Autoimmune Hemolytic Anemia

Richard C. Friedberg

Vandita P. Johari

CLASSIFICATION

In autoimmune hemolytic anemia (AIHA), pathologic antibodies (autoantibodies) attach to and lead to the destruction (hemolysis) of endogenous erythrocytes (red blood cells, RBCs) resulting in anemia. By definition, both autoantibodies and associated RBC consumption must be identified. AIHA is readily subclassified according to the characteristic temperature activity of the responsible antibodies (Table 29.1). Cold-active antibodies typically have little, if any, activity at body temperature but have greater affinity for RBCs as the temperature decreases toward 0°C. Conversely, warm-active antibodies have their greatest affinity at 37°C. Generally speaking, cold-active antibodies are typically IgM, fix complement, and lead to immediate intravascular RBC destruction or hepatic-mediated clearance. In contrast, warm-active antibodies are typically IgG, may or may not fix complement, and primarily lead to RBC loss by splenicmediated clearance of sensitized cells. Patients who express both (mixed) cold- and warm-active antibodies are particularly troublesome clinically because of the dual impact from severe RBC destruction and characteristically poor response to therapy.¹ Drug-induced immune hemolytic anemia (DI-IHA) is caused by warm-active antibodies that may be clinically and serologically indistinguishable from the idiopathic warm autoimmune type (α-methyldopa type), or may be dependent on the presence of the drug in serologic studies to demonstrate attachment of the antibody to the RBC. The clinical spectrum of drug-induced antibody attachment to RBCs ranges from asymptomatic positive serologic studies to life-threatening massive hemolysis.² Finally, a different type of immune-mediated hemolytic anemia can occur as a complication of organ transplantation. Because the antibodies are generated from donor-derived lymphocytes, the disorder is not truly of “auto”-immune origin, but can be thought of as a graft-versus-host disorder.

Etiology of the Immune Response in Autoimmune Hemolytic Anemia

Immunologic tolerance is a state in which the individual is incapable of developing an immune response to a specific antigen. Self-tolerance refers to lack of responsiveness to an individual’s own (self) antigens, which is the normal state. Autoimmunity results from a loss of self-tolerance leading to T-cells or antibodies reacting against self-antigens and the consequent tissue injury. In AIHA, autoantibodies are directed against targets on the individual’s own RBCs (“self-antigens”), leading to their enhanced clearance through F_c-receptor-mediated phagocytosis (“extravascular hemolysis”) or complement-mediated breakdown (“intravascular hemolysis”). AIHA may be in large part due to self-reactive antibodies against erythrocyte band 3, an anion transporter found in the RBC membrane that is involved in RBC senescence.³, ⁴

Central Tolerance

Central tolerance refers to the normal deletion of self-reactive T-and B-lymphocyte clones during their maturation in the central lymphoid organs (thymus for T-cells; bone marrow for B-cells).⁵ Central tolerance prevents widespread autoimmunity by preferentially selecting nonautoreactive (i.e., “normal”) T-cells for circulation into the periphery (“intrathymic negative selection”). Central tolerance is not complete, and a population of T-cells with intermediate avidity for self-antigens invariably escapes into the circulation. Under certain conditions these cells can become activated and lead to organ-specific or systemic autoimmune disease.⁶, ⁷

Peripheral Tolerance

The mechanisms by which self-reactive T-cells that escape intrathymic negative selection and are deleted in the peripheral tissues constitute peripheral tolerance, including anergy, suppression by regulatory T-cells, and clonal deletion by activation-induced cell death (see Fig. 29.1).

Anergy refers to prolonged or irreversible functional inactivation of lymphocytes. Activation of antigen-specific T-cells requires two signals: (i) recognition of peptide antigen in association with self-MHC (major histocompatibility complex) molecules on the surface of antigen-presenting cells and (ii) a set of costimulatory signals provided by antigen-presenting cells (the co-stimulators B7-1 and B7-2). In the absence of costimulators, a negative signal is delivered, and the T-lymphocyte becomes anergic. Anergic lymphocytes cannot be activated even if the relevant antigen is presented by antigen-presenting cells (e.g., dendritic cells) that can deliver co-stimulation. Anergy also affects B-cells as they encounter antigen in the absence of
specific helper T-cells. They become unable to respond to subsequent antigenic stimulation and may be excluded from lymphoid follicles.⁶

TABLE 29.1 CLASSIFICATION OF IMMUNE HEMOLYTIC ANEMIAS

Cold-active antibodies
	Cold agglutinin disease (CAD)
		Primary or idiopathic Secondary
			Lymphoproliferative diseases Autoimmune disorders Infections
				Mycoplasma pneumoniae Infectious mononucleosis Other viruses
	Paroxysmal cold hemoglobinuria (PCH)
		Syphilis
		Measles, mumps, other viruses
Mixed cold- and warm-active antibodies
Warm-active antibodies
	Idiopathic autoimmune hemolytic anemia Secondary autoimmune hemolytic anemia
		Lymphoproliferative disorders
		Autoimmune and immunodeficiency disorders Malignancy
		Viral infections
	Drug-induced immune hemolytic anemia (DI-IHA)
		Drug adsorption type (penicillin)
		Neoantigen type (quinidine/stibophen)
		Autoimmune type (α-methyldopa)
		Nonimmune type (first-generation cephalosporins)
Transplant-associated hemolytic anemia
	Hematopoietic stem cell transplant
		Minor ABO group mismatch
		Major ABO group mismatch
		Passive antibody transfer
	Solid organ transplant
		Passenger lymphocyte syndrome
		Passive antibody transfer

FIGURE 29.1. Schematic illustration of the mechanisms involved in central and peripheral tolerance. The principal mechanisms of tolerance in CD4⁺ T-cells are shown. APC: antigen-presenting cell. (Used with permission from Abbas AK, Diseases of immunity. In: Kumar V, Abbas A, Fausto N, eds. Robbins and Cotran pathologic basis of disease, 7th ed. Philadelphia, PA: Elsevier Saunders, 2005:223-225.)

