there is problem in flow cytometer analysis of platelet microparticles
Dear Hasnaa,
The following publications cover the answer to your question:
1- Vasc Health Risk Manag. 2010; 6: 1125–1133.
Published online 2010 Dec 6. doi: 10.2147/VHRM.S13236
PMCID: PMC3004516
Detection of circulating microparticles by flow cytometry: influence of centrifugation, filtration of buffer, and freezing
Emily Dey-Hazra,1,* Barbara Hertel,1,* Torsten Kirsch,1 Alexander Woywodt,2 Svjetlana Lovric,1 Hermann Haller,1Marion Haubitz,1 and Uta Erdbruegger1,3
Author information ► Article notes ► Copyright and License information ►
This article has been cited by other articles in PMC.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3004516/
Abstract
The clinical importance of microparticles resulting from vesiculation of platelets and other blood cells is increasingly recognized, although no standardized method exists for their measurement. Only a few studies have examined the analytical and preanalytical steps and variables affecting microparticle detection. We focused our analysis on microparticle detection by flow cytometry. The goal of our study was to analyze the effects of different centrifugation protocols looking at different durations of high and low centrifugation speeds. We also analyzed the effect of filtration of buffer and long-term freezing on microparticle quantification, as well as the role of Annexin V in the detection of microparticles. Absolute and platelet-derived microparticles were 10- to 15-fold higher using initial lower centrifugation speeds at 1500 × gcompared with protocols using centrifugation speeds at 5000 × g (P < 0.01). A clear separation between true events and background noise was only achieved using higher centrifugation speeds. Filtration of buffer with a 0.2 μm filter reduced a significant amount of background noise. Storing samples for microparticle detection at −80°C decreased microparticle levels at days 28, 42, and 56 (P < 0.05 for all comparisons with fresh samples). We believe that staining with Annexin V is necessary to distinguish true events from cell debris or precipitates. Buffers should be filtered and fresh samples should be analyzed, or storage periods will have to be standardized. Higher centrifugation speeds should be used to minimize contamination by smaller size platelets.
Keywords: circulating microparticles, detection method, flow cytometry
2- Platelet microparticles in transfusion
L. H. Boudreau,
G. Marcoux and
E. Boilard*
Article first published online: 13 APR 2015
DOI: 10.1111/voxs.12142
© 2015 International Society of Blood Transfusion
http://onlinelibrary.wiley.com/doi/10.1111/voxs.12142/abstract?systemMessage=Wiley+Online+Library+will+have+be+unavailable+on+Saturday+5th+December+from+10%3A00-14%3A00+GMT+%2F+05%3A00-09%3A00+EST+%2F+18%3A00-22%3A00+SGT+for+essential+maintenance.+Apologies+for+the+inconvenience.
Keywords:
microparticles;
platelet components;
platelet concentrates
Platelets are active participants in maintaining haemostasis. Upon activation, platelet shed vesicles from their cytoplasmic membrane known as platelet microparticles. These platelet microparticles and their unique cargo are thought to participate in intercellular communication. Platelet transfusion is frequently required for patients with low platelet counts or platelet disorders to restore normal functions. Unfortunately, adverse transfusion reactions are most commonly associated with platelet transfusion than any other type of labile blood products. In this review, we raise the potential implications of platelet microparticles in adverse transfusion reactions.
3-IJBC 2015; 7(3): 119-128
http://www.ijbc.ir/files/site1/user_files_6e79e6/dr_karimi-A-10-9-49-2cda659.pdf
An Overview on Platelet-derived Microparticles in
Platelet Concentrates: blood collection, method
preparation and storage
Nasiri S1
*
1. Blood Transfusion Research Center, High Institute for Research and Education in Transfusion
Medicine, Tehran, Iran.
*Corresponding Author: Nasiri S, Email: [email protected]
Submitted: 12-03-2014 , Accepted: 29-05-2014
Abstract
Preparations of platelet concentrates (PCs) that are stored under blood bank conditions and used for transfusion purposes, appear to be enriched in platelet derived-microparticles (PMPs) with high coagulant activity that may
change platelet efficacy and safety issues. High shear stress could cause shedding of PMPs from the platelet plasma membrane, platelet aggregation, and activation of the coagulation cascade by increasing the catalytic phospholipid surface. These stresses may be prompted by processing and storage of blood and platelet rich plasma through various variables that has been fully described in this review. Depending on different rates of shear stress during processing and storage of PC, different quantities of MPs might be shed from platelets. On the other hand, the therapeutic effect of high levels of PMPs in PC has been reported for some patients. By using more sensitive and standardized methods for PMP measurement and change of platelet preparation process, further studies are required to monitor
PMP generation during blood collection, processing and storage of PC to improve quality of PC and also in recipient’s reactions to transfusion.
Keywords: Platelet-derived microparticle, Platelet concentrate, Blood collection, Platelet storage
Hoping this will be helpful,
Rafik
I
Dear Hasnaa,
The following publications cover the answer to your question:
1- Vasc Health Risk Manag. 2010; 6: 1125–1133.
