Measuring Myeloma in the LAB. BL Ferry Clinical Lead Sept 15 th 2014
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1 Measuring Myeloma in the LAB BL Ferry Clinical Lead Sept 15 th 2014
2 Oxford Immunology Laboratory Clinical Immunology Churchill
3 WHAT DO WE MEASURE AND HOW? Clinical Immunology Churchill
4 Serum proteins Proteins in serum, urine Intact immunoglobulins Serum Free lite chains Hevy lite Methods used: 1. Nephelometry 2. Electrophoresis and imunofixation 3. viscosity and cyroglobulins Clinical Immunology Churchill
5 LAB JOURNEY for a: Myeloma Screen RESULT Currently, SERUM + URINE should arrive 1. Serum arrives in lab Arrival in LAB 2. Use nephelometry to find Ig Level in g/l. AT SAME TIME, sample is run on electrophoresis to detect paraprotein These two results tell us Ig levels + if paraprotein present 3. If Paraprotein present--- Immunofix, densitometry to measure how much, 4. Urine electrophoresis, urine fix and urine densitometry, freelite
6 NEPHELOMETRY How much Immunoglobulin does patient have? Does patient have low or high levels of IgA, IgG and IgM? Does patient have a paraprotein in Serum? in Urine?
7 1. Nephelometry Measurement of serum proteins e.g. Immunoglobulins, M protein complement, α1-antitrypsin, β 2 microglobulin The more paraprotein in the patients serum, the more light is scattered and the higher the measurement Clinical Immunology Churchill
8 1. Nephelometry OXFORD LAB ---- Normal range IgG 6-16g/L IgA 0.5-3g/L IgM g/L Done every day, very quick result and very accurate.
9 1. Nephelometry WESTGARD RULES FOR QUALITY CONTROL ASK YOUR LAB IF THEY APPLY THESE RULES!!!
10 2 Electrophoresis and Immunofixation 1. Take patients serum and run on an electrophoresis gel to separate out serum proteins 2. If a paraprotein in present in serum, then 3. Immunofix that gel by adding in a detection marker to detect whether paraprotein is IgA, IgG, IgM or IgD or E.
11 ELECTROPHORESIS GEL: Normal Gamma and Hypogamma regions Alpha 1 Alpha 2 Beta 1 Beta 2 Gamma Region Normal Gamma region Hypogamma
12 2. Electrophoresis QUESTIONS Alpha and beta regions Immunoglobulins Is a paraprotein there? What proteins are missing? Where is the PP? If PP is present, gives an idea of how big 5. If PP is present, indicates if other Igs are decreased Normal Serum No protein Or clear urine?band in Beta Band in Gamma Normal? Band in beta Immunoparesis Clinical Immunology Churchill
13 Abnormal Electrophoresis Gels Paraproteins in beta,gamma Hypogamma, Hypergamma and other regions
14 2. Electrophoresis Alpha and beta regions Immunoglobulins Immunofix Normal Serum No protein Or clear urine?band in Beta Band in Gamma Normal? Band in beta Immunoparesis Clinical Immunology Churchill
15 3. Immunofixation Serum Urine Clinical Immunology Churchill
16 Immunofixation Serum Urine Clinical Immunology Churchill
17 IMMUNOFIXATION
18 4. DENSITOMETRY HOW LARGE IS THE paraprotein? Are other immunoglobulins decreased?
