EXCITATION study: Unexplained in-custody deaths: Evaluating biomarkers of stress and agitation

Journal of Forensic and Legal Medicine 66 (2019) 100–106

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Research Paper

EXCITATION study: Unexplained in-custody deaths: Evaluating biomarkers of stress and agitation

T

Gary M. Vilkea,∗, Deborah C. Mashb, Marta Pardob, William Bozemanc, Christine Halld, Christian Sloanea, Michael P. Wilsone, Christopher J. Coynea, Xiaobin Xieb, Edward M. Castilloa a

University of California, San Diego Medical Center, Department of Emergency Medicine, San Diego, CA, USA University of Miami Miller School of Medicine, Department of Neurology and Molecular and Cellular Pharmacology, Miami, FL, USA c Wake Forest University School of Medicine, Department of Emergency Medicine, Winston Salem, NC, USA d University of British Columbia, Department of Emergency Medicine, Island Health, Victoria, Canada e University of Arkansas, Department of Emergency Medicine, USA b

A R T I C LE I N FO

A B S T R A C T

Keywords: Excited delirium syndrome Agitation Biomarker Sudden death, arrest related death Stress

Background: Law enforcement personnel often confront violent and dangerous individuals suffering from Excited Delirium Syndrome (ExDS) who need emergent medical evaluation and treatment to optimize the best outcomes for this potentially lethal medical emergency. These subjects typically require physical restraint and use of force measures to control them. We sought to determine if stress-related biomarkers can differentiate ExDS subjects when compared with agitation and stress under other circumstances, including agitation and extreme physical exhaustion and restraint coupled with emotional stressors. Methods: This was a prospective multi-center study enrolling a convenience sample of patients who presented with agitation or ExDS. Patients were enrolled from three academic emergency departments (ED), two in the United States and one in Canada. Three study groups (SG) included: SG1) patients brought to the ED with ExDS based on the use of standardized clinical criteria; SG2) ED patients with acute agitation who were not in a clinical state of ExDS but required sedation; SG3) a laboratory control group of subjects exercised to physical exhaustion, restrained, and psychologically stressed with threat of Conducted Energy Device (CED) activation. We examined a panel of stress-related biomarkers, including norepinephrine (NE), cortisol, copeptin, orexin A, and dynorphin (Dyn) from the blood of enrolled subjects. Results: A total of 82 subjects were enrolled: 31 in the agitation group, 21 in the ExDS group, and 30 in the laboratory control group. Data were analyzed, comparing the findings between ExDS and the two other groups to determine if specific stress-related biomarkers are associated with ExDS. Biomarker comparisons between subjects identified with ExDS, agitation, and control groups demonstrated that cortisol levels were more elevated in the ExDS group compared with the other groups. Orexin was only significant in ExDs (with Agitated tendency but lot of variability in the group). NE and Dyn increased as response to stress in Agitated and ExDS. Conclusions: Cortisol levels were more elevated in subjects in the ExDS group compared with the other comparison groups and orexin was elevated in ExDS compared to controls, a trend that did not reach statistical significance in the agitated group. The clinical or diagnostic significance of these difference have yet to be defined and warrants further study.

1. Background Law enforcement personnel often confront violent and dangerous individuals suffering from Excited Delirium Syndrome (ExDS) who need emergent medical evaluation and treatment to optimize the best outcomes for this potentially lethal medical emergency. These subjects commonly are sympathomimetic abusers or patients with psychiatric

∗

disorders, such as schizophrenia and bipolar disorder, who may be noncompliant with their medications. ExDS is common enough that it is seen in emergency departments across the country, but not so common that good epidemiologic studies can be performed. The bigger concern is that ExDS is associated with reported fatality rates up to 11%.1 Although patients presenting in this state of ExDS have an increased propensity to die, the majority of cases are not fatal. Current challenges

Corresponding author. 200 West Arbor Drive, Mailcode 8676, San Diego, CA, 92103, USA. E-mail address: [email protected] (G.M. Vilke).

https://doi.org/10.1016/j.jflm.2019.06.009 Received 3 April 2019; Received in revised form 5 June 2019; Accepted 16 June 2019 Available online 21 June 2019 1752-928X/ © 2019 Published by Elsevier Ltd.

