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
SIR of 2 Channels EI+ 666.281.04e5
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
%
SIR of 2 Channels EI+ 664.281.20e5
RT
ASI12 Sm (Mn, 2x3)
SIR of 2 Channels EI+ 666.281.20e5
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
http://www.dgkl-rfb.de
http://www.dgkl-rfb.de
http://www.dgkl-rfb.de
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
(%)
http://www.dgkl-rfb.de
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 -
http://www.dgkl-rfb.de
-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
http://www.dgkl-rfb.de
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
http://www.dgkl-rfb.de
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)
http://www.dgkl-rfb.de
http://www.dgkl-rfb.de
http://www.dgkl-rfb.de
http://www.dgkl-rfb.de
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
IDMS / Aldosterone [nmol/l]
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
IDMS / Aldosterone [nmol/l]
KIT
-S /
Ald
os
tero
ne
[n
mo
l/l]