Discuss the Relevance of IgM and IgG Antibodies in Transfusion Science IgM and IgG are two isotypes

of immunoglobulins. IgM molecules are in the form of a pentamer with 10 antigen binding sites. IgG molecules are flexible, and sub-classes have different properties and effector functions. Naturally occurring antibodies are predominantly IgM, whereas immune antibodies are predominantly IgG. All antibodies exert their biological effects by binding to the appropriate red blood cell antigens. An antibody is said to be clinically significant when it has the potential to initiate accelerated destruction of red cells carrying the appropriate antigen. Therefore, alloantibodies, whether naturally occurring or immune or autoantibodies are clinically important. IgM and IgG are of importance in certain clinical situations; haemolytic transfusion reaction (IgM and IgG), haemolytic disease of the newborn (IgG) and haemolytic anaemia (IgG and IgM). Antibodies may be naturally occurring or immune in nature. The term naturally occurring is used for blood group antibodies produced in individuals who have never been transfused with red cells carrying the relevant antigen or been pregnant with a foetus carrying the relevant antigen, and are predominantly IgM. Explanations for the existence of these antibodies include the possibility that some cells may be capable of making specific antibody in the absence of antigenic stimulus or that antibodies, such as anti-A and anti-B are produced as an immune response to glycoproteins on the surface of gut bacteria. Examples of IgM red blood cell antibodies are those against the ABO system and the Lewis system. Naturally occurring antibodies are only clinically important if they are reactive at 37C. Immune antibodies are those produced in response to a foreign antigen as a result of blood transfusion or pregnancy, and are predominantly IgG. There is a much greater variety of IgG antibodies, for example antiD, auto anti-Pi, anti-Duffy (anti-Jka and anti-Jkb) anti-Kidd (anti-Jka and anti-Jkb, anti-Kell (anti-K, anti-k, antiKpa, anti-Jsa etc). Complement has a role in both IgM and IgG mediated immune response to red blood cell antigens. The classical pathway of complement can be initiated through the binding of two adjacent antigen sites by IgM or IgG (only IgG1, IgG2, and IgG3). The IgM-antigen complex is particularly efficient at binding the first component of complement due to the 10 antigen binding sites, thereby activating the complement cascade, which leads to red blood cell lysis. Two molecules of IgG must be close enough together on the red cell membrane to form a doublet before they can activate the complement cascade. There are differences in the ability of IgG subtypes to activate complement. For example the longer hinge region of IgG3 contributes to the flexibility of the molecule and makes it more efficient at complement-fixing. Red cell alloantibodies are predominantly IgG1 and IgG3. Haemolytic transfusion reaction may result from intravascular haemolysis or extravascular haemolysis. Intravascular red cell destruction presents as haemoglobinuria and anaemia due to the haemolysis of the transfused cells. High titre IgM anti-A and -B are most often implicated, and cause immediate haemolysis due to the high concentration of A and/or B antigens on red blood cells. When red blood cells are coated with IgM, antigen-antibody complexes are formed which activate the first component of complement (C1). With sufficient antibody density, complement activation may be robust enough to overwhelm the inhibitory activity of the complement regulatory proteins DAF and MIRL on the red blood cell surface. Defects occur in the red cell membrane if the activation proceeds sequentially through to the C5b-C9 insertion of the MAC complex. The membrane defects allow ions to enter the cell, which eventually swells and ruptures, causing haemolysis.

