Cell Biology, Immunology and Biochemistry (basic and preclinical research)
Melinda M. Dean, B.App.Sc.PhD (she/her/hers)
School of Health, University of the Sunshine Coast, Petrie, Australia; Research and Development, Australian Red Cross Lifeblood, Brisbane, Australia; Faculty of Health, Queensland University of Technology, Brisbane, Australia
Petrie, Queensland, Australia
Background: Intraoperative cell salvage (ICS) is an autologous blood conservation technique that allows the collection and reinfusion of a patient's blood that is lost during surgery. ICS can reduce the need for allogenic blood and has advantages including: i) reduced risk of transfusion reactions, ii) improved immune competence, and iii) reduced post-operative infection risk. However, ICS is not a passive process and blood undergoes processing, centrifugation and filtration. Extracellular vesicles (EVs) are small, lipid bound particles that are released by cells and are present in biological fluids including blood. The number, cellular origin and function of EVs have been characterised in allogenic blood components, but our understanding of the changes in EVs during the ICS process remains limited.
Study
Design/Methods:
Methods: Nineteen elective orthopaedic patients, with the potential for significant blood loss, were recruited and consented to receive ICS (ethics: RBWH [HREC/17/QRBW/685). Thirteen of these patients received autologous ICS blood. ICS blood was sampled following initial collection and processing (pre-filtration) and before reinfusion into the patient (post-filtration). For characterisation of EVs, samples of ICS blood were centrifuged twice (3000g, 15 minutes) to obtain the supernatant which was stored at -80°C. Samples of thawed ICS supernatant were diluted in phosphate buffered saline, then analysed using the Nanosight NS300 to determine the concentration and size distribution of EVs(). EV concentration was determined, and pre- and post- filtration compared using Wilcoxon matched pairs test. Mean and mode of particle size were determined, and pre- and post- filtration results compared using paired T-test. n=10/13 patients analysed.
Results/Findings:
Results: ICS blood contained a median of 7.37 x 109 EVs/mL (IQR 5.51 x 109 – 9.89 x 109) in the pre-filtered product and 5.90 x 109 EVs/mL (IQR 4.34 x 109 – 1.19 x 1010) in the filtered product for reinfusion into the patient. Overall, there was no difference in EV concentration before and after filtration, although considerable differences in the EV concentration were observed between individuals. The mean particle size and distribution was also similar before and after filtration of the ICS blood (prefiltration size 149 nm (SD 11) and post-filtration size 148 nm (SD 10); mode size distribution 129nm (SD 11) for pre-filtered ICS blood and 127nm (SD 14)).
Conclusions:
Conclusions: EVs were present in ICS blood. Our preliminary results indicate standard filtration of the ICS blood prior to reinfusion did not result in significantly altered EV size, distribution nor concentration, although considerable inter-individual differences were evident. In the next phase of the project, the cellular origin of the EVs will be determined by flow cytometry and the profile of EVs in ICS will be compared to allogenic blood during routine storage.
Importance of research: Extracellular vesicles (EVs) are small, lipid bound particles that are released by cells and are present in biological fluids including blood. EVs play a role in cell-to-cell communication by transferring biological content comprising of proteins, lipids, nucleic acids to recipient cells. The clinical significance of EVs in cell salvaged and allogeneic blood is not fully understood, and limited knowledge exists about their cellular origin and types.