Michael, you are right you will see aggregation occurring with this method and yes it can result in a loss of events/failure in method. However, the numbers of platelets in Tomer's methods are relatively low, suspended in citrated plasma and diluted 100 fold further for flow cytometry. So only in a few cases will you observe aggregation. When I was using this method for looking at platelet activation by non-HIT anti-platelet antibodies a few samples aggregated all the time, and some dependent upon platelet donor. If you want to avoid this occuring then I suggest that using an anti-GPIIbIIIa agent such as GPRP is best. It does add to the cost though, so you could reserve this for the samples that clot. Craig Turner ---------- From: MSuter@omlabs.com To: cyto-inbox Subject: RE: Platelet activation studies Date: 29 January 2002 09:01 <<File Attachment: ALTERNATIVE.HTM>> I'm grateful to the many people who took time to reply so quickly and carefully to my question about technical details involved with platelet activation studies. To summarize the responses: * For inducing platelet activation, a number of people recommended ADP (20 mM) over calcium ionophore (which Stephen Benoit described as a 'blunt instrument' for this purpose). Other suggestions were thrombin (with peptide GPRP to inhibit fibrin polymerization), TRAP, and ADP + epinephrine. After warning me against use of calcium ionophore, many folks then described how to make this work (apparently CaI is very labile). CaI is also expensive, so I'm thinking ADP which we stock anyway for platelet aggregation studies will be our agonist of choice for creating a positive control for platelet activation studies. * I recieved consistent advice to consider Annexin V over CD62p as a marker of platelet activation. * Washing of platelets was discouraged, to avoid platelet clumping. I will list further details below. Particular thanks to: Craig Turner (CDL, UK); Stephen Benoit; Suha Kasey (Beckman Coulter); and Michael Koratich (Birmingham,AL). As an additional question: The flow cytometry assay for HIT begins like the platelet aggregation method. Normal platelets + heparin + patient plasma are incubated. In patients with HIT, won't the platelets aggregate? If so, won't this impair the flow cytometry measurement of platelet activation? -------------------------------------------------------------------------- -- ------------------ Post 1: Michael, with regard to your problem with ionophore and platelet activation. In my experience A23187 can be very labile under certain conditions. Here are some of the standard methods I use to ensure consistent activation. I use the free acid form from Calbiochem (supplied by CN biosciences cat:100105). Make up a stock 1mg/ml solution in DMSO Store in 50 microlitre aliquots at -80. I have stored at -20 at the bottom of a chest freezer. For use remove from the -80 and leave to thaw at room temperature in the dark. DO NOT warm in the hand or waterbath. For around concentration 2x10^9/L you need about 5-10 microlitres/100 microlites of platelets of a 1 in 10-20 dilution depending on the effect you are looking for (MIX WELL). These values will give you significant activation and consistent binding of FITC annexin V (>90% positive events). Incubation times vary with final concentration, 5-15 min with those described above. However, I suggest you optimize for your system. I wouldn't recommend a wash step, because unless you have really well washed platelets or GPIIbIIIa blocker the platelets will clump in a mass at the bottom of your tube. I keep the unused stock in the fridge for about a week and the -80 stock for about 2 months as per data sheet. As an alternative, have you thought about using thrombin or TRAP to induce P-selectin expression? Both of these are used regularly in the literature (and in Purdue archives), and I have used both for positive controls of P-selectin expression. -------------------------------------------------------------------------- -- ------------------ Post 2: I did some platelet work a few years ago and was using ADP as my positive control for activation. It worked like a charm. I believe we were using Dr. Ault's method but I don't have a specific reference for it at this time. I hope this helps. -------------------------------------------------------------------------- -- ------------------ Post 3: Calcium ionophore is a rather blunt instrument for getting activated platelets. You might try Biodata's ADP combined with their Epinephrine or some of John Fenton's thrombin (include the peptide GPRP to inhibit fibrin polymerization). Although I haven't read Tomer's paper, I would be encourage you to look at some papers by Michelson AD, Barnard M, and Benoit SE, and others for methods of studying platelet function using flow cytometry. -------------------------------------------------------------------------- -- ------------------ Post 4: Your email regarding the question regarding HIT Assay was forwarded to me, I was one of the people who developed it. First I would like to ask why are using CD62 instead of Annexin V, second I found that working with commercially available CD62 that activation of platelets should be done with ADP instead of ionophore. I am sorry to say that I just moved to San Diego and all my papers regarding this subject are in transit. I am afraid that I will be leaving today to go to Miami until Fed 5, I don't have my address book with me, but I am trying to get the email of the author -I just emailed Atlanta to see if I can get before I leave today-. As I said working with commercially available CD62 I found that it is activated with ADP (20 mM) and not IONO. I would urge however to see if you can get your hands on some Annexin V because the assay works just perfectly with it. The IONO used in the experiment is Sigma C9400 Calcium Ionophore III hemimagnesuim salt. I am sorry that I can't remember my exact numbers, like I said all my notes are in transit, but and I am hoping that they still make it in a sealed vial, inject 1 ml methanol into a 5mg vial and it is important that the vial stays sealed. I draw 200 ml aliquot from the sealed vial, using a syringe, as needed into a small centrifuge tube and discard it after a few experiments, you can actually smell the IONO when first taken out of the vial, and after a time any IONO in the centrifuge tube looses that distinctive smell. Take 5.3 ml of the stock IONO and add that to 95 ml of PBS, the final concentration in the 50 ml reaction tube should be 5 mM. As for the reaction after incubating the platelets with the heparin you take an aliquot into a prepared tube with your CD41, Annexin V (or CD62) and PBS and incubate. Also you use the IONO only as an internal control and is not added to all the tubes and it is incubated with the cocktail -DO NOT WASH THE PLATELETS AT ANY STAGE-. If you insist on using CD62 I would suggest titration of the CD62 with Activated platelets -ADP or IONO- to see how much is needed to effectively see a reaction. -------------------------------------------------------------------------- -- ------------------ +++++++++++++++++++++++++++++++++++++++++++++++++++++++ The National Blood Service. Do something amazing today - Give Blood. Please call 0845 7 711 711. You can visit us at www.blood.co.uk, or on BBC2, Ceefax page 465. +++++++++++++++++++++++++++++++++++++++++++++++++++++++ The views expressed in this e-mail are those of the sender, and not necessarily those of the National Blood Service. This text confirms that this e-mail message and its attachments have been swept for the presence of computer viruses by the National Blood Service, however we cannot guarantee that they are virus free. All e-mails and their attachments to and from the nbs.nhs.uk domain are archived, and their contents may be monitored. +++++++++++++++++++++++++++++++++++++++++++++++++++++++
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:25:57 EST