Suppression by regulatory T-cells involves CD4⁺ cells that co-express CD25, the α chain of the interleukin-2 (IL-2) receptor, but some CD4⁺ cells that lack CD25 may also induce peripheral tolerance by suppression. These T-cells can suppress (inhibit) lymphocyte activation and effector functions in part by the secretion of cytokines such as interleukin-10 (IL-10) and transforming growth factor-β (TGF-β). However, the precise mechanism of their action is unknown.

Clonal deletion by activation-induced cell death refers to the process by which CD4⁺ T-cells that are activated by self-antigens may receive signals that cause apoptosis. Lymphocytes express Fas (CD95), a member of the tumor necrosis factor (TNF)-receptor family, and activated lymphocytes express FasL, a membrane protein that is structurally homologous to the cytokine TNF. The engagement of Fas by FasL induces apoptosis of activated autoreactive T-cells. Self-antigens that are abundant in peripheral tissues cause repeated and persistent stimulation of self-antigen-specific T-cells, leading eventually to their elimination via Fasmediated apoptosis. FasL on T-cells engaging Fas on the B-cells may also delete self-reactive B-cells.⁶

Factors Affecting Initiation of Autoimmunity

Autoimmunity can be affected by a number of different factors, including the nature of the autoantigens, genetic associations, and environmental factors. In AIHA, autoantigenic T-cell epitopes have been mapped for the RhD autoantigen.⁸ Although these autoantigenic sites may be a potential target for novel therapeutic interventions, the precise mechanism of disease initiation remains unclear. In addition, MHC class I and class II genes may predispose individuals to certain types of autoimmune disease. For example, the human HLA-DQ-6 molecule has been associated with AIHA. However, the genetic association is multifactorial as with most other autoimmune diseases. Finally, inflammatory stimuli such as viral and bacterial infections have
been implicated as environmental triggers of autoimmunity, possibly because of antigenic mimicry leading to tolerance breakdown, i.e., environmental or infectious agents may have molecular structures similar to self-antigens. Other possible mechanisms involve production of interferon γ during viral infection that causes up-regulation of F_cRI. Alternatively, viral infection may cause a change in the expression pattern of F_c receptors as a result of transcriptional activation or other mechanisms (discussed further under section “IgG-Mediated Red Blood Cell Destruction”).⁹

Mechanism of Immune-mediated Red Blood Cell Lysis

The most important features of RBC destruction by IgM and IgG antibodies are summarized in Table 29.2.

IgM-mediated Red Blood Cell Destruction

Destruction of erythrocytes sensitized with IgM antibodies is mediated by the complement system, either directly by cytolysis or indirectly via interaction of RBC-bound activation and degradation fragments of C3 with specific receptors on reticuloendothelial cells, principally liver macrophages (Kupffer cells).

The pentameric structure of IgM enables efficient complement activation. High-titer IgM antibodies can cause direct intravascular hemolysis by generating the cytolytic membrane attack complex (MAC) of complement on the RBC surface. With sufficient antibody density, complement activation may be robust enough to overwhelm the inhibitory activity of the complement-regulatory proteins DAF (CD55) and MIRL (CD59) on the RBC surface and result in hemolysis.¹⁰ However, in most clinical situations, IgM antierythrocyte antibodies are present in sublytic quantities. Under these conditions, DAF (CD55) and MIRL (CD59) are able to prevent direct RBC lysis. Nonetheless, some C3b is deposited on the RBC surface as a consequence of the IgM-induced complement activation, and interactions of C3b and its ligand iC3b with their specific complement receptors (CR) on liver macrophages (Kupffer cells) are ultimately responsible for the immune destruction of RBCs under sublytic conditions. Table 29.3 summarizes the characteristics of various complement receptors.¹⁰, ¹¹, ¹²

Although ligation of erythrocyte-bound C3b to CR1 on Kupffer cells may mediate some of the clearance, interaction between RBC-bound iC3b and macrophage CR3 is probably the principal mediator of extravascular destruction of complementsensitized erythrocytes. Clearance of the complement-sensitized RBCs is likely mediated by phagocytosis, because the liver lacks the unique anatomy of the spleen and is thus unable to sequester cells. Once RBC-bound iC3b has been converted to C3dg (ligand for CR2), the RBCs are no longer subject to immune destruction because phagocytic cells do not express the specific receptor for C3dg. Thus, erythrocytes bearing only C3dg have a normal life span.¹³, ¹⁴

TABLE 29.2 RED CELL DESTRUCTION BY IgM AND IgG ANTIBODIES

Antibody	Intravascular Clearance	Extravascular Clearance—Liver	Extravascular Clearance—Spleen	Complement Dependency	Hemoglobinuria	Bilirubinemia	Specificity of Antiglobulin Test
IgM Low-titer^a	+	+	–	+	±	+	Complement
IgM High-titer^a	+	–	–	+	+	+	Complement
IgG Low-titer^b	–	–	+	–	–	+	Immunoglobulin
IgG High-titer^b	–	±	+	–	±	+	Immunoglobulin
^aAnti-A or anti-B blood group antibodies are examples of IgM antibodies that can be present at low or high concentrations. ^bAnti-Rh_o(D) is an example of an IgG antibody that can be present at low or high concentration.

IgG-mediated Red Blood Cell Destruction

IgG is a relatively ineffective initiator of activation of the classical complement pathway. Consequently, direct complementmediated cytolysis of RBCs induced by IgG antibodies is unusual (a notable exception is the D-L antibody of paroxysmal cold hemoglobinuria [PCH]). In the absence of complement activation, clearance of IgG-sensitized erythrocytes is primarily splenic. Two distinct processes appear to be involved. First, binding to F_c receptors expressed by tissue macrophages in the red pulp of the spleen can mediate direct and complete phagocytosis. Second, partial phagocytosis, in which the phagocytes remove a portion of the membrane, results in a decrease in the surface area-to-volume ratio and the consequent generation of spherocytes, the classic morphologic hallmark of immune hemolytic anemia. The loss of deformability as a consequence of spherocyte formation results in sequestration of the abnormal RBCs in the red pulp because the consequent rigidity limits their ability to traverse the splenic cords into the sinuses. The trapped spherocytes are vulnerable to phagocytosis by macrophages that are found in abundance in the splenic cords. In addition, the life span of the sequestered RBCs is shortened by the unfavorable metabolic environment found in the splenic cords (splenic conditioning). Once trapped, RBC destruction is complete within minutes.

