0709_coverstory

8/2/2019 0709_coverstory

1/7

c o v e r s t o r y

10 July 2009 MLO www.mlo-online.com

Detecting monoclonal gammopathies, or plasma cell dis-orders, usually involves serum protein electrophoresis(SPEP) and immunoelectrophoresis (IFE) to test bothserum and urine. But the growing clinical acceptance o a

serum ree light chain assay has all but eliminated urine tests

in identiying such plasma cell disorders as multiple myeloma

(MM), smoldering myeloma, monoclonal gammopathy o

undetermined signifcance (MGUS) and primary systemic

amyloidosis (AL); and because the assay has proven to be

more sensitive than IFE or detecting ree or unbound im-

munoglobulin light chains when it is used in conjunction with

SPEP, up to 99% o 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 o the immunoglobulin, but also identifes each

plasma cell clone.

Five types o immunoglobulin heavy chains have been

identifed: gamma, alpha, mu, delta and epsilon. Light chainsare identifed as either kappa or lambda. When a heavy chain

combines with a light chain, they produce molecules o IgG,

IgA, IgM, IgD, or IgE. Because plasma cells produce a larger

quantity o light chains than heavy chains, the excess light

chains enter the bloodstream as ree light chains (FLC).

In instances where plasma cell clones prolierate 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 o ree light chains led U.K.-based The BindingSite Ltd. to begin work on a new assay, explains Graham Mead,

PhD, director o research and development. Starting in the

1970s, there have been many studies published looking at di-

sum fligh hainaa:Ding plama ll didBy Richard R. Rogoski

To earn CEUs, see current test on page 22 or at

www.mlo-online.comunderthe CE Tests tab.

LEARNING OBJECTIVES

Upon completion of this article, the reader will be able to:Name fve types o immunoglobulin heavy chains and1.

two types o immunoglobulin light chains.

State specifc advantage(s)/disadvantage(s) o serum2.

ree light chain assessment.

Identiy three platorms on which the serum ree light3.

chain assay currently can be run.

State three pathologies detectable by serum ree light4.

chain assessment.Identiy a possible additional use or serum ree light5.

chain assessment.

c o N t I N U I N G e D U c A t I o N
http://www.mlo-online.com/http://www.mlo-online.com/http://www.mlo-online.com/http://www.mlo-online.com/http://www.mlo-online.com/


2/7

c o v e r s t o r y

www.mlo-online.com

erent methods or measuring serum ree

light chains. For most o these experimen-

tal assays, a lack o suitable specifcity

was apparent (i.e., they cross-reacted with

intact immunoglobulin), and none were

adopted or routine clinical use.Work on developing our own assays

was started in 1996. Our sta already had

a number o years experience producing

highly specifc polyclonal antisera or

measuring IgG subclasses. We hoped to

build on this experience to develop se-

rum ree light chain assays and improve

upon the studies previously published. It

took several years o trial beore we wereable to produce antibodies adequate or

developing nephelometric assays, and

our frst study o their application was

not published until 2001.

So ar, the product, Freelite, is the only

Food and Drug Administration (FDA) ap-

proved FLC assay on the market, although

a similar product manuactured by an

Italian company is being used in a ewEuropean countries to test urine samples.

New guidelines in the United States, how-

ever, specifcally recommend the use o

FLC serum tests or diagnosis.

Since the serum FLC test involves

the action o antibodies, the underlying

technology is simple and is the same as

many other nephelometric/turbidimetric

immunoassays, Mead says. The anti-

bodies orm immune complexes with

the ree light chains in a test serum and

the size/speed o complex ormation is

detected by a laser shone through the

reaction vessel. To ampliy the signal,

the antibodies are bound to microscopic

polystyrene particles (requently calledlatex). The main challenge o develop-

ing and producing the assays is the

production o suitable antibodies which

must have:

a high degree o specifcity, so they

recognize immunoglobulin light

chains when they are ree but not when

they are bound to heavy chains in in-

tact immunoglobulin molecules; and

a balanced response against the va-

riety o dierent monoclonal FLCs

produced by patients.

