1. 1 ANALYTICAL AND CLINICAL EFFECTS OF CREATININE STANDARDISATION Prof. J. Delanghe, MD, PhDProf. J. Delanghe, MD, PhD Dept. Clinical ChemistryDept. Clinical Chemistry Ghent UniversityGhent University

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3. 3 Creatinine according to Jaffe…. Analysis following deproteinisationAnalysis following deproteinisation pseudochromogens time windowpseudochromogens time window Lloydss reagentLloydss reagent optimalisationoptimalisation extremely cheap extremely cheap

4. 4 Jaffe reaction

5. 5 VARIATION BETWEEN METHOD- GROUPS

6. 6 Variability in Jaffés method (I) (Hanser et al, Ann Biol Clin 2001;59:737-42)

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8. 8 Technical evolution 1954 Skeggs: dialysis membrane1954 Skeggs: dialysis membrane 1970 centrifugal analyzer no longer dialysis step! protein error introduced1970 centrifugal analyzer no longer dialysis step! protein error introduced 1980 random acess analyzer1980 random acess analyzer

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10. 10 The protein error got into the determination! 1970 - 2000: Jaffé rules the creatinine market in Europe and VS1970 - 2000: Jaffé rules the creatinine market in Europe and VS Protein error leads to underestimation of GFR! (CrCl << GFR)Protein error leads to underestimation of GFR! (CrCl << GFR) some enzymatical determinations recalculated to Jaffé-equivalence!some enzymatical determinations recalculated to Jaffé-equivalence!

11. 11 What happened with creatinine? Protein error got into the determination!Protein error got into the determination! Only ref. values were adapted!!!!Only ref. values were adapted!!!! Physiology books, derived formulas,Physiology books, derived formulas, pharmacokinetics??????pharmacokinetics??????

12. 12 Pseudochromogens Enzymatic methods theoretically eliminate effect of pseudochromogensEnzymatic methods theoretically eliminate effect of pseudochromogens Not widely used (cost price)Not widely used (cost price)

13. 13 Creatinine clearance urine collection often difficult in practiceurine collection often difficult in practice Intra individual CV 10 %Intra individual CV 10 % practical formulas:practical formulas: e.g. Cockroft & Gault Nephron 1976;16:31-41e.g. Cockroft & Gault Nephron 1976;16:31-41

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16. 16 COCKROFT & GAULT Cl = (140 -L) (year) x W(kg) S (mg/dl) x 72 for women result x 0.85 MDRD (classical) GFR (mL/min/1.73 m2) = 186 x (Scr)-1.154 x (Age)-0.203 x (0.742 if female) x (1.210 if African American) SCHWARTZ practical in children and infants practical in children and infants GFR (ml/min/1.73 m2) = 0.55 x L (cm)/P (mg/dl) (Pediatrics 1976; 58:259)

17. 17 7.12.2003 CE GUIDELINES CE Guideline 98/79/EG on in-vitro diagnosticsCE Guideline 98/79/EG on in-vitro diagnostics PROBLEMS:PROBLEMS: Calibration using standard of higher orderCalibration using standard of higher order link with literature?link with literature? What with derived calculations?What with derived calculations?

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19. 19 REALITY AND THEORY IN DISAGREEMENT !

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21. 21 Creatinine in children

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24. 24 CREATININE IN CHILDREN

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27. 27 Clinical impact of a change in CrCl, reference range Fields of potential riskFields of potential risk drugs eliminated by renal excretion aminoglycosides/glycopeptides/carboplatine Important for borderline Clcr for antibioticsImportant for borderline Clcr for antibiotics management for some antineoplastic agents management for some antineoplastic agents for clinical trialsfor clinical trials

28. 28 LOOKING INTO THE FUTURE

29. 29 Cystatin C vs. creatinine Advantages:Advantages: 1/[Cys C] ~ GFR1/[Cys C] ~ GFR independent from muscle mass, dietindependent from muscle mass, diet urinary determination not neededurinary determination not needed valuable in the blind range zone of creatininevaluable in the blind range zone of creatinine

30. 30 NIST SRM 967 Commutable reference materialCommutable reference material Submitted bij JCTMLSubmitted bij JCTML IDMSIDMS 0.8 mg/dl0.8 mg/dl 4 mg/dl4 mg/dl Revised MDRDRevised MDRD Release postponed!!Release postponed!!

