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Letters to the Editor |
1 Laboratory Medicine, Immunology, University Hospital Leuven, Herestraat 49, B-3000 Leuven, Belgium
2 Analis Inc., Rue Dewez 14, B-5000 Namur, Belgium
aAuthor for correspondence. Fax 32-16-34-7931; e-mail Xavier.Bossuyt{at}uz.kuleuven.ac.be.
To the Editor:
Capillary zone electrophoresis (CZE) is documented to have high sensitivity and specificity for the detection of monoclonal (M) proteins. In two large (>1500 participants) prospective studies in which CZE was compared with "immunofixation/immunoelectrophoresis", the sensitivity of CZE was 95% (1)(2). This is superior to the sensitivity of agarose gel electrophoresis (AGE), which Katzmann et al. (1) found to be 91% and Meunier (3) found to be 92.2% (evaluation of 2060 sera containing an M-protein). Especially in the ß-globulin region, CZE with Paragon CZE 2000 (Beckman-Coulter) is superior to AGE for detection of low-concentration M-proteins (IgA and light chains) (3)(4). The specificity of CZE is reported to be high (98.6%) and comparable to that of AGE (1)(5).
In a prospective study (2), we described three M-proteins with high pI values that migrated in the slow 
region on AGE but were not detected with Paragon. In addition, one high-concentration IgG|gl with a pI 
7 that migrated in the mid- region on AGE was not separated by CZE. The Paragon has recently been upgraded [modified buffer, higher voltage (10.3 kV), more efficient cooling, adapted software (1.6.02)], and according to the manufacturer, this should have improved resolution of certain rarely occurring M-proteins. With the updated system, ß-lipoprotein and fibrinogen were also resolved, which was not the case under earlier operating conditions.
The aim of the study was twofold: (a) we evaluated whether the upgrade enhanced resolution of previously unresolved M-proteins and in increased overall sensitivity; and (b) we evaluated whether the increased resolution affected specificity. One could hypothesize that because of the higher resolution, clinically unimportant heterogeneity in the -globulin zone will be observed more frequently.
To address the question of whether the upgrade increased resolution of previously unresolved M-proteins, we reanalyzed four sera that we described to contain a M-protein that could be separated by AGE but not by CZE (2). An additional similar sample with a M-protein migrating in the slow region with AGE but not with CZE was analyzed as well. Of the four post--migrating M-proteins, one M-protein was detected with the new buffer system, one generated an error code, and two were missed. The high-concentration IgG|gl M-protein with a pI 7 that migrated in the mid- region on AGE (2) was not separated by CZE under software version 1.6.02 but gave an error code.
Collectively, the recently introduced new buffer system for CZE with the Paragon CZE system has a slightly increased sensitivity for detecting M-proteins that were previously missed. Some rare M-proteins still remain unrecognized under these new operating conditions.
In the second part, we assessed the sensitivity and specificity in a prospective study. CZE analysis (Paragon CZE 2000, software version 1.602) and immunofixation (Hydrasys; Sebia) were performed on 1106 consecutive samples submitted to our laboratory to screen for the presence of a M-protein or to reevaluate a known M-protein. The results are shown in Table 1
. The sensitivity of CZE for the detection of M-proteins was 95%, which is similar to the previously reported sensitivity of 95% (1)(2). The M-proteins missed were present in low concentrations except for the IgG M-protein. The specificity of CZE 1.6 to detect M-proteins was calculated to be 78%. This is appreciably lower than the specificity of 98.6% published in an earlier report (2). The most frequently observed abnormalities in immunofixation-negative samples are slight changes in the morphology of the anodal part of the -globulin fraction. This might be attributable to complement degradation in aging samples or to fibrinogen in incompletely clotted serum samples.
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In conclusion, the adapted system did show somewhat improved resolution of certain (but not all) rarely occurring M-proteins that were missed by CZE operating under previous software versions and buffer conditions. Because the frequency of these rare slow--migrating M-proteins is extremely low, overall sensitivity (95%) was not influenced. There remain uncommon samples in which AGE is able but CZE is unable to separate the M-protein correctly. Efforts to improve the resolution of CZE for certain rarely occurring M-proteins did not produce an overall increased sensitivity for the detection of M-proteins but did decrease the specificity. The decrease in specificity (to 78%) may have consequences, such as increased costs for follow-up immunofixation analyses. It should be mentioned, however, that false-positive rates should decrease as operators become more familiar with the new pattern of enhanced resolution.
References
3 Beckman Coulter, Inc., 200 S. Kraemer Blvd., Brea, CA 92822
bAuthor for correspondence.
To the Editor:
We wish to address two points relative to the above letter by Mariën et al.:
(a) Mariën et al. state that the recent operating changes incorporated in Version 1.6.02 of Beckman Coulter’s Paragon CZE 2000 have adversely affected specificity, and claim that specificity has decreased to 82%.
In fact, CZE Version 1.6.02 offers improved specificity through enhanced resolution of the serum protein pattern. This enhanced resolution often detects minor degradation proteins that formerly were not readily observable. The enhanced operating conditions also produce a faster migrating gamma zone that appears as a taller, narrower band and, in some cases, may appear as a peak to operators trained on previous patterns. As operators become more familiar with the enhanced CZE patterns, their ability to interpret the patterns improve and false-positive rates decrease.
(b) Mariën et al. note that one of their previous monoclonal samples gave a suppressed result on the CZE, and they considered this to be a false negative.
In fact, this appears to have resulted from the very high protein concentrations in the sample. The "Instructions For Use" state that samples giving a suppressed result flag should be diluted and rerun. Had the sample been diluted and rerun, we believe that an accurate result would have been obtained.
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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K. Gijbels, G. Marien, and X. Bossuyt Ethanol Precipitation Is Not Reliable for Selectively Removing Nonmonoclonal Peaks Seen in the Fibrinogen Region on Capillary Zone Electrophoresis of Serum Proteins Clin. Chem., October 1, 2004; 50(10): 1880 - 1881. [Full Text] [PDF]
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G. Marien and X. Bossuyt Response to the Comments of K. Day and J. Zakowski on "Clinical Capillary Zone Electrophoresis of Serum Proteins: Balancing High Sensitivity and High Specificity" Clin. Chem., October 1, 2003; 49(10): 1711 - 1712. [Full Text] [PDF]
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