1

ANALYTICAL AND CLINICAL EFFECTS OF ANALYTICAL AND CLINICAL EFFECTS OF CREATININE STANDARDISATIONCREATININE STANDARDISATION

• Prof. J. Delanghe, Prof. J. Delanghe, MD, PhDMD, PhD

• Dept. Clinical Dept. Clinical Chemistry Chemistry

• Ghent University Ghent University

2

3

Creatinine according to Jaffe….Creatinine according to Jaffe….

• Analysis following Analysis following deproteinisationdeproteinisation

• pseudochromogens pseudochromogens time windowtime window

• Lloyds’s reagentLloyds’s reagent

• optimalisationoptimalisation

• extremely cheapextremely cheap

4

Jaffe reactionJaffe reaction

5

VARIATION BETWEEN METHOD-VARIATION BETWEEN METHOD-GROUPSGROUPS

6

Variability in Jaffé’s method (I)Variability in Jaffé’s method (I) (Hanser et (Hanser et al, Ann Biol Clin 2001;59:737-42)al, Ann Biol Clin 2001;59:737-42)

7

8

Technical evolutionTechnical evolution

• 1954 Skeggs: dialysis membrane1954 Skeggs: dialysis membrane

• 1970 centrifugal analyzer1970 centrifugal analyzer→ no longer → no longer dialysis step!→ protein error dialysis step!→ protein error introducedintroduced

• 1980 random acess analyzer1980 random acess analyzer

9

10

The protein error got into the The protein error got into the determination!determination!

• 1970 - 2000: Jaffé rules the creatinine 1970 - 2000: Jaffé rules the creatinine market in Europe and VSmarket in Europe and VS

• Protein error leads to underestimation Protein error leads to underestimation of GFR! (CrCl << GFR)of GFR! (CrCl << GFR)

• some enzymatical determinations some enzymatical determinations recalculated to Jaffé-equivalence! recalculated to Jaffé-equivalence!

11

What happened with creatinine?What happened with creatinine?

• Protein error got into the Protein error got into the determination!determination!

• Only ref. values were adapted!!!!Only ref. values were adapted!!!!

• Physiology books, derived formulas,Physiology books, derived formulas,• pharmacokinetics??????pharmacokinetics??????

12

PseudochromogensPseudochromogens

• Enzymatic methods theoretically Enzymatic methods theoretically eliminate effect of pseudochromogens eliminate effect of pseudochromogens

• Not widely used (cost price)Not widely used (cost price)

13

Creatinine clearanceCreatinine clearance

• urine collection often difficult in urine collection often difficult in practicepractice

• Intra individual CV 10 %Intra individual CV 10 %

• practical formulas:practical formulas:• e.g. Cockroft & Gault Nephron e.g. Cockroft & Gault Nephron

1976;16:31-411976;16:31-41

14

15

16

COCKROFT & GAULTCl = (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)GFR (ml/min/1.73 m2) = 0.55 x L (cm)/P (mg/dl)(Pediatrics 1976; 58:259)(Pediatrics 1976; 58:259)

17

7.12.2003 CE GUIDELINES7.12.2003 CE GUIDELINES

• CE Guideline 98/79/EG on in-vitro diagnostics CE Guideline 98/79/EG on in-vitro diagnostics

• PROBLEMS:PROBLEMS:

• Calibration using standard of “higher order”Calibration using standard of “higher order”

• link with literature?link with literature?

• What with derived calculations?What with derived calculations?

18

0

20

40

60

80

100

120

140

160

180

200

0 50 100 150 200

Creatinine Clearance (Jaffé and Jaffé compensated)

Cre

ati

nin

e C

lea

ran

ce

(en

zym

ati

c)

Jaffé Jaffé compensated

19

REALITY AND THEORY IN REALITY AND THEORY IN DISAGREEMENTDISAGREEMENT!!

