How to detect and correct interferences

The best way to detect false-positive results caused by the interferents described in this chapter is to observe the appearance of the discordant sample. At the interferent concentrations that may cause significant interference, hemolysis, icterus (caused by increased bilirubin), and turbidity (caused by lipids) are easily detectable visually. However, in practice the collection tubes have so many labels and barcode stickers outside, it is often difficult to inspect the specimen within. Many automated analyzers can measure the degree of hemolysis, icterus and turbidity in the sample, and post alert in the results. The degree of interference, if any, is noted by an "index." The index for lipids is generated mostly by spiking specimens with IntraLipid. For example, Table 3 presents the representative system flag indices corresponding to the specimen's approximate interferent (hemoglobin, bilirubin, and triglyceride) concentrations in an auto-analyzer.

There have been several reports about the accuracy of such indexes. Sonntag and Glick (10) found that the Hitachi serum index for hemolysis correlated well with the hemoglobin concentration. Dahlin found a linear relationship between serum indices and interferent concentrations: hemoglobin, R = 0.9976 (range 0.0-9.9 g/dL), and bilirubin, R = 0.9851 (range < 1.0-10.6 mg/dL) (21). However, because of the hetero-genic nature of serum lipids (as described earlier), lipid indexes often do not correlate with the actual triglyceride levels of the specimen, which is often measured by Fossati's method of forming Trinder chromophore from the glycerol that is generated by triglyceride hydrolysis (22). Thus, the lipemic index of an auto-analyzer, which is measured by blank absorbance at 700 nm (scores 1-6 to correspond to IntraLipid concentrations of 0-3000 mg/dL) correlated poorly with the triglyceride concentrations in 1115 patients (23).

Sample blanking is a very effective way to minimize interferences from bilirubin, lipid, and hemoglobin in homogeneous assays. In this method, applicable ideally for two reagent methods, the sample is treated with the first reagent, followed by signal measurement—the "sample blank." Reagent 2 is then added and final signal reading is

Table 3

System Flags Commonly Used for Bilirubin, Hemoglobin, and Lipid Interference in

Autoanalyzers

Interferent Concentration

Table 3

System Flags Commonly Used for Bilirubin, Hemoglobin, and Lipid Interference in

Autoanalyzers

Interferent Concentration

Interferent

System Flag Index

Common Unit (mg/dL)

SI Unit

Bilirubin

+

1.7

29 pmol/L

++

6.6

113 pmol/L

+ + +

16.0

274 pmol/L

+ + ++

>30

513 pmol/L

Hemoglobin

+

50

0.5 g/L

++

150

1.5 g/L

+ + +

250

2.5 g/L

+ + ++

>500

5.0 g/L

Lipids (triglyceride, as triolein)

+

24

0.27 mmol/L

++

65

0.73 mmol/L

+ + +

280

3.16 mmol/L

+ + ++

>650

7.35 mmol/L

taken. The difference between the two readings is the actual signal arising from analyte reaction and is converted to equivalent analyte concentration. Thus, when sample blanking was introduced to the turbidimetric determinations of immunoglobulin A, G, or M, there was significant improvement in interferences observed (24,25).

Modification of reagents such that assay signal can be generated at wavelengths farther away from where the interferents absorb (or fluoresce) is another way to reduce these interferences. An example of this approach is reformulations of triglyceride and uric acid reagents on an auto-analyzer, which moved the absorbance measurement wavelength from 520/600 to 660/800 nm (primary/secondary wavelengths) to reduce interferences (26).

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