Published online 2010 Dec 6. doi: 10.2147/VHRM.S13236
PMCID: PMC3004516
Detection of circulating microparticles by flow cytometry: influence of centrifugation, filtration of buffer, and freezing
Emily Dey-Hazra,1,* Barbara Hertel,1,* Torsten Kirsch,1 Alexander Woywodt,2 Svjetlana Lovric,1 Hermann Haller,1Marion Haubitz,1 and Uta Erdbruegger1,3
Author information ► Article notes ► Copyright and License information ►
This article has been cited by other articles in PMC.
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3004516/
Abstract
The clinical importance of microparticles resulting from vesiculation of platelets and other blood cells is increasingly recognized, although no standardized method exists for their measurement. Only a few studies have examined the analytical and preanalytical steps and variables affecting microparticle detection. We focused our analysis on microparticle detection by flow cytometry. The goal of our study was to analyze the effects of different centrifugation protocols looking at different durations of high and low centrifugation speeds. We also analyzed the effect of filtration of buffer and long-term freezing on microparticle quantification, as well as the role of Annexin V in the detection of microparticles. Absolute and platelet-derived microparticles were 10- to 15-fold higher using initial lower centrifugation speeds at 1500 × gcompared with protocols using centrifugation speeds at 5000 × g (P < 0.01). A clear separation between true events and background noise was only achieved using higher centrifugation speeds. Filtration of buffer with a 0.2 μm filter reduced a significant amount of background noise. Storing samples for microparticle detection at −80°C decreased microparticle levels at days 28, 42, and 56 (P < 0.05 for all comparisons with fresh samples). We believe that staining with Annexin V is necessary to distinguish true events from cell debris or precipitates. Buffers should be filtered and fresh samples should be analyzed, or storage periods will have to be standardized. Higher centrifugation speeds should be used to minimize contamination by smaller size platelets.
Keywords: circulating microparticles, detection method, flow cytometry
2- Platelet microparticles in transfusion
L. H. Boudreau,
G. Marcoux and
E. Boilard*
Article first published online: 13 APR 2015
DOI: 10.1111/voxs.12142
© 2015 International Society of Blood Transfusion
http://onlinelibrary.wiley.com/doi/10.1111/voxs.12142/abstract?systemMessage=Wiley+Online+Library+will+have+be+unavailable+on+Saturday+5th+December+from+10%3A00-14%3A00+GMT+%2F+05%3A00-09%3A00+EST+%2F+18%3A00-22%3A00+SGT+for+essential+maintenance.+Apologies+for+the+inconvenience.
Keywords:
microparticles;
platelet components;
platelet concentrates
Platelets are active participants in maintaining haemostasis. Upon activation, platelet shed vesicles from their cytoplasmic membrane known as platelet microparticles. These platelet microparticles and their unique cargo are thought to participate in intercellular communication. Platelet transfusion is frequently required for patients with low platelet counts or platelet disorders to restore normal functions. Unfortunately, adverse transfusion reactions are most commonly associated with platelet transfusion than any other type of labile blood products. In this review, we raise the potential implications of platelet microparticles in adverse transfusion reactions.
3-IJBC 2015; 7(3): 119-128
http://www.ijbc.ir/files/site1/user_files_6e79e6/dr_karimi-A-10-9-49-2cda659.pdf
An Overview on Platelet-derived Microparticles in
Platelet Concentrates: blood collection, method
preparation and storage
Nasiri S1
*
1. Blood Transfusion Research Center, High Institute for Research and Education in Transfusion
Medicine, Tehran, Iran.
*Corresponding Author: Nasiri S, Email: [email protected]
Submitted: 12-03-2014 , Accepted: 29-05-2014
Abstract
Preparations of platelet concentrates (PCs) that are stored under blood bank conditions and used for transfusion purposes, appear to be enriched in platelet derived-microparticles (PMPs) with high coagulant activity that may
change platelet efficacy and safety issues. High shear stress could cause shedding of PMPs from the platelet plasma membrane, platelet aggregation, and activation of the coagulation cascade by increasing the catalytic phospholipid surface. These stresses may be prompted by processing and storage of blood and platelet rich plasma through various variables that has been fully described in this review. Depending on different rates of shear stress during processing and storage of PC, different quantities of MPs might be shed from platelets. On the other hand, the therapeutic effect of high levels of PMPs in PC has been reported for some patients. By using more sensitive and standardized methods for PMP measurement and change of platelet preparation process, further studies are required to monitor
PMP generation during blood collection, processing and storage of PC to improve quality of PC and also in recipient’s reactions to transfusion.
Keywords: Platelet-derived microparticle, Platelet concentrate, Blood collection, Platelet storage
Hoping this will be helpful,
Rafik
I
Dear Hasnna
You may also try CD41 and look at the following paper
Detection and quantification of microparticles from different cellular lineages using flow cytometry. Evaluation of the impact of secreted phospholipase A2 on microparticle assessment. Rousseau et al. PMID: 25587983
You could try CD41 and annexin V. CD41 will identify tge orgin of the mircoparticles and annexin V can be used to distiguish from platelets.
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