American Journal of Hematology 69:258271 (2002)

Autoimmune Hemolytic Anemia

1

Bradley C. Gehrs1 and Richard C. Friedberg2*

Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama 2 Department of Pathology, Baystate Medical Center, Springeld, Massachusetts

Red blood cell (RBC) autoantibodies are a relatively uncommon cause of anemia. However, autoimmune hemolytic anemia (AIHA) must be considered in the differential diagnosis of hemolytic anemias, especially if the patient has a concomitant lymphoproliferative disorder, autoimmune disease, or viral or mycoplasmal infection. Classications of AIHA include warm AIHA, cold agglutinin syndrome, paroxysmal cold hemoglobinuria, mixed-type AIHA, and drug-induced AIHA. Characteristics of the autoantibodies are responsible for the various clinical entities. As a result, diagnosis is based on the clinical presentation and a serologic work-up. For each classication of AIHA, this review discusses the demographics, etiology, clinical presentation, laboratory evaluation, and treatment options. Am. J. Hematol. 69:258271, 2002. 2002 Wiley-Liss, Inc. Key words: autoimmune hemolytic anemia; red blood cell autoantibodies

INTRODUCTION

Immune hemolytic anemia (IHA) is the clinical condition in which IgG and/or IgM antibodies bind to RBC surface antigens and initiate RBC destruction via the complement system and the reticuloendothelial system. IHA is classied as either autoimmune, alloimmune, or drug-induced based on the antigenic stimulus responsible for the immune response. Autoimmune hemolytic anemia (AIHA) is characterized by the production of antibodies directed against self RBCs. Since the autoantibodies usually are directed against high-incidence antigens, they often exhibit reactivity against allogeneic RBCs as well. AIHA is a fairly uncommon disorder, with estimates of the incidence at 13 cases per 100,000 per year [1,2]. In contrast, alloimmune hemolytic anemia requires exposure to allogeneic RBCs, and the resulting alloantibodies show no reactivity toward autologous RBCs. Sources of allogeneic RBC exposure include pregnancy, blood product transfusion, and transplantation. The principal manifestations of RBC alloimmunization are hemolytic transfusion reactions and hemolytic disease of the newborn (HDN). The incidence of acute hemolytic transfusion reactions has been estimated to be 0.0030.008%, while 0.050.07% of transfused patients develop a clinically recognized delayed hemolytic transfusion reaction [35]. Drug-induced IHA is the nal classication of
2002 Wiley-Liss, Inc.

IHA. Drug-induced antibodies can recognize either intrinsic RBC antigens or RBC-bound drug. Antibodies that react with intrinsic RBC antigens are serologically indistinguishable from autoantibodies. In contrast, antibodies that react against RBC-bound drug require the drug for hemolysis. The pathogenesis of IHA ultimately overlaps for these three classications. The degree of hemolysis depends on characteristics of the bound antibody (e.g., quantity, specicity, thermal amplitude, ability to x complement, ability to bind tissue macrophages) as well as the target antigen (density, expression, patient age). IgG antibodies are relatively poor activators of the classical complement pathway, but they (in particular IgG1 and IgG3 antibodies) are recognized readily by Fc receptors on various phagocytic cells [68]. Therefore, IgG-sensitized RBCs generally are eliminated by phagocytes of the reticuloendothelial system. Since reticuloendothelial cells also have
*Correspondence to: Richard C. Friedberg, MD, PhD, Chairman, Department of Pathology, Baystate Medical Center, 759 Chestnut Street, Springeld, Massachusetts 01199. Received for publication 15 October 2001; Accepted 15 November 2001. Published online in Wiley InterScience (www.interscience.wiley. com). DOI: 10.1002/ajh.10062

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receptors for complement factors C3b and iC3b, these complement components, if present, can potentiate the extravascular hemolysis [9]. On the other hand, IgM-sensitized RBCs generally are associated with a combination of intravascular and extravascular hemolysis. Intravascular hemolysis occurs because IgM antibodies, unlike IgG antibodies, readily activate the classical complement pathway and produce cytolysis. However, due to the presence of regulatory RBC proteins such as decay accelerating factor (DAF, CD55) and membrane inhibitor of reactive lysis (MIRL, CD59), overwhelming complement activation usually is required to produce clinically evident intravascular hemolysis, e.g., as seen with ABO-incompatible blood transfusions. More commonly, IgM-sensitized RBCs undergo extravascular hemolysis. While reticuloendothelial cells do not have receptors for the Fc fragment of IgM antibodies with comparable activity to the receptors directed against the Fc fragment of IgG, they do have receptors for the abundant RBC-bound C3b and iC3b resulting from complement activation. Whereas the spleen is the principal site of IgG-associated extravascular hemolysis, Kuper cells in the liver are the principal eectors of IgM-associated extravascular hemolysis. The remainder of this article concentrates on AIHA. Initial focus is placed on the basic classication scheme as well as the shared laboratory ndings and theoretical etiologies. Later, more detailed background, laboratory ndings, and treatment options are presented for each distinct clinical subtype of AIHA. These subclassications include warm AIHA, cold agglutinin syndrome, paroxysmal cold hemoglobinuria, mixed-type AIHA, and drug-induced AIHA.
AUTOIMMUNE HEMOLYTIC ANEMIA Classications Cases of AIHA generally are classied according to the characteristic temperature reactivity of the RBC autoantibody (Table I). Warm autoantibodies react most strongly near 37C and exhibit decreased anity at lower temperatures. Cold autoantibodies, on the other hand, bind to RBCs most strongly near 04C and typically show little anity at physiologic temperature. Occasionally, patients have a combination of warm and cold autoantibodies. Cases of AIHA can be subdivided further on the basis of etiology. By denition, idiopathic or primary AIHA shows no apparent association with an underlying disorder. Depending on the patient population studied, a suspected secondary cause of AIHA has been determined in 2080% of reported series; these include lymphoproliferative disorders, autoimmune disorders, infec-

