Postmortem Toxicology: Challenges and Interpretive Considerations

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1

Introduction

Abstract

Toxicology is one of the oldest scientific disciples. History is replete with the evolution of toxicology, early challenges being the identification of poisons or foods that had been adulterated with or naturally contain a poison. The development of various analytical techniques over time improved one’s ability to detect drugs and poisons. In fact, the development of various medical examiner systems evolved out of the eventual detection of poisoners.

There are no absolute rules for the interpretation of postmortem toxicological results. The proper interpretation of toxicological results requires a global awareness of the challenges and pitfalls associated with the test results and the inclusion of the totality of circumstances surrounding the death.

Death investigations can be affected by a variety of unusual complexities. Confounding factors or artifacts impacting the interpretation of results may arise from conclusions drawn from or due to preanalytical factors, the specimen container and/or storage conditions, prior medical therapy or medical intervention. Postmortem changes or artifacts introduced due to organ or tissue procurement, specimens collected from an embalmed body prior to burial, or after exhumation may also lead to misinterpretation of the toxicological results.

The focus of this treatise is a discussion of the challenges and pitfalls that may impact the interpretation of postmortem toxicological findings.

Keywords

Analytical detection; arsenic; history of poisoning; King of Poisons; LaFarge; medical examiner; postmortem interpretation; postmortem artifacts; Traite des Poisons

Toxicology is one of the oldest scientific disciples. History is replete with the evolution of toxicology, early challenges being the identification of poisons or foods that had been adulterated with or naturally contain a poison.

Toxicology is derived from the ancient Greek word toxicon ν] [1]. The term relates to bow poison. Poisons were occasionally placed on the tips of arrows to make them more lethal. The word toxicos is derived from this meaning, leading to the word intoxicated which meant making one sick by poison arrows.

Toxicology encompasses multiple scientific areas of study; analytical chemistry, biochemistry, clinical chemistry, medicinal chemistry, anatomy, biology, physiology, pharmacology, and pharmacy. The role of the toxicologist is twofold: characterize and identify substances in the body and provide an interpretation of what their presence or in some cases absence means; relating to the behavior of an individual and/or was the detected drug or poison a causative factor in the individual’s death.

Merriam-Webster dictionary defines the noun forensic, meaning an argumentative exercise. The English word was derived from a Latin word forensic meaning of the market place or form, public, which in turn comes from the Latin word forum, meaning market place, forum [2]. Simply put, the forensic aspect of forensic toxicology is bringing toxicological data and interpretations into a legal setting.

The knowledge of poisons predates recorded history. The written history of toxicology may be traced back as far as 2700 BC with information in the Chinese journals discussing plant and fish poisons [7]. The Ebers papyrus is an ancient papyrus scroll dating back to c.1500 BC, which contained descriptions of various poisons and proposed antidotes. Between 50 and 70 AD Pedanius Dioscorides, a Greek physician wrote De Materia Medica (Latin translation: On Medical Material), which classified over 600 plant, animal, and mineral poisons; from which 1000 medicines had been made from them [3]. From 250 to 400 AD, the Romans had used poisons for executions and assassinations [4]. One of the most notable assassins was Locusta, employed by Nero as his personal poisoner, dispatching several of his wives and a brother [5]. Anyone, who has taken a course in philosophy, recognizes another victim of note during this time was the philosopher Socrates who was executed with hemlock for teaching radical ideas to the youth of the time [6].

The Roman Empire passed the first law against poisoning in c.82 AD called lex Cornelia de sicariis et veneficis or the Cornelian Law (against) of Assassins and Poisoners [7]. Arsenic was the poison of choice by Roman assassins; it became known as the King of Poisons. Due to the frequent use of arsenic as a poisoning agent, Roman Emperors and families of nobility hired food testers to protect themselves from surreptitious poisoning. These food testers could have unwillingly been the first toxicologists used to identify poisons.