19 Densitometry Electrophoresis
20 Electrophoresis Densitometry Quantifies paraprotein
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22 ONE SERUM SAMPLE can be tested for: Nephelometry, Electrophoresis, Immunofixation & Densitometry IgA 0.2 g/l IgM 0.2 g/l IgG 24.5 g/l
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24 Serum Paraprotein Negative Do nephelometry and Electrophoresis tally? YES (and urine is clear) Unusual in gel?, any relevant clinical details? NO RESULT NO paraprotein. Serum Paraprotein Negative Do nephelometry and Electrophoresis tally? YES (and no urine received) Unusual in gel?, any relevant clinical details? NO RESULT NO paraprotein, BUT ASK FOR URINE Urine Paraprotein Negative YES? ( Currently, we do not perform free lite) RESULT- No paraprotein Urine Paraprotein Positive YES RESULT- Positive, refer to haematology
25 Serum Paraprotein Positive Do nephelometry and Electrophoresis tally? YES (and urine is clear) Unusual in gel?, any relevant clinical details? irrelevant Do Free lite by nephelometry RESULT Positive paraprotein. Refer to haematology Serum Paraprotein Positive Do nephelometry and Electrophoresis tally? YES (NO urine received) Unusual in gel?, any relevant clinical details? Irrelevant Do FreeLite by nephelometry RESULT Positive paraprotein, ASK FOR URINE. Refer to haematology Urine Paraprotein Negative YES Freelite result important RESULT- No paraprotein in Urine Urine Paraprotein Positive Free lite result important RESULT- Positive, refer to haematology YES
26 Serum Free Light Chain Measurements: Key to Early Stage Detection of Monoclonal Gammopathies
27 Freelite Analysis Immunoglobulin Production Kappa FLC Lambda FLC Normal range: mg/l mg/l κ/λ ratio Median = 0.6 (Range = ) Katzmann et al., Clin Chem (2002); 48:
28 Glomerular Filtration of Light Chains κflc - 25 kda Glomerulus kda filters λflc - 50 kda Does not filtrate as much as kappa, So more Lambda in serum and a longer half life Urine FLCs
29 Renal reference range for κ/λ ratio Serum lambda FLC (mg/l) 1,000 Normal κ/λ ratio AKI - Myeloma (λ) AKI - Myeloma (κ) AKI - No MG Normal sera Renal range κ/λ = ,000 Serum kappa FLC (mg/l) Immunology use both normal and renal ranges
30 Clinical Utility of the Serum Free Light Chain Assay: 1. Screening for monoclonal gammopathies: More sensitive/specific than urine BJP tests 2. Prognosis: MGUS, SMM and MM Risk stratification and optimal follow up 3. Monitoring: Assessment of progresssion/response 2009 IMWG MM and AL amyloidosis guidelines 2010 IMWG MGUS/SMM guidelines 2010 BCSH/UKMF guidelines
31 1. Screening for Monoclonal Gammopathies g/l mg/l Monoclonal Intact Immunoglobulins Serum electrophoresis Serum electrophoresis or Hevylite Monoclonal Free Light Chains Urine electrophoresis Urine electrophoresis or Freelite
32 Freelite Assay vs Urine Analysis sflc advantages: A. Analytically more sensitive than UPE/uIFE B. Biologically more specific than UPE/uIFE C. Serum analysis
33 A. Sensitivity of light chain analysis techniques Light chain concentration (mg/l) SPE CZE sife Normal range in serum Freelite UPE uife
34 Freelite Assay vs Urine Analysis sflc advantages: A. Analytically more sensitive than UPE/uIFE B. Biologically more specific than UPE/uIFE C. Serum analysis
35 B. Biological Specificity κflc Glomerulus λflc Proximal tubule 0.5-1g/day FLC produced by normal healthy individuals 10-30g/day FLC reabsorption & breakdown Urine FLCs = Production minus Reabsorption
36 Freelite Assay vs Urine Analysis sflc advantages: A. Analytically more sensitive than UPE/uIFE B. Biologically more specific than UPE/uIFE C. Serum analysis
37 C. sflc is an assay performed on serum Monoclonal Free Light Chains mg/l X Serum Urine Freelite electrophoresis Analysis 1. Patient cant or wont produce urine
38 Freelite Assay vs Urine Analysis sflc advantages: A. Analytically more sensitive than UPE/uIFE 10 fold or more B. Biologically more specific than UPE/uIFE Light chains not always deposited in urine C. Serum analysis Urine not always supplied
39 GP will request Produced by Karthik and the immunology lab for GPs LAB will? *
40 GP will request Produced by Karthik and the immunology lab for GPs LAB will automatically? *
41 THANKS TO Clinical Immunology laboratory Fred Gamper Dawn Simpson James Hoy
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54 Paraprotein measurement Paraprotein is the specific antibody that is produced by and is unique to an individual patient s myeloma cells. Measurement of paraprotein levels is important both in diagnosing myeloma and as an indicator of changes in it s activity. For this reason, paraprotein measurements are done regularly to see how well treatment is working and to check that the myeloma is remaining stable during periods when you are not receiving active treatment. Paraprotein levels will therefore fall with successful treatment and increase when the myeloma becomes active again. What tests are used to measure paraprotein? Paraprotein measurement can be made on either a blood and / or urine sample. A test called protein electrophoresis is carried out to measure the levels of paraprotein present. Immunofixation is a similar test to electrophoresis but it is more sensitive. This test may be carried out to investigate the results of an electrophoresis test in more detail and it can be used to identify the type of paraprotein the myeloma cells are producing. The different types of paraprotein are described in more detail in the Types of myeloma information sheet. You can get a copy of this information sheet from your doctor or nurse.
