Postmortem Blood

Introduction

Postmortem blood samples may be used by forensic toxicologists as an aid to determining whether a deceased person might have been affected by a drug or poison at the time of death or whether the drug or poison detected could have been directly responsible for death. Recent research has shown, however, that the use of postmortem blood drug concentrations for interpretative purposes must be undertaken circumspectly, and their comparison against published data collections should be undertaken with extreme caution and only after careful consideration of all of the factors that could potentially affect the analytical results.

Sample Collection

Following a sudden or unexpected death the forensic toxicologist is often requested to undertake toxicolo-gical analysis to determine whether drugs or other poisons may have contributed to or caused death. The common circumstances where analysis may be requested are:
• after a suspected drug overdose;
• after a suspected poisoning;
• in accident investigation – to determine whether drugs may have been a contributory factor to an accident;
• after a death linked with a possible criminal offence (e.g. assault/murder).
In order to investigate whether drugs and/or poisons may have contributed to the cause of death, or whether the deceased may have been under the influence of drugs at the time of death, blood, among other tissue samples, can provide valuable information. A general outline concerning blood as a tool for toxico-logical analysis is presented elsewhere in this work and the reader is referred to the section dealing with antemortem blood samples for a general discussion on the matrix of blood. There are, however, a number of significant considerations that must be borne in mind when analysing postmortem tissues that are unique to these samples; a different approach to interpretation is warranted than that which is applied to blood samples obtained from living subjects.
In most instances it is possible to collect a range of tissues at postmortem examination and this should be the recommended procedure whenever possible. It is the duty of the toxicologist to ensure that the pathologist is provided with advice concerning the samples required and appropriate containers. Whenever possible, the laboratory should provide a sample collection kit containing suitable containers, labels and packaging to enable the pathologist to collect and submit a full range of samples. It is better to collect more samples than may be required at the first postmortem examination, rather than having to try to go back for more later.
The postmortem specimen collection kit should include, at a minimum:
• three 10 ml screw-cap containers and containing oxalate for peripheral blood samples;
• one 30 ml universal container with screw cap for a urine sample;
• two 5 ml fluoride oxalate vials with septum caps for blood alcohol/volatiles analysis;
• one 5 ml fluoride oxalate vial for urine alcohol analysis;
• two large screw-cap containers or jars for the collection of liver and stomach contents;
• three 20 ml syringes with needles;
• set of tie-on labels (sufficient for all containers);
• laboratory submission/information form;
• secure transportation box;
• instruction sheet outlining collection requirements.
There is no universal test for a drug or poison. So, when the availability of tissue/samples is limited, the analyst should have all of the information available to assist in conserving valuable samples for the most appropriate analysis. Access to a full case history may not only help to save valuable samples but can also save considerable time and expense in carrying out unnecessary tests. The investigating officer should therefore be required to submit as much information about the case as possible, as this may influence the type and extent of analysis undertaken as well as the interpretation of analytical results. Submitting officers should be required to provide, at a minimum, the following information:
• name of deceased;
• age;
• occupation;
• race (Caucasian, Chinese, Afro Caribbean, etc.);
• date/time of discovery of body;
• date/time of death;
• date/time of postmortem;
• time between death and postmortem;
• full details/case history and circumstances surrounding/leading up to death;
• details of any signs or symptoms noted prior to death (e.g. diarrhoea, loss of weight, delirium, drunkenness, convulsions, hallucinations, etc);
• temperature of body surroundings/environment where body was found;
• medical history of deceased;
• availability to deceased or relatives of drugs/ medicines/poisons;
• whether deceased was a known or suspected drug user;
• whether the body was embalmed prior to autopsy;
• if death occurred following admission to hospital, the survival period between admission and death;
• the treatment/drugs/resuscitation procedures carried out before death;
• if death occurred in hospital, details of any ante-mortem blood samples collected (if any of these are available they should be requested for analysis);
• whether the stomach was washed out on admission to hospital (if so, access to the stomach wash, if it is available, should be requested).
All too often a seemingly straightforward and uncomplicated death can turn into a serious case that will culminate in some form of criminal proceedings. It is far better to have all of the information at hand while planning how to manage the case than to have to revisit it in hindsight or at the request of the defense lawyer. The more information that is at hand before undertaking any work, the more reasoned the approach to analysis and interpretation will be. With the increasing availability of sensitive bench-top mass spectrometry to almost all laboratories in recent years, there has been a trend towards undertaking blood analysis alone in many cases, rather than performing analysis on a range of tissues. Historically, blood, stomach contents, liver and urine were all used to provide a composite picture in postmortem toxicology cases. This was partly necessitated because analytical methods were relatively insensitive and necessitated a plentiful supply of material for drug extraction. With the advent of gas chromatography and inexpensive bench-top mass spectrometry, toxicologists have been able to develop analytical methods that enable blood analyses to be undertaken for a wide range of drugs in small blood samples, often at concentrations significantly lower than in liver, stomach content and urine. The introduction of these new technologies led to the publication of many data compilations listing the concentrations of drugs in blood following the consumption of therapeutic quantities, in addition to those measured where patients had taken toxic or overdose quantities of drugs. The increasing availability of data relating to concentrations of drugs in blood led many toxicologists to direct their analysis towards blood only, particularly when this could be accompanied by minimizing the costs and time spent on undertaking multiorgan toxicology. The concept that whole blood analysis would provide an estimation of the concentration of a drug at the time of death is one that was held in wide belief and led to a widespread fall in laboratories undertaking multiorgan toxicology. During recent years our knowledge concerning what happens to drugs post mortem, between death and the time of sampling, has grown and led many forensic toxicologists to view with caution the way that analytical results are used. If blood samples could be collected within minutes of death, the philosophy that a postmortem blood sample provides a ‘snapshot’ of the blood concentration of a drug at the time of death might be supportable. In reality, however, it is usually impossible to collect blood samples within minutes of death. In most cases a considerable amount of time elapses between the moment of death and postmortem blood sampling. In the majority of cases encountered by the forensic toxicologist, the time of death and the interval elapsing between death and sampling is a totally unknown entity.