Swiss physician Paracelsus (1493–1541), born as Philippus Theophrastus Aureolus Bombastus von Hohenheim, is credited with being the father of modern toxicology [8]. He received his baccalaureate in medicine in 1510 and was awarded his doctorate in 1516 from the University of Ferrara (main university in the city of Ferrara, Northern Italy). It was around this time he took the name Paracelus; being derived from para: beside, beyond and Celsus: a noted Roman physician. He wrote (loosely translated from German): All things are poison and nothing is without poison, only the dose permits something not to be poisonous. Thus, a basic tenant in toxicology is "Dose makes the Poison"; substances considered toxic may be harmless in small doses and conversely an ordinarily harmless substance can be deadly if overconsumed, for example, water or salt (sodium chloride).

Spanish Physician Mathieu J.B. Orfila (1787–1853) established toxicology as a distinct scientific discipline. He wrote, in 1813, a treatise entitled Traite des Poisons, which in part described techniques for the identification of arsenic in biological sample. Orfila is best known for his expert witness role in the LaFarge arsenic poisoning case in France; where in 1840 Marie LaFarge was tried for murder of her husband using arsenic [9], thus bringing this scientific discipline into a court of law and establishing him as the father of forensic toxicology.

In addition to the Romans recognizing poisons being used as agents for murder, England [10] and other European countries had an awareness as well. In 1851, the United Kingdom passed legislation entitled An Act to Regulate the Sale of Arsenic; also referred to by its short title Arsenic Act of 1851 [11]. The Act defined arsenic as Arenious acid and the Arsenites, arsenic acid and the arsenates, and all other colourless poisonous preparations of arsenic. This Act required documentation which identified who the purchaser was, the amount sold and the purpose of use of the arsenic; similar to various pseudoephedrine statutes currently in the United States to address the misuse of the drug as a precursor for methamphetamine production. The Act further required that the arsenic have a colorant added such as soot or indigo dye; unless it would cause the chemical to be ineffective for use in medicinal or agricultural products. The Act called for fines to be levied if the terms of the Act were violated. The Arsenic Act of 1851 was repealed by parliamentarian action by passing the Pharmacy and Poisons Act of 1933 [12].

The detection of poisons and the identification of poisoners has been the challenge to science, medicine, and law enforcement. A poisoned victim was generally identified by circumstantial evidence, with limited, to no scientific evidence to support the cause of death. Joseph Plenck (Josephi Iacobi Plenk) was the first to suggest that the proof of poisoning was by the identification of the poison in biological tissues; published in his work Elementa medicinae et chirurgiae forensis, published in 1786 [13].

The development of various analytical techniques; such as the Marsh test for arsenic in 1836 [14], the separation of alkaloidal drugs from biological matrixes in 1850 [15], and instrumental analysis began to evolve in the 1960s. More recently, spectrophotometric methods and chromatographic methods, such as gas and liquid chromatograph along with various hyphenated instrumental platforms, for example, gas chromatography–mass spectrometry and liquid chromatography–tandem mass spectrometry has led to a rapid increase in the ability to detect poisons in decedents [16].

Although detection methods of drugs and poisons were improving, the application of these in forensic work in the United States did not begin to evolve until 1804 [17]. The errors of the coroner system, as brought over from England, were being recognized, which lead to the formation of the office of the Chief Medical Examiner for the state of Massachusetts in 1877. The Chief Medical Examiner’s Office of New York saw its beginnings in 1918, with the toxicology laboratory founded under the direction of Dr. Alexander Gettler.

In fact, the development of various medical examiner systems throughout the United States, evolved out of the eventual detection of poisoners and the need for a more sophisticated investigation of death. Nannie Doss was a prolific serial killer in Tulsa, Oklahoma, USA from the late 1920’s until the mid-1950’s [18,19]. She dispatched several of her husbands, children, sisters, mother, mother-in-law, and a grandson with arsenic. Her final discovery led to legislation in the state of Oklahoma (USA) forming what is now known as the Office of the Chief Medical Examiner (personal communication: the author spent several years working for the office as the Chief and Deputy Chief Toxicologist).