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60 Serum Free Light Chain Measurements: Key to Early Stage Detection of Monoclonal Gammopathies
61 Monoclonal Gammopathies Plasma cell Kappa FLC MGUS and SMM Lambda FLC 80% 15-20% 1-2% Intact Immunoglobulin Light chain only Nonsecretory AL Amyloidosis LCDD Multiple myeloma
62 Freelite Analysis Immunoglobulin Production Kappa FLC Lambda FLC Normal range: mg/l mg/l κ/λ ratio Median = 0.6 (Range = ) Katzmann et al., Clin Chem (2002); 48:
63 Glomerular Filtration of Light Chains κflc - 25 kda λflc - 50 kda Glomerulus kda filters Urine FLCs
64 Polyclonal sflc increase as GFR decreases Kappa FLC Lambda FLC Hutchison Clin J Am Soc Nephrol 3: , 2008
65 κ/λ ratio increases as kidney function decreases Renal reference range κ/λ ratio: Hutchison Clin J Am Soc Nephrol 3: , 2008
66 Renal reference range for κ/λ ratio Serum lambda FLC (mg/l) 1,000 Normal κ/λ ratio AKI - Myeloma (λ) AKI - Myeloma (κ) AKI - No MG Normal sera Renal range κ/λ = ,000 Serum kappa FLC (mg/l) Hutchison et al. BMC Nephrology 2008, 9:11
67 Clinical Utility of the Serum Free Light Chain Assay: 1. Screening for monoclonal gammopathies: More sensitive/specific than urine BJP tests 2. Prognosis: MGUS, SMM and MM Risk stratification and optimal follow up 3. Monitoring: Assessment of progresssion/response 2009 IMWG MM and AL amyloidosis guidelines 2010 IMWG MGUS/SMM guidelines 2010 BCSH/UKMF guidelines
68 MGs: monoclonal component distribution MG s with a monoclonal intact Ig ~80% (Require SPE/CZE or Hevylite for detection) Detectable with: SPE/CZE ~70% or UPE ~95% (if supplied) or Freelite ~99% MG s with monoclonal free light chains ONLY ~15% MG s with a monoclonal free light chain ~97% MG s with monoclonal Intact Ig ONLY ~5% Only detectable with: SPE/CZE ~90% or Hevylite ~ 99% (Require Urine or MG s with BOTH Freelite for Detectable with monoclonal detection) either: Intact Ig AND SPE/CZE ~90% or monoclonal free Hevylite ~ 99% or light chains UPE ~95% (if ~80% supplied) or Freelite ~99%
69 1. Screening for Monoclonal Gammopathies g/l mg/l Monoclonal Intact Immunoglobulins Serum electrophoresis Serum electrophoresis or Hevylite Monoclonal Free Light Chains Urine electrophoresis Urine electrophoresis or Freelite
70 Freelite Assay vs Urine Analysis sflc advantages: A. Analytically more sensitive than UPE/uIFE B. Biologically more specific than UPE/uIFE C. Serum analysis
71 A. Sensitivity of light chain analysis techniques Light chain concentration (mg/l) SPE CZE sife Normal range in serum Freelite UPE uife
72 Freelite Assay vs Urine Analysis sflc advantages: A. Analytically more sensitive than UPE/uIFE B. Biologically more specific than UPE/uIFE C. Serum analysis
73 B. Biological Specificity κflc Glomerulus λflc Proximal tubule 0.5-1g/day produced by normal healthy individuals 10-30g/day reabsorption & breakdown Urine FLCs = Production minus Reabsorption
