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By Richard R. Rogoski
Detecting monoclonal gammopathies, or plasma cell disorders, usually involves serum protein electrophoresis (SPEP) and immunoelectrophoresis (IFE) to test both serum and urine. But the growing clinical acceptance of a serum free light chain assay has all but eliminated urine tests in identifying such plasma cell disorders as multiple myeloma (MM), smoldering myeloma, monoclonal gammopathy of undetermined significance (MGUS) and primary systemic amyloidosis (AL); and because the assay has proven to be more sensitive than IFE for detecting free or unbound immunoglobulin light chains when it is used in conjunction with SPEP, up to 99% of myelomas can be detected.
Each clonal plasma cell undergoes heavy and light chain rearrangements to produce an immunoglobulin molecule. And it is this rearrangement that determines not only the antigen binding site of the immunoglobulin, but also identifies each plasma cell clone.
Five types of immunoglobulin heavy chains have been identified: gamma, alpha, mu, delta and epsilon. Light chains are identified as either kappa or lambda. When a heavy chain combines with a light chain, they produce molecules of IgG, IgA, IgM, IgD, or IgE. Because plasma cells produce a larger quantity of light chains than heavy chains, the excess light chains enter the bloodstream as "free" light chains (FLC). In instances where plasma cell clones proliferate too rapidly, immunoglobulin concentrations increase. These molecules are then called monoclonal immunoglobulins and are directly related to malignant or potentially malignant disorders such as MM and MGUS.
The significance of free light chains led U.K.-based The Binding Site Ltd. to begin work on a new assay, explains Graham Mead, PhD, director of research and development. "Starting in the 1970s, there have been many studies published looking at different methods for measuring serum free light chains. For most of these experimental assays, a lack of suitable specificity was apparent (i.e., they cross-reacted with intact immunoglobulin), and none were adopted for routine clinical use.
"Work on developing our own assays was started in 1996. Our staff already had a number of years’ experience producing highly specific polyclonal antisera for measuring IgG subclasses. We hoped to build on this experience to develop serum free light chain assays and improve upon the studies previously published. It took several years of trial before we were able to produce antibodies adequate for developing nephelometric assays, and our first study of their application was not published until 2001."
So far, the product, Freelite, is the only Food and Drug Administration (FDA) approved FLC assay on the market, although a similar product manufactured by an Italian company is being used in a few European countries to test urine samples. New guidelines in the United States, however, specifically recommend the use of FLC serum tests for diagnosis.
... serum free light chain elevations were associated with an increased risk of progression to lymphoma among HIV-positive patients.
Since the serum FLC test involves the action of antibodies, the underlying technology is simple and is the same as many other nephelometric/turbidimetric immunoassays, Mead says. "The antibodies form immune complexes with the free light chains in a test serum and the size/speed of complex formation is detected by a laser shone through the reaction vessel. To amplify the signal, the antibodies are bound to microscopic polystyrene particles (frequently called latex). The main challenge of developing and producing the assays is the production of suitable antibodies which must have:
"With regard to specificity, simply immunizing with free light chains and absorbing with intact immunoglobulin does not produce antisera with adequate avidity or titer. We use proprietary techniques to focus the antibody production on the parts of the light chain molecule which are hidden in intact immunoglobulin but exposed on free light chains.
"Producing antisera with a balanced response against the free light chains from all patients is a formidable challenge and one of the reasons that monoclonal antibodies are not suitable for these assays. Careful control of the range of proteins used for immunizations and antibody purifications as well as the use of antisera pools of >100 liters, has allowed us to optimize the assay response."
As with any lab test, there are advantages and disadvantages. Mead stresses that the major advantage of running a FLC test is that it measures concentrations in serum rather than in urine. "One of the important functions of the kidneys is to re-absorb and catabolize small proteins, such as free light chains, which have been filtered from the blood in the glomeruli. It is only when this capacity for re-absorption is overwhelmed that significant quantities of free light chains can pass through the kidney tubules and into the urine. Therefore, many patients with small plasma cell tumors are found to have abnormal serum free light chain results while their urine appears normal."
“With regard to specificity, simply immunizing with free light chains and absorbing with intact immunoglobulin does not produce antisera with adequate avidity or titer.”
