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Electrophoresis may be defined as the separation of charged particles in a uniform electric field. For a particular system of electrophoresis, the voltage is held constant as are the pH and ionic strength of the suspending medium.
Tiselius, using a moving boundary liquid system, separated serum proteins by electrophoresis into four components in 1937.1 Paper electrophoresis, popular in the 1950s, provided the rst solid electrophoresis support. The fragility of paper as a support medium saw the introduction of the more robust cellulose acetate a decade later. An improvement in resolution was subsequently gained by using agarose gel, which, in serum samples, gave 5 bands of separation.2,3 In the late 1980s, high resolution agarose gels were introduced which produced at least 6 bands, and depending on the system, as many as 17 bands in serum.4,5
Fully automated serum electrophoresis commenced in the 1990s with the introduction of capillary electrophoresis (CE), a reintroduction of a liquid medium but with exquisite resolution compared to Tiselius’ procedure. Using CE instrumentation it is possible to program a sequence of samples and leave them overnight to be processed.
Amalgamation of laboratories with an increasing number of patient samples was probably the reason for the semi-automation of gel electrophoresis. The introduction of the Helena SPIFE and Sebia Hydrasys gel systems provided ways of electrophoresing over a hundred serum samples per day. There is certainly a role for such instrumentation in electrophoresis laboratories today.
The primary reason for performing serum protein electrophoresis is to discover a paraprotein or B cell dyscrasia. An irregularity in the gamma region can be due to a small monoclonal band, free light chains or oligoclonal IgG. Other findings of clinical significance include increased alpha-1 and alpha-2 globulins indicative of an acute phase response, a decrease in alpha-1 globulins suggestive of alpha-1 antitrypsin (A1AT) deficiency (that can be followed up with phenotyping to check for a clinically significant A1AT variant), an increase in the beta-1 region suggestive of increased transferrin and iron deficiency, a polyclonal increase in gamma globulins indicative of in ammation or infection or of liver disease.
The main reason for performing urine protein electrophoresis is to find a light chain myeloma producing an excess of free light chains (Bence Jones protein), an important part of a myeloma screen. A band in the urine protein electropherogram may also result from an intact monoclonal immunoglobulin, especially if the patient has poor renal function. Immunofixation is important in defining the nature of the band and in distinguishing between Bence Jones protein and an intact monoclonal protein originating from the serum. From the urine electropherogram we can also tell if the proteinuria is of glomerular origin with a predominance of albumin, or if it has tubular components with excretion of smaller molecular weight proteins such as retinol binding protein and alpha-1 microglobulin. Fragmented albumin in urine is occasionally seen but is of unknown significance.6
Historically, urine has been concentrated by either removal of water from the specimen leaving the proteins in higher concentration, or by centrifugation whereby the proteins are spun away from the majority of the water. Demonstration of the protein components of urine from concentrated specimens was originally performed on cellulose acetate and later on agarose and high-resolution agarose gel. The use of CE for urine analysis has not been accomplished to date by instrument manufacturers such as Beckman or Sebia, although Sebia have promoted a method which involves dialysis followed by a centrifugation step. An alternative urine protein method using CE has been published.7
High-resolution agarose gel electrophoresis whether commercial or in-house has been used routinely for over 20 years and with serum delivers a separation of between 6 and 17 bands. The technique has been shown to give reproducible quantification of monoclonal bands provided the buffer pH, voltage and type of stain are meticulously reproduced. Immunofixation of high-resolution gels has been successfully used for demonstrating low-level monoclonal IgA and IgM paraproteins. High-resolution agarose gel electrophoresis together with immunoblotting can also be used to separate the various isoforms of transferrin such as asialotransferrin or beta-2-transferrin. This is important in the detection of such proteins in CSF and in other leakage fluid samples.
The separation of serum proteins by CE was first demonstrated in the early 1990s. CE is a technique that gives excellent separation of serum proteins while reducing the hands-on time required by means of automation.
With CE, the pH of the buffer used must be constant for a particular system whether a turnkey commercial or research instrument is used. The applied voltage of CE of between 8 and 17 kV is very much higher than the 250–400 V used with agarose gels (Table 1). Use of wavelengths in the far UV to detect light absorption by peptide bonds avoids variable staining of proteins with conventional detection techniques (Table 1). In this regard, it has been demonstrated that the sensitivity of detection at 200 nm is three times that at 215 nm.
