Dear Mallikajun,

activated platelets present P-selectin on their surface (CD62P) which is presented on some PMPs. Platelet derived MPs (PMPs) have platelets specific integrin CD61 or CD61/CD41 complex. I also used CD42 complex (CD42b - Ib integrin - which is restricted to platelets and megakaryocytes). You have many variants of PMP staining for flow cytometry.

Dear Mallikajun,

activated platelets express P-Selectin on their surface. So, the best way to measure the activation of platelet is by using fluorescent labelled (like FITC, PE etc) CD62P marker.  Platelet derived MPs (PMPs) will be also positive to this surface marker and can be detected in the lower FSC -SSC region of the flow cytometry.

you can also use washed platelets (like Sepharose 2B coloumn wash) to detect PMPs.

Agree with both answers above - look for P-selectin expressing particles by gating on the small (

Dear Mallikajun,

You can also include Annexin-V (APC) dye that stains the memberane Phosphatidylserine to determine the early-stage apoptotic cells.

All of the above are correct and relevant answers.

In our platelet micropartcile protcol we tried to increase specificty by using two gates

- a platelet specific marker f.e. CD61 (FITC)

- an activation marker f.e. CD62P (PE)

To define the size we used calbrated beads (providers such as bang or biocytex are good) just as Leah described. We had 1um beads to define the upper cutoff. We also used a lower cutoff (0.2um; this was totally arbitrary) as resolution and unsepcific signals are alsways an issue. With control experiments we thought to be able to show that we could actually measure down to this size while elimination a lot of unspecific signals.

One caveat is, you need really clean sheath fluid (SF). We microfiltered (Millipore) and ran the flow cytometer on microfiltered SF for >15 minutes prior to starting our experiments. You can see the difference by running control samples and seeing all the "dirt" in your microparticle gate reduce as time under microfiltered SF progresses.

There will always be background. So you need to run adequate control samples and subtract the ackground signal from your emasured values

Tanusri is right in pointing out the similarities to apoptosis. There is some literature on that.

Beware that at congresses people will criticze your microparticle work becuase of the theoretic resolution power of flow cytometry. For mciroparticles Bruce Furie and his brother I belioeve invented an expensive machine, with better resolution, but none of us have access to that. If you do the right controls, f.e. showing micropartciles by electron microscopy and counting them absolutely, you will have a better stand.

Thank you for all the valuable suggestions..I will try as suggested.

Measuring platelet or other circulating microparticles is not an easy task. Some flow cytometry technologies have limitations due the size od MPs. In fact, measuring microparticles with flow cytometry is an object of debate. Please refer to the following references:

Perez-Pujol S, Marker PH, Key NS. Platelet microparticles are heterogeneous and highly dependent on the activation mechanism: studies using a new digital flow cytometer. Cytometry A. 2007 Jan;71(1):38-45. PubMed PMID: 17216623.

 Xiong G, Aras O, Shet A, Key NS, Arriaga EA. Analysis of individual platelet-derived microparticles, comparing flow cytometry and capillary electrophoresis with laser-induced fluorescence detection. Analyst. 2003 Jun;128(6):581-8. PubMed PMID: 12866871.

 Jy W, Horstman LL, Jimenez JJ, Ahn YS, Biró E, Nieuwland R, Sturk A, Dignat-George F, Sabatier F, Camoin-Jau L, Sampol J, Hugel B, Zobairi F, Freyssinet JM, Nomura S, Shet AS, Key NS, Hebbel RP. Measuring circulating cell-derived microparticles. J Thromb Haemost. 2004 Oct;2(10):1842-51. PubMed PMID: 15456497.

 Ayers L, Kohler M, Harrison P, Sargent I, Dragovic R, Schaap M, Nieuwland R, Brooks SA, Ferry B. Measurement of circulating cell-derived microparticles by flow cytometry: sources of variability within the assay. Thromb Res. 2011 Apr;127(4):370-7. doi: 10.1016/j.thromres.2010.12.014. Epub 2011 Jan 22. PubMed PMID: 21257195.

  Freyssinet JM, Dignat-George F. More on: Measuring circulating cell-derived microparticles. J Thromb Haemost. 2005 Mar;3(3):613-4. PubMed PMID: 15748272.  

Gines is right.

What you can do is

- set up your flow cytometry protocol (I can send you one by email, if you like)

- test it with in vitro generated platelet derived microparticles (add 1uM TRAP to PRP)

- calibrate your system with size beads AND counting beads

- verify your system by electron  microscopy (you'll see what you are measuring), you can count the MP absolutely as well

- double check your system by eliminating the MP either by ultracentzrifugation or by passing them through an 0.2um filter; MP are sticky you will filter them out very efficiently; this way you prove you are noz measuring artefact

Ref Valsami S et al. Thromb Res 2012

Greetings

Lars

Good bet to look for PMP. These are the easiest (or the less difficult) to study in plasma samples.

