Bilirubin is derived from hemoglobin of aged or damaged red blood cells (RBC). Bilirubin does not have iron and is rather a derivative of the heme group. Some part of serum bilirubin is conjugated as glucuronides ("direct" bilirubin); the unconjugated bilirubin is also referred as indirect bilirubin. In normal adults, bilirubin concentrations in serum are from 0.3 to 1.2 mg/dL (total) and <0.2 mg/dL (conjugated) (5). In different forms of jaundice, total bilirubin may increase to as high as 20 mg/dL, but the ratio of direct versus indirect bilirubin also varies. In obstructive jaundice, the increase in total bilirubin is contributed mainly by direct bilirubin. In hemolytic and neonatal jaundice, the increase is mostly in indirect bilirubin. Both fractions of bilirubin increase in hepatitis.

Elevated bilirubin causes interference, proportional to its concentration. The interference of bilirubin in TDM/DAU assays is mainly caused by bilirubin absorbance at 454 or 461 nm. Thus, it may interfere in colorimetric enzyme-linked immunosorbent assay (ELISA) that use alkaline phosphatase label and p-nitro phenol phosphate substrate (measured at 405 nm measured at). However, if the assay is enzymatic or colorimetric, bilirubin may interfere also by reacting chemically to the reagents (6).

In one case study (7), a severely jaundiced 17-year-old male patient (total bilirubin 19.8 mg/dL) with abdominal pain and increased serum transaminase results was suspected of acetaminophen overdose, although the patient himself denied using any medications containing acetaminophen within the previous week. The apparent plasma acetaminophen concentration by an enzyme method was found to be 3.4 mg/dL. In this method, acetaminophen is enzymatically (by arylacylamidase) hydrolyzed to p-aminophenol, which is condensed with o-cresol in the presence of periodate to form the blue indophenol chromophore. The method was run on the Roche Modular chemistry analyzer, with absorbance measurement at 600 nm (2-point rate) and background correction at 800 nm. To investigate false-positive results from elevated bilirubin, the authors spiked twelve hyperbilirubinemic plasma samples and bilirubin linearity calibrators with various levels of acetaminophen concentrations and measured them in the acetaminophen assay. Plasma specimens with bilirubin (range: 15.9-33.8 mg/dL), but without any acetaminophen spiking (the patients from whom these specimens were collected had no recent acetaminophen exposure), showed false-positive acetaminophen (0.6-1.8 mg/dL) results. The false-positive acetaminophen results plateaued at 2.5-3.0 mg/dL at total bilirubin concentrations of 23-35 mg/dL. The acetaminophen dilution profiles of these samples were non-linear, reaching to undetectable levels (the expected results) after fourfold dilution with the assay diluent. Because the background correction failed to correct the bilirubin interference in this assay, the authors hypothesized that bilirubin, with substantial reducing activity, might have reacted with periodate to produce a product that absorbed more strongly at 600 nm than did unreacted bilirubin. The authors also found that accuracy of spiked (5.0-15.0mg/dL) acetaminophen results was not affected by high bilirubin (92-97%

recovery), suggesting that the nominal cross-reactivity of bilirubin with the aryla-cylamidase or periodate-catalyzed reaction was at a competitive disadvantage in the presence of acetaminophen.

Another example of bilirubin interference was noted in the acetaminophen assay but utilizing different assay technology (8). Fifteen serum samples, none containing acetaminophen, but with total bilirubin concentrations between 2.2 and 16.7mg/dL, when tested in an acetaminophen assay involving the reaction of the analyte with ferric-2,4,6-tripyridyl-s-triazine, demonstrated false-positive results between 0.7 and 13.6mg/dL. This is critical, because detoxification treatment of acetaminophen is indicated when the serum levels of the drug exceed 5.0mg/dL. The authors found that the interference could be minimized by using the protein-free ultrafiltrate because bilirubin is mostly bound to proteins, but acetaminophen is not.

Wood et al. (9) reported a case where increased bilirubin (22.6mg/dL), specially consisting of high percentage of conjugated fraction (82%), caused negative interference in a fluorescence polarization immunoassay (FPIA) for vancomycin. In their study, the authors first compared 28 plasma samples with total bilirubin <5.9mg/dL, between two different Abbott's vancomycin assays, by using a TDx analyzer and AxSYM analyzer. Vancomycin, a glycopeptide antibiotic used in treating serious infections, is toxic with plasma concentration >20 ^g/mL (trough) and >80 ^g/mL (peak). The method used in the TDx analyzer is a homogeneous FPIA, using a polyclonal sheep antibody and fluorescein-labeled antigen. The assay on the AxSYM analyzer also uses the same assay principle but utilizes a different, monoclonal mouse antibody. The vancomycin results from these 28 samples, ranging from 2.0 to 34.5 ^g/mL, were in close agreement between the assays performed using two different analyzers (correlation coefficient r2 = 0.996). When the authors analyzed plasma specimens containing abnormal bilirubin, they observed discordant results between the two vancomycin assays. For example, in specimen containing 22.6 mg/dL of total bilirubin, the vancomycin concentration observed by using the TDx analyzer was 2.6 ^g/mL but the corresponding value obtained by the AxSYM analyzer was 8.0 ^g/mL (9).

Suspecting the elevated bilirubin as the source of discordance between the two vancomycin methods, the authors spiked vancomycin in 10 plasma specimens from jaundiced patients (total bilirubin ranging from 9.5 to 28.2 mg/dL; direct bilirubin ranging from < 1.0 to 16.0 mg/dL) not receiving vancomycin and measured the samples using both vancomycin assays. By the Wilcoxon rank-sum test, they found vancomycin recoveries using the assay on the TDx analyzer significantly were lower than with the recoveries using the assay on the AxSYM (p < 0.001) analyzer (mean TDx and AxSYM recoveries were 79.7 ± 13.1% and 102.2 ± 6.4%, respectively). The lower recovery of the assay using the TDx analyzer was inversely related to the direct (conjugated) bilirubin concentration in the specimens (r2 = 0.54, p < 0.005). No such correlation was found between the recovery and the total bilirubin. The negative interference in the assay using the TDx analyzer was probably caused by direct bilirubin generating falsely increased fluorescence blanks. The authors noted that for the assay method for the TDx analyzer, the package-insert reported interference of <5% for bilirubin concentrations of 15 mg/dL. This suggested a possibility that the package insert data were generated using unconjugated bilirubin, which as the authors' data demonstrated, does not interfere with the assay. The authors concluded that the assay on the AxSYM

analyzer somehow was not affected by high direct bilirubin (because of either the antibody difference, or method difference), whereas the assay on the TDx analyzer demonstrated false-negative results for such samples (9).

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