Mechanism of interference

Most TDM immunoassays involve analytes of small molecular size, and these assays employ the competition format where the analyte molecules compete with limited number of specific binding sites, e.g., on specific antibodies, with labeled analyte molecules in a reaction (details described in Chapter 3). Signals generated by the bound label are converted into the analyte concentration in the assay. The signals are mostly optical in nature including absorbance, fluorescence, or chemiluminescence. The assays can be homogeneous or heterogeneous. In the former format, the bound label has different properties than the free label, thus the method is simpler, requiring no separation and the difference in signal between bound and free labels is utilized to quantify the analyte. In heterogeneous immunoassays, the bound label is physically separated from the unbound labels and then the signal is measured.

As explained in Table 1, most of the interference from bilirubin, hemoglobin, lipids, or paraproteins are caused by the fluorimetric or photometric properties of the interferents, which interfere with the generation of signal. Thus, such interference is more common in homogeneous than in heterogeneous assays. In certain assay formats, the interference enhances the signal. As in most competitive immunoassays the signal is inversely proportional to the reported specimen analyte concentration, such interference generates false-negative results. In some other assay formats, the increased signal causes false-positive results.

Bilirubin absorbs around 450-460 nm. Hemoglobin begins to absorb from 340 to 560 nm and absorbance peak is observed at 541 nm (oxyhemoglobin). Therefore, hemolysis affects assays that use the absorbance properties of nicotinamide adenine dinucleotide (NADH) or nicotinamide adenine dinucleotide phosphate (NADPH) (1) at 340 nm. Selecting a longer wavelength (>650 nm) for detection may minimize the interference from bilirubin or hemolysis in a turbidimetric immunoassay. But because lipid interferes mostly by light scattering, lipid may still interfere with the method.

Bilirubin and hemoglobin can also interfere in assays through unintended side reactions. Both participate in redox reactions, commonly used in TDM or DAU assays that are not immunoassays. The Package Inserts from most commercial assay kits normally lists the effect of these interferents. Most manufacturers follow the EP7-P protocol from National Committee for Clinical Laboratory Standardization (NCCLS, currently called Clinical Laboratory Standards Institute or CLSI), where the interference is studied at two different steps (2). In the "Screening" protocol, serum pools containing a clinically important concentration of the analyte are spiked with various concentrations of the interferent, and the assay results are compared with a suitable control sample (without spiked interferent). Interference is judged significant when the results of a spiked sample are statistically different from the control sample and the difference between the two means is >10%. The protocol also recommends plotting analyte versus interferent concentrations to find the pattern of interference, if any. If there is more than single clinically relevant analyte concentration, then the interference should be studied with serum pools containing each of those analyte concentrations (3).

Table 2

Clinical Laboratory Standards Institute (CLSI) (NCCLS)-Recommended Interference Levels

Highest Interferent Concentration

Interferent NCCLS Recommended Manufacturer Reported

Common Unit (mg/dL) SI Unit Common Unit (mg/dL) SI Unit

Bilirubin 20 342 |imol/L 30 513 |xmol/L

Hemoglobin 500 5.00 g/L 2000 20.00 g/L

Lipida 1000 11.3 mmol/L 2000 22.6mmol/L

aAs triolein.

The CLSI-recommended levels of interfering substances up to which interference should be studied are summarized in Table 2. However, many package inserts report results beyond those levels.

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