Human platelet activation in vitro?

You want to measure Platelet Aggregation??

I think 3.8% sod. citrate is better than others.

3.8 % citrate : whole blood = 1 : 9  (v/v)

If you want to measure platelet aggregation in platelet-rich plasma then definitely use sodium citrate at the concentration Hyun-Hong Kim mentioned.

Hi Hyun-Hong and Carol,

Thank you for your suggestions.

I will be measuring activation via expression of surface markers (e.g. CD62P) by flow cytometry. Because I'm activating them in vitro, I want them to be drawn into a solution that will still allow them to function.

Thank you,

In my experience, taking into account , I reccomend to you that Since platelets are activated easily, it is recommended that samples :

It is drawn in syringes or as in vacuum tubes. He

preferred anticoagulant is sodium citrate 3.2%,

It is acting chelate calcium ions (Ca ++). is

recommends a ratio blood / citrate

9: 1; a higher concentration of citrate on chelation

Ca ++ and subsequently interfere with subsequent

studies of platelet function. The samples

should be kept at room temperature (20-25 C)

during transport and storage. the tubes

They are transported vertically and should be

careful not to stir or shake the samples; manual transport is preferred over pneumatic tube systems.

Try hirudin. For many platelet functional aspects this anticoagulant has many advantages. 

And when using citrate, never test blood immediately after venepuncture. A resting time of at least 15 minutes gives more reproducible results.

sodium citrate is the best solutiion

Dear Joshua,

First question is : how many platelets are you going to evaluate. I suppose you will use platelets with plasma (PRP) , therefore what will be G, and time of centrifugation? And also the time span between the sample collection and start of analysis. As Juan Carlos, we suggest using 3.2% citrate , with citrate / blood ratio 1:9 and with accurate preanalytical phase. For example see article: Sticky Platelet Syndrome : History and Future Perspectives in Semin Thromb Hemost 2014; 40:523-534

Best Regards

Peter

Hi Joshwa,

You may use the following:

1.  Sodium citrate 3.2%

2.  Hirudin

3.  TRAP.

Iqbal

Dear Omer,

TRAP is inducer of platelet aggregation and is used as positive control , for example in testing the effect of DAPT by P2Y12 inhibitors.

See also : Platelets. 2010;21(7):515-21.

Kind Regards

Peter

We recently published a review on platelet flow cytometry. Perhaps you can find some useful tips there. Among others we mention some different pros and cons on anticoagulants. (Platelet Function Determined by Flow Cytometry: New Perspectives? Seminars in Thrombosis and Hemostasis 42(3) · February 2016)

I general I would recommend hirudin (or heparin altough this might be a bit controvesrial) because you don't disturb the calcium balance. If you wish to use thrombin for activation this will however not work and you have to use citrate. 

ACD should only be used when isolating platelets.

Don't use EDTA in any form because it induces shape change.