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.

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Thank you for your attention!

AIST Tsukuba Headquarters / AIST Tsukuba

http://www.nmij.jp/english/

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