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Technical Briefs |
1 Laboratory Medicine, Immunology, University Hospital Leuven, Herestraat 49, B-3000 Leuven, Belgium
aauthor for correspondence: fax 00-32-16-34-7931, e-mail Xavier.Bossuyt{at}uz.kuleuven.ac.be
Over the last 5 years, capillary zone electrophoresis (CZE) with the use of fused-silica capillaries has been increasingly introduced in clinical laboratories for routine serum-protein electrophoresis (1). The multichannel, automated Paragon CZE 2000 instrument (seven capillaries in parallel; Beckman Coulter) represented an especially attractive alternative to time-consuming manual techniques. CZE has been documented to perform reliably for the analysis of serum proteins and for the detection of monoclonal components (2)(3). We reported (4) that the sensitivity of the Paragon CZE 2000 system for the detection of monoclonal components (93%) was superior to the sensitivity of cellulose acetate gel electrophoresis (74%) and agarose gel electrophoresis (86%). In a prospective study, Katzman et al. (5) reported sensitivities of 95% and 91% for seven-capillary electrophoresis and agarose gel electrophoresis, respectively. When compared with agarose gel electrophoresis, CZE was able to detect more low-concentration IgA monoclonal components or light chains that were hidden in agarose gel electrophoresis because of comigration with transferrin or C3 (6). Paraproteins that are missed by CZE are typically very low-concentration monoclonal components that are also missed by agarose gel electrophoresis, but not by immunofixation.
Problems with the detection of monoclonal components by CZE have been described (7)(8). Using a single-capillary Beckman P/ACE instrument with a capillary and buffer different from the Paragon CZE 2000 system, Jenkins and Guerin (7) performed a prospective study on 5500 specimens in which they compared CZE with agarose gel electrophoresis for the detection of monoclonal components. The authors identified six paraproteins that did not separate correctly on CZE. On agarose gel electrophoresis, all of these proteins migrated in the very slow 
region. The pI was >8.5 for the IgG paraproteins and >6.9 for the IgM paraproteins. When the ionic strength and the pH of the buffer were increased, they were able to detect all paraproteins by CZE. Henskens et al. (8) reported missing a high-concentration, 20 g/L monoclonal IgM component (with a rather cathodal migration on agarose gel) by their method using a P/ACE system. By changing the ionic strength and the pH of the buffer, they were able to detect the paraprotein.
In the present study, we evaluated the Paragon CZE 2000 system (Beckman-Coulter) for detection of serum protein abnormalities in a prospective study. CZE analysis, as well as immunofixation, was performed on 2629 samples from 1692 different individuals submitted to our laboratory to screen for the presence of a paraprotein or to reevaluate a known paraprotein. Immunofixation was performed with the Hydrasys Automate (Sebia) according to the manufacturer’s instructions with the use of Hydragel 4 immunofixation gels. Immunofixation revealed the presence of a distinct paraprotein in 481 of the 1692 patients: an IgG-type paraprotein (315 cases; 65%); an IgA-type paraprotein (74 cases; 15.4%); an IgM type-paraprotein (71 cases; 14.8%); an IgD-type paraprotein (1 case); and a light chain disease (20 cases; 4.1%). In 14 cases, a double paraprotein was present (IgA in combination with IgM or IgG in 9 cases and IgG in combination with IgM in 5 cases).
CZE failed to detect an abnormality in 24 (5%) of the 481 samples in which immunofixation revealed a paraprotein. This sensitivity of CZE to detect monoclonal proteins (95%) agrees with that reported by Katzman et al. (5).
The most commonly missed paraproteins were present in low concentrations. CZE failed to detect the IgD paraprotein. No abnormalities were found by CZE in samples in which immunofixation revealed monoclonal free light chains (five samples), low-concentration monoclonal IgA proteins (total IgA concentration, <3.2 g/L; six samples), low-concentration monoclonal IgM paraproteins (total IgM concentration, <2.1 g/L; five samples), and low-concentration monoclonal IgG paraprotein (three samples). All IgA paraproteins that were overlooked by CZE migrated in the ß region and were hidden under the transferrin or C3 peak, whereas the low-concentration IgM paraproteins migrated in the region, except for one that comigrated with C3. The low-concentration IgG paraproteins that were undetected by CZE were situated in the ß and regions.
