Clinical Chemistry
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Clinical Chemistry 44: 760-764, 1998;
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(Clinical Chemistry. 1998;44:760-764.)
© 1998 American Association for Clinical Chemistry, Inc.

Enzymes and Protein Markers

Detection and classification of paraproteins by capillary immunofixation/subtraction

Xavier Bossuyta, Ann Bogaerts, Gilberte Schiettekatte, and Norbert Blanckaert

a Author for correspondence. Fax 32 16 332896; e-mail xavier.bossuyt{at}uz.kuleuven.ac.be.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results and Discussion
 References
 
A selection of 58 specimens with a monoclonal component identified by immunoelectrophoresis and/or immunofixation was analyzed with the immunosubtraction procedure on the Paragon 2000 capillary electrophoresis system. The capillary system detected 93% of the paraproteins and, using immunosubtraction, correctly identified 91% of the paraproteins. Paraproteins that were detected by immunofixation and/or immunoelectrophoresis but not by capillary electrophoresis were also missed by agarose electrophoresis and cellulose acetate electrophoresis. Cellulose acetate electrophoresis was the least sensitive method for detection of paraproteins. Only 74% of the monoclonal components were detected by this technique, whereas 86% were revealed by agarose electrophoresis. In addition to monoclonal paraproteins, we also studied biclonal paraproteins and oligoclonal banding. Capillary electrophoresis and immunosubtraction correctly detected and identified three specimens containing biclonal paraproteins. In one specimen, capillary zone electrophoresis detected only one band, whereas agarose gel electrophoresis detected two bands. The sensitivity for detection and identification of oligoclonal banding by capillary electrophoresis was inferior to immunofixation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results and Discussion
 References
 
Detection and classification of paraproteins is important for the diagnosis of multiple myeloma, Waldenström's disease, and monoclonal gammopathy of undetermined severity. The latter condition occurs in >3% of the population 70 years or older (1). Paraproteins are classically detected by agarose gel electrophoresis (AGE)1 or cellulose acetate electrophoresis (CAE) and appear as spikes on the serum protein profile. Correct classification is important for diagnosis and treatment and is accomplished by immunoelectrophoresis or immunofixation, both of which are established methods. Both techniques, however, are technically demanding, and immunoelectrophoresis is especially lengthy. Immunofixation has proven to be the most sensitive method (2)(3)(4).

Over the last few years, capillary zone electrophoresis (CZE) has emerged as a novel technique for the rapid and effective separation of serum proteins (see accompanying paper) (5)(6)(7)(8)(9). A process similar to immunofixation but adapted for capillary electrophoresis systems has recently been developed (10). This process, called immunofixation electrophoresis/subtraction (IF-ES), involves incubation of patient serum with Sepharose beads to which a specific binder for a heavy (IgG, IgA, or IgM) or a light ({kappa} and {lambda}) chain is attached. During the incubation period, proteins that bind specifically to the bead-linked antibodies are retained by the solid phase. After the beads settle, capillary electrophoresis is performed on the supernatant. The monoclonal protein underlying the abnormal peak is identified by comparing the subtracted capillary electropherogram with the control electropherogram (incubated with solid phase without linked antibody). The specificity of the immunospecific binder that caused the disappearance of the monoclonal peak identifies the paraprotein.

Recently, a multichannel automated system for CZE of human serum proteins (Paragon 2000 clinical capillary electrophoresis system, Beckman Instruments) became commercially available. The system is designed for routine analysis in clinical laboratories and allows rapid, reproducible, and sensitive separations. The system is also available with an automated IF-ES-based procedure for classifying paraproteins. We compared the Paragon 2000 CZE with AGE and CAE for the detection and identification of paraproteins. To gauge the capacity of the IF-ES procedure to classify paraproteins, the procedure developed for the Paragon 2000 CZE system was compared with immunofixation and/or immunoelectrophoresis, which are considered reference methods.


