CHAPTER 11: Acquired Hemolytic Anemias

Hemolytic anemias are encountered less often than anemias due to decreased red cell production or blood loss. By far the most common hemolytic anemia in both pediatric and adult medicine is sickle cell disease (Chapter 9). Next in prevalence are acquired hemolytic anemias. Familiarity with the pathophysiology of this group of disorders is essential because these patients often pose formidable challenges in both diagnosis and management.

An overview of the hemolytic anemias is presented in Chapter 3 and summarized in Table 3-2. As indicated in Table 11-1, with the exception of the rare disorder paroxysmal nocturnal hemoglobinuria, all of the acquired hemolytic anemias are extracorpuscular. This means that red cells from a compatible donor are hemolyzed as readily as the patient's own red cells. Acquired hemolysis is most often caused by immune-mediated destruction or traumatic mechanical damage of red cells.

Immune hemolysis can be triggered by:

• Autoantibodies binding to antigens on the patient's red cells.

• Alloantibodies binding to antigens on transfused red cells.

This chapter will deal with autoimmune hemolytic anemia. Alloimmune hemolysis will be covered in Chapter 25 (Transfusion Medicine).

Autoimmune hemolytic anemia is conveniently divided into two categories based on the temperature dependence of autoantibody binding to red cells, summarized in Table 11-2.

WARM AUTOANTIBODIES

Warm autoantibodies bind avidly to the patient's red cells at body temperature. They are immunoglobulin G (IgG) antibodies with specificity for Rh group antigens found on red cells of nearly all individuals. IgG-coated red cells are cleared primarily in the spleen, where they are engulfed by resident macrophages that have receptors for the constant region (Fc) of the heavy (H) chain of IgG. Figure 11-1A shows a macrophage from a patient with warm antibody hemolytic anemia that has just ingested two red cells and is about to destroy them. Alternatively, the patient's IgG-coated red cells may bind to the surface of the macrophage but escape total engulfment. Under these circumstances, the macrophage nibbles at the red cell membrane and removes a small portion of it. As shown in Figure 11-1B, this depletion of the red cell surface area results in the formation of a spherocyte. Spherocytes are the predominant morphologic feature in two disorders: warm antibody autoimmune hemolytic anemia (Fig. 11-2A) and hereditary spherocytosis (Chapter 10). Even though these two types of anemia have very different pathogenic mechanisms, they produce identical morphologic alterations in red cells.

Patients with warm antibody immune hemolysis usually present with fatigue, shortness of breath, and general symptoms of anemia. Physical examination often reveals slight icterus and splenomegaly.

COLD AUTOANTIBODIES

Cold autoantibodies bind to the patient's red cells with much higher affinity at low temperature than at body temperature. As a result, binding of cold autoantibodies to red cells is restricted to cool areas of the body, such as the extremities and the ear lobes. In the vast majority of cases, they are IgM macroglobulins that have specificity for the I antigen, which (like the Rh complex) is found on nearly all adult red cells. The large pentameric IgM binds the C3 component of complement much more efficiently than does IgG. As red cells return from the periphery and the blood warms, the IgM falls off the surface, but complement remains attached. C3-coated red cells are prey to two modes of destruction. If complement components are fully assembled, the cell will lyse and release its cytosolic contents into the plasma. More often, however, full fixation of complement does not occur, but sufficient C3b is deposited on the red cell surface for it to be recognized by C3 receptors on macrophages in the liver and elsewhere, which clear the cells from the circulation.

The clinical presentation of patients with cold-type autoantibodies is fully in accord with the pathogenic events described earlier. Most patients have extravascular hemolysis and a clinical presentation similar to that of patients with warm autoantibody hemolysis. Others, however, have a more dramatic clinical picture, in which vascular sludging in parts of the body exposed to low temperature (toes, fingers, ear lobes) results in pain, cyanosis, and even gangrene. In another subset of patients with cold-type autoantibody, the full fixation of complement, particularly following cold exposure, results in acute intravascular hemolysis with hemoglobinuria and a sudden drop in hemoglobin level.

Table 11-3 classifies autoimmune hemolytic anemias according to etiology. In about half of cases, the cause is unknown (idiopathic). In a small fraction of patients the autoimmune process is incited by drugs, some of which act as haptens. More often, drugs, particularly the cephalosporins, trigger autoantibody formation by an unknown mechanism. Patients with idiopathic or drug-induced disease usually have warm IgG autoantibodies. In the remainder of patients, an underlying disease is responsible for the development of autoimmune hemolysis. Lymphoproliferative disorders, particularly chronic lymphocytic leukemia and non-Hodgkin lymphoma (Chapter 22), account for most of these cases. Figure 11-2B shows a blood film of a patient with chronic lymphocytic leukemia who has developed autoimmune hemolysis. Patients with systemic lupus erythematosus are also at risk of developing immune hemolysis. In both lymphomas and systemic lupus erythematosus, the hemolysis is often due to a combination of warm and cold autoantibodies. Patients with either lymphoma or systemic lupus erythematosus are also at high risk of developing autoimmune thrombocytopenia (Chapter 14). Those with mycoplasma pneumonia and infectious mononucleosis occasionally develop transient acute hemolysis secondary to cold autoantibodies.

