Certified Reference Materials for ensuring Metrological ...€¦ · contributes to industry as a...
Transcript of Certified Reference Materials for ensuring Metrological ...€¦ · contributes to industry as a...
Certified reference materials for
ensuring traceability: From experience
with steroids and peptide
Akiko Takatsu
National Metrology Institute of Japan (NMIJ),
National Institute of
Advanced Industrial Science and Technology (AIST)
1
Outline
1. Introduction of our institute and production
of CRMs
2. Our experience in standardization of clinical
measurements with CRM technology
- cortisol and aldosterone
3. Toward the standardization of peptide
measurements
2
Copyright © National Institute of Advanced Industrial Science and Technology (AIST). All rights reserved.
July 2014
AIST Hokkaido
AIST Tohoku
AIST Tokyo Waterfront AIST Chubu
AIST Kansai
AIST Chugoku
AIST Shikoku
AIST Kyushu
Sapporo, Hokkaido
Sendai, Miyagi
Koriyama, Fukushima
Koto, Tokyo
Nagoya, Aichi
Ikeda, Osaka
Takamatsu, Kagawa
Higashi-hiroshima, Hiroshima
Tosu, Saga Fukushima Renewable Energy Institute
AIST Tsukuba Tsukuba, Ibaraki
National Institute ofAdvanced Industrial Science and Technology (AIST)
Researchers (International) 2,316
Administrative employees 696
Executives 13
Postdoctoral researchers 228
Technical staff 1,491
・established by the integration of 15 national research institutes under the
supervision by Ministry of Economy, Trade and Industry (METI) in 2001
National Metrology
Institute of Japan
(NMIJ)
Research Institute for Material
and Chemical Measurement
Research Promotion Division
of NMIJ
Research Institute for
Engineering Measurement
Research Institute for Physical
Measurement
Research Institute for
Measurement and Analytical
Instrumentation
Center for Quality Management
of Metrology
4
Organization of NMIJ/AIST
7 Specialized Research Fields in AIST
NMIJ performs functions of NMI, but also
contributes to industry as a member of AIST.
Mission of NMIJ
█ Development of new national measurement
standards and dissemination of measurement
standards to industrial and social users
█ Improvement on basic national measurement
standards
█ Contribution to the global metrology system
In physics, engineering, and chemistry (and bio) field
5
Production & Supply of NMIJ CRMs
Total 289 CRMs & RMs
Industrial
Material 58
Organic 71
Environment &
Food 38
Green
Procurement
16 High Purity
Inorganic 16
Gas
10 Thermo-physical
Properties
10
Primary STD
for JCSS 70
6
https://www.nmij.jp/english/service/C/
Metrological Traceability
Calibration Hierarchy : ISO 17511 Material Calibration Procedure Implementation
Value Assignment
SI Unit
Primary Reference
Measurement Procedure Primary Calibrator:
Pure materials
Manufacturer’s
Working Calibrator Manufacturer’s Standing
Measurement Procedure Manufacturer’s
Product Calibrator
Und-User’s Routine
Measurement Procedure Routine Sample
Result
BIPM, NMI
End-user
ML
ML
Secondary Calibrator
Serum materials
Secondary Reference
Measurement Procedure NMI, ARML
Manufacturer’s Selected Measurement Procedure
BIPM, NMI
Traceability
7
NMIJ CRMs for Clinical Chemistry (1)
Pure substance
Metabolites & Lipids
Cholesterol, Creatinine, Urea, Uric acid, Triolein
Amino Acids
L-Alanine, L-Leucine, L-Isoleucine, L-Phenylalanine,
L-Valine, L-Proline, L-Arginine,
L-Lysine monohydrochloride, L-Tyrosine, L-Threonine,
L-Serine, Glycine, L-Methionine, L-Histidine, L-Cystine
L-Glutamic acid, L-Aspartic acid
Steroids
Testosterone, Progesterone, 17b-Estradiol,
Hydrocortisone (Cortisol)
8
Serum Material
Cortisol in human serum (4 concentration levels)
Aldosterone in human serum (3 concentration levels)
Protein & peptide standard solution
• C-reactive protein solution
• C-peptide
• Human serum albumin
• Human insulin solution
Nucleic acid (DNA & RNA)
• RNA solution for quantitative analysis (Synthetic RNA)*
• DNA solution for quantitative analysis (Synthetic DNA)*
*) Collaboration with Biomedical Research Institute, AIST
NMIJ CRMs for Clinical Chemistry (2)
9
Network in Japan for Standardization
in Laboratory Medicine