31. 31 PREPARATION OF SRM 967 CREATININE IN HUMAN SERUMPREPARATION OF SRM 967 CREATININE IN HUMAN SERUM Prepared at Solomon Park Research Institute, Kirkland, WashingtonPrepared at Solomon Park Research Institute, Kirkland, Washington Human serum with the following characteristics:Human serum with the following characteristics: Master Pool comprised of units drawn from postmenopausal female donorsMaster Pool comprised of units drawn from postmenopausal female donors to yield pool with creatinine of 0.8 mg/dL or lower.to yield pool with creatinine of 0.8 mg/dL or lower. Collected and handled following NCCLS C37-ACollected and handled following NCCLS C37-A All units tested and found negative for viral markersAll units tested and found negative for viral markers Pooled and split into 2 sub-pools:Pooled and split into 2 sub-pools: Low level, High level, second sub-pool spiked with crystalline creatinineLow level, High level, second sub-pool spiked with crystalline creatinine to bring the concentration to 4.0 mg/dL.to bring the concentration to 4.0 mg/dL. LC/MS METHOD FOR SERUM CREATININELC/MS METHOD FOR SERUM CREATININE [P. Stokes, G. OConnor, J Chromatog B 794, 125-136 (2003)][P. Stokes, G. OConnor, J Chromatog B 794, 125-136 (2003)] Spike serum with creatinine-d3, Precipitate proteins with cold ethanol, Centrifuge, Decant supernatant and dry under nitrogenSpike serum with creatinine-d3, Precipitate proteins with cold ethanol, Centrifuge, Decant supernatant and dry under nitrogen Reconstitute in water and filter, Dilute with 10 mM NH 4 acetateReconstitute in water and filter, Dilute with 10 mM NH 4 acetate LC/MSLC/MS Phenomenex LUNA C-18; Gradient: 10 mM NH 4 acetate for 7 min,Phenomenex LUNA C-18; Gradient: 10 mM NH 4 acetate for 7 min, Then acetonitrile:10 mM NH 4 acetate (80:20) and hold for 13 minThen acetonitrile:10 mM NH 4 acetate (80:20) and hold for 13 min Electrospray ionization – positive mode monitoring (M+H)+ at 114/117Electrospray ionization – positive mode monitoring (M+H)+ at 114/117 Calibration from standard curve using SRM 914a and creatinine- d3Calibration from standard curve using SRM 914a and creatinine- d3

32. 32 NIST SRM 967 2006-2009:Adaptation by IVD industry2006-2009:Adaptation by IVD industry 2010: problem solved?2010: problem solved? In the mean time MDRD is pushing!!!!In the mean time MDRD is pushing!!!! Risks for confusion ! Risks for confusion !

33. 33 Some Statistics (United States) End-stage renal disease (ESRD)End-stage renal disease (ESRD) Prevalence (2003): 452,957 U.S. residents were under treatmentPrevalence (2003): 452,957 U.S. residents were under treatment Resulting from these primary diseases:Resulting from these primary diseases: Diabetes: 165,113 Hypertension: 109,642 Glomerulonephritis: 74,444 Cystic kidney: 20,409 All other: 83,349Diabetes: 165,113 Hypertension: 109,642 Glomerulonephritis: 74,444 Cystic kidney: 20,409 All other: 83,349 Number of kidney transplants performedNumber of kidney transplants performed 2003: 16,043 2000: 14,557 1995: 12,021 1990: 10,012 1988: 7,5012003: 16,043 2000: 14,557 1995: 12,021 1990: 10,012 1988: 7,501

34. 34 MDRD: THE SOLUTION??MDRD: THE SOLUTION?? Limitations (age, CrCl value, ethnicity,..)Limitations (age, CrCl value, ethnicity,..) Confusion still ungoingConfusion still ungoing Some unwanted side effectsSome unwanted side effects Drug dosage schemes!Drug dosage schemes! Pediatrics?Pediatrics?

