To identify the characteristics of xylazine poisoning cases in clinical and forensic practice, “Xylazine poisoning” in PubMed and CNKI, excluded the cases without humans involved, and identified 20 papers which included more than 160 cases; the related information is summarized in Table 1 [8,10,11,13,15,28,29,30,34,35,36,37,38,39,40,41,42,43,44,45]. As shown in Table 1, most of the involved cases were accidental; however, homicide and suicide were also seen in the xylazine poisoning cases. It was reported that it may occur in persons who accidentally ate meat that was injected with xylazine, and due to the rather small dose of xylazine, the victim may be rescued [28,29]. This was associated with mass poisoning incidents, and it may occur in homicide cases. In 2023, Xu et al. reported that a criminal purchased xylazine on the internet and poisoned the breakfast in a hotel; the scene investigation revealed that the criminal had been fired a few ago and he did this for revenge. Recently, injection was a common exposure of xylazine in the USA, and it was also combined with fentanyl or heroin in those illicit drug overdoses [3,4]. However, inhalation was a new approach in the exposure of xylazine and it may become popular as necrotizing skin ulceration has followed the injection. This related skin injury is different from the wound common in injection drug users and can occur at or away from the injection site regardless of the exposure route [3,4].

Authors and Publication Year	Age/Sex	Cause	Exposure Route	Primary Symptoms	Time Interval between Oral and Symptom	Drug Concentration
Yao et al. 2023, [29]	Unknown, 5 victims	Accident	Oral	Drowsiness, hypokalemia	15–40 min Mean 25 min	Blood 4.8–11.3 ng/mL, Urine 6.7–218.1 ng/mL, Cooked beef 440.0 µg/kg
	Unknown, 5 victims	Accident	Oral	Dry mouth, dizziness, drowsiness	30–60 min Mean 45 min	Blood 8.6–46.8 ng/mL, Venison 193.0 µg/kg Cooked venison 5.0 mg/kg
Xu et al. 2023, [28]	Unknown, 12 victims	Homicide	Oral	Dizzy	Unknown	9.6~139.5
Gill EL et al. 2023, [34]	8/M	Unknown	Unknown	loss of consciousness, hypopneic with pinpoint pupils, headache	Unknown	Xylazine and fentanyl positive
Krongvorakul et al. 2018, [8]	73/F	homicide	Oral	Coma, bradycardia	Unknown	Gastric content, positive. Serum, negative
	71/F	homicide	Oral	Coma, bradycardia	Unknown	Urine, 0.294 μg/mL Serum, 0.0057 μg/mL
	76/M	homicide	Oral	Coma, bradycardia	Unknown	Urine, 0.533 μg/mL
Liu et al. 2017, [30]	unknown	Accident	Oral	Unknown	Unknown	Peripheral blood xylazine and 2,6-Dimethylaniline positive
Forrester MB. 2016, [35]	76 victims, 41M, 33F, mean age 37	49 unintentional, 24 were intentional, 1 misuse; 1 adverse reaction, 1 unknown	39 injection, 21 ingestion, 12 dermal route, 11 ocular route, 2 inhalation, and 2 unknown, 9 multiple.	Drowsiness/lethargy [36], Bradycardia [15], Hypotension [8], Hypertension [7] Puncture/wound [6], Slurred speech [6], Coma [5], Ocular irritation/pain [5], Respiratory depression [4]	Unknown	Positive
Wang et al. 2014, [36]	Unknown, 1 male, 4 females	Suicide	Oral	Unknown	Unknown	200–1000 mg, one death
Hou et al. 2013, [37]	Unknown, more than 50 victims	Accident	Oral	Unknown	Unknown	Peripheral blood, cooked meat xylazine positive
Meyer et al. 2013 [38]	14/M	Accident	Injection	Coma, bradycardia	Unknown	Plasma 0.3 mg/L of xylazine and 0.1 mg/L of ketamine
Zhang et al. 2010, [39]	31/M	Accident	Inhaled	Dyspnea	Unknown	Unknown
Liu et al. 2007, [40]	19/M	Accident	Oral	Coma, 35.5 °C, bradycardia	Unknown	Urine 582 mg/L of ketamine, 448 mg/L of norketamine, 745 mg/L of phenobarbital, and 762 mg/L of xylazine
Velez et al. 2006, [13]	38/M	Accident	Unintentional irrigation of both eyes	Bradycardia, hypotension to 90/60 mm Hg, consciousness impairment,	2 h	Unknown
Xia et al. 2006, [41]	7 victims, 1 male, 6 females, 28–48 years old	homicide	Oral	Dizziness, drowsiness, palpitations, impaired consciousness	0.5–2 h	Heart blood 12.86 μg/mL
Elejalde et al.2003, [42]	18/M	Suicide	inhaled	Blood pressure 90/60 mmHg, heart rate 45 bpm, disorientation, dysarthria, dysmetria, ataxia, sinus bradycardia	Unknown	urine for common toxins was negative
Capraro et al. 2001, [15]	16/M	Suicide	inhaled	Coma, bradycardia	Unknown	Blood 0.54 μg/mL perhaps 3 mg of dose
Samanta et al. 1990, [43]	19/M	Accident	Injection	80/60 mm Hg, small pupils	30 min	Unknown