The liver clears IgG-coated RBCs less efficiently than the spleen. Nevertheless, the liver plays a clinically significant role in RBC destruction. The quantity of antibody fixed to the RBC roughly correlates with the site of destruction (smaller amounts of antibody lead mainly to splenic sequestration whereas larger amounts of antibody lead to increased sequestration within the liver).¹⁵, ¹⁶ The more rapid clearance of RBCs sensitized with a higher density of IgG antibodies and the shift in clearance from the spleen to the liver are due to complement activation.¹⁶ Although IgG alone can mediate RBC clearance, the concomitant presence of RBC-bound C3 fragments greatly enhances the rate of immune-mediated destruction.

Phagocytosis of IgG-coated RBCs occurs in the spleen and is mediated by surface receptors for the F_cγ region of the IgG molecule. There are three different classes of F_cγ receptors. F_cγRI mediates in vitro cytotoxic activity. F_cγRII inhibits B-lymphocyte and mast-cell activation. F_cγRIII is responsible for phagocytosis, endocytosis, and antibody-dependent cell-mediated cytotoxicity and therefore plays a key role in hemolysis. The characteristics of F_cγR are summarized in Table 29.4.

Of the four subclasses of IgG, IgG₃ has the highest affinity for the F_cγR and therefore is most efficient at causing extravascular
hemolysis (IgG₃> IgG₁> IgG₄>>> IgG₂).¹⁷, ¹⁸ The critical role of F_cγ receptors in immune destruction in vivo is further demonstrated by the therapeutic approach to management of AIHA and immune thrombocytopenic purpura (ITP). Treatment with corticosteroids, intravenous IgG (IVIG), anti-D, and/or splenectomy is aimed at reducing the capacity of reticuloendothelial cells to mediate immune clearance of IgG-sensitized RBCs.¹⁹ Administration of monoclonal antibody 2.4G2 in mice, which binds to and blocks mouse F_cγRII and F_cγRIII, allows rapid recovery after induction of AIHA, suggesting that alteration of the balance of stimulatory to inhibitory F_cγ receptors has a marked effect on disease progression and susceptibility. Understanding the detailed structure of the F_cγ receptors may lead to the development of novel therapeutic strategies. For example, molecules that inhibit the binding of the F_cγR to an IgG-coated RBC or those that might inhibit the F_cγ receptor signaling at the different steps leading to phagocytosis may be useful therapeutic tools. Treatments that decrease expression of the activating F_cγ receptor or increase expression of the inhibitory F_cγ receptor may also be effective.⁹

TABLE 29.3 CHARACTERISTICS OF COMPLEMENT RECEPTORS

Receptor	Characteristics	Complement Ligand	Cellular Distribution	Function
CR1 (CD35)	210-330 kDa Four allotypes Single-chain glycoprotein 30 SCRs	C3b (high affinity) C4b iC3b (weak affinity)	RBCs; neutrophils; monocytes; macrophages; B- and some T-cells, follicular dendritic cells; Langerhans cells; Kupfer cells	Regulates C3, C4, and C5 convertase of classic and alternative pathways of complements; factor 1 cofactor, RBC CR1, phagocyte CR1
CR2 (CD21)	145 kDa; integral membrane glycoprotein 15 SCRs	C3dg, C3d	B-cells; follicular dendritic cells	Immune modulation; cellular receptor for the Epstein-Barr virus
CR3 (CD11b/CD18)	165 kDa (CD11b); 95 kDa (CD18); heterodimer	iC3b	Neutrophils; monocytes; macrophages; NK cells; cytotoxic T-cells	Adherence and phagocytosis of opsonized RBCs
CR4 (CD11c/CD18)	150 kDa (CD11c); 95 kDa (CD18); heterodimer	iC3b	Neutrophils; monocytes; macrophages; NK cells; cytotoxic T-cells	Undefined
RBCs, red blood cells; SCR, short consensus repeat.

TABLE 29.4 CHARACTERISTICS OF F_Cγ RECEPTORS

Receptor	Characteristics	Affinity for IgG	Cellular Distribution	Function
F_cγRI (CD64)	72 kDa; integral membrane glycoprotein IgG	High (1-3 × 10^-8 L/M)	Monocytes; tissue macrophages neutrophils	Binds monomeric IgG interferon-γ stimulated ADCC and resetting of IgG-coated RBCs; not essential for phagocytosis
F_cγRII (CDw32)	40 kDa; integral membrane glycoprotein	Low (2 × 10^-5 L/M)	Monocytes; tissue macrophages; neutrophils platelets; B-cells	Binds aggregated IgG; mediates ADCC; weak mediator of resetting; important for phagocytosis
F_cγRIII (CD16)	50-80 kDa; both integral membrane (F_cγRIIIa) and glycosyl phosphatidylinositol-anchored (F_cγRIIIb) forms	Low (5 × 10^-5 L/M)	Neutrophils (F_cγRIIIb); tissue macrophages; NK cells (F_cγIIIa)	Binds aggregates IgG; important for clearance of IgG-sensitized RBCs
ADCC, antibody-dependent cell-mediated cytotoxicity; NK, natural killer; RBCs, red blood cells.

Laboratory Diagnosis

Two criteria must be met to diagnose AIHA: clinical or laboratory evidence of hemolysis and serologic evidence of an autoantibody.

