Metrological Traceability andAssay Standardization in

Laboratory Medicine

Metrological Traceability andAssay Standardization in

Laboratory Medicine

STRESA, ITALYMay 24, 2013

Standardization of cTnI:Is there a light at the end of

the tunnel?

Standardization of cTnI:Is there a light at the end of

the tunnel?

Jill TatePathology QueenslandBrisbane, AUSTRALIA

Joint Committee for Traceability in Laboratory Medicine

Talk OutlineTalk Outline

• Background• IFCC cTnI Pilot Study

– Serum pool as surrogate SRM– Harmonisation capability– Commutability across all assays– Stability

• Next steps– Preparation of candidate SRM– Value assignment and uncertainty budget– Value transfer to manufacturer’s calibrator– Harmonisation / commutability testing phase

SI unit

Pure substance certified reference material

Reference measurement procedureRP-LC and amino acid analysis

Matrix certified reference material

Materials Procedures

Reference measurement procedure

Manufacturer’s master calibrator set

Value Transfer Procedure(Reference Laboratory)

Value Transfer Protocol

Manufacturer’s assay calibrators

Routine clinical assay

Patient

Traceability

Patient serum sample

Measurem

ent Uncertainty

Purified reference material:NIST SRM 2921

Serum-based (commutable) reference material: cTnI-positive pool

Candidate RMP: ELISA or MS orMethod harmonisation consensus approach

using commercial cTnI assays

cTnI reference measurement systemcTnI reference measurement system

Reference materials for cTnIReference materials for cTnI

• NIST SRM 2921– Purified ICT complex– Value assignment: RP-LC & amino acid analysis – Not commutable in ~50% commercial assays

• Serum-based certified SRM– Commutable in all commercial assays– Lack of interferences

e.g. cTnI autoantibodies, heterophile antibodies

– Stable over long-term– Standardised procedures in place for value

assignment and value transfer to manufacturers’master calibrators

Requirements for equivalent cTnI measurements

Requirements for equivalent cTnI measurements

• Measurand is defined– unique, invariant part of molecule common to all

components of the mixture present in serum • Antibody specificity is defined

– Abies preferably recognise epitopes located in the stable part of cTnI molecule

– all plasma cTnI forms have equal reactivity or the difference in reactivity is not clinically relevant

• Assays are capable of being harmonised• SRM is commutable across majority of assays• Manufacturers have calibration traceability to

SRM

cTnI isoforms and assay recognitioncTnI isoforms and assay recognition

Changes in cTnI ratio Accu:Ultra over time post chest pain onset

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

0 5 10 15 20 25 30 35 40 45 50 55

Time (h)

Rat

io A

ccu:

Utlr

a cT

nI

AMI - Patient A

AMI - Patient B

AMI - Patient M

Bates KJ et al. Clin Chem 2010; 56: 952-8

cTnIC + T

cTnIC + cTnITC

+ T

cTnIC + cTnITC

+ T

cTnIC + T

• Pools to consist of a blend of clinically relevant cTnI forms and act as “surrogate SRM for cTnI” rather than reflecting the cTnI composition of each individual clinical sample

• Pools are commutable with patient samples covering the clinical cTnI concentration range

• Pools lead to equivalent cTnI measurement values

PROOF of PRINCIPLE: Serum pools as SRM for cTnI

PROOF of PRINCIPLE: Serum pools as SRM for cTnI

• Validation of the immunoassay reference measurement procedure for cTnI

• Current status of commercial cTnI assays

• Assessment of the commutability of “blended”serum pooled cTnI candidate reference materials

• Evaluation of the stability of serum reference materials for cTnI

cTnI Pilot Study in 2010-2012: AIMS

cTnI Pilot Study in 2010-2012: AIMS

cTnI Pilot Study SamplescTnI Pilot Study Samples

• Collection of samples from >90 patients with suspected AMI– cTnI concentrations in range ≈0.05-20 µg/L– Collected from patients up to 72 h post presentation

• 30 samples per low, medium and high level – ≈20 mL serum (≈50 mL blood) collected per patient– Aliquotted within 4 h of collection & stored at ≤ -70 °C

• Preparation of pools & sample kits at NIST

• Testing by NIST and Diagnostic Industry (NPL)– January to May 2012 (1 lab in December 2012)

