Apr 18, 2023

Public workspaceSkin Biopsy Protocol (Mammals): Non-lethal Sampling V.5

DOI
dx.doi.org/10.17504/protocols.io.n2bvj64nxlk5/v5
  • 1Viagen Pets, Cedar Park, TX, USA;
  • 2U.S. Fish and Wildlife Service;
  • 3Smithsonian Conservation Biology Institute, National Zoological Park, Front Royal, VA, USA;
  • 4Conservation Science Wildlife Health, San Diego Zoo Wildlife Alliance, Escondido, CA, USA;
  • 5Zoo New England;
  • 6Revive & Restore, Sausalito, CA, USA;
  • 7Smithsonian National Zoological Park and Conservation Biology Institute, Front Royal, VA, USA;
  • 8USDA, ARS, NLGRP, National Animal Germplasm Program, Fort Collins, CO, USA
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DOI: dx.doi.org/10.17504/protocols.io.n2bvj64nxlk5/v5
Protocol Citation: sanaz.arenivas, justin_bohling, comizzolip, mhouck, Rachel A Johnston, Jacquelyn Mountcastle, Budhan Pukazhenthi, phil.purdy, brian_small, seth_willey 2023. Skin Biopsy Protocol (Mammals): Non-lethal Sampling. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj64nxlk5/v5Version created by Revive and Restore
Manuscript citation:

License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: In development
We are still developing and optimizing this protocol
Created: April 18, 2023
Last Modified: April 18, 2023
Protocol Integer ID: 80660
Keywords: biobanking, cryopreservation, living tissue, skin biopsy, cryobanking, ear punch
Abstract
Version date: April 2023

The following protocol illustrates how to collect and ship living tissue from a mammalian species under field conditions for long-term cryopreservation. Skin tissues are optimal for harvesting live fibroblast cells that can either be immediately cryobanked at -196°C, or processed for cell culture and later cryobanked at -196°C.

To print this protocol: Click the arrow to the right of the "MORE" button at the top of this page and select the option to print or download as PDF version.
Guidelines
Skin biopsies can be taken when animals are immobilized for veterinary exams, radio-collaring, relocation, capture and release for captive breeding, and ear tagging. The collection of samples should be opportunistic and follow all applicable regulations.

Biopsies must be larger than 3mm3 to yield enough living fibroblasts cells for the eventual creation of cell lines. For large mammals, 5 or 6 mm biopsies are recommended. For very small mammals, 2 or 3 mm biopsies are recommended. Collect at least two biopsies per individual from different locations on the body. Collecting two samples per individual increases the chances of successful biobanking in case one sample is not successful. Only for very small mammals should one sample per individual be collected.

Biopsied tissues are stored in vials containing a nutrient-rich media to keep the cells alive and a mixture of antibiotics to prevent bacterial growth. Include only one biopsy sample per vial. If you are running low on materials, up to two biopsy can be including in one vial. DO NOT mix biopsies from different individuals in the same vial.

For urgent questions regarding protocol steps or collection guidelines, please contact the Revive & Restore Biobanking Team at informedbiobanking@reviverestore.org
Materials
Included in Commercial Sampling Kit:
Biopsy vials with media
Biopsy tool or ear punch
  • Large Mammal: 4, 5, or 6 mm Integra Biopsy Punch Dermal #33-34, 33-35, or 33-36
  • Small Mammal: 2, 3, or 4 mm Integra Biopsy Punch Dermal #33-31, 33-32,or 33-34
Parafilm
Insulated Tupperware 
Vinyl lunch bag
Ice packs
Styrofoam container
Biopsy form
Pre-paid FedEx shipping label

User-supplied Materials:
Sterile scalpel
Disposable gloves
Battery powered or electric clippers (optional)
Gauze
Rubbing alcohol
Disposable tweezers
Sterile scissors (optional)
CELOX hemostatic agent
Neosporin 
Field notebook 
Pencil/Pen
Permanent/alcohol-resistant marker
Newspaper or bubble wrap
Safety warnings
Steps for personal safety must be considered before going into the field. Some wildlife diseases are transmissible to humans. Refer to your agency’s health and safety guidelines for personal protective equipment (PPE). At a minimum, field personnel should wear disposable gloves and a fresh pair should be used between handling different specimens to avoid cross contamination. Sampling instruments and equipment should be thoroughly cleaned and disinfected or disposed of after use.


