- Email: [email protected]
Analysis of DNA profiles extracted from degraded samples from archival of formalin fixed tissue included in paraffin (FFTIP) and hairs
Forensic Science International: Genetics Supplement Series 2 (2009) 167–168
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Research article
Analysis of DNA profiles extracted from degraded samples from archival of formalin fixed tissue included in paraffin (FFTIP) and hairs Edna S. Miazato Iwamura a,*, Jose´ Arnaldo Soares-Vieira b, Marcelo Souza Silva a, ˜ oz b Karina S. Funabashi a, Carla D. Godoy a, Daniel Romero Mun a b
Paulista Medical School, Federal University of Sa˜o Paulo – EPM/UNIFESP, Department of Pathology, Sa˜o Paulo, Brazil Faculty of Medicine Sa˜o Paulo University, Department of Legal Medicine, Medical Ethics, Social and Occupational Health, Sa˜o Paulo, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 August 2009 Accepted 14 August 2009
The possibility of studying DNA extracted from archival of formalin fixed tissue included in paraffin (FFTIP) enables valuable retrospective investigations. However, according to some authors it is difficult to obtain genomic DNA of good quality, since the process of fixation often results in fragmentation of DNA. In order to evaluate the quality and quantity of DNA extracted, necropsy samples of FFTIP (spleen/ lung) and hairs, with or without bulbs, were analyzed using three methods of extraction (QIAamp DNA mini, QIAamp DNA micro-kit and phenol–chloroform followed by microcon YM-30). The amount of DNA recovered was quantified by spectrophotometer. The b-actin, amelogenin gene and the profiles of STR were analyzed. Based on experimental results, a general guideline concerning the appropriate extraction method according to the tissue and the quantity of the starting material for the analysis of DNA from FFTIP and hairs could be suggested. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Paraffin Hairs DNA PCR Short amplicons STR
1. Introduction Tissues embedded in paraffin are an extraordinary source for DNA molecular studies because of the availability of large pathology archives of tissues related to clinical cases in almost all hospital pathology departments. In addition to biopsy and surgical paraffin embedded tissues, postmortem tissues are an important resource, especially for rare diseases, neuropathology or molecular epidemiology studies, because both pathological and normal tissues can be analyzed. The major difficulty in using these tissues is the degradation of nucleic acids [1]. During formalin fixation, cross-links of protein–protein and protein–DNA interactions are formed. In addition, the formaldehyde in the tissue gradually changes into formic acid, hydrolyzing the DNA. The fixative used, inclusion and storage conditions can contribute to the degradation of DNA [2,3]. Another source of DNA, easily found and particularly important in forensics, is hair samples. However, the small DNA quantity in hair is usually degraded. Another problem is PCR inhibitors in the hair. In particular hair pigments, the melanins, are known to inhibit PCR. To increase the chances of correct typing of hair, the small amount of DNA must be successfully isolated and the inhibitors have to be removed or neutralized [4–6]. Today, the
* Corresponding author. E-mail address: [email protected] (E.S.M. Iwamura). 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.096
short tandem repeat (STR) typing of FFTIP and hair shafts is regarded as a challenge. The first aim of this study is to evaluate the quality and quantity of DNA extracted from FFTIP and hairs with or without bulbs using different methods of extraction. The second aim is to analyze the best protocol for each type of sample, in order to obtain the profile of STR typing for human identification. 2. Materials and methods Tissue samples fixed in formalin included in paraffin (FFTIP) in 2002: Fresh tissue specimens of spleen and lung from an AIDS patient, who died from miliary tuberculosis, positive for acid-fast bacilli, were subjected to tissue fixation and paraffin embedding. As negative control, a non-infected block from spleen was tested. Fixation was carried out at room temperature, using formalin phosphate-buffered at pH 7.0 (formaldehyde 37%, monosodic phosphate 47 mM, disodic phosphate 28 mM) or 10% formalin in distilled water. Embedding was performed using paraffin (Retrowax-Parafinas Nordeste). Pubic hair samples: Pubic hair samples were obtained from one individual. For each sample the morphological analysis was performed with light microscope. The first segment of the hair, including the root, was 0.5 cm in length, cut with a razor blade. The hair samples without bulb were obtained by cutting the bulb and following proximal 0.5 cm portion. Tissue and patient data were obtained and used after advice of the Medical Ethics Committee.
