23.12.2020
Universiteit van Amsterdam
Master Forensic SciencePost-mortem biochemistry in sudden cardiac death and
diabetes mellitus
__________________________________
Tim Stölting
12748781
Supervisor: Dr Francisco Javier Defez Torán
Examiner: Prof Dr Roelof-Jan Oostra
Index
Abstract ... 1
1. Introduction ... 2
2. Results ... 4
2.1 Sudden Cardiac Death ... 5
2.1.1 Cardiac Troponins ... 5
2.1.2 CK-MB ... 6
2.1.3 BNP and NTproBNP ... 6
2.1.4 Ischemia Modified Albumin ... 7
2.2 Diabetes Mellitus ... 8
2.2.1 Glucose ... 8
2.2.2 Lactate ... 8
2.2.3 Ketone Bodies ... 9
2.2.4 Glycated Hemoglobin and 1,5-AG... 9
2.3 Complications of Diabetes Mellitus ... 10
2.3.1 Ante-Mortem Hypoglycemia ... 10
2.3.2 Diabetic Ketoacidosis ... 10
2.3.3 Alcoholic Ketoacidosis ... 11
2.3.4 Hyperosmolar Hyperglycemic State... 12
3. Discussion ... 13
3.1 Interpretation of Biomarker Profiles ... 13
3.2 Limitations ... 15 4. Conclusion ... 16 5. References ... 17 Appendix ... 21 A1 Search Strategy ... 21 A2 Abbreviations ... 21
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Abstract
During autopsy, the forensic doctor aims to establish the cause and course of death in forensically relevant, unexpected deaths. Morphological and histopathological findings of tissues most often lead to conclusive results about the cause of death of a patient. In some cases, however, complications develop so rapidly that tissue damage is not reflected macro- and microscopically, but by the release of tissue-specific biomarkers. In these cases, post-mortem biochemistry offers an auxiliary approach to assist the forensic doctor in determining the cause of death. While biochemical analyses are used on a daily basis in clinical diagnostics, no standard protocols and validated reference values have been established in post-mortem biochemistry and it is used inconsistently during autopsy. Cut-off values from clinical diagnostics are not transferable to the framework of post-mortem biochemistry, as post-mortem changes affect the concentrations of biomarkers. In this literature thesis, the current state of knowledge on post-mortem biochemistry is reviewed and guidance on how to interpret post-post-mortem biochemical profiles is provided.
Sudden cardiac death after acute myocardial infarct and lethal complications of diabetes mellitus are complex cases most often encountered by the forensic doctor. In sudden cardiac death, morphological findings of the heart and histopathological anomalies of the myocardium are limited or even missing due to the rapid progression of the disease. Post-mortem biochemistry of myocardial markers in pericardial fluid can assist in identifying peri-mortem stress on the heart and indicate sudden cardiac death. Elevations of cardiac troponins cTnI and cTnT in pericardial fluid above 86.2 ng/mL and 8.025 ng/mL, respectively have been proposed to indicate sudden cardiac death. Furthermore, elevations in creatine-kinase MB, brain natriuretic peptide (BNP) and NTproBNP can be used to support the diagnosis of sudden cardiac death. Ischemia modified albumin has recently been proposed as another cardiac-specific biomarker and elevations are correlated to acute myocardial infarct.
Sudden death due to diabetes mellitus can occur when the disease is undiagnosed or mistreated. Diabetic ketoacidosis and hyperosmolar hyperglycemic state are complications resulting from acute hyperglycemia with a rapid and often lethal progression. Macro- and microscopical findings during autopsy are limited, so post-mortem biochemistry can be utilized to establish the exact cause of death. Relevant biomarkers for post-mortem analysis are vitreous glucose and lactate levels, vitreous ketone body concentrations, especially beta-hydroxybutyrate (bHB), 1,5-AG and glycated hemoglobin. In both diabetic ketoacidosis and hyperosmolar hyperglycemic state, vitreous glucose is elevated due to peri-mortem hyperglycemia with a cut-off value of > 10 mmol/L. To distinguish, vitreous bHB concentration is essential. In diabetic ketoacidosis, vitreous bHB is strongly elevated with a cut-off value of around 2500 µmol/L, while in hyperosmolar hyperglycemic state, vitreous bHB concentration is normal. Alcoholic ketoacidosis is unrelated to diabetes mellitus and is caused by prolonged alcohol abuse and malnutrition. It shows similar post-mortem biochemical profiles and is therefore discussed as well. Alcoholic ketoacidosis shows normal vitreous glucose levels of around 3.5 mmol/L and elevated vitreous bHB levels of around 2500 µmol/L. By looking at the complete biochemical profile in combination with morphological findings, excluding criteria and clinical data, valid inferences can be made on the cause of death from post-mortem biochemical analyses. In this review, an interpretation model is proposed for the diagnosis of sudden death due to diabetes mellitus.
As post-mortem inferences and methodology highly influence biomarker concentrations, laboratories need to make the effort to establish their own reference ranges based on autopsy data and the available equipment rather than to find the one gold standard for each biomarker. Forensic research and practice have to detach post-mortem biochemistry from the clinical framework and view it as its own discipline. No death is the same, so procedures and analytical methods must be both standardized and flexible at the same time to maximize the benefit of post-mortem biochemistry as an auxiliary tool to establish the cause of death.
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1. Introduction
Forensic doctors are involved in medical cases that are situated at the nexus of medicine and law. They are required to apply their medical knowledge within a legal context and most often perform medico-legal examinations into the cause of death and the circumstances surrounding death. These examinations are also referred to as autopsies. During autopsy, the forensic doctor aims to establish the cause of death based on morphological, histological and toxicological findings. The cause of death is defined as the disease or injury initiating the sequence of events that eventually lead to the death of the individual1.
Other disciplines, such as molecular genetics, port-mortem biochemistry or microbiology, are less often employed by forensic doctors as scientific knowledge about post-mortem processes is limited, consistent reference values as they exist in clinical medicine are not available and standardized protocols are missing. While biochemical analysis in clinical diagnostics has been part of routine examinations for years, post-mortem biochemistry is used inconsistently during autopsy. The strength of post-mortem biochemistry lies within its potential to reinforce morphological, histopathological or toxicological findings. More importantly, it can give insight into the cause of death when other findings are inconclusive or even absent. In addition, post-mortem biochemistry also enables investigation into the process of death, the systemic and tissue-specific pathophysiological changes that take place in the body after death, also referred to as pathophysiological vital reactions2.
