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Lothar Siekmann Institute of Clinical Biochemisstry, University of Bonn

Challenges for International Standardization and Traceability - HORMONES - ______________________________________________. Lothar Siekmann Institute of Clinical Biochemisstry, University of Bonn Reference Institute of Bioanalysis, DGKL. http://www.dgkl-rfb.de.

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Lothar Siekmann Institute of Clinical Biochemisstry, University of Bonn

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  1. Challenges for International Standardization and Traceability- HORMONES -______________________________________________ Lothar Siekmann Institute of Clinical Biochemisstry, University of Bonn Reference Institute of Bioanalysis, DGKL http://www.dgkl-rfb.de

  2. Target Concentrations for Steroid Hormones in a Commercial Control Serum __________________________________________________________________ Aldosterone Cortisol Progesterone Estradiol pmol/l nmo/l nmo/l pmol/l _________________________________________________________________________________________________________ ABBOTT 121.9 AMERSHAM 113.1 BAXTER DADE DIR 104.8 2.16 396.4 BAXTER DADE AG ER 244.1 BAXTER DADE AD EXT 196.0 BIOMERIEUX 2.54 539.6 BIOTEX PREMIX 99.4 70.6 3.72 759.9 CAMBRID´GE MEDICAL 120.8 0.86 CIBA CORNING 110.3 CLINICAL ASSAYS 99.3 CYBERFLUOR FIAGEN 88.2 DIAGNOSTIC PRODUCTS 113.1 3.12 119.3 EURODIAGNOSTICS 115.8 FARMOS DIAGN. 99.3 4.67 394.9 IMMUNCHEM COV. COAT 110.3 5.41 348.7 LEECO 113.1 2.99 144.2 MALLINCKRODT 96.6 NMS PHARMACEUTICALS 3.18 205.5 PANTEX IMMUNO 118.6 4.13 190.8 PANTEX IMMUNOCOAT 132.4 7.00 154.9 PHARMACIA DELFIA 99.9 790.0 RSL 169.2 4.77 117.4 SCLAVO LISO PHASE 277.4 3.82 SERONO 112.0 SIBAR ELISA 121.3 1.27 SORIN 165.9 68.9 2.86 139.5 SYVA EMIT 137.9 TECHLAND RIA 4.77 VITEK SYSTEMS 110.0

  3. In Vitro Diagnostica DirectiveEuropean Union__________________________________________ • “.... the traceability of values assigned to calibrators and control materials must be assured through available reference measurement procedures and/or reference materials of higher order ...”

  4. Measurement Traceability _______________________________________ property of the result of a measurement or the value of a standard whereby it can be related to stated references, usually national or international standards, through an unbroken chain of comparisons all having stated uncertainties

  5. SI-Unit (definition) BIPM µc(y) BIPM: International Institute of Weights and Measures NMI: National Metrological Institute ACL: Accredited Calibration Laboratory MCL: Manufacturer´s Calibration Laboratory ML: Manufacturer´s Laboratory Mf: Manufacturer NMI, ACL Primary reference measurement procedure NMI, ACL Primary calibrator Secondary reference measurement procedure NMI, ACL Secondary calibrator ACL, MCL Traceability Mf.´s selected measurement procedure MCL Mf.´s working (master) calibrator ML Mf.´s standing measurement procedure ML Mf.´s product calibrator Mf.-> End-user End-user´s routine measurement procedure Mf. and/or End-user Routine sample End-user RESULT End-user

  6. Reference Systems Laboratory Medicine_____________________________________________ • Reference Materials • Reference Measurement Procedures • Reference Laboratories

  7. MEASUREMENT OF ESTRADIOL-17ß BY ISOTOPE DILUTION MASS SPECTROMETRY ___________________________________________________________ Addition of [14C]Estradiol-17ß SERUM Extraction with Dichloromethane Column chromatography on Sephadex LH-20 Formation of the Heptafluorobutyric ester Calculation of the Estradiol-17ß Concentration from Isotope Ratios in Samples and Standard Mixtures Selected Ion Recording at m/z 664 and 666

  8. Injection Vacuum Valve Magnet Mass Spectrometer Carrier gas Ion Source Gas Chromatograph Detector M+ • m/z Mass Spectrum min Selected ion recording

