Selection Of Postmortem Specimens

The choice of specimens available in a postmortem forensic toxicology investigation can be numerous and variable. Specimens may be selected based on case history, institutional policy, and availability for a given case. Generally, the specimens routinely collected from cases in which an autopsy was performed include blood from both peripheral and cardiac sources, all urine and bile available, vitreous humor, all available gastric contents, and tissues (particularly liver).2 However, because the autopsy allows a one-time opportunity to collect as many specimens as may be needed to complete the toxicological investigation, it is suggested that as many specimens be obtained as is feasible for the institution.

In cases where no autopsy is performed, only peripheral blood, urine, and vitreous humor are collected. Heart blood should be avoided due to the potential of contamination by esophageal contents when performing a blind stick.31,32

On some occasions a medical examiner or coroner's case may have had a significant survival time in the hospital prior to death. In cases where hospital survival time exceeds 24 to 48 h, the value of postmortem specimens diminishes considerably. This is especially true if there are allegations that a death may be drug related. Under these circumstances, hospital admission specimens (blood and urine) taken prior to significant therapeutic intervention can be invaluable in the documentation and support of this history. It is important that the postmortem toxicology laboratory physically obtain these specimens (under COC) for reanalysis, since the results available from the hospital are frequently unconfirmed screening results.

Decomposed, skeletonized, or embalmed cases present unique challenges for the forensic toxicologist. The possibilities for specimens in some of these cases are limited only by availability, analytical capabilities, and sometimes imagination, of the toxicology laboratory. Some of these unusual specimens are discussed later in this section.

2.4.1 Blood

Blood is the specimen of choice for detecting, quantifying, and interpreting drug concentrations in postmortem toxicology. Historically, most of the meaningful data derived from the literature was determined in blood.33 Despite concerns about postmortem redistribution of drugs from tissues to blood, some aspects of interpretation remain straightforward. A negative result below a defined limit of detection for a given analyte can be readily interpreted as lack of acute exposure to that analyte or noncompliance in the case of therapeutic agents. Conversely, blood drug concentrations that exceed therapeutic (or, for some drugs, toxic) concentrations by 10 to 20 times are still consistent with intoxication or death (barring an obvious contamination problem). In addition, the higher the parent drug-to-metabolite ratio, the more likely acute intoxication is a factor. This is especially the case when multiple drug analytes are involved, and in cases involving ethanol.

Interpretation becomes especially difficult in cases where drug analytes known to undergo postmortem redistribution are determined to be present in a heart blood specimen at concentrations ranging between the upper therapeutic limit and the lower limit where intoxication or death has been reported. In these cases, analysis of a peripheral blood specimen may be critical in determining the role that the drug may have played in the decedent. Although drug concentrations in cardiac blood generally rise due to postmortem drug redistribution, and peripheral blood drug concentrations tend to remain constant, this is not always the case.13,34,35 Thus, results from a single postmortem blood specimen, whether cardiac or peripheral, may be difficult if not impossible to interpret. Since cardiac blood is usually more plentiful than peripheral blood, many laboratories perform initial toxicological tests on cardiac blood, reserving the peripheral blood specimens for cases where additional context is needed for interpretation. Regardless of the blood samples available for analysis, it is important for the toxicologist to appreciate that the material collected as "blood" at autopsy is not the same specimen collected in an ante-mortem venipuncture and clinically based pharmacokinetic principles may not be applicable to postmortem cases.

Following injury or trauma to the head, blood clots from the brain cavity (subdural, subarachnoid, and/or epidural) should be collected in properly identified containers and saved for the laboratory. These materials are potential "time capsules," which are generally poorly perfused, and may reflect drug or alcohol concentrations closer to the time of injury. These specimens become more important as accurate knowledge of the post-injury survival time increases. Blood clots may also be useful for documenting preexisting drug use prior to hospital therapy. Most laboratories routinely analyze alcohol on these specimens, reserving analysis for additional drugs if indicated. Thoracic and abdominal cavity blood should be collected for analysis only if blood or uncontaminated blood clots cannot be obtained from any other area. These "blood" specimens tend to be contaminated by and contain large numbers of microbes. Additional contamination from gastric contents is also possible. Nevertheless, qualitative documentation of presence of given analytes is of importance and value in death investigations with respect to compliance and exposure issues.

