- Email: [email protected]
Toxicological findings in three cases of suicidal asphyxiation with helium
Accepted Manuscript Title: Toxicological findings in three cases of suicidal asphyxiation with helium Author: Roelof Oosting Rogier van der Hulst Leo Peschier Miranda Verschraagen PII: DOI: Reference:
S0379-0738(15)00263-7 http://dx.doi.org/doi:10.1016/j.forsciint.2015.06.028 FSI 8053
To appear in:
FSI
Received date: Revised date: Accepted date:
17-3-2015 4-6-2015 28-6-2015
Please cite this article as: R. Oosting, R. van der Hulst, L. Peschier, M. Verschraagen, Toxicological findings in three cases of suicidal asphyxiation with helium., Forensic Science International (2015), http://dx.doi.org/10.1016/j.forsciint.2015.06.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Page (with authors and addresses)
Toxicological findings in three cases of suicidal asphyxiation with helium. Roelof Oosting1, Rogier van der Hulst1, Leo Peschier2, Miranda Verschraagen1. 1Netherlands Forensic Institute (NFI), Department of Forensic Medicine, The Hague (The Netherlands). 2NFI, Department of Chemical Identification Research. [email protected]
Ac
ce pt
ed
M
an
us
cr
ip t
Email address corresponding author:
Page 1 of 9
Highlights (for review)
Toxicological findings in deaths by asphyxiation due to helium are rare. Helium is easily lost if sampling is not performed properly. Tissue sampling directly in head space vials is suitable to detect helium.
Ac
ce pt
ed
M
an
us
cr
Semi-quantitative gas analyses using HS-GC/TCD enables to detect helium.
ip t
Sampling air from the lungs in foil bags is also suitable to detect helium.
Page 2 of 9
Toxicological findings in three cases of suicidal asphyxiation with helium. Abstract
Ac
ce pt
ed
Keywords. Helium, suicide, HS-GC/MS.
M
an
us
cr
ip t
Introduction. Toxicological findings in deaths by asphyxiation due to a pure inert gas like helium are rare. We present three suicide cases of asphyxial death attributed to anoxia caused by inhalation of helium in a plastic bag positioned over the head. Methods. In one case, lung tissue, brain tissue and heart blood were obtained during standard autopsy procedures. In two cases, samples were obtained differently: heart blood, femoral blood, brain tissue, lung tissue and/or air from the lungs were directly sealed into headspace vials during autopsy. Air from the lungs was collected using a syringe and transferred into an aluminum gas sampling bag which was heat sealed as soon as possible. Semi-quantitative gas analyses were performed using headspace gas chromatographythermal conductivity detection (HS-GC/TCD) with a molsieve column capable of separating permanent gasses. Nitrogen was used as carrier gas. Results. In the first case no helium was detected in lung tissue, brain tissue and heart blood. In the second case the presence of helium was detected in lung tissue (approximately 5 % helium in gaseous phase) but not in femoral blood. In the third case the presence of helium was detected in air from the lungs (0.05 %), lung tissue (0.4 %), brain tissue (0.1 %) and heart blood (0.04 %). Conclusions. Helium is easily lost if sampling is not performed properly. The presented cases suggest that quick sample collection of various matrices during autopsy is suitable to detect gasses like helium in postmortem cases. Use of HS-GC/TCD enables to detect an inert gas like helium.
1
Page 3 of 9
1. Introduction
2. Case history
ce pt
ed
M
an
us
cr
ip t
Since the start of the new millennium, there has been an increase in the number of suicides involving helium [1,2]. On the internet information can be found about euthanasia or committing suicide by inhalation of helium gas without the intervention of a doctor [3]. Helium is a colorless, odorless, tasteless and biological inert noble gas. Inhalation of pure helium which is guided inside a plastic bag or in a mask can cause suffocation by expelling oxygen in the inhaled air. Physiological effects and symptoms that may occur when breathing air with reduced oxygen are increased heart rate and fatigue (at 21-14 % oxygen), hampered moving and thinking (at 14-11 % oxygen), development of headaches, dizziness and fainting after a short amount of time, fainting within 8-6 minutes, resuscitation possible if carried out directly (at 11-8 % oxygen) and almost immediately fainting, death or severe brain damage (at 6-0 % oxygen)[4]. Observation of two suicides by helium inhalation in a prefilled environment confirmed rapid loss of consciousness and sudden death [5]. As excess of carbon dioxide (instead of lack of oxygen) triggers the breathing reflex and since carbon dioxide is also expelled by helium, the feeling of suffocation and urge to breathe is not present [6]. Unfortunately, especially cases involving inert gases like helium are often presented without detailed toxicological findings [7]. This is probably because gasses like helium can easily be lost during sampling, storage and sample preparation. During autopsy non-specific signs of intoxication may be observed by the pathologist such as brain edema and pulmonary edema. Usually the assignment of suicide by asphyxiation caused by inhalation of helium as the cause of death is based on the presence of paraphernalia such as a plastic bag over the head (or a mask) of the deceased and a commercially available helium cylinder and the absence of a more plausible cause of death. It is however possible that the evidence or paraphernalia are removed from the scene by a third party, complicating the pathological and toxicological investigation [8]. A reason for this can for instance be financial, when the life-insurance does not cover a suicide. In cases where the paraphernalia were removed, it may actually be of great importance to prove presence of helium in the body of the deceased. We report toxicological findings in three suicides committed by use of helium.
