[Cytometry] monocyte-platelet aggregates
Marc.Barnard at umassmed.edu
Fri Apr 19 11:13:40 EDT 2013
EDTA is the correct choice for avoiding platelet binding to monocytes (or any other leukocyte) because the P-selectin/PSGL-1 (CD62P/CD162) interaction required is calcium dependent. Most of the time people are trying to avoid it!
Citrate should be used, assure a clean blood draw in a 19g or larger needle. Mix the blood a as little as possible and label (or FACSLyse if Abs fix-stable) for MPAs ideally within 15 min of blood draw. It is very easy to cause >90% MPA, usually a difficult draw, blood taken off a badly cleared line, aged or vigorously mixed blood are all ways to get there.
Flow Cytometry Core Facility, Rm. S5-322
University of Massachusetts Medical School
55 Lake Avenue North
Worcester, MA 01655
From: cytometry-bounces at lists.purdue.edu [cytometry-bounces at lists.purdue.edu] on behalf of D. Robert Sutherland [rob.sutherland at utoronto.ca]
Sent: Friday, April 19, 2013 10:01 AM
To: Adeeb Rahman
Cc: Cytometry at lists.purdue.edu
Subject: Re: [Cytometry] monocyte-platelet aggregates
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.
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?
> Adeeb Rahman
> Postdoctoral Fellow
> Division of Liver Diseases
> Mount Sinai School of Medicine
> New York, NY
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