Comprehensive characterization and resolution of discrepant spectrophotometric bilirubin results in patients on eltrombopag therapy

Abstract

BackgroundEltrombopag is a thrombopoietin receptor agonist used for the treatment of thrombocytopenic conditions. It can cause pH-dependent discoloration of plasma/serum. Eltrombopag is potentially hepatotoxic. It can affect the assessment of hyperbilirubinemia because of its (i) absorbance at ~450 nm (bilirubin), (ii) absorbance at ~550 nm (diazo-bilirubin) and (iii) it can cause yellowish discoloration of the eyes at normal circulating bilirubin levels.MethodsWe collected 66 samples from patients on a range of eltrombopag dosages up to 150 mg daily. Bilirubin was measured using multiple routine spectrophotometric analyzers, the Doumas reference method and high-performance liquid chromatography (HPLC). Plasma/serum eltrombopag concentrations were determined using liquid chromatography tandem mass spectrometry (LC-MS/MS). Spike-in and admixture experiments delineated the effects of eltrombopag and its metabolites.ResultsForty-nine of 52 samples from patients on ≥50 mg daily eltrombopag therapy showed significantly discrepant inter-analyzer total bilirubin results, a difference up to 64 μmol/L (3.7 mg/dL). In one sample, total bilirubin varied from 8 to 65 μmol/L (0.4–3.8 mg/dL) by different routine analyzers, with direct bilirubin ≤4 μmol/L (0.2 mg/dL). There was a positive correlation between total bilirubin difference and plasma eltrombopag concentration (r = 0.679), and spike-in experiments demonstrated that Beckman AU and Doumas reference methods were susceptible to positive interference. HPLC can quantify bilirubin after separating eltrombopag, and results suggest different analyzers are affected to varying degrees by eltrombopag and its metabolites.ConclusionsEltrombopag and its metabolites can cause positive interference to the spectrophotometric measurements of total bilirubin. Accurate measurements of total bilirubin may improve our understanding of the prevalence of hyperbilirubinemia in patients on eltrombopag therapy.

Interference of eltrombopag with bilirubin measurements on the Vitros 5600 analyzer

“…The values in Table 1 that are in bold exceed these limits for total allowable error. For all the groups, the values exceeding the total allowable error corresponded to eltrombopag concentrations above the approximate maximal therapeutic concentrations in plasma (<45 µg/ml) 5 …”

“…Several studies have assessed the interference of eltrombopag on bilirubin measurements, showing a variety of results ranging from negative interference, no interference, to positive interference. These variations seem to be due to several factors including sample pH, analyzer used, and drug concentration under study 5,12,13 . The aim of our study was to determine the concentrations of the drug at which interference with bilirubin measurements appears, using the VITROS ® TBILI and VITROS ® BuBc slides on the Vitros 5600 automated chemistry platform.…”

“…The drug can inhibit UDP glucuronosyltransferase (UGT)1A1 and organic anion‐transporting polypeptide (OATP)1B1, which can result in indirect hyperbilirubinemia 2,4 . Additionally, eltrombopag's yellow to reddish color in solution has been shown to positively interfere with bilirubin assessment on spectrophotometric measurements 5 . These two confounding factors pose a challenge in the results interpretation and require additional laboratory testing to further confirm whether high bilirubin results are physiological or due to drug interference.…”

An Eltrombopag Red (Plasma) Alert

“…It was subsequently recognized that the discoloration was due to initiation of the thrombopoietin receptor agonist eltrombopag 4 days earlier at 150 mg daily. Eltrombopag can turn plasma reddish-brown (and sclerae icteric-appearing at normal bilirubin levels) at the high doses used to treat aplastic anemia [1]. This may cause false alarm (as in this case) and can also interfere with common spectrophotometric bilirubin assays, resulting in falsely low bilirubin measurements [1, 2].…”

“…Eltrombopag can turn plasma reddish-brown (and sclerae icteric-appearing at normal bilirubin levels) at the high doses used to treat aplastic anemia [1]. This may cause false alarm (as in this case) and can also interfere with common spectrophotometric bilirubin assays, resulting in falsely low bilirubin measurements [1, 2]. It may also interfere with measurement of other metabolites, and interference for both bilirubin and other metabolites is assay and instrument-specific.…”