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Technical Briefs |
Department of Clinical Pathology, University Hospital Leuven, Kapucijnenvoer 33, B-3000 Leuven, Belgium
a author
for correspondence: fax 00 32 16 332896, e-mail xavier.bossuyt{at}uz.kuleuven.ac.be
Capillary zone electrophoresis (CZE) has emerged as a novel technique for the rapid and effective separation of serum proteins (1)(2)(3)(4)(5)(6)(7)(8). Recently, a multichannel automated system for CZE of human serum proteins (Paragon 2000 clinical capillary electrophoresis system; Beckman Instruments) became commercially available; this system offers a clinically reliable alternative to cellulose acetate and agarose electrophoresis. CZE has the advantage of automation, improved precision, and a faster turnaround time (6)(8).
In the conventional methods, quantification of the protein fractions is based on dye binding, whereas CZE uses ultraviolet detection at 214 nm for direct protein quantification via the peptide bonds. We asked whether intravascular agents such as radio-contrast media that absorb at 214 nm would simulate a monoclonal component on CZE.
We performed high resolution agarose electrophoresis with the Hydrasys
analyzer (Sebia), using Hydragel 15 HR gels (Sebia) according to the
manufacturer's instructions. The serum proteins were separated
into the following fractions: prealbumin, albumin,
1-antitrypsin, 1-lipoprotein,
haptoglobin plus 2-macroglobulin, ß- lipoprotein,
transferrin, complement C3, and the gamma globulins. Spectrophotometric
analysis was done with a Varian Cary 3 biospectrophotometer (Varian).
We combined sera from five adults for the addition experiments. The
capillary zone and agarose electropherograms of each of the five sera
were normal.
The CZE electropherograms (Beckman Paragon CZE 2000, Ver. 1.08),
determined using conditions as described by Bossuyt et al.
(8), from patients who received intravascular radio-contrast
medium 2–4 h before specimen collection for electrophoretic analysis
are shown in Fig. 1
. The radio-opaque media were Urografin® (Schering
AG; Fig. 1A
), Telebrix® ( Guerbet; Fig. 1B
), and
Omnipaque® (Nycomed Imaging AS; Fig. 1C
). In these
electropherograms, the 2-globulin fraction showed a peak
("spike") suggestive of the presence of a monoclonal component. The
peak was at the anodal side of the 2-globulin fraction
in the electropherogram for Urografin, in the middle of the
2 fraction in the electropherogram for Telebrix, and at
the cathodal side of the 2 fraction in the
electropherogram for Omnipaque. In each of these cases, however, no
peak was discerned on agarose electrophoresis (not shown). The latter
observation provided strong evidence against the presence of a
monoclonal component.
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The unexpected peaks on the CZE electropherograms appeared to represent interference by the radio-contrast agents. First, each of the three contrast agents, Urografin, Telebrix, and Omnipaque, administered to the patients absorbs light at 214 nm (data not shown), which is the wavelength used for quantification of proteins in CZE. Second, no abnormal peak was found when CZE analysis was performed on a specimen collected 2–6 days after intravenous administration of the contrast medium (data not shown). After this time period, the contrast had been cleared from the blood stream. The elimination half-life is 121 min for Iohexol® (the radio-opaque medium in Omnipaque) (9), 120 min for Telebrix, and 60–120 min for Urografin. Third, addition to a serum pool of 7.5 g/L Urografin, 4.7 g/L Telebrix, or 7.5 g/L Omnipaque, concentrations that can be expected after bolus injection for radiographic examination, in each case led to the appearance in the capillary zone electropherograms of abnormal peaks with the same shapes and in the same locations as the extra peaks found in the electropherograms from patients injected with contrast agents. Addition of the contrast agents to a serum pool did not affect electropherograms of samples submitted to agarose electrophoresis (data not shown).
The CZE elution times of the three radio-opaque agents were similar in serum and in 9 g/L NaCl. This indicates that there is no evidence of actual binding of the radio-opaque agents to a specific protein and that the observed interference is purely a result of the coincidence of their elution times and absorption spectra with the elution times and absorption spectra of the proteins.
At our institution, interference by radio-opaque agents is encountered in ~4 of every 1000 specimens submitted for protein electrophoresis. Thus, in high-throughput laboratories, interference by radio-opaque media might be seen in a substantial number of samples.
We conclude that interference by radio-opaque media may be confused with monoclonal proteins and that instructions should be given to not collect blood for protein electrophoresis by CZE shortly after a patient receives contrast media.
References
The following articles in journals at HighWire Press have cited this article:
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  P. Vermeersch, G. Marien, and X. Bossuyt Pseudoparaproteinemia Related to Iomeprol Administration after Angiocardiography: Detection in the {beta} Fraction by Capillary Zone Electrophoresis Clin. Chem., December 1, 2006; 52(12): 2312 - 2313. [Full Text] [PDF]
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G. van der Watt and P. Berman Pseudoparaproteinemia after Iopamidol Infusion for Coronary Angiography Clin. Chem., January 1, 2005; 51(1): 273 - 274. [Full Text] [PDF]
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X. Bossuyt, J. Verhaegen, G. Marien, and N. Blanckaert Effect of Sulfamethoxazole on Clinical Capillary Zone Electrophoresis of Serum Proteins Clin. Chem., February 1, 2003; 49(2): 340 - 341. [Full Text] [PDF]
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X. Bossuyt and W. E. Peetermans Effect of Piperacillin-Tazobactam on Clinical Capillary Zone Electrophoresis of Serum Proteins Clin. Chem., January 1, 2002; 48(1): 204 - 205. [Full Text] [PDF]
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D. F. Keren, R. Gulbranson, J. L. Carey, and J. C. Krauss 2-Mercaptoethanol Treatment Improves Measurement of an IgM{{kappa}} M-Protein by Capillary Electrophoresis Clin. Chem., July 1, 2001; 47(7): 1326 - 1327. [Full Text] [PDF]
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R. Claeys, C. Groven, and F. K. Gorus Capillary Zone Electrophoresis of Proteins in Body Fluids: Comparison of Capillary and Agarose Gel Electrophoresis Clin. Chem., May 1, 2001; 47(5): 967 - 970. [Full Text] [PDF]
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M. L. Arranz-Pena, M. Gonzalez-Sagrado, A. M. Olmos-Linares, N. Fernandez-Garcia, and F. J. Martin-Gil Interference of Iodinated Contrast Media in Serum Capillary Zone Electrophoresis Clin. Chem., May 1, 2000; 46(5): 736 - 737. [Full Text] [PDF]
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C. R. Blessum, N. Khatter, and S. C. Alter Technique to Remove Interference Caused by Radio-Opaque Agents in Clinical Capillary Zone Electrophoresis Clin. Chem., August 1, 1999; 45(8): 1313 - 1313. [Full Text] [PDF]
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