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

Abstract

This study provides the first estimates of microparticle numbers in platelet-rich plasma (PRP) from normal individuals, closer to in-vivo levels, using higher-resolution flow cytometry. We measured platelet (CD41+) and annexin V+ microparticles in fresh and frozen aliquots of PRP, platelet-poor plasma, platelet-free plasma (PFP), and microparticles isolated by high-speed centrifugation. PRP from healthy individuals contained 730,000/μl total microparticles based on light-scattering measurements. A median of 27,000/μl microparticles in PRP were of platelet origin and 120,000/μl annexin V+, and of these, 24,000/μl were dual-positive procoagulant platelet microparticles. Double centrifugation of PRP removed 99% of platelets, but also 80% of annexin V+ CD41+, 93% of annexin V+ CD41-, and 58% of annexin V- CD41+ microparticles. Loss of microparticles with centrifugation varied from individual to individual. Microparticle counts after isolation by centrifugation and double washing were not significantly different than counts in the original PFP sample, but lower than in PRP. Freeze-thawing of PFP had no effect on platelet microparticle counts, but slightly increased annexin V+, CD41- counts. Freeze-thawing of isolated washed microparticles resulted in a 30-50% increase in annexin V+ microparticles. PRP contains large numbers of cellular microparticles, including platelet and annexin V+ microparticles, which are lost to varying degrees when PRP is double centrifuged to remove platelets. Microparticles remaining in PFP can be recovered by high-speed centrifugation without loss compared to the original PFP sample. Freeze-thawing has variable effects on microparticle counts depending on the sample preparation used.

Measurement of microvesicle levels in human blood using flow cytometry

“…On fresh samples, a second centrifugation at 13,000g has been reported to decrease endothelial and annexin V1 microvesicles by about 50% in some studies (22,23), but not others (14,20,24). If plasma samples are going to be frozen, a single centrifugation step is inadequate to remove all platelets from the sample, double centrifugation is required to prevent 100% to 1,500% increases in platelet and annexin V1 microvesicle counts due to fragmentation of residual platelets in the sample (15,20,(25)(26)(27).…”

“…To prepare platelet free plasma the sample is typically spun twice at 2,000g for 15 min, a total of 60,000g-min of sedimentation, 60 times more than the preparation of PRP. This additional centrifugation of whole blood or platelet rich plasma removes cells and platelets but also reduces red cell, platelet, and annexin V1 microvesicles to a variable extent from sample to sample (16,17,20). Figure 1 shows total light scatter particles and platelet-derived (CD411) microvesicles in platelet rich plasma versus platelet microvesicles in platelet free plasma.…”

“…Figure 1 shows total light scatter particles and platelet-derived (CD411) microvesicles in platelet rich plasma versus platelet microvesicles in platelet free plasma. It can be seen that essentially all of the light scatter events in the platelet peak are platelets, but only a fraction of microparticle light scatter events are platelet microvesicles, and centrifugation to remove platelets also removes many platelet microvesicles (20,21). Whole blood or platelet rich plasma contains 10,000/lL and 100,000/lL platelet, annexin, and red cell microvesicles compared to 1,000/lL to 10,000/lL after centrifugation.…”

“…If plasma samples are going to be frozen, a single centrifugation step is inadequate to remove all platelets from the sample, double centrifugation is required to prevent 100% to 1,500% increases in platelet and annexin V1 microvesicle counts due to fragmentation of residual platelets in the sample (15,20,(25)(26)(27). Ultracentrifugation to pellet and collect microvesicles recovers 70 2 80% of microvesicles from fresh samples, but less from frozen/thawed samples (15,20).…”

“…Some reported no change (14,20), some found modest increases after freeze/thawing (15,25,28), while others reported large changes (22,29). Studies reporting the largest changes used methodologies that count only a small percentage of the microvesicles present.…”