Whole blood storage and transport - (Apr/16/2012 )

Hi,

I'm undertaking a research project involving getting blood from around 60 ewes over one day and looking at storing in an eski with wet ice. I've got the s-monovettes (EDTA KE/9ml) likely using the vacuum method. I might not be able to make it back the campus laboratory before it closes at 5pm, however I need to conduct Sambrook's (2006) "Rapid isolation of mammalian DNA" protocol with the whole blood which I then plan to sequence and genotype if polymophisms found.

The issue is I'm a little confused on how I need to store the blood for what I'm trying to do and if I can't make it to the laboratory till the next day would storing the samples in my new refrigerator at home be a problem, as I was planning on freezing the samples at the campus in a freezer in the laboratory. Plus I'm not 100% certain how long it will take me to complete the protocol by myself and therefore if I may need to freeze samples, are there any steps of the protocol that are unsuitable for freezing? and how long can I freeze the samples for? what temperature ranges are suitable?

If I undertake genotyping will that determine the genetic relatedness for siblings?

I was also considering cobalt and chromium tests as an appended research project submission if I proceeded to genotyping, but unsure whether frozen whole blood is suitable?

Regards,

Jesse

If you're using vacutainer capture of the blood into EDTA then storage overnight at +4oC is absolutely fine. Can you stort them in a coolbox with ice and put in the fridge?

As for the genotyping question - it depends on exactly what you are genotyping. If it's for one gene, say, then probably not. What that would be able to do it tell you how unrelated some animals were but not about relatedness itself.

Astilius on Mon Apr 16 16:15:42 2012 said:

When you say +4oC, can the temperature be either side of +4oC in the fridge? Say if I needed to take the samples home overnight, but my home fridge only has the cooler and warmer dial, what temperature range is best i.e. Between 0 degrees C to +4oC?

I was planning on putting 5 - 10kg of wet ice into a large plastic cooler to place the samples within immediately after whole blood withdrawal is performed upon each animal, keeping lid closed. I might be able to borrow some test tube racks from the uni to keep the samples upright although I believe they are air tight. It will probably take the practical 4 hours to get through the 60 sheep, including the following glucose tests. After blood withdrawal is finished students will move to a classroom to undertake glucose/d-beta-hydroxybutyrate tests using handheld human diabetic glucometers. I will instruct fellow 2nd yr students to handle each sample within a minimum period of time to undertake the glucose/ketone tests, handling one sample at a time by the top of the lid and placing sample back into cooler after use, with cooler lid closed constantly.

I might investigate using a micropipette tip to extract the required blood from the vials, otherwise dipping the glucose strips into the whole blood will increase the chances of contaminating the samples with foriegn DNA.

After the 2nd year students are finished, it should only take me 15-30 minutes to get back to the campus and straight into fridge.

Or

If I go straight home instead, then it is 1 hours journey with samples placed into smaller coolers that I could have pre-chilled and then storing samples in these in my home fridge, which I would then need to transfer back to the larger cooler in the morning. I plan to purchase extra bags of ice and store a couple in the freezer at home for the Friday morning trip back to the university campus.

Once back at uni, I will put the samples into the fridge in the laboratory, maybe taking out small batches over the day to conduct the protocol in stages and placing any complete protocol samples back into the fridge. Once the protocol is finished for all samples, I will then prepare to have the samples sent to the Australian Genome Research Facility in Melbourne for 'unpurified PCR product (PD+) service'. I haven't determined the genotyping pathway to follow yet.
.

For your purposes I'd avoid freezing your samples. Freezing will cause cell rupturing and although that shouldn't affect your tests I'd avoid changing the composition of your bloods.
+4oC is ideal for your purposes but anywhere from +1oC and +8oC should be just fine for short term (less than a couple of days) storage and transport.

Since your home fridge has a cooler/warmer dial. Stick a thermometer in your fridge, let it equilibrate for a few hours and quickly take readings. Remembering that opening the door will let cold air out and warm air in so you need to euilibrate between readings. You should easily get your fridge down to +3 or +4oC over the space of an evening or two.

Micropipetting the bloods out for testing is better practice than dipping the test stips in. I'd say that the likelihood of contamination from the test strip is low but it's not good practice just to stick the strip into your stock bloods. So, yes, your thinking here is good.

So, aim for +4oC when transporting and storing. Wet ice should be okay but avoid freezing your samples. Do a dummy run first with a vial of water - it's not perfect but should be suitable for an initial ballpark guess of the conditions you'll achieve.

i shouldn't imagine you'll run into any real issues.

Thankyou very much. Your replies are greatly appreciated.

Just a question- can you not ask for after hours access to the lab? Or find someone who has access and won't mind meeting you there or waiting back for you?