Common Laboratory Features

The basic tests to evaluate hemolysis include a complete blood count (CBC) with peripheral smear, bilirubin, lactate dehydrogenase (LDH, particularly isoenzyme 1), haptoglobin, and urine hemoglobin or hemosiderin. Universal signs of hemolysis include decreased serum hemoglobin and hematocrit, increased serum LDH and unconjugated bilirubin, and polychromasia and reticulocytosis given adequate hematopoietic reserve. Haptoglobin is typically reduced, although sequential levels should be assessed because the protein is an acute-phase reactant and thereby dependent on both hepatic function and systemic stress. Intravascular hemolysis further manifests with increased free hemoglobinemia, increased hemoglobinuria, hemosiderinuria, methemalbuminemia, and decreased serum haptoglobin. Hemoglobinemia is usually evident in the specimen collection tube as a distinctly red- or pink-tinged serum or plasma. Hemolysis in AIHA can be either intravascular or extravascular. Typically, intravascular hemolysis is rapid and aggressive, whereas extravascular hemolysis is milder. The peripheral blood smear may reveal spherocytes in warm AIHA or RBC agglutination in cold AIHA.

Serologic Investigation

The demonstration of RBC surface-bound immunoglobulin or evidence of complement fixation supports the diagnosis of immunemediated RBC destruction. IgM-coated RBCs may spontaneously agglutinate because the pentameric antibody can directly cross-link RBCs. The capability for IgM antibodies to agglutinate salinesuspended RBCs without additional reagents led to the traditional terminology of “complete” antibodies. In contrast, IgG antibodies typically require antihuman globulin (AHG) as a co-factor to agglutinate saline-suspended RBCs and are thus termed “incomplete” antibodies.

The explanation for this serologic difference lies in the physical properties of the RBCs and the antibody molecules involved. Erythrocytes have a strong net negative surface charge (“zeta potential”) produced by the sialoglycoprotein coat, such that the shortest separation attainable between two RBCs is approximately 18 nm. Other factors may also play a role in maintaining the separation distance, such as water that is tightly bound to the surface of the RBCs. IgM molecules, with their large pentameric structure, create a 30-nm distance between adjacent binding sites and can therefore bridge two RBCs. The smaller IgG can accommodate a span of only 12 nm between antigen-recognition sites and thus usually cannot lead to agglutination alone.²⁰ Exogenous AHG can bridge IgG molecules, which explains the term “incomplete” above. However, some IgG antibodies can indeed agglutinate RBCs (e.g., anti-A, anti-B, and anti-M), revealing the influence on agglutination of the number of antigen sites per RBC²¹ and how far the antigenic determinants project from the surface of the RBC. The blood group-defining A and B oligosaccharides extend well beyond the lipid bilayer membrane.²² In addition to their relative abundance, they are also close together on the RBC surface, potentiating agglutination. The critical number of IgG molecules required to agglutinate RBCs is approximately 7,000 to 20,000 per RBC, whereas the requisite number of IgM molecules is only 25 to 50 per RBC.²³, ²⁴ Although serologic tricks such as enzyme treatment, centrifugation, and addition of substances such as albumin, polyvinylpyrrolidone (PVP), and dextran have been used to enhance agglutination by IgG antibodies, the most common way to determine the presence of immune system components on RBCs is with the direct antiglobulin test (DAT, direct Coombs).

Direct Antiglobulin Test (Direct Coombs Test)

In 1908, Moreschi described antiglobulin reactions as an aid to RBC agglutination.²⁵ This work remained largely unnoticed until 1945, when the investigation of anti-RBC antibodies was revolutionized with the development of the antiglobulin test by Coombs et al.²⁶ and its role in the identification of maternal IgG on fetal RBCs in hemolytic disease of the newborn (HDN) in 1946.²⁷ The principle of the DAT is quite simple. In order to ascertain if RBCs carry surface-bound immunoglobulin and/or complement, antisera with reactivity to human immunoglobulin and/or complement molecules is added to a suspension of the RBCs in question, providing the necessary cross-link to elicit agglutination. Figure 29.2 illustrates the principle of the test.²⁸ An ethylenediaminetetra-acetic acid (EDTA)-collected sample from the patient is used to prevent subsequent complement adherence to the RBC membrane in vitro. The test is performed by first washing the RBCs to remove nonspecifically adhered proteins. Following the addition of AHG, the mixture is centrifuged to enhance agglutination. The result is interpreted by resuspending the RBCs gently and observing carefully for clumps. Magnifying mirrors or lowpower microscopy may aid in discerning weak reactions. Negative reactions are further incubated at room temperature, centrifuged, and read again, as this additional incubation promotes positive reactions in the presence of complement.²⁹ Initial testing is done with polyspecific AHG (antisera), which contain anti-IgG, anti-C3d, and, occasionally, some anti-light-chain activity. Monospecific reagents differentiate between IgG and C3d to further define the specificity. Other monospecific antisera are available for C3b, C4b, C4d, and IgG heavy chain.³⁰ Specific antiserum for IgM or IgA is rarely used, as IgM is not usually found attached to the RBC surface ex vivo, and IgA is very rarely a cause of immunoglobulin coating by itself.³¹, ³² The U.S. Food and Drug Administration (FDA) has licensed monoclonal reagents, the most common of which is anti-C3d, which do not cross-react with other complement components.³⁰

FIGURE 29.2. Direct antiglobulin test (DAT) and indirect antiglobulin test (IAT). The DAT reflects in vivo antibody sensitization of RBCs. Erythrocytes are washed to remove any unbound antibodies, and anti-IgG AHG reagent (AHG: antihuman globulin) is then added. IgG antibodies cannot cause direct RBC agglutination, but IgG-coated RBCs will agglutinate in the presence of AHG containing anti-IgG. This test can also be performed using anticomplement AHG reagent. If it is present, RBC-bound IgG can be eluted for specificity determination. The IAT is used to detect the presence of IgG antibodies in serum (in vitro sensitization). Reagent RBCs are incubated in the presence of serum that may contain antibodies. If they are present, antibodies bind to their target antigens on the reagent RBCs. After incubation, the RBCs are washed to remove unbound antibodies. Anti-IgG AHG reagent is added and will cause IgG-coated erythrocytes to agglutinate. (From Zarandona JM, Yazer MH. Teaching case report. The role of the Coombs test in evaluating hemolysis in adults. Can Med Assoc J 2006;174:305-307.)

