Hi Sailesh,

Maybe your platelets become activated during the preparation. Which protocol are you using?

Best,

Simon

I had similar problems when I started with platelet functional analysis. How do you do your platelet separation out of the blood? Maybe your centrifugation speed is too high, so that you loose many platelet during preparation because of activation? We first also tried to use purified platelets for our activation analyses by FACS, but then changed to measuring the activation directly in whole blood. You don't need to purify the platelets, so you don't loose them. So, you also don't activate them by too much centrifugation or pipetting etc. Simply give 10-20 µL of whole blood to your antibodies and after washing one time with PBS directly take them to the cytometer and activate them during measurement. If you don't want to see the whole activation process but only the state before and after activation, take two samples of the whole blood, activate one but not the other, stain both the same way and wash and fixate them with 0,1% paraformaldehyde, then you even don't have to measure it directly but can do this some other day. Good luck!

If you need a more detailed description, I can also send you a copy of my manuscript, where I used this protocol: http://www.ncbi.nlm.nih.gov/pubmed/23382103

At what speed are you obtaining your PRP? You might be losing platelets during centrifugation. Have you tried to perform your flow cytometry using whole blood?

In addition to the questions asked above, which monoclonal antibodies are using as a primary platelet specific markers (e.g CD41a, PAC-1, CD42b,or CD31). If you activating platelets and using either CD41a or CD42b as your platelet specific marker then you might be getting low platelet counts due to the conformational changes that occur in these receptors upon agonist induced activation. Hence your monoclonal antibodies might not bind to these platelets with have undergone such changes on their surface receptors. Another aspect that shouldn't be ignored is that if you using whole blood, activated platelets may interact with leukocytes and form complexes and this may result in a population of platelets that may not fall in our normal platelet gate based on side scatter and forward scatter properties.

lastly, do you perform platelet counts using an alternative method (using a hematology analyzer) on each patient sample?

Dear all,

Thank you all for the answer.

I have attached my protocol. Please view it and you can give me suggestion in more detail.

Julia Frobel, I couldn't get access to your link, so could you send me pdf file of that article.

Regards,

Sailesh

All the comments above are valid points that you should question and address. One of the most important will be the consistency of your platelet rich plasma (PRP) preps. The volume of PRP you are going to get from 4mL will be fairly low, so centrifugation procedures will be critical (eg. the amount of braking following the run will affect how clean your separation is). Even your post-centrifugation harvest of the PRP from the blood will need to be done carefully to ensure you are getting the majority of platelets. Blood from males or subjects with high hematocrits will result in lower PRP volumes which might make it difficult to harvest a sufficient volume of PRP. However, as pointed out, you could use whole blood for these studies to minimize changes to your platelets due to handling since it is the direct in-vitro state that you want to measure.

I fully agree with Simon, platelets may easily aggregate when they get activated during preparation . Of note, in certain patients the level of activated platelets in circulating blood may be very high- for example if they suffer from certain heart diseases. In consequence such platelets become invisible in gates set for small objects you use on your FACS. Full blood analysis may help because you don't need to manipulate samples that extensively, just dilute them with PBS but remember to take a large group of healthy donors for comparison. Good luck.

Arkadiusz

Washed platelets for ETP assays Reagents

• HEPES-buffered saline (HBS; 150mM NaCl; 5mM KCl; 1mM MgSO4; 10mM HEPES),

- pH 7.4 and pH 6.0

- filtered with 0.2m filter

• PGI1 Sigma P6188; (20mg/mL stock in dH2O = 100X conc.)

• ACD anticoagulant (0.085M tri-sodium citrate; 0.071M citric acid; 0.11M glucose)

Method

1. Collect blood into citrate vacutainer tubes and immediately mix with 1/6th volume ACD anticoagulant (draw off 3ml into an EDTA tube first and use for a full blood count).

2. Prepare PRP by centrifugation at RT at 900rpm (143 g) for 20 min.

3. While the blood is spinning, prepare a 1 in 100 dilution of 100X conc PGI1 stock. Keep on ice throughout.

4. Transfer PRP to a clean round bottomed tube (LP4) and add 1uL diluted PGI1 per 1mL PRP. Mix gently by inversion.

5. Sediment platelets by centrifugation at RT at 1750 rpm (600 g) for 15 min.

6. While the sample is spinning, prepare another 1 in 100 dilution of PGI1 stock, and keep on ice.

7. Take centrifuged tubes and remove supernatant. Resuspend platelets gently, without forming bubbles, using a pastette, in a small volume (~0.5ml) HBS pH6.

