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Synovial fluid: An alternative source for forensic DNA
Forensic Science International: Genetics Supplement Series 3 (2011) e323–e324
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Synovial fluid: An alternative source for forensic DNA Kornkiat Vongpaisarnsin a,*, Wisut Charito a, Achara Boonlert b, Nat Tonsrisawat a, Teerachote Jongsakul a a b
Department of Forensic Medicine, Faculty of Medicine, Chulalongkorn University, 1873 Rama 4 Road, Pathumwan, Bangkok 10330, Thailand Forensic Serology and DNA Laboratory, Forensic Medicine Unit, King Chulalongkorn Memorial Hospital and Thai Red Cross Society, Thailand
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 August 2011 Received in revised form 11 September 2011 Accepted 15 September 2011
In human remains that have undergone decomposition, blood and other biological tissues are recognized as sources for forensic DNA identification, yet the success rate is influenced by the extent of decomposition to the subcellular structures and nucleic acids. Thus, synovial fluid may be considered as an alternative source for forensic DNA recovery, as compared to other decomposed tissues. In this study, 24 samples (12 synovial, 8 blood, and 4 muscle tissue samples) were obtained from 12 decomposed bodies. Our results show that synovial fluid is a suitable source for forensic DNA recovery compared to decomposed blood or muscle tissue. In addition, no mixture or contamination of DNA was detected in synovial sources. Further study is required to determine whether an additional pre-treatment of the synovial sample with hydrolysis can potentially recover more DNA. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Synovial fluid Forensic DNA Hydrolysis
1. Introduction Many degraded human samples, blood or tissues, have been studied for their success in forensic DNA recovery [1]. The alternative source of material, vitreous humor, is generally used for forensic toxicology detection. However, few studies have reported its use for DNA identification because the limit of the collected volume is rather low due to postmortem dehydration [2]. Synovial fluid is another source of tissue sample that should be examined for DNA identification purposes considering that it is highly viscous, and under normal conditions, a knee joint contains leukocytes approximately 200 cells/mm3. Moreover, bacteria or autolytic enzymes are not detected in healthy human joints [3]. 2. Materials and methods The knee joint, sub-patellar bursa, was approached while the body was positioned in a knee–hip flexion position. After extensively washing the skin remnant with running water, the slipped skin was subsequently cleansed with 90% alcohol. A 30-mL syringe and an 18-gauge needle were chosen for the aspiration of the synovial fluid, which was then transferred to a 1.5-mL microcentrifuge tube. The reference DNA was collected from the heart blood (3–5 mL) or the deep muscle from the upper thigh
* Corresponding author. Tel.: +66 2256 4296x204; fax: +66 2652 5150. E-mail addresses: [email protected], [email protected] (K. Vongpaisarnsin). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.09.024
(approximately 8 cm3). These samples were kept in a sterile container and stored at 20 8C until the extraction was performed. The finely minced muscle tissue and synovial samples were incubated with proteinase K solution at 56 8C overnight or until completely liquidified. Two different kits, the QIAamp DNA Micro Kit by Qiagen1 and the DNA IQTM System by Promega1, were used for the DNA extraction and purification according to the manufacturers’ protocols. A total of 2 mL of extracted synovial sample was quantified using the Quantifiler1 kit running on the 7500 Real-Time PCR System by Applied Biosystems following the manufacturer’s recommendations. Amplification was performed using the AmpFlSTR1 IdentifilerTM kit with 5 mL of the DNA sample and a modified protocol using one-half volume of the PCR master mix (7.5 mL/reaction). The thermal cycling was performed using the GeneAmp PCR 9700 Applied Biosystems for 28 cycles, and the DNA typing was performed using capillary electrophoresis with the ABI 3130xl Genetic Analyzer. The alleles were determined using GeneMapper ID software version 3.2. 3. Results Of the 12 synovial fluid samples, 6 (50%) synovial samples (E7, E9, E25, E27, E35 and E42 with concentration 0.461, 2.199, 0.678, 0.241, and 0.022 ng/mL, accordingly, except E42 that had limited sample volume) successfully provided a DNA profile, whereas 6 blood or tissue samples did not reveal any DNA profiles. All of the synovial samples had a positive IPC (internal PCR control), indicating no effect of any PCR inhibitors in each reaction (data not shown). The DNA profile in E35 and E42 samples shows a successful recovery from both sources.