168
E.S.M. Iwamura et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 167–168
2.1. Extraction of archival of FFTIP and pubic hairs from a female From each paraffin-embedded specimen, 3–10-mm thick sections were cut, using a new blade for each sample. Paraffin was removed using four 30 min incubations with xylol (Merck, Germany) followed by four washes with 100% ethanol. Tissue was digested with proteinase K 600 mg/mL (final concentration) in TE (Tris–HCl 1 mM, EDTA 0.5 mM pH 8) and Tween 20 0.5% for 16 h at 37 8C. Samples were subjected to heat-shock in liquid nitrogen, for 1 min, and incubated for 10 min at 95 8C. DNA was extracted with: (a) phenol–chloroform–isoamylic alcohol 25:24:1 and precipitated with 10% sodium acetate 3 M pH 4.8 and 2.5 volumes of absolute ethanol (Merck). The precipitate was resuspended in TE and concentrated in microcon YM-30 (Millipore); (b) QIAamp Mini Kit (Qiagen) extraction according to the manufacturer. We collected 1, 2, 3 and 6 hair samples in each tube (with and without bulbs) in triplicates. DNA was extracted from those segments of hairs using: (a) 100 ml of the following solution: 29 ml of Tween 201 (USB), 38.5 ml PCR buffer 10 (Invitrogen), 38.5 ml MgCl2 (25 mM) Invitrogen; 3.8 ml of H2O and 0.5 ml Proteinase K 20 mg/ml (Qiagen). The samples were incubated at 60 8C for 15 min and 95 8C for 15 min. Subjected to heat-shock (2) in nitrogen liquid, for 1 min, and incubated for 10 min at 100 8C (Layr -J.Ryal, Brasil); (b) QIAamp micro-kit (Qiagen) according to the manufacturer. After the extraction of DNA, concentration was determined in NanoDrop spectrophotometer at 260 nm and purity was evaluated by analysis of readings at 260 and 280 nm. Amplification of human b-actin gene: A 135 bp fragment of the human b-actin gene was amplified using 2.5 ml (0.5 mM of primers b5: AGCGGGAAATCGTGCGTG and bR: GGTGATGACCTGGCCGTC) in reactions containing 12 ml of Taq PCR Master Mix (Qiagen), 8.0 ml of H2O and 2.0 ml DNA (20 ng) was amplified with one cycle at 96 8C for 5 min, 40 cycles at 96 8C for 2 min, 60 8C for 2 min and 72 8C for 2 min and a final extension cycle at 72 8C for 7 min. Amplification of amelogenin gene (Promega) and Identifiler kit (Applied Biosystems): The samples were amplified according to manufacturer’s recommendations. PCR products were performed in GeneAmp PCR System 9700 (Applied Biosystems) and visualized in 2% agarose gels in buffer TBE (Tris–borate 45 mM, EDTA 1 mM) under UV light, after electrophoresis at 100 V/min and gel red staining. The DNA typing was performed using the AmpFlSTR IdentifilerTM Kit. Fragment size analysis was performed using the GeneMapper v.3.1 software after electrophoresis on the ABI 3130 Avant Genetic Analyzer (Applied Biosystems). Control supplied with AmpFlSTR Kits [7].
Hairs: The QIAamp DNA micro-kit (a and b) and the PCR buffer DNA extraction (c–g) results are as follows in ng/ml: (a) 6 bulbs: 8.64 0.73 (n = 3); (b) 6 shafts without bulbs: 2.99 0.66 (n = 3); (c) 6 bulbs: 33.29 13.89 (n = 3); (d) 6 shafts without bulbs: 12.78 4.50 (n = 3); (e) 3 shafts without bulbs: 13.23 7.74 (n = 6); (f) 2 shafts without bulbs: 9.26 1.13 (n = 5); (g) 1 shaft without bulb: 8.79 1.02 (n = 5). PCR: (a, c, and d) b-actin positive and (b, e, f, and g) negative, in the agarose gel. Amelogenin gene and Identifiler loci were negative, except a and c, in the ABI 3130 Avant sequencer. To avoid intra-individual variation, hairs were collected from one individual, by herself, resulting in a total of 100 plucked pubic hairs. Each sample contained 1, 2, 3 or 6 hair segments, with or without bulbs (in triplicates). The 6 segments with bulbs served as a source for DNA control for DNA typing in the Identifiler kit. The procedures for extraction and quantification were repeated at least three times. 4. Conclusions For FFTIP, the best PCR performance was the QIAamp DNA mini. However in cases in which there is a minute amount of tissue, for example biopsy material, the QIAamp DNA micro-kit could be better. For the hairs, with or without bulbs, the best strategy was extraction directly in the reagent buffers from PCR. Our analysis shows that, despite degradation, amplification of short specific DNA sequences (including those from single copy genes) is still possible. The pubic hair DNA extraction approach has shown to be a very useful strategy for alternative protocols when the goal is to analyze low copy numbers of DNA. This preliminary results has been very important, in both autopsy and biopsy specimen studies. The techniques illustrated here should be applicable to FFTIP sample investigations for any disease where short amplicons (bacterial, viral, and parasitic agents or endogenous alterations at the DNA level) are thought to play a causative role, including those of genetic diseases. However, for PCR in the STR multiplex analysis, the need of improving the purity and balance of DNA quantity is the goal and challenge in regard to both FFTIP and hairs. Conflict of interest None. Acknowledgement We would like to thank Joaquim Soares de Almeida for his technical assistance. This work was supported by grants from FAPESP 2008/11233-8, CAPES and LIM HC FMUSP.