In three other samples, paraproteins in the slow region (7)(8) did not separate correctly on CZE. For one sample, the CZE result was suppressed and gave an error code (Fig. 1C
). High-resolution agarose gel electrophoresis (Hydrasys) of this sample revealed a high-concentration paraprotein (22 g/L) that migrated in the slow region (Fig. 1A
). Isoelectric focusing disclosed that the pI of the monoclonal IgG protein was >8.5. We hypothesize that the problem is related to the high pI of the monoclonal protein and the pH and ionic strength of the buffer used, as suggested previously (7)(8). Analysis of the same sample with CEofixTM high-resolution capillary electrophoresis reagent set (Analis) according to the manufacturer’s instructions on a P/ACE 5000 (Beckman-Coulter) system revealed the monoclonal protein. In a second sample, a low-concentration IgG paraprotein migrated in the slow region on agarose gel electrophoresis and gave a normal CZE electropherogram (data not shown). CZE also failed to correctly separate an IgM paraprotein (total IgM concentration, 5.6 g/L). The monoclonal protein was revealed by agarose gel electrophoresis and by the CEofix high-resolution capillary electrophoresis reagent set (data not shown). Isoelectric focussing of this sample revealed several bands migrating between pI 
6 and 8.5. These three cases illustrate that the widely used Paragon 2000 system may miss some paraproteins that typically migrate in the slow region, as has been described previously by Henskens et al. (8) and by Jenkins and Guerin (7) for CZE methods on a P/ACE system. We believe that modifying the pH and ionic strength of the buffer used with the Paragon CZE 2000 system should enable the system to correctly separate monoclonal proteins with a high pI.
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Finally, in one sample, CZE completely failed to detect the presence of a high-concentration paraprotein. CZE showed a normal electropherogram (Fig. 1D
), whereas agarose gel electrophoresis revealed a distinct spike in the mid- region (18 g/L; Fig. 1B
). Immunofixation identified an IgG 
band. This paraprotein is different from the others because (a) it migrated in the mid region and not in the slow region on agarose gel electrophoresis, and (b) its pI (by isoelectric focusing) was 7. Moreover, analysis of the same sample with the CEofix high-resolution capillary electrophoresis reagent set on a P/ACE 5000 instrument also failed to detect the monoclonal component. No cryoglobulins were present in the sample. Lipoprotein electrophoresis showed a normal pattern, excluding the possibility of the presence of protein-lipid complexes that might interfere with CZE. The reason this protein was missed by CZE was unclear. Perhaps it was related to the optical absorbance characteristics of this specific protein. With CZE, protein is detected by absorbance measurement, whereas with conventional methods, binding of dyes is used to quantify proteins.
In summary, this prospective study showed that, when compared with immunofixation, the sensitivity of CZE to detect monoclonal proteins was 95%, thereby confirming an earlier report (5). The monoclonal proteins that are missed by CZE are typically low-concentration paraproteins that can be detected only by immunofixation. We also illustrated that, although uncommon, the Paragon 2000 CZE may fail to correctly separate high-concentration monoclonal components. These monoclonal components typically have a high pI value and migrate in the slow region on agarose gel electrophoresis. In addition, we demonstrated for the first time that CZE may also fail to detect high-concentration monoclonal components with a pI 7 that migrate in the mid region. The reason for this failure is not known. It is not related to the presence of cryoglobulins or lipid-protein complexes. In view of these results, we routinely perform CZE analysis as well as immunofixation analysis on all samples that are submitted for the first time to our laboratory with the specific question to search for the presence of paraproteins.
Acknowledgments
We are indebted to Guido Vranken an Anja De Saeleer for helpful discussions and for performing the analyses on the P/ACE instrument. We thank Z. Zaman for helping with the isoelectric focusing.
References
The following articles in journals at HighWire Press have cited this article:
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  X. Bossuyt and G. Marien Detection of Monoclonal Proteins by Capillary Zone Electrophoresis: Comparison of 2 Multichannel Automated Systems Clin. Chem., January 1, 2007; 53(1): 152 - 153. [Full Text] [PDF]
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I. Infusino, P. Luraschi, M. Panteghini, and C. Franzini Pretreatment of serum with penicillamine: effects on capillary electrophoresis patterns and on immunonephelometric measurement of immunoglobulins. Clin. Chem., April 1, 2006; 52(4): 772 - 774. [Full Text] [PDF]
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G. Marien, G. Verhoef, and X. Bossuyt Detection of Heavy Chain Disease by Capillary Zone Electrophoresis Clin. Chem., July 1, 2005; 51(7): 1302 - 1303. [Full Text] [PDF]
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P. Luraschi, I. Infusino, I. Zorzoli, G. Merlini, C. Fundaro and, and C. Franzini Heavy Chain Disease Can Be Detected by Capillary Zone Electrophoresis Clin. Chem., January 1, 2005; 51(1): 247 - 249. [Full Text] [PDF]
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H. Zetterberg and H. Nilsson-Ehle False-Negative Result in the Detection of an IgM Monoclonal Protein by Capillary Zone Electrophoresis Clin. Chem., October 1, 2004; 50(10): 1878 - 1880. [Full Text] [PDF]
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G. Marien, G. Vranken, M. Demuylder, N. Blanckaert, X. Bossuyt, K. Day, and J. Zakowski Clinical Capillary Zone Electrophoresis of Serum Proteins: Balancing High Sensitivity and High Specificity * Representatives of Beckman Coulter respond: Clin. Chem., August 1, 2003; 49(8): 1419 - 1421. [Full Text] [PDF]
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G. Marien, E. Oris, A. R. Bradwell, N. Blanckaert, and X. Bossuyt Detection of Monoclonal Proteins in Sera by Capillary Zone Electrophoresis and Free Light Chain Measurements Clin. Chem., September 1, 2002; 48(9): 1600 - 1601. [Full Text] [PDF]
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