Reference materials

Reference materials (RMs) are applied in a variety of analytical procedures to perform harmonized quantifications of analytes. Methods can be standardized by the use of common RMs [30]. The use of international RMs ensures world wide uniformity in most analytical measurements and provides users with reliable and comparable quantitative information based on operationally defined "conventional true values" in the sense of a "gold standard" of the certified analytes. The agreement of a measured average value with a given "conventional true value" displays the systemic error component or the trueness of the measurement expressed as a bias, i.e., a deviation from a "conventional true value" [31]. Thus, RMs are an important tool for quality control and quality assurance, and are crucial for the determination ofthe accuracy of a measurement determined by both its trueness and precision. In conjunction with reference procedures, RMs are the best guarantee for correct calibration and thus trueness of results [32].

Besides these benefits, the application of appropriate RMs is the basis for the cross validation of technologies and methods. The use of RMs enables the diligent evaluation of pre-analytical influences, e.g., sampling, storage and processing, and analytical influences. These aspects are of definite importance for the objectives of the HUPO PPP [1, 2]. An example of their use is provided by Favaloro et al. [33] who showed that refrigerated storage of blood samples can have an effect on the measurement of Factor VIII and von Willebrand Factor proteins. These values were shown to be lowered, when compared to reference sample results, as a result of storage under refrigerated conditions and such measurements could potentially result in the misdiagnosis of von Willebrand disorder or hemophilia A.

RMs can also aid in the determination of reference limits [30]. This can be helpful in the discovery of new diagnostic markers, and can also affect their validation, which is a crucial step towards their clinical application [34]. In this respect, the use of RMs or related standards may also be leveraged in proteomics research, as one major goal of proteomics' activities is the discovery of new markers for diagnostic applications. This will necessarily apply to HUPO PPP efforts in the initial phase and also for its long term goal.

RMs are standards developed by international and national organizations that contain certified quantities of substances. The working group on Plasma Protein Standardization of the International Federation of Clinical Chemistry (IFCC) established a Certified Reference Material (CRM) for plasma proteins called CRM 470 [35-37 and http://www.irmm.jrc.be/mrm.html]. CRM 470 contains 15 proteins with certified concentrations; these are listed in Tab. 5. The CRM 470 preparation has been obtained by a very well described protocol encompassing sample collection, pooling, processing, stability testing, accelerated degradation, and finally, value assignment. Molecular characteristics of all the proteins in CRM 470 were established by electrophoretic and immunochemical techniques. The values of the 15 constituents are within the reference intervals for those of healthy individuals, and are detailed in Tab. 5.

Based on primary and secondary international RMs like CRM 470, industry standards, calibrators, and controls are produced that serve laboratories as references. For example, among other calibrators from Dade Behring used in the immunoassay analysis ofthe HUPO PPP reference specimens, Dade Behring's N Protein Standard SL was applied [18]. The N Protein Standard SL is based on CRM 470, WHO 67/97, WHO 80/578, IRP Code 75/502, and highly purified proteins in case international RMs are not available. N Protein Standard SL contains 25 proteins with certified concentrations, listed in Tab. 5.

Undoubtedly, the use of common RMs involves compromises and falls short of an ideal solution, in particular for proteomics solutions as only a limited number of proteins have certified concentrations, and serum or plasma is processed to gen-

Tab. 5 List of proteins with certified concentrations for CRM 470 and Dade Behring's N Protein Standard SL (Lot #083629)

Protein

Concentrations [g/L]

CRM

Dade Behring N Protein

470a)

Standard SL Lot

No. 083629b)

Total protein

72.8c)

Albumin

39.7

47.80d)

IgG

9.68

9.33d)

IgG1

6.06e)

Transferrin

2.45

2.95d)

IgG 2

2.75e)

Ig/L chain type kappa

2.24b)

IgA

1.96

1.85d)

Alpha-2-macroglobulin

1.64

1.60d)

Alpha-1-antitrypsin (alpha-1-proteinase-inhibitor)

1.206

1.57d)

Haptoglobin

0.893

1.41d)

Complement 3c

1.091

1.40d)

Ig/L chain type lambda

1.23b)

Hemopexin

0.943f)

Alpha-1-acid glycoprotein (orosomucoid)

0.656

0.889d)

IgM

0.797

0.735d)

IgG 4

0.553e)

IgG 3

0.368e)

Prealbumin (transthyretin)

0.243

0.314d)

Ceruloplasmin

0.205

0.31d)

Alpha-1-antichymotrypsin

0.245

Complement 4

0.151

0.241d)

Retinol-binding protein

0.045f)

C-reactive protein

0.0392

Beta-2-microglobulin

0.0013f)

Soluble transferrin receptor

0.0012f)

Ferritin

0.000348g)

IgE

568 IU/mLh)

b) Dade Behring N Protein Standard SL, product data sheet c) Measurement byTCA method-Dade Behring BN

d) IFCC/BCR/CAP (International Federation of Clinical Chemistry/Community Bureau of Reference/College of American Pathology)- CRM 470= RPPHS (Reference Preparations for Proteins in Human Serum) Lot 5

e) WHO (World Health Organization) 67/97

f) Based on highly purified protein hemopexin (Lot No. 070792/IA), retinol binding protein (Lot No. 100990/IA), ß2-microglobulin (Lot No. 111093/IA), soluble transferrin receptor (Lot No. 97.2)

g) SRP WHO 80/578

h) IRP (International Reference Preparation) Code 75/502

b) Dade Behring N Protein Standard SL, product data sheet c) Measurement byTCA method-Dade Behring BN

d) IFCC/BCR/CAP (International Federation of Clinical Chemistry/Community Bureau of Reference/College of American Pathology)- CRM 470= RPPHS (Reference Preparations for Proteins in Human Serum) Lot 5

e) WHO (World Health Organization) 67/97

f) Based on highly purified protein hemopexin (Lot No. 070792/IA), retinol binding protein (Lot No. 100990/IA), ß2-microglobulin (Lot No. 111093/IA), soluble transferrin receptor (Lot No. 97.2)

g) SRP WHO 80/578

h) IRP (International Reference Preparation) Code 75/502

erate RMs. The latter aspect indicates that RMs do not reflect a native serum or plasma composition. Nevertheless, quality control and quality assurance by RMs provide more than just information on the trueness of measurements of certified proteins. They also help in the assessment ofthe quality and comparison of data regarding proteins that are not certified. Thus, RMs and derived standards will allow for the systematic inter-laboratory and inter-technology comparisons that will be part ofthe continuing HUPO-PPP initiative. Clinical chemists, clinicians, and scientists working in proteomics should follow the reference values strategy [38], as it will improve standardization in preparation for the subsequent phases of HUPO PPP efforts of larger, population-based studies.

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