Steps.

The physical stress of these centrifugation steps often leads to
Steps. The physical stress of these centrifugation steps often leads to platelet activation and subsequent granule content release which alters results if looking at platelet contents via protein or RNA. The dog has quickly gained recognition as an important animal model for many human diseases including cancer. Not only are dogs of veterinary importance from a companion animal standpoint, but also develop many of the same naturally occurring cancers and diseases as humans [8-10].Our goal was to provide a fast and reliable method for producing ultra-pure platelet samples suitable for proteomic and transcriptomic analysis from clinically relevant (3? mL) whole blood volumes with minimal platelet activation.barrier centrifugation resulted in a 24,574-fold decrease of the leukocyte to platelet ratio from whole blood.Reduction of leukocyte contamination by bead separationThroughout optimization of the OptiPrep density barrier purification technique, CD45-labeled Dynabeads were tested as a method to remove additional residual leukocytes. An average 95-fold decrease in leukocyte contamination was seen for samples containing high levels of contaminating 3-(2,2,2-Trifluoroethoxy)aniline hydrochloride cells. In samples containing an already low amount of contaminating leukocytes such as those seen with the OptiPrep density barrier centrifugation, there was no added benefit of continuing with the additional purification step using the leukocyte-specific beads due to additional platelet loss from the bead separation process.Purity, contamination, and assessment of activation by flow cytometryResultsDecreased leukocyte contamination using OptiPrep density barrierFollowing centrifugation of whole blood with a 1.063 g/mL density barrier, distinct layering was observed (Figure 1). After optimization of the OptiPrep density barrier centrifugation and platelet layer extraction steps, collection of 1 mL of the cloudy second layer consistently yielded a highly concentrated platelet fraction with an average purity of 99.99 ?0.01 (mean ?standard deviation) by manual count, and average platelet recovery of 51.56 ?9.66 . These platelet fractions were highly pure with only 100.07 ?48.77 leukocytes per 108 platelets. The original whole blood samples had 1-phenyl-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole cell counts, on average, of 2,457,405 ?746,380 leukocytes per 108 platelets. Therefore, on average, optimized densityFlow cytometric analysis of the platelet samples obtained after OptiPrep density barrier centrifugation indicated, on average, 99.47 ?0.21 CD61-positive events and 0.19 ?0.04 CD45-positive events. After incubation with CD45-labeled Dynabeads the samples had, on average, 98.84 ?0.03 CD61-positive events and 0.27 ?0.03 CD45-positive events (Figures 2 and 3). Platelet sample activation was evaluated using Annexin V and P-selectin PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/13867361 labeling by flow cytometry (Figures 4 and 5). After initial centrifugation with the 1.063 g/mL density barrier, the platelet sample had 0.66 annexin positivity and 0.4 PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/14445666 P-selectin positivity, and after incubation with CD45labeled beads there was 3.00 Annexin positivity. Because of the platelet function inhibitory effect of the PECT anticoagulant, whole blood was collected in citrate to acquire PRP for use as positive controls by activatingFigure 1 Density barrier layering before and after centrifugation with whole blood.Trichler et al. BMC Veterinary Research 2013, 9:144 http://www.biomedcentral.com/1746-6148/9/Page 3 ofFigure 2 Flow cytometric analyses of optimized platelet samples after density barrier centrifu.