2.4.2 Urine

Urine collected at autopsy has the greatest potential of any specimen to provide the toxicologist with qualitative ante-mortem drug-exposure information. The urine matrix is generally devoid of circulating serum proteins, lipids, and other related large-molecular-weight compounds due to the renal filtration process, simplifying preparation of the specimen for analysis. The accumulation of drugs and their metabolites in urine results in relatively high drug concentrations, facilitating detection of an exposure to a potential poison. Immunoassays and non-instrumental spot tests can be performed directly on the urine specimen for the analysis of certain drug classes. Detection times for drugs in urine can vary from 24 h to as long as a month, depending on the drug. Thus, except for acute drug deaths where survival time is less than an hour and drugs may not yet have been excreted into the urine, urine provides an ideal matrix for the detection for the widest variety of compounds.

Positive identification of drugs in the urine indicates past drug use, but does not indicate when or how much drug was ingested. To interpret the context of exposure, blood should be tested for the analytes found in the urine. In cases where death is suspected to have occurred rapidly due to drug ingestion, as might be suggested by the presence of a needle in the decedent's arm at the time of death, negative urine drug findings may be consistent if blood drug concentrations are very high.

2.4.3 Bile

Bile is another fluid that should be collected at autopsy as a matter of course since many drugs and drug metabolites have been demonstrated to accumulate in this specimen.1,9,21,36,37 The qualitative finding of the presence of drug and/or metabolites in this specimen is important for documentation of historic exposures to specific agents and chronic drug-use history. In the absence of urine, bile has also been useful as an alternative specimen for alcohol analysis38 and has been used in immunoassay screening after sample pretreatment39 or without pretreatment using enzyme-linked immunosorbent immunoassays.40 Historically, bile has most often been used in the determination of opiates in general and morphine in particular.1,36,37 More recently, it has been noted that many drugs are found to accumulate at concentrations significantly higher then those in blood.39 With an appropriate sample cleanup, bile is a useful specimen for the analysis of a wide variety of drugs and their metabolites, including benzodiazepines.

2.4.4 Vitreous Humor

Vitreous humor plays an important role in helping resolve many issues in a postmortem examination. Because of this, it should be collected in all cases when possible, including cases where no autopsy is performed. Vitreous humor, by virtue of its protected environment inside the eye, is less subject to contamination and bacterial decomposition. As a result it may be used to distinguish ante-mortem alcohol ingestion from postmortem alcohol formation and may provide the only opportunity to establish an ante-mortem ethanol concentration in embalmed bodies.23,24,41-43 Additionally, because vitreous humor is contained in a peripheral compartment, there is a delay in both the uptake of drugs and alcohol into this fluid as well as a delay in the excretion process.

It has been observed that vitreous drug concentrations often reflect circulating blood concentrations 1 to 2 h prior to death and that any drug found in the blood will be detected in the corresponding vitreous specimen given analytical techniques of sufficient sensitivity.9 Although these findings suggest that vitreous humor analysis following positive blood findings might be useful to aid in the interpretation of blood drug concentrations, more research needs to be performed. Studies comparing vitreous humor and blood ethanol concentrations yielded a wide variety of ratios of vitreous humor to blood ethanol.42 If such diversity is seen for an analyte that demonstrates minimal postmortem redistribution effects, attempting to use vitreous humor drug concentrations to aid in interpreting heart blood drug concentrations may prove difficult.11 Despite these concerns, vitreous humor is the best specimen from which to evaluate postmortem digoxin concentrations.21 However, the analysis of femoral blood in addition to vitreous humor is still recommended to provide the best context for an appropriate interpretation of ante-mortem digoxin toxicity.44

Vitreous humor lacks the esterases that hydrolyze certain drugs and metabolites in blood and may be the specimen of choice to detect the metabolite of heroin, 6-acetylmorphine.45-48 Because

6-acetylmorphine is quickly hydrolyzed in vivo and in vitro (especially without the presence of sodium fluoride) to morphine in blood, the analysis of vitreous humor may be helpful in establishing whether a death occurred after heroin use. Similarly, cerebrospinal fluid may also be useful.48,49