In most medico-legal cases, the cause of death is unknown to the forensic doctor, so they have to employ a screening approach when investigating the body of a deceased. Post-mortem biochemistry offers an additional approach for an initial screening to enable a more targeted investigation into the cause of death. Post-mortem biochemical profiles allow inferences about multiple variables surrounding death, such as pre-existing medical conditions, survival period, complications and cause of death. However, it is important to note that post-mortem biochemical profiles are affected by a multitude of variables that are often difficult to assess. These include the post-mortem interval (PMI), decompositional changes of tissues and biomarkers, diffusion and redistribution of biomarkers, chosen sampling site and analytical method and many more.2 In order to make valid inferences from
post-mortem biochemical profiles, these variables have to be taken into account and the biochemical profiles have to be evaluated in conjunction with morphological, histopathological and toxicological findings. Biomarkers for systemic conditions, such as metabolic disorders, including diabetes mellitus, and tissue-specific biomarkers that reflect tissue or organ damage, such as myocardial infarct, are utilized in post-mortem biochemistry. Biomarkers for systemic conditions, but also tissue-specific markers are often also used in clinical diagnostics, so clinical reference values and criteria may be partly transferable to
post-mortem diagnostics3. The reason why the framework of clinical diagnostics does not fully apply to
post-mortem biochemical diagnostics lies within the variability of the post-mortem processes. After the death of an individual, energy metabolism stops, cell-cell contacts part and selective membrane permeability ceases. Biomarkers diffuse through tissues and body fluids along their concentration gradient leading to altered concentrations not representing the state when death occurred, also called post-mortem redistribution4. Therefore, selection of sampling site and specimen is a key factor in
post-mortem biochemistry. A variety of specimens have been established as appropriate, including blood, urine, vitreous humor, pericardial fluid and cerebrospinal fluid5,6. Each of these specimens has its
preferred application depending on the suspected cause of death and the investigated biomarkers, but also the laboratory equipment that is available. The decompositional stage and integrity of the body is usually the limiting factor when deciding which specimen to sample. In most autopsy cases, the forensic doctor has to performing a screening examination to investigate all kinds of possible causes of death, so multiple specimens are collected and examined. Sample choice and collection can be coordinated alongside the toxicology department to ensure sufficient specimen recovery.
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Cases where the cause of death is not obvious become forensically relevant. Usually, autopsy is requested by either the family of the deceased or the judicial system. Establishing the cause of death based on morphological and histopathological findings and existing medical records most often leads to conclusive and reliable results. When morphological and histopathological findings are missing and medical records pointing towards a lethal condition do not exist, defining a definite cause of death becomes extremely difficult. Unexpected deaths with no obvious cause are often referred to as sudden deaths. The death of the individual is the first clinical symptom of an underlying disease of the believed to be healthy subject and hence, autopsy is the only way to identify the underlying condition and the
resulting cause of death7. The two most frequent conditions that lead to unexpected, sudden death are
coronary heart disease8 and diabetes mellitus9.
Sudden cardiac death in the context of coronary heart disease most often occurs either during or immediately after acute ischemic myocardial infarction8. In the clinical context, sudden cardiac death is
defined as unexpected, nontraumatic death occurring within one hour of the onset of new or progressing
symptoms10. In a forensic context, however, the death of the individual occurs without any witnesses,
for instance during sleep, and the body is usually discovered at a later point in time with an unknown PMI. In this setting, ‘sudden’ refers to the individual being in a healthy state 24h before death11. Sudden
cardiac death in the context of coronary heart disease occurs either during or immediately after acute
ischemic myocardial infarction8. The term sudden cardiac death implies that a patient does not survive
this condition. If the patient survives, it is called sudden cardiac arrest12. In case that autopsy is requested,
the forensic doctor needs to establish whether the death is of cardiac nature or whether other causes might be responsible. Morphological and histopathological examination of the heart might help in defining the underlying cardiac condition, whether it was ischemic or non-ischemic, arrhythmic or mechanical. Toxicology can offer insights into toxin or illicit drug abuse and resulting cardiac arrest and clinical records can help in identifying risk factors or other contributing conditions7.
Sudden death in the context of diabetes occurs when complications arise in undiagnosed or mishandled cases. Diabetes mellitus is characterized by either absolute or relative insulin insufficiency, resulting in the body not being able to use glucose in tissues for energy metabolism. Type I is defined by the absolute lack of insulin due to autoimmune-induced degradation of insulin-producing beta cells in the pancreas. The cause of this autoimmune response is still unknown. Type II diabetes is defined by the relative lack of insulin due to the body building up resistance against produced insulin and target cells fail to respond to insulin stimulus13. Both type I and type II diabetes can, if undiagnosed or mishandled, result in fatal
hypo- and hyperglycemic states. Often, these critical states can develop rapidly, leading to unexpected and sudden death that has to be examined by the forensic doctor. During autopsy, a screening of the body is performed to determine the cause of death. Similar to sudden cardiac death, rapidly progressing complications in diabetes mellitus are difficult to diagnose post-mortem, as macro- and microscopical findings are not present in all cases, especially in early stages of the disease. During autopsy, the forensic doctor can examine the morphology of the pancreas, especially its size. Studies have shown that the degradation of insulin-producing beta-cells in type I diabetes can lead to a diminished size of the pancreas14. Histopathological examination of the pancreas tissue, specifically the Langerhans isles,
where the beta cells are located, can identify a decrease in number of beta cells. The forensic doctor can quantify the present beta-cells by immunolabeling in order to establish a possible cell degradation15.
These morphological findings are often found in later stages of diabetes mellitus. Acute complications, however, often arise in undiagnosed cases, where medical records regarding diabetes do not exist and an early stage of the disease can be assumed. These acute complications are often related to hypo-and hyperglycemic states. The two hyperglycemic states most often related to sudden death due to diabetes mellitus are diabetic ketoacidosis (DKA) and hyperosmolar hyperglycemic state (HHS). Both can occur
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in undiagnosed or mishandled diabetes mellitus and can result in sudden death with few or no morphological characteristics16. Alcoholic Ketoacidosis (AKA) is unrelated to diabetes mellitus but
shows similar post-mortem biochemical characteristics and is discussed alongside DKA for this reason. In both sudden cardiac death and sudden death due to diabetes mellitus, disease progression is very rapid with non-specific to no symptoms at all, resulting in the unexpected death of the individual17. It is the
responsibility of the forensic doctor to examine and define the cause of death that will eventually decide whether a criminal investigation will follow.
