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M-Protein by Capillary Electrophoresis
1
Warde Medical Laboratory, 5025 Venture Dr., Ann Arbor, MI 48108
2
Henry Ford Hospital, Department of Pathology, Detroit, MI 48202-2689
3
St. Joseph Mercy Hospital, Ypsilanti, MI 48197
aAuthor for correspondence. Fax 734-665-0668; e-mail dfkeren{at}yahoo.com.
To the Editor:
A recent consensus conference on monoclonal gammopathies recommended the use of high-resolution electrophoresis, which achieves crisp separation of the major ß-1 (transferrin) and ß-2 (C3) bands (1)(2). Capillary electrophoresis (CE) provides this degree of resolution and has an additional advantage of automating both the analysis and the presentation of serum protein electrophoresis results (3)(4)(5). We report a rare discrepancy in the CE measurement of an IgM M-protein in the serum.
The patient, a 59-year-old man, was first referred in August 1999 for evaluation of alcohol dependence, thrombocytopenia, and splenomegaly. Hemoglobin was 140 g/L, the white blood cell count was 4.7 x 109/L, and the platelet count was 54 x 109/L. A bone marrow aspirate was almost entirely plasmacytoid lymphocytes consistent with Waldenström macroglobulinemia. In addition to the lymphoproliferative process, the patient suffered from alcoholic hepatitis. On the day the sample was collected, his blood alcohol was 4.5 g/L.
CE was performed using the Beckman CZE 2000 with software version 1.5.02 (Beckman Coulter) and the standard Paragon CZE 2000 Capillary Electrophoresis Borate Buffer-100 (part no. 446280). Undiluted serum was aspirated into the instrument. The M-protein was quantified by integrating the area under the 214 nm peak (6). Immunosubtraction was also performed on the Beckman CZE 2000.
The Sebia semiautomated agarose gel electrophoretic system, with the Beta-1,2 gel, which provides crisp resolution of the transferrin (ß-1) and C3 (ß-2) bands, was used for all gel-based studies. Immunofixation (IFE) was performed using the Sebia IFE gel, buffers, and antisera. The Beckman Array was used to measure serum immunoglobulins.
Immunoglobulins measured on the present sample included IgG (9.9 g/L), IgA (2.4 g/L), IgM (15 g/L), 
(16 g/L), 
(4.8 g/L), and the : ratio (3.39). By CE, the ß-region M-protein was 2.6 g/L (Fig. 1A
). Immunosubtraction identified the M-protein as an IgM. Because of the disparity between the total IgM measurement by nephelometry and the tiny M-protein peak on the CE, we performed a second protein electrophoresis using the Sebia semiautomated agarose system. On this system, the M-protein measured by densitometric scan was 15.6 g/L, consistent with the nephelometric measurement of 15.2 g/L for total IgM (Fig. 1B
). An IFE using the Sebia system confirmed the identification of the M-protein as IgM. Because of these results, the CE was repeated after treatment with 2-mercaptoethanol (2-ME; 5 µL in 1 mL of serum at 37 °C for 1 h) to render the monoclonal IgM into monomeric structure. This produced a large band in the 
region that measured 9.0 g/L (Fig. 1C
).
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Despite favorable reviews of CE in comparison with gel-based protein electrophoresis (3)(4), both false-positive and false-negative results have been reported. Because CE measures absorbance at 214 nm, substances other than proteins have been reported to produce suspicious bands. Radio-contrast media that absorb at this wavelength produce discrete bands that may be mistaken for M-proteins (7). Fortunately, this may be removed by desalting (8). Difficulties in detecting M-proteins in serum from six patients by CE were reported by Jenkins and Guerin (9). They noted difficulty in separating five IgM M-proteins that had slow -region migration. A modification in the boric acid buffer system and pH allowed detection of these M-proteins.
In the present case, the M-protein was detected, but at a much lower concentration than indicated by nephelometry or high-resolution agarose gels. Treatment with 2-ME produced an M-protein peak that was more consistent with the other results. With immunoelectrophoresis, 2-ME treatment improved identification of IgM M-proteins because IgM monomers migrate more readily through the agarose than the IgM pentamer. The characteristics of this IgM M-protein that may have interfered with its passage through the capillary or that imparted an affinity for the capillary wall are not known. However, the 2-ME treatment was able to ameliorate this and effect a more reasonable measurement of the M-protein.
We recommend that laboratories using CE consider the use of 2-ME to evaluate M-proteins when the nephelometric concentrations or clinical features are inconsistent with the CE electropherogram measurement.
References
The following articles in journals at HighWire Press have cited this article:
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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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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 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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