Impact of sample processing on the measurement of circulating microparticles: storage and centrifugation parameters
Background:
Microparticles (MPs) have been shown to be markers of cellular activation and interactions. Pre-analytical conditions such as the centrifugation protocol and sample storage conditions represent an important source of variability in determining MPs values. The objectives of this study were to evaluate the influence of sample storage conditions and centrifugation speed and temperature on the determination of MPs in plasma.
Methods:
Citrate-anticoagulated blood samples obtained from 21 healthy subjects were centrifuged under four different protocols involving different speeds (2500 g or 1500 g) and temperatures (4 °C or 20 °C) to isolate platelet-poor plasma (PPP). The number of MPs in fresh and frozen-thawed PPP were analyzed by flow cytometry, and MPs-mediated procoagulant activity was determined by a thrombin generation test and phospholipid-dependent procoagulant tests.
Results:
The number of MPs and their procoagulant activity were affected by freeze-thaw cycling and centrifugation speed but not by centrifugation temperature. Sample freezing increased MPs number (six-fold) and thrombin generation (four-fold), and decreased clotting time (two-fold). Low centrifugation speed caused an increase in MPs number and a parallel increase in MP-mediated procoagulant activity.
Conclusions:
Sample storage conditions and centrifugation speed are important processing conditions affecting MPs number and activity. Before any study, the protocol for MPs isolation should be optimized to ensure a reliable characterization of MPs, which could provide important information for diagnostic purposes and for understanding the pathogenesis of diseases.
Insights
Pre-analytical conditions significantly impact microparticle (MP) measurements. Freezing samples and using lower centrifugation speeds increase MP numbers and activity, highlighting the need for optimized protocols for reliable diagnostic and research findings.
Area of Science:
- Hematology
- Biotechnology
- Clinical Chemistry
Background:
- Microparticles (MPs) are key indicators of cellular activation and interactions.
- Pre-analytical variables like centrifugation and storage critically influence MP quantification.
- Standardizing MP measurement is crucial for accurate clinical and research applications.
Purpose of the Study:
- To investigate the impact of sample storage conditions on MP determination.
- To evaluate the effects of centrifugation speed and temperature on MP analysis.
- To assess how pre-analytical factors influence MP number and procoagulant activity.
Main Methods:
- Plasma samples from 21 healthy subjects were processed using varied centrifugation speeds (1500g vs 2500g) and temperatures (4°C vs 20°C).
- Platelet-poor plasma (PPP) was analyzed for MP count via flow cytometry.
- MPs-mediated procoagulant activity was assessed using thrombin generation and phospholipid-dependent assays.
Main Results:
- Freeze-thaw cycles significantly increased MP counts (6-fold) and thrombin generation (4-fold), while reducing clotting time (2-fold).
- Lower centrifugation speed (1500g) resulted in higher MP numbers and enhanced procoagulant activity.
- Centrifugation temperature did not significantly affect MP levels or activity.
Conclusions:
- Sample storage (freezing) and centrifugation speed are critical pre-analytical factors influencing MP quantification and activity.
- Optimized isolation protocols are essential for reproducible and reliable MP characterization.
- Accurate MP analysis holds potential for diagnostic advancements and disease pathogenesis understanding.
