Serum Free Light Chain Measurement
Encyclopedia
Measurement of immunoglobulin free light chains in serum has recently become possible as an alternative to the analysis of Bence Jones proteins in urine. These tests are used as an aid in the diagnosis and monitoring of multiple myeloma
and related disorders. There are two types of immunoglobulin light chain produced in humans, designated by the Greek letters kappa (κ) and lambda (λ). Comparing the ratio of kappa / lambda free light chains in the serum of an individual with that measured in a number of subjects known to be disease-free, indicates whether the individual might have a plasma cell
tumour such as multiple myeloma or AL amyloidosis
.
Each domain comprises two β-pleated sheets
. The sheets are linked by a disulfide bridge and together form a roughly barrel-shaped structure known as a β-barrel
.
The variable (V) domain of light chains has a high degree of structural diversity, particularly the antigen binding region. In addition, the first 23 amino acids of the 1st variable domain framework region have a number of variations known as subgroups. Four kappa (Vκ1 -Vκ4) and 6 lambda subgroups (Vλ1 -Vλ6) can be identified. The specific subgroup structures influence the potential of the free light chains to polymerise such that AL amyloidosis is associated with Vλ6 and light chain deposition disease with Vκ1 and Vκ4.
Light chains are incorporated into immunoglobulin molecules during B-cell development and are expressed initially on the surface of pre B-cells. Production of light chains occurs throughout the rest of B-cell development and in plasma cells, where secretion is highest.
with basement membrane
pores that allow filtration of immunoglobulin light chains and other small molecules from the blood into the proximal tubule
of the nephron.
Filtered molecules are either excreted in the urine or may be specifically re-absorbed. Protein molecules that pass through the glomerular pores are either absorbed unchanged (such as albumin
), degraded in the proximal tubular cells and absorbed (such as free light chains) or excreted as fragments. This re-absorption is mediated by a receptor complex (megalin
/cubulin) and prevents the loss of large amounts of protein into the urine. It is very efficient and can process between 10-30g of small molecular weight proteins per day, so under normal conditions, no light chains pass beyond the proximal tubules.
If immunoglobulin light chains are produced in sufficient amounts to overwhelm the proximal tubules’ absorption mechanisms (usually due to the presence of a plasma cell tumour) the light chains enter the distal tubules and can appear in the urine. The passage of large amounts of immunoglobulin light chains through the kidneys may cause inflammation or blockage of the kidney tubules.
The distal tubules
of the kidneys secrete large amounts of uromucoid (Tamm-Horsfall protein
). This is the dominant protein in normal urine and is thought to be important in preventing ascending urinary infections. It is a relatively small glycoprotein (80kDa) that aggregates into polymers of 20-30 molecules. It contains a short amino-acid sequence that can specifically bind to some free light chains. Together they can form an insoluble precipitate which blocks the distal part of the nephrons. This is termed "cast nephropathy" or "myeloma kidney" and is typically found in patients with multiple myeloma. This can block the flow of urine causing the death of the respective nephrons. Rising concentrations of light chains are filtered by the remaining nephrons leading to a cycle of accelerating renal damage with rising concentrations of free light chains in the blood. At the same time, the amount of free light chains entering the urine will be decreased and will be zero if the patient stops producing urine (anuria
). Conversely, urine concentrations of free light chains could increase if renal function improved in a multiple myeloma patient receiving treatment. This could account for the poor correlation frequently seen when urine and serum free light chain concentrations are compared.
The 500 mg of FLCs produced per day by the normal lymphoid system
, however, flows through the glomeruli and is completely processed by the proximal tubules. If the proximal tubules of the nephrons are damaged or stressed (such as in hard exercise), filtered FLCs may not be completely metabolised and small amounts may then appear in the urine.