3. Results and discussion
References
FFTIP: The amount of DNA extracted from FFTIP after seven years of storage with phenol–chloroform followed by microcon YM-30 (a and b) and QIAamp DNA mini kit (c and d) extraction: (a) spleen (n = 5): 67.55 0.02; (b) lung (n = 4): 21.81 0.09; (c) spleen (n = 4): 60.08 0.03; (d) lung (n = 5): 57.93 0.11. Purity analysis of readings at 260/280 nm: 1.69–1.90 (a and b) and 1.89–2.0 (c and d), respectively. PCR: b-actin gene amplified 60% (a and b) and 90% (c and d); amelogenin gene 25% (a and b) and 50% (c and d); Identifiler kit none. Because we had previously (2002) utilized the PCR, in FFTIP sections, to detect the presence of IS6110 (123 bp), specific from the Mycobacterium tuberculosis complex [8], we chose to use the same material as a model system to ascertain the possibility of studying archival tissue specimens. The success of PCR amplification of b-actin gene (135 bp) was 90% from lung and spleen (FFTIP), however the failure of Identifiler kit may be critically dependant on a variety of factors such as handling of specimens before tissue fixation, storage, imbalance and purity of DNA.
[1] S. Bonin, F. Petrera, B. Niccolini, G. Stanta, PCR analysis in archival postmortem tissues, J. Clin. Pathol.: Mol. Pathol. 56 (2003) 184–186. [2] A. Gillio-Tos, L. De Marco, V. Fiano, F. Garcia-Bragado, R. Dikshit, P. Boffetta, F. Merletti, Efficient DNA extraction from 25-year-old paraffin-embedded tissues: study of 365 samples, Pathology 39 (2007) 345–348. [3] Z. Budimlija, C. Lu, G. Axler-DiPerte, J. Seifarth, D. Popiolek, F. Fogt, M. Prinz, Malignant tumors and forensics—dilemmas and proposals, Croat. Med. J. 50 (2009) 218–227. [4] L. Bourguignon, B. Hoste, T. Boonen, K. Vits, F. Hubrecht, A fluorescent microscopyscreening test for efficient STR-typing of telogen hair roots, Forensic Sci. Int. Genet. 3 (2008) 27–31. [5] R. Szibor, I. Plate, H. Schmitter, H. Wittig, D. Krause, Forensic mass screening using mtDNA, Int. J. Legal Med. 120 (2006) 372–376. [6] A. Hellmann, U. Rohleder, H. Schmitter, M. Wittig, STR typing of human telogen hairs—a new approach, Int. J. Legal Med. 114 (2001) 269–273. [7] E.S.M. Iwamura, J.A. Soares-Vieira, J.C. Ponce, D.F. Zamarrenho, A.C. Cruvinel, M.G. Pradal, D.R. Mun˜oz, Paternity investigation analysis in artificial mother-deficient cases using STR loci in the Brazilian population, Forensic Sci. Int. Genet. Supp. Ser. 1 (2008) 516–517. [8] D. Barcelos, M.F. Franco, S.C. Lea˜o, Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffinembedded samples, Rev. Inst. Med. Trop. Sao Paulo 50 (2008) 321–326.