Although it seems that autopsies for forensically relevant cases should be subject to regulation and standardization, there exists no consensus on a general approach, the applied methodology and analytical instruments. Especially in post-mortem biochemistry, where influencing variables are difficult to assess, reference ranges are uncertain and a cluster of available studies and meta-reviews exist, there needs to be clarification on what is scientifically reliable, what biomarkers and techniques are available and most importantly, how forensic doctors can interpret post-mortem biochemical profiles.
In this literature thesis, an overview of diabetes mellitus and coronary heart disease-related biomarkers in the context of sudden death is given. Moreover, this review was designed specifically for the forensic doctor to illustrate the current state of knowledge, the possibilities and strengths of post-mortem biochemistry as well as its limitations in actual casework. The most common complications in diabetes mellitus and coronary heart disease resulting in sudden death will be discussed and for each and reference ranges and cut-off values for related biomarkers will be recommended to provide the forensic doctor with a guide for the interpretation of post-mortem biochemical profiles. The aim of this literature thesis is to evaluate how cardiovascular and metabolic disease-related biomarker parameters from post-mortem samples can be interpreted by the forensic doctor during autopsy.
2. Results
Post-mortem processes and their influence on biomarker concentrations in different body fluids are key factors when interpreting post-mortem biochemical profiles. Hence, the forensic doctor needs to consider differences between specimens and sampling sites when performing biochemical analyses. When using blood or serum, it is especially important to consider the sampling site, as the chemical composition of blood varies depending on where it was sampled. For forensic purposes, blood is mainly sampled from the left and right heart, central arteries or the periphery, preferably the femoral vein6.
Sampling and processing blood for biochemical analysis is the gold standard in clinical diagnostics. In a forensic setting, however, blood is not always the optimal choice, as it is sensitive to post-mortem
changes caused by autolysis and bacterial decomposition, contamination and biomarker redistribution18.
The vitreous humor has become the specimen of choice for most post-mortem biochemical investigations. As opposed to blood, it is protected from contamination and post-mortem degradation, the blood retinal barrier, which usually regulates solute transport, minimizes passive diffusion even after death, resulting in relatively stable biomarker concentrations19. In addition, it is easy to sample during
autopsy and research has shown no significant differences between right and left eye, meaning that, if necessary, samples can be pooled20. Pericardial fluid and cerebrospinal fluid, which are essentially
ultra-filtrations of plasma, have similar properties to vitreous humor, as they are protected from degradation and contamination, the biomarker concentration appears to be relatively stable and their production stops after death21,22. Pericardial fluid is of great interest especially in cases involving heart injury. In the
context of sudden cardiac death and sudden death due to diabetes mellitus, both tissue-specific body fluids, such as pericardial fluids, and systemic body fluids, such as serum or vitreous humor have to be considered to measure both systemic and tissue-specific changes.
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2.1 Sudden Cardiac Death
Necrotic areas of the myocardium and hemorrhages of heart muscle tissue are indicative for death due to myocardial infarct, especially in patients with a medical history of coronary heart disease7. In sudden
cardiac death, however, two thirds of sudden cardiac deaths with coronary heart disease as the underlying condition occur in patients with either no previous heart-related medical conditions or low-risk cardiac conditions. This means that in most cases clinical data is either not available or not sufficiently informative23. Thus, the biggest challenge in sudden cardiac death is that morphological,
histopathological and toxicological findings, as well as medical records, are often missing due to acute disease progression24. In these cases, tissue damage is not reflected macro- and microscopically, but by
the release of tissue-specific biochemical markers. To analyze these biomarkers, different body fluids can be used. The preferred choice is the pericardial fluid, as myocardium-specific biomarker concentrations appear to be higher than in serum due to its close proximity to the heart. In addition, pericardial fluid is overall less sensitive to degradation and contamination than blood25. In the clinical
context, a lot of biomarkers are available to diagnose myocardial damage due to ischemic infarct. The most common are cardiac troponins (cTn), creatine kinase MB (CK-MB), Brain Natriuretic Peptide (BNP) and N-terminal pro BNP (NTproBNP)26. Some of these markers are also valuable for
post-mortem diagnosis. Clinical reference values, however, are not transferable to post-post-mortem biochemistry for these biomarkers. For example, the concentration of these biomarkers in adjacent tissues and body fluids depend on the PMI and the severity of myocardial damage. Also, post-mortem redistribution affects the concentrations in different body locations and fluids27.
2.1.1 Cardiac Troponins
Cardiac troponin is a regulatory protein found in striated muscle myofibrils involved in the contraction process. It has three subunits, cardiac troponin T (cTnT), cardiac troponin I (cTnI) and cardiac troponin C (cTnC). cTnT and cTnI are isoforms that are specific for cardiac muscle tissue. They are highly sensitive and are therefore used in clinical and post-mortem diagnostics of cardiac muscle damage28,29.
Cardiac troponin serum base levels in healthy patients are low, with a range of about 0-1.5 ng/mL30 and
they can be detected in serum after about 4 hours of myocardial infarct31. A variety of studies have
shown that post-mortem serum cTnT and cTnI levels taken from different sampling sites were elevated compared to ante-mortem values, however, the elevation in cardiac troponins was found to be independent from the cause of death. Studies investigating post-mortem cTnI and cTnT levels in pericardial fluids have shown significant elevations that seem to be more specific for myocardial damage. In addition, varying serum and PCF cardiac troponin concentrations for different PMIs and
sampling sites have been reported, as reviewed by Barberi and Hondel32, making it difficult to define
specific post-mortem cut-off values for cTnT and cTnI. However, research has shown that cTnI and cTnT levels in pericardial fluid can indeed indicate sudden death due to acute myocardial infarct. Cao et al. set out to define reference values for both biomarkers by performing a meta-analysis on the available literature and found highly variable cut-off values between different studies, mainly due to different PMIs and analytical methods. They concluded a cut-off value of 86.2 ng/mL in pericardial fluid for cTnI and 8.025 ng/mL in pericardial fluid for cTnT to indicate sudden cardiac death due to acute myocardial infarct33. Inferences on the cause of death should never be made solely based on a
single biomarker concentration. Instead, all available biomarkers should be evaluated together with macroscopic and microscopic findings.
