The Centre for The Centre for Metrological Traceability Metrological Traceability in Laboratory Medicine in Laboratory Medicine (CIRME): scope and (CIRME): scope and activitiesactivitiesMauro PanteghiniCIRME Director

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OutlineOutline

•• Introductory remarksIntroductory remarks•• Scope of CIRMEScope of CIRME•• CIRME activitiesCIRME activities•• Examples of ongoing projectsExamples of ongoing projects

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Comparability in Laboratory Comparability in Laboratory Medicine Medicine

•• Interchangeability of results over time and Interchangeability of results over time and space would significantly contribute to space would significantly contribute to improvements in healthcare, since results of improvements in healthcare, since results of clinical studies undertaken in different clinical studies undertaken in different locations or times could be universally locations or times could be universally appliedapplied

Standardize clinical decision limits (i.e., cutpoints for intervention)

Standardize clinical decision limits Standardize clinical decision limits (i.e., (i.e., cutpointscutpoints for intervention)for intervention)

Effective application of evidence-based medicineEffectiveEffective applicationapplication of of evidenceevidence--basedbased medicinemedicine

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Why comparability?Why comparability?

•• Use of clinical guidelines is becoming more and Use of clinical guidelines is becoming more and more prominent in the clinical practicemore prominent in the clinical practice

•• AnalytesAnalytes in these guidelines (glucose, HbAin these guidelines (glucose, HbA1c1c, PSA, , PSA, creatininecreatinine ((eGFReGFR), etc.) have specific cutoffs that ), etc.) have specific cutoffs that are independent of the assay usedare independent of the assay used

•• To globally utilize these cutoffs, the assay results To globally utilize these cutoffs, the assay results for the for the analyteanalyte in question must be comparable: in question must be comparable: TO BE COMPARABLE THEY MUST BE TO BE COMPARABLE THEY MUST BE ““TRACEABLETRACEABLE””

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NOTESNOTES1.1. The The conceptconcept isis oftenoften expressedexpressed byby the the adjectiveadjective ““traceabletraceable””2.2. The The unbrokenunbroken chainchain of of comparisonscomparisons isis calledcalled a a ““traceabilitytraceability

chainchain””

WhatWhat isis metrologicalmetrological traceabilitytraceability??Property of the result related to nationalProperty of the result related to national

or international standards through anor international standards through anunbroken chain of comparisons all havingunbroken chain of comparisons all having

stated stated uncertaintiesuncertaintiesObjectiveObjective →→ ToTo enableenable the the resultsresults obtainedobtained byby the the calibratedcalibrated routine procedure routine procedure toto bebe expressedexpressed in in termsterms of the of the valuesvalues obtainedobtained at the at the highesthighestavailableavailable levellevel of the of the calibrationcalibration hierarchyhierarchy

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Reference Measurement SystemReference Measurement System

Primary Ref. ProcedurePrimary Ref. Procedure

Secondary Ref. Secondary Ref. ProcedureProcedure

ManufacturerManufacturer’’ssInternal ProcedureInternal Procedure

EndEnd--useruser’’s Routines RoutineProcedureProcedure

Primary Reference MaterialPrimary Reference Material(e.g. pure (e.g. pure analyteanalyte))

Secondary Ref. MaterialSecondary Ref. Material(serum(serum--based)based)

ManufacturerManufacturer’’s Calibrators Calibrator

Routine SampleRoutine Sample

Test ResultTest Result

UncertaintyUncertaintyincreasesincreases SI UnitsSI Units

TraceabilityTraceability

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Legal Background for the Use of Legal Background for the Use of MetrologicallyMetrologicallyCorrect Measurement Systems in Laboratory Correct Measurement Systems in Laboratory

MedicineMedicineRequirement of theRequirement of theEU 98/79/ECEU 98/79/EC--IVD Directive: IVD Directive:

The traceability of values The traceability of values assigned to calibrators and/or assigned to calibrators and/or control materials must be assured control materials must be assured through available reference through available reference measurement procedures and/or measurement procedures and/or available reference materials of a available reference materials of a higher orderhigher order

[[Annex I Annex I -- Essential Requirements (Part A. General Essential Requirements (Part A. General Requirements)]Requirements)]Official Journal of European Communities (1998)Official Journal of European Communities (1998)

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HowHow to to fulfillfulfill thesethese essential essential requirementsrequirements??

