Dear all,
I would like to thank all of you in the evoldir community for your very
helpful replies to my question on sending blood samples in ethanol
internationally. We are still deciding exactly what to do, but I had some
very good suggestions to send the blood+ethanol by courier (or even normal
post) without the dry ice / ice packs, which would mean the packaging would
be much smaller and lighter and hence cheaper. Provided IATA guidelines are
followed*, some people had good experience taking samples as checked
luggage, however it was pointed out to me that in the end the pilot has a
final decision of whether to accept the luggage, even if all of the
paperwork is in order (which could end up with quite a stressful experience
at the airport!). Others had suggestions for alternative storage solutions
including lysis buffer and RNAlater. And I had many people emphasising the
importance of checking the regulations and ensuring that all the paperwork
at both ends of the journey is prepared thoroughly.
I have compiled all the replies below, following a copy of my initial
message.
Thank you again for your excellent ideas!
Very best wishes,
Anna
asanture@gmail.com

*from my understanding, in my case where we have eppendorfs of ~1mL of
blood, these should be put in a hard container (i.e. an eppendorf box),
sealed in a plastic bag, an absorbant material wrapped around and then
sealed inside another plastic bag, and then up to 10 of these boxes (i.e.
<1L total ethanol) can be packaged together to form one package for either
posting via airmail or checking in as checked luggage. Airline's own
guidelines may differ.

*Original email: *
I would very much appreciate some pointers for how to transport blood
samples stored in ethanol internationally (from UK to New Zealand). I had
been intending to bring a chilly bin full of samples on dry ice* / ice
packs on a flight from the UK to New Zealand with me, however I have
recently become aware that many (all?) airlines classify ethanol as a
dangerous good** and restrict the total volume that can be checked in.
According to FAA guidelines (which I'm not sure apply globally, but Air New
Zealand's guidelines seem very similar) I think you are not allowed more
than 1L of total ethanol.

>From what I have read, the alternative is to send as a dangerous good with
one of the international carriers e.g. Fedex, however this is likely to be
prohibitively expensive. An online quote suggests the chilly bin we have in
mind would cost around NZD $2,500 to transport, and this is before I've
even mentioned the 'hazardous' contents.

Any help would be very much appreciated!

With many thanks,
Anna Santure
University of Auckland, New Zealand

*also a dangerous good...
**apparently litres of duty free gin, vodka, wine and rum are not dangerous
goods though!

*Responses - thank you all again!*
# I can only tell you something you probably won't like so much: my
experience is to best leave it to a carrier, which is indeed expensive. I
use World Courier a lot, much more reliable than FedEx or DHL etc. they are
known to loose a lot of their packages, I would not risk that. Dry ice/ ice
packs are usually not permitted on flights and I can only say the very best
about World Courier, they are fast and professional, which you can see in
their prices :( contact their office and ask about prices, but you can
expect about double than FedEx. I guess it really depends on how valuable
those samples are, but they know how to handle transport and customs.

# I've flown with ethanol samples several times before. The easiest thing
to do is to pour off as much of the ethanol as possible before transporting
them and then top them up once you arrive at your destination. Of course,
this is a lot easier if the sample is tissue - I'm not quite sure how it
would work with blood. Perhaps you could spin them down and pour the top
ethanol layer off and then just transport them as blood samples'?

# About your question, depending on how serious the checks you think will
be and how important/unique are the samples, you could decide to just put
them in your checked luggage (maybe paying also for an extra luggage) and
cross your fingers.
In case the material you're transporting needs some sort of permit to be
exported from the UK or imported in NZ, make sure you have one so that in
case of problems you can always show them the documents. Otherwise, you can
always have some sort of official letter. I doubt they'll be willing to
stick their face in blood samples to check if they're in ethanol. At that
point they'll be more concerned if you're bringing some sort of bio-hazard
rather than the ethanol. So the idea is: you put them in your luggage and
then you have some official document stating that those samples are safe
and you're allowed to transport them.
I know this is not exactly sticking to the rules but I know some people who
have done this in inter-continental flights.

# Here, for DNA, ethanol and freezing are alternatives: we certainly don't
do both - not even in the lab (though I know others do). We haven't tested
every tissue in every taxon, of course, but ethanol's certainly good for
nucleated blood cells and surely penetrates most tissues fairly rapidly.
We're shipping samples globally in ethanol all the time because it's so
cheap and convenient. One litre is a lot of ethanol and a lot of samples
(typically about 1 ml each), so we never exceed it. But you could obviously
use separate consignments. We're more worried about leakage spoiling labels
so always use polypropylene tubes with screw lids with seals - typically
screw-top microfuge tubes. We pack with absorbent paper and in a plastic
bag, just in case.
We have less experience with RNA, but RNAlater offers the same convenience,
though the samples should be frozen for storage.