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2.1.2 CK-MB
Creatine kinase is a phosphokinase found in the intracellular space primarily in skeletal and heart muscle and the brain. It is involved in the regeneration of ATP by transferring a phosphate group from phosphor-creatine to ADP. It is made up of two subunits: subunit M and subunit B. The combination of MB is highly specific for the myocardium34. In clinical diagnostics, CK-MB is used as a biomarker to diagnose
cardiac abnormalities, such as acute myocardial infarction35, and its concentration elevates in serum
about 4 hours after myocardial infarct36. Base levels in healthy individuals are variable and depend on
the analytical method employed, but usually range from 5-25 IU/L37. Pericardial fluid and heart blood
is the preferred specimen for post-mortem CK-MB concentration analysis, as concentrations are higher than in peripheral blood due to direct leakage of the biomarker into the adjacent tissues and body fluids38.
Xu et al. performed a meta-analysis on studies investigating the post-mortem concentration of CK-MB in cardiac death and concluded that CK-MB concentration in pericardial fluid was significantly higher in acute myocardial infarction groups than in control groups33. In serum, however, multiple studies
suggest no significant difference between the cardiac death group and the control group35,39. Due to the
high variability of CK-MB elevation in sudden cardiac death due to differences in PMI, post-mortem interference and analytical methods, reliable cut-off values for CK-MB in post-mortem biochemistry do not exist.
2.1.3 BNP and NTproBNP
Brain Natriuretic Peptide (BNP) and NTproBNP, which is a by-product of BNP generation, are part of the atrial natriuretic peptide family. BNP was first isolated from pig brain, hence the name, however, it is mainly produced in cardiomyocytes of the left ventricle. BNP is not continuously expressed by cardiomyocytes, but rather upon mechanical stress on the heart or during ischemic states and hypoxia. Its purpose is to relieve the heart during elevated afterload periods by having diuretic, natriuretic and
vasodilatatory effects40. In clinical diagnostics, BNP and NTproBNP are routinely used in diagnosing
acute heart insufficiency and cardiovascular dysfunctions. Physiological serum BNP concentration is
around 35 pg/mL and physiological serum concentration of NTproBNP are around 125 pg/mL41. BNP
and NTproBNP are interesting for post-mortem biochemistry, as production and excretion is limited to stress reactions of the heart, including ischemic episodes, making it very specific for cardiac death. BNP and NTproBNP levels in post-mortem peripheral blood showed no significant elevation in cardiac death groups compared to control groups, as reviewed by Cao et al40. In pericardial fluid, however,
post-mortem levels of BNP and NTproBNP were significantly elevated in sudden cardiac death groups with and without observable myocardial necrosis. The latter finding is especially important, as post-mortem biochemistry becomes particularly valuable for the forensic doctor when morphological findings are missing. NTproBNP appears to be more suitable for post-mortem analysis than BNP as studies have shown that is more stable and less sensitive to temperature and storing conditions prior to analysis. Studies have shown that cardiopulmonary resuscitation has no effect on post-mortem values of both
BNP and NTproBNP42,43. Elevations in pericardial BNP and NTproBNP concentrations can therefore
indicate death due to acute heart failure or acute myocardial infarct. In order to differentiate between the two, biochemical findings should be used in combination with morphological and histopathological findings and other biomarker analyses.
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2.1.4 Ischemia Modified Albumin
Ischemia modified albumin (IMA) is a metabolic variant of serum albumin that is produced when circulating albumin comes into contact with infarcted tissue, making it very specific for ischemic cardiac
lesions44. Serum IMA levels are currently evaluated using an albumin cobalt binding assay, which was
approved by the Food and Drug Administration in 2003 and has since been used in clinical medicine to diagnose acute myocardial infarction. It showed strong discriminative power in differentiating between ischemic and non-ischemic patients and a high sensitivity (78.0%) and specificity (82.2%) for the detection of acute coronary syndrome. Given these properties, IMA has become the marker that is used in routine clinical diagnostics45. A recent study performed by Yağmur et al. investigated the potential of
IMA as a biomarker for sudden cardiac death in post-mortem biochemistry. They measured serum IMA using the albumin cobalt binding assay during medico-legal autopsies of patients that died from cardiovascular disease compared to patients that died from blunt trauma, asphyxia, cerebral hemorrhage, bleeding and poisoning and found that serum IMA concentrations were significantly elevated in the
cardiovascular disease group compared to the other groups44. More research has to be done to evaluate
the diagnostic potential of IMA in post-mortem biochemical examinations in the context of sudden cardiac death, but the clinical properties of IMA and recent findings regarding post-mortem concentrations seem promising.
It is evident and cannot be emphasized enough that these values can only indicate sudden cardiac death and a diagnosis should not be made solely based on individual myocardial biomarker levels alone. Table 1 can aid in the interpretation of myocardial biomarkers when sudden cardiac death is suspected. Conclusions and inferences on the cause of death have to be made based on the full biochemical profile, including exclusionary criteria, as well as morphological and histopathological findings during autopsy.
Table 1 – Post-mortem biochemical profile in the context of sudden cardiac death
Biomarker Body Fluid Cut-off Analytical Method
cTnT Pericardial Fluid > 8.025 ng/mL Immunoassay46,47 cTnI > 86.2 ng/mL Immunoassay46,47 CK-MB elevated Immunoassay47 BNP elevated Immunoassay40
NTproBNP elevated Immunoassay40,46
IMA* Serum elevated Albumin Cobalt
Binding Assay44
Abbreviations – cTnT: Cardiac troponin T, cTnI: Cardiac troponin I, CK-MB: Creatine kinase MB, BNP: Brain natriuretic peptide, NTproBNP: N-terminal pro brain natriuretic peptide, IMA: Ischemia modified albumin
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2.2 Diabetes Mellitus
In cases of rapidly progressing complications of diabetes mellitus with no morphological or histopathological findings and no conclusive clinical patient data, post-mortem biochemistry offers an additional approach to determining the cause of death by measuring the post-mortem concentrations of different biomarkers in various specimens. The post-mortem diagnosis of diabetic ketoacidosis is a model showcase of how biochemical analysis can aid and even be the decisive factor in the determination of the cause of death.
The biomarkers used in post-mortem biochemistry are mostly used in clinical diagnosis as well, so reference ranges are partly transferable. However, post-mortem degradation and biomarker redistribution need to be considered. For post-mortem diagnosis of diabetes mellitus and lethal complications, the concentrations of glucose, lactate, ketone bodies, 1,5-anhydroglucitol and glycated hemoglobin can be utilized, with glucose, lactate and ketone bodies being the most specific biomarkers.
