Cell Destruction

Tests of Increased Erythropoiesis

Laboratory Tests to Determine Specific Hemolytic Processes

Differential Diagnosis

Objectives

After completion of this chapter, the reader will be able to:

  1. Define hemolysisand recognize its hallmark clinical findings.
  2. Differentiate a hemolytic disorder from hemolytic anemia by definition and recognition of laboratory findings.
  3. Discuss methods of classifying hemolytic anemias and apply the classification to an unfamiliar anemia.
  4. Describe the processes of fragmentation (intravascular) and macrophage-mediated (extravascular) hemolysis, including sites of hemolysis, catabolic products, and time frame for the appearance of those products after hemolysis.
  5. Describe protoporphyrin catabolism (bilirubin production), including metabolites and their sites of production and excretion.
  6. Describe the mechanisms that salvage hemoglobin and heme during fragmentation hemolysis.
  7. Describe changes to bilirubin metabolism and iron salvage systems that occur when the rate of fragmentation or macrophage-mediated hemolysis increases.
  8. Identify, explain the diagnostic value, and interpret the results of laboratory tests that indicate increased hemolysis and erythropoiesis.
  9. Differentiate between hemolytic anemias and other causes of increased erythropoiesis given laboratory or clinical information.
  10. Differentiate between hemolytic anemias and other causes of bilirubinemia given laboratory or clinical information.

CASE STUDY

After studying the material in this chapter, the reader should be able to respond to the following case study:

A 34-year-old woman was admitted to the hospital for a vaginal hysterectomy. Except for excessive menstrual bleeding, she was in otherwise good health, and all of her preoperative laboratory test results were within their respective reference intervals. There was no excessive blood loss during or after surgery, and recovery was uneventful except for some expected pain, for which the patient received ibuprofen.

Three days after surgery, the patient began to experience abdominal pain and passed “root beer”-colored urine. A CBC at that time revealed a hemoglobin level of 5.8 g/dL.

  1. What process is indicated by the root beer–colored urine?
  2. What laboratory tests can be used to differentiate the cause of the hemolysis?
  3. Based on the patient’s clinical presentation, predict the results expected for each test listed for question 2.

This chapter presents an overview of the hemolytic process and provides a foundation that is applicable in the following chapters on red blood cell (RBC) disorders. The term hemolysis orhemolytic disorder refers to increased rate of destruction (i.e., lysis) of RBCs, shortening their life span. The reduced number of cells results in reduced tissue oxygenation and increased erythropoietin production by the kidney. When the patient is otherwise healthy, the bone marrow responds by accelerating erythrocyte production, which leads to reticulocytosis. A hemolytic process is present without anemia if the bone marrow is able to compensate by accelerating RBC production sufficiently to replace the RBCs lost through hemolysis. Healthybone marrow can increase its production of RBCs by six to eight times normal;1 therefore, significant RBC destruction must occur before an anemia develops. A hemolytic anemia results when the rate of RBC destruction exceeds the increased rate of RBC production.

Classification

Many anemias have a hemolytic component, including the anemia associated with vitamin B12 or folate deficiency and the anemia of chronic inflammation, renal disease, and iron deficiency. In these conditions, the hemolysis alone does not cause anemia, and so they are not typically classified as hemolytic disorders. Rather, these anemias develop as a result of the inability of the bone marrow to increase production of RBCs. Because hemolysis is not the primary underlying cause, these disorders are considered anemias with a secondary hemolytic component.

When hemolysis is the primary feature, the anemias can be classified as follows:

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

Every hemolytic condition can be classified according to each of these descriptors. Table 23-1 shows this and provides a noncomprehensive list of hemolytic anemias. This chapter focuses on the mechanism of hemolysis—that is, the distinction between fragmentation and macrophage-mediated hemolytic conditions. The other classifying schemes are summarized here briefly for application in the chapters that follow.

TABLE 23-1

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

 

Predominantly Fragmentation (Intravascular) Hemolysis

Predominantly Macrophage-Mediated (Extravascular) Hemolysis

 

 

Extrinsic defects

Agents from Outside the RBC

Acquired conditions

 

Immune hemolysis: cold antibody 
Microangiopathic hemolysis 
Infectious agents, as in malaria 
Thermal injury 
Chemicals/drugs 
Venoms 
Prosthetic heart valve

Immune hemolysis: warm antibody 
Drugs

 

Membrane Abnormalities

 

Spur cell anemia of severe liver disease 
Paroxysmal nocturnal hemoglobinuria

Hereditary membrane defects

 

Intrinsic defects

 

Abnormalities of the RBC Interior

Hereditary conditions

 

Enzyme defects such as G6PD deficiency 
Globin abnormalities such as sickle cell, thalassemia

 

 

Green text indicates acute or episodic hemolysis.Red text indicates chronic hemolysis.Some conditions may exhibit mixed presentations under certain circumstances. It is evident that most hereditary conditions lead to chronic hemolysis, whereas acquired conditions are more often acute. Furthermore, the intrinsic red blood cell defects typically are due to hereditary conditions, whereas extrinsic factors typically lead to acquired hemolytic disorders.G6PD, Glucose-6-phosphate dehydrogenase; RBC, red blood cell.

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

Chronic hemolysis may not be evident if the bone marrow is able to compensate, but it 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 red blood cell production is brisk, each cell possesses an inadequate complement of one type of globin chain, and functional hemoglobin production is decreasedoverall. 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 erythroid cells, which leads to hemolysis and exacerbates chronically reduced hemoglobin production.

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 disorders 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 the RBC membrane, 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 that can damage RBCs. In extrinsic hemolysis, cross-transfusion studies have shown that the patient’s RBCs have a normal life span in the bloodstream of a healthy 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 (Table 23-1). A few exceptions exist, such as paroxysmal nocturnal hemoglobinuria, an acquired disorder involving an intrinsic defect (Chapter 24).

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 (Table 23-1).

Another classification scheme is based on the site of hemolysis and related to the general mechanism of lysis. Intravascular hemolysis occurs by fragmentation. Although this takes place most often within the bloodstream, RBCs can lyse by fragmentation in the spleen and bone marrow as well. Macrophage-mediated 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 vasculature. Commonly the terms fragmentation and intravascular are used interchangeably, as are macrophage-mediated and extravascular. The mechanistic classifying scheme is useful because screening laboratory tests rely on the differences in the hemolytic processes. However, the exact cause of the hemolysis must still be determined by targeted testing for appropriate treatment to be implemented.

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. There is also a process for recycling the amino acids of the globin chains to build new proteins. The protoporphyrin component, however, is catabolized and excreted but facilitates dietary fat absorption in the process. Bilirubin is the excretory product derived from 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 (Chapter 8), creating a macrophage-mediated hemolytic process. The organs involved include the spleen, bone marrow, liver, lymph nodes, and circulating monocytes, but it is primarily the macrophages in spleen and liver that process senescent RBCs.

