Standardization of. S.M.Boutorabi DCLS,PhD

Transcription

1 Standardization of Creatinine Assay S.M.Boutorabi DCLS,PhD

2 Chronic kidney disease (CKD) is a major public health problem in developed countries

3 Why measure serum creatinine?

4 Jaffe Reaction Creatinine +picric acid alkaline l ph Orange red red (Janovski) complex 510 nm

5 The alkaline picrate (Jaffe) method interference by glucose, fructose, pyruvate, acetoacetate, uric acid, ascorbic acid, cephalosporins, 5-aminolevulinic acid, bilirubin, and exogenous and endogenous substances. The assay may overestimate serum creatinine by up to 25%, depending di on the severity of renal ldysfunction

6 Interferences from glucose and acetoacetate are particularly important because diabetic persons are a high-risk population to develop CKD

7 Several modifications, including optimization of kinetic assays, have been made to improve method specificity and minimize susceptibility to interfering substances

8 Jaffé Reaction - Modifications Kinetic Interference Rapid e.g. acetoacetate within 20 secs Slow e.g. protein secs Measure absorbance change between 20 and 80 secs Compensated Interference Protein adjust calibrator set point to take account of pseudo-creatinine contribution of protein i.e. subtract approx 27 umol/l Combination of the above

9 Compensated Methods In the compensated Jaffe method, 26.5 micromol/l (0.3 mg/dl) /dl)is subtracted t dfrom the Jaffe method to match the enzymatic method results.

10 the Roche Integra compensated AP assay a concentration of 0.2 mg/dl is automatically subtracted from each result data show that this approach may undoubtedly improve the overall results, especially ill at creatinine i concentrations ti where the non specificity effect is more evident application of this offset may create a paradoxical effect on higher creatinine concentrations, because of a combined effect with the slope of calibration curve, with a significant underestimation at these still clinicallyrelevant creatinine values

11 In a study to evaluate the effect of a compensated Jaffe method on estimated GFR, the MDRD Study equation overestimated GFR by 50% in individuals with serum creatinine concentrations 155 mol/l (1.75 mg/dl)

12 For children who generally present with higher non-creatinine chromogens and very low serum creatinine concentrations, as well as for adults who have low protein and low creatinine concentrations in serum, such as elderly, pregnant women or cancer patients, then poor trueness for compensated assays is to be expected

13 In addition, as there is no non-creatinine chromogens present in urine, which interfere with the alkaline picrate reaction, compensation is basically not necessary with creatinine measurements in urine. Thus, if serum and urine are measured on the same instrument channel using a compensated method, the results for urine will show a basic negative bias due to the automatic subtraction of the offset

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17 Enzymatic Creatinine Assay Enzymatic creatinine methods have less interference than the alkaline picrate methods, but they are affected by 5-fluorocytosine, ethamsylate, dopamine, dobutamine, monoclonal IgM, nitromethane, and other substances.

18 The only alternative methods that have been widely adopted for routine clinical laboratory use are enzymatic creatinine methods

19 Not only trueness, but also the precision of creatinine measurements may significantly improve when enzymatic methods are employed

20 HPLC Methods More than 50 methods for the analysis of creatinine have been described, including cation-exchange, normal-phase, reversed-phase, and reversed-phase ion-pair i chromatography h Sample deproteinization i ti improves the specificity of creatinine measurement by HPLC by removing many protein-bound endogenous and exogenous compounds without altering the quantification of creatinine

21 HPLC Methods Interference studies have demonstrated that HPLC methods have greater analytical specificity than conventional methods

22 Since 2006, the Laboratory Working Group of the National Kidney Disease Education Program(NKDEP) have described recommendations for improving GFR estimation with guidelines for measuring serum creatinine highlighting the need for traceable and reproducible methods for scr measurement

23 The Laboratory Working Group recommended the recalibration of scr methods in order to be traceable to IDMS. After recalibration, the desirable total error goal should be less than 7.6%, at a scr concentration of 88.4 µmol/l (1 mg/dl), to ensure a maximum relative 10% increase in the root mean square error of estimated GFR.