2.2.1 Glucose
In clinical medicine, blood glucose level is the defining parameter when diagnosing diabetes mellitus. Serum glucose levels in healthy individuals range from about 60 to 99 mg/dL (3.5 to 5.5 mmol/L), depending on the last meal of the subject. Diabetes mellitus is diagnosed if blood glucose levels exceed 126 mg/dL (7.0 mmol/L) when fasting or 200 mg/dL (11.1 mmol/L) 2h post-prandial (after the last meal)48. In post-mortem blood and serum, however, these reference values cannot be used. After the
suspension of vital organ functions, such as heart and lungs, some cells remain viable for a limited amount of time. Surviving cells keep metabolizing glucose in anaerobic glycolysis to produce ATP, resulting in a decrease of blood glucose concentration and an increase in lactate levels49. Hypoglycemic
states are therefore extremely difficult to identify using post-mortem glucose levels, as low glucose
concentration post-mortem does not necessarily mean low glucose levels ante-mortem50. On the basis
of this phenomenon and its dependency on the PMI, using serum glucose levels in post-mortem analyses is not recommended. Vitreous humor is the specimen of choice for post-mortem diagnosis of diabetes mellitus. Studies have shown stable concentrations of glucose in vitreous humor after an initial decrease within the first 6 hours due to glycolysis49.
2.2.2 Lactate
Lactate is produced by the reduction of pyruvate to generate ATP in anaerobic glycolysis as mentioned. In healthy individuals, lactate concentration ranges from about 0.2 to 2.2 mmol/L in serum and
2.88-5.06 mmol/L in vitreous humor51. After death, lactate values increase significantly in serum and leak
into the vitreous humor due to the loss of selective membrane permeability in the retinal-blood-barrier52.
Traub53 has proposed a formular based on the fact that one glucose molecule produces two lactate
molecules during anaerobic glycolysis to estimate ante-mortem glucose levels by combining glucose and lactate concentration values. A multitude of studies and researchers have supported or questioned the validity of the method, as lactate is also produced by microbes in the cadaver, and its use in post-mortem biochemistry is disputed.
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2.2.3 Ketone Bodies
Ketone bodies include Acetoacetate (AcAc), β-Hydroxybutyrate (bHB) and acetone and are a side product of the beta-oxidation of fatty acids. Ketone bodies are mainly produced by fat cells in the liver during increased fat metabolization as an additional source of energy when glucose is not present in sufficient amounts54. In patients with diabetes mellitus, ketone body production is a result of the inability
to utilize glucose due to either insulin deficiency or resistance. Other states of increased ketone body production are extensive fasting periods, high-fat diets or endurance exercise, where glucose availability
is low9. AcAc is a by-product of fatty acid oxidation, bHB is formed by enzymatic reduction of AcAc
in the liver and acetone is made spontaneously from AcAc. Elevated ketone body production in the body can result in ketonemia and subsequently and more dangerously, ketoacidosis. Common causes of pathological ketoacidosis are diabetic ketoacidosis (DKA) and alcoholic ketoacidosis (AKA). While DKA is caused by diabetes type I or II, alcoholic ketoacidosis is caused by prolonged alcohol intake and malnutrition. AKA shows similar post-mortem biochemical profiles, which is why it is discussed in this context as well. For forensic biochemistry, vitreous bHB is the most specific biomarker to identify ketoacidosis and is therefore preferred when performing biochemical analyses. In healthy patients,
serum levels of bHB are usually lower than 0.25 mmol/L55.
2.2.4 Glycated Hemoglobin and 1,5-AG
In addition to the above-mentioned biomarkers, 1,5-Anhydroglucitol (1,5-AG) and glycated hemoglobin can be used to support a diagnosis of ante-mortem diabetes. 1,5-AG is naturally occurring monosaccharide found in food and, therefore, also in the human body. Its concentration in serum is usually above 14 mg/mL. It competes with glucose for re-absorption in the kidney, which qualifies it as a useful biomarker. The anti-proportional relationship enables inferences about ante-mortem glucose levels from post-mortem glucose levels, as low vitreous 1,5-AG concentrations are associated with extreme hyperglycemia56.
Glycated hemoglobin in serum allows inferences about long-term glycemic control, as glycation levels depend on the blood glucose concentration over the lifetime of an erythrocyte. Glycation of hemoglobin occurs spontaneously and the reaction rate depends on glucose concentration57. For post-mortem
biochemistry, glycated hemoglobin can be used to estimate the metabolic status of the individual in the weeks prior to death. Elevated glycated hemoglobin levels for instance could indicate prolonged hyperglycemia prior to death. Glycated hemoglobin is relatively stable in post-mortem blood, but the
effect of hemolysis has to be taken into account when measuring blood concentrations9.
The above-mentioned biomarkers can help the forensic doctor in determining the cause of sudden death when diabetes mellitus is the underlying condition. Especially in undiagnosed patients that experienced unexpected death and show small to no morphological findings, these markers can be used to identify the exact cause of death based on their concentrations and the combination of marker concentrations. Sampling blood, vitreous humor or pericardial fluid and measuring biomarker concentrations is standard practice for most forensic doctors. Interpreting these findings and putting them into the correct context, however, can be difficult. The following section will provide a manual for forensic doctors that can help in interpreting biomarker concentrations in sudden diabetic death in the context of diabetic ketoacidosis, alcoholic ketoacidosis and hyperosmolar hyperglycemic state.
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2.3 Complications of Diabetes Mellitus
2.3.1 Ante-Mortem Hypoglycemia
Hypoglycemia prior to death can be challenging to diagnose for the forensic doctor, as low blood or vitreous glucose concentrations do not necessarily indicate low blood sugar concentrations ante-mortem. The underlying phenomenon is the continuing metabolization of glucose even after death. Surviving cells continue to produce ATP via anaerobic glycolysis for hours after death. Post-mortem serum glucose levels are therefore decreasing rapidly and the reported serum glucose concentration is highly dependent on the PMI. Karlovsek proposed some criteria for the diagnosis of ante-mortem hypoglycemia. He postulated that low vitreous glucose levels immediately after death (< 3.5 mmol/L), a combined glucose and lactate concentration in vitreous humor below 8.9 mmol/L, low glycated hemoglobin levels in serum due to prolonged hypoglycemic states and other toxicological findings that could indicate abuse of an anti-diabetic drug58.
