Enrichment of plasma in platelets and extracellular vesicles by the counterflow to erythrocyte settling

Abstract

In order to prepare optimal platelet and extracellular vesicle (EV)-rich plasma for the treatment of chronic temporal bone inflammation, we studied effects of centrifugation parameters on redistribution of blood constituents in blood samples of 23 patients and 20 volunteers with no record of disease. Concentrations of blood cells and EVs were measured by flow cytometry. Sample content was inspected by scanning electron microscopy. A mathematical model was constructed to interpret the experimental results. The observed enrichment of plasma in platelets and EVs after a single spin of blood depended on the erythrocyte sedimentation rate, thereby indicating the presence of a flow of plasma that carried platelets and EVs in the direction opposite to settling of erythrocytes. Prolonged handling time correlated with the decrease of concentration of platelets and larger EVs in platelet and EV-rich plasma (PVRP), R = −0.538, p = 0.003, indicating cell fragmentation during the processing of samples. In further centrifugation of the obtained plasma, platelet and EV enrichment depended on the average distance of the sample from the centrifuge rotor axis. Based on the agreement of the model predictions with observations, we propose the centrifugation protocol optimal for platelet and EV enrichment and recovery in an individual sample, adjusted to the dimensions of the centrifuge rotor, volume of blood and erythrocyte sedimentation rate.

Variability in Platelet-Rich Plasma Preparations Used in Regenerative Medicine: A Comparative Analysis

“…The other six studies processed the collected blood with centrifugation using diverse types of centrifuges (horizontal or fixed angle rotors) and conditions (temperature or nontemperature controlled). One study used single centrifugation with variable G values [ 17 ]; three studies used double centrifugation with variable G values [ 9 , 13 , 18 ]; another study used single centrifugation while using a commercial kit [ 13 ]. All these methods followed pipetting to collect the PRP from centrifuged samples.…”

“…The goal is still in distant sight. We lack complete and transparent large-scale data [ 1 , 9 ]. The major reason for variations in the data and its comparability is due to variable approaches in preparation methodology such as the volume of blood collected for processing, method of collection (apheresis vs venous blood), source of sample (venous blood vs umbilical cord blood), different equipment, single centrifugation vs double centrifugation methods, normal temperature processing vs temperature-controlled settings, automation vs manual aspects, studies using fresh PRP vs Freeze-thawed PRP, usage of anticoagulants, and additives.…”

“…It proposed a mathematically extrapolated model of obtaining enriched PRP and extracellular vesicles suspension by centrifugation after considering the variables. Its recommended procedure could manufacture a product high enough to merit the criteria for moderately concentrated PVRP by the PAW classification [ 9 ]. This study has numerous limitations.…”

“…This consequently adds variability to the outcomes of therapeutic procedures. The variability can be attributed to two main factors, PRP preparation procedure and patient-related factors [2,[5][6][7][8][9]. However, the preparatory procedures can be standardized, bringing them closer to the level of pharmaceuticals, to bring the product variability into an acceptable range.…”

“…The difference in the quality of PRP preparations arises from the fact that various setups tend to use variations of the same procedure to increase the quality of their PRP. The variations are seen in the amount of venous blood collected for processing, tubes used for collection, use of anticoagulant, centrifugation instruments, amount of G force used, time of centrifugation, temperature control, usage of additives/activators in PRP, pipetting methods (there is a human error involved here), the volume of PRP, storage methods, and duration of storage [2,3,[9][10][11]. We have seen significant efforts in the direction of standardization of biologics, especially by the mechanization of the preparatory procedure using robotics and standardized kits.…”