Usually the mixture in most tubes is in the region of 1ml 3.8% citrate is addred to an 8ml tubes but this standard amount originates from the amount used in transfusion bags where the blood has to be anticoagulated for several weeks. In reality 0.5ml will effectively anticoagulate 8ml blood resulting in significantly less dilution of the PRP fraction after centrifugation.
Are there any disadvantages of using less citrate, for example increased platelet activation during centrifugation?
Please, refer to: Recommendations for the Standardization of Light Transmission Aggregometry: A Consensus of the Working Party from the Platelet Physiology Subcommittee of SSC/ISTH.M Cattaneo, C Cerletti, P Harrison, C P M Hayward, D Kenny, D Nugent, P Nurden, A K Rao, A H Schmaier, S P Watson, F Lussana, M T Pugliano, A D Michelson: Journal of Thrombosis and Haemostasis 04/2013
I assume that you are preparing PRP for therapeutic uses? Then for us the best option is to use the light blue tubes for coagulation assays, so that you don't have to handle the blood or add any additive, just calcium chloride when you want to prepare a clot. I'm not aware of any untoward reaction, we have used them for dental, trauma and plastic surgery.
Amino Acids (2013) 44:1159-1165
A novel approach in potential anticoagulants from peptides
epitope 558–565 of A2 subunit of factor VIII
C. Anastasopoulos, Y. Sarigiannis & G. Stavropoulos*
Blood from healthy individuals was collected
into plastic tubes containing 3.8 % trisodium citrate as
anticoagulant (blood/trisodium citrate 9:1). Citrated blood
was immediately centrifuged at 13,000 rpm for 10 min at
RT to obtain platelet poor plasma (PPP). Citrated samples
can be run within 2 h of sample collection.
Hi, Chris,
If you need to get platelet rich plasma, follow this:
Peripheral venous blood was collected in polypropylene tubes with 3.8 sodium citrate (9:1) by clean antecubital venipuncture for clotting tests. To obtain PRP, blood was centrifuged at 300 x g for 10 min., and the platelet rich supernatant was collected and pooled into another polypropylene tube. The remaining plasma was re-centrifuged at 1300 x g for 15 min to obtain platelet poor plasma (PPP).
Depending what you have to do with PRP, remember this one:
In case of samples with very large platelets, PRP should be prepared by blood sedimentation. Aggregometric studies in PRP with low platelet count (
According to our experience, I completely agree with the procedure described by Adriana Ines Woods
Yours sincerely
Michel Guichardant
We used the procedure similar to Adriana Ines Woods except for minor difference
To study the platelets aggregation we used phosphate buffered saline with calcium to dilute PRP to obtain desired platelet count.
If you need non-activated, non damaged PRP, you could do it in the way that we perform Pt separation (in order to label them with radionuclide In-111, re-inject them to the patient and discover the patophysiological mechanism of thrombocytopenia). I have investigated separated platelets (labelled with In-111 oxinate) in vivo and there was no activation, nor the shortening of their lifespan (in healthy voluntiers) with this procedure.
Besides ACD, centrifugation speed is very important in order not to have activated platelets.
You will find the procedure (in text and as a ppt together with references, enclosed), as well as the composition and volumes of ACD that we use enclosed.
Best regards,
Mila
it has been investigated that half the citrate concentration is enough to prevent clotting, providing adequate mixing during collection. However, in these studies (Hogman for example, 0.5CPD/0.5CP2D) only the general appearance of platelets is investigated, not in detail if there was mayb a slight, reversible, activation of the platelets.
If you add the citrate to blood the final citrate concentration depends on the hematocrit. There are patients with high or with low hematocrit that result in higher or lower citrate concentrations than for healthy donors. For re-calcification of any plasma final concentrations of about 20-25 mM calcium ions are ideal in nearly undiluted plasma.
In general practice we put 9 ml of whole blood to 1 ml of sodium citrate (3.8%) solution and spin the blood samples at 800 rpm for 20 minutes to obtain platelet rich plasma. As you might be aware that the international scientific community has a consensus to use of 3.2% sodium citrate instead of 3.8%. This is for your kind information.
I am sorry that I did not address your other question:
Are there any disadvantages of using less citrate, for example increased platelet activation during centrifugation?
The real disadvantage of using less citrate is development of clots. The ration of 1 part of sodium citrate to 9 parts of whole blood should be strictly followed. Using less citrate will cause clots to develop and obviously causes more platelet activation during centrifugation.
Omer is correct you should really be using the internationally accepted citrate % of 3.2 for a start. The standard anticoagulation of 1 part citrate to 9 parts blood is still the way to go with your collection. Seriously the difference in your collected PRP is not going to be affected to a big degree by the citrate to blood ratio. The harvest of PRP is more so affected by the platelet count of the donor and their Hb. As Omer has also suggested decreasing the citrate to blood ratio may place your collection in jeopardy from clots forming and the subsequent activation of your platelet fraction.
According to ISTH/2015,: ,The citrate concentration affects the results of coagulation tests. On the basis of these data, it is recommended that 3.2% citrate be used for all coagulation test.
what's the purpose of your PRP isolation, functional testing or use in PRP dressings or something else? You can easily concentrate your PRP by adding 1 part of ACD-A to 9 parts of PRP (lowering pH to a value between 6 and 6.5), pellet them down and resuspend in plasma or any buffy you want.
I agree with the other propositions :
The standard anticoagulation of 1 part citrate (3.2 %) to 9 parts blood is still the way to go with your blood collection. However if you want to prevent platelet aggregation I suggest to use EDTA as anticoagulant instead of citrate.
In our experience blood collected in citrate tubes (blue cap) works right for most dental and surgical procedures. Platelet rich plasma can be obtained with any anticoagulant you want to use, it only depends on the centrifugation protocol. However, citrated plasma is the best if you want to analyse platelets or prepare a platelet gel later on. Then, a blue cap tube is the best, because it will save you all the trouble gettin sterile tubes, etc etc.
Our local protocol has changed and now we use low volume blood collection bags. Whole blood is collected in the blood bank and separated in parts like in blood donation. The platelet rich plasma is divided in three aliquots to be used at different times. But in the end the anticoagulation doesnt change: CPDA citrate, phosphate, dextrose and adenine. The ratio citrate: whole blood is adapted to the blood volume you collect.
I too can read :))
3.2% citrate is more related to standardising anticoagulation for coagulation studies. In reality the exact amount of citrate is probably far less important for PRP preparation since when injected the platelets are activated by contact with collagen (as indeed is fibrin formation)
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