Ac
2.1 Case 1 A 48 year old man was found alive in the bedroom by family members. Around his head was a (not tightly closed) plastic bag with a plastic tube coming out attached to a gas cylinder with helium. In the room a manual for suicide on the basis of helium was found, as well as a letter with a wish not to be treated in the case of "early discovery". The family informed the doctor approximately 1.5 hours after death. One of the family members was a physician. Autopsy took place approximately one day after death. The first results of the autopsy were the following. The heart showed showed a distended and congested right ventricle. Autopsy and further research showed signs of generalized lack of oxygen such as petechia in lungs and renal pelvis. Non-specific signs of intoxication like pulmonary edema, brain edema and a filled bladder were seen. No anatomical cause of death was found.
2
Page 4 of 9
Results toxicological routine analyses (see description 3.1): Ethanol (alcohol) in femoral blood (0.65 g/L) and urine (0.96 g/L). No evidence was obtained for the presence of drugs, medicines or pesticides.
us
cr
ip t
2.2 Case 2 An 81 year old woman died as a result of asphyxiation by inhalation of helium in a plastic bag fixed over the head. She was wheelchair dependent. An assisted suicide was suspected. Autopsy took place approximately 3 days after death. The first results of the autopsy were as follows: pulmonary edema, advanced generalized arteriosclerosis and no identifiable anatomical cause of death. Results toxicological routine analyses (see description 3.1): Ethanol in urine (approximately 0.11 g/L), no ethanol in femoral blood, possibly omeprazole in heart blood. No evidence was obtained for the presence of other drugs, medicines or pesticides.
ce pt
ed
M
an
2.3 Case 3 A 41 year old woman was found lying on a couch in a house by the main occupant, in serious need of resuscitation. According to statements , a gas cylinder would have been present, and possibly a bag around the head. She was said to be resuscitated and the duration of this was not known. Autopsy took place approximately one day after death. The first results of the autopsy were as follows: The head and neck showed some congestion above the level of the clavicles. The conjunctiva of the left eye contained petechiae. The surface of both lungs showed some petechiae. These findings are non-specific but could indicate a sudden failure of the heart function. Also the finding of lung edema is non-specific. Autopsy revealed no anatomical cause of death. Results toxicological routine analyses (see description 3.1): Citalopram or escitalopram in femoral blood (0.41 mg/L) and in heart blood (0.46 mg/L); the performed analysis was not able to differ between the isomers of citalopram. No evidence was obtained for the presence of other drugs, medicines or pesticides.
3. Material and methods
Ac
3.1 Toxicological routine analysis In toxicological investigation of autopsy cases at the Netherlands Forensic Institute (NFI), four toxicological analyses are performed routinely to investigate the presence of drugs of abuse, prescription drugs, pesticides and ethanol (alcohol). This was also done for the three cases. A screening for drugs of abuse, medicines and pesticides was performed in whole blood (heart blood) after solid phase extraction (SPE) by using high performance liquid chromatography with diode array detection (HPLC-DAD) and gas chromatography with mass spectrometric detection (GC-MS) after derivitization with bis(trimethylsilyl)trifluoroacetamide (BSTFA) [9]. Secondly, the identification and quantification of 57 substances in whole blood (heart and femoral blood or vitreous humor), including both medicinal and illicit drugs, was performed using a validated UPLC-MS/MS (ultra performance liquid chromatography-tandem mass spectrometry) method [10]. Thirdly, the presence and concentration of GHB in three matrices (femoral blood, heart blood, vitreous humor and/or urine) was determined separately by using GC-MS (gas chromatography mass spectrometry)[11]. 3
Page 5 of 9
And finally, concentrations of alcohol (i.e. ethanol) in the post-mortem cases were determined in two matrices (femoral blood, vitreous humor and/or urine) by head-space gas chromatography with flame ionization detection. The limits of quantification (LOQ) are 0.001 or 0.005 mg/L for all drugs using the UPLC-MS/MS method and 5 mg/L for GHB.
