January 2016

A 60 year old man with abdominal pain, shortness of breath, weakness, and nausea/vomiting

Derrick Green, MD and Danyel Tacker, PhD

Overview

A 60 year old man with incomplete past medical history was admitted to the hospital for abdominal pain, shortness of breath, weakness, and nausea/vomiting. His liver enzymes were elevated, his total bilirubin was elevated at 5.9 mg/dL [0.3-1.3 mg/dL], and his conjugated bilirubin was elevated at 3.9 mg/dL [<0.3 mg/dL]. His serum creatinine was within normal limits. The patient was recently treated with Levaquin for a urinary tract infection. A computed tomography (CT) scan of the abdomen and pelvis with and without contrast was performed at admission for an acute abdomen.

Gross Description

Serum and urine protein electrophoresis (SPEP/UPEP) studies were performed as part of the laboratory workup. The SPEP showed the following:

The serum albumin and serum total protein at the time of the SPEP were measured at 2.5 g/dL [3.4 to 4.8 g/dL] and 5.1 g/dL [5.6 to 7.6 g/dL], respectively.
A patient sample with normal SPEP tracing overlay

Noting an unusual band that appeared to be in the pre-beta region (white arrow), an immunotyping study was performed (unusual band, white arrowheads):

A random urine albumin and random urine total protein were measured as 3.8 mg/dL [<30 mg/dL] and 17 mg/dL [<30 mg/dL], respectively. Immunotyping was added and showed the following:

Diagnosis

What is the best explanation for these findings ?

Answer

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Discussion

Serum and urine protein electrophoresis (SPEP and UPEP, respectively) are used primarily to detect circulating monoclonal immunoglobulins. SPEP can also be utilized early in the diagnostic approach when auto-immune, acute inflammatory, and chronic inflammatory disease states are suspected due to alterations the expression of circulating antibodies, which can yield abnormal bands (aka, “spikes”).UPEP is recommended in patients that have known monoclonal gammopathy or otherwise unexplained renal dysfunction (6).

Capillary electrophoresis was used in this case. This is a liquid based method that uses small bore fused silica tubes (ie, capillaries) for protein separation rather than a gel medium. The sample passes through the capillaries, carried in a buffer solution, and the proteins in the sample are separated within an electrical charge gradient that is applied to the capillary; as protein fractions elute from the capillary, they pass a spectrophotometric detector emitting UV light. Peptide bonds have spectral activity in the UV range (approximately 200 nm) and the absorbance pattern (ie, tracing) is generated by a computer that processes the activity at the detector (4). Consistent with Beer’s Law, as protein concentration increases, so does absorbance of the light at 200 nm; the resulting optical density is viewed as peaks on the tracing.

Immunotyping is an automated method which employs different capillaries for the SPEP and antisera-complexed specimen treatments; software integrates the resulting tracings into an on-screen mosaic (visible in figures 3 and 5 in the Background section). With immunotyping, the antigen-antibody complexes alter the migration of the immunoglobulins, thus “subtracting” them from the tracing; if the SPEP tracing is overlaid onto each tracing generated after antisera treatment, monoclonal bands can be “typed”.

Though similar protein migrations may be observed in a urine specimen with abundant protein in the specimen, most normal UPEPs will show minimal protein or trace albumin and virtually absent immunoglobulins. Urine immunotyping is usually unnecessary, unless an abnormal fraction is detected on UPEP or serum studies suggest that immunotyping should be conducted.

In our case, after the abnormality within the β fraction was recognized, an immunotyping study was performed; it did not reveal any significant abnormalities and excluded monoclonal gammopathy. The SPEP pattern did not fit neatly within any commonly observed electrophoretic result; therefore, the possibility of an interfering agent was considered. After immunofixation, this was interpreted “reactive”.

The findings on the UPEP with subsequent immunotyping were initially interpreted as having a suspicious strong band near β, inconsistent with measured protein content and accounting for 80% of protein detected by electrophoresis which prompted immunotyping. Because the immunotyping result was negative, the band present was interpreted as tubular protein.

Literature search revealed several articles describing aberrant peaks within the α-2 and β-1 zones in relation to the recent administration of radiopaque contrast dyes used in imaging studies (8). The manufacturer of the electrophoresis platform was contacted to discuss this possibility; their expert agreed that the findings were very likely due to the presence of such a dye in the specimen.

The patient had received radiocontrast as part of a CT scan performed to evaluate his abdomen, a few hours prior to the collection of the serum specimen used for the SPEP. Urine collection occurred several hours later than the serum specimen. We concluded that the patient’s aberrant SPEP and UPEP results were a result of the recent IV administration of radiocontrast dye, cleared through the kidney and hence strongly visible in the urine.

Given the frequent use of imaging studies using radiocontrast, it is important to be aware of the potential for this finding in samples taken from patients who have recently received these agents as part of their care. In general, a patient should not have received contrast in the past 12 hours (preferably 24 hours) prior to sample collection for the performance of protein electrophoresis studies.

Bibliography

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  3. Bossuyt X, Mewis A, Blanckaert N. Interference of radio-opaque agents in clinical capillary zone electrophoresis. Clin Chem 1999;45:129-131.
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  5. Brody, J.R., Kern, S.E. (2004): History and principles of conductive media for standard DNA electrophoresis. Anal Biochem. 333(1):1-13
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