Capillary Zone Electrophoresis in the Evaluation of Serum Protein Abnormalities

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1 Capillary Zone Electrophoresis in the Evaluation of Serum Protein Abnormalities David F. Keren, MD Key Words: Capillary zone electrophoresis; M-protein; Serum protein electrophoresis; monoclonal gammopathy; a,-antitrypsin; Albumin; a,-globulin; a2-globulin; p-globulin; y-globulin Serum protein electrophoresis has been an integral part of the diagnostic process for more than half a century. It is used principally to evaluate patients for the presence of a monoclonal gammopathy, but it is also useful for detecting humoral immunodeficiency, liver disease, a,-antitrypsin deficiencies, acute phase reaction, chronic inflammation, and a variety of other conditions.1 During the past decade, a new technique, capillary zone electrophoresis (CE), has been developed for use in clinical laboratories.2-3 To appreciate the advantages of this new technology, as well as some concerns about unanswered questions, it is useful to place this technique in the context of the evolution of serum protein electrophoresis. The technology has evolved from the moving boundary electrophoresis, first performed in the U-shaped Tiselius apparatus, to the solid support zone electrophoresis (originally filter paper now cellulose acetate or agarose) widely used today. Through technical advances in production of capillaries, sampling devices, optical devices, and software, automated CE is now a practical alternative for the clinical laboratory. In many respects, CE resembles the original Tiselius technology. In 1932, Tiselius4 reported the use of a U-shaped electrophoretic cell in the analysis of colloidal mixtures. Tiselius layered the solution to be analyzed on 1 end of a buffer-filled U-shaped tube. When current was applied to electrodes at each end of the tube, the substances to be analyzed separated on the basis of their isoelectric points. Separation of the substances was confirmed by the sensitive Schlieren band optical system that detected changes in light refraction as the molecules moved past afixedpoint. By using this moving boundary electrophoresis device, early workers were able to document that serum contained at least 5 electrophorefically defined fractions: albumin, a,-globulin, Oj-globulin, P-globulin, and y-globulin. Unfortunately, in moving boundary electrophoresis, when the current was turned off, the protein fractions would intermingle and the individual fractions could not be visual248 Am J Clin Pathol 1998,110: ized readily or purified. During the late 1930s, zone electrophoresis was developed. This allowed visualization of protein fractions on a support medium. Between 1939 and 1960, a variety of support media were used that facilitated the identification and measurement of the major protein fractions. Filter paper, cellulose acetate, and agarose were all suitable for this purpose. After electrophoresis, protein fractions could be fixed in place and stained, permitting study of the proteins, as well as providing a permanent record. Cellulose acetate was easy to work with and provided a clear background that allowed for quantification of stained protein bands by densitometry. Although agarose was somewhat more difficult to work with than cellulose acetate, it provided a resolution superior to that of the early acetate products. All 3 zone electrophoresis support media were affected by a new variable endosmotic flow. Endosmosis results when the positively charged molecules in the buffer appose themselves to the negative charges on the support media. The media cannot move, but the cations flow toward the negatively charged cathode. Because most proteins are negatively charged at ph 8.6 (typical for serum protein electrophoresis), they are attracted, by the voltage, toward the anode. However, the endosmotic flow exerts an opposite effect, pulling the proteins toward the cathode. In most zone electrophoretic systems, the y-globulins migrate toward the cathode because of their relatively weak negative surface charge compared with more anodal proteins under the typical buffer conditions. The amount of endosmosis that occurs depends on the amount of negative charge on the support medium and the buffer conditions. With CE, owing to the narrow bore (creating a large surface area) and negative charge of the capillaries, endosmotic flow plays a major part in the migration of proteins. Zone electrophoresis has been a standard in the clinical laboratory since the 1950s. Some companies have automated sample application and staining steps for zone electrophoresis.