Table I. Classication of Autoimmune Hemolytic Anemia Warm autoimmune hemolytic anemia Idiopathic Secondary (lymphoproliferative disorders, autoimmune disorders) Cold autoimmune hemolytic anemia Cold agglutinin syndrome Idiopathic Secondary Acute transient (infections) Chronic (lymphoproliferative disorders) Paroxysmal cold hemoglobinuria Idiopathic Secondary Acute transient (infections other than syphilis) Chronic (syphilis) Mixed-type autoimmune hemolytic anemia Idiopathic Secondary (lymphoproliferative disorders, autoimmune disorders) Drug-induced immune hemolytic anemia Autoimmune type Drug adsorption type Neoantigen type

tions, immunodeciency disorders, and tumors [1,1013]. Lymphoproliferative disorders account for approximately half of the cases of both secondary warm and cold AIHA. Autoimmune disorders are the next leading cause of secondary warm AIHA, whereas infections are the next leading cause of secondary cold AIHA. Idiopathic disease is more common in women and has a peak incidence in the fourth and fth decades of life. The demographics of secondary disease correspond with those of the various underlying illnesses.
Laboratory EvaluationSerology Two criteria must be met to diagnose AIHA: serologic evidence of an autoantibody and clinical or laboratory evidence of hemolysis. Serologic evidence of an autoantibody is provided by positive autocontrol and direct antiglobulin test (DAT, direct Coombs test) results and subsequent identication of an autoantibody in the RBC eluate and possibly the serum. Serum reactivity with autologous RBCs generally indicates the presence of an autoantibody, but it does not exclude the presence of an alloantibody. In addition, if the patient has been transfused recently, alloantibodies bound to the transfused cells can produce a mixed-eld positive autocontrol. While the autocontrol measures in vitro binding of antibodies to RBCs, the DAT indicates whether there is in vivo RBC binding of either IgG or C3d. The DAT involves mixing the individuals anticoagulated RBCs with polyspecic anti-human globulin (AHG) with anti-IgG and anti-C3d activities; if positive, the

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sample can be tested separately (``split'') with reagents specic for anti-IgG or anti-C3d. A positive DAT is non-specic for an RBC autoantibody. Additional primary causes of a positive DAT include an acute or delayed hemolytic transfusion reaction, hemolytic disease of the newborn, transplantation, drug-induced antibodies, and administration of various therapeutics including intravenous immunoglobulin (IVIG), Rh immune globulin (RhIg), antilymphocyte globulin, and antithymocyte globulin. A positive DAT also may arise secondary to sickle cell disease, b-thalassemia, renal disease, multiple myeloma, autoimmune diseases, AIDS, and other diseases with elevated serum globulin [14,15]. Given the broad dierential diagnosis for a positive DAT, supplemental serologic testing is necessary to ascertain the cause. The antibody screen detects both unbound autoantibodies and alloantibodies. Antibody bound to RBCs can be eluted o the cell surface and subsequently identied in the eluate. Both the RBC eluate and the serum sample can be examined for soluble autoantibodies and/or alloantibodies using diagnostic RBC panels and indirect antiglobulin testing. Drug-dependent antibodies, on the other hand, may not be detected unless the drug is added for in vitro testing. Generally autoantibodies react with all panel RBCs (i.e., panreactive), whereas alloantibodies exhibit antigen specicity, only reacting with specic antigen positive cells. Other techniques may be necessary to distinguish between the two types. For example, an alloantibody to a high-incidence antigen in a post-transfusion setting can mimic an autoantibody with a positive DAT (mixed-eld) and reactions with all panel cells. In addition, autoantibodies may exhibit apparent specicity. Autoadsorption and antigenic phenotyping can help dierentiate autoantibodies and alloantibodies, especially if the patient has not been transfused recently. Autoadsorption uses autologous RBCs to adsorb autoantibodies prior to repeating serum testing for alloantibodies. If an antibody exhibits specicity, then demonstration that autologous RBCs are negative for the corresponding antigen

conrms that it is an alloantibody; in the absence of a recent transfusion, a positive result suggests that it is an autoantibody. Autoadsorption can also be used to cross-match donor RBC units for patients with warm autoantibodies (only for patients who have not been transfused recently). In addition to the customary serum antibody identication, an RBC eluate should be tested for antibodies if the positive DAT shows anti-IgG reactivity. IgM autoantibodies generally detach from the RBC surface in vivo, so they are not detected directly in the DAT and are not present in the eluate. Elution may also concentrate any existing bound antibodies and thus improve sensitivity. Autoantibodies are present in both the eluate and the serum in approximately 80% of the cases of AIHA [16]. Together with the DAT, results from serum and RBC eluate antibody studies help distinguish the forms of AIHA (Table II).
Laboratory EvaluationHemolysis The diagnosis of AIHA also requires manifestations of hemolytic anemia. From 0.007% to 0.1% of healthy blood donors and from 0.3% to 8% of hospital patients have a positive DAT without clinical evidence of IHA [1720]. Hemolysis in AIHA can be either extravascular or intravascular. Typically, intravascular hemolysis has a rapid and aggressive presentation, whereas extravascular hemolysis is milder. A CBC with peripheral smear, bilirubin, LDH (in particular isoenzyme 1), haptoglobin, and urine hemoglobin are the basic tests used to evaluate and dierentiate intravascular and extravascular hemolysis. Autoantibody Etiology The etiology of most RBC autoantibodies is not well understood. Given the association between AIHA and other autoimmune disorders, generalized immune system dysfunction likely plays a role. The relationship between AIHA and lymphoproliferative

Table II. Serologic Characteristics of Autoimmune Hemolytic Anemias* Ig type Warm AIHA CAS PCH Mixed-type AIHA Drug-induced AIHA IgG (IgA or IgM) IgM IgG IgG, IgM IgG DAT IgG and/or C3 C3 C3 IgG + C3 IgG C3 RBC eluate IgG Nonreactive Nonreactive IgG IgG Specicity Panreactive; (Rarely Rh ) others) I > i >> Pr P Panreactive + unclear > I > others Often Rh-related

*Ig, immunoglobulin; DAT, direct antiglobulin test; RBC, red blood cell; AIHA, autoimmune hemolytic anemia; CAS, cold agglutinin syndrome; PCH, paroxysmal cold hemoglobinuria.