Nice to hear/read so many motivated "PMP hunters" on this forum. ISTH SSC vascular biology (Pr Dignat-George, Pr N. Key) has been running 2 successive international workshops (one finished in 2009 and one just recently closed) trying to harmonize PMP counts on various flow cytometers (see profiles on RG for F. Dignat-George and/or R. Lacroix for original and review papers, as well as mine for additionnal  posters). A major point to know is which flow cytometer you are using, (you do not need the magic "ghost" impedance flow cytometer from boston).

Can be either:

1)  FSC-optimized (mainly Beckman-Coulter Gallios/Navios and possibly BD LSR or Fortessa with PMT-FSC, plus possibly Stratedigm) or rather

2) SSC-optimized (mainly all BD FCMrs + several other brands e.g. Miltenyi MACSQuant, Partec CY-Flow, Stratedigm as well ... list available upon request).

Depending on which subtype you should use different reference bead sizes :

1) from 0.3 to 0.9 µm e.g. BioCytex Megamix-Plus FSC beads (also including 0.1 µm for future lower thresholds) for case 1 = FSC

2) from 0.16 to 0.5 µm e.g. BioCytex Megamix-Plus SSC beads for case 2 = SSC.

Indeed the same PMP (similar counts and subsets) will appear after gating between 0.3 µm-eq. to 1 µm-eq. in FSC or between 0.17 µm-eq to 0.6 µm-eq. in SSC (see a few of my posters available on my personal RG page).

For staining, CD41, CD42 are specific and highly expressed markers (Sinauridze et al Thr Hemost 2007, 97:425-34). If, as suggested, you want to use more colours and more (not totally platelet-specific) markers, CD9 and CD31 are also very well expressed on PMP (although one running dogma says that CD9 - a tetraspannin- is somehow specific for exosomes, not true in my experience). Adding Annexin V is very useful since most (but not all) PMP are AnnV+ . We currently do AnnV-FITC / CD41-PE staining but sometimes also CD41-PC7 (for multi-color staining of all 3 major subsets + one free PE stain available) or CD41 APC (to avoid compensation issues with AnnV-FITC).

When using AnnV (and the associated Ca+ binding buffer) you should be aware that (micro-)clots may quickly appear esp. in the most procoagulant plasma samples. Obviously those include plasma with MP-TF from some some cancer patients, or sepsis or ... but more trivial, these may also include normal plasma samples that you may have stored a few hours in the fridge before staining (or after thawing) to keep them under hands until your FCMr becomes available for MPs, since cold exposure activates many coag factors including FVII. Our suggested trick? Systematically add Hirudin 2 ATU in your binding buffer (see attached file, poster presented at ISEV 2014).  

Hope that helps. Philippe

Happy to share more of our experience in this field if interest. A few additionnal refs.:

Iversen et al, JIM 2013 (suggest Heparin as anti-coagulant to avoid micro-clots but I don't like odd effects of heparin and this anti-coagulant artificially increases [PMP] after phlebotomy, see Lacroix et al JTH 2011).

Aass et al, Cytometry A, 2011: always "ultra-centrifuge" (at least 20,000 g 5 min) MAb-conjugates prior to use, since fluorescent MAb aggregates mimick the size of MPs, enter MP gates and may be confounded with stained MPs.

Robert et al, ATVB 2012 (for FSC-optimized Gallios/Navios, standardized cut-off at 0.3 µm-eq.)

Lacroix et al (several papers related to both Pre-Analytics and Analytics of PMP in the 5 last years). 

Dear Lars M Asmis,

I would like to have detailed protocol for flow cytometer. I use beckman coulter Navios for analysis. You can send  protocol copy to [email protected].

Thank you

Dear Mallikajun,

you can achieve useful info from this article

Dey-Hazra E, Hertel B, Kirsch T, Woywodt A, Lovric S, Haller H, et al. Detection of circulating microparticles by flow cytometry: influence of centrifugation, filtration of buffer, and freezing. Vascular health and risk management. 2010;6:1125.

i do this protocol for PMP isolation from plt concentrate:

- 200g -15 min for -cell removal

-4200 rpm 15 min 2 times- for plt removal

-16000g 20 min for pmp 

good louk

Philippe:

I've spotted antibody aggregates by just adding my antibody mix to the buffer and running this through the flow cytometer. I was thinking of solving the problem by including an "antibody only" control in every experiment as the baseline, and subtract the false-positive MP event number from that measured in the sample tubes. This would be easier than centrifuging and transferring supernatant from 9 antibodies prior to each experiment. What do you think of this?

I do agree to Lars and suggest for SEM as well as DLS for morphological characterisation as well as size estimation after performing microparticle expression under FACs. Although after FACs ,cell sorting must be done under FACs cell sorter to isolate platelets and microparticles by selecting the qudarant of respective optimization and collecting both of them in separate chambers and then performing the said experiments.Good luck

Is CD61 a better marker for PMP .... what is the best?

Please contact Dr. Fabian Eckstein for request about platelet microparticles and flow cytometry analysis.