I have read a few articles about reticulated platelet (RP) detection in whole blood or PRP. I have found that Thiazole Orange is the dye of choice for measuring RP percentage by flow cytometry, but that it is not 100% specific for RNA, also binding granule components.
Does anyone have a hint on how to make a trustworthy measurement of RP in whole blood by flow cytometry? (I intend to sort this platelets afterwards).
Hi Joao. Thiazole orange (TO) seems to be the stain of choice for platelets. Another stain auromine O is used in a commercial analyser (made by Sysmex) but I know little about its staining properties. It is excited by a blue (488nm) laser. TO binds to ADP in platelets and TO staining is reduced in patients with Hermansky Pudlak Syndrome.
My good friend Paul Harrison has a review on reticulated platelets and staining here on Research Gate:
https://www.researchgate.net/profile/Paul_Harrison2/publication/6990218_Reticulated_platelets/links/0deec52a84b60ad6c6000000.pdf
There's also this relatively recent paper:
Hoffmann, J. Clin Chem Lab Med 2014; 52(8): 1107–1117.
Sorting platelets? Now that's an interesting approach.
I've sorted virtually everything else from blood but them!! You would have to be very careful not to activate them by just putting them through the FACS. Are you using a FACSAria? If so - and I know this sounds a counter-intuitive way to sort small cell fragments - but I'd use the 100um nozzle so you are using the lowest pressure you can on the instrument (OK 130um is lower but it's an SOB to stabilise the stream with). You could also try dropping the pressure further but you would have to optimise the Aria with Accudrops. Also I'd use a flow rate of 1.0, the lowest possible.
These guys recommend adding apyrase to your suspension:
http://uic.igc.gulbenkian.pt/docs/flowcytometry/Poster%20CYTO%202015_Platelets.pdf
I can only add that a prostacyclin analogue or PGE2 might work as well or even better in combination.
Oh just found this from a FACS maestro, Ian Dimmick:
https://lists.purdue.edu/pipermail/cytometry/2008-April/034958.html
If Ian says do it this way then it works!!
Best of luck!
Article Reticulated platelets
Thank you very much for your help. I am still planning the whole experiment, but to know the sorting is feasible is great.
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