False-negative urine protein electrophoresis by semiautomated gel electrophoresis.

Urine is evaluated by electrophoretic methods both to gather information about the location and degree of damage within the nephron and to detect and quantify monoclonal free light chains (Bence Jones proteins). For many years, urine protein electrophoresis consisted of concentrating the urine (usually 50-fold) followed by manual application of the concentrated sample to a gel. Recently, semiautomated methods have become available that allow for a more efficient application of urine to the gels. The Sebia Hydragel 12 15/30 (Sebia, Inc.) method provides urine gel results with crisp separation of 12 as recommended by the guidelines for clinical and laboratory evaluation of patients with monoclonal gammopathies (1 ). However, in a recent urine sample, despite the presence of 2288 mg/24 h of total protein, the urine protein electrophoresis gel failed to demonstrate the proteinuria although the urine had been concentrated 50-fold (Fig. 1A). The technologist had observed sediment in the concentrated urine specimen. Because of the discrepancy between the total protein concentration and the appearance of the urine on electrophoresis, the electrophoresis on this sample was repeated on the next run. Before this run, the urine was centrifuged for 5 min in a fixed-head Sero-fuge (Clay Adams). Studies on urine samples have shown that this centrifugation has no effect on serum protein migration (data not shown). After centrifugation, the clear supernatant was placed in the applicator well. The second electrophoresis demonstrated a glomerular and tubular proteinuria with a suspicious broad -region band (Fig. 1B). The difference in the two patterns indicated that no sample had been applied to the gel during the first analysis. The Sebia semiautomated agarose gel electrophoresis method uses plastic applicators (product no. 4199; Sebia) to apply the urine to the gel (Fig. 1C). Samples are pipetted into a plastic well, the bottom of which is a microporous membrane (Sebia) that leads to a straight edge. The liquid is drawn by wick effect into the microporous membrane and then to its edge. The applicator touches the edge of the microporous membrane to the gel, which places a small amount of the urine at the point of origin for the electrophoresis. The manufacturer’s instructions advise that urine with a high salt content may cause deformation of the gel. The instructions also note that use of old, improperly stored urine samples, in which enzymatic degradation of proteins might occur, should be avoided. During customer training, the company instructs