Coma, bradycardia and hypotension were the primary symptoms in xylazine poisoning cases and they may occur within 30 min and last for several days. Hypokalemia and small pupils were also reported. The time interval between xylazine exposure and the primary symptom in Table 1 was as soon as 15 min or as late as 2 h, and due to the unavailable information on the time interval between xylazine exposure and sample collected, the association link between clinical characteristics and blood or urine concentrations was hard to build. The signs and symptoms of xylazine poisoning are common to various disease states and/or toxicological exposures; therefore, xylazine cannot be detected rapidly in real time and can be a barrier to treatment, and should be added to the differential diagnosis if a toxicological cause is suspected.

Generally, central nervous system depression was common in such cases and respiratory depression was reported in people taking xylazine; the underlying mechanism may be attributed to the increase in the occurrence of respiratory depression induced by opioids. It is a common view that naloxone does work on respiratory depression caused by opioid whereas it does little to xylazine-induced respiratory depression [3,4]. However, xylazine poisoning may mimic an opioid overdose, and due to the prevalence of opioid overdoses, naloxone is still needed in such cases. Additional therapeutic support is required even if naloxone administration has been given. This additional therapeutic support includes keeping control of the airways, supplemental oxygen, and other treatment as needed. To date, there are still no approved drugs that can reverse the effect of xylazine in humans [3,4].

Generally, pure xylazine overdose death cases were uncommon in forensic practice. Although numerous studies indicated the increasing incidence of xylazine in forensic practice, most of them just focused on the detection method of xylazine and other characteristics of xylazine-related deaths and the pathological changes in such cases were rarely reported. The reasons may be the non-specific pathological changes in such cases, and xylazine contributes little to the cause of death in most of cases. 

Although Sibbesen et al. retrospectively studied 3292 drug deaths from 2019 to 2021 in West Virginia and identified 117 cases involving xylazine, they found that xylazine-related death always involved other drugs such as opioids, stimulants, benzodiazepines, and antidepressants/antipsychotics. It also indicated that liver diseases were more common in xylazine-related deaths. Injection was still the leading exposure of xylazine in xylazine-related deaths. Other exposure routes include snorting, smoking; oral and inhaling were also reported.

Skin ulcers are a primary health problem in chronic xylazine users that arouse public attention. These skin ulcers can occur far from the injection site, which could distinguish them from other chronic drug users. The underlying mechanisms were attributed to the oxygenation response to xylazine intoxication of the skin, and the chronic use of xylazine may limit the mobility of the limbs and lead to amputation in severe cases [9,46,47,48,49,50]. Generally, the injection site in such cases should be examined carefully and distinguished from iatrogenic injection injuries. Therefore, it is critical to emphasize the scene investigation, case history and toxicological test results.

In 2007, the abuse of xylazine in humans was first reported in Puerto Rico [51,52]. Since then, xylazine abuse has been recorded in other states (mainly in the Northeast), as well as an increasing number of drug-related deaths [53]. It was confirmed as a fentanyl adulterant as it prolonged the duration of fentanyl effects and increased the duration of brain hypoxia [51]. When used with opioids, xylazine has a synergistic effect and it could increase the level of brain hypoxia that may increase the risk of overdose or death [54]. Bradycardia, a depressed central nervous system, and hypotension were the most common side effects associated with human xylazine poisoning. To date, there is no clear definition of xylazine toxic in humans and the lethal concentrations of xylazine in humans have been absent. The role of xylazine in xylazine-related death is far from confirmed as xylazine and fentanyl were the common findings in such cases. The toxic mechanism of xylazine and the combination of xylazine and fentanyl still need further study to be confirmed.

The exact contribution of xylazine to the process of death is still unknown, although its pharmacological effects were reported to enhance the respiratory and central nervous system depressive effects of other substances. The toxicity and lethal concentrations of xylazine are still unclear and there is considerable overlap in the blood concentration in patients who suffered xylazine poisoning and xylazine overdose death cases [1]. It is reported that the blood concentration of xylazine ranged from 5 to 49 ng/mL in xylazine overdose death cases, whereas blood or plasma concentrations ranging from 30 to 460 ng/mL were also detected in non-lethal xylazine overdose cases [1]. The concentration of xylazine summarized in Table 1 showed that the blood concentration of xylazine can reach 540 ng/mL in non-lethal xylazine overdose cases. Although there is not a significant difference in xylazine concentrations in the number of co-intoxicants, rather higher concentrations of xylazine were seen in decedents who took only one other drug compared to those who took more drugs. Indeed, xylazine plays an important role in the death mechanisms of other co-intoxicants.

