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Detection of metallic elements from paraffin-embedded tissue blocks by energy dispersive X-ray fluorescence spectrometry
Legal Medicine 12 (2010) 102–103
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Brief Communication
Detection of metallic elements from paraffin-embedded tissue blocks by energy dispersive X-ray fluorescence spectrometry Motonori Takahashi a,*, Hiroshi Kinoshita b, Minori Nishiguchi a, Hajime Nishio a a b
Department of Legal Medicine, Hyogo College of Medicine, Hyogo 663-8501, Japan Department of Forensic Medicine, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan
a r t i c l e
i n f o
Article history: Received 19 November 2009 Received in revised form 11 December 2009 Accepted 11 December 2009 Available online 27 January 2010 Keywords: Element analysis Paraffin-embedded tissue block Gunshot wound Energy dispersive X-ray fluorescence spectrometry
a b s t r a c t Energy dispersive X-ray fluorescence spectrometry (EDX) enables rapid, non-destructive, multi-elemental analysis. Using EDX, lead and some other metallic elements were detected in paraffin-blocks in which skin samples from a gunshot wound were embedded. Lead was not identified in control samples from non-injured cases. These findings indicate that EDX is a useful method for rapid non-destructive analysis of paraffin-embedded blocks. This technique can provide scientific evidence for identification of a firearm even after storing the sample for a long time. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In the field of forensic medicine, identification of the weapon used to inflict a wound is an important subject. Such identification is usually performed according to the morphology and characteristics of the wound. However, metallic residues adhered to the surface of the wound should also be investigated. In the case of gunshot wounds, two methods have been routinely used for detection of the metallic components of gunshot residues [1–3]: bulk analysis, such as atomic absorption spectrometry (AAS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES); and quantitative morphological analysis, such as scanning electron microscopy with energy dispersive Xray spectrometry (SEM–EDX). Although these methods can detect metallic elements with high sensitivities, their sample preparation methods are degenerative and extensive. Energy dispersive X-ray fluorescence spectrometry (EDX) is a simple bulk analysis method that can be used to detect various elements in different types of samples. It only needs a small desktop spectrometer and sample preparation is not necessary for analysis. We have applied EDX analysis to the detection of bromide in seawater [4] and bromide in blood samples from cases of poisoning with bromide containing drugs [5].
* Corresponding author. Address: Department of Legal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. Tel.: +81 798 45 6578; fax: +81 798 49 3279. E-mail address: [email protected] (M. Takahashi). 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.12.003
Histological examinations of paraffin-embedded tissues are important for routine analysis in forensic medicine and pathology. In the present study, we examined the usefulness of EDX to detect metallic elements in paraffin-embedded samples from a gunshot wound. 2. Materials and methods 2.1. Case history A male committed suicide by gunshot and left a suicide note [6]. In autopsy, the entrance and exit holes were found in the pharynx and the occipital region of the head, respectively. A metallic foreign body was found in the gunshot wound in the occipital region. Subsequent EDX analysis of the metallic foreign body indicated that it was a piece of bullet containing lead and antimony. The cause of death was determined to be a brain contusion from the gunshot. The skin of the gunshot wound in the occipital region was collected and embedded in paraffin, and this was analyzed by EDX at a later date. The paraffin-embedded blocks were stored at room temperature for approximately 4 years. 2.2. EDX analysis The spectrometer used in this work for EDX (JSX-3220; JEOL, Tokyo, Japan) was operated as follows: target, rhodium anode; operating voltage and current, 30 kV and 300 lA; X-ray path, vacuum; detector, silicon (lithium); measurement time, 300 s. Two
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EDX enables semi-quantitative analysis by calculating the area of the peak [4,5], it may be possible to differentiate the entrance from the exit of gunshot wound by detecting metallic elements using EDX. EDX spectra showed additional peaks for nickel and iron in both the samples from the gunshot wound and control samples. This result suggests that peaks for these elements are not specific to gunshot wounds. We determined that these metals arise from the container used for the paraffin-embedding procedure, as no peaks for these elements were detected by EDX analysis (data not shown) when we used a nylon mesh pack, which does not contain these metals, for paraffin-block preparation. Identification of metals on skin samples has been performed by various methods, such as AAS, ICP-AES and SEM–EDX. AAS and ICP-AES are simple methods to perform; however their sample preparation methods are degenerative and extensive. Although it is possible to use SEM–EDX to detect characteristic particles from gunshot residues, it can take a long time to obtain results. In comparison, no sample preparation is required for EDX. Although the sensitivity of EDX is approximately 1/1000th or less than that of ICP-AES, it is useful as a screening test when rapid analysis is desired and sample preservation is required for evidence. In this study, we showed that metallic elements in paraffinembedded tissue blocks could be detected by EDX in only 300 s. Further applications of EDX in the fields of forensic medicine and pathology are expected. Acknowledgments The authors have no conflicts of interest to declare. This study was supported by a Grant-of-Aid from Kobe University School of Medicine Alumni Association. We would thank Mrs. Takako Minami for her technical support in paraffin-embedding procedure. References
Fig. 1. EDX spectra of paraffin-embedded skin samples. (A) Paraffin-embedded skin samples from a gunshot wound (n = 1) showing peaks for lead (Pb-La, b, c) and other elements, such as nickel (Ni-Ka, b), iron (Fe-Ka), calcium (Ca-Ka, b), sulfur (SKa) and phosphorus (P-Ka) but not antimony or barium. (B) Control samples of non-injured skin (n = 10) showing no peaks for lead.