ed
M
an
us
cr
ip t
3.2 Semi-quantitative analyses of helium The method of analysis of helium was largely similar to the assay as published by Schaff et al [12]. Semi-quantitative gas analyses were performed using headspace gas chromatography-thermal conductivity detection (HS-GC/TCD) with a molsieve column capable of separating permanent gasses. Nitrogen was used as carrier gas. This method is not a routinely used method in forensic toxicological cases at the NFI. In comparison to Schaff et al. we used a combined column (Varian CP7429, with two columns in parallel, of which only the CP-Molsieve 5A column was used for the helium analysis) with a different column flow and slightly different oven temperature (30 °C instead of 35 °C). Detector settings were almost identical (make-up flow 7 ml /min instead of 5 ml /min). Injector temperature was 50 °C instead of 200 °C). Schaff et al. used a small split ratio. We used a split/splitless injector with a total flow of 50 ml/min which limits the residence time in the liner and accounts for a sharp injection pulse. As sample preparation, the HS vials were incubated /shaken at 70 °C in the oven of the headspace sampler for 20 minutes. The limit of detection is estimated to be approximately 50 ppm, based on the signal to noise ratio in a 100 ppm standard helium in nitrogen. A semi-quantitative result of helium (in gaseous phase) was determined by calibration with (diluted) helium.
ce pt
3.3 Breath experiment To get an impression of the helium concentrations in exhaled breath after inhaling pure helium gas, the experiment described by Auwerter et al was repeated [13]. A healthy volunteer deeply exhaled and then deeply inhaled helium once from a balloon. The subsequent exhaled breaths were sampled and analyzed.
Ac
3.4 Sample collection In the first case lung tissue, brain tissue and heart blood were analyzed for the presence of helium. During autopsy approximate 10 gram of brain and lung tissue where collected in separate airtight glass jars. The heart blood was collected in a plastic container. These samples were stored at -20 °C. At the time of toxicological analyses a subsample of approximate 1 gram of frozen tissue and blood is brought over in a headspace vials for further analysis for helium as described above. The samples in case two and three were obtained differently. The presence of helium was analyzed in case 2 in lung tissue and femoral blood and in case 3 in lung tissue, brain tissue, heart blood and air from the lungs. During autopsy the obtained tissue and blood samples were directly were placed in 10 ml headspace vials (Chromacol 10-CV P238) and immediately sealed with a composite magnetic crimpcap with a silicone/PTFE seal (Chromacol MCBC-ST3). The air from the lungs was collected using a syringe and transferred into an PET/aluminum/EVA multilayer gas sampling foil bag, which was prepared to have only a small opening left through which the sample could be entered using the syringe. The bag was temporary closed in the autopsy room 4
Page 6 of 9
by folding and applying a piece of tape and was sealed by heat-sealing (using a Lovero Impulse bag sealer, type 510) upon arrival in the laboratory (during or immediately after autopsy). This simple and direct sampling procedure insures a minimal loss of potentially present helium in the samples.
4. Results
Table 1. Results breath experiment: concentrations helium in exhaled air
an
ed
5. Discussion
Concentration helium (% v/v) 56 31 19 0.6 0.05
M
Number of exhalations 1 2 3 10 20
us
cr
ip t
In the first case no helium was demonstrated in lung tissue, brain tissue and heart blood. In the second case helium was demonstrated in lung tissue (approximately 5 % helium in gaseous phase) but not in the femoral blood. In the third case helium was demonstrated in the expired air from the lungs (approximately 0.05 %), lung tissue (approximately 0.4 %), brain tissue (approximately 0.1 %) and heart blood (approximately 0.04 %). The results of the breath experiment are listed in Table 1.
Ac
ce pt
In the first case an anatomical cause of death was not found. The toxicological research demonstrated the presence of ethanol (alcohol) but showed no evidence for the presence of other drugs, medicines and pesticides. Helium was not demonstrated in lung tissue, brain tissue and heart blood. A possible explanation that helium was not detected in the samples may be that helium was lost at the moment the containers were opened with the purpose to take a subsample for toxicological analysis, even though the lung tissue and brain tissue samples were kept in airtight glass containers (-20°C) and the blood sample was kept in a glass tube (-20°C) . In the second and third case no anatomical cause of death was found either. Helium was demonstrated in the second case in the lung tissue (approximately 5 % helium in gaseous phase) and in the third case in air from the lungs (approximately 0.05 %), lung tissue (approximately 0.4 %), brain tissue (approximately 0.1 %) and heart blood (approximately 0.04 %). In both cases the demonstrated concentrations helium well exceeded the concentrations of helium normally present in air (0.0005 %). The second case shows that even after 3 days after death helium can still be demonstrated. The third case shows that even after resuscitation helium can be demonstrated, although we don’t know how long the resuscitation was maintained. In all three cases the pathologist concluded that the onset of death can be well explained by inhalation of helium and consequently lack of oxygen.