Early DAT methodology suffered from relative insensitivity. More recent developments have improved the sensitivity for
RBC-bound immunoglobulin and complement. The traditional tube method described above detects a lower limit of 150 to 200 IgG molecules per RBC.³³ With PVP enhancement and an autoanalyzer, the detection limit decreases to as few as 8 IgG per RBC producing 5% agglutination. If bromelin is added as well, the sensitivity increases even further, with 1 IgG per RBC producing 5% agglutination and 3 IgG per RBC producing 50% agglutination.³⁴ Additional acceptable techniques include flow cytometry, enzymelinked antiglobulin tests (ELISA), radioimmunoassays (RIA) using ¹²⁵I-labeled anti-IgG or staphylococcal protein A, solid-phase techniques using microtiter plates, and gel testing.³⁵ However, only the tube test, solid-phase test, and gel tests are in common use. Of these, the solid-phase and gel tests are the most standardized and have largely replaced older tube testing technology.

A positive DAT does not always mean decreased RBC survival. Most patients with a positive DAT have no obvious clinical signs of hemolytic anemia. DAT interpretation must consider the context of clinical history and other laboratory findings. Outside of AIHA, a positive DAT may also be seen with (i) alloantibodies in a recipient of RBC or plasma transfusion, (ii) antibodies from maternal circulation that cross the placenta and coat the fetal RBCs, (iii) antibodies directed against certain drugs that bind to the RBC membrane (e.g., penicillin), (iv) nonspecifically adsorbed proteins including immunoglobulins and Wharton’s jelly, (v) RBC-bound complement, and (vi) antibodies produced by passenger lymphocytes in transplanted organs or hematopoietic components. Further evaluation of a positive DAT in a patient with clinical and laboratory evidence of hemolysis includes testing for clinically significant antibodies to RBC antigens and testing the eluate. Figure 29.3 illustrates an approach to the evaluation of a positive DAT.²⁹ A negative DAT, on the other hand, does not exclude AIHA. Possible causes of a negative DAT with clinical evidence of hemolysis include an IgA or IgM autoantibody, insufficient antibody molecules for detection, and low-affinity autoantibodies.³⁵ Repeat testing, enhancement techniques, and the use of nonroutine reagents may be required when the clinical suspicion is strong.²⁹

FIGURE 29.3. Flowchart suggesting a rational clinical laboratory approach for investigating a positive DAT. AIHA: autoimmune hemolytic anemia; CAD: cold agglutinin disease; DI-IHA: drug-induced immune hemolytic anemia; HTR: hemolytic transfusion reaction; PCH: paroxysmal cold hemoglobinuria. (Modified from Brecher ME, ed. Technical manual, 15th ed. Bethesda, MD: American Association of Blood Banks, 2005:480.)

Indirect Antiglobulin Test (Indirect Coombs Test)

Approximately 80% of patients with AIHA have autoantibodies in their serum as well as on their RBCs.³⁶ The antibodies in their serum or plasma and the antibodies eluted from their RBCs (the “eluate”) are detected by the indirect antiglobulin test (IAT; more commonly known as the antibody screen). Unlike the DAT, which uses patient RBCs with reagent serum, the IAT tests patient serum against reagent RBCs. Immunoglobulin from the patient serum attaches to the reagent RBCs and is detected with antiglobulin sera, which cross-link the RBCs together and produce agglutination, as in the DAT. See Figure 29.2.²⁸

Warm autoantibodies are typically panagglutinins, reacting with all cells in the diagnostic RBC panel (i.e., panreactive). Formerly, panagglutinins were believed to react with a basic determinant of the Rh antigen system as they do not react with the very rare Rh_null RBCs which do not express Rh antigens.³⁷ However, as more eluates are studied with a broader population of rare null phenotypes, it has been shown that, in addition to Rh, autoantibodies react to LW antigens, glycophorins A, B, C, and D, and, very rarely, Kidd or Kell blood group system antigens.³⁸ A small number of case reports exist describing AIHA associated with ABO antigens.³⁹, ⁴⁰

Elution

If the DAT is positive for RBC-bound antibody, that antibody can be eluted (removed) from the RBC with the aid of acid or xylene, and any binding specificities can be further investigated with a reagent red cell panel. Elutions are not typically performed on DAT specimens positive only for complement, as these molecules do not exhibit antigen specificity. Occasionally, however, when antibody presence is suspected but perhaps at too low a concentration to be detected on the RBCs, eluates may show reactivity. Elution tends to produce a more concentrated antibody solution, so reactions are often stronger. Once the antibody is in solution, indirect antiglobulin techniques may help define the antibody characteristics.²⁹

Other Serologic Techniques

Generally, autoantibodies are panreactive, whereas alloantibodies exhibit antigen specificity, reacting only with specific antigenpositive RBCs. In certain situations an autoantibody may mimic an alloantibody. For example, an alloantibody to a high-incidence antigen in a post-transfusion setting can mimic an autoantibody with a positive DAT (mixed field) and reactions with all panel cells. In addition, autoantibodies may exhibit apparent specificity. Autoadsorption and antigenic phenotyping can help differentiate 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 specificity, demonstration that autologous RBCs are negative for the corresponding antigen confirms the 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 who have not been recently transfused.²⁹

IMMUNE HEMOLYTIC ANEMIAS CAUSED BY COLD-ACTIVE ANTIBODIES

Cold-active antibodies exhibit greater titer and RBC-binding activity as the temperature decreases toward 0°C. Two different clinical syndromes are manifested from cold autoimmune antibodies. Cold agglutinin disease (CAD) is associated with IgM antibodies usually directed at the RBC I antigen. CAD typically occurs in adult patients and may be primary or secondary to another disease process, usually infectious. In contrast, PCH is caused by an IgG hemolysin, the Donath-Landsteiner (D-L) antibody.⁴¹ Both PCH and CAD are less common than warm AIHA and make up approximately 20% or less of AIHAs. See Tables 29.5 and 29.6.

