Does Bilirubin Cause Interference in Roche Creatinine Methods?

Bilirubin interference in kinetic alkaline picrate (Jaffe) creatinine assays has been well documented (1)(2). The exact mechanism of interference is still unclear, however, and both conjugated and unconjugated bilirubin have been implicated. Enzymatic assays, which reportedly suffer less interference, are an alternative, but the extra reagent cost has restricted their use. Historical data continue to be used to estimate the extent of bilirubin interference, although modifications to these assays or instruments may have taken place since the original interference studies were performed. We used liquid chromatography tandem mass spectrometry (LC-MS/MS) as the reference method to investigate the extent of bilirubin interference in 2 automated creatinine assays. We have previously shown that this LC-MS/MS assay compares well to the 2 automated methods at creatinine concentrations of <150 μmol/L (3) by analyzing >100 samples with bilirubin concentrations within reference intervals. We added varying amounts of creatinine (Sigma) to phosphate buffered saline (PBS) pH 7.4, containing 40 g/L bovine serum albumin, to give concentrations of 37.5–1000 μmol/L. One liter of PBS contained 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, and 0.24 g potassium dihydrogen phosphate. Unconjugated bilirubin (Sigma) was then added to give bilirubin concentrations up to 511 μmol/L. Anonymized icteric sera (n = 73) with creatinine concentrations <150 μmol/L were stored at −20 °C for ≥2 weeks, and total and conjugated bilirubin were determined by the Roche liquid diazonium ion and Jendrassik-Grof based assays, respectively. All samples were analyzed by the following 3 different creatinine methods. The automated creatinine assays were the rate-blanked, compensated Jaffe method and the creatinine plus enzymatic assay performed on the Roche Modular according to the manufacturer’s instructions (Roche Diagnostics). The reagent set insert states that there is no significant interference by bilirubin for concentrations ≤171 μmol/L and 428 μmol/L, respectively. The LC-MS/MS assay employed was used as described by Owen et al. (3). The comparative LC-MS/MS method shows no significant interference by bilirubin (see Fig. 1 in the Data Supplement that accompanies the online version of this Letter at http//www.clinchem.org/content/vol53/issue2). Surprisingly, the Jaffe method (Fig. 1A1 ) did not show significant interference in most PBS samples; however, the samples with the lowest creatinine concentrations showed a 10% reduction in creatinine at bilirubin concentrations >220 μmol/L. In the majority of samples analyzed by the enzymatic method (Fig. 1B1 ), a 10% reduction in creatinine was seen at bilirubin concentrations of >400 μmol/L, and even at lower creatinine concentrations the effect was seen when bilirubin was >200 μmol/L. The Jaffe method showed no significant interference at total bilirubin concentrations of <700 μmol/L in serum samples (Fig. 1C1 ). The mean difference between the Jaffe and LC-MS/MS assays (0.74 μmol/L) was not significant (P = 0.27, 95% confidence interval = −0.58–2.05 μmol/L, paired t-test). The enzymatic assay showed values lower than the LC-MS/MS (Fig. 1D1 ), with a significant mean difference of 10.71 μmol/L (P= <0.0001, 95% confidence interval; 9.04–12.38 μmol/L, paired t-test). The difference also increased at higher total bilirubin concentrations and is greater than would be expected when comparing these two methods in normal serum. When the effects of unconjugated and conjugated bilirubin on the enzymatic assay were examined separately, both species appeared to exert an effect, but the effect of unconjugated bilirubin appeared to be slightly greater (data not shown), possibly because it is more difficult for conjugated bilirubin to oxidize under alkaline conditions. The enzymatic assay, although often recommended as the method of choice, nevertheless showed greater interference than the Jaffe method. This interference may be attributable to the consumption of peroxide in the initial reaction mixture, as previously suggested (4). Our results show that the Jaffe assay performs well in the presence of icterus; however, the effects of bilirubin in this assay are variable and poorly reproducible. Despite this we found no statistically significant difference in serum creatinine values obtained by the LC-MS/MS and Jaffe methods. Serum samples with bilirubin concentrations >700 μmol/L are rarely seen, and users of this assay can feel confident in the accuracy of the creatinine values obtained from the vast majority of icteric samples. When percentage differences were considered, the greatest effect in the Jaffe method was seen in PBS samples containing creatinine concentrations within reference intervals. Although these PBS samples are not clinical samples, and the alternative matrix may behave differently to serum, this exaggerated effect is in agreement with previous reports (5). In conclusion, our data show that the Roche Jaffe method performs well in the presence of icterus despite often being considered to suffer from significant interference. The observed percent change in creatinine concentration as bilirubin concentration increases in PBS-based samples analyzed with the Jaffe method (A) and the enzymatic assay (B). Each line in Fig. 1A and Fig. 1B represents a different creatinine concentration: 1000 μmol/L (a), 600 μmol/L (b), 300 μmol/L (c) (shown as a broken line in Fig. 1B for clarity), 150 μmol/L (d), 75 μmol/L (e), and 37.5 μmol/L (f). The absolute difference (μmol/L) between Jaffe (C) and enzymatic assays (D) compared with the LC-MS/MS assay in serum samples with increasing bilirubin concentration. We thank the Department of Biochemistry, Selly Oak Hospital for providing samples and Roche Diagnostics for providing the enzymatic reagent set used in this study.