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IFCC Standardization of Enzyme Measurements: a T Fth C Tsit Ti IFCC Standardization of Enzyme Measurements: AtfthCtSittiAssessment of the Current Situation IXth Czech National Congress of Clinical Biochemistry Gerhard Schumann Institute for Clinical Chemistry, Medical University Hannover September 22, 2009 Area of the Medical University Hannover Institute for Clinical Chemistry, Medical University Hannover September 22, 2009 Reference Syyystems for Enzymes Essential components Ö Primary reference measurement procedures (IFCC / C-RSE) Ö Certified reference materials (IRMM) Ö Official accreditation for reference laboratories (BIPM) Ö International ring trials for reference laboratories (IFCC / DGKL) Ö Common reference intervals and decision limits (IFCC / C-RIDL) Ö Network of reference laboratories (IFCC / C-RSE) Objectives Traceability - how to verify, how to control? IFCC - ALP IFCC - Lipase Institute for Clinical Chemistry, Medical University Hannover September 22, 2009 In-vitro-diagnostic Medical Devices (IVD) Directive 98/79/EC of the European parliament Appendix 1 General Requirements Traceability of calibration materials and control materials has to be assured by reference measurement procedures and reference materials of higher order. Time frame Dec. 1998: Directive published Dec. 2003: End of transition period Dec. 2005: End of transition for putting into service Traceability and international standardization International standardization = improved inter-laboratory comparability Traceability = links to the components of a reference system = stable relation of results (with and without pathological findings) = reliable decision limits = definitive reference intervals Traceability - how to verify since 2006? Internal quality control Æ 100 % recovery Manufacturer of a CE-labeled test kit shall have complete responsibility for traceability. External quality assessment Æ certificate Limits of acceptance are wide Commutability of the control material is not guaranteed German Society for Clinical Chemistry and Laboratory Medicine 23 % External Quality Assessment German Society for Clinical Chemistry and Laboratory Medicine Ex terna l Q ualit y A ssessment (2006 versus 2008/2009) 10 9 8 7 6 % 5 VV C 4 3 2 1 0 CK AST ALT LDH GGT AMY Enzyme Traceability / Uncertainty True value (human serum) >REFERENCE METHODOLOGY< Primary calibrator Master calibrator Kit calibrator Result (human serum) >ROUTINE LABORATORY< ideal Traceability / Uncertainty True value (human serum) Primary calibrator Sorry for the Master calibrator restandardization Kit calibrator Result (human serum) ideal real Problems with processed, lyophilized calibrators in the calibration hierarchie for enzyme measurements Very often non-commutable Secondary Reference Material: = Pooled human sera Collection of human sera Tailored target concentrations Standardized freezinggg and thawing of the specimens 1 ml aliquots stored below - 75 °C Determination of the reference method value Considerations for the composition of pooled sera ¾ The more single sera in the pool the better ¾ Exclusion of sera with intensiv lipemia, hemolysis and bilirubinemia ¾ Exclusion of sera with elevated concentration of monoclonal immunoglobulins ¾ Avoid unusual composition of isoenzymes (e.g. CK-BB) ¾ No high concentration of the “wrong” isoenzyme (e.g. salivary α-amylase) Stability of the secondary reference materials (example: AST) 106 104 (%) .. 102 100 . conc tt 98 el. ca 96 RR 94 200 400 600 Day 95 % tolerance interval (100 % ± 1,4 %) Measurement result of one measurement day (mean of three single values) ZeitlicherTime course Verlauf of derthe relativerelativen catalytic katalytischen concentration ALT-Konzentration of AST in pooled human serum im Humanserumpool 108 n % oo 106 ntrati 104 ee 102 e Konz 100 hh 98 lytisc aa 96 ive kat ive 94 tt { = Mittelwert aus Dreifachbestimmung an einem Messtag 92 Rela 0 200 400 600 800 1000 1200 1400 Tage Control material: Pooled human sera with certified RMVs applied to routine procedures from Roche Diagnostics (Modular, Hitachi 917) CK (U/L) 45,7 101,1 204,7 975,8 GGT (U/L) 18,7 51,1 102,6 179,2 277,9 521,2 AMY (U/L) 24,1 121,1 601,7 ALT (U/L) 14,9 43,9 108,0 192,4 299,6 677,2 AST U(/L) 16,6 38,1 109,5 172,9 264,2 LDH (U/L) 121,5 138,8 195,6 279,4 286,8 404,1 509,2 545,0 ALP (()U/L) 72,6 237,1 639,3 CK in lot 6, level 3: deviation from the reference value 10 RMV = 204,7 U/L 5 % 0 iation vv De -5 -10 12345 Relative combined expanded (k=2) Measurement day uncertainty of the reference method value MdlModular P Hitachi-917 GGT in lot 6, level 3: deviation from the reference value 10 RMV = 102,6 U/L 5 % 0 iation vv De -5 -10 12345 Relative combined expanded (k=2) Measurement