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Quality Assurance in Medical Laboratories Protein standards traceable to SI-units Prof. Dr. med. Reinauer

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Traceability chain SI units

Definitive methods

Primary reference materials

 Reference measurement procedure development  Production of high accuracy secondary reference materials

Reference measurement procedures

 Routine method development and evaluation  Preparation of routine working reference materials  Internal and external quality assurance Borovetz_Sept.10 14.09.2010 15:23

Secondary reference materials CRM

Routine methods

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SI base units Quantity

Name of Unit

Symbol for unit

length

meter

M

mass

kilogram

kg

time

second

s

electric current

ampĂŠre

A

thermodynamic temperature

kelvin

K

luminous intensity

Candela

Cd

amount of substance

mole

mol

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SI - Units Base units: Seven units have been selected as the basis of the SI-System Name of quantity

Definition

Unit

substance concentration

amount of substance of a solute divided by volume of solution

mol/l

molality

amount of substance of a solute divided by mass of solvent

mol/kg

mass concentration

mass of a component (e.g., solute divided by volume of system (e.g., solution)

kg/l

substance content

amount of substance of a component divided by mass of system (mixture)

mol/kg

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IVDMD Directive 98/79/EC ESSENTIAL REQUIREMENTS The traceability of values assigned to calibrators and/or control materials must be assured through available reference

measurement

procedures

and/or available reference materials of a higher order. Borovetz_Sept.10 14.09.2010 15:23

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Reference Measurement Procedure thoroughly

investigated

measurement

procedure shown to yield values having an uncertainty of measurement commensurate with its intended use, especially in assessing the

trueness

of

other

measurement

procedures for the same quantity and in characterizing reference materials EN 12286:1998, 3.7 ISO 15193:2007 (E) Borovetz_Sept.10 14.09.2010 15:23

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Definitions (International Vocabulary of Basic and General Terms in Metrology - VIM Uncertainty of measurement: Parameter, result

of

associated a

with

the

measurement,

that

characterizes the dispersion of the values that could reasonably be attributed to the measurand. Borovetz_Sept.10 14.09.2010 15:23

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Relevant ISO Standards ISO 17511:2003

In vitro diagnostic medical devices – Metrological traceability of values assigned to calibrators and control materials.

ISO 18153:2003

Metrological traceability of values for catalytic concentration of enzymes assigned to calibrators and control materials.

ISO FDIS 15193:2008 Requirements, content, and format of reference measurement procedures.

ISO EN 15194:2009

Requirements for reference materials of higher order and the content of supporting documentation.

EN ISO 15195:2003

Requirements for reference measurement laboratories.

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Certified Reference Material Reference material, accompanied by a certificate, one or more of whose property values are certified by a procedure which establishes metrological traceability to an accurate realization of the unit in which the property values are expressed, and for which each certified value is accompanied by an uncertainty at a stated level of confidence. ISO 17511:2003; VIM:1994 To produce useful and reliable results of measurement, whether in science, technology, or routine service, it is necessary that they are supported by a Reference Measurement System so as to be comparable and ultimately traceable to measurement standards and/or measurement procedures of the highest metrological level. ISO/WD 15194:2006 Suppliers: - NIST (USA) - IRMM (Geel, Belgium) - WHO Borovetz_Sept.10 14.09.2010 15:23

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Certified reference materials

Definition:

Certified concentrations of substances in aqua dest. or in certain matrices. Calibration and verification of measurement process.

Use:

Verification of reference measurement procedures. Validation of new methods of measurement. WHO – NIBSC

Distribution:

National Institute of Standards and Technology (NIST) Institute for Reference Materials and Measurements (IRMM)

Examples: Borovetz_Sept.10 14.09.2010 15:23

NIST SRM 909 – lyophilized human serum 11


Protein Standards

1) Consensus Standards CRM 470 2) Standards traceable to SI demonstrated with HbA1c

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Certified reference materials for proteins CRM 470 RPPHS Lot 5

• albumin

• IgA

• α1-antitrypsin

• IgG

• C3-complement

• IgM

• C4-complement

• α2-macroglobulin

• coeruloplasmin

• prealbumin

• C-reactive protein/CRP

• transferrin

• α1-acid-glycoprotein

• haptoglobin

(Orosomucoid)

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• α1-antichymotrypsin

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Quality management of diabetes DCCT study

Diabetes Control and Complication Trial

HbA1c Standardisation of intercomparison studies NGSP - Goldstein

National Glycohemoglobin Standardisation Program

Reference measurement procedure Reference method of IFCC of IFCC for HbA1c

International International comparison comparison on on highest highest level level of of accuracy accuracy Borovetz_Sept.10 14.09.2010 15:23

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ß-chain of haemoglobin

HbA0 HbA1c Borovetz_Sept.10 14.09.2010 15:23

Val – His – Leu – Thr – Pro – Glu – Glu – Lys – Glc – Val – His – Leu – Thr – Pro – Glu – Glu – Lys – 15


IFCC reference method for determination of HbA1c in blood with HPLC-ESI-MS Principle of measurement: Determination of the ratio of glycated and non-glycated Ă&#x;-Nterminal hexapeptides of haemoglobin

1.1. blood sample 1. Haemolysis Haemolysis Haemolysisof of of whole whole whole blood bood sample sample

2.

