Challenges for International Standardization and Traceability - HORMONES -...

Challenges for International Standardization and Traceability

- HORMONES -______________________________________________

Lothar Siekmann

Institute of Clinical Biochemisstry, University of Bonn

Reference Institute of Bioanalysis, DGKL

http://www.dgkl-rfb.de

Target Concentrations for Steroid Hormones in a Commercial Control Serum __________________________________________________________________

Aldosterone Cortisol Progesterone Estradiol

pmol/l nmo/l nmo/l pmol/l_________________________________________________________________________________________________________

ABBOTT 121.9AMERSHAM 113.1BAXTER DADE DIR 104.8 2.16 396.4BAXTER DADE AG ER 244.1BAXTER DADE AD EXT 196.0BIOMERIEUX 2.54 539.6BIOTEX PREMIX 99.4 70.6 3.72 759.9CAMBRID´GE MEDICAL 120.8 0.86CIBA CORNING 110.3CLINICAL ASSAYS 99.3CYBERFLUOR FIAGEN 88.2DIAGNOSTIC PRODUCTS 113.1 3.12 119.3EURODIAGNOSTICS 115.8FARMOS DIAGN. 99.3 4.67 394.9IMMUNCHEM COV. COAT 110.3 5.41 348.7LEECO 113.1 2.99 144.2MALLINCKRODT 96.6NMS PHARMACEUTICALS 3.18 205.5PANTEX IMMUNO 118.6 4.13 190.8PANTEX IMMUNOCOAT 132.4 7.00 154.9PHARMACIA DELFIA 99.9 790.0RSL 169.2 4.77 117.4SCLAVO LISO PHASE 277.4 3.82SERONO 112.0SIBAR ELISA 121.3 1.27SORIN 165.9 68.9 2.86 139.5SYVA EMIT 137.9TECHLAND RIA 4.77VITEK SYSTEMS 110.0

In Vitro Diagnostica DirectiveEuropean Union

__________________________________________

“.... the traceability of values assigned to

calibrators and control materials must be

assured through available reference

measurement procedures and/or reference

materials of higher order ...”

Measurement Traceability

_______________________________________ property of the result of a measurement or the

value of a standard whereby it can be related to

stated references, usually national or international

standards, through an unbroken chain of

comparisons all having stated uncertainties

SI-Unit (definition)

BIPM

Primary calibrator

Secondary calibrator

Mf.´s working (master) calibrator

Mf.´s product calibrator

Secondary referencemeasurement procedure

NMI, ACL

NMI, ACL

Mf.´s selectedmeasurement procedure

Mf.´s standingmeasurement procedure

End-user´s routinemeasurement procedure

Routine sample

RESULT

NMI, ACL

ACL, MCL

ML

Mf.-> End-user

Mf. and/or End-user

MCL

Tra

ceab

ilit

yBIPM: International Institute of Weights and Measures

NMI: National Metrological Institute

ACL: Accredited Calibration Laboratory

MCL: Manufacturer´s Calibration Laboratory

ML: Manufacturer´s Laboratory Mf: Manufacturer

End-user

End-user

Primary reference measurement procedure

ML

µc(y)

Reference Systems Laboratory Medicine_____________________________________________

• Reference Materials

• Reference Measurement Procedures

• Reference Laboratories

SERUM Addition of [14C]Estradiol-17ß

Extraction withDichloromethane

Column chromatographyon Sephadex LH-20

Formation of theHeptafluorobutyric ester

Selected Ion Recordingat m/z 664 and 666

MEASUREMENT OF ESTRADIOL-17ß BY ISOTOPE DILUTION MASS SPECTROMETRY

___________________________________________________________

Calculation of the Estradiol-17ßConcentration from Isotope Ratiosin Samples and Standard Mixtures

M+

m/z

Carrier gas

Injection Vacuum

Valve

Ion Source

Magnet

Detector

Mass Spectrum

Mass Spectrometer

min

Selected ion recording

Gas Chromatograph

Acquired on 22-Jun-1998 at 23:02:15Sample ID: 6/ Pool -772.05-

2.500 3.000 3.500 4.000 4.500 5.000 5.500 6.000 6.500 7.000 7.500 8.000 8.500 9.000 9.500rt0

100

%

0

100

%

8.129

6.862

SIR of 2 Channels EI+ 664.281.04e5

RT

ASI12 Sm (Mn, 4x4)

8.127

7.765 8.420


RT

ASI12 Sm (Mn, 4x4)

m/e 666

[4-14] Estradiol-17ß-3,17ß-di-heptafluorobutyric ester

m/e 664

Estradiol-17ß-3,17ß-di-heptafluorobutyric ester

Acquired on 22-Jun-1998 at 23:02:15Sample ID: 6/ Pool -772.05-

7.940 7.960 7.980 8.000 8.020 8.040 8.060 8.080 8.100 8.120 8.140 8.160 8.180 8.200 8.220rt0

100

%

0

100

%


RT

ASI12 Sm (Mn, 2x3)


RT

ASI12 Sm (Mn, 2x3)

m/e 664

Estradiol-17ß-3,17ß-diheptafluorobutyric ester

m/e 666

[4-14C] Estradiol-17ß-3,17ß-diheptafluorobutyric ester

Implementation of the Traceability Concept___________________________________________

by:

- Diagnostic Kit Manufacturers

- Organisers of External Quality Assessment Schemes

DGKL Reference Procedures • Electrolytes: Calcium

Chloride Lithium

Magnesium

Potassium Sodium

• Metabolites and Substrates: Cholesterol

Creatinine Glucose Total Glycerol

Uric Acid UreaBilirubineLactate

• Enzmes;

AST GGT ALT AMYLASECK

• Hormones:

Aldosterone Cortisol Estradiol-17ß Estriol Progesterone

17-Hydroxy-progesteroneTestosterone

Thyroxine

• Drugs: Theophylline Digoxin Digitoxin

• Total Protein

PROGESTERONE

MEDIANS IN RING TRIALS

y = 2,98 + 0,87 xn = 40, r = 0,681

Median (x) = 14,2Median (y) = 15,75Median (y-x)% = 10,3

PROGESTERONE

PATIENT SAMPLES

y = 1,84 + 0,96 xn = 42, r = 0.99

Median (x) = 19,2Median (y) = 17,9Median (y-x)% = 17,4

0

10

20

30

40

50

60

70

0 10 20 30 40 50 60 70

IDMS / Progesteron [nmol/l]

KIT

-D-R

IA /

Pro

ges

tero

n

[

nm

ol/l

]

0

10

20

30

40

50

60

70

0 10 20 30 40 50 60 70

ID-GCMS (nmol/l)

KIT

- D

-RIA

(n

mo

l/l)

PROGESTERONE

MEDIANS IN RING TRIALS

PROGESTERONE

PATIENT SAMPLES

-1000

100200300400500600700800900

1000

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Progesteron [nmol/l]

Devi

atio

n

(%)

-1000

100200300400500600700800900

1000

0 2 4 6 8 10 12 14 16 18 20 22 24 26 28 30

Progesteron [nmol/l]

Devi

atio

n

(%)

y = 1,17 x + 6,04 n = 38, r = 0,998Median (y-x)% = 18,4

y = 1,33 x - 7,25n = 54, r = 0,98Median (y-x)% = 30,1

0

200

400

600

800

1000

1200

1400

1600

0 200 400 600 800 1000 1200 1400 1600

IDMS / Cortisol [nmol/l]

KIT

-04

- FIA

[nm

ol/l

]

Cortisol - Patient Samples- Method Comparison-

0

200

400

600

800

1000

1200

1400

1600

0 200 400 600 800 1000 1200 1400 1600

IDMS [nmol/l]

KIT

-04

-FIA

[nm

ol/l

]

Cortisol - Ring Trials- Method Comparison -

-50

0

50

100

150

200

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6

De

via

tio

n

(

%)

Aldosterone nmol/l

Aldosterone - Ring Trials- Medians and Distributions (16-84%) -

Aldosterone – Ring Trials - Method Comparison -

Aldosterone – Patient Samples - Method Comparison -

Y = 1.13 X + 0.09n = 38, r = 0.939Median (y-x)% = 32.3

Y = 1.97 X + 0.06n = 50, r = 0.873Median (y-x)% = 69.0

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8

IDMS / Aldosterone [nmol/l]

KIT

-A /

Ald

ost

erone

[nm

ol/l

]

Estriol, unconjugatedDGKL -2005/01

Selected Participants: 54(93)Test – KIT 44

Ref.Values A: 44,3 nmol/l B: 35,4 nmol/l

Limits A: 25.6 – 62.1 nmol/lB: 21,2 – 49,6 nmol/l


0

20

40

60

80

100

0 20 40 60 80 100

Ref.Method IDMS nmol/l

Imm

unooassay Test Ki 44 n

mo/l 4

4

Unconjugated Estriolin Serumof Pregnant Women

Method Comparison

SI-Unit (definition)

BIPM

Primary calibrator

Secondary calibrator

Mf.´s working (master) calibrator

Mf.´s product calibrator

Secondary referencemeasurement procedure

NMI, ACL

NMI, ACL

Mf.´s selectedmeasurement procedure

Mf.´s standingmeasurement procedure

End-user´s routinemeasurement procedure

Routine sample

RESULT

NMI, ACL

ACL, MCL

ML

Mf.-> End-user

Mf. and/or End-user

MCL

Tra

ceab

ilit

yBIPM: International Institute of Weights and Measures

NMI: National Metrological Institute

ACL: Accredited Calibration Laboratory

MCL: Manufacturer´s Calibration Laboratory

ML: Manufacturer´s Laboratory Mf: Manufacturer

End-user

End-user

Primary reference measurement procedure

ML

µc(y)

Strategy to establish Reference Systemsfor non-SI Traceable Quantities

____________________________________________

• Definition of the Analyte

• Development of a Reference Procedure

• Development of a Reference Material

Definition of Proteo-Hormone Analytes __________________________________________

• Decision on the sub-unit to be measured

according to clinical relevance

• Decision on the epitope(s) to be detected

by mono- or poli-clonal antibodies

• Decision on the glycosidic structure to be

measured

Y = 0.974 X + 14.6n = 40, r = 0.998Median (y-x)% = 0.7

Y = 1.064 X + 15.4n = 40, r = 0.954Median (y-x)% = 11.6

0

200

400

600

800

1000

1200

1400

1600

0 200 400 600 800 1000 1200 1400 1600

IDMS [nmol/l]

KIT

-28

-EIA

[nm

ol/l

]

Cortisol - Ring Trials- Method Comparison -

Aldosterone – Ring Trials - Method Comparison -

Aldosterone – Patient Samples - Method Comparison -

Y = 1,127 X + 0.073n = 40, r = 0.980Median (y-x)% = 29

Y = 2.845 X - 0.042n = 50, r = 0.831Median (y-x)% = 165

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2


KIT

-S /

Ald

os

tero

ne

[n

mo

l/l]

0

0,2

0,4

0,6

0,8

1

1,2

1,4

1,6

1,8

2

0 0,2 0,4 0,6 0,8 1 1,2 1,4 1,6 1,8 2


KIT

-S /

Ald

os

tero

ne

[n

mo

l/l]

Challenges for International Standardization and Traceability - HORMONES -...

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