24 Resources for Standardization of Serum Creatinine Measurement reference materials commutable tbl reference materials il commutable PT / EQAP materials reference measurement procedures

25 Reference materials NIST Standard Reference Material (SRM) 914a crystalline creatinine, is intended for use in calibration of reference measurement procedures intended d primarily il for use in high-order reference measurement procedures (e.g., GCIDMS and LC-IDMS) and are not generally suitable for direct assay by routine clinical analyzers NIST (SRM 909b-1 and -2) and IRMM, BCR (573, 574, and 575) multilevel lyophilized human serum based certified reference materials with GC-IDMS assigned values intended as trueness control products for high-order reference measurement procedures

26 commutable reference materials NKDEP, CAP, and NIST NIST SRM 967 human serum-creatinine reference material with acceptable commutability with native clinical specimens in routine methods fresh-frozen human serum pool prepared according to Clinical i l and Laboratory Standards d Institute (CLSI) guideline C-37A value-assigned by NIST with the GC-IDMS and LC- IDMS reference measurement procedures

27 Commutable PT / EQAP materials EQAS and PT providers should make available commutable materials for regularly recurring assessment of serum creatinine measurement performance in routine clinical laboratories Target values are assigned by NIST, using IDMS. This external PT program uses frozen off-the-clot human serum pools prepared according to CLSI C-37A EQA and PT surveys will provide IVD manufacturers and individual clinical laboratories an excellent way to validate the traceability of their clinical measurement procedures.

28 Reference measurement procedures Three GC-IDMS methods nominated by the University of Ghent (Belgium) DGKC NIST have been approved as reference measurement procedures for serum creatinine require a separation step to remove creatine very time-consuming procedures with limited sample throughput LCIDMS method much simpler and faster sample preparation than do GC- IDMS methods

29 Traceability chain for Creatinine Measurement NIST SRM 914a GC IDMS & LC IDMS SRM 967 Manufacturer RMP Working Calibrator Clinical Laboratory QC Patient Result

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32 Creatinine Standardization Recommendations Clinical laboratories

33 for countries or regions that are introducing or have completed standardization of creatinine calibration 1. Use a creatinine method that has calibration traceable to an IDMS reference measurement procedure. Methods based on either enzymatic or Jaffe method principles should have calibration traceable to IDMS 2. Use a validated IDMS traceable equation such as the Modification of Diet in Renal Disease (MDRD) Study equation or the Chronic Kidney Disease Epidemiology Collaboration (CKD EPI) equation for estimating GFR for adults using creatinine results from a method that has calibration traceable to IDMS. Methods that produce results that have acceptable bias 3. Use the IDMS traceable version of the Schwartz equation for estimating GFR for children 4. Report Proficiency Testing and External Quality Assessment (PT/EQA) results for serum and urine creatinine using the correct instrument/method peer group for IDMS traceable calibration. 5. If desired, monitor calibration performance of routine methods that have calibration traceable to an IDMS reference measurement procedure through participation in the College of American Pathologists' for countries or regions that are introducing or have not completed standardization of creatinine calibration 1. Continue using the Original Modification of Diet in Renal Disease (MDRD) Study equation for routine methods that havenot been calibrated to be traceable to IDMS. It is appropriate to use this equation because most methods in this category will produce creatinine results that have a bias similar to that of the method used in developing the Original MDRD Study equation. Contact the reagent and/or calibrator manufacturer with questions about the traceability of the calibration for the method used in your laboratory. 2. Ensure immediate use of the CKD EPI or IDMS traceable MDRD Study equation for estimating GFR in adults, and the IDMS traceable Schwartz equation for children, once your laboratory begins using a creatinine method that has its calibration traceable to IDMS. Methods that produce results that have acceptable bias 3. Communicate the following to health care providers, when using a serum creatinine method that has its calibration traceable to an IDMS reference method 4. Report Proficiency Testing and External Quality Assessment (PT/EQA) results for serum and urine creatinine using the correct instrument/method peer group for IDMS traceable calibration. 5. If desired, monitor calibration performance of routine methods that have calibration traceable to an IDMS reference method by participating in an EQA program that uses commutable samples and IDMS target values

