Free Calcium in Serum. II.Rigor of Homeostatic - Clinical Chemistry
Understanding the Serum Free LightUnderstanding...
Transcript of Understanding the Serum Free LightUnderstanding...
Understanding the Serum Free LightUnderstanding the Serum Free Light Chain Test
Linda Traylor, Ph.D.Technical Director
The Binding Site, Inc.San Diego, CA
ObjectivesObjectives
• Plasma Cell Involvement andPlasma Cell Involvement and Immunoglobulin Proteins
• Importance of Serum Free Light Chains in• Importance of Serum Free Light Chains in Plasma Cell DyscrasiasS F Li ht Ch i T t F lit• Serum Free Light Chain Test - Freelite
• Provide Patient Examples of Monitoring Disease
• Practical Considerations – Lab Results
Multiple Myeloma And Amyloidosis Are B th C Of Th Pl C llBoth Cancers Of The Plasma Cell
• Cancerous plasma cells can accumulate in the bone marrow and cause:– Low blood counts (e g anemia)Low blood counts (e.g., anemia)– Bone damage– Abnormally high protein levels in the blood and/or urine– Increased risk of infection– Increased risk of infection– In amyloidosis, proteins can deposit on critical organs (e.g.,
heart, kidneys) and cause damage to those organs
• Testing is important to monitor the disease so that it can be treated before any of these things occur
Bone Marrow AspirateBone Marrow Aspirate
Plasma Cells Produce Immunoglobins ( l ll d ib di )(also called antibodies)
Plasma CellPlasma CellPlasma CellPlasma CellAntibodies Antibodies
(Immunoglobulins)(Immunoglobulins)
There Are Three Immunoglobulins Commonly Associated withCommonly Associated with
Myeloma/Amyloidosis
IgAIgA IgGIgG IgMIgM
Plasma Cells Produce Fully Formed I l bl li d “F Li ht Ch i ”Immunogloblulins and “Free Light Chains”
ImmunoglobulinsImmunoglobulins
SPE and IFE
Free light chain
Plasma CellPlasma Cell
Free light chain test = Freelite
Free Light ChainsFree Light ChainsPlasma CellPlasma Cell
Methods to Detect and/or Quantify Immunoglobulins and Free LightImmunoglobulins and Free Light
Chains• Protein electrophoresis• Protein electrophoresis
– Serum (SPEP)– Urine (UPEP)Urine (UPEP)
• Immunofixation electrophoresis (IFE)• Capillary zone electrophoresis (CZE)• Nephelometry and turbidimetry
– Quantitative immunoglobulins: measure levels of I G I A I M I E I DIgG, IgA, IgM, IgE, IgD
– Serum free light chain assays: measure levels of kappa, lambda, and their ratiokappa, lambda, and their ratio
Serum Protein ElectrophoresisSerum Protein Electrophoresis
1 2Alb α1 α2 β γ
Normal Monoclonal Disease
Serum Immunofixation Electrophoresis (IFE)
N l Monoclonal DiseaseNormal Monoclonal Disease
Immunoglobulins Are Made Up Of g pHeavy Chains And Light Chains
Light ChainsLight ChainsImmunoglobulinImmunoglobulin
Light ChainsLight Chains
H Ch iH Ch iHeavy ChainsHeavy Chains
There Are Two Types Of Free Light Chains – Kappa And Lambda
Kappa Free Kappa Free Light ChainsLight Chains
Lambda Free Lambda Free Light ChainsLight Chains
Normal Levels of Polyclonal I l b li d F Li ht Ch iImmunoglobulins and Free Light Chains
IgG: 7 000 16 000 mg/L• IgG: 7,000 – 16,000 mg/L• IgA: 700 – 4,000 mg/L• IgM: 400 – 2 300 mg/LIgM: 400 2,300 mg/L• IgD: 0 – 80 mg/L• IgE: 0.01 mg/Lg g• Free κ: 3.3 – 19.4 mg/L• Free λ: 5.7 – 26.3 mg/L
In multiple myeloma, the concentration of monoclonal proteins may exceed 100,000 mg/L, or the level of monoclonal proteins may be so low as to be hidden within the polyclonal proteins or the tumorso low as to be hidden within the polyclonal proteins, or the tumor may be truly nonsecretory
Monoclonal Plasma Cells in Bone Marrow by Immunoflourescence with Specific Anti-lambda or u o ou esce ce t Spec c t a bda o
Anti-kappa Antibodies
Free Light Chain Assays: Polyclonal Ab to Sequestered Light Chain Epitopes
S f
Kappa
Sequestered surface
Antibody
targetAntibody
targetHeavy chain
Light chainLambda
The FreeLite Test Measures Serum Free Light Chains
• Latex Reagent: consisting ofLatex Reagent: consisting of monospecific polyclonal antibody coated onto polystyrene latexonto polystyrene latex
• Mixed with serum sample and analyzed using a nephelometer (light scatter) orusing a nephelometer (light scatter) or turbidimeter (light absorption)Q tifi d k t ti l bd• Quantified: kappa concentration, lambda concentration and kappa/lambda ratio
Manufacturing of Sheep Antibodies
• Expose American Sheep to HumanExpose American Sheep to Human Proteins (Antigens) using specific immunization and tolerization protocolimmunization and tolerization protocol– Queens Award for Innovation
Patented and Trade Secret– Patented and Trade Secret– Specific Polyclonal Sheep Antibodies to
Human ProteinsHuman Proteins • Collect sheep plasma in USA and ship to
UK for processingUK for processing• Purification and Rolling Pool Process
Why is it important to measureWhy is it important to measure free light chains?g
The Free Light Chain TestThe Free Light Chain Test
• Serum test specific for free light chainsSerum test specific for free light chains
• More sensitive for detection of free light chains than SPE or IFEchains than SPE or IFE
• May indicate if therapy is working, sometimes earlier than SPE or IFEsometimes earlier than SPE or IFE
• May detect relapse of disease earlier than other diagnostic tests.t a ot e d ag ost c tests
Sensitivity of Assays forLi h Ch iLight Chains
1000
on, m
g/L
100SPEP
CZEIFE
cent
ratio
10
sIFE
UPEP
tein
con
c 10uIFENormal range in serum
M-p
rot
1sFLCNormal range in urine
Adapted from Bradwell et al. Serum Free Light Chain Analysis. 3rd ed. 2005. The Binding Site, Ltd.
