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canīt get serum from blood samples - (May/19/2010 )

Hi everyone!
I can not get serum from mice blood samples (bleeding from retro orbital plexus). The clot donīt form properly . The same mice were bled some weeks ago with no problems. any clues on why this could happen? Thanks :(

-clutz-

Hi there,

How much blood are you getting? What kind of blood tubes are you using? How long are you waiting for the blood to clot? What speed are you centrifuging? I haven't used serum for my rodents as of yet just plasma, but in my vet clinic experience for dogs and cats, we used serum separator tubes (they have a red top or marble top usually), inverted about 5 times, let it sit about 10 minutes to clot, and then you centrifuge the tubes.

http://www.bd.com/vacutainer/pdfs/plus_pla...uide_VS5229.pdf This site from BD goes over tubes and additives.

http://www.bd.com/vacutainer/pdfs/plus_pla...mple_VS5177.pdf Procedure for BD serum tubes


Hope this helps. Let me know how it works out for you.

Stacy

-Stace-

Stace on May 21 2010, 08:12 AM said:

Hi there,

How much blood are you getting? What kind of blood tubes are you using? How long are you waiting for the blood to clot? What speed are you centrifuging? I haven't used serum for my rodents as of yet just plasma, but in my vet clinic experience for dogs and cats, we used serum separator tubes (they have a red top or marble top usually), inverted about 5 times, let it sit about 10 minutes to clot, and then you centrifuge the tubes.

http://www.bd.com/vacutainer/pdfs/plus_pla...uide_VS5229.pdf This site from BD goes over tubes and additives.

http://www.bd.com/vacutainer/pdfs/plus_pla...mple_VS5177.pdf Procedure for BD serum tubes


Hope this helps. Let me know how it works out for you.

Stacy


Hi Stacy! thanks for your reply.
I'm getting about .3- .5 ml of blood. We use regular eppendorf tubes and this time we waited like 1 h at 37šC without getting the clot (normally 10-15 min). Then, we tried to centrifuge at 2000 rpm and still not serum! The very small amount of serum obtained is with hemolisis. The only difference between previous sampling and this one is that we recently change the mice beds using a comercial product (sawdust) from a petshop. We suspect that this bed may contain some toxic resins and that could cause problems with the liver and coagulation factors (they are in cages into a closed ventilated cabinet). I would appreciate your opinion.
Otherwise the procedure and material are the same as usual...

-clutz-

Hi,

I'm not sure if the sawdust could be a major problem in getting serum, but if it is, someone please correct me on it. I would consider using a serum blood tube for the samples. The serum blood tube contains a gel in it which should help in separating the layers in the blood. You might even get more serum using a tube like that one rather than a regular eppendorff tube. I also found this on the BD website regarding their tubes and centrifugation speeds/times:

"non-gel blood collection tubes, including those that contain heparin, EDTA and non-gel serum tubes can be centrifuged at ≤1300 RCF for 10 minutes. The BD VacutainerŪ SST™ and PST™ gel tubes should be spun at room temperature at a speed of 1000 to 1300 RCF for 10 minutes in a swinging bucket centrifuge and 15 minutes in a fixed-angle centrifuge"

I'm really not sure what the conversion between rcf and rpm is. I believe that it depends on the size of the rotor, but hopefully, this will help you a bit more.

-Stace-

I know the phenomena in human blood and this was most of the time correlated with high protein content >>100 g/l and/ or monoclonal protein (most of the time IgM-class). Most of the time we switched to plasma to get results.
Also cryoglobulines with coagulation temperatures above 30 degrees celsius are a possibility, centrifuging at 37 degrees could help.

-Gerard-

Thanks a lot for your comments! Iīll try the serum tubes and centrifugation at 37šC.
greetings!

-clutz-

You could also try using Microtainer serum separator tubes.

VWR

I allow the blood to clot for at least an hour than spin them for 5 min at 10,000 rpm, 22C. always have more than enough serum

-toxo-