paraffin-embedded skin samples from the gunshot wound case (n = 1) and 10 samples from control non-injured cases (n = 10) were analyzed without any further sample preparation. 3. Results and discussion Bullets usually contain lead, antimony and barium in the primer, and copper and zinc in the metal jacket [7–9]. Iron may come from the barrel of the firearm or from the bullet if it had a European-type metallic jacket [9,10]. For positive identification of a gunshot wound it is necessary to detect lead and at least one other element out of antimony or barium [2,3]. In this study, paraffinembedded skin samples were examined by EDX. Peaks for some metallic elements, such as lead, nickel and iron, were identified in the skin samples from the gunshot wound, while antimony and barium were not detected (Fig. 1). This may be because not enough antimony or barium had adhered to the skin around the exit hole to allow detection by EDX. Lead concentrations around entrance holes are reported to be higher than those around the corresponding exit holes [11]. As
[1] Saverio Romolo F, Margot P. Identification of gunshot residue: a critical review. Forensic Sci Int 2001;119(2):195–211. [2] Molina DK, Martinez M, Garcia J, DiMaio VJ. Gunshot residue testing in suicides: Part I: Analysis by scanning electron microscopy with energydispersive X-ray. Am J Forensic Med Pathol 2007;28(3):187–90. [3] Molina DK, Castorena JL, DiMaio VJ. Gunshot residue testing in suicides: Part II: Analysis by inductive coupled plasma-atomic emission spectrometry. Am J Forensic Med Pathol 2007;28(3):191–4. [4] Takahashi M, Kinoshita H, Nishiguchi M, Ouchi H, Minami T, Matsui K, et al. Application of energy dispersive X-ray fluorescent spectrometry (EDXRF) for the quantification of bromide in water. Curr Study Environ Med Sci 2008;1:11–3. [5] Takahashi M, Kinoshita H, Nishiguchi M, Kasuda S, Ouchi H, Minami T, et al. Application of energy dispersive X-ray fluorescent spectrometry (EDXRF) in drug-related cases. Leg Med (Tokyo) 2009;11:S411–2. [6] Kinoshita H, Nishiguchi M, Kasuda S, Matsui K, Ouchi H, Minami T, et al. Application of energy dispersive X-ray fluorescent spectrometry (EDXRF) in the field of forensic medicine: identification of the lethal weapon. Med Biol 2008;152:108–10. [7] Wunnapuk K, Durongkadech P, Minami T, Ruangyuttikarn W, Tohno S, Vichairat K, et al. Differences in the element contents between gunshot entry wounds with full-jacketed bullet and lead bullet. Biol Trace Elem Res 2007;120(1–3):74–81. [8] Wunnapuk K, Minami T, Durongkadech P, Tohno S, Ruangyuttikarn W, Moriwake Y, et al. Discrimination of bullet types using analysis of lead isotopes deposited in gunshot entry wounds. Biol Trace Elem Res 2009;129(1– 3):278–89. [9] Brazeau J, Wong R. Analysis of gunshot residues on human tissues and clothing by X-ray microfluorescence. J Forensic Sci 1997;42:424–8. [10] Miyauchi H, Kumihashi M, Shibayama T. The contribution of trace elements from smokeless powder to post firing residues. J Forensic Sci 1998;43:90–6. [11] Ohtsuji M, Rohwer J, Oehmichen M, Ohshima T. Inorganic lead concentration analysis at the gunshot wounds for differentiation of entrance from exit hole. Acta Crim Japon 1998;64(6):213–8.
