[Cytometry] monocyte-platelet aggregates

Bruce Davis brucedavis at trilliumdx.com
Mon Apr 22 07:44:08 EDT 2013


I suggest you look up the literature the work of Ken Ault and platelet
aggregation studies.  In order to get a true view of the in vivo
situation, one must minimize the pre-analytical activation of platelets
and monocytes, which is only done with certainty by doing immediate
fixation or processing of blood samples at the bedside.  Whether the use
of blood stabilizers now available commercially (Caltag Medsystemes in UK
and Streck or R&D Systems in U.S) has been validated for monocyte-plt
aggregates would require a literature search.

Bruce


Bruce H. Davis, MD

President, Trillium Diagnostics, LLC
Phone:  +1-207-945-0900
FAX: +1-207-942-0346
Mobile: +1-207-653-5247




On 4/19/13 10:56 AM, "Matthew Linden" <matthew.linden at uwa.edu.au> 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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