TITLE: Field Data Collection Protocols for NIJ Grant The Impact of Drugs on Human Decomposition  and the Postmortem Interval: Insect, Scavenger and Microbial Evidence
AUTHORS: Hayden S. McKee-Zech, Erin A. Patrick, Allison R. Mason, Katha Hoeland, Charity Owings, Sarah T Schwing, Thomas Delgado, Amanda May, Mary C. Davis, Jennifer Debruyn, Shawn Campagna, Dawnie W Steadman

[DESCRIPTION]
The following is the data collection protocols for the National Institute of Justice funded grant "The Impact of Drugs on Human Decomposition and the Postmortem Interval: Insect, Scavenger and Microbial Evidence" (DOJ-NIJ-2018-DU-BX-0180). These protocols include those for anthropology, soil sciences, and chemistry field sampling events. This project was designed to examine the range of effects on decomposition of common drugs ingested by human donors through prime decomposers (e.g., scavengers, insects and microbes).  


 

[BEFORE_START]
Definitions: 
Donor is an deceased individual who has donated their body to the Forensic Anthropology Center's body donation program at the University of Tennessee-Knoxville.

Soil sampling in advance: 
Prepare 10cc syringes (Norm-ject Henke-ject REF: 19E16C8) to use as disposable corers (2 per donor): wearing gloves and using a sterile syringe, cut the end off of a 10cc syringe.  Store prepped syringe corer in a clean plastic bag. This creates a 15 cm diameter soil corer.
Fill dewer/CryoPod with liquid N

Decomposition fluid sampling in advance:
In advance:
Label a 4 oz. whirlpak bag with donor and date information (2 per donor). 
Label 4 2mL cryovials with donor and date information (4 per donor). (Bring extra sterile 2mL cryovials and a sharpie in case there is extra fluid). 
Fill dewar/CryoPod with liquid N

[GUIDELINES]
In cases of extreme weather, data collection at the outdoor Anthropology Research Facility was suspended for safety measures of the researchers.

[STEPS]
SECTION: Intake
2. Photographs - A standard sequence of photographs should be taken at intake for each enrolled donor, including: an overhead view of the exact condition in which the donor arrived, an overhead view with  clothing and coverings removed, overhead views of the donor's body in overlapping thirds (head to nipples, shoulders to mid-thigh, hips to toes), frontal and lateral photos of the head, photos of hands and feet to record presence or absence of digits, and any distinguishing marks (scars, tattoos, discoloration, medical devices, etc.).
SECTION: Intake
4. Aorta (central) and femoral (peripheral) blood sticks should be performed. Approximately 4 mL of each blood type should be collected. 

SECTION: Placement
14. Control site- Identify a site approximately one meter away from the donor placement site. Control site should be located uphill if donor resides on a slope, and should have soil available for regular sampling. Soil samples will be taken at the same ADH intervals as the donor soils for a baseline comparison  throughout decomposition.
SECTION: Field 2x Daily observations ( AM + PM)
26. Photographs 

 




SECTION: Field 2x Daily observations ( AM + PM)
27. Record arthropod documentation, photographs and field notes of arthropod activity at each observation time. All samples must contain a label inside of the collection vial with the following information: date, time, case number, sample number, and collector’s initials. 


SECTION: Field 2x Daily observations ( AM + PM)
28. Scavenger Form 
SECTION: Field 2x Daily observations ( AM + PM)
29. Decomposition Fluid Form 
SECTION: Field 2x Daily observations ( AM + PM)
30. Tiny Tag Temperature log reading 
SECTION: Field 2x Daily observations ( AM + PM)
31. Game Camera 
SECTION: PM Daily Observations
46. Photographs 
SECTION: PM Daily Observations
47. Insect Form 
SECTION: PM Daily Observations
48. Scavenger Form
SECTION: PM Daily Observations
49. Decomposition Fluid Form 
SECTION: PM Daily Observations
50. Tiny Tag Temperature log reading 
SECTION: Field 2x Daily observations ( AM + PM)
27.1. Arthropod Observation Form 
This includes adult arthropod arrival, presence of immature arthropods (e.g., eggs, larvae, pupae), formation of larval aggregations, and larval dispersal behavior. Absence of arthropods will also be documented. See attached form. 
SECTION: Placement
12. Climate monitor- A Tiny Tag data logger should be placed in the field near each donor, approximately 1 meter off the ground and within 1 meter of the donor. Localized, hourly recordings of temperature and relative humidity must be collected to allow for the calculation of accumulated degree hours (ADH).
SECTION: Placement
13. Game Camera- Mount or secure a wildlife trail camera with the focus encompassing the entirety of the subject to the best of your ability. Settings should include 3 burst images to be taken at 1 second intervals when movement is detected. Regularly check game cameras to ensure batteries are charged and the memory SD card is not full. 
SECTION: Placement
15. Placement photographs - all photographs include a North arrow when possible to provide spatial orientation. 
 
