TITLE: A protocol to prevent plastic contamination during sample collection of cadaveric tissues
AUTHORS: Kara Coffman-Rea, Alvise Vianello, Karen E. Samonds

[DESCRIPTION]
Research on microplastic pollution and its effects on atmospheric, aquatic, and terrestrial environments has grown rapidly as potential risks for human health are recognized. Recently, microplastic particles have been detected in a variety of human tissue samples (e.g., blood, placenta, lung tissue), but studies use different methods of collection, preparation, and analysis, making results difficult to compare. In particular, the risk of microplastic contamination during sample collection is high and can potentially produce misleading results. Here we present a set of guidelines to mitigate all sources of potential plastic contamination while collecting cadaveric tissue samples. We discuss specific protocols for the collection of three tissue types: (1) internal body fluids; (2) soft tissue; and (3) tissue lining body orifices. Guidelines for each tissue type include sterilization of equipment and specimen, extraction of the sample, and containment of the cadaveric specimen after removal. Each methodology has the potential to be modified to collect samples from any anatomical region within the human body. Representatives from each tissue type (intestinal contents, lung tissue, and contents from the nasal cavity) are presented.

[BEFORE_START]
Before start

1. Wear a 100% cotton lab coat throughout the entire procedure.
2. Wear 100% cotton under lab coats.
3. Nitrile gloves should be worn during tissue collection.
4. The color of lab coats, clothing, and gloves should be documented.
5. No plastic tools or products should be used throughout the entirety of the procedure.
6. Cover pre-cleaned glassware with aluminum foil and muffle all at 450° overnight.
7. Dissection tools used per sample should either be new or pre-cleaned, disinfected, and autoclaved.
8. Filter all ethanol and water that will be used throughout the procedure.

 


 


[GUIDELINES]
Guidelines

We recommend storing filtered ethanol and water in BOD bottles as screw cap bottles have been shown to shed MPs from the plastic cap (1). We also recommend allowing dissection tools to dry in a laminar flow hood or clean bench as it has been shown to reduce the risk of exposure to airborne particulates (2).

References

1. Giese A, Kerpen J, Weber F, Prediger J. A Preliminary Study of Microplastic Abrasion from the Screw Cap System of Reusable Plastic Bottles by Raman Microspectroscopy. ACS ES&T Water. 2021;1(6):1363-8.

2. Wesch C, Elert AM, Wörner M, Braun U, Klein R, Paulus M. Assuring quality in microplastic monitoring: About the value of clean-air devices as essentials for verified data. Scientific Reports. 2017;7(1):5424.

[STEPS]
SECTION: Sterilization of tools
2. Rinse dissection trays, scalpel handles, forceps, hemostats, stainless steel biopsy punch, and glass syringes with particle-free ethanol.
SECTION: Sterilization of tools
1. Rinse tools with particle-free water 3 times.
SECTION: Sterilization of tools
1.1. Rinse dissection trays, scalpel handles, forceps, hemostats, stainless steel biopsy punch, and glass syringes with particle-free water. (1/3)
SECTION: Sterilization of tools
1.2. Rinse dissection trays, scalpel handles, forceps, hemostats, stainless steel biopsy punch, and glass syringes with particle-free water. (2/3)
SECTION: Sterilization of tools
1.3. Rinse dissection trays, scalpel handles, forceps, hemostats, stainless steel biopsy punch, and glass syringes with particle-free water. (3/3)
 

 

SECTION: Sterilization of tools
3. Place the tools in a laminar flow hood or clean bench to dry.
 
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SECTION: Sterilization of tools
4. Once all tools are dry, place them in the dissection tray(s) and cover the tray(s) with aluminum foil until needed.
 

