TITLE: Skin Biopsy Protocol (Mammals): Non-lethal Sampling
AUTHORS: sanaz.arenivas, comizzolip, mhouck, racheljohnston, Jacquelyn Mountcastle, Budhan Pukazhenthi, phil.purdy

[DESCRIPTION]
Version date: 10 November 2022

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: Select the arrow to the right of the protocol title (above) and select the option to print or download as PDF version. 

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

[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 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 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. 

[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 small mammals, 3 to 4 mm biopsies are recommended. Biopsied tissues are stored in vial containing a nutrient-rich media to keep the cells alive and a mixture of antibiotics to prevent bacterial growth. 

[STEPS]
SECTION: Preparation
3. For general sedation, anesthetize according to standard practices. Instructions for local anesthesia are in the proceeding steps (Refer to Handbook of Wildlife Chemical Immobilization by Terry Kreeger).
SECTION: Preparation
4. 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 step if the area (e.g. ear) is too small to trim.
SECTION: Preparation
5. Soak gauze with rubbing alcohol and rub shaved sampling area to thoroughly disinfect. Discard used gauze. Reserve some clean gauze for use later.
SECTION: Preparation
7. Use a sterile scalpel and manually shave off all remaining hair in the area. Skip this step if the area (e.g. ear)  is too small. 
SECTION: Preparation
8. Use a new piece of alcohol-drenched gauze to clean the shaved area again very well. Discard used gauze. 
SECTION: Tissue Collection
9. Remove the biopsy vials with media from the kit and have them readily available. There are different methods for obtaining skin biopsies. Select the method below that is most appropriate for the species being handled:
SECTION: Tissue Preservation
11. Using the disposable forceps, 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. 

SECTION: Tissue Preservation
12. Using an alcohol-resistant marker, label the biopsy vial with: 
Scientific name of animal
Sex of individual denoted as ♂ (male) or ♀(female)
Date of collection
Identification number of individual
Tissue type
SECTION: Tissue Preservation
14. 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**
SECTION: Tissue Preservation
11. 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. 

SECTION: Tissue Preservation
11. Using the disposable forceps, 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. 
SECTION: Tissue Preservation
10. Using a permanent marker, label the biopsy vial with: 
Scientific name of animal
Common name of animal
Sex of individual 
Date of collection
Identification number of individual
Biopsy location on body 
SECTION: Tissue Preservation
14. Place samples on cold ice packs in an insulated cooler for transport back to your facility. You may use newspaper or bubble wrap to ensure that the tubes are not in direct contact with the ice packs. Store samples upright in a refrigerator until you are ready to ship. Do not freeze samples. 

  
  
     

SECTION: Wound Care
15. Apply antibiotic ointment to sampling area and styptic powder to stop bleeding. 
SECTION: Tissue Preservation
12. Using an alcohol-resistant marker, label the biopsy vial with: 
Scientific name of animal
Sex of individual denoted as ♂ (male) or ♀(female)
Date of collection
Identification number of individual
Tissue type
SECTION: Tissue Preservation
14. 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**
SECTION: Shipping
15. 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:
 

SECTION: Shipping
17. 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. 

  


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

   



SECTION: Shipping
17. Close the container with packing tape and use the pre-paid FedEx label to ship overnight. 

  

SECTION: Tissue Preservation
13. Add a thin layer of Parafilm around the vial covering where the cap meets the vial.  
An example of how to apply Parafilm can be found here: https://www.youtube.com/watch?v=u0AuSUvgXoo
SECTION: Tissue Preservation
13. Add a thin layer of Parafilm around the vial covering the seal.  

How to apply Parafilm: 
 

SECTION: Tissue Preservation
13. Add a thin layer of Parafilm around the vial covering the seal.  

How to apply Parafilm: 
  

SECTION: Preparation
6. If using local anesthesia, apply a topical anesthetic such as lidocaine, bupivacaine, or ropivacaine to temporarily relieve pain. Consult a veterinarian for recommended products. 
SECTION: Preparation
2. Record all information indicated in the biopsy form, including a picture of the animal for identification and GIS  location where the animal was found. 
SECTION: Shipping
16. 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
 
  

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

  

SECTION: Shipping
20. Close the container with packing tape and use the pre-paid FedEx label to ship overnight. 

  

SECTION: Shipping
19.  Put the lid on the styrofoam container and place the completed biopsy forms on top. 

  

SECTION: Shipping
18. Put an icepack on top of the lunchbag. Do not put the frozen icepack inside the lunchbag. 

  





SECTION: Shipping
17. Slide the tupperware into the lunchbag and load into the styrofoam shipping container horizontally so that the vials remain upright.

  
  

SECTION: Shipping
16. When you are ready to ship the samples, place the biopsy vials upright into the provided tupperware. Secure the lid. 

  

SECTION: Tissue Collection
10. Apply CELOX hemostatic agent to stop the bleeding and antibiotic ointment to sampling area. 
SECTION: Tissue Collection
10. Apply CELOX hemostatic agent to stop the bleeding and antibiotic ointment to sampling area. 
SECTION: Tissue Preservation
12. 
SECTION: Shipping
20. Place the second frozen icepack on top of the vinyl lunch bag. 

  

SECTION: Shipping
21. 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. 

  



SECTION: Shipping
15. 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:
 

SECTION: Shipping
16. 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
 
  

SECTION: Shipping
17. 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. 

  


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

   



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

  

SECTION: Shipping
20. Place the second frozen icepack on top of the vinyl lunch bag. 

  

SECTION: Shipping
21. 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. 

  



SECTION: Tissue Preservation
11. 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. 

SECTION: Tissue Collection
10. Apply CELOX hemostatic agent to stop the bleeding and antibiotic ointment to sampling area. 
SECTION: Tissue Preservation
12. Using an alcohol-resistant marker, label the biopsy vial with: 
Scientific name of animal
Sex of individual denoted as ♂ (male) or ♀(female)
Date of collection
Identification number of individual
Tissue type
SECTION: Tissue Preservation
13. Add a thin layer of Parafilm around the vial covering the seal.  

How to apply Parafilm: 
 


SECTION: Tissue Preservation
14. 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**
SECTION: Shipping
21. 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. 

  



SECTION: Shipping
20. Place the second frozen icepack on top of the vinyl lunch bag. 

  

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

  

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

   



SECTION: Shipping
17. 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. 

  


SECTION: Shipping
16. 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
 
  

SECTION: Shipping
15. 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:
 

SECTION: Preparation
1. Pre-chill icepacks in the freezer the day before planning to collect and ship samples.
SECTION: Shipping
22. Contact the receiving facility when you have shipped your sample and provide the tracking number so that personnel know when to expect your shipment. 
SECTION: Shipping
22. Contact the receiving facility when you have shipped your sample and provide the tracking number so that personnel know when to expect your shipment. 
SECTION: Shipping
22. Contact the receiving facility when you have shipped your sample and provide the tracking number so that personnel know when to expect your shipment.