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disorders and other neoplasms likewise suggests generalized dysfunction of immune surveillance. The immune system has many control points that keep a balance between the need to tolerate self-antigens and the need to respond appropriately to foreign antigens. Immune self-tolerance occurs centrally with developing lymphocyte precursors via clonal deletion or clonal anergy, and peripherally with mature T and B cells via down-regulation of the immune response. Disruption of any of these processes may be a potential cause of autoimmune disease. Less generalized autoimmune processes also exist. For example, autoimmune disease can arise from the response to a foreign antigen if the foreign antigen shows sucient homology with a self-antigen. Another theoretical source of autoantibodies is a malignant B cell clone, but RBC autoantibodies generally are polyclonal. On the basis of studies with other autoimmune disorders, such as ankylosing spondylitis and multiple sclerosis, both genetic and environmental factors likely play a role in the production of RBC autoantibodies.
WARM AUTOIMMUNE HEMOLYTIC ANEMIA Background Warm autoantibodies are responsible for 4870% of AIHA cases [13,21]. Lymphoproliferative disorders such as CLL, Hodgkins disease, non-Hodgkins lymphoma, and Waldenstroms macroglobinemia are the leading causes of secondary cases. Rare cases have been associated with multiple myeloma [22]. Other secondary causes include autoimmune disorders (e.g., SLE, rheumatoid arthritis, scleroderma, and ulcerative colitis), non-lymphoid neoplasms (e.g., ovarian dermoid cysts, teratomas, Kaposis sarcoma, and carcinomas), immunodeciency disorders (e.g., AIDS, dysglobulinemia, and hypogammaglobulinemia), and childhood viral illnesses. Due to these secondary causes, the incidence of warm AIHA increases starting around 40 years of age. Children have a peak incidence in the rst 4 years of life. There is an approximate 2:1 female predilection, at least partially due to the association with autoimmune disease, and no racial predilection is evident. Occasional familial cases have been reported, and there is conicting data concerning association with HLA-A1, B7, and B8 [2330]. Warm AIHA has a highly variable clinical presentation. Typically patients insidiously develop anemic symptoms such as weakness, dizziness, fatigue, and dyspnea on exertion; other less specic symptoms include fever, bleeding, coughing, abdominal pain, and weight loss [31]. Cases with insidious onset generally follow a chronic waxing and waning course. In patients with fulminant hemolysis, jaundice, pallor, edema, dark urine (hemoglobinuria), splenomegaly,

hepatomegaly, and lymphadenopathy accompany the anemia. This more acute, potentially life-threatening presentation is usually associated with viral infections, especially in children. In cases of secondary disease, the symptoms of AIHA may precede the recognition of the underlying illness by months to years, but ultimately the symptoms of the underlying disorder dominate. When presentation includes massive splenomegaly or lymphadenopathy, an underlying lymphoproliferative disorder should be considered. Symptoms can be precipitated by trauma, surgery, infection, pregnancy, and psychological stress. Pregnancy also carries a 5-fold risk of developing autoantibodies compared to an age-matched control population [32]. Some infants born in the setting of maternal AIHA are unaected, but others suer transient hemolysis due to passively acquired autoantibody [3335].
Laboratory Evaluation The laboratory evaluation shows a hemoglobin and hematocrit that varies widely, from normal in the compensating patient with indolent hemolysis to severely decreased in the patient with fulminant hemolysis. The MCV usually is elevated reecting reticulocytosis, but reticulocytopenia can exist early in the disorder or secondary to autoimmune hemolysis of the reticulocytes or an inadequate bone marrow response [36]. The WBC count typically exhibits a mild leukocytosis representing predominantly neutrophils, but neutropenia may also be seen. The platelet count is normal to low. Sometimes there is concurrent leukopenia and/or thrombocytopenia due to an immune-mediated process, such as Evans syndrome [37]. The peripheral blood smear generally displays polychromasia and macrocytosis from the reticulocytosis as well as nucleated RBCs. Erythrophagocytosis and microspherocytes also are indicative of autoimmune hemolysis. The bone marrow typically reveals erythroid hyperplasia, and increased lymphocytes or plasma cells suggest the presence of an underlying lymphoproliferative disorder or collagen vascular disease. Hemolysis also causes other laboratory ndings. Mild to moderate indirect bilirubinemia and an increased LDH are common. In cases of fulminant hemolysis, the released hemoglobin quickly depletes haptoglobin and produces hemoglobinemia. Because haptoglobin is an acute phase reactant, its level may be normal or even increased if the hemolysis is mild and there is adequate hepatic function. Mild cases increase urobilinogen, and severe cases create hemoglobinuria and urine hemosiderin. As previously mentioned, by denition warm autoantibodies react more strongly at 37C than at