# Regarding blood samples preserved in ethanol, I'm
guessing it's whole blood (that may or may not be on filter paper, or
similar)? In the past, I've brought in whole blood on filter paper that
had been stored in ethanol from Canada, but had my colleagues pour
off/evaporate the ethanol before shipping it. But if NZ isn't happy with
that option these days (at the time, I'm sure they decided that the RBCs
have been well and truly desiccated/fixed in the ethanol that they didn't
pose a risk), is it an option to completely evaporate the ethanol and then
add RNA later, or similar? I've never tried it, but might be worth an ask?

# Why don't you use RNA later or any other conservative for blood. Is there
a specific need for Ethanol?

# Why do you need to keep the samples cold if they are already stored in
ethanol?  It is the fact that the ethanol draws all the water out of the
blood and dessicates it that preserves the blood.  In the field and in
transport we keep our samples at room temperature.  As long as you have had
at least 5 x ETOH to blood the samples will be well preserved. At that
point you can pour off the ethanol and just keep the dried blood in the
eppendorf.  Thus negating the need to fly around with lots of ethanol.  You
can always add more ethanol when the sample arrive if you feel worried. We
have done this numerous times and the DNA we extract is still excellent.

# This may come too late, but for future reference, I can recommend that
you preserve and ship the samples in RNAlater instead of ethanol. We have
shown that it does a better job than ethanol at preserving DNA's and RNA's
integrity and will probably not be considered a hazardous substance. See
our paper discussing these benefits, which also includes the recipe for an
effective homemade RNAlater: MIGUEL CAMACHO-SANCHEZ, PABLO BURRACO, IVAN
GOMEZ-MESTRE and JENN I FER A. LEONARD (2103) Preservation of RNA and DNA
from mammal samples under field conditions. Molecular Ecology Resources 13,
663"673 doi: 10.1111/1755-0998.12108

#In Canada ethanol is a dangerous good but falls under the limited
quantities exemption, which means that if it is in individual quantities of
<1L it is exempt from many of the regulations. This limit applies to each
container (e.g. each individual tube/vial; as long as they are each <1L you
can have total >1L). However, there are still a number of regulations that
must be followed for it to be appropriate (e.g. gross mass of container
<30kg, in a suitable means of containment, labelled as limited quantities
along with the UN number, etc.).
Obviously this may differ in the UK and NZ, and you must meet criteria for
both countries. The implementation of the Globally Harmonized System may
make them quite similar. Also, each carrier can also set its own
regulations and decline any shipment even if it meets the criteria.
If you are planning to use the blood samples for genetic analysis, I would
suggest storing in lysis buffer (generally not a dangerous good, can be
shipped at room temp) or FTA cards (dry, room temp). As you noted dry ice
is also a dangerous good and so these methods would eliminate that as well.
The other thing to keep in mind is that transport via air can sometimes
cause small vials to leak (due to pressure changes in the cargo), so it
would be best to use O-ring caps and/or wrap lids tightly in parafilm.
Also, alcoholic beverages are a dangerous good, but their limited quantity
index is 5L, which would be an inordinately large bottle of whiskey. :)

# I haven't shipped samples internationally cold before (although I will
hopefully do it next week). I have shipped dried blood, tissues in ethanol
and other samples from Costa Rica to the US via FedEx, though. I've been
really satisfied by how they handle things but it is expensive and you have
to make sure you have all of your paperwork in perfect order. My samples
contain ethanol, animal products and potentially hazardous biologicals, so
they have to get cleared though the US Fish and Wildlife Service before
entering the country and can't go on passenger planes. I don't know what
kind of blood you're working with -- I think health human, dog, cat and
laboratory animal blood is under fewer restrictions about travel.
In general, the guidelines in question are the IATA guidelines and I'm
pretty sure you can have more than 1L of ethanol but not in any single
container (so if you split your shipment into 2 boxes, you could have up to
2L -- up to 1L in each box). I ship my samples via FedEx and it hasn't been
that expensive (but perhaps that's because my samples are going a shorter
distance). I think technically ethanol isn't allowed on passenger planes
(and I agree it's stupid b/c you can bring your rum but not your samples).
I know many people who've just stuck stuff on the plane with them and
nothing's happened but I'm always too afraid of losing my samples to do
that (and as I said, mine might be more regulated than yours). I have
traveled with just plain ethanol tubes that I wasn't afraid to lose and
literally nothing happened.
If you want to be absolutely certain nothing will happen, you might need to
bite the bullet and go the international carrier route (but as you said
it's very expensive and you're under higher scrutiny b/c the samples are
going through all the proper channels and out of your control). If you
decide to go that route, here's a blog post that can help:
http://www.anoleannals.org/2011/05/30/traveling-with-ethanol-think-twice/

