Glycosylation Profiling of the Neoplastic Biomarker Alpha Fetoprotein through Intact Mass Protein Analysis.

Elevated serum alpha-fetoprotein (AFP) can be observed in liver cirrhosis and hepatocellular carcinoma (HCC). The glycosylation patterns of AFP have been shown to differentiate these conditions, with AFP glycoforms with core fucosylation (AFP-L3) serving as a malignancy risk predictor for HCC. We have developed a method to detect endogenously present AFP proteoforms and to quantify the relative abundance of AFP-L3 glycoforms (AFP-L3%) in serum samples. This method consists of immune enrichment of endogenous AFP, followed by liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS) intact protein analysis of AFP. Data are available via ProteomeXchange with identifier PXD038606. Based on the AFP profiles in authentic patient serum samples, we have identified that the frequently observed AFP glycoforms without core fucosylation (AFP-L1) are G2S2 and G2S1, and common AFP-L3 glycoforms are G2FS1 and G2FS2. The intensities of glycoforms in the deconvoluted spectrum are used to quantify AFP-L3% in each sample. The method evaluation included reproducibility, specificity, dilution integrity, and comparison of AFP-L3% with a lectin-binding gel shift electrophoresis (GSE) assay. The AFP-L1 and AFP-L3 proteoforms were reproducibly identified in multiple patient serum samples, resulting in reproducible AFP-L3% quantification. There was considerable agreement between the developed LC-HRMS and commercial GSE methods when quantifying AFP-L3% (Pearson r = 0.63) with a proportional bias.

Impact of the Opioid Epidemic on Drug Testing.

BACKGROUND: This review provides a description of how the opioid epidemic has impacted drug testing. METHODS: Four major service areas of drug testing were considered, including emergency response, routine clinical care, routine forensics, and death investigations. RESULTS: Several factors that the opioid epidemic has impacted in drug testing are discussed, including specimens, breadth of compounds recommended for testing, time to result required for specific applications, analytical approaches, interpretive support requirements, and examples of published practice guidelines. CONCLUSIONS: Both clinical and forensic laboratories have adapted practices and developed new testing approaches to respond to the opioid epidemic. Such changes are likely to continue evolving in parallel with changes in both prescription and nonprescription opioid availability and use patterns, as well as emerging populations that are affected by the "waves" of the opioid epidemic.

Collection Tubes Can Cause False Elevations in Occupational and Clinical Evaluation of Antimony Exposure.

Occupational exposure to antimony has become rare in the past decades due to antimony mine closures and technological improvement in antimony processing plants in the USA. Although antimony's ubiquitous presence in plasticwares does not pose known health risk, it can present as a potential contaminant to antimony analysis for occupational exposure assessment. To understand the level of antimony contamination from plastic collection devices, we evaluated two different whole-blood plastic collection tubes that are routinely used for trace and toxic element assessment: royal blue BD Vacutainer® EDTA tube and Greiner VACUETTE® trace elements sodium heparin tube. We analyzed how different fill volumes may impact the concentrations of antimony detected. Although both collection tubes can introduce antimony contaminations to nitric acid and neutral buffer rinse, the Greiner heparin tube introduces a significantly lower amount of antimony to freshly collected whole-blood samples compared to the BD EDTA tube. When patients' samples are collected with BD EDTA tubes, they would exhibit elevated antimony concentrations that can be interpreted as potential antimony exposure. We conclude that the royal blue BD EDTA plastic tube is not suitable to evaluate blood antimony levels, and laboratories need to validate their own alternative sources when the glass tubes are not available.

Have you caught that outlier yet? Evaluate the utility of repeat testing in nutritional and toxic element assessment.

Repeat testing is routinely required by regulatory bodies as a measure to rule out contamination in trace elements and heavy metal analysis, especially when the initial analysis result is outside the reference interval. However, its clinical utilities in detecting analytical measurement outliers have not been systematically evaluated in different clinical testing scenarios. In this study, we present an extensive evaluation of repeat testing and its comparison with the initial analysis in four serum and plasma trace element assays performed by inductively coupled plasma mass spectrometry. We demonstrate that the patient population distributions for these elements differ significantly from the reference interval established by healthy individuals. Accordingly, a significant proportion of the patient specimens would require repeat testing when using reference intervals as the threshold to perform repeat analysis. Crucially, comparison of the first analysis and repeat analysis reveals the limited utility of performing repeat measurements. The relative differences between the first and second measurements are consistent with the observed analytical imprecision of the assay and the likelihood of detecting actual analytical outliers is very low.

Evaluate the comparability of two automated liquid handling systems for clinical toxicology assays.

Automated liquid handling (ALH) platforms are increasingly implemented in clinical laboratories to improve analytical reproducibility and replace manual handling during sample analysis. In clinical toxicology laboratories, ALH platforms are primarily utilized to perform sample preparation and extraction prior to subsequent analysis by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). When performing analysis with complex human biological matrices, verifying the performance characteristics of ALH platforms is required to ensure the assays' accuracy and reproducibility. Here, we evaluated and compared the analytical performances of Perkin Elmer JANUS® and Tecan Fluent® ALH systems in parallel, based on their performance in two toxicology assays designed to identify and quantify various opiates, semisynthetic opiates, and their metabolites. The comparability of the instrument platforms was evaluated by comparing assay analytical measuring range, total analytical imprecisions, and patient samples measurement when the ALH platforms are incorporated as part of the clinical assay's workflow. We have shown that both ALH platforms meet quality and performance criteria suitable for clinical toxicology assays. Nevertheless, the two platforms exhibit biases when measuring unknown patient samples. Such variations in their analytical performances may cause discrepancies when comparing results obtained from two different ALH platforms. In conclusion, it is important to consider how variations in ALH platform performances can affect patient results interpretation when implementing them in clinical laboratories.

Demand for Interpretation of a Urine Drug Testing Panel Reflects the Changing Landscape of Clinical Needs; Opportunities for the Laboratory to Provide Added Clinical Value.

BACKGROUND: The direct detection of drugs and metabolites in urine using a targeted panel offers sensitive and specific detection in comparison to the traditional approach to urine drug testing (screen with reflex of samples with positive results to confirmation testing). The purpose of this study was to evaluate changes in clinical demand for the laboratory to provide interpretation of patient adherence and abstinence, based on reconciling laboratory results and individual patient medication information provided by the clinician. The shifts in toxicology testing likely reflect the inherent complexity of the data and associated interpretation. METHODS: Retrospective testing results associated with a targeted urine drug panel and its related interpretation were collected from our laboratory. We examined the associated testing volume and positivity rates of each reported analyte over 5 consecutive years (2015-2019). Requests from clinicians for consultation regarding this test and use of interpretive comments for the most recent year (2019), as well as access to publicly available educational resources over two years (2018-2019) were collected. RESULTS: The changes in test ordering patterns demonstrate shifting of clinical demands for toxicology testing, by increased adoption of a targeted panel for which laboratory-based interpretation is provided. Positivity rates reflect national shifts in controlled substance prescriptions. Several consultative services were accessed by clinicians suggesting interest and need. CONCLUSION: The value of clinical urine drug testing is improved by providing laboratory-based result interpretation and consultative services.