35. 35 Conventional Calibration MDRD EquationConventional Calibration MDRD Equation This equation should be used only with those creatinine methods that have not been recalibrated to be traceable to IDMS. If you have any question about the traceability of the calibration for the method, NKDEP recommends that you contact the reagent and/or calibrator manufacturer for assistance.This equation should be used only with those creatinine methods that have not been recalibrated to be traceable to IDMS. If you have any question about the traceability of the calibration for the method, NKDEP recommends that you contact the reagent and/or calibrator manufacturer for assistance. The equation requires 4 variables:The equation requires 4 variables: Serum, or plasma, creatinine (Scr)Serum, or plasma, creatinine (Scr) Age in years (18 years or older)Age in years (18 years or older) SexSex Race (African American or not)Race (African American or not) When Scr is in mg/dL (conventional units): GFR (mL/min/1.73 m2) = 186 x (Scr)-1.154 x (Age)-0.203 x (0.742 if female) x (1.210 if African American) When Scr is in mg/dL (conventional units): GFR (mL/min/1.73 m2) = 186 x (Scr)-1.154 x (Age)-0.203 x (0.742 if female) x (1.210 if African American)

36. 36 CONFUSION…… Conventional Calibration MDRD EquationConventional Calibration MDRD Equation should be used only with those creatinine methods that have not been recalibrated to be traceable to IDMS. For more information, visit NKDEP's Laboratory Professionals section. should be used only with those creatinine methods that have not been recalibrated to be traceable to IDMS. For more information, visit NKDEP's Laboratory Professionals section.NKDEP's Laboratory ProfessionalsNKDEP's Laboratory Professionals GFR (mL/min/1.73 m2) = 186 x (Scr)-1.154 x (Age)- 0.203 x (0.742 if female) x (1.210 if African American) (conventional units)GFR (mL/min/1.73 m2) = 186 x (Scr)-1.154 x (Age)- 0.203 x (0.742 if female) x (1.210 if African American) (conventional units) IDMS-Traceable MDRD EquationIDMS-Traceable MDRD Equation should be used only with those creatinine methods that have been recalibrated to be traceable to IDMS. For more information, visit NKDEP's Laboratory Professionals section.should be used only with those creatinine methods that have been recalibrated to be traceable to IDMS. For more information, visit NKDEP's Laboratory Professionals section.NKDEP's Laboratory ProfessionalsNKDEP's Laboratory Professionals GFR (mL/min/1.73 m2) = 175 x (Scr)-1.154 x (Age)-0.203 x (0.742 if female) x (1.210 if African American) (conventional units)GFR (mL/min/1.73 m2) = 175 x (Scr)-1.154 x (Age)-0.203 x (0.742 if female) x (1.210 if African American) (conventional units)

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38. 38 IDMS-Traceable MDRD EquationIDMS-Traceable MDRD Equation This equation should be used only with those creatinine methods that have been calibrated to be traceable to IDMS. If you have any question about the traceability of the calibration for the creatinine method, NKDEP recommends that you contact the reagent and/or calibrator manufacturer for assistance.This equation should be used only with those creatinine methods that have been calibrated to be traceable to IDMS. If you have any question about the traceability of the calibration for the creatinine method, NKDEP recommends that you contact the reagent and/or calibrator manufacturer for assistance. The equation requires 4 variables:The equation requires 4 variables: Serum, or plasma, creatinine (Scr)Serum, or plasma, creatinine (Scr) Age in years (18 years or older)Age in years (18 years or older) SexSex Race (African American or not)Race (African American or not) When Scr is in mg/dL (conventional units): GFR (mL/min/1.73 m2) = 175 x (Scr)-1.154 x (Age)- 0.203 x (0.742 if female) x (1.210 if African American) When Scr is in mg/dL (conventional units): GFR (mL/min/1.73 m2) = 175 x (Scr)-1.154 x (Age)- 0.203 x (0.742 if female) x (1.210 if African American)

39. 39 Conclusions (I) Compensated Jaffé method results in a more correct approximation of GFRCompensated Jaffé method results in a more correct approximation of GFR Cockroft & Gault, MDRD : only valid for enzymatic or compensated methodsCockroft & Gault, MDRD : only valid for enzymatic or compensated methods Schwartz formula not usable for compensated (or enzymatic) method!!!Schwartz formula not usable for compensated (or enzymatic) method!!! Adaptation of reference values is insufficient !!!!!Adaptation of reference values is insufficient !!!!!

40. 40 CONCLUSIONS (II) Careful when using nomograms for drug dosage!Careful when using nomograms for drug dosage! Creatinine standardisation: situation is still confuse! Vigilance necessaryCreatinine standardisation: situation is still confuse! Vigilance necessary MDRD has limitations (CrCl value, age, standardisation) which should be respectedMDRD has limitations (CrCl value, age, standardisation) which should be respected Task for industry, laboratories, clinicians, pharmacistsTask for industry, laboratories, clinicians, pharmacists