Recovery and composition of microparticles after snap-freezing depends on thawing temperature

Abstract

Consent regarding the correct processing and storage of blood microparticles is lacking and different protocols for the freeze-thaw cycle exist. Therefore, three different thawing procedures were evaluated regarding their influence on recovery and composition of microparticles. Microparticles were prepared by TRAP-6 or A23187 stimulation of platelet-rich plasma from smokers and nonsmokers (n = 8), from an endothelial cell line or directly obtained from platelet-free plasma of septic patients (n = 5). After snap-freezing in liquid nitrogen platelet-free samples were thawed at 37 degrees, at room temperature or on ice and staining of microparticles was carried out with Annexin V-Cy5 as well as fluorescein isothiocyanate (FITC) or phycoerythrin (PE) labelled antibodies or isotype controls. Microparticle concentrations were determined by means of Trucount tubes. Recovery of platelet microparticles was significantly reduced when samples were thawed on ice (P = 0.001 for all antigens) compared with the two other techniques (P = 0.6 for 37 degrees and P = 0.7 for room temperature, respectively) whereas microparticles of endothelial origin appeared to be less influenced. There was a strong trend towards altered microparticle composition as microparticle counts detected by CD41 staining showed a stronger decrease on ice than Annexin V enumeration (P = 0.07). For microparticle detection thawing of snap-frozen, platelet-free plasma samples should be carried out at room temperature or at 37 degrees C in a water bath but not on ice.

Multicolor Flow Cytometry and Nanoparticle Tracking Analysis of Extracellular Vesicles in the Plasma of Normal Pregnant and Pre-eclamptic Women1

“…Some studies suggest changes in NormP compared to NonP [31,32,45,55,57], and in PE compared to NormP [28,31,32,45,55,[57][58][59][62][63][64][65], whereas others report no change [27,28,31,32,45,55,57,58]. These inconsistencies may reflect preanalytical methodological differences that are thought to affect quantification of EVs in plasma [36,[66][67][68][69][70][71][72].…”

Extracellular Vesicles in Renal Diseases

“…Several studies have looked at the effect of preanalytic steps, including blood and urine collection, anticoagulant use, sample centrifugation, sample freezing, thawing, and storage. 30,[35][36][37][38] Societies for EV research have provided guidelines and recommendations for proper processing of EVs. 39,40 There are specific challenging issues regarding the isolation of uEVs.…”

Application of Flow Cytometry in the Studies of Microparticles

“…Another aspect of MP isolation that has a major impact on MP quality is sample collection, preparation and storage [Dey-Hazra et al, 2010, Trummer et al, 2009, Shah et al, 2008. Unfortunately, here again no uniform protocols exist that would state the proper way to address these issues.…”

“…As with different isolation protocols, the sample collection, preparation and/or storage depend on individual needs and settings characteristic of the study. However, there seems to be a general understanding that freshly obtained samples are the best to work with and that multiple "freezing and thawing" has a substantial impact on both the MP number as well as the surface determinant expression, which suggest that, if possible, should be avoided (see section 2.1.1) [Dey-Hazra et al, 2010, Trummer et al, 2009, Shah et al, 2008.…”