Unexpected Test Results in a Patient with Multiple Myeloma

IMMUNOGLOBULIN EXCESS AND THE PROZONE EFFECT

Quantitative immunoglobulin concentrations in this case were suspiciously low considering the marked increases in total protein, the large monoclonal band identified on serum electrophoresis, and the patient's anemia and thrombocytopenia, all of which suggested the presence of active disease.

Immunoglobulins IgA, IgM, and IgG were measured using polyethylene glycol-enhanced immunoturbidimetric methods in which antihuman immunoglobulin in the reagent binds to immunoglobulin in the patient sample to generate a precipitate, thereby increasing the turbidity of the solution (3). The resulting absorbance is then used to calculate the immunoglobulin concentration in the patient sample from the calibration curve.

Aberrant immunoglobulins expressed in monoclonal gammopathies may cause interference in routine immunoassay methods. Reagent immunoglobulins used in methods for immunoglobulin quantification are optimized to recognize the diverse constellation of circulating polyclonal immunoglobulins found in physiological states other than monoclonal gammopathy. The presence of an overabundance of a single clone has been shown to cause unexpected behavior in routine immunoassay methods, resulting in erroneously high or low immunoglobulin measurements (4). It has been hypothesized that expression of atypical epitopes or large concentrations of single clones causes incomplete reaction of the reagent antibodies with the aberrant immunoglobulins, resulting in inaccurate measurements when using routine immunoassay methods.

Immunoturbidimetric and nephelometric methods are susceptible to an additional type of interference known as antigen excess or prozone effect. This phenomenon occurs when excessive quantities of antigen inhibit stable antigen–antibody complex formation. The subsequent decrease in detected absorbance leads to a falsely low measured concentration (3, 4). Detection of the prozone effect requires dilution of the sample to remove the antigen excess and allow appropriate formation of the antigen–antibody complex, resulting in an accurate measurement (3, 5). The prozone effect is more often observed when measurements are obtained using an immunoturbidimetric method; modern nephelometers are typically less susceptible to this effect because they employ an automatic dilution step to detect antigen excess (5).

Testing was repeated with a 1:100 dilution (1 part serum to 99 parts normal saline) and an IgG concentration of 14 400 mg/dL (144.0 g/L) was obtained, which was consistent with the serum protein electrophoresis results and the patient's overall clinical presentation. Repeat measurements of IgA and IgM were not performed due to sample volume constraints.

IMMUNOGLOBULIN EXCESS AND THE VOLUME DISPLACEMENT EFFECT

The patient's initial serum sodium was 124 mmol/L, but he lacked symptoms of hyponatremia. The low sodium concentration was found to be a result of pseudohyponatremia, an artificially low measurement of sodium concentration produced by the presence of severe hyperproteinemia.

In healthy individuals, the plasma volume is comprised of approximately 93% water, which contains dissolved electrolytes and other compounds, whereas the remaining plasma volume consists of lipids and proteins (2, 3). Substantial increases in plasma proteins and/or lipids can cause displacement of the aqueous component and its dissolved solutes in a phenomenon called volume displacement. Although the proportion of plasma water occupies a relatively smaller percentage of the total plasma volume, the solute concentrations within the plasma water are physiologically normal.

For measurement of serum electrolytes, many laboratories employ ion-selective electrodes (ISE) that utilize indirect potentiometry, in which patient samples are diluted before the measurement process. Prediluting the sample prevents excessive protein exposure to the ISE membrane, thus minimizing electrode degradation and calibration drift (6). Instruments that utilize a predilution step assume a normal ratio exists between the plasma water volume and the total plasma volume (2). This assumption may be invalid when analyzing samples containing abnormal increases in proteins and/or lipids and can lead to falsely low measurements of solute, with sodium being most commonly affected. The volume displacement effect can be avoided in these samples by employing ion-selective electrodes that utilize direct potentiometry, in which electrolyte measurements are evaluated in an undiluted plasma sample (6).

The original serum sodium result of 124 mmol/L, as measured by indirect potentiometry, was inconsistent with the patient's clinical presentation. In this case, the volume displacement effect was suspected as the most likely etiology of the pseudohyponatremia. The patient's serum sodium concentration was repeated utilizing direct potentiometry on the NOVA CCX analyzer, and a sodium concentration of 138.5 mmol/L, which is within the reference interval, was obtained.

IMMUNOGLOBULIN EXCESS AFFECTING MEASUREMENT OF PHOSPHOROUS

On initial measurement the patient's serum inorganic phosphate was markedly increased, but the calcium concentration was within the reference interval. Serum inorganic phosphate and calcium concentrations are maintained within a tight physiologic range via closely associated mechanisms regulated by the kidney, bone, and gut (1). An interfering substance producing factitious hyperphosphatemia, or “pseudohyperphosphatemia,” was suspected owing to the profoundly high inorganic phosphate concentrations in a relatively asymptomatic patient with calcium concentrations within the reference interval.

Spectrophotometric quantification of serum inorganic phosphate concentration is based on the reaction of phosphate ions with ammonium molybdate to form a phosphomolybdate complex (1, 7). Absorbance of the unreduced phosphomolybdate complex is measured and indexed to a calibration curve to determine analyte concentration (3, 7). The formation of the complex is pH dependent and uses an acid buffer (3, 7). The low pH environment of the reaction mixture can lead to precipitation of immunoglobulins and result in increased turbidity and light scattering (5, 7). Resolution of the interference is obtained by diluting the sample or removing the plasma proteins via various deproteinization methods (5, 7).

The initial measurement of inorganic serum phosphate was 14.49 mg/dL (2.50–4.60 mg/dL) [4.68 mmol/L (0.81–1.49 mmol/L)], which exceeded the absorbance limit for the assay. A 1:5 dilution (1 part serum to 4 parts normal saline) was performed and a serum phosphate concentration within the reference interval [4.4 mg/dL (1.41 mmol/L)] was obtained.

SUMMARY

Multiple myeloma is a hematologic disease that often results in increased production of monoclonal immunoglobulins. As the paraprotein concentration rises, the potential for erroneous results increases. This case illustrates how unusually high concentrations of serum immunoglobulins can result in multiple laboratory interferences, including the prozone effect, the volume displacement effect, and precipitation interference. Laboratorians should remain attentive to potential sources of discrepant results in patients with high plasma protein concentrations and should take appropriate action to prevent the release of erroneous results.