Vitreous humor has been shown to be particularly useful for the postmortem analysis of glucose, urea nitrogen, uric acid, creatinine, sodium, and chloride. Measuring these analytes is important for documenting diabetes, degree of hydration, electrolyte balance, and the state of renal function prior to death. A recent article has reviewed the extent and breadth of chemistry analyte analysis applied to vitreous fluid, among other postmortem specimens.27

2.4.5 Gastric Contents

Oral ingestion is still the major route of drug administration for prescribed drugs and therefore a major compartment for the investigation of a potential poisoning. Drug overdoses, whether by accident or by intent, may readily be discovered through the analysis of gastric contents. In many cases undissolved capsules or tablets may be discovered, which may be useful for identification. In addition, illicit drugs are frequently smuggled by ingestion of balloons or condoms filled with the contraband. If these devices burst and an acute drug death occurs, evidence of these items may be seen in the gastric contents at autopsy.50

A large quantity of the parent drug in the gastric content, relative to a prescription dose, is indicative of an oral drug overdose when supported by blood and/or tissue findings.51 However, the toxicologist is cautioned that low concentrations of drug in the stomach, especially drug metabolites and weak bases, may represent passive diffusion and/or ion trapping from the blood back into the stomach contents and may not be indicative of a recent oral ingestion of these agents. It is important to make a record of the total volume of gastric contents present in the decedent in order to calculate the total amount of the analyte present in the stomach. Since the gastric content is not a homogeneous specimen, ideally the entire specimen should be submitted to the toxicology laboratory for mixing before aliquoting.

The odor of gastric contents can potentially point to a specific agent that might otherwise elude routine detection in the toxicology laboratory. Cyanide ingestions produce stomach contents with the odor of bitter or burned almonds. Although not everyone is able to discern this odor, its presence is almost certainly indicative of a cyanide intoxication, and may be potentially hazardous in close quarters. Other characteristic odors include the "fruity-like" odor of ethanol and its congeners; the odor of airplane glue (xylene, toluene); cleaning fluid (halogenated hydrocarbons); carrots (eth-chlorvynol);32 and garlic (organophosphate insecticides).32

2.4.6 Tissues

Tissues commonly collected for postmortem study include liver, kidney, brain, lung, and spleen. Tissues provide the best and most useful context with which to interpret blood findings. They may also be the only specimens available in decomposed cases. A large amount of data for drug findings in tissue exists, primarily for liver and kidney, and, to a lesser degree, brain and lung.33 Comparison is most often between heart blood findings and those in the liver, the site where many drugs are metabolized and for which the greatest amount of reference data is available. For example, in cases where the concentration of basic drugs in blood is high and ratios of liver-to-blood drug concentration exceed 10, a drug fatality is strongly suggested if no other interceding cause of death is present. Smaller ratios, even with high heart blood concentrations, tend to suggest a greater potential for postmortem redistribution of drugs into the blood.

Analysis of tissue may be more appropriate than analysis of biological fluids for some analytes. In cases of heavy metal poisoning, kidney is a very useful specimen as heavy metals concentrate in it. In addition, structural damage to the kidney due to heavy metal or ethylene glycol exposure may be documented histologically. Spleen, an organ rich in blood, is useful for the analysis of compounds that bind to hemoglobin, such as carbon monoxide and cyanide. Frequently, in fire deaths where extensive charring is present, spleen may be the only useful specimen available to perform these assays. Lung tissue is particularly useful in cases where inhalation of volatile substances, such as solvents or Freon, is suspected. In addition, air may be collected directly from the trachea with a syringe and injected into a sealed vial to be used for headspace analysis.52 Brain, due to its high fat content, tends to accumulate lipophilic substances, such as chlorinated hydrocarbons, and other organic volatiles. Additionally, there is evidence to suggest that ratios of brain to blood cocaine are high in cocaine fatalities; thus cocaine deaths may be more readily interpreted through the analysis of both matrices.53 Finally, because the brain is in a protected environment, it also tends to be more resistant to postmortem decomposition.

