From: Robert Pyle (pyle@smtpgw.kfshrc.edu.sa)
Date: Wed Feb 11 1998 - 05:00:28 EST
Without having your detailed procedure in front of me I find it difficult to
successfully trouble shoot what you did. However, we have done platelet
glycoprotein measurements for years and seldom have a platelet recovery problem.
Here is what we do:
1. Blood is collected in NaEDTA and centrifuged at a very low speed for 20
minutes to get a platelet rich plasma preparation. In our centrifuge the
centrifugal force is around 80 x g.
2. The platelet rich plasma is then removed (use plastic disposable pipets)and
spun at 200 x g in a round bottom polystyrene ( I guess polypropylene would
work too but avoid glass due to its tendency to activate the platelets and
lead to loss/clumping) test tube. Conical tubes seem to pack the platelets
too hard and indeed can cause irreconcilable clumps.
3. We remove the plasma and wash twice with an EDTA-PBS buffer. The buffer
uses 2.79 grams of NaEDTA per 500 mls of PBS. Usually takes heat to get the
EDTA into solution and about 1 hours of constant stirring. pH to 7.2 with
NaOH.
4. You can resuspend the platelets in the EDTA-PBS to a desired concentration
and we have successfully left them overnight at room temperature this way.
We have been able to recover sufficient platelet concentrations in extremely
thrombocytopenic patients using only 5-7 ml of EDTA anticoagulated blood.
Patients reporting platelet counts of less than 20,000 have not proven to be a
problem.
I hope this helps.
Haywood Pyle
KFSH, MBC#3
Box 3354, Riyadh 11211
Saudi Arabia
tel/fax:966-1-464-7272 ext. 32860
e:pyle@kfshrc.edu.sa
_______________________________________________________________________________
Subject: Platelet preps
From: Ray Hicks <rh208@cus.cam.ac.uk> at INTERNET-MAIL
Date: 10/02/98 10:47
Hi,
I've been asked to post this message to the list, has anyone got any
reliable preps for platelets? Any ideas with what might have gone wrong
with the cited method?
TIA
Ray
>A method for preparing isolated platelets for FACS analysis (we will be
>looking to see if a particular protein is expressed on the surface of these
>cells).
>
>We will be using blood from humans and from pigs in relatively small
>volumes (tens of mls rather than litres; individual samples not pooled) and
>have access to cold rooms, centrifuges, etc. etc.
>We followed the method given in 'Flow Cytometry - A Practical Approach' by
>Ormerod 2nd edition; but this gave us un-resuspendable platelet pellets and
>relatively high red blood cell contamination.
>
>If anyone has a reliable prep method, or knows where we are likely to have
>gone wrong in following the textbook method mentioned we will be very
>grateful.
Ray Hicks
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