Chapter 54: Hemolytic Anemia Resulting from Immune Injury

The two main features of immune red blood cell (RBC) injury are (1) shortened RBC survival in vivo and (2) evidence of host antibodies reactive with autologous RBCs, most frequently demonstrated by a positive direct antiglobulin test (DAT), also known as the Coombs test. Most cases in adults are mediated by warm-reactive autoantibodies. A smaller proportion of patients exhibit cold-reactive autoantibodies or drug-related antibodies.

By the early 20th century, reticulocytes, spherocytes, and osmotic fragility of RBCs had been described. Clinicians could diagnose hemolytic anemia, but the distinction between congenital and acquired forms was imprecise. Some clinicians even doubted the existence of acquired hemolytic anemia.¹ The sera of some patients with hemolytic anemia directly agglutinated saline suspensions of normal or autologous human RBCs. These serum factors, later shown to be specific antibodies (largely of the immunoglobulin [Ig] M class), were termed direct or saline agglutinins. In a smaller proportion of cases, the patients’ sera could mediate lysis of the test RBCs in the presence of fresh serum as a complement source. The heat-stable factors (antibodies) necessary for in vitro complement-mediated lysis were called hemolysins. However, in the majority of cases of hemolytic anemia, neither direct agglutinins nor hemolysins could be demonstrated. In 1945, Coombs and colleagues² reported that RBCs coated with nonagglutinating Rh antibodies (now known to be of the IgG isotype) could be agglutinated by rabbit antiserum to human γ-globulin. That is, the rabbit antiglobulin serum crosslinked IgG antibody-coated RBCs to produce visible agglutination. Addition of rabbit antiglobulin serum to a suspension of washed RBCs isolated from patients with suspected acquired hemolytic anemia produced agglutination in many cases, including those patients lacking saline agglutinins or hemolysins. RBCs from patients with congenital hemolytic anemia did not agglutinate.^3,4 This procedure now is termed the direct antiglobulin (Coombs) test. Subsequent studies established that positive direct antiglobulin reactions in autoimmune hemolytic anemia (AHA) are attributable to coating of the RBCs with immunoglobulins (mainly IgG) and/or complement proteins. When the RBCs are coated chiefly with complement proteins, a positive DAT depends upon the presence of anticomplement (principally anti-C3) in the antiglobulin reagent. In warm-antibody AHA, the autoantibodies, chiefly of IgG isotype, bind optimally to RBCs at 37°C. Warm antibodies may or may not activate complement binding to RBCs.

Cryopathic hemolytic syndromes are caused by autoantibodies that bind RBCs optimally at temperatures less than 37°C and usually less than 31°C. Two major types of “cold antibody” may produce AHA. Cold agglutinins, which directly agglutinate RBCs, mediate cold agglutinin disease. The Donath-Landsteiner autoantibody, which is not an agglutinin but a potent hemolysin, mediates paroxysmal cold hemoglobinuria. In both cryopathic syndromes, the complement system plays a major role in RBC injury (Chap. 19); as such, much greater potential exists for direct intravascular hemolysis than in warm-antibody–mediated AHA.

Cold agglutinins were first described by Landsteiner⁵ in 1903. However, recognition of the connection among cold agglutinins, hemolytic anemia, and Raynaud-like peripheral vascular phenomena evolved slowly. In 1918, Clough and Richter⁶ detected cold agglutinins in a patient with pneumonia. In 1925 and 1926, Iwai and MeiSai^7,8 reported two patients with cold agglutinins and Raynaud phenomenon and showed that flow of blood through capillary tubes in vitro or in superficial capillaries in vivo was impeded at low temperatures. During the late 1940s and early 1950s, the observations of many investigators gradually established the pathogenic importance of cold agglutinins in RBC injury. Schubothe⁹ introduced the term cold agglutinin disease in 1953 and clearly distinguished the disorder from other acquired hemolytic syndromes.

In current usage, cold agglutinin disease pertains to patients with chronic AHA in which the autoantibody directly agglutinates human RBCs at temperatures below body temperature, maximally at 0 to 5°C. Fixation of complement to a patient’s RBCs by cold agglutinins in vivo occurs at higher temperatures but generally less than 37°C. Cold agglutinins typically are IgM, although occasionally they may be immunoglobulins of other isotypes. The cold agglutinins in chronic cold agglutinin disease generally are monoclonal. Most cold agglutinins have specificity for oligosaccharide antigens (I or i) of the RBC (see “Origin of Cold Agglutinins” below).

Donath and Landsteiner first described the cold hemolysin that bears their name in 1904. The Donath-Landsteiner antibody is responsible for complement-mediated hemolysis in paroxysmal cold hemoglobinuria, a rare form of AHA in adults. The disorder is characterized by recurrent episodes of massive hemolysis following cold exposure.^10,11 A related form of hemolytic anemia occurs much more commonly in children (or young adults) as an acute, self-limited hemolytic process following several types of viral syndromes.^{10,11,12,13,14,15,16} The disease was recognized during the latter half of the 19th century, when the disease was more common because of its association with congenital or tertiary syphilis. With the advent of effective therapy for syphilis, this cause of paroxysmal cold hemoglobinuria has almost disappeared. Now, recurrent paroxysmal cold hemoglobinuria occurs very rarely in a chronic idiopathic form.^10,11 An increasing proportion of Donath-Landsteiner autoantibody-mediated hemolytic anemias occurs as a single postviral episode in children, without recurrent attacks (paroxysms). The prognosis for such cases is excellent. Thus, rather than paroxysmal cold hemoglobinuria, a proposed term for this latter entity is Donath-Landsteiner hemolytic anemia.^13,14

The first example of drug-related immune blood cell destruction was Ackroyd’s description of sedormid purpura in 1949.¹⁷ In 1953, Snapper and coworkers¹⁸ described a case of immune hemolysis and pancytopenia in a patient treated with mephenytoin (Mesantoin). Hemolysis ceased upon withdrawal of the drug. In 1956, Harris¹⁹ reported what are now classic studies of a patient who developed immune hemolytic anemia during a second course of stibophen administered for treatment of schistosomiasis. Since then, many drugs have been implicated in the production of positive DATs and accelerated RBC destruction.

CLASSIFICATION

Warm-Reactive versus Cold-Reactive Red Cell Antibody

AHA can be classified in two complementary ways (Table 54–1). The majority of cases (80 to 90 percent in adults) are mediated by warm-reactive autoantibodies^10,11,20 or antibodies displaying optimal reactivity with human RBCs at 37°C. A smaller proportion of cases is attributable to cold-reactive autoantibodies exhibiting greater affinity for RBCs at temperatures less than 37°C. The distinction is important, not only because of differences in the pathophysiology of RBC injury but also in the therapeutic approaches required. An even smaller proportion of patients with AHA exhibit both cold-reactive and warm-reactive autoantibodies,^21,22 which apparently recognize different antigens on the RBC membrane.²³ RBC destruction is generally more severe in mixed cases.

Absence or Presence of an Associated Disease

Classification of AHA based on the presence or absence of underlying diseases also is useful (see Table 54–1). When no recognizable underlying disease is present, the AHA is termed primary or idiopathic. When AHA appears to be a manifestation or complication of an underlying disorder, the term secondary AHA is applied. Lymphocytic malignancies, particularly chronic lymphocytic leukemia (CLL) and lymphomas, account for approximately half of all secondary AHA cases and for the majority of AHA cases mediated by cold agglutinins.²⁴ Systemic lupus erythematosus (SLE) and other autoimmune diseases account for a lesser but considerable proportion of secondary AHA cases. A large proportion of patients with mixed cold and warm autoantibodies have SLE.^21,22 Infectious mononucleosis and Mycoplasma pneumoniae occasionally are associated with cryopathic AHA. Despite the frequent occurrence of immune thrombocytopenia and positive DATs in patients infected with HIV, AHA is relatively rare in these patients.^25,26,27 Table 54–1 lists other associated diseases that are less-commonly reported. The etiologic and pathogenic significance of these associations is poorly understood, but most of the associated diseases involve components of the immune system, either by neoplasia or by aberrant immunopathologic responses.

Drug-Mediated Cases

Certain drugs also mediate immune injury to RBCs, and three general mechanisms are recognized (see Table 54–1 and Fig. 54–1). This classification is based on the effector mechanism of RBC injury, because the induction mechanism for formation of drug-related RBC antibodies is unknown. Two of the mechanisms, hapten-drug adsorption and ternary complex formation, involve drug-dependent antibodies. In the third mechanism, the drugs in question appear to induce formation of true autoantibodies capable of reacting with human RBCs in the absence of the inciting drug. These types of drug-mediated immune injury to RBCs often are referred to collectively as drug-immune hemolytic anemia to distinguish them from de novo AHA. Distinguishing among the mechanisms is not always possible, and some cases involve a combination of mechanisms. In addition, drug-related non-immunologic protein adsorption by RBCs may result in a positive DAT without actual RBC injury. This phenomenon should be distinguished from the three forms of drug-immune RBC injury. Table 54–2 lists drugs documented to cause either immune injury or a positive DAT.

The annual incidence of warm-antibody AHA is 1 per 75,000 to 80,000 population.¹¹ Estimates of the frequency of primary (idiopathic) AHA vary from 20 to 80 percent of all types of AHA, depending on the referral patterns of the reporting center.^11,20,118 In general, AHA is considered secondary (1) when AHA and the underlying disease occur together with greater frequency than can be accounted for by chance alone; (2) when the AHA reverses simultaneously with correction of the associated disease; or (3) when AHA and the associated disease are related by evidence of immunologic aberration.¹¹ Using these criteria, the frequency of primary warm-antibody AHA probably is closer to 50 percent of all cases. Careful followup of patients with primary AHA is essential, because hemolytic anemia may be the presenting finding in a patient who subsequently develops overt evidence of an underlying disorder. For example, in one series, 18 of 107 patients with AHA developed a malignant lymphoproliferative disorder at a median of 26.5 months after diagnosis of the AHA.¹¹⁹

Warm-antibody AHA has been diagnosed in people of all ages, from infants to the elderly. The majority of patients are older than 40 years of age, with peak incidence around the seventh decade. This age distribution probably reflects, in part, the increased frequency of lymphoproliferative malignancies in the elderly, resulting in an age-related increase in the frequency of secondary AHA. Although multiple cases are occasionally observed in families,^120,121,122 most cases of primary AHA arise sporadically. Development of AHA does not have an apparent association with any particular human leukocyte antigen (HLA) haplotype or other genetic factor.

Cold agglutinin disease is less common than warm-antibody AHA, with a prevalence of approximately 14 per 1 million population,²⁴ accounting for only 10 to 20 percent of all cases of AHA.^10,11,123 Women are affected more commonly than men.^10,11 No genetic or racial factors are known to contribute to the pathogenesis of this disease.

Secondary cold agglutinin disease is seen most commonly in adolescents or young adults as a self-limited process associated with M. pneumoniae infections or infectious mononucleosis and, rarely, in children with chickenpox. The term also has been used to describe a chronic disorder occurring in older patients with known malignant lymphoproliferative diseases. On the other hand, idiopathic (primary) chronic cold agglutinin disease has its peak incidence after age 50 years. This disorder, with its characteristic monoclonal IgM cold agglutinins, may be considered a special form of monoclonal gammopathy (Chap. 106). Nearly all of these patients exhibit clonal B lymphocyte proliferation.²⁴ As with other “essential” or idiopathic monoclonal gammopathies, some patients in this group gradually develop features of a B-cell lymphoproliferative disorder resembling Waldenström macroglobulinemia, CLL, or a B-cell lymphoma. Thus, the distinction between primary and secondary types of chronic cold agglutinin disease is not absolute.

