Hello all
I am looking for a suitable method for preserving neonatal blood specimens for future use. these will be collected over a two year study period. the grant provider wants the sample to be kept but they do not know what they are going to use it for. They are most likely to give another grant in future for finding biomarker for infection. so, the objective of this storage is to preserve the blood in a state as close as to the original.
My target is to preserve the cytokines, nucleic acids, soluble proteins and cell surface markers. it is ok if the cells die but do not lyse.
Hope someone will be able to help me.
Since you are not too sure of what they want to use the sample for then it will be difficult give specific advice, meanwhile you get this publication :Biological sample collection and processing for
molecular epidemiological studies
Nina T. Holland∗, Martyn T. Smith, Brenda Eskenazi, Maria Bastaki:Mutation Research 543 (2003) 217–234
Yesterday, I got the result of an experiment. I drew my own blood in citrate tube. i dipped the tube directly in liquid nitrogen and then stored the tube at -80 C one week ago. I brought the tube out yesterday and kept at room temp to thaw. Then I centrifuged the tube. All the cells were haemolysed.
Is zap freezing responsible for the extensive haemolysis? I can not keep a -80 C freezer in the field.
Should I try separating the cells and plasma, then adding DMSO to 10%? I want to freeze the two tubes in liquid nitrogen vapour (slow cooling rate), then keep those in that chamber (without dipping in liquid nitrogen).
What do you think of this method?
Dry ice is not available in that division/state.
It will take at least 12 hours to take to the field.
can you tell me - how long will dry ice last inside the box? a resupply schedule of more than twice a week may not be feasible.
in that case, I will have to budget for another -80 freezer. however, do you suggest i send a man to the field with the dry ice? he will put the specimen tubes in the dry ice and head back to the lab.
will that be cheaper than liquid nitrogen? it stays in the cylinder for more than a month with virtually no loss. do you think shipment in liquid nitrogen vapour will be difficult?
Can you tell me if there is any portable freezer/refrigerator that can run 5 to 6 hours on its own (without power from car or anything)?
Is there any renowned dry ice chamber in the market (wherever it is) that can hold dry ice for 3 to 4 days?
I am making the SOP with option for both liquid nitrogen and dry ice. the sites will decide which one they want to use depending on availability and cost.
The specimen transports are within countries (remote to town) only. will need special measures only when we send those out to another country (to reference labs). we will sort it out as the study kicks off (after getting the grant).
You have given a very important vision. we may not ever need the erythrocytes. I will check if density gradient centrifugation is possible. Is there any commercial kit for fully removing the erythrocytes from blood? what anticoagulant does it recommend?
Thank you
Irfan
Hello Grant
I got your point on dry ice. I will give a trial soon.
Please send the information regarding the separation process. though we plant to collect blood in citrate based Bd vacutainer tube, the volume of blood will be at most 2 ml. for banking, a maximum of .5 to 1 ml will be left. will the separation tubes work with such low volume? I will search BD site today. please send me the information on separation.
Irfan
so far I have studied, haemolysis spoils the scope of all other analysis except PCR. freezing will cause haemolysis to some extent. so, the best way is to separate the cells and also erythrocyte if possible. the problem is, the site laboratories are not in favour of separating blood components. they will not be able to do any centrifugation. it feels like i will have to be satisfied only with adding DMSO to the blood tubes.
we are using this kind of a centrifuge in our lab. ours is from eppendorf.
anyway, thank you for the help. i would love to have the protocol you make. who knows, this may not be the end of the road.
regards
Irfan
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