I have received a couple of requests for more information about defibrination. I tried to reply off-list, but one of the requestors email address won't work for me (Dr. Hollmann) so I decided to post my reply here. Sorry to use the bandwidth for such an obscure purpose but here goes: It has been so many years ago that I no longer have a written protocol, but I'll try to describe what we did. The basic idea is to collect the blood with no anticoagulant and then rapidly stir the blood for about 10 minutes while it clots. If rapidly stirred, the fibrin forms as long strings on the stirrer and separates cleanly from the rest of the blood. After about 10 minutes, you are left with nice blood that will no longer clot and is nearly totally devoid of platelets. We were able to layer this blood onto Ficoll and get very nice separation of mononuclear cells at the interface. We used two methods. For small volumes of blood (a few ml.) you can put the blood into a tube or beaker and stir with a glass or wooden stick - I used to use the long end of a wooden swab. You have to stir quite fast and not stop at all until the clotting is finished. The fibrin will wind up on the stick. I think it works better if you use two sticks held together. For large volumes of blood (100ml or more) and to keep the cells sterile, we would place glass beads in a flask. The beads were about 3-4mm in diameter and we would put about 30ml of beads into a 250ml flask. The flask could then be autoclaved before use. We would put in 100-200ml of blood and rotate the flask rapidly so that the beads swirled around inside. After 10 minutes of rapid rotation the fibrin would be wound up on the beads in one big sticky mass and the blood would no longer clot. The only disadvantages of this method are that you can get some hemolysis if you are too vigorous, and, of course, you are activating the coag cascade thus activating platelets and perhaps leukocytes. However, I used to get perfectly functional lymphocytes and monocytes this way - I don't believe I ever looked at the neutrophils using this method. I hope this is helpful, Ken Ault
This archive was generated by hypermail 2b29 : Sun Jan 05 2003 - 19:01:41 EST