I am using whole blood to obtain platelets via PRP. Most seem to use a 200 x g spin to separate the red cells, then a 1000 x g the PRP to pellet the platelets. I have also seen use of a density gradient, which one would yield the higher amount of platelets/least contamination? I usually spin the 50ml samples.
Many thanks in advance!
Dear kindly read this.
The first step of this procedure includes obtaining the whole blood and preparing platelet-rich plasma (PRP) by centrifugation. The centrifugation conditions can vary widely (e.g. from 800 x g for 5 min to 100 x g for 20 min), but give consistently good separations.
A phlebotomist should draw blood into a Becton Dicinson Vacutainer® (containing ACD, yellow cap) or into a plastic syringe containing 1/10 volume of CPD buffer. The volume of blood needed depends on the experiment. A volume of 40–45 ml blood results in average 1–3 x 109 platelets. The platelet number can vary depending on the individual the blood is drawn from.
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Platelet isolation
Recently I optimized my own protocol and obtain very good results now. Maybe this will help you.
I draw blood into a 7,5ml EDTA tube and add 21µl PPACK, 13µl Apyrase, 5µl PGE. Before centrifugation I split the volume into two 15ml tubes and fill the tube with Hepes/Tyrode buffer (pH 6,5).
Centrifugation: 20min, 120g
The total supernatant is split into six 5ml tubes (3-4ml each).
Centrifugation: 10min, 1400g
Discard the supernatant. Resuspend the pellet with 0,5ml buffer (containing 5% BSA and 13µl Apyrase, 5µl PGE per 10ml). Merge the platelets in a new 5ml tube. If you need a higher platelet concentration, repeat the centrifugation (10min, 1400g). Resuspend the pellet as needed.
With this procedere you have low platelet activation, high platelet count and low contamination. Depending on what you need the platelets for, you should adapt this protocol. I would be happy, if you can give a feedback, how it works for you.
Thank you, will consider this and will let you know the results if i use it
Production of PRP with minimal impact, if your aim is to test platelet function, is described here: https://www.tandfonline.com/doi/abs/10.3109/09537104.2012.672780 or https://doi.org/10.3109/09537104.2012.672780
In short, we used sodium heparin tubes and 140 x g for 20 min at RT.
Dear the best and easiest procedure for platelets measurement in a whole blood is by use of automation.
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