8. Dilute platelets in a larger volume (~10ml or to top of tube) of HBS pH6 and add 1uL diluted PGI1 per 1mL buffer.

9. Centrifuge platelets at 1750 rpm (600 g) for 20 min.

10. Remove supernatant.

11. Resuspend platelets in small volume (1-2ml) of HBS pH 7.4) TBS and perform cell count, using the AcT Diff.

12. Adjust platelet count to 200 x 103/uL, in HBS pH 7.4.

13. Use platelets as soon as possible but ensure that platelets have had >30minutes since PGI1 was last added.

Clearly you should precise the isolation protocol you're using. With such amount of blood you should harvest about 1-1.5ml of platelet rich plasma with at least 200 000 000 plt/ml. There is obviously a problem with your centrifugation protocol.

What type of anticoagulant are you using?

If you're having problems of activation of your samples (as the cause of the poor number of cells you harvest), you probably have a high CD62P signal in the small number of events you're able to detect. How is your CD62P signal?

Alternatively you could use, as it was mentioned on the message above, whole blood using 5µL blood in you antibody and add 0.5-1ml PBS after incubation and before running the samples on the cytometer. You need to dilute the whole blood to be able to detect platelet events on the FS/SS plot.

I hope this will be helpful!

- Good luck -

I have had good luck with washed platelets (but you have to be even more careful not to activate them than with just PRP) in FACS for P-selectin expression and PAC-1 binding assays to evaluate effect of platelet activation inhibitors. Usually, I start with at least 20-30 mL of blood drawn into 3.8% sodium citrate in a 60-mL syringe and add ACD before first spin. See attached protocol. Good luck!

Hi Sailesh,

For me, 4 mL of blood is good enough to get the enough platelets for receptor expression analysis through Flowcytometry. Platelet isolation is very simple and sensitive method. As above mentioned, platelet easily get activated even with mishandling and temperature fluctuation. They love being on 370C throughout the procedure. I would suggest you to spin the blood at 100g for 7 minutes to get the platelet rich plasma (PRP). If you want to use PRP for your analysis then you are good otherwise wash platelet twice with modified Tyrode’s buffer (137 mM NaCl, 0.3 mM Na2HPO4, 2 mM KCl, 12 mM NaHCO3, 5 mM N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid, 5 mM glucose, pH 7.3) in the presence of PGI2 by centrifugation at 700g, and resuspended in Tyrode’s buffer at concentrationof 4 x108 platelets/mL. Good Luck

I think that for your study, when you would like to find whether platelet activation occurs in chronic urticaria patients (allergic disease) or not, you should employ the simple protocol for staining platelets in whole blood. You can apply a protocol from Becton Dickinson webpage (www.bdbiosciences.com) or can find any guidance in publications from our laboratory . In flow cytometry you may analyse platelets in whole blood without isolation of PRP or cells. Then you should use the gated antibodies for platelets like CD61, and monitor the interested antigens (like P selectin) in CD61-positive objects. This protocol allows to avoid the platelet activation related with PRP isolation and monitor platelets in a small blood volume.

The major problem with your method is in the centrifugation. You cannot centrifuge the sample for 20 min. for 2 reasons: 1. you are going to loose the platelets 2. you might activate the platelets which will lead to false results. We centrifuge the platelets for 2 minutes by 2000 rpm to obtain PRP. Try to analyse your PRP you get after the first centrifugation by a standard blood cell counter to find out how many platelets are left in it.

Thank you Mr. Peter for your suggestion.

Try using a PDQ centrifuge for PRP prep

Platelet-rich plasma is obtained by centrifugation of the blood at 140 (or 180)g for 15 min. If you use higher speed you will obtain platelet poor plasma. Faster spin = shorten time.

The PRP should be acidified to pH 6.5 (with 1 M citric acid), and latter centrifuge at 2,000 g for 20 min to obtain a pellet which can be resuspended in a Ca2+-free Tyrode's buffer. You should easily see the swirling effect in resulting suspension