Field Museum of Natural History

Prepared by: Kendall Fitzgerald

Collaborators: Emily Ruhs, Gwen Kettenberg, Cara Brook
Date: April 2023

Disease-based Sampling Suggestions

OVERVIEW
This document details disease-based sample collection suggestions for vertebrate zoology field
teams. These samples can be used for disease studies, conservation studies, hormone assays,
and more to provide information on individual and/or population health. We suggest collecting as
many of these samples as possible. This may involve adding this type of sampling to your
permits. Regardless of whether your group plans to use these samples, we encourage you to
collect them due to their great potential for future study. ​

This document will first review the types of samples we recommend adding to your collection
protocol, followed by recommendations on which samples to collect based on your cold chain.
We do not intend this to be a document that lectures collectors on protocol, but rather to provide
useful suggestions in the case that adding these types of samples to your protocol seems
feasible.

Incorporation of disease ecology field protocols into future collecting trips will help establish the
Field Museum as a pioneering repository for infectious disease resources.

Out of scope
This document will not recommend ways to analyze this data, but point out previous types
analyses and studies these samples have been used in. This document is general, and therefore
will not provide details on how to collect samples given the range of organisms collected by the
FMNH teams. For example, the amount of blood to draw depends on organisms’ size and weight,
among other factors. If you will be collecting these samples you should inquire as to the specific
procedures for your species of interest (SOI).

SAMPLE TYPES AND USES

Blood Samples
Blood samples can be used for a variety of purposes ranging from DNA/RNA extraction for
sequencing, antibody detection, immune function, and more. As little as 2-4uL of serum is
needed to provide information on antibodies to a number of viruses, so even very small blood
samples are immensely useful in disease studies! The following are a variety of ways to take
these useful samples.

1. “Whole Blood” sample

a. Typically taken with a syringe, lancet or capillary tubes for smaller amounts, whole
blood samples are useful for sequencing, barcoding, blood metabolites,
hormones, and more.
b. Ideally collected in two different samples:
i. One: a plain Eppendorf tube that is then centrifuged and aliquoted for
serum and red blood cell pellet samples
ii. Two: in a cryovial tube with RNA protect buffer to preserve viral
sequences.
c. Please see “Sample Collection” for more information.
2. Plama, serum and red blood cell pellet
a. It is useful for disease and immune studies to take a whole blood sample, allow
the blood to rest for a short period, and then proceed to separate serum and RBC
pellet (non-cellular from cellular contents) via centrifuge before freezing. Serum is
the portion of blood/plasma that contains proteins, antibodies, hormones and
other molecules, and no cells, platelets or clotting factors. Having a separated
serum sample (distinct from a whole blood sample) is enormously useful for
immune studies, like antibody assays with ELISA or Luminex tools (for serology).
Likewise, having a separated RBC pellet sample is useful for blood cell studies
and PCR for parasites.
b. You can alternatively collect plasma by collecting blood samples in a tube coated
with anticoagulants and spin the sample down later. (Plasma, as opposed to
serum, is the liquid portion of the blood mixed with an anticoagulant.) Plasma
samples are also useful for antibody assays, immune functions studies, measuring
complement proteins, and hormone assays.
c. Today you can purchase battery powered, portable centrifuges to separate serum
from RBC pellets in the field (like this one). These do not need to be stored in a
buffer and need only be stored at -20ºC.
3. Dry blood spots
a. Collected typically by means of a small lancet and capillary tube, small spots of
blood blotted onto Whatman Filter Paper can be eluted for serum and used for
many studies, including antibody and hormone detection. There are widely
available protocols for eluting serum from blood spots (this can be done long after
collection).
 b. While freezing DBS after first collection can increase the lifespan of the sample, it
is not necessary. This is a dry sample that requires no cold chain. Additionally, this
sample does not require any excess biosafety protocols for handling or storage
(e.g. does not have to be stored in a biosafety cabinet)
4. Blood smear slides
a. These can be used for macro-pathogen detection and identification, like protozoa
identification, as well as red and white blood cell studies. Blood smear slides
should be air-dried and are typically fixed in methanol afterwards.
b. This is a dry sample that requires no cold chain. Note: Take care not to let the
slides get moldy if you are working in a humid environment.

Swab Samples
Swab samples are used for many methods in health, disease, and diet studies of wild animals.
Metabarcoding and other sequencing techniques now allow for wide cast simultaneous detection
of pathogens in these samples. Swab samples also provide opportunities to isolate viruses and
other pathogens. Collecting different swabs is recommended as some pathogens are shed only
through certain bodily fluids and not others. For example, lyssaviruses, like rabies, are shed
through saliva (throat swab).

