Proteins and Paraproteins

Interference from proteins is possible both in hypo- and in hyper-proteinemia (normal serum protein concentration is about 6-8g/dL, and in plasma fibrinogen adds to total protein by about 0.2-0.4 g/dL). Such interference is most apparent where the specimen is used in assay without pretreatment, e.g., in immunoassays. Although the calibrator matrix for an immunoassay is often pooled serum or plasma, or protein-containing buffered solutions, specimens vary in plasma protein concentration from subject to subject and even in a same subject over time or during the day. Plasma protein concentrations change as people age or in various physiological states, such as pregnancy or in various disease states. Often, such kinds of effects are included in the vaguely defined "matrix effect" in an immunoassay. Ezan et al. (18) attempted to estimate such effect on TDM assays in plasma samples. The authors spiked a seven-amino-acid peptide experimental drug for the treatment of chronic diarrhea in pooled plasma to generate the calibration curve in their experimental immunoassay for the drug. When they compared the recovery of the spiked drug at two different concentrations in plasma from 25 different subjects, the recoveries ranged from 70 to 152% at the low concentration spike and from 79 to 114% at the high concentration spike. The authors suggested that the differences arose from differences in plasma proteins.

Plasma specimens, which have been refrigerated for prolonged periods or which have undergone freeze-thaw cycles, demonstrate another facet of protein interference. Fibrins precipitate under such conditions. These fibrin clots may block auto-analyzer sample probes, generating incorrect results. Such samples should be centrifuged to remove any precipitates before assay. However, most modern auto-analyzers include clot-detection and alert systems to flag results suspected to be subject of clot interference.

Manufacturers of commercial assays normally report the effects of hypo- (up to 3g/dL) and hyper-proteinemia (up to 12.5 g/dL) in the package inserts. Hyper-proteinemia may increase the viscosity of the specimen, thus interfering with accurate sampling for the assay.

Paraproteins circulate because of multiple myeloma or similar diseases. The concentrations of a specific class and idiotype of immunoglobulins (Ig) are greatly increased. There have been many literature examples of paraproteins interfering with all kinds of clinical chemistry assays, including immunoassays. In most cases, the mechanism of paraprotein interference in colorimetric or turbidimetric assays is their precipitation when the specimen is treated with the first reagent in acidic or alkaline reaction conditions. Such condition causes turbidity. This occurs especially in methods using sample blanking, because turbidity causes larger blanking, mostly generating false-negative results.

Hullin reported a case study, where a 77-year-old man whose plasma samples had 500mg/dL of paraprotein (IgMK monoclonal component) ingested 100 tablets of acetaminophen before 18 h (19). Serum acetaminophen concentration, using a commercial enzyme assay kit, was 5.3mg/dL (toxic >5-20mg/dL, depending on ingestion time before sampling). But the sample-blanking absorbance (absorbance measured after the addition of sample to the enzyme reagent, but before adding the chromogen) was very high (0.145, compared with <0.01 for normal sera). Suspecting this acetaminophen result, the authors assayed the sample by a high-performance liquid chromatography (HPLC) method for acetaminophen, the result being 8.6 mg/dL. The sample showed non-linear dilution in the enzymatic assay. Presence of paraprotein in the sample was indicated by the formation of flocculent precipitate when a drop of serum was added to water (the Sia water test) (20).

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