After collecting approximately 800ul of mouse blood at the end of a terminal experiment in a 1.5ml centrifuge tube with a dab of "serum goop" from a red top vacutainer, we allow the blood to clot for 45min at room temp. We then spin at 5,000xg for 5min at 4°C and transfer the serum top layer to a fresh tube and store at -80°C until analysis by ELISA. When the tube is thawed an "additional clot" is present in the serum. Any ideas as to what the "additional clot" is and how I can prevent it from forming in my sample?
I concur with Madelyn - the clot is most likely due to residual fibrinogen surviving the initial clotting event. Be it mouse, rat, rabbit or human, it is always a good idea to chill the clotted sample to allow the clot to contract and hopefully any residual fibrogen to be converted to fibrin.
Whether you need to aliquot or not depends on what proteins you are studying. Antibodies/immunoglobulins are pretty hardy; I have used 20+ year-old serum samples that had been stored at -20oC and been thawed and refrozen numerous times with little or no reduction in antibody titre. But cytokines like gamma-interferon are more labile and can be destroyed by multiple freeze-thaw cycles.
What you are seeing is probably a fibrin clot. Incubate your blood for 1 hour at 37 degrees and then chill at 4 degrees for at least an hour before processing. Freezing at -20 or -80 should not make a difference but aliquots are definitely needed because of freeze thaw issues.
I concur with Madelyn - the clot is most likely due to residual fibrinogen surviving the initial clotting event. Be it mouse, rat, rabbit or human, it is always a good idea to chill the clotted sample to allow the clot to contract and hopefully any residual fibrogen to be converted to fibrin.
Whether you need to aliquot or not depends on what proteins you are studying. Antibodies/immunoglobulins are pretty hardy; I have used 20+ year-old serum samples that had been stored at -20oC and been thawed and refrozen numerous times with little or no reduction in antibody titre. But cytokines like gamma-interferon are more labile and can be destroyed by multiple freeze-thaw cycles.
In your case the coagulation of the blood before you are start to prepare the serum is incomplete. You still have fibrinogen and active thrombin in your "serum".
Either you are going for an complete anticoagulation to receive plasma (either EDTA 1 mmol/L or citrate or Heparin). In some conditions it's also helpful to add some protease inhibitors (like aprotinin or PMSF).
If you would like to receive serum, let it at room temperature at least 4 - 8 h, overnight at 4 °C, than go for centrifugation. There are special "serum tubes" with a clot activator (it's a silicate which increases the surface which is important for initiating the coagulation process).
Both, serum and plasma can have some precipitates after thawing. These are mostly proteins with a large MW (vWF, alpha2macroglobulin). They can removed by high speed centrifugation as well.
Strange this behavior, but we will try to raise some possibilities, which the activator of coagulation in this tube, try to investigate this event by submitting a simple comparison, collect the blood in a vacuum tube to the BD and vest in a tube completely dry with no additive, may find the answer to that happenstance
I am agree with Stephan Kiessig, as my observation is that these clots are more often in Human plasma of malaria patients than Human serum of same patients. I am collecting both to analyze differences in platelet related factors.
We see the same with many plasma collected from children with malaria in heparin tubes and after storage in -80. My thought is that these are just some plasma proteins that precipitate out on freezing. What we do is we vortex the sample and do a short spin to remove the clot as it might interfere with the assay. The degree of clot formation seems varied also, with some having none and some having much more. I have also seen these with malaria naive plasma so i think it has nothing to do with the infection.
As mentioned by most of the researchers the clots are most often seen in the human plasma samples post freezing. As suggested, post thawing you can fiter it with the 0.22 micron filter. Also i would suggest you can some sodium citrate soln before filteration and then you can freeze it down. This should take care of the clots completely.
Do you guys think this could be because of the insufficient time of clotting (45 min (as done by Bruce) versus 60 min- as recommended by the BD for red cap tubes) and can be avoided if let it sit for 60 minutes in an upright position?
I just want to know so if it is an easy fix or do we have to go through 1 hour inucubation at 37 C and chill for 1 hour at 4 C before centrifugation? The later stage is not practical when you are handling lot of samples. Thanks.
- Badges
- Science method
- Similar topics
- Medicine
- Immunology
- Immunology Techniques
- Immunoassay
- ELISA