The majority of RBC degradation occurs inside macrophages as enzymes of the macrophage granules lyse the phagocytized erythrocytes (Figure 23-1). 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 needy cells. The remaining protoporphyrin is degraded through a series of biochemical reactions in different tissues and organs.

 
FIGURE 23-1 Normal catabolism of hemoglobin. Macrophages lyse ingested red blood cells (RBCs) and separate hemoglobin (Hb) into globin chains and heme components. The amino acids from the globin chains are reused. Heme is degraded to iron and protoporphyrin. Iron is returned to the plasma to be reused. Protoporphyrin is degraded to bilirubin.

Figure 23-2 illustrates protoporphyrin catabolism. While protoporphyrin is inside the macrophage, heme oxygenase acts on it, breaking the protoporphyrin 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 23-1). This form of bilirubin is called unconjugated for reasons that will be evident shortly. Because it is hydrophobic, this form of bilirubin must bind to albumin to be transported in plasma to the liver. In the liver sinusoid, the bilirubin dissociates from the albumin. The bilirubin is then transported across the hepatocyte membrane by a process that is not yet clear. It may be carrier independent or involve organic anion transporter (OAT) proteins.2,  3 Once inside the hepatocyte, the unconjugated bilirubin is joined (i.e., conjugated) with two molecules of glucuronic acid by glucuronyl transferase to form bilirubin diglucuronide. The addition of the two sugar acid molecules makes the molecule polar and water soluble. Bilirubin diglucuronide is also called conjugated bilirubin or direct bilirubin (Box 23-2). Thus the bilirubin originally released from macrophages that lacks these sugars is termedunconjugated bilirubin or indirect bilirubin (Box 23-2).

 
FIGURE 23-2 Catabolism of heme to bilirubin. In cells containing heme oxygenase, iron is removed from heme, and the protoporphyrin ring is opened up to form an intermediate, biliverdin. Biliverdin is converted to unconjugated bilirubin by biliverdin reductase. The unconjugated bilirubin is secreted into the plasma and binds to albumin for transport to the liver. When unconjugated bilirubin enters the hepatocyte, glucuronyl transferase adds two molecules of glucuronic acid to form bilirubin diglucuronide, also called conjugated bilirubin.

BOX 23-1

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.

BOX 23-2

Laboratory Testing for Serum Bilirubin

Because bilirubin occurs in two forms, conjugated and unconjugated, the total bilirubin level in serum is the total of the two forms: total serum bilirubin = unconjugated serum bilirubin + conjugated serum bilirubin (each expressed in mg/dL).

Normally, there is relatively little total bilirubin, and most of it is composed of the unconjugated form in transit from the macrophages, where it was produced, to the liver. The small amount of direct bilirubin in the plasma has been absorbed from the intestine by the portal circulation.

Typical reference intervals are as follows:

Total serum bilirubin level = 0.5–1.0 mg/dL

Direct (conjugated) serum bilirubin level = 0–0.2 mg/dL

Indirect (unconjugated) serum bilirubin level = 0–0.8 mg/dL

Conjugated bilirubin is a polar molecule and reacts well in the water-based spectrophotometric assay that uses diazotized sulfanilic acid. Unconjugated bilirubin does not react well in this system unless alcohol is added to promote its solubility in water. Conjugated bilirubin also is called direct bilirubin because it reacts directly with the reagent, and unconjugated bilirubin is called indirect because it has to be solubilized first.* When alcohol is added to the test system, however, both the direct and indirect forms react. In practice, the total bilirubin level is measured by adding a solubilizing reagent. In a separate test, the direct bilirubin is measured alone without addition of the solubilizing agent. The indirect bilirubin level is calculated by subtracting the direct value from the total value: total serum bilirubin − direct serum bilirubin = indirect serum bilirubin.

*Van den Bergh AA, Muller P: Über eine direkte und eine indirekte diazoreaktion aus bilirubin, Biochem Z 77:90, 1916; and Hutchinson DW, Johnson B, Knell AJ: The reaction between bilirubin and aromatic diazo compounds, Biochem J 127:907-908, 1972.

Conjugated bilirubin is excreted into the hepatic bile duct, continues down the common bile duct, and goes into the intestines (Figure 23-3). 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.

 
FIGURE 23-3 Normal macrophage-mediated hemolysis. 1, In a macrophage, hemoglobin is degraded to heme, the iron is released, and the protoporphyrin ring is converted to unconjugated bilirubin. 2, Macrophages release unconjugated bilirubin into the plasma, where it binds to albumin for transport to the liver. 3, Unconjugated bilirubin enters the hepatocyte. 4, The hepatocyte converts unconjugated bilirubin to conjugated bilirubin. 5, Conjugated bilirubin leaves the liver in the bile and enters the small intestine. 6, Bacteria convert conjugated bilirubin to urobilinogen, most of which is excreted in the stool. 7, Some of the water-soluble urobilinogen is reabsorbed in the portal circulation, and most is recycled through the liver for excretion. 8, A small component of the reabsorbed urobilinogen is filtered and excreted in the urine.  Source:  (Adapted from Brunzel NA. Fundamentals of Urine and Body Fluid Analysis. 3e. St. Louis, Elsevier, 2013.)

Because they are water soluble, some conjugated bilirubin and urobilinogen molecules are absorbed from the intestines into the plasma by osmosis (Figure 23-3). The portal circulation (the blood vessels that surround the intestines to absorb nutrients) collects these bile products. 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. Conjugated bilirubin is virtually undetectable in urine, but a measurable amount of urobilinogen can be expected normally. The yellow color of urine is not due to the urobilinogen, which is colorless. It is due to urobilin, a derivative of urobilinogen that is also water-soluble.

Normal plasma hemoglobin salvage during fragmentation hemolysis

Fragmentation hemolysis is the result of trauma to the RBC membrane that causes a breach sufficient for the cell contents, chiefly hemoglobin, to spill directly into plasma (Box 23-3). Approximately 10% to 20% of normal RBC destruction is via fragmentation,4 secondary to turbulence and anatomic restrictions in the vasculature.

BOX 23-3

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 (Chapters 14 and 15).

Because hemoglobin is filtered by the kidney, some iron could be lost daily with even normal amounts of fragmentation hemolysis. In addition, free hemoglobin, and especially free heme, can cause oxidative damage to cells. Several mechanisms exist to salvage hemoglobin iron and prevent oxidation reactions (Box 23-4) and are collectively called the haptoglobin-hemopexin-methemalbumin system (Figure 23-4).