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ethnicity or sexual preference. In the consenting process the subject was told of the study protocol and that they would be receiving a 5 s TASER X26 CED activation in drive stun mode to the thigh. This was done to induce a psychological stress response. However, no actual contact between subject and the device was made during the implemented study protocol. The subjects had an intravenous catheter placed into one of the upper extremities from which to draw blood using standard sterile technique. Blood sample was taken to determine basal levels of the biomarkers focus of interest (time point 1, pre exercise). The subject then performed a maximal 600 m treadmill run until maximal oxygen consumption was achieved. Immediately following exercise, another blood sample was obtained (time point 2, post exercise). The subject was then immediately placed in the hobble restraint position defined as prone position on an exercise mat, with wrists restrained behind the back, ankles secured by standard law enforcement hobble cuffs, and attached to the wrist restraint at a distance such that the knees are flexed at 90°. Subjects remained in this position for 10 min. Blood sample was taken after being placed in the restraint position (time point 3, post restraint). At 5 min into the restraint position, subjects were informed that a TASER ECD activation would be deployed on their thigh. Blood sample was taken before receiving the TASER (time point 4, pre exposure to TASER). The TASER X26 CED was then brandished in front of the subject and spark activated within the visual range of the subject. After this, the subject was advised they would receive a 5 s application to the thigh. The handle of the TASER X26 CED was pressed against the subject's skin, the operator announced “Clear, TASER, TASER” and then activated the device while not touching the subject. This procedure was designed to give subjects the emotional stress of an expected activation, without the physical pain. Following this event, a blood sample was drawn (time point 5, post exposure to TASER. At the 10-min point in restraints, a final blood sample was drawn (time point 6, post restraint TASER). All venous blood samples were processed and stored as per established protocol. Processing at the study site includes centrifugation of four vacutainer sample tubes and extraction of 0.5 ml aliquots of serum into 1.25 ml vials for shipping and analysis. The vials were placed into a −20C or −80C freezer for temporary storage, then shipped using refrigerated containers to the University of Miami Biorepository and Laboratory Data Site in Miami for analysis. Subjects on SG3 who completed the study received $100 compensation for their participation. Biomarker panel assays were performed at the Biorepository and Laboratory at the University of Miami. All assays were performed using protocols as established by the manufacturer. Norepinephrine (NE) was measured in plasma using the Human Norepinephrine ELISA Kit (Ct. # MBS9363244, MyBiosource, LLC). The sensitivity of this kit is 1.0 ng/ ml. The detection range is 3.12 ng/ml- 100 ng/ml. Cortisol was measured in serum using KGED008B Cortisol Assay (R&D Systems). The detection range of cortisol is 0.156 ng/ml-100 ng/ml. Detection and quantitation of Orexins A in serum were measured using Human Orexin A ELISA Kit (Cat. # MBS263266, MyBioscource, LLC). The detection range of orexin is 15.6 pg/ml-1000 pg/ml with the minimum detectable human orexin up to 5 pg/ml. Dynorphin (Dyn) was measured in serum using human dynorphin, Dyn ELISA Kit (Cat. # MBS701322, MyBioscource, LLC). Detection range of the kit is 3.12 pg/ml-200 pg ml with a minimum detection of 0.78 pg/ml. Quantitation of Concentration of copeptin in plasma was measured using human copeptin ELISA Kit (Cat. # 606841, Phoenix Pharmaceuticals, Inc). Detection range of this kit is 0 ng/ml-100 ng/ml. Quality control (QC) samples intra and inter-assay variation was established by measuring each QC five times per assay and in three different assays. Intra-assay and between-assay data variations were tested for all biomarkers with part of samples by assays performed in multiple wells of same plate, or in multiple ELISA plates. Data for all blood biomarkers measured were analyzed using a commercial

