Centrifugation is a crucial step impacting microparticle measurement

Dear Editor

We thank Drs. Françoise Dignat-George, Jean-Marie Freyssinet, and Nigel S. Key (Françoise Dignat-George, Jean-Marie Freyssinet, Nigel S. Key. Centrifugation is a crucial step impacting microparticle measurement. ultracentrifugation would be quite challenging in the hospital setting. Platelets 2009; CPLA-2009–0006) for their comments regarding our study [1]. Blood collection, sample processing, transportation, and centrifugation conditions have significant impact on microparticle measurement and have been insufficiently addressed in the literature. We appreciate their argument that this is of high importance for proper microparticle enumeration in basic research and clinical studies.

We would like to respond to the authors concerns that our modifications to the pre-analytical steps may have resulted in the loss of microparticles, possibly accounting for the very low microparticle counts reported. During our initial review of the literature, we found a range of centrifugation conditions used to pellet microparticles: 10,000 × g for 10 minutes [2], 14,000 × g for 2 minutes [3], through 100,000 × g for 1 hour [4],[5]. Unable to find a consensus, we chose to use 13,000 × g for 2 minutes to sediment microparticles for two reasons. First, in developing our protocol modifications, we were interested in microparticles as vesicular or lamellar structures released by several types of blood cells and having procoagulant activity [6],[7]. Exosomes are a particular subtype of microparticles produced by the endocytic-lysosomal system of several cell lines after activation, but their contribution to procoagulant activity remains unclear [8]. Using a relatively low-speed centrifugation (13, 000 × g for 2 minutes) would reduce exosome contamination, given the published literature reports isolation of exosomes at centrifugation conditions of 70,000 × g for 1 hour to 100,000 × g for 1 hour [9],[10]. In fact, a review by Shet [11] acknowledges that the high-speed centrifugation may, in fact, contain exosomes. Second, the goal of conducting the original study was to identify a method feasible for clinical application, particularly in multi-center studies. Any method requiring ultracentrifugation would be quite challenging in the hospital setting.

Although we are confident that our protocol collects microparticles as determined by annexin V positivity and particle size gating (Figure 5 [1]), the low-speed centrifugation, as the authors point out, may be insufficient in collecting all microparticles. As a result, our microparticle counts, as reported in our study, may be lower than previously reported. However, we believe that the absolute microparticle count may be less significant as compared to the relative increase (or decrease) in disease conditions. For this reason, we strongly advocate including normal controls in each specific protocol. For each parameter tested in our study, data analyses were performed on parallel samples obtained from the same donor but tested under different conditions. Furthermore, we also believe that the concerns about potential platelet contamination (as a confounding factor) at 13,000 × g centrifugation have been addressed by the size and granulation gating (Figure 5 [1]). Finally, the low microparticle counts observed in our study may also be due to the fact that all samples were obtained from healthy adult volunteers.