The Importance of Commutability of Reference Materials Used as Calibrators: The Example of Ceruloplasmin

I. Zegers, R. Beetham, T. Keller, J. Sheldon, D. Bullock, F. MacKenzie, S. Trapmann, H. Emons, and H. Schimmel

September 2013

www.clinchem.org/content/59/9/1322.full

© Copyright 2013 by the American Association for Clinical Chemistry

© Copyright 2009 by the American Association for Clinical Chemistry

Ceruloplasmin measurementsCeruloplasmin measurements

Ceruloplasmin a major serum protein, about 150 kDa binds 6 copper ions

Clinical use decreased concentrations of ceruloplasmin may

indicate aceruloplasminemia, Menkes disease, or Wilson disease

Normal concentrations about 0.2 – 0.5 g/L

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 1. The standardisation of ceruloplasmin measurements was based on the use of the common reference material ERM-DA470 (certified for 15 major serum proteins). The traceability chain involves also the manufacturer’s working calibrators and kit calibrators.

Traceability Traceability chain for chain for ceruloplasmin ceruloplasmin measurementsmeasurements

Routine sample result

Immunoassay

US National Reference Preparation

ERM-DA470 (matrix reference material)

Reference procedure, immunoassay ring trial

Value transfer 1: LC-MS, immunoassay

Value transfer 2: immunoassay

Immunoassay

Pure ceruloplasmin

Manufacturer’s working calibrator

Manufacturer’s kit calibrator

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 2. In 2002, Beetham et al. wrote a Letter to the Editor of Clinical Chemistry. Observations from the UK National External Quality Assessment Service (NEQAS) showed that for ceruloplasmin discrepancies remained between results from different methods. This was the case despite the fact that all measurement results were traceable to value of the reference material ERM-DA470 (formerly called CRM 470). For other proteins certified in ERM-DA470 discrepancies were reduced after its generalised use.

R. Beetham, P. White, P. Riches,D. Bullock, F. MacKenzie, and for UK NEQAS Protein Assays Specialist Advisory Group. Use of CRM 470/RPPHS Has Not Achieved True Consensus for Ceruloplasmin Measurement. Clin Chem 2002; 48: 2293-2294.

UK NEQAS observes discrepanciesUK NEQAS observes discrepancies

© Copyright 2009 by the American Association for Clinical Chemistry

CommutabilityCommutability

Commutability of reference materials (RM) is an essential requirement to achieve comparability of patient results

It is required when a RM is used as calibrator or as EQAS sample

Commutability is a property of a reference materialsuch that values measured for that reference materialand for representative clinical samples have the samerelationship between two, or more, measurementprocedures for the same measurand.

© Copyright 2009 by the American Association for Clinical Chemistry

Materials and MethodsMaterials and Methods Sets of samples containing 3 reference materials (RMs)

and 30 serum samples from healthy people were measured

The following RMs were evaluated: ERM-DA470 (freeze-dried)

certified for 15 serum proteins, distributed from 1994 to 2008 the use of this material led to a significant reduction of

between-method variability for proteins like IgG, albumin, etc.

ERM-DA470k/IFCC (freeze-dried) certified for 12 serum proteins, distributed since 2008 not certified for ceruloplasmin concentration

ERM-DA472/IFCC (liquid frozen) certified for CRP, replaced by ERM-DA474/IFCC in 2011

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Table 1. Methods used to evaluate the commutability of reference materials for ceruloplasmin measurements.

Methods usedMethods usedPlatform Reagents Laboratory code

Abbott Architect c16000 Abbott Ceruloplasmin REF 6K9101 Lot 70185

Sentinel Diagnostics, Milano, IT Abbott

BN ProSpec Siemens reagents (N Antiserum to Human Ceruloplasmin)

Siemens Healthcare Diagnostic Products GmbH, Marburg, DE

ProSpec I

BN ProSpec Dade Behring anti-ceruloplasmin reagent lot: 155289, Dade Behring standard SL lot:083678

St. Georges Hospital, London, UK ProSpec II

Beckman Immage Beckman calibrant 2 lot:704575, Beckman anti-ceruloplasmin reagent lot:706567

