RBC hemolysis - (Dec/15/2009 )

dear friends

i have problem in getting clear serum from mice blood. i collect the blood from orbital sinus (eye), keep it at 37 C for 1 hr. then keep it in fridge (4 degree C) for 3-4 hrs. but when i centrifuge it, blood gets hemolysed. this prob doesn't occur with rabbit blood. whats the reason for the hemolysis, because i handle the sample very gently? please help..

I think this can happen during the eyebleed. you might try for a larger bore capillary tube? sometimes that can help, although you need very precise technique or this is harder on the mice.

I'd also be concerned about the time at 37C; that will only accelerate the hemolysis.

you might try bleeding via saphenous leg vein stick; there seems to be less trauma to the tissue and less hemolysis.

http://www.nc3rs.org.uk/bloodsamplingmicro...page.asp?id=390

why do U have to keep the sample for such a long time before U centrifuge?

one hr incubation at 37 C is recommended before centrifugation. there is no specific reason for putting clotted blood samples in fridge.i do it because i dont have to collect serum immediately.

Nabi on Dec 15 2009, 06:12 PM said:

Why can't you use a serum separation tube?

First, did you check the hemolysis is not present in vivo?
Otherwise there is the possibility the celmembrane is very fragile by nature and then you can try to use less G's by centrifugation.

no..how can i check it?

Gerard on Dec 18 2009, 01:40 PM said:

After clotting you should see bloodserum that is the yellow liquid apart from the clot, when there is strong hemolysis there is almost no difference in color between serum and clot.

sgt., the serum-separator tubes are often not very effective with really small volumes of blood, like when you want to do serial bleeds on the same mouse.

I often had hemolysis on eye bleeding. And the worst was I made a number of poor creatures blind after serial bleeds.
Then I had much less hemolysis and spent less time with tail bleeding. Prewarm mice 3-5min before bleeding. No anaesthesia required by my institution animal ethics. Use a mouse strainer to fix the animal except the tail. I got ~50uL serum per mouse this way every fortnight. Of course you could get more but the animal ethics may not allow you to. In general I believe tail bleeding is less stressful both for me and mice.