Due to the ever increasing number of therapeutic agents, illicit/designer drugs that are prevalent in our society today; toxicological examinations continue to play a significant role in the evaluation of a death investigated by a Coroner or Medical Examiner system. Although the ability to detect and quantitate drugs and poisons in biological material has significantly improved over the last century; the questions remains the same: Did a drug or combination of drugs alter the decedents behavior, play a direct casual role in the death or contribute to the death or in some instances was the absence of a medication, for example, an antiseizure medication, a factor in the death or circumstances leading up to the death?

There are no absolute rules for the interpretation of postmortem toxicological results. The proper interpretation of toxicological results requires a global awareness of the challenges and pitfalls associated with the test results and the inclusion of the totality of circumstances surrounding the death. The first and foremost consideration of postmortem toxicological results is that they cannot and should not be interpreted in a vacuum; nor can one just pick up The Red Book or a table listing toxic or lethal drug concentrations to form a conclusion on what role a drug played in the death. The determination of how the toxicological results will impact the ultimate opinion of the cause and/or manner of death must incorporate not only the laboratory findings, but also include the decedent’s medical history, predeath clinical findings, scene investigation, and a thorough autopsy examination. The challenge is that in some medical examiner/coroner cases some or all of this information may be incomplete or not available at all.

Death investigations can be affected by a variety of unusual complexities. These confounding factors or artifacts impacting the interpretation of results may arise from preanalytical factors, the specimen container, and/or storage conditions, prior medical therapy may mislead the investigation as well as medical intervention prior to the death. Artifacts leading to misinterpretation may also be introduced due to organ or tissue procurement. There also arise situations where the pathological specimens are collected from an embalmed body prior to burial or after exhumation. The embalming process may hinder the detection of drugs and/or interpretation of the toxicological findings.

The dynamic nature of the body, not only during life but after death as well may impact the toxicological findings as well. Many of the challenges arise from artifacts that occur due to a plethora of changes that occur just prior to or during the postmortem interval, such as the postmortem redistribution of drugs. Decompositional changes may alter findings; production of target analytes, such as ethyl alcohol or gamma hydroxybutyrate and/or the formation of other compounds that may obscure the laboratory results. These all have the potential to lead to a wrong conclusion; be that an incorrect Cause or Manner of Death or it could be the nondeduction (detection) or undue suspicion of a crime.

In addition to the various artifacts presenting challenges in the interpretation of toxicological findings in a death case; given the increasing frequency of polypharmacy cases, the interactions of the drugs found are just as important. Did the drug combination have additive or synergistic effects or did one drug impede the clearance of another, thus allow for the buildup of a toxic concentration of the drug?

The practice of forensic toxicology is continually advancing and evolving; the ever increasing number of drugs being developed or rediscovered that can lead to toxicity and the development of new analytical techniques used to characterize, identify, and quantitate these compounds in complex biological matrixes.

The focus of this treatise is a discussion of these factors that may impact the interpretation of the toxicological findings and their bearing on the cause and manner of death.

References

1. Askitopoulou HE, Ramoutsaki IA, Konsolaki E. Analgesia and anesthesia: etymology and literary history of related Greek eords. Anesth Analg. 2000;91:486–491.

2. https://www.merriam-webster.com/dictionary/forensic [accessed 22.10.18].

3. De Vos P. European materia medica in historical texts: longevity of a tradition and implications for future work. J Ethnopharmacol. 2010;132:28–47.

4. Smith S. Poisons and poisoners through the ages. Med Leg J. 1952;20:153–167.

5. Cilliers L, Retief FP. Poisons, poisoning and the drug trade in ancient Rome. Akroterion. 2000;45:88–100.

6. Hotti H, Rischer H. The killer of Socrates: coniine and related alkaloids in the plant kingdom. Molecules. 2017;22:e1962 https://doi.org/10.3390/molecules22111962.

7. Gilbert SG. A small dose of toxicology hisotry or an introduction to the hisoty of toxicology and lessons learned. In: Gilbert SG, ed. A small dose of toxicology - the health effects of common chemicals. 2nd ed. Seattle: Healthy World Press; 2012;41–42.