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lower temperatures. They generally are polyclonal. Studies have showed that over 95% of warm AIHA cases have a positive DAT. Consistent with the high prevalence of IgG among the warm autoantibodies, the DAT positive cases include 2066% with only IgG detected on the RBC surface, 2463% with both IgG and C3 on the surface, and 714% with only C3 on the surface [13,21,38]. The vast majority of the IgG autoantibodies are in the IgG1 subclass; the IgG3 subclass is the next most common, but it is found alone in less than 7% of warm AIHA patients [39,40]. The small percentage of negative DAT cases may represent patients with either IgG autoantibodies in quantities lower than the detectable threshold for the DAT, IgA autoantibodies, or IgM autoantibodies [4146]. The characteristics that distinguish a pathologic warm autoantibody from a clinically harmless one are not clearly delineated. As a result, there is no diagnostic test to determine whether a warm autoantibody produces in vivo RBC destruction. The presence of bound IgG1 and especially IgG3 autoantibodies [68,47,48], the presence of concomitantly bound IgA and/or IgM autoantibodies [49], and the quantity of bound RBC autoantibodies and the resulting strength of the DAT [5053] all play a role in the risk of hemolysis. Other variables aecting hemolytic potential include the quantity of bound complement, characteristics of the RBC antigens, the anity of the autoantibodies for those antigens, the number of IgG Fc and complement receptors on the macrophages, and the functional status of the mononuclear phagocytic system [5557]. Warm autoantibodies are typically panagglutinins, reacting with all cells on the diagnostic antibody panel. Recent studies employing immunoblotting have variably implicated Rh antigens, membrane protein band 4.1, protein band 3, and glycophorin A as universal RBC targets [5759]. When apparent, specicity is usually directed against Rh antigens such as e, E, or C. Other reported specicities include A, B, K, Jka, Fyb, M, N, S, LW, U, Wrb, Ena, and Ge [16,6064].
Treatment Therapy generally depends on the severity of the hemolysis, though folic acid supplementation is recommended for all. If the bone marrow can compensate, then the patient can continue to be monitored. However, once anemia develops, glucocorticoids are the rst-line treatment. Patients generally show some improvement within a week, and 7080% improve within 3 weeks. In responders, the steroid dose can be tapered gradually as long as clinical improvement is maintained. Among new cases, 1520% will achieve

complete remission and can discontinue steroids, but more typically, patients require a maintenance dose [65]. If the patient has no initial response to steroids, then the next line of therapy includes splenectomy and cytotoxic drugs. Because of side eects, these additional therapies are reserved for patients who still require high-dose steroids after several months of treatment. Splenectomy is usually the second line treatment in surgical candidates who fail glucocorticoid therapy. Removal of the spleen theoretically has a 2-fold eect. First, because IgG antibodies predominantly mediate warm AIHA, it removes the primary site of extravascular hemolysis. Less importantly, the spleen is a site of antibody production. Studies indicate that splenectomy has a response rate of approximately 6075%, but many of these patients require maintenance with lower doses of steroids, and other patients relapse months to years later [21,6769]. The procedure has a low morbidity and mortality rate. Due to the infection risk from encapsulated bacteria, splenectomized patients should be vaccinated for pneumococcus and meningococcus. Cytotoxic drugs are another treatment option for patients who have failed glucocorticoids and/or splenectomy. Studies suggest that the response rate in that population is approximately 4060% [66,68,69]. Cyclophosphamide is the agent most commonly used, although azathioprine also is employed. One potential regimen involves starting the patient with a cytotoxic drug and a steroid, tapering the steroid over 3 months if clinically responsive and tapering the cytotoxic drug after 6 months of treatment [65]. The possibility of bone marrow suppression from these agents necessitates careful WBC monitoring. These agents also can produce secondary malignancies, hemorrhagic cystitis, alopecia, and sterility. Cyclosporine A also has reportedly produced a response in some patients [70]. Recently, a case of warm AIHA refractory to steroids and the commonly used chemotherapeutics has been reported to respond to rituximab (anti-CD20 monoclonal antibody) treatment [71]. Other therapies have been tried with variable success. Plasmapheresis has shown a limited response, but in the case of fulminant hemolysis it may be transiently benecial until drug therapy takes eect. Some reports suggest that IVIG is benecial, other reports show no benet [7278]. Danazol has also been noted to be eective for some patients [79,80]. Another treatment providing anecdotal success is vincristine-loaded platelets; selective macrophage injury is the suspected mechanism of its activity [81,82]. Although RBC transfusion is not contraindicated for AIHA, it is generally limited to cases of lifethreatening anemia or a high risk of cardiac or cere-

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brovascular ischemic events. Panagglutinating warm autoantibodies complicate the serologic work-up on the transfusion service. A panreactive autoantibody can mask an existing alloantibody by making all donor units appear cross-match incompatible, regardless of whether an alloantibody is present. If the patient has not been recently transfused and does not have a high titer autoantibody, autoadsorption techniques can eliminate the confounding autoantibody and reduce this risk. When transfusion is necessary, the least incompatible units should be issued, and the infusion should be slow and carefully monitored. Donor RBCs are destroyed at the same rate as autologous RBCs [83] unless the autoantibodies exhibit specicity, in which case antigen-negative units should be transfused if available. Transfusions may induce further autoantibody production [84].
COLD AGGLUTININ SYNDROME Background Cold-reactive autoantibodies cause two distinct clinical entities: cold agglutinin syndrome (CAS, cold hemagglutinin disease) and paroxysmal cold hemoglobinuria (PCH). CAS represents approximately 1632% of AIHA cases [13,21]. Primary CAS generally aects older adults, with a peak incidence at approximately 70 years of age [85]. There appears to be a slight female predilection. Infection and lymphoproliferative disorders are the predominant causes of secondary cases. The typical case of an infectious etiology involves mycoplasmal pneumonia or infectious mononucleosis in an adolescent or young adult. Other infectious agents associated with CAS include adenovirus, CMV, inuenza viruses, varicella zoster virus, human immunodeciency virus, Escherichia coli, Listeria monocytogenes, and Treponema pallidum [86]. While infectious etiologies may produce transient CAS, lymphoproliferative disorders (e.g., CLL, lymphomas, and Waldenstroms macroglobulinemia) typically produce a more chronic course. Approximately 40% of these patients with a B cell clone have a karyotype of trisomy 3 and/or trisomy 12. These karyotypes also are seen in some cases of idiopathic CAS. Other neoplasms have been reported in association with CAS, including pulmonary squamous cell carcinoma, metastatic colonic adenocarcinoma, metastatic adrenal adenocarcinoma, basal cell carcinoma, and a mixed parotid tumor [87]. Patients with primary CAS and CAS secondary to a lymphoproliferative disorder commonly have a mild, chronic hemolytic anemia producing pallor and fatigue. The baseline disease is often stable, with any progression being insidious. However, a cold environment may exacerbate the condition. Therefore,