34 Creatinine Standardization Recommendations IVD Manufacturers

35 for countries or regions that are introducing or have completed standardization of creatinine calibration 1. Calibrate serum creatinine methods to be traceable to an isotope dilutionmassspectrometry spectrometry (IDMS) reference measurement procedure. Standardization of method calibration will reduce the interlaboratory bias in results and yield more accurate estimated GFR (egfr) 2. Coordinate with customer laboratoriesso so that, upon switching to a method with IDMS traceable calibration, they immediately begin using the IDMS traceable MDRD Study 3. Provide serum and urine creatinine reference intervals appropriate it for the method. 4. Describe the relationship between creatinine results when measured by a method's original calibration and its updated version with IDMS traceable calibration. Include detailed descriptions i (including mathematical conversion factors, equations, or functions) of the impact of a calibration change for both serum and urine creatinine values 5. Be prepared to communicate to customers important changes related to using a creatinine method with calibration traceable to IDMS. for countries or regions that are introducing or have not completed standardization of creatinine calibration 1. Continue using or recommending the original Modification of Diet in Renal Disease (MDRD) Study equationand and the originalschwartz equation for routine methods that have not been calibrated to be traceable to IDMS. 2. Change the calibration of creatinine methods to be traceable to an isotope dilution mass spectrometry (IDMS) reference measurement procedure. Standardization of method calibration will reduce the interlaboratory bias in results and yield more accurate estimated GFR (egfr) 3. Coordinate with customer laboratories lb so that, t upon switching to a method with IDMS traceable calibration, they immediately begin using the IDMS traceable versions 4. Provide serum and urine creatinine reference intervals appropriate for the method. 5. Describe the relationship between creatinine results when measured by a method's original calibration and its updated version with IDMS traceable calibration. Include detailed descriptions (including mathematical conversion factors, equations, or functions) of the impact of a calibration change for both serum and urine creatinine values 6. Be prepared to communicate to customers important changes related to using a creatinine method with calibration traceable to IDMS.

36 for countries or regions that are introducing or have completed standardization of creatinine calibration for countries or regions that are introducing or have not completed standardization of creatinine calibration 6. Where necessary, improve the bias and precision of creatinine 7. Emphasize that creatinine measurements at the lower concentrations methods to meet the total error goal for serum creatinine measurement 7. Where necessary, method bias and imprecision at concentrations 2 with newer estimating equations. 8. Design instruments that report serum creatinine values as mg/dl to two decimal places, or as µmol/l to the nearest whole number. This will reduce the contribution of a rounding error when estimating GFR from a creatine result. 9. Address analytical nonspecificity issues in routine serum creatinine methods usually observed in pediatric patients have greater measurement variability than for values seen in adults. Estimates of kidney function based on these values also will have greater variability than for adults. 8. Where necessary, improve the bias and precision of creatinine methods to meet the total error goal for serum creatinine measurement 9. Where necessary, method bias and imprecision at concentrations < 1.00 mg/dl (88.4 µmol/l) should be addressed to reduce the uncertainty in egfr for pediatric populations, and to allow extension of reporting egfr to values 2 with newer estimating equations. 10. Design instruments that report serum creatinine values as mg/dl to two decimal places, or as µmol/l to the nearest whole number. This will reduce the contribution of a rounding error when estimating GFR from a creatinine result. 11. Address analytical nonspecificity issues in routine serum creatinine methods 12. Communicate with Proficiency Testing and External Quality Assessment Scheme (PT/EQA) providers to inform them when a revised creatinine calibration will become effective, and work with PT/EQA providers to develop appropriate instrument/method peer groups for participants to be evaluated appropriately.

