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Suspected Myeloma: Investigation of Paraproteinaemia

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Myeloma - Paraproteins

Myeloma is a condition that most often presents with symptoms related to bone pain, hypercalcaemia, renal disease, or infection. In about 10% of cases the disease is found by chance when an unexplained high ESR is investigated. The frequency of myeloma in the population is in the order of 50 per million and like many other malignancies it peaks in the latter decades; however, it can present in any age group. There is some evidence for an increased incidence in the Hunter Valley. Multiple myeloma results from the uncontrolled proliferation of plasma cells, and diagnosis is based on the triad of increased and abnormal plasma cells in the bone marrow, osteolytic lesions on skeletal x-ray, and detection of a paraprotein on EPG of serum and/or urine. If myeloma is suspected, the assessment of possible paraproteinaemia is an important early step.

The term "paraprotein", or abnormal protein, historically referred to the detection of an additional band on protein electrophoresis and has become synonymous with the production of monoclonal immunoglobulins. The paraprotein is characterised by identification of the heavy and light chain isotype of the immunoglobulin clone, which has prognostic significance. The most important clinical setting for the detection of a paraprotein is in the diagnosis of myeloma; however, paraproteins also occur in clinical settings other than myeloma (Table 1). The central question regarding any paraprotein is "is this paraprotein malignant?". While a number of clinical and laboratory parameters enable this question to be answered in the majority of situations, only progress assessment of all paraproteins will detect all malignant disease. Low levels of monoclonal paraprotein that occur in "Monoclonal Gammopathy of Uncertain Significance" (MGUS) will evolve into myeloma in 10% of patients. Paraprotein typing is the diagnostic criterion for the rare "Heavy Chain Disease", where the clone produces only heavy chains.

Electrophoresis

Serum electrophoresis (EPG) is a sensitive screening test for the detection of monoclonal immunoglobulin bands of greater than 0.5 g/L. Serum and urine EPG should be requested initially on all patients suspected of having myeloma, as 20% of myeloma patients secrete monoclonal light chains only, and the light chains will only appear in serum in the presence of renal dysfunction. A distinct narrow band may be visible on EPG (Figure 1), suggestive of a paraprotein. However, immunoglobulin clonality can only be confirmed by immunoelectrophoresis (IEP) or isoelectric focusing/immunofixation (IEF-IF). The serum EPG may also indicate immune paresis, and investigation for light-chain-only myeloma should be undertaken. Progress measurement of the paraprotein using EPG analysis is usually the best way to monitor progress of disease.

Figure 1: Serum Protein Electrophoresis.
Identification of Monoclonal Immunoglobulins

Immunoelectrophoresis

Immunoelectrophoresis (IEP) combines the principles of EPG with immunoprecipitation against specific antisera. Immunoprecipitation arcs formed against heavy and light chain specific antisera are used to characterise the paraprotein (Figure 2). IEP is also helpful in excluding other non-immunoglobulin bands such as haemoglobin (haemolysed sample) and fibrinogen (plasma sample). IEP is not a good screening test, as low concentration monoclonal bands may be missed, especially IgM and IgA paraproteins. Typing of the corresponding light chains and identification of free light chains can also be difficult by this method and may require clarification by isoelectric focusing.

Figure 2: Immunoelectrophoresis tying of monoclonal immunoglobulins
using specific antisera (IgG Kappa).

Isoelectric Focusing/Immunofixation

Isoelectric focusing (IEF) combined with immunofixation (IF) is used to clarify a monoclonal band when IEP results are inconclusive. IEF-IF is a highly sensitive test, detecting paraproteins as low as 0.01 g/L. Characteristic banding patterns occur for monoclonal paraproteins and can be used to differentiate monoclonal from oligoclonal and polyclonal banding patterns. Immunofixation with specific antisera allows identification of the abnormal band, serving to clarify clonality and to identify heavy and light chain typing (Figure 3). IEF-IF is not a good screening test as it can be difficult to differentiate between oligoclonal and monoclonal patterns without the aid of an EPG, especially for urinary free light chains.

EPG/IF on agarose is an alternative method to IEF-IF, but has lower sensitivity and specificity for determining clonality.

Figure 3: Isoelectricfocussing typing of monoclonal immunoglobulins
with immunofixation using specific antisera (IgG Lambda)

Immunoglobulins

Immunoglobulin quantitations can provide the first clue to a paraprotein disorder by revealing immune paresis. The baseline level of immunoglobulins is also useful for subsequent monitoring of the paraprotein level during treatment. The immunoglobulin level is a more accurate method for monitoring than the EPG paraprotein band estimate when the monoclonal band migrates in the beta or beta-gamma region, as is the case with most IgA paraproteins. However, extremely elevated levels of monoclonal immunoglobulins can not be accurately quantitated for the purposes of monitoring and in this situation the EPG paraprotein band estimate is more appropriate.

The measurement of immunoglobulins is also necessary for the laboratory preparation of appropriate serum dilutions to be used in the IEP and IEF-IF paraprotein typing. Knowing the immunoglobulin levels improves the turn around time for results as it reduces the number of repeat tests required.

Summary

An appropriate combination of these tests will establish the presence or absence of a monoclonal paraprotein with a high degree of certainty (Table 2). The best screening tests are serum and urine EPG. If any abnormality appears on the EPG, immunoglobulin quantitations and an IEP should be requested to further clarify clonality and exclude non-immunoglobulin bands. In some cases the laboratory will proceed to IEF-IF to accurately define the presence or absence of a paraprotein or to confirm the heavy and light chain typing. However, it should be remembered that a small proportion (~1%) of myeloma patients will have no detectable serum or urine paraproteins.

Patients classified as MGUS should be monitored by serum and urine EPG and immunoglobulin quantitation six-monthly for a period of two years and then annually, with skeletal x-ray survey and bone marrow examination if any doubt exists.

Testing Information

EPG, IEF-IF and immunoglobulins are assayed daily. IEP requires an overnight incubation and are assayed Monday-Thursday. Urine samples require concentration prior to testing and are usually processed for EPG and IEP the day following receipt in the laboratory.

EPGs and immunoglobulins are reported daily. Reporting of IEP and IEF-IF is a consultative process by senior scientific and medical staff. The confirmation of clonality and paraprotein typing usually takes 3-4 days for straightforward cases but may take 2-3 weeks for difficult cases.

Sample Requirements

It is essential that 2.0 mL of clotted serum is provided for these tests. Plasma, from anticoagulated blood, and haemolysis can interfere with the immunoglobulin quantitations and will produce artefacts on serum EPG. Heat treatment will denature the proteins, making samples unsuitable for these tests.

A spot urine specimen or a 24 hour urine collection, collected without any preservatives, are both suitable for the investigation of paraproteins. A 24 hour urine collection estimate of the paraprotein level is more suitable for monitoring treatment.

References

1. Longo DL. Plasma cell disorders. In: Harrison's Principles of Internal Medicine. Eds. Wilson JD et al 12th Edition 1991: p1410-1417.
2. Whicher JT et al. The laboratory investigation of paraproteinaemia. Ann Clin Biochem 1987:24:119-132.
3. Young B and Gleeson M. Audit of investigation and follow-up of patinets with raised monoclonal proteins. Aust Clin Rev 1989:8:217-220.

Written by:                     Glenn Reeves, Maree Gleeson, Robert Clancy

Last Reviewed:             10.5.2001