episodes of acute hemolysis with hemoglobinemia and hemoglobinuria are more common in the winter months. Because blood is more susceptible to the eects of the environmental temperature in the extremities, patients also present with acrocyanosis during exacerbations. Some patients experience Raynauds phenomenon, and rarely the RBC agglutination becomes signicant enough to produce vascular occlusions with resulting necrosis. These additional complications may be potentiated by concurrent cryoglobinemia [8890]. The clinical presentation of the transient form of CAS usually corresponds with the immune response to an infectious agent. As such, the symptoms appear 23 weeks after the infection starts (corresponding to a peaking cold agglutinin titer), and they resolve spontaneously an additional 23 weeks later. Typically, the patients present with pallor and jaundice, but severe cases can result in signicant hemoglobinuria and transient renal failure. Acrocyanosis can result from exposure to cold temperatures.
Laboratory Evaluation Often the laboratory evaluation provides the rst evidence of CAS. Clumping from the cold agglutinins complicates both the peripheral blood smear and the calculation of the cell counts and red cell indices. RBC clumping artifactually increases the MCV while decreasing the apparent RBC count. Dissolution of the clumping upon warming indicates the presence of a cold agglutinin rather than rouleaux formation or brin clumping. In the absence of an acute exacerbation, hemoglobin and hematocrit are mildly decreased. The reticulocyte count is mildly elevated unless bone marrow failure is present. The WBC and platelet counts are usually normal. The peripheral smear may show agglutination, polychromasia, anisocytosis, poikilocytosis, and occasionally spherocytosis. Other common laboratory ndings secondary to the hemolysis include mildly elevated indirect bilirubinemia and LDH. As with any cause of intravascular hemolysis, severe exacerbations produce decreased haptoglobin, hemoglobinemia, and hemoglobinuria. As a group, patients with CAS have more homogeneous DAT results than patients with warm AIHA. Since the pathophysiology of CAS typically involves IgM autoantibodies and complement, patients almost exclusively have a positive DAT with anti-C3 and polyspecic reagents and a negative result with antiIgG. The IgM autoantibodies dissociate from the RBCs subsequent to C3 binding, so they generally are not detected in vitro. In rare cases, cold autoantibodies are IgG or IgA [21,64]. The cold autoantibodies in idiopathic CAS and CAS secondary to

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lymphoproliferative disorders typically are IgM j monoclonal antibodies, while they are polyclonal IgM j or k in CAS secondary to infection. By denition cold autoantibodies react more strongly at 04C than at higher temperatures. Because cold agglutinins can be detected in most healthy individuals if testing is performed at 04C, the majority of cold autoantibodies clearly are benign. Pathologic cold autoantibodies are characterized by a large thermal amplitude or a high titer, with thermal amplitude as the better predictor of hemolysis [91]. Thermal amplitude refers to the temperature range at which in vitro agglutination is detectable. Activity at 37C always corresponds with a clinically signicant cold autoantibody. In addition, pathologic cold autoantibodies generally have a titer of greater than 1:1,000 at 04C. Primary CAS and CAS secondary to lymphoproliferative disorders usually exhibit higher titers than CAS secondary to infection. The pathophysiology of CAS is highly temperaturedependent. The thermal amplitude determines whether and where initiation occurs. Hemolysis is initiated when cold autoantibodies bind to RBCs in the cooler peripheral circulation. Since the temperature in the distal extremities ranges from 28 to 31C, cold autoantibodies with a signicant titer at 30C or more can cause chronic hemolysis. Cold agglutinins with a lower thermal range require excessively cold environments to bind RBCs. Subsequent to binding RBCs, IgM autoantibodies x C1 and initiate the classical complement cascade. Warmer temperatures of the central circulation then facilitate hemolysis. First, the higher temperatures maximize complement xation and activation. In addition, the higher temperatures cause dissociation of the cold agglutinins, which allows them to bind additional RBCs back in the colder peripheral circulation and repeat the cycle. If the complement cascade progresses to the membrane attack complex, intravascular hemolysis results. Otherwise, bound C3b leads to extravascular hemolysis. Cold autoantibodies commonly show specicity against the Ii blood group system, with approximately 90% directed against the I antigen and most of the remaining ones directed against the i antigen [21]. The I/i antigens are closely related, high-frequency carbohydrates that also are related to the ABO antigens. The RBC surface densities of I/i are inversely proportional to one another: neonatal RBCs almost exclusively express large amounts of i antigen on their surface, but antigen switching occurs during infancy, so that I antigen is predominant by 18 months of age. As a result, antibody panels with adult RBCs will detect anti-I agglutinins, and cord RBCs are needed to detect anti-i agglutinins. Only rare adults never express I antigen on their RBCs [92]. Anti-I specicity