The analysis of tissue is performed by weight. Usually, 1 to 5 g of tissue is shredded and homogenized with four parts of water (or saline) to generate a final dilution factor of 5. Recovery of drug from this homogenate has been found to be consistent with recovery of drug from postmortem blood.54 Drilling through the tissue with a cork-borer allows tissue to be sampled and weighed while frozen.55 This method is easier and more precise than sampling wet tissue and is less malodorous when handling decomposed specimens.

2.4.7 Hair

Hair has a long history as a useful specimen in forensic toxicology. Traditionally, hair, along with fingernails, was the specimen of choice in determining chronic heavy metal poisoning such as arsenic, mercury, and lead. Heavy metals bind to sulfhydryl groups on the cysteine molecule to form a covalent complex. Keratin, found in large amounts in hair and nails, is an excellent source of cysteine, and therefore an ideal specimen for determining chronic arsenic and mercury poisoning. Interpretation of positive findings can be augmented by segmentation of the hair strands to assist in determining the time of exposure.56-58

More recently, hair has been successfully used as a specimen from which chronic drug use may be determined.59 Numerous drugs have been identified in hair including drugs of abuse60,61 and, more recently, various therapeutic agents.62,63 The usefulness of hair analysis in determining compliance remains controversial.64 However, in postmortem toxicology, segmental analysis can offer a temporal mapping of the drug abuse pattern, and such information may prove useful to identify possible hazardous drug combinations and to detect periods of abstinence that might indicate reduced tolerance to particular groups of drugs. In extremely decomposed or skeletonized cases where no other specimens remain, positive findings for drugs in hair may at least corroborate a history of drug use. The use of hair in workplace drug testing is controversial due to issues such as environmental contamination,65 washing techniques,66 sex or ethnic bias,67,68 the difficulty in performing quantitative analysis,69 and establishing cutoff concentrations.70 In the postmortem setting these issues are not critical, and drug analysis for both pharmaceutical and illicit drugs in hair will most likely become more frequently applied following a growing appreciation of the supplemental information that such analysis may add to the interpretation of the routine toxicological results.

2.4.8 Bone and Bone Marrow

Bone marrow has not received a great deal of consideration as an alternative specimen in postmortem toxicology. Because it is protected by bone, the highly vascularized tissue may be particularly useful when contamination of blood specimens is suspected in trauma cases. Research studies have been performed, primarily on rabbits, showing that linear relationships exist between bone marrow and peri-mortem blood drug concentrations for up to 24 h for many substances including tricyclic antidepressants, barbiturates, benzodiazepines, and ethyl alcohol.71-74 However, these studies were performed when the bone marrow was still fresh and moist. Although putrefaction is delayed in bone marrow, usually bone marrow is not considered as an alternative specimen in postmortem toxicology unless other specimens are unavailable. Typically, at this stage of decomposition the bone marrow has transformed from spongy red marrow to a brown viscous liquid or paste-like substance, and it is unknown if any interpretation can be made from quantitative data.75 However, drugs have even been identified in the bone marrow of skeletonized remains,7677 and heavy metals have been identified in the bone itself.78

2.4.9 Skeletal Muscle

Skeletal muscle is an often-overlooked specimen with many potential applications in postmortem forensic toxicology. It meets many of the criteria of an ideal forensic specimen: it is relatively homogeneous, almost always available, and not easily contaminated. Studies have shown that drug concentrations in thigh muscle reflect drug concentrations in blood for many common basic drugs and ethyl alcohol, except in cases of an acute drug death where muscle drug concentrations may be lower than blood due to inadequate time for tissue equilibration.79 The analysis of thigh muscle may be especially useful in cases where drugs suspected of undergoing postmortem release are detected in the heart blood.79 It is important that extremity muscle be collected, where possible, as drug concentrations in other muscles, such as abdominal muscle, increase with time while remaining constant in thigh muscle.80,81

Because skeletal muscle is often well preserved despite advanced decomposition of other tissue, it may be useful as an indicator of postmortem blood concentrations even in decomposed cases, although more studies need to be performed.81,82 Surprisingly, even parent cocaine, which is known to be unstable in blood, has been identified in numerous cases of decomposed, dried skeletal muscle.83

The potentially useful data that may be obtained from the analysis of skeletal muscle have prompted some toxicologists to recommend that skeletal muscle be collected in all cases where drugs may be implicated in the cause of death.81 One disadvantage to skeletal muscle is the need to homogenize the sample prior to analysis. However, this is true of many traditional specimens as well, such as liver and kidney. As more laboratories analyze skeletal muscle leading to the availability of additional data to aid in the interpretation of results, its potential advantages will outweigh any disadvantages.

