Due to the limited sensitivities of stool-based microscopy and/or culture techniques for Strongyloides stercoralis, the detection of antibodies to this intestinal nematode is relied upon as a surrogate for determining exposure status or making a diagnosis of S. stercoralis infection. Here, we evaluated three immunoassays, including the recently released InBios Strongy Detect IgG enzyme-linked immunosorbent assay (ELISA) (InBios International, Inc., Seattle, WA), the SciMedx Strongyloides serology microwell ELISA (SciMedx Corporation, Denville, NJ), and the luciferase immunoprecipitation system (LIPS) assay performed at the National Institutes of Health (NIH), for their detection of IgG antibodies to S. stercoralis. A total of 101 retrospective serum samples, previously submitted for routine S. stercoralis antibody detection using the SciMedx assay, were also evaluated by the InBios and LIPS assays. The qualitative results from each assay were compared using a Venn diagram analysis, to the consensus result among the three assays, and each ELISA was also evaluated using the LIPS assay as the reference standard. By Venn diagram analysis, 65% (66/101) of the samples demonstrated perfect agreement by all three assays. Also, the numbers of samples considered positive or negative by a single method were similar. Compared to the consensus result, the overall percent agreement of the InBios, SciMedx, and LIPS assays were comparable at 87.1%, 84.2%, and 89.1%, respectively. Finally, the two ELISAs performed analogously but demonstrated only moderate agreement (kappa coefficient for the two assays, 0.53) with the LIPS assay. Collectively, while the two commercially available ELISAs perform equivalently, neither should be used independently of clinical evaluation to diagnose strongyloidiasis.
Background Therapeutic drug monitoring (TDM) for immunosuppressive (ISP) drugs is an important component of organ and tissue transplantation and chemotherapy management. Whole blood is the specimen type for the quantitative analysis of cyclosporine A, everolimus, sirolimus, and tacrolimus. Some alternatives to venous whole blood samples have the potential to reduce blood volume requirements and simplify sample collection and transport. Methods The feasibility of ISP drug (cyclosporine A, everolimus, sirolimus, and tacrolimus) monitoring via microsampling device (MitraTM, Neoteryx) was assessed by comparing venous samples collected and extracted using microsampling device to conventional extraction procedure. Analysis was performed by LC-MS/MS. Results All analytes were found to be linear across the measurement range of 22.7–937.0 ng/mL (18.9–779.1 nmol/L) for cyclosporine A, 2.3–44.2 ng/mL (2.4–46.1 nmol/L) for everolimus, 2.2–47.2 ng/mL (2.4–51.6 nmol/L) for sirolimus, and 2.2–41.3 ng/mL (2.7–51.4 nmol/L) for tacrolimus. Imprecision was evaluated at concentrations within the therapeutic range and was found to be 10.1% and 5.8% for cyclosporine A, 10.0% and 10.0% for everolimus, 15.0% and 11.9% for sirolimus, and 6.8% and 8.5% for tacrolimus. Method comparison (n = 30 for each analyte, using Deming regression) indicated slopes of 1.08, 1.02, 0.90, and 1.15 and intercepts of −12.8 ng/mL (−10.7 nmol/L), 0.8 ng/mL (0.8 nmol/L), 1.5 ng/mL (1.7 nmol/L), and −0.3 ng/mL (−0.3 nmol/L) for cyclosporine A, everolimus, sirolimus, and tacrolimus, respectively. Conclusions This feasibility study demonstrates that precision and bias of ≤15% can be achieved for microsampling-based ISP monitoring.
Background When choosing an analog internal standard (IS) in a quantitative LC-MS/MS assay, careful selection and thorough verification are important for developing an accurate quantitative assay. The IS is a critical component in quantitative mass spectrometry because it is used to normalize results by compensating for variations in sample preparation and instrument performance. Here we present the results of our investigation in the selection process for a structural analog IS (SA-IS) to be used in the quantification of 6-methylmercaptopurine (6-MMP) in cytolysed red blood cell (RBC). Methods A cocktail solution of 9 SA-ISs including the isotopically labeled structural isomer and the 6-MMP stable isotope-labeled IS (SIL-IS) was spiked into cytolysed RBC controls and patient samples. Linearity, accuracy, sensitivity, precision, run stability, method comparison, and reinjection reproducibility experiments were performed. Ion suppression was also assessed by T-infusing the cocktail solution. Results All analogs were linear from 100 to 1200 ng/mL 6-MMP with acceptable precision and sensitivity by use of a spiked blank lysate. Method comparison plots of 6-MMP concentrations in patient samples had excellent agreement for 2 of the SA-ISs (i.e., the isotopically labeled structural isomer and an SA-IS with an added methyl group) when compared to the SIL-IS. Halogen-substituted analogs (i.e., Cl and Br) also met the criteria as an acceptable IS. However, 2 of the selected SA-ISs having substituted amine moieties showed unacceptable performance, with ≥15% bias when compared to the SIL-IS. Conclusion There are many parameters to consider when determining if an analog will be a good IS choice, and the approaches highlighted in this article can be applied to the selection of SA-IS in the development of other LC-MS/MS assays.
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