Assessing the Preanalytical Variability of Plasma and Cerebrospinal Fluid Processing and Its Effects on Inflammation-Related Protein Biomarkers

Jesse Huang,Tomas Olsson,Gunn Jönsson,Ingrid Kockum,Mohsen Khademi,Fredrik Piehl,Örjan Lindhe

doi:10.1016/j.mcpro.2021.100157

Jesse Huang, Tomas Olsson + Show 5 more

Open Access

https://doi.org/10.1016/j.mcpro.2021.100157

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Abstract

Proteomics studies are important for the discovery of new biomarkers as clinical tools for diagnosis and disease monitoring. However, preanalytical variations caused by differences in sample handling protocol pose challenges for assessing biomarker reliability and comparability between studies. The purpose of this study was to examine the effects of delayed centrifuging on measured protein levels in plasma and cerebrospinal fluid (CSF). Blood from healthy individuals and patients with multiple sclerosis along with CSF from patients with suspected neurological disorders were left at room temperature for different periods (blood: 1, 24, 48, 72 h; CSF: 1 and 6 h) prior to centrifuging. Ninety-one inflammation-related proteins were analyzed using a proximity extension assay, a high-sensitivity multiplex immunoassay. Additional metabolic and neurology-related markers were also investigated in CSF. In summary, many proteins, particularly in plasma, had increased levels with longer delays in processing likely due in part to intracellular leakage. Levels of caspase 8, interleukin 8, interleukin 18, sirtuin 2, and sulfotransferase 1A1 increased 2-fold to 10-fold in plasma after 24 h at room temperature. Similarly, levels of cathepsin H, ectonucleoside triphosphate diphosphohydrolase 5, and WW domain containing E3 ubiquitin protein ligase 2 differentiated in CSF with <6 h delay in processing. However, the rate of change for many proteins was relatively consistent; therefore, we were able to characterize biomarkers for detecting sample handling variability. Our findings highlight the importance of timely and consistent sample collection and the need for increased awareness of protein susceptibility to sample handling bias. In addition, suggested biomarkers may be used in certain situations to detect and correct for preanalytical variation in future studies.

Highlights

Proteomics studies are important for the discovery of new biomarkers as clinical tools for diagnosis and disease monitoring
Certain hemolytic factors may be autocatalytic as shown by the increased levels of intracellular axis inhibitor 1 (AXIN-1) and signal-tranducing adaptor molecule binding protein [14] while the resulting accumulation of inflammatory mediators may cause increased expression of inflammatory cytokines in immune cells, such as IL-8 and macrophage inflammatory protein-1 alpha [7]
The results showed relative reliability as markers of processing delay, there was a general trend of overestimation, which could be due to differences in other handling parameters between studies

Summary

Introduction

Proteomics studies are important for the discovery of new biomarkers as clinical tools for diagnosis and disease monitoring. The complex composition of blood, the diverse cellular proteomes that are susceptible to contaminating blood-related media, increases the risk for sample handling bias further amplified by highsensitivity technologies [2, 5] These challenges are common in multicenter studies where sample processing can be delayed, often inconsistently, because of site-specific logistical restrictions. We examine the preanalytical effects of handling time prior to sample processing on the levels of inflammation-related proteins in both plasma and CSF using high-sensitivity proximity extension technology [3, 13]. Changes are predictable and we have suggested models for correcting discrepancies in protein levels because of sample handling, for the purpose of limiting false positives and reducing cross-study variation

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