Table 3 also shows that the blood concentration of xylazine in homicide cases was higher than in suicide cases, and was much higher in pure xylazine poisoning cases compared to combined with other toxics. To date, only a homicide case was reported and xylazine was the only reported toxic; whether the type of death and the cause of death had a role in the concentration of xylazine in different samples still needs further study. The blood concentration of xylazine was the lowest in most cases, while the urine concentration of xylazine was much higher. The urine concentration of xylazine can reach 533 ng/mL even in non-lethal xylazine overdose cases. The concentration of xylazine in the heart blood was lower than in the peripheral blood; considering the existence of postmortem redistribution in toxic overdose death cases, the heart blood may not be the most suitable sample in such cases. And due to the existence of putrefaction and autolysis, blood samples may be unable to be collected.

The organ and fluid distribution of xylazine in xylazine overdose death cases has rarely been reported in forensic practice. To date, only two research articles published the organ distribution of xylazine in xylazine overdose death cases with paradoxical results. In 1985, Poklis et al. reported that a 36-year-old male injected xylazine to commit suicide and the organ concentration of xylazine was as follows: lung (1100 ng/mL), liver (900 ng/mL), kidney (600 ng/mL), brain (400 ng/mL) [11]. In 2003, Moore et al. reported that a 42-year-old male injected xylazine to commit suicide and the concentration of xylazine in the kidney (780 ng/mL) was higher than in the liver (610 ng/mL) [45]. This may be attributed to the intake dose of xylazine as the intake dose of other drugs revealed the distribution characteristics in vivo are different in various lethal doses. Due to the existence of postmortem dispersion and other factors, the postmortem redistribution of xylazine was an inevitable phenomenon.

The affected factors included diffusion distance, postmortem gavage time and dose. It is prone to disperse into tissues and organs close to the gastrointestinal tract, whereas it did have some impact on the heart blood, bile, and peripheral blood, and never affected the brain and muscles. Therefore, to identify the concentration of xylazine in forensic practice, not only the routine examination materials in forensic toxicological practice such as the heart blood, liver and stomach content should be collected, but also the less affected tissues and organs such as the brain and muscle should be taken for examination.

When it comes to the xylazine overdose death cases whose exposure route was injection, the injection site was the ideal sample for the detection of xylazine. Therefore, it should be carefully examined, taken fixed in formalin for histopathological examination and taken stored at −80 °C for subsequent analysis. Generally, the routine sample for the detection of xylazine was not identified in forensic practice, and it still needs further study to confirm the ideal sample in such cases.

It has been reported that in almost all xylazine deaths, 98.3%, the patient also took fentanyl, and most of the decedents were young white men [9]. Previous studies have documented this high fentanyl prevalence, consistent with the identification of xylazine as a fentanyl adulterant [46,47,48,49,50]. Xylazine is increasingly involved in drug overdose decedents that ingest more toxicants, and xylazine abuse is an emerging public health problem [46,47,48,49,50].

It has been reported that drug or alcohol abuse history increases the incidence of xylazine-related death [9]. Xylazine-related deaths are more common in individuals who take more than two drugs, including xylazine. In 2022, Sibbesen et al. retrospectively reviewed 3292 drug deaths in West Virginia from 2019 to mid-2021, identified the characteristics of xylazine-related death, found that approximately 90% of the xylazine-related decedents took more than three drugs, and that 40% of the xylazine-related decedents had taken more than five drugs [9]. Opioids, stimulants, benzodiazepines, and antidepressants/antipsychotics are commonly associated with xylazine-related deaths. Fentanyl analogues, heroin and other opioids are found in about a quarter of xylazine-related deaths and nearly one in five of non-xylazine deaths.

Few studies focus on the interaction between xylazine and fentanyl. In 2023, Smith et al. used C57BL/6 mice which were 42 days of age to quantify the lethal effects of fentanyl and xylazine, found that 56 mg/kg fentanyl administration could shift the LD50 of xylazine from 157.2 mg/kg to 32.0 mg/kg and 100 mg/kg xylazine administration could shift the LD50 of fentanyl from 131.3 mg/kg to 1.27 mg/kg, suggesting the synergistic manner in their interaction and that the combination of them can lead to more rapid death than alone [54]. Choi et al. used adult male Long–Evans rats that weighed 450 ± 50 g to evaluate the neuro effects of xylazine alone or combined with fentanyl and heroin and found that xylazine alone not only can decrease temperature and locomotor activity, but also prolong the lower oxygen levels in the brain [55].