The cluster of differentiation (CD) is a protocol used for the identification and investigation of cell surface molecules present on leukocytes. CD molecules can act in numerous ways, often acting as receptors or ligands (the molecule that activates a receptor) important to the cell. The CD nomenclature was proposed and established in the 1st International Workshop and Conference on Human Leukocyte Differentiation Antigens (HLDA) (Zola et al. 2005; Zola and Swart 2005). This system was intended for the classification of the many monoclonal antibodies (mAbs) generated by different laboratories around the world against epitopes on the surface molecules of leukocytes (white blood cells). Since then, its use has expanded to many other cell types, and more than 320 CD unique clusters and subclusters have been identified. The human leukocyte antigen (HLA) is expressed as cell surface molecules/antigens on various immune cells. Through flow cytometry, the CD markers or HLA molecules are identified using mAbs directed against a specific cell surface antigen. Under the current naming system, antigens that are well characterized are assigned an arbitrary number (e.g., CD1, CD2, etc.), whereas molecules that are recognized by just one monoclonal antibody are given the provisional designation “CDw.” Physiologically, CD antigens do not belong in any particular class of molecules, with their functions ranging from cell surface receptions to adhesion molecules. Although initially used for just human leukocytes, the CD molecule naming convention has now been expanded to cover both other species (e.g., mouse) as well as other cell types. Human CD antigens are currently numbered up to CD363. Before discussing RIT in detail, it is helpful to provide a brief introductory discussion on antibodies, antigens, and lymphoma and their roles in RIT.

Antibodies are immune system-related proteins called immunoglobulins. A typical antibody molecule is made up of four polypeptide chains. The four chains are divided into two identical light chains and two identical heavy chains (Fig. 9.1). An antibody molecule is depicted as Y-shaped molecules with two identical antigen-binding sites at the ends of the arms of the Y. The light and heavy chains contribute to the antigen-binding sites to receptors or specific “antigens.” Each antibody molecule can bind to two identical antigenic determinants. Where the arms meet, the stem of the Y is known as the hinge region. The hinge region allows segmental flexibility of the antibody molecule. The two antigen-binding ends (or amino-terminal ends) of the antibody molecule are called the Fab fragments (for fragment antigen binding), whereas the stem (or carboxyl-terminal end) of the Y is considered to be the Fc fragment (for fragment crystallizable). The Fc region of the antibody molecule is responsible for its biologic properties.

The amino-terminal end of an antibody is called the variable, or V, region and the carboxyl-terminal end is called the constant, or C, region. The C region is about the same size as the V region in the light chain and three to four times larger than the V region in the heavy chain. The V regions of light and heavy chains form the antigen-binding sites. Light and heavy chains consist of repeating, similarly folded homology units or domains. The light chain has one V region (VL) domain and one C region (CL) domain, whereas the heavy chain has one V region (VH) and three or four C region (CH) domains. The most variable parts of the V regions are limited to several small hypervariable regions or complementarity-determining regions (CDRs). The light- and heavy-chain V regions contain three CDRs. The CDRs come together at the amino-terminal end of the antibody molecule to form the antigen-binding site, which determines specificity. The invariant regions of amino acids, between the CDRs, make up about 85 % of the V regions and are designated framework residues. The three functions which explain antibody chemical structure include binding versatility, binding specificity, and biological activity.

