We want to do whole blood assay in the field. Blood in cryovial will be stimulated with antigens. The other day, the tube with clot at below and the supernatant above is to be frozen UPRIGHT and UNDISTURBED. We have liquid nitrogen, but can simply throw the tube into the vessel. We need to freeze it slowly. And then throw into liquid nitrogen. We will not be able to use fridge in the field. I think if we can get something like a DC run small freezer??
well, it depends on the volume of blood sample, and whatever other materials you would have in the field...
using a box with ice (and maybe added salt to stimulate temperature drop due to melting) or something alike could slowly freeze low-volume samples... and the you could use liquid nitrogen slowly added to the mixture, for example, to gradually lower the temp of the ice until eventually deciding to put the samples in the pure liq nitrogen...
you need to freeze it slowly just so you maintain the separation clot vs supernatant, in an upright manner? or for some other reason...?
flashfreezing directly in N2 would be the best for conservational purposes... and you can achieve that by pouring the liquid nitrogen in a smaller container where you can literally, for example, using forceps, carefully submerge the tubes/criovials...
As Rafael said, it depends on the volume of sample.
It also depends on what type of Anticoagulants are used, for example if EDTA is used instead of CPDA then EDTA forms crystals at low temperature and can rupture the RBC's which might increase the potassium levels in whole blood. Freezing cells is not a good idea according to my knowledge and while thawing the cells mostly will be ruptured.
If you can say what amount of blood you are trying to freeze and is it with or without anticoagulant, then it would be easy to suggest.
kirankumar,
is the blood not already coagulated as a result of antibody precipitation? Or is there more coagulation to occur from fibrinogen and other blood components? I guess they're coagulating through immunoprecipitation, so they know in field what bloodtype they're collecting, and then they need cold for conservational purposes, but I don't know anything about EDTA and other agents normally used with blood samples and now I'd like to hear your explanation :)
best regards
Thanks!! great replies!!
1. Rafael, We had thought cooling salt solution in a wide cup with liquid nitrogen. This forms a hard upper surface of crystallized salt. We have to break it and put the cryovials in. This is good but I fear this will shake the vial and mix the RBCs below with the plasma, reddening it.
2.Dear Kirankumar and Killol, we use heparin (in the blood collection tube). We use 1ml of blood. We put prescribed amount of antigen and incubate at 37C. The next day the RBCs have precipitated to the bottom, not clotted. We plan to do the ELISA. Killols method of seperating the plasma is good. That may be done. Is there any method by which I can still keep the RBCs and plasma together? but without making a chance of mixing them, which can affect ELISA. After some months of freezing, we thaw and are not going to use the RBCs but the plasma only.
@Rafael Félix
Although coagulation can be done for antibody precipitation, I would not recommend it, because when you coagulate the sample, there is a good chance that you are loosing the antibody in the clot. When fibrin starts to form after 5-6 min of blood collection, calcium also plays a role in clotting (clotting factors) so the clump you will have will contain the antibodies and you will not get a good yield, hence anti-coagulated blood is more preferable in my opinion.
@Divya Rana
The only way which I can think of keeping plasma separated from cell yet keeping both together is by centrifugation, this way (as your sample is having anticoagulant) the cells will settle down and just above the cells buffy coat is formed and plasma will stay well clear of cells but the centrifugation may cause all your antibody to settle down and probably they will be in the buffy coat and you need to very careful aspirating buffy coat (you need a little bit of practice to do it). Once the buffy coat is formed its easy to freeze and thaw, even if RBC's rupture during this process the plasma will not be affected.
Divya,
I'm sorry for such a strange question, but if nowhere in the experiment will you need the RBCs, why so much care for them? Or is it all care for the plasma that you want preserved?
Why don't you guys remove the supernatant in the field (carrying with you stuff like pipettes, micropipettes, whatever) and discard the pellet, flash freezing the aliquot of plasma removed? Something you can try to ease that up is the lettuce-dryer as a "DIY field centrifuge":
http://bitesizebio.com/3200/how-to-build-a-plate-centrifuge-for-25/
hehe
best of lucks!
and thanks fro the clarifications of you all!
Haha, thanks Rafael.
Removing the supernatant is most reasonable.
All these experiments I have said was prepared by a bigger expert. I am just following the protocol made by him.
May be that is because we may not have a centrifuge in the field.
Your link is helpful. We have to see if we can prepare that.
Thanks Kirankumar about making me aware of the minor things I have to be careful.
Sincerely,
Divya
We use Mr Frosty's box for slow freezing our cells. Not sue if that will be helpful for your case. Put enough iso-propyl alcohol in the inside chamber then store the vial in designated slots. Transfer entire box to Liquid nitrogen. Thermoscientific Cat No. 5100-0001 for the box (Link below)
http://www.thermoscientific.com/content/tfs/en/product/mr-frosty-freezing-container.html
Dear Susmita
Please say something more.
A Mr. Frosty we have is about 10 cm diameter. This cant be inserted into our Liquid nitrogen jar (mouth 5 cm).
We have to pour the the liq. nitrogen into another vessel. The thickest of our plastic jar had broken last time when we put the liquid nitrogen into it. It cant resiist.
Inspite of that, will the liquid nitrogen "slowly" freeze our tube with the LIQUID NITROGEN?
Sincerely,
Divya
Well Divya,
Our N2 Dewar (tank rather than flask) is way wide mouthed so no problem inserting Mr.Frosty in it. I think iso propyyl alcohol does the slow freezing trick and frosty box of yours are same as ours. You may try similar trick in any Liq N2 friendly tube (which will fit into your Dewar) into which you can dunk your sample tube and pre-fill the outer tube with cold isopropyl alcohol. Just one of my wild ideas.
Good luck.
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