[Cytometry] monocyte-platelet aggregates (*tech support response*)

Roy Edward roy at biostatus.com
Sat Apr 20 05:49:39 EDT 2013


Dear Adeeb,

The problem may be associated with RBC lysis, introducing enormous amounts
of membraneous fragments into the cell suspension.

This has been addressed in a number of publications.  I have picked out a
couple ..

E Arellano-Rodrigo (2006) Haematologica 91 (2) 169-175 (thrombosis risk in
essential thrombocythemia)
J Kraan et al. (2012) J. Thromb. Hemostasis 10 (5) 931-939 (in search of
circ. endothelial cells (CEC))

..where the far-red DNA dye DRAQ5 has been employed to obviate RBC lysis,
and thereby also simplify sample preparation. The protocols rely upon a
simple dilution (e.g. With PBS) and gating on the nucleated cell fraction
to exclude platelets and RBCs from analysis, whilst avoiding the
generation of additional debris.  Due to the far-red property of the dye
it is possible to perform multi-colour experiments, such as the gating
cascade required for an unequivocal identification of CEC in peripheral
blood (this used FITC, PE, PerCP and APC antibodies with DRAQ5 on a BD
FACS Canto (4+2 geometry)).  This is equally possible on the BC FC500 (and
later platforms). See Bjornsson et al. (2008) Cytometry Part B 74B:91­103
for a suitable fluorophore panel.

I have also had anecdotal reports on this which have not been published
but support the strategy of DRAQ5+ nucleated cell gating in place of RBC
lysis for robust leukocyte-platelet aggregation assessment.

I hope this might help.
Please contact me off-line or visit www.biostatus.com
<http://www.biostatus.com> for full technical and ordering information.

Kind regards,
Roy

Roy Edward
E  roy(at)biostatus(dot)com

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=============================================================
On 18/04/2013 20:11, "Adeeb Rahman" <adeeb_r at yahoo.com> 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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