Although the majority of patients with mycoplasma pneumonia have significant cold agglutinin titers, they only infrequently develop clinical hemolytic anemia.^124,125,126 However, subclinical RBC injury may occur. In M. pneumoniae infections, weakly positive direct antiglobulin reactions and/or mild reticulocytosis are often noted in the absence of anemia in a substantial number of cases.¹²⁴ Cold agglutinins occur in more than 60 percent of patients with infectious mononucleosis, but again, hemolytic anemia is rare.^127,128,129

Medical centers that receive many referrals report that paroxysmal cold hemoglobinuria constitutes 2 to 5 percent of all cases of AHA.^10,11 Among children, however, Donath-Landsteiner hemolytic anemia accounted for 32.4 percent of 68 immune hemolytic syndromes diagnosed over a 4-year period.¹⁵ Commonly, the diagnosis is missed because of lack of physician awareness or failure to perform the proper serologic studies (see “Serologic Features” below).^12,15 Thus, the true incidence may be higher. Although familial occurrence has been reported, no racial or genetic risk factors are known.¹⁰ As noted, most childhood cases follow either specific viral infections or upper respiratory infections of undefined etiology.^{10,11,12,13,14,15}

Older series report that drug-induced immune hemolytic anemia accounts for 12 to 18 percent of immune hemolytic anemias.¹¹ The disorder is much less common now that α-methyldopa and megaunit doses of penicillin rarely are used. The current incidence of drug-induced immune hemolytic anemia is estimated at 1 per 1 million population, approximately 88 percent of which result from the second- and third-generation cephalosporins, cefotetan, and ceftriaxone.^89,130 Fludarabine has replaced α-methyldopa as the most common cause of drug-induced autoantibodies.⁸⁹

ETIOLOGY

Warm-Antibody Autoimmune Hemolytic Anemia

The etiology of AHA is unknown. In warm-antibody AHA, the autoantibodies that mediate RBC destruction are predominantly (but not exclusively) IgG globulins possessing relatively high binding affinity for human RBCs at 37°C. As a result, the major share of plasma autoantibody is bound to the patient’s circulating RBCs. Eluates prepared from the patient’s washed, autoantibody-coated RBCs constitute an important source of purified autoantibody for investigation of specificity, immunoglobulin structure, or other properties. In addition, sera from patients with warm AHA often are used in blood banks for crossmatching and for general screening of antibody specificity. The quantity of such autoantibody in serum may be low and in some cases may not reflect the full spectrum of anti-RBC specificity revealed in concurrently prepared RBC eluates.¹²⁴

In patients with primary AHA, erythrocyte autoantibodies are the only recognizable immunologic aberration. Furthermore, the autoantibodies of any one patient often are specific for only a single RBC membrane protein (see “Serologic Features” below). The narrow spectrum of autoreactivity suggests the mechanism underlying AHA development in such patients is not secondary to a generalized defect in immune regulation. Rather, these patients may develop warm-antibody AHA through an aberrant immune response to a self-antigen or to an immunogen that mimics a self-antigen.

In patients with secondary AHA, the disease may be associated with a fundamental disturbance in the immune system, for example, when in the setting of lymphoma, CLL, SLE, primary agammaglobulinemia (common variable immunodeficiency), or hyper-IgM immunodeficiency syndrome. In these settings, warm-antibody AHA most likely arises through an underlying defect in immune regulation, although the contribution of an aberrant immune response to self-antigen cannot be excluded. AHA seems especially frequent in patients with low-grade lymphoma or CLL treated with fludarabine ^88,89 or 2-chlorodeoxyadenosine (cladribine).⁹⁰ The T-lymphocytopenia induced by these drugs may exacerbate the preexisting tendency of patients to form autoantibodies.

A long-recognized but poorly understood phenomenon, the development of AHA or a positive DAT following RBC transfusion, has received renewed interest lately.^132,133 Although generally transient, the positive DAT may persist for up to 300 days in some transfusion recipients, long after any transfused RBCs have disappeared.^134,135 It is not clear whether this represents true autoimmunity or some other mechanism, for example, microchimerism resulting from temporary engraftment of passenger memory lymphocytes from the RBC donor.¹³²

A still unexplained observation is that certain drugs, such as α-methyldopa, can induce warm-reacting IgG anti-RBC autoantibodies in otherwise normal persons. The autoantibodies induced by α-methyldopa have Rh-related serologic and immunochemical¹³⁶ specificity similar to that of autoantibodies arising in many patients with “spontaneous” AHA. A critical difference is that the drug-associated autoantibodies subside when the drug is discontinued, suggesting that (1) the latent potential to form this type of anti-RBC autoantibody is present in many immunologically normal individuals, and (2) the steps required to generate such autoantibodies do not necessarily create a sustained autoimmune state. On the other hand, maintenance of chronic idiopathic AHA may be either secondary to a continuing (but unknown) stimulus or induced by a short stimulus to which the patient continues to respond.

Normal subjects sometimes have a positive DAT when they volunteer to donate blood.^137,138 The positive DAT in these normal donors often results from warm-reacting IgG autoantibodies, similar in serologic specificity¹³¹ and in IgG subclass¹³⁷ to the autoantibodies occurring in AHA. Although many of these donors remain DAT-positive without developing overt hemolytic anemia, a few have been documented to develop AHA.^137,138 The prevalence of positive DATs in normal blood donors is approximately 1 in 10,000 donors.^137,139 Because blood donation per se likely does not contribute to an increased risk of developing autoantibodies, the 1 in 10,000 proportion likely is the approximate frequency of positive DATs in the entire population. A proportion of patients who present with clinically overt primary AHA may come from a subset of asymptomatic individuals who are DAT-positive, but this notion is not established.

Several concepts have been developed to explain immunologic tolerance to self-antigens.^{140,141,142,143} Relevant to warm-antibody AHA, membrane-bound antigens expressed in a multivalent array at high concentration may induce tolerance by effecting clonal deletion of autoreactive B cells.¹⁴⁴ Both the Rh-related and the non-Rh types of RBC antigens targeted by AHA autoantibodies (see “Serologic Features” below) are expressed normally by human fetal erythrocytes, as early as 10 to 12 weeks of life.¹⁴⁵ However, because new B cells develop daily in the marrow throughout life and because B cells may somatically mutate their Ig receptors, self-tolerance in the B cell compartment is never assured. Analogy to observations in NZB (New Zealand black) mice^146,147 suggests the peritoneal cavity is a privileged compartment that shelters autoreactive B cells from host RBCs, allowing them to escape deletion, later to produce anti-RBC autoantibodies with appropriate T-cell help. The strong predominance of IgG antibodies in AHA suggests B-cell isotype switching, which is consistent with the idea of an antigen-driven process. Moreover, because T-cell help is necessary for inducing B-cell isotype switching, the pathway(s) to autoantibody induction in AHA also may involve an abnormal or unique mode of antigen presentation to T cells.¹⁴⁸

Origin of Cold Agglutinins

A high proportion of monoclonal IgM cold agglutinins with either anti-I or anti-i specificity have heavy-chain variable regions encoded by IGHV4–34 (immunoglobulin heavy chain variable region), formerly designated IGHV4.21.^{143,149,150,151} IGHV4–34 encodes a distinct idiotype identified by the rat monoclonal antibody 9G4. This idiotype is expressed both by the cold agglutinins themselves and on the surface immunoglobulin of B cells synthesizing cold agglutinins or related immunoglobulins possessing IGHV4–34 sequences.¹⁵² Using the 9G4 monoclonal antibody as a probe, this idiotype was found not only in a very high proportion of circulating B cells and marrow lymphoplasmacytoid cells of patients with lymphoma-associated chronic cold agglutinin disease, but also in a smaller proportion of B cells in the blood and lymphoid tissues of normal adult donors and in the spleens of 15-week human fetuses.¹⁵² These data suggest B cells expressing the IGHV4–34 gene (or a closely related sequence) are present throughout ontogeny. Therefore, chronic cold agglutinin disease may represent a marked, unregulated expansion of a subset (clone) of such B cells.

Light-chain V-region gene use in anti-I cold agglutinins is highly selective. A strong bias toward use of the κ III variable region subgroup (Vκ-III) is observed.^{150,151,152,153} Light-chain selection among anti-i cold agglutinins, however, is much more variable and includes the λ type.^{150,151,152,153,154}

Observations that pathologic cold agglutinins are synthesized with distinct and highly selected V-region sequences must be viewed against the background of two other subsequent observations. First, IGHV4–34 or related IGHV genes also may encode the heavy-chain variable regions of other types of antibodies, such as rheumatoid factor autoantibodies and alloantibodies to a variety of blood group antigens, including polypeptide determinants such as Rh.¹⁵⁵ Second, normal human antibodies to an exogenous carbohydrate antigen, Haemophilus influenzae type b capsular polysaccharide, also are encoded by a restricted set of IGHV genes¹⁵⁶ and Ig light-chain V genes.¹⁵⁷ Thus, regulation of Ig gene use for production of anti-I or anti-i cold agglutinins may not differ fundamentally from normal antibody formation to other carbohydrate antigens.

In the setting of B-cell lymphoma or Waldenström macroglobulinemia, cold agglutinins may be produced by the malignant clone itself. Two patients with lymphoma and monoclonal cold agglutinin were identified as having a karyotypically abnormal B-cell clone that produced a cold agglutinin identical to that found in their sera.^158,159 Trisomy 3 has been the most frequently observed karyotypic abnormality in patients with non-Hodgkin lymphoma and cold agglutinins.^158,160

Normal human sera generally have naturally occurring polyclonal cold agglutinins in low titer (usually 1/64 or less).¹⁰ Otherwise healthy persons may develop elevated titers of cold agglutinins specific for I/i antigens during certain infections (e.g., M. pneumoniae, Epstein-Barr virus, cytomegalovirus). In contrast to other forms of cold agglutinin disease, hyperproduction of these postinfectious cold agglutinins is transient. Some evidence indicates postinfectious cold agglutinins may be less clonally restricted than those occurring in chronic cold agglutinin disease,¹⁶¹ but this finding is not universal.¹⁶² Whether IGHV4–34 also encodes most heavy-chain variable regions of all naturally occurring or postinfectious cold agglutinins remains to be determined.

The increased production of cold agglutinins in response to infection with M. pneumoniae may be secondary to the fact that the oligosaccharide antigens of the I/i type serve as specific Mycoplasma receptors.¹⁶³ This process may lead to altered antigen presentation involving a complex between a self-antigen (I/i) and a non–self-antigen (Mycoplasma). Alternatively, the anti-i cold agglutinins may arise as a consequence of polyclonal B cell activation, as occurs in infectious mononucleosis (Chap. 82).

The mechanism(s) whereby dissimilar infectious agents (e.g., spirochetes and several types of virus) induce the immune system to produce Donath-Landsteiner antibodies with specificity for the human P blood group antigen (see “Serologic Features” below) is not known.

PATHOGENESIS

Pathogenic Effects of Warm Antibodies

Warm autoantibodies to RBCs in AHA are pathogenic. In contrast to autologous RBCs, labeled RBCs lacking the antigen targeted by the autoantibodies may survive normally in patients with warm-antibody AHA.^10,164,165 Furthermore, transplacental passage of IgG anti-RBC autoantibodies from a mother with AHA to the fetus can induce intrauterine or neonatal hemolytic anemia.¹⁶⁶ Finally, despite notable exceptions and differences related to IgG subclass of the autoantibody, in general, an inverse relationship between the quantity of RBC-bound IgG antibody and RBC survival is noted in serial studies performed on animals and patients.^{167,168,169,170,171,172}

In warm-antibody AHA, the patient’s RBCs typically are coated with IgG autoantibodies with or without complement proteins. Autoantibody-coated RBCs are trapped by macrophages in the Billroth cords of the spleen and, to a lesser extent, by Kupffer cells in the liver (Chap. 68).^{164,167,168,170,171,172,173,174} The process leads to generation of spherocytes and fragmentation and ingestion of antibody-coated RBCs.^175,176 The macrophage has surface receptors for the Fc region of IgG, with preference for the IgG₁ and IgG₃ subclasses^177,178 and surface receptors for opsonic fragments of C3 (C3b and C3bi) and C4b.^179,180,181 When present together on the RBC surface, IgG and C3b/C3bi appear to act cooperatively as opsonins to enhance trapping and phagocytosis.^{170,171,180,181,182,183,184} Although RBC sequestration in warm-antibody AHA occurs primarily in the spleen,^{164,171,172,173} very large quantities of RBC-bound IgG^167,168,174 or the concurrent presence of C3b on the RBCs^167,170,171 may favor trapping in the liver.

