Hemolysis is initiated when an autoantibody binds to the one or several RBC membrane antigens and promotes antibody dependent, cell-mediated cytotoxicity, and/or recruits and activates complement. Destruction of the RBC can occur directly in the circulation (intravascular hemolysis) and/or by removal of the cell by macrophages in the spleen, liver, or both (extravascular hemolysis) (Fig. 1). Several immunoglobulin (Ig) subclasses can fix complement and activate the classical complement (Ct) path way: IgG3 activates Ct more efficiently than does IgG1, whereas IgM, which is a potent Ct activator, is usually eluted ex vivo and therefore not found on RBC membrane by the direct antiglobulin test (DAT). Macrophages recognize opsonized erythrocytes via receptors specific for the Fc fragment of IgG, namely Fcγ receptors (FcγRs) and for C3d. RBCs coated with IgG or complement alone are destroyed in the spleen and liver, IgG-coated cells in the spleen, and IgM-coated cells in the liver. This has major implications for treatment, particularly for the effect of steroids and splenectomy.
Fig1. MECHANISM OF EXTRAVASCULAR HEMOLYSIS IN AUTOIMMUNE HEMOLYTIC ANEMIA. (A) Macrophage encounters an IgG coated erythrocyte and binds to it via its Fc receptors. Thus entrapped, the red blood cell (RBC) loses bits of its membrane as a result of digestion by the macro phage’s ectoenzymes. The discoid erythrocyte transforms into a sphere. (B) RBCs lightly coated with IgG (and therefore incapable of activating the complement cascade) are preferentially removed in the sluggish circulation of the spleen. (C) RBCs with a heavy coat of IgG; thus, C3b (black circles) can be removed both by the spleen and the liver. (Courtesy Cunningham MJ, Silberstein LE. Autoimmune hemolytic anemia. In: Hoffman R, Benz EJ Jr., Shattil SJ, et al., eds. Hematology: Basic Principles and Practice. 4th ed. Philadelphia, PA: Elsevier; 2005.)
Warm Antibody Hemolytic Anemia
wAIHAs represent 60% to 80% of all AIHAs. The RBC anti bodies in wAIHAs are mostly polyclonal IgG1 or IgG3, which are able to activate the complement system (Table 1). The DAT in wAIHAs is positive either with IgG (37%) or IgG + C3d (43%). Rarely, the DAT is only positive with C3d (when the amount of IgG on the RBCs is very small or in case of “warm,” IgM autoantibody). Patients with IgG antibodies may have also IgA antibodies, but IgA antibodies without IgG antibodies are a very rare cause of wAIHAs. wAIHAs are often directed against Rh antigens, but also against other RBC’s membrane antigens (non-Rh–related autoantibodies) such as band 3 protein or glycophorin A. The antibodies fix complement and bind tightly to the RBCs at 37°C. Therefore, only a small amount of antibody is detectable in the serum. The antibody-coated RBCs are removed from the circulation by splenic (to a lesser degree also by hepatic) macrophages via FcγRIII receptors. IgG3 and IgG1 have the highest affinity for the Fc receptors of macrophages. Erythrocytes that are only partially phagocytosed by macrophages become spherocytes, which are removed in the splenic cords because of their rigid structure and can be easily detected on peripheral blood smear. Destruction of RBCs may also be caused by other mechanisms such as antibody-dependent cellular cytotoxicity as well as complement-dependent cytotoxicity when complement activation is involved.
Table1. Properties and Specificities of Red Blood Cell Autoantibodies
IgM wAIHAs are a very rare cause of AIHA. This type of AIHA can be suspected if RBC autoagglutination occurs at room temperature. The DAT result is positive with C3d alone (65%) or with IgG (24%), there are no detectable cold agglutinins (CA) in the serum. Using sensitive methods, IgM on the RBCs can be detected in 71%.19 Non-Hodgkin lymphoma (NHL) is the underlying disease in some of these cases. This AIHA is often life-threatening and refractory to steroids and splenectomy.
Cold Antibody Hemolytic Anemia
Cold antibodies in primary or secondary chronic forms of cAIHA (known as cold agglutinin disease or CAD) are usually monoclonal IgM. The IgM has two binding sites for C1q and fixes complement easily. The targets are polysaccharides (I, IT, I, or Pr antigens). The “i” antigen is a nonbranched polysaccharide in the cord blood and the “I” antigen is a similar but a branched molecule expressed in the RBCs of adults. Cold antibodies, more commonly known as “CAs,” bind to the RBCs at low temperatures and cause their lysis at temperatures above 22°C. The DAT is typically positive with C3d alone. When CAs are present at high titers, they may activate the complement system directly, produce a membrane attack complex, and produce intra vascular hemolysis with hemoglobinuria. Usually complement-coated RBCs are sequestered by liver macrophages.
Some forms of transient cAIHA triggered by an infection may rarely occur, they can classically be associated with a Mycoplasma pneumoniae infection or yet with some viral infections (mostly EBV or CMV). The onset is usually abrupt and the degree of anemia may be severe with transfusion requirement, but the outcome is most often good within few weeks.
Lastly, paroxysmal cold hemoglobinuria (PCH) is a very rare form of AIHA caused by the DL antibody. This is a rare, usually poly clonal IgG cold antibody to P antigen (glycosphingolipid globoside), which binds to the RBCs at 4°C. The cells are lysed at higher temperatures. The DAT is positive with C3d, no CAs are present, and the diagnosis can only be ascertained by means of the DL test in a specialized lab. In this test, normal RBCs and patient and normal serum are incubated at 4°C. Agglutination occurs after warming to 37°C. The prominent clinical feature of PCH is a brisk, immediate but sometimes also delayed hemoglobinuria after cold exposure even in patients with low antibody titers. In the past, it has been associated with viral infections in children and secondary or tertiary syphilis in adults. Now, two types can be distinguished clinically: (1) an acute, severe form (often associated with hemoglobinuria) but self-limiting AIHA after (respiratory) infections in children, and (2) a rare, chronic AIHA in nonsyphilitic persons with various underlying conditions, including NHL. Patients with chronic PCH respond poorly to steroids and splenectomy.
Mixed Warm and Cold Autoimmune Hemolytic Anemia
A small number of patients have an AIHA mixed with a positive DAT for both IgG and C3d, indicating coexistence of warm IgG autoantibodies and high-titer CAs (titer >1/64). Mixed AIHAs have a severe course of disease and lower median hemoglobin (Hb) values (5.8 g/dL) that frequently require multiple therapy.
Atypical Autoimmune Hemolytic Anemia
The term characterizes DAT-negative, IgA-driven, or warm-IgM forms. They represent a diagnostic challenge, resulting in delayed treatment.