LABORATORY DIAGNOSIS

Patients with autoimmune hemolytic anemia generally have straightforward hemolysis with elevation of the reticulocyte count as well as of unconjugated bilirubin and lactate dehydrogenase in the serum. Serum haptoglobin is usually undetectable. As mentioned earlier, the peripheral blood film often reveals spherocytes.

The diagnosis is definitively established by the direct antiglobulin or Coombs test, which is depicted in Figure 11-3A. Following the addition of an animal anti-human IgG antibody, red cells will form visible clumps if they are coated with IgG immunoglobulin. The direct antiglobulin test can also utilize anti-C3 to detect complement on the surface of red cells of patients with a cold autoantibody. If cold autoantibodies are present, clumps of red cells may be seen in the blood film (agglutination) due to the fall in temperature between the time the blood is drawn and when the film is prepared. Usually, cold agglutinins are of no clinical significance, but some are associated with the cold-induced symptoms and signs described earlier.

The indirect antiglobulin test, shown in Figure 11-3B, detects antibody that can bind to a cognate red cell antigen. Patients with warm-type hemolysis usually have a high titer of autoantibody in the serum, which, as mentioned earlier, has specificity for the Rh group of red cell antigens. However, as explained in Chapter 25, the primary application of the indirect antiglobulin test is in blood banking, where it is used to identify and characterize the specificity of alloantibodies in serum of patients who are about to receive transfused red cells.

TREATMENT

Similar to that of other autoimmune disorders, the mainstay of therapy is immunosuppression. Initial treatment usually entails the administration of high-dose corticosteroids, with gradual tapering once the hemolysis abates. Patients with warm autoantibodies who fail to respond or require a high dose of steroids to remain in remission are treated either with splenectomy or with rituximab, a monoclonal antibody directed against CD20 on B lymphocytes. This treatment is virtually identical to that used for the treatment of immune thrombocytopenia (Chapter 14). Patients with cold-type immune hemolysis are usually more difficult to treat. A simple but critically important measure is avoidance of exposure to cold, including the warming of blood prior to transfusion. Occasionally, a patient needs to move to a warmer climate before the hemolysis is controlled. Steroids and splenectomy are much less effective in cold antibody disease. However, some impressive remissions have been achieved with rituximab therapy.

Although the red cell is remarkably sturdy and can tolerate considerable stretching and deformation during 120 days of travel through the microcirculation, extreme turbulence or other mechanical stresses can cause red cells to fracture into small fragments called schistocytes or become irreversibly deformed into shapes resembling a triangle or a helmet, as illustrated in Figure 11-4.

The most prevalent and challenging types of traumatic hemolysis are thrombotic thrombocytopenic purpura and hemolytic uremic syndrome. The formation of platelet microthrombi is critical in the pathogenesis of these disorders, which are covered in detail in Chapter 14. In addition, similar red cell morphology is observed in a sizable minority of patients with an important acquired bleeding disorder, disseminated intravascular coagulation, which is covered in Chapter 16. However, hemolysis is seldom a dominant feature of disseminated intravascular coagulation. Severe thrombocytopenia is a critical feature in all of these disorders.

Grossly turbulent blood flow can also cause traumatic damage to red cells without affecting platelet function or number. The primary example is traumatic hemolysis owing to a malfunctioning artificial heart valve. As shown in Figure 11-4, the blood film reveals striking abnormalities in red cell shape, but, in contrast to the low platelet counts in other disorders mentioned above, the platelet count is normal.

Infectious pathogens and toxins can cause severe intravascular hemolysis. By far the most important of these worldwide is malaria. This protozoal infection is transmitted by Anopheles mosquitoes. Sporozoites injected into the dermis travel to the liver, where they develop into schizonts, which release multiple merozoites into the bloodstream. After infecting red cells (Fig. 11-5A), the parasite undergoes further multiplication. Falciparum is the most severe form of malaria because it elicits the formation of knobs on the red cell surface that promote adherence to receptors on venule and capillary endothelium, resulting in sequestration of the parasite in certain venous beds and the spleen. Along with the constitutional symptoms of fever and malaise, patients infected with falciparum malaria can have life-threatening complications such as encephalopathy due to sequestration of parasites in cerebral vessels, lactic acidosis, and loss of renal function. The pathogenesis of anemia in malaria is complex, but premature destruction of infected red cells in the spleen plays a major role. Some patients have severe intravascular hemolysis and hemoglobinuria (blackwater fever).

Babesiosis is a protozoan infection with a reservoir in animals and transmission to humans by tick bite. Most cases in the United States are localized to the northeast coastline, particularly the islands off of Cape Cod. Like malarial organisms, Babesia enters red blood cells (Fig. 11-5B) and causes a debilitating illness with fever, chills, malaise, and mild hemolytic anemia. Asplenic individuals are at particularly high risk of developing severe and potentially fatal infections.

