Interference

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  A new method should be tested for possible interfering substances. At a minimum, the effect of hemolysis, lipemia, and hyperbilirubinemia should be evaluated. Quality control or patient samples, containing an analyte at the upper and lower reference range limits are spiked with a final concentration of interfering substance that approximates the highest level expected in a clinical specimen. The volume of interfering substance added to the sample should be < 10% of the sample volume, so that the matrix is minimally disrupted. An unadulterated sample should be prepared by adding a similar volume of the solvent used to dissolve each interfering substance. Unadulterated samples should have the following concentrations of interfering substances: 0 mg/dL hemoglobin, < 1.5 mg/dL total bilirubin, and < 150 mg/dL triglyceride.Hemolysis may be simulated by freezing and thawing packed red cells to obtain a stock solution of free hemoglobin at an approximate concentration of 5000 mg/L. The concentrated hemoglobin solution is centrifuged to pellet debris and added to test samples to achieve final concentrations of 100, 300, and 500 mg/dL.
Lipemic samples are prepared by adding a lipid suspension, such as Liposyn, to saline to prepare a stock solution of ~5000 mg/L. This stock solution is added to test samples to a final concentration of 300, 500, and 700 mg/dL.

Unconjugated bilirubin is dissolved in dimethyl sulfoxide to a concentration of ~2500 mg/L and this solution is added to a sample to obtain a concentration of ~200 mg/L. This bilirubin solution is then added to the test samples to achieve concentrations of 5, 10, and 15 mg/dL.

Adulterated and unadulterated samples should be analyzed in triplicate and averaged. The difference between the averages of the adulterated and unadulterated samples is attributed to interference.