Contents lists available at ScienceDirect
Forensic Science International: Genetics Supplement Series journal homepage: www.elsevier.com/locate/FSIGSS
Research article
Analysis of DNA profiles extracted from degraded samples from archival of formalin fixed tissue included in paraffin (FFTIP) and hairs Edna S. Miazato Iwamura a,*, Jose´ Arnaldo Soares-Vieira b, Marcelo Souza Silva a, ˜ oz b Karina S. Funabashi a, Carla D. Godoy a, Daniel Romero Mun a b
Paulista Medical School, Federal University of Sa˜o Paulo – EPM/UNIFESP, Department of Pathology, Sa˜o Paulo, Brazil Faculty of Medicine Sa˜o Paulo University, Department of Legal Medicine, Medical Ethics, Social and Occupational Health, Sa˜o Paulo, Brazil
A R T I C L E I N F O
A B S T R A C T
Article history: Received 14 August 2009 Accepted 14 August 2009
The possibility of studying DNA extracted from archival of formalin fixed tissue included in paraffin (FFTIP) enables valuable retrospective investigations. However, according to some authors it is difficult to obtain genomic DNA of good quality, since the process of fixation often results in fragmentation of DNA. In order to evaluate the quality and quantity of DNA extracted, necropsy samples of FFTIP (spleen/ lung) and hairs, with or without bulbs, were analyzed using three methods of extraction (QIAamp DNA mini, QIAamp DNA micro-kit and phenol–chloroform followed by microcon YM-30). The amount of DNA recovered was quantified by spectrophotometer. The b-actin, amelogenin gene and the profiles of STR were analyzed. Based on experimental results, a general guideline concerning the appropriate extraction method according to the tissue and the quantity of the starting material for the analysis of DNA from FFTIP and hairs could be suggested. ß 2009 Elsevier Ireland Ltd. All rights reserved.
Keywords: Paraffin Hairs DNA PCR Short amplicons STR
1. Introduction Tissues embedded in paraffin are an extraordinary source for DNA molecular studies because of the availability of large pathology archives of tissues related to clinical cases in almost all hospital pathology departments. In addition to biopsy and surgical paraffin embedded tissues, postmortem tissues are an important resource, especially for rare diseases, neuropathology or molecular epidemiology studies, because both pathological and normal tissues can be analyzed. The major difficulty in using these tissues is the degradation of nucleic acids [1]. During formalin fixation, cross-links of protein–protein and protein–DNA interactions are formed. In addition, the formaldehyde in the tissue gradually changes into formic acid, hydrolyzing the DNA. The fixative used, inclusion and storage conditions can contribute to the degradation of DNA [2,3]. Another source of DNA, easily found and particularly important in forensics, is hair samples. However, the small DNA quantity in hair is usually degraded. Another problem is PCR inhibitors in the hair. In particular hair pigments, the melanins, are known to inhibit PCR. To increase the chances of correct typing of hair, the small amount of DNA must be successfully isolated and the inhibitors have to be removed or neutralized [4–6]. Today, the
* Corresponding author. E-mail address: [email protected] (E.S.M. Iwamura). 1875-1768/$ – see front matter ß 2009 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.fsigss.2009.08.096
short tandem repeat (STR) typing of FFTIP and hair shafts is regarded as a challenge. The first aim of this study is to evaluate the quality and quantity of DNA extracted from FFTIP and hairs with or without bulbs using different methods of extraction. The second aim is to analyze the best protocol for each type of sample, in order to obtain the profile of STR typing for human identification. 2. Materials and methods Tissue samples fixed in formalin included in paraffin (FFTIP) in 2002: Fresh tissue specimens of spleen and lung from an AIDS patient, who died from miliary tuberculosis, positive for acid-fast bacilli, were subjected to tissue fixation and paraffin embedding. As negative control, a non-infected block from spleen was tested. Fixation was carried out at room temperature, using formalin phosphate-buffered at pH 7.0 (formaldehyde 37%, monosodic phosphate 47 mM, disodic phosphate 28 mM) or 10% formalin in distilled water. Embedding was performed using paraffin (Retrowax-Parafinas Nordeste). Pubic hair samples: Pubic hair samples were obtained from one individual. For each sample the morphological analysis was performed with light microscope. The first segment of the hair, including the root, was 0.5 cm in length, cut with a razor blade. The hair samples without bulb were obtained by cutting the bulb and following proximal 0.5 cm portion. Tissue and patient data were obtained and used after advice of the Medical Ethics Committee.
