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10 July 2009 MLO www.mlo-online.com
D
etecting monoclonal gammopathies, or plasma cell dis-
orders, 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 signifcance (MGUS) and primary systemic
amyloidosis (AL); and because the assay has proven to be
more sensitive than IFE for detecting free or unbound im-
munoglobulin 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 rear-
rangements to produce an immunoglobulin molecule. And
it is this rearrangement that determines not only the antigen
binding site of the immunoglobulin, but also identifes each
plasma cell clone.
Five types of immunoglobulin heavy chains have been
identifed: gamma, alpha, mu, delta and epsilon. Light chains
are identifed 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 signifcance 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 dif-
serum free
light chain
assays:
Detecting plasma cell disorders
By Richard R. Rogoski
To earn CEUs, see current test on page 22 or at
www.mlo-online.com under the CE Tests tab.
LEARNING OBJECTIVES
Upon completion of this article, the reader will be able to:
Name fve types of immunoglobulin heavy chains and 1.
two types of immunoglobulin light chains.
State specifc advantage(s)/disadvantage(s) of serum 2.
free light chain assessment.
Identify three platforms on which the serum free light 3.
chain assay currently can be run.
State three pathologies detectable by serum free light 4.
chain assessment.
Identify a possible additional use for serum free light 5.
chain assessment.
c o N t I N U I N G e D U c A t I o N
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ferent methods for measuring serum free
light chains. For most of these experimen-
tal assays, a lack of suitable specifcity
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 specifc polyclonal antisera for
measuring IgG subclasses. We hoped to
build on this experience to develop se-
rum 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 frst study of their application was
not published until 2001.
So far, the product, Freelite, is the only
Food and Drug Administration (FDA) ap-
proved 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, how-
ever, specifcally recommend the use of
FLC serum tests for diagnosis.
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 anti-
bodies 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 develop-
ing and producing the assays is the
production of suitable antibodies which
must have:
a high degree of specifcity, so they
recognize immunoglobulin light
chains when they are free but not when
they are bound to heavy chains in in-
tact immunoglobulin molecules; and
a balanced response against the va-
riety of different monoclonal FLCs
produced by patients.
With regard to specifcity, simply
immunizing with free light chains and
absorbing with intact immunoglobulin
does not produce antisera with adequate
avidity or titer. We use proprietary tech-
niques to focus the antibody production
on the parts of the light chain molecule
which are hidden in intact immunoglobu-
lin 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 anti-
bodies are not suitable for these assays.
Careful control of the range of proteins
used for immunizations and antibody
Electrophoresis Simplied
Open door. Insert sample. Walk away.
The MINICAP is so simple to use,
its like having a new employee.
Electrophoresis of serum and urine is recommended
to screen for and monitor monoclonal gammopathies.
(800) 835-6497 U www.sebia-usa.com
... serum free light chain
elevations were associated
with an increased risk of
progression to lymphoma
among HIV-positive patients.
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purifcations 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 advan-
tages 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 fl-
tered from the blood in the glomeruli. It is
only when this capacity for re-absorption
is overwhelmed that signifcant quantities
of free light chains can pass through the
kidney tubules and into the urine. There-
fore, many patients with small plasma cell
tumors are found to have abnormal serum
free light chain results while their urine
appears normal.
The sensitivity of the assay, however,
is largely disease specifc (i.e., for only
those diseases with higher likelihoods
of having free light chains will the as-
say be more sensitive than IFE alone).
For example, Katzmann, et al, evaluated
1877 patients with monoclonal gammo-
pathy using fve 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%; macro-
globulinemia 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
Groups published guidelines recom-
mend 24-hour urine samples for some
patients. First published online in Leu-
kemia 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 gam-
mopathy 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 Memo-
rial 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
With regard to specifcity,
simply immunizing with free
light chains and absorbing with
intact immunoglobulin does not
produce antisera with adequate
avidity or titer.
F L c A s s A y s
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they are not getting 24-hour urine.
The rationale for emphasizing the
need for the 24-hour urine protein elec-
trophorsis in following patients with light
chain myeloma is that there is a poor cor-
relation between the serum FLC and the
urinary monoclonal protein as measured
by urine PEL
3
and for patients with amy-
loidosis, serial 24-hour urine measure-
ments are critical for monitoring the status
of a patients 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, process-
ing, urine immunofxation, time for in-
terpretation, or the Medicare reimburse-
ment costs, however, there are benefts
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 reimburse-
ment 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 al
4
, and Hill, et al.
5
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 thousand-
fold 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 main-
tained, 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; Beck-
man 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 systems software that
fgures out the ratio. The entire assay
takes between fve 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 larg-
er safety margin for normal patients.
Validating studies
When I first saw the data, I was
skeptical, admits Dr. Keren, adding
When costing analysis has
included storage, processing,
urine immunofxation, time for
interpretation, or the Medicare
reimbursement costs, however,
there are benefts to using the
serum assays.
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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 oligose-
cretory 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 immu-
noassay then being studied could be
used to assay FLC concentrations in a
routine clinical laboratory setting.
7
Other studies also have supported the
use of Freelite for screening, diagnosing,
and monitoring patients.
In a 2007 article published in Clini-
cal Lymphoma & Myeloma, Sundar
Jagannath looked at free light chain
measurements at short sampling inter-
vals 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 diag-
nostic 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.
... 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.
F L c A s s A y s
July 2009 MLO www.mlo-online.com
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A Division of IRIS International, Inc.
B00-7B2-B774 vvv.snsn.con
New developments
While it is a certainty that other indepen-
dent 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 eleva-
tions 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 prod-
uct, it became apparent that abnormalities
of the free light chain ratio (kappa/lambda)
could provide a more sensitive indication
of monoclonal disease than simple eleva-
tions 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 frst
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 felds of
cardiology and cardiac surgery; imaging technol-
ogy; information technology; and the business side of
healthcare. Contact him at rogoski@aol.com.
References:
Katzmann JA, Kyle RA, Benson J, Larson DR, 1.
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, 2.
et al. International Myeloma Working Group guide-
lines for serum free light chain analysis in multiple
myeloma and related disorders. Leukemia. 2009.
Dispenzieri A, Zhang L, Katzmann JA, et al. Ap- 3.
praisal of immunoglobulin free light chain as a
marker of response. Blood. 2008;111:4908-4915.
Katzmann JA, Dispenzieri A, Kyle RA, Snyder MR, 4.
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 Immunofxation and
Free Light Chain Assays. Mayo Clin Proc. 2006.
Hill PG, Forsyth JM, Rai B, Mayne S. Serum Free 5.
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, 6.
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, 7.
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 Test- 8.
ing for the Diagnosis and Monitoring of Monoclonal
Gammopathies in Hematology. Clinical Lymohoma
& Myeloma; 2007;7(8).
Hutchison CA, Plant T, Drayson M, Cockwell P, 9.
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.
www.mlo-online.com MLO July 2009 43
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