Platelet Activation Studies in Pheresis -Reply

Darren Hickerson dhickerson at brody.med.ecu.edu
Mon Feb 10 11:04:51 EST 1997


We have stained platelets many different ways.   Adding prediluted
antibody to 1:100 diluted PRP (platelet rich plasma, prepared by a
gentle spin to settle out RBC & leuks) has been the most satisfactory
to us.  Some people like to fix the cells, but we have noticed changes
in some glycoprotein expression, and only fix when absolutely
necessary.  A fellow lab simply dilutes 5 - 8 ul whole blood in
buffer and adds stock Ab, inc. 30 min., and runs on cytometer.

A few other hints (for the new to platelets):  washing and lysing
protocols can sometimes activate the platelets, as you may know, so
we try to avoid this.  Not washing away excess Ab can lead to higher
nonspecific fluorescence, so antibodies should be titered to optimum
concentration per platelet number in your lab prior to establishing a
routine protocol.  Always add negative control Ab at the same ug
amount as positive Ab, and verify the same isotype and molar ratio of
dye to protein.  BD makes a good PE platelet neg CTL (cat # 340013)
that has reduced non-bound PE in the suspension, but we have used
others that seem to work as well.  Also, try to avoid using EDTA as
the anticoagulant.  GPIIbIIIa expression is Ca dependent to some
degree, as well as some activation markers.  Na3Citrate is best. 
Miller and Bode have shown that triggering on linear side scatter
(visualizing on log forward scatter) gives the best platelet
distribution.

I'm sure there are other opinions in the field, but the above has
worked well for us.  I hope you find it useful.




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