Clinical Laboratory
IVD-MDD Manufacturer
Reference Laboratory
Accredited Producer of RMs
Government
(METI, MHLW)
Academic
JSCC
(Japan society for
clinical chemistry)
JSLM (Japanese society
for laboratory
medicine)
etc
Industry
JACRI (Japan
association of
clinical reagents
industries)
JAIMA (Japan analytical
instruments
manufactures’
association)
etc
NMIJ/AIST
Japanese Committee for
Clinical Laboratory Standards
(JCCLS)
10
Outline
1. Introduction of our institute and production
of CRMs
2. Our experience in standardization of clinical
measurements with CRM technology
- cortisol and aldosterone
3. Toward the standardization of peptide
measurements
11
Metrological Traceability
Calibration Hierarchy : ISO 17511 Material Calibration Procedure Implementation
Value Assignment
SI Unit
Primary Reference
Measurement Procedure Primary Calibrator:
Pure materials
Manufacturer’s
Working Calibrator Manufacturer’s Standing
Measurement Procedure Manufacturer’s
Product Calibrator
Und-User’s Routine
Measurement Procedure Routine Sample
Result
BIPM, NMI
End-user
ML
ML
Secondary Calibrator
Serum materials
Secondary Reference
Measurement Procedure NMI, ARML
Manufacturer’s Selected Measurement Procedure
BIPM, NMI
Traceability
12
Process of Standardization Study Medical Needs (from diagnostics, etc) for standardization.
Request for cooperation: Clinical personnel, Manufacturers, Reference
material producers etc.
Collaboration study
Understanding of the difference between current measurement kits.
Confirmation of calibration effect by reference materials
Production of Serum based certified reference materials
Establishment of traceability by manufacturer
Establishment of high order measurement method (such as isotope
dilution mass spectrometry: IDMS) and material preparation method
Maintenance and monitoring of standardization by EQA
13
Reference Materials for Serum Cortisol Measurement
End Users
Secondary Reference Materials
(Calibrators, Control Materials)
Cortisol in Human Serum (4 Concentration Levels) (CRM 6401-b)
Hydrocortisone
(Cortisol) (CRM 6007-a)
Certified value (kg/kg)
Purity:0.993±0.002
2009.03
Isotope Dilution Mass Spectrometry
Concentration (mg/L)
Certified
value
Expanded
uncertainty
Level 1 21.0 0.6
Level 2 48.3 1.1
Level 3 91.4 2.2
Level 4 188.8 4.4
14
For low level cortisol
measurement.
Serum Cortisol Measurement:
Preparation of materials
IDMS vs. routine method
15
5.00
10.00
F E D C A G H B
測定1
測定2
測定3
測定4
下限(-20%)
上限(+20%)
参照値
3.00
8.00
C E A F G H B D
測定1
測定2
測定3
測定4
下限(-20%)
上限(+20%)
参照値
Serum (untreated) Serum treated with charcoal
Concen
trat
ion µ
g/dL
Immunoassay kit
IDMS
+20 %
- 20 %
Immunoassay kit
Conc
entr
atio
n µg
/dL
Standardization of aldosterone
• Participants: manufacturers of clinical test reagent kits (commercial
radioimmunoassay kit, 3 kits under development (CLEIA, ELISA))
& testing laboratory (LC/MS/MS).
• We distributed serum samples (candidate RMs, and patient samples),
and evaluated the degree of agreement between the measured value
and the reference value.
• For candidate RMs reference values were determined using isotope
dilution mass spectrometry at NMIJ.
• For patient samples LC/MS/MS values were used as (tentative)
reference values.
Aldosterone: the main
mineralocorticoid hormone
Collaboration study for standardization of low concentration serum
aldosterone measurement for development of reference materials
Nishikawa et. al; Endocr J. 2016, 63(12):1065-1080.
16
IDMS result (pg/mL)
result o
f each m
eth
od (
pg/m
L)
LC/MSMS
Method B
Method C
Method D
Method E
Measurement of candidate reference materials
17
Before calibration After calibration
LC/MS/MS result (pg/mL) LC/MS/MS result (pg/mL
Is calibration with CRM effective?
Method B
Method C
Method D
Method E
Calibration is effective in some of the commercial kits.
Results of 5 patient samples were re-calculated with calibration curve.
Calibration curve was made from results of candidate CRMs.
18
Rela
tive d
iffe
rence fro
m L
C/M
S/M
S r
esult (
%)
Fig. Relative difference between obtained value and reference value.