The sensitivity of the assay, however, is largely disease specific (i.e., for only those diseases with higher likelihoods of having free light chains will the assay be more sensitive than IFE alone). For example, Katzmann, et al, evaluated 1877 patients with monoclonal gammopathy using five assays: serum and urine protein electrophoresis (PEL), serum and urine IFE, and the serum FLC assay. For all comers, the sensitivity of the serum IFE and the FLC were 87% and 74%, respectively. If one breaks down the sensitivity analysis by disease, however, the respective sensitivities are as follows: multiple myeloma 94% vs. 97%; macroglobulinemia 100% vs. 73%; smoldering myeloma 98% vs. 81%,; MGUS 93% vs. 42%; plasmacytoma 72% vs. 55%; AL amyloidosis 74% vs. 88%; and light chain deposition disease 56% vs. 78%.1
The International Myeloma Working Group’s published guidelines recommend 24-hour urine samples for some patients. First published online in Leukemia in November 2008, the article by Dispenzieri, et al, states that "for the purpose of screening for monoclonal proteins for all diagnoses except AL, the FLC can replace the 24-hour urine IFE. Once a diagnosis of monoclonal gammopathy is made, however, the 24-hour protein IFE should be performed. For AL screening, however, the urine IFE should still be done in addition to the serum tests, including the serum FLC."2
Howard Robin, MD, medical director of laboratory services at Sharp Memorial Hospital in San Diego, CA, says he has been using the FLC assay for more than two years for screening, diagnoses, and prognostications. Yet, he says urine collections are a problem. "One of the problems with urine is that we seldom get a true 24-hour urine. Mostly, it is random or spot urine samples. And labs do not like working with urine because they are not getting 24-hour urine."
The rationale for emphasizing the need for the 24-hour urine protein electrophorsis in following patients with light chain myeloma is that there is a poor correlation between the serum FLC and the urinary monoclonal protein as measured by urine PEL3 and for patients with amyloidosis, serial 24-hour urine measurements are critical for monitoring the status of a patient’s nephrotic syndrome.
As for any disadvantages in using an FLC assay, Mead points to two. "The material cost of running serum Freelite assays is greater than that of a simple urine electrophoresis gel. When costing analysis has included storage, processing, urine immunofixation, time for interpretation, or the Medicare reimbursement costs, however, there are benefits to using the serum assays. Freelite has been shown to be more cost effective than urine testing. This is evidenced by the fact that the Medicare reimbursement costs for the urine panel of tests is higher than for the alternative serum panel which includes serum Freelite." This cost analysis was substantiated by separate studies published in 2006 by Katzmann et al4, and Hill, et al.5
When costing analysis has included storage, processing, urine immunofixation, time for interpretation, or the Medicare reimbursement costs, however, there are benefits to using the serum assays.
Another issue that is sometimes raised is the accuracy of FLC assays compared to other tests. "Some aspects of analytical performance have been criticized; and it is true that the precision and accuracy does not equal that of a C3 assay, for example," says Mead. "This is understandable when you consider that free light chains are monoclonal proteins that can vary by more than a thousandfold in concentration and may exist in different polymeric forms."
David Keren, MD, medical director of Warde Medical Laboratory, a private reference lab in Ann Arbor, MI, says he uses FLC assays every day with excellent results. But he agrees there can sometimes be a computation problem. "In a few cases, we have gotten a falsely low value because of higher levels of antigen. It is uncommon, but it is an issue."
For the laboratory professional, running a FLC assay is simple. "As long as the nephelometer/turbidimeter is correctly programmed and appropriately maintained, running Freelite assays is as easy as running other serum tests," says Mead. "No special training is required to run the tests but a basic understanding of the biology of free light chains is helpful when interpreting results."
Dr. Keren concurs: "It is an automated test that can be run on the same machines used to measure IgG, IgA, and IgM."
Currently, these machines include Dade Behring BNII and ProSpec; Beckman IMMAGE; Roche/Hitachi 911, 912, 917, and Modular P; Olympus AV400, 640, 2700, 5400; Radim Delta; and Bayer Advia. And since it is the kappa/lambda ratio which is read, Dr. Robin notes that it is the system’s software that figures out the ratio. The entire assay takes between five and 18 minutes to run, depending on the analyzer used.