1. Nasopharyngeal swabs, throat/saliva swab, or skin mucus

a. These samples can be analyzed for pathogen detection and physiological
parameters associated with metabolism, stress, immune system activities, and
inflammation.
b. Skin mucus: Skin mucus samples for amphibian and fish studies have been used
to study fungal pathogens, stress, and skin microbiome. See if there is a
pre-existing protocol for collecting skin mucus for your SOI.
c. These samples should be stored in a buffer that maintains DNA and RNA integrity.
Please see the Sample Collection section for more information.
2. Fecal swabs or feces
a. Feces are widely used to study disease and diet in wild animals. This sample is
especially useful for endoparasite detection, and has also been widely used for
steroid studies. Validated immunoassays on feces can reveal information about
reproductive cycle, stress, and other physiological parameters. This sample can
also be used for gut microbial studies.
b. Feces can often be collected from small animals via swabs or collected from the
environment for wider population-level study.
c. When collecting this sample, try to avoid contamination with urine where possible
as metabolite levels could be overestimated and viral shedding could be confused
or overestimated.
 d. These samples should be stored in a buffer that maintains DNA and RNA integrity.
Please see the Sample Collection section for more information.
3. Urine swabs
a. Urine can be analyzed for pathogen detection and physiological parameters
associated with metabolism, stress, reproduction, immune system activities, and
inflammation.
b. Urine can often be collected from small animals via swabs or collected from the
environment for wider population-level study.
c. When collecting this sample, try to avoid contamination with feces as metabolite
levels could be overestimated and viral shedding could be confused or
overestimated.
d. These samples should be stored in a buffer that maintains DNA and RNA integrity.
Please see the Sample Collection section for more information.

Ectoparasites
Collecting ectoparasites individually or collectively in tubes can be used for general ectoparasite
studies on the species, seasonal variation in ectoparasites, and disease dynamics between those
ectoparasites and the host species.

When processing an individual animal, one should remove all ectoparasites into vials specific to
that individual animal’s identification number, as with all other samples. It is best to collect all
species of ectoparasites into their own tube (i.e. ticks in one tube, fleas in another), but if this is
not possible in terms of time, storage, or identification ability, you can also collect one individual’s
ectoparasites into one “ecto” tube. If you are only interested in DNA pathogen studies, you can
store this sample in 100% EtOH. However, if you are interested in RNA pathogens, we
recommend storing these samples in DNA/RNA Shield or VTM/UTM. Store these samples
according to the buffer protocols.

Hair or feather clipping

Hair clipping can be used for toxicology, mineral, and hormone assays including
corticosterone/glucocorticoid levels for stress analysis. Another common assay with this sample
are CNH (carbon-nitrogen-hydrogen) isotope ratios for diet analysis. Hair samples can typically be
stored in a coin envelope or similar package and do not have to be frozen. If collecting feathers,
it is recommended that the same feather be taken from each individual.

Organ tissue samples

Organ tissue samples can be used in a myriad of studies regarding health, diet, disease and
more. In the case of an opportunistic necropsy or lethal sampling, we suggest you take the
following samples: brain, intestines, kidney, spleen, liver, and gonads. For each of these organs
we suggest you take some or all of the following: a small piece of the organ in a cryovial with
DNA/RNA shield or VTM/UTM, and another section of the organ in a plain cryovial. If the museum
has space to store the following, then it is also good to collect: a thin slice of the organ containing
as many areas as possible (i.e. mirroring what a microtome would do, but course not as thin; for
brain: include the brain stem when slicing) laid flat in aluminum foil, and the rest of the organ in
aluminum foil. Immediately freeze all samples.

Epigenetic aging tissue biopsy

Many species have, or have in development, an epigenetic clock whereby DNA methylome
measurements of a specific tissue sample are used to determine age. If determining molecular
age is something that you are interested in, we advise that you research whether an epigenetic
clock has been created for your SOI and collect the tissue sample used to model the clock. This
tissue sample can then be analyzed for the age of that individual. This information can be used to
build population viability analysis, for conservation purposes, and be paired with disease
information for further disease dynamic studies. This sample is typically stored in 100% ethanol.
Please note: storing this sample in DMSO will degrade the methylome and render the sample
unusable for epigenetic aging.

Bat-specific wing punch biopsy: Now standard in bat sampling, wing punch biopsies can be
used in methylome age studies. These are taken using a small (~2mm) “punch biopsy” tool to
take a sample of wing tissue between 4th and 5th finger (the dactylopatagium). This sample is
typically stored in 100% EtOH and frozen immediately. Please note: storing this sample in DMSO
will degrade the methylome and render the sample unusable for epigenetic aging.

