After fluid and tissue samples have been collected and analyzed for the presence of xenobiotics, the process of interpreting these results begins. This complex task attempts to account for the clinical effects of a xenobiotic at the time of death by integrating medical history, autopsy, death-scene findings, and toxicologic reports. Multiple confounding variables may affect the sample concentrations of xenobiotics from the time of death to the time of testing after the autopsy. Variables include the nature, metabolism, and distribution of the xenobiotic; the state of health of the decedent prior to death; physical and environmental variables during the postmortem interval; the techniques of analysis; and other findings of the autopsy (Tables 34–6 and 34–7).
TABLE 34–6.Considerations in Interpreting Postmortem Xenobiotic Concentrations ||Download (.pdf) TABLE 34–6. Considerations in Interpreting Postmortem Xenobiotic Concentrations
|Xenobiotic Dependent ||Decedent Dependent ||Autopsy Dependent ||Other |
State of absorption or distribution at time of death
Expected clinical effects
Postmortem xenobiotic stability during
Handling and preservation
Postmortem interval: State of preservation or decomposition
Evidence at scene
Previously published tissue concentrations
Variables Relating to the Xenobiotic
The xenobiotic concentration in blood may be higher during the time of sampling at autopsy than at the actual time death occurred if significant postmortem redistribution occurs.54,81,104, 105, 106, 107, 108, 109, 110, and 111 Redistribution typically occurs with xenobiotics that have large volumes of distribution and when decomposition results in release of intracellular xenobiotic into the extracellular compartment.86 For example, amitriptyline may be released from tissue stores into the blood as autolysis progresses, resulting in a significantly higher blood concentration during the autopsy than at the time of death. If postmortem redistribution is not considered, xenobiotic concentrations obtained during the autopsy may be misinterpreted as being supratherapeutic or even toxic, and the cause of death may be inappropriately attributed to this xenobiotic.
Less commonly, xenobiotic concentration may decrease secondary to postmortem metabolism. For example, cocaine continues to be degraded after death by endogenous enzymes such as cholinesterases in the blood, which continue to function in postmortem tissue and in vitro. Unless blood is collected immediately after death and placed in tubes containing enzyme inhibitors such as sodium fluoride, the concentration of cocaine will continue to decrease, and the analysis will not accurately reflect the concentration of the drug at the time of death.59,71,98,102 All available information regarding postmortem redistribution or metabolism of a specific xenobiotic must be considered for the proper interpretation of the toxicologic results.
State of Absorption and Distribution.
Both in the living and deceased, the state of absorption, distribution, and other toxicokinetic principles affect the apparent concentration of a sampled xenobiotic. For a xenobiotic with minimal postmortem metabolism or redistribution, the phase of absorption is suggested by the relative quantity of the xenobiotic in different fluids and solid organs. For example, a high concentration of xenobiotic in the gastric contents, with progressively lower concentrations in the liver, blood, vitreous, and brain, suggests an early phase of absorption at the time of death. When a xenobiotic is orally administered and the tissue concentration is highest in the liver, the relationship suggests a postabsorption phase but a predistribution concentration. A concentration found to be highest in the urine suggests that the xenobiotic was in an elimination phase at the time of death. Although this approach has limitations, it may be important for correlating the state of absorption and the expected clinical course of the xenobiotic. Unfortunately, multiple samples may not always be available at the time of autopsy or the interpretation of reports, and opportunities for subsequent sampling are often limited.
Xenobiotic stability refers to the ability of a xenobiotic to maintain its molecular integrity despite postmortem changes such as decomposition of the body, adverse storage conditions, or the lack of preservatives.5,13,15,56,57,62,92,100,107,109,110 Postmortem xenobiotic stability was assessed in homogenized liver tissue infused with various concentrations of xenobiotics.100 The samples were allowed to putrefy outdoors, and sequential sampling of xenobiotic concentrations was performed. The xenobiotics that decreased in concentration as putrefaction progressed were considered labile, and samples with a constant concentration were considered stable. The authors proposed that the chemical characteristics of a xenobiotic determine its stability. For example, labile xenobiotics share the molecular configuration of an oxygen–nitrogen bond, thiono groups, or aminophenols. Conversely, chemical structures that enhance stability include single-bonded sulfur groups, carbon–oxygen and carbon–nitrogen bonds, and sulfur–oxygen and hydrogen–nitrogen bonds. Although not explicitly studied in otherwise intact but putrefying bodies, logically, a less stable xenobiotic may be recovered in a lower concentration than the actual concentration at the time of death. This must be considered when information regarding stability is available and the body of a decedent is in an advanced stage of decomposition.