With regard to specifcity, simplyimmunizing with ree light chains and

absorbing with intact immunoglobulin

does not produce antisera with adequate

avidity or titer. We use proprietary tech-

niques to ocus the antibody production

on the parts o the light chain molecule

which are hidden in intact immunoglobu-

lin but exposed on ree light chains.

Producing antisera with a balanced

response against the ree light chains rom

all patients is a ormidable challenge and

one o the reasons that monoclonal anti-bodies are not suitable or these assays.

Careul control o the range o proteins

used or immunizations and antibody

Electrophoresis Simplified

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 recommendedto screen for and monitor monoclonal gammopathies.

(800) 835-6497 www.sebia-usa.com

... serum ree light chain

elevations were associated

with an increased risk o

progression to lymphoma

among HIV-positive patients.
http://www.mlo-online.com/http://www.mlo-online.com/


3/7www.mlo-online.com

purifcations as well as the use o antisera

pools o >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 o running a FLCtest is that it measures concentrations in

serum rather than in urine. One o the

important unctions o the kidneys is to re-

absorb and catabolize small proteins, such

as ree light chains, which have been fl-

tered rom the blood in the glomeruli. It is

only when this capacity or re-absorption

is overwhelmed that signifcant quantities

o ree light chains can pass through the

kidney tubules and into the urine. There-

ore, many patients with small plasma cell

tumors are ound to have abnormal serumree light chain results while their urine

appears normal.

The sensitivity o the assay, however,is largely disease specifc (i.e., or only

those diseases with higher likelihoods

o having ree 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 o the serum

IFE and the FLC were 87% and 74%,

respectively. I one breaks down the

sensitivity analysis by disease, however,the respective sensitivities are as ollows:

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 or some

patients. First published online in Leu-

kemia in November 2008, the article byDispenzieri, et al, states that or the

purpose o screening or monoclonal

proteins or all diagnoses except AL, the

FLC can replace the 24-hour urine IFE.

Once a diagnosis o monoclonal gam-

mopathy is made, however, the 24-hour

protein IFE should be perormed. For AL

screening, however, the urine IFE should

still be done in addition to the serumtests, including the serum FLC.2

Howard Robin, MD, medical director

o laboratory services at Sharp Memo-

rial Hospital in San Diego, CA, says he

has been using the FLC assay or more

than two years or screening, diagnoses,

and prognostications. Yet, he says urine

collections are a problem. One o the

problems with urine is that we seldomget 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 ree

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
http://www.mlo-online.com/mailto:[email protected]://www.a2la.org/http://www.mlo-online.com/


4/7

c o v e r s t o r y

www.mlo-online.com

they are not getting 24-hour urine.

The rationale or emphasizing the

need or the 24-hour urine protein elec-

trophorsis in ollowing patients with light

chain myeloma is that there is a poor cor-

relation between the serum FLC and theurinary monoclonal protein as measured

by urine PEL3and or patients with amy-loidosis, serial 24-hour urine measure-

ments are critical or monitoring the status

o a patients nephrotic syndrome.

As or any disadvantages in using an

FLC assay, Mead points to two. The

material cost o running serum Freelite

assays is greater than that o a simple

urine electrophoresis gel. When costing

analysis has included storage, process-

ing, urine immunofxation, time or 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 eective

than urine testing. This is evidenced by

the act that the Medicare reimburse-

ment costs or the urine panel o tests

is higher than or 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

Another issue that is sometimes

raised is the accuracy o FLC assayscompared to other tests. Some aspects

o analytical perormance have been

criticized; and it is true that the precision

and accuracy does not equal that o a C3

assay, or example, says Mead. This is

understandable when you consider that

ree light chains are monoclonal proteins

that can vary by more than a thousand-

old in concentration and may exist in

dierent polymeric orms.