Cold Agglutinin Disease

Although Landsteiner first described cold agglutinins in 1903,⁴² it was not until the late 1940s and early 1950s that the connection between cold autoantibodies and RBC destruction was firmly made. In the 1950s, Schubothe coined the term “CAD,” and the disorder became recognized as a separate entity from other acquired hemolytic processes.⁴³ The responsible pathologic IgM antibodies are distinguished from naturally occurring cold autoantibodies by their titer and thermal amplitude, a term describing the range of temperatures over which the antibody is reactive. Natural cold autoantibodies occur with titers less than 1:64 at 4°C and have little to no activity at higher temperatures. However, pathologic cold agglutinins typically have titers well over 1:512 and may react at 28°C to 31°C (peripheral body temperature) or even up to 37°C.³¹ See Table 29.6 and Figure 29.4.

TABLE 29.5 COLD AUTOANTIBODIES

	Primary Cold Agglutinin Disease	Secondary Cold Autoantibodies	Paroxysmal Cold Hemoglobinuria
Immunoglobulin	IgM	IgM	IgG
Clonality	Monoclonal	Monoclonal or polyclonal	Polyclonal
Direct antiglobulin test	C3	C3	C3
Hemolysis	Chronic, mild	Self-limited, mild to severe	Episodic, self-limited, mild to severe
Target RBC antigen	I	I, i	P
RBC, red blood cell.

Primary versus Secondary Cold Agglutinin Disease

Primary or idiopathic CAD is typically an affliction of older adults, with a peak incidence around age 70.⁴³ Both sexes are affected, but women predominate.⁴⁴ A monoclonal IgMk antibody is the usual culprit, and, as with other monoclonal gammopathies of unknown significance, may be a harbinger of future B-cell neoplasms. Most commonly, patients tolerate a relatively benign, waxing and waning hemolytic anemia.

Patients with Waldenstrom macroglobulinemia or other B-cell neoplasms may produce monoclonal anti-RBC antibodies with cold reactivity. As in primary CAD, they are nearly always IgMκ. This type of secondary cold agglutinin may be effectively treated with antineoplastic chemotherapy. A return of hemolysis may herald a tumor relapse. Other non-B-cell tumors reported in association with cold antibody production include squamous cell carcinoma of the lung, metastatic adrenal adenocarcinoma, metastatic adenocarcinoma of the colon, basal cell carcinoma, and a mixed parotid tumor.⁴⁵

Another scenario of secondary cold autoantibody hemolysis occurs after Mycoplasma pneumoniae infection or infectious mononucleosis and is more commonly seen in younger adults. This transient, self-limited process is mediated by polyclonal IgM (κ or λ), lasts a few weeks, and seldom requires more than supportive care. In rare cases, massive intravascular hemolysis and acute renal failure may be seen. See Table 29.7.

Antibody Characteristics

Immunochemistry and Origin

As stated earlier, nearly all cold agglutinins are IgM. A few reports of IgG or IgA agglutinins are recorded, and a mixed IgM-IgG has been seen in infectious mononucleosis and angioimmunoblastic lymphadenopathy.⁴⁶ Those patients with non-IgM antibodies are more likely to have cold autoantibody hemolysis secondary to another disease and are less likely to display specificity for the I antigen.⁴⁴, ⁴⁷

TABLE 29.6 SEROLOGIC OVERVIEW OF HEMOLYTIC ANEMIAS

	Cold Agglutinin Disease	Paroxysmal Cold Hemoglobinuria	Mixed Warm and Cold Autoimmune Hemolytic Anemia	Warm Autoimmune Hemolytic Anemia	Drug-Induced Hemolytic Anemia
Percentage of cases	16-32%	32% (children); rare in adults	7-8%	40-70%	12-18%
Direct antiglobulin test	C3	C3	IgG ± complement	IgG ± C3; rarely C3 alone	IgG or C3; occasionally IgG ± C3
Ig	IgM	IgG	IgG, IgM	IgG, occasionally with IgA or IgM	IgG
RBC eluate	Nonreactive	Nonreactive	IgG	IgG	IgG or nonreactive
Antibody specificity	I > i >> Pr	P	Panreactive	Panagglutinin	Rh-related
			Unclear > I> others	Rarely Rh	Drug-dependent
RBC, red blood cell.

Anti-idiotypic antibodies and direct nucleotide sequencing of the rearranged immunoglobulin variable-region genes have revealed significant cross-reactivity and homologies among cold autoantibodies with similar specificity.⁴⁸, ⁴⁹ For instance, the monoclonal anti-idiotypic antibody 9G4 recognizes an idiotypic determinant present on the heavy chains of both anti-I and anti-i cold agglutinins as well as the responsible neoplastic B-cells.⁵⁰ Essentially all pathologic anti-I and anti-i cold agglutinins are derived from a distinct subset of heavy-chain variable-region genes called V_H4 family genes, specifically V_H4-21.⁵¹ In ˜40% of the patients, a circulating B-cell clone can be identified with a distinctive karyotypic marker (trisomy 3q11-q29; trisomy 12; or 48XX+3+12). The chromosomal abnormalities were associated with chronic idiopathic cold agglutinin syndrome as well as with monoclonal cold agglutinins secondary to a neoplasm.⁵², ⁵³, ⁵⁴ In addition, the cold agglutinins have the same serologic specificity and isoelectric focusing spectrotype and are therefore likely derived from a pre-neoplastic or neoplastic B-cell clone.⁷

