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Department of Forensic Medicine, Saitama Medical University, 38 Morohongo, Moroyama, Saitama 350-0495, Japan
Forensic Medicine, Juntendo University School of Medicine, 2-1-1Hongo, Bunkyo-Ku, Tokyo 113-8421, Japan
Department of Forensic Medicine, Hirosaki University Graduate School of Medicine, 5 Zaifu-cho, Hirosaki, Aomori 036-8562, Japan
d
Department of legal Medicine, Osaka Medical College, 2-7 Daigaku-machi, Takatsuki, Osaka, 569-8686, Japan
b
c
A R T I C L E I N F O
A B S T R A C T
Article history:
Received 20 August 2015
Accepted 7 September 2015
Available online 10 September 2015
The effects of different storage methods on both DNA degradation and the results of preliminary blood
tests were evaluated. Bloodstains stored at room temperature, 4 C, 20 C, and 80 C for 20 years; whole
blood samples stored at 20 C and 80 C for 20 years; and fresh whole blood samples were analyzed.
DNA degradation was evaluated by quantifying the ratios of 305 and 129 base pair (bp) fragments to 41 bp
fragments. Preliminary testing included leuco-malachite-green staining, anti-human hemoglobin (Hb)
testing by immunochromatography, and the detection of the hemoglobin-beta (HBB) mRNA. Bloodstains
stored at room temperature and 4 C were more highly degraded than fresh blood, as indicated by the
ratio of 129:41 bp DNA fragments and 305:41 bp DNA fragments. All samples tested positive using leucomalachite-green staining and anti-human Hb assays. HBB was not detected in any whole blood samples
stored at 20 C or 80 C, although HBB was detected in all bloodstain samples. Therefore, to prevent
DNA degradation during long-term storage, it is recommended that blood samples be stored at below
20 C. In addition, stored bloodstains are suitable for preliminary tests of blood, rather than storing
blood.
2015 Elsevier Ireland Ltd. All rights reserved.
Keywords:
Storage condition
DNA degradation
Leuco-malachite-green test
Anti-human Hb test
mRNA detection
1. Introduction
When seeking a resolution to a long-unsolved crime, short
tandem repeat (STR) genotyping of old bloodstains is evidentially
important. However, forensic data may be difcult to obtain if
samples have been improperly stored. In the present study, we
explored the effects of different storage conditions on DNA
degradation and preliminary blood tests.
2. Materials and method
2.1. Samples
Bloodstains that adhered to cotton gauze and were stored at
room temperature, 4 C, 20 C, and 80 C for 20 years (n = 6,
each); whole blood samples stored at 20 C and 80 C for
20 years (n = 6, each); and fresh whole blood samples (n = 6) were
e40
M. Hara et al. / Forensic Science International: Genetics Supplement Series 5 (2015) e39e41
Fig. 1. The DNA degradation ratio in bloodstains stored at room temperature, 4 C, 20 C, and 80 C for 20 years; of whole blood samples stored at 20 C and 80 C for
20 years; and of fresh whole blood. Mean values SD for six samples each are shown. Dark gray bars represent the 129:41 bp ratio, whereas the light gray bars represent the
305:41 bp ratio. *Signicant differences were calculated using the t-test (P < 0.05), compared with fresh whole blood.
Table 1
The number of positive reaction of preliminary tests in various storage conditions.
Bloodstain RT
Bloodstain 4 C
Bloodstain 20 C
Bloodstain 80 C
Whole blood 20 C
Whole blood 80 C
Fresh whole blood
Anti-human Hb test
mRNA detection
HBB
18S
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
6
0
0
6
6
6
6
6
6
6
6
M. Hara et al. / Forensic Science International: Genetics Supplement Series 5 (2015) e39e41
e41
References
[1] M. Hara, H. Nakanishi, S. Takahashi et al. Relationship between DNA degradation
ratios and the number of loci detectable by STR kits in extremely old seminal
stain samples, Legal Med. In press.