A skilled observer can distinguish Babesia parasites within infected red cells from malaria organisms by subtle differences in morphologic appearance.

Most disorders of the red cell membrane are inherited, and these topics are covered in the preceding chapter. There are two rare but illuminating acquired membrane abnormalities that cause hemolytic anemia: spur cell anemia and paroxysmal nocturnal hemoglobinuria.

SPUR CELL ANEMIA

Patients with liver disease, particularly those with biliary obstruction, have red cells that appear on the blood film as targets, with a puddle of red hemoglobin in the center of the cell rather than an area of relative pallor (see Fig. 3-7 and Fig. 11-6). These cells have an increased ratio of surface area to volume, owing to the passive accumulation of extra cholesterol and phospholipid in the lipid bilayer of the red cell membrane. Target cells have normal pliability and a normal life span in the circulation. In contrast, an occasional patient, usually with severe alcoholic liver disease, will develop more marked increases of lipid on red cell membranes, resulting in bizarre red cells with spikes and thorny projections on the cell surface. In contrast to target cells, these so-called spur cells are rigid and are rapidly destroyed in the circulation. This is an extracorpuscular abnormality. If normal red cells are transfused into a patient with spur cell anemia, they too become spiculated and assume the same abnormal morphology.

PAROXYSMAL NOCTURNAL HEMOGLOBINURIA

Paroxysmal nocturnal hemoglobinuria (PNH) is one of a small and distinguished group of disorders in which the investigators who study it may outnumber the patients who are afflicted. Teachers are appropriately chary about foisting very rare diseases upon overloaded students. However, PNH is a disorder of sufficient scientific and medical interest that an understanding of its pathogenesis, clinical features, and treatment is well worth pursuing.

During the last 100 years, occasional previously healthy patients have been reported to develop severe anemia accompanied by passage of red-brown urine following bed rest. Clinical investigation over the ensuing decades established that these patients had intravascular hemolysis and that the pigment in their urine was free hemoglobin. One astute early investigator wondered if the nocturnal hemoglobinuria could be due to the slight drop in pH resulting from hypoventilation during sleep. This hypothesis was tested by asking a patient to sleep through the night with mechanical ventilatory assistance from a crude, external vacuum-driven respirator (iron lung). This maneuver proved to be fully effective in preventing night-time hemolysis. The significance of this finding was clarified by the demonstration that the fixation of complement on red cells is enhanced by a small drop in pH and that PNH red cells are much more sensitive to the lytic action of complement than are normal cells. A diagnostic blood test was developed, based on the enhanced lysis of PNH red cells at low pH.

As these laboratory studies were underway, hematologists began to appreciate a broader clinical spectrum of PNH. Many patients with hemolytic anemia had a positive acid lysis test without any history of hemoglobinuria, nocturnal or otherwise. In some, the anemia was due to impaired red cell production rather than hemolysis. Most of these patients also had leukopenia and thrombocytopenia, and a few had bone marrow aplasia. Finally, it was appreciated that a dominant clinical feature of the disease was a high risk of developing thromboembolic complications, often life-threatening.

These confusing revelations came into focus through the discovery that PNH is an acquired clonal disorder of the hematopoietic pluripotent stem cell. A variable fraction of a PNH patient's red cells, white cells, and platelets were shown to be hypersensitive to complement and to have arisen from the same abnormal clone. More recently, the gene PIG-A was found to be mutated in the hematopoietic cells, but not in the other cells, of patients with PNH. PIG-A is located on the X chromosome, meaning that any cell in a male or female that acquires a somatic inactivating mutation in the single active PIG-A gene will be unable to express the normal PIG-A protein. PIG-A catalyzes the transfer of a glycosyl phosphatidyl inositol group to certain proteins, a modification that is necessary for their expression on the plasma membrane of cells. Thus, the surface of cells derived from PIG-A defective hematopoietic clone is deficient in glycosyl phosphatidyl inositol-linked proteins. Among these are CD55 and CD59, two proteins that play a key role in down-regulating activated complement. Figure 11-7 shows flow cytometry results demonstrating uniform expression of CD55 and CD59 on the red cells of a normal individual, whereas the red cells from a patient with PNH include a substantial fraction that is deficient in these two surface proteins.

The remarkable advances in the molecular pathogenesis of PNH have led to a partial understanding of the clinical features of the disease, particularly the enhanced sensitivity of the clonal hematopoietic progeny to complement. The mechanisms underlying the development of marrow aplasia and thrombosis are less clear. However, recent experience with a novel therapy has provided additional clues to pathogenesis. The administration of a monoclonal antibody directed against the terminal portion of the complement cascade has been shown to be remarkably effective not only in reducing, and in many cases eliminating, hemolysis but also in preventing thrombotic complications.¹