IFCC Standardization of Enzyme Measurements:
A t f th C t Sit tiAssessment of the Current Situation
IXth Czech National Congress of Clinical Biochemistry
Gerhard Schumann
Institute for Clinical Chemistry,Medical University HannoverSeptember 22, 2009
Area of the Medical University Hannover
Institute for Clinical Chemistry,Medical University HannoverSeptember 22, 2009
Reference Systems for Enzymesy y
Essential components
Primary reference measurement procedures (IFCC / C-RSE)
Certified reference materials (IRMM)Certified reference materials (IRMM)
Official accreditation for reference laboratories (BIPM)
International ring trials for reference laboratories (IFCC / DGKL)
Common reference intervals and decision limits (IFCC / C-RIDL)
Network of reference laboratories (IFCC / C-RSE)
ObjectivesObjectives
Traceability - how to verify, how to control?
IFCC ALPIFCC - ALP
IFCC LipaseIFCC - Lipase
Institute for Clinical Chemistry,Medical University HannoverSeptember 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 materialshas to be assured by reference measurement procedures
and reference materials of higher order.
Time frameTime frameDec. 1998: Directive publishedDec. 2003: End of transition periodDec. 2003: End of transition period
Dec. 2005: End of transition for putting into service
Traceability and international standardizationTraceability 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 100 % recoveryInternal quality control 100 % recovery
Manufacturer of a CE-labeled test kit shall have complete responsibility for traceabilitytraceability.
External quality assessment certificate
Limits of acceptance are widep
Commutability of the control material is not guaranteed
German Society for Clinical Chemistry and Laboratory Medicine
E t l Q lit A tExternal Quality Assessment(2006 versus 2008/2009)
10
8
9
V % 5
6
7
CV
3
4
0
1
2
EnzymeCK AST ALT LDH GGT AMY
0
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 theMaster calibrator
Sorry for therestandardization
Kit calibrator
Result (human serum)
ideal real
Problems with processed, lyophilized calibrators in the calibration hierarchie for enzyme measurements
Very oftennon-commutable
Secondary Reference Material:Secondary Reference Material:
= Pooled human sera
Collection of human seraCollection of human sera
Tailored target concentrationsg
Standardized freezing and thawing of the g gspecimens
1 ml aliquots stored below 75 °C1 ml aliquots stored below - 75 °C
Determination of the reference method valueDetermination of the reference method value
Considerations for the composition of pooled sera
The more single sera in the pool the betterThe more single sera in the pool the better
Exclusion of sera with intensiv lipemia, hemolysisExclusion of sera with intensiv lipemia, hemolysisand bilirubinemia
Exclusion of sera with elevated concentrationof monoclonal immunoglobulins
Avoid unusual composition of isoenzymes (e.g. CK-BB)
No high concentration of the “wrong” isoenzyme(e.g. salivary α-amylase)
Stability of the secondary reference materials(example: AST)
106
(example: AST)
. (%
)
102
104
106
t. co
nc.
100
102
Rel
. cat
96
98
200 400 600
R
94
Day200 400 600
95 % tolerance interval (100 % ± 1,4 %)
Measurement result of one measurement day (mean of three single values)
Zeitlicher Verlauf der relativen katalytischen ALT-Konzentration im Humanserumpool
Time course of the relative catalytic concentration of ASTin pooled human serumim Humanserumpool
on
% 108
p
entr
atio
104
106
he K
onze
100
102
alyt
isch
98
100
tive
kata
94
96
0 200 400 600 800 1000 1200 1400
Rel
at
92= Mittelwert aus Dreifachbestimmung an einem Messtag
Tage
Control material:Pooled human sera with certified RMVs
applied to routine procedures from Roche Diagnostics (Modular, Hitachi 917)
521,2277,9179,2102,651,118,7GGT (U/L)
975,8204,7101,145,7CK (U/L)
521,2277,9179,2102,651,118,7GGT (U/L)
975,8204,7101,145,7CK (U/L)