A series of studies, principally from the Mayo Clinic
, have indicated that patients with an abnormal free kappa / free lambda ratio have an increased risk of progression to active myeloma from precursor conditions including Monoclonal gammopathy of undetermined significance
(MGUS), Smoldering myeloma and solitary plasmacytoma
of the bone. Abnormal free light chain production has also been reported to be prognostic of a worse outcome in multiple myeloma and chronic lymphocytic leukaemia.
and serum immunofixation electrophoresis is sufficient to screen for pathological monoclonal plasmaproliferative disorders other than AL amyloidosis which requires all the serum tests as well as 24 h urine immunofixation electrophoresis.
Other guidelines for the use of serum free light chain measurement in the management of AL amyloidosis, plasmacytoma and the comparison of treatment responses in clinical trials have also been published.
Technical/clinical reviews of serum free light chain assays have recently been written by Pratt and Jagannath.
Multiple myeloma
Multiple myeloma , also known as plasma cell myeloma or Kahler's disease , is a cancer of plasma cells, a type of white blood cell normally responsible for the production of antibodies...
and related disorders. There are two types of immunoglobulin light chain produced in humans, designated by the Greek letters kappa (κ) and lambda (λ). Comparing the ratio of kappa / lambda free light chains in the serum of an individual with that measured in a number of subjects known to be disease-free, indicates whether the individual might have a plasma cell
Plasma cell
Plasma cells, also called plasma B cells, plasmocytes, and effector B cells, are white blood cells which produce large volumes of antibodies. They are transported by the blood plasma and the lymphatic system...
tumour such as multiple myeloma or AL amyloidosis
AL amyloidosis
Amyloid Light-chain amyloidosis, primary systemic amyloidosis or just primary amyloidosis is the most common form of systemic amyloidosis in the US. The disease is caused when a person's antibody-producing cells do not function properly and produce abnormal protein fibers made of components of...
.
Structure
Each immunoglobulin light chain molecule contains approximately 220 amino acids in a single polypeptide chain that is folded to form constant and variable region domains.Each domain comprises two β-pleated sheets
Beta sheet
The β sheet is the second form of regular secondary structure in proteins, only somewhat less common than the alpha helix. Beta sheets consist of beta strands connected laterally by at least two or three backbone hydrogen bonds, forming a generally twisted, pleated sheet...
. The sheets are linked by a disulfide bridge and together form a roughly barrel-shaped structure known as a β-barrel
Beta barrel
A beta barrel is a large beta-sheet that twists and coils to form a closed structure in which the first strand is hydrogen bonded to the last.Beta-strands in beta-barrels are typically arranged in an antiparallel fashion...
.
The variable (V) domain of light chains has a high degree of structural diversity, particularly the antigen binding region. In addition, the first 23 amino acids of the 1st variable domain framework region have a number of variations known as subgroups. Four kappa (Vκ1 -Vκ4) and 6 lambda subgroups (Vλ1 -Vλ6) can be identified. The specific subgroup structures influence the potential of the free light chains to polymerise such that AL amyloidosis is associated with Vλ6 and light chain deposition disease with Vκ1 and Vκ4.
Synthesis
Kappa light chain molecules are constructed from approximately 40 functional Vκ gene segments (chromosome 2), five Jκ gene segments and a single Cκ gene. Lambda molecules (chromosome 22) are constructed from about 30 Vλ gene segments and four pairs of functional Jλ gene segments and a Cλ gene.Light chains are incorporated into immunoglobulin molecules during B-cell development and are expressed initially on the surface of pre B-cells. Production of light chains occurs throughout the rest of B-cell development and in plasma cells, where secretion is highest.