Xenobiotic Chemical Interactions.
An artifact may result from a chemical interaction with a xenobiotic added during the postmortem interval, such as embalming fluid.39 In a study of xenobiotic-spiked blood and formalin in test tubes, amitriptyline was formed by the methylation of nortriptyline.27,110 Identification of amitriptyline, which was not present at the time of death, could confuse the interpretation of toxicologic analyses.
Expected Clinical Effects of the Xenobiotic.
For a fatality to be attributed to a xenobiotic, the expected clinical course from the exposure should be consistent with the autopsy findings. For example, what are the implications of a person found dead minutes after having been seen ingesting pills, if a large concentration of acetaminophen (APAP) is identified in both the gastric contents and blood but not in other tissues at autopsy?91 Although suicidal intent may be supported by this finding, the onset of death within minutes is inconsistent with a fatality due to an APAP overdose. Thus, another cause of death must be sought. Interpretation of postmortem toxicology must also incorporate clinically relevant consequences of xenobiotic interactions. For example, the combined ingestion of phenobarbital and ethanol can cause fatal respiratory depression. Although neither may be fatal alone, their additive effects must be acknowledged during toxicological interpretation.
Variables Related to the Decedent
The clinical response to a xenobiotic may be affected by acquired and inherited physiologic conditions that are not always identified or identifiable on autopsy. A thorough medical history is important and may assist in interpreting the clinical effects of a xenobiotic exposure. Similarly, certain clinical conditions may produce substances that interfere with postmortem laboratory assays. For example, an individual with a critical illness may produce digoxinlike immunoreactive substances (DLIS), which may cross-react with the postmortem digoxin assay.6 Without knowledge of DLIS production, the results may confound toxicologic analysis (Chap. 65).
Tolerance is an acquired condition in which increasingly higher xenobiotic concentrations are required to produce a given clinical effect. It is an important consideration for deaths in the presence of opioids, ethanol, and sedative–hypnotics. For example, respiratory depression and death from methadone may be easily diagnosed in an opioid-naive individual with a history of methadone exposure and methadone-positive postmortem samples. However, the same methadone concentrations in a patient on chronic methadone maintenance therapy will not produce the same outcome. Unfortunately, no autopsy markers are available to indicate tolerance, and no biochemical or histologic markers are available during autopsy that may be used to predict clinically dangerous xenobiotic concentrations in a tolerant individual.28 Complex postmortem assays analyzing opioid receptors are not routinely used.36 Postmortem assessment of tolerance ultimately depends on knowledge of the patient, pharmacokinetics of the xenobiotic, and the best judgment of the investigator.
There is genetic variability in the expression of certain metabolic enzymes. For example, pharmacogenetic differences in metabolic enzymes, such as CYP2D6, predispose some individuals to fatal hypotension from an inability to metabolize debrisoquine.
Similarly, deaths in young children have been reported from the use of therapeutic codeine. Postmortem blood analysis may reveal an elevated concentration of morphine, the codeine metabolite, and raise suspicion for a malicious overdose. Postmortem genotyping may provide an alternate explanation. Individuals with duplicate alleles for CYP2D6 may be ultrarapid metabolizers of codeine, rendering them susceptible to morphine toxicity despite generally acceptable dosing of codeine. Such distinctions are not routinely identifiable on autopsy.17,29,30
Variables Relating to the Autopsy
In decedents in advanced stages of decomposition, xenobiotics may diffuse from depot compartments such as the stomach or bladder into adjacent tissues and blood vessels and secondarily affect their sample concentrations.23,38,66,76, 77, and 78,85, 86, and 87
During putrefaction, bacteria cause fermentation of endogenous carbohydrates, resulting in ethanol formation. In decedents without gross evidence of putrefaction, especially those in cool, dry environments, endogenous ethanol production is minimal.18,22 With a longer postmortem interval or in an environment that is more conducive to ethanol production, the distinction between endogenous and exogenous sources of ethanol becomes more difficult. Sampling from multiple sites is often useful in making the distinction.101 A comprehensive review of interpreting postmortem ethanol concentrations is available elsewhere.66
Handling of the Body or Samples.
Inappropriate handling of the body may result in artifacts.91,93 In one reported case, methanol was detected in the vitreous humor of a decedent after embalming.12 The methanol was subsequently traced to a spray cleanser that likely settled on the surface of open eyes during washing of the body.
In addition, inappropriate handling of samples may affect xenobiotic concentrations. In one study, autopsy blood was obtained from a diabetic man who died of bronchopneumonia. The samples were stored at 40°F (4°C) and tested at 2 and 5 days postmortem. The blood ethanol increased from 0.4 to 3.5 g/L because of an inadequate addition of fluoride preservative to the samples. The combination of inadequate preservation, hyperglycemia (vitreous glucose of 996 mg/dL), and bacterial sepsis created an ideal environment for ethanol production via fermentation.66
In the United States, preservatives containing metals are currently banned for use in embalming because they may contaminate subsequent evaluation for metal poisoning. Formalin may also affect stability or quantitative identification of some xenobiotics. When necessary, an analysis of embalming fluid used by the mortician or soil sampling around disinterred bodies may facilitate the toxicologic investigation.19,34,107,109,110
In many xenobiotic-related deaths, the anatomic findings are nonspecific.108 In some cases, the autopsy reveals confirmatory or supportive findings. A large quantity of undigested pills in the stomach is consistent with an intentional overdose, and suicide should be considered. Centrilobular hepatic necrosis may be found in decedents with a history of APAP overdose. The autopsy may also reveal other findings such as coronary artery narrowing, chronic hypertension, renal abnormalities, or a clinically silent myocardial injury. Such information may be useful to assess the potential effects of a xenobiotic in a patient with previously undiagnosed conditions. In other cases, the absence of a chronic condition may be strongly suggestive of a xenobiotic-related death. For example, a decedent with an autopsy finding of aortic dissection in the absence of chronic hypertensive findings or other predisposing conditions may suggest a xenobiotic-induced hypertensive crisis as may occur from use of cocaine or other sympathomimetics.
Artifacts Related to Sampling Sites.
Site-specific differences in postmortem xenobiotic blood concentrations are common.35 For example, blood obtained from femoral vessels may have low glucose concentrations because of postmortem glycolysis, but the blood glucose concentration removed from the right heart chambers may be high as a result of perimortem release of liver glycogen stores. As noted above, hyperglycemic conditions are more reliably assessed from sampling the vitreous humor as vitreous is a relatively protected environment in the early postmortem interval. An elevated vitreous glucose concentration suggests antemortem hyperglycemia. The individual interpreting the toxicologic report must know the exact site from which the sample was taken.20,60
Ideally, samples from more than one site would be available for comparison; unfortunately, multiple blood samples are not often routinely obtained. Comparison of concentrations from different sites may reveal important information regarding the extent of xenobiotic absorption at the time of death and acute versus chronic exposure.9, 10, and 11,20,26,30,52,67,72,80,82,85,88, 89, and 90,95,96,101,103
Published therapeutic, toxic, and fatal postmortem xenobiotic concentrations are available to aid in the interpretation of postmortem specimens.4,70 However, the conditions associated with reported concentrations do not necessarily permit comparisons with the concentrations of the particular case under investigation. Thus, these resources are valuable but should be used mainly as representative of prior attempts at rigorous analysis and not accepted as absolute values that define either toxic or therapeutic concentrations. Similarly, formulas available for assessing xenobiotic doses or concentrations in the living are not usually applicable when analyzing postmortem samples.
Although there are generalized standards of practice in forensic investigations, specimen collection and laboratory methodologies may vary.2,4 Some xenobiotic concentrations may be falsely elevated or depressed depending on the chosen methodology.32,72 Descriptions of specific toxicology laboratory techniques are beyond the scope of this chapter, but these variables must also be given consideration in the interpretations of results. Other limitations may include a lack of information relating to the circumstances of death and possible compromises in specimen handling because of the required protocols for the maintenance of proper chain of custody often of paramount importance in forensic autopsies.37,38