5
Page 7 of 9
an
us
cr
ip t
There are only a few published cases in literature of suicidal asphyxiation by helium in which helium was actually demonstrated in toxicological findings [13,14]. Quick sampling during autopsy in airtight containers may make the difference between demonstrating presence of helium or not. Direct reason to investigate helium as a possible cause of death (suicidal asphyxiation) in these 3 cases was situational evidence found at the death-scene, particularly a helium cylinder, plastic bag, instructional literature (especially on the computer) and a suicide note [4]. As detailed how-to information is increasingly readily available we expect that the number of cases of suicidal asphyxiation with helium will rise. In some of these cases no situational evidence at the death scene may be found because of assisted suicide and these cases may only be solved when suitable samples (e.g. lung tissue in a HS-vial) have indeed been secured during autopsy. Gasses like argon, propane, nitrogen and methane should also be kept in mind. The results of the breath experiment showed that in the first 10 exhalations the subsequent exhalations contain 40 % less helium in comparison with the exhalation before. After the 20th exhalation the measured concentration of helium was higher than expected but still very well measurable. Therefore one may wonder if analyzing helium in body material still makes sense (that is helium is still detectable) in cases the involved person is found, resuscitated but nevertheless died more than roughly one hour later. 6. Conclusion
Ac
ce pt
ed
M
Helium is easily lost if sampling is not performed properly. The presented cases suggest that quick sample collection of various matrices during autopsy, i.e. blood and lung or brain tissue sampled directly into head space vials or air from lungs in a foil bag is suitable to detect gasses like helium in postmortem cases. This sampling method can easily be incorporated in standard sampling procedures. Use of HS-GC/TCD enables to detect an inert gas like helium.
6
Page 8 of 9
Reference list
Ac
ce pt
ed
M
an
us
cr
ip t
1. Austin A. et al. Recent trends in suicides utilizing helium. J Forensic Sci. 2011; 56(3): 649-51 2. Gunnell D. et al. Suicide by gases in England and Wales 2001-2011: Evidence of the emergence of new methods of suicide. J Affect Disord. 2014; 170C: 190-195. 3. e.g. www.patientsrightscouncil.org, www.finalexit.org or www.eenwaardiglevenseinde.nl 4. Auwärter V. et al. Analytical investigations in a death case by suffocation in an argon atmosphere. Forensic Sci Int. 2004; 143(2-3): 169-75 5. Ogden R.D. Observation of two suicides by helium inhalation in a prefilled environment. Am J Forensic Med Pathol. 2010; 31(2): 156-61. 6. Grassberger M., Krauskopf A. Suicidal asphyxiation with helium: report of three cases. Wien Klin Wochenschr. 2007; 119(9-10): 323-5. 7. Musshoff F. et al Two cases of suicide by asphyxiation due to helium and argon. Forensic Sci Int. 2012; 223(1-3): e27-30. 8. Schön C.A., Ketterer T. Asphyxial suicide by inhalation of helium inside a plastic bag. Am J Forensic Med Pathol. 2007; 28(4): 364-7. 9. Chen X.H. et al. Solid-phase extraction for the screening of acidic, neutral and basic drugs in plasma using a single-column procedure on Bond Elut Certify. J Chromatogr. 1990; 529:161-166. 10. Boone C. et al. Determination of the most frequently used drugs in whole blood by using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Toxichemical Krimtechnology. 2010; 77:219. 11. Elian A.A. GC-MS determination of gamma-hydroxybutyric acid (GHB) in blood. Forensic Sci. Int. 2001; 122: 43-47. 12. Schaff J.E. et al. A gas chromatography-thermal conductivity detection method for helium detection in postmortem blood and tissue specimens. J Anal Toxicol. 2012; 36(2): 112-5. 13. Auwaerter V. et al. Toxicological analysis after asphyxial suicide with helium and a plastic bag. Forensic Sci Int. 2007; 170(2-3): 139-41. 14. Howard M.O. et al. Suicide by asphyxiation due to helium inhalation. Am J Forensic Med Pathol. 2011; 32(1): 61-70.
