Candidate High-Resolution Mass Spectrometry-Based Reference Method for the Quantification of Procalcitonin in Human Serum Using a Characterized Recombinant Protein as a Primary Calibrator

Abstract

Procalcitonin (PCT) is a widely used biomarker for rapid sepsis diagnosis and antibiotic stewardship. Variability of results in commercial assays has highlighted the need for standardization of PCT measurements. An antibody-free candidate reference measurement procedure (RMP) based on the isotope dilution mass spectrometry and protein calibration approach was developed and validated to quantify PCT in human serum. The method allows quantification of PCT from 0.25 to 13.74 μg/L ( R > 0.998) with extension up to 132 μg/L after dilution of samples with PCT concentration above 13.74 μg/L. Intraday bias was between −3.3 and +5.7%, and interday bias was between −3.0 and −0.7%. Intraday precision was below 5.1%, and interday precision was below 4.0%. The candidate RMP was successfully applied to the absolute quantification of PCT in five frozen human serum pools. A recombinant PCT used as a primary calibrator was characterized by high-resolution mass spectrometry and amino acid analysis to establish traceability of the results to the SI units. This candidate RMP is fit to assign target values to secondary certified reference materials (CRMs) for further use in external quality assessment schemes to monitor the accuracy and comparability of the commercially available immunoassay results and to confirm the need for improving the harmonization of PCT assays. The candidate RMP will also be used to evaluate whether the correlation between the candidate RMP and immunoassays is sufficiently high. Overall, this candidate RMP will support reliable sepsis diagnosis and guide treatment decisions, patient monitoring, and outcomes.

Immunoaffinity LC–MS/MS Quantification of the Sepsis Biomarker Procalcitonin Using Magnetic- and Polystyrene-Bead Immobilized Polyclonal Antibodies

“…The standard material was considered sufficiently pure for this study with a focus on method development. A more stringent characterization may be required for future clinical sample analysis as demonstrated by Huynh et al 29 The choice of peptides is a critical factor in ensuring that quantitative data relate to intact endogenous PCT and that the quantified level is not affected by the presence of degradation products or by processing into the known PCT-derived hormones calcitonin and katacalcin. For the selection of peptides that are representative of intact PCT, an initial comparison of different proteolytic enzymes was undertaken.…”

“…Interestingly, in a similar comparison between an LC−MS/MS-based assay and an immunoassay, the immunoassay data were found to provide PCT concentrations 18 to 55% higher. 29 ■ CONCLUSIONS Using an immunoaffinity LC−MS/MS quantitative method with a polyclonal antibody, a highly sensitive and robust method for detecting PCT in human serum has been developed. A comparison of Latex and magnetic beads for antibody immobilization demonstrated the superior performance of magnetic beads, particularly regarding the lower propensity for nonspecific binding of matrix proteins.…”

“…The employed monoclonal antibodies were generated for capturing selected tryptic PCT peptides with an LLOQ of 0.1 ng/mL for recombinant PCT. 27 Huynh et al developed an antibody-free LC−MS/MS method on an Orbitrap Q-Exactive utilizing PRM mode with the capture of tryptic PCT peptides by twostage solid-phase extraction (SPE) and with peptide 28 and protein 29 calibrators to adjust for variations in SPE recoveries. An LLOQ of 0.25 ng/mL was reported.…”

Development of Certified Reference Material for Amino Acids in Dried Blood Spots and Accuracy Assessment of Disc Sampling

“…Clinical applications require the use of matrix CRMs based on the intended human samples, such as serum, plasma, etc., for the validation and quality assurance of specific measuring systems. 2 − 4 …”

“…Among various types of CRMs, matrix CRMs are used to verify measurement procedures and quality control materials, in addition to playing a role as secondary calibrators. Clinical applications require the use of matrix CRMs based on the intended human samples, such as serum, plasma, etc., for the validation and quality assurance of specific measuring systems. − …”