Contents lists available at ScienceDirect
Legal Medicine journal homepage: www.elsevier.com/locate/legalmed
Brief Communication
Detection of metallic elements from paraffin-embedded tissue blocks by energy dispersive X-ray fluorescence spectrometry Motonori Takahashi a,*, Hiroshi Kinoshita b, Minori Nishiguchi a, Hajime Nishio a a b
Department of Legal Medicine, Hyogo College of Medicine, Hyogo 663-8501, Japan Department of Forensic Medicine, Faculty of Medicine, Kagawa University, Kagawa 761-0793, Japan
a r t i c l e
i n f o
Article history: Received 19 November 2009 Received in revised form 11 December 2009 Accepted 11 December 2009 Available online 27 January 2010 Keywords: Element analysis Paraffin-embedded tissue block Gunshot wound Energy dispersive X-ray fluorescence spectrometry
a b s t r a c t Energy dispersive X-ray fluorescence spectrometry (EDX) enables rapid, non-destructive, multi-elemental analysis. Using EDX, lead and some other metallic elements were detected in paraffin-blocks in which skin samples from a gunshot wound were embedded. Lead was not identified in control samples from non-injured cases. These findings indicate that EDX is a useful method for rapid non-destructive analysis of paraffin-embedded blocks. This technique can provide scientific evidence for identification of a firearm even after storing the sample for a long time. Ó 2009 Elsevier Ireland Ltd. All rights reserved.
1. Introduction In the field of forensic medicine, identification of the weapon used to inflict a wound is an important subject. Such identification is usually performed according to the morphology and characteristics of the wound. However, metallic residues adhered to the surface of the wound should also be investigated. In the case of gunshot wounds, two methods have been routinely used for detection of the metallic components of gunshot residues [1–3]: bulk analysis, such as atomic absorption spectrometry (AAS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES); and quantitative morphological analysis, such as scanning electron microscopy with energy dispersive Xray spectrometry (SEM–EDX). Although these methods can detect metallic elements with high sensitivities, their sample preparation methods are degenerative and extensive. Energy dispersive X-ray fluorescence spectrometry (EDX) is a simple bulk analysis method that can be used to detect various elements in different types of samples. It only needs a small desktop spectrometer and sample preparation is not necessary for analysis. We have applied EDX analysis to the detection of bromide in seawater [4] and bromide in blood samples from cases of poisoning with bromide containing drugs [5].
* Corresponding author. Address: Department of Legal Medicine, Hyogo College of Medicine, 1-1 Mukogawa-cho, Nishinomiya, Hyogo 663-8501, Japan. Tel.: +81 798 45 6578; fax: +81 798 49 3279. E-mail address: [email protected] (M. Takahashi). 1344-6223/$ - see front matter Ó 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.legalmed.2009.12.003
Histological examinations of paraffin-embedded tissues are important for routine analysis in forensic medicine and pathology. In the present study, we examined the usefulness of EDX to detect metallic elements in paraffin-embedded samples from a gunshot wound. 2. Materials and methods 2.1. Case history A male committed suicide by gunshot and left a suicide note [6]. In autopsy, the entrance and exit holes were found in the pharynx and the occipital region of the head, respectively. A metallic foreign body was found in the gunshot wound in the occipital region. Subsequent EDX analysis of the metallic foreign body indicated that it was a piece of bullet containing lead and antimony. The cause of death was determined to be a brain contusion from the gunshot. The skin of the gunshot wound in the occipital region was collected and embedded in paraffin, and this was analyzed by EDX at a later date. The paraffin-embedded blocks were stored at room temperature for approximately 4 years. 2.2. EDX analysis The spectrometer used in this work for EDX (JSX-3220; JEOL, Tokyo, Japan) was operated as follows: target, rhodium anode; operating voltage and current, 30 kV and 300 lA; X-ray path, vacuum; detector, silicon (lithium); measurement time, 300 s. Two
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EDX enables semi-quantitative analysis by calculating the area of the peak [4,5], it may be possible to differentiate the entrance from the exit of gunshot wound by detecting metallic elements using EDX. EDX spectra showed additional peaks for nickel and iron in both the samples from the gunshot wound and control samples. This result suggests that peaks for these elements are not specific to gunshot wounds. We determined that these metals arise from the container used for the paraffin-embedding procedure, as no peaks for these elements were detected by EDX analysis (data not shown) when we used a nylon mesh pack, which does not contain these metals, for paraffin-block preparation. Identification of metals on skin samples has been performed by various methods, such as AAS, ICP-AES and SEM–EDX. AAS and ICP-AES are simple methods to perform; however their sample preparation methods are degenerative and extensive. Although it is possible to use SEM–EDX to detect characteristic particles from gunshot residues, it can take a long time to obtain results. In comparison, no sample preparation is required for EDX. Although the sensitivity of EDX is approximately 1/1000th or less than that of ICP-AES, it is useful as a screening test when rapid analysis is desired and sample preservation is required for evidence. In this study, we showed that metallic elements in paraffinembedded tissue blocks could be detected by EDX in only 300 s. Further applications of EDX in the fields of forensic medicine and pathology are expected. Acknowledgments The authors have no conflicts of interest to declare. This study was supported by a Grant-of-Aid from Kobe University School of Medicine Alumni Association. We would thank Mrs. Takako Minami for her technical support in paraffin-embedding procedure. References
Fig. 1. EDX spectra of paraffin-embedded skin samples. (A) Paraffin-embedded skin samples from a gunshot wound (n = 1) showing peaks for lead (Pb-La, b, c) and other elements, such as nickel (Ni-Ka, b), iron (Fe-Ka), calcium (Ca-Ka, b), sulfur (SKa) and phosphorus (P-Ka) but not antimony or barium. (B) Control samples of non-injured skin (n = 10) showing no peaks for lead.