7
Page 9 of 9
S0379-0738(15)00263-7 http://dx.doi.org/doi:10.1016/j.forsciint.2015.06.028 FSI 8053
To appear in:
FSI
Received date: Revised date: Accepted date:
17-3-2015 4-6-2015 28-6-2015
Please cite this article as: R. Oosting, R. van der Hulst, L. Peschier, M. Verschraagen, Toxicological findings in three cases of suicidal asphyxiation with helium., Forensic Science International (2015), http://dx.doi.org/10.1016/j.forsciint.2015.06.028 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Title Page (with authors and addresses)
Toxicological findings in three cases of suicidal asphyxiation with helium. Roelof Oosting1, Rogier van der Hulst1, Leo Peschier2, Miranda Verschraagen1. 1Netherlands Forensic Institute (NFI), Department of Forensic Medicine, The Hague (The Netherlands). 2NFI, Department of Chemical Identification Research. [email protected]
Ac
ce pt
ed
M
an
us
cr
ip t
Email address corresponding author:
Page 1 of 9
Highlights (for review)
Toxicological findings in deaths by asphyxiation due to helium are rare. Helium is easily lost if sampling is not performed properly. Tissue sampling directly in head space vials is suitable to detect helium.
Ac
ce pt
ed
M
an
us
cr
Semi-quantitative gas analyses using HS-GC/TCD enables to detect helium.
ip t
Sampling air from the lungs in foil bags is also suitable to detect helium.
Page 2 of 9
Toxicological findings in three cases of suicidal asphyxiation with helium. Abstract
Ac
ce pt
ed
Keywords. Helium, suicide, HS-GC/MS.
M
an
us
cr
ip t
Introduction. Toxicological findings in deaths by asphyxiation due to a pure inert gas like helium are rare. We present three suicide cases of asphyxial death attributed to anoxia caused by inhalation of helium in a plastic bag positioned over the head. Methods. In one case, lung tissue, brain tissue and heart blood were obtained during standard autopsy procedures. In two cases, samples were obtained differently: heart blood, femoral blood, brain tissue, lung tissue and/or air from the lungs were directly sealed into headspace vials during autopsy. Air from the lungs was collected using a syringe and transferred into an aluminum gas sampling bag which was heat sealed as soon as possible. Semi-quantitative gas analyses were performed using headspace gas chromatographythermal conductivity detection (HS-GC/TCD) with a molsieve column capable of separating permanent gasses. Nitrogen was used as carrier gas. Results. In the first case no helium was detected in lung tissue, brain tissue and heart blood. In the second case the presence of helium was detected in lung tissue (approximately 5 % helium in gaseous phase) but not in femoral blood. In the third case the presence of helium was detected in air from the lungs (0.05 %), lung tissue (0.4 %), brain tissue (0.1 %) and heart blood (0.04 %). Conclusions. Helium is easily lost if sampling is not performed properly. The presented cases suggest that quick sample collection of various matrices during autopsy is suitable to detect gasses like helium in postmortem cases. Use of HS-GC/TCD enables to detect an inert gas like helium.
1
Page 3 of 9
1. Introduction
2. Case history
ce pt
ed
M
an
us
cr
ip t
Since the start of the new millennium, there has been an increase in the number of suicides involving helium [1,2]. On the internet information can be found about euthanasia or committing suicide by inhalation of helium gas without the intervention of a doctor [3]. Helium is a colorless, odorless, tasteless and biological inert noble gas. Inhalation of pure helium which is guided inside a plastic bag or in a mask can cause suffocation by expelling oxygen in the inhaled air. Physiological effects and symptoms that may occur when breathing air with reduced oxygen are increased heart rate and fatigue (at 21-14 % oxygen), hampered moving and thinking (at 14-11 % oxygen), development of headaches, dizziness and fainting after a short amount of time, fainting within 8-6 minutes, resuscitation possible if carried out directly (at 11-8 % oxygen) and almost immediately fainting, death or severe brain damage (at 6-0 % oxygen)[4]. Observation of two suicides by helium inhalation in a prefilled environment confirmed rapid loss of consciousness and sudden death [5]. As excess of carbon dioxide (instead of lack of oxygen) triggers the breathing reflex and since carbon dioxide is also expelled by helium, the feeling of suffocation and urge to breathe is not present [6]. Unfortunately, especially cases involving inert gases like helium are often presented without detailed toxicological findings [7]. This is probably because gasses like helium can easily be lost during sampling, storage and sample preparation. During autopsy non-specific signs of intoxication may be observed by the pathologist such as brain edema and pulmonary edema. Usually the assignment of suicide by asphyxiation caused by inhalation of helium as the cause of death is based on the presence of paraphernalia such as a plastic bag over the head (or a mask) of the deceased and a commercially available helium cylinder and the absence of a more plausible cause of death. It is however possible that the evidence or paraphernalia are removed from the scene by a third party, complicating the pathological and toxicological investigation [8]. A reason for this can for instance be financial, when the life-insurance does not cover a suicide. In cases where the paraphernalia were removed, it may actually be of great importance to prove presence of helium in the body of the deceased. We report toxicological findings in three suicides committed by use of helium.