8. Borzelleca JJ. Paracelsus: herald of modern toxicology. Toxicol Sci. 2000;53:2–4.

9. Campbell WA. The history of the chemical detection of poisons. Med Hist. 1981;25:202–203.

10. Bartrip PA. Pennurth of arsensic for rat poison: the Arsenic Act, 1851 and the prevention of secret poisoning. Med Hist. 1992;36:53–69.

11. UK National Archives: http://www.legislation.gov.uk/ukpga/1851/13/contents/entacted; retrieved 22 Oct 2017.

12. Analyst. 1933;58:548–50.

13. Eckert WG. Historical aspects of poisoning and toxicology. Am J Forensic Med Pathol. 1980;1:261–264.

14. Marsh J. Account of a method of separating small quantities of arsenic from substances with which it may be mixed. Edinburgh New Philosophical J. 1836;21:229–236.

15. Wennig R. Back to the roots of modern analytical toxicology: Jean Servais Stas and the Bocarmé murder case. Drug Test Anal. 2009;1:153–155.

16. Pappas AA, Massoll NA, Cannon DJ. Toxicology: past, present and future. Ann Clin Lab Sci. 1999;29:253–262.

17. Niyogi SK. Historic development of forensic toxicology in America up to 1978. Am J Forensic Med Pathol. 1980;1:249–264.

18. Litchfield Y. Cheerful Tulsan Nannie Doss was poison on husbands. TulsaWorld 1993; Available from: http://www.tulsaworld.com/archives/cheerful-tulsan-nannie-doss-was-poison-on-husbands/article_8bed9fc4-f1d5-5c89-8bf0-8125b6b282ed.html [accessed 01.11.17].

19. Curtis G. Only in Oklahoma: Black widow enjoyed the limelight. TulsaWorld 2007; Available from: http://www.tulsaworld.com/archives/only-in-oklahoma-black-widow-enjoyed-the-limelight/article_72d236aa-b108-5a0f-bec7-d21e5197193d.html [accessed 01.11.17].

2

Preanalytical Considerations

Abstract

Toxicological analyses play a critical role in the determination of the cause and manner of death. However, as critical as the toxicological testing phase is, the preanalytical phase that is the specimen collection method and/or storage can have a significant impact on the toxicological results, thus adding additional complexities to the interpretation. Drug concentrations may change due to the specimen collection process, the storage container the specimen is held in and the storage conditions for the specimen(s). The storage container may add, form or remove target analytes to or from the specimen. The storage conditions can influence not only the stability of the biological matrix itself, but also the stability of the drug in the specimen. Changes in drug concentrations and/or target analytes found in the specimen could lead to misinterpretation of the toxicological test results and potentially change the determination of the cause and manner of death.

Keywords

Agonal aspiration; blind stick; phthalate plasticizers; serum separator tubes (SST); specimen container; specimen storage conditions; tricyclic antidepressants (TCA); vacutainer effect

Toxicological analyses play a critical role in the determination of the cause and manner of death. The presence or absence of a drug and/or poison may support or refute a purported cause of death. The analytical findings may also be used to substantiate a manner of death, for example, suicide. In many instances, the measured concentration of the drug plays a pivotal role in the interpretation of the case.

As critical as the analytical phase is, that is, the testing, the toxicological results can be impacted in the preanalytical phase; drug concentrations may be artificially changed due to the collection process during the autopsy, the storage conditions of the specimen and/or the storage container itself. Targeted analytes may be added to the specimen from the storage container.

In many death investigations, an important question to be answered is Was the decedent under the influence of alcohol at the time of his/her death? Thus blood alcohol analyses are one of the most commonly requested toxicological examination performed in death investigations. In approximately one-third to one-half of these, cases are positive for ethyl alcohol.

The collection of a heart blood sample via a blind-stick through the chest is routinely performed in some jurisdictions for the procurement of a blood sample. A blind-stick is performed by the insertion of a large gauge needle through the chest wall at an approximate 45° angle in the fourth intercostal space, targeting the ventricle of the heart. The training and skill of the specimen collector vary widely from jurisdiction to jurisdiction, some of the collections are obtained by medically trained or supervised individuals; however, there are many instances that the heart blood sample is collected by untrained individuals such as funeral home staff and law enforcement.