2 The resolution achieved by early automated zone electrophoresis systems was similar to the low-resolution manual methods. More recent automated techniques have provided excellent resolution. When put to the test on an interlaboratory survey, relatively few participants who used lower resolution equipment were able to detect a subtle monoclonal gammopathy, compared with the participants using high-resolution manual techniques.5 Capillary Zone Electrophoresis During the past few years, high-performance CE has been used to provide information about the concentration and electrophoretic migration of the major protein fractions in serum.2,3 CE does not provide a gel pattern and resembles the earlier Tiselius technique in that respect. The technological advances of CE provide high-resolution and detailed information about the major protein fractions and allow automation using tiny sample volumes. As with zone electrophoresis, migration in CE depends on the net charge carried by the protein. This, in turn, depends on buffer conditions and the strength of the electrical field. Buffer conditions differ in CE from gel electrophoresis because the barbital buffer most often used in gel electrophoresis has high absorbance at wavelengths near 200 nm (the absorbance of peptide bonds).6,7 Therefore, CE systems use borate or other buffers that do not interfere with the detection system. Migration under conditions of CE is strongly influenced by endosmosis owing to the relatively large surface area charge within the narrow-bore fused-silica capillaries. The surface charge results from the negative charge of the fused silica that is used to make the capillaries. Further, the surface area inside the capillaries is relatively large owing to the narrow lumen (typically about 50 urn in diameter). This results in a migration of buffer cations toward the anode similar to but much stronger than the endosmosis that occurs during zone electrophoresis. The combined effects of the charge on the capillaries and the large surface area result in a movement of proteins toward the cathode that is considerably greater than the effect of the voltage to attract the proteins toward the anode. As a result, the proteins are separated into about 10 recognizable fractions by these 2 forces, similar to the results obtained by high-resolution zone techniques. A detector located at the cathodal end of the capillary records the optical density at about 200 nm (peptide bond absorption) as the proteins migrate past it. The y-globulins pass by the detector first, followed by p-, a 2 -, and a,-globulins, then albumin and transthyretin (prealbumin). Because the y and (3 region proteins pass through the detector before the other components, they are exposed to the pull of the voltage for a shorter time than the more anodal components of serum. This may account for the relatively narrower y and (3 regions and a relatively broader albumin through a 2 region by CE than obtained with highresolution agarose gels IFigure II. Therefore, although CE provides excellent resolution of proteins, the visualization of the protein fractions is better at the anodal than at the cathodal end of the pattern. CE provides sufficient resolution in the a, region to allow distinction of a, acid glycoprotein (orosomucoid) from ctj-antitrypsin (both are acute phase reactants) (Figure 1). The excellent resolution in the 3 region facilitates detection of monoclonal proteins in that region (Figure 1); however, the narrow y region, requires careful attention to avoid missing subtle monoclonal gammopathies IFigure 21. Because of this, I recommend that even modest asymmetries in the y region be studied by immunofixation. In addition, when there is a massive polyclonal increase in y globulins, the narrower y region may be confusing to the unwary, and one may be tempted to overinterpret a polyclonal increase in y globulins as a monoclonal gammopathy. By being aware of these possibilities, false-positive and false-negative reports can be avoided. Studies have compared the ability of CE with standard gel electrophoresis to detect monoclonal gammopathies in serum. Clark et al8 found that results obtained by CE on the analysis of 305 serum specimens were equivalent, or superior (especially in the P region), to those obtained from a lowresolution agarose method. Similarly, in a study performed on 200 samples, Wijnen and van Dieijen-Visser9 noted good reproducibility of migration times for the 5 major serum protein fractions with the CE. Their CE method gave slightly higher variation in relative peak areas than those obtained by densitometric scans of their low-resolution agarose gel electrophoresis method.9 However, a good correlation was found between the 2 methods for all 5 major protein fractions. Jenkins et al10 compared the detection of monoclonal gammopathies by CE and high-resolution agarose electrophoresis in 1,000 prospective serum specimens. Their CE system used a 72-cm x 50-Lim (inner diameter) fused-silica capillary and detected absorbance of proteins at 200 nm. They reported an overall correlation between the 2 methods of 0.96 in the detection of 362 monoclonal proteins.10 The high-resolution agarose method gave slightly higher values for the monoclonal bands than did their CE method. In another study, Jenkins and Guerin" claimed that CE provided an advantage over the high-resolution agarose method in detecting P migrating IgA monoclonal bands owing to the crisp resolution in the P region of their CE system. Advantages and Disadvantages of CE Notable technical advantages of CE include high-resolution, ease of automation, and no need for a densitometer. Yet, Am J Clin Pathol 1998;110:

3 Keren / CAPILLARY ZONE ELECTROPHORESIS IN THE EVALUATION OF SERUM PROTEIN ABNORMALITIES A Albumin B Albumin P Transferrin Figure I I Virtual gel image displayed by the Beckman CZE 2000 monitor (Beckman Instruments, Fullerton, Calif). Evaluations of 6 serum sample are shown. The major protein fractions are labeled in the top serum sample: A is to the left of the albumin band; 1 overlies a,-acid glycoprotein with the a,-antitrypsin band just to its right; 2 is just to the left of the a2-globulin band; t (small arrowhead) points to the transferrin band in the P1 region; c (large arrowhead) points to the C3 band in the P2 region; and G is in the y-globulin region. The arrow in the second serum sample from the top denotes a dense monoclonal band in the (3 region. The y region of this sample is decreased from normal. A subtle monoclonal band is indicated by an arrow in the p region of the fifth serum sample from the top. The albumin band in the fifth sample is decreased and shows anodal slurring due to the effect of heparin on the a-lipoprotein band.17 Figure 21 A, A capillary zone electrophoresis pattern from a patient with a small y migrating IgA X monoclonal gammopathy. The major protein fractions are labeled. The arrow indicates the slight asymmetry at the anodal end of the y region where the IgA X monoclonal protein migrated. Data obtained with the Beckman CZE 2000 (Beckman Instruments, Fullerton, Calif). B, A densitometric scan of the high-resolution agarose gel from the same serum sample, but assayed 2 days later. The arrow indicates the small, monoclonal gammopathy. Note the loss of the (32 region band (C3) during the 2-day delay (Beckman SPE-2, stained with Paragon blue). CE is still a relatively new technology, and much still needs to be learned about its limitations. For instance, with CE an actual gel is not examined. The bands represent substances that have absorbancy at about 200 nm. Substances other than proteins absorbing at that wavelength have the potential to produce a band that may be confused with a band that suggest monoclonal gammopathy. Such interference has been noted in a couple of patients receiving antibiotic therapy (Cynthia Blessum, Beckman Instruments, Fullerton, Calif, oral communication, May 1998), and my laboratory has seen 2 similar cases. Some mysteries remain about CE technology. For instance, we do not detect any band for fibrinogen, even on plasma samples tested on the Beckman CZE 2000 (Beckman 250 Am J Clin Pathol 1998; 110:

4 Instruments). Similar observations have been made in other laboratories.12 Why not? It certainly has peptide bonds. No one seems to know. I prefer not to see the residual fibrinogen in incompletely clotted serum, or in a heparinized sample. Fibrinogen will produce a band on traditional gel electrophoresis that may be confused with a band suggesting monoclonal gammopathy. Yet, I am uncomfortable with not understanding why it is absent. If it is unknown why fibrinogen eludes detection by CE, how are we to be certain that some monoclonal gammopathies may not be missed if CE is used as the screening technique? Although such undetected monoclonal gammopathies are rare, the issue is of more than a theoretical concern. Jenkins and Guerin,13 using CE as their primary detection method, reported the failure to adequately identify 8 monoclonal gammopathies out of approximately 6,500 specimens. They noted that when these monoclonal proteins were evaluated by high-resolution agarose electrophoresis, they all migrated in the very slow y region. In my laboratory, I have seen 1 monoclonal gammopathy migrating in the slow y region that was not detectable in our CE method. Was it influenced by hyperlipidemia, the position of the monoclonal protein, or by binding of a relatively cationic monoclonal protein to the walls of the capillaries?14 When Jenkins and Guerin13 modified the boric acid buffer system they used from a 50-mmol concentration, ph 9.7, to a 75-mmol concentration, ph 10.3, they were able to detect and quantify all of the monoclonal bands. These concerns must be put in perspective. All techniques have false-negative and false-positive results. Usually the fibrinogen band is a needless worry that obscures an important part of the (3-y region on gel electrophoresis. Also, as mentioned, detection of monoclonal gammopathies by CE is at least the equivalent of traditional zone electrophoresis methods. An additional advantage of CE technology is the use of digitized information to enhance unusual gel patterns. Further, one instrument, the Beckman CZE 2000, provides a software-generated virtual gel image from the absorbancy information. At the present time, the Beckman CE 2000 is the only CE equipment approved by the US Food and Drug Administration. It is approved only for study of the serum. Some studies have shown that CE may be a practical method to study cerebrospinal fluid (CSF)'5 or urine, as well