168
E.S.M. Iwamura et al. / Forensic Science International: Genetics Supplement Series 2 (2009) 167–168
2.1. Extraction of archival of FFTIP and pubic hairs from a female From each paraffin-embedded specimen, 3–10-mm thick sections were cut, using a new blade for each sample. Paraffin was removed using four 30 min incubations with xylol (Merck, Germany) followed by four washes with 100% ethanol. Tissue was digested with proteinase K 600 mg/mL (final concentration) in TE (Tris–HCl 1 mM, EDTA 0.5 mM pH 8) and Tween 20 0.5% for 16 h at 37 8C. Samples were subjected to heat-shock in liquid nitrogen, for 1 min, and incubated for 10 min at 95 8C. DNA was extracted with: (a) phenol–chloroform–isoamylic alcohol 25:24:1 and precipitated with 10% sodium acetate 3 M pH 4.8 and 2.5 volumes of absolute ethanol (Merck). The precipitate was resuspended in TE and concentrated in microcon YM-30 (Millipore); (b) QIAamp Mini Kit (Qiagen) extraction according to the manufacturer. We collected 1, 2, 3 and 6 hair samples in each tube (with and without bulbs) in triplicates. DNA was extracted from those segments of hairs using: (a) 100 ml of the following solution: 29 ml of Tween 201 (USB), 38.5 ml PCR buffer 10 (Invitrogen), 38.5 ml MgCl2 (25 mM) Invitrogen; 3.8 ml of H2O and 0.5 ml Proteinase K 20 mg/ml (Qiagen). The samples were incubated at 60 8C for 15 min and 95 8C for 15 min. Subjected to heat-shock (2) in nitrogen liquid, for 1 min, and incubated for 10 min at 100 8C (Layr -J.Ryal, Brasil); (b) QIAamp micro-kit (Qiagen) according to the manufacturer. After the extraction of DNA, concentration was determined in NanoDrop spectrophotometer at 260 nm and purity was evaluated by analysis of readings at 260 and 280 nm. Amplification of human b-actin gene: A 135 bp fragment of the human b-actin gene was amplified using 2.5 ml (0.5 mM of primers b5: AGCGGGAAATCGTGCGTG and bR: GGTGATGACCTGGCCGTC) in reactions containing 12 ml of Taq PCR Master Mix (Qiagen), 8.0 ml of H2O and 2.0 ml DNA (20 ng) was amplified with one cycle at 96 8C for 5 min, 40 cycles at 96 8C for 2 min, 60 8C for 2 min and 72 8C for 2 min and a final extension cycle at 72 8C for 7 min. Amplification of amelogenin gene (Promega) and Identifiler kit (Applied Biosystems): The samples were amplified according to manufacturer’s recommendations. PCR products were performed in GeneAmp PCR System 9700 (Applied Biosystems) and visualized in 2% agarose gels in buffer TBE (Tris–borate 45 mM, EDTA 1 mM) under UV light, after electrophoresis at 100 V/min and gel red staining. The DNA typing was performed using the AmpFlSTR IdentifilerTM Kit. Fragment size analysis was performed using the GeneMapper v.3.1 software after electrophoresis on the ABI 3130 Avant Genetic Analyzer (Applied Biosystems). Control supplied with AmpFlSTR Kits [7].
