For a successful toxicological analysis, samples need to be selected with the desired analytical test in mind and collected carefully. This paper describes the best practice methods for sampling and the effects of post-mortem redistribution on sampling.
Introduction
Sampling is of the utmost importance in forensic toxicology. The reliability and accuracy of any result is usually determined by the nature and integrity of the samples provided. Proper sample selection and collection is important for the results to be accurately interpreted with scientific validity.
The best places to get samples for testing are the locations where chemicals enter the body, concentrate within the body, and along the routes of elimination. Thus blood, stomach contents, and tissues around injection sites may possess high concentrations of the drug. Analyses of liver, brain and other tissues can reveal where a drug or its metabolites accumulated and urine analysis can indicate where the drug and its metabolites are concentrated for final elimination.
Samples from living subjects
Toxicological analysis is usually required on samples from living subjects in cases of drug facilitated crimes or attempted poisonings. In all situations, samples should be collected as quickly as possible and properly labelled. The best containers for liquid samples are disposable hard plastic or glass tubes. When a preservative is needed, sodium fluoride should be used at a concentration of approximately 2% weight by volume. Unless otherwise noted, samples must be maintained at temperatures not greater than 4°C to ensure sample integrity.
Post-mortem Samples
Cases of mysterious deaths tend to require forensic toxicologists to perform extensive toxicological analyses. The availability of autopsy samples in post-mortem toxicology allow for a more flexible approach to the analysis. Some samples have more value than others when specific drugs or poisons are involved in the death.
Autolysis and putrefactive processes that occur after death, as well as post-mortem redistribution, can have a profound effect on drugs and poisons that are present in the body prior to death. For this reason, post-mortem samples should be collected as quickly as possible and in separate containers. For most samples, disposable hard plastic or glass tubes are recommended. Each sample must be labelled appropriately. Samples should be stored at a maximum of 4°C when being analyzed soon after autopsy, otherwise at -20°C.
Samples in Detail
Blood
Blood is the sample of choice for quantifying and interpreting concentrations of drugs and their metabolites. Blood provides a list of toxic substances present in the subject’s body at the time of collection. Concentrations within the blood correlate well with levels of intoxication. A quantity of 10 – 15 ml of blood is required to screen and confirm the presence of most toxic substances.
In post-mortem cases, blood shows what was going on in the body at the time of death. At least two blood specimens should be collected, 30 ml of central blood for qualitative analysis and 10 ml peripheral blood for quantitative analysis.
Urine
Urine is used for comprehensive drug and poison screening. Urine is an easy sample to obtain and is relatively rapid and non-invasive. The accumulation of toxins in urine results in high concentrations that aid their detection. Because the kidneys are situated along one of the body’s major drug and toxin elimination routes, substances can often be found in greater concentrations in urine than in blood.
Urine samples don’t necessarily reflect the toxins the subject was influenced by at the time of the sample collection as it can show substances even several weeks after their ingestion. For example, cannabis can be detected two weeks after use. It can also take as long as 8 hours until a given substance can be detected. This is a disadvantage where death occurs very rapidly after exposure to a drug or poison. In these cases, the urine specimen may be negative for the causative agent.
A minimum of 30 ml is required for thorough screening in living subjects. In post-mortem cases all urine available should be collected.
Hair
Toxins in the bloodstream can transfer to growing hair and provide an intoxication timeline. Head hair grows at rate of approximately 1 to 1.5 cm a month and so cross sections of the hair at different intervals will give a rough estimate of when the drug was ingested.
Approximately 100-200 mg of hair should be collected from the vertex posterior on the back of the head by cutting as close to the scalp as possible, ensuring that it is clearly marked which end is closest to the scalp and stored at room temperature.
Vitreous Humour
Vitreous humour (VH) is an isolated and protected area of the body. Coupled with its good stability as a biological fluid, these features make this specimen more resistant to putrefactive changes than other post-mortem samples.
The VH and blood maintain equilibrium, meaning that any water soluble chemical in the blood is also in the VH. However substance levels in the VH lag behind levels found in the blood by one to two hours, so testing the VH reflects the concentration of the toxin in the blood one to two hours earlier.
All available vitreous fluid from each eye should be collected separately.
Gastric Contents
Oral ingestion remains the most popular means of exposure to drugs and poisons. Therefore, gastric contents are essential for screening tests. The inspection of gastric contents must be part of every post-mortem examination if possible. It allows the detection of undigested pills or liquids that were ingested just before death. It may also provide qualitative information concerning the nature of the last meal and the presence of abnormal constituents.
All of the available sample should be collected without the addition of a preservative. Undigested pills and tablets should be separated and placed into plastic pillboxes for analysis.
Liver
Since most drugs and poisons are metabolized in the liver, both the parent compound and its metabolites may be present in high concentrations in this tissue, making it a valuable sample in post-mortem cases.
Many drugs can be measured in liver and bile, even when blood tests show no traces of them. The liver may reflect levels of a drug during the hours before death, and the bile may indicate what drugs were in the system during the past three to four days.
Approximately 25-50g of tissue should be collected.
Other possible samples
Other common organs used are the brain, spleen, lungs and kidneys. Nails can be used in a similar way to hair samples. Insects that feed on dead bodies can also be tested for drugs. Collection of scene samples such as drug paraphernalia, cups or bottles, and suspicious household products will aid in a thorough toxicological analysis.
Dealing with post-mortem redistribution
Post-mortem redistribution (PMR) refers to the changes that occur in drug concentrations after death. The drug redistributes into blood from solid organs. PMR is not just limited to blood. The movement of drugs from the gastrointestinal tract into neighbouring tissues has also been shown to contribute to redistribution. PMR is time dependent and occurs via diffusion from a site of high concentration to one of low concentration. Drug properties such as volume of distribution, lipophilicity, and pKa are important factors in PMR.
When post-mortem cardiac blood samples have been compared with samples taken ante-mortem or shortly after death, drug concentrations have been seen to increase up to 10-fold. Femoral blood is also subject to redistribution after death, but less so than cardiac or centrally collected blood.
The anatomical location of blood sampling can influence the drug concentration. The ideal site for sampling is a clamped femoral vein. The liver is also a valuable sample for drugs that undergo PMR. Forensic toxicologists carrying out tests involving drugs likely to undergo PMR must be aware of its potential contribution to the post-mortem drug concentration. Correlation with laboratory data and any available ante-mortem or peri-mortem clinical information is necessary to decipher the cause of death.
Conclusion
The most suitable samples for each forensic case should be taken depending on the type of case. In death investigations two peripheral blood specimens, vitreous humour, a section of liver, stomach contents and a hair sample is recommended. In living subject cases blood, urine and hair is recommended.
The detection of drugs and poisons in post-mortem samples can be more difficult than in clinically derived samples. This is due to the presence of putrefactive compounds and the often-altered nature of specimens in the post-mortem setting.
Toxicologists should take PMR into consideration when choosing the type of samples taken for analysis.
References
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