 
FIGURE 23-4 Normal fragmentation hemolysis. 1, Normally a small number of red blood cells lyse within the circulation, forming schistocytes and releasing hemoglobin (Hb) into the plasma, mostly as αβ dimers. 2, The plasma protein haptoglobin (Hpt) binds a hemoglobin dimer in a complex. 3, The hemoglobin-haptoglobin complex binds to CD163 on the surface of macrophages in various organs. 4, The complex is internalized into the macrophage, where the hemoglobin dimer is released. The hemoglobin dimer is degraded to heme, the iron is released, and the protoporphyrin ring is converted to unconjugated bilirubin. 5, The haptoglobin is degraded. 6, The unconjugated bilirubin released into the plasma is bound to albumin and processed through the liver as in Figure 23-3 (steps 2-8). 7, When free hemoglobin is released into the plasma with fragmentation, the iron is rapidly oxidized, forming methemoglobin, and the heme (metheme) molecule dissociates from the globin. 8, The plasma protein hemopexin (Hpx) binds free metheme into a complex. 9, The hemopexin-metheme complex binds to CD91 on the surface of hepatocytes. 10, The complex is internalized into the hepatocyte. 11, The iron is released from the metheme, and the protoporphyrin ring is converted to unconjugated bilirubin, ready for conjugation and further processing, as in Figure 23-3 (steps 4-8). 12, Hemopexin is recycled to the plasma. Note that metheme can also bind to albumin, forming metheme-albumin (not shown), but this complex is temporary because metheme is rapidly transferred to hemopexin.  Source:  (Adapted from Brunzel NA. Fundamentals of Urine and Body Fluid Analysis. 3e. St. Louis, Elsevier, 2013.)

BOX 23-4

Therapeutic Applications of Haptoglobin and Hemopexin

During severe fragmentation hemolysis, the oxidizing capabilities of hemoglobin and heme cause serious tissue damage. Both readily transfer into extravascular spaces, damaging cells and causing organ dysfunction, such as renal failure. When hemoglobin binds to haptoglobin and heme binds to hemopexin, they are retained in the vasculature, diminishing tissue damage. As a result, the two proteins are gaining serious consideration as therapeutic agents that could be used during instances of fragmentation hemolysis to reduce organ damage. Haptoglobin has been used in Japan since 1985. Hemopexin has not yet been used clinically, but Schaer and colleagues report that both salvage proteins are under investigation by U.S. and European pharmaceutical firms.5

When free in the plasma, hemoglobin exists mostly as α/β dimers5 that rapidly complex 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 the iron is saved from urinary loss. Haptoglobin binds a hemoglobin dimer in a conformation that is very similar to the complementary dimer in the native hemoglobin structure, so the conformation of the complex has been termed a pseudotetramer.6,  7 In this complex, the hemes are sequestered, as they are in intact hemoglobin, so that cells are protected from their oxidative properties.6 As the plasma is carried to various tissues, the complex is taken up by macrophages, principally those in the liver, spleen, bone marrow, and lung.8 In these tissues, the macrophages express CD163, the haptoglobin scavenger receptor, on their membranes. Once the hemoglobin-haptoglobin complex binds to CD163, the entire complex is internalized into the macrophage in a lysosome.8,  9 Inside the lysosome, iron is salvaged, the globin is catabolized, and the protoporphyrin is converted to unconjugated bilirubin, just as though an intact RBC had been ingested by the macrophage. The haptoglobin is also degraded within the lysosome.

The level of haptoglobin in plasma is typically adequate to salvage only the small amount of plasma hemoglobin generated each day. If hemolysis is accelerated, haptoglobin is depleted because the liver’s production does not increase in response to the increased consumption of haptoglobin. See Excessive Fragmentation (Intravascular) Hemolysis below.

A secondary mechanism of iron salvage and oxidation prevention involves hemopexin (Figure 23-4). The iron in free plasma hemoglobin rapidly becomes oxidized, forming methemoglobin. The heme molecule (actually metheme or hemin) dissociates from the globin and binds to another liver-produced plasma protein, hemopexin.10,  11 This binding also saves the iron from urinary loss and prevents oxidant injury to cells and tissues. Hemopexin-metheme binds to hepatocyte CD91 receptors, the lipoprotein receptor-related protein (LRP1),12and is internalized.13 The fate of the internalized heme remains an area of research because some studies suggest that intact heme can be incorporated into needed proteins within the hepatocyte, like cytochromes, and others suggest it is broken down to bilirubin with reuse of the iron.13 It appears that under normal circumstances, the bulk of the hemopexin is recycled to the plasma from the hepatocyte.14

A third mechanism of iron salvage is the metheme-albumin system. Albumin acts as a carrier for many molecules, including metheme. This is just a temporary holding state for the metheme, merely by virtue of the high concentration of albumin in plasma. But metheme is rapidly transferred to hemopexin, which has a higher binding affinity for metheme than does albumin.15 The hemopexin-metheme complex then travels to the liver for processing.

If the previous systems are overloaded, the excess (met) hemoglobin and metheme will be filtered into the urine. Normally, this is a negligible amount so that the kidney is not significantly involved in normal iron salvage. Yet, it has been known for some time that the proximal tubular cells can reabsorb iron, since iron staining of tubular cells in urinary sediment during periods of excessive hemolysis demonstrates the presence of hemosiderin. The full picture of renal handling of iron and related proteins is an area of active research yet to be fully elucidated. However, an emerging picture suggests that under normal circumstances, a small amount of filtered transferrin is salvaged by transferrin receptor on the apical surface of proximal tubular cells.16 This may be the normal source of iron for those cells’ metabolic needs. Ferroportin has been identified on the basolateral membrane of proximal tubular cells, suggesting that renal cells are then able to transfer additional salvaged iron back into the plasma.17 Once again, these systems evolved to manage the amount and “form” of iron present in normal urinary filtrate. During excessive fragmentation hemolysis, other forms of iron are presented to and processed by the kidney.

Excessive macrophage-mediated (extravascular) hemolysis

Many hemolytic anemias are a result of increased macrophage-mediated hemolysis (Figure 23-5), 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 (Chapter 8). Pathologic processes also lead to expression of the same markers, so cells are recognized 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 excessive oxidation of hemoglobin causes increased formation of Heinz bodies, 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.

 
FIGURE 23-5 Excess macrophage-mediated hemolysis. 1, More than the usual number of red blood cells are ingested each day by macrophages. 2, An increased amount of unconjugated bilirubin is produced, released into the plasma, and binds to albumin. 3, When increased unconjugated bilirubin is presented to the liver, an increased amount of conjugated bilirubin is made and excreted into the intestine. 4, When an increased amount of conjugated bilirubin is present in the intestine, an increased amount of urobilinogen is formed and excreted in the stool. 5, Increased urobilinogen in the intestine results in increased urobilinogen reabsorbed into the plasma. 6, Increased urobilinogen in the plasma results in increased urobilinogen filtered and excreted in the urine.  Source:  (Adapted from Brunzel NA. Fundamentals of Urine and Body Fluid Analysis. 3e. St. Louis, Elsevier, 2013.)

When an RBC is ingested by a macrophage, it is lysed within a phagolysosome, and the contents are processed entirely within the macrophage as described previously. The contents of the RBC are not detected in plasma because it is lysed inside the macrophage, and the contents are degraded there—hence the designation extravascular hemolysis. Since defective cells display markers like those of senescent RBCs, macrophage-mediated hemolysis of defective cells occurs most often in the spleen and liver, where the macrophages possess receptors for those markers.