20

21

Creatinine in childrenCreatinine in children

00,5

11,5

22,5

33,5

0 0,5 1 1,5 2 2,5 3 3,5

serum creatinine enzymatisch

se

rum

cre

ati

nin

e

Ja

ffé

co

mp

en

sa

ted

Ja

ffé

22

23

24

CREATININE IN CHILDRENCREATININE IN CHILDREN

y = -0,9926x3 + 2,2024x2 - 0,3023x

R2 = 0,9932

-0,2

0

0,2

0,4

0,6

0,8

1

1,2

0 0,5 1 1,5

creatinine Jaffé niet gecompenseerd

cre

ati

nin

e J

aff

é c

om

pe

nsate

d

25

26

27

Clinical impact of a change in CrCl, Clinical impact of a change in CrCl, reference rangereference range

• Fields of potential risk Fields of potential risk

• drugs eliminated by renal excretionaminoglycosides/glycopeptides/carboplatine

• Important for borderline Clcr for antibioticsImportant for borderline Clcr for antibiotics

• management for some antineoplastic agentsmanagement for some antineoplastic agents

• for clinical trialsfor clinical trials

28

LOOKING INTO THE FUTURELOOKING INTO THE FUTURE

29

Cystatin C vs. creatinineCystatin C vs. creatinine

• Advantages:Advantages:

• 1/[Cys C] ~ GFR 1/[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 valuable in the “blind range” zone of

creatininecreatinine

30

NIST SRM 967NIST 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

• 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 Master Pool comprised of units drawn from postmenopausal female

donorsdonors• 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 markers All 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 Low level, High level, second sub-pool spiked with crystalline

creatininecreatinine• 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. O’Connor, J Chromatog B 794, 125-136 (2003)][P. Stokes, G. O’Connor, J Chromatog B 794, 125-136 (2003)]• Spike serum with creatinine-d3, Precipitate proteins with cold ethanol, Spike serum with creatinine-d3, Precipitate proteins with cold ethanol,

Centrifuge, Decant supernatant and dry under nitrogenCentrifuge, Decant supernatant and dry under nitrogen• Reconstitute in water and filter, Dilute with 10 mM NHReconstitute in water and filter, Dilute with 10 mM NH4 4 acetateacetate• LC/MSLC/MS• Phenomenex LUNA C-18; Gradient: 10 mM NHPhenomenex LUNA C-18; Gradient: 10 mM NH44 acetate for 7 min, acetate for 7 min,• Then acetonitrile:10 mM NHThen acetonitrile:10 mM NH44 acetate (80:20) and hold for 13 min 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

NIST SRM 967NIST 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

Some Statistics (United States)Some Statistics (United States)

• End-stage renal disease (ESRD)End-stage renal disease (ESRD)• PrevalencePrevalence (2003): 452,957 U.S. residents were under treatment  (2003): 452,957 U.S. residents were under treatment 

• Resulting from these primary diseases:Resulting from these primary diseases: • Diabetes:Diabetes: 165,113 165,113

Hypertension:Hypertension: 109,642 109,642Glomerulonephritis:Glomerulonephritis: 74,444 74,444Cystic kidney:Cystic kidney: 20,409 20,409All other:All other: 83,349 83,349

• Number of kidney transplants performedNumber of kidney transplants performed  • 2003:2003:  16,043  16,043

2000:2000:  14,557  14,5571995:1995:  12,021  12,0211990:1990:  10,012  10,0121988:1988:  7,501   7,501

34

• MDRD: THE SOLUTION??MDRD: THE SOLUTION??• Limitations (age, CrCl value, Limitations (age, CrCl value,

ethnicity,..)ethnicity,..)• Confusion still ungoingConfusion still ungoing• ““Some unwanted side effects”Some unwanted side effects”• Drug dosage schemes!Drug dosage schemes!• Pediatrics?Pediatrics?

