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Technical Briefs |
1 Department of Clinical Chemistry, Vrije Universiteit Medical Centre, Amsterdam, Postbus 7057, 1007 MB Amsterdam, The Netherlands
2 Clinical Laboratory, St-Annaziekenhuis, Postbus 90, 5660 AB Geldrop, The Netherlands
aaddress correspondence to this author at: Clinical Laboratory, St-Annaziekenhuis, Postbus 90, 5660 AB Geldrop, The Netherlands; fax 31-40-2854945, e-mail m.thelen{at}st-anna.nl
The College of American Pathologists has recently defined and introduced guidelines for clinical and laboratory evaluation of patients with monoclonal gammopathies (1)(2)(3)(4). One of these guidelines states that all patients with multiple myeloma, Waldenström macroglobulinemia, amyloidosis, and related disorders should be assessed for the presence of urinary monoclonal free light chains by immunofixation electrophoresis (3). Because free light chains are associated with more aggressive disease, the clinical relevance lies with the quantities of free monoclonal light chains excreted and not with the intact immunoglobulins, which also may be present in the urine (2)(5). Here we demonstrate that in some cases the intact immunoglobulins comigrate with the free light chains in immunofixation electrophoresis, and we provide a method to disclose this interference.
In the diagnostic work-up of a patient suffering from multiple myeloma, we performed immunofixation electrophoresis on serum and concentrated urine using a immunofixation reagent set (Beckman) with anti-human immunoglobulin antibodies (Dako), according to the manufacturer’s protocol. Briefly, electrophoresis of 5 µL of 1:10 diluted serum (2 µL of serum plus 18 µL of buffer) and 5 µL of 50-fold concentrated urine was followed by immunofixation with 80 µL of antiserum. Unprecipitated proteins were washed away before staining of the precipitated complexes with Amido black.
Immunofixation electrophoresis of our patient’s urine revealed the same monoclonal pattern as was found in the patient’s serum; an IgG heavy chain with the same mobility as the 
light chain (Fig. 1A
, lanes 2 and 3). However, there was an unexpected discrepancy between the IgG heavy chain and the light chain signal in favor of the latter.
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To explore whether part of the light chain excess might be explained by the presence of free light chains, we used an immunosubtraction technique. We preincubated 10 µL of the concentrated urine with 20 µL of anti-IgG serum for 30 min at 37 °C and centrifuged the mixture (15 min at 12 000g) before applying 5 µL of the supernatant to the gel. The anti-IgG signal was not seen in the subsequent immunofixation. The anti- signal, however, was only partly affected by the immunosubtraction, indicating that free light chains indeed were responsible for part of the single monoclonal band in the electrophoretic pattern (Fig. 1A
, lanes 5 and 6). Fig. 1B
shows immunofixation of the urine of a control patient, with free light chains and intact immunoglobulins migrating at different positions on the gel, demonstrating that heavy chain immunosubtraction affects only the intact immunoglobulins and not the free light chain signals. Preincubation with anti-IgA did not affect the anti- signal. Using this technique, we also identified more patients with a similar comigration phenomenon (Fig. 1C
).
Our results demonstrate that in immunofixation electrophoresis, free light chains indeed can be obscured by comigrating intact immunoglobulins. The presented immunosubtraction method provides a means to disclose this problem. Although the use of antibodies that specifically recognize only free light chains theoretically also can reveal such problems, reported problems with the sensitivities and specificities of such antibodies make results unpredictable and unsuitable for diagnostic use (1). The method we describe here combines the specificity of immunosubtraction with the sensitivity of immunofixation, making it an attractive addition to the arsenal of laboratory tests for gammopathies in cases where there is some doubt about the stoichiometry of immunoglobulin heavy and light chains.
References
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light chain serum; lane 5, preincubation with anti-IgG antiserum before electrophoresis and incubation with anti-IgG serum after electrophoresis; lane 6, preincubation with anti-IgG antiserum before electrophoresis and incubation with anti-