for emergency physicians treating ExDS patients as well as for forensic investigators evaluating subjects after in custody deaths include the lack of an easily obtainable test to evaluate for and confirm a diagnosis of ExDS.1–3 Currently, the only tests that can be performed to assist medical examiners in identifying anatomical changes for ExDS is on postmortem brains.4 Currently here are no biochemical or genetic tests for ExDS subjects who have not died. The purpose of this study was to determine if stress-related biomarkers can differentiate and possibly diagnose ExDS in subjects when compared with agitation and stress under other clinical circumstances. 2. Methods This was a prospective multi-center study enrolling a convenience sample of patients who presented to one of three hospitals with agitation or ExDS comparing with subjects enrolled to participate in a controlled laboratory model of stress. Subjects for the first two study groups were enrolled from three academic emergency departments, two in the United States and one in Canada. Subjects for the third study group were recruited from a college campus. The three study groups (SG) included: SG1) ED patients with acute agitation who were not in a clinical state of ExDS but required sedation; SG2) included ED patients brought with signs and symptoms meeting established clinical criteria for ExDS; and SG3) a laboratory control group of subjects exercised to physical exhaustion, restrained, and psychologically stressed with threat of Conducted Electrical Device (CED) activation. The selection criteria for SG2 was based on the subject meeting 6 or more of the 10 clinical criteria defined by Hall as: pain tolerance, tachypnea, sweating, agitation, tactile (or measured) hyperthermia, non-compliance with police (or medical personnel) directions, lack of tiring, unusual strength, inappropriately clothed, and mirror or glass attraction.5 All groups had blood drawn to examine a panel of stress-related biomarkers, including norepinephrine (NE), cortisol, copeptin, orexin A, and dynorphin (Dyn) on the blood obtained from the subjects. Blood was drawn in SG 1 and SG2 in the emergency department as soon as possible by nursing during the acute agitation episode. IRB approval was obtained at all enrollment sites including North Carolina, San Diego, Miami, Florida, and Victoria, Canada. The initial consenting of the blood draws for SG1 and SG2 was considered exempt and patients were subsequently consented when the acute agitation episode had passed and the patient had capacity to give written informed consent to use the blood for the study. SG3 participants gave informed written consent to participate. Blood draws were performed according to a standard operating procedure to ensure reliable sampling across sites. The SG3 subjects served to determine the impact of physiological as well as emotional stress on the biomarkers in a drug free and psychologically stable population. The blood draws for SG3 were done at different time points that are explained below. Subjects in SG3 were enrolled in a prospective trial to study stress associated with exercise, physical exhaustion, and restraint with an induced psychological stress simulating field settings of an ExDS event. Twenty-five healthy adults 18–45 years of age were recruited to participate in the study. Subjects underwent an exercise period followed by being placed into a hobble restraint with induced psychological stress as described below. Before and after the exercise stress period, blood was collected. Subject exclusion criteria for the SG3 control group portion of the study included: pregnancy, recent illicit drug use or history of previous chronic illicit drug use, current illness, chronic psychiatric medical diagnosis requiring medical therapy, inability to be handcuffed behind the back, inability to exert themselves on a running treadmill machine, a negative response to PAR-Q questionnaire utilized to self-screen individuals for exercise testing (www.csep.ca/pdfs/par-q.pdf), or refusal of consent. No exclusion was made on the basis of gender, race, 101

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agitated patients, and 2.15 ± 0.92 pmol/L in ExDs patients. However, one-way ANOVA analysis for orexin A yielded significant differences (F (2, 62) = 3.03, p = 0.05; Fig. 1C). Orexin concentrations were 0.79 ± 0.37 ng/ml in the control group, 0.84 ± 0.50 ng/ml for agitated patients, and 1.09 ± 0.39 ng/ml in ExDs patients. Post-hoc analyses showed significant effect between control and ExDS group (p < 0.05) as well as a clear trend towards significance between agitated and ExDS condition (p = 0.07). An overall significant correlation between orexin A levels and race was found (Table 3). One-way ANOVA analysis did not reach statistically significance for dynorphin (F(2,67) = 2.27, p = 0.11; Fig. 1D). Dyn concentrations were 343.5 ± 174.7 ng/ml in the control group, 493.5 ± 307.5 ng/ml for agitated patients, and 481.4 ± 298.9 ng/ml in ExDs patients. However, individual t-tests yielded significant increase in the agitated group compared to control (t(47) = 2.088, p < 0.05) as well as a strong increase trend in the ExDS group compared to control (t (43) = 1.92, p = 0.06). Finally, significant effects of experimental group were found for NE (F (2, 53) = 3.23, p < 0.05; Fig. 1E). NE concentrations were 2.68 ± 0.51 ng/ml in the control group, 3.58 ± 1.63 ng/ml for agitated patients, and 3.35 ± 1.17 ng/ml in ExDs patients. Post-hoc analyses yielded significant differences between control and agitated (p < 0.05) as well as control and ExDS (p < 0.05).