Ac
2.1 Case 1 A 48 year old man was found alive in the bedroom by family members. Around his head was a (not tightly closed) plastic bag with a plastic tube coming out attached to a gas cylinder with helium. In the room a manual for suicide on the basis of helium was found, as well as a letter with a wish not to be treated in the case of "early discovery". The family informed the doctor approximately 1.5 hours after death. One of the family members was a physician. Autopsy took place approximately one day after death. The first results of the autopsy were the following. The heart showed showed a distended and congested right ventricle. Autopsy and further research showed signs of generalized lack of oxygen such as petechia in lungs and renal pelvis. Non-specific signs of intoxication like pulmonary edema, brain edema and a filled bladder were seen. No anatomical cause of death was found.
2
Page 4 of 9
Results toxicological routine analyses (see description 3.1): Ethanol (alcohol) in femoral blood (0.65 g/L) and urine (0.96 g/L). No evidence was obtained for the presence of drugs, medicines or pesticides.
us
cr
ip t
2.2 Case 2 An 81 year old woman died as a result of asphyxiation by inhalation of helium in a plastic bag fixed over the head. She was wheelchair dependent. An assisted suicide was suspected. Autopsy took place approximately 3 days after death. The first results of the autopsy were as follows: pulmonary edema, advanced generalized arteriosclerosis and no identifiable anatomical cause of death. Results toxicological routine analyses (see description 3.1): Ethanol in urine (approximately 0.11 g/L), no ethanol in femoral blood, possibly omeprazole in heart blood. No evidence was obtained for the presence of other drugs, medicines or pesticides.
ce pt
ed
M
an
2.3 Case 3 A 41 year old woman was found lying on a couch in a house by the main occupant, in serious need of resuscitation. According to statements , a gas cylinder would have been present, and possibly a bag around the head. She was said to be resuscitated and the duration of this was not known. Autopsy took place approximately one day after death. The first results of the autopsy were as follows: The head and neck showed some congestion above the level of the clavicles. The conjunctiva of the left eye contained petechiae. The surface of both lungs showed some petechiae. These findings are non-specific but could indicate a sudden failure of the heart function. Also the finding of lung edema is non-specific. Autopsy revealed no anatomical cause of death. Results toxicological routine analyses (see description 3.1): Citalopram or escitalopram in femoral blood (0.41 mg/L) and in heart blood (0.46 mg/L); the performed analysis was not able to differ between the isomers of citalopram. No evidence was obtained for the presence of other drugs, medicines or pesticides.
3. Material and methods
Ac
3.1 Toxicological routine analysis In toxicological investigation of autopsy cases at the Netherlands Forensic Institute (NFI), four toxicological analyses are performed routinely to investigate the presence of drugs of abuse, prescription drugs, pesticides and ethanol (alcohol). This was also done for the three cases. A screening for drugs of abuse, medicines and pesticides was performed in whole blood (heart blood) after solid phase extraction (SPE) by using high performance liquid chromatography with diode array detection (HPLC-DAD) and gas chromatography with mass spectrometric detection (GC-MS) after derivitization with bis(trimethylsilyl)trifluoroacetamide (BSTFA) [9]. Secondly, the identification and quantification of 57 substances in whole blood (heart and femoral blood or vitreous humor), including both medicinal and illicit drugs, was performed using a validated UPLC-MS/MS (ultra performance liquid chromatography-tandem mass spectrometry) method [10]. Thirdly, the presence and concentration of GHB in three matrices (femoral blood, heart blood, vitreous humor and/or urine) was determined separately by using GC-MS (gas chromatography mass spectrometry)[11]. 3
Page 5 of 9
And finally, concentrations of alcohol (i.e. ethanol) in the post-mortem cases were determined in two matrices (femoral blood, vitreous humor and/or urine) by head-space gas chromatography with flame ionization detection. The limits of quantification (LOQ) are 0.001 or 0.005 mg/L for all drugs using the UPLC-MS/MS method and 5 mg/L for GHB.
