3.1. Immunoglobulin structureFigure 3.1).
Immunoglobulin heavy and light chains each have constant and variable regions. A pair of heavy and light chain variable regions together forms the antigen-binding site. The variable regions exhibit enormous structural diversity, particularly of antigen-binding contacts, allowing the recognition of a huge variety of antigens.. (Figure 3.2). In humans, light chains are encoded by two different gene loci, resulting in the serologically distinguishable light chain types, κ and λ. Immunoglobulin molecules are assembled in plasma cells with exclusively κ or λ light chain types, never both. (Figure 3.3).
In B-cells, the heavy chains, κ light chains and λ light chains are each encoded by independent chromosomal loci containing multiple copies of analogous gene segments. The gene segments within each locus are rearranged stochastically by somatic recombination and RNA processing mechanisms, ultimately resulting in the expression of functional immunoglobulin proteins. The light chain variable domain is constructed from variable (V) and joining (J) gene segments, whilst the constant domain is encoded by a separate constant (C) gene segment (Figure 3.4). The heavy chain variable domain is constructed from three gene segments: V, D (diversity) and J.
3.2. Immunoglobulin diversity. Such diversity is generated in four main ways:
Firstly, different combinations of gene segments are used in the rearrangement of heavy and light chain genes during early B-cell development. κ light chains are constructed from one of approximately 40 functional Vκ gene segments, one of 5 Jκ gene segments and a single Cκ gene. λ light chains are constructed from one of approximately 30 Vλ gene segments, and one of four (or more) pairs of functional Jλ gene segments and Cλ genes  (Figure 3.5) . The heavy chain variable region is formed from one of around 60 VH, one of 30 DH, and one of six JH gene segments . This combinational diversity accounts for a substantial amount of variable region diversity. Secondly, diversity arises from the addition or removal of nucleotides at the junctions between V (D) and J gene segments during recombination. A third source of diversity arises from the many different combinations of heavy and light chains, and finally, somatic hypermutation introduces point mutations in the variable region genes of light and heavy chains in mature activated B-cells .
In light chains, variations are also found in a region of the variable domain corresponding to the first 23 amino acids of the first framework region (a region not associated with antigen binding). Using monoclonal antibodies, four κ (Vκ I - Vκ IV) and six λ subgroups (Vλ I - Vλ VI) have been identified  (Figure 3.6A). The specific subgroup structures influence the potential of free light chains (FLCs) to polymerise. For example, AL amyloidosis is associated with Vλ VI, and light chain deposition disease (LCDD) with Vκ I and Vκ IV.
3.3. Isotypic and allotypic variation of light chain constant domains(Figure 3.6B and C). The human genome contains a variable number of λ constant genes, giving rise to multiple λ chain isotypes, which can be distinguished serologically by the expression of Mcg, Kern and Oz markers. Additionally, the genome contains a single κ constant gene for which three serologically-defined allotypes have been identified, designated Km1, Km2 and Km3 . These allotypes define three Km alleles, which differ in two amino acids, as presented in Table 3.1. There is no evidence that these constant region variants affect FLC measurements using Freelite® assays, which are based on polyclonal antisera (Chapter 5).
|Km allele||Amino acids|
Table 3.1. Amino acid substitutions in κ light chain constant domains.
3.4. Immunoglobulin and FLC production, and this excess of FLCs is thought to favour accurate assembly of intact immunoglobulin molecules. Light chains which remain unbound from their heavy chain partner are secreted into the blood as FLCs. Secretion of FLC is highest from plasma cells, with twice as many producing κ-chains than λ-chains. κ FLCs are normally monomeric, while λ FLCs tend to be dimeric, joined by disulphide bonds; however, higher polymeric forms of both FLCs may occur (Figure 3.7). Tumours associated with the different stages of B-cell maturation may secrete monoclonal FLCs and/or monoclonal intact immunoglobulins into the serum (Figure 3.8).. The molecules enter the blood and are rapidly partitioned between the intravascular and extravascular compartments. The normal plasma cell content of the bone marrow is about 1%, whereas in multiple myeloma (MM) this can rise to over 90%. In chronic infections and autoimmmune diseases the bone marrow may contain 5 - 10% plasma cells, and may be associated with hypergammaglobulinaemia and corresponding increases in polyclonal serum FLC (sFLC) concentrations. Identification of monoclonal plasma cells in the bone marrow by histology or flow cytometry is an essential part of MM diagnosis, and is frequently based on identifying intracellular κ and λ light chains by direct immunofluorescence techniques (Figure 3.9).