computer software (GraphPad Prism6). Concentrations were analyzed using ANOVA analysis with ad hoc comparisons. Correlation analyses were performed using Pearson correlation coefficient. Data were expressed as mean ± standard error (SEM), and a p value of less than 0.05 represents the statistical significance. For the ED field SGs, the primary data analysis was designed to determine if any differences in the biomarker blood panels exist between subjects in SG2 (ExDS) with the agitated subjects in SG1(agitated). For the laboratory physiologically stressed subjects in SG3, the primary data analysis was designed to determine if there were any differences in the biomarker blood panels between time points based on the condition using an ANOVA with ad hoc comparisons. Data were expressed as mean ± standard error (SEM), and a p value of less than 0.05 represents the statistical significance. 3. Results A total of 82 subjects were enrolled: 31 in the agitation group (SG1), 21 in the ExDS group (SG2), and 30 in the laboratory control group (SG3). The demographic data has been grouped based on the analyses. Due to availability of drawing blood samples, the N between conditions can vary. The demographics for all control subjects that were part of the laboratory study are presented in the top of Table 1. The overall demographics for all groups that were part of the blood biomarkers analysis including patients of the emergency department are presented in Table 1 grouped by biomarker analysis. The gender distribution between groups as well as the average age are represented. Biomarker comparisons were performed with the blood drawn in the emergency department at the time of the acute agitation episode for SG1 and SG2. Blood draw number five, after the exercise and sham TASER activation, was used for comparison with the SG3 group. Biomarker comparisons for SG3, laboratory group, are presented in Table 2. There was a significant effect of time for each biomarker. Individual ANOVAs were performed. One-Way ANOVA for cortisol showed significant effect of time of blood drawn (F (5, 135) = 5.041, p < 0.01). Planned comparisons yielded increased cortisol levels pre exposure to TASER (time 4) (p < 0.05). Additionally, One-Way ANOVA for Orexin A also showed significant effect of time of blood drawn (F(5, 137) = 4.473, p < 0.01). However, increased of orexin observed at time 4 did not reach statistical significance when compared to time 1 (starting time, pre exercise). Dyn levels followed same tendency (One-Way ANOVA (F(5, 130) = 4.24, p < 0.01)). Planned comparisons revealed significant increase in Dyn levels at time 4 of blood drawn (p < 0.05). In the opposite direction, independent OneWay ANOVAs for copeptin and NE, yielded significant effects of time of blood drawn (Copeptin: F (5135) = 11.60, p < 0.01; NE: F (5, 117) = 8.57, p < 0.01). Planned comparisons showed reduced levels of copeptin at time 3 (post restraint) and 4 (p < 0.05) and reduced levels of NE in tine 2 (post exercise) and 3 (p < 0.05). Biomarker comparisons between SG1, SG2 and SG3 are presented in Fig. 1. Correlation analysis for each biomarker with age, sex and race are presented in Table 3. We tested levels of cortisol in serum of controls, agitated and ExDS patients. One-way ANOVA analyses showed significant effect of experimental condition (F(2,73) = 7.88, p < 0.01; Fig. 1A). Planned post hoc analyses yielded significant increases in blood cortisol in ExDS cases (p < 0.05 compared to control and agitated conditions). Cortisol concentrations were 9.3 ± 2.7 μg/dL in the control group, 8.9 ± 3.5 μg/dL for agitated patients, and 12.7 ± 4.6 μg/dL in ExDs patients. Individual data from each cohort are reported in Fig. 1A. Importantly, no significant correlations were observed between measured blood cortisol level and major demographic factors, including age, gender and race in all subjects from the three cohorts (Table 3). No significant differences were found between groups for copeptin (F (2, 78) = 2.011, p = 0.14; Fig. 1B). Copeptin concentrations were 1.92 ± 0.45 pmol/L in the control group, 1.81 ± 0.41 pmol/L for

