Proteomic Characterization of Transfusable Blood Components: Fresh Frozen Plasma, Cryoprecipitate, and Derived Extracellular Vesicles via Data-Independent Mass Spectrometry.

Extracellular vesicles (EVs) are a heterogeneous collection of particles that play a crucial role in cell-to-cell communication, primarily due to their ability to transport molecules, such as proteins. Thus, profiling EV-associated proteins offers insight into their biological effects. EVs can be isolated from various biological fluids, including donor blood components such as cryoprecipitate and fresh frozen plasma (FFP). In this study, we conducted a proteomic analysis of five single donor units of cryoprecipitate, FFP, and EVs derived from these blood components using a quantitative mass spectrometry approach. EVs were successfully isolated from both cryoprecipitate and FFP based on community guidelines. We identified and quantified approximately 360 proteins across all sample groups. Principal component analysis and heatmaps revealed that both cryoprecipitate and FFP are similar. Similarly, EVs derived from cryoprecipitate and FFP are comparable. However, they differ between the originating fluids and their derived EVs. Using the R-package MS-DAP, differentially expressed proteins (DEPs) were identified. The DEPs for all comparisons, when submitted for gene enrichment analysis, are involved in the complement and coagulation pathways. The protein profile generated from this study will have important clinical implications in increasing our knowledge of the proteins that are associated with EVs derived from blood components.

Extracellular vesicles in fresh frozen plasma and cryoprecipitate: Impact on in vitro endothelial cell viability.

BACKGROUND: Transfusion-related acute lung injury (TRALI) remains a major contributor to transfusion-associated mortality. While the pathogenesis of TRALI remains unclear, there is evidence of a role for blood components. We therefore investigated the potential effects of fresh frozen plasma (FFP), cryoprecipitate, and extracellular vesicles (EVs) derived from these blood components, on the viability of human lung microvascular endothelial cells (HLMVECs) in vitro. METHODS: EVs were isolated from FFP and cryoprecipitate using size-exclusion chromatography and characterized by nanoparticle tracking analysis, western blotting, and transmission electron microscopy. The potential effects of these blood components and their EVs on HLMVEC viability (determined by trypan blue exclusion) were examined in the presence and absence of neutrophils, either with or without prior treatment of HLMVECs with LPS. RESULTS: EVs isolated from FFP and cryoprecipitate displayed morphological and biochemical properties conforming to latest international criteria. While FFP, cryoprecipitate, and EVs derived from FFP, each reduced HLMVEC viability, no effect was observed for EVs derived from cryoprecipitate. CONCLUSION: Our findings demonstrate clear differences in the effects of FFP, cryoprecipitate, and their respective EVs on HLMVEC viability in vitro. Examination of the mechanisms underlying these differences may lead to an improved understanding of the factors that promote development of TRALI.

Investigation of the effect of pre-analytical factors on particle concentration and size in cryoprecipitate using nanoparticle tracking analysis.

BACKGROUND: Cryoprecipitate is used primarily to replenish fibrinogen levels in patients. Little is known about the presence of micro- or nano-sized particles in cryoprecipitate. Therefore, we aimed to quantify these particles and investigate some pre-analytical considerations. MATERIALS AND METHODS: Particle concentration and size distribution were determined in 10 cryoprecipitate units by nanoparticle tracking analysis (NTA). The effects of freeze-thawing cryoprecipitate and 0.45 µm filtration with either regenerated cellulose (RC) or polytetrafluoroethylene (PTFE) filters before sample analysis were examined. RESULTS: Neither the size nor concentration of particles were affected by two freeze/thaw cycles. PTFE filtration, but not RC filtration, significantly reduced particle mean and mode size compared to RC filtration and mode size compared to unfiltered cryoprecipitate. The 10 cryoprecipitate units had an average particle concentration of 2.50 × 1011 ± 1.10 × 1011 particles/mL, a mean particle size of 133.8 ± 7.5 nm and a mode particle size of 107.9 ± 11.1 nm. CONCLUSION: This study demonstrated that preanalytical filtration of cryoprecipitate units using RC filters was suitable for NTA. An additional freeze/thaw cycle did not impact NTA parameters, suggesting that aliquoting cryoprecipitate units prior to laboratory investigations is suitable for downstream analyses.

Mobilizing serum factors and immune cells through exercise to counteract age-related changes in cancer risk.

An increasing body of evidence suggests that age-related immune changes and chronic inflammation contribute to cancer development. Recognizing that exercise has protective effects against cancer, promotes immune function, and beneficially modulates inflammation with ageing, this review outlines the current evidence indicating an emerging role for exercise immunology in preventing and treating cancer in older adults. A specific focus is on data suggesting that muscle- derived cytokines (myokines) mediate anti-cancer effects through promoting immunosurveillance against tumourigenesis or inhibiting cancer cell viability. Previous studies suggested that the exercise-induced release of myokines and other endocrine factors into the blood increases the capacity of blood serum to inhibit cancer cell growth in vitro. However, little is known about whether this effect is influenced by ageing. Prostate cancer is the second most common cancer in men. We therefore examined the effects of serum collected before and after exercise from healthy young and older men on the metabolic activity of androgen-responsive LNCaP and androgen-unresponsive PC3 prostate cancer cells. Exercise-conditioned serum collected from the young group did not alter cell metabolic activity, whereas post-exercise serum (compared with pre-exercise serum) from the older men inhibited the metabolic activity of LNCaP cancer cells. Serum levels of candidate cancer-inhibitory myokines oncostatin M and osteonectin increased in both age groups following exercise. Serum testosterone increased only in the younger men postexercise, potentially attenuating inhibitory effects of myokines on the LNCaP cell viability. The data from our study and the evidence in this review suggest that mobilizing serum factors and immune cells may be a key mechanism of how exercise counteracts cancer in the older population.