Participating LaboratoriesParticipating Laboratories

• Beckman Access (AccuTnI) Roche Elecsys cobas e601• Biomerieux VIDAS TnI Ultra Roche Elecsys cobas e411• Siemens ADVIA Centaur (Ultra) Siemens Immulite 1000 TPI• Siemens Immulite 2000/Xpi Siemens Dimension Vista• Siemens Dimension EXL w/LM Siemens Dimension RxL• Siemens Stratus CS Abbott Architect STAT hsTnI• PATHFAST cTnI (PF 1011-K) Abbott Architect i2000SR• PATHFAST cTnI-II (PF 1101-K) Abbott AxSYM cTnI-ADV• OCD Vitros 5600 cRMP (at NIST)

Preparation of Serum PoolsPreparation of Serum Pools

• Patient pools prepared in three ways by:– addition of individual cTnI-positive native patient

samples– dilution of high cTnI concentration pool with low and

medium concentration pools– dilution of high & medium pools with a normal pool– final concentration range ≈ 0.2-10 µg/L

• Normal Pool– 500 mL pool from ~5-10 female donors (<30 y,

BMI <25, & no reported history of heart disease)– pre-screened for cTnAAs – none detected – all participating labs also screened an aliquot.

Pool DescriptionA 18 low cTnI patient samples pooled using

volumes which ranged from 1.25 mL to 8.0 mL

B 21 medium cTnI patient samples pooled using volumes which ranged from 1.5 mL to 6.0 mL

C 21 high cTnI patient samples pooled using volumes which ranged from 0.75 mL to 10.75 mL

D 28.0 mL Pool A and 7.0 mL Pool C

E 14.0 mL Pool C and 21.0 mL Pool B

F 4.0 mL Pool C and 36.0 mL Pool NORM

G 4.0 mL Pool B and 36.0 mL Pool NORM

cTnI Candidate Serum PoolscTnI Candidate Serum Pools

• Imprecision– Duplicate measurements for 90 patient samples and

7 duplicate vials of pools

• Current status of commercial cTnI assays and cRMP– Between-method variation

• Commutability– Pools vs 90 patient samples

• Harmonisation capability– Between-method agreement

cTnI Pilot Study:data analysis and results

cTnI Pilot Study:data analysis and results

cTnI Pilot Study - imprecisioncTnI Pilot Study - imprecision

0.0

2.0

4.0

6.0

8.0

10.0

12.0

0.0 0.1 1.0 10.0 100.0

Mean cTnI for 17 assays ( µµµµg/L)

Mea

n im

prec

isio

n fo

r 17

ass

ays

(%C

V)

Low patient samples - 4.1% CV

High patient samples - 2.1% CV

Medium patient samples - 2.2% CV

Serum pools - 2.2% CV

L12

L16L21

Assay 2 vs. Assay 4

0

5

10

15

20

0 5 10 15 20 25

Assay 2 [cTnI], μg/L

Ass

ay

4

[cT

nI]

, μ

g/

L

Patient Samples

serum pools

Assay 2 and 4 from same manufacturer

Paired comparisons

Commutability Assessment of PoolsCommutability Assessment of Pools

Paired comparisons

Assay 14 and 15 from different manufacturer

Assay 14 vs. Assay 15

0

2

4

6

8

10

12

14

0 5 10 15 20

Assay 14 [cTnI], μg/L

Ass

ay

15

[cT

nI]

, μ

g/

L

Patient Samples

serum pools

Commutability Assessment of PoolsCommutability Assessment of Pools

Most of the 136 paired comparisons looked like these

Assay 9 vs. Assay 11

0

2

4

6

8

10

12

14

0 1 2 3 4

Assay 9 [cTnI], μg/L

Ass

ay

11

[cT

nI]

, μ

g/L

Patient Samples

serum pools

Paired comparisonsAssay 5 vs. Assay 7

0

2

4

6

8

10

12

0 10 20 30 40

Assay 5 [cTnI], µµµµg/L

Ass

ay

7 [

cTn

I],

?g

/L

Patient Samples

serum pools

Assay 3 vs. Assay 9

0

0.5

1

1.5

2

2.5

3

3.5

4

0 5 10 15 20 25

Assay 3 [cTnI], µµµµg/L

Ass

ay

9 [

cTn

I], µg

/L

Patient Samples

serum pools

Patient Samples

serum pools

Commutability Assessment of PoolsCommutability Assessment of Pools

Assay 6 vs. Assay 11

0

2

4

6

8

10

12

14

0 5 10 15 20 25 30

Assay 6 [cTnI], µµµµg/L

Ass

ay

11

[cT

nI]

, µg

/L

Nearly all paired comparisons of the cRMP vs. commercial assays looked like this

Assay 7 vs. cRMP (NIST)

0

1

2

3

4

5

6

7

8

9

0 2 4 6 8 10 12

Assay 7 [cTnI], μg/L

cRM

P (

NIS

T)