Sometimes the macrophage ingests a portion of the membrane, leaving the remainder to reseal. Little, if any, cytoplasmic volume is lost, but with less membrane, the cell becomes aspherocyte, the characteristic shape change associated with macrophage-mediated hemolysis. Although the spherocyte may enter the circulation, its survival is shortened because of its rigidity and inability to traverse the splenic sieve during subsequent passages through the red pulp. It may become trapped against the basement membrane of the splenic sinus and be fully ingested by a macrophage, or it may lyse mechanically due to its rigidity and in so doing contribute a fragmentation component to what is otherwise a macrophage-mediated process.

In macrophage-mediated hemolytic anemias, the total plasma bilirubin level rises as RBCs lyse prematurely. The rise of the total bilirubin is due to the increase of the unconjugated fraction (Figure 23-5). As long as the liver is healthy, it processes the increased load of unconjugated bilirubin, producing more than the usual amount of conjugated bilirubin that enters the intestine. Increased urobilinogen forms in the intestines and is subsequently absorbed by the portal circulation and excreted by the kidney. As a result, increased urobilinogen is detectable in the urine. Although there is an increase in unconjugated bilirubin in the plasma, none of it appears in the urine because it is bound to albumin and cannot pass through the glomerulus. These findings are summarized in Table 23-2.

TABLE 23-2

Comparison of Laboratory Findings Indicating Accelerated Red Blood Cell Destruction in Fragmentation Versus Macrophage-Mediated Hemolysis

Test Specimen

Result

Fragmentation

Macrophage-Mediated

Serum

Total bilirubin

Indirect (unconjugated) bilirubin

Direct (conjugated) bilirubin

WRI

WRI

Lactate dehydrogenase activity (RBC fraction)

sl ↑

Haptoglobin

sl ↓

Free hemoglobin

sl ↑

Hemopexin

sl ↓

Urine

Urobilinogen

Free hemoglobin

Positive

Negative

Methemoglobin

Positive

Negative

Prussian blue staining of urine sediment

Positive

Negative

Anticoagulated whole blood

Hemoglobin, hematocrit, RBC count

Schistocytes

Often present

 

Spherocytes

 

Often present

Glycated hemoglobin

Special tests

Endogenous carbon monoxide

Erythrocyte life span

↑, Typically increased; sl ↑, typically only slightly increased, minor component; ↓, typically decreased; RBC, red blood cell; WRI, within the reference interval.

Excessive fragmentation (intravascular) hemolysis

Although fragmentation hemolysis is a minor component of normal RBC destruction, it can be a major feature of pathologic processes. Dramatic examples of fragmentation hemolysis are the traumatic, physical lysis of RBCs caused by prosthetic heart valves and the exit of mature intracellular RBC parasites, such as malaria protozoa, by bursting out of the cell. In these instances, the fragmentation destruction of RBCs can cause profound anemias.

Excessive fragmentation hemolysis is characterized by the appearance in the plasma of the contents of the red blood cell, chiefly hemoglobin, and thus the development of (met) hemoglobinemia. As a result, the salvage proteins form complexes with their ligands (Figure 23-6A, B), and hemoglobin-haptoglobin, metheme-hemopexin, and metheme-albumin are detectable, if measured. The levels of free haptoglobin will drop, since more than the usual amounts of the complex will form and be taken up by macrophages (Table 23-2). The endocytosed protein is not recycled to the plasma and there is no compensatory increase in production, so the plasma is depleted of haptoglobin. The levels of free hemopexin can also decrease even though it is normally recycled. It appears that the hepatic recycling system can become saturated when there are high levels of metheme to be salvaged.12 During these circumstances, hemopexin then gets degraded within the hepatocyte, and plasma levels fall. Still the drop in hemopexin is not as dramatic as the decline of haptoglobin,4 since some recycling continues.

 
 
 
 
FIGURE 23-6 A, Excess fragmentation hemolysis: the role of macrophages. 1, When an increased number of red blood cells lyse by fragmentation, more than the usual amount of hemoglobin (Hb) is released into the plasma, mostly as αβ dimers. 2, Haptoglobin (Hpt) binds the increased hemoglobin dimers, forming more than usual numbers of complexes. 3, The hemoglobin-haptoglobin complexes are taken up by macrophages bearing the CD163 receptor in various organs. 4, An increased amount of hemoglobin dimers is released from the complexes. The hemoglobin is degraded to heme, the iron is released, and the protoporphyrin ring is converted to unconjugated bilirubin. The increased amount of unconjugated bilirubin is then transported to the liver and processed as with excess macrophage mediated hemolysis (Figure 23-5, steps 2-6). 5,Degradation of haptoglobin is accelerated as compared to normal. B, Excess fragmentation hemolysis: the role of the liver. 1, If the amount of hemoglobin released from lysing red blood cells exceeds the capacity of haptoglobin, the unbound free hemoglobin is rapidly oxidized, forming methemoglobin, and the metheme molecule dissociates from the globin. 2, Hemopexin binds to metheme, and the complex is captured by the CD91 receptor on hepatocytes. 3, The complex is internalized by the hepatocyte, the iron is released from the metheme, and the protoporphyrin ring is converted to unconjugated bilirubin and ultimately to conjugated bilirubin to be processed, as in Figure 23-5, steps 3-6. 4, Although a small of amount of hemopexin is recycled to the plasma, most is degraded. Metheme can also temporarily bind to albumin, forming metheme-albumin (not shown), but metheme is rapidly transferred to hemopexin C, Excess fragmentation hemolysis: the role of the kidney. 1, When excess red blood cells lyse by fragmentation and other systems are saturated, free (met)hemoglobin enters the urinary filtrate. 2, Cubilin (Cb) on the luminal side of the proximal tubular cells binds proteins for reabsorption, including hemoglobin. 3, Cubilin carries hemoglobin into the proximal tubular cells. 4, The hemoglobin is degraded to heme, the iron is released, and the protoporphyrin ring is converted to unconjugated bilirubin and secreted into the plasma (5) in the same manner as in the macrophages (Figure 23-1). 6, When the amount of hemoglobin exceeds the capacity of the proximal tubular cells to absorb it from the filtrate, hemoglobinuria occurs. D, Fate of iron removed from salvaged hemoglobin in the kidney. 1, Iron (Fe) salvaged from absorbed hemoglobin can be transported into the circulation by ferroportin on the basolaminal side of the tubular cell. In the plasma it will be bound to transferrin (Tf) for transport. 2, Iron in excess of what can be transported into the circulation is stored as ferritin, and some is converted to hemosiderin. If the tubular cell is sloughed into the filtrate and appears in the urine sediment, the hemosiderin can be detected using the Prussian blue stain.  Source:  (A-C, Adapted from Brunzel NA. Fundamentals of Urine and Body Fluid Analysis. 3e. St. Louis, Elsevier, 2013.)