Production
Production of free immunoglobulin light chains in normal individuals is approximately 500 mg/day from bone marrow and lymph node cells. There is approximately 40% excess immunoglobulin light chain production over immunoglobulin heavy chain synthesis. Possibly this is simply to allow proper conformation of the intact immunoglobulin molecules but an immunological role for the free light chains has also been proposed. There are approximately twice as many kappa producing plasma cells as lambda plasma cells. Kappa free light chains are normally monomeric, while lambda free light chains tend to be dimeric, joined by disulphide bonds. Polymeric forms of both types of free light chain can also occur.Metabolism
In normal individuals, free light chains are rapidly cleared from the blood and catabolised by the kidneys. Monomeric free light chains are cleared in 2–4 hours while dimeric light chains are cleared in 3–6 hours. Removal may be prolonged to 2–3 days in people with complete renal failure. Human kidneys are composed of approximately half a million nephrons. Each nephron contains a glomerulusGlomerulus
A glomerulus is a capillary tuft that is involved in the first step of filtering blood to form urine.A glomerulus is surrounded by Bowman's capsule, the beginning component of nephrons in the vertebrate kidney. A glomerulus receives its blood supply from an afferent arteriole of the renal...
with basement membrane
Basement membrane
The basement membrane is a thin sheet of fibers that underlies the epithelium, which lines the cavities and surfaces of organs including skin, or the endothelium, which lines the interior surface of blood vessels.- Composition :...
pores that allow filtration of immunoglobulin light chains and other small molecules from the blood into the proximal tubule
Proximal tubule
The proximal tubule is the portion of the duct system of the nephron of the kidney which leads from Bowman's capsule to the loop of Henle.-Structure and appearance:...
of the nephron.
Filtered molecules are either excreted in the urine or may be specifically re-absorbed. Protein molecules that pass through the glomerular pores are either absorbed unchanged (such as albumin
Albumin
Albumin refers generally to any protein that is water soluble, which is moderately soluble in concentrated salt solutions, and experiences heat denaturation. They are commonly found in blood plasma, and are unique to other blood proteins in that they are not glycosylated...
), degraded in the proximal tubular cells and absorbed (such as free light chains) or excreted as fragments. This re-absorption is mediated by a receptor complex (megalin
LRP2
Low density lipoprotein-related protein 2 also known as LRP2 or megalin is a protein which in humans is encoded by the LRP2 gene.-Function:...
/cubulin) and prevents the loss of large amounts of protein into the urine. It is very efficient and can process between 10-30g of small molecular weight proteins per day, so under normal conditions, no light chains pass beyond the proximal tubules.
If immunoglobulin light chains are produced in sufficient amounts to overwhelm the proximal tubules’ absorption mechanisms (usually due to the presence of a plasma cell tumour) the light chains enter the distal tubules and can appear in the urine. The passage of large amounts of immunoglobulin light chains through the kidneys may cause inflammation or blockage of the kidney tubules.
The distal tubules
Distal convoluted tubule
The distal convoluted tubule is a portion of kidney nephron between the loop of Henle and the collecting duct system.- Physiology :It is partly responsible for the regulation of potassium, sodium, calcium, and pH...
of the kidneys secrete large amounts of uromucoid (Tamm-Horsfall protein
Tamm-Horsfall protein
The Tamm-Horsfall glycoprotein also known as uromodulin is a glycoprotein that in humans is encoded by the UMOD gene. Up to 150 mg/dl of uromodulin may be excreted in the urine, making it the most abundant protein in normal urine.- Gene :...
). This is the dominant protein in normal urine and is thought to be important in preventing ascending urinary infections. It is a relatively small glycoprotein (80kDa) that aggregates into polymers of 20-30 molecules. It contains a short amino-acid sequence that can specifically bind to some free light chains. Together they can form an insoluble precipitate which blocks the distal part of the nephrons. This is termed "cast nephropathy" or "myeloma kidney" and is typically found in patients with multiple myeloma. This can block the flow of urine causing the death of the respective nephrons. Rising concentrations of light chains are filtered by the remaining nephrons leading to a cycle of accelerating renal damage with rising concentrations of free light chains in the blood. At the same time, the amount of free light chains entering the urine will be decreased and will be zero if the patient stops producing urine (anuria
Anuria
Anuria means nonpassage of urine, in practice is defined as passage of less than 50 milliliters of urine in a day. Anuria is often caused by failure in the function of kidneys. It may also occur because of some severe obstruction like kidney stones or tumours. It may occur with end stage renal...