Electro-osmotic flow is an all-important phenomenon in CE. If the buffer is above pH 2, the internal surface of the fused silica capillary is negatively charged due to exposed silanol ions. In an electric field, hydrated cations in the diffuse double-layer adjacent to the silica wall migrate towards the cathode, dragging solvent with them. This is termed electro-osmotic flow. The order of migration of proteins past the detector will reflect the balance between the electrophoretic and electro-osmotic forces within the capillary. By adjusting the pH of the buffer, electro-osmotic flow can, in principle, either enhance or oppose electrophoretic migration. In analysis of serum proteins, the pH used is markedly alkaline (Table 1) and the anodal electrophoretic migration is dominated by cathodal electro-endosmosis.
Immunosubtraction, a concept proposed originally by Aguzzi and Poggi,8 is used in CE in an analogous way to immunofixation on agarose gels - it enables the identification of paraprotein found in serum by using immunoglobulin-specific antibodies. By having the antibodies attached to solid supports such as beads, any component binding to the antibody will become attached to the support and removed from solution. If the paraprotein seen in CE is an IgG(kappa) for example, binding (and removal) of the paraprotein with IgG and kappa antibodies will cause a diminution of the band, whereas antibodies to IgA, IgM and lambda components will show no such effect.
There are several techniques, which can be used in conjunction with serum and urine electrophoresis in determining the size and identity of the plasma cell dyscrasia which the patient may have. These techniques include isoelectric focusing of the patient’s serum and urine,9 immunofixation, either of serum protein electrophoresis to identify particularly low level IgA and IgM bands, or of isoelectric focusing, to distinguish between a monoclonal protein, a prominent IgG(kappa) or IgG(lambda) clone, oligoclonal IgG or free light chains. The resolution of immunofixation of isoelectric focusing is approximately five times better than the resolution of immunofixation of electrophoresis. There is also a paper in this issue by FN Cornell on these complementary techniques. Comparison of the techniques of capillary electrophoresis, high resolution electrophoresis and isoelectric focusing are shown in Figures 1–4.
Quantification of the paraprotein in the patient’s serum should be from the electropherogram if a CE method is used, or by densitometric analysis if gel electrophoresis is used. Somewhat arbitrarily, myeloma is diagnosed by the finding of a monoclonal IgG of more than 20 g/L or a monoclonal IgA of more than 10 g/L. Patients with monoclonal bands of lower concentration may be classed as MGUS (monoclonal gammopathy of undetermined significance). A monoclonal IgM is usually indicative of Waldenström’s macroglobulinaemia, and only in rare cases does it indicate myeloma.
Quantification of immunoglobulins IgG, IgA or IgM, by either immunonephelometric or immunoturbidometric methods, will show if the patient’s residual gamma globulins are diminished or within the reference interval.10 When there is a proliferation of a monoclonal protein, a decrease in the residual gamma globulins is significant.
In the past three years, quantification of free kappa and free lambda light chains using the Free Lite™ kit from The Binding Site (Birmingham, UK) has taken the detection of light chains in serum down to the mg/L range. As a result, the ratio of free kappa to free lambda has a defined reference interval. Results outside this interval suggest an increased amount of free light chains in the patient’s circulation and that further haematology investigations may be required. Quantification of light chains forms part of a protein work-up on a patient suspected of myeloma and is ideally reported together with the patient’s serum and urine protein electrophoresis results.
After working in a laboratory for a number of years I have observed an increasing complexity of monoclonal bands in patients’ sera. Ten years ago we usually found only one monoclonal band, whereas recently we have been finding an increasing number of patients with two monoclonal bands with differing heavy chains, or three or four monoclonal bands, often with differing heavy and light chain types. Whether this finding originates from environmental factors, genetic factors or better detection is yet to be determined. From a laboratory perspective this should be kept in mind when examining the serum of a patient for myeloma.
The techniques used for serum and urine protein electrophoresis have improved significantly in both detection and resolution during the past 70 years. The more sophisticated techniques of isoelectric focusing, immunofixation and quantification of immunoglobulins are important in a work-up of a patient suspected of myeloma. Assay of serum free light chains provides an additional tool which can assist the laboratory in this process.
Competing Interests: None declared.