2.3.2 Diabetic Ketoacidosis
Diabetic ketoacidosis is a severe complication of both type I and type II diabetes that is caused by either the complete absence of insulin (type 1) or the resistance to insulin (type II). Complete insulin absence mostly occurs in patients with undiagnosed diabetes mellitus but can also occur when patients do not take their insulin medication according to their treatment plan or refuse to take it at all59. In addition,
excretion of insulin counter-regulatory hormones increases during absolute and also relative insulin deficiency, especially when stress is involved. The general insulin deficiency and the increase in insulin counter-regulatory hormones leads to a shift in the metabolism of the individual increasing the metabolism of fatty acids and decreasing the metabolism of carbohydrates60. The increase in fat
metabolism leads to the subsequent production of ketone bodies leading to a decrease in blood pH. Acetoacetate, β-hydroxybutyrate and acetone are the most relevant ketone bodies adding to increasing acidosis of blood. The body is capable of buffering early ketonemia but is unable to continue to do so if the condition remains untreated. In diabetic ketoacidosis, ketone body concentration is so high that metabolic acidosis occurs. This process extremely rapid, often developing within 24 hours and has a mortality rate of 2-5%61. DKA is the most frequent cause of sudden death in patients with type I diabetes
mellitus and about 50% of patients under the age of 24 die due to rapidly onsetting DKA62. For the
forensic doctor, DKA is difficult to diagnose, as the death usually is unexpected, morphological and histopathological findings are missing and medical records often do not point towards diabetes mellitus. In cases of diabetic ketoacidosis, performing post-mortem biochemical analyses can be the key to determining the cause of death. The specimen of choice for this analysis is the vitreous humor.
Multiple studies have defined cut-off values for vitreous glucose and bHB levels and most studies coincide with the postulated values to identify DKA. A vitreous humor glucose level exceeding 10 mmol/L (>180 mg/dL) indicates extreme hyperglycemia prior to death63. A vitreous humor bHB
concentration exceeding 2500 µmol/L (>250 mg/L) indicates pathological ketoacidosis64. The
combination of vitreous glucose and bHB above these cut-off values can be used to identify DKA as the cause of death. Additional measurements can include glycated hemoglobin in post-mortem blood and vitreous 1,5-AG levels. High levels of glycated hemoglobin and vitreous 1,5-AG levels below 3.5 mg/mL indicate hyperglycemia in the days prior to death and support the diagnosis of DKA.
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Table 2 – Post-mortem biochemical profile in the context of diabetic ketoacidosis
Biomarker Body Fluid Concentration Validated Analytical
Method Glucose Vitreous Humor > 10 mmol/L (180 mg/dL) Amperometric Glucose Biosensor63 bHB > 2500 µmol/L (> 250 mg/dL) GC-MS 64 1,5-AG < 3.5 mg/mL LC-MS 56 Enzymatic Assay65 Glycated
Hemoglobin Serum elevated HPLC
66
Abbreviations – bHB: beta-Hydroxybutyrate, 1,5-AG: 1,5-Anhydro-d-glucitol
2.3.3 Alcoholic Ketoacidosis
Although diabetes mellitus is not the underlying condition of alcoholic ketoacidosis, it is mentioned in this context because it shows very similar post-mortem biochemical profiles and it can be challenging for the forensic doctor to distinguish between DKA and AKA. AKA occurs in patients with a history of alcohol abuse. Regular intake of significant amounts of alcohol together with malnutrition causing carbohydrate deficiency can lead to increased fat metabolism and subsequent increase of ketone bodies67. Ketoacidosis caused by alcohol intake is often accompanied by hypoglycemia, as ethanol
consumption inhibits glucose production68. Post-mortem blood ethanol levels above 300 mg/100 mL
indicate contribution of ethanol to death. It is important to keep in mind that bacterial metabolism in the deceased body can result in ethanol levels of up to 190 mg/100 mL independent of ante-mortem alcohol
consumption69. In AKA, vitreous levels of bHB above 2500 µmol/L (250 mg/L) again indicate
ketoacidosis. Vitreous glucose levels in AKA, however, are low to normal and not elevated. as they are in DKA.
Table 3 – Post-mortem biochemical profile in the context of alcoholic ketoacidosis
Biomarker Body Fluid Concentration Validated Analytical
Method
Ethanol Serum > 200 mg/ 100 mL Gas Chromatography with flame ionizing detector70 Glucose Vitreous humor < 3.5 mmol/L Amperometric Glucose
Biosensor63
bHB Vitreous humor > 2500 µmol/L
(> 250 µg/L) GC-MS
64
1,5-AG Vitreous humor > 14 mg/mL LC-MS
56
Enzymatic Assay65
glycated
Hemoglobin Serum normal HPLC
12
To distinguish between DKA and AKA, both vitreous glucose and vitreous bHB have to be measured. High vitreous glucose in combination with high vitreous bHB levels indicate death due to DKA, while low to normal vitreous glucose levels in combination with high vitreous bHB levels indicate death due to AKA. To further support the diagnosis of DKA, glycated hemoglobin and vitreous 1,5-AG levels should be measured.
2.3.4 Hyperosmolar Hyperglycemic State
Hyperosmolar hyperglycemic state most often occurs in patients with type II diabetes mellitus. As the name suggests, it is a condition characterized by hyperglycemia, hyperosmolarity due to extreme dehydration and mild ketonemia. Ketonemia in HHS is limited, as the amount of available pancreatic insulin is sufficient to suppress extreme lipolysis, preventing enhanced ketone body production and
subsequent metabolic acidosis71. Progression of HHS is slower than in DKA, developing over days or
even weeks. Dehydration and prolonged osmotic diuresis cause severe electrolyte imbalances that can result in arrhythmia, renal failure, cardiovascular collapse and, ultimately, death16. The mortality rate of
HHS lies at about 15%. The post-mortem profile of HHS shows elevated vitreous glucose levels, indicating ante-mortem hyperglycemia, but no significant elevation of vitreous bHB levels. A cut-off value of 10 mmol/L for vitreous glucose is recommended. Normo-metabolic individuals have shown base vitreous bHB concentrations of up to 480 µmol/L (<50 mg/dL)64. High levels of glycated
hemoglobin and vitreous levels of 1,5-AG below 3.5 mg/mL would support ante-mortem hyperglycemia. In combination with high vitreous glucose levels and normal vitreous bHB levels, death due to HHS is likely.