4. Discussion The term Excited Delirium Syndrome (ExDS) had traditionally been used in the forensic literature to describe findings in a subgroup of patients with delirium who suffered lethal consequences from their untreated severe agitation.3,5–7 Although the exact signs and symptoms are difficult to define precisely, clinical findings often include many of the following: tolerance to significant pain, rapid breathing, sweating, severe agitation, elevated temperature, delirium, non-compliance or poor awareness to direction from police or medical personnel, lack of fatiguing, unusual or “superhuman strength”, and inappropriate clothing for the current environment.1–3 Historically, subjects exhibiting such signs and symptoms were often encountered by law enforcement, and not infrequently the subject would suffer a sudden cardiac arrest and die. Over the decades, restraint position, weight placed on the torso, CED's and other less lethal technologies have been reported as the cause of death in many of these cases, though there is still controversy.8–10 Examining the relationship between physiologic stress and unexplained in-custody deaths was the focus of this study. Since sudden deaths in police custody have clearly occurred in individuals placed in the hobble, hogtie or prone maximal restraint (PMR) position, some have argued that the PMR position prevents adequate chest wall, abdominal, and diaphragmatic movement, leading to hypoventilatory respiratory compromise and risk for so-called positional asphyxia.11 However, multiple case series of the sudden deaths of restrained individuals do not indicate a specific mechanism.11–15 Historical and autopsy evidence is often unhelpful as there is no anatomic cause of death determined in most cases. Importantly, similar sudden deaths in custody have been reported in patients who were not restrained in the PMR position, but in the prone, side and even sitting positions.16,17 Deaths from the application of weight to the torso have also been described in the medical literature.18 The term “traumatic” or “mechanical asphyxiation” have been applied to cases in which extreme force was applied to individuals, such as when an automobile runs over the torso of an individual. Research evaluating position and weight on the back in normal subjects showed that there was no evidence supporting the theory of compressional asphyxia as evidenced by hypoxia (oxygen desaturation) or hypercapnia (CO2 retention).19–22 A constellation of clinical symptoms and findings is referred to as ExDS.1,2,5 A task force was sponsored by the American College of 102

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Table 1 Demographics study groups SG1, SG2 and SG3 used for blood biomarker analysis. CORTISOL

Controls (n = 30)

Agitated (n = 25)

ExDS (n = 21)

M = 23

W=7

M = 21

W=4

M = 17

W=4

Age (yrs)

30.61 ± 9.1

22.4 ± 1.1

38.1 ± 11.0

40.5 ± 16.9

36.2 ± 10.4

33.8 ± 9.8

Race White Hispanic Asian Black Other

11 3 6 1

5 0 2 0

13 3 0 5

2 0 0 2

13 2 0 1 1

4 0 0 0

Controls (n = 21) M = 15 27.5 ± 5.2

W=6 22.3 ± 1.2

Agitated (n = 23) M = 19 39.3 ± 10.5

W=4 40.5 ± 1.2

6 3 5 1

5 0 1 0

11 3 0 5

2 0 0 2

Controls (n = 28) M = 21 31.5 ± 9.3

W=7 22.4 ± 1.1

Agitated (n = 25) M = 21 38.1 ± 11.0

W=4 40.5 ± 16.9

11 4 5 1

5 0 2 0

13 3 0 5

2 0 0 2

Controls (n = 29) M = 22 30.6 ± 9.3

W=7 22.4 ± 1.1

Agitated (n = 31) M = 24 37.7 ± 11.1

W=7 48.3 ± 10.6

10 4 7 1

5 0 2 0

14 4 0 6

2 0 0 5

Controls (n = 23) M = 18 26.8 ± 5.1

W=5 22.1 ± 1.3

Agitated (n = 21) M = 19 35.3 ± 11.1

W=2 53 ± 12.7

8 3 6 1

4 0 1 0

11 3 0 5

1 0 0 1

OREXIN A Age (yrs) Race White Hispanic Asian Black Other DYNORPHIN Age (yrs) Race White Hispanic Asian Black Other COPEPTIN PHRINE Age (yrs) Race White Hispanic Asian Black Other NOREPINE Age (yrs) Race White Hispanic Asian Black Other

ExDS (n = 21) M = 17 36.2 ± 10.4

13 2 0 1 1 ExDS (n = 21) M = 17 36.2 ± 10.4

13 2 0 1 1 ExDS (n = 20) M = 17 36.2 ± 10.4

13 2 0 1 1 ExDS (n = 12) M = 10 33.7 ± 8.5

8 0 0 1 1

W=4 33.8 ± 9.8

4 0 0 0

W=4 33.8 ± 9.8

4 0 0 0

W=3 35.7 ± 11.0

3 0 0 0

W=2 37.5 ± 13.4

2 0 0 0

and Baldwin et al. who found ExDS in 2% and 1.5% of police use of force subjects respectively, using the same criteria, in a total of over 8 million police-public interactions.27,28 Ross et al. reported that twelve percent of a cohort of 635 arrestees who exhibited symptoms of ExDS had seven or more of the recognized symptoms associated with ExDS.29 Subsequent anatomic and molecular characterization of fatal ExDS patients has focused primarily on postmortem brain examination findings. Mash et al.4,30,31 demonstrated that there are discrete neurochemical pathology changes in certain brain circuits associated with ExDS. The biological signature of excited delirium includes dysregulated dopamine transporters (hyperdopaminergic state), elevated heat shock proteins (hyperthermia), and immediate early gene activation as a marker of paranoid aggression (c-fos protein). Stress is defined as any influence that throws the body out of homeostatic balance.32 Significant stress induces changes in higher nervous system/sleep wakefulness, fear, autonomic function and