day uncertainty of the reference method value MdlModular P Hitachi-917 Amylase in lot 5, level 3: deviation from the reference value ALT in lot 6, level 4: deviation from the reference value 10 10 RMV = 601, 7 U/L 5 5 ion % ion % tt 0 tt 0 Devia Devia -5 -5 RMV = 192,4 U/L -10 -10 12345 12345 Relative combined expanded (k=2) Measurement day Relative combined expanded (k=2) Measurement day uncertainty of the reference method value uncertainty of the reference method value Modular P Modular P Hitachi-917 Hitachi-917 LDH in lot 7, level 3: deviation from the reference value AST in lot 6, level 4: deviation from the reference value 10 15 RMV = 286,8 U/L 10 5 5 0 ation % ation % ation 0 Devi Devi -5 -5 RMV = 172,9 U/L -10 -10 12345 12345 Relative combined expanded (k=2) Measurement day Relative combined expanded (k=2) Measurement day uncertainty of the reference method value uncertainty of the reference method value Modular P Modular P Hitachi-917 Hitachi-917 Recovery experiments: 5 measurements days, 3-6 pooled sera 110 115 Hitachi-917 Hitachi-917 105 Modular P 110 Modular P 105 100 100 95 95 overy % overy overy % overy cc 90 cc 90 Re Re 85 85 80 80 0 200 400 600 800 1000 0 200 400 600 RMV - CK U/L RMV - GGT U/L 105 120 Modular P Modular P Hitachi-917 Hitachi-917 115 100 % % 110 95 105 Recovery Recovery 90 Recovery 100 85 95 0 200 400 600 0 200 400 600 RMV - AMY U/L RMV - ALT U/L 105 Modular P Hitachi-917 % 100 very very oo 95 Rec 90 200 400 600 RMV - LDH U/L 120 Modular P Hitachi-917 115 % 110 very very oo 105 Rec 100 95 0 100 200 300 RMV - AST U/L 105 Modular P Hitachi-917 100 % 95 very very oo Rec 90 85 200 400 600 RMV - ALP U/L Allowable bias considering intra individual and inter individual biological variation S-Amylase 7,4 % S-ALT 12, 0 % S-AST 5,4 % S-CK 11,5 % S-GGT 10,8 % S-LDH 4,3 % *) CRiC. Ricos e t. a lCl. Curren tdtbt databases on bilbiolog ica l var itiiation: pros, cons an d progress. Scand J Clin Lab Invest 1999; 59: 491-500. Pooled human sera with certified RMVs applied to routine procedures from Roche Diagnostics (Modular, Hitachi 917) Mean recovery (Modular) Mean recovery (Hitachi 917) CK 94,2 % 95,3 % GGT 97, 7 % 100, 1 % AMY 94,4 % 95,5 % ALT 104,6 % 103,6 % AST 108,9 % 5,4 % 110,2 % LDH 94,0 % 4,3 % 98,4 % ALP 90, 7 % 99, 4 % Pooled human sera with certified RMVs Use for manufacturers: - Calibration concepts based on commutable calibration material - Control material sufficient number of aliquots sufficient stable at – 75 °C Benefit for routine laboratories: - Control of commercial routine procedures easy handling airtight results Reaction Principle for ALP measurements Decision for a reference procedure for ALP using the substrate AMP ALP 4-Nitrop heny lphhthosphate + AMP 4-Nitrop henox ide+AMP-phhthosphate ALP 4-Nitrophenylphosphate + H2O 4-Nitrophenoxide + Phosphate Reasons for a decision on ALP-AMP IFCC proposal for ALP-AMP at 30 °C Many routine procedures are using AMP. However, this procedure was never endorsed by IFCC. Measurement parameters of the proposed IFCC reference measurement procedure for ALP (1) Concentrations in the Final Complete Reaction Mixture: 2-Amino-2-methyl-1-propanol 750 mmol/l pH (37 °C) 10.20 ± 0.05 4-Nitrop heny lp hosph at e 16 mmo l/l Zinc sulfate 1 mmol/l Magnesium acetate 2 mmol/l HEDTA 2 mmol/l Volume fraction of sample 0.0222 (1 : 45) Measurement parameters of the proposed IFCC reference measurement procedure for ALP (2) Measurement Conditions: Temperature 37.0 °C ± 0.1 °C Wave length 405 nm ± 1 nm Band width ≤ 2 nm Light path 10. 00 mm ± 001mm0.01 mm Incubation time 60 s Delay time 60 s Measurement interval 120 s Readings (measurement points) ≥ 6 ALP-AMP: pH optimum 100 Serum 1 Serum 2 90 Serum 3 80 Serum 4 Serum 5 (%) 70 Optimum pH and Serum 6 range of uncertainty (k=2) ALP 60 Serum 7 50 Serum 8 Serum 9 40 Serum 10 9,6 9,8 10,0 10,2 10,4 10,6 10,8 pH pppH optima of ALP isoforms 120 120 100 100 80 80 ALP % ALP 60 % ALP 60 40 40 20 20 9,6 9,9 10,2 10,5 10,8 9,6 9,9 10,2 10,5 10,8 pH pH commercial calibrator commercial control material 4 commercial control material 1 ring trial material 1 commercial control material 2 ring trial material 2 commercial control material 3 ring trial material 3 human serum human serum Method comparison ALP: RfReference proced ure vs Roc he Diagnos tics (MdlP)(Modular P) 1000 Number of values 99 Slope 0,908 Slope, lower limit 95 % 0,898 800 Slope, upper limit 95 % 0,917 Intercept 1,75 Intercept, lower limit 95% -0,03 ) U/l 600 In tercep t, upper li m it 95% 4074,07 Coefficient of correlation 0,9982 Ratio mean 0,921 400 P (Roche LL A ALP measurements 200 y = x Regression line (Passing/Bablok) 0 0 200 400 600 800 1000 ALP (IFCC) U/l Nearly no intercept.
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