Atypical findings of protein studies using capillary zone electrophoresis

Atypical findings of protein studies using capillary zone electrophoresis

Cutlan, Robert T

ABBREVIATIONS: CZE = capillary zone electrophoresis; HRE = high-resolution agarose gel electrophoresis.

INDEX TERMS: Beta-lipoproteins; capillary zone electrophoresis; high resolution electrophoresis; immunofixation; monoclonal gammopathy.

Clin Lab Sci 1999;12(6):325

We present two atypical cases of protein studies that were evaluated by immunofixation and immunosubtraction using capillary zone electrophoresis and high-resolution agarose gel electrophoresis. The first study showed an abnormal peak in the beta region by capillary zone electrophoresis that was located in the gamma region of the high-resolution agarose gel electrophoresis. Further investigation showed that this monoclonal protein was displaced due to binding with beta-lipoproteins. In the second case, a large peak was detected in the alpha-2 region and was shown by capillary zone electrophoresis to be a non-proteinaceous material that mimicked a paraprotein.

Capillary zone electrophoresis (CZE) has become a commonly used method to evaluate human serum proteins using a narrow-bore uncoated fused silica capillary. The proteins are separated by differences in their electrophoretic mobility, enhanced by capillary chromatography technology. At alkaline pH most proteins are negatively charged and their migration is based on their isoelectric point, tertiary structure, charge to mass ratio at specific voltage, electrolyte composition, and pH. The negatively charged silica surface and the positively charged ions in the buffer along with applied voltage create electro-osmotic flow that carries cations toward the cathode. When high voltage is applied the electro-osmotic flow exceeds the electrophoretic mobility of the proteins and carries them toward the cathode. As proteins elute off the column, their absorbance at 214 run is measured and an electropherogram is produced, showing the amount of protein versus migration time. This method allows the clinical laboratorian to detect monoclonal gammopathies or other pathophysiological states associated with various protein quantitations.

Identification of the monoclonal paraprotein is accomplished by incubating the patient’s serum with SepharoseTM beads that have either antibodies against IgG, IgA, IgM, Kappa, or Lambda light chains attached. This is called immunosubtraction. The proteins bind specifically to the solid phase and the supernatant is run on the CZE. These electropherograms are compared with the one run without incubation with the solid phase. The disappearance of the peak on an electropherogram incubated with a specific antibody identifies the type of paraprotein.

CZE has been shown to have greater resolution compared with densitometric scans of traditional agarose gel electrophoresis, resulting in greater sensitivity.’,The increase in resolution has led to the clinical assessment of the two major components of the beta region, transferrin and complement.’ The increase in the sensitivity along with the infancy of this technology has also led to the detection of new artifacts. Bilirubin, hemoglobin, triglycerides, and fibrinogen have been previously reported not to cause interference.4 However, we recently identified two aberrant serum protein studies which required further investigation and are presented as case studies.

CASE 1

A 70-year-old African American man diagnosed with multiple myeloma two and one-half years ago was being treated with cyclic chemotherapy and followed with periodic serum protein electrophoresis. Initial studies on gel electrophoresis had revealed a monoclonal gammopathy, which had been defined as IgG Kappa by immunofixation studies. Serum for protein electrophoresis was submitted to the clinical laboratory for testing. In this evaluation, an abnormal peak appeared in the beta region of the electropherogram using CZE (Beckman Coulter, Brea CA) (Figure 1). However, high-resolution agarose gel electrophoresis (HRE) (Beckman Coulter) showed that the paraprotein was located in the gamma region (Figure 2). Serum was further evaluated and the peak clearly shown to be IgG Kappa by immunofixation and immunosubtraction by CZE. Another six-month follow-up study revealed similar results. Based on the previous reports on lipoprotein binding, it was determined that this shifting of the paraprotein from the gamma region in the FIRE to the beta region with CZE was due to changes in protein charge with the beta-lipoprotein binding. The presence of the peak in the beta region of the CZE electropherogram therefore reflected an artifact of such binding.