3.5. Clearance and metabolism
Serum concentrations of FLCs and intact immunoglobulins reflect the balance between their production and clearance rates. An understanding of immunoglobulin clearance mechanisms in both normal and pathological conditions is important when considering the utility of sFLCs and intact immunoglobulins as tumour markers in monoclonal gammopathies.
3.5.1. Half-life of sFLCs(Figure 3.10), while larger polymers are cleared more slowly . In contrast, IgG has a half-life of approximately 21 days with minimal renal clearance (Section 5.3). Although κ FLC production rates are estimated to be twice that of λ, their faster removal ensures that actual serum concentrations are approximately 50% lower (Chapter 5). The half-life of FLCs is dependent upon kidney function, so that FLC removal may be prolonged to 2 - 3 days in MM patients with complete renal failure . In patients with chronic kidney disease (CKD), κ and λ sFLC concentrations increase due to reduced renal clearance . When renal clearance is reduced, a greater proportion of sFLC are removed through pinocytosis by cells of the reticuloendothelial system . This mechanism removes κ and λ sFLC at the same rate so the relative FLC concentrations change to reflect more closely the higher rate of κ production and there are minor increases in the κ/λ sFLC ratio .
3.5.2. Renal clearance of FLCsFigure 3.11 shows the glomerular filtration and metabolism of FLCs within a kidney nephron. Each nephron contains a glomerulus with basement membrane fenestrations, which allow filtration of serum molecules into the proximal tubules. Pore sizes are variable, with restricted filtration of molecules that are greater than 20 kDa in size, and a molecular weight cut-off of around 60 kDa. Protein molecules that pass through the glomerular pores are bound by the multi-ligand megalin and cubulin receptors on proximal tubule epithelium; these are then absorbed unchanged, degraded in the proximal tubular cells into their constituent amino acids, or excreted as fragments . This megalin/cubulin absorption pathway is designed to prevent loss of large amounts of proteins and peptides into urine. It is very efficient and can process between 10 and 30 g of small molecular weight proteins daily. Therefore, the 500 mg of FLCs produced each day by the normal lymphoid system are filtered by the glomeruli and completely processed in the proximal tubules .
Because of the huge metabolic capacity of the proximal tubule, the amount of FLCs in urine (even when production is considerably increased in a patient with MM), is more dependent upon renal function than synthesis by the tumour. As a consequence, serum and urine FLC concentrations may differ during the evolution of light chain MM (LCMM) (Figure 3.12). From low initial starting concentrations, sFLCs increase steadily with growing tumour mass, while concentrations in the urine show little change until the proximal tubular metabolism is exceeded and overflow proteinuria develops. Hence, early disease and oligo-secretory disease are not identified from urine tests. Subsequently, urine FLCs rise rapidly as overflow occurs, to reach a maximum. Concentrations then decrease as renal impairment occurs, and are low in complete renal failure. By contrast, sFLC levels increase as renal impairment develops due to the lengthening half-life of FLCs that are no longer cleared by the kidneys. Because of the biphasic urine curve, decreasing concentrations may indicate response to treatment or deterioration of renal function. Urine measurements are therefore unreliable during disease monitoring. Serum levels, however, rise or fall in correct relationship to worsening or improving disease status. The merits of serum over urine testing are further discussed in Chapter 24.