Emergency Physicians (ACEP) in 2009 to provide consensus on the issues of definition, diagnosis, and treatment of ExDS. The ACEP joined the National Association of Medical Examiners (NAME) in officially recognizing ExDS as a medical condition.3 Although knowledge of the etiology and pathophysiology of ExDS is limited, basic science and clinical studies have provided some insight.23–26 Over a two-year study, ten potential clinical features of ExDS were recorded by Canadian police for cases seen in over one million police-public interactions.5 The features of ExDS included pain tolerance, tachypnea, sweating, agitation, tactile hyperthermia, non-compliance with police, lack of tiring, unusual strength, inappropriately clothed, and mirror or glass attraction. Of the 698 encounters involving use of force, 24 (3.4%) probable ExDS cases were identified, based on the presence of abnormal behavior and at least 6 of the 10 clinical criteria for ExDS. Only 18 (2.7%) subjects in the cohort had seven or more features including tactile hyperthermia. These findings are echoed by further work by Hall et al. 103

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magnitude and degree of involvement of stress-related mediators in ExDS. Therefore, we examined a multi-biomarker panel based on several parameters that each mirror different pathophysiologic aspects of the stress response. This panel included a number of well-established mediators of stress, including: NE, corticosteroids (cortisol – canonical stress hormone), orexin (flight or fight response and stress-induced hyperthermia), dynorphin (stress-related dysphoria), and copeptin (hormone biomarker of cardiovascular stress). Each of these stress responses are integrated with significant crosstalk between them. These markers were chosen given their known association with stress and physiologic importance. As ExDS represents what appears to be a centrally driven physiologic response that is atypical, though many of the markers chosen have specific patterns of rise and fall in “normal” subjects, individuals in as a state of ExDS are not normal or typical and thus these markers may be abnormally high or have elevations that are prolonged compared with other groups. Any stressor that activates the HPA axis leads to an increase in concentrations of the adrenal stress hormone cortisol. Our study confirms increases in cortisol but only in the ExDS group. One of the major hypothalamic stress hormones, stimulated by different stressors is vasopressin (AVP).32,33 However, measurement of circulating AVP levels is challenging because it is released in a pulsatile pattern, it is unstable and it is rapidly cleared from plasma. AVP derives from a larger precursor peptide (pre-provasopressin) along with copeptin, which is released in an equimolar ratio to AVP. Copeptin is more stable in the circulation and easy to determine.34 Copeptin levels are associated with risk of death and cardiac stress.35 Since ExDS patients are intrinsically at high risk for cardiac arrest, measures of copeptin may prove important for identifying cardiac stress in ExDS cases. Unexpectedly, no significant differences were found in copeptin levels between controls, agitated and ExDS patients. NE is rapidly released shortly after a stressful event and can be measured in blood and urine using published HPLC methods.36–38 Catecholamines together with catecholamine metabolites can be correlated with other hormone mediators of the stress response. Stress releases a burst of cortisol on top of circadian rhythm, which integrates the Central Nervous System (CNS) and peripheral responses to stress over hours to days.39 Our data suggests that NE levels are significantly increased in response to stress in agitated and ExDS patients, with no

Table 2 Biomarker analysis for the laboratory study group (SG3). Time point 1 Cortisol N 22 Mean 7.047 Std Dev 3.220 SEM 0.6866 Orexin A N 24 Mean 19.69 Std Dev 14.17 SEM 2.892 Dynorphin N 21 Mean 1.985 Std Dev 1.349 SEM 0.2943 Copeptin N 22 Mean 1.827 Std Dev 0.4382 SEM 0.09343 Norepinephrine N 20 Mean 19.63 Std Dev 4.375 SEM 0.9784