 
 
SECTION: Intake
3. Anthropometric data- Cadaver stature (centimeters) and weight (ideally, kilograms) should be documented at intake for each donor. Body fat percentage should be estimated using calipers for a skin fold test. Loose skin at the top of the iliac crest and the base of the rib cage should be pinched together for measurements. Calipers should measure the thickness at the base of the skin pinch. The measurement at each site should be repeated until the same measurement is obtained twice.
SECTION: Field 2x Daily observations ( AM + PM)
27.2. Arthropod Collection
SECTION: Field 2x Daily observations ( AM + PM)
29.1. Decomposition Fluid Collection
SECTION: Unenrollment Criteria
51. Unenrollment Criteria - The donor's abdomen should cease to release fluid, with most tissues desiccated, marking the end of the active decomposition phase. 
SECTION: Enrollment Criteria
1. Human remains with enrollment criteria: 
 Known date of death 
 Obtainable blood serum 
 Known medical information including drugs used, medical conditions, treatments 7-30 days prior to death 
 No outward trauma
SECTION: Serum Preparation
5. Draw whole blood into tube(s) containing no anticoagulant. Draw approximately 2 1⁄2 times the volume needed for use.
SECTION: Field Soil Sampling Procedure
16. Soil Sample collection- Initial collection occurs at placement (0 ADH). Subsequent samples should be collected at time stamps of 100, 250, 500, 750, 1000, 2000 ADH, and then every 500 ADH until unenrollment. At each sampling timepoint, soils should be collected from control sites and within decomposition-impacted areas. PPE (Gloves, pants, shoe covers) should be worn during all steps of the soil sampling protocol.
SECTION: Serum Preparation
6. Incubate in an upright position at room temperature for 45 minto allow clotting. 

SECTION: Serum Preparation
7. Centrifuge for 15 min at 2000 rcf

SECTION: Serum Preparation
8. The resulting supernatant is designated serum. Carefully aspirate the supernatant (serum) at room temperature and pool into a centrifuge tube, taking care not to disturb the cell layer or transfer any cells. 
 

SECTION: Serum Preparation
9. Inspect serum for turbidity
SECTION: Serum Preparation
9.1. Turbid samples should be centrifuged for 15 min at 2000 rcf again and aspirated again to remove remaining insoluble matter. 
SECTION: Serum Preparation
10. Aliquot into triplicate samples 200 µLor 1 mLserum into 3 cryovials with a minimum for a single replicate ~100 uL.
 


SECTION: Serum Preparation
11. Store cryovials of serum at -80 °C
SECTION: Intake
4.1. The samples are split, with half of each sample stored as whole blood and the other half spun into serum (see next section). 
1 cryovial - Central - whole blood 1 mL stored at -80 °C 
1 cryovial - Femoral - whole blood 1 mL stored at -80 °C 
3 cryovials - Central - serum  200 µL each stored at -80 °C 
3 cryovials - Femoral - serum  200 µL each stored at -80 °C 

Total of 8 cryovials per donor

SECTION: Field Soil Sampling Procedure
17. At placement:  Identify a control site for each donor.  Should be 1 – 1.5 m uphill (if on slope) from the donor, and not in another decomp zone. 
SECTION: Field Soil Sampling Procedure
18. Label one 7 oz bag and one 4 oz whirlpak bag with donor number, date, and "donor".  
SECTION: Field Soil Sampling Procedure
20. Homogenize sample by hand: gently press the bag between your thumb and index/pointer fingers to ‘mix’ the soil and break up individual soil cores. Pour the mixed soil into a large sterile weigh boat and remove any roots, rocks, hair, maggots or other foreign (non-soil) material using sterile tweezers as best as possible.
SECTION: Field Soil Sampling Procedure
21. Using a sterile spatula or scoop, take a 20 g subsample of soil and place in a 4 oz whirlpak bag labeled “DNA/Enzymes”, donor number, date, and sample type (donor or control). Next take a 5 g subsample of soil and place in a 4 oz whirlpak bag labeled “Met”, donor number, date, and sample type (donor or control).  There should be at least 10 g of soil left in the 7 oz whirlpak.  Close bags. 
SECTION: Field Soil Sampling Procedure
22. Put both 4 oz whirlpak bags into dewar/CryoPod‱ Carrier of liquid N, ensuring they are submerged.  The 7 oz whirlpak bags can be transported at ambient temperature, after which they should be stored at 4° C.
SECTION: Field Soil Sampling Procedure
23. Label one 7 oz bag and one 4 oz bag with donor number, date, and “control”.  At control site (1 to 1.5 m uphill, marked with a flag), remove large debris to expose the soil surface and use a clean corer to repeat steps 18-22.
SECTION: Field Soil Sampling Procedure
24. Repeat steps 18-22 with additional donors, using a new corer for each donor.  Dispose of corers and PPE in autoclave bags.
SECTION: Field Soil Sampling Procedure
25. Transport samples from field to lab. Samples in 4 oz bags, labeled "DNA" or "MET" are stored in a -80°C freezer until analysis while 7 oz bags are stored in the 4° C fridge.
 