BIO-SAFETY
Depending on your SOI, you may have to take bio-safety protocols into consideration when
transporting and storing blood, organ, and swab samples. Sometimes countries require samples
to be heat-treated (common for serum), inactivated with TRIZOL or RIPA buffer (sequencing still
viable with these buffers), or similarly neutralized for export. Please refer to the export regulations
in the countries you fieldwork is based in, and consult with museum cryogenic and lab staff
regarding sample storage and processing procedures.

SAMPLE COLLECTION

Buffer information
The best and most common field practice buffer for these samples is DNA/RNA Shield which
does not have to be frozen immediately. According to Zymo Research, samples in DNA/RNA
Shield can remain at 35-40ºC for up to 7 days, 5°C – 30°C (ambient) for 30 days, and at -20ºC
indefinitely. An alternative is RNAlater, a buffer which also preserves DNA and RNA in samples.
According to Sigma-Aldrich, this buffer can be kept at 37 °C for 1 day, 25 °C for 1 week, 4 °C for up
to 1 month and long term at -20 °C. Some researchers have reported trouble with RNA extraction
from RNAlater samples. With all samples, reduce thaw-refreeze as much as possible as each
thaw and refreeze degrades quality.

For disease studies, a viral or universal transport medium (VTM/UTM) buffer is especially useful
for later isolating viruses. It is best to flash-freeze VTM/UTM samples immediately with liquid
nitrogen and then store them at -80ºC1,2,3 with minimal thaw/refreeze. Please note, samples in
VTM will degrade if kept at -20ºC.

The golden standard for freezing medium for cells is a mixture of 10% DMSO with 90% of some
type of nutrient-containing cell medium buffer like DMEM. These can be used for RNA analysis
but only if the samples are frozen slowly in something like a Mr. Frosty container or a step down
freezer, which is not common in field work, this is an option if you prefer.

On the whole in fieldwork, DMSO, EtOH and EDTA are suitable for DNA extraction and studies
but do not maintain RNA well, and as such are less useful buffers for disease studies, particularly
RNA viral studies. (Especially 100% DMSO, which will degrade RNA and DNA). Whatever buffer is
most suitable and reasonable for you to use will still be helpful for DNA and disease studies.

Whichever buffer you choose, we encourage you to include this information in your field notes
and datasheets on each sampling trip. Knowing in which buffer samples are stored is essential for
processing samples down the line.

Please note: As you probably know, it is best to store all frozen samples in screw-top cryovials.
Storing samples in Eppendorph tubes will leave them liable to burst open or “explode” upon
freezing and/or thawing.

Strong Cold Chain

The strongest cold-chain will flash-freeze samples in liquid nitrogen immediately and maintain
samples at at least -80ºC at nearly all times. If you are able to bring liquid nitrogen to your field
site, and have a reliable, back-up-generator supported -80ºC freezer at your research base, we
suggest you collect the following samples in the following buffers. These methods yield the best
RNA extraction results specifically, which is useful for viral disease studies.

Blood samples: For each individual, we suggest you take 3 frozen blood samples.

1. A “whole blood” sample in a cryovial with RNAprotect buffer

 2. Another “whole blood” sample that is then centrifuged before freezing (portable
centrifuge example: this one) and aliquoted into . . .
a. Serum samples stored in a cryovial with no buffer
i. It is best to have multiple serum aliquots per individual; for example, one
20µL serum sample and two 60µL serum samples, depending on how
much blood you can draw from your SOI.
b. RBC pellet samples (at least 2) stored in a cryovial with no buffer.

Freeze the serum and pellet samples immediately by flash freezing in LN2, and keep at -80ºC.
Follow instructions for the RNAprotect buffer regarding freezing protocol. Serum and RBC pellet
samples can alternatively be kept at -20ºC. We recommend you also take filter paper blood spots
and blood smear slides which do not require freezing (but freezing filter paper blood spots does
increase the sample's lifespan!).

Please note: the amount of blood to take depends on factors like individual
weight. For serum, as little as 2-20µL can be run on assays like ELISA, Luminex,
and “VirScan” to provide information on antibodies to a number of viruses, so
even very small blood samples are immensely useful in disease studies!

Swab samples: Nasopharyngeal/throat, fecal, and urine swabs in cryovials with VTM/UTM or
DNA/RNA Later. Freeze immediately. VTM/UTM samples must be stored at -80ºC at all times.
DNA/RNA Shield samples must be kept at at least -20ºC.

Ectoparasites: Stored in cryovials with VTM/UTM or DNA/RNA Shield. Freeze immediately.

VTM/UTM samples must be stored at -80ºC at all times. DNA/RNA Shield samples must be kept
at at least -20ºC.