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K. Vongpaisarnsin et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e323–e324
Fig. 1. The DNA profiles were compared between decomposed blood/tissue and synovial fluid samples. The blood sample (A) revealed a partial DNA profile with extremely low peak height. The synovial sample (B) provided more information regarding alleles but exhibited a low peak height. The synovial sample with alkaline hydrolysis (C) recovered DNA with a balance peak height for each locus. Moreover, a dropout phenomenon (arrows) was detected in the synovial sample (B).
4. Discussion Our results show that synovial fluid is a suitable source for forensic DNA recovery compared to decomposed blood or muscle tissue. In addition, no mixture or contamination of DNA was detected in synovial sources. In general, the high viscosity of synovial fluid is not amenable to DNA extraction with magnetic resins. To facilitate the extraction, some procedures involve the reduction of the viscosity using alkaline hydrolysis, which breaks down polysaccharide into monosaccharide, and NaOH has been tested for its ability to hydrolyze and neutralize PCR inhibitors [4]. The synovial sample (E35) was divided into two conditions with and without an alkaline hydrolysis solution by adding 0.2 mL of 0.4 M NaOH to hydrolyze the sample into a liquid solution followed by a general PCR protocol, and the DNA profile was recovered completely. Moreover, dropout alleles occurred in the nonhydrolyze sample (Fig. 1). The well-balanced peak height in the heterozygous locus was not lower than the recommended level of
60%. Even though the synovial sample was a robust source, we recommend that pre-PCR concentrations should be greater than 0.1 ng/mL to increase the success of the DNA profile recovery. Conflict of interest None. References [1] P.H. Olsen, S. Jacobsen, B. Mevag, et al., Microsatellite stability in human postmortem tissues, Forensic Sci. Int. 119 (July (3)) (2001) 273–278. [2] I. Soltyszewskil, J.A. Niemcunowicz, W. Pepin˜ski, et al., Vitreous humour as a potential DNA source for postmortem human identification, Folia Histochem. Cytobiol. 45 (2007) 135–136. [3] B. Madea, C. Kreuser, S. Banaschak, Postmortem biochemical examination of synovial fluid – a preliminary study, Forensic Sci. Int. 118 (April (1)) (2001) 29–35. [4] M.T. Bourke, C.A. Scherczinger, C. Ladd, et al., NaOH treatment to neutralize inhibitors of Taq polymerase, J. Forensic Sci. 44 (September (5)) (1999) 1046–1050.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Synovial fluid: An alternative source for forensic DNA Kornkiat Vongpaisarnsin a,*, Wisut Charito a, Achara Boonlert b, Nat Tonsrisawat a, Teerachote Jongsakul a a b
Department of Forensic Medicine, Faculty of Medicine, Chulalongkorn University, 1873 Rama 4 Road, Pathumwan, Bangkok 10330, Thailand Forensic Serology and DNA Laboratory, Forensic Medicine Unit, King Chulalongkorn Memorial Hospital and Thai Red Cross Society, Thailand
A R T I C L E I N F O
A B S T R A C T
Article history: Received 19 August 2011 Received in revised form 11 September 2011 Accepted 15 September 2011
In human remains that have undergone decomposition, blood and other biological tissues are recognized as sources for forensic DNA identification, yet the success rate is influenced by the extent of decomposition to the subcellular structures and nucleic acids. Thus, synovial fluid may be considered as an alternative source for forensic DNA recovery, as compared to other decomposed tissues. In this study, 24 samples (12 synovial, 8 blood, and 4 muscle tissue samples) were obtained from 12 decomposed bodies. Our results show that synovial fluid is a suitable source for forensic DNA recovery compared to decomposed blood or muscle tissue. In addition, no mixture or contamination of DNA was detected in synovial sources. Further study is required to determine whether an additional pre-treatment of the synovial sample with hydrolysis can potentially recover more DNA. ß 2011 Elsevier Ireland Ltd. All rights reserved.