An antigen is defined as “any foreign substance that elicits an immune response (e.g., the production of specific antibody molecules) when introduced into the tissues of a susceptible animal and is capable of combining with the specific antibodies formed.” Antigens are generally of high molecular weight and are commonly proteins, carbohydrates, or polysaccharides, lipids, nucleic acids, or even small molecules like neurotransmitters which can act as antigens. These molecules elicit an immune response involving the production of specific antibody molecules when introduced into the tissues of a susceptible animal or human. The antigens are capable of combining with the specific antibodies which are formed. Specific antibodies can interact with only a small region of an antigen, and in the case of a polypeptide, this is generally about 5–12 amino acids. This region can be continuous or it can be distributed in different regions of a primary structure that are brought together because of the secondary or tertiary structure of the antigen. The antigen region which is recognized by an antibody is called an “epitope” and usually consists of one to six monosaccharides or 5–8 amino acid residues on the antigen surface. Because antigen molecules exist in space, the epitope recognized by an antibody may be dependent upon the presence of a specific three-dimensional antigenic conformation. This may be represented by a unique site formed by the interaction of two native protein loops or subunits. In addition, the epitope may correspond to a simple primary sequence region. The range of possible binding sites is enormous, with each potential binding site exhibiting its own structural properties derived from covalent bonds, ionic bonds, and hydrophilic and hydrophobic interactions. For efficient interaction to occur between the antigen and the antibody, the epitope must be readily available for binding. If the target molecule is denatured, e.g., through fixation, reduction, pH changes, or during preparation for gel electrophoresis, the epitope may be altered, and this may affect its ability to interact with an antibody. The epitope may be present in the antigen’s native, cellular environment, or only exposed when denatured. In their natural form, they may be cytoplasmic (soluble), membrane associated, or secreted. The number, location, and size of the epitopes depend on how much of the antigen is presented during the antibody-making process.

There are a large variety of important characteristics of biologic antigens. The most striking feature of antigen–antibody interactions is the high specificity and affinity. It is accepted that almost all the antigens are identified by specific antibodies, but very few have the ability to stimulate the antibodies. Often, it is useful to illicit the formation of antibodies in response to small molecules of interest. However, in contrast to macromolecular molecules which can often illicit the formation of antibodies, small molecules cannot independently provoke an immune response and thus antibody formation. To overcome this, immunologists can attach several copies of small molecules of interest, called “haptens,” to a carrier protein prior to immunization. In this manner, the antibodies which are formed are specific to the hapten/carrier–protein complex. Each antibody binds to a particular part of the antigen called the antigenic determinant (or epitope), which is the specific site on an antigen to which the specific antibody binds. Epitopes are hence also called as antigenic determinants. The random structure on the antigenic molecule that are identified by the antibody as an antigenic binding site thus form the epitope of that antigen. The strength of the antibody–antigen binding is important, and the binding strength between an antigenic determinant in an antigen (epitope) and an antigen-binding site in an antibody (paratope) is referred to as the affinity. Different epitopes can be organized on a single protein molecule in such a manner that their spacing may affect the binding of antibody molecules in various ways. There are a variety of specific terms used in RIT which include the following.

Lymphomas are malignancies of the lymphoid tissue and are broadly classified into Hodgkin’s lymphoma (HD) and non-Hodgkin’s lymphomas (NHL, 85 %). The hallmark of Hodgkin’s lymphoma (HL) is the presence of large, mononucleated Hodgkin and multinucleated Reed/Sternberg cells. These cells represent the tumor cells, but usually comprise less than 1 % of the cellular infiltrate in the lymphoma tissue (Weiss et al. 1999). Due to the rarity of the Hodgkin and Reed/Sternberg (HRS) cells and their unusual phenotype, the origin of these cells from germinal center (GC) B cells in both the lymphocyte predominant (LP) and the classical subtype of HL has only recently been clarified (Küppers 2002). Only in very rare cases do the HRS cells of classical HL represent transformed T cells (Müschen et al. 2000; Seitz et al. 2000).

In contrast, non-Hodgkin’s lymphomas are a heterogeneous group of lymphoreticular malignancies with a wide range of aggressiveness. The majority of NHL are B-cell lymphomas, with the follicular and diffuse large B-cell lymphomas constituting up to 50 % of NHL. NHL can also be classified as indolent (i.e., slow progression; 40 %) or aggressive lymphomas (60 %). B-cell CLL/small lymphocytic lymphoma, marginal zone lymphoma, lymphoplasmacytoid, and follicle center lymphoma constitute the indolent types, whereas diffuse large B-cell, mantle cell, Burkitt’s, and precursor B-cell leukemia constitute the aggressive types. NHL accounts for 4 % of all malignancies and 4 % of all cancer-related deaths (Anand et al. 2008).