2.4.10 Larvae

In cases of suspected poisoning where decomposition prevents traditional specimens from being obtained, homogenized fly larvae, usually of Calliphorid genus (blowfly), have proved to be useful alternative specimens in which drugs may be identified. Depending on temperature, larvae may be present as soon as 1 to 2 days after death. The first reported use of fly larvae in drug analysis occurred as recently as 1980 and involved a phenobarbital case.84 Since then numerous drugs have been identified in fly larvae including barbiturates, benzodiazepines, and tricyclic antidepressants,85 opiates,86 cocaine,87 and the organophosphate, malathion.88

The choice of where larvae are best collected from the body needs further study. Interpretation of positive findings seem to be most useful if the larvae are collected at the site of their food source, such as any remaining muscle or liver, under the premise that drugs detected in fly larvae feeding on a body can only have originated from the tissues of that body.89 This assumption seems to be supported by one study where a quantitative relationship was suggested between the morphine concentrations in the larvae and the livers on which they fed.90 By contrast, other studies suggest that the analysis of fly larvae provides only qualitative data.85,86,91 However, in these studies larvae were collected from multiple sites and pooled before analysis. If larval drug concentrations are based on the tissue on which they fed,89 these results are not surprising.

Studies using Calliphorid larvae have shown that the age of the larvae may also play a major role in determining whether drugs may be identified in them.91,92 By collecting larvae over a period of up to 11 days in cases of known suicidal drug overdoses, it was demonstrated that drugs were readily detectable in larvae through the third instar stage, but a precipitous fall in drug concentration was associated with pupariation after their food ingestion ceases. Similarly, larvae that had been feeding on drug-laden muscle for 5 days demonstrated a significant loss in drug concentration within 1 day of being transferred to drug-free tissue. This suggests that Calliphorid larvae readily eliminate drugs when removed from a food source. Thus it appears to be critical that larvae collected for drug analysis from a decomposed body be frozen and analyzed as soon as possible after collection. Even under refrigerated conditions, when larvae are in a state of diapause, slow bioelimination of drugs still occurs over the course of several weeks.93 In addition, to eliminate surface contamination as a possible source of interpretive error, larvae should be washed with deionized water prior to analysis.

2.4.11 Meconium

Meconium, the first fecal matter passed by a neonate, has recently been given much attention because it is a useful specimen in which to determine fetal drug exposure. Issues relating to the screening and confirmation of most drugs of abuse in meconium have been reviewed.94 While meconium analysis has principally been performed to assess in utero drug exposure in newborns so that treatment may begin as early after birth as possible, it may also be useful in determining drug exposure in stillborn infants. One study demonstrated the presence of cocaine in the meconium of a 17-week-old fetus, suggesting that fetal drug exposure can be determined early in gestation.95

Unlike urine, which allows the detection of fetal drug exposure for only 2 to 3 days before birth, meconium extends this window to about 20 weeks. Most postmortem toxicology laboratories are not currently performing meconium analysis. While potentially useful, there are several issues that must be considered. Because meconium forms layers in the intestine as it is being deposited, it is not a homogeneous specimen. As with other nonhomogeneous specimens, such as gastric contents, it is important that all available specimens be collected and thoroughly mixed before sampling. Consideration should also be given to the fact that infants do not metabolize some drugs the way adults do. If commercial immunoassay screening kits are used that target metabolites found in adult urine, the ability to detect some drugs in meconium may be compromised.96

2.4.12 Other Specimens

Many other specimens have been used in toxicological investigations. Any item with which a body or bodily fluid has been in contact is a potential candidate for the identification of drugs or poisons. Examples include tracheal air,52 blood stains on clothing,97 soil samples collected at the site of a skeleton or decomposed body,98 and even cremation ash.99 Even though positive findings in these specimens are qualitative, there is at least the potential that this information can be useful in determining the circumstances of a death.

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