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LEARNING OBJECTIVES
Upon completion of this article, the
reader will be able to:
- Name five types of immunoglobulin heavy chains and two types of
immunoglobulin light chains. - State specific advantage(s)/disadvantage(s) of serum free light
chain assessment. - Identify three platforms on which the serum free light chain assay
currently can be run. - State three pathologies detectable by serum free light chain
assessment. - Identify a possible additional use for serum free light chain
assessment.
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.
The light chain connection
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.
Homing in on FLC
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:
- a high degree of specificity, so they recognize immunoglobulin light
chains when they are free but not when they are bound to heavy chains in
intact immunoglobulin molecules; and - a balanced response against the variety of different monoclonal FLCs
produced by patients.
“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.”
A simple test to run
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.
Validating studies
“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.
New developments
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
[email protected].
References:
- Katzmann JA, Kyle RA, Benson J, Larson DR, Snyder MR, et al.
Screening Panels for Detection of Monoclonal Gammopathies Clin Chem.
June 2009; doi:10.1373/clinchem.126664. - Dispenzieri A, Kyle R, Merlini G, Miguel JS, Ludwig H, et al.
International Myeloma Working Group guidelines for serum free light
chain analysis in multiple myeloma and related disorders.
Leukemia. 2009. - Dispenzieri A, Zhang L, Katzmann JA, et al. Appraisal of
immunoglobulin free light chain as a marker of response. Blood.
2008;111:4908-4915. - Katzmann JA, Dispenzieri A, Kyle RA, Snyder MR, Plevak MF, Larson
DR, Abraham RS, Lust JA, Melton III LJ, Rajkumar SV. Elimination of the
Need for Urine Studies in the Screening Algorithm for Monoclonal
Gammopathies by Using Serum Immunofixation and Free Light Chain Assays.
Mayo Clin Proc. 2006. - Hill PG, Forsyth JM, Rai B, Mayne S. Serum Free Light Chains: An
Alternative Test to Urine Bence Jones Proteins When Screening for
Monoclonal Gammopathies. Clin Chem. 2006. - Katzmann JA. Clark RJ, Abraham RS, Bryant S, Lymp JF, Bradwell AR et
al. Serum Reference Intervals and Diagnostic Ranges for Free Kappa and
Free Lambda Immunoglobulin Light Chains: Relative Sensitivity for
Detection of Monoclonal Light Chains. Clin Chem.
2002. - Bradwell AR, Carr-Smith HD, Mead GP, Tang LX, Showell PJ, Drayson
MT, Drew RL. Highly sensitive, automated immunoassay for immunoglobulin
free light chains in serum and urine. Clin Chem.
2001. - Jagannath S. Value of Serum Free Light Chain Testing for the
Diagnosis and Monitoring of Monoclonal Gammopathies in Hematology.
Clinical Lymohoma & Myeloma; 2007;7(8). - Hutchison CA, Plant T, Drayson M, Cockwell P, Kountouri M, Basnayke K,
Harding S, Bradwell AR, Mead G. Serum free light chain measurement aids the
diagnosis of myeloma in patients with severe renal failure. BMC
Nephrology. 2008.