ed
M
an
us
cr
ip t
3.2 Semi-quantitative analyses of helium The method of analysis of helium was largely similar to the assay as published by Schaff et al [12]. Semi-quantitative gas analyses were performed using headspace gas chromatography-thermal conductivity detection (HS-GC/TCD) with a molsieve column capable of separating permanent gasses. Nitrogen was used as carrier gas. This method is not a routinely used method in forensic toxicological cases at the NFI. In comparison to Schaff et al. we used a combined column (Varian CP7429, with two columns in parallel, of which only the CP-Molsieve 5A column was used for the helium analysis) with a different column flow and slightly different oven temperature (30 °C instead of 35 °C). Detector settings were almost identical (make-up flow 7 ml /min instead of 5 ml /min). Injector temperature was 50 °C instead of 200 °C). Schaff et al. used a small split ratio. We used a split/splitless injector with a total flow of 50 ml/min which limits the residence time in the liner and accounts for a sharp injection pulse. As sample preparation, the HS vials were incubated /shaken at 70 °C in the oven of the headspace sampler for 20 minutes. The limit of detection is estimated to be approximately 50 ppm, based on the signal to noise ratio in a 100 ppm standard helium in nitrogen. A semi-quantitative result of helium (in gaseous phase) was determined by calibration with (diluted) helium.
ce pt
3.3 Breath experiment To get an impression of the helium concentrations in exhaled breath after inhaling pure helium gas, the experiment described by Auwerter et al was repeated [13]. A healthy volunteer deeply exhaled and then deeply inhaled helium once from a balloon. The subsequent exhaled breaths were sampled and analyzed.
Ac
3.4 Sample collection In the first case lung tissue, brain tissue and heart blood were analyzed for the presence of helium. During autopsy approximate 10 gram of brain and lung tissue where collected in separate airtight glass jars. The heart blood was collected in a plastic container. These samples were stored at -20 °C. At the time of toxicological analyses a subsample of approximate 1 gram of frozen tissue and blood is brought over in a headspace vials for further analysis for helium as described above. The samples in case two and three were obtained differently. The presence of helium was analyzed in case 2 in lung tissue and femoral blood and in case 3 in lung tissue, brain tissue, heart blood and air from the lungs. During autopsy the obtained tissue and blood samples were directly were placed in 10 ml headspace vials (Chromacol 10-CV P238) and immediately sealed with a composite magnetic crimpcap with a silicone/PTFE seal (Chromacol MCBC-ST3). The air from the lungs was collected using a syringe and transferred into an PET/aluminum/EVA multilayer gas sampling foil bag, which was prepared to have only a small opening left through which the sample could be entered using the syringe. The bag was temporary closed in the autopsy room 4
Page 6 of 9
by folding and applying a piece of tape and was sealed by heat-sealing (using a Lovero Impulse bag sealer, type 510) upon arrival in the laboratory (during or immediately after autopsy). This simple and direct sampling procedure insures a minimal loss of potentially present helium in the samples.
4. Results
Table 1. Results breath experiment: concentrations helium in exhaled air
an
ed
5. Discussion
Concentration helium (% v/v) 56 31 19 0.6 0.05
M
Number of exhalations 1 2 3 10 20
us
cr
ip t
In the first case no helium was demonstrated in lung tissue, brain tissue and heart blood. In the second case helium was demonstrated in lung tissue (approximately 5 % helium in gaseous phase) but not in the femoral blood. In the third case helium was demonstrated in the expired air from the lungs (approximately 0.05 %), lung tissue (approximately 0.4 %), brain tissue (approximately 0.1 %) and heart blood (approximately 0.04 %). The results of the breath experiment are listed in Table 1.
Ac
ce pt
In the first case an anatomical cause of death was not found. The toxicological research demonstrated the presence of ethanol (alcohol) but showed no evidence for the presence of other drugs, medicines and pesticides. Helium was not demonstrated in lung tissue, brain tissue and heart blood. A possible explanation that helium was not detected in the samples may be that helium was lost at the moment the containers were opened with the purpose to take a subsample for toxicological analysis, even though the lung tissue and brain tissue samples were kept in airtight glass containers (-20°C) and the blood sample was kept in a glass tube (-20°C) . In the second and third case no anatomical cause of death was found either. Helium was demonstrated in the second case in the lung tissue (approximately 5 % helium in gaseous phase) and in the third case in air from the lungs (approximately 0.05 %), lung tissue (approximately 0.4 %), brain tissue (approximately 0.1 %) and heart blood (approximately 0.04 %). In both cases the demonstrated concentrations helium well exceeded the concentrations of helium normally present in air (0.0005 %). The second case shows that even after 3 days after death helium can still be demonstrated. The third case shows that even after resuscitation helium can be demonstrated, although we don’t know how long the resuscitation was maintained. In all three cases the pathologist concluded that the onset of death can be well explained by inhalation of helium and consequently lack of oxygen.
