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

23 %

External Quality Assessment

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

105Modular P

% 100

Hitachi-917

over

y 100

Rec

o

95

90

RMV - LDH U/L200 400 600

120Modular P

%

115 Hitachi-917

over

y

105

110

Rec

o

100

105

95

100

RMV - AST U/L0 100 200 300

105Modular P

%

100Hitachi-917

over

y

95

Rec

o

90

85

RMV - ALP U/L200 400 600

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

Rainer KlaukeRenate Strache

Calibration laboratory(Reference laboratory)

DKD-K-20602