ThroughThrough thethe availabilityavailability of:of:→→ ReferenceReference materialsmaterials→→ ReferenceReference methodsmethods→→ ReferenceReference laboratorylaboratory servicesservices

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OtherOther keykey stakeholdersstakeholders::••ProducersProducers of of ReferenceReference MaterialsMaterials••RegulatoryRegulatory BodiesBodies••IVD IVD IndustryIndustry••EQAS EQAS OrganizationsOrganizations

ObjectivesObjectives and and PurposePurpose

To support comparability and equivalence of measurement results in Laboratory Medicine for the purpose of improvinghealthcare, through worldwide accepted traceability effort

following the principles of metrology

To support comparability and equivalence of measurement To support comparability and equivalence of measurement results in Laboratory Medicine results in Laboratory Medicine for the for the purposepurpose of of improvingimprovinghealthcarehealthcare, through worldwide accepted traceability effort , through worldwide accepted traceability effort

following the principles of metrologyfollowing the principles of metrology

To support IVD manufacturers in registration and licensing the CE label conforming with the EU directive

To support IVD manufacturers in registration and licensing To support IVD manufacturers in registration and licensing the CE label conforming with the EU directivethe CE label conforming with the EU directive

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WhatWhat has JCTLM has JCTLM delivereddelivered??

The WorldThe World’’s only qualitys only quality--assured database of:assured database of:

a)a) Higher Order Reference MaterialsHigher Order Reference Materialsb)b) Higher Order Reference Measurement ProceduresHigher Order Reference Measurement Proceduresc)c) Laboratory Reference Measurement Services Laboratory Reference Measurement Services

http://http://www.bipm.org/en/committees/jc/jctlm/jctlmwww.bipm.org/en/committees/jc/jctlm/jctlm--db/db/

For use by (primarily):For use by (primarily):a) IVD industry (to ensure that results produced by a) IVD industry (to ensure that results produced by IVDsIVDs are traceable to)are traceable to)b) Regulators (to verify that results produced by b) Regulators (to verify that results produced by IVDsIVDsare traceable to)are traceable to)

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Traceability implementationTraceability implementation

Reference Reference Measurement Measurement Service Service ProvidersProviders

ISO 17511:2003. In vitro diagnostic medical devices ISO 17511:2003. In vitro diagnostic medical devices -- Measurement of quantities in biological samples Measurement of quantities in biological samples ––Metrological traceability of values assigned to calibrators and Metrological traceability of values assigned to calibrators and control materials. control materials.

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ReferenceReference MeasurementMeasurement Service ProvidersService Providers

Laboratories have been assessed according to the following Laboratories have been assessed according to the following criteriacriteria::

a) metrological level of the employed reference measurement a) metrological level of the employed reference measurement procedure (compliance with the JCTLM list). procedure (compliance with the JCTLM list).

b) accreditation to ISO 17025/15195 standards (b) accreditation to ISO 17025/15195 standards (for for eacheachindividualindividual measurandmeasurand asas calibrationcalibration laboratorylaboratory)) or link to a or link to a national metrology institute. national metrology institute.

c) demonstration of their competence by regular comparative c) demonstration of their competence by regular comparative measurements in specific EQAS. measurements in specific EQAS.

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A A ClearerClearer ImplementationImplementation of of thethe IVD IVD DirectiveDirective

Ref. Materials, Ref. Materials, MethodsMethods, Labs , Labs

IVD IndustryIVD IndustryConformityConformity and Traceability and Traceability

Uniform Uniform CalibrationCalibrationComparabilityComparability of Patient of Patient ResultsResults

EU EU -- IVD IVD -- DirectiveDirective

The Centre for The Centre for Metrological Traceability Metrological Traceability in Laboratory Medicine in Laboratory Medicine (CIRME)(CIRME)created on June 2006 with the scope to join in a sole entity scientists and activities of various Departments of the University of Milan interested in the development of reference methods and calibration materials of high metrological order in the field of biomedical diagnostics.

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CIRME reference measurement services in the JCTLM listCIRME reference measurement services in the JCTLM list

CIRME reference measurement services in the JCTLM listCIRME reference measurement services in the JCTLM list

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CIRME reference measurement services in the JCTLM listCIRME reference measurement services in the JCTLM list

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Participation to the IFCC ring trials for Participation to the IFCC ring trials for Reference Laboratories (RELA)Reference Laboratories (RELA)

ResultsResults of IFCC ring of IFCC ring trialstrials are are availableavailable at: at: http://www.dgklhttp://www.dgkl--rfb.de:81rfb.de:81

Result from CIRME laboratoryResult from CIRME laboratory

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Participation to the IFCC ring trials for Participation to the IFCC ring trials for Reference Laboratories (RELA)Reference Laboratories (RELA)

Result from CIRME laboratoryResult from CIRME laboratory

ResultsResults of IFCC ring of IFCC ring trialstrials are are availableavailable at: at: http://www.dgklhttp://www.dgkl--rfb.de:81rfb.de:81

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In addition, to warrant continuity, stability and further In addition, to warrant continuity, stability and further reliability, the CIRME reference laboratories reliability, the CIRME reference laboratories participate participate in the IFCC network studiesin the IFCC network studies

CIRME MSCIRME MS

CIRME CECIRME CE

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SERVICESSERVICES

CertificationCertification ofof TargetTarget--settingsetting for for ValidationValidation ofofReference Materials Reference Materials EQAS EQAS DiagnosticDiagnostic

KitsKits//InstrumentsInstruments

In cooperation withIn cooperation with In cooperation with In cooperation In cooperation with In cooperation withwithproducersproducers of of RMsRMs EQAS EQAS organizersorganizers manufacturersmanufacturers

0.90

0.92

0.94

0.96

0.98

1.00

1.02

1.04

0 50 100 150 200 250 300

AST IFCC Reference Procedure, U/L

AST r

outine

met

hod/

IFCC

Ref

. Pr

oc.

