[Cytometry] monocyte-platelet aggregates

D. Robert Sutherland rob.sutherland at utoronto.ca
Fri Apr 19 11:01:33 EDT 2013


Hi Matthew,
thanks for that clarification. When we were assessing anti-coagulants for the studies we were engaged in, we chose the one (EDTA) that minimised platelet binding to monocytes, as we were trying to assess CD36 expression on monocytes, rather than on platelets that had adhered to monocytes. 

best
rob

On 2013-04-19, at 10:56 AM, Matthew Linden wrote:

> Hi Rob and Adeeb,
> 
> EDTA is too potent a chelator of calcium, and will dissociate the GPIIb-IIIa receptor on platelets, making it not ideal for such studies of platelet function. Heparin binds to and activates platelets, causing P-selectin expression and an artificial increase in monocyte platelet aggregates (MPAs).
> 
> Citrate is better in this regard. However you must work quickly! Citrated blood left on the bench will begin to form monocyte-platelet aggregates within 15 minutes of collection, so samples should be fixed as soon as possible after collection. Leaving the blood too long is a common cause of elevated MPAs when testing.
> 
> The blood collection protocol itself might be responsible for your elevated MPAs. You mentioned tubes later in the draw sequence, which is good. However shear activates platelets, so even too high a gauge needle can alter your results - though 90% MPAs would certainly be unusual.
> 
> These protocols might be of help.
> 
> http://link.springer.com/protocol/10.1007/978-1-62703-339-8_18
> 
> https://www.thieme-connect.com/ejournals/abstract/10.1055/s-2004-835671
> 
> http://www.currentprotocols.com/WileyCDA/CPUnit/refId-cy0615.html
> 
> Regards,
> 
> Matt
> 
> 
> Matthew Linden, PhD
> Associate Professor and Head - Flow Cytometry Technique Group
> The Centre for Microscopy, Characterisation and Analysis
> (an Australian Microscopy and Microanalysis Research Facility)
> University of Western Australia
> 
> Tel: +61-8-9346-4525
> Fax: +61-8-6488-1087 
> Email: matthew.linden at uwa.edu.au
> Web: http://www.cmca.uwa.edu.au and http://www.ammrf.org.au/
> Mail: M510, 35 Stirling Highway
> Office: Room 1.21, M Block, QEII Medical Centre
> Nedlands, WA, 6009
> 
> 
> 
> 
> -----Original Message-----
> From: cytometry-bounces at lists.purdue.edu [mailto:cytometry-bounces at lists.purdue.edu] On Behalf Of D. Robert Sutherland
> Sent: Friday, 19 April 2013 10:02 PM
> To: Adeeb Rahman
> Cc: Cytometry at lists.purdue.edu
> Subject: Re: [Cytometry] monocyte-platelet aggregates
> 
> Adeeb,
> I am not an expert in tis area, but I believe EDTA is the anti-coagulant of choice for the types of studies you are doing.
> HTH
> best
> rob
> 
> D. Robert Sutherland
> Toronto General Hospital/University Health Network
> 
> On 2013-04-18, at 4:11 PM, Adeeb Rahman wrote:
> 
>> Dear flow folk,
>> 
>> Do any of you have experience detecting monocyte-platelet aggregates by flow? I've been trying to quantify MPAs using antibodies against CD14 (clone HCD14) and CD42b (clone HIP1) to stain whole blood, followed by RBC lysis/fixation with BD FACSLyse.Using this protocol, I'm finding that a very high frequency of monocytes are CD42b+ (over 90%), which is much higher than the frequency reported in various publications (typically ~10%). 
>> 
>> 
>> I've looked at blood collected in citrate tubes and sodium heparin tubes and have looked at tubes drawn later in the draw sequence (i.e. not the first tube drawn) but am still seeing the same thing. Does anyone have any thoughts about what may be causing this high frequency of MPAs? 
>> Thanks,
>> 
>> Adeeb
>> 
>> 
>> ___________________________
>> 
>> 
>> Adeeb Rahman
>> Postdoctoral Fellow
>> Division of Liver Diseases
>> Mount Sinai School of Medicine
>> New York, NY
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> 
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