). Conversely, urine concentrations of free light chains could increase if renal function improved in a multiple myeloma patient receiving treatment. This could account for the poor correlation frequently seen when urine and serum free light chain concentrations are compared.
The 500 mg of FLCs produced per day by the normal lymphoid system
Lymphatic system
The lymphoid system is the part of the immune system comprising a network of conduits called lymphatic vessels that carry a clear fluid called lymph unidirectionally toward the heart. Lymphoid tissue is found in many organs, particularly the lymph nodes, and in the lymphoid follicles associated...
, however, flows through the glomeruli and is completely processed by the proximal tubules. If the proximal tubules of the nephrons are damaged or stressed (such as in hard exercise), filtered FLCs may not be completely metabolised and small amounts may then appear in the urine.
Clinical Use
Serum free light chain assays have been utilised in a number of published studies which have indicated superiority over the urine tests, particularly for patients producing low levels of monoclonal free light chains, as seen in nonsecretory multiple myeloma and AL amyloidosis. This is primarily because of the re-absorption of free light chains in the kidneys, creating a "threshold" of light chain production which must be exceeded before measurable quantities overflow into the urine. While there are a number of publications indicating that serum free light chain analysis is preferable to urine analysis at diagnosis, there is currently no consensus on whether urine tests for monitoring should be replaced.A series of studies, principally from the Mayo Clinic
Mayo Clinic
Mayo Clinic is a not-for-profit medical practice and medical research group specializing in treating difficult patients . Patients are referred to Mayo Clinic from across the U.S. and the world, and it is known for innovative and effective treatments. Mayo Clinic is known for being at the top of...
, have indicated that patients with an abnormal free kappa / free lambda ratio have an increased risk of progression to active myeloma from precursor conditions including Monoclonal gammopathy of undetermined significance
Monoclonal gammopathy of undetermined significance
Monoclonal gammopathy of undetermined significance , formerly benign monoclonal gammopathy, is a condition in which a paraprotein is found in the blood during standard laboratory tests...
(MGUS), Smoldering myeloma and solitary plasmacytoma
Plasmacytoma
Plasmacytoma refers to a malignant plasma cell tumor growing within soft tissue or within the skeleton. The skeletal forms usually have other occult tumors and frequently disseminate to multiple myeloma over the course of 5–10 years. The soft tissue forms most often occur in the upper respiratory...
of the bone. Abnormal free light chain production has also been reported to be prognostic of a worse outcome in multiple myeloma and chronic lymphocytic leukaemia.
Guidelines
The International Myeloma Working Group have recently published guidelines making recommendations of when serum free light chain analysis should be used in the management of multiple myeloma.Diagnosis
The serum free light chain assay in combination with serum protein electrophoresisSerum protein electrophoresis
Serum protein electrophoresis is a laboratory test that examines specific proteins in the blood called globulins. Blood must first be collected, usually into an airtight vial or syringe...
and serum immunofixation electrophoresis is sufficient to screen for pathological monoclonal plasmaproliferative disorders other than AL amyloidosis which requires all the serum tests as well as 24 h urine immunofixation electrophoresis.
Prognosis
The serum free light chain assay should be measured at diagnosis for all patients with MGUS, smoldering or active multiple myeloma. Solitary plasmacytoma and AL amyloidosis.Monitoring
Serial FLC ascertainment should be routinely performed in patients with AL amyloidosis and multiple myeloma patients with oligosecretory disease. It should also be done in all patients who have achieved a complete response to determine whether they have attained a stringent complete response.Other guidelines for the use of serum free light chain measurement in the management of AL amyloidosis, plasmacytoma and the comparison of treatment responses in clinical trials have also been published.
Technical/clinical reviews of serum free light chain assays have recently been written by Pratt and Jagannath.