In roughly the same time frame that hemoglobin appears in the plasma, it can also appear in the urine (hemoglobinuria) (Table 23-2) if the amount of liberated hemoglobin and heme exceeds the salvage capacity of the plasma proteins. Increased amounts of iron-containing proteins are then absorbed into the proximal tubular cells (Figure 23-6C).

The mechanisms by which renal cells are able to reabsorb more than usual amounts of iron, heme, or iron-containing proteins are still emerging. At least one mechanism is the megalin-cubilin receptor endocytosis system.18 These receptors are not specific for heme/iron-containing compounds. They are responsible for nonspecific but very efficient reabsorption of proteins from the urinary filtrate in the proximal tubule. Each has been shown to bind hemoglobin and myoglobin.18 Additionally, megalin can bind lactoferrin, and cubilin can bind transferrin.18 The kinetics of such nonspecific competitive reabsorption favors the iron-containing proteins when they are present in high concentrations. A mechanism for reabsorption of free (met)heme has not been clearly identified. Proximal tubular cells are able to dismantle heme from hemoglobin via heme-oxygenase-1,19 contributing to elevations of unconjugated bilirubin in plasma and thus freeing the iron for export to the plasma by ferroportin. It is reasonable to expect that a specific heme receptor in the kidney may yet be identified.

Proximal tubular cell iron in excess of what can be transported into the circulation is stored as ferritin, and some is converted to hemosiderin (Figure 23-6D). If the tubular cell is sloughed into the filtrate and appears in the urine sediment, the hemosiderin can be detected using the Prussian blue stain. This provides evidence that hemoglobin has been salvaged from the filtrate.

Elevated levels of plasma indirect bilirubin and urinary urobilinogen are also measurable, although not immediately, because time is needed to produce these products. The time course of these findings assists with the differential diagnosis. Following an acute onset, a rise in reticulocytes several days later would also be seen.

Clinical features

The clinical findings typical of hemolysis may be prominent if the hemolytic process is the primary cause of anemia. For patients in whom hemolysis is secondary, however, other clinical features may be more noticeable. If hemolysis is sufficient to result in anemia, patients experience the general symptoms of fatigue, dyspnea, and dizziness to a degree that is consistent with the severity and rate of development of the anemia. The associated signs of pallor and tachycardia can be expected.

Increase in plasma bilirubin gives a yellow tinge, not only to the plasma but also to body tissues. It is readily evident in the sclera of the eyes and the skin of fair-skinned individuals.Jaundice refers to the yellow color of the skin and sclera, whereas icterus describes plasma and tissues. An increase in plasma bilirubin and subsequent jaundice can occur in other conditions besides hemolysis, such as hepatitis or gallstones. If the jaundice is the result of hemolysis, it is called hemolytic jaundice or prehepatic jaundice, which reflects the predominance of unconjugated bilirubin in plasma. The lipid solubility of unconjugated bilirubin also leads to deposition in the brain when hemolysis affects newborns (Chapter 26), since the blood-brain barrier is not fully developed. This can lead to a type of brain damage called kernicterus, which refers to the yellow coloring (icterus) of the brain tissue.

The frequency or constancy of jaundice provides clues to the cause. In glucose-6-phosphate dehydrogenase deficiency, for example, jaundice is periodic, appearing following a crisis. In thalassemia, jaundice is chronic. Jaundice may not be present at all if hemolysis is minimal and the liver is able to process the additional bilirubin, as is often the case in hereditary elliptocytosis.

Some signs differentiate chronic from acute hemolysis. Splenomegaly can develop, particularly with chronic macrophage-mediated hemolytic processes. Gallstones (cholelithiasis) can occur whenever hemolysis is chronic; the constantly increased amount of bilirubin in the bile leads to the formation of the stones.20 When hemolysis is chronic in children, the persistent compensatory bone marrow hyperplasia can lead to bone deformities because the bones are still forming (Figure 28-3). For patients in whom an acquired, acute hemolytic process develops, the associated malaise, aches, vomiting, and possible fever may cause it to be confused with an acute infectious process. Profound prostration and shock may develop, particularly with acute fragmentation hemolysis. Flank pain, oliguria, or anuria develops, which leads to acute renal failure.

Other clinical features may offer a clue as to whether hemolysis is macrophage-mediated or due to fragmentation. In particular, brown urine, associated with (met)hemoglobinuria, points to a fragmentation hemolytic process.

Laboratory findings

In patients with the clinical features of hemolytic anemia, laboratory tests typically show evidence of increased erythrocyte destruction and the compensatory increase in the rate of erythropoiesis. Other tests that are specific to a particular diagnosis also may be indicated.

Tests of accelerated red blood cell destruction

Visual examination of the plasma and urine may suggest fragmentation hemolysis. The presence of methemoglobin, methemalbumin and hemopexin-heme impart a coffee-brown color to plasma, strongly suggestive of fragmentation hemolysis. When these compounds are present in urine, the color is more often described as root beer- or beer-colored. In a properly collected blood specimen, the normal physiologic fragmentation hemolysis produces a plasma hemoglobin level of less than 1 mg/dL.21 Plasma does not become visibly red/brown until the plasma hemoglobin level is at 50 mg/dL.21 Typical values during hemolytic processes may be as low as 15 mg/dL, so an increase in plasma hemoglobin may not always be visible.22

Hemoglobin/heme from fragmentation hemolysis can be detected in urine when the capacity of the plasma salvage systems is exceeded and the hemoglobin/heme is filtered into the urine. The urinalysis test strip for blood can be positive even when the hemoglobin is not present in a high enough concentration to change the color of the urine significantly. Since the product entering the urine is free hemoglobin/heme, the sediment will be negative for red blood cells. However, renal tubular cells sloughed into the filtrate during the period of hemoglobinuria can demonstrate deposits of hemosiderin (iron), resulting from absorbed hemoglobin, when stained with Prussian blue stain.

For a patient with a hemolytic process, a complete blood count (CBC) may provide clues to the cause. Spherocytes can be expected to be seen with macrophage-mediated hemolysis (Table 23-2), whereas fragmented cells, or schistocytes, are noted with fragmentation hemolysis. Other clues to the particular cause of the hemolysis may be present in the morphology, such as ring forms in malaria, sickle cells in sickle cell anemia, target cells and microcytes in thalassemia, and spherocytes in hereditary spherocytosis or immune-related hemolysis.

In either fragmentation or macrophage-mediated hemolysis, the increased rate of hemoglobin catabolism results in increased amounts of plasma unconjugated bilirubin and carbon monoxide. If liver function is normal, conjugated bilirubin is formed and excreted as urobilinogen in the stool, and the serum level of conjugated bilirubin remains within the reference interval. No bilirubin is detected in the urine because unconjugated bilirubin is bound to albumin and the complex is not filtered by the glomerulus. The urinary urobilinogen level may be increased, however, because there is increased urobilinogen in the stool, and more than usual amounts are absorbed by the portal circulation. In some patients, serum indirect (unconjugated) bilirubin values can be misleadingly low because the amount of bilirubin in the blood depends on the rate of RBC catabolism as well as hepatic function. If the rate of hemolysis is low and liver function is normal, the total serum bilirubin level can be within the reference interval. Quantitative measurements of fecal urobilinogen, however, would demonstrate an increase.

