Forensic Science International 151 (2005) 233237 www.elsevier.

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Evaluation of a novel tagging and tissue preservation system for potential use in forensic sample collection
Martin Grassberger *, Christina Stein, Stefan Hanslik, Manfred Hochmeister
Medical University of Vienna, Department of Forensic Medicine, Sensengasse 2, A-1090 Vienna, Austria Received 15 December 2004; received in revised form 13 February 2005; accepted 22 February 2005 Available online 16 March 2005

Abstract The authors describe a new, easy-to-use barcode-based tissue collection, preservation and body tracking system, which might prove instrumental in the containment of mass fatalities such as aircraft accidents, war related accidents, environmental disasters (e.g. earthquakes, hurricanes, and oods) terrorist bombings or mass murders. # 2005 Elsevier Ireland Ltd. All rights reserved.
Keywords: DNA analysis; Tissue preservation; Disaster identication; TypixTM

1. Introduction Tissue preservation is a critical issue in forensic investigations where human remains are collected for DNA analysis following plane crashes, terrorist bombings, homicides, and mass murders [17]. The subsequent maintenance of a forensically sound chain of custody is also a critical part of eld as well as laboratory practice. Low ambient temperatures and rapid recovery of human remains are ideal conditions to ensure successful DNA analysis of human remains [1]. However, such conditions are difcult to achieve in disaster areas, or in geographically remote regions of the world. The new ear-tag system TypiFixTM (Agrobiogen, Hilgertshausen, Germany) recently developed for large-scale tissue sampling and tagging of animals, works simply by using a clamp-like applicator (Fig. 1). By operating the
* Corresponding author. Tel.: +43 699 113 26 708. E-mail address: martin.grassberger@meduniwien.ac.at (M. Grassberger).

loaded applicator, a tissue sample is punched out by a collection stud and automatically introduced into a selfsealing sample container (Figs. 2 and 3). In the tightly sealed sample container, the tissue and its DNA are preserved through desiccation in the presence of molecular sieve beads consisting of sodiumaluminium-silicate. The ear-tag and sample container are preprinted with the same identication number as well as a barcode (Fig. 2d). They are attached to each other until the sample is introduced into the sample container. Because of the simultaneous barcoding of the remains and the tissue sample at the point of recovery, sample switch is excluded. This tissue collection and body tagging system provides potential benets for the forensic community, because it is convenient, it enables sample preservation in an ambient temperature environment, i.e., no requirement for a refrigerator/freezer or any other additional device; the DNA integrity can be maintained for a long period of time, particularly during the stress and demands of a mass disaster or other related event.

0379-0738/$ see front matter # 2005 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.forsciint.2005.02.012

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Fig. 1. Loaded TypiFixTM applicator clamp (dashed line: sample container).

A feasibility study on DNA analysis was conducted using short tandem repeat (STR) methodology to determine the usefulness and the limitations of this device in a forensic setting and to evaluate the effect of long term storage of tissues in the sealed TypiFixTM container.

2. Material and methods 2.1. Sampling procedure Ten bodies (3 females, 7 males; time since death ranging between 3 and 25 days) were selected from the morgue for

Fig. 2. TypiFixTM Application procedure. (a) Before application at the interdigital skinfold; (b) after successful application of the tag; (c) palmar view after application; (d) removal of the sample container, the tag remains on the sampled body region. Note, that each unit bears the same ID and barcode.

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Fig. 3. Tissue sample in the self-sealed sample container, plug and socket unit. (a) Plug; (b) sample container (socket); (c) punch-out conus with steele sleeve; (d) tissue sample.

this study. Tissue sampling with simultaneous tagging was performed at the interdigital fold between the thumb and the index nger of either hand using the TypiFixTM applicator (Fig. 3). Each body was sampled twice.

2.5. Autosomal STR analysis Autosomal STR analysis was carried out with 1 ng of genomic DNA using the AmpFLSTR1 SGM Plus1 PCR amplication kit (Applied Biosystems). All analyses were performed in accordance with the manufacturers instructions.