Table 4 – Post-mortem biochemical profile in the context of hyperosmolar hyperglycemic state
Biomarker Body Fluid Concentration Validated Analytical
Method
Glucose Vitreous Humor > 10 mmol/L (> 180 mg/dL)
Amperometric Glucose Biosensor63
bHB Vitreous Humor < 480 µmol/L
(< 50 mg/dL) GC-MS
64
1,5-AG Vitreous Humor < 3.5 mg/mL LC-MS
56
Enzymatic Assay65
Glycated
Hemoglobin Serum elevated HPLC
66
In the following section, a strategy on how to properly employ post-mortem biochemistry in the context of metabolic disease and sudden cardiac death in forensic laboratories is recommended. In post-mortem biochemistry, efficient coordination of sampling, storing and analysis is crucial and often an overlooked factor and can improve the validity of inferences made from post-mortem biochemical profiles.
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3. Discussion
In summary, every course of death is unique and therefore every forensic examination is different and has to be approached from different angles by the forensic doctor. Unlike clinical diagnostics, where standard procedures are in place for each test and set reference ranges are being used, in forensic medicine the forensic doctor has to make strategic decisions based on the PMI, the condition of the body and the available laboratory infrastructure. Nevertheless, it is an absolute necessity in forensic medicine to establish standard procedures and protocols that can guide the forensic doctor in order to obtain evidence-based and, above all, reproducible results. In the context of post-mortem biochemistry, these protocols need to start at the sampling process. To maximize the potential of post-mortem biochemistry, the collection of specimens already has to be performed in consideration of multiple variables. In general, the forensic doctor should try to sample sufficient amounts of all available body fluids, including bilateral heart blood (~30 mL), vitreous humor (2-5 mL), peripheral femoral or subclavian vein blood (~10 mL), pericardial and cerebrospinal fluid (~10 mL) and urine (~30 mL)72. Sampling
should be performed with an aseptic syringe to avoid any sort of contamination. The availability and also usability of these body fluids depends on the PMI and the overall condition of the cadaver. Body fluids of closed compartments, such as vitreous humor and pericardial fluid, are less sensitive to
decompositional changes and contamination, while blood composition is severely altered by hemolysis3.
Depending on the case, the forensic doctor needs to make decisions on which specimens to sample and should always aim to recover as much usable material as possible. Sampled probes can also be used by toxicology for investigation of toxins or drugs. Analogous to the selection of specimens, the sampling site has to be considered. This relates mainly to the collection of blood, as different sampling sites can yield different results. Nevertheless, a systemic sampling approach is recommended in routine forensic autopsy to maximize the possible number of tests.
To ensure reliable results, samples should be processed for storage immediately. Samples of
low amounts or poor quality can be diluted73. If serum or plasma is needed, these should be separated
before storage. If low amounts of vitreous humor are recovered from each eye, samples can be pooled74.
It is important to keep in mind that even sampled body fluids still undergo post-mortem changes and degradation, so it is crucial to properly store all collected samples. Samples that are stored short-term only can be stored in sealed containers at 4°C. Samples that have to be stored for longer periods of time
should be stored at -20°C or even -80°C72. The analytical methods used for determining post-mortem
biochemical concentrations and patterns highly depend on the available laboratory infrastructure. In general, the analytical phase of post-mortem biochemical examination can be split into two phases. Phase one involves the screening for general conditions, including hematology, the overall electrolyte and mineral status of the individual and possible metabolic diseases. Phase two involves the investigation into tissue-specific markers, including myocardial markers or pulmonal markers3. A
strategy for all different kinds of forensic cases would exceed the scope of this review article, so the next section will focus on the two most relevant but also challenging cases encountered by the forensic doctor and will provide a decision-making model. Advice on how to interpret post-mortem biochemical profiles in the context of sudden death due to diabetes mellitus and sudden cardiac death is given.
3.1 Interpretation of Biomarker Profiles
The number of diagnosed cases of diabetes mellitus is steadily increasing with no end in sight, making it one of the biggest challenges to not only the medical community but also the general public75.
Epidemiological studies and statistics even underestimate the dimension of its prevalence, as cases of diabetes mellitus are only recorded when diagnosed, which usually only happens when the disease
14
shows complications or symptoms. Lethal complications of diabetes mellitus are difficult to evaluate during autopsy, highlighting the importance of standardized and validated routine post-mortem biochemical analyses.
For a first systemic screen, blood composition should be analyzed. Acetone levels in blood are easily identified during routine analysis of blood and the resulting concentration can already assist the forensic doctor in establishing a targeted sampling strategy. Research has shown that elevated serum acetone concentrations are strongly correlated to elevated bHB concentrations, indicating ketoacidosis. Serum acetone concentration > 2 mg/dL indicate elevated overall ketone bodies and indicate a possible underlying metabolic disease64. If acetone concentration in serum is above 2 mg/dL, the forensic doctor
should determine both vitreous glucose and vitreous bHB levels to further narrow down possible causes of deaths. If serum acetone concentration is normal, the forensic doctor should still determine vitreous glucose levels to identify or rule out ante-mortem hyperglycemia. Both elevated vitreous glucose levels (> 10 mmol/L) and vitreous bHB levels (< 2500 µmol/L) indicate death due to diabetic ketoacidosis. Normal to low glucose levels (< 3.5 mmol/L) but high bHB levels (< 2500 µmol/L) indicate death due to alcoholic ketoacidosis. High glucose levels (> 10 mmol/L) but normal to low bHB levels (< 480 µmol/L) indicate death due to hyperosmolar hyperglycemic state. Additional measurements to support the results are vitreous 1,5-AG and serum glycated hemoglobin determination. As the given cut-off values depend on the PMI and the analytical method employed, laboratories should make the effort and establish their own cut-off values based on their analytical methods and instruments. Final conclusions on the cause of death should always be made based on the combination of morphological, histopathological, toxicological and biochemical findings, not on post-mortem biochemical profiles alone. Figure 1 shows a simplified, schematic overview of the proposed decision model. The steps shown do not represent individual steps during sampling and analysis but propose a way of reasoning to correctly interpret post-mortem biochemical profiles in the context of complications of diabetes mellitus.