Interaction of a trapped RBC with splenic macrophages may result in phagocytosis of the entire cell. More commonly, a type of partial phagocytosis results in spherocyte formation. As RBCs adhere to macrophages via the Fc receptors, portions of RBC membrane are internalized by the macrophage. Because membrane is lost in excess of contents, the noningested portion of the RBC assumes a spherical shape, the shape with the lowest ratio of surface area to volume.^175,176,185 Spherical RBCs are more rigid and less deformable than normal RBCs. As such, spherical RBCs are fragmented further and eventually destroyed in future passages through the spleen. Spherocytosis is a consistent and diagnostically important hallmark of AHA,¹⁸⁶ and the degree of spherocytosis correlates well with the severity of hemolysis.¹⁰

Direct complement-mediated hemolysis with hemoglobinuria is unusual in warm-antibody AHA, even though many warm autoantibodies fix complement. The failure of C3b-coated RBCs to be hemolyzed by the terminal complement cascade (C5–C9) has been attributed, at least in part, to the ability of complement regulatory proteins (factors I and H) in plasma and C3b receptors on the RBC surface to alter the hemolytic function of cell-bound C3b and C4b.¹⁸⁷ Glycosylphosphatidylinositol-linked erythrocyte membrane proteins, such as decay-accelerating factor (DAF; CD55) and homologous restriction factor (HRF; CD59), may limit the action of autologous complement on autoantibody-coated RBCs.^188,189,190 DAF inhibits the formation and function of cell-bound C3-converting enzyme,¹⁸⁸ thus, indirectly limiting formation of C5-converting enzyme. HRF, on the other hand, impedes C9 binding and formation of the C5b–9 membrane attack complex.¹⁸⁹

Cytotoxic activities of macrophages and lymphocytes also may play a role in the destruction of RBCs in warm-antibody AHA. Monocytes can lyse IgG-coated RBCs in vitro independently of phagocytosis.^191,192 Cell-bound complement is neither necessary nor sufficient for such cytotoxicity, but bound C3b/C3d can potentiate the effects of IgG.¹⁹² In one study, cytotoxicity, but not phagocytosis, was inhibited by hydrocortisone in vitro.¹⁹¹ Lymphocytes also can lyse IgG antibody-coated RBCs in vitro.^193,194,195 The relative contribution of antibody-dependent monocyte- and lymphocyte-mediated cytotoxicity to RBC destruction in patients with warm-antibody AHA is not known.

Pathogenic Effects of Cold Agglutinins and Hemolysins

Most cold agglutinins are unable to agglutinate RBCs at temperatures higher than 30°C. The highest temperature at which these antibodies cause detectable agglutination is termed the thermal amplitude. The value varies considerably among patients. Generally, patients with cold agglutinins with higher thermal amplitudes have a greater risk for cold agglutinin disease.⁹ For example, active hemolytic anemia has been observed in patients with cold agglutinins of modest titer (e.g., 1:256) and high thermal amplitudes.¹⁹⁶

The pathogenicity of a cold agglutinin depends upon its ability to bind host RBCs and to activate complement.^{10,182,197,198} This process is called complement fixation. Although in vitro agglutination of the RBCs may be maximal at 0 to 5°C, complement fixation by these antibodies may occur optimally at 20 to 25°C and may be significant at even higher physiologic temperatures.^10,196,197 Agglutination is not required for the process. The great preponderance of cold agglutinin molecules are IgM pentamers, but small numbers of IgM hexamers with cold agglutinin activity are found in patients with cold agglutinin disease. Hexamers fix complement and lyse RBCs more efficiently than do pentamers, suggesting that hexameric IgM plays a role in the pathogenesis of hemolysis in these patients.¹⁹⁹

Cold agglutinins may bind to RBCs in superficial vessels of the extremities, where the temperature generally ranges between 28 and 31°C, depending upon ambient temperature.²⁰⁰ Cold agglutinins of high thermal amplitude may cause RBCs to aggregate at this temperature, thereby impeding RBC flow and producing acrocyanosis. In addition, the RBC-bound cold agglutinin may activate complement via the classic pathway. Once activated complement proteins are deposited onto the RBC surface, the cold agglutinin need not remain bound to the RBCs for hemolysis to occur. Instead, the cold agglutinin may dissociate from the RBCs at the higher temperatures in the body core and again be capable of binding other RBCs at the lower temperatures in the superficial vessels. As a result, patients with cold agglutinins of high thermal amplitude tend toward a sustained hemolytic process and acrocyanosis.²⁰¹ In contrast, patients with antibodies of lower thermal amplitude require significant chilling to initiate complement-mediated injury of RBCs. This sequence may result in a burst of hemolysis with hemoglobinuria.²⁰¹ Combinations of these clinical patterns also occur. Cold agglutinins of the IgA isotype, an isotype that does not fix complement, may cause acrocyanosis but not hemolysis.²⁰² Thus, the relative degree of hemolysis or impeded RBC flow is influenced significantly by the properties and quantity of the cold agglutinins in a given patient.

Complement fixation by cold agglutinins may affect RBC injury by two major mechanisms: (1) direct lysis and (2) opsonization for hepatic and splenic macrophages. Both mechanisms probably operate to varying degrees in any patient. Direct lysis requires propagation of the full C1 to C9 sequence on the RBC membrane. If this process occurs to a significant degree, the patient may experience intravascular hemolysis leading to hemoglobinemia and hemoglobinuria. Intravascular hemolysis of this severity is relatively rare because phosphatidylinositol-linked RBC membrane proteins (DAF and HRF) protect against injury by autologous complement components. Thus, the complement sequence on many RBCs is completed only through the early steps, leaving opsonic fragments of C3 (C3b/C3bi) and C4 (C4b) on the cell surface. The fragments provide only a weak stimulus for phagocytosis by monocytes in vitro.^184,203 However, activated macrophages may ingest C3b-coated particles avidly.²⁰⁴ Accordingly, RBCs heavily coated with C3b (and/or C3bi) may be removed from the circulation by macrophages either in the liver or, to a lesser extent, the spleen.^{171,197,205,206} The trapped RBCs may be ingested entirely or released back into the circulation as spherocytes after losing plasma membrane.

In vivo studies of the fate of ⁵¹Cr-labeled C3b-coated RBCs^{170,197,205,206} indicate many of the erythrocytes trapped in the liver or spleen gradually may reenter the circulation. The released cells generally are coated with the opsonically inactive C3 fragment C3dg. Conversion of cell-bound C3b or C3bi to C3dg results from the action of the naturally occurring complement inhibitor factor I in concert with factor H or CR1 receptors.¹⁸¹ The surviving C3dg-coated RBCs circulate with a near-normal life span^{170,197,205,206} and are resistant to further uptake of cold agglutinins or complement.^197,205,207 However, C3dg-coated RBCs also may react in vitro with anticomplement (anti-C3) serum in the DAT. In fact, most of the antiglobulin-positive RBCs of patients with cold agglutinin disease are coated with C3dg.

In paroxysmal cold hemoglobinuria, the mechanism of hemolysis probably parallels in vitro events (see “Serologic Features” below). During severe chilling, blood flowing through skin capillaries is exposed to low temperatures. The Donath-Landsteiner antibody and early acting complement components presumably bind to RBCs at the lowered temperatures. Upon return of the cells to 37°C in the central circulation, the cells are lysed by propagation of the terminal complement sequence through C9. The Donath-Landsteiner antibody itself dissociates from the RBCs at 37°C. Erythrocyte membrane proteins that restrict C5b–9 assembly (e.g., HRFs) may be less effective in controlling Donath-Landsteiner antibody-initiated complement activation than that initiated by cold agglutinins.

Pathogenesis of Drug-Mediated Immune Injury

Table 54–3 summarizes the three mechanisms of drug-mediated immune injury to RBCs. Drugs also may mediate protein adsorption to RBCs by nonimmune mechanisms, but RBC injury does not occur.

Hapten or Drug Adsorption Mechanism

This mechanism applies to drugs that can bind firmly to proteins, including RBC membrane proteins. The classic setting is very-high-dose penicillin therapy,^{28,29,30,31,32,33,34} which is encountered less commonly today than in previous decades.

Most individuals who receive penicillin develop IgM antibodies directed against the benzylpenicilloyl determinant of penicillin, but this antibody plays no role in penicillin-related immune injury to RBCs. The antibody responsible for hemolytic anemia is of the IgG class, occurs less frequently than the IgM antibody, and may be directed against the benzylpenicilloyl,³¹ or, more commonly, nonbenzylpenicilloyl determinants.^28,29,30,32 Other manifestations of penicillin sensitivity usually are not present.

All patients receiving high doses of penicillin develop substantial coating of RBCs with penicillin. The penicillin coating itself is not injurious. If the penicillin dose is very high (10 to 30 × 10⁶ units per day, or less in the setting of renal failure) and promotes cell coating, and if the patient has an IgG antipenicillin antibody, the antibody binds to the RBC-bound penicillin molecules and the DAT with anti-IgG becomes positive (see Fig. 54–1A).^{29,31,32,51,208} Antibodies eluted from patients’ RBCs or present in their sera react in the indirect antiglobulin test (IAT) only against penicillin-coated RBCs. This step is critical in distinguishing these drug-dependent antibodies from true autoantibodies.

Not all patients receiving high-dose penicillin develop a positive DAT reaction or hemolytic anemia because only a small proportion of such individuals produce the requisite antibody. Destruction of RBCs coated with penicillin and IgG antipenicillin antibody occurs mainly through sequestration by splenic macrophages.^30,209 In some patients with penicillin-induced immune hemolytic anemia, blood monocytes and presumably splenic macrophages may lyse the IgG-coated RBCs without phagocytosis.²¹⁰ Hemolytic anemia resulting from penicillin typically occurs only after the patient has received the drug for 7 to 10 days and ceases a few days to 2 weeks after the patient discontinues taking the drug.

Low-molecular-weight substances, such as drugs, generally are not immunogenic in their own right. Induction of antidrug antibody is thought to require firm chemical coupling of the drug (as a hapten) to a protein carrier. In the case of penicillin, the carrier protein involved in antibody induction need not be the same as the erythrocyte membrane protein to which penicillin is coupled in the effector phase, that is, when the IgG antipenicillin antibodies bind to penicillin-coated RBCs. In contrast to evidence on the ternary complex mechanism, no evidence indicates the drug-dependent antibodies responsible for RBC injury in this hapten/drug adsorption mechanism also recognize native erythrocyte membrane structures.

Cephalosporins have antigenic cross-reactivity with penicillin^211,213 and bind firmly to RBC membranes, as do semisynthetic penicillins.^33,34 Hemolytic anemia similar to that seen with penicillin has been ascribed to cephalosporins^{35,36,37,38,39} and some semisynthetic penicillins.^33,34 Tetracycline^40,41 and tolbutamide^44,45 also may cause hemolysis by this mechanism. Carbromal causes positive IgG antiglobulin reactions by a similar mechanism,⁴³ but hemolytic anemia has not been described.

Ternary Complex Mechanism: Drug–Antibody–Target Cell Interaction

Many drugs can induce immune injury not only of RBCs but also of platelets or granulocytes by a process that differs in several ways from the mechanism of hapten/drug adsorption (see Table 54–3). First, drugs in this group (see Table 54–2) exhibit only weak direct binding to blood cell membranes. Second, a relatively small dose of drug is capable of triggering destruction of blood cells. Third, cellular injury appears to be mediated chiefly by complement activation at the cell surface. The cytopathic process induced by such drugs previously has been termed the innocent bystander or immune complex mechanism. The terminology reflected the prevailing notion that, in vivo, drug–antibody complexes formed first (immune complexes) and then became secondarily bound to target blood cells as “innocent bystanders,” either nonspecifically or possibly via membrane receptors (e.g., Fcγ receptors on platelets or C3b receptors on red cells), with the potential for subsequent activation of complement by bound complexes.