2.2. Sample processing Samples were stored at room temperature (approximately 22 8C) and processed after 2 weeks and again after 5 months after collection. Using a special extractor clamp provided by the manufacturer of TypiFixTM, the bottom of each sealed sample container was removed, and the dried tissue samples were transferred to 1.5 ml eppendorf-cups.

3. Results Using the easy to handle TypiFixTM applicator, the tags were afxed at the interdigital skinfolds between the thumb and the index nger of each body without difculty (Fig. 2). The average amount of dried tissue recovered from the TypiFixTM sample containers was 10 1.3 mg (mean S.D.). On average 8 5.7 and 15.4 8.3 mg DNA (mean S.D.) were puried from each sample after 2 weeks and after 5 months, respectively (Table 1). Although not signicant, the yield of DNA on average was greater after 5 months. The success rate of STR genotyping at 2 weeks and after 5 months was 100%. The DNA proles after 5 months of storage were identical to those obtained after 2 weeks. An example of an AmpFLSTR1 SGM PlusTM prole generated from DNA extracted from a biopsy sample stored at room temperature for 5 months is shown in Fig. 4. Tissue samples processed after 5 months of storage at ambient temperature in TypiFixTM containers generated allele peak heights/areas that generally were balanced across the ten STR loci included in the AmpFLSTR1 SGM PlusTM kit. For

2.3. DNA extraction The isolated tissue samples (diameter approximately 1 mm) were subjected to DNA extraction using the QIAamp DNA Mini Kittissue protocol (Qiagen Inc., Valencia, CA).

2.4. DNA quantication Quantication of human genomic DNA was determined using real-time PCR (ABI PRISM1 7000 Sequence Detection System, Applied Biosystems), the QuantilerTM Human DNA quantication kit (Applied Biosystems), and by following the manufacturers recommendations.

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Table 1 DNA yield/TypiFixTM and effects of long-term storage in TypiFixTM at ambient temperature on STR DNA analysis Sample no. DNA yield/TypiFixTM (mg) after storage at room temperature for 2 weeks 1 2 3 4 5 6 7 8 9 10 10 19.3 5.4 16.9 6.4 2.8 7 3 5.9 3.5 5 months 11.7 17.1 35.6 12 15.4 14.5 13.7 9.6 20.2 4 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Full AmpFLSTR1 SGM Plus1 proles after 5 months

4. Discussion Currently, the most commonly used method of preserving tissues for subsequent DNA analyses is freezing. Very few alternative approaches have been developed to preserve soft-tissue samples for forensic DNA analysis at room temperature [810]. An alternative fast and reliable tissue labeling and preservation procedure of human remains in difcult forensic recovery situations such as disaster areas has always been needed and has been challenging to develop. We present a new sample collection/tagging system, originally developed for animal ear-tagging and identication, which was evaluated for its utilization with human remains and its ability to preserve DNA for subsequent forensic STR analysis. The results indicate that storage of samples at ambient temperature for 5 months in TypiFixTM containers did not compromise the STR typing analysis of the tissue samples tested. The observed greater DNA yield after 5 months is probably a result of varying tissue composition of the biopsy samples (i.e. relative amount of muscle, fat or cutis). Further DNA analyses after 1 and 2 years are intended, as well as storage of mock challenged samples. Using the described system keeps the collection costs low, provides fast and reliable collection of DNA tissue samples

all STR loci that presented a heterozygous prole, both alleles within each prole were balanced. No allelic dropout or extraneous bands were detected in proles generated from the DNA of the tissue preserved in TypiFixTM containers. No inhibitory effect on the PCR process was noted. All sets of sampled tissues examined in this study showed the same trend (Table 1).

Fig. 4. AmpFLSTR1 SGM PlusTM prole generated from DNA extracted from a biopsy sample stored at room temperature for 5 months.