When IgM is present in sub-lytic amounts DAF and MIRL are able to prevent direct RBC lysis, however some C3b is deposited on the red blood cell membrane as a consequence of the IgM-induced complement activation, and interaction of C3b and its ligand iC3b and specific complement receptors on liver macrophages (Kupffer cells) cause the immune destruction of red blood cells. Although ligation of RBCbound C3b to complement receptor 1 on Kupffer cells may mediate some of the clearance, interaction between iC3b and macrophage complement receptor 3 is probably the main mediator of extravascular red cell destruction. Extravascular red cell destruction is usually associated with IgG antibodies, which fail to activate complement or fix complement only up to the C3 stage of the cascade. Consequently, direct complemented-mediated lysis of RBCs induced by IgG is unusual. Extravascular red cell destruction also presents as haemoglobinuria and anaemia due to the breakdown of transfused cells. IgG anti-D and other Rh antibodies are most often implicated. If the antibody does not fix complement, such as virtually all IgG anti-D and some IgG anti-K, -S, and -Fya, the cells are mainly destroyed in the spleen. In the absence of complement activation, clearance of IgG-sensitized cells primarily occurs in the spleen. Two processes seem to be involved. First red cells coated with IgG1 and/or IgG3 adhere at the hinge region of the antibody molecule to Fc receptors on tissue macrophages and this can mediate direct and complete phagocytosis. Second, partial phagocytosis of the red cell membrane results in a reduction of surface area to volume ratio, and the remainder of the red cell circulates as a spherocyte, which is a classical feature of an immune haemolytic anaemia. Free IgG in the circulation prevents significant destruction at other sites because the circulating IgG molecules compete for the binding sites on macrophages. Cells heavily coated with IgG1 and/or IgG3 antibodies usually trigger destruction by the antibody-dependent cytotoxicity mechanism. Lysosomal enzymes released by cells such as NK cells cause the destruction; however, both phagocytosis and cytotoxic mechanisms can occur at the same time. The liver clears IgG-coated RBCs less efficiently than the spleen, but there is a rough correlation between the amount of antibody fixed to the RBC membrane and the site of destruction; smaller amounts lead to splenic phagocytosis, and larger amounts to liver phagocytosis. The cornerstone of antenatal care is regular testing for antibodies of potential importance and monitoring an alloimmunised pregnancy accordingly. IgM antibodies, whilst efficient at provoking complement activation and so responsible for most cases of transfusion-related intravascular red cell destruction, do not cross the placenta and are therefore not implicated in HDFN. IgG is able to cross the placenta due to the presence of Fc receptors on the cell membrane. The most severe manifestation of HDFN is caused by IgG antibodies directed against D, c, or K antigens on the foetal red cells. Foetal cells sensitised by IgG1 and/or IgG3 antibody are destroyed in the foetal spleen. In autoimmune HA autoantibodies react with all normal red cells, including the patients own. Warm autoantibodies are responsible for 48-70% of AIHA cases. The incidence of AIHA has been reported to be in the range of 1 per 50,000 to 1 per 75,000 which rises with age. Most antibodies are IgG with preponderance of IgG1, and to a lesser extent IgG3. The sub-types vary in their efficiency at causing haemolysis because of the higher affinities of the macrophage Fc receptors for IgG1 and IgG3. Various combinations of IgG subclasses may occur together, though it is uncommon for IgA or IgM to occur as well as IgG. Warm autoantibodies are panagglutinins, which means that they react with all the RBCs in the diagnostic panel. Of the reported specificities, Rh is by far the most common (70%), including all but the Rh null RBCs. Occasionally an autoantibody is detected which have specificity for a particular Rh antigen such as e, but it is extremely rare for antibodies of true specificity to occur in the absence of broader specificity. Therefore,

there is little to be gained by transfusing antigen-negative blood to these patients. Other blood group specificities that have been reported include Wra, Fyb, Gerbich, JKa, K, Lutheran, LW, M, N, S, Pr, U. In cold antibody HA, the autoantibody tends to be IgM and can activate complement. The peripheral blood sample generally shows the presence of C3d on the red cells. Paroxysmal cold haemoglobinuria (PCH) is most often associated with post-viral infection in children. The antibody is a biphasic haemolysin, IgG in nature and with P specificity. Atypical HA are known to occur, for example, combining warm and cold antibody involvement and HA in which the DAT is negative. In conclusion, both IgM and IgG have great importance in transfusion. IgM mediates the acute haemolytic transfusion reactions that occur when an ABO incompatible unit is transfused, and IgG mediates haemolytic disease of the newborn, both conditions can be fatal.