The “immune complex” and “innocent bystander” terminology now seems less appropriate because of models developed from research on analogous drug-dependent platelet injury^214,215,216 (Chap. 117) and a series of relevant serologic observations on drug-mediated immune hemolytic anemia. These studies suggest blood cell injury is mediated by a cooperative interaction among three reactants to generate a ternary complex (see Fig. 54–1B) involving (1) the drug (or drug metabolite in some cases), (2) a drug-binding membrane site on the target cell, and (3) antibody. For example, several patients possessed drug-dependent antibodies that exhibited specificity for RBCs bearing defined alloantigens such as those of the Rh, Kell, or Kidd blood groups. That is, even in the presence of drug, the antibodies were selectively nonreactive with human RBCs lacking the alloantigen in question.^{58,84,217,218,219} In each case, high-affinity drug binding to cell membrane could not be demonstrated. The drug-dependent antibody is thought to bind, through its Fab domain, to a compound neoantigen consisting of loosely bound drug and a blood group antigen intrinsic to the red cell membrane. Elegant studies on quinidine- or quinine-induced immune thrombocytopenia have demonstrated the IgG antibodies implicated in this disorder bind through their Fab domains, not by their Fc domains to platelet Fcγ receptors.^220,221

The data elucidate how one patient with quinidine sensitivity may have selective destruction of platelets and another may have selective destruction of RBCs. This process occurs because the pathogenic antibody recognizes the drug only in combination with a particular membrane structure of the RBC (e.g., a known alloantigen) or of the platelet (e.g., α domain of the glycoprotein Ib complex). Therefore, at least in these cases, the target cell does not appear to be purely an innocent bystander. Binding of the drug itself to the target cell membrane is weak until the attachment of the antibody to both drug and cell membrane is stabilized. Yet the binding of the antibody is drug dependent. Such a three-reactant interdependent “troika” is unique to this mechanism of immune cytopenia.

The foregoing discussion depicting drugs as creating a “self + non-self” neoantigen on the target cell applies to the effector phase as opposed to the induction phase of the process. However, the same drug-binding membrane protein appears to be involved in forming the immunogen that induces the antibody, as evidenced by drug-dependent antibodies exhibiting selective reactivity with defined red cell alloantigens (carrier specificity).^{58,84,217,218,219} How this process is accomplished in the absence of evidence for strong, covalent binding of the drugs in this group to a host membrane protein remains to be elucidated.

RBC destruction by this mechanism may occur intravascularly after completion of the whole complement sequence, resulting in hemoglobinemia and hemoglobinuria. Some destruction of intact C3b-coated RBCs may be mediated by splenic and liver sequestration via the C3b/C3bi receptors on macrophages. The DAT is positive usually only with anticomplement reagents, but exceptions occur. Sometimes, however, the drug-dependent antibody itself can be detected on the RBCs if the offending drug (or its metabolites) is included in all steps of the antiglobulin test, including washing.²²²

Autoantibody Mechanism

A variety of drugs induce the formation of autoantibodies reactive with autologous (or homologous) RBCs in the absence of the instigating drug (see Tables 54–2 and 54–3). The most studied drug in this category has been α-methyldopa, an antihypertensive agent that no longer is commonly used.^73,74,75,76 Levodopa and several unrelated drugs also have been implicated.^{39,46,59,61,68,77,78,79,80,81,82,83,84,85,86} Patients with CLL treated with pentostatin,⁸⁷ fludarabine,⁸⁸ or cladribine⁹⁰ are particularly predisposed to autoimmune hemolysis, which usually is severe and sometimes fatal.

Positive DAT reactions (with anti-IgG reagents) in patients taking α-methyldopa vary in frequency from 8 to 36 percent. Patients taking higher doses of the drug develop positive reactions with greater frequency.^73,75,76 A lag period of 3 to 6 months exists between the start of therapy and development of a positive antiglobulin test. The delay is not shortened when the drug is administered to patients who previously had positive antiglobulin tests while taking α-methyldopa.⁷⁵

In contrast to the frequent observation of positive antiglobulin reactions, less than 1 percent of patients taking α-methyldopa exhibit hemolytic anemia.⁷⁴ Development of hemolytic anemia does not depend on drug dose. The hemolysis usually is mild to moderate and occurs chiefly by splenic sequestration of IgG-coated RBCs. α-Methyldopa has been proposed to suppress splenic macrophage function in some patients, and normal survival of antibody-coated RBCs in such patients may be related, in part, to this effect of the drug.²²³

The DAT reaction usually is positive only for IgG.¹¹ Occasionally, weak anticomplement reactions also are encountered.¹¹ Patients with immune hemolytic anemia resulting from α-methyldopa therapy typically exhibit strongly positive DAT reactions and serum antibody, evidenced by the IAT reaction.¹¹ Antibodies in the serum or eluted from RBC membranes react optimally at 37°C with unaltered autologous or homologous RBCs in the absence of drug (see Fig. 54–1C).^74,76,224 Frequently the autoantibodies are reactive with determinants of the Rh complex,^74,76,224 and at least some appear to target the same 34-kDa Rh-related polypeptide targeted by the autoantibodies in many cases of “spontaneously arising” AHA.¹³⁶ Thus, distinguishing these drug-induced antibodies from similar warm-reacting autoantibodies in idiopathic AHA currently is not possible.

The mechanism by which a drug induces formation of an autoantibody is unknown. Radiolabeled α-methyldopa does not react directly with the membranes of intact human RBCs.^76,225 However, both α-methyldopa and levodopa reportedly bind to isolated RBC membranes. Binding of the drug to membranes of intact RBCs is inhibited by RBC superoxide dismutase and probably by hemoglobin.^225,226 Although not formally demonstrated, these drugs probably bind to membrane antigens of cells that are relatively hemoglobin free, for example, cells at the early proerythroblast stage or RBC stroma. In any case, the resulting altered membrane antigens then may induce autoantibodies. The concept that a drug–membrane compound neoantigen could lead to production of an autoantibody is supported by studies of patients receiving drugs unrelated to α-methyldopa. Patients simultaneously developed a drug-dependent antibody and an autoantibody, both of which showed specificity for the same RBC alloantigen.⁸⁴ Another hypothesis is that α-methyldopa interacts with human T lymphocytes, resulting in loss of suppressor cell function.²²⁷ Subsequent studies, however, have failed to demonstrate any evidence for such a mechanism.²²⁸

Patients with CLL treated with the purine analogues fludarabine^88,229,230 or cladribine⁹⁰ may develop AHA. Risk factors for hemolysis include previous therapy with a purine analogue, high β₂-microglobulin, a positive DAT prior to therapy, and hypogammaglobulinemia. Purine analogues are potent suppressors of T lymphocytes. These drugs may accelerate the preexisting T-cell immune suppression that normally occurs during progression of CLL, exacerbating the underlying tendency to autoimmunity in CLL. However, the degree of depletion of T-cell subsets is similar in patients who develop hemolysis and in patients who do not.

Nonimmunologic Protein Adsorption

Less than 5 percent of patients receiving cephalosporin antibiotics develop positive antiglobulin reactions¹¹ as a result of nonspecific adsorption of plasma proteins to their RBC membranes.^93,94,231 This process may occur within 1 to 2 days after the drug is instituted. Multiple plasma proteins, including immunoglobulins, complement, albumin, fibrinogen, and others, may be detected on RBC membranes in such cases.^231,232 Hemolytic anemia resulting from this mechanism has not been reported. The clinical importance of this phenomenon is its potential to complicate crossmatch procedures unless the drug history is considered. Cephalosporin antibiotics also may induce RBC injury by the hapten mechanism, by the ternary complex mechanism, and by the autoantibody mechanism. The latter reactions are more serious but apparently occur less frequently than nonimmunologic protein adsorption.

WARM-ANTIBODY AUTOIMMUNE HEMOLYTIC ANEMIA

Presenting complaints of warm-antibody AHA usually are referable to the anemia itself, although occasionally jaundice is the immediate cause for the patient to seek medical advice. Symptom onset usually is slow and insidious over several months, but occasionally a patient has sudden onset of symptoms of severe anemia and jaundice over a period of a few days. In secondary AHA, the symptoms and signs of the underlying disease may overshadow the hemolytic anemia and associated features.

In idiopathic AHA with only mild anemia, results of physical examination may be normal. Even patients with relatively severe hemolytic anemia may have only modest splenomegaly. However, in very severe cases, particularly those of acute onset, patients may present with fever, pallor, jaundice, hepatosplenomegaly, hyperpnea, tachycardia, angina, or heart failure.

Clinical warm-antibody AHA may be aggravated or first become apparent during pregnancy.^166,233,234 Most cases are mild, however, and the prognosis for the fetus is generally good, provided the mother is treated early.²³³

COLD-ANTIBODY AUTOIMMUNE HEMOLYTIC ANEMIA

Most patients with cold agglutinin hemolytic anemia have chronic hemolytic anemia with or without jaundice. In other patients, the principal feature is episodic, acute hemolysis with hemoglobinuria induced by chilling (see discussion of thermal amplitude in “Pathogenic Effects of Cold Agglutinins and Hemolysins” above). Combinations of these clinical features may occur. Acrocyanosis and other cold-mediated vasoocclusive phenomena affecting the fingers, toes, nose, and ears are associated with sludging of RBCs in the cutaneous microvasculature. Skin ulceration and necrosis are distinctly unusual. Hemolysis occurring in M. pneumoniae infections is acute in onset, typically appearing as the patient is recovering from pneumonia and coincident with peak titers of cold agglutinins. The hemolysis is self-limited, lasting 1 to 3 weeks.¹¹ Hemolytic anemia in infectious mononucleosis develops either at the onset of symptoms or within the first 3 weeks of illness.¹²⁸

Other physical findings are variable, depending upon the presence of an underlying disease. Splenomegaly, a characteristic finding in lymphoproliferative diseases or infectious mononucleosis, may be observed in idiopathic cold agglutinin disease.

In paroxysmal cold hemoglobinuria, constitutional symptoms are prominent during a paroxysm. A few minutes to several hours after cold exposure, the patient develops aching pains in the back or legs, abdominal cramps, and perhaps headaches. Chills and fever usually follow. The first urine passed after onset of symptoms typically contains hemoglobin. The constitutional symptoms and hemoglobinuria generally last a few hours. Raynaud phenomenon and cold urticaria sometimes occur during an attack; jaundice may follow.

DRUG-INDUCED IMMUNE HEMOLYTIC ANEMIA

A careful history of drug exposure should be obtained from all patients with hemolytic anemia and/or a positive DAT. As in idiopathic AHA, the clinical picture in drug-induced immune hemolytic anemia is quite variable. The severity of symptoms largely depends upon the rate of hemolysis. In general, patients with hapten/drug adsorption (e.g., penicillin) and autoimmune (e.g., α-methyldopa) types of drug-induced hemolytic anemia exhibit mild to moderate hemolysis, with insidious onset of symptoms developing over a period of days to weeks. In contrast, the ternary complex mechanism (e.g., cephalosporins or quinidine) often causes sudden, severe hemolysis with hemoglobinuria. In the latter setting, hemolysis can occur after only one dose of the drug in a patient previously exposed to the drug. Acute renal failure may accompany severe hemolysis by the ternary complex mechanism.^{39,56,58,62,63,86} Several reports indicate that second- and third-generation cephalosporins may cause severe, even fatal, hemolysis by the ternary complex mechanism.^37,38,39,64

GENERAL FEATURES

By definition, patients with AHA present with anemia, the severity of which ranges from life-threatening to very mild. Patients with warm-antibody AHA may present with hematocrit levels less than 10 percent or may have compensated hemolytic anemia and a near-normal hematocrit. For the latter patients, the predominant laboratory features are an increased reticulocyte count and a positive DAT. Occasionally, the patient has leukopenia and neutropenia.^10,235 Platelet counts typically are normal. Rarely, severe immune thrombocytopenia is associated with warm-antibody AHA. This constellation is termed Evans syndrome.²³⁶ In this syndrome, the RBC and platelet antibodies are apparently distinct.²³⁷

Patients with classic chronic cold agglutinin disease exhibit mild to moderate, fairly stable anemia, with hematocrit levels only occasionally as low as 15 to 20 percent. In contrast, patients with paroxysmal cold hemoglobinuria have hematocrit levels that decrease rapidly during a paroxysm. During a paroxysm, leukopenia is noted early, followed by leukocytosis. Complement titers frequently are depressed because of consumption of complement proteins during hemolysis.