Katzmann, et al, determined in 2002 the normal range using both fresh and frozen sera from 282 individuals aged 21 to 90. By including 100% of donors, the normal diagnostic range for FLC kappa/lambda was set at 0.26 mg/L to 1.65 mg/L. Normal kappa FLC levels are 3.3 mg/L to 19.4 mg/L, and normal lambda FLC levels are 5.7 mg/L to 26.3 mg/L. Patients with kappa/lambda ratios greater than 1.65 mg/L have higher levels of kappa FLC. Those ratios less than 0.26 mg/L have higher levels of lambda FLC.6
The normal range for kappa/lambda ratios also is greater than those used for most other tests in order to provide a larger safety margin for normal patients.
"When I first saw the data, I was skeptical," admits Dr. Keren, adding that he wanted to see more empirical data than just those in the original Katzmann study. To date, there have been more than 200 published studies using Freelite as an FLC assay. As a result, this assay is now recommended for the initial evaluation of suspected myeloma; for prognosis of plasma cell dyscrasias; and for monitoring oligosecretory myeloma and AL amyloidosis. Although the 2002 Katzmann study remains the landmark, another study he published in 2006 concluded that urine analysis was not necessary if only serum was analyzed using an FLC assay combined with PE and IFE. Even as far back as 2001, research by Bradwell, et al, published in Clinical Chemistry, concluded that the automated immunoassay then being studied could be used to assay FLC concentrations "in a routine clinical laboratory setting."7
... this assay is now recommended for the initial evaluation of suspected myeloma; for prognosis of plasma cell dyscrasias; and for monitoring oligosecretory myeloma and AL amyloidosis.
Other studies also have supported the use of Freelite for screening, diagnosing, and monitoring patients.
In a 2007 article published in Clinical Lymphoma & Myeloma, Sundar Jagannath looked at free light chain measurements at short sampling intervals — given that the half-life of FLC is less than six hours. He said such testing could "allow real-time measurement of treatment-induced tumor kill and could possibly provide prompt indications of chemosensitivity, dose adequacy and the need for alternative approaches."8
Hutchison, et al, in a 2008 study published in BMC Nephrology, looked at myeloma patients with renal failure. The researchers concluded: "The diagnostic accuracy of these assays and their rapid laboratory turnaround time should aid nephrologists in their assessment of acute renal failure."9
Interestingly, Dr. Robin recommends that patients over 50 with renal failure should also be screened for monoclonal gammopathies.
While it is a certainty that other independent studies will be done using Freelite, Mead says his company will continue to focus on free light chains as well as heavy chains. "The current focus of research is looking at different applications for serum free light chain analysis. For example, a presentation at a recent conference reported that serum free light chain elevations were associated with an increased risk of progression to lymphoma among HIV-positive patients. Here in the U.K., we are currently providing support for a trial of extended dialysis to remove free light chains and improve the prognosis for myeloma patients with acute renal failure (caused by light chain cast nephropathy). We are also investigating whether free light chains contribute to renal damage in patients without myeloma or other plasma cell tumors," he says.
"From the early studies with our product, it became apparent that abnormalities of the free light chain ratio (kappa/lambda) could provide a more sensitive indication of monoclonal disease than simple elevations of one light chain. This is because a tumor will usually express a surplus of one light chain but also suppress the production of the alternate light chain. As a result of this observation, we are now developing assays that will determine these ratios for intact immunoglobulins (e.g., the IgGkappa/IgGlambda ratio). Our first full report of these assays has now been accepted for publication. The preliminary results indicate that these assays will be useful for diagnosis, prognosis and monitoring of plasma cell tumors. They will be complementary to our serum free light chain assays because they provide a sensitive marker for those tumors which produce little free light chain."
Richard R. Rogoski is a freelance journalist based in Durham, NC. His extensive list of published articles have dealt with new developments in the fields of cardiology and cardiac surgery; imaging technology; information technology; and the business side of healthcare. Contact him at firstname.lastname@example.org.