I/i Blood Group System Specificity

More than 90% of cold-active antibodies have the I antigen as their target on the RBC, and the i antigen is the binding site for a significant portion of the remaining 10%.³¹ The closely related I/i antigens are high-frequency carbohydrates similar to the ABO antigens. The RBC surface densities of I and i are inversely proportional, with neonatal RBCs exclusively expressing large amounts of i antigen, usually converting to exclusively I antigen by 18 months of age. Consequently, adult RBCs are used to detect anti-I agglutinins and cord RBCs are needed to detect anti-i agglutinins. Extremely rare adults have been described who never express I antigen on the RBCs. Other uncommon but reported antigen
targets include Pr. Anti-Pr cold agglutinins tend to be high-titer, with a wide thermal range, and cause symptomatic anemia.⁵⁵, ⁵⁶ Other infrequent targets are Gd, Fl, Vo, Li, Sa, Lud, M, N, Me, Om, D, Sd^x, and P.³⁸, ⁴⁴, ⁵⁷ The fact that M. pneumoniae induces anti-I antibodies in the majority of patients is potentially related to the finding that sialylated I/i antigens serve as specific Mycoplasma receptors.⁵⁸ Minor modification of this antigen may incite autoantibodies. Another theory suggests that an I-like antigen appears on the organism itself, and cross-reacting antibodies lead to RBC lysis.⁵⁹ Despite the high rate of antibody production, clinically significant hemolysis occurs in very few patients.⁶⁰ Infectious mononucleosis is also associated with CAD, but to a much lesser degree than Mycoplasma. Only 0.1% to 3.0% of mononucleosis patients have clinical hemolysis,⁶¹ although anti-i is present in 8% to 69% of sera post-infection.⁶², ⁶³, ⁶⁴ Therefore, the majority of patients with antibodies are asymptomatic. Anti-I activity is usually noted as well, but not to the same degree. Also, anti-Pr and anti-N have been reported.⁶⁵ Both IgM and IgG antibodies as well as IgM rheumatoid-like factors reacting with IgG may act as cold agglutinins after infectious mononucleosis.⁶⁶, ⁶⁷ See Table 29.7 for a list of other infectious diseases associated with cold agglutinins, most of which are anti-I, although anti-i has been seen in cytomegalovirus infections and in lymphomas.⁶⁸

FIGURE 29.4. Temperature ranges for cold agglutinin fixation and lytic complement action. (From Schubothe H. The cold hemagglutinin disease. Semin Hematol 1966;3:27-47, with permission.)

TABLE 29.7 SECONDARY COLD AGGLUTININ DISEASE

Neoplasms
	Waldenstrom macroglobulinemia
	Angioimmunoblastic lymphoma
	Other lymphomas
	Chronic lymphocytic leukemia
	Kaposi sarcoma
	Myeloma
	Nonhematologic malignancy (rare)
Infections
	Mycoplasma pneumoniae
	Mononucleosis (Epstein-Barr virus)
	Adenovirus
	Cytomegalovirus
	Encephalitis
	Influenza viruses
	Rubella
	Varicella
	Human immunodeficiency virus
	Mumps
	Ornithosis
	Legionnaires’ disease
	Escherichia coli
	Subacute bacterial endocarditis
	Listeriosis
	Syphilis
	Trypanosomiasis
	Malaria
	Other
	Autoimmune diseases
	Tropical eosinophilia

Functional Characteristics

Cold agglutinins attach to the RBC in the cooler peripheral circulation. As the blood returns to the warmer core circulation, the antibody dissociates from the RBC. Antibodies that attach, fix complement, and then dissociate are free to attack another erythrocyte and begin the process again.⁶⁹ Complement fixation and activation, which are responsible for the destruction of the RBCs, are far more efficient at the warmer core temperatures. However, with a high antibody titer and a wide thermal amplitude, there may be sufficient temperature overlap to produce hemolysis at 22±10°C.⁴³ See Figure 29.4. Because of this diversity of temperature requirements for optimal activity of the antibody and complement, RBC destruction is usually not particularly severe with cold autoantibodies. Quite impressive exceptions occur, and these are typically the antibodies with either high titers (>1:1,000) or activity up to 37°C even in the face of modest titers. Thermal amplitude is a better predictor of hemolysis than titer.⁴⁶, ⁷⁰ High-titer cold agglutinins with a narrow thermal amplitude may produce a clinical picture with bursts of hemolysis associated with exposure to cold, often manifested as intermittent hemoglobinuria between quiescent periods.⁶⁹

A frequent misconception about cold agglutinins is the assumption that they are cryoglobulins, whereas in fact they are two distinct disease processes. Both may cause cyanosis and Raynaud phenomenon in cooler temperatures. However, cryoglobulins do not fix complement on the RBCs or lead to hemolysis.

Clinical Manifestations

Mild, chronic hemolytic anemia with exacerbations in the winter is the general rule for CAD. Rarely does the hemoglobin drop below 7 g/dl.⁷¹ Pallor and jaundice may occur if the rate of hemolysis is greater than the endogenous capability to metabolize bilirubin.⁴⁴ Some patients have intermittent bursts of hemolysis associated with hemoglobinemia and hemoglobinuria on exposure to cold and may be forced to move to warmer climates to minimize attacks. Acrocyanosis can occur from agglutination of RBCs in the cooler vessels of the hands, ears, nose, and feet.³¹, ⁴⁴ Digits may become cold, stiff, painful, or numb and may turn purplish. Limbs may manifest livedo reticularis, a mottled appearance that is readily reversible upon warming of the affected area. Only rarely does actual gangrene of digits develop, and nearly all of these cases have an associated cryoglobulin.⁷² A minority of chronic CAD patients have mild splenomegaly or hepatomegaly. The spleen may be enlarged or more frequently palpable in secondary cold agglutinins due to lymphoma or infectious mononucleosis.⁴⁴

If hemolysis does occur after Mycoplasma infections, it typically begins during the post-pneumonia recovery period when cold autoantibody titers are at peaking. The process, even if severe, resolves spontaneously within 1 to 3 weeks.³¹ Hemolytic anemia after infectious mononucleosis may begin with the onset of illness or within the next 3 weeks.⁴⁴ The self-limited, post-infectious CAD tends to affect younger patients, whereas the chronic idiopathic form is a disease of the elderly, with peak incidence at ˜age 70 years.⁴³