In drug-induced immune hemolytic anemia of the hapten/drug adsorption and true autoantibody types, the hematologic findings are similar to those described for spontaneously occurring warm-antibody AHA. Most patients exhibit anemia and reticulocytosis. Leukopenia and thrombocytopenia may be noted in cases of ternary complex-mediated hemolysis.

Evaluation of the blood film can reveal several features related to all types of AHA (Fig. 54–2). Polychromasia indicates a reticulocytosis, reflecting an increased rate of reticulocyte egress from the marrow. Spherocytes are seen in patients with moderate to severe hemolytic anemia. If hereditary spherocytosis can be excluded, this finding suggests an immune hemolytic process. RBC fragments, nucleated RBCs, and occasionally erythrophagocytosis by monocytes may be seen in severe cases (Fig. 54–2). Most patients have mild leukocytosis and neutrophilia. Additionally, patients with cold-antibody AHA may exhibit RBC autoagglutination in the blood film and in chilled anticoagulated blood (Fig. 54–3).

The reticulocyte count usually is elevated. Nevertheless, early in the course of the disease, more than one-third of all patients may have transient reticulocytopenia despite a normal or hyperplastic erythroid marrow.^{238,239,240,241} The mechanism is unknown, but autoantibodies reactive against antigens on reticulocytes are speculated to lead to their selective destruction.²³⁹ One unusual patient with warm-antibody AHA, reticulocytopenia, and marrow erythroid aplasia had a serum autoantibody that inhibited erythroid colony formation in vitro.²⁴² The aplastic crisis remitted after the serum IgG level was lowered by immunoadsorption. Reticulocytopenia also may be seen in patients with marrow function compromised by an underlying disease, parvovirus infection, toxic chemicals, or nutritional deficiency. Marrow examination usually reveals erythroid hyperplasia and may provide evidence of an underlying lymphoproliferative disorder.

Hyperbilirubinemia (chiefly unconjugated) is highly suggestive of hemolytic anemia, although its absence does not exclude the diagnosis. Total bilirubin is only modestly increased (up to 5 mg/dL) and, with rare exceptions, the conjugated (direct) fraction constitutes less than 15 percent of the total. Urinary urobilinogen is increased regularly, but bile is not detected in the urine unless serum conjugated bilirubin is increased. Usually, serum haptoglobin levels are low, and lactate dehydrogenase levels are elevated. Hemoglobinuria is encountered in rare patients with warm-antibody AHA and hyperacute hemolysis, more commonly in patients with cold agglutinin disease, and characteristically in patients with paroxysmal cold hemoglobinuria and with drug-induced immune hemolytic anemia mediated by the ternary complex mechanism.

Direct Antiglobulin Test Pattern

Diagnosis of AHA or drug-induced immune hemolytic anemia requires demonstration of immunoglobulin and/or complement bound to the patient’s RBCs. As a screening procedure, use of a “broad-spectrum” antiglobulin (Coombs) reagent—that is, one that contains antibodies directed against human immunoglobulin and complement components (principally C3)—is customary. If agglutination is noted with a broad-spectrum reagent, antisera reacting selectively with IgG (the “gamma” Coombs) or with C3 (the “nongamma” Coombs) are used to define the specific pattern of RBC sensitization. Monospecific antisera to IgM or IgA also have been used in selected cases.

Three possible major patterns of direct antiglobulin reaction in AHA and drug-induced immune hemolytic anemia exist: (1) RBCs coated with only IgG, (2) RBCs coated with IgG and complement components, and (3) RBCs coated with complement components without detectable immunoglobulin.^{10,123,243,244} In patterns 2 and 3, the complement components most readily detected are C3 fragments (mainly C3dg). Each pattern is associated with accelerated RBC destruction. Positive antiglobulin reactions with anti-IgA or anti-IgM are encountered less commonly, often in association with bound IgG and/or complement.^{245,246,247,248,249,250,251} Table 54–4 summarizes the diagnostic significance of each of these major patterns (see “Serologic Features” below).

SEROLOGIC FEATURES

Warm-Antibody Autoimmune Hemolytic Anemia

Free versus Bound Autoantibody

The autoantibody molecules in patients with warm-antibody AHA exist in a reversible, dynamic equilibrium between RBCs and plasma.^252,253 In addition to the major portion of autoantibody bound to the patient’s RBCs (detected by the DAT), “free” autoantibody may be detected in the plasma or serum of these patients by the IAT. In the IAT, the patient’s serum or plasma is incubated with normal donor erythrocytes at the appropriate temperature (in this case, 37°C). The cells are washed, suspended in saline solution, and then tested for agglutination by antiglobulin serum. The presence of unbound autoantibody in plasma depends upon the total amount of antibody being produced and the binding affinity of the antibody for RBC antigens. In general, patients whose RBCs are heavily coated with IgG more likely exhibit plasma autoantibody. Protease-modified RBCs are more sensitive than native RBCs in detecting plasma autoantibody, but such data must be interpreted with caution, because alloantibodies, naturally occurring antibodies to cryptic antigens, and other serum components may interact with enzyme-modified RBCs. Patients with a positive IAT as a result of a warm-reactive autoantibody should also have a positive DAT. A patient with a serum anti-RBC antibody (positive IAT) and a negative DAT probably does not have an autoimmune process but rather an alloantibody stimulated by prior transfusion or pregnancy.

Quantity, Affinity, and Isotype of Red Blood Cell–Bound Autoantibody

Direct Antiglobulin Test–Negative Autoimmune Hemolytic Anemia

Figure 54–4 relates the intensity of the direct antiglobulin reaction, using specific anti-IgG serum, to the number of IgG molecules bound per RBC. The latter was determined by a sensitive antibody-consumption method.²⁵⁴ A trace-positive antiglobulin reaction (read macroscopically) detects 300 to 400 molecules of IgG per cell.^254,255 In another laboratory, a trace-positive antiglobulin reaction with anti-C3 was obtained with 60 to 115 molecules C3 per cell.¹⁸²

Sometimes patients with warm-antibody AHA and many of its hallmarks, for example, anemia, reticulocytosis, spherocytes, elevated lactate dehydrogenase (LDH), low or absent haptoglobin, present with a negative DAT. There are three principal causes for the negative DAT: IgG or complement sensitization below the threshold of detection of commercial antiglobulin (Coombs) reagents; low affinity IgG sensitization with loss of cell-bound antibody during the cell washing steps before the direct antiglobulin reaction; sensitization with IgA or IgM antibodies which many commercial DAT reagents cannot detect because they contain only anti-IgG anti-C3.

More sensitive methods for quantifying RBC-bound IgG allow identification of AHA patients having all the usual features of warm-antibody AHA but a negative DAT with antiimmunoglobulin and anticomplement reagents.^254,255,256 In these cases, specialized methods (e.g., anti-IgG consumption assays, automated enhanced agglutination techniques, enzyme-linked immunoassays, radioimmunoassays, flow cytometry) detect very small quantities of cell-bound IgG. In such cases, studies with highly concentrated RBC eluates confirm these IgG molecules are warm-reacting anti-RBC autoantibodies.²⁵⁴ Patients generally have relatively mild hemolysis and often respond favorably to glucocorticoid therapy. By these specialized methods, subthreshold IgG also may be detected in a significant number of patients exhibiting the “complement alone” pattern of direct antiglobulin reaction in the absence of drug sensitivity or cold agglutinins. Studies with concentrated RBC eluates suggest subthreshold quantities of bound IgG antibodies are capable of fixing much larger quantities of C3 to the cell membrane.²⁵⁴ In cases of low-affinity IgG sensitization, detection of IgG bound to RBCs may be accomplished by cold washing (0 to 4°C) or by use of low ionic strength saline in the wash steps prior to the DAT reaction. Cell Bound IgA and IgM may be detected by means of antisera to IgA and IgM. DAT-negative AHA has been reviewed extensively elsewhere.^257,258,259

Nature of the Autoantibodies and Red Blood Cell Target Antigens

In any series of warm-antibody AHA patients, the correlation between the strength of the antiglobulin reaction (IgG molecules per RBC) and the rate of RBC destruction is variable. The IgG subclass of warm autoantibodies influences the degree to which these antibodies shorten RBC survival. IgG₁ is the most commonly encountered subclass, either alone or in combination with other IgG subclasses.^245,260 IgG₁ and IgG₃ autoantibodies appear to be more effective in decreasing RBC life span than do those of the IgG₂ or IgG₄ subclass.^245,261

The difference may result from the greater affinity of macrophage Fc receptors for IgG₁ and IgG₃^177,178 and the higher complement-fixing activity of IgG₁ or IgG₃ antibodies relative to the activity of IgG₂ or IgG₄ antibodies.¹⁸⁷

Autoantibodies eluted from patients’ RBCs or present in their plasma typically bind to all the common types of human RBCs represented in test panels used by blood banks and thus might appear to be nonspecific. However, the antibodies of any one patient typically recognize one or more antigenic determinants (epitopes) that are common to almost all human RBCs, that is, “public” antigens. These antibodies have been useful for evaluating RBC membrane structures and for identifying rare RBC phenotypes, namely, RBCs that lack a common blood group antigen(s). Nearly half of all AHA patients have autoantibodies specific for epitopes on Rh proteins.^{10,11,131,262,264} The autoantibodies of such patients commonly do not react with human Rh_null RBCs, which lack expression of the Rh complex. Occasionally, the anti-Rh autoantibodies have anti-e, anti-E, or anti-c (or, more rarely, anti-D) specificity. Patients who have autoantibodies with selective specificity (e.g., anti-e) nearly always have other autoantibodies reactive with all human RBCs, except Rh_null. Autoantibodies with such specificity are designated collectively as Rh related.^136,264

The remaining patients with warm-antibody AHA have IgG autoantibodies that are fully reactive with Rh_null RBCs.^{10,11,131,262,263,264} The exact specificity of the autoantibodies for many of these patients is undefined. However, in other instances, autoantibody specificity for serologically defined blood group antigens outside the Rh system has been identified (using RBCs of appropriate antigen-deficient phenotype) including anti-Wr^b,131 anti-En^a,265 anti-LW,²⁶⁶ anti-U,²⁶⁷ anti-Ge,^250,268 anti-Sc1,²⁶⁹ or antibodies to Kell blood group antigens.²⁷⁰ For ease of reference, the entire group of autoantibodies is designated non-Rh related.^136,264

Immunochemical studies indicate the autoantibodies from almost any AHA patient react with individual membrane proteins. The major target of Rh-related autoantibodies is a 32- to 34-kDa nonglycosylated polypeptide lacking on Rh_null RBCs.^136,271 This polypeptide is similar, if not identical, to the polypeptide expressing the Rh(e) alloantigen. Many α-methyldopa–induced autoantibodies also react with this polypeptide.¹³⁶ Autoantibodies with non-Rh serologic specificity react with the band 3 anion transporter^136,272 or with both band 3 and glycophorin A.¹³⁶ The latter autoantibodies may react with an epitope formed through the interaction of these two proteins on the RBC membrane.²⁷³ It is interesting to note that anti-RBC autoantibodies in NZB mice exhibit anti–band 3 specificity.²⁷⁴ Furthermore, naturally occurring anti–band 3 IgG autoantibodies are found in almost all humans.^275,276 These autoantibodies may play a role in the clearance of senescent RBCs by reacting with neoantigens formed on these cells by proteolytic alteration²⁷⁵ or aggregation²⁷⁶ of band 3 proteins. Such neoantigens are not found on younger RBCs. An important but unanswered question concerns the possible relationship between naturally occurring and pathologic anti–band 3 autoantibodies.