   Materials and Methods
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 Abstract
 Introduction
 Materials and Methods
Results and Discussion
 References
 
electrophoretic methods
For conventional AGE, we used the Paragon SPE kit (Beckman Instruments) according to the manufacturer's instructions. CAE was performed on Sepharose cellulose polyacetate electrophoresis strips for the Microzone system (Gelman Sciences). The gels were equilibrated in diethylbarbital equilibration buffer, pH 8.6. We applied approximately 1 µL of each sample and carried out the electrophoresis at 220 V for 25 min in equilibration buffer. Proteins were stained by incubating the gels in Ponceau S (Analis) for 10 min. The individual fractions were quantified by densitometry (Beckman Appraise, Beckman Instruments). CZE and IF-ES were performed with the Paragon CZE(TM) 2000 clinical capillary electrophoresis system (Beckman Instruments). Proteins were measured by direct absorption at 214 nm through a small optical window in the capillary. The fused-silica capillaries were 20 cm long x 20 µm (i.d.). Instrument settings for serum protein electrophoresis and IF-ES were as follows: 1 min conditioning time, 1 s injection time, 5 min separation time, 0.5 min wash time, 0.5 min rinse time, 8000 V, and 24 °C. Sample dilution was 1:2, 1:7, or 1:15. The sample buffer was a borate buffer, and the capillary rinse between samples was with <1% NaOH cleaning solution (Beckman Instruments). The software used was Paragon, Ver. 1.08 (Beckman Instruments). For conventional immunofixation, the Paragon IFE kit (Beckman Instruments) was used. Detection of monoclonal bands was by direct inspection of the gels. Immunoelectrophoresis was performed as described by Grabar and Williams (11), using barbital buffer, pH 8.6, and antibodies from Sanofi Diagnostics Pasteur. The gels were from Kallestad Laboratories.

other analyses
IgG, IgA, IgM, and {lambda}- and {kappa}-light chains were determined by endpoint nephelometry on a Behring BNA instrument (Behringwerke). All reagents and calibrators were from Behring (Behringwerke).


   Results and Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
References
 
Specimens with monoclonal components that had been detected and characterized by immunoelectrophoresis and/or immunofixation were analyzed by the immunosubtraction procedure on the Paragon 2000 CZE System. The distribution of the monoclonal components used for the study is shown in Table 1 . This distribution does not follow the distribution usually seen in the clinical laboratory. The group encompassed 5 specimens from patients with light chain disease (4 {lambda} and 1 {kappa}), 9 with IgA (3 IgA{kappa} and 6 IgA{lambda}) monoclonal paraproteins, 40 with IgG (25 IgG{kappa} and 15 IgG{lambda}) monoclonal paraproteins, and 4 with IgM (3 IgM{kappa} and 1 IgM{lambda}) monoclonal paraproteins. In addition to the monoclonal proteins, free monoclonal light chains were present in 1, 3, and 12 specimens of the IgM, IgA, and IgG paraprotein groups, respectively.


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  		Table 1. Comparison of methods for detection and identification of paraproteins.

The results of the method comparison between CZE and conventional electrophoresis for paraprotein detection and identification is summarized in Table 1Up . Nephelometric quantitation was performed on each sample. Although such quantitations do not distinguish between monoclonal and polyclonal immunoglobulins, they allow the division of a group of known paraproteins into subgroups and provide an estimation of the amount of paraprotein in individual cases. Overall detection of paraproteins was 93% for CZE, 86% for AGE, and 74.5% for CAE. Correct identification by IF-ES was achieved in 91% of the cases studied. Discrepancies between the different techniques pertained to: (a) the detection of "hidden" paraproteins, i.e., paraproteins that cannot be detected by conventional CAE and AGE, but that can be revealed by immunoelectrophoresis and/or immunofixation; (b) the detection in serum of free monoclonal light chains of the same type as the characterized paraprotein; and (c) the electrophoretic migration of the paraproteins. For each paraprotein group, discordances between the different techniques are reviewed below.

The IgA paraprotein series consisted of nine specimens. The group included two specimens with hidden paraproteins not detected by either CAE or AGE. Both paraproteins were small paraproteins, as illustrated by the nephelometric quantitation, that showed <5 g/L. One of these paraproteins was unambiguously detected and identified by CZE. Immunoelectrophoresis revealed the presence of free monoclonal light chains in three of the nine specimens. AGE and CAE failed to detect these free light chains. With CZE, the free light chains were suggested in one specimen. In this specimen, incubation with the light chain binder, but not with the heavy chain binder, caused the complete disappearance of the monoclonal peak (data not shown). In another specimen, we found that the electrophoretic migration of the IgA paraprotein differed between CZE and the traditional electrophoretic techniques. In CZE, the monoclonal protein migrated between transferrin and the {gamma}-globulins (Fig. 1 C), whereas it migrated between the {alpha}1-globulin and {alpha}2-globulin fraction with AGE and CAE (Fig. 1 , A and B).



