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1 Department of Clinical Biochemistry, North Bristol NHS Trust, Frenchay Hospital, Bristol BS16 1LE United Kingdom
2 Department of Immunology, PO Box 894, Sheffield S5 7YT United Kingdom
3 Protein Reference Unit, St. Georges, PO Box 10 295, London SW17 0NH, United Kingdom
4 UK NEQAS, Wolfson EQA Laboratory, Queen Elizabeth Medical Centre, Birmingham B15 2UE, United Kingdom
aAuthor for correspondence. Fax 44-117-9571-866; e-mail robert.beetham{at}north-bristol.swest.nhs.uk.
To the Editor:
The use of primary protein reference material CRM 470/RPPHS (1) was intended to lead to reduced method-dependent variation in specific protein analyses. Observations from UK NEQAS for Specific Proteins indicate that this is true for most proteins, but not for ceruloplasmin. Because the measurement of ceruloplasmin is an important part of the initial screening procedure for Wilson disease, we deemed it important to publicize this anomaly to the clinical chemistry community, including both analysts and the diagnostic industry.
UK NEQAS distributes specimens for chosen analytes to be analyzed by participating laboratories by their usual laboratory methods as though they were patient specimens. For the Specific Proteins Scheme, monthly distributions are made of material derived from pooled human donor serum stored at 4 °C; the pools reported here contained sodium azide (1 g/L) as preservative. The method used by each laboratory is recorded by UK NEQAS so that results may be studied for method-related differences. Data are analyzed by both method principle (e.g., turbidimetry) and then by the instrument or reagent used. All participants classified under nephelometric method A used the Beckman Array, whereas all those classified under method B used the Behring Nephelometer II. The turbidimetric group was extremely heterogeneous, with at least nine different manufacturers/suppliers represented according to calibrant used. One-third of this group used a calibrant from Roche and probably, although not certainly, an antiserum from this supplier.
The initial observation was of unexpectedly large differences among method-related mean values for the different methods such that for the January 1999 distribution, the overall mean for ceruloplasmin was 0.418 g/L (SD, 0.068 g/L; n = 90). The highest mean value (nephelometric method A) was 0.479 g/L (SD, 0.043 g/L; n = 28), whereas the lowest mean value (the turbidimetric group) was 0.368 g/L (SD, 0.061 g/L; n = 29; P <0.0001, unpaired t-test). However, when the same pool was redistributed 4 months later, these values had converged and were now 0.418 g/L (SD, 0.050 g/L; overall mean), 0.449 g/L (SD, 0.033 g/L; nephelometric method A), and 0.413 g/L (SD, 0.055 g/L; turbidimetric group; P <0.01 for nephelometric A v turbidimetric). We have now confirmed that this phenomenon is consistent for 17 pools, each of which was distributed on two occasions, separated by periods of 2–6 months, in the period January 1999 until October 2000 (Table 1
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The overall mean concentrations were relatively constant, and there was no significant difference in concentration between distributions. The mean for the turbidimetric method increased significantly, whereas the mean for nephelometric method A decreased significantly and, on average, to the same degree that the mean value for turbidimetry increased. That this is not purely attributable to the method principle is indicated by the increase of the mean for another nephelometric method (method B; n = 10) between distributions (mean difference, +0.017 g/L; P <0.001).
The degree of this phenomenon appears to be time-dependent because the difference between nephelometry method A and the turbidimetry group mean values was virtually abolished for all specimens where the second distribution was at least 5 months after the first distribution (mean difference, 0.005 g/L; range, -0.009 to 0.022 g/L; 0.1 < P < 0.5). That this is not simply a property of the specimens used in the scheme has been demonstrated previously by examining data for complement component C4, with concentrations similar to those of ceruloplasmin (2).
Specimens are generally circulated for the first time within 1–2 weeks of preparation (usually within 4 weeks of donation). They are subsequently stored at 4 °C until the second distribution.
We believe that the observations are best explained by an alteration in ceruloplasmin structure on storage that lead to changes in antigen–antibody reaction characteristics. When fresh, the antigen in the pools interacts with antiserum in a manner that is related to the characteristics of the antiserum. After storage and alteration in structure, certain epitopes recognized by individual antisera are altered, giving rise to more homogeneous values. Because calibration and internal quality-control procedures will, in general, be performed with "aged" material, assays appear to be in control and appropriately aligned to CRM 470. We acknowledge that our hypothesis would be more secure if monoclonal rather than polyclonal antibodies were involved and that its continuing manifestation over the time period is dependent on a supply of antisera with constant behavior toward ceruloplasmin. Nevertheless, we find no better explanation. The phenomenon cannot be ascribed to changes in calibrators or the primary standard because we describe a temporal effect that occurs subsequent to the preparation and distribution of each new serum pool. As 17 new pools were prepared and circulated during the period under study, approximately one each month with new and aged pools constantly overlapping, such an alternative explanation is not tenable.
This phenomenon is not new, having been described more than 20 years ago and ascribed to the process described above (3). The exact mechanism remains to be elucidated, but our preliminary experiments (not shown here) using isoelectric focusing suggest that it is not attributable to alterations in glycosylation with age; we are investigating further the lability of ceruloplasmin with respect to copper binding and proteolytic degradation (4).
Overall, the importance of our findings is that a universal reference interval based on a calibrant traceable to CRM 470 clearly cannot be used for ceruloplasmin in fresh specimens from patients.
References
The following articles in journals at HighWire Press have cited this article:
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  P J Twomey, A S Wierzbicki, T M Reynolds, and A Viljoen The copper/caeruloplasmin ratio in routine clinical practice in different laboratories J. Clin. Pathol., January 1, 2009; 62(1): 60 - 63. [Abstract] [Full Text] [PDF]
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 C. M. Mak, C.-W. Lam, and S. Tam Diagnostic Accuracy of Serum Ceruloplasmin in Wilson Disease: Determination of Sensitivity and Specificity by ROC Curve Analysis among ATP7B-Genotyped Subjects Clin. Chem., August 1, 2008; 54(8): 1356 - 1362. [Abstract] [Full Text] [PDF]
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P. J Twomey, A. Viljoen, I. M House, T. M Reynolds, and A. S Wierzbicki Copper:caeruloplasmin ratio J. Clin. Pathol., April 1, 2007; 60(4): 441 - 442. [Abstract] [Full Text] [PDF]
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P J Twomey, A Viljoen, I M House, T M Reynolds, and A S Wierzbicki Adjusting copper concentrations for caeruloplasmin levels in routine clinical practice J. Clin. Pathol., August 1, 2006; 59(8): 867 - 869. [Abstract] [Full Text] [PDF]
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