Postmortem Change

The living person comprises millions of microscopic cells grouped together as tissues or organs, each with specific functions to perform. Each cell is a highly organized structure of chemical membranes, containing and controlling numerous complex chemical processes in a highly organized manner. The cells are bathed by a fluid, the interstitial fluid, that provides a link with the circulating blood, the function of which is to transport oxygen and essential substances around the body for the cells and to carry waste products and toxins away from the cells to specific sites in the body from where they may be eliminated (e.g. liver and kidneys). When death occurs, the mechanisms for controlling the chemical reactions within the cells cease to function. Failure of the heart to circulate blood deprives the body tissues of oxygen and other nutrients and leads to the accumulation of waste materials in and around the cells, resulting, within a short time, in cell death. Cell death is characterized by chemical disorganization, failure of cell metabolism and cell function, and eventually a disintegration of the cell structure. As the cell walls begin to lose integrity, leakage of the cell contents into the surrounding environment takes place and ‘out of control’ enzymes, formerly contained within the cells, start to destroy the cells and tissues in a self-destructive process known as autolytic decomposition.
After death, the temperature at which the body is stored can alter the rate of decomposition. In a body stored under refrigerated conditions the process of autolysis and decomposition is slowed down, whereas a body maintained at an elevated temperature will be likely to decompose at a faster rate. After death, the processes that combat and protect the body from microbial infection become ineffective, hence micro-bial action also contributes to the process of decomposition. As decomposition advances, drugs may be released from tissues and intracellular binding sites and diffuse from areas of higher concentration to areas of lower concentration. This offers an explanation as to why postmortem heart blood concentrations may be several orders of magnitude higher than blood collected from the femoral vein in the upper leg.
Following the consumption of a drug overdose, the liver concentrations of drugs can become very high, as this organ is one of the principal organs in the body where drugs are metabolized in preparation for their elimination. Likewise, capillary-rich lung tissue containing lipoproteins may also accumulate high concentrations of drugs. After death, drugs diffuse from the lung, liver and other tissues where they are present in high concentrations into the pulmonary artery and vein and also into the vena cava. The diffusion process continues over time, resulting in significant variations in blood drug concentrations.
The observation that drugs are found in different concentrations at different sites in the body after death, was first recorded in I960. This observation prompted the recommendation that, ideally, peripheral blood samples should be used for toxicological analyses. It was not, however, until the mid to late 1980s that toxicologists really began to realize the full significance of postmortem change. Several groups published a number of significant observations that have led toxicologists to express caution in interpreting postmortem blood drug concentrations. One group demonstrated that postmortem redistribution of basic drugs can occur rapidly after death and that attempts to resuscitate a corpse using cardiac massage, or even movement of the body from the site of death to the postmortem room, may influence drug distribution. As a result of their observations, the authors judged that it is unsafe to attempt to apply pharmacokinetic calculations, based on the analysis of a postmortem blood analysis, to deduce the quantity of drug taken. It is also unsafe to assume that blood drawn from a peripheral site is unaffected by postmortem redistribution. Pathologists or postmortem room technicians must be aware that simply withdrawing blood down the femoral vein may result in drawing blood down from the major vessels in the torso, such as the vena cava, where the process of diffusion from the liver or heart may be well advanced.
The ability to obtain a satisfactory peripheral blood sample at post mortem may be affected by the state of the body and blood vessels. Ideally, the femoral vein should be dissected and cut and the portion closest to the abdomen tied off with a ligature. Blood should then be collected from the leg portion of the vein, either using a syringe or by gently massaging the vein along the leg. Many pathologists prefer to collect blood samples from the vein in the upper arm rather than the leg. However, since the subclavian vein is much closer to the heart and pulmonary blood vessels than the femoral vein, there is an increased chance that blood drawn from this site may have been affected by postmortem diffusion from the heart.