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  		Figure 1. Electropherograms from a patient with an IgA paraprotein.

The electropherograms obtained with CAE, AGE, and CZE are represented by A, B, and C, respectively. The arrow indicates the position of the monoclonal protein located between the {alpha}1-globulin and {alpha}2-globulin fractions in AGE and CAE (A and B) and between transferrin and the {gamma}-globulins with CZE (C).

Forty specimens containing IgG paraproteins were studied. The group included two hidden paraprotein samples that were not detected by CZE, AGE, or CAE. Because these paraproteins could not be identified by CZE, they could not be characterized by IF-ES. In four other specimens, a small monoclonal band that was obvious with CZE and AGE could not be unambiguously detected by CAE. This shows that CZE and AGE are more sensitive methods than CAE for detection of monoclonal bands. In another specimen, the paraprotein could be detected with CZE, but the light chain type could not be identified with the immunosubtraction technique. In 12 of the 40 IgG monoclonal paraprotein specimens studied, immunoelectrophoresis revealed the presence of free monoclonal light chains. With CZE, these free light chains were detected in only one specimen. In this sample, the electropherogram showed a second small peak (Fig. 2 A, arrow) that disappeared after incubation with the light chain binder (Fig. 2C ) but not after the incubation with the heavy chain binder (Fig. 2B , arrow).



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  		Figure 2. CZE and IF-ES electropherograms from a patient with an IgG monoclonal component and excess free light chains in serum.

A, control electropherogram; B, electropherogram after incubation with the IgG binder; C, electropherogram after incubation with the light chain binder. The arrow indicates the free light chains, which can be noted in the control (A) and the electropherogram after incubation with the IgG binder (B) but not in the electropherogram after incubation with the light chain binder (C).

The IgM paraproteins studied in all four specimens studied could be detected and identified by CZE. CAE failed to detect paraproteins in two specimens. In one specimen, additional free light chains were present. These free light chains could not be detected by CZE, AGE, or CAE.

CZE could not detect or identify four of the five light chain diseases that were studied. A representative CZE electropherogram is shown in Fig. 3 . It shows an abnormally high peak at the complement position (A) that disappeared after incubation with the light chain binder (B). Immunoelectrophoresis confirmed the presence of light chains. AGE detected light chains in only one of these four specimens. None of the light chain diseases was detected by CAE. These findings suggest that CZE is more sensitive than AGE for the detection of light chain disease.



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  		Figure 3. CZE and IF-ES electropherograms from a patient with light chain disease.

A, control electropherogram; B, electropherogram obtained after incubation with the light chain binder. The arrow in A indicates the monoclonal light chains that disappear after incubation with the light chain binder.

In addition to monoclonal paraproteins, we also investigated four biclonal disease specimens. Three biclonal paraproteins (an IgG{lambda} in combination with an IgA{kappa}, an IgG{lambda} in combination with an IgM{lambda}, and an IgG{kappa} in combination with an IgA{kappa}) were detected and correctly identified by CZE (data not shown). In one specimen, AGE detected two bands, immunofixation detected two IgG{kappa} bands, and CZE/IF-ES detected only one IgG{kappa} peak. The CZE electropherogram of this specimen is shown in Fig. 4 , and the immunofixation is shown in Fig. 5 .



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  		Figure 4. CZE electropherogram from a patient with a monoclonal gammopathy.

A, control electropherogram; B, electropherogram after incubation with the IgG heavy chain binder; C, electropherogram obtained after incubation with the {kappa}-light chain binder. The arrow in A indicates the monoclonal protein that disappeared after incubation with the heavy and light chain binder.



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  		Figure 5. Immunofixation of the specimen presented in Fig. 4Up .

The sample was applied to six different lanes on an agarose gel, and proteins were separated by electrophoresis. Monospecific antiserum was then applied to five of the electrophoresis patterns (IgG, IgA, IgM, {kappa}, and {lambda}). After fixation, the electrophoresis gel was washed, and the proteins were stained. The immunofixation revealed a biclonal IgG{kappa} paraprotein.