Laboratory Features

Mild chronic anemia is the rule, but the hemoglobin may fall to 5 to 6 g/dl, especially in the winter months in cold climates. The peripheral smear, if not obtained from a carefully collected prewarmed specimen properly maintained warm until spread on a warm slide, may show significant agglutination and RBC clumping under magnification. Occasionally, clumping is so extensive as to be grossly visible without magnification and may even preclude an adequate smear examination. Agglutinates are frequently visible in the specimen tube and can appear to be a large clot. Dissolution with warming demonstrates that the clumped and clotted appearance is a result of a cold agglutinin rather than Rouleaux formation or fibrin strands. Often, the first suspicion of a cold agglutinin comes from a failed attempt to obtain a valid RBC count and indices on an automated CBC. The initially reported RBC count is often artifactually low and the MCV artifactually high, producing a spuriously high MCHC. The reticulocyte count is modestly elevated except in rare cases of concomitant marrow failure, such as those due to parvovirus B19 infection.⁷³ Spherocytosis is not pronounced as in warm AIHA. WBC and platelet counts are usually normal, but low levels of both have been reported, as has leukocytosis.⁴⁴ Bilirubin is mildly elevated, rarely >3 mg/dl. LDH may be increased (reflecting RBC destruction), and complement and haptoglobin are often low or absent. During brisk hemolysis, hemoglobinuria and hemoglobinemia are manifest. The DAT is positive with polyspecific and anticomplement antisera. As above, IgM has dissociated and is not detectable. In extremely rare cases, the antibody involved is IgG or IgA, either alone or in addition to IgM.³¹, ⁴⁴ Mixed warm and cold autoantibodies are not rare (discussed later). Titers measured at 4°C may range from 1:1,000 to 1:1,000,000, although typical values are between 1:1,000 and 1:500,000. Much lower levels can be clinically significant if activity is measurable at 37°C. Post-infectious CAD titers are lower (<1:4,000) than the chronic idiopathic or lymphoma-associated varieties. In patients with monoclonal IgM, evaluation of serum proteins frequently reveals an M spike, shown by serial observations to be stable in the chronic idiopathic disease.⁷⁴

Management

Primary Cold Agglutinin Disease

Because of the mild chronic nature of the anemia, the majority of patients need no specific therapy other than the general principle of avoiding temperatures below those at which their antibody shows activity. In some cases this may necessitate a move to a warmer climate. For patients with more severe anemia and cardiovascular compromise, aggressive therapy is indicated.

Immunosuppressive Therapy: The ideal therapy would only suppress production of the pathologic antibody. Until that becomes an option, a common approach in treating symptomatic CAD is to use cyclophosphamide or chlorambucil. A minority of patients respond, and in these cases transfusions can be avoided. One alternative is chlorambucil, beginning with 2 to 4 mg/day and increasing by 2 mg every 2 months until either a response is
obtained or unacceptable myelotoxicity results. Twice-weekly blood counts plus reticulocyte count should be monitored for toxicity.³¹, ⁷⁵ Pulse therapy with cyclophosphamide (250 mg/day) and prednisone (100 mg/day ×4 days) every 2 to 3 weeks, blood counts permitting, or a large dose regimen of cyclophosphamide (1,000 mg), and intravenous methylprednisolone (500 mg) may adequately control hemolysis.⁷⁶ In addition, α-interferon has reportedly produced significant remissions.⁷⁷ However, recent studies have shown that rituximab is the most effective form of treatment and can be used as first line.

Rituximab: Rituximab, a chimeric human/murine monoclonal CD20 antibody, has been used with success in CAD in several case reports and series. The proposed mechanism of action involves complement-dependent cytotoxicity, antibody-dependent cellular cytotoxicity, direct apoptotic effects, and inhibition of B-cell proliferation. The most effective and best-evaluated treatment is rituximab in standard lymphoma dose (375 mg/m² each week for 4 weeks). Berentsen et al., in an open, uncontrolled prospective phase 2 study of rituximab in CAD showed that 20 of 27 patients responded with a median duration of response of 11 months.⁷⁸ Most relapsed patients responded to retreatment with rituximab. Similar results were obtained by other studies.⁷⁹ Some pediatric patients were successful with just two infusions.⁸⁰ This evidence suggests that rituximab can be used as first-line treatment in symptomatic CAD.

Plasmapheresis: Given the predominantly intravascular distribution of IgM, a logical conclusion would be that plasmapheresis or plasma exchange should provide rapid relief from hemolysis due to cold autoantibodies. Unfortunately, the results have been somewhat disappointing.⁸¹, ⁸² Simply removing the circulating antibody does not diminish ongoing endogenous antibody production, so improvements are transient at best. In patients with chronic CAD, plasmapheresis should probably be combined with immunosuppressive therapy in an attempt to decrease antibody production.⁸³ Successful use of plasmapheresis to temporarily decrease cold agglutinin titers in order to permit safe coronary artery bypass surgery has been reported.⁸⁴ Other reports of successful cardiac surgery involving warm cardioplegia have circumvented the need for cold exposure and thus the risk of hemolysis.⁸⁵, ⁸⁶ Cryofiltration apheresis has been used for acute exacerbations and for surgical procedures requiring hypothermia, such as coronary artery bypass surgery with cardioplegia. In one small study, two of five such patients who received cryofiltration apheresis had a favorable response with a reduction in titer.⁸⁷

Blood Transfusion: Although most patients with CAD have mild anemia and do not need transfusion, RBC support may be required in patients who are clinically symptomatic or severely anemic.88 Patients with high-titer or wide-thermal-amplitude antibodies can pose extremely difficult serologic problems for the blood bank laboratory. Testing needs to be performed carefully at 37°C to minimize the effects of the cold agglutinin so that a search for alloantibodies may be properly performed.³¹ This still does not eliminate interference from some particularly pesky autoantibodies. Time-consuming and technically challenging cold autoadsorptions may be necessary to rule out the presence of underlying alloantibodies. Occasionally, incompatible units may need to be issued because of residual agglutination from the cold autoantibody. Most cold agglutinins are directed at the I antigen, which is present on nearly all adult donor RBCs. Locating i adult RBCs (i.e., I-negative) is not practical because of their extreme rarity. Reports have documented i adult RBCs effectiveness and lack thereof.⁸⁹, ⁹⁰ If transfusion is necessary to treat significant cardiovascular compromise, RBC infusion through an in-line blood warmer at 37°C is recommended.⁹¹ Uncontrolled heating of RBC products is quite dangerous because of the damaging effects of excessive heat and should be avoided. If blood warmers are not available, transfusion may still be accomplished by the slow infusion of room temperature RBCs into a large vein while keeping the patient warm.³¹, ⁴⁴

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