Hello,
Hopefully, someone can please help me out with my misunderstandings surrounding prp. I have been told that the standard protocol for prp Iisolation from whole blood is 160xG for 15 minutes. This is easy enough, however what I am unclear on is which layer to take! I seems easy enough, but I keep seing pictured and videos of only three layers, then some with more than three layers. The last time I asked about prp isolation, someone gave me the same protocol and said to aspirate the supernatant, being careful not to extract the white blood cells.
Please let me provide some pictures and you tell me which will be the product of the protocol 160xG for 15minutes no ficoll just 30ml's of whole blood.
Picture 1:
http://g03.s.alicdn.com/kf/HTB1.8T9FVXXXXbCaXXXq6xXFXXXI/200429328/HTB1.8T9FVXXXXbCaXXXq6xXFXXXI.jpg
Picture 2:
http://www.histology.leeds.ac.uk/blood/assets/blood_ttube.gif
Picture 3:
http://clinical.diabetesjournals.org/content/25/2/43/F2.large.jpg
Which one can I expect and which layer do I aspirate?
If I'm not mistaken, it will be neither. To me, the second and the third scheme seems to be the result of the same protocol, and on the first glance, it looks more like platelet poor plasma (PPP) separation centrifugation with higher Gs. Then you'd get (as indicated in those images) a platelet poor plasma on top, a white layer (buffy coat) containing platelets and white blood cells (more or less separate), and a large pack of red blood cells on the bottom. The first image has a separation gel, but otherwise it's somewhere between the true PRP and PPP plasma settings because the top part of plasma is clear and the bottom part contains platelets, so the centrifugation settings have been somewhat harder.
I don't have my protocol at hand, but if I remember correctly, we used 120g for 20 minutes, then we have basically two layers - PRP on top and larger cells on bottom, and you should take the top layer leaving a good centimeter above the erythrocyte layer. Then you are sure to have cleaner PRP preparation and less chance of picking a white blood cell too.
Thanks guys! Coen, you are the one I got that protocol from! :) So now I have two protocols... 160xG 15minutes or 120G for 20minutes. According to Ilze, there will be two layers after centrifugation of the 120xG protocol. Will this be true with the 160xG protocol? Can I expect the same thing with both protocols? Thanks
I would venture a guess that the final result might be similar: in both cases it's low centrifugal speed for a relatively longer time. Though, as I said, my settings are from memory only. If you have time and blood to spare, you could see what comes out of both protocols. Ideally you shouldn't get a clearer layer on top (like in picture 1), but get a more-or-less uniformly cloudy top layer.
P.S. and no brake, of course.
Ok, thanks! I guess I will try the 160xg for 15 minutes and hope it turns out in two layers. Either way, I will aspirate the top layer. Thanks
Ok guys thanks a ton to both of you! And thanks to Coen for answering my other questions that I've posted. I missed the chance to thank you before so thanks so much!:)
You will find my latest version of platelet separation protocol (as a result of more than 20 years of experience in patients with thrombocytopenia), together with ppt presentation (for better explaining the procedure) and references. This protocol fulfills the criteria not to harm the cells that are labelled and thereafter intravenously injected to the patient, in order to be able to diagnose the problem that exists in patient (inadequate Pt production, or augmented Pt destruction, or disturbed Pt distribution).
dear Elli
take a 50ml blue cap with 50ml fresh whole blood anticoagulatet with 20ml NaCitr., zentrifugation by 380g, 15min
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