35

• Conventional Calibration MDRD EquationConventional Calibration MDRD Equation• This equation should be used only with those creatinine This equation should be used only with those creatinine

methods that have not been recalibrated to be traceable methods that have not been recalibrated to be traceable to IDMS. If you have any question about the traceability of to IDMS. If you have any question about the traceability of the calibration for the method, NKDEP recommends that the calibration for the method, NKDEP recommends that you contact the reagent and/or calibrator manufacturer you contact the reagent and/or calibrator manufacturer for assistance.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) • Sex Sex • Race (African American or not) Race (African American or not) •

When Scr is in mg/dL (conventional units):When Scr is in mg/dL (conventional units):GFR (mL/min/1.73 m2) = 186 x (Scr)-1.154 x 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 (Age)-0.203 x (0.742 if female) x (1.210 if African American) American)

36

CONFUSION……CONFUSION……

• Conventional Calibration MDRD EquationConventional Calibration MDRD Equation• should be used should be used onlyonly with those creatinine methods that with those creatinine methods that

have have not been recalibratednot been recalibrated to be traceable to IDMS. For to be traceable to IDMS. For more information, visit more information, visit NKDEP'sNKDEP's LaboratoryLaboratory Professionals Professionals section.section.

• GFR (mL/min/1.73 m2) = 186 x (Scr)-1.154 x (Age)-0.203 x 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) (0.742 if female) x (1.210 if African American) (conventional units)(conventional units)

• IDMS-Traceable MDRD EquationIDMS-Traceable MDRD Equation• should be used should be used onlyonly with those creatinine methods that with those creatinine methods that

have been recalibratedhave been recalibrated to be traceable to IDMS. to be traceable to IDMS. For more For more information, visit information, visit NKDEP'sNKDEP's LaboratoryLaboratory Professionals Professionals section. section.

• GFR (mL/min/1.73 m2) = GFR (mL/min/1.73 m2) = 175175 x (Scr)-1.154 x (Age)-0.203 x x (Scr)-1.154 x (Age)-0.203 x (0.742 if female) x (1.210 if African American) (conventional (0.742 if female) x (1.210 if African American) (conventional units)units)

37

38

• IDMS-Traceable MDRD EquationIDMS-Traceable MDRD Equation• This equation should be used only with those This equation should be used only with those

creatinine methods that have been calibrated to be creatinine methods that have been calibrated to be traceable to IDMS. If you have any question about the traceable to IDMS. If you have any question about the traceability of the calibration for the creatinine traceability of the calibration for the creatinine method, NKDEP recommends that you contact the method, NKDEP recommends that you contact the reagent and/or calibrator manufacturer for assistance.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) • Sex Sex • Race (African American or not) Race (African American or not) •

When Scr is in mg/dL (conventional units):When Scr is in mg/dL (conventional units):GFR (mL/min/1.73 m2) = 175 x (Scr)-1.154 x (Age)-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)0.203 x (0.742 if female) x (1.210 if African American)

•

39

Conclusions (I)Conclusions (I)

• Compensated Jaffé method results in a Compensated Jaffé method results in a more correct approximation of GFRmore correct approximation of GFR

• Cockroft & Gault, MDRD : only valid for Cockroft & Gault, MDRD : only valid for enzymatic or “compensated” methodsenzymatic or “compensated” methods

• Schwartz formula not usable for Schwartz formula not usable for compensated (or enzymatic) method!!!compensated (or enzymatic) method!!!

• Adaptation of reference values is Adaptation of reference values is insufficient !!!!!insufficient !!!!!

40

CONCLUSIONS (II)CONCLUSIONS (II)

• Careful when using nomograms for drug dosage!Careful when using nomograms for drug dosage!

• Creatinine standardisation: situation is still Creatinine standardisation: situation is still confuse! Vigilance necessaryconfuse! Vigilance necessary

• MDRD has limitations (CrCl value, age, MDRD has limitations (CrCl value, age, standardisation) which should be respectedstandardisation) which should be respected

• Task for industry, laboratories, cTask for industry, laboratories, clinicians, linicians, pharmacistspharmacists