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Liquid blood and RBC fractionation

Plasma or serum are frequently used in biomarker discovery for the detection of signalling proteins such as cytokines. Our discoveries show that in concentration terms, the plasma or serum component provides a minor part of the signalling involved in inflammation. We are taking the unique position of looking at plasma in conjunction with a variety of red blood cell isolated components, and well as additional cellular components such as PBMCs.

By investigating the RBC lysate, the RBC membranes, or the protein release from intact RBCs or isolated membranes, we have found that RBCs are a major reservoir of more than 80 signalling and inflammatory proteins in blood. Of note, the levels of many of these proteins are higher in RBCs than they are in the plasma (Table 1). In addition, the proteins that we have identified as associated with RBCs have a wide range of functions, from pro-inflammatory, regulating inflammation (anti-inflammatory), hormones, receptors, and enzymes (Table 2).

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Table 1.   Concentration of a few example cytokines in plasma and red blood cells respectively per millilitre of whole blood.

We are aiming to use this research to explore the clinical utility of our novel discoveries involving red blood cells (RBCs) as reservoirs for signalling molecules. Proteins such as cytokines are candidate markers for diagnostics and for monitoring therapeutic responses because of their roles in inflammation, immune response, and repair. Our research indicates that these RBCs are acting as a ‘buffer’ of signalling proteins against extremes of concentration, thereby modulating immune activity. Dysfunction in this system during disease may contribute to progression and may also be an ideal target for diagnostic development.

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Table 2.   Number of detected proteins in RBC lysates per functional category out of total assayed analytes.

Cancer

Inflammatory biomarkers are associated with worse outcomes in numerous cancers and have been a focus of cancer research in many studies.  We are initially assessing plasma and RBC biomarker levels (cytokines and cancer specific markers) in male and female cancer patients.  We have observed differences in the RBC lysate protein profile isolated from pre-treatment cancer patients compared to healthy controls. A number of these differences were not detectable in the plasma fraction, but were observed in the RBC lysate (Figure 1). These results highlight the value in analysing multiple fractions of blood in disease. We are planning to compare these marker profiles with the distribution of inflammatory cells including T-cell subsets including CD8+ T-cells, CD4+ T-cells, natural killer cells, regulatory T cells, and immature myeloid cells by flow cytometry.

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Figure 1. Concentration of cytokines in the plasma or red blood cell lysates in one millilitre of whole blood collected from healthy individuals or individuals with cancer.

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