A substantial decline in the serum haptoglobin level indicates fragmentation hemolysis. In what is mostly a macrophage-mediated hemolysis, there can still be a minor component of fragmentation lysis involving spherical cells that are fragile, so a modest decline in haptoglobin level can be seen. In short, whenever the level of hemoglobin in the plasma increases, the haptoglobin level declines. In one study, a low haptoglobin level indicated an 87% probability of hemolytic disease.23 Haptoglobin measurement is, however, prone to both false-positive and false-negative results. Low values suggest hemolysis but may be due instead to impaired synthesis of haptoglobin caused by liver disease. Alternatively, a patient with hemolysis may have a relatively normal haptoglobin level if there is also a complicating infection or inflammation, because haptoglobin is an acute phase reactant. Although quantitation of hemopexin may also demonstrate a low value, it is not often measured, relying instead on the more dramatic haptoglobin decline to detect fragmentation hemolysis.

Tests to determine the rate of endogenous carbon monoxide production have been developed, because carbon monoxide is produced in the first step of heme breakdown by heme oxygenase. Values of 2 to 10 times the normal rate have been detected in some patients with hemolytic anemia,24 but testing for carbon monoxide production is not typically required for clinical diagnosis.

Other laboratory test results are incidentally abnormal. Serum lactate dehydrogenase activity is often increased in patients with fragmentation hemolysis due to the release of the enzyme from ruptured RBCs, but other conditions, such as myocardial infarction or liver disease, also can cause increases. Although enzyme isoform fractionation could be used to identify lactate dehydrogenase of RBC origin, this test is generally not needed. Rather, when other results point to fragmentation hemolysis, one should expect an increase in the level of serum lactate dehydrogenase and other RBC enzymes and should not be misled into assuming that there is liver damage (Table 23-2).

General evidence of reduced RBC survival can be gleaned by measuring glycated hemoglobin (by the Hb A1c test).25 Glycated hemoglobin increases over the life of a cell as it is exposed to plasma glucose. Glycated hemoglobin level is usually decreased in chronic hemolytic disease because the cells have less exposure to plasma glucose before lysis. The mean reference interval for glycated hemoglobin in one report was 6.7%; in a hemolytic process, the mean value decreased to 3.9%.25 The magnitude of the decrease is related to the magnitude of the hemolytic process over the previous 4- to 8-week period. Glycated hemoglobin level is not a reliable indicator of shortened RBC survival in patients with diabetes mellitus because of the increased rate of glycation with elevated blood glucose levels. Thus glycated hemoglobin measurement is more useful for diagnosis or monitoring blood glucose control in diabetic patients, rather than detection of hemolysis.

A more exact RBC survival assay uses random labeling of blood with chromium radioisotope. This is the reference method for RBC survival studies published by the International Committee for Standardization in Haematology.26 A sample of blood is collected, mixed with the isotope, and returned to the patient. The labeled cells are of all ages, reflecting normal peripheral blood. This method differs from cohort labeling in which RBCs are labeled with radioactive iron or heavy nitrogen as they are produced in the bone marrow, so the labeled cells are generally of the same age. In both methods, the disappearance of the label from the blood is measured over time. As measured using the random chromium labeling technique, the normal half-time of chromium is 25 to 32 days.26 A half-time of 20 to 25 days suggests mild hemolysis; 15 to 20 days, moderate hemolysis; and less than 15 days, severe hemolysis.26There are clinical instances when an estimation of red cell survival would be useful for assessing erythropoietin treatment.27 However, both cohort and random techniques have significant limitations,28 in addition to being time consuming, expensive, and requiring the use of radioactive isotopes. Therefore, these methods are not often used clinically but are used for research, particularly in the search for improved methods of determining red blood cell survival.

Tests of increased erythropoiesis

If the bone marrow is healthy, the hypoxia associated with hemolysis leads to increased erythropoiesis. Recognition of this increase may be a first clue to the presence of a hemolytic process. Laboratory findings indicating increased erythropoiesis include an increase in circulating reticulocytes and, in severe cases, nucleated red blood cells (Table 23-3). These findings are persistently present in chronic hemolytic disease and are evident within 3 to 6 days after an acute hemolytic episode. Increased erythropoiesis is not unique to hemolytic anemias and is not diagnostic. Similar results are expected after hemorrhage and with successful specific therapy for anemia caused by iron, folate, or vitamin B12 deficiency. An assessment of erythropoiesis can determine the effectiveness of the bone marrow response, however, and should be factored into the differential diagnosis (see Differential Diagnosis).

TABLE 23-3

Hematologic Findings Indicating Accelerated Red Blood Cell Production

Specimen

Findings

Anticoagulated peripheral blood

Increased absolute reticulocyte count, immature reticulocyte fraction, reticulocyte production index Rising mean cell volume (compared with baseline) 
Polychromasia, nucleated RBCs

Bone marrow

Erythroid hyperplasia

RBC, Red blood cell.

Complete blood count and morphologic features

Peripheral blood film evaluation is crucial. An increase in polychromatic RBCs (reticulocytes) and nucleated RBCs represents bone marrow compensation for hemolysis or blood loss. Schistocytes are expected with excessive fragmentation hemolysis, while spherocytes may be seen with macrophage-mediated processes. Additional morphologic changes to red blood cells may point toward the etiology of the hemolysis (see below).

An increase in the mean cell volume (MCV) is usually seen with extreme compensatory reticulocytosis resulting from the larger, prematurely released “shift” reticulocytes. The increase must be assessed by comparison with the value seen early in hemolysis, before the shift reticulocytes have emerged. The MCV may not increase above the reference interval but rather may be above the baseline value for that patient. Exceptions occur if the hemolytic condition itself involves smaller cells that counter the increased volume of the reticulocytes. In hereditary spherocytosis, microspherocytes are the cause of the anemia, and the MCV may be within the reference interval even when larger shift reticulocytes are generated—hence the importance of a baseline value for comparison. In other circumstances, such as severe burns, numerous schistocytes or microspherocytes may outnumber the reticulocytes so that the MCV remains low.

Leukocytosis and thrombocytosis may accompany acute hemolytic anemia and are considered reactions to the hemolytic process. Conversely, conditions that directly cause leukocytosis, such as sepsis, might cause hemolysis. Low platelet counts in association with other signs of hemolysis may indicate a platelet-consuming microangiopathic process, such as disseminated intravascular coagulopathy.