5
Page 7 of 9
an
us
cr
ip t
There are only a few published cases in literature of suicidal asphyxiation by helium in which helium was actually demonstrated in toxicological findings [13,14]. Quick sampling during autopsy in airtight containers may make the difference between demonstrating presence of helium or not. Direct reason to investigate helium as a possible cause of death (suicidal asphyxiation) in these 3 cases was situational evidence found at the death-scene, particularly a helium cylinder, plastic bag, instructional literature (especially on the computer) and a suicide note [4]. As detailed how-to information is increasingly readily available we expect that the number of cases of suicidal asphyxiation with helium will rise. In some of these cases no situational evidence at the death scene may be found because of assisted suicide and these cases may only be solved when suitable samples (e.g. lung tissue in a HS-vial) have indeed been secured during autopsy. Gasses like argon, propane, nitrogen and methane should also be kept in mind. The results of the breath experiment showed that in the first 10 exhalations the subsequent exhalations contain 40 % less helium in comparison with the exhalation before. After the 20th exhalation the measured concentration of helium was higher than expected but still very well measurable. Therefore one may wonder if analyzing helium in body material still makes sense (that is helium is still detectable) in cases the involved person is found, resuscitated but nevertheless died more than roughly one hour later. 6. Conclusion
Ac
ce pt
ed
M
Helium is easily lost if sampling is not performed properly. The presented cases suggest that quick sample collection of various matrices during autopsy, i.e. blood and lung or brain tissue sampled directly into head space vials or air from lungs in a foil bag is suitable to detect gasses like helium in postmortem cases. This sampling method can easily be incorporated in standard sampling procedures. Use of HS-GC/TCD enables to detect an inert gas like helium.
6
Page 8 of 9
Reference list
Ac
ce pt
ed
M
an
us
cr
ip t
1. Austin A. et al. Recent trends in suicides utilizing helium. J Forensic Sci. 2011; 56(3): 649-51 2. Gunnell D. et al. Suicide by gases in England and Wales 2001-2011: Evidence of the emergence of new methods of suicide. J Affect Disord. 2014; 170C: 190-195. 3. e.g. www.patientsrightscouncil.org, www.finalexit.org or www.eenwaardiglevenseinde.nl 4. Auwärter V. et al. Analytical investigations in a death case by suffocation in an argon atmosphere. Forensic Sci Int. 2004; 143(2-3): 169-75 5. Ogden R.D. Observation of two suicides by helium inhalation in a prefilled environment. Am J Forensic Med Pathol. 2010; 31(2): 156-61. 6. Grassberger M., Krauskopf A. Suicidal asphyxiation with helium: report of three cases. Wien Klin Wochenschr. 2007; 119(9-10): 323-5. 7. Musshoff F. et al Two cases of suicide by asphyxiation due to helium and argon. Forensic Sci Int. 2012; 223(1-3): e27-30. 8. Schön C.A., Ketterer T. Asphyxial suicide by inhalation of helium inside a plastic bag. Am J Forensic Med Pathol. 2007; 28(4): 364-7. 9. Chen X.H. et al. Solid-phase extraction for the screening of acidic, neutral and basic drugs in plasma using a single-column procedure on Bond Elut Certify. J Chromatogr. 1990; 529:161-166. 10. Boone C. et al. Determination of the most frequently used drugs in whole blood by using ultra performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS). Toxichemical Krimtechnology. 2010; 77:219. 11. Elian A.A. GC-MS determination of gamma-hydroxybutyric acid (GHB) in blood. Forensic Sci. Int. 2001; 122: 43-47. 12. Schaff J.E. et al. A gas chromatography-thermal conductivity detection method for helium detection in postmortem blood and tissue specimens. J Anal Toxicol. 2012; 36(2): 112-5. 13. Auwaerter V. et al. Toxicological analysis after asphyxial suicide with helium and a plastic bag. Forensic Sci Int. 2007; 170(2-3): 139-41. 14. Howard M.O. et al. Suicide by asphyxiation due to helium inhalation. Am J Forensic Med Pathol. 2011; 32(1): 61-70.
7
Page 9 of 9