Hairs: The QIAamp DNA micro-kit (a and b) and the PCR buffer DNA extraction (c–g) results are as follows in ng/ml: (a) 6 bulbs: 8.64 0.73 (n = 3); (b) 6 shafts without bulbs: 2.99 0.66 (n = 3); (c) 6 bulbs: 33.29 13.89 (n = 3); (d) 6 shafts without bulbs: 12.78 4.50 (n = 3); (e) 3 shafts without bulbs: 13.23 7.74 (n = 6); (f) 2 shafts without bulbs: 9.26 1.13 (n = 5); (g) 1 shaft without bulb: 8.79 1.02 (n = 5). PCR: (a, c, and d) b-actin positive and (b, e, f, and g) negative, in the agarose gel. Amelogenin gene and Identifiler loci were negative, except a and c, in the ABI 3130 Avant sequencer. To avoid intra-individual variation, hairs were collected from one individual, by herself, resulting in a total of 100 plucked pubic hairs. Each sample contained 1, 2, 3 or 6 hair segments, with or without bulbs (in triplicates). The 6 segments with bulbs served as a source for DNA control for DNA typing in the Identifiler kit. The procedures for extraction and quantification were repeated at least three times. 4. Conclusions For FFTIP, the best PCR performance was the QIAamp DNA mini. However in cases in which there is a minute amount of tissue, for example biopsy material, the QIAamp DNA micro-kit could be better. For the hairs, with or without bulbs, the best strategy was extraction directly in the reagent buffers from PCR. Our analysis shows that, despite degradation, amplification of short specific DNA sequences (including those from single copy genes) is still possible. The pubic hair DNA extraction approach has shown to be a very useful strategy for alternative protocols when the goal is to analyze low copy numbers of DNA. This preliminary results has been very important, in both autopsy and biopsy specimen studies. The techniques illustrated here should be applicable to FFTIP sample investigations for any disease where short amplicons (bacterial, viral, and parasitic agents or endogenous alterations at the DNA level) are thought to play a causative role, including those of genetic diseases. However, for PCR in the STR multiplex analysis, the need of improving the purity and balance of DNA quantity is the goal and challenge in regard to both FFTIP and hairs. Conflict of interest None. Acknowledgement We would like to thank Joaquim Soares de Almeida for his technical assistance. This work was supported by grants from FAPESP 2008/11233-8, CAPES and LIM HC FMUSP.
3. Results and discussion
References
FFTIP: The amount of DNA extracted from FFTIP after seven years of storage with phenol–chloroform followed by microcon YM-30 (a and b) and QIAamp DNA mini kit (c and d) extraction: (a) spleen (n = 5): 67.55 0.02; (b) lung (n = 4): 21.81 0.09; (c) spleen (n = 4): 60.08 0.03; (d) lung (n = 5): 57.93 0.11. Purity analysis of readings at 260/280 nm: 1.69–1.90 (a and b) and 1.89–2.0 (c and d), respectively. PCR: b-actin gene amplified 60% (a and b) and 90% (c and d); amelogenin gene 25% (a and b) and 50% (c and d); Identifiler kit none. Because we had previously (2002) utilized the PCR, in FFTIP sections, to detect the presence of IS6110 (123 bp), specific from the Mycobacterium tuberculosis complex [8], we chose to use the same material as a model system to ascertain the possibility of studying archival tissue specimens. The success of PCR amplification of b-actin gene (135 bp) was 90% from lung and spleen (FFTIP), however the failure of Identifiler kit may be critically dependant on a variety of factors such as handling of specimens before tissue fixation, storage, imbalance and purity of DNA.
[1] S. Bonin, F. Petrera, B. Niccolini, G. Stanta, PCR analysis in archival postmortem tissues, J. Clin. Pathol.: Mol. Pathol. 56 (2003) 184–186. [2] A. Gillio-Tos, L. De Marco, V. Fiano, F. Garcia-Bragado, R. Dikshit, P. Boffetta, F. Merletti, Efficient DNA extraction from 25-year-old paraffin-embedded tissues: study of 365 samples, Pathology 39 (2007) 345–348. [3] Z. Budimlija, C. Lu, G. Axler-DiPerte, J. Seifarth, D. Popiolek, F. Fogt, M. Prinz, Malignant tumors and forensics—dilemmas and proposals, Croat. Med. J. 50 (2009) 218–227. [4] L. Bourguignon, B. Hoste, T. Boonen, K. Vits, F. Hubrecht, A fluorescent microscopyscreening test for efficient STR-typing of telogen hair roots, Forensic Sci. Int. Genet. 3 (2008) 27–31. [5] R. Szibor, I. Plate, H. Schmitter, H. Wittig, D. Krause, Forensic mass screening using mtDNA, Int. J. Legal Med. 120 (2006) 372–376. [6] A. Hellmann, U. Rohleder, H. Schmitter, M. Wittig, STR typing of human telogen hairs—a new approach, Int. J. Legal Med. 114 (2001) 269–273. [7] E.S.M. Iwamura, J.A. Soares-Vieira, J.C. Ponce, D.F. Zamarrenho, A.C. Cruvinel, M.G. Pradal, D.R. Mun˜oz, Paternity investigation analysis in artificial mother-deficient cases using STR loci in the Brazilian population, Forensic Sci. Int. Genet. Supp. Ser. 1 (2008) 516–517. [8] D. Barcelos, M.F. Franco, S.C. Lea˜o, Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffinembedded samples, Rev. Inst. Med. Trop. Sao Paulo 50 (2008) 321–326.