Reticulocyte count

The reticulocyte count is the most commonly used test to detect accelerated erythropoiesis and is expected to rise during hemolysis or hemorrhage. Assuming the bone marrow is healthy and there are adequate raw materials, all measures of reticuloycyte production should rise: absolute reticulocyte count, relative reticulocyte count, reticulocyte production index, and the immature reticulocyte fraction. The association of reticulocytosis with hemolysis is so strong that if an anemic patient has an elevated reticulocyte count and hemorrhage is ruled out, a cause of hemolysis should be investigated. The reticulocyte increase usually correlates well with the severity of the hemolysis. Exceptions occur during aplastic crises of some hemolytic anemias and in some immunohemolytic anemias with hypoplastic marrow, which suggests that the autoantibodies were directed against the bone marrow RBC precursors and circulating erythrocytes.21 Chapters 14 and 19 describe the interpretation of reticulocyte indices in patients with anemia.

Bone marrow examination

Bone marrow examination is usually not necessary to diagnose hemolytic anemia. If conducted, however, bone marrow examination will reveal erythroid hyperplasia that results in peripheral blood reticulocytosis. As the erythroid component (the denominator) of the myeloid-to-erythroid ratio increases, the overall ratio decreases. (The myeloid-to-erythroid ratio is defined in Chapter 17.) As always, the cellularity of the bone marrow should be determined on a core biopsy specimen, rather than an aspirated specimen, for a more accurate judgment.

Laboratory tests to determine specific hemolytic processes

As noted above, the appearance of spherocytes or schistocytes on a peripheral blood film can point to a hemolytic cause for anemia. Other abnormalities found on the film, such as elliptocytes, acanthocytes, burr cells, sickle cells, target cells, agglutination, erythrophagocytosis, or parasites, may help reveal the specific disorder causing the hemolysis (Table 23-4).

TABLE 23-4

Morphologic Abnormalities Associated with Hemolytic Anemia

RBC Morphology

Hemolytic Disorders

Spherocytes

Hereditary spherocytosis, IgG-mediated immune hemolytic anemia, thermal injury to RBCs

Elliptocytes (ovalocytes)

Hereditary elliptocytosis

Acanthocytes

Abetalipoproteinemia, severe liver disease (spur cell anemia)

Burr cells

Pyruvate kinase deficiency, uremia

Schistocytes

Microangiopathic hemolytic anemia, macroangiopathic (traumatic cardiac hemolytic anemia), IgM-mediated immune hemolytic anemia

Erythrophagocytosis

Damage to RBC surface, especially due to complement-fixing antibodies

RBC agglutination

Cold agglutinins, immunohemolytic disease

RBC, Red blood cell.

Other tests dealing with specific types of hemolytic anemia are discussed in subsequent chapters. They include the direct antiglobulin test, osmotic fragility test, eosin-5′-maleimide binding test, Heinz body test, RBC enzyme studies, serologic tests, and immunophenotyping.

Differential diagnosis

The differential diagnosis of hemolytic anemias incorporates several intersecting lines of deduction. The first is to establish the hemolytic nature of the anemia. A rapid decrease in hemoglobin concentration (e.g., 1 g/dL per week) from levels previously within the reference interval can signal hemolysis when hemorrhage and hemodilution have been ruled out. Jaundice and reticulocytosis provide additional confirmation of a hemolytic cause for an anemia of at least several days’ duration. When only the indirect (unconjugated) fraction of the total serum bilirubin is elevated, hemolytic jaundice is confirmed. An elevated urinary urobilinogen level strengthens the conclusion.

The rapid decrease in hemoglobin during an acute hemolytic episode, however, usually is apparent before reticulocytosis and bilirubinemia develop. For acute hemolysis, hemoglobinemia and hemoglobinuria are expected with fragmentation causes; therefore, their absence suggests a macrophage-mediated cause. RBC morphology and haptoglobin levels can assist in differentiating fragmentation from a macrophage-mediated cause. Figure 23-7 is a graphic representation of the general time line of the events in acute fragmentation and macrophage-mediated hemolysis. In chronic hemolysis, persistence of hemoglobinemia, hemoglobinuria, decreased serum haptoglobin level, indirect bilirubinemia, and reticulocytosis can be expected, depending on the mechanism of the hemolysis.

 
FIGURE 23-7 A, Fragmentation (intravascular) hemolysis time line. During fragmentation hemolysis, hemoglobin is detectable in plasma and urine for a period of time very soon after the hemolysis occurs. Haptoglobin levels will drop as the plasma hemoglobin rises. When hemolysis ends, the hemoglobin will disappear from plasma and urine as the haptoglobin returns to normal. However, the protoporphyrin of salvaged hemoglobin will be processed to bilirubin that will rise after the hemolytic event. Assuming the hemolysis has resulted in anemia, reticulocyte indices will also rise several days after the hemolytic event. B, Macrophage-mediated (extravascular) hemolysis time line. With macrophage-mediated hemolysis, the contents of the red blood cells do not enter the plasma, so evidence of hemolysis is delayed until bilirubin and reticulocytes rise.

Hemolytic anemias must be differentiated from other anemias associated with bilirubinemia, reticulocytosis, or both. Anemia with reticulocytosis but without bilirubinemia is expected during recovery from hemorrhage not treated with transfusion or with effective treatment of deficiencies such as iron deficiency. Anemia that results from hemorrhage into a body cavity is characterized by reticulocytosis during recovery and bilirubinemia due to catabolism of the hemoglobin in the hemorrhaged cells. The RBC morphology should remain normal throughout the event. Anemias associated with ineffective erythropoiesis, such as megaloblastic anemia, are essentially hemolytic, with the cell death occurring in the bone marrow. Bilirubinemia and elevated serum lactate dehydrogenase levels are to be expected, but the reticulocyte count is low. Because most of the RBCs never reach the periphery, such anemias are typically classified as anemias of diminished production rather than as hemolytic anemias. As summarized in Table 23-5, the differential diagnosis in each of these instances may rely on negative results of tests for increased cell destruction or accelerated production.

TABLE 23-5

Differential Diagnosis of Hemolytic Anemias Versus Other Causes of Indirect Bilirubinemia and Reticulocytosis

 

Hemoglobin Level

Indirect Bilirubinemia

Reticulocytosis

Spherocytes or Schistocytes

Hemolytic anemia—acute, fragmentation

Rapidly dropping

Delayed

Delayed

Schistocytes

Hemolytic anemia—acute, macrophage-mediated

Rapidly dropping

Delayed

Delayed

Spherocytes

Hemolytic anemia—chronic, fragmentation

Persistently low

Persistent

Persistent

Schistocytes

Hemolytic anemia—chronic, macrophage-mediated

Persistently low

Persistent

Persistent

Spherocytes

Acute hemorrhage

Rapidly dropping

Absent

Absent

Absent

Hemodilution

Rapidly dropping

Absent

Absent

Absent

Recovery from hemorrhage

Rising

Absent

Present

Absent

Treated anemia (iron, vitamin B12, or folate deficiency)

Rising

Absent

Present

Absent

Hemorrhage into a body cavity

Rapidly dropping

Delayed

Delayed

Absent

Ineffective erythropoiesis (e.g., megaloblastic anemia)

