Collection and preservation of biological samples for toxicological analysis

In handling the Medicolegal autopsy cases, certain standard guidelines are necessary to be laid down to assist in the selection of appropriate specimens of the body fluids and tissue for postmortem biochemical and toxicological analysis. After death there is a rapid change in the cellular level biochemistry due to autolysis. The drugs and other poisons may be released from the binding sites in tissues and major organs.

The unabsorbed drug may diffuse from stomach, care should be taken in selection of blood and tissue sampling sites. Many a times the autopsy is conducted before all the circumstantial evidences are collected and investigated. Hence, it is vital to preserve all the necessary samples at the time of autopsy. Ideally the samples for toxicological or biochemical analysis should be collected before the postmortem. However it may not be possible for all the samples and there may be dificulty in sampling without opening the body.

Biological fluids

1. Blood

In all medico legal investigation cases a blood specimen should be obtained when blood is available. It is used as a reference sample for identification in unidentified cases and also for toxicological analysis. Peripheral blood concentration have been shown to be more reliable for toxicological analysis than the conventional heart blood. Therefore, in all suspected poisoning deaths or in all cases of unknown causes of death a femoral blood specimen should be collected.

2. Urine

Urine specimen is of great value even in small amount especially in screening of unknown drug or poison, particularly substance of abuse since the concentrations are generally higher than in blood and a number of metabolites may also be present. Urine specimen are also valuable in the quantitative analysis of alcohol, where there is uncertainity over the validity of a blood specimen.

3. Bile

Bile is helpful in estimating the drugs, which are concentrated by liver and excreted into the gall bladder like opiates and acetaminophen ( paracetamol) . It is not routinely preserved, but only in selected cases. It is preserved in 30 ml glass screw capped container. A 20 ml of bile is adequate for toxicological analysis. It can be collected directly by incising the gall bladder into a glass bottle. It is a viscous fluid, which makes it difficult to be sucked by needle and syringe.

4. Vitreous Humor

The vitreous humor specimen is particularly useful for alcohols, or in diabetes and insulin related deaths. It is also very useful where the body has decomposed. The fluid in the eye resists putrefaction longer than other body fluids as it is sterile and remains well protected in eye. It is useful for certain biochemical tests such as urea, creatinie, glucose, lactose and alcohol. Vitreous humor must be collected from both eyes in separate vials of 10 ml. It is preserved with sodium fluoride (10 mg/ml).

5. Cerebrospinal fluid

The cerebrospinal fluid sample is rarely required for toxicological analysis. If needed it should be collected by cisternal puncture.It is difficult to collect CSF at medicolegal autopsy by conventioal lumbar punture. It is relatively easier to obtain by cisternal punture. The CSF sample has to be preserved in sodium fluoride.

6. Other body fluids

In cases where blood and urine are not available other available body fluids like pericardial and synovial fluids can be used for toxicological analysis like alcohol.

BIOLOGICAL TISSUES

1. Liver

Body tissues are often used for toxicological analysis. Liver is the most important tissue because it concentrates many substances. It can contain large amount of drugs and metabolites and may in some difficult cases help establish whether acute or chronic toxicity has occurred. Ideally the part of the liver retained should be fresh unfixed, taken from the periphery of right lobe, away from the stomach,major vessels and gall bladder. A minimum of 100 gm is sufficient for toxicological analysis.

2. Stomach contents

The other routinely preserved viscera are stomach and small intestine with its contents and kidney. The sample is useful when drugs have been taken orally as the concentrations will be many times higher than in other fluids. It can also be helpful to determine the amount of drug present in stomach if blood concentration is difficult to interpret. The stomach should be ligated on both ends (oesophagus and pylorus) and dissected out. Then the greater curvature should be opened up, so that, the contents directly pour onto the wide mouthed jar.About 30 cm of small intestine are preserved with the contents. One half of each kidney is preserved. The stomach and intestine with its contents are preserved in one bottle.

3. Other tissues

Other tissue samples may be useful for investigating deaths where volatile substances e.g solvents or gases, are implicated. Brain, fat tissue, lung and kidney are the most useful. Ideally a wet unfixed tissue should be collected into separate glass containers. In case of lung, the sample has to be collected from the apex of the lung. The whole lung may have to be preserved in case of solvent abuse or volatile substance poisoning.

4. Bone and Muscle tissue

In case of decomposed, exhumed, burnt or skeletonized body it becomes difficult and challenging due to absence of blood or scarcity of solid tissues.But ,whatever remains are available we have to collect all the relevant samples though it may not be the routine sample. If bones are available the whole long bone should to be collected and preserved. It has to be dried in normal temperature and sealed in plastic bag. Bone marrow samples may be useful in drug identification ( qualitative and also quantitative ) in cases where all soft tissue has degenerated. The skeletal muscle is also useful for toxicological analysis. A 100 gm muscle tissue (preferably quadricep muscle ) has to be preserved in saturated solution of common salt in a plastic or glass container.

5. Hair and Nail

Hair and nails are useful samples for analysing chronic poison ( heavy metals) or drug of abuse (opioids). These should be sent if chronic poisoning is suspected, particularly to distinguish between episodic or continuous exposure, or for those poisons which may have already been eliminated from the body by the time of death. Hair should be plucked from the scalp with the entire root, shaft and tip. About 500 ug (20 – 30 hairs ) of hair should be collected and laid aligned by rolling into a clean plastic or foil sheet with an indication of the scalp ends on the attached label. The whole nail from one toe or fingers can be lifted and collected in a plastic packet.

6. Maggots

In decomposed body, if maggots are present 20 gms of maggots can be collected in a plastic or glass container with saturated common salt as the preservative. If drugs or intoxicants are detected they could only have originated from tissues upon which the larvae were feeding. However the correlations between the level in the larvae and the human has not been established. It only provides qualitative information about drug use.

7. Injection sites or snake bite

In case of death due to injection of drugs or suspected snake bite the sample from the injection site has to be preserved. The skin sample with the underneath muscle tissue around the injection site area must be preserved along with a control sample of similar composition from the opposite normal site in saturated solution of common salt

8. Tablets, powders and syringes

These samples should be packed with care and any needle protected by a suitable shield to avoid injury. These items may be particularly useful in deaths in medical personal or drug addicts who may use agents which are difficult to detect once they have entered the body.

The use of disposable, hard plastic or glass containers are recommended for preservation. The plastic containers ( especially of polypropylene) are increasingly used and have the advantage of not smashing when dropped and also much lighter. The ideal samples are best sent in their original state without adding any preservative in a refrigerated storage (4°C) within few hours. But generally it is not possible to send in this ideal state due to lack of good autopsy facilities, cold storage facilities, quick transport arrangements, legal formalities and quick forensic laboratory services. It usually gets delayed. Therefore, sample has to be put in ideal preservatives to provide optimal conditions till they reach the laboratory. The specimen are generally preserved at 4^oC during the time until they are analysed. For long term storage it has to be kept in freezer (- 10^oC) until analysed and disposed off. The most commonly used preservative for viscera tissues are saturated solution of common salt. It is the most easily available, cheap and effective preservative. It is important that the solution should be prepared using pure sodium chloride in distilled water to avoid any contaminants. The other option is rectified spirit except in cases of poisoning due to alcohol, chloral hydrate, chloroform, phenol, formaldehyde, ether, and phosphorus. In acid or alkali poisoning rectified spirit is the prescribed preservative. The blood for toxicological analysis has to be preserved in NaF at the concentration of 10 mg/ml of blood and potassium oxalate, 30 mg/10ml of blood . Fluoride should be added to urine, vitreous humor if alcohol estimations are required.