Microparticle counts in platelet-rich and platelet-free plasma, effect of centrifugation and sample-processing protocols.

Author(s): Chandler WL

Publication: Blood Coagul Fibrinolysis, 2013, Vol. 24, Page 125-32

PubMed ID: 23249614 PubMed Review Paper? No

Purpose of Paper

Conclusion of Paper

Studies

1. Study Purpose

   The purpose of this study was to determine the effects of centrifugation steps in the production of PFP and microparticle pellets and a single freeze-thaw cycle on microparticle counts. PRP was obtained from citrated blood by centrifugation at 100 g for 10 min. PPP was obtained from PRP by centrifugation at 1500 g for 20 min. PFP was obtained from PPP by centrifugation at 13,000 g for 2.3 min (PFP1) or 1500 g for 20 min (PFP2). Microparticles and microparticle poor plasma were obtained by centrifugation of PFP2 at 18000 g for 30 min. Microparticles were washed twice with HEPES buffer and centrifuged at 18,000 g for 30 min. An aliquot of PRP, PPP, PFP1, PFP2, microparticle poor plasma, and pre-wash and post-washing microparticles were assayed immediately and again after overnight storage at -80 degrees C.

   Summary of Findings:

   The median microparticle count in PRP was 733,000/uL with microparticles ranging in size from 0.35 um to more than 1 um. The intensity of the annexin V and/or CD41 staining of microparticles in PRP increased with increasing size of the microparticles. Centrifugation of PRP to obtain PPP resulted in loss of 95% of the platelets, 79% of the annexin V and CD41 positive microparticles (Procoagulant microparticle, PrPMPs), 90% of the annexin V positive CD41 negative microparticles (annexin microparticles, AMPs) and 36% of the annexin V negative and CD41 positive microparticles (platelet microparticles, PMPs). Centrifugation at 13,000 g for 2.3 min to produce PFP1 or 1500 g for 20 min to produce PFP2 led to the loss of 99% of the platelets, 84% of the PrPMPs, 93% of the AMPs, and 58% of the PMPs compared to PRP, with only slightly larger losses observed in PFP1 than PFP2. Further, centrifugation steps to produce microparticles or wash microparticles led to non-significant loss of PrPMPs, AMPs, and PMPs. Subjecting PRP to a single freeze-thaw cycle led to 20-, 9- and 4-fold higher PrPMP, AMP and PMP counts, respectively, and a single centrifugation step was not sufficient to overcome the freeze-thaw effect. The effects of a single freeze-thaw cycle on PRP were smaller with only a 2-fold increase in PrPMP, a 60% increase in AMP, and no change in PMP. Freeze-thawing of PFP1 or PFP2 only led to a 20% increase in AMP counts, and freeze-thawing of microparticles increased PrPMP and AMP counts by 30-50%.

   Biospecimens

   • Bodily Fluid - Plasma

   Preservative Types

   • None (Fresh)
   • Frozen

   Diagnoses:

   • Normal

   Platform:

   Analyte	Technology Platform
   Cell count/volume	Flow cytometry

   Pre-analytical Factors:

   Classification	Pre-analytical Factor	Value(s)
   Storage	Freeze/thaw cycling	0 cycles 1 cycle
   Biospecimen Aliquots and Components	Blood and blood products	Platelet-poor plasma Platelet-rich plasma Microparticle free plasma Microparticles Platelet free plasma
   Biospecimen Preservation	Type of fixation/preservation	Frozen None (fresh)
   Biospecimen Aliquots and Components	Centrifugation	Multiple durations compared Multiple speeds compared

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