Keywords: Synovial fluid Forensic DNA Hydrolysis
1. Introduction Many degraded human samples, blood or tissues, have been studied for their success in forensic DNA recovery [1]. The alternative source of material, vitreous humor, is generally used for forensic toxicology detection. However, few studies have reported its use for DNA identification because the limit of the collected volume is rather low due to postmortem dehydration [2]. Synovial fluid is another source of tissue sample that should be examined for DNA identification purposes considering that it is highly viscous, and under normal conditions, a knee joint contains leukocytes approximately 200 cells/mm3. Moreover, bacteria or autolytic enzymes are not detected in healthy human joints [3]. 2. Materials and methods The knee joint, sub-patellar bursa, was approached while the body was positioned in a knee–hip flexion position. After extensively washing the skin remnant with running water, the slipped skin was subsequently cleansed with 90% alcohol. A 30-mL syringe and an 18-gauge needle were chosen for the aspiration of the synovial fluid, which was then transferred to a 1.5-mL microcentrifuge tube. The reference DNA was collected from the heart blood (3–5 mL) or the deep muscle from the upper thigh
* Corresponding author. Tel.: +66 2256 4296x204; fax: +66 2652 5150. E-mail addresses: [email protected], [email protected] (K. Vongpaisarnsin). 1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2011.09.024
(approximately 8 cm3). These samples were kept in a sterile container and stored at 20 8C until the extraction was performed. The finely minced muscle tissue and synovial samples were incubated with proteinase K solution at 56 8C overnight or until completely liquidified. Two different kits, the QIAamp DNA Micro Kit by Qiagen1 and the DNA IQTM System by Promega1, were used for the DNA extraction and purification according to the manufacturers’ protocols. A total of 2 mL of extracted synovial sample was quantified using the Quantifiler1 kit running on the 7500 Real-Time PCR System by Applied Biosystems following the manufacturer’s recommendations. Amplification was performed using the AmpFlSTR1 IdentifilerTM kit with 5 mL of the DNA sample and a modified protocol using one-half volume of the PCR master mix (7.5 mL/reaction). The thermal cycling was performed using the GeneAmp PCR 9700 Applied Biosystems for 28 cycles, and the DNA typing was performed using capillary electrophoresis with the ABI 3130xl Genetic Analyzer. The alleles were determined using GeneMapper ID software version 3.2. 3. Results Of the 12 synovial fluid samples, 6 (50%) synovial samples (E7, E9, E25, E27, E35 and E42 with concentration 0.461, 2.199, 0.678, 0.241, and 0.022 ng/mL, accordingly, except E42 that had limited sample volume) successfully provided a DNA profile, whereas 6 blood or tissue samples did not reveal any DNA profiles. All of the synovial samples had a positive IPC (internal PCR control), indicating no effect of any PCR inhibitors in each reaction (data not shown). The DNA profile in E35 and E42 samples shows a successful recovery from both sources.
e324
K. Vongpaisarnsin et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e323–e324
Fig. 1. The DNA profiles were compared between decomposed blood/tissue and synovial fluid samples. The blood sample (A) revealed a partial DNA profile with extremely low peak height. The synovial sample (B) provided more information regarding alleles but exhibited a low peak height. The synovial sample with alkaline hydrolysis (C) recovered DNA with a balance peak height for each locus. Moreover, a dropout phenomenon (arrows) was detected in the synovial sample (B).
4. Discussion Our results show that synovial fluid is a suitable source for forensic DNA recovery compared to decomposed blood or muscle tissue. In addition, no mixture or contamination of DNA was detected in synovial sources. In general, the high viscosity of synovial fluid is not amenable to DNA extraction with magnetic resins. To facilitate the extraction, some procedures involve the reduction of the viscosity using alkaline hydrolysis, which breaks down polysaccharide into monosaccharide, and NaOH has been tested for its ability to hydrolyze and neutralize PCR inhibitors [4]. The synovial sample (E35) was divided into two conditions with and without an alkaline hydrolysis solution by adding 0.2 mL of 0.4 M NaOH to hydrolyze the sample into a liquid solution followed by a general PCR protocol, and the DNA profile was recovered completely. Moreover, dropout alleles occurred in the nonhydrolyze sample (Fig. 1). The well-balanced peak height in the heterozygous locus was not lower than the recommended level of
60%. Even though the synovial sample was a robust source, we recommend that pre-PCR concentrations should be greater than 0.1 ng/mL to increase the success of the DNA profile recovery. Conflict of interest None. References [1] P.H. Olsen, S. Jacobsen, B. Mevag, et al., Microsatellite stability in human postmortem tissues, Forensic Sci. Int. 119 (July (3)) (2001) 273–278. [2] I. Soltyszewskil, J.A. Niemcunowicz, W. Pepin˜ski, et al., Vitreous humour as a potential DNA source for postmortem human identification, Folia Histochem. Cytobiol. 45 (2007) 135–136. [3] B. Madea, C. Kreuser, S. Banaschak, Postmortem biochemical examination of synovial fluid – a preliminary study, Forensic Sci. Int. 118 (April (1)) (2001) 29–35. [4] M.T. Bourke, C.A. Scherczinger, C. Ladd, et al., NaOH treatment to neutralize inhibitors of Taq polymerase, J. Forensic Sci. 44 (September (5)) (1999) 1046–1050.