74 sflc is a Biologically More Specific Way of Measuring Light Chain Production Than Urine Analysis Urine Kappa BJP (g/24hr) Severe osteolytic lesions Serum Kappa FLC (mg/l) Urine Kappa BJP (g/24hr) HDC ASCT Serum Kappa FLC (mg/l) Time (months) Patient Time (months) Patient 2 sflc 24 hr urine BJP excretion Alyanakian Am J. Hematol. 2004; 75:
75 Absolute Sensitivity: sflc (Freelite) vs Urine Electrophoresis Screening algorithm Diagnostic sensitivity (%) SPE + sife + uife SPE + sflc All PCDs(n=1877) MM (n=467) AL (n=581) All PCDs MM AL Freelite provides increased sensitivity over urine analysis for symptomatic patients Katzmann, J.A., et al., Clin Chem, 55: 8; 2009
76 Freelite Assay vs Urine Analysis sflc advantages: A. Analytically more sensitive than UPE/uIFE B. Biologically more specific than UPE/uIFE C. Serum analysis
77 C. sflc is an assay performed on serum Monoclonal Free Light Chains mg/l X Serum Urine Freelite electrophoresis Analysis
78 Urine Compliance in the UK Study No. of sera Urine compliance Hill et al Holding et al Beetham et al Abadie et al Robson et al % % % - 35% 653 <5%
79 Serum Electrophoresis alone is an insensitive Monoclonal Gammopathy screen Screening algorithm Diagnostic sensitivity (%) SPE MM AL sife + uife MM AL SPE + sflc MM AL Katzmann, J.A., et al., Clin Chem, 55: 8; 2009
80 Freelite Assay vs Urine Analysis sflc advantages: A. Analytically more sensitive than UPE/uIFE 10 fold or more B. Biologically more specific than UPE/uIFE Light chains not always deposited in urine C. Serum analysis Urine not always supplied
81 MGs: monoclonal component distribution MG s with a monoclonal intact Ig ~80% (Require SPE/CZE or Hevylite for detection) MG s with monoclonal free light chains ONLY ~15% SPE/CZE plus UPE/uIFE ~97% MG s with a monoclonal free light chain ~97% MG s with monoclonal Intact Ig ONLY ~5% (Require Urine or MG s with BOTH Freelite for monoclonal detection) Intact Ig AND monoclonal free light chains ~80%
82 MGs: monoclonal component distribution MG s with a monoclonal intact Ig ~80% (Require SPE/CZE or Hevylite for detection) MG s with monoclonal free light chains ONLY ~15% SPE/CZE plus plus UPE/uIFE Freelite Taking in to account Urine collection issues only ~20% collected in this context ~85% ~99% MG s with monoclonal Intact Ig ONLY ~5% MG s with BOTH monoclonal Intact Ig AND monoclonal free light chains ~80% MG s with a monoclonal free light chain ~97% (Require Urine or Freelite for detection)
83 Screening Sensitivity in Routine Practice: Freelite vs Urine Electrophoresis (All Query MG requests to a laboratory with or without a urine) SPE/CZE+Urine vs SPE/CZE+Freelite Hill et al. Clin Chem (2006); 52: months: 3 extra symptomatic malignancies detected by using Freelite Holding et al. Clin Chem Lab Med (2011); 49: months: 4 extra symptomatic malignancies detected by using Freelite McTaggart et al. Am J Clin Pathol (2013); 140: months: 1 extra symptomatic malignancy detected using Freelite 3-20 symptomatic patients missed per year using urine analysis but that are picked up by Freelite analysis
