Abstract:
INTRODUCTION:Flow cytometry (FCM) is the most commonly used method for detection of platelet-derived microparticles (PDMPs), but it is poorly standardized and mainly used for "bedside" analyses in fresh samples. If PDMPs could be analyzed in previously frozen samples it would increase the usefulness of the method. However, cell membrane fragments from contaminating cells created during freezing/thawing may cause artifacts and disturb measurements. MATERIALS AND METHODS:PDMPs were labeled with monoclonal antibodies directed against CD42a and CD62P, or CD42a and CD142. The PDMP gate was determined using forward scatter (FSC) and CD42a expression. The mean fluorescence intensities (MFIs) of CD62P or CD142 positive particles were translated into MESF -values (Molecules of Equivalent Soluble Fluorochrome) using a standard curve. FITC-labeled phalloidin (which binds to intracellular actin) was used to detect destroyed cells/cell fragments. RESULTS:Phalloidin-positive particles were significantly more common in supernatants of frozen/thawed platelet rich and platelet poor plasma samples compared with supernatants of platelet free plasma. High-speed centrifugation was then used to obtain PDMP samples with low contamination of cell fragments. Electron microscopy showed that these samples contained numerous round stained particles with cellular membranes of a size of 100-700 nm. Reproducibility experiments using plasma samples from healthy individuals showed that the coefficients of variation (CVs) of MESF values of CD62P and CD142 (both intra- and interassay) were <10%, and the variation between two cytometers in two different laboratories was <5%. We also found that PDMP expression of CD142 (i.e. tissue factor [TF]) and CD62P (i.e P-selectin) was around two times higher in samples from type 1-diabetes patients compared with those from healthy controls (p<0.001). CONCLUSIONS:The use of MESF values to quantify PDMP expression of P-selectin and TF yields reproducible data and enables comparison of data between laboratories. If high-speed centrifugation is performed, contamination of cell fragments is low in frozen/thawed samples.
journal_name
Thromb Resjournal_title
Thrombosis researchauthors
Mobarrez F,Antovic J,Egberg N,Hansson M,Jörneskog G,Hultenby K,Wallén Hdoi
10.1016/j.thromres.2009.10.006subject
Has Abstractpub_date
2010-03-01 00:00:00pages
e110-6issue
3eissn
0049-3848issn
1879-2472pii
S0049-3848(09)00456-3journal_volume
125pub_type
杂志文章abstract::Extended half-life (EHL) products have shown robust efficacy in clinical trials, whilst allowing for less intense treatment regimens when compared with standard half-life products. Regimen optimisation with EHL products could lead to further improvements in bleeding rates, quality of life and reductions in treatment b...
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pub_type: 临床试验,杂志文章
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doi:10.1016/0049-3848(90)90136-z
更新日期:1990-07-15 00:00:00
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doi:10.1016/s0049-3848(03)00288-3
更新日期:2003-04-15 00:00:00
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pub_type: 杂志文章
doi:10.1016/j.thromres.2012.04.009
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journal_title:Thrombosis research
pub_type: 杂志文章,随机对照试验
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pub_type: 杂志文章
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journal_title:Thrombosis research
pub_type: 杂志文章
doi:10.1016/s0049-3848(99)00004-3
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journal_title:Thrombosis research
pub_type: 杂志文章,随机对照试验
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journal_title:Thrombosis research
pub_type: 临床试验,杂志文章,随机对照试验
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更新日期:2002-02-01 00:00:00
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journal_title:Thrombosis research
pub_type: 临床试验,杂志文章,随机对照试验
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更新日期:1998-03-15 00:00:00
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journal_title:Thrombosis research
pub_type: 杂志文章
doi:10.1016/j.thromres.2014.07.025
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更新日期:2000-10-01 00:00:00