601,7121,124,1AMY (U/L)
521,2277,9179,2102,651,118,7GGT (U/L)
601,7121,124,1AMY (U/L)
521,2277,9179,2102,651,118,7GGT (U/L)
264,2172,9109,538,116,6AST U(/L)
677,2299,6192,4108,043,914,9ALT (U/L)
264,2172,9109,538,116,6AST U(/L)
677,2299,6192,4108,043,914,9ALT (U/L)
639,3237,172,6ALP (U/L)
545,0509,2404,1286,8279,4195,6138,8121,5LDH (U/L)
639,3237,172,6ALP (U/L)
545,0509,2404,1286,8279,4195,6138,8121,5LDH (U/L)
,,,( ) ,,,( )
CK in lot 6, level 3: deviation from the reference value10
RMV = 204,7 U/L
%
5
viat
ion
0
Dev
-5
-10
Measurement day1 2 3 4 5
Relative combined expanded (k=2)uncertainty of the reference method valueM d l PModular PHitachi-917
GGT in lot 6, level 3: deviation from the reference value10
RMV = 102,6 U/L
%
5
viat
ion
0
Dev
-5
-10
Measurement day1 2 3 4 5
Relative combined expanded (k=2)uncertainty of the reference method valueM d l PModular PHitachi-917
Amylase in lot 5, level 3: deviation from the reference value10
RMV = 601 7 U/L
ALT in lot 6, level 4: deviation from the reference value10
tion
%
0
5
RMV = 601,7 U/L
tion
%
0
5
Dev
iat
-5
0
Dev
iat
-5
0
RMV = 192,4 U/L
Measurement day1 2 3 4 5
-10
Relative combined expanded (k=2)uncertainty of the reference method valueModular PHitachi-917
Measurement day1 2 3 4 5
-10
Relative combined expanded (k=2) uncertainty of the reference method valueModular PHitachi-917
LDH in lot 7, level 3: deviation from the reference value10
RMV = 286 8 U/L
AST in lot 6, level 4: deviation from the reference value15
atio
n %
0
5
RMV 286,8 U/L
atio
n % 5
10
Dev
i
-10
-5
Dev
i
-10
-5
0
RMV = 172,9 U/L
Measurement day1 2 3 4 5
-10
Relative combined expanded (k=2)uncertainty of the reference method valueModular PHitachi-917
Measurement day1 2 3 4 5
-10
Relative combined expanded (k=2)uncertainty of the reference method valueModular PHitachi-917
Recovery experiments: 5 measurements days, 3-6 pooled sera
105
110Hitachi-917Modular P 110
115Hitachi-917Modular P
cove
ry
%
90
95
100
cove
ry
%
95
100
105
0 200 400 600 800 1000
Rec
80
85
90
0 200 400 600
Rec
80
85
90
RMV - CK U/L0 200 400 600 800 1000
RMV - GGT U/L0 200 400 600
% 100
105Modular PHitachi-917
%
110
115
120Modular PHitachi-917
Rec
over
y
90
95
Rec
over
y
100
105
110
RMV - AMY U/L0 200 400 600
85
RMV - ALT U/L0 200 400 600
95
Allowable bias consideringintra individual and inter individual biological variation
S-Amylase 7,4 %
S ALT 12 0 %S-ALT 12,0 %
S-AST 5,4 %,
S-CK 11,5 %
S-GGT 10,8 %
*) C Ri t l C t d t b bi l i l i ti d
S-LDH 4,3 %
) C. Ricos et. al. Current databases on biological variation: pros, cons andprogress. Scand J Clin Lab Invest 1999; 59: 491-500.
Pooled human sera with certified RMVsapplied to routine procedures from Roche Diagnostics (Modular, Hitachi 917)applied to routine procedures from Roche Diagnostics (Modular, Hitachi 917)
Mean recovery (Modular) Mean recovery (Hitachi 917)
100 1 %97 7 %GGT
95,3 %94,2 %CK
100 1 %97 7 %GGT
95,3 %94,2 %CK
95,5 %94,4 %AMY
100,1 %97,7 %GGT
95,5 %94,4 %AMY
100,1 %97,7 %GGT
110,2 %108,9 %AST
103,6 %104,6 %ALT
110,2 %108,9 %AST
103,6 %104,6 %ALT
5,4 %
99 4 %90 7 %ALP
98,4 %94,0 %LDH
99 4 %90 7 %ALP
98,4 %94,0 %LDH 4,3 %
99,4 %90,7 %ALP 99,4 %90,7 %ALP
Pooled human sera with certified RMVs
Use for manufacturers:Use for manufacturers:
- Calibration concepts based on commutable calibration material
- Control material
sufficient number of aliquots sufficient stable at – 75 °Csufficient 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 measurementsReaction Principle for ALP measurements
Decision for a reference procedurefor ALP using the substrate AMP
4 Nit h l h h t AMP ALP 4 Nit h id AMP h h t4-Nitrophenylphosphate + AMP ALP 4-Nitrophenoxide + AMP-phosphate
4-Nitrophenylphosphate + H2O ALP 4-Nitrophenoxide + Phosphate