2

3

4

5

6

24 7.089 4.006 0.8177

22 7.324 2.551 0.5440

25 10.94* 3.069 0.6139

24 9.729 3.901 0.7963

24 8.583 3.961 0.8085

24 17.85 15.06 3.075

22 8.167 7.854 1.674

25 26.90 21.34 4.269

25 22.73 18.47 3.694

23 12.88 10.37 2.162

23 2.459 1.405 0.2929

24 2.327 1.218 0.2486

21 4.132* 3.046 0.6646

23 3.055 1.516 0.3160

24 3.233 1.436 0.2932

25 1.793 0.5971 0.1194

23 1.276* 0.3722 0.07761

23 0.8970* 0.2339 0.04877

24 1.476 0.8051 0.1643

24 1.849 0.5719 0.1167

21 16.60* 4.760 1.039

20 14.00* 2.732 0.6109

20 20.16 3.676 0.8219

21 17.18 4.234 0.9239

21 14.60* 3.010 0.6569

Time points: 1 (pre exercise), 2 (post exercise), 3 (post restraint), 4 (pre exposure to TASER), 5 (post exposure to TASER), 6 (post restraint TASER). *p < 0.05 compared to 1 (pre exercise).

activation of the hypothalamo-pituitary-adrenal (HPA) axis.32 There are multiple influences on stress, including duration, type of stress (physical vs. psychological), context of the encounter, age, gender, and genetic make-up. Stress releases various substances in the brain and blood including neurotransmitters (monoamines), neuropeptides, and hormones.32 Each mediator of stress is affected by type of stress, genetic background, age and gender. Because a stress response takes place on timescales that span milliseconds to days, we wanted to evaluate multiple neurotransmitter, peptides and hormones to determine the

Fig. 1. Blood biomarker level from control, Agitated and ExDS group. Average value comparison (Mean ± SEM), (horizontal bar represents average value). *p < 0.05. Individual dots represents individual subject data. Fig. 1a-e. Biomarker boxplot comparisons by Emergency Department Study Group. 104

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Table 3 Correlations between biomarker level and demographic parameters. Control

Cortisol

Orexin A

Dynorphin

Copeptin

Norepinephrine

Age Gender Race Age Gender Race Age Gender Race Age Gender Race Age Gender Race

Agitated

ExDS

Total

r

p

r

p

r

p

r

p

0.174 −0.136 −0.256 0.325 0.009 0.198 0.003 0.014 0.000 0.032 0.000 0.091 0.024 0.062 0.028

0.358 0.474 0.173 0.007* 0.687 0.043* 0.770 0.547 0.918 0.354 0.942 0.111 0.480 0.251 0.443

−0.168 −0.292 −0.136 0.038 0.003 0.095 0.065 0.005 0.067 0.026 0.020 0.005 0.060 0.255 0.017

0.421 0.157 0.516 0.369 0.795 0.152 0.219 0.727 0.212 0.383 0.451 0.706 0.284 0.020* 0.571

0.105 −0.256 −0.067 0.009 0.001 0.003 0.033 0.146 0.010 0.084 0.080 0.122 0.000 0.020 0.131

0.652 0.263 0.771 0.686 0.902 0.815 0.433 0.087 0.673 0.216 0.226 0.131 0.983 0.660 0.248

0.042 −0.120 −0.203 0.032 0.001 0.074 0.000 0.018 0.005 0.000 0.023 0.003 0.063 0.002 0.015

0.720 0.084 0.079 0.153 0.804 0.028* 0.953 0.253 0.531 0.948 0.178 0.650 0.062 0.718 0.373

Values represent correlation coefficient (r) and statistical significance (p value). *p < 0.05.

those at higher risk of sudden cardiac arrest and guide clinical care or indicate the potential need for extended monitoring or additional studies such as echocardiography in this subgroup.