Proteolytic the haemolysate Proteolyticcleavage cleavageofof the haemolysate

3. Borovetz_Sept.10 14.09.2010 15:23

HPLC-ESI-MS analysis

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Proteolytic cleavage of the haemolysate

HbA0

HbA1c

Val – His – Leu – Thr – Pro – Glu – Glu – Lys –

Glc – Val – His – Leu – Thr – Pro – Glu – Glu – Lys –

Glu

–L ys –– Glu – Lys

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IFCC reference measurement procedure for determination of HbA1c in blood with HPLC-ESI-MS Principle of measurement:

1. Haemolysis of whole blood sample

2. Proteolytic cleavage of the haemolysate

3. HPLC-ESI-MS analysis

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HPLC-ESI-MS analysis HPLC (Shimadzu) pump A pump B solvent mixer autosampler column heater degasser switching valve controller

MS (Applied Biosystems) API 4000

triple quadrupole mass spectrometer

ESI : Software:

TurboVTM source with Turboion SprayTM probe AnalystTM

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Ă&#x;-chain N-terminal hexapeptide

Unlabelled

Labelled

VHLTPE

m/z 348.3

D7 m/z 351.8

deoxyfructosylVHLTPE

m/z 429.3

D7 m/z 432.8

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Determination of a reference measurement procedure value by isotope dilution mass spectrometry Certified Reference Material (CRM) (unlabelled standard)

HD0 and GD0

EQA sample

calibrator

certified accuracy control

internal standard (isotope labelled standard)

HD7 and GD7 Reference Measurement Procedure value


Results: Calibration systems for HbA1c determination based on liquid chromatography-isotope dilution-mass spectrometry (LC-ID-MS) and on the IFCC reference measurement

procedure

were

compared.

A

linear

regression analysis demonstrated a correlation of r2 = 1.00 between the 2 different calibration systems. Mean deviation was 5.5 % for the calibration and 3.3 % for hemolysate samples, with a mean expanded uncertainty of 4.9 %. Borovetz_Sept.10 14.09.2010 15:23

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HbA1c results of IFCC reference method vs. NGSP-Goldstein

12

NGSP (%HbA1c)

NGSP = (0.915 * IFCC) + 2.15 10 8 6 4 2 2

4

6

8

10

12

IFCC (%HbA1c)

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International intercomparison study of the IFCC Working Group on HbA1c Standardisation

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Conversion of HbA1c values from NGSP- to IFCC-calibration 1

IFCC (mmol/mol) = (10,93 x HbA1c%) - 23,5

2

IFCC (mmol/mol) = (HbA1c% - 2,15) x 10,929

3

(HbA1c% - 2,15) IFCC (mmol/mol) =

0,0915

mmol HbA1c / mol total Hb Borovetz_Sept.10 14.09.2010 15:23

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Example: HbA1c = 6,8% (NGSP)

1

IFCC (mmol/mol) = (10,93 x 6,8%) - 23,5 = 50,82

2

IFCC (mmol/mol) = (6,8% - 2,15) x 10,929 = 50,82

3

6,8% - 2,15 IFCC (mmol/mol) =

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0,0915

= 50,82

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n = 551

target value

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Start of HbA1cStandardization in EQAS

2009, June 1st

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2009, January 1st

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Global standardization of HbA1c – results reporting 2009 – IFCC numbers

NGSP numbers

USA

no

yes

Sweden

yes

yes

Swedish numbers

Japan

yes

no

JDS numbers

Country

RMP 2008 August

UK

yes

yes

2009 June 1st (dual reporting up to 31 March 2011)

Germany

yes

no

2009 January 1st

IFCC Network

yes

no

2008 August

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Reference measurement procedures an protein standards traceable to SI units HbA1c

Kaiser P. et al. Clin.Chem. 56, 750-754, 2010

HbA2

in progress (Mosca A. et al.)

HbAo

in progress (Kaiser P. et al.)