paraffin-embedded skin samples from the gunshot wound case (n = 1) and 10 samples from control non-injured cases (n = 10) were analyzed without any further sample preparation. 3. Results and discussion Bullets usually contain lead, antimony and barium in the primer, and copper and zinc in the metal jacket [7–9]. Iron may come from the barrel of the firearm or from the bullet if it had a European-type metallic jacket [9,10]. For positive identification of a gunshot wound it is necessary to detect lead and at least one other element out of antimony or barium [2,3]. In this study, paraffinembedded skin samples were examined by EDX. Peaks for some metallic elements, such as lead, nickel and iron, were identified in the skin samples from the gunshot wound, while antimony and barium were not detected (Fig. 1). This may be because not enough antimony or barium had adhered to the skin around the exit hole to allow detection by EDX. Lead concentrations around entrance holes are reported to be higher than those around the corresponding exit holes [11]. As
[1] Saverio Romolo F, Margot P. Identification of gunshot residue: a critical review. Forensic Sci Int 2001;119(2):195–211. [2] Molina DK, Martinez M, Garcia J, DiMaio VJ. Gunshot residue testing in suicides: Part I: Analysis by scanning electron microscopy with energydispersive X-ray. Am J Forensic Med Pathol 2007;28(3):187–90. [3] Molina DK, Castorena JL, DiMaio VJ. Gunshot residue testing in suicides: Part II: Analysis by inductive coupled plasma-atomic emission spectrometry. Am J Forensic Med Pathol 2007;28(3):191–4. [4] Takahashi M, Kinoshita H, Nishiguchi M, Ouchi H, Minami T, Matsui K, et al. Application of energy dispersive X-ray fluorescent spectrometry (EDXRF) for the quantification of bromide in water. Curr Study Environ Med Sci 2008;1:11–3. [5] Takahashi M, Kinoshita H, Nishiguchi M, Kasuda S, Ouchi H, Minami T, et al. Application of energy dispersive X-ray fluorescent spectrometry (EDXRF) in drug-related cases. Leg Med (Tokyo) 2009;11:S411–2. [6] Kinoshita H, Nishiguchi M, Kasuda S, Matsui K, Ouchi H, Minami T, et al. Application of energy dispersive X-ray fluorescent spectrometry (EDXRF) in the field of forensic medicine: identification of the lethal weapon. Med Biol 2008;152:108–10. [7] Wunnapuk K, Durongkadech P, Minami T, Ruangyuttikarn W, Tohno S, Vichairat K, et al. Differences in the element contents between gunshot entry wounds with full-jacketed bullet and lead bullet. Biol Trace Elem Res 2007;120(1–3):74–81. [8] Wunnapuk K, Minami T, Durongkadech P, Tohno S, Ruangyuttikarn W, Moriwake Y, et al. Discrimination of bullet types using analysis of lead isotopes deposited in gunshot entry wounds. Biol Trace Elem Res 2009;129(1– 3):278–89. [9] Brazeau J, Wong R. Analysis of gunshot residues on human tissues and clothing by X-ray microfluorescence. J Forensic Sci 1997;42:424–8. [10] Miyauchi H, Kumihashi M, Shibayama T. The contribution of trace elements from smokeless powder to post firing residues. J Forensic Sci 1998;43:90–6. [11] Ohtsuji M, Rohwer J, Oehmichen M, Ohshima T. Inorganic lead concentration analysis at the gunshot wounds for differentiation of entrance from exit hole. Acta Crim Japon 1998;64(6):213–8.