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Svårigheter att bestämma HbA1c

Svårigheter att bestämma HbA1c. Jan-Olof Jeppsson Klinisk Kemi Universitetssjukhuset MAS Malmo. HbA1c + Glukos. Pre-HbA1c. HbA1c. Glykering av Hemoglobin. Maillard reaction. Agenda. Kortfattad biokemisk bakgrund IFCC referenssystem Internationella jämförelser.

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Svårigheter att bestämma HbA1c

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  1. Svårigheter att bestämma HbA1c Jan-Olof Jeppsson Klinisk Kemi Universitetssjukhuset MAS Malmo

  2. HbA1c + Glukos Pre-HbA1c HbA1c Glykering av Hemoglobin Maillard reaction

  3. Agenda • Kortfattad biokemisk bakgrund • IFCC referenssystem • Internationella jämförelser

  4. Glycation sites of Hemoglobin N-terminal valinered HbA1c Lysinegreen Glycated Hemoglobin Glutathione HbA3 or HbA1d

  5. Importtant landmarks • Diabetes Control and Complications Trial (DCCT)NEJM 1993;329:977-86 • UK Prospective Diabetes Study Group (UKPDS)BMJ 2000;321:405-11

  6. HbA1c och risk för komplikationer % HbA1c (IFCC)

  7. Harmonisation of HbA1cDesignated Comparison Methods (DCM) • National Glycohemoglobin Standardization Program(NGSP) for DCCT in USA • Mono S ion exchange chromatography (EQUALIS) in Sweden

  8. BioRex 70 Ion Exchange Chromatography HbA1c HbA0 HbA1c HbA0 Reference interval 4-6% Goldstein et al in Clark WL,Larner J, Pohl SL eds Methods of Diabetes Research Vol 2; 1986:475-504

  9. Ion Exchange Chromatography of HbA1c • Mono S chromatography • Reference interval3.6 - 5.0 % < 50 years4.0 - 5.3 % > 50 years HbA0 HbA1c

  10. Contents of the HbA1c peak ( Mono S) 99

  11. Correlation between HbA1c and MPG Rohlfing C et alDiabets Care 2002;25:S33-49

  12. Correlation between HbA1c och fB-Glucose 20-88 years, n=255 <50 y >50y fB-Glucose mmol/l % HbA1c Bäck S-E et al CCLM 1999;37:573-92

  13. Biological Variation of HbA1cIntra- and inter individual variation at non-diabetes Rohlfing C et al Clin Chem 2002;48:111618

  14. Årstidsvariation av HbA1c Data från Dr Bent Lind KPLL, Köpenhamn

  15. Hb A1c response over time Tamara Y, Shima K. Diabetes Care 1995;18:440-7

  16. 70-årig kvinna med diabetes % HbA1c Socialstyrelsens rekommenderade behandlingsmål Icke diabetiker Månader

  17. Filter paper for HbA1c produced in SwedenDiabetes Care 1996; 19(2):142-45

  18. Referenssystem för HbA1cIFCC arbetsgrupp • Definition av analyt • Preparation av rent HbA1c och HbA0 • Utveckling av referensmetod • Etablering av nätverk, 14 internationella laboratorier • Jämförande studier mellan USA, Sverige och Japan samt alla tillverkare av reagenser

  19. Major Glycation sites of Hemoglobin b-Chains: Yellow a-Chains: Blue N-terminal Valine: Red Hexapeptide Red/Green

  20. Electrospray ionisation mass spectrometry of HbA1c 15126 Da Non-glycated a-chain 16028 Da Mono-glycated b-chain Finke et al Clin Chem Lab Med. 1998;36(5):299-308

  21. Electrospray ionisation mass spectrometry of HbA0 15125 Da Non-glycated a-chain 15866 Da Non-glycated b-chain

  22. Hemolysate EnzymaticCleavage HPLC-Capillary Electrophoresis HPLC-Mass Spectrometry Flow Chart for Reference Methods Clin Chem Lab Med 2002;40:78-89 Accepted by Scientific Division, IFCC, July 2001

  23. Proteolytic Cleavage of b-chainby Endoproteinase Glu-C VHLTPE E KSAVTALWGKVNVDE VGGE ALGRLLVVYPWTQRFFE SFGDLSTPDAVMGNPKVKAHGKKVLGAFS DGLAHLDNLKGTFATLSE LHCDKLHVDPE NFRLLGNVLVCVLAHHFGKE FTPPVQAAYQKVVAGVANALAHKYH

  24. Proteolytic cleavage of b-chain (146 amino acids) Glu-C HbAo-peptide ……... Val His Leu Thr Pro Glu Glu Lys Ser HbA1c-peptide ……... Glc Val His Leu Thr Pro Glu Glu Lys Ser The Analytical Challenge

  25. Photometric detection of peptides

  26. Two-dimensional separation ofN-terminal hexapeptides of hemoglobin Capillary Electrophoresis C 18 Chromatography Non-glycated hexapeptide Glycated hexapeptide

  27. IFCC Working group for standardization of HbA1c

  28. Comparison between the IFCC reference method and three designated comparison methods 12,00 NGSP = 0,92xIFCC + 2,15 10,00 8,00 NGSP Mono-S DSM JDS 6,00 Mono S = 0,989xIFCC+0.88 4,00 JDS = 0,927xIFCC+1,73 2,00 0,00 0,00 2,00 4,00 6,00 8,00 10,00 12,00 % HbA1c IFCC method Hölzel et al Clin Chem 2004;50:166-74

  29. Kvalitetssäkring av HbA1c i USACollage of American Pathologist

  30. IFCC Monitoring Program for HbA1c Year 2006

  31. Sammanfattning • IFCC systemet utgör en ny global HbA1c standard för tillverkare av reagenser • DCM nätverken NGSP och EQUALIS ska vara spårbara till IFCC nätverkslaboratorier • Tillverkare och användare behåller sina nuvarande enheter tills studien - sambandet mellan HbA1c och MBG är utvärderad

  32. Acknowledgements Clinical Chemistry, UMAS Malmö Anna Arnetorp, Benny Larsson Ann-Christin Mårtensson, May-Lill Svensson EQUALIS, Uppsala Marie Lundberg Arne Mårtensson Gunnar Nordin

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