significant differences between both groups of patients. Multiple lines of evidence indicate that hypocretin/orexin (HCRT) participates in the regulation of arousal and arousal-related process.40–42 Orexin plays a role in consolidation of waking and/or coupling metabolic state with behavioral state.43 Additionally, substantial evidence suggests a potential involvement of HCRT in high-arousal conditions, including stress. Orexin is reliably measured in plasma and circulating levels can act in brain44. Orexin neurons contribute to cardiovascular, respiratory and analgesic components of the fight-orflight response against stressors45 as well as play an important role in the regulation of appetite, arousal and motivation. . Orexins also have been shown to be involved in stress-mediated hyperthermia.46 Published methods using ELISA in plasma or serum demonstrate that this marker can be reliably measured.47,48 Our data supports that ExDS patients present increase Orexin A levels, levels that differ significantly from control subjects. Agitated patients do not show significant differences between controls, nor ExDS, demonstrating a bigger variability in terms of Orexin A levels in the agitated group. Recent reports have shown increases in orexin A related to drugs of abuse and periods of drug abuse abstinence.49 Land et al.50 first described a mechanism of dysphoria in which corticotropin-releasing factor (CRF) provokes Dyn release. Dyn, a member of the opioid peptide family that binds to kappa opiod receptor, has been related to epilepsy, addiction, depression and schizophrenia. Dynorphin-A-(1–8), is present in vasopressin-containing neurosecretory cells terminating in the neural lobe of the pituitary gland. Dyn is released in the pituitary concomitantly with vasopressin during the antidiuretic response. Exposure to stress induces a rapid dyn release. The intersection of stress-induced CRF and the dynorphin/kappa opioid receptor (KOR) system in the amygdala suggests that CRF and dynorphin/KOR systems may coordinate stress-induced anxiety behaviors.51 As seen in NE, our data suggests that dyn levels act as a marker of stress response, since we found significant increases in agitated and ExDS patients. We found in our evaluation of the panel of five biomarkers, that cortisol blood levels were significantly elevated in ExDS cases compared to controls and agitated patients that underwent through similar stress conditions compared to ExDS patients. In the present study, the addition of an agitated group demonstrated the sensitivity and specificity of cortisol as an abnormal mediator in ExDS. This is the first study of this kind assessing biomarkers for ExDS and agitation. Thus, there are no other studies with which to compare these findings. The potential clinical utility of identifying a clinical biomarker of ExDS could one day lead to a blood test to help clinicians distinguish patients with ExDS from less lethal forms of agitation, and to identify

4.1. Limitations This study has a number of limitations. Although established criteria were used to define ExDS patients compared with agitated patients, it is possible that patients could have been mis-categorized. As with all studies involving multiple sites and multiple blood draws, it is possible that samples could have been mislabeled or processed in error. However, standardized procedures of multi-checks were implemented to minimize these risks. The healthier college campus recruited SG3 may have had different baseline physiology and pathology compared with subjects in SG1 and SG2, creating the possibility of varying results of the biomarker panels. Sample size is a limitation in this study. With a total of 21 patients in the ExDS study group and 31 in the agitation group, it is possible that a statistically significant difference between biomarkers other than cortisol has not been detected. While we have documented a significant difference in serum cortisol between the ExDS group and other agitated persons, further study is needed to describe this difference with the precision needed for clinical utility or diagnostic criteria. Demonstration of this difference in our study will aid in sample size calculations for future work. Further larger scale study is also necessary to document whether other important markers of severe stress are significantly different between those in a state of ExDS vs other agitated states. 5. Conclusions Biomarker comparisons between subjects identified with ExDS, agitation, and control groups demonstrated that mean cortisol levels were significantly different between study groups, more elevated in the ExDS group compared with the other groups. Additionally, orexin was significantly increased only in the ExDS group. Given the limitations of this preliminary study and associated comparisons between groups, the clinical or diagnostic significance of these differences has yet to be defined and warrants further study. Conflict of statement Gary Vilke, Christine Hall, Christian Sloane and Michael Wilson are paid legal consultants. There are no other conflicts of interest to report. 105

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G.M. Vilke, et al.

CRediT authorship contribution statement 23.

Gary M. Vilke: Conceptualization, Data curation, Funding acquisition, Methodology, Writing - original draft. Deborah C. Mash: Conceptualization, Data curation, Funding acquisition, Methodology, Writing - review & editing. Marta Pardo: Data curation, Validation, Writing - review & editing. William Bozeman: Conceptualization, Data curation, Funding acquisition, Methodology, Writing - review & editing. Christine Hall: Conceptualization, Data curation, Funding acquisition, Methodology, Writing - review & editing. Christian Sloane: Methodology, Project administration, Data curation, Writing - review & editing. Michael P. Wilson: Data curation, Writing - review & editing. Christopher J. Coyne: Data curation, Writing - review & editing. Xiaobin Xie: Data curation, Validation, Writing - review & editing. Edward M. Castillo: Project administration, Supervision, Validation, Writing - review & editing.

24. 25.

26. 27.

28.

29. 30. 31.

Acknowledgment 32.

National Institute of Justice Funding Award Number: 2012-R2-CXK006.

33.

References

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