Forensic Science International: Genetics Supplement Series 3 (2011) e339–e340

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Forensic Science International: Genetics Supplement Series

journal homepage: www.elsevier.com/locate/FSIGSS

Morphological and DNA analysis in human skeletal remains exposed to

environmental conditions in Brazil
M.P. Soler a,*, M.T.S. Alves a,c, M.S. Silva a, M.A. Guimarães b, M.L.A.P.O. Sousa a,
J.S. Almeida a, E.S.M. Iwamura a
a
Laboratory of Molecular Pathology, Department of Pathology – Escola Paulista de Medicina, São Paulo Federal University, Brazil
b
Center of Legal Medicine, Department of Pathology and Legal Medicine, Faculdade de Medicina de Ribeirão Preto, São Paulo University, Brazil
c
Center of Forensic Pathology, São Paulo Forensic Medicine Institute, Brazil

A R T I C L E I N F O A B S T R A C T

Article history: Investigations of genetic kinship and human identification through DNA analysis of human skeletal
Received 23 August 2011 remains have been required for several types of cases. A molecular study of this kind of sample is a
Received in revised form 7 September 2011 challenge because of the small amount of cells available. In Brazil, there is a dominating humid tropical
Accepted 15 September 2011
climate which can exert a direct influence on the microscopic morphology of bones and consequently on
the DNA. The objective of this study was to analyze the microstructure of femoral compact bone tissue
Keywords: samples that were exposed to harsh environmental conditions in Brazil, correlating it with the amount of
Bones
human DNA extracted. Compact bone fragments were used from femoral diaphysis of 20 skeletonized
Human nuclear DNA
corpses found in the period 1998–2009 in Ribeirao Preto, São Paulo, Brazil. Samples were processed,
Morphology, Brazil
stained with H&E and from the densest cellular area, 10 consecutive fields were selected by a pathologist
and software Image Tool (UTSCH-USA) and Image J (NHI-USA) were used for cellular analysis. DNA
extraction was performed through commercial kit and quantification with Quantifiler Duo DNA kit
(Applied Biosystems). Osteocytes were observed in all cases ranging from 1 to 40 and DNA amount was
correlated with the morphology observed. These results indicate preservation of osteocytes in bone
material exposed to tropical environmental conditions, signifying the feasibility of obtaining DNA for
genetic studies. A preliminary morphology analysis in skeleton samples can predict success in extracting
DNA from these samples, since a significant correlation was found between these two variables.
ß 2011 Elsevier Ireland Ltd. All rights reserved.

1. Introduction
Human identification and investigation of genetic ties are
essential in many circumstances and DNA analysis is fundamental
for conclusive results. But when skeletonized human remains are
used as a biological source the analyses are often complex and
expensive (common in mass war casualties and civil cases with
exhumation samples) [1].
Despite the fact that long bone diaphysis has a thick layer of
cortical tissue in which the osteocytes are ‘‘protected’’ within
lacunae, external factors such as chemical, physical and biological
agents can reach bone cells, affecting the nuclear DNA and
consequently the molecular studies [2]. Thus, post-mortem bone
material whose cell numbers were already reduced during the
* Corresponding author at: Dpto. Patologia, Universidade Federal de São Paulo,
Rua Botucatu 740, 04023 062 São Paulo, SP, Brazil. Tel.: +55 11 5576 4266;
fax: +55 11 5571 9295.
E-mail address: mipsoler@gmail.com (M.P. Soler).
physiological process of cell death has its numbers further reduced
during exposure to environment. In addition, some regions of
Brazil have high rainfall, high temperature, more acid soil and high
solar irradiation, which can damage even more the bone
microstructure. Therefore, it is important to verify the morpho-
logical changes that hinder DNA analysis, since a prior evaluation
of bone material could indicate sample viability in relation to
genetic studies.
2. Materials and methods
Compact bone fragments were used from femoral diaphysis of
20 skeletonized corpses that had been exposed to Brazilian
environmental conditions, found in the period 1998–2009,
provided by Center of Forensic Medicine, Faculty of Medicine of
Ribeirão Preto-USP. For DNA analysis, fragments were sanded with
a rotary tool (Dremel) and powdered with a metal cup blender
(Waring Product, CT, USA) and extraction was performed with
QIAamp DNA Mini Kit (QIAGEN) adding 200 ml of EDTA in lysis step
with 100 mg of bone powder. DNA quantification was employed in

1875-1768/$ – see front matter ß 2011 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.fsigss.2011.09.032
e340 M.P. Soler et al. / Forensic Science International: Genetics Supplement Series 3 (2011) e339–e340

Table 1 including the presence of nuclei with well-defined nuclear

Number of nuclei observed and DNA quantity per case.
membrane. DNA amount ranged from 0 to 0.02 ng/ml and was
Sample Nuclei number DNA (ng/ml) significantly correlated (p = 0.027) with osteocytes nuclei number
1 1 0.0000 observed by microscopy (Table 1).
2 16 0.0009 This is the first study that quantitatively describes DNA
3 1 0.0004 extracted from bone samples that had been exposed to tropical
4 40 0.0094 environmental conditions, using image analysis software. This
5 3 0.0207
analysis helps to explain why the amount of human DNA is so
6 4 0.0024
7 4 0.0127 scarce. The fact that many osteocytes nuclei were pycnotic or in
8 5 0.0223 karyolysis, is another issue involved in the difficulty of obtaining
9 1 0.0000 DNA in good enough conditions to be studied. When working with
10 4 0.0050
materials that contain few viable cells, it is essential to develop
11 2 0.0000
12 3 0.0058
strategies for highly efficient extraction of DNA [3]. In conclusion,
13 12 0.0007 the image analysis of bone fragments qualifies this type of sample
14 3 0.0003 and is consistent with the poor results obtained in DNA analysis in
15 3 0.0026 forensic laboratories.
16 10 0.0093
17 2 0.0000
18 3 0.0009
19 7 0.0022 Role of funding
20 5 0.0000
This study was financially supported by the ‘‘Fundação de
Amparo à Pesquisa do Estado de São Paulo 2010/19127-2’’ and by
‘‘Coordenação de Aperfeiçoamento de Pessoal de Nı́vel Superior’’.
7500 Real Time PCR System (Applied Biosystems) with Quantifiler
Duo DNA kit (Applied Biosystems). For the histological study,
fragments were fixed in formalin, immersed in decalcification Conflict of interest
solution of 8% nitric acid for 4 days, following dehydration, paraffin
embedding, cutting and H&E staining. Despite being more None.
aggressive, nitric acid ensures a faster decalcification, without
forming artifacts. Per case, 10 consecutive fields were photo-
graphed with Q Capture Pro 6.0 program from the area with the Acknowledgements
largest number of nuclei. Images were captured in a 40 objective
for the purpose of gaining greater accuracy for visualization. The authors would like to thank the technical team from the
Captured images were improved with Photoshop CS4 software; ‘‘Departamento de Patologia da Universidade de São Paulo’’.
bone area was calculated using software Image Tool (UTSCH-USA)
and number of lacunae and nuclei were calculated by Image J
software (NIH, USA). Review of all results was performed by 2 References
observers. Results were analyzed in Prism 3.0 software with
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(2009) 822–828.
[3] O.M. Loreille, T.M. Diegoli, J.A. Irwin, M.D. Coble, T.J. Parsons, High efficiency DNA
The mean bone area analyzed was 173,735 mm2 per case. extraction from bone by total demineralization, Forensic Sci. Int. Genet. 1 (2)
Osteocytes were observed in all cases, ranging from 1 to 40, (2007) 191–195.