Cold-Antibody Hemolytic Anemia

Cold agglutinins are distinguished by their ability to directly agglutinate saline-suspended human RBCs at low temperature, maximally at 0 to 5°C. The reaction is reversible by warming. In chronic cold agglutinin disease, serum titers are commonly 1:1000 or higher and may reach 1:512,000 or more.¹¹ Cold agglutinins are characteristically IgM. IgA or IgG cold agglutinins have been reported,^11,202,277 sometimes in combination with IgM.²⁷⁸ In mixed warm- and cold-antibody AHA, warm-reactive IgG autoantibodies are found in association with IgM cold agglutinins.²¹

The DAT result is positive with anticomplement reagents. The antibody itself, however, is not detected by the DAT because the cold agglutinins readily dissociate from the RBCs both in vivo and during the washing steps of the standard antiglobulin procedure. In contrast, C4b and C3b are covalently bound to target RBCs via thioester linkages. In one unusual case, a low-titer IgG cold agglutinin could be detected by washing the patient’s RBCs in ice-cold saline solution and performing the DAT at 4°C.²⁷⁷

The majority of cold agglutinins are reactive with oligosaccharide antigens of the I/i system, which are precursors of the ABH and Lewis blood group substances.^279,280,281 The I/i determinants are bound to erythrocyte membrane glycoprotein (band 3 anion transporter) or to glycolipids.^280,281 Anti-I and anti-i reportedly bind solubilized RBC glycoproteins at 37°C, suggesting the temperature dependence of cold agglutination of intact RBCs may be a function of temperature-induced conformational effects on the cell surface.^282,283

I antigens are expressed strongly on adult RBCs but weakly on neonatal (cord) RBCs. The converse is true of i antigens, indicating I/i antigen expression is developmentally regulated.²⁸⁰ The differences between adult and cord blood RBCs allow evaluation of the serologic specificity of cold agglutinins.^10,11,202 I/i antigens, or structurally related analogues, occur in human saliva, milk, amniotic fluid, and hydatid cyst fluid,²⁰² and are expressed on human lymphocytes, neutrophils, and monocytes.²⁸⁴

Anti-I is the predominant specificity of cold agglutinins in idiopathic cold agglutinin disease, in patients with M. pneumoniae, and in some cases of lymphoma. Cold agglutinins with anti-i specificity are found in patients with infectious mononucleosis and in some patients with lymphoma. A small percentage of cold agglutinin-containing sera react equally well with adult and neonatal RBCs. These antibodies recognize antigens outside the I/i system, including Pr antigens, consisting of carbohydrate epitopes of glycophorins that are inactivated by protease treatment²⁰² and, less commonly, the M or P blood group antigens.^285,286 Most cold agglutinins associated with chickenpox exhibit anti-Pr specificity. A single case with anti-I specificity has been observed.²⁸⁷ Hemolysis resulting from a cold agglutinin with anti-Pr specificity occurred following an allogeneic marrow transplant.²⁸⁸

In hemolytic anemia associated with infectious mononucleosis, the patient’s serum may contain IgM anti-i cold agglutinins or cold-reactive nonagglutinating IgG anti-i with IgM cold-reactive anti-IgG antibodies (“rheumatoid factors”) that may crosslink the IgG-coated RBCs to produce agglutination.²⁸⁹

In paroxysmal cold hemoglobinuria, the direct antiglobulin reaction usually is positive during and briefly following an acute attack because of the coating of surviving RBCs with complement, primarily C3dg fragments. The Donath-Landsteiner antibody is responsible for complement deposition on the cells; it is a nonagglutinating IgG that binds RBCs only in the cold. It readily dissociates from the RBCs at room temperature. In adults subject to recurring episodes in association with cold exposure, the DAT result remains negative between attacks. The antibody is detected by the biphasic Donath-Landsteiner test, in which the patient’s fresh serum is incubated with RBCs initially at 4°C and the mixture is then warmed to 37°C.¹¹ Intense hemolysis occurs. Addition of fresh guinea pig serum or ABO-compatible human serum may be necessary to serve as a source of fresh complement if the patient’s serum has been stored or is complement depleted. Antibody titers rarely exceed 1:16. The Donath-Landsteiner antibody typically has specificity for the P blood group antigen, a glycosphingolipid structure.²⁸¹ The P antigen also occurs on lymphocytes and skin fibroblasts.¹⁶ The finding of P antigen on skin fibroblasts might be related in some way to the occurrence of cold urticaria in paroxysmal cold hemoglobinuria, a phenomenon that may be transferred passively by serum to normal skin.¹⁰ Antibody specificities for RBC antigens other than the P blood group have been noted.²⁹⁰

Drug-Induced Immune Hemolytic Anemia

In the hapten/drug adsorption mechanism of immune injury associated with cephalosporins or penicillin, the patient’s drug-coated RBCs bind drug-specific IgG antibody and exhibit positive DAT reactions with anti-IgG. Rarely, both anti-IgG and anti-C3d antisera produce positive DAT reactions. Such cases could have superficial resemblance to warm-antibody AHA. The key serologic difference is that, in this form of drug-induced immune hemolytic anemia, the antibodies in the patient’s serum or eluted from the patient’s RBCs react only with drug-coated RBCs. In contrast, the IgG antibodies in warm-type AHA react with unmodified human RBCs and may show preference for certain known blood groups (e.g., within the Rh complex). Such serologic distinction and the history of exposure to high blood levels of penicillin or a cephalosporin should be instructive.

In hemolysis mediated by the ternary complex mechanism, the DAT is positive with anticomplement serum. Immunoglobulins are only rarely detectable on the patient’s RBCs. This pattern is similar to that encountered in AHA mediated by cold agglutinins. Moreover, the brisk type of hemolysis in the ternary complex mechanism also is seen in certain cases of cold-antibody AHA. In the drug-induced cases, however, the cold agglutinin titer and the Donath-Landsteiner test result are normal, and demonstration of serum antibody acting on human RBCs depends upon the presence of the drug in the test system. Thus, the IAT reaction with anticomplement serum may be positive only if the incubation mixture permits interaction of (1) normal RBCs; (2) antidrug antibody from the patient’s serum; (3) the relevant drug, either still in the patient’s serum or added in vitro in appropriate concentration; and (4) a source of complement, that is, fresh normal serum or the patient’s own serum if freshly obtained. A negative result does not necessarily absolve the suspected drug because the critical determinant may be a metabolite of the drug in question. In some cases, use of urine or serum (of the patient or a volunteer taking the drug) as a source of drug metabolite has permitted successful demonstration of a drug-dependent mechanism.^{61,218,222,291}

In patients with true autoantibodies as a result of α-methyldopa, the DAT reaction is strongly positive for IgG, but complement only rarely is detected on the patient’s RBCs. Autoantibody to RBCs is regularly present in the serum of patients and mediates a positive IAT reaction with unmodified human RBCs, often showing specificity related to the Rh complex. No presently available specific serologic test can separate idiopathic warm-reacting IgG autoantibodies with Rh-related specificities from those induced by α-methyldopa administration. The evidence must be circumstantial, with the helpful knowledge that discontinuation of α-methyldopa, without any form of immunosuppressive therapy, consistently permits a slow recovery from anemia and a gradual disappearance of anti-RBC antibodies.

Drugs now not known to cause immune RBC injury will be implicated in the future. In any patient with a clinical picture compatible with drug-related immune hemolysis, a reasonable approach is stopping any drug that is suspect while serologic studies are being performed. The patient should be monitored for improvement in hematocrit level, decrease in reticulocytosis, and gradual disappearance of the positive DAT. Repeat challenge with the suspected drug may confirm the diagnosis, but this measure is seldom necessary in patient management and may be unsafe. Therefore, rechallenge to exclude a drug-induced immune hemolytic anemia should be undertaken only for compelling reasons, such as the need to use the specific drug for the patient’s illness.

Several nonautoimmune diseases may result in spherocytic anemia, such as hereditary spherocytosis (HS), Zieve syndrome, clostridial sepsis, and the hemolytic anemia preceding Wilson disease. Among the hereditary hemolytic anemias, HS can resemble acquired AHA most closely because the spherocytic anemia associated with HS may be detected first in adulthood (Chap. 46). In addition, splenomegaly may be prominent in both HS and AHA. Family studies of patients with HS, however, usually can identify other affected individuals. Most important, in hereditary hemolytic anemia the DAT is negative.

In hemolytic anemia accompanied by a positive DAT, serologic characterization of the autoantibody may distinguish warm-antibody AHA from cold-reacting autoantibody syndromes. Diagnosis of a drug-induced immune hemolytic anemia depends upon a history of appropriate drug intake supported by compatible serologic findings. In patients who recently received a transfusion, a positive DAT reaction may reflect the binding of a newly formed alloantibody to donor RBCs in the patient’s circulation (delayed transfusion reaction; Chap. 138). This finding could lead to a false impression of an autoimmune process.

The venom of the brown recluse spider (Loxosceles recluse) may cause severe life-threatening hemolysis.^292,293 The DAT may be positive for IgG and or complement^292,293 and spherocytes and RBC fragments are present on the blood film.²⁹³ The diagnosis should be considered when there is history or evidence by physical examination of a spider bite. The role of IgG and complement in hemolysis is unclear, but the terminal complement inhibitor, eculizumab, inhibits lysis in vitro. To date, the clinical use of eculizumab in this population has not been reported.

Recent recipients of allogeneic blood stem cell or solid-organ transplantations may develop autoimmune hemolysis.²⁹⁴ In the former, antibodies are produced by the stem cell graft against RBCs also produced by the stem cell graft; that is, both antibodies and RBCs are of donor origin. In the case of solid-organ transplantations, the recipient’s own lymphocytes make antibody against recipient RBCs. In both situations, the autoimmunity is thought to arise from immunosuppressive therapy causing delayed reconstitution or dysfunction of T-cell immunity, leading to development of antibodies autologous to the offended immune system.

Recipients of transplantations may also develop an alloimmune hemolytic anemia that mimics warm-antibody AHA. The problem is seen in kidney, liver, or hematopoietic stem cell transplantations and usually occurs when an organ from a blood group O donor is transplanted into a blood group A or B recipient. B lymphocytes present in the donated organ or stem cell product form alloantibodies against recipient RBCs.^{295,296,297,298,299} Patients of blood group O who receive a stem cell transplant from a donor of blood group A or B may develop a transiently positive DAT and hemolysis of RBCs made by the marrow graft because of temporary persistence of previously synthesized host anti-A or anti-B.³⁰⁰ Furthermore, some group O stem cell transplantation recipients exhibit mixed hematopoietic chimerism with persistence of host B lymphocytes that can make alloantibodies directed against RBCs made by the stem cell graft.³⁰⁰ In these settings, the findings of hemolysis and a positive DAT as a result of anti-A and anti-B probably are diagnostic of an alloimmune process, because autoantibodies directed against the major blood group antigens A and B are extremely rare.

Other acquired types of hemolytic anemia are less easily confused with either warm- or cold-antibody AHA because spherocytes are not prominent on the blood film and the DAT is negative. Patients with paroxysmal nocturnal hemoglobinuria (PNH) may complain of dark urine (hemoglobinuria). This finding is unusual in patients with warm-antibody AHA but can occur in patients with the cold-antibody syndromes. Decreased levels of CD55 and CD59 on blood cells, detected by flow analysis, are characteristic of PNH but not AHA (Chap. 40). Microangiopathic hemolytic disorders, such as thrombotic thrombocytopenic purpura and hemolytic uremic syndrome, can be distinguished from AHA by examining the blood film. In the microangiopathic hemolytic diseases, the blood film displays marked RBC fragmentation and minimal spherocytosis. In addition, microangiopathic hemolytic anemias more frequently are associated with thrombocytopenia than is either warm- or cold-antibody AHA.

