And a Protocol for Collecting and Preserving Arthropods

It is recommended some cares to participate on the investigation and on collection of entomological evidence especially at the death scene. FE experts should wear protective clothing, gloves and shoe covers or boots mainly to avoid any contamination of the scene with fibres or other material (Amendt et al. 2007).

All material or equipment used to collect sample and for toxicological analysis must be clean and, if possible washed in hot water. A plastic film (magi packĀ®) or aluminium paper can be put in a lid of glass to avoid contamination of the sample.

Tissue, fluid and insects must be in natura and collected in vials free of preservatives or any external contamination. In most cases a refrigerated storage before the analysis is recommended. If possible, should be also stored a part of individuals of each sample in different vials under refrigerated condition to keep as reference sample.

Samples can be collect on the corpse (natural orifices, eyes, traumatic wounds or everywhere on the body), clothes, shoes and around the corpse (Amendt et al. 2007).

Though many samples for analysis are best sent in their original state, others require additives to maintain them in optimum conditions until they reach the laboratory (Knight 1991). Entomological samples can be collected by using forceps and spoons for collecting immature stages of flies, fine paintbrush for collecting eggs or handheld insect capture net for flying adults (Amendt et al. 2007). The toxicological analysis should be done as soon as possible to guarantee the integrity of samples.

When insects were collected from a carcass or corpse, they must be sampled in individual glass vials labelled with proper information (see protocol sheet for the collection of entomological evidence illustrated in Table 9.2). They must be taken to the laboratory, where larvae (if alive can be killed by freezing prior the analysis) must be rinsed in distilled water and pupae/puparia must be rinsed with methanol prior to extraction to avoid contaminants. Then, the sample must be prepared for analysis. The methods for toxicological analysis take into account the substances to be analysed, inorganic (metals) or organic (drugs and pesticides) as well as major and minor affinities to organic solvents, which depend on lipid, protein and cartilaginous components (Gagliano-Candela and Aventaggiato 2001). It is important to emphasize that all material used for analysis must be of glass to avoid contamination of phthalate present on plastic vials. Adult flies also can be sent to analysis although the drug concentration is less than immature stages.

Contaminations of entomological samples used in forensic investigations to determine the PMI or to detect toxic compounds can occur when arthropods collected from a body or body discovery site originate from a source other than the deceased (Archer et al. 2006). Some contaminating insects can be collected in the mortuary due accidental transfer of insects between corpses or infestation of bodies by insects living in the mortuary. For example, in a case of drug related death, if contaminating maggots are collected, the result can be negative once these maggots are free of drug and the toxicological research is affected.

Besides, some errors caused by post-mortem changes before the sample is removed cannot be avoided. These are variable, depending partly on the conditions

Table 9.2 Protocol of collecting samples for toxicological analysis

Case no:----Collection date:

Case no:----Collection date:

Table 9.2 Protocol of collecting samples for toxicological analysis

Collector Name:

Date found:

Body details:

Date reported

missing:

Age:

Gender:

Height:

Weight (kg)

City:

Region

Urban

Country side

Wildland

Death scenario:

Indoors

Outdoors

Field

River

Ocean

Road

Dense vegetation

Burned

Temperature:

Ambient

Relative humidity

Precipitation

Decomposition

Fresh

Bloated

Early

Advanced

Skeletonized

stage:

decomposition

decomposition

Number of sp/body

Evidence of

part:

Scavengers:

Insects

Imatures:

Living

Egg

Maggot

Pupae

Dead

Egg

Maggot

Pupae

Adults:

Living

Dead

Site of collection:

Skin

Head,neck

Thorax, abdomen

Hands, foot

Internal

Brain

Liver, kidneys,

Heart, lungs

organs

intestines

Soil, coffin

Samples for

Immatures

Adults

analysis:

Others:

Drug vials

Syringes

Prescriptions

Probable substance:

of post-mortem environment and partly on the microbiological population on corpse (Knight 1991).

There are two alternatives to obtain samples for toxicological investigations in animal experimentation. The first method is used an animal model to administrate the drug and then their samples are offered to immature insects to complete the development. Experimentation on animal models is usually necessary when trying to detect, quantify and evaluate drug effects on arthropod development. The domestic rabbit is the most indicated animal for this kind of research; for it is of easily handled and satisfactory results can be obtained with fair drug dosages. We would recommend using ten 3.0-4.0 kg rabbits of same sex for each experiment (five treatment replicates and five controls), minding that the animals should be kept in separate cages provisioned with water and food for as long as 5 days before the procedures, to avoid excessive stress. The drugs must be in contaminant-free saline solution for intravenous administration. As such, lethal, sub-lethal or LD50 dosages of each drug can be tested and determined. Past 30 min from the administration of the drugs, any surviving rabbits must be killed without inflicting external injuries, to avoid exposing blood and other liquids. Samples of the blood, liver, spleen, brain, urine, heart are then obtained through immediate necropsy and stored in labelled vials for posterior analysis. After that, additional samples from the rabbit's tissues must be taken for directly exposure to larvae of insects of forensic importance.

Each insect species must be separately placed with a tissue sample. Larval development time, pupation period until emergence, longevity length and mortality rates are to be recorded from both control and treatment conditions, and then compared to evaluate any drug alterations over development. Toxicological analysis may be performed on samples from these insects, usually by the same manner as is done with samples from mammals, as explained below.

For preparing the samples for toxin extraction, about 1.0 g of tissue, 1.0 mL for fluids and 10 insect samples (maggot, pupae or adult) are mixed separately with a solvent with homogenizing equipment. There are several techniques for extracting each different drug or chemical, and the methods employed may vary according to the laboratory or Institution of Legal Medicine standards and the available materials.

The extracted material can then be injected into GC-MS equipment to obtain a chromatogram (Fig. 9.1). A curve of calibration is prepared to quantify the drug and compare it to other concentrations. An appropriate standard solution is mixed to the sample, if necessary. Several substances involved in drug-related deaths have been already detected in previous studies by researchers of different countries (Table 9.3). The identity of each substance must be verified by comparison of their retention time and mass spectra with authentic standards, when possible (Casale and Moore 1994).

The second method, immature insects can be directly reared on artificial diets containing known concentrations of the drug. However, this method is restricted for lacking the metabolic processes on the drug that would have occurred after the previous ingestion and incorporation by a living organism.

Herein is suggested a protocol for collecting arthropod necrofauna aiming to facilitate and standardize procedures in investigations and toxicological analyses.

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