Figure 1 – Schematic decision-making model when interpreting post-mortem biochemical profiles in the context of diabetes mellitus
15
Cardiovascular diseases remain the leading cause of death worldwide accounting for approximately 17
million deaths every year. About a quarter of these deaths are sudden cardiac deaths76. Coronary heart
disease is responsible for about 80% of all sudden cardiac deaths and is therefore the main underlying condition77. In acute lethal cases, post-mortem biochemistry can help the forensic doctor by either
excluding or indicating sudden cardiac death, but the complex interference of post-mortem processes on myocardial biomarkers impedes the interpretation of post-mortem profiles.
Elevation of biomarkers in adjacent tissues and body fluids after acute myocardial infarct are less specific and depend on the PMI in general, post-mortem redistribution and the severity of myocardial damage2. Since macroscopic findings are more likely in cases of severe myocardial damage,
post-mortem biochemistry is especially valuable when myocardial damage is less severe and not observable. The proper interpretation of biomarker concentrations is particularly important in these cases, but also particularly difficult. In addition to the concentration of a biomarker in one specific body fluid, topological concentration differences can give insight into survival time and PMI. To make valid inferences from biochemical profiles in the context of sudden cardiac death, all myocardium-specific biomarkers should be measured and their concentrations should be checked against laboratory-specific reference ranges. Elevations in cardiac troponins (cTnI and cTnT), CK-MB, BNP and NTproBNP indicate ante-mortem myocardial damage. Morphological and histopathological anomalies should always be prioritized when determining the cause of death, while post-mortem biochemistry should be considered as an auxiliary tool.
3.2 Limitations
In theory, the potential of post-mortem biochemical diagnostics is endless. In practice, however, the forensic doctor deals with challenges when employing post-mortem biochemistry. Deconstructing all the limitations associated with post-mortem biochemical analyses would exceed the scope of this review. The limitations mentioned in this chapter are especially important for the forensic doctor when trying to interpret post-mortem biochemical profiles.
One limitation that is especially evident when comparing post-mortem biochemical diagnostics and clinical diagnostics is sample quality and availability. In post-mortem biochemistry, samples, when available, are of poor quality due to decompositional changes, autolysis and damage. Contamination of specimens in post-mortem analysis is almost inevitable, and analytical procedures need to be able to handle contaminations caused by hemolysis or autolysis to a certain degree3.
The PMI plays a crucial role when interpreting post-mortem biochemical profiles, as few biomarkers are stable over time and almost all biomarkers undergo changes. Especially in unexpected, unwitnessed deaths, the PMI can extend to multiple days. The validity of post-mortem biochemical analysis during autopsy in cases where the PMI is longer than 48 hours is disputed. Some biomarkers are more sensitive than others in regard to PMI and the forensic doctor needs to be careful when choosing biomarkers for post-mortem biochemical analysis. When choosing sampling site and specimen, body fluids from closed compartments, like vitreous humor, pericardial fluid and cerebrospinal fluid should be the preferred specimen of choice, as they are less sensitive to post-mortem changes and contamination than heart and peripheral blood.
In the context of sudden death due to diabetes mellitus, it is important for the forensic doctor to note that glucose is still metabolized in post-mortem blood. Therefore, low serum glucose levels do not necessarily indicate hypoglycemia. The proposed cut-off values for glucose, bHB, acetone, etc. are recommendations based on a variety of studies and primarily depend on the sampling protocols and analytical methods employed by individual laboratories.
In the context of sudden cardiac death, it is important for the forensic doctor to be aware of the specificity of biomarkers. Post-mortem elevations cardiac troponin, for instance, have been proven not only in acute
16
myocardial infarct but also in other myocardial dysfunctions unrelated to ischemia46. It is crucial that
the forensic doctor interprets individual biomarker concentrations in the context of specimen and sampling site, PMI and other biomarker concentrations, as well as morphological and histopathological findings.
4. Conclusion
The potential of post-mortem biochemistry to establish the cause and course of death is undisputed. However, the current state of both theoretical knowledge and practical experience is characterized by more questions than answers. In order to establish post-mortem biochemical diagnostics, the medical community must stop trying to apply the framework of clinical diagnostics to post-mortem biochemical examinations and start treating it as its own field of expertise with its own variables and rules. A standardized approach is needed and forensic doctors must be educated about strengths and weaknesses and especially the limits within post-mortem biochemistry.
To ensure valid interpretation of biochemical results, the acquisition of these results needs to be standardized first. The forensic doctor should be aware that specimens and sampling sites need to carefully be chosen based on PMI, integrity of the body and available analytical possibilities. To prevent further degradation of biomarkers, samples need to be sealed and stored properly. In cases of advanced decomposition, samples should be actively investigated for contaminations to ensure sufficient quality. The method of choice when determining the cause of death in sudden deaths is a general screening approach. Time and money are limiting factors in routine forensic analyses, so a two-step approach has been proposed, including a first phase called systemic screening, where analyses regarding the general ante-mortem condition of the body are performed. This includes electrolyte concentration assessment and metabolic state assessment. A second phase can be valuable when phase one has proven to be inconclusive. In this phase tissue-specific screening is performed. Biomarkers for myocardial damage, lung diseases or brain damages can be investigated to further close in on the cause of death. The key to making valid inferences about the cause of death from biomarker concentrations are validated reference and cut-off values. Cut-off values have been used in clinical diagnostics for decades, but forensic researchers struggle to identify reference values in a forensic setting. A multitude of variables, such as the PMI, mortem redistribution, contamination or the chosen analytical method affect post-mortem biochemical profiles. Forensic research needs to shift the focus from finding the gold standard cut-off value for each biomarker to looking for more variable reference ranges. No death process is the same, peri-mortem conditions vary from case to case and the quality of post-mortem samples cannot reach clinical standards, which is why databases must be established where all these variables are taken into account. To set up these databases, more data is required. Studies on animal models are not sufficient in these cases. Data from medicolegal autopsies is needed to establish databases for more flexible cut-off ranges that are dependent on the chosen specimen and the PMI. Furthermore, forensic laboratories should aim to establish their own databases based on their equipment and their procedures. Post-mortem biochemistry has gained a lot of attention over the last years as more and more people acknowledge its potential. However, both forensic researchers and forensic doctors need to detach it from the framework of clinical diagnostics, as post-mortem biochemistry requires its own protocols, methods and databases. Recent findings have shown the potential of post-mortem biochemistry and with both standardized protocols and flexible reference ranges it can significantly benefit the forensic doctor when establishing the cause of death of an individual.
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