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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.

Interference = adulterated concentration - unadulterated concentration

For example, the effect of bilirubin on a glucose method could be determined in the following manner.A specimen containing 100 mg/dL of glucose is spiked with 0, 5, 10, and 15 mg/dL of bilirubin and the glucose concentration is measured.

Bilirubin in adulterated sample

Adulterated sample glucose (mg/dL)

Unadulterated sample glucose (mg/dL)

Interference (mg/dL)

















As seen in the table, increasing amounts of bilirubin caused increasing interference with glucose measurement. If the degree of interference (constant error) is less than the total allowable error for that analyte then the effect of the potential interfering substance is considered inconsequential (see CLIA Acceptable Test Performance Criteria document).

The same data can also be analyzed by graphing the averages of the triplicate measurements in Excel using an XY plot. Unadulterated samples are plotted on the X axis and adulterated samples on the Y axis. Deviation from a straight line indicates interference.

The following graph illustrates the effect of increasing degrees of hemolysis on the measurement of four different chemistry analytes; LDH, AST, potassium and total bilirubin.


Screenshot 2016 06 19 17.23.58


Lactate dehydrogenase, AST and potassium values increased with increasing free hemoglobin levels. All of these analytes are present in higher concentration within red blood cells and are released into plasma during hemolysis. Increasing concentrations of hemoglobin caused total bilirubin measurements to be falsely decreased, due to interference with the analytical method.

Modern chemistry analyzers can detect these interfering substances in patient samples and alert the operator when a specimen is grossly hemolyzed, icteric or lipemic. The medical director should determine if these results should be reported with a cautionary disclaimer or rejected.


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