37 Creatinine Standardization Recommendations PT/EQAP Provider

38 for countries or regions that are introducing or have completed standardization of creatinine calibration All major manufacturers of creatinine measurement procedures in the USA have standardized calibration of creatinine methods to be traceable to IDMS. These differences in calibration may need to be accommodated by creating appropriate instrument/method peer groups(traditional or IDMS traceable calibration) for evaluation of results that reflect the calibration status of particular methods. NKDEP recommends introducing a regularly recurring PT/EQA program that uses commutable serum materials with target values traceable to an IDMS reference measurement procedure for creatinine. Such a program will allow individual laboratories and IVD manufacturers, on an on going basis, to assess the performance of routine clinical laboratory methods and the success of calibration traceability for each of their methods. for countries or regions that are introducing or have not completed standardization of creatinine calibration NKDEP recommends introducing a regularly recurring PT/EQA program that uses commutable serum materials with target values traceable to an IDMS reference measurement procedure for creatinine. Such a program will allow individual laboratories and IVD manufacturers, on an ongoing basis, to assess the performance of routine clinical laboratory methods and the success of calibrationtraceability for each of their methods. 1. Advise participant laboratories that you will collaborate with IVD manufacturers to ensure appropriate grading of PT/EQA data during a transition period for implementation. 2. necessary changes in participant grading within your respective survey programs during the transition of routine creatinine methods to revised calibrations that are traceable to an IDMS reference method. PT/EQA providers to collaborate with IVD manufacturers to create new instrument/method peer groups (traditional or IDMStraceable calibration) 3. Inform participant laboratories that they will need to choose the correct instrument/method peer group for the creatinine calibration in use by their laboratory for a given PT/EQA challenge. 4. Request IVD manufacturers' expected dates for introduction of IDMS traceable calibrations for creatinine for each of their methods, and the anticipated timeframe to achieve completion of the transition

39 For serum creatinine in the range of mg/dl Non IDMS Creat (mg/dl)=idms Creat(mg/dl) x

40 GFR Estimation

41 The MDRD Equation Original GFR (ml/min/1.73 m 2 ) = 186 (S (Age) cr ) (0.742 if female) (1.212 if African American) IDMS Traceable GFR (ml/min/1.73 m 2 ) = 175 (S cr ) (Age) (0.742 if female) (1.212 if African American)

42 The CKD-EPI Equation GFR = 141 min (S cr /κ, 1) α max(s cr /κ, 1) ( cr, ) ( cr, ) Age [if female] [if black]

43 The Bedside Schwartz equation GFR (ml/min/1.73 m 2 ) = (0.41 Height in cm) / Creatinine in mg/dl

44 Are alkaline picrate assays still suitable for clinical usefulness?

45 even if the imprecision is low and the assay is standardized to an IDMS reference measurement procedure, if analytical non specificity bias remains, then errors in estimated GFR for individual patients will occur

46 standardization through traceability implementation does not solve the analytical interferences related to an assay's non-specificity. The ISO standard clearly states that, if the reference measurement procedure and or responding lower-order routine methods have not identical, specificities iti for the measurand, or at least very similar, il traceability cannot be obtained.

47 current EQAS are not appropriate to evaluate the traceability of results obtained in clinical laboratories. The control materials are often not commutable and EQAS too often use consensus-based (i.e., the peer group mean or other indicators for the central tendency) instead of accuracy-based criteria (i.e., target values assigned by the reference procedure) to evaluate the performance of participating laboratories

48 Cr Enzymatic Cr- Jaffe P-1 Cr - Jaffe P-2 Number of values Minimum % Percentile Median % Percentile Maximum Mean Std. Deviation Std. Error Lower 95% CI of mean Upper 95% CI of mean Sum Data Cr 0 r Enzymatic Cr- Jaffe P-1 Cr - Jaffe P-2

49 Parameter Table Analyzed Data 1 One way analysis of variance P value P value summary *** Are means signif. if different? (P < 0.05) 05) Yes Number of groups 3 F R square Bartlett's test for equal variances Bartlett's statistic (corrected) P value P value summary ns Do the variances differ signif. (P < 0.05) No ANOVA Table SS df MS Treatment (between columns) Residual (within columns) Total Tukey's Multiple Comparison Test Mean Diff. q Significant? P < 0.05? Summary 95% CI of diff Cr Enzymatic vs Cr Jaffe P No ns to Cr Enzymatic vs Cr Jaffe P Yes *** to Cr Jaffe P 1 vs Cr Jaffe P No ns to

50 Histogram of Cr Enzymatic, Cr- Creatinine Jaffe P-1, Cr - Jaffe P-2 Frequency Variable Cr Enzy matic Cr- Jaffe P-1 Cr - Jaffe P-2 Mean StDev N Data

51 Recommendation for IRAN Transition period from current situation to standardizes situation IVD Manufacturer use SRM 967 Installation ti of Reference Rf measuring system EQAP with commutable material

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