The Free Light Chain TestThe Free Light Chain Test
• Serum test specific for free light chainsSerum test specific for free light chains
• More sensitive for detection of free light chains than SPE or IFEchains than SPE or IFE
• May indicate if therapy is working, sometimes earlier than SPE or IFEsometimes earlier than SPE or IFE
• May detect relapse of disease earlier than other diagnostic tests.t a ot e d ag ost c tests
Patient: sFLC and IgG Levels During Treatment
IgG κ Multiple Myeloma
250
300
n,
60
70Kappa FLC Monoclonal IgG
CVAMP
g p y
150
200
250
ncen
trat
io/L
40
50
IgG
, g/L
HDM/PBSCT Dex
0.1% Plasma cells
50
100
150
sFLC
κC
om
g
10
20
30
onoc
lona
l
0
50
0 200 400 600 800 1000 1200 1400Ti f P t ti (d )
s
0
10 Mo
Time from Presentation (days)
Mead et al BJH 2004 126:348
The Free Light Chain TestThe Free Light Chain Test
• Serum test specific for free light chainsSerum test specific for free light chains
• More sensitive for detection of free light chains than SPE or IFEchains than SPE or IFE
• May indicate if therapy is working, sometimes earlier than SPE or IFEsometimes earlier than SPE or IFE
• May detect relapse of disease earlier than other diagnostic tests.t a ot e d ag ost c tests
Serum Free Light Chains: An Earlier I di ti f R l Th IFEIndication of Relapse Than IFE
, mg/
L4-6-mo Earlier Indication of Relapse
Mosbauer et al. Haematologica 2007:92:275-276.
Practical ConsiderationsPractical Considerations
Normal Ranges for Serum FreeNormal Ranges for Serum Free Light Chains
Units (mg/L) Units (mg/dl)
Kappa:3.3–19.4 mg/L
Kappa:.33–1.94 mg/dL
Lambda: 5.7–26.3 mg/L
Lambda:.57–2.63 mg/dL
k/l ratio:0.26–1.65
k/l ratio:0.26–1.65
ExamplesExamplesMay - Lab A
385 mg/L385 mg/L
July – Lab B
Laboratory VariabilityLaboratory Variability
• Absolute values in the serum free light chain assay can vary from lab to labchain assay can vary from lab to lab
– Different chemistry instrumentsDifferent chemistry instruments– Different kits for different instruments– Normal lab to lab variation
Laboratory VariabilityLaboratory Variability
If you change the laboratory where your serumIf you change the laboratory where your serum free light chains are measured:
– If possible, obtain some of the last sample and run it along with the new sample orE t bli h b li f f li ht– Establish a new baseline for your serum free light chain levels.
The change in your absolute sFLC level should not effect the clinical interpretation.p
International guidelines for sFLC ganalysis in MM and related disorders
• Assessment of responseSerial sFLC should be routinely performed in– Serial sFLC should be routinely performed in• AL amyloidosis
Oli t MM• Oligosecretory MM • All MM patients to define a stringent CR
Negative serum and urine IFE Absence of clonal cells in BMNormal sFLC ratio
Dispenzieri et al. Leukemia epub 20 Nov 2008
International guidelines for sFLC analysis in MM and related disorders
• Screening– Recommended in combination with serum
electrophoresis– sFLC can replace 24 h urine IFE*
*For screening for AL amyloidosis, 24 h uIFE still recommended
Dispenzieri et al. Leukemia epub 20 Nov 2008
Interpretation of sFLC Results:
• sFLC testing is considered abnormal when the clonal
Updates to Guidelinesg
free light chain is increased above normal and the ratio is abnormal (Durie BG, Harousseau JL, Miguel JS, et al. International uniform response)International uniform response)
• Measurable disease with free light chain testing hasMeasurable disease with free light chain testing has been defined as a clonal free light chain concentration of at least 100 mg/L with an abnormal ratio (Durie BG, Harousseau JL Miguel JS et al International uniformHarousseau JL, Miguel JS, et al. International uniform response)
International guidelines for sFLC analysis in MM and related disorders
• Prognosis– sFLC should be measured at diagnosis for g
all patients with MGUS, SMM or MM, Solitary plasmacytoma and AL amyloidosis
Dispenzieri et al. Leukemia epub 20 Nov 2008
Q ti ?Questions?
Thank you!y