SECTION: Sterilization of the cadaveric specimen
5. Locate area where tissue extraction will occur. Thoroughly scrub the area.
SECTION: Sterilization of the cadaveric specimen
6. Using a razor, shave off external body hair from the area. Use the razor in multiple directions to ensure that all the hair is removed. 
SECTION: Sterilization of the cadaveric specimen
7. Using a glass syringe, flush the target area, in a downward direction, with particle-free water (3 times) followed by flushing it with particle-free ethanol. Repeat the flushing sequence 4 times. 
SECTION: Sterilization of the cadaveric specimen
7.1. Flush with particle-free water. (1/12)
SECTION: Sterilization of the cadaveric specimen
7.2. Flush with particle-free water. (2/12)
SECTION: Sterilization of the cadaveric specimen
7.3. Flush with particle-free water. (3/12)
SECTION: Sterilization of the cadaveric specimen
7.4. Flush with particle-free ethanol. (1/4)
SECTION: Sterilization of the cadaveric specimen
7.5. Flush with particle-free water. (4/12)
SECTION: Sterilization of the cadaveric specimen
7.6. Flush with particle-free water. (5/12)
SECTION: Sterilization of the cadaveric specimen
7.7. Flush with particle-free water. (6/12)
SECTION: Sterilization of the cadaveric specimen
7.8. Flush with particle-free ethanol. (2/4)
SECTION: Sterilization of the cadaveric specimen
7.9. Flush with particle-free water. (7/12)
SECTION: Sterilization of the cadaveric specimen
7.10. Flush with particle-free water. (8/12)
SECTION: Sterilization of the cadaveric specimen
7.11. Flush with particle-free water. (9/12)
SECTION: Sterilization of the cadaveric specimen
7.12. Flush with particle-free ethanol. (3/4)
SECTION: Sterilization of the cadaveric specimen
7.13. Flush with particle-free water. (10/12)
SECTION: Sterilization of the cadaveric specimen
7.14. Flush with particle-free water. (11/12)
SECTION: Sterilization of the cadaveric specimen
7.15. Flush with particle-free water. (12/12)
7.16. Flush with particle-free ethanol. (4/4)
SECTION: Extraction of Tissue: Internal body fluids (representative body region: intestinal contents)
8. Using a scalpel (size 10 blade), make a 7.5 cm vertical incision from the umbilicus running toward the xiphoid process. 
 

SECTION: Extraction of Tissue: Internal body fluids (representative body region: intestinal contents)
10. Using forceps and your fingers, locate the preferred section of the intestine and pull it out of the abdominal cavity without additional incisions, leaving any other viscera in place.
 

SECTION: Extraction of Tissue: Internal body fluids (representative body region: intestinal contents)
11. After the desired segment of the intestine is removed from the abdominal cavity, use the linear stapler to secure contents within the intestine. Staple 2 sites, 3 cm from each other, on both the proximal and distal ends of the intestinal segment.
SECTION: Extraction of Tissue: Internal body fluids (representative body region: intestinal contents)
12. Use scalpel to cut between the staple sites on both ends.
SECTION: Extraction of Tissue: Internal body fluids (representative body region: intestinal contents)
13. Remove the segment of intestine so that contents can later be removed and analyzed.
SECTION: Extraction of Tissue: Soft tissue (representative body region: lung tissue)
14. Using a scalpel (10 blade), make a 5 cm incision in the intercostal space, cutting through skin and musculature.
 

SECTION: Extraction of Tissue: Soft tissue (representative body region: lung tissue)
15. Using your fingers, locate the lung tissue. Insert forceps or a hemostat into the incision, using the tool to grab and stabilize the lung segment.
SECTION: Extraction of Tissue: Soft tissue (representative body region: lung tissue)
16. Still holding the lung segment, push the biopsy punch through the tissue.
 

SECTION: Extraction of Tissue: Soft tissue (representative body region: lung tissue)
17. Remove the biopsy punch from the thoracic cavity and use forceps or a probe to push tissue through the biopsy punch and into a glass container.
SECTION: Extraction of Tissue: Soft tissue (representative body region: lung tissue)
18. Repeat until you have collected the desired amount of tissue.
SECTION: Extraction of Tissue: Tissues lining body orifices (representative body region: nasal cavity)
19. 
Insert the scalpel blade into one or both nostrils and gently scrape the lateral and medial walls, removing portions of the mucosal membrane.
SECTION: Extraction of Tissue: Tissues lining body orifices (representative body region: nasal cavity)
20. Insert glass collection jar into nares.
 

SECTION: Extraction of Tissue: Tissues lining body orifices (representative body region: nasal cavity)
21. Use a 16-gauge syringe needle to pierce the lateral nostril wall just superior to the inferior edge.
SECTION: Extraction of Tissue: Tissues lining body orifices (representative body region: nasal cavity)
22. Using a new needle, draw particle-free water into a glass syringe and insert the needle into the nostril through the piercing. With the needle pointing in a superior direction, flush the nasal cavity. Allow the particle-free water and nasal contents to flow into the glass collection jar.
SECTION: Containment of Tissue Samples
23. Seal the glass containment jar(s) with metal lid(s), aluminum foil, or glass lid(s).
SECTION: Containment of Tissue Samples
24. Human tissues should be frozen at -20 ̊ C until they are ready for analysis preparation. Leave additional space at the top of the collection jar to account for the expansion of water within the tissue.
 


SECTION: Extraction of Tissue: Internal body fluids (representative body region: intestinal contents)
9. At both the superior and inferior points of the incision, use the scalpel to cut laterally in either direction. The lateral incision should not exceed 5 cm.