Human seracRM 1cRM 2

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The feasibility results combined with the results of the homogenThe feasibility results combined with the results of the homogeneity eity study allow us to expect an expanded uncertainty of around 4% fostudy allow us to expect an expanded uncertainty of around 4% for r the assigned value of the candidate reference material, which isthe assigned value of the candidate reference material, which issimilar to the expanded uncertainty of the other IRMM reference similar to the expanded uncertainty of the other IRMM reference materials for enzymes.materials for enzymes.

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Cooperation with IRMM and the IFCC Committee on Plasma Cooperation with IRMM and the IFCC Committee on Plasma Proteins for the preparation of the new reference Proteins for the preparation of the new reference material for plasma proteins [ERMmaterial for plasma proteins [ERM--DA470]:DA470]:

-- serum collection, serum collection, completedcompleted;;-- selection of 18 laboratories participating in the value selection of 18 laboratories participating in the value transfer, transfer, completedcompleted;;-- lab feasibility study, lab feasibility study, completedcompleted;;-- value transfer campaign for 14 plasma proteins value transfer campaign for 14 plasma proteins (Autumn 2007);(Autumn 2007);-- availability of the certified material (June 2008).availability of the certified material (June 2008).

ReferenceReference material ERMmaterial ERM--DA470 DA470 Human Serum ProteinsHuman Serum Proteins

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IFCC Project for IFCC Project for StandardizationStandardization of of MyoglobinMyoglobin ImmunoassaysImmunoassays

PURPOSE PURPOSE →→ toto determinedetermine the the suitabilitysuitability of 5 candidate of 5 candidate secondarysecondaryRMsRMs in relation in relation toto linearitylinearity, , recoveryrecovery rate and rate and commutabilitycommutability

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31.7

25.8

14.2

28.5

14.4

19.7

05

1015

202530

35R

elat

ive

stan

dard

de

viat

ion

Before

calib

ration

cRM 1

cRM 2

cRM 3

cRM 4

cRM 5

ResultsResults obtainedobtained fromfrom 11 11 differentdifferent platformsplatforms

Selected materialIRMM/IFCC 458

SelectedSelected materialmaterialIRMM/IFCC 458IRMM/IFCC 458

Panteghini M et al., Panteghini M et al., ClinClin ChimChim ActaActa 2004 2004

IFCC Project for IFCC Project for StandardizationStandardization of of MyoglobinMyoglobin ImmunoassaysImmunoassays

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Current status of the projectCurrent status of the project•• After production, the material was stored at After production, the material was stored at

IRMM and part of the batch was stored at IRMM and part of the batch was stored at different temperatures under isochronous different temperatures under isochronous conditions for a stability study (short and longconditions for a stability study (short and long--term). These samples are now ready for term). These samples are now ready for analysis as well as some samples foreseen for analysis as well as some samples foreseen for homogeneity testing. These studies are homogeneity testing. These studies are performed by CIRME.performed by CIRME.

•• After this phase, the value assignment of the After this phase, the value assignment of the material will be performed using an LCmaterial will be performed using an LC--MSMS--MS MS method developed in a collaborative project method developed in a collaborative project with IRMM. with IRMM.

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PrimaryPrimary::-- Pure HbAPure HbA00 and HbAand HbA22 (three batches produced so (three batches produced so far)far)

Reference Materials for Reference Materials for HemoglobinsHemoglobins AA0 0 and Aand A22Available c/o CIRME (Dept. Science and Biomedical Technology)Available c/o CIRME (Dept. Science and Biomedical Technology)

SecondarySecondary::-- Lyophilized Lyophilized hemolysateshemolysates;;-- Production process started on May 2007;Production process started on May 2007;-- Preliminarily tested for stability and Preliminarily tested for stability and commutability for 3 HPLC methods;commutability for 3 HPLC methods;-- In cooperation with IRMM and IFCCIn cooperation with IRMM and IFCC

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PrinciplePrincipleHHbAbA22 ratio to whole hemoglobin is determined as ratio of a delta ratio to whole hemoglobin is determined as ratio of a delta chain specific peptide to an alpha chain specific peptide. chain specific peptide to an alpha chain specific peptide. Peptides are obtained by treating total red blood cell Peptides are obtained by treating total red blood cell lysatelysatewith with trypsintrypsin. . Peptide mixture is analyzed by HPLCPeptide mixture is analyzed by HPLC--ESI/MS. ESI/MS.