# It's a little bit complicated to ship ethanol samples legally, and the
penalties for doing it wrong are substantial.  Best bet if you can is to
spin down the cells, decant and allow the alcohol to flash off.  Now the
containers are "evacuated by normal means" and your shipment becomes
non-hazardous.  There might still be restrictions on shipping animal
products, depending on species, etc.
If you must ship alcohol,  all air freight falls under IATA regulations.
Fedex hazmat folks can be very helpful in preparing your shipment even if
you don't use their service.  There are restrictions on volume and on how
many inner containers are required/allowed (e.g., individual snap-cap
microcentrifuge tubes).  It is also required to have secondary containment
like a plastic bag with adsorbent packing material inside.  No matter how
careful you are, the pilot has ultimate authority and can reject your
package for any reason.  Try to get help from a local expert in the UK if
you absolutely must ship ethanol.  The regs actually require that you have
training to ship hazmats.  Our lab has been audited and individuals were
required to produce proof of training.  Again, penalties can be 10s of Ks
of USD.

#I think you will find that EtOH has a volume threshold before it becomes a
*dangerous good for postal purposes (I believe that is a total volume of
1L, but could easily be wrong).  Because of that, and because it is much
easier to import samples preserved in EtOH using the NZ import permit for
non viable samples (IHS), I tend to just get samples sent on EtOH (some
people like to preserve them and then pour off the EtOH from tissue etc,
not necessarily a goer for blood!). If you have a really really large
number of them, then I would consider separating them into more than one
package (maybe keeping the total volume of the samples <1L each time).  But
using just the plain old post and the paperwork for the import of non
viable samples (IHS) I have moved dozens of packages of samples into NZ. So, I
would just post them (they need to be contained in a couple of layers of
ziplocks and the likes) and include the IHS with each package. That said,
for irreplaceable samples, maybe a courier is a good idea!
I think it is very wise to subsample them and leave some in the UK (just in
case).
The triple layer packaging thing is as simple as it sounds ... the tube is
one layer, then a bunch of tubes with paper towel in a zip lock, then paper
towel in a second ziplock and you have triple containment ... that is all
that is required for posting. So, I think if you aimed for some not too big
packages (I think the issue here is just the total number of samples!), and
put a copy of the IHS in the parcel (even highlighting section 6.3 for the
customs people at this end), and another attached to the outside of the
parcel, and triple contained the samples you should be fine.  I would put
something like "Non-viable scientific samples (bird blood in EtOH), no
commercial value" on the Customs form (and maybe even note "Import Health
Standard attached").  They will sometimes inspect them even though they are
coming in the IHS ... normally just to charge you ~$30 before they release
them.  With the post you can just go for it that way ... with a courier you
probably have to state that you have triple contained a minimal amount of
EtOH (I would just state <1L if pressed).

# I feel for you! We had similar problems in a previous lab and often the
designated countries' regulations differed widely. A similar transport to
your intended one would have been our shipments between the UK and Japan.
Here we sent plasma samples (not in ethanol though, just in anticoagulant).
We designated them as "diagnostic" and shipped them on dry ice. We used DB
Schenker, a subsidery of "Deutsche Bahn". They were much cheaper than the
likes of Fedex and really easy to deal with. We never accompanied the
samples, they were sent by freight. Just make sure you put in enough dry
ice to allow for travel time and potential hold ups in customs as you
cannot guarantee cold storage at customs.
Rules and regulation differ between carriers and designated countries, so
you have to find out about both sets! And supply requested copies of the
import/customs/shipping forms (I can't remember the offical term for this
form but the carrier will ask for it) that states sender, receiver and
contents info.
Also speak in detail to your New Zealand colleagues to get all the info
they may be able to dig up from their side. Have they received such samples
before?
If you decide to ship without the dry ice / icepacks, just make sure
everything will be okay with your blood after it thaws and make sure you
have done some tests in the lab that it doesn't 'go
off'.
It reminds me of my PhD samples which were soil samples preserved
with 100% ethanol, which worked fine initially but then I went back to some
unused
bottles after a couple of years for another project and I couldn't get any
DNA out of them anymore. We think the ethanol was too concentrated after
all and destroyed the DNA...

asanture@gmail.com