Field Data Collection Protocols for NIJ Grant The Impact of Drugs on Human Decomposition and the Postmortem Interval: Insect, Scavenger and Microbial Evidence

Human remains with enrollment criteria:

1. Known date of death
2. Obtainable blood serum
3. Known medical information including drugs used, medical conditions, treatments 7-30 days prior to death
4. No outward trauma

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.).

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.

Aorta (central) and femoral (peripheral) blood sticks should be performed. Approximately 4mL of each blood type should be collected.

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 1mL stored at -80°C

1 cryovial - Femoral - whole blood 1mL stored at -80°C

3 cryovials - Central - serum 200µL each stored at -80°C

3 cryovials - Femoral - serum 200µL each stored at -80°C

Total of 8 cryovials per donor

Draw whole blood into tube(s) containing no anticoagulant. Draw approximately 2 1⁄2 times the volume needed for use.

Incubate in an upright position at room temperature for 0h 45m 0sto allow clotting.

Centrifuge for 0h 15m 0s at 2000rcf,0h 0m 0s

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.

Inspect serum for turbidity

Turbid samples should be centrifuged for 0h 15m 0s at 2000rcf,0h 0m 0s again and aspirated again to remove remaining insoluble matter.

Aliquot into triplicate samples 200µLor 1mLserum into 3 cryovials with a minimum for a single replicate ~100 uL.

Store cryovials of serum at -80°C

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).

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.

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.

Placement photographs - all photographs include a North arrow when possible to provide spatial orientation.

A
Detail photos - areas of interest
Lower third of the body (hips, legs, feet)
Genitalia
Upper third of the body (head, neck, chest)
Head striaght on
Identification Number
L Axilla
L elbow
L Foot
L hand
L Hip
L Knee
L Profile of head
L Torso Ground Body Interface
Oblique of body
Overview
R Axilla
R Elbow
R Foot
R hand
R Hip
R Knee
R Profile of head
R Torso Ground Body Interface
Middle third of the body (torso)
Weather Station - same photo of the trail at every observation - to document evironmetal conditions

List of photographs to be taken at every daily observation.

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.

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.

Mark the control site with a flag labelled with donor number and “control”.

Label one 7 oz bag and one 4 oz whirlpak bag with donor number, date, and "donor".

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.

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.

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.

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.

Put both 4 oz whirlpak bags into dewar/CryoPod^TM 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.

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.

Repeat steps 18-22 with additional donors, using a new corer for each donor. Dispose of corers and PPE in autoclave bags.

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.

Photographs

A
Weather Station - same photo of trail every observation - to document environmental conditions.
Middle third of the body (torso)
R Torso Ground Body Interface
R Profile of head
R Knee
R Hip
R hand
R Foot
R Elbow
R Axilla
Overview
Oblique of body
L Torso Ground Body Interface
L Profile of head
L Knee
L Hip
L hand
L Foot
L elbow
L Axilla
Upper third of the body (head, neck, chest)
Head striaght on
Genitalia
Lower third of the body (hips, legs, feet)
Detail photos - areas of interest
Identification Number

List of photographs to be taken at every daily observation.

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.

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.

Arthropod Collection

Scavenger Form

Decomposition Fluid Form

Decomposition Fluid Collection

Tiny Tag Temperature log reading

Game Camera

Conduct visual observations and note the presence or absence of various arthropod life stages on

the data collection form.

Do five aerial sweeps with a butterfly net to collect flying arthropods.

Place captured flying arthropods in 70% EtOH in 20mL scintillation vials with appropriate labels.

Using forceps or another collection device (e.g., plastic spoon, spatula), collect immature arthropods from at least three locations and document on collection form.  

Collected larvae will be divided into two samples for either metabolic analysis or morphological species identification, with each sample containing roughly 50 individuals.

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 0h 1m 0sbefore 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.

For metabolic analysis: NEED TO BE FROZEN IN LIQUID NITROGEN in 1-2 ml cryo vials

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”.

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.

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.

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.

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.

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.

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.

Seal the whirlpak bag and place it in liquid nitrogen immediately.

If there are other locations with adequate fluid pooling, repeat steps 36-43 for that donor.

Repeat steps 36-43 for each donor.

Store collected decomposition fluid samples at -80°C

Photographs

Insect Form

Scavenger Form

Decomposition Fluid Form

Tiny Tag Temperature log reading

Unenrollment Criteria - The donor's abdomen should cease to release fluid, with most tissues desiccated, marking the end of the active decomposition phase.