Determination of temporal reproducibility and variability of cancer biomarkers in serum and EDTA plasma samples using a proximity extension assay

Abstract

Background Proximity extension assay (PEA) is a novel antibody-based proteomic technology. Sparse data have been published concerning the matrix effect of serum vs. ethylenediamine tetraacetic acid (EDTA) plasma and the reproducibility of results obtained using PEA technology. Methods We analyzed samples with the PEA-based 92-plex Olink® immuno-oncology (I-O) assay. To estimate the matrix effect, we analyzed paired serum and EDTA plasma samples from 12 patients with biliary tract cancer. To evaluate the reproducibility, we used data from 7 studies, where 6–8 serum samples from patients with pancreatic cancer were used as bridging samples on 3 versions of the panel over a 2.5-years period. Results For the study of serum vs. plasma, 80 proteins were evaluable. The mean serum to EDTA plasma ratio ranged from 0.41–3.01. For 36 proteins, the serum and plasma values were not comparable due to high variability of the ratio, poor correlation, or possible concentration effect. For the bridging samples, the mean intra-study inter-assay coefficient of variation (CV) ranged from 11.3% to 26.1%. The mean inter-study CV was 42.0% before normalization and 26.2% after normalization. Inter-study results were well correlated (r ≥ 0.93), especially for studies using the same version of the panel (r ≥ 0.99). Conclusion For 44 of 92 proteins included in the Olink® I-O panel, the variation between results obtained using serum and EDTA plasma was constant and results were well correlated. Furthermore, samples could be stored for several years and used on different versions of the same PEA panel without it effecting results.

Protein Signatures and Individual Circulating Proteins, including IL-6 and IL-15, Associated with Prognosis in Patients with Biliary Tract Cancer

“…Two different blood matrices were used in the discovery cohort (serum) and first-line validation cohort (EDTA plasma). This can affect the protein level measured using the PEA platform [51,52]. Therefore, it is also a strength of the study that we were able to replicate our results across blood matrices.…”

Development and validation of circulating protein signatures as diagnostic biomarkers for biliary tract cancer

“…To compare serum and plasma samples, plasma results were adjusted using a previously identified serum/plasma ratio. 33 …”

“…34 The primary set of protein signatures was identified using the 81 proteins + CA19-9. The second set of signatures was generated using CA19-9 + 42 proteins previously identified to have a consistent serum to plasma variation 33 . The signatures were generated using a multi-step strategy based on a similar approach previously employed by our group in a cohort of patients with pancreatic ductal adenocarcinoma.…”

The Edge Effect in High-Throughput Proteomics: A Cautionary Tale

“…Here we sought to investigate technical batch effects within multiwell plates in a high-throughput plasma protein digestion protocol. Interplate variation has previously been characterized in LC-MS/MS and other biomarker discovery platforms such as as Olink (Uppsala, Sweden) and SomaLogic (Colorado, USA); − however, intraplate variation is underreported in comparison. Čuklina and colleagues evaluated intraplate variation introduced by a liquid handling system and methods to reduce this effect in silico , but to our knowledge, no previous work has investigated intraplate temperature gradients as a potential source of technical variation in proteomics.…”