[MARMAM] Gas bubble sampling and analysis
mmoore at whoi.edu
Tue Jun 5 08:56:10 PDT 2012
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
<#_ENREF_1>). 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.
ØAdaptation to US material
In our method published in Scientific Reports (Bernaldo de Quirós et
al., 2011 <#_ENREF_1>), 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 *MONOJECT^TM 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).
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
*US Material list:*(This is the basic material you might need):
ØTubes: *MONOJECT^TM 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
ØOne Use Tube Holders: BD Vacutainer (ref # 36481), or equivalent in
other brand. Price according to Fishersci: Case of 1000 for $50.09
ØDouble 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
ØDisposable 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.
ØThe dissection should be done very carefully in order to avoid cutting
large vessels. No organs should be removed before gas sampling.
ØSample 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.
ØSample 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.
ØSample subcapsular gas (emphysema) if found. A location of interest is
the peri-renal area. Sample the subcapsular gas as described for the
ØStore 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 <http://www.nature.com/protocolexchange/labgroups/636>".
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 at whoi.edu
<mailto:ybernaldodequiros at 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
When we ship the pressure housing chamber to you, it will come with
return FedEx authorization.
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
Bernaldo De Quirós, Y., González-Díaz, Ó., Saavedra, P., Arbelo, M.,
Sierra, E., Sacchini, S., Jepson, P.D., Mazzariol, S., Di Guardo, G.,
and Fernández, A. (2011). Methodology for in situ gas sampling,
transport and laboratory analysis of gases from stranded cetaceans.
/Scientific Reports/ 1.
Woods Hole Oceanographic Institution
508 289 3228
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