Aims: An automated method for the measurement of blood tacrolimus on volumetric absorptive microsampling (VAMS) devices was developed. Materials & methods: VAMS devices prepared by the automated method were compared with those prepared by the existing manual method (n = 284; mean concentration: 8.0 μg/l; range: 0.6-18.1). Results: The performance of both methods was comparable. Passing-Bablok regression demonstrated an acceptable correlation (y = -0.449 + 1.06x). Bland-Altman analysis demonstrated acceptable agreement (mean bias: -0.007 μg/l; standard deviation: 1.536). Automation reduced operator touch time by 40 min (48-sample batch). Conclusion: Automated preparation of VAMS devices reduced touch time and improved process consistency, facilitating high-throughput testing and transformation of existing laboratory workflows. Automation did not improve precision for VAMS devices but did so for liquid blood samples.

Background: We analyzed differences in protein concentrations in human blood serum depending on the tube material and the immunoassay platform used. Materials & methods: Blood samples from study participants were collected in glass and polypropylene tubes (n = 292). Serum concentrations of six proteins (BDNF, IGF-1, VEGF-A, TGF-β1, MCP-1 and IL-18) were assessed by using ELISAs (all biomarkers), as well as a novel fully automated immunoassay platform (all but IGF-1, n = 211). Bland-Altman analyses were conducted to investigate intrasample variability of protein concentrations. Results: Tube comparison resulted in mean biases of between -0.45 and -70.64%. Platform comparison revealed mean biases of between 21.04 and -128.10%. Conclusion: Protein concentrations can vary significantly depending on the types of tube and immunoassay used, with protein-specific differences.

Aim: Pharmacokinetic evaluation of cefotaxime in neonates is currently a challenge due to the large volume requirement of blood for its analysis by existing methods. A dried blood spot (DBS) based method is the best alternative. Materials & methods: We validated an HPLC method for estimation of cefotaxime from DBS and plasma. Extraction employed a simple procedure using acetonitrile and buffer. Selective separation of cefotaxime was achieved on a C8 column using gradient programming. Results & conclusion: The linearity of the method ranged from 2 to 200 μg/ml with acceptable precision and accuracy for both plasma and DBS. Hematocrit was not affecting the assay accuracy. A strong correlation and interchangeability observed with the plasma method proves its clinical validity for application to PK evaluations.

Aim: We present a novel methodology to compare results between distinct immunogenicity assays, performed by two laboratories, for the same biotherapeutic. Materials & methods: Human serum pools from clinical trials were generated to provide representative immunogenicity titers. Pools were evaluated at two laboratories in a blinded fashion to assess the effect of assay format and laboratory change on clinical interpretation of immunogenicity results. Results: The laboratories validated two different assay formats and demonstrated comparable sensitivity and drug tolerance. Overall, the comparisons in assay format and laboratory ensured a comparable ability to detect treatment-emergent antidrug antibodies for a biotherapeutic. Conclusion: We have established an approach, using pooling of patient samples, that allows for the interlaboratory comparisons without creating duplicative results.

Aim: The aim of this study was to detect misfolded Cu/Zn SOD1 as a potential biomarker for amyotrophic lateral sclerosis (ALS). Materials & methods: Two ultrasensitive immunodetection assays were developed for the quantification of total and misfolded SOD1. Results: The detection of total and misfolded SOD1 was possible in human serum and cerebrospinal fluid. Total SOD1 was increased in cerebrospinal fluid from ALS patients. Misfolded SOD1 had low and variable expression in both control and ALS patient samples. Conclusion: These assays hold promise for improving our understanding of ALS and its detection, and could lead to more effective treatment options in the future. Further studies in larger cohorts are now required.

Background: Measurement of plasma oxalate (POx) is challenging, but critical, for management of patients with primary hyperoxaluria type 1. A novel LC-MS/MS assay was developed, validated and used to quantify POx in patients with primary hyperoxaluria type 1. Methods: Samples (100 μl of plasma in K2EDTA) were spiked with internal standard (13C2-labeled oxalic acid), acidified and cleaned by protein precipitation before analysis using anion HPLC-ESI-MS/MS. The assay was validated with a quantitation range of 0.500-50.0 μg/ml (5.55-555 μmol/l). All parameters successfully met acceptance criteria, including 15% (20% at lower limit of quantification) for accuracy and precision. Conclusion: This assay has advantages over previously published POx quantitation methods, was validated in accordance with regulatory guidelines and accurately determined POx levels in humans.

Previously designed flow cytometry panels have provided a framework to analyze T-cell activation; however, few provide an extensive view of lymphocyte populations, and none are optimized for murine models. This article describes a panel designed specifically to assess the expression of activation and exhaustion markers in expanding lymphocyte populations in tumor-bearing mice across two distinct genetic backgrounds: BALB/c and C57BL/6. This comprehensive panel enables the assessment of multiple functional states and immune checkpoint markers across cytotoxic CD8+ T cells, helper and regulatory CD4+ T cells and natural killer cells in murine whole blood, lymph nodes and tumor.

Aim: Isobutyrylcarnitine (IBC) is a possible biomarker for hepatic OCT1, as IBC plasma concentrations are reduced when OCT1 is inhibited. An accessible, characterized assay is needed to quantitate IBC in human plasma. Materials & methods: A triple quadrupole MS surrogate matrix assay for the quantitation of IBC was characterized to support a first-in-human study. Results: An assay for IBC quantitation was fully characterized for accuracy, precision, selectivity and parallelism. IBC was measured in a clinical study and the data were correlated to the in vitro model prediction. Conclusion: A triple quadrupole-based assay for IBC should broaden the monitoring of IBC for OCT1 inhibition in early clinical trials, generating the data needed to establish IBC as a valid biomarker.

Background: Pharmacokinetic evaluation is essential for the precise dosing of ceftriaxone in neonates. There is a need for developing a sensitive, affordable and convenient analytical method that can estimate ceftriaxone from dried blood spot (DBS) samples of neonates. Method: An HPLC-UV method was developed and validated as per ICH M10 for ceftriaxone from DBS and plasma using an Inertsil-ODS-3V column with gradient elution. DBS samples were extracted with methanol. Clinical validation was performed using neonatal samples. Results: The developed plasma- and DBS-based-HPLC method were linear from 2-700 μg/ml and 2-500 μg/ml, respectively, for ceftriaxone. Bland-Altman analysis indicated a strong interconvertibility between the plasma and DBS assays. Conclusion: Observed concentrations in clinical samples were comparable to the predicted concentrations, proving the clinical validity of the method.