Serum Reference Material for Aldosterone
Measurement
Aldosterone in Human Serum (3 Concentration Levels)
(CRM 6402-a)
Purity evaluated
Aldosterone
reagent Purity:0.95±0.02 (kg/kg)
Isotope Dilution Mass Spectrometry
Concentration (pg/mL)
Certified
value
Expanded
uncertainty
Level 1 201 26
Level 2 411 54
Level 3 792 76
19
End Users
Secondary Reference Materials
(Calibrators, Control Materials)
Outline
1. Introduction of our institute and production
of CRMs
2. Our experience in standardization of clinical
measurements with CRM technology
- cortisol and aldosterone
3. Toward the standardization of peptide
measurements
20
Traceability in Protein Quantification
Serum CRMs
C-reactive Protein
Solution (CRM 6201-c)
Amino Acid CRMs (Pure)
Alanine: CRM 6011-a
Leucine: CRM 6012-a
Isoleucine: CRM 6013-a
Phenylalanine: CRM 6014-a
Amino Acid Analysis
Amino
Acid
CRMs
Protein,
Peptide
Standard
Solutions
(CRMs)
C-peptide (CRM 6901-b)
Tyrosine: CRM 6019-a
Threonine : CRM 6020-a
Serine : CRM 6021-a
Glycine: CRM 6022-a
Histidine : CRM 6024-a
Glutamic acid: CRM 6026-a
Aspartic acid: CRM 6027-a
Methionine : CRM 6023-a
Cystine : CRM 6025-a
Valine: CRM 6015-a
Proline: CRM 6016-a
Arginine: CRM 6017-a
Lysine(HCl) : CRM 6018-a
21
End Users
Secondary Reference Materials (Calibrators, Control Materials)
Human Insulin (CRM 6209-a) Human serum
albumin(CRM 6202-a)
Profile of Insulin related peptides
Insulin: Insulin is a peptide hormone , produced by beta cells of the
pancreatic islets. It is responsible for regulating the body‘s glucose levels.
C-peptide: A byproduct of insulin production, usually by the pancreas.
C-peptide is useful as a marker of insulin production, since the pancreas
typically releases C-peptide and insulin in about equal amounts.
C-peptide
31 amino acid peptide Structure of Proinsulin
C-peptide
Insulin B-chain
A-chain
Insulin
two polypeptide chains
are linked together by
disulfide bonds.
A-chain: 21 amino acid
B-chain: 30 amino acid
22
Material: synthetic peptide having human C-peptide sequence
Single Peptide Solution CRMs
• NMIJ CRM 6901-b C-peptide
Lyophilized material of C-peptide in phosphate buffer solution.
Certified Concentration (for prepared solution)
C-peptide: 100 ± 5 (mg/L)
Total C-peptide (Mixture of C-peptide, deamidated
C-peptide, and pyroglutamylated C-peptide): 102 ± 5 (mg/L)
• NMIJ CRM 6209-a Human Insulin Solution
Purified recombinant human insulin in solution.
Certified Mass Concentration
(77.9± 2.3) mg/L
23
Traceability in Protein Quantification
Serum CRMs
Amino Acid CRMs (Pure)
Amino Acid Analysis
Amino
Acid
CRMs
Serum CRMs?
IDMS for peptide in serum
24
End Users
Secondary Reference Materials (Calibrators, Control Materials)
Protein,
Peptide
CRMs
Serum
Development of IDMS for serum insulin
GIVEQCCTSICSLYQLENYCN
FVNQHL(d10)CGSHL(d10)VEAL(d10)YL(d10)VCGERGFFYTPKT
Human insulin-D40
A chain
B chain
FVNQHL(d10)CGSHL(d10)VE AL(d10)YL(d10)VCGE
Derivatization & LC-MS/MS analysis
+
B1-13 B14-21
Enzymatic digestion with Glu-C
Reduction reaction for SH
Target peptides
Sakaguchi Y, et al., Anal. Biochem. 2017, 537:26-32.
25
y = 0.8713x - 0.0079 R² = 0.9943
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 0.5 1 1.5 2
y = 0.8558x - 0.0041 R² = 0.9944
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
0 0.5 1 1.5 2
Further research will be necessary to establish as a higher order
reference method.
IDMS for serum insulin IDMS vs immunoassay
Immunoassay result Immunoassay result
IDM
S r
esu
lt
IDM
S r
esu
lt
26
Summary
• NMIJ/AIST is producing and maintaining primary
level reference materials.
• CRM is not a goal for standardization.
• There are many steps for ensuring traceability to
clinical lab.
• Cooperation among clinical communities, NMIs,
manufactures, etc. is very important.
27
Thank you for your attention!
AIST Tsukuba Headquarters / AIST Tsukuba
http://www.nmij.jp/english/
28