[cT

nI]

, μ

g/

L

Patient Samples

serum pools

Paired comparisons

Commutability Assessment of PoolsCommutability Assessment of Pools

• For commercial assays ~10-fold difference in concentration between assays

• cRMP shows poor correlation with all routine assays

• Passing-Bablok analysis indicates overlap of the 95% confidence intervals of the regression slopes of patient samples and all pools indicating that all the serum pools are commutable for all routine assays

• PCA also indicates pools are commutable

Current status of cTnI assays in 2012Current status of cTnI assays in 2012

Data analysis of cTnI harmonisationData analysis of cTnI harmonisation

• Slope correction was determined for each assay– using Passing-Bablok regression analysis against mean

cTnI for 17 assays for 90 patient samples

• Mathematical recalibration/recalculation was applied

– correction factor (CF) determined as [1/regression slope] – recalculated cTnI = measured cTnI x CF

• Between-method agreement (CV) post recalibration for:

– all 17 assays– 16 assays (1 assay excluded)

Intercept : -0.0112 [ -0.0327 to 0.0074 ]Slope : 1.011 [ 0.964 to 1.045 ]

Passing-Bablok regression N = 880 4 8 12 16

Mean cTnI 17 assays0

4

8

12

16AxSYM cTnIafter recalibration

Mean difference : -0.00726 [ -0.0234 to 0.00887 ]Logarithmic difference plot N = 88

-1.6-1.2-0.8-0.4 0.0 0.4 0.8 1.2Mean cTnI

-0.3

-0.2

-0.1

0.0

0.1

0.2Log Difference(AxSYM minus mean cTnI)

Intercept : -0.0024 [ -0.0254 to 0.0146 ]Slope : 1.034 [ 0.996 to 1.070 ]

Passing-Bablok regression N = 880 4 8 12 16

Mean cTnI 17 assays0

4

8

12

16AccuTnI after recalibration

Mean difference : 0.000835 [ -0.0168 to 0.0185 ]Logarithmic difference plot N = 88

-1.2 -0.8 -0.4 0.0 0.4 0.8 1.2

Mean cTnI-0.6

-0.4

-0.2

0.0

0.2Log Difference(AccuTnI minus mean cTnI)

cTnI post recalibrationcTnI post recalibration

Mean difference : 0.00918 [ -0.0155 to 0.0338 ]Logarithmic difference plot N = 88

-1.2 -0.8 -0.4 0.0 0.4 0.8 1.2Mean cTnI

-0.4

-0.2

0.0

0.2

0.4

0.6

Log Difference(Dimension VISTA minus mean cTnI)

Mean difference : 0.0577 [ 0.0327 to 0.0826 ]Logarithmic difference plot N = 88

-1.2 -0.8 -0.4 0.0 0.4 0.8 1.2Mean cTnI

-0.4

-0.2

0.0

0.2

0.4

0.6

0.8

Log Difference(Dimension EXL minus mean cTnI)

Mean difference : 0.00838 [ -0.0105 to 0.0272 ]Logarithmic difference plot N = 88

-1.2 -0.8 -0.4 0.0 0.4 0.8 1.2Mean cTnI

-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4

Log Difference(Dimension RxL minus mean cTnI)

Mean difference : -0.0095 [ -0.0283 to 0.00932 ]Logarithmic difference plot N = 88

-1.6 -1.2 -0.8 -0.4 0.0 0.4 0.8 1.2

Mean cTnI-0.3

-0.2

-0.1

0.0

0.1

0.2

0.3

0.4Log Difference(Stratus CS minus mean cTnI)

Assays with same antibody specificityAssays with same antibody specificity

L9L23

M25M27

L9

L23M25

M27

M27M25

L23L9 H15

H15

L12L33

L19

Low cTnI Patient Samples

0.0

20.0

40.0

60.0

80.0

100.0

120.0

0.00 0.20 0.40 0.60

Mean cTnI for 17 assays (µg/L)

Bet

wee

n-m

etho

d va

riatio

n (%

) po

st r

ecal

ibra

tion

L9

L23

L16 & 21

Medium cTnI Patient Samples

0.0

10.0

20.0

30.0

40.0

50.0

60.0

0.00 1.00 2.00 3.00 4.00Mean cTnI for 17 assays (µg/L)

Bet

wee

n-m

etho

d va

riatio

n (%

) po

st r

ecal

ibra

tion

M25M27

M23

High cTnI Patient Samples

0.0

10.0

20.0

30.0

40.0

50.0

0.00 5.00 10.00 15.00Mean cTnI for 17 assays (µg/L)

