ABSTRACT
Conditions of routine blood tube transportation may significantly vary in terms of generated agitation. One recent study investigated the impact of gentle and strong agitation by comparison with com-plete absence of any agitation.28 There was no evidence of a signifi-cant impact of gentle agitation on MV counts and procoagulant ac-tivity. In contrast, strong agitation induces a critical, although highly variable, increase in MV counts and procoagulant potential. To bet-ter mimic the real situation in hospitals, blood tubes transported ei-ther unsupported as usual in double plastic bags or purposely kept immobile using transportation boxes were compared to reference
tubes that remained motionless in the lab (in both horizontal or vertical positions). In contrast to what is observed when red blood cells are vortexed,53 results of this study based on high-sensitivity FCM did not demonstrate a significant impact on the generation of EryMVs by gentle agitation. Importantly, the impact of agitation in-duced by a common hospital transportation system was similar to the effect of strong agitation. Interestingly, transporting blood tubes in a vertical rather than a horizontal position, via the use of special transportation boxes, also limited the extent of in vitro MV genera-tion.28 The data identify agitation during transportion as a crucial step and suggest that such transportation boxes should be used for MV studies when patients’ samples are not directly collected in the local environment. In this study, as blood samples were collected from healthy volunteers directly in the laboratory, it was also possible to study the impact of delay between blood sampling and the first centrifugation step with a short delay of 5 min serving as the reference control sample. In such conditions, results showed that a 1 h time delay before the first centrifugation influences total and PMV counts as well as procoagulant activity, consistent with a PMV increase already described in other studies.50,52,54 However, the increase during the two first hours remains moderate in comparison to other preanalytical parameters such as centrigution speed or agitation during transportation, suggesting that a short delay, which remains compatible with current laboratory practice, may be acceptable, provided control and test samples are handled in an identical manner. Noteworthy, we did not evidence a significant change on EryMVs with such a short time delay, contrary to what may happen during storage of blood units for several days.53 5. 5 Plasma Processsing and Microvesicle
Preparation An appropriate centrifugal speed should be applied for plasma preparation in order to eliminate cells, including platelets, to freeze the samples regardless of the potential of cellular fragmentation during thawing and consequent artifactual formation of MVs. According to the Clinical and Laboratory Standards Institute (CLSI), plasma with a platelet count less than 10 × 109/L is obtained by centrifuging blood
at 1,500 g for 15 min at room temperature (RT).55 To obtain platelet-poor plasma (PPP), the CLSI also recommends recentrifuging the plasma for another 10 min at 1,500 g. However, platelet-free plasma (PFP) obtained by performing two-step centrifugation, each at 2,000 g for 10 min, can also significantly reduce the level of any residual platelets.20 Some studies indicate that MV counts measured by conventional FCM are lower in PFP than in PPP, suggesting that a percentage of MVs could also be lost during this step.29,56 However, careful removal of the majority of platelets is also required prior to freezing the samples. The ideal equilibrium remains to be found. The literature indicates that centrifugation conditions, speed, and time all vary widely among different studies and laboratories (see Table 5.1, adapted from Ref. 20). The initial centrifugation intended to generate cell-free plasma is 1,500-2,500 ¥ g for 15-20 min in most studies. However, a significant number of platelets also persist after a single centrifugation step.18 An additional centrifugation step of 13,000 ¥ g for 2 min ensures the generation of PFP.2,57,58 Following this second centrifugation, the plasma should be carefully aspirated but leaving the bottom 1 cm undisturbed.59 Because this high-speed centrifugation is not always easily available in some laboratories, a recent study compared the currently recommended protocol of 1,500 g 15 min + 13,000 g 2 min57,60 to a more routine laboratory-adapted protocol, that is, 2 ¥ 2,500 g 15 min.61 The effect of these two different centrifugation protocols, both intended to entirely remove platelets, was therefore compared on healthy donors. When fresh samples were analyzed, no significant difference was observed between both protocols. In contrast, after a freeze-thaw cycle, data showed a significant increase in MV counts and procoagulant activity with the protocol of 1,500 g 15 min + 13,000 g 2 min compared to the 2 ¥ 2,500 g 15 min protocol.28 This was also confirmed by another team.62 Although both protocols create PFP with very low levels of contaminating platelets, the routine laboratory-adapted protocol generates less artifactual PMV after freezing, due to a lower level of residual platelets. The more complex tube handling involved in the high-speed centrifugation may partly explain such a difference. Altogether, these studies demonstrated the best efficiency of the routine laboratory-adapted protocol in terms of platelet removal efficiency and thus stability after deep-freeze storage.