Detection and identification of oligoclonal banding was evaluated in three samples. One sample had three bands on AGE but only one, unidentifiable band on CZE. In two other specimens, CZE detected and identified three bands, whereas immunofixation revealed and characterized four small bands.

We have also compared paraprotein detection by CZE and CAE in 524 specimens from hospitalized patients. Of the 524 specimens, 50 monoclonal components were detected by both electrophoretic methods. CZE revealed 10 additional patients with an M-component that were missed by CAE. This group included patients with non-Hodgkin's lymphoma (two patients), Hodgkin's lymphoma (two patients), Still's disease (one patient), infections (three patients), and cardiovascular problems (two patients, >68 years of age). This again illustrates that CAE is less sensitive than CZE for the detection of faint monoclonal bands.

The performance of CZE for paraprotein screening is superior to the performance of CAE and comparable with the performance of AGE. CZE, therefore, constitutes a good alternative to AGE because it has the advantages of automation and high precision (see accompanying paper) (5).

Paraproteins detected by CZE were correctly classified as IgG, IgA, IgM, {kappa}, or {lambda} by the IF-ES procedure. No reagents are available for identification of the rare cases of monoclonal IgD and IgE by the IF-ES procedure. A small percentage of faint monoclonal bands was missed by CZE. These bands were also missed by AGE and CAE and were revealed only by immunofixation. The latter technique remains the most sensitive (reference) method. Although small monoclonal components are commonly part of a monoclonal gammopathy of undetermined significance or reflect the presence of an infectious disease, they may also be part of clinically important processes such as B-cell lymphoma, leukemia, chemical immunosuppression, or myeloma (light-chain disease). Early detection of malignant B-cell proliferation requires the greatest sensitivity. Small monoclonal components can also be the cause of amyloidosis or polyneuropathy, or they may be the clue to autoimmune or immune complex diseases (12). Therefore, in situations involving clinical suspicion, immunofixation is the method of choice because a small monoclonal band can hide in normal bands.

CZE also missed the presence in serum of free monoclonal light chains of the same type as of the characterized paraprotein. However, the detection of these free light chains as an indicator of clinical activity, organ involvement, or relapse of disease is of no value (13). Moreover, the preferred means of detecting free light chains is to analyze urine rather than serum samples (14). Therefore, a failure to detect free light chains in serum is not clinically relevant. It should be pointed out, however, that the Paragon 2000 cannot be used for urine analysis at the present time, and that an immunofixation method must be maintained for the analysis of urine specimens.

The sample incubation and running temperature in the Paragon 2000 was 24 °C. Therefore, monoclonal cryoglobulins that precipitate at temperatures >24 °C might be missed. Presently, there is no means of handling samples to avoid the loss of cryoglobulins when the Paragon 2000 is used.

Introduction of the automated CZE for paraprotein detection and identification in the clinical laboratory will result in labor savings and improved turnaround time, especially when CZE replaces immunoelectrophoresis. When equipped with a bar code reader and a bidirectional interface, CZE allows positive sample identification, thus avoiding sample identification errors in the laboratory.


   Acknowledgments
 
We acknowledge M. Artoos and H. Raveschot for expert technical assistance. We thank G. Marien and E. Stevens of the Laboratory of Immunology UZ Leuven for their contribution in the generation of the nephelometric, immunoelectrophoresis, and immunofixation data. We thank Analis for providing the instrumentation and reagents for CZE and AGE.


   Footnotes
 
Central Clinical Laboratory, Department of Clinical Pathology, University Hospital of Leuven, Kapucijnenvoer 33, B-3000 Leuven, Belgium.

1 Nonstandard abbreviations: AGE, agarose gel electrophoresis; CAE, cellulose acetate electrophoresis; CZE, capillary zone electrophoresis; and IF-ES, immunofixation electrophoresis/subtraction.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results and Discussion
 References
 

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  12. Aguzzi F, Merlini G, Whicher JT, Chir B. Serum monoclonal components. In: Ritchie RF, Navolotskaia O, eds. Serum proteins in clinical medicine, 1st ed. Vol. I. Laboratory section. Brea, CA: Beckman Instruments and Scarborough, MA: Foundation for Blood Research, 1996:11.03-1–03-12..
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