CytoFlex Flow Cytometer Application Notes

Without centrifuging the blood sample, it is reasonable to expect a large percentage of intact RBCs in the RBC units after three months of refrigerated storage. However, the percentage of particles identified as microparticles indicates that the cells in storage were releasing exocytic microparticles, either before or on the way to hemolysis. The functional significance of these RBC-derived microparticles has been studied, and the microvesicles are implicated in poor patient prognoses. 1 Tips for success • This protocol does not require ultracentrifugation • To minimize photobleaching of PE and FITC conjugates, perform all incubation steps in the dark • Preservation of RBCs for varying amounts of time may yield more/fewer microparticles Conclusions Banking blood is medically necessary, and as the need for human blood products increases, the strain on the blood supply will also increase. This evidence, generated with the CytoFLEX Flow Cytometer, indicates that RBCs stored under refrigeration have a finite usable lifespan, after which the cells release microparticles which may adversely impact patients. Detecting these >150 nm microvesicles with flow cytometry is a true advance in the field, and enables deeper research into the genesis of bioactive microparticles and methods to prevent the deterioration of the banked blood supply. Notes The results shown here represent data generated on the Beckman Coulter CytoFLEX Flow Cytometer. Due to differences in the performance of makes and models of flow cytometers, the authors cannot guarantee similar results with the use of other flow cytometers.

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References 1.

Rubin, O., Canellini, G., Delobel, J., Lion, N., & Tissot, J.-D. (2012). Red Blood Cell Microparticles: Clinical Relevance. Transfusion Medicine and Hemotherapy, 39(5), 342–347. http://doi.org/10.1159/000342228

2. Barteneva, N. S., Fasler-Kan, E., Bernimoulin, M., Stern, J. N., Ponomarev, E. D., Duckett, L., & Vorobjev, I. A. (2013). Circulating microparticles: square the circle. BMC Cell Biology, 14, 23. http://doi.org/10.1186/1471-2121-14- 23 3. Donadee, C., Raat, N. J. H., Kanias, T., Tejero, J., Lee, J. S., Kelley, E. E., … Gladwin, M. T. (2011). Nitric Oxide Scavenging by Red Cell Microparticles and Cell Free Hemoglobin as a Mechanism for the Red Cell Storage Lesion. Circulation, 124(4), 465–476. http://doi.org/10.1161/CIRCULATIONAHA.110.008698 4. Wisgrill, L., Lamm, C., Hartmann, J., Preißing, F., Dragosits, K., Bee, A., Hell, L., Thaler, J., Ay, C., Pabinger, I., Berger, A. and Spittler, A. (2016), Peripheral blood microvesicles secretion is influenced by storage time, temperature, and anticoagulants. Cytometry, 89: 663–672. doi:10.1002/cyto.a.22892

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