Dropping

Persistent

Absent

Absent

Summary

  • A hemolytic disorder is a condition in which there is increased destruction of erythrocytes and a compensatory acceleration in erythrocyte production by the bone marrow.
  • A hemolytic anemia develops when the bone marrow is unable to compensate for the shortened survival of the RBCs.
  • Hemolytic anemias can be classified as acute or chronic, intravascular or extravascular, acquired or inherited, intrinsic or extrinsic, and fragmentation or macrophage-mediated.
  • Normally, most erythrocyte death occurs via macrophages of the spleen and liver. A small amount occurs by fragmentation due to mechanical trauma.
  • Hemoglobin from the RBCs is converted to heme and globin within macrophages. Heme is further degraded to iron, carbon monoxide, and unconjugated bilirubin. The bilirubin is secreted into the plasma, where it binds to albumin and is transported to the liver. In the liver, the bilirubin is conjugated with glucuronic acid, excreted as bile into the intestines, and converted to urobilinogen. Some urobilinogen is reabsorbed into the portal circulation and reexcreted through the liver. A small amount of urobilinogen remains in the plasma, and it is excreted by the kidney into the urine.
  • In macrophage-mediated (extravascular) hemolytic anemia, there is a delayed increase in unconjugated bilirubin in the plasma and an increase in urobilinogen in the stool and urine. Spherocytes may be seen on the blood film.
  • Signs of fragmentation (intravascular) hemolysis include (met)hemoglobinemia, (met)hemoglobinuria, and hemosiderinuria. Serum haptoglobin is markedly decreased or absent. Schistocytes may be seen on the blood film.
  • Jaundice can result from increased serum unconjugated bilirubin during any hemolytic anemia.
  • The major clinical features of chronic inherited hemolytic anemia are varying degrees of anemia, jaundice, splenomegaly, and the development of cholelithiasis. In children, bone abnormalities may develop as a result of accelerated erythropoiesis.
  • Laboratory studies providing evidence of hemolytic anemia include tests for increased erythrocyte destruction and compensatory increase in the rate of erythropoiesis. Elevated serum indirect bilirubin level with a normal serum direct bilirubin level suggests accelerated RBC destruction. A moderate to marked decrease in serum haptoglobin level suggests a fragmentation cause of hemolysis. The reticulocyte count is the most commonly used laboratory test to identify accelerated erythropoiesis, including an elevation of the immature reticulocyte fraction. Other tests that are specific to a particular diagnosis also may be needed.
  • Hemolytic anemias must be differentiated from other anemias with reticulocytosis, including the post-acute hemorrhage state and recovery from iron, vitamin B12, or folate deficiency, and from those with bilirubinemia, such as with internal bleeding.

Now that you have completed this chapter, go back and read again the case study at the beginning and respond to the questions presented.

Review questions

Answers can be found in the Appendix.

  1. The term hemolytic disorderin general refers to a disorder in which there is:
  2. Increased destruction of RBCs after they enter the bloodstream
  3. Excessive loss of RBCs from the body
  4. Inadequate RBC production by the bone marrow
  5. Increased plasma volume with unchanged red cell mass
  6. RBC destruction that occurs when macrophages ingest and destroy RBCs is termed:
  7. Extracellular
  8. Macrophage-mediated
  9. Intra-organ
  10. Extrahematopoietic
  11. A sign of hemolysis that is typically associated with both fragmentation and macrophage-mediated hemolysis is:
  12. Hemoglobinuria
  13. Hemosiderinuria
  14. Hemoglobinemia
  15. Elevated urinary urobilinogen level
  16. An elderly white woman is evaluated for worsening anemia, with a decrease of approximately 0.5 mg/dL of hemoglobin each week. The patient is pale, and her skin and eyes are slightly yellow. She complains of extreme fatigue and is unable to complete the tasks of daily living without napping in midmorning and midafternoon. She also tires with exertion, finding it difficult to climb even five stairs. Which of the features of this description points to a hemolytic cause for her anemia?
  17. Pallor
  18. Yellow skin and eyes
  19. Need for naps
  20. Tiredness on exertion
  21. Which of the following tests provides a good indication of accelerated erythropoiesis?
  22. Urine urobilinogen level
  23. Hemosiderin level
  24. Reticulocyte count
  25. Glycated hemoglobin level
  26. A 5-year-old girl was seen by her physician several days prior to this visit and was diagnosed with pneumonia. Her mother has brought her to the physician again because the girl’s urine began to darken after the first visit and now is alarmingly dark. The girl has no history of anemia, and there is no family history of any hematologic disorder. The CBC shows a mild anemia, polychromasia, and a few schistocytes. This anemia could be categorized as:
  27. Acquired, fragmentation
  28. Acquired, macrophage-mediated
  29. Hereditary, fragmentation
  30. Hereditary, macrophage-mediated
  31. A patient has a personal and family history of a mild hemolytic anemia. The patient has consistently elevated levels of total and indirect serum bilirubin and urinary urobilinogen. The serum haptoglobin level is consistently decreased, whereas the reticulocyte count is elevated. The latter can be seen as polychromasia on the patient’s blood film, along with spherocytes. Which of the findings reported for this patient is inconsistentwith a classical diagnosis of fragmentation hemolysis?
  32. Elevated total and indirect serum bilirubin
  33. Elevated urinary urobilinogen
  34. Decreased haptoglobin
  35. Spherocytes on the peripheral film
  36. Select the statement that is trueabout bilirubin metabolism.
  37. Indirect bilirubin is formed in the liver by the addition of two sugar molecules to direct bilirubin.
  38. Macrophages of the spleen liberate bilirubin during hemoglobin catabolism.
  39. Urobilinogen is not water soluble and is not excreted in the urine.
  40. Normally, the major fraction of bilirubin in the blood is the direct (conjugated) form released from macrophages.
  41. A patient has anemia that has been worsening over the last several months. The hemoglobin level has been declining slowly, with a drop of 1.5 g/dL of hemoglobin over about 6 weeks. Polychromasia and anisocytosis are seen on the blood film, consistent with the elevated reticulocyte count and RBC distribution width (RDW). Serum levels of total bilirubin and indirect fractions are normal. Urinary urobilinogen level also is normal. When these findings are evaluated, the conclusion is drawn that the anemia does not have a hemolytic component. Based on the data given here, why was hemolysis ruled out as the cause of the anemia?
  42. The decline in hemoglobin is too gradual to be associated with hemolysis.
  43. The elevation of the reticulocyte count suggests a malignant cause.
  44. Evidence of increased protoporphyrin catabolism is lacking.
  45. Elevated RDW points to an anemia of decreased production.
  46. Which of the following sets of test results is typically expected with chronic fragmentation hemolysis?
 

Serum Haptoglobin

Urine Hemoglobin

Urine Sediment Prussian Blue Stain

a.

Increased

Positive

Positive

b.

Decreased

Negative

Negative

c.

Decreased

Positive

Positive

d.

Increased

Positive

Negative

References

  1.   Crosby W. H, Akeroyd J. H. The limit of hemoglobin synthesis in hereditary hemolytic anemiaAm J Med; 1952; 13:273-283.
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