RESUMO
The introduction of tandem mass spectrometry (MS/MS) to clinical laboratories and the advent of expanded newborn screening (NBS) were crucial changes to public health programs worldwide. Speed, robustness, accuracy, selectivity, and specificity of analysis are all requirements of expanded NBS and are needed to minimize false positive results risks, to possibly eliminate false negatives, and to improve the positive predictive value of NBS. In this study, we firstly evaluated the analytical performances of the RenataDX Screening System, a fully integrated flow-injection MS/MS (FIA-MS/MS) IVD system for high-throughput dried blood spot (DBS) analysis in a routine NBS laboratory. Since a choice of several commercial NBS kits is available, we sought to compare NeoBaseTM 2 (PerkinElmer®) and MassChrom® (Chromsystems) non-derivatized kits on the RenataDX platform by evaluating their analytical performances. Moreover, we verified the degree of correlation between data obtained by the two different NBS MS/MS kits by FIA-MS/MS of over 500 samples. Our data suggest that both methods correlate well with clinically insignificant differences that do not impact the NBS result. Finally, while NeoBase™ 2 offers an easier and faster sample preparation, MassChrom® provides a cleaner sample extract which empirically should improve instrument reliability.
RESUMO
Bile acids (BA) play a pivotal role in cholesterol metabolism. Their blood concentration has also been proposed as new prognostic and diagnostic indicator of hepatobiliary, intestinal, and cardiovascular disease. Liquid chromatography tandem mass spectrometry (LC-MS/MS) currently represents the gold standard for analysis of BA profile in biological samples. We report here development and validation of a LC-MS/MS technique for simultaneously quantifying 15 BA species in serum samples. We also established a reference range for adult healthy subjects (n = 130) and performed a preliminary evaluation of in vitro and in vivo interference. The method displayed good linearity, with high regression coefficients (>0.99) over a range of 5 ng/mL (lower limit of quantification, LLOQ) and 5000 ng/mL for all analytes tested. The accuracies were between 85-115%. Both intra- and inter-assay imprecision was <10%. The recoveries ranged between 92-110%. Each of the tested BA species (assessed on three concentrations) were stable for 15 days at room temperature, 4 °C, and -20 °C. The in vitro study did not reveal any interference from triglycerides, bilirubin, or cell-free hemoglobin. The in vivo interference study showed that pools obtained from hyper-cholesterolemic patients and hyper-bilirubinemic patients due to post-hepatic jaundice for benign cholestasis, cholangiocarcinoma and pancreatic head tumors had clearly distinct patterns of BA concentrations compared with a pool obtained from samples of healthy subjects. In conclusion, this study proposes a new suitable candidate method for identification and quantitation of BA in biological samples and provides new insight into a number of variables that should be taken into account when investigating pathophysiological changes of BA in human diseases.