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from a large number of individuals in a short time and ensures a forensically solid chain of custody from the point of recovery in the eld to the DNA-analysis in the forensic laboratory. The major advantages of the system are:  The collection of tissue samples for DNA analyses can easily be achieved under eld conditions.  In case of mass fatalities, it enables investigating authorities to collect numerous specimens for DNA analysis and to simultaneously label the remains. Barcodes can be manufactured according to customer needs.  The system is fail-safe and fraud-proof.  The use of barcode technology enhances the rate and accuracy at which information can be collected and eliminates the opportunity for error in recording data.  The specimen container is contamination-proof since only the single-use parts come in contact with biological materials.  The storage of tissue samples for DNA analysis is possible without the need for refrigeration or freezing.  According to the manufacturer, tissues stored over 4 years in the TypiFixTM system are still suitable for amplication of long fragments by PCR. Our ndings support this based on 5 months storage at ambient temperature and 100% success with STR typing. A limitation is that the sampling device is not specically adapted for use in forensic settings or for hard tissue such as bone. Therefore, the application is restricted to certain anatomical regions, such as interdigital skinfolds, tendons and the edges of amputated limbs as well as small body parts. Shelf-stable preservation (for at least 5 months; possibly several years) and uncomplicated storage possibilities is the primary value of using TypiFixTM containers. This process of sample collection and storage allows convenient and inexpensive shipment of samples at ambient temperatures even from geographically remote regions. Additionally, the introduced system effectively reduces the labor associated with specimen collection and processing, decreases the number of errors in the eld as well as in the laboratory that could occur with specimen labeling, and improves the integrity of specimen handling throughout the steps of specimen processing. Therefore, the TypiFixTM system provides a new, reliable and useful tool for the collection, labeling, and storage of human remains and tissue samples in mass fatalities.

Acknowledgements The authors wish to acknowledge the assistance of M. Fichtinger and R. Jeitler in the laboratory and R. Langer in the morgue.

References
[1] J. Ballantyne, Mass disaster genetics, Nat. Genet. 15 (1997) 329331. [2] T.M. Clayton, J.P. Whitaker, C.N. Maguire, Identication of bodies from the scene of a mass disaster using DNA amplication of short tandem repeat (STR) loci, Forensic Sci. Int. 76 (1995) 715. [3] D. Corach, A. Sala, G. Penacino, Sotelo, Mass disasters: rapid molecular screening of human remains by means of short tandem repeats typing, Electrophoresis 16 (1995) 1617 1623. [4] T. Kahana, M. Freund, J. Hiss, Suicidal terrorist bombings in Israelidentication of human remains, J. Forensic Sci. 42 (1997) 260264. [5] B. Ludes, A. Tracqui, H. Ptzinger, P. Kintz, F. Levy, M. Disteldorf, Medico-legal investigations of the Airbus, A320 crash upon Mount Ste-Odile, France, J. Forensic Sci. 39 (1994) 11471152. [6] V.O. McCarty, A.P. Sohn, R.S. Ritzlin, J.H. Gauthier, Scene investigation, identication, and victim examination following the accident of Galaxy 203: disaster preplanning does work, J. Forensic Sci. 32 (1987) 983987. [7] B. Olaisen, M. Stenersen, B. Mevag, Identication by DNA analysis of the victims of the August 1996 Spitsbergen civil aircraft disaster, Nat. Genet. 15 (1997) 402405. [8] C.J. Fregeau, H. Vanstone, S. Borys, D. McLean, J.A. Maroun, H.C. Birnboin, AmpFlSTR Proler Plus and AmpFlSTR COler analysis of tissues stored in GenoFix, a new tissue preservation solution for mass disaster DNA identication, J. Forensic Sci. 46 (2001) 11801190. [9] K. Muralidharan, C. Wemmer, Transporting and storing eldcollected specimens for DNA without refrigeration for subsequent DNA extraction and analysis, Biotechniques 17 (1994) 420422. [10] C.L. Schultz, Y. Akker, J. Du, H. Ratech, A lysis, storage, and transportation buffer for long-term, room-temperature preservation of human clinical lymphoid tissue samples yielding high molecular weight genomic DNA suitable for molecular diagnosis, Am. J. Clin. Pathol. 111 (1999) 748 752.