as serum.16 For instance, in a study of 30 randomly selected samples of CSF, Cowdrey et al15 were able to detect between 20 and 25 peaks, some of which were only identified in CSF and not in the corresponding serum. Similarly, Jenkins et al16 reported a strong correlation between CE and high-resolution agarose for quantifying Bence Jones protein and albumin. Despite these encouraging results, no method is currently approved for evaluation of routine clinical CSF or urine samples. Further studies must be performed to determine whether CE will be able to detect oligoclonal bands (they often migrate in the slow y region), or if small polypeptides, drug metabolites, or microbial products present in urine will interfere with interpretation of the CE patterns. Monoclonal gammopathies may be characterized by performing immunosubtraction on CE. This involves performing CE before and after incubating the serum sample with solid particles coated with antibodies against specific immunoglobulin isotypes and then performing CE on the serum sample. Removal of a peak by beads coated with specific antibodies indicates that the monoclonal component is that isotype. Because there is relatively little published information comparing immunosubtraction to immunofixation or immunoelectrophoresis, it is premature to recommend use of this technique on clinical samples. Potential problems, such as antigen excess, detection of small monoclonal proteins, monoclonal proteins that migrate in the (3 region (where transferrin, C3, and (3-, lipoprotein may obscure the band), and detection of heavy chain or light chain disease require extensive study. Summary CE techniques offer a highly efficient alternative to traditional zone electrophoresis for performing serum protein electrophoresis. CE is most useful in laboratories that have a relatively large daily workload (at least 40 serum samples per day). Because no equipment is currently approved for the study of urine or CSF samples, gel techniques must be maintained in the laboratory. Immunosubtraction could be an attractive method to identify monoclonal gammopathies; however, detailed reports about the advantages and disadvantages of this technique in a large number of clinically well-characterized monoclonal gammopathies are required to clarify the role of immunosubtraction in the clinical laboratory. Overall, CE is an exciting new method that may improve the detection of monoclonal gammopathies and other abnormalities, while improving the efficiency of the protein chemistry workload. From the Warde Medical Laboratory, Ann Arbor, Michigan. Address reprint requests to Dr Keren: Warde Medical Laboratory; 5025 Venture Dr, Ann Arbor, MI References 1. Keren DF. Approaches to pattern interpretation in serum. In: High-Resolution Electrophoresis and Immunofixation: Techniques and Interpretation. 2nd ed. Boston, Mass: Butterworth-Heinemann; 1994: Landers JP. Clinical capillary electrophoresis. Clin Chem. 1995;41: Am J Clin Pathol 1998,110:

5 Keren / CAPILLARY ZONE ELECTROPHORESIS IN THE EVALUATION OF SERUM PROTEIN ABNORMALITIES 3. Karger BL, Chu YH, Foret F. Capillary electrophoresis of proteins and nucleic acids. Annu Rev Biophys Biomol Struct. 1995;24: Tiselius A. A new apparatus for electrophoretic analysis of colloidal mixtures. Trans Faraday Soc. 1932;33: College of American Pathologists. CAP Survey Report EC-07, Northfield, 111: College of American Pathologists; Dolnik V. Capillary zone electrophoresis of serum proteins: study of separation variables. J Chromatogr A. 1995; 709: Chen FT, Sternberg JC. Characterization of proteins by capillary electrophoresis in fused-silica columns: review on serum protein analysis and application to immunoassays. Electrophoresis. 1994;15: Clark R, Katzmann JA, Weigert E, et al. Rapid capillary electrophoretic analysis of human serum proteins: qualitative comparison with high-throughput agarose gel electrophoresis. JChromatorA. 1996;744: Wijnen PA, van Dieijen-Visser MP. Capillary electrophoresis of serum proteins: reproducibility, comparison with agarose gel electrophoresis and a review of the literature. Eur J Clin Chem Clin Biochem. 1996;34: Jenkins MA, Kulinskaya E, Martin HD, et al. J Chromatogr B BiomedAppl ;672; Am J Clin Pathol 1998; 110: Jenkins MA, Guerin MD. Quantification of serum proteins using capillary electrophoresis. Ann Clin Biochem. 1995; 32:493^ Jellum E, Dollekamp H, Blessum C. Capillary electrophoresis for clinical problem solving: analysis of urinary diagnostic metabolites and serum proteins. J Chromatogr B BiomedAppl. 1996;683: Jenkins MA, Guerin MD. Optimization of serum protein separation by capillary electrophoresis. Clin Chem. 1996;42: Cordova E, Gao J, Whitesides GM. Noncovalent polycationic coatings for capillaries in capillary electrophoresis of proteins. Anal Chem. 1997;69: Cowdrey G, Firth M, Firth G. Separation of cerebrospinal fluid proteins using capillary electrophoresis: a potential method for the diagnosis of neurological disorders. Electrophoresis. 1995;16: Jenkins MA, O'Leary TD, Guerin MD. Identification and quantitation of human urine proteins by capillary electrophoresis. J ChromatogrB BiomedAppl. 1995;662: Pearson JP, Keren DF. The effects of heparin on lipoproteins in high-resolution electrophoresis of serum. Am J Clin Pathol. 1995;104: