What is immunofixation?

What is Immunofixation Electrophoresis (IFE)?

Immunofixation electrophoresis is a laboratory technique that identifies and characterizes specific proteins—particularly monoclonal immunoglobulins—by combining electrophoretic separation of proteins with immunoprecipitation using specific antisera against immunoglobulin heavy chains (IgG, IgA, IgM) and light chains (κ or λ). 1

Technical Methodology

• IFE separates proteins by electrophoresis, then applies specific antibodies directly onto the gel to precipitate and identify the target proteins in situ. 2, 3
• The technique works by layering antiserum over the agarose gel immediately after electrophoretic separation, resulting in antigen-antibody precipitation that creates visible bands. 4
• IFE can be performed on serum, concentrated urine specimens (typically from 24-hour collections), or other low-viscosity body fluids. 1, 2

Clinical Applications and Diagnostic Utility

Superior Sensitivity to Standard Electrophoresis

• IFE is significantly more sensitive than standard protein electrophoresis (SPEP) and is essential for detecting small amounts of monoclonal immunoglobulin that may be missed by electrophoresis alone. 1, 5
• In a recent study, IFE achieved 100% detection of monoclonal gammopathies, whereas SPEP identified only 69.6% of cases. 5
• IFE detected and identified paraproteins in cases where immunoelectrophoresis failed, with no monoclonal bands observed by electrophoresis or immunoelectrophoresis that were not detected by IFE. 4

Essential Diagnostic Functions

• IFE is necessary for definitive identification and typing of monoclonal immunoglobulins, determining complete response to treatment, and confirming the presence of monoclonal proteins when SPEP shows abnormal patterns. 1
• The test identifies which specific immunoglobulin heavy chain (IgG, IgA, or IgM) and light chain (κ or λ) is being produced by the clonal plasma cells. 1, 6
• Both serum and urine immunofixation should be performed because this combined approach is more sensitive than protein electrophoresis alone for detecting monoclonal proteins. 1

Specific Clinical Contexts

Multiple Myeloma and Plasma Cell Disorders

• IFE is a fundamental component of the diagnostic workup for multiple myeloma, required to confirm the presence and type of monoclonal protein. 1, 7, 6
• Serum immunofixation may be more helpful than serum free light-chain assays in diseases associated with an intact monoclonal immunoglobulin, such as proliferative glomerulonephritis with monoclonal immunoglobulin deposits (PGNMID). 1
• IFE should be performed even if there is no measurable protein and even if there is no peak on urine electrophoresis, as some cases produce only small amounts of monoclonal protein. 1

Monoclonal Gammopathy of Renal Significance (MGRS)

• Immunofixation of both serum and concentrated urine from a 24-hour collection is essential for evaluating MGRS because the culprit monoclonal immunoglobulin detected must match that found in kidney tissue. 1
• The test is critical for diagnosis, prognostication, and response assessment in patients with kidney disease related to monoclonal proteins. 1

Peripheral Neuropathy Evaluation

• Serum protein immunofixation electrophoresis has the highest yield among screening tests for identifying monoclonal gammopathies in patients with distal symmetric polyneuropathy of unknown etiology. 1
• IFE is more sensitive than standard serum protein electrophoresis (SPEP), especially for detecting small or non-malignant monoclonal gammopathies—identifying 30% of IgM monoclonal gammopathies under 5 g/L that SPEP missed. 1

Important Technical Considerations

Specimen Requirements

• For serum testing, the sample can be analyzed directly, but proper dilution is critical—the optimal paraprotein concentration for immunofixation is 28 to 35 g/dL. 4
• For urine testing, a concentrated aliquot from a 24-hour collection must be used; random urine samples with analytes corrected relative to creatinine concentration cannot replace 24-hour collections. 1
• Urine can be concentrated by membrane filtration or desiccation, with desiccation providing comparable or better results at minimal cost. 8

Interpretation Nuances

• Using antisera specific to free light chains (FLC) rather than conventional antisera provides more sensitive detection and avoids obscuration of monoclonal free light chains by co-migration with intact immunoglobulin monoclonal proteins. 8
• The technique can detect deletion of the first constant domain, which is the hallmark of heavy-chain deposition disease and AH amyloidosis. 1

Clinical Decision-Making Algorithm

When to Order IFE

1. Order serum and urine IFE when SPEP shows an abnormal pattern, monoclonal spike, or when there is strong clinical suspicion of plasma cell disorder even with normal SPEP. 1, 7, 5
2. In patients with unexplained peripheral neuropathy, renal dysfunction with proteinuria, or systemic symptoms suggesting plasma cell disorder, order IFE as part of initial screening. 1
3. For monitoring known monoclonal gammopathies, IFE is essential for determining complete response, as it can detect residual disease not visible on standard electrophoresis. 1

Common Pitfalls to Avoid

• Do not rely solely on SPEP to rule out monoclonal gammopathy—a normal SPEP does not exclude multiple myeloma or other plasma cell disorders, particularly in light-chain-only disease. 6, 5
• Avoid ordering IFE, SPEP, and serum free light chain assays simultaneously without clinical indication, as this leads to excessive and potentially unnecessary testing. 5
• Do not use urine free light-chain assays to quantify Bence Jones protein—these assays have not been validated for this purpose; use urine protein electrophoresis instead. 1
• Ensure the same IFE methodology is used for serial monitoring of individual patients, as different antisera and techniques may have variable reactivity to monoclonal proteins. 8