The clinical and laboratory features of chronic cold agglutinin disease are sufficiently distinctive so that the diagnostic possibilities are limited. In general, a high-titer cold agglutinin (>1:512) and a positive DAT with anticomplement serum (but not with anti-IgG) are consistent with cold agglutinin disease. In many instances of drug-induced immune hemolytic anemia, the DAT result also is positive only for complement. The drug history and a low (or absent) cold agglutinin titer, however, help to distinguish drug-induced immune hemolytic anemia from cold agglutinin disease. If the patient has an elevated cold agglutinin level and a positive DAT result with both anti-IgG and anti-C3, then the patient may have a mixed-type AHA. Warm-antibody AHA, hereditary hemolytic disorders, and PNH should be excluded in cases exhibiting primarily a chronic hemolytic anemia. The pattern of antiglobulin reaction, family history, the result of analysis of CD55/CD59 on blood cells provide additional help in difficult cases. When the hemolysis is episodic, paroxysmal cold hemoglobinuria, march hemoglobinuria, and PNH also should be considered. When cold-induced peripheral vasoocclusive symptoms are predominant, the differential diagnosis should include cryoglobulinemia and Raynaud phenomenon, with or without an associated rheumatic disease. Infectious mononucleosis, M. pneumoniae infection, and lymphoma can be considered in appropriate clinical settings.

Paroxysmal cold hemoglobinuria must be distinguished from the subset of cases of chronic cold agglutinin disease manifesting episodic hemolysis and hemoglobinuria. This distinction is made primarily in the laboratory. In general, patients with paroxysmal cold hemoglobinuria lack high titers of cold agglutinins. Furthermore, the Donath-Landsteiner antibody is a potent in vitro hemolysin, in contrast to most cold agglutinins, which are weak hemolysins. Warm-antibody AHA, march hemoglobinuria, myoglobinuria, and PNH can be distinguished through the history and appropriate laboratory studies.

Immune hemolysis caused by drugs should be distinguished from (1) the warm- or cold-antibody types of idiopathic AHA, (2) congenital hemolytic anemias such as HS, and (3) drug-mediated hemolysis resulting from disorders of red cell metabolism, such as glucose-6-phosphate dehydrogenase deficiency. Patients with drug-induced immune hemolytic anemia have a positive DAT that distinguishes this group from patients with inherited RBC defects.

GENERAL

Transfusion

The clinical consequences of AHA or drug-induced immune hemolytic anemia are related to the severity of the anemia and acuity of its onset. Many patients develop anemia over a period sufficient to allow for cardiovascular compensation and hence do not require RBC transfusions. However, RBC transfusions may be necessary and should not be withheld from a patient with an underlying disease complicating the anemia, such as symptomatic coronary artery disease, or a patient who rapidly develops severe anemia with signs and/or symptoms of circulatory failure, as in paroxysmal cold hemoglobinuria or ternary complex drug-induced immune hemolysis.

Transfusion of RBCs in immune hemolytic anemia presents two difficulties: (1) crossmatching and (2) the short half-life of the transfused RBCs (Chap. 138). Finding truly serocompatible donor blood is nearly always impossible except in rare cases when the autoantibody is specific for a defined blood group antigen (see “Serologic Features” above).

It is most important to identify the patient’s ABO type so as to avoid a hemolytic transfusion reaction mediated by anti-A or anti-B. This part of the matching process allows for selection of either ABO-identical or -compatible blood for transfusion. With respect to compatibility, the more difficult technical issue relates to the detection of RBC alloantibodies which may be masked by the presence of the autoantibody.

Clinicians often speak of “least incompatible” blood for transfusion, but this term has lately fallen into disrepute because it lacks a precise definition.^301,302 In fact all units will be serologically incompatible but units that are incompatible because of the presence of autoantibody are less dangerous to transfuse than those units that are incompatible because of an alloantibody.

Before transfusing an incompatible unit, the patient’s serum must be tested carefully for an alloantibody that could cause a severe hemolytic transfusion reaction against donor RBCs, especially in patients with a history of pregnancy, abortion or prior transfusion.^{264,303,304,305} Patients who have been neither pregnant nor transfused with blood products are unlikely to harbor an alloantibody. Early consultation between the clinician and the blood bank physician is essential. An understanding of the basic aspects of blood compatibility testing, coupled with the knowledge of a patient’s pregnancy and transfusion history allow for informed discussion and confident transfusion of mismatched blood if the situation demands.

Once selected, the packed RBCs should be administered slowly and in the case of cold hemolysis syndromes should be brought at least to room temperature. During the transfusion, the patient should be monitored for signs of a hemolytic transfusion reaction (Chap. 138). The transfused cells may be destroyed as fast as or perhaps even faster than the patient’s own cells. However, the increased oxygen-carrying capacity provided by the transfused cells may be sufficient to maintain the patient during the acute interval required for other modes of therapy to become effective.

For patients with AHA who require chronic transfusion support, use of prophylactic antigen-matched donor RBCs for transfusion has been proposed as a means of preventing alloimmunization.³⁰⁶ This process is feasible only in institutions with access to a good selection of phenotyped RBC units and a reference laboratory.³⁰⁷

THERAPY OF WARM-ANTIBODY AUTOIMMUNE HEMOLYTIC ANEMIA

Glucocorticoids

Therapy with glucocorticoids has reduced the mortality associated with severe idiopathic warm-antibody AHA. Glucocorticoids were first used for this disorder more than 60 years ago.³⁰⁸ Glucocorticoids can cause dramatic cessation or marked slowing of hemolysis in about two-thirds of patients.^{10,11,123,309,310} Approximately 20 percent of treated patients with warm-antibody AHA achieve complete remission. Approximately 10 percent show minimal or no response to glucocorticoids. The best responses are seen in idiopathic cases or in those related to SLE.

Most patients should be treated with oral prednisone at an initial daily dose of 1 to 1.5 mg/kg (e.g., 50 to 100 mg). Critically ill patients with rapid hemolysis may receive intravenous methylprednisolone 100 to 200 mg in divided doses over the first 24 hours. High doses of prednisone may be required for 10 to 14 days. When the hematocrit stabilizes or begins to increase, the prednisone dose can be decreased in rapid-step dose reductions to approximately 30 mg/day. With continued improvement, the prednisone dose can be further decreased at a rate of 5 mg/day every week, to a dose of 15 to 20 mg/day. These doses should be administered for 2 to 3 months after the acute hemolytic episode has subsided, after which the patient can be weaned from the drug over 1 to 2 months or treatment switched to an alternate-day therapy schedule (e.g., 20 to 40 mg every other day). Alternate-day therapy reduces glucocorticoid side effects but should be attempted only after the patient has achieved stable remission on daily prednisone in the range of 15 to 20 mg/day. Therapy should not be stopped until the DAT becomes negative. Although many patients achieve full remission of their first hemolytic episode, relapses may occur after the glucocorticoids are discontinued. Consequently, patients should be followed for at least several years after treatment. A relapse may require repeat glucocorticoid therapy, splenectomy, or immunosuppression.

Occasionally, patients who present with only a positive DAT, minimal hemolysis, and stable hematocrit require no treatment. However, these patients should be observed for clinical deterioration because the rate of RBC destruction may increase spontaneously.

Glucocorticoids may influence hemolysis in warm-antibody AHA by several mechanisms. Earlier investigators noted that hematologic improvement was often, but not always, accompanied by reduction in the strength of the DAT.¹⁰ The subsequent observation of a decrease in cell-bound and/or free serum autoantibody during stable glucocorticoid-induced remission suggested improved RBC survival following treatment with glucocorticoids resulted from a decrease in synthesis of anti-RBC autoantibodies.^169,252 However, this finding cannot explain why glucocorticoid-treated patients often improve within 24 to 72 hours, a time much shorter than the half-life of anti-RBC autoantibody. Rather, glucocorticoids may suppress RBC sequestration by splenic macrophages.^{171,172,183,311} A quantitative decrease in one of the three known classes of Fcγ receptors^170,171 has been observed in the blood monocytes of AHA patients during glucocorticoid therapy.³¹²

Splenectomy

Nearly one-third of patients with warm-antibody AHA require prednisone chronically in doses greater than 15 mg/day to maintain an acceptable hemoglobin concentration. These patients are candidates for laparoscopic splenectomy.

Splenectomy removes the primary site of RBC trapping. Investigations in human¹⁶⁹ and animal¹⁷¹ subjects confirm that maintenance of a given rate of RBC destruction requires 6 to 10 times more RBC-bound IgG in splenectomized subjects than in nonsplenectomized subjects. Continuation of hemolysis after splenectomy is partly related to persisting high levels of autoantibody, favoring RBC destruction in the liver by hepatic Kupffer cells.^169,171,174

Several investigators noted the amount of RBC-bound autoantibody decreased in AHA patients following splenectomy.^10,309,313 However, a significant proportion of patients show no change in cell-bound autoantibody following splenectomy. The processes determining the rate of autoantibody production are poorly understood. The beneficial effect of splenectomy may be related to several factors interacting in complex fashion.³¹⁴

A patient’s clinical data constitute the best selection criteria for splenectomy. Attempts to select potential responders by ⁵¹Cr RBC sequestration studies have been disappointing.^10,309,315 In most cases, a reasonable approach is to continue glucocorticoids for 1 to 2 months while waiting for a maximal response. However, if no response is noted within 3 weeks, the patient’s condition deteriorates, or the anemia is very severe, splenectomy should be performed sooner.

Results of splenectomy are variable. Approximately two-thirds of AHA patients have a partial or complete remission following splenectomy.^309,314 However, the relapse rate is disappointingly high. Many patients require further glucocorticoid therapy to maintain acceptable hemoglobin levels, although often at a lower dose than required prior to splenectomy.^10,123,309 Alternate-day therapy is preferable to daily therapy in these cases if adequate control of the anemia can be achieved.

The immediate mortality and morbidity from splenectomy depend upon the presence of underlying disease and the preoperative clinical status but generally are quite low.³¹⁶ Following splenectomy, children, more than adults, have an increased risk for developing sepsis as a result of encapsulated organisms.³¹⁷ Vaccination against H. influenzae type b and pneumococcal and meningococcal organisms is recommended at least 2 weeks prior to surgery (Chap. 56).³¹⁸

Rituximab

Rituximab is a monoclonal antibody directed against the CD20 antigen expressed on B lymphocytes and is used for treatment of B-cell lymphoma. Its use for treatment of AHA is based on the antibody’s ability to eliminate B lymphocytes, including, presumably, those making autoantibodies to RBCs. However, the mechanism of action is more complex than that, as the effect of rituximab can occur very early, before the autoantibodies can recede. In fact, sometimes in responding patients, autoantibody levels are not significantly affected.^319,320 Opsonized B lymphocytes may decoy effector monocytes and macrophages from autoantibody complexes and normalize autoreactive T lymphocyte responses.³¹⁹

Rituximab was used initially in refractory AHA either unresponsive to or relapsed after oral glucocorticoid therapy. In a prospective series,³²¹ 13 of 15 children with warm-antibody AHA responded to rituximab 375 mg/m² weekly for 2 to 4 weeks, intravenously. Other case series support the use of similar doses of rituximab in adults, with response rates ranging from 40 to 100 percent.^320,322 Another prospective study in adults exhibited a 100 percent response using rituximab 100 mg/m² weekly for 4 weeks along with a short course of oral glucocorticoid as first- or second-line therapy.³²³ Sustained responses were observed at 3 years in more than two-thirds of the cases.³²⁴

A phase 3 randomized controlled trial compared glucocorticoid monotherapy versus glucocorticoid and rituximab 375 mg/m² as first-line therapy in patients with warm-antibody AHA.³²⁵ The complete and partial response rates were similar (approximately 50 percent) in the two groups at 3 and 6 months. At 12 and 36 months after randomization the relapse-free survival was superior for the combination of glucocorticoids and rituximab.