Factor VIII

in progress (Kaiser P. et al.)

Insulin, Proinsulin

Kippen DA et al. J.Biol.Chem. 272, 12513-12522,1997

C-peptide

Fierens, C. et al. Clin.Chemistry, 49, 992-994, 2003

Somatotropin

Pritchard C. et al. Clin.Chemistry, 55, 1984-1990, 2009 Arsene C. et al. Analyt.Biochem., 401, 228-235, 2010

Thyroglobulin

Borovetz_Sept.10 14.09.2010 15:23

Hoofnagle A.N. et al. Clin.Chemistry, 54, 1796-1804, 2008

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Summary The quantification of proteins with the best accuracy possible is realised by quantitative mass spectrometry using the LC-ID-MS procedure. The protein to be analysed is treated with trypsin. Characteristic peptides are selected for analysis with LC-MS procedure. The selected peptides are synthesized in labelled and unlabelled forms. The main calibrators are these synthesized peptides the purity of which must be determined by amino acid content after hydrolysis with LC-ID-MS. The amino acid analyses are traced back to certified pure amino acids. The principle of the reference measurement procedure is demonstrated with the analyte haemoglobin A1c. Borovetz_Sept.10 14.09.2010 15:23

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Literature 1) Arsene CG, Ohlendorf R, Burkitt W, Pritchard C, Henrion A, O’Connor G et al., Protein Qualification by Isotope Dilution Mass Spectrometry of Proteolytic Fragments: Cleavage Rate and Accuracy: Anal.Chem.; 80,4154-60 (2008). 2) Burkitt W, Pritchard C, Arsene C, Henrion A, Bunk D, O’Connor G. Toward amino acids to proteins. Anal.Biochem;176:242-51 (2008). 3) Nakanishi T, Iguchi K, Shimizu A. Method for hemoglobin A1c measurement based on peptide analysis by electrospray ionization mass spectrometry with deuterium-labelled synthetic peptides as internal standard Clin.Chem.;49,829-31(2003). 4) Kaiser P, Akerboom Th, Dux L, Reinauer H. Modified HPLCElectrospray Ionization. Mass Spectrometry Method for HbA1c Based on IFCC Reference Measurement Procedure. Clin.Chem.;54,1018-1022 (2008). Borovetz_Sept.10 14.09.2010 15:23

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5) Kaiser P, Akerboom Th, Ohlendorf R, Reinauer H.: Liquid Chromatography-Isotope Dilution-Mass Spectrometry as a New Basis for the Reference Measurement Procedure for Hemoglobin A1c Determination Clin.Chem.56,750-754 (2010).

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Thank you for your attention

www.instand-ev.de Borovetz_Sept.10 14.09.2010 15:23

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Selection of peptides after trypsin treatment

Trypsinisation products T32 T71 T183 T184 -185

AS-Sequence

NNEEAEDY*DDDLTDSEMDVVR NTGDY*Y*EDSY*EDIASAY*LISK TTLQSDQEEIDY*DDTISVEMK KEDFDIY*DEDENQSPR Y*

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Tyrosin-Sulfate

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LC-MS Chromatogram of Faktor VIII after Trypsin-treatment

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LC-MS/MS Chromatogram of Factor VIII after trypsin treatment neutral loss mode

T 184-185 m/z 1040

T 183 m/z 1271

T 71 m/z 1293

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Molmassen von Aminosäuren Name

Abkürzung

monoisotopische Masse

Name

Abkürzun monoisog topische Masse

Alanin

Ala A

71.037

Leucin

Leu L

113.084

Arginin

Arg R

156.101

Lysin

Lys K

128.095

Asparagin

Asn N

114.043

Methionin

Met M

131.041

Asparaginsäu re

Asp D

115.027

Phenylalanin

Phe F

147.068

Cystein

Cys C

103.009

Prolin

Pro P

97.053

Glutaminsäur e

Glu E

129.043

Serin

Ser S

87.032

Glutamin

Gln Q

128.059

Threonin

Thr T

101.048

Glycin

Gly G

57.021

Tryptophan

Trp W

186.079

Histidin

His H

137.059

Tyrosin

Tyr Y

163.063

Isoleucin

Ile I

113.084

Valin

Val V

99.068

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Factor VIII T 71 (AS 714-733)

Thrombin Borovetz_Sept.10 14.09.2010 15:23

T 184-185 (AS 1674-1689)