Reasons for a decision on ALP-AMPReasons 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/lpH (37 °C) 10.20 ± 0.054 Nit h l h h t 16 l/l4-Nitrophenylphosphate 16 mmol/lZinc sulfate 1 mmol/lMagnesium acetate 2 mmol/lMagnesium acetate 2 mmol/lHEDTA 2 mmol/lVolume 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 °CWave length 405 nm ± 1 nmBand width ≤ 2 nmLight path 10 00 mm ± 0 01 mmLight path 10.00 mm ± 0.01 mmIncubation time 60 s Delay time 60 syMeasurement interval 120 sReadings (measurement points) ≥ 6
ALP-AMP: pH optimum
100 Serum 1
90
100 Serum 1
Serum 2
Serum 3
(%)
70
80 Serum 4
Serum 5
ALP
60
70Serum 6
Serum 7
Optimum pH andrange of uncertainty(k=2)
40
50 Serum 8
Serum 9
Serum 10
H9,6 9,8 10,0 10,2 10,4 10,6 10,8
40 Serum 10
pH
pH optima of ALP isoforms
120 120
p p
100 100
ALP
%
60
80
ALP
%
60
80
40 40
pH9,6 9,9 10,2 10,5 10,8
20
pH9,6 9,9 10,2 10,5 10,8
20
commercial calibratorcommercial control material 1commercial control material 2commercial control material 3
commercial control material 4ring trial material 1ring trial material 2ring trial material 3
human serum human serum
Method comparison ALP:R f d R h Di ti (M d l P)Reference procedure vs Roche Diagnostics (Modular P)
1000 Number of values 99Slope 0,908
) U
/l
800Slope, lower limit 95 % 0,898Slope, upper limit 95 % 0,917Intercept 1,75Intercept, lower limit 95% -0,03I t t li it 95% 4 07
LP (R
oche
400
600 Intercept, upper limit 95% 4,07Coefficient of correlation 0,9982Ratio mean 0,921
AL
200 ALP measurementsy = xRegression line (Passing/Bablok)
ALP (IFCC) U/l0 200 400 600 800 1000
0
Nearly no intercept. Recalibration is only a matter of the slope
Network of reference laboratories performing a feasibility study
Francesca Canalias SpainF Ceriotti ItalyPFH Franck NetherlandsFJ Gella SpainFJ Gella SpainPJ Jørgensen DenmarkR. Klauke GermanyR Nagel (Roche Diagn.) GermanyM Panteghini ItalyG Schumann GermanyG Schumann Germany
Investigation of pooled human sera,gprocessed control material,a candidate reference material
Co-operation of two IFCC committeesp
Committee Reference Systems for Enzymes(C-RSE)(C RSE)
Committee Reference Intervals and Decision Limits(C-RIDL)
Publication of the IFCC reference measurement procedure andPublication of the IFCC reference measurement procedure and reference intervals in preparation
R f P d f LiReference Procedure for Lipase
Measurement principlep p
Titrimetryy
Spectrometryp y
Reference measurement procedurecloser to routine procedures
Candidate for a Reference Procedurefor Lipase
((DGGR)DGGR)
(Beisson (Beisson et alet al., Eur. J. Lipid Technol., 2000)., Eur. J. Lipid Technol., 2000)
Candidate for a Reference Procedurefor Lipase
PancreasPancreas
for Lipase
1,21,2--Dioleoylglycerol + HDioleoylglycerol + H22O O 22--Monooleoylglycerol + Oleic acidMonooleoylglycerol + Oleic acid
Pancreas Pancreas LipaseLipase
2-Monooleoylglycerol + H2O Glycerol + Oleic acid
Glycerol + ATP ADP + Glycerol-3-P
MGLP
GKGlycerol + ATP ADP + Glycerol 3 P
ADP + D-Glucose D-Glucose-6-P + AMP
GK
ADP-HK
D-Glucose-6-P + NADP+ D-Glucono-δ-lactone-6-P + NADPH + H+
G6PDH
66--PGLPGLD-Glucono-δ-lactone-6-P + H2O 6-Phosphogluconate
MGLP: Monoglyceride lipase, GK: Glycerol kinase , ADP-HK: ADP-dependent hexokinase, G6PDH: Glucose -6-phosphate dehydrogenase, 6-PGL: 6-Phosphogluconolactonase
Reference measurement procedure for pancreatic lipase
C-RSE is working on a concept for the development of a reference measurement procedure (RMP) for lipase.p ( ) p
C-RSE is in favour of spectrophotometry as the measurement principle forC RSE is in favour of spectrophotometry as the measurement principle for the RMP for lipase.
The decision on a single substrate (see two choices) for the RMP needs consensus with the corporate members of IFCC.
C-RSE needs common consent or even a mandant from the corporate f CC f fmembers of IFCC for the choice of a suited measurement principle and the
selected substrate for reference methodology.
Committee Reference Systems for Enzymes: C-RSE
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