Calibration is performed against primary ref. materials (mixtureCalibration is performed against primary ref. materials (mixtures s of HbAof HbA22 and HbAand HbA00 materials).materials).

The SOP has been defined and implemented in 2 reference The SOP has been defined and implemented in 2 reference laboratories: one is from CIRME.laboratories: one is from CIRME.

Reference Measurement Procedure for Reference Measurement Procedure for HbAHbA22

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Reference Measurement Procedure Reference Measurement Procedure for Alkaline for Alkaline PhosphatasePhosphatase (ALP)(ALP)

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IFCC/IRMM studyIFCC/IRMM studyTesting transferability of the candidate reference Testing transferability of the candidate reference

procedure for measurement of the catalytic procedure for measurement of the catalytic concentration of ALPconcentration of ALP

•• The state of the evaluation of the IFCC The state of the evaluation of the IFCC reference procedure for ALP at 37 reference procedure for ALP at 37 °°C requires C requires now a now a multicentremulticentre study. study.

•• For that purpose, 7 laboratories with For that purpose, 7 laboratories with experiences in spectrometric kinetic reference experiences in spectrometric kinetic reference analysis of catalytic concentration of enzymes analysis of catalytic concentration of enzymes have been recruited to take part in the study. have been recruited to take part in the study.

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Evaluation of commutability of Evaluation of commutability of EQAS materialsEQAS materials

+1,35

+3,85

-1,15

-12.5-10.0-7.5-5.0-2.50.02.55.07.5

10.012.5

4 5 6 7 8 9 10 11 12HPLC HbA1c, %

Bia

s C

OB

AS -

HPL

C, %

+1,35

+3,85

-1,15

-12.5-10.0-7.5-5.0-2.50.02.55.07.5

10.012.5

4 5 6 7 8 9 10 11 12HPLC HbA1c, %

Bia

s C

OB

AS -

HPL

C, %

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IFCC Committee on Reference Intervals and IFCC Committee on Reference Intervals and Decision Limits (CDecision Limits (C--RIDL)RIDL)

MulticentreMulticentre Reference Interval Study Reference Interval Study for AST, ALT & for AST, ALT & γγGTGT

•• Preparation of a protocol for Preparation of a protocol for collaborative experiments on the collaborative experiments on the establishment of reference values using establishment of reference values using assays traceable to reference systemsassays traceable to reference systems

•• Production of Production of ““standardizedstandardized”” reference reference intervals for AST, ALT, and intervals for AST, ALT, and γγGTGT

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1.1. Preparation of the material for Preparation of the material for trueness/traceability controltrueness/traceability control

–– 3 serum pools at borderline concentrations, 3 serum pools at borderline concentrations, minimally processed (filtering, freezing at minimally processed (filtering, freezing at --80 80 °°C);C);

–– value assignment by the reference method value assignment by the reference method by three reference laboratories according by three reference laboratories according to a defined protocol (three batches, two to a defined protocol (three batches, two or three replicate measurements per or three replicate measurements per batch). batch).

IFCC Committee on Reference Intervals and IFCC Committee on Reference Intervals and Decision Limits (CDecision Limits (C--RIDL)RIDL)

MulticentreMulticentre Reference Interval Study Reference Interval Study for AST, ALT & for AST, ALT & γγGTGT

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Verification of trueness of the participating laboratoriesVerification of trueness of the participating laboratories

GGTy = 1,0788x - 2,7019

R2 = 0,9989

y = 0,9343x - 2,3946R2 = 0,9998

y = 0,97x - 2,9626R2 = 0,9997

0,0

20,0

40,0

60,0

80,0

100,0

120,0

0 20 40 60 80 100 120

Reference method (U/L)

Rou

tine

met

hods

(U/L

)

Lab 1Lab 2Lab 3

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AST (U/L)AST (U/L)F 15F 15––3434M 18M 18––4242

ALT (U/L)ALT (U/L)F 7F 7––4141M 9M 9––5555

GGT (U/L)GGT (U/L)F 13F 13––4343M 14M 14––5959

PremisePremiseUse of commercial assays that provide traceable results permits Use of commercial assays that provide traceable results permits to employ to employ ““commoncommon”” reference intervals (at least within reference intervals (at least within homogeneous ethnic groups)homogeneous ethnic groups)

Traceability as a mean to obtain worldwide Traceability as a mean to obtain worldwide useful reference intervalsuseful reference intervals

IFCC Committee on Reference Intervals and Decision Limits (CIFCC Committee on Reference Intervals and Decision Limits (C--RIDL)RIDL)

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