VWF 39


SI base units Quantity

Name of derived unit

Symbol for unit

area

square meter

m2

volume

cubic meter

m3

speed

meter per second

m/s

acceleration

meter per second squared

m/s2

substance concentrtion

mole per cubic meter

mol/m3

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Evaluation of uncertainty: Type A: Repeated measurement of the measurand and calculation of average measured vale (x) and the standard deviation (sX or SEM) in a Gaussian distribution. Type B: Systematic error (bias) and random error values are assessed (total error concept). Bias ∆: = Xi – X0 X0 -> reference measurement Xi -> mean of the measurements

Uncertainty of the bias: Borovetz_Sept.10 14.09.2010 15:23

U∆ =

U2 U2 Xi + X0 41


Hierarchy of the methods according to the Guidelines of the Federal Medical Association for Quality Assurance in Medical Laboratories

Methods

Determination

Method Principles

Supervision

Definitive method

Certified concentration in standards

Isotope dilution/ mass spectrometry

National Reference Institutions

Reference measurement procedure

Standards, target values for IQA and EQAS

GC, ID-MS, HPLC, AAS, GC-MS, LC-MS/MS

EQAS organizers Manufacturers

Assigned value

EQA target values

Routine methods

Manufacturers EQAS organizers

Routine laboratory

Specimens of patients

Routine methods

Federal Medical Association, Calibration Offices

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Ă&#x;-chains of different haemoglobins (N-Term. AA 1-9) 

Val-His-Leu-Thr-Pro-Glu-Glu-Lys-Ser HbA

1

3

6

9



Val-His-Leu-Thr-Pro-Val-Glu-Lys-Ser HbS



Val-His-Leu-Thr-Pro-Val-Glu-Lys-Ser HbC



Gly-His-Phe-Thr-Glu-Glu-Asp-Lys-Ala HbF

1 Borovetz_Sept.10 14.09.2010 15:23

3

6

9 AA 43


HPLC-ESI-MS analysis HPLC conditions: Zorbax SB-CN, 5 µ 2.1 mm x 150 mm

50 °C, 2 µl

eluent A:0.025 % TFA in water eluent B:0.023 % TFA in acetonitril gradient: 300 µl/min time [min] %B

0 0

3 0

9 5

13.5 5

13.6 100

17 100

17.1 0

23 stop

MS conditions: Scan type: Mass: Dwell time: Temperature: Borovetz_Sept.10 14.09.2010 15:23

Q1 multiple ions 348.3 amu (non-glycated hexapeptide) 429.3 amu (glycated hexapeptide) 1s 500 °C 44


HPLC-ESI-MS analysis LC-MS chromatogram of a haemolysate containing 6.3 % HbA1c

Calibration curve HbA1c 0,18 Ratio area HbA1c/HbA0

0,16 0,14 0,12 0,1

y = 1,0289x + 0,0003 2

0,08

R = 0,9999

0,06 0,04 0,02 0 0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

0,16

0,18

Ratio Konz. [HbA1c/HbA0]

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Harmonisation of HbA1c calibration

Calibration according to

ADA/EASD/IDF working group

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Federal medical chamber of Germany

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1st Version

Development of the RMP for HbA1C with HPLC and LC-MS

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2nd Version

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Formic acid-gradient



Sensibility much better



Stability better / Robustness



Reproducibility



Voting of the members

But:

Traceability ?

48


3rd Version

Isotope-dilution-mass-spectrometry



Highest metrological order



Traceability to pure amino acids

UNIT: mmol HbA1C/mol HbA0 + HbA1C

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Traceable ID-MS-Procedure Analyte Tracer Synthetic unlabelled and labelled hexapeptide unglycated and glycated

HbA1c

glycated hexapeptide unglycated hexapeptide

Analysis of purity and hexapeptide content after hydrolysis and LC-ID-MS of the single amino acids traceable to certified pure amino acids

LC – ID - MS Borovetz_Sept.10 14.09.2010 15:23

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Results: Calibration systems for HbA1c determination based on liquid chromatography-isotope dilution-mass spectrometry (LC-ID-MS) and on the IFCC reference measurement

procedure

were

compared.

A

linear

regression analysis demonstrated a correlation of r2 = 1.00 between the 2 different calibration systems. Mean deviation was 5.5 % for the calibration and 3.3 % for hemolysate samples, with a mean expanded uncertainty of 4.9 %. Borovetz_Sept.10 14.09.2010 15:23

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The IFCC reference measurement procedure for determination of HbA1c in blood allows the international comparison on highest level of accuracy



The international harmonisation of the HbA1c calibration at the highest level of accuracy is unfortunately not guaranteed for the next few years



The LC-ID-MS reference measurement procedure is now traceable to SI.



The actual reference measurement procedure has to be adapted to the values of the LC_ID_MS/MS procedure (Kaiser et al.-2010)

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6c_08