Hello Karina,

I don't want to be the bearer of bad news, but using any "stale" platelet concentrate from the hospital to test your anti-aggregation agent will produce a plethora of artifacts.  Platelets should be diluted with aliquots of platelet poor plasma originating from the original sample to a standard concentration of platelets.  This is important for two reasons- first due to differences in plasma clarity, path length of your sample cuvette, and various other reasons you cannot rely on using simple base absorbency to normalize your samples.  For example, is it 0.5 Abs after blanking the reading to your dilution medium, or just total Abs- similar to normalizing protein adjusting concentrations of Buffer A (the one the platelets came in from the hospital) and Buffer B (your dilution buffer) will cause a constantly shifting background level of absorbance which will then distort your readings.  Second, if you do originate with different absolute numbers of platelets then the level of drug they each see will be different, adding another variable.  

     To do the assay correctly fresh blood samples must be taken in blue top citrated tubes, a volume platelet rich plasma spun out at low speed, then the tube spun at high speed to produce platelet poor plasma.  You can then use a CBC machine or coultur counter to normalize the platelet levels using the plasma as the dillutent.  

    You should also remember that aggregation of platelets requires them to be completely functional.  Platelet concentrates obtained from the hospital will undoubtably be "stale", as they are used on a first in first out manner.  If you do  a quick pubmed search on "platelet storage lesion" you will find that this issue has been plaguing the field of transfusion medicine for years.  You may be able to judge relative differences but these will not relevant on a molecular biology level as the receptors involved in platelet aggregation are adversely effected by preparation of and storage of platelet concentrates.  If your antithrombotic agents are not effecting the platelet receptors then their targets will be in the plasma itself, and you will still miss your effects.  

With the invention of whole blood aggregometry all these issues have been solved, but if you do not have access to an aggregometer then I suggest you obtain fresh samples even time you run these experiments.  Ill include a link that is more detailed than my explanation from a publicly available website.  Good Luck, and if you have any other questions please let me know.

Jason

http://www.practical-haemostasis.com/Platelets/platelet_function_testing_lta.html

hi Jason. thank you so much for your answer. I am really new at this. and i live in a country where english is not our first language so there might be some english words that im not used to lol. there are further question i'd like to ask.

first is, what do you mean by "first in first out manner"?

second is that, is there a difference between PRP or platelet rich plasma and thrombocyte concentrate (the one i bought in the hospital)? and what do u mean by stale? is it inactive? wouldnt ADP activate it in order to test its aggregation activity?

Dear Karina,

To summarise that exhausting and profound answer from Dr, Newton, You definintely should not use thrombocyte concentrate, since platelets need to be functional, especially for testing antiplatelet treatment.

Depending on the assay, you should use platelet rich plasma or whole blood (i.e. in impedance aggregometry). Most convenient assays are Light transmission aggregometry (LTA), multiplate impedance aggregometry, VASP-P flow cytometric (prefferably) or ELISA assay and VerifyNow

Not to mention that buying thrombocyte concentrate is unnecessary financial burden, which will not yield results in your work.

Best Regards

Peter

See some of our papers such as :

Article Sticky Platelet Syndrome

Article Sticky Platelet Syndrome: History and Future Perspectives

Article Monitoring the efficacy of ADP inhibitor treatment in patien...

 Hi Karena,

Sorry I missed your additional questions once the conversation moved to the messenger.  First in first out refers to the practice of using the oldest products first in order to prevent the expiration of the concentrates as much as possible, the first one prepared is the first on distributed.  I believe I answered the "stale question" in the messenger.  

If you need anything else feel free to send me a message.

Best of luck, 

Jason

Dr. Kubisz,

I am unsure why a gentleman with such a lengthy publication record and prestigious position would begin a comment by disparaging my answer then completely agree with that same answer.  I guess it would have been possible for me to simply reply that she should pursue another route and then link to a few unrelated publications, but I chose to go a different route.

Ill break my own rule, and post a link to wikipedia.  I think trolling research gate is very unprofessional, and sets a very poor example for this young lady who is just beginning her career in science.

https://en.wikipedia.org/wiki/Internet_troll

Dear Jason

Be calm. Your platelets become sticky if you are nervous.

The articles, I attached are regarding our experience and know-how with LTA procedures. Im sure Karina can use these methods. They are related to question as far as I know

Feel free to visit us and discuss personally, and do not feel hurt via social networks.

I will send you adress in Private message.

Peter