The heart is often a source of a plentiful supply of blood. However, if heart blood is collected and analyzed, peripheral blood samples must also be collected and analyzed to demonstrate whether significant differences in concentrations are present. The lungs are served with a plentiful supply of blood that can contain drugs in very high concentrations. After death, diffusion of drugs may occur from the lung into the left ventricle, resulting in blood drug concentrations in the left ventricle that are up to 10 times higher than those in the right ventricle. Particular caution should be exercised in the interpretation of results when blood samples have been collected from babies. In small infants, the volume of available blood may be limited because of the body size. There is also an increased risk that samples may contain blood drawn from the major blood vessels in the torso, where the risk of contamination by hepatic blood is high. In these cases consideration must be given to the fact that hepatic blood may contain drugs in concentrations that are orders of magnitude higher than in peripheral blood samples.
This reinforces the necessity for toxicologists to insist that all samples are clearly labeled with respect to the site and time of sampling. Where the site of sampling is in doubt, this should be stated in the toxicology report and the interpretation should be covered by appropriate caveats. Fluid scooped from the body cavity is occasionally submitted under the description of ‘blood’. Such a sample is unsuitable for interpretative purposes, as it is seldom an authentic blood sample and there is a high probability that it is contaminated with stomach content, liver blood, bile, urine and intestinal contents.
If significant variations in drug concentrations may be encountered in blood drawn from different sites in the body, the blood drug concentrations published in the literature and used in reference collections may also have been subject to postmortem change phenomena. Data compilations of blood drug concentrations rarely provide detailed case histories and many were compiled before the extent of the implications of postmortem change were realized. Those tasked with interpreting postmortem blood concentrations should therefore exercise extreme caution before finally expressing their views in a report or before a court of law. Caution must also be exercised when, in the case of an extremely decomposed or an exhumed body, no blood is available and tissue drug concentrations are used for interpretative purposes. The practice of attempting to deduce a postmortem blood drug concentration from a tissue concentration by using tissue:blood ratios published in data collections is scientifically unsound and should not be undertaken.
Postmortem artifacts may also influence the ratios of metabolites to their parent drug in blood. For example, it has been suggested that the ratios of unconjugated to conjugated morphine might assist in indicating whether death had occurred within a short time of the entry of the drug into the body. This philosophy proposed that, if death occurred rapidly after entry of morphine or heroin into the body, the concentration of unconjugated morphine present in blood would be higher than the conjugated morphine concentration. The volume of distribution of morphine is, however, large compared with that of morphine glucuronides. Hence, while only a small proportion of morphine is present in the circulating blood, a significant proportion of the morphine glu-curonides are present in blood. Any factors that could change the distribution of morphine, or materialize the conversion of some or all of the morphine glucur-onides back into morphine, could grossly affect the interpretation of results based on morphine:morphine glucuronide ratios and subsequently provide an erroneous conclusion with respect to time of dosing in relation to death.
A list of concentrations for commonly encountered drugs or drug metabolites that have been widely associated with fatal poisoning is provided as Table 1. It must always be remembered, however, that in many cases blood drug concentrations cannot be interpreted in isolation and the task of interpretation should only be undertaken after full discussion has taken place between the toxicologist and the pathologist. Most experienced toxicologists will have encountered cases where so called ‘fatal blood concentrations’ have been measured in a deceased person but the cause of death was not directly associated with the drug detected. Drug addicts and palliative care patients can build up considerable tolerance to drugs such as opiates and appear to behave relatively normally, while the concentrations of opiates in their blood would be capable of causing death in a nontolerant subject. It is also well known that in many cases the cause of death may be directly attributed to gunshot wounds, stabbing or road traffic accidents but the concentrations of drugs in blood fall within the range of concentrations associated with fatal poisoning.
Our knowledge of postmortem change and the effects that may be associated with it is still far from being complete. However, an awareness that such phenomena exist should provide a warning to toxi-cologists and pathologists that the process of interpreting postmortem blood results is not simple. Because of the problems associated with drug tolerance and postmortem movement of drugs, all that can be safely deduced from a blood drug concentration alone is that the drug or its metabolites are present. A more elaborate interpretation should only be reached after all of the factors in a case have been drawn together, including the histological evidence, the gross pathology, the known circumstances of the case and the medical history of the deceased.

Table 1 Postmortem blood concentrations of 50 common drugs and poisons that may be associated with fatal poisoning