David Keren, MD, medical director

o Warde Medical Laboratory, a private

reerence lab in Ann Arbor, MI, says he

uses FLC assays every day with excellentresults. But he agrees there can sometimes

be a computation problem. In a ew

cases, we have gotten a alsely low value

because o higher levels o antigen. It is

uncommon, but it is an issue.

A simple test to run

For the laboratory proessional, running

a FLC assay is simple. As long as the

nephelometer/turbidimeter is correctly

programmed and appropriately main-

tained, running Freelite assays is aseasy as running other serum tests, says

Mead. No special training is required to

run the tests but a basic understanding o

the biology o ree light chains is helpul

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; andBayer Advia. And since it is the kappa/

lambda ratio which is read, Dr. Robin

notes that it is the systems sotware 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 resh and

rozen sera rom 282 individuals aged 21

to 90. By including 100% o donors, the

normal diagnostic range or FLC kappa/

lambda was set at 0.26 mg/L to 1.65 mg/L.

Normal kappa FLC levels are 3.3 mg/Lto 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 o kappa

FLC. Those ratios less than 0.26 mg/L

have higher levels o lambda FLC.6

The normal range or kappa/lambda

ratios also is greater than those used or

most other tests in order to provide a larg-

er saety margin or normal patients.

Validating studies

When I irst saw the data, I was

skeptical, admits Dr. Keren, adding

When costing analysis has

included storage, processing,

urine immunofxation, time or

interpretation, or the Medicare

reimbursement costs, however,

there are benefts to using the

serum assays.


5/7www.mlo-online.com MLO July 2009

Bio-Rad Laboratories C L I N I C A L D I A G N O S T I C S

MONOLISA

,all that and more

With outstanding performance and standardized procedures,the MONOLISA Hepatitis product line is only one of the

great buys you get shopping with Bio-Rad.

MONOLISA

HEPATITIS ASSAYS

AND MORE

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 recommendedor the initial evaluation o suspected

myeloma; or prognosis o plasma cell

dyscrasias; and or 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 i only

serum was analyzed using an FLC assay

combined with PE and IFE. Even as ar

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 o Freelite or screening, diagnosing,

and monitoring patients.

In a 2007 article published in Clini-

cal Lymphoma & Myeloma, Sundar

Jagannath looked at ree light chain

measurements at short sampling inter-

vals given that the hal-lie o FLC is

less than six hours. He said such testing

could allow real-time measurement o

treatment-induced tumor kill and could

possibly provide prompt indications ochemosensitivity, dose adequacy and the

need or alternative approaches.8

Hutchison, et al, in a 2008 study

published in BMC Nephrology, looked

at myeloma patients with renal ailure.

The researchers concluded: The diag-

nostic accuracy o these assays and their

rapid laboratory turnaround time should

aid nephrologists in their assessment o

acute renal ailure.9

Interestingly, Dr. Robin recommends

that patients over 50 with renal ailureshould also be screened or monoclonal

gammopathies.

... this assay is now recommended

or the initial evaluation o

suspected myeloma; or prognosis

o plasma cell dyscrasias;

and or monitoring oligosecretory

myeloma and AL amyloidosis.

F L c A s s A y s


6/7July 2009 MLO www.mlo-online.com

c o v e r s t o r y

Many labs are moving to Lean processing and single piece flow. Now you dont need to wait for samples tospin in a slow conventional centrifuge. The StatSpin Express family of small high speed centrifuges is youranswer to rapid sample processing.

The high speed horizontal StatSpin Express 4 provides flat gel separation in only 3 minutes. Samples can beput directly on the analyzer. Both the StatSpin Express 3 and StatSpin Express 4 hold 8 tubes up to 10 mLThey have a rugged brushless motor for added reliability and the entire unit is backed by a 2-year warranty.Plus they are incredibly easy to use. Just push the button and walk away.

Anywhere a STAT sample isneeded, the StatSpin Express 2provides Platelet Poor Plasma injust 2 minutes. The small foot-print lets you put it anywhere inthe lab.