Other Immunosuppressive Drugs

Cytotoxic drugs such as cyclophosphamide, 6-mercaptopurine, azathioprine, and 6-thioguanine have been given to patients with AHA to suppress synthesis of autoantibody. Direct evidence of such an effect is lacking. Although immunosuppressive therapy is not universally accepted, beneficial responses to immunosuppressive drugs have been observed in some patients who did not respond to glucocorticoids.^11,326 Importantly, the majority of patients with warm-antibody AHA respond to glucocorticoids and/or splenectomy and usually are not candidates for immunosuppressive therapy. At present, immunosuppressive therapy should be reserved primarily for patients who do not respond to glucocorticoids, rituximab, or splenectomy, or for patients who are poor surgical risks.³²⁶

The most successful approach used high-dose cyclophosphamide 50 mg/kg ideal body weight per day for 4 consecutive days, intravenously, with granulocyte colony-stimulating factor support.³²⁷ Of nine patients, eight of whom had warm autoantibodies, all became transfusion independent. All patients had prolonged severe cytopenias and required hospitalization for a median of 21 days. Cyclophosphamide may cause severe hemorrhagic cystitis and sodium 2-mercaptoethanesulfonate (mesna) 10 mg/kg was given at 3, 6, and 8 hours following each cyclophosphamide dose, to minimize the effect of cyclophosphamide on the bladder.

For patients who may not tolerate prolonged cytopenias, the drugs of choice are cyclophosphamide 60 mg/m² or azathioprine 80 mg/m² given daily. If the patient tolerates the drug, continue treatment for up to 6 months while waiting for a response. When response occurs, the patient can be weaned slowly from the drug. If no response is observed, the alternative drug can be tried. Because cyclophosphamide and azathioprine suppress hematopoiesis, blood counts, including reticulocyte count, must be monitored with extra care during therapy. Treatment with either agent increases the risk of subsequent neoplasia.

Patients with refractory AHA have been treated effectively with the purine analogue 2-chlorodeoxyadenosine (cladribine)³²⁸ and with mycophenolate mofetil.^329,330 Alemtuzumab was successfully used to treat 5 patients with refractory AHA associated with CLL.³³¹

Other Therapies

For patients with chronic compensated hemolysis, treatment with folate at 1 mg/day, orally, is recommended to satisfy the increased demands for the vitamin because of increased red cell production. Plasma exchange or plasmapheresis has been used in patients with warm-antibody AHA. Improvement has been reported in a few cases, but use of the method is controversial.^332,333 A patient with life-threatening warm IgG-mediated AHA refractory to glucocorticoids and splenectomy exhibited rapid clinical improvement following manual whole blood exchange.³³⁴ A patient with severe warm IgM-mediated AHA was successfully treated with C1-esterase inhibitor (C1-inh) resulting in attenuation of complement deposition on RBCs and hemolysis, along with improved recovery of transfused RBCs.³³⁵ Another patient with refractory warm IgM- mediated AHA responded to rituximab and eculizumab.³³⁶ Thymectomy has been reported useful in a few children who were refractory to glucocorticoids and splenectomy.³²⁶ Selective injury to splenic macrophages by administration of vinblastine-loaded, IgG-sensitized platelets reportedly was successful in a few patients.³³⁷ Several anecdotal reports and a case series indicate short-term successful treatment of patients with AHA using high-dose intravenous γ-globulin.^{338,339,340,341} Uncontrolled studies indicate danazol, a nonvirilizing androgen, may be useful in patients with AHA.^342,343 Danazol may eliminate the need for splenectomy when combined with prednisone and may allow for a shorter duration of prednisone therapy.³⁴³ Some patients with ulcerative colitis and AHA unresponsive to glucocorticoids and splenectomy may respond to colectomy.³⁴⁴ In patients with AHA associated with an ovarian dermoid cyst, cyst removal produces remission of the hemolysis.³⁴⁵

THERAPY OF COLD-ANTIBODY HEMOLYTIC ANEMIA

Keeping the patient warm, particularly the patient’s extremities, is moderately effective in providing symptomatic relief. This action may be the only measure required in patients with mild chronic hemolysis. In symptomatic patients, rituximab is effective and well tolerated. In two prospective trials, approximately half the patients responded to rituximab 375 mg/m² weekly for 4 weeks.^346,347 Patients who relapsed responded to a second course of rituximab at about the same rate. In a prospective trial of rituximab and fludarabine, the response rate was 76 percent, including complete responses in 21 percent with a median response duration of 66 months.³⁴⁸ In another small study, 60 percent of patients responded to low dose rituximab, 100 mg/m², a rate comparable to that seen with rituximab 375 mg/m².³²³ Chlorambucil or cyclophosphamide may be helpful for patients with symptomatic chronic cold agglutinin disease.^{9,10,11,349,350} In patients with lymphoma, treatment of the underlying disorder usually results in control of the cold agglutinin disease. Single patients with refractory cold agglutinin disease have exhibited responses to eculizumab^351,352 and bortezomib.³⁵³ Results of splenectomy^10,11,354 or use of glucocorticoids^10,11 generally have been disappointing, although exceptions have been reported,^{10,196,277,278} particularly in atypical cases. Experimental¹⁷¹ and clinical¹⁹⁶ bases exist for considering very high doses of glucocorticoids in seriously ill patients. RBC transfusions generally are reserved for patients with severe anemia of rapid onset who are in danger of cardiorespiratory complications. Washed RBCs often are used to avoid replenishing depleted complement components and reactivating the hemolytic process. In critically ill patients, plasma exchange (with replacement by albumin-containing saline solution) may provide transient amelioration of hemolysis.^355,356,357

In patients with cold agglutinin disease secondary to infection, spontaneous resolution of the hemolysis is expected in all patients after resolution of the infection. RBC transfusion may be required as a temporizing measure and in severe cases, plasma exchange may be beneficial. There is little evidence to support the use of glucocorticoids or antiviral therapy.

Most contemporary cases of paroxysmal cold hemoglobinuria are self-limited. Acute attacks in both chronic and transient forms of paroxysmal cold hemoglobinuria may be prevented by avoiding cold exposure. Glucocorticoid therapy and splenectomy have not been useful. When paroxysmal cold hemoglobinuria is associated with syphilis, effective treatment of the infection may result in complete remission. Antihistaminic and adrenergic agents may relieve symptoms of cold urticaria.

THERAPY OF DRUG-INDUCED IMMUNE HEMOLYTIC ANEMIA

Discontinuation of the offending drug often is the only treatment needed. This measure is essential and may be lifesaving in patients with severe hemolysis mediated by the ternary complex mechanism.

In the past, high-dose penicillin was not necessarily discontinued because of a positive DAT alone. A change in therapy was considered mainly in the presence of overt hemolytic anemia. For example, lowering the penicillin dose and coadministering other antibiotics sometimes allowed continuation of the drug, particularly if hemolysis was not severe. For other drugs causing only mild hemolysis by the hapten-drug adsorption mechanism, in the unlikely event that no alternatives are available, a similar approach may be effective.

In patients taking α-methyldopa in the absence of hemolysis, a positive DAT has not necessarily been an indication for stopping the drug. However, given all the choices available, it is prudent to consider alternative antihypertensive therapy. Because less information on the natural history of autoantibodies induced by drugs other than α-methyldopa is available, discontinuation of the offending drug is advisable unless no suitable alternative exists.

Glucocorticoids are generally unnecessary, and their efficacy is questionable. However, prednisone is effective in patients with CLL and autoimmune hemolysis caused by purine analogues,^88,89 as are cyclosporine, rituximab, and intravenous immunoglobulin.²³⁰ For treatment of CLL, combination of cyclophosphamide with fludarabine, with or without rituximab, seems to reduce the frequency of fludarabine-induced AHA.^229,230 Transfusions should be given in the unusual circumstance of severe, life-threatening anemia. Problems with crossmatching, similar to those encountered in warm-antibody AHA, may occur in patients with a strongly positive IAT, for example, in α-methyldopa–related cases. Patients with hemolytic anemia resulting from the hapten/drug adsorption mechanism should have a compatible crossmatch because the serum antibody reacts only with drug-coated cells. However, if therapy with the offending drug is still in progress, transfused cells may be destroyed at an increased rate as they become coated with drug in vivo. Patients with ternary complex–mediated hemolysis will also hemolyze transfused RBCs until the offending drug clears from the plasma.

Several cases of transfusion-associated graft-versus-host disease as a result of purine analogues have been reported in CLL patients transfused for hemolysis.^90,358,359 Such patients, who have an immunodeficiency state secondary to CLL, impairing their ability to eliminate the transfused lymphocytes, should receive irradiated blood products.

Patients with idiopathic warm-antibody AHA have unpredictable clinical courses characterized by relapses and remissions. No particular feature of the illness has been a consistent predictor of outcome. Despite a rather high initial rate of response to glucocorticoids and splenectomy, the overall mortality rate was significant (up to 46 percent) in several older series, but much lower in more recent studies.^{10,11,309,360,361} The actuarial survival at 10 years reportedly is 73 percent.³⁶⁰

Pulmonary emboli, infection, and cardiovascular collapse are causes of death. Thromboembolic episodes in the form of deep vein thrombosis or splenic infarcts are relatively common during active phases of the disease.^309,361 In one series, 8 of 30 patients with AHA developed venous thromboembolism; 19 of the patients had antiphospholipid antibodies, including six of the eight patients with thromboembolism.³⁶² In another retrospective analysis of 36 exacerbations of severe AHA in 28 patients, only six of whom were tested and found negative for antiphospholipid antibodies, venous thromboembolism occurred in 5 of 15 exacerbations without anticoagulation and in 1 of 21 with anticoagulation.³⁶³ The contribution of antiphospholipid antibodies to morbidity and mortality in AHA is not clear from these data. However, it seems prudent to consider prophylactic anticoagulation for patients with AHA and antiphospholipid antibodies or other risk factors for venous thromboembolism.

The prognosis in secondary warm-antibody AHA largely depends upon the course of the underlying disease.

In children, warm-antibody AHA frequently follows an acute infection or immunization.^313,364,365 Most of these patients exhibit a self-limited course and respond rapidly to glucocorticoids. Those who recover from the initial hemolytic episode have a good prognosis and are unlikely to relapse, although exceptions are known. Children with chronic AHA tend to be older.^364,365 The overall mortality rate is lower than in adults, ranging from 4 to 30 percent,^{313,364,365,366,367,368,369} with higher mortality rates in those with chronic AHA^313,368,369 and associated autoimmune thrombocytopenia (Evans syndrome).^369,370 Evans syndrome was noted in 37 percent of children with AHA in one large study, a much higher frequency than observed in adults.³⁶⁹

Patients with idiopathic cold agglutinin disease often have a relatively benign course and survive for many years.^9,10,11 Occasionally, death results from infection or severe anemia or, not uncommonly, from an underlying lymphoproliferative process.

The postinfectious forms of cold agglutinin disease typically are self-limited. Recovery generally occurs in a few weeks. A few cases with massive hemoglobinuria have been complicated by acute renal failure, requiring temporary hemodialysis.

Postinfectious forms of paroxysmal cold hemoglobinuria terminate spontaneously within a few days to weeks after onset,^12,13,14,15 although the Donath-Landsteiner antibody may persist in low titer for several years.¹⁰ Most patients with chronic idiopathic paroxysmal cold hemoglobinuria survive for many years despite occasional paroxysms of hemolysis.

Immune hemolysis in response to drugs usually is mild, and the prognosis is good. Occasional episodes of exceptionally severe hemolysis with renal failure or death have been reported, usually because of drugs operating through the ternary complex mechanism or purine analogues in patients with CLL.^{39,56,58,62,63,64,65,67,86,108,109} In hemolysis resulting from ternary complex or hapten/drug adsorption mechanisms, the DAT becomes negative shortly after the drug is discontinued, that is, soon after the drug clears from the circulation. In addition, the hemolysis associated with α-methyldopa–induced autoantibodies ceases promptly after drug cessation. However, a positive DAT of gradually diminishing intensity may remain for weeks or months.