Impact of sample processing on the measurement of circulating microparticles: storage and centrifugation parameters.
Author(s): Vila-Liante V, Sánchez-López V, Martínez-Sales V, Ramón-Nuñez LA, Arellano-Orden E, Cano-Ruiz A, Rodríguez-Martorell FJ, Gao L, Otero-Candelera R
Publication: Clin Chem Lab Med, 2016, Vol. 54, Page 1759-1767
PubMed ID: 27155005 PubMed Review Paper? No
Purpose of Paper
This paper investigated the effects of centrifugation protocol and frozen storage on microparticle counts and function in platelet-poor plasma (PPP).
Conclusion of Paper
Microparticle counts and thrombin levels were higher and clotting times were shorter in both fresh and frozen PPP isolated at 1500 x g for 30 min when compared to PPP isolated by double-centrifugation at 2500 x g for 10 min. In addition, phospholipid levels in frozen PPP were higher when isolated at 1500 x g for 30 min than by double-centrifugation at 2500 x g for 10 min. Centrifugation temperature (4˚C versus 20˚C) did not affect microparticle counts, thrombin levels, or clotting time. Analysis of microparticle-free plasma (MPFP) revealed that only 6% of thrombin remained after additional centrifugation, indicating that coagulant activity is dependent on the presence of microparticles.
Studies
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Study Purpose
This study investigated the effects of centrifugation protocol and frozen storage on microparticle counts and function in platelet-poor plasma (PPP). Venous blood obtained with a 21-gauge needle was collected into a sodium-citrate Vacutainer after discarding the first 3 mL from 21 healthy donors (10 male, 11 female) who had not taken medication within the last 15 days. Platelet-poor plasma (PPP) was obtained by centrifugation at 1500 x g for 30 min or double-centrifugation at 2500 x g for 10 min at 4°C and 20°C. To avoid cell contamination, collection of PPP was stopped at 1 cm above the buffy coat. PPP was either analyzed immediately for microparticle counts and coagulant activity or stored at –80°C for 1 month. Thrombin levels and clotting time were measured for all specimens using an automated analyzer and phospholipid concentration was determined by auto analyzer in frozen/thawed PPP. Additional aliquots (500 µl) of PPP were further centrifuged at 25,000 x g for 120 min at 4°C to obtain microparticle-free plasma (MPFP) as a negative control.
Summary of Findings:
Centrifugation at 4°C rather than at 20°C did not affect microparticle counts, thrombin levels, or clotting time in fresh or frozen PPP. Fresh PPP isolated by centrifugation at 1500 x g for 30 min rather than by double-centrifugation at 2500 x g for 10 min had higher microparticle counts and thrombin levels and shorter clotting time when centrifuged at either 4°C (P<0.05, P<0.001, and P<0.01, respectively) or 20°C (P<0.05, P<0.01, and P<0.05, respectively). Similarly, frozen PPP isolated by centrifugation at 1500 x g for 30 min rather than by double-centrifugation at 2500 x g for 10 min had higher thrombin levels, regardless of centrifugation temperature (P<0.01 for 4°C and P<0.01 for 20°C) and higher phospholipid levels when centrifuged at -20°C (P<0.01). Microparticle counts and thrombin levels were higher and clotting times shorter in frozen PPP than fresh PPP, regardless of centrifugation protocol (P<0.001, all). Analysis of MPFP revealed that only 6% of thrombin remained after centrifugation at 25,000 x g for 120 min at 4°C, indicating that coagulant activity is dependent on the presence of microparticles.
Biospecimens
Preservative Types
- None (Fresh)
- Frozen
Diagnoses:
- Normal
Platform:
Analyte Technology Platform Cell count/volume Flow cytometry Protein Clinical chemistry/auto analyzer Lipid Clinical chemistry/auto analyzer Pre-analytical Factors:
Classification Pre-analytical Factor Value(s) Storage Storage temperature Room temperature
Frozen
Biospecimen Aliquots and Components Centrifugation Multiple speeds compared
Multiple temperatures compared
Different number of centrifugation steps compared