1. 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

2. © Copyright 2009 by the American Association for Clinical Chemistry Ceruloplasmin 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

3. © 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 chain for ceruloplasmin measurements 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

4. © 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 discrepancies

5. © Copyright 2009 by the American Association for Clinical Chemistry Commutability  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 material such that values measured for that reference material and for representative clinical samples have the same relationship between two, or more, measurement procedures for the same measurand.

6. © Copyright 2009 by the American Association for Clinical Chemistry Materials 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

7. © Copyright 2009 by the American Association for Clinical Chemistry Table 1. Methods used to evaluate the commutability of reference materials for ceruloplasmin measurements. Methods used PlatformReagentsLaboratorycode 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 # 5576Olympus Clare (now Beckman Coulter Biomedical Ltd.), Clare, IE AU2700

8. © 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 commutability 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

9. © 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. a 1 indicates commutability, 0 indicates a lack of commutability. Commutability of ERM-DA470

10. © 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:

11. © 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?

12. © 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 samples

13. © 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 discrepancies US National Reference Preparation ERM-DA470 (matrix reference material Pure ceruloplasmin Manufacturer’s working calibrator Manufacturer’s kit calibrator External quality assessment sample

14. © Copyright 2009 by the American Association for Clinical Chemistry Conclusions  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.

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