84 Adapted from Kariyawasan C, et al. Q J Med 2007; 100: Delayed Diagnosis Results in Higher Chance of Increased Clinical Complications Speciality Interval (months) All > 6 GP Haematologist Rheumatologist Nephrologist Neurologist Orthopedic Urgent Care Homeopath Oncologist ENT Physician Total Complication No Complication % 50% 50% 75% 100%
85 Delayed Diagnosis Leads to Increased Incidence of Renal Impairment, Anemia and Bone Disease Months 3-6 Months > 6 Months Infection Neurological Renal Disease Bone Disease Anemia Adapted from Kariyawasan C, et al. Q J Med 2007; 100:
86 Major presenting symptoms of Multiple Myeloma Bone Pain Fatigue Dyspnea (breathing disorder) Asthenia (general feeling of weakness/ill-ness) Weight Loss Major presenting symptoms in ninety two de novo myeloma patients. Adapted from Kariyawasan C, et al. Q J Med 2007; 100:
87 Light Chain-Mediated Renal Insufficiency Never an initial symptom of myeloma (and related disorders) However at Diagnosis >50% of myeloma patients will have renal impairment (Wirk BMT 2011; 46:771-83) 10 20% myeloma patients will have acute renal failure requiring dialysis (Wirk BMT 2011; 46:771-83) Acute tubular necrosis Fanconi s syndrome AL amyloid LCDD Cast nephropathy
88 Light Chain-Mediated Renal Failure: 80% of patients will never recover renal function following treatment resulting in decreased survival. Median OS ~7m
89 Factors predicting independence of dialysis for patients with Light Chain-derived Renal Failure 67 patients (16 multinational renal units): ~ 90% Dialysis-Dependent as a Result of Cast Nephropathy Treated with Various Chemotherapy Regimes Factors Predicting Independence of Dialysis No. of Patients Achieving Independence of Dialysis Time to Initiation of therapy >/=7d <7d 30% 71% Degree of Light Chain Reduction by Day 12 <25% 25-75% >75% 40% 70% 81% Hutchison et al., Nephrol Dial Transplant (2012)
90 Delayed Diagnosis Leads to Poorer Patient Outcomes Medical emergencies such as Renal Failure or Spinal Lesions as a result of a monoclonal gammopathy, are rarely, if ever, an initial reported symptom of the disorder They generally only develop as a result of delayed or missed diagnosis Thus: 1.Rapid, accurate diagnosis at an earlier point of presentation to the NHS, followed by rapid treatment would decrease the incidence/development of these extra, more significant, sometimes life-threatening complications and therefore improve outcomes. 2.Furthermore rapid diagnosis and treatment is also indicated for patients who have unfortunately developed these extra, more significant, sometimes life-threatening complications, as this has also been shown to improve outcomes.
91 2009 IMWG Screening Guidelines Clinical Suspicion sflc SPE sife Recommend Freelite to replace 24 h urine IFE in a general screening algorithm Dispenzieri et al. Leukemia 2009: 23,
92 2012 International Kidney and Monoclonal Gammopathy Research Group Recommendations for Monoclonal Gammopathy Investigation * Freelite Analysis Hutchison et al. Nat Rev Neph (2012) 8:43-51
93 2. Prognosis: MGUS/SMM 1% risk per year 10% risk per year MGUS/SMM WM IgM lymphoma CLL? MM AL amyloid LCDD Solitary plasmacytoma Benign MGUS versus early MM? Follow up MGUS patients: How frequently?