Organ tissue samples: Stored in cryovials with VTM/UTM or DNA/RNA Shield and aluminum foil.
Freeze immediately and store at -80ºC for VTM/UTM, or -20ºC for DNA/RNA Shield.

Epigenetic aging tissue: tissue for epigenetic aging studies in cryovials with 100% EtOH, frozen
immediately and stored at -80ºC at all times.

Hair or feather sample: this sample requires no freezing.

Weak Cold Chain (most common)

This type of cold chain is much more common in field studies and still yields very useful disease
data! If you are able to bring freezer blocks, some other kind of cooler into the field, or even if
you have no way of freezing samples in the field, but your samples can be stored at -20ºC at your
research base within a week, you have what we call a “weak” cold chain. We suggest you collect
the following samples in the following buffers for the most reliable results.
Blood samples: For each individual, we suggest you take 3 frozen blood samples.

3. A whole blood sample in a cryovial with RNAprotect buffer

4. Another whole blood sample in an Eppendorf tube that is then centrifuged (portable
centrifuge like this one) and aliquoted into . . .
a. Serum samples stored in a cryovial with no buffer
i. It is best to have multiple serum aliquots per individual; for example, one
20µL serum sample and two 60µL serum samples, depending on how
much blood you can draw from your SOI.
b. RBC pellet samples (at least 2) stored in a cryovial with no buffer.

Immediately place and keep the serum and pellet samples at 4-8ºC, and store at -20ºC within 7
days4. Follow instructions for the RNAprotect buffer regarding freezing protocol. Serum and RBC
pellet samples can be kept at -20ºC. We recommend you also take filter paper blood spots and
blood smear slides which do not require freezing (but freezing filter paper blood spots does
increase the sample's lifespan!).

Please note: the amount of blood to take depends on factors like individual
weight. For serum, as little as 2-20µL can be run on assays like ELISA, Luminex,
and “VirScan” to provide information on antibodies to a number of viruses, so
even very small blood samples are immensely useful in disease studies!

Swab samples: Nasopharyngeal/throat, fecal, and urine swabs in cryovials with DNA/RNA Shield.
Freeze at 20ºC (or colder) within 7 days.

Ectoparasites: Stored in cryovials with DNA/RNA shield. Freeze at 20ºC within 7 days.

Organ tissue samples: Stored in cryovials with DNA/RNA Shield and aluminum foil. Freeze at
20ºC within 7 days.

Epigenetic aging tissue: tissue for epigenetic aging studies in cryovials with EtOH. Freeze at
20ºC within 7 days.

Hair or feather sample: this sample requires no freezing.

No Cold Chain
If you have no way to keep samples cold, no access to a freezer, etc. you can still collect useful
samples for health- and disease-specific studies.

1. Swab samples for hormone studies: Fecal, urine and throat swabs stored in 100% EtOH
can be kept at ambient temperatures for up to 3 weeks before freezing at 20ºC. These
 samples will maintain metabolites used in hormone studies, but are not viable for DNA or
RNA studies5.
2. Blood spots on Whatman Filter paper: this sample does not need to be frozen. DNA
pathogens can still be detected down the road in this sample without freezing.
3. Blood smears: this sample does not need to be frozen.
4. Hair or feather clipping: this sample does not need to be frozen.

Cold Chain Notes

Please note, as an alternative to liquid nitrogen (LN2), if you have dry ice and pour acetone over
it, you can create something of similar temperature to liquid nitrogen to flash freeze. This may still
be difficult to bring into the field but it is another option.

References
1. World Health Organization . World Health Organization; 2020. Laboratory testing for
coronavirus disease 2019 ( COVID-19) in suspected human cases: interim guidance, 2
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of herpes simplex virus, respiratory syncytial virus, and adenovirus. Diagn Microbiol Infect
Dis. 1994 Jul 1;19(3):137–142. [PubMed] [Google Scholar]
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and specimen transport conditions for optimal detection of viruses by PCR. J Clin
Microbiol. 2012 Mar;50(3):1064–1065. [PMC free article] [PubMed] [Google Scholar]
4. Surveillance Guidelines for Measles, Rubella and Congenital Rubella Syndrome in the
WHO European Region. Geneva: World Health Organization; 2012 Dec. Annex 3,
Collection, storage and shipment of specimens for laboratory diagnosis and interpretation
of results. Available from: https://www.ncbi.nlm.nih.gov/books/NBK143256/
5. Pappano DJ, Roberts EK, Beehner JC. Testing extraction and storage parameters for a
fecal hormone method. Am J Primatol. 2010 Nov;72(11):934-41. doi: 10.1002/ajp.20859.
PMID: 20623500.