Dear Colleagues,
As some of you already know, analyses
of gas bubbles
by gas chromatography has been able to show that gases in acute
and chronic gas
embolism-affected cetaceans that were minimally decomposed had
high or very
high nitrogen contents in bubbles (Bernaldo
de Quirós et al., 2011). This technique allows us to distinguish between
putrefaction and
decompression related gases. However, putrefaction gases are
always produced postmortem,
and may mask decompression related gases if the necropsy and gas
sampling is
not performed soon after death.
Until very recently, the method
described by
Bernaldo de Quiros et al. (2011) could not be applied in the US
because of the
impossibility of acquiring the right sampling materials. Now, we
are pleased to
announce that we have adapted our method for gas sampling in
stranded cetaceans
to materials available in the US, as well as the availability of
a shipping
method of the samples without altering the original gas
composition of the
samples. Therefore, we would like you to know that if you now
face a gas
embolism case you can collect the samples properly following the
step-by-step
protocol found in http://dx.doi.org/10.1038/protex.2012.002, and ship
the samples to us
for gas analysis if you so wish. Below we describe the new
adaptations and
shipping method that will enable you to sample and send us gas
samples from
stranded marine mammals.
For colleagues in other countries we
are also keen
to receive gas bubble samples for analysis. We have been given a
determination
that they are NOT subject to CITES import into the US as long as
the samples
are taken from a dead animal. Please consult your suppliers for
the equivalent
sampling equipment as described for the US below.
Ø Adaptation to
US material
In our method
published in
Scientific Reports (Bernaldo
de Quirós et al., 2011), we used 5ml BD vacutainers for sampling and storage of
the gas
sample. Unfortunately BD vacutainers are no longer produced but
we have found
an alternative that is commercially available in the US. This is
the MONOJECTTM
Red Stopper (Silicone
Coated) glass tubes of 2 mL (ref #8881301116). These tubes
have
successfully undergone the tests described for the 5ml BD
vacutainers described
in Bernaldo de Quirós et al. (2011).
Ø Shipping
method
We have
acquired a plastic
housing resistant to negative pressures. We would be pleased to
send you the
housing as soon as you let us know that you might have some gas
samples that
you would like us to analyze. This housing allows air freight
shipping without
pressure changes in its interior, assuring that your samples
won’t suffer
modifications because of changes in pressure during the flight,
as well as
enabling a fast transportation for immediate analysis of the gas
samples in our
lab.
In addition, we could send you all
the necessary
material for gas sampling together with our housing if
requested. However, we
encourage you to acquire the sampling supplies in order to do
the necropsy and gas
sampling as soon as possible. As previously mentioned,
post-mortem time is
crucial if you would like to use the gas analyses as an
additional diagnostic
technique. Thus ideally
you should buy
the materials for sampling, to be ready for a case where there
are bubbles of
potential interest. They are all useful for more than just
bubble sampling. We have
a limited number of sampling kits we
can supply if you wish.
Please find bellow a list of
materials and a brief
description of the protocol.
US Material list: (This is the basic material you might need):
Ø Tubes: MONOJECTTM Red Stopper (Silicone Coated)
glass tubes of 2 mL
(ref #8881301116). It has to be this exact tube. Price
according to
Fishersci: Pack of 100 for $20.61
Ø One Use Tube
Holders: BD
Vacutainer (ref # 36481), or equivalent in other brand. Price
according to
Fishersci: Case of 1000 for $50.09
Ø Double
pointed needles: BD
Vacutainer Blood Collection Needles (ref # 367210), or its
equivalent in other
brand, but the tube puncture side of the needles must have a
rubber barrier covert.
Price according to Fishersci: Pack of 100 for $20.88
Ø Disposable
Insulin syringes:
BD Plastipak U-100
insulin (ref #
329651). It has to be this exact syringe. Price according
to Fishersci:
Pack of 100 for $35.93.
PROTOCOL
Ø The
dissection should be
done very carefully in order to avoid cutting large vessels. No
organs should
be removed before gas sampling.
Ø Sample up to
5 bubbles from
each of these locations: mesenteric veins, renal veins and
coronary veins. Take
extra samples if you observe bubbles in other locations of
interest (each case might
be different). Sample the bubbles using the insulin syringe and
inject the
content promptly into the monoject tubes.
Ø Sample
intestinal gas from
the following locations: duodenum or nearby intestine sections,
middle intestine
and rectum. Do so by using the monoject tubes together with the
double pointed
needles coupled to the one-use tube holders.
Ø Sample
subcapsular gas
(emphysema) if found. A location of interest is the peri-renal
area. Sample the
subcapsular gas as described for the intestinal gas.
Ø Store tubes
at room
temperature, preferably with the stopper downwards. Store blanks
together with
the samples (a minimum of 3 is required).
You can find a step by step protocol
for gas
sampling in the following link:
http://dx.doi.org/10.1038/protex.2012.002. The Protocol
Exchange is an open
resource where the communities of scientists pool their
experimental know-how
to help accelerate research. It
enables
researchers to more readily reproduce or adapt the methodology
described. Protocols
Exchange is organized
around the concept of Laboratory Groups. All Protocols are
associated with a
Lab Group and authors cannot share protocols except as members
of a Lab Group.
This protocol belongs to an “open” lab Group. It is open by
invitation and upon
request. We will be happy to answer all your questions by email
if required,
but we encourage you to use this tool, since your questions
might be helpful to
other groups. Therefore if you wish to participate, or make comments or suggestions
concerning this
protocol, you should request to become a member of the following
lab Group,
where this protocol belongs: “Institute
of
Animal Health, University of Las Palmas de Gran Canaria”.
However, we would be glad
also to answer
your questions in a more individual way if preferred. Questions
should be
addressed to the following e-mail address: ybernaldodequiros@whoi.edu
If you
wish to send us
samples, you could contact Yara Bernaldo de Quirós on the
following phones:
working hours 508-289-3651, emergencies 508-274-5964 and we will
discuss
personally how is the best way to proceed on each specific case.
We recommend that
you contact us as soon as possible in case you need specific
material to be
sent to you, if you need some recommendations in your specific
case or simply
to send you the housing chamber as fast as possible. Gas samples
should not be
stored longer than a week; therefore we encourage you to send
the samples the
day after the necropsy as latest. Thank you for your
cooperation.
When we ship the
pressure housing chamber to you, it
will come with return FedEx authorization.
Sincerely,
Yara Bernaldo de Quirós Miranda, PhD
Postdoctoral
Investigator
Michael Moore, Vet MB, PhD Senior
Research
Specialist
Biology Department, Woods Hole
Oceanographic
Institution,
Woods Hole, MA 02543 USA
REFERENCE
-- Michael Moore Woods Hole Oceanographic Institution 508 289 3228