SECTION: Field Soil Sampling Procedure
17.1. Mark the control site with a flag labelled with donor number and “control”.
SECTION: Field Entomology Sampling
32. Conduct visual observations and note the presence or absence of various arthropod life stages on
the data collection form. 
SECTION: Field Entomology Sampling
33. Do five aerial sweeps with a butterfly net to collect flying arthropods. 
SECTION: Field Entomology Sampling
34. Using forceps or another collection device (e.g., plastic spoon, spatula), collect immature arthropods from at least three locations and document on collection form.  
SECTION: Field Entomology Sampling
33.1. Place captured flying arthropods  in 70% EtOH in 20 mL scintillation vials with appropriate labels.

SECTION: Field Entomology Sampling
34.1. Collected larvae will be divided into two samples for either metabolic analysis or morphological species identification, with each sample containing roughly 50 individuals. 
SECTION: Field Entomology Sampling
34.2. Larvae intended for species identification will be killed via parboiling. An electric kettle containing distilled water will be allowed to reach boiling point (100 °C). Water will then be decanted into each 20mL glass scintillation vial containing live larvae and allowed to incubate for 1 minbefore being strained to remove water. Larvae will be placed back into their respective vials, which will be filled with 70 - 85% ethanol for indefinite preservation.

SECTION: Field Entomology Sampling
34.3. For metabolic analysis: NEED TO BE FROZEN IN LIQUID NITROGEN in 1-2 ml cryo vials
SECTION: Field Collection of Decomposition Fluid
35. In the field, observe donor to determine if there is fluid visibly pooling around the body. Record observations in field notes.  If there is enough (~15 ml of collectable fluid), proceed with sampling.  If not, note “not enough fluid”.
SECTION: Field Collection of Decomposition Fluid
37. Open a sterile 30cc luer-lock syringe and ensure the plunger is fully depressed. Gently place the tip into the pooling fluid, holding the syringe at ~ 45 degree angle and ensuring the tip is not pressing into the soil. 
SECTION: Field Collection of Decomposition Fluid
38. Slowly pull the plunger to draw fluid into the syringe. If the fluid is viscous and will not draw into the syringe, cut the tip back with sterile scissors to enlarge the tip. 
SECTION: Field Collection of Decomposition Fluid
39. Clean the syringe tip by wiping with a clean paper towel. Dispense fluid into 50 ml falcon tube.  Take at least 15 ml of fluid if possible.  Cap and gently invert the 50 mL tube 5 times to mix the fluid.
 

SECTION: Field Collection of Decomposition Fluid
40. Label 2mL cyrovials (at least 4) with donor number, date, sample type (DNA or MET), and location of collection (2 will be labeled DNA and 2 MET). Use a clean syringe to aliquot 1.75 mL of fluid into each 2mL cryovial to leave room for expansion when frozen.
SECTION: Field Collection of Decomposition Fluid
41. Screw the cap on the cryovials and place in the corresponding labelled 4oz whirlpak bag.  All cryovials from one donor and analysis type should go into one bag.
SECTION: Field Collection of Decomposition Fluid
42. Seal the whirlpak bag and place it in liquid nitrogen immediately. 
SECTION: Field Collection of Decomposition Fluid
44. Repeat steps 36-43 for each donor. 
SECTION: Field Collection of Decomposition Fluid
45. Store collected decomposition fluid samples at -80 °C 

 



SECTION: Field Soil Sampling Procedure
19. Using a clean corer, collect approximately 5-8 soil cores from multiple locations around the body, avoiding areas that have already been sampled.  If there is a visible decomposition island (dark soils), collect from these soils.  
SECTION: Field Soil Sampling Procedure
19.1. To take one soil sample: Identify the location of the soil sample and gently remove any large debris (leaves, twigs, etc.). Remove the sterile syringe (with the tip cut off) from its covering and remove the plunger from the syringe. Press the end of the corer into soil vertically to a depth of ~5 cm. Carefully reinsert the plunger to push soil cores into a 7 oz whirlpak bag (one bag per donor).  Repeat this process until at least 35 g (use kitchen scale to check) of soil have been collected (~5-8 cores).  Dispose of the corer. If the corer breaks or becomes damaged due to hard, dry soil, rocks, etc., use a new sterile corer.
SECTION: Field Collection of Decomposition Fluid
36. At each sampling, decomposition fluid samples will be collected for both DNA extraction and untargeted metabolomics, therefore the fluid sample will be aliquoted into respective 4 oz bags. Begin by labelling 2 4 oz whirlpak bags with donor number, date, analysis type (DNA or MET), and location of collection. 
SECTION: Field Collection of Decomposition Fluid
43. If there are other locations with adequate fluid pooling, repeat steps 36-43 for that donor.