FORENSIC SCIENCES: CRIMINALISTICS - Bio Medical Forensics

sequencing results obtained from a single source of bone sample. For
the purposes of this study, a tibia was obtained from an anthropological
research facility. This tibia had been buried for a period of approximately
three years prior to dry storage at room temperature at the facility for an
unknown period of time. Prior to distribution, the tibia was assessed for
suitability and verification of mtDNA sequence. The tibia was sectioned
by the organizing laboratory and distributed to the twenty-one
participating laboratories. Extraction, amplification, and sequencing of
the bone sections were performed according to each laboratory’s
standard protocols. Results were submitted from nineteen of the
participants with concordant results for mtDNA sequencing. For
laboratories submitting results for autosomal and Y STRs, concordant
results were also obtained.
In addition to submitting typing results, participating laboratories
submitted their standard operating procedures which contained details of
their extraction methodologies as well as amplification and sequencing
strategies. These details are presented. Despite variation in the cleaning
methods of these bone portions, as well as variations in extraction
methods (including decalcification, if applicable), quantity of sample
used, amplification parameters, post-amplification quantification,
sequencing chemistries and instrumentation, all methods proved reliable
and the results obtained were concordant. Comparison of these results
highlights the robust nature of forensic typing methodologies.
Although the results obtained from the current study demonstrated
the reliability of forensic testing, the next generation of this interlaboratory
bone exchange study will include a more environmentally
challenged sample to more closely mimic the type of samples
encountered in a forensic context.
This study also displays the willingness of the forensic community
to advance the knowledge of the field through collaborative studies.
Mitochondrial DNA, Skeletal Remains, Inter-Laboratory Study
B52 Dieletrophoretic (DEP) Separation
