ד"ר יעל דויטשר-צ'רטקוב,

Transcription

1 הביולוגי ועקרון המדידה של הבסיס Free Light בסרום Chains רמות ד"ר יעל דויטשר-צ'רטקוב, המעבדה ההמטולוגית, רמב"ם - הקריה הרפואית לבריאות האדם

2 Free light chains: Facts and Numbers Plasma cells 2 Kappa : 1 Lambda producing cells Heavy chain Kappa: ~215 amino acids Mainly monomer MW 22.5kDa Serum half life: 2-4hr Lambda: ~215 amino acids Mainly Dimer MW 45kDa Serum half life: 3-6hr Light chain

3 Measurement of FLC: Serum vs Urine 1 1. Serum is a more sensitive measurement of FLC levels than urine. increased levels of free light chains will be observed earlier in serum than in urine due to their reabsorption and catabolism in renal tubular cells. 2. While increasing serum concentrations indicate worsening disease, falling urine g g g concentrations may falsely suggest disease stabilization or improvement. 2

4 Brief history of FLCH measuring 1845 Detection of Bence Jones proteins 1956 Light chains are located on immunoglobulin molecules The monoclonal light chains immunoglobulin are identified as Bence Jones proteins

5 Brief history of FLCH measuring 1845 Detection of Bence Jones proteins 1956 Light chains are located on immunoglobulin molecules The monoclonal light chains immunoglobulin are identified as Bence Jones proteins Antibody against total light chain immunoglobulin 1972 Structure of the antibody molecule 1975 Generation of the first monoclonal antibodies (Georges Köhler) and (César Milstein) 1975 Size-separation column chromatography, was used to isolate FLCH from intact immunoglobulins, prior to analysis Commercial kit for quantifying FLCH using latex-enhanced turbidimetric / nephelometric assays using polyclonal Ab

6 Brief history of FLCH measuring 2012 Potential monoclonal antibodies for immunoassays - Clinical comparison of new monoclonal antibody-based nephelometric assays for free light chain kappa and lambda to polyclonal antibody-based assays and immunofixation electrophoresis. Hoedemakers, Rein M.J.et al. Clin Chem Lab Med 2012;50(3): Diagnostic accuracy of monoclonal antibody based serum immunoglobulin free light chain immunoassays in myeloma cast nephropathy Colin A Hutchison, Paul Cockwell and Mark Cook BMC Clinical i l Pathology 2012, 12:12 doi: / To be continued

7 Serum free light chain immunoassay Heavy chain Kappa Variable domain Light chain Constant domain Hidden surface Exposed surface Lambda

8 Considerations in FLCH measuring Sensitivity Normal ranges Free kappa, Lambda 3-25 mg/l IgG gr/l IgA 7-35 gr/l IgM 4-28 gr/l IgD mg/l IgE mg/l 1000 mg/l) concent tration ( Ligh ht chain Adapted from: Serum free light chain analysis, 3 rd edition AR Bradwell 2005 SPE CE IF UPE FLC Normal range in serum

9 Considerations in FLCH measuring Specificity Cross reactivity with intact Immunoglobulin and other serum proteins Reference intervals Total 1 Free 2 KAPPA mg/L mg/L LAMBDA mg/L mg/L RATIO (K/L) Reference: 1) Clin Lab Sci. 2011; 24: ) Clin Chem.2002; 48: Current data indicates that normal serum immunoglobulin levels cause overestimation of only ~10% (3 4 mg/l, representing a cross-reactivity of about 0.05%) at normal FLC concentrations. From: Quantitative Serum Free Light Chain Assay Analytical Issues.Clin Biochem Rev Jill Tate et al.

10 Considerations in FLCH measuring Specificity Structural heterogeneity : Monomers, dimmers and higher polymers.

11 FLCH measuring The A, B, C Ab-Ag reaction The role of Latex Reaction kinetics: practical considerations Nephlometric-turbidometric instruments

12 Ab-Ag reaction Serum proteins including FLCH Ab-Ag complexes are formed with FLCH LATEX Ab-Ag complexes are formed with FLCH

13 LATEX bead Latex agglutination procedures in Immunodiagnosis F. Javier Gella at al. Pure&Appl. Chem. 1991,Vol.63, No.8, pp

14 Heidelberger-Kendall Curve Kinetics of immunoprecipitation Antibody excess Equivalence Antigen excess Antibody precipitation / light scattering Antigen concentration

15 Heidelberger-Kendall Curve Kinetics of immunoprecipitation Antibody precipitation / light scattering Antibody Equivalence Antigen excess excess Antigen (FLCH) concentration

16 Standard curve Protein Standardization III: Method Optimization. Basic Principles for Quantitative Determination of Human Serum Proteins on Automated Instruments Based on Turbidimetry or Nephelometry. Søren Blirup-Jensen. Clin Chem Lab Med 2001; 39(11):

17 Heidelberger-Kendall Curve Kinetics of immunoprecipitation Antibody precipitation / light scattering Antibody excess Equivalence Antigen excess Antigen (FLCH) concentration

18 Heidelberger-Kendall Curve Kinetics of immunoprecipitation Antibody precipitation / light scattering Antibody excess Equivalence Antigen excess Antigen (FLCH) concentration

19 Heidelberger-Kendall Curve Kinetics of immunoprecipitation Antigen excess FLC concentrations can range from <1 mg/l to >100,000 mg/l.

20 Aprox. Normal range Waldenström s Protein Standardization III: Method Optimization. Basic Principles for Quantitative Determination of Human Serum Proteins on Automated Instruments Based on Turbidimetry or Nephelometry. Søren Blirup-Jensen. Clin Chem Lab Med 2001; 39(11):

21 When antigen excess is not detected automatically

22 Nephelometry vs Turbidimetry Detector Incident monochromatic light Turbidimetry Detector Nephelometry

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24 Laboratory instruments categorized by the principle i detection ti Turbidimetric ubd instruments stu ts Beckman IMMAGE Binding Site SPAPLUS (detect antigen excess) COBAS Integra 400 (detect antigen excess) Roche Hitachi Modular P Siemens ADVIA 1650 Beckman Coulter AU 400 Roche Cobas c501 and c502 Nephelometric instruments Siemens BNTMII Siemens BN ProSpec Binding Site MININEPHPLUS Radim Delta

25 תודה... דרר דר''ר שריג גלית יהודית הודת גשייט מארש אפרת

26 The End