commonly is observed with primary CAS and CAS secondary to lymphoproliferative disorders and mycoplasmal pneumonia. While the majority of patients infected with Mycoplasma pneumoniae transiently produce anti-I agglutinins, most are not clinically signicant. On the other hand, anti-i agglutinins are typically associated with infectious mononucleosis, and they also have been identied during CMV infections and with lymphomas [93]. As with mycoplasmal pneumonia, the majority of anti-i cold autoantibodies secondary to infectious mononucleosis are benign. Studies have noted that the sera of 869% of infectious mononucleosis patients have anti-i, but only 0.13% exhibit hemolysis [9498]. Rarely do patients with infectious mononucleosis and angioimmunoblastic lymphadenopathy develop CAS from an IgG anti-i autoantibody [99]. These patients also produce IgM anti-IgG antibodies that ultimately initiate the agglutination and hemolysis. Occasionally cold autoantibodies are identied with specicity against the Pr antigen. Anti-Pr activity should be suspected when a cold agglutinin reacts equally strongly with both cord and adult RBCs, and disappearance of the agglutination with papain pretreatment further suggests anti-Pr specicity. AntiPr cold agglutinins often are pathologic due to a high titer and a high thermal amplitude [100]. Other reported specicities include Gd, Sa, Lud, Fl, Vo, M, N, D, and P [63,64,101].
Treatment Treatment for CAS is dependent on its etiology and severity. With primary CAS most patients only have mild anemia. Therefore, avoidance of cold exposure is the primary therapy. For some patients this necessitates moving to a warmer climate. Folic acid supplementation also is recommended for these patients. In cases with severe hemolysis, immunosuppression with chlorambucil or cyclophosphamide may be benecial [102]. a-Interferon also reportedly has produced signicant responses [103]. While steroids are eective for WAIHA, they are rarely helpful for CAS. Steroids only have been noted to be benecial for patients with either low titer or high thermal amplitude IgM cold agglutinins or with IgG cold agglutinins. Likewise, since extravascular hemolysis in CAS generally occurs in the liver, splenectomy has only beneted those patients with IgG cold agglutinins [104]. Plasmapheresis can provide a temporary improvement in cases of severe hemolysis, and it may be used prophylactically for surgeries requiring cold exposure. However, because plasmapheresis has no eect on the underlying production of cold agglutinins, it should be combined with concomitant immunosuppressive therapy.

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Similarly, treatment of the hemolysis often is not necessary for secondary CAS. However, when signicant hemolysis occurs, treatment of secondary CAS is directed at the underlying cause. Thus, for CAS resulting from a lymphoproliferative disorder or another malignancy, treatment typically encompasses chemotherapeutic agents. In the context of CD20-positive chronic lymphoproliferative disorders producing monoclonal IgM, cold AIHA has been successfully treated with rituximab [105,106]. For CAS secondary to non-viral infections, antibiotics can be helpful. Because infection-associated CAS is a transient disorder, immunosuppressives are rarely indicated. In cases with severe hemolysis, plasmapheresis may be necessary. Supportive measures also can include transfusion and hydration to maintain adequate renal blood ow. RBC transfusions should be limited for two reasons. First, as mentioned previously, cold agglutinins cause serologic diculties during the blood bank work-up. Thus, the blood bank may have to release least incompatible RBC units that have a higher risk of containing an undetected alloantibody. Second, transfused units can potentiate the hemolysis. Most cold autoantibodies are directed against the I antigen, and I antigen-negative donor units are extremely rare. In addition, if low complement levels limit hemolysis, then exogenous donor plasma complement can exacerbate the hemolysis. As a result of these factors, transfusions typically are reserved for patients with signicant underlying cardiovascular or cerebrovascular disease. If necessary, washing RBC units will reduce the exogenous complement load. The risk of additional, transfusion-related hemolysis can be reduced by using an in-line blood warmer at 37C and by keeping the patient warm [107].
PAROXYSMAL COLD HEMOGLOBINURIA Background Paroxysmal cold hemoglobinuria (PCH) is a relatively uncommon form of AIHA, with studies noting an incidence of 210% among cases of hemolytic anemia [21,108,109]. Both idiopathic and secondary forms of the disease exist. Both idiopathic PCH and PCH secondary to late-stage or congenital syphilis are chronic processes that have become increasingly uncommon. Most present-day cases of PCH have an acute transient pathology secondary to infection. These acute cases predominantly aect children, and they represent one of the leading causes of acute hemolytic anemia in children [109,110]. While most cases involve an antecedent upper respiratory infection, the causative agent often is not identied. Infectious agents associated with PCH include measles,

mumps, EBV, CMV, varicella zoster virus, adenovirus, inuenza A, M. pneumoniae, Haemophilus inuenzae, and E. coli. No racial or gender predilection is evident. Clinically, PCH is characterized by an acute attack of high fever, chills, back and/or leg pain, and abdominal cramping. Other symptoms may include headache, nausea, vomiting, and diarrhea. Hemoglobinuria typically occurs, producing dark red to black urine. A preceding exposure to cold temperatures may be identied retrospectively. Cold urticaria, tingling of the extremities, and Raynauds phenomenon have been reported in cases precipitated by cold exposure. Constitutional symptoms generally subside within a few hours. However, the hemolysis can be severe and even life-threatening and can require up to several weeks to recover. The chronic forms of PCH are characterized by recurrent episodes of hemolysis precipitated by cold exposure.
Laboratory Evaluation Hematologic ndings include hematocrit and hemoglobin that may be severely decreased. Reticulocytopenia is common in the acute phase, but ultimately reticulocytosis develops. Similarly, initial leukopenia is followed in the recovery phase by a normal to high WBC count. The platelet count generally is unremarkable. Examination of the peripheral blood smear can show agglutination, polychromasia, nucleated RBCs, anisocytosis, poikilocytosis, spherocytes, and erythrophagocytosis. Additional evidence of intra- and extravascular hemolysis is provided by increased LDH, indirect bilirubinemia, decreased haptoglobin, hemoglobinemia, and hemoglobinuria. The urine also contains methemoglobin. Since PCH can produce acute renal failure, elevated BUN and creatinine may be present. PCH is caused by a biphasic IgG autoantibody (DonathLandsteiner antibody) that xes complement at low temperatures but ultimately dissociates at higher temperatures. As a result, the DAT is positive with anti-C3, but it is generally negative with anti-IgG unless performed at colder temperatures. Biphasic IgG autoantibodies bind RBCs eciently at 04C and subsequently x complement C1 at that temperature. However, the other complement components bind more eciently and cause lysis at temperatures nearer normal body temperature. While the maximal thermal amplitude of the autoantibody typically does not extend above 20C in vitro with acute transient PCH, for unknown reasons hemolysis can still occur in the absence of cold exposure. Occasionally DonathLandsteiner antibodies appear to be monophasic because their thermal amplitude extends to