A blind-stick collection always begs the question as to whether a valid sample is collected. Given the nature of the collection technique, the question arises as to whether or not the heart was missed and the sample originated from the esophagus, where even a small amount of gastric alcohol contaminated the sample due to agonal regurgitation. In heavily traumatized cases, the issue of the stomach and diaphragm being ruptured and thus leading to contamination by the gastric contents is a possibility.

Logan and Lindholm [1] reported a case of gastric contamination of a postmortem blood sample during a blind-stick. A 45-year-old female was found dead at home. The decedent had a history of atherosclerotic coronary cardiovascular disease and noninsulin-dependent diabetes. Her physician was willing to sign the death certificate. The local corner requested that a blood sample be drawn. The specimen was drawn by local funeral home staff by a blind-stick through the chest cavity wall.

The sample was collected in a gray top tube (potassium oxalate/sodium fluoride) and was submitted for toxicological analyses. The laboratory personnel described the sample as brown-discolored and largely immobile. The toxicological analyses revealed an ethyl alcohol concentration of 0.22 g% with an amitriptyline concentration of 6.16 mg/L and a nortriptyline concentration of 0.35 mg/L.

The Coroner opined that the reported concentrations were consistent with a combined drug and alcohol overdose. The family of the decedent expressed concern over the ruling and requested that an autopsy be performed. The autopsy was performed 4 days later on the decedent.

The external examination of the chest noted four postmortem needle-puncture wounds; two near the sternum and two above the left breast; suggesting several attempts at sample collection. The autopsy examination revealed that the larynx, trachea, and bronchi were over lain with a yellow-green-bile stained material and there was granular gastric material noted in the respiratory track, mouth, and nares. Additional toxicological specimens were collected and analyzed. Table 2.1 reveals the results of the initial toxicological tests as well as the results from the specimen (subclavian blood) collected from the autopsy.

Table 2.1

The qualitative results were the same, thus ruling out a specimen mix-up. The amitriptyline results could be explained by postmortem redistribution. The described discoloration of the autopsy blood suggests gastric contamination. A cytological examination of the blind-stick sample revealed the presence of columnar cells, ciliated columnar cells, and squamous cells and cooked meat fibers. This suggests the strong probability that the blood was contaminated by unabsorbed pill fragments that were in the gastric contents. These observations suggest that the blood sample was contaminated by gastric contents and/or aspiration of the gastric contents and the blind-stick hitting the esophagus during the sampling.

It is not uncommon for cases presenting to the Medical Examiner to be heavily traumatized. In such cases of extreme trauma, blood may not be available from the heart, femoral, or carotid vasculature. In these cases, blood is sometimes scooped from the chest cavity and submitted for analyses. This type of specimen has a high potential for contamination from the gastric contents. This usually arises from the stomach and/or diaphragm being ruptured due to the traumatic event.

As a result of medical intervention, the traumatized patient may have had a chest tube inserted and blood is collected from the draining tube. The contamination issues from either specimen sources; that is, scooped or drainage from a chest tube are similar to those of a blind-stick in that the measure alcohol or drug concentration(s) may be elevated due to contamination from gastric contents.

Budd [2] reported a retrospective study which he compared paired samples of chest cavity fluid and blood collected directly from the heart for alcohol content. This study covered an 8-year period and evaluated 25 cases of paired samples. There were 15 cases which had positive alcohol findings. These cases were divided into two groups; uncompromised, which some no evidence of stomach or diaphragm perforation and compromised, which some evidence of stomach/diaphragm perforation, decomposition or the sample was otherwise questionable by the examining pathologist. The results, as given in Table 2.2, suggest that a cavity blood sample is suitable for alcohol analyses if there is no compromise of the integrity of the stomach/diaphragm or other factors that may affect the integrity of the barriers between the cavity and stomach, that is,