94 Monoclonal Gammopathy of Undetermined Significance (MGUS) Risk Factors for Progression: SPE Serum M Protein >15g/l Serum IFE Serum M Protein NOT IgG Freelite Abnormal sflc ratio Kyle R. NEJM 2002; 346: Rajkumar Blood 2005; 106:
95 MGUS risk stratification model incorporating M-protein size, type and Freelite ratio Risk of progression No. of abnormal risk factors No. patients Absolute risk of progression at 20 years* Low (No risk factors) Low-Intermediate (Any 1 risk factor) High-Intermediate (Any 2 risk factors) High (All 3 risk factors) % % % % * Accounting for death as a competing risk Rajkumar et al. Blood 2005; 106:
96 2010 IMWG Guidelines Risk of progression Recommended Follow-up Low (No risk factors) 6 months then every 2-3 years or at progression Low-Intermediate (Any 1 risk factor) High-Intermediate (Any 2 risk factors) 6 months then annually High (All 3 risk factors) Kyle et al. Leukemia (2010): 1-7
97 Cost Savings associated with using Freelite to Risk Stratify IgG MGUS Patients Standard Practice IMWG Guidelines Asymptomatic Patient Monoclonal IgG <30g/L Asymptomatic Patient Monoclonal IgG <30g/L MGUS or SMM? MGUS or SMM? (~40% of pts) Normal FLCr Abnormal FLCr BM aspirate/bm biopsy Skeletal Survey BM aspirate/bm biopsy Skeletal Survey <10% Clonal BM MGUS >10% Clonal BM SMM Low Risk MGUS <10% Clonal BM Int-High Risk MGUS >10% Clonal BM SMM Monitor Regularly with Haem Appointment Discharge Monitor Regularly with Haem Appointment
98 3. Monitoring Patients with Freelite Which patients can be monitored with sflc following diagnosis? All patients with a LC component to their disease Oligosecretory Intact Immunoglobulin (<10g/L by SPE and <200mg/24h by UPE) Light chain only Nonsecretory AL Amyloidosis LCDD 89% of IIMM also have abnormal Freelite ratio Mead et al. Br J Haematol 2004;126 : Intact Immunoglobulin
99 Monitoring IIMM patients with Freelite 89% of IIMM also have abnormal Freelite ratio at diagnosis Mead et al. Br J Haematol 2004;126 : Intact Immunoglobulin MM Why monitor sflc in IIMM patients: sflc and intact Ig s are independent markers of tumour burden
100 sflc and intact immunoglobulins are independent markers of tumour burden n=164 IgGκ MM R 2 = Freelite κ sflc (mg/l) IgG (g/l) IFM data courtesy of H. Avet-Loiseau
101 ...and may be produced by different plasma cell clones IgGκ multiple myeloma: IgGκ κ FLC Ayliffe Haematologica 2007; 92:
102 Monitoring IIMM patients with Freelite 89% of IIMM also have abnormal Freelite ratio at diagnosis Mead et al. Br J Haematol 2004;126 : Intact Immunoglobulin MM Why monitor sflc in IIMM patients: sflc and intact Ig s are independent markers of tumour burden a. Progression/relapse may be indicated by one but not the other
103 a. Light Chain Escape, Intact Ig Escape and Clonal Tiding FLC κ/λ ratio HLC IgA κ/λ ratio FLC κ/λ ratio 10 1 HLC upper NR FLC upper NR HLC IgA κ/λ ratio Time (days) Time (days) FLC κ/λ ratio 1 FLC upper NR HLC upper NR 10 HLC IgA κ/λ ratio FLC κ/λ ratio 10 1 HLC upper NR FLC upper NR 10 1 HLC IgA κ/λ ratio Time (days) Time (days) 0.1 sflc mig Ludwig et al., 2011 ASH Poster
104 Light chain escape Rising monoclonal free light chain production at relapse without increased monoclonal intact immunoglobulin Brioli et al. Blood 2014 Myeloma IX trial: LCE at relapse in 10.4% - 6.5% IgG 19.9% IgA 85% increase in iflc >200mg/L - classification of relapse requiring treatment despite lack of clinical symptoms IMWG GUIDELINES Light chain escape: Without doing periodic urinary evaluations or serum FLC measurements, this phenomenon can be missed
105 a. For example light chain escape: IgAλ paraprotein Serum lambda FLC Velcade Serum lambda FLC (mg/l) IgA paraprotein (g/l) Time (days) Courtesy of Effie Liakopoulou, Christies Hospital
106 2009 IMWG Guidelines Freelite in patient Monitoring Use dflc (iflc-uflc) (NB NOT FLC ratio) Uniform Response Criteria CR Stringent CR Negative S/U IFE BM plasma cells 5% Negative S/U IFE Normal sflc ratio Absence of clonal cells in BM Routine practice: AL Amyloidosis, LCDD, Oligosecretory MM, LCE Durie et al Leukemia , Dispenzieri et al Leukemia ,
107 How to Effectively Monitor with Freelite Treatment
108 How to Effectively Monitor with Freelite Treatment
109 Any Questions? Keep up-to-date at:
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