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body temperature. DonathLandsteiner antibodies are potent, so even small titers can produce hemolysis. As with cold agglutinins, biphasic IgG autoantibodies often exhibit specicity. Usually they are directed against the P antigen, which is found on the RBCs of most individuals [111]. The DonathLandsteiner test was developed to detect the biphasic IgG autoantibody responsible for PCH. It involves in vitro testing for hemolysis with a patients serum, washed group O cells expressing the P antigen, and fresh normal serum (complement source) [112]. One group of samples is incubated only at 04C, another group is incubated only at 37C, and the third group is incubated rst at 04C for 30 min and then at 37C for 60 min. The diagnosis of PCH is indicated when hemolysis only is observed in samples with patients serum which have been incubated rst at 04C for 30 min and then at 37C for 60 min.

Laboratory Evaluation The laboratory work-up shows that the DAT is positive for both IgG and C3. As with the separate diseases, the mixed-type AIHA produces diculties with the antibody screen and the cross-match. The RBC eluate typically indicates panreactive warm IgG autoantibody. The cold autoantibody usually exhibits specicity against the I antigen, but reactivity against i has been reported [114116]. Donor units have to be released as cross-match least-incompatible due to the autoantibodies.

Treatment Mixed-type AIHA appears to respond to treatment in a similar manner as warm AIHA. Patients generally respond to steroids, and immunosuppressive agents and splenectomy have been employed successfully as well. Associated diseases, if present, also need to be treated to optimize recovery. DRUG-INDUCED AUTOIMMUNE HEMOLYTIC ANEMIA Background Drugs can produce hemolysis by both immune and non-immune mechanisms. Historically, a-methyldopa and high-dose penicillin were responsible for the majority of cases of drug-induced IHA. Studies published in the early 1980s, when these two agents were more commonly used, indicated that 1218% of AIHA cases were drug-induced [13,21]. While the incidence of drug-induced IHA has likely decreased since then, second- and third-generation cephalosporins, especially cefotetan and ceftriaxone, have been associated increasingly with cases of drug-induced IHA [117,118]. Rarely, these cases of cephalosporininduced IHA are fatal. Drug-induced IHA results from several types of interactions between a drug, antibodies, and RBC membrane components. The three major mechanisms include induction of autoantibodies, neoantigen (immune complex) formation, and drug adsorption onto the RBCs. IHA secondary to neoantigen formation occurs when a drug binds weakly to a normal RBC component, and the immune system perceives the drug+RBC component complex or the conformationally altered RBC component as foreign. IHA due to the drug adsorption mechanism results when antibodies largely directed against the drug interact with RBCs to which the drug has strongly bound. These mechanisms are not mutually exclusive. For example, while cephalosporins generally are associated with IHA due to the neoantigen formation and/or drug

Treatment Since most cases of PCH are self-limited, treatment is usually symptomatic, including keeping the patient warm. While needed blood transfusions would ideally consist of P antigen-negative RBCs, the rarity of these units normally precludes their use. P antigenpositive RBCs typically are benecial when a blood warmer is employed [113]. Although glucocorticoids have not been shown to be eective routinely, they often are given to severely anemic children. Splenectomy typically is not helpful. In cases of life-threatening PCH, plasmapheresis can temporarily dampen the hemolysis. Syphilis should be ruled out in patients with chronic PCH. If syphilis is present, treatment generally eliminates the concurrent hemolysis. MIXED-TYPE AUTOIMMUNE HEMOLYTIC ANEMIA Background Some patients with warm AIHA also possess a cold agglutinin. While the majority of these cold agglutinins are not clinically signicant, occasionally they have a sucient thermal amplitude (greater than 30C) or high titer (greater than 1:1,000 at 04C) to indicate CAS. Similar to the separate entities, mixedtype AIHA can be either idiopathic or secondary to lymphoproliferative disorders or SLE. Patients usually have a chronic course interrupted by severe exacerbations, which can result in a hemoglobin level below 5.0 g/dL. These exacerbations do not appear to be associated with cold exposure, and they do not result in acrocyanosis or Raynauds phenomenon [114].

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adsorption mechanisms, they also can induce autoantibody formation [118120].

Laboratory Evaluation The three mechanisms can be distinguished on the basis of serologic reactions of the serum and the eluate (Table III). Drug-induced IHA secondary to neoantigen formation or drug adsorption has a positive DAT and can be serologically distinguished from the autoantibody mechanism because of the requirement for exogenous drug to detect the antibody. In contrast, with drug-induced AIHA, the serum and eluate antibodies react with RBC panels even if drug is absent. Drug-induced AIHA is serologically indistinguishable from warm AIHA; a presumptive diagnosis can be made only if the patient responds to withdrawal of the drug. a-Methyldopa is the prototypical drug operating by the induction of autoantibodies, producing a positive DAT in 1136% of patients (dose-dependent) within 36 months of initiation [121,122]. The DAT is positive due to bound IgG, and occasionally complement may also be present. These autoantibodies are typically panreactive, though documented specicities include c, e, Wrb, Jka, and U [16,123125]. As mentioned previously, second- and third-generation cephalosporins occasionally induce RBC autoantibodies [118]. Other drugs that reportedly can induce autoantibodies include levodopa, mefenamic acid, procainamide, and diclofenac [127130].

evident hemolysis, a positive DAT alone is not an indication to discontinue drug usage. If the hemolysis is clinically signicant, use of the suspected drug should be discontinued, especially when alternative therapies exist. Drug-induced AIHA usually resolves within several days of discontinuing the medication but occasionally requires months to fully resolve [132]. In cases with severe hemolysis, corticosteroids may aid in recovery. RBC transfusions also can be given, but the transfused RBCs will be hemolyzed at a similar rate as the endogenous RBCs in the typical case where the donor unit of RBCs is crossmatch incompatible.
SUMMARY