Immunoglobulin binding to lipoproteins has been well documented. Multiple myeloma has been associated with xanthomatosis.5 The monoclonal paraprotein binds to lipoproteins, forming immune complexes which interfere with lipid transport and accumulate in dermal macrophages, forming foam cells. Patients may have hyperlipidemia or normal lipidemia.7 Immunoglobulin binding to lipoproteins has also been associated with an increased risk for atherosclerosis. Lipoproteins can be modified to be immunogenic. One example of this is seen in patients with diabetes who undergo glycation of lipoproteins. These lipoproteins are susceptible to oxidation and binding to immunoglobulins. These immune complexes bind to the Fc receptor located on macrophages resulting in the formation of foam cells and the release of other proatherogenic factors.” The patient presented did have hyperlipidemia, but did not have clinical evidence of xanthomatosis.

CASE 2

A 47-year-old African American woman presented to the hospital for the evaluation of ischemic rest pain in her right foot. Her past medical history was significant for severe peripheral vascular and coronary artery disease, several strokes, non-insulin dependent diabetes, and hypercoagulable syndrome. The day after admission she developed acute chest pain and had emergent coronary artery angioplasty. Her post-operative course was complicated with an acute intracerebral bleed and gastrointestinal bleeding. She continued to deteriorate despite receiving maximal clinical support in an intensive care setting. While in the intensive care unit the patient received numerous medications including antibiotics and vasopressors, and also CT contrast material. She expired six days following her admission.

A serum sample for serum protein immunoelectrophoresis was obtained several days prior to death. This sample was evaluated for monoclonal proteins by immunofixation and immunosubtraction with CZE. As shown in the original CZE protein study, a large, well-defined peak was seen in the alpha-2 region of the electropherogram (Figure 3). Immunosubtraction studies using the CZE showed that the peak was not diminished by treatment with antibody-coated beads directed against IgG, IgM, IgA, Kappa, or Lambda (Figure 4). A sample of the serum was then desalted and reanalyzed (Figure 5). The removal of the large peak following desalting along with its absence on agarose gel electrophoresis is evidence for a non-proteinaceous material. The non-proteinaceous material must have a charge compatible with that of the alpha-2 proteins and an absorbance at 214 nm, since it appears in this region. In all likelihood, the sample was obtained shortly after a bolus of radiological contrast media or other medication was given intravenously. Unfortunately, a follow-up sample was not available for evaluation since the patient expired.

The use of CZE presents an automated mechanism for a procedure that has become commonplace in the clinical laboratory and provides a semiautomated mechanism for detecting serum protein abnormalities. However, new artifacts not usually seen by standard cellulose acetate, agarose gel, or high-resolution electrophoresis are being observed. The two cases presented show that variations of artifacts can be determined readily and that CZE continues to be an improvement over current methods of protein analysis 2. As these new methods are placed in the laboratory, artifacts and aberrations need to be shared with others to optimize use of the methods. HRE along with desalting procedures can be used to help in the determination of unknown peaks. Desalting cartridges and columns are available commercially, the former being convenient for single samples.

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5 Taylor JS, Lewis LA, Battle JD,and others. Plane xanthoma and multiple myeloma with lipoprotein-paraprotein complexing. Arch Dermatol 1978;114:425-31.

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7. Buezo GF, Porras JI, Fraga J, and others. Coexistence of diffuse plane normolipaemic xanthoma and amyloidosis in a patient with monoclonal gammopathy. Br J Dermatol 1996;135:460-2.

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The peer-reviewed Clinical Practice section seeks to publish case studies, reports, and articles that are immediately useful, of practical nature, or demonstrate improvement in the quality of laboratory care. Direct all inquiries to Susan Leclair CLS Clinical Practice Editor Department ofMedical Laboratory Science, University ofMassachusetts Dartmouth, 285 Old Westport Road, Dartmouth MA 027472300; or send e-mail to sleclair@umassd. edu

Robert T Cutlan MD is Chief Resident, Department of Pathology, University of Tennessee, Memphis TN.

David L Smalley PhD is Director, Diagnostic Immunology, Baptist Regional Laboratories, and Professor in the Department of Pathology, University of Tennessee, Memphis TN.

Robin P Mayer is a Supervisor in Diagnostic Immunology at Baptist Regional Laboratories, Memphis TN.

Jerald E Mullersman MD PhD is an Associate Professor in the Department of Patholop University of Tennessee, Memphis TN. Cynthia R Blessum is a Technical Specialist for Beckman Coulter Inc, Brea CA.

Address for correspondence: Dr David L Smalley Baptist Regional

Laboratories, 22 N Pauline Street, Memphis TN38105. (901) 2277645, (901) 227-8477 (fax). david smalley@bmhcc.org

Copyright American Society for Clinical Laboratory Science Nov/Dec 1999

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