Abstract A034: Addressing the challenge of pre-analytical variables in circulating protein biomarker analysis for liquid biopsy
Authors: Miller Rachel M , Li Jing 
Published: November 2024

Abstract
While liquid biopsy using plasma proteins is a promising avenue for early cancer detection, diagnosis, prognosis, and disease monitoring, the clinical translation of plasma proteomics has been limited due to the complexity of the proteome and the impact of pre-analytical variation on its integrity. In recent years, advancements in plasma proteomic technologies, such as Olink, have helped address the challenge of proteome complexity. However, challenges with pre- analytical variables have persisted. During blood collection and whole blood storage, ex vivo blood cell activation and lysis can occur, releasing proteins into the plasma and obscuring relevant in vivo abundances. This is especially true for cancer where many key biomarkers are expressed in white blood cells and platelets. To address these issues, Streck developed Protein Plus BCT, which stabilizes blood cells and minimizes plasma proteome changes following prolonged whole blood storage. To demonstrate its functionality and compatibility with Olink assays, blood was collected from 5 donors into EDTA, ACD-A, and Protein Plus BCT. Plasma was isolated at draw time (&lt; 2 hours) and following 24, 72, and 120 hours of ambient whole blood storage using a double spin protocol (per Instructions for Use). Plasma samples were analyzed using an Olink Explore 384-plex panel. After just 24 hours of whole blood storage, plasma isolated from EDTA or ACD-A had 4.2 and 2.2 times more proteins that are significantly elevated in abundance relative to draw time, respectively, than plasma isolated from Protein Plus BCT. For the key cancer biomarker IL-8, which is associated with worse clinical outcomes and poor response to immunotherapy, Protein Plus BCT maintains draw-time levels such that the mean NPX difference relative to draw time never exceeds -0.18, or a 0.88-fold change for up to 120 hours. Comparatively, samples collected into EDTA and ACD-A show dramatic increases in IL-8 levels with mean NPX differences of 6.34 and 6.62, respectively, corresponding to 80.1- and 98.3-fold increases after 120 hours of whole blood storage. For Galectin-3, an inflammatory biomarker elevated in many cancer types (i.e., lung, breast, and ovarian), the mean NPX difference relative to draw time for samples drawn into EDTA is 1.46 after 24 hours and increases to 2.27 after 120 hours of whole blood storage (a 2.75-fold and 4.8-fold increase, respectively). In samples collected into Protein Plus BCT, the mean NPX difference, relative to draw time, for Galectin-3 never exceeds 0.05 (a 1.04-fold increase) even after 120 hours of whole blood storage. Taken together, these data suggest that Protein Plus BCT extends the storage of whole blood at ambient temperature without compromising the integrity of the sample or the proteomic results, providing researchers and assay developers valuable flexibility in the transport, storage, and processing of their samples. Protein Plus BCT is for Research Use Only. Not for use in diagnostic procedures. Protein Plus BCT should only be used for research or the development of new assays.
Citation Format: Rachel M Miller, Jing Li. Addressing the challenge of pre-analytical variables in circulating protein biomarker analysis for liquid biopsy [abstract]. In: Proceedings of the AACR Special Conference: Liquid Biopsy: From Discovery to Clinical Implementation; 2024 Nov 13-16; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2024;30(21_Suppl):Abstract nr A034.