Treatment Despite the high incidence of a positive DAT associated with a-methyldopa usage, the responsible autoantibody results in hemolytic anemia in less than 1% of patients [131]. Therefore, without clinically

Diagnosis of AIHA requires both serologic evidence of an autoantibody and clinical and laboratory evidence of hemolysis. Cases of AIHA can be classied on the basis of the characteristic temperature reactivity of the RBC autoantibody. The autoantibodies associated with warm AIHA and drug-induced AIHA react most strongly near body temperature, while CAS and PCH are characterized by autoantibodies that react most strongly near 04C. Rarely patients develop a mixed-type AIHA with both warm and cold autoantibodies. Cases of AIHA also can be classied on the basis of etiology as idiopathic or secondary. While lymphoproliferative disorders are the most common secondary cause, autoimmune disorders sometimes produce warm AIHA, and infections occasionally lead to cold AIHA. When clinically indicated, treatment of warm AIHA and mixed-type AIHA includes glucocorticoids, splenectomy, and immunosuppressive agents. If drug-induced AIHA is suspected, the initial treatment is discontinuation of the drug. The primary treatment for cold AIHA is warming the

Table III. Serologic Characteristics of Drug-Induced Hemolytic Anemias* AutoAb formation DAT Polyspecic IgG C3 Serum Ab Routine Soluble drug Drug-treated RBC's RBC eluate Ab Routine Soluble drug Drug-treated RBC's + + Usually ) + + + Drug adsorption + + Usually ) ) ) + ) ) + Neoantigen formation + Usually ) + ) + + ) ) )

*Ab, antibody; DAT, direct antiglobulin test; RBC, red blood cell.

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17. Garratty G. The signicance of IgG on the red cell surface. Transfus Med Rev 1987;1:4757. 18. Lau P, Haesler WE, Wurzel HA. Positive direct antiglobulin reaction in a patient population. Am J Clin Pathol 1976;65: 368375. 19. Okuno T, Germino F, Newman B. Clinical signicance of autologous control. Am Soc Clin Pathol 1984 (abstr). 20. Judd WJ, Barnes BA, Steiner EA, Oberman HA, Averill DB, Butch SH. The evaluation of a positive direct antiglobulin test (autocontrol) in pretransfusion testing revisited. Transfusion 1986;26:220224. 21. Petz LD, Garratty G. Acquired immune hemolytic anemias. New York: Churchill Livingstone; 1980. 22. Vaiopoulos G, Kyriakou D, Papadaki H, Fessas P, Eliopoulos GD. Multiple myeloma associated with autoimmune hemolytic anemia. Haematologica 1994;79:262264. 23. Fialkow PJ, Fudenberg H, Epstein WV. Acquired antibody hemolytic anemia and familial aberrations in gamma globulins. Am J Med 1964;36:188199. 24. Dodds CE. Familial auto-immune hemolytic anemia. Arch Intern Med 1965;116:273276. 25. Cordova MS, Baez-Villasenor J, Mendez JJ, Campos E. Acquired hemolytic anemia with positive antiglobulin (Coombs test) in mother and daughter. Arch Intern Med 1966;117:692695. 26. Pirofsky B. Hereditary aspects of autoimmune hemolytic anemia: a retrospective analysis. Vox Sang 1968;14:334347. 27. Da Costa JAG, White AG, Parker AC, Grigor GB. Increased incidence of HL-A 1 and 8 in patients showing IgG or complement coating on their red cells. J Clin Pathol 1974;27:353355. 28. Clauvel JP, Marcelli-Barge A, Gautier Coggia I, Poirier JC, Benajam A, Dausset J. HL-A antigens and idiopathic autoimmune hemolytic anemias. Transplant Proc 1974;6:447448. 29. Abdel-Khalik A, Paton L, White AG, Urbaniak SJ. Human leucocyte antigens A, B, C and DRW in idiopathic warm autoimmune haemolytic anaemia. Br Med J 1980;280:760761. 30. Toolis F, Parker AC, White A, Urbaniak S. Familial autoimmune haemolytic anaemia. Br Med J 1977;1:1392. 31. Pirofsky B. Clinical aspects of autoimmune hemolytic anemia. Semin Hematol 1976;13:251265. 32. Sokol RJ, Hewitt S, Stamps B. Erythrocyte autoantibodies, autoimmune haemolysis and pregnancy. Vox Sang 1982;43: 169176. 33. Starksen NF, Bell WE, Kickler TS. Unexplained hemolytic anemia associated with pregnancy. Am J Obstet Gynec 1983;146: 617622. 34. Burt RL, Prichard RW. Acquired hemolytic anaemia in pregnancy: report of a case. Obstet Gynecol 1957;10:444450. 35. Yam P, Wilkinson L, Petz LD, Garratty G. Studies on hemolytic anemia in pregnancy with evidence for autoimmunization in a patient with a negative direct antiglobulin (Coombs) test. Am J Hematol 1980;8:2329. 36. Liesveld JL, Rowe JM, Lichtman MA. Variability of the erythropoietic response in autoimmune hemolytic anemia: analysis of 189 cases. Blood 1987;69:820826. 37. Evans RS, Takahashi K, Duane RT, Payne R, Liu C-K. Primary thrombocytopenic purpura and acquired hemolytic anaemia. Evidence for a common etiology. Arch Intern Med 1951;87:4865. 38. Chaplin H Jr. Clinical usefulness of specic antiglobulin reagents in autoimmune hemolytic anemias. Prog Hematol 1973;7:2549.

patient. Glucocorticoids and splenectomy have limited benet in patients with CAS and PCH. RBC transfusions are not absolutely contraindicated, though they should be limited to patients with lifethreatening anemia or signicant cardiovascular or cerebrovascular disease due to the increased risk of possessing undetected alloantibodies and the risk of exacerbating the hemolysis.
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