Before start
Tissue Handling
Biopsied tissues are stored at 4°C in vials containing a nutrient-rich media to keep the cells alive and a mixture of antibiotics to prevent bacterial growth. If vials with media are not readily available, biopsies can be placed in a tube containing sterile saline and kept at 4°C. All samples must be collected under aseptic conditions to avoid contamination. Using sterile tweezers, scalpels, and scissors, as well as cleaning the sampling site with alcohol or surgical scrub (e.g. chlorhexidine) will decrease the chances of contamination. Collect as many samples as possible.
Cold Storage
Avoiding temperature fluctuations is very important for preserving the tissue samples. Use ice packs for transport of specimens from the field and ensure refrigeration is available immediately after returning from the field site. Do not use wet ice or other commercial therapeutic packs. Chill ice packs in the freezer the night before collection. Frozen ice packs will remain cold in an insulated container for up to 24 hours. If the field site is more than 24 hours from refrigeration, plan to bring a portable electric refrigerator. Vials containing tissue should not have direct contact with the ice packs. Never freeze tissue biopsies before shipping to a biobanking facility.

Shipping
Samples must be shipped to a biobanking laboratory via overnight express. You must use an insulated container and ice packs to maintain a temperature between 4-8°C. Considering that samples may be in queue at the receiving facility for a period of time before they are processed, ship your samples as soon as possible. Shipment Monday through Wednesday will guarantee arrival at the receiving facility before the weekend. Do not ship samples on Friday. Most facilities will not be available to receive shipments on weekends. If samples are collected Thursday through Sunday, samples must be stored in the fridge until Monday. Consult with the receiving facility for any questions regarding shipping timelines before sending samples.
Preparation
Preparation
Pre-chill icepacks in the freezer the day before planning to collect and ship samples.
Record all information indicated in the biopsy form, including a picture of the animal for identification and GPS location where the animal was found.
Proper protective equipment must be worn (gloves, etc). Sterility must be maintained as much as possible.
For general sedation, anesthetize according to standard practices (refer to Handbook of Wildlife Chemical Immobilization by Terry Kreeger). For local anesthesia, use what is most appropriate for the species at hand. Avoid use of injection or topical lidocaine. Bupivacine is recommended.
Shave sampling area with hair trimmer, removing as much hair as possible. Ear punches are most preferred for collection, but biopsies can also be collected from the neck, flank, or lower abdomen. Skip this shaving if the area (e.g. ear) is too small to trim.
Soak gauze with rubbing alcohol and rub shaved sampling area to thoroughly disinfect. Chlorhexidine may also be used in addition to alcohol to disinfect.

Discard used gauze. Reserve some clean gauze for use later.
Remove the biopsy vials with media from the kit and have them readily available. There are different methods for obtaining skin biopsies. The table below indicates the recommend size and number of biopsies for small and large mammals. Select the biopsy size that is safest for your species:

Biopsy sizeNo. biopsies PER individual
Small Mammals2, 3, or 4 mm1 or more
Large Mammals4, 5, or 6 mm2 or more
Next, select the method below that is most appropriate for the species being handled:
Step case

Skin biopsy tool
13 steps

A 4mm biopsy tool is preferred. However, a 2 or 3 mm biopsy tool can be used for small mammals. A 4 mm or larger biopsy tool is ideal for large mammals. Collect at least 2 biopsy samples from different areas of the body per individual. For very small mammals, one sample per individual may be collected.

Remove biopsy tool from sterile packaging. Keep the sharp metal tip sterile by avoiding contact with non-sterile surfaces. With the biopsy tool perpendicular to the skin, gently press down onto the skin in the shaved area.

Photo credit: Coyner, 2011
Use a gentle twisting motion in one direction with slight downward pressure to cut through all the layers of skin including epidermis, dermis, and the most superficial parts of the subcutaneous fat. Gently pull out the tool at a 45 degree angle, avoiding damage to the sample.