Bet

wee

n-m

etho

d va

riatio

n (%

) po

st r

ecal

ibra

tion

H15

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

0.00 2.00 4.00 6.00 8.00 10.00

Mean cTnI for 17 assays (µg/L)

Bet

wee

n-m

etho

d va

riatio

n (%

) af

ter

reca

libra

tion

C

F

D & B

G

A

E

D

cTnI harmonisation post recalibrationcTnI harmonisation post recalibration

Overall mean variation~26%

Overall mean variation~18%

Overall mean variation~14%

Serum Pools

cTnI between-method agreementcTnI between-method agreement

0

10

20

30

40

50

60

70

1 2 3 4 5 6 7 8 9 10 11 12 13 14

Serum Pools A to G (duplicate series)

Bet

wee

n-m

etho

d va

riatio

n (%

)

PRE recalibration of 17 assays

POST recalibration of 17 assays

POST recalibration of 16 assays

cTnI Pilot Study: CONCLUSIONScTnI Pilot Study: CONCLUSIONS

• Serum pools behave better than most of the patient samples with lower inter-assay variability

• All serum pools are commutable with all routine assays

• Some assays correlated to the mean value better than other assays

• A high between-assay correlation for some assays from same manufacturer

• After calibration differences are removed method agreement was ~8 to 15 %CV in range 1-8 µg/L cTnI

Next StepsNext Steps

• Production of SRM for cTnI• Value assignment and commutability

testing of SRM• Uncertainty budget determined for SRM• Value transfer to manufacturers’ master

calibrators• Phase 3: harmonisation testing in a round

robin

Production of SRM 2922 for cTnIProduction of SRM 2922 for cTnI

• Minimum of 20 patient serum samples (min. vol 20 mL each)

• cTnI in range 5-20 µg/L• Stored at ≤ -70 °C• Prepare a serum pool from

– ≥20 patient sera (min. vol 610 mL) and – Dilute 5-fold with normal pooled human serum (min.

vol 2,440 mL)

• Aliquot diluted serum pool (0.5 mL) into 2 mL PP vials to be stored at ≤ -70 °C

• 6,000 vials to be stored at NIST

Consensus value assignment for cTnIConsensus value assignment for cTnI

• Method harmonisation consensus approach using all commercial cTnI assays– mean or weighted mean value

• Use another panel of individual patient samples to confirm correlation at the time of value-assignment measurements– similar to the pilot study but scaled down– fewer patient samples and narrower concentration

range

• Also use calibrant samples prepared from dilutions of SRM 2921 in cTnI negative serum to “re-calibrate” the manufacturers’ data sets of the patient serum panel

Performance criteria for cTnI: measurement uncertainty

Performance criteria for cTnI: measurement uncertainty

CVintraindividual 9.7%; CVinterindividual 56.8%* TE = Bias goal + 1.96xCVa

Performance goal

Imprecision goal

Bias goal Total error goal *

Minimum 7.3% 21.6% 36%

Desirable 4.9% 14.4% 24%

Optimum 2.4% 7.2% 12%

Panteghini M. In: Laboratory and Clinical Issues Affecting the Measurement and Reporting of Cardiac Troponins: A Guide for Clinical Laboratories. Alexandria: AACB; 2012. p. 53-61.

Value transfer to manufacturers’ calibratorsValue transfer to manufacturers’ calibrators

• Compare with value transfer of cystatin C ERM-DA471/IFCC

• Consensus method process uses a standardised value transfer RMP consisting of dilutions of master calibrator for cTnI and candidate SRM– Within and between day runs– Number of replicates to depend on a predetermined

precision goal

• Phase 3 Round Robin: – Harmonisation testing using patient samples

IFCC WG Standardization of Troponin I

IFCC WG Standardization of Troponin I

Labs that participated in

cTnI Pilot Study

ABBOTT DIAGNOSTICS BECKMAN COULTERBIOMERIEUXMITSUBISHI CHEMICAL MED COORTHO-CLINICAL DIAGNOSTICROCHE DIAGNOSTICS GmbHSIEMENS DIAGNOSTICSNISTNPL

WG-TNI Membership

Name Affiliation

J Tate (Chair) (AU) IFCC

J Barth (UK) ACB

D Bunk (US) NIST

R Christenson (US) AACC

A Katrukha (FI) HyTest Ltd.

M Panteghini (IT) CIRME

R Porter (UK)J Noble (UK)

NPL

H Schimmel (BE) IRMM

L Wang (US) NIST

I Young IFCC SD Liaison