Tired of waiting for samplesto spin? Place any Express

centrifuge next to youranalyzer and get results fast!

To get up and runningcontact Iris Sample Processing or your Iris distributor .

Tired of waiting for samples to spin?In just 3 minutes a StatSpin

Express can get you up and running!

A Division of IRIS International, Inc.

New developments

While it is a certainty that other indepen-

dent studies will be done using Freelite,

Mead says his company will continue to

ocus on ree light chains as well as heavy

chains. The current ocus o research islooking at dierent applications or serum

ree light chain analysis. For example,

a presentation at a recent conerence

reported that serum ree light chain eleva-

tions were associated with an increased

risk o progression to lymphoma among

HIV-positive patients. Here in the U.K.,

we are currently providing support or a

trial o extended dialysis to remove ree

light chains and improve the prognosis or

myeloma patients with acute renal ailure

(caused by light chain cast nephropathy).We are also investigating whether ree

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

o the ree light chain ratio (kappa/lambda)

could provide a more sensitive indication

o monoclonal disease than simple eleva-

tions o one light chain. This is because

a tumor will usually express a surplus

o one light chain but also suppress theproduction o the alternate light chain. As

a result o this observation, we are now

developing assays that will determine these

ratios or intact immunoglobulins (e.g.,

the IgGkappa/IgGlambda ratio). Our frst

ull report o these assays has now been

accepted or publication. The preliminary

results indicate that these assays will

be useul or diagnosis, prognosis and

monitoring o plasma cell tumors. They

will be complementary to our serum ree

light chain assays because they provide asensitive marker or those tumors which

produce little ree light chain.

Richard R. Rogoski is a reelance journalist based inDurham, NC. His extensive list o published articleshave dealt with new developments in the felds ocardiology and cardiac surgery; imaging technol-ogy; inormation technology; and the business side ohealthcare. Contact him at [email protected].

Reerences:

Katzmann JA, Kyle RA, Benson J, Larson DR,1.Snyder MR, et al. Screening Panels or Detectiono 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 or serum ree light chain analysis in multiplemyeloma and related disorders. Leukemia. 2009.

Dispenzieri A, Zhang L, Katzmann JA, et al. Ap-3.praisal o immunoglobulin ree light chain as amarker o response. Blood. 2008;111:4908-4915.

Katzmann JA, Dispenzieri A, Kyle RA, Snyder MR,4.Plevak MF, Larson DR, Abraham RS, Lust JA, MeltonIII LJ, Rajkumar SV. Elimination o the Need or UrineStudies in the Screening Algorithm or MonoclonalGammopathies by Using Serum Immunofxation andFree Light Chain Assays. Mayo Clin Proc. 2006.

Hill PG, Forsyth JM, Rai B, Mayne S. Serum Free5.Light Chains: An Alternative Test to Urine Bence

Jones Proteins When Screening or MonoclonalGammopathies. Clin Chem. 2006.

Katzmann JA. Clark RJ, Abraham RS, Bryant S,6.Lymp JF, Bradwell AR et al. Serum ReerenceIntervals and Diagnostic Ranges or Free Kappaand Free Lambda Immunoglobulin Light Chains:Relative Sensitivity or Detection o MonoclonalLight Chains. Clin Chem. 2002.

Bradwell AR, Carr-Smith HD, Mead GP, Tang LX,7.Showell PJ, Drayson MT, Drew RL. Highly sensitive,automated immunoassay or immunoglobulin reelight chains in serum and urine. Clin Chem. 2001.

Jagannath S. Value o Serum Free Light Chain Test-8.ing or the Diagnosis and Monitoring o MonoclonalGammopathies 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 ree light chain measurement aids

the diagnosis o myeloma in patients with severerenal ailure. BMC Nephrology. 2008.
http://www.mlo-online.com/mailto:[email protected]://www.mlo-online.com/mailto:[email protected]


7/7www.mlo-online.com MLO July 2009 43

Documents

0709_coverstory