Photo credit: Coyner, 2011
Use tweezers or a 25-gauge needle to gently grasp the biopsy and pull the sample up and out. Do not crush the sample with the tweezers. If it does not lift, do not tug to tear it off. Use a razor blade or sterile scissors to cut as low as possible at the underlying fat. Discard/replace instruments between individual animals.

Photo credit: Coyner, 2011
Apply CELOX hemostatic agent to stop the bleeding and antibiotic ointment to sampling area. For larger biopsies, a suture may be needed.
Tissue Preservation
Tissue Preservation
Using the disposable tweezers, immediately transfer skin piece to biopsy vial. Ensure that tissue is fully submerged in liquid. Close the cap tightly. Each biopsy should be placed in a separate vial.

Using an alcohol-resistant marker, label the biopsy vial with an identifier that matches exactly what is indicated on the specimen form. Check to make sure that each vial is easy to identify with the information provided on the form.
Required information to include on the form:
  • Scientific name of animal
  • Sex of individual denoted as ♂ (male) or ♀(female)
  • Date of tissue collection
  • Tissue type
  • Any other identification number of individual
Add a thin layer of Parafilm around the vial covering the seal.

How to apply Parafilm:
https://www.youtube.com/embed/u0AuSUvgXoo


Place samples on cold ice packs in an insulated cooler for transport back to your facility. Use the insulated Tupperware to transport your vials. If you do not have the Tupperware on hand, use about 3 inches of newspaper or bubble wrap to ensure that the tubes are not in direct contact with the ice packs. Do not freeze samples.

**Do not ship samples via overnight express on Thursday-Sunday. For samples collected on these days, store samples upright in a refrigerator until you are ready to ship them**
Shipping
Shipping
When you are ready to ship the samples, make sure the ice packs are frozen ahead of time.
Below are video instructions on how to pack your box:
https://www.youtube.com/embed/TADF7s-N4X4

Open the shipping box and place one frozen ice pack at the bottom of the styrofoam container inside.
Photo credit for the following photos: ViaGen Pets & Equine, 2021

Place the biopsy vials inside the insulated Tupperware container included in your kit. If you do not have the insulated Tupperware container, wrap each vial in bubble wrap or newspaper and secure with tape to avoid direct contact with the icepacks.



Close the lid and place the Tupperware container into the vinyl lunch bag provided in your kit.




Place the vinyl lunch bag into the styrofoam container, on top of the first ice pack.


Place the second frozen icepack on top of the vinyl lunch bag.


Close the lid to the styrofoam container. Enclose all paperwork to the top, including the Biopsy Form. Close the shipping box and place the prepaid shipping label to the top.




Contact the receiving facility when you have shipped your sample and provide the tracking number so that personnel know when to expect your shipment.
Protocol references
 Coyner, K.S. (2011). Skills Laboratory: How to perform a skin biopsy. DVM306. https://www.dvm360.com/view/skills-laboratory-how-perform-skin-biopsy

Hackworth, C. (2021). Skin Punch Biopsy: An overview for the Veterinary Nurse. Kansas State Veterinary Diagnostic Laboratory. https://www.ksvdl.org/resources/news/diagnostic_insights_for_technicians/august2019/skin-punch-biopsy.html

Houck, M. L., Lear, T. L., & Charter, S. J. (2017). Animal cytogenetics. In The AGT Cytogenetics Laboratory Manual (pp. 1055–1102). https://doi.org/10.1002/9781119061199.ch24

Trans Ova Genetics. (2022). Trans Ova Livestock Cloning. https://transova.com/service/cloning-services/

ViaGen Pets & Equine. (2021). Emergency Pets Protocol – U.S. Clients. https://www.viagenpets.com/emergency-pets/

White, C. L., & Dusek, R. J. (2015). Wildlife specimen collection, preservation, and shipment. In J. C. Franson, M. Friend, S. E. J. Gibbs, & M. A. Wild (Eds.), Techniques and Methods. US Geological Survey. https://doi.org/10.3133/tm15c4