Toxicology Testing: Interpreting What That Number Means

Introduction

When someone dies, toxicology testing may be obtained to determine the cause of death. Toxicologic testing is an important part of both clinical care and forensics. Unlike what may be portrayed on television, this process is a lot more complex. Using reliable and specific analytical methods is imperative. Inappropriate selection of testing modality or misinterpretation of laboratory findings may result in faulty conclusions, which depending on the context (e.g., legal), could have devastating consequences. Information obtained from testing must be interpreted in the context of the circumstances of death; for example, a person who died while sleeping is unlikely to have died from stimulant toxicity, even if amphetamines are identified in their blood. Additionally, it is important to understand how the testing was conducted and what biologic matrix (e.g., blood, serum, urine) was used to properly interpret the results. Testing modalities and biologic matrices each have their benefits and limitations. Collection, preparation, and storage of samples, in addition to the choice of biologic matrix, also plays an important role in correctly interpreting the results. Finally, postmortem redistribution, the idea that toxicology concentrations (“levels”) may change after death, is important to consider when interpreting toxicology testing.

Types of Toxicology Testing

Immunoassays are often used as an initial screening test, with positive results generally followed by a more specific form of confirmatory testing, especially when used for forensics. There are situations, particularly in clinical medicine, where confirmation is not necessary (e.g., patient confirms that the immunoassay is accurate). Immunoassays use an antibody directed at a specific part of the compound to identify it. The antibodies can cross-react with similar structures allowing them to identify multiple members of a drug class or mistakenly interact with compounds of a different drug class resulting in a false positive test.¹ They provide rapid and cost-effective results for common substances but have significant limitations.^1,2

In hospitals, the urine drug screens that are ordered and result quickly are almost always immunoassays. Reference and send-out laboratories, which are used for forensic testing, often utilize immunoassays as their screening test. However, the immunoassays used by the hospital and reference laboratory are basically the same technology used in direct-to-consumer drug tests that are available for purchase in drug stores or pharmacies. The result of an immunoassay is typically reported as positive or negative. Reporting a “positive” or “negative” result for a family of substances (e.g., opioids) can be misleading, however. Immunoassays generally only identify a subset of substances in a drug class, yielding false negative results (e.g., only identifying a few amphetamines but not all amphetamines), as well as false positives due to cross-reactivity with similar substances.^1,2 Immunoassays for opiates generally test for morphine, which is a metabolite of heroin and codeine. Therefore, opiate testing by immunoassay will only identify substances with very similar chemical structures to or that are metabolized to morphine. This is why separate immunoassays are required to identify other opioids such as oxycodone, methadone, fentanyl, and buprenorphine.^2,3

Gas chromatography-mass spectrometry (GC/MS) and liquid chromatography mass-spectrometry (LC/MS) combine the separation power of chromatography with the high specificity and definitive identification power of mass spectrometry and are generally used to confirm positive findings from the immunoassay.^1,2 Chromatography uses properties of the substance (e.g., how lipophilic it is) to separate out the various compounds that require identification. There are multiple forms of chromatography, including liquid or gas, which indicates the phase of the substance or compound when it is being separated. In LC, unknown compounds move up a column which causes them to be separated before being placed in a spectrometer. Following the separation, spectrometry is a method that uses an ionization source to generate molecular fragments that create a characteristic molecular signature that can be used to analyze and identify substances.³ The most common forms of spectrometry includes mass (for molecular weight), nuclear magnetic resonance (for identifying structure), optical (for determining chemical bonds and electronic states), in addition to emission and absorption-based methods. In some situations, chromatography is used without spectrometry to identify the substance of concern based on how far up the column the compound traveled, referred to as the retention time. The correct choice of biologic sample, in addition to preparation of the sample, is imperative when using MS due to its limitations with analyzing drugs or compounds that are highly polar, large, or heat-sensitive/thermally unstable.^1–3 Some advantages of GC/MS or LC/MS modalities include their multiplexing capabilities: analyzing hundreds of substances simultaneously and providing structural information i.e., molecular weight and structural confirmation of unknown compounds.¹

With the increasing frequency of novel synthetic substances in the illicit drug supply worldwide, there is a demand for unknown substance screening and identification. This is particularly true in forensics when attempting to identify substances that contributed to the cause of death.