St. Georges Hospital, London, UK Immage

Hitachi 917 Tina-quant C4-2, Id. 11 875 051, Lot 696 008: C4, ACN 032

Roche Professional Diagnostics, Penzberg, DE

Roche Hitachi

Roche Integra Ceruloplasmin, Id. 20 764 663, Lot 696825: CERU3, Test-Id. 0-666

Roche Professional Diagnostics, Penzberg, DE

Roche Integra

Hitachi 917 DAKO (Antibody: Q0121 Lot 00012259 Ceruloplasmin)

DAKO, Glostrup, DK DAKO Hitachi

Olympus systems analyser AU2700 OA28

Olympus OSR6164 # 5576 Olympus Clare (now Beckman Coulter Biomedical Ltd.), Clare, IE

AU2700

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 3. Example of a pair-wise commutability assessment for ceruloplasmin (CER) measurements in sera from 30 healthy individuals (CS), ERM-DA470, ERM-DA470k, and ERM-DA472. The plotted values are the means per sample. The continuous black line is the results of a Deming regression, the dotted black lines correspond to the limits of the prediction interval at the 95% confidence level.

Results: example Results: example commutability commutability assessment assessment

The recovery of ceruloplasmin in ERM-DA470 is reasonable with both the DAKO Hitachi and Beckman Immage methods (certified concentration 205 ± 11 mg/L)

ERM-DA470 is not commutable for this combination of methods

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Table 1. Comparison of results for serum samples and commutability of reference materials. The upper-right part of the table contains results from the evaluation of the commutability of ERM-DA470. The lower-left half of the table contains the slope of Passing-Bablok regression. a1 indicates commutability, 0 indicates a lack of commutability.

Commutability of ERM-DA470Commutability of ERM-DA470

© Copyright 2009 by the American Association for Clinical Chemistry

The slopes of the correlation lines vary from 0.56 to 1.42, showing that there are large discrepancies

The largest deviation from unity is between results from DAKO Hitachi and Beckmann Immage

ERM-DA470 is not commutable for many combinations of methods

ERM-DA470 is commutable for clusters of methods: - Abbott Architect and BN ProSpec- AU270, DAKO Hitachi and Roche Integra

Main findings:Main findings:

© Copyright 2009 by the American Association for Clinical Chemistry

Reference materials are stabilised materials stored for long times. The stabilisation of ERM-DA470 included the addition of stabilising agents like sodium azide.

Ceruloplasmin is a complex molecule that requires the binding of 6 copper ions. It can undergo structural changes.

Crossed immunoelectrophoresis shows that in ERM-DA470 a second form of ceruloplasmin is present that is not found in fresh serum. At present biochemical data on are not conclusive about the nature of differences between the forms.

What causes the lack of commutability?What causes the lack of commutability?

© Copyright 2009 by the American Association for Clinical Chemistry© Copyright 2009 by the American Association for Clinical Chemistry

Figure 4. Results from measurements of samples distributed by UK NEQAS. Fresh samples: Results from different methods were very discrepant. Aged Samples (distributed after 6 months storage in the presence of sodium azide): Consistent results. ALTM stands for all-laboratory trimmed mean, Roche stands for Roche Integra, Dade for BN ProSpec and BN II, Array for the Beckmann Array and Immage for the Beckman Immage.

Effect of prolonged storage of EQAS samplesEffect of prolonged storage of EQAS samples

© Copyright 2009 by the American Association for Clinical Chemistry

The reference material ERM-DA470 behaved like an aged serum sample

All methods gave results that were traceable to the ERM-DA470 value

Methods gave equivalent results for aged serum samples, like UK NEQAS samples stored in the presence of sodium azide

The methods gave discrepant results for fresh samples

Use of a non-commutable RM causes discrepanciesUse of a non-commutable RM causes discrepancies

US National Reference Preparation

ERM-DA470 (matrix reference material

Pure ceruloplasmin

Manufacturer’s working calibrator

Manufacturer’s kit calibrator

External quality assessment sample

© Copyright 2009 by the American Association for Clinical Chemistry

ConclusionsConclusions The reference material ERM-DA470 was not commutable for

ceruloplasmin. Therefore, its generalised use did not lead to equivalent results for ceruloplasmin (in contrast to most of the other parameters certified in ERM-DA470).

A reference material format may be appropriate for certain analytes and not for others.

UK-NEQAS has developed procedures for preparing commutable EQAS samples. These might also be used for preparing a commutable reference material for ceruloplasin.

Commutability of reference materials is an essential requirement to achieve comparability of patient results.

© Copyright 2009 by the American Association for Clinical Chemistry

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