  9. Implementation of the Traceability Concept___________________________________________ by: - Diagnostic Kit Manufacturers - Organisers of External Quality Assessment Schemes

  10. Electrolytes:Calcium Chloride Lithium Magnesium Potassium Sodium Metabolites and Substrates:Cholesterol Creatinine Glucose Total Glycerol Uric Acid Urea Bilirubine Lactate Enzmes; AST GGT ALT AMYLASE CK Hormones: Aldosterone Cortisol Estradiol-17ß Estriol Progesterone 17-Hydroxy-progesterone Testosterone Thyroxine Drugs: Theophylline Digoxin Digitoxin Total Protein DGKL Reference Procedures

  11. http://www.dgkl-rfb.de

  12. http://www.dgkl-rfb.de

  13. http://www.dgkl-rfb.de

  14. PROGESTERONE MEDIANS IN RING TRIALS y = 2,98 + 0,87 x n = 40, r = 0,681 Median (x) = 14,2 Median (y) = 15,75 Median (y-x)% = 10,3 PROGESTERONE PATIENT SAMPLES y = 1,84 + 0,96 x n = 42, r = 0.99 Median (x) = 19,2 Median (y) = 17,9 Median (y-x)% = 17,4

  15. PROGESTERONE MEDIANS IN RING TRIALS PROGESTERONE PATIENT SAMPLES

  16. http://www.dgkl-rfb.de

  17. y = 1,17 x + 6,04 n = 38, r = 0,998 Median (y-x)% = 18,4 y = 1,33 x - 7,25 n = 54, r = 0,98 Median (y-x)% = 30,1

  18. http://www.dgkl-rfb.de

  19. Aldosterone – Ring Trials - Method Comparison - Aldosterone – Patient Samples - Method Comparison - Y = 1.13 X + 0.09 n = 38, r = 0.939 Median (y-x)% = 32.3 Y = 1.97 X + 0.06 n = 50, r = 0.873 Median (y-x)% = 69.0

  20. Estriol, unconjugated DGKL -2005/01 Selected Participants: 54(93) Test – KIT 44 Ref.Values A: 44,3 nmol/l B: 35,4 nmol/l Limits A: 25.6 – 62.1 nmol/l B: 21,2 – 49,6 nmol/l http://www.dgkl-rfb.de

  21. Unconjugated Estriol in Serum of Pregnant Women Method Comparison

  22. http://www.dgkl-rfb.de

  23. SI-Unit (definition) BIPM µc(y) BIPM: International Institute of Weights and Measures NMI: National Metrological Institute ACL: Accredited Calibration Laboratory MCL: Manufacturer´s Calibration Laboratory ML: Manufacturer´s Laboratory Mf: Manufacturer NMI, ACL Primary reference measurement procedure NMI, ACL Primary calibrator Secondary reference measurement procedure NMI, ACL Secondary calibrator ACL, MCL Traceability Mf.´s selected measurement procedure MCL Mf.´s working (master) calibrator ML Mf.´s standing measurement procedure ML Mf.´s product calibrator Mf.-> End-user End-user´s routine measurement procedure Mf. and/or End-user Routine sample End-user RESULT End-user

  24. http://www.dgkl-rfb.de

  25. http://www.dgkl-rfb.de

  26. http://www.dgkl-rfb.de

  27. http://www.dgkl-rfb.de

  28. Strategy to establish Reference Systemsfor non-SI Traceable Quantities ____________________________________________ • Definition of the Analyte • Development of a Reference Procedure • Development of a Reference Material

  29. Definition of Proteo-Hormone Analytes __________________________________________ • Decision on the sub-unit to be measured according to clinical relevance • Decision on the epitope(s) to be detected by mono- or poli-clonal antibodies • Decision on the glycosidic structure to be measured

  30. Y = 0.974 X + 14.6 n = 40, r = 0.998 Median (y-x)% = 0.7 Y = 1.064 X + 15.4 n = 40, r = 0.954 Median (y-x)% = 11.6

  31. Aldosterone – Ring Trials - Method Comparison - Aldosterone – Patient Samples - Method Comparison - Y = 1,127 X + 0.073 n = 40, r = 0.980 Median (y-x)% = 29 Y = 2.845 X - 0.042 n = 50, r = 0.831 Median (y-x)% = 165

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