Identifying substances on GC/MS or LC/MS can involve either directed (targeted) or undirected testing. Directed screening is obtained by matching the resulting spectra (the compound of interest) to a spectrum library, which corresponds to compounds representing various pharmaceuticals, illicit drugs, and their metabolites.^1–3 Drugs such as amphetamines, cocaine, opioids, benzodiazepines, neuroleptics, and their respective metabolites are quantified using LC-MS in what is called multiple-reaction-monitoring (MRM) mode. This is essentially a highly sensitive ‘fingerprint’ to target and quantify specific molecules of the desired drug. Some substances like ethanol and various toxic alcohols (e.g., methanol) are volatile, requiring heating and subsequent vaporization. The vapor sample is then injected into a GC which separates the ethanol from other volatile compounds. All results are compared to the internal standard to determine the exact concentration of the sample. In undirected testing, the laboratory uses what is plotted by the spectrometer to identify an unknown substance.

Given the frequency of novel psychoactive substances adulterating the drug supply, it is important to know if the laboratory is using directed or undirected testing. For instance, in the case of death from a suspected opioid overdose, if the opioid is one that has not previously been detected in the drug supply, the MS may not identify it if it is only matching the substance to what is in the laboratory’s known library (directed testing).

While chromatography and spectrometry are considered the gold standard in identifying most substances, these modalities still have limitations. The testing requires greater technical expertise and has longer turnaround times compared to immunoassays.³ Many hospitals and even state forensic laboratories send specimens to reference labs to obtain GC/MS or LC/MS testing. More importantly as will be discussed, a positive postmortem screen does not indicate cause of death, and therefore, it is imperative that the clinician evaluating these results incorporates medical history, previous exposures and tolerance, and circumstantial information before making conclusions.^4–6

When “Positive” and “Negative” Aren’t Absolute

While a test may be reported as positive or negative, it actually depends on exceeding a threshold to be considered “positive.” Immunoassays and chromatography/spectrometry are all generally based on a cutoff when resulting as either positive or negative. The established cutoff must be high enough to minimize false positives but also low enough to prevent false negatives, particularly in patients with a history of chronic use of the xenobiotic in question.⁷ Essentially, the cutoff that is used is a tradeoff balancing sensitivity and over-detection. Higher thresholds can result in false negative testing; there is not enough of the substance in the sample to turn the test positive, although the substance is technically in the sample. This can be problematic in forensic testing as someone may have substances in their blood or urine, but the test is reported as negative. Cutoffs at lower concentrations can lead to a more sensitive immunoassay or LC-MS/MS confirmatory analysis but may also complicate interpretation. The test will be positive at lower concentrations of the drug but may cause a positive test for a concentration that is clinically insignificant.⁸ While manipulating the threshold may seem problematic, it may also be done in a way that is beneficial.^7,8 For instance, by raising the cutoff threshold, immunoassays are incredibly unlikely to be positive for opioids or cannabinoids from eating a poppyseed bagel or being exposed to second-hand cannabis smoke, respectively. Again, the laboratory director or test manufacturer can manipulate the threshold resulting in a positive test depending on their goals of detection. While there are predetermined concentration cutoffs, the presence of a detectable amount of the xenobiotic does not necessarily imply functional impairment—particularly in patients with tolerance.

Additionally, interpretation of drug concentrations must also be done in the context of the pharmacodynamics and kinetics (e.g., mechanism of action, metabolism, potency, efficacy or therapeutic index) of the substance. Also, sampling may not be witnessed and is easily susceptible to tampering, although this is less likely an issue in forensic testing which entails specific chain-of-custody procedures. Finally, the matrix tested must also be considered as blood or serum sampling has a narrower detection window compared to urine.

What is Postmortem Redistribution?

In forensic toxicology when someone dies under suspicion of a drug or medication overdose, it is not appropriate to draw a concentration (a “level”) and compare it to an antemortem sample. Immediate postmortem or perimortem samples may be interpreted similarly to antemortem samples (i.e., the sample is drawn very close following death). However, when there is a delay in obtaining the samples, interpretation of the concentration must take into consideration the concept of postmortem redistribution. Since drug concentrations and distribution continue to change after circulation stops (i.e., death), the interpretation of postmortem toxicology findings is heavily affected by the time elapsed since death.⁹ Sometimes, the exact time of death is uncertain and “last known alive” estimates may be imprecise. Bodies are sometimes discovered hours or days later, leading to marked variability in the extent of decompensation and postmortem changes at the time samples are collected.^10,11 In forensic toxicology, postmortem redistribution describes the measurement of drug and toxin concentrations after death, which may be different from the antemortem value.

Postmortem redistribution makes interpretation of drug concentrations challenging because substances diffuse from organs with high tissue concentrations (e.g. liver, lungs, myocardium) into the bloodstream once circulation ceases.^12,13 This can lead to falsely elevated concentrations in central blood samples (e.g., from the heart or major thoracic vessels) that do not reflect antemortem (prior-to-death) concentrations.^12,14 Peripheral blood, especially femoral vein samples, is therefore preferred, as it is less affected by redistribution, and therefore, more closely reflects antemortem circulating concentrations.^13,14 Results are best interpreted by comparing concentrations across multiple specimens, including peripheral and central blood, solid tissues, urine, and vitreous humor, to assess consistency and identify redistribution patterns (i.e., how likely it is that a particular substance undergoes postmortem redistribution).^14,15 While hair and nail testing may help document long-term or prior exposure, they are not reliable indicators of acute intoxication at the time of death.¹⁶

Gastric postmortem redistribution occurs when drugs that remain in the gastrointestinal tract at the time of death diffuse across the stomach or intestinal wall into nearby tissues and blood. This can produce artificially elevated drug concentrations in central blood samples after death.^9,12,14 Unabsorbed drug in the stomach or intestines effectively acts as a postmortem reservoir, contributing to delayed redistribution and further complicating the interpretation of postmortem drug concentrations.⁹ In addition, gut bacteria may continue to metabolize or degrade certain drugs after death, leading to differences between antemortem and postmortem concentrations and altering parent drug–metabolite ratios.^9,11,17 For example, ethanol concentrations may change after death as a result of microbial fermentation, meaning measured concentrations do not always reflect antemortem exposure.¹⁸ Cocaine and heroin are also vulnerable to postmortem chemical breakdown; cocaine can hydrolyze to benzoylecgonine, while heroin rapidly degrades to 6-monoacetylmorphine and morphine, which can significantly alter parent drug–metabolite ratios.^13,19 In addition, some benzodiazepines and opioids may show postmortem degradation or redistribution, particularly when there is prolonged gastrointestinal stasis or bacterial overgrowth.⁹

Other factors can both increase and decrease postmortem concentrations. Ongoing diffusion of drugs between tissues and blood, along with postmortem metabolism of certain drugs, may also result in lower parent drug concentrations and different metabolite patterns than would have been present antemortem.^9,11 Additionally, environmental factors, particularly temperature and humidity, further affect the rate of decomposition and can accelerate redistribution and drug breakdown.^9,17 As a result, certain drug concentrations measured at the time of discovery of the body may differ substantially from drug concentrations at the actual time of death. Therefore, since both time and environmental conditions influence postmortem changes, they should be considered when interpreting the role of drugs in death.^9,11

Interpreting postmortem toxicology results also requires an understanding of drug-specific pharmacokinetics, since properties such as lipophilicity, protein binding, and volume of distribution influence the degree and pattern of postmortem redistribution.^9,13 Highly lipophilic drugs with large volumes of distribution tend to accumulate in adipose tissue and well-perfused organs during life. After death, these tissue stores can release drugs back into the bloodstream, often resulting in elevated concentrations in central blood samples.^9,12,14 In contrast, hydrophilic drugs with low volumes of distribution and minimal tissue binding undergo less redistribution, so measured blood concentrations more closely reflect antemortem concentrations.^11,13 Protein binding also plays a role, as only unbound drug is able to diffuse across tissue and vascular compartments after death.⁹ Body habitus further modifies these effects; for instance, in individuals with significant adipose tissue, fat can act as a larger drug reservoir, prolonging postmortem release and altering concentration gradients.^9,12,14 These pharmacokinetic factors should therefore be considered when evaluating postmortem drug concentrations and determining toxicologic contribution to death.

When Chemistry Misleads: Analytical Artifacts After Death

Analytical pitfalls can further complicate interpretation in postmortem toxicology, as certain compounds may be produced or altered after death, creating results that do not reflect true antemortem conditions.^10,11 Microbial activity during decomposition, for example, can generate ethanol through fermentation of glucose in blood or tissues, potentially leading to a false impression of alcohol ingestion.^18,20 This is why vitreal sampling for alcohol is preferred.²⁰ Ongoing cellular breakdown and bacterial metabolism may also shift concentrations of organic acids, ketones, and other endogenous substances, sometimes mimicking metabolic disturbances or toxic exposures.¹⁷ Additionally, hemolysis, tissue autolysis, and extended postmortem intervals may interfere with assay performance or produce cross-reactivity in analytical methods.^10,11 Hence, careful specimen handling, use of preservatives, selection of appropriate matrices (such as vitreous humor for ethanol confirmation), and correlation with scene and clinical history are essential to distinguish true toxicologic findings from postmortem artifacts.^15,20

It’s More Than Just a Number

Estimating antemortem drug concentrations from postmortem specimens is challenging and should be done with caution, if at all. Postmortem measurements cannot be reliably back-calculated to precise antemortem concentrations; however, they may be used to assess whether antemortem concentrations were likely low, within a therapeutic range, or potentially toxic.^9–11 It is important to remember that even this simplification is nuanced. Once a tested sample yields a positive result or concentration (“level”), the interpreter must consider the dose-response relationship, which refers to the quantitative association between the amount of drug consumed and the intensity of its effect. Tolerance of the user and the time between exposure and collection can affect quantitative results and interpretation.

For instance, when interpreting postmortem opioid concentrations, tolerance must be considered. In someone without tolerance (no chronic use), opioid concentrations may be low or in a therapeutic range but still be toxic, while for someone with an opioid use disorder, a moderately high concentration might not be expected to be harmful. Several pharmacokinetic variables are usually unknown, including the timing of ingestion, the exact dose taken, and whether the formulation was immediate or extended-release. If known, these are all important considerations when interpreting the concentration.¹³

Medical interventions prior to death may introduce confounders which may add an additional layer of uncertainty when interpreting the test.²¹ For example, intravenous fluids may dilute blood drug concentrations, vasopressors can change tissue perfusion and drug distribution, and cardiopulmonary resuscitation can cause unpredictable redistribution of drugs between compartments. Emergency treatments such as activated charcoal or antidotes may also alter absorption, metabolism, or elimination. This indicates that postmortem concentrations may reflect post-intervention rather than true pre-collapse concentrations.^11,21 Therefore, these factors make precise calculation of antemortem drug concentrations from postmortem samples unreliable.

Conclusion

Toxicology testing is a common component of determining if drugs contributed to a fatality. Knowing the biologic matrix and understanding the form of testing, and its limitations, are imperative to properly contextualize the laboratory value. Immunoassays and chromatography have their benefits as well as limitations. Reports may be issued with both qualitative and quantitative values. It is particularly important to know the cutoff for qualitative testing when interpreting the result as either positive or negative. Substances may also undergo postmortem redistribution which can cause them to elevate after death. Additionally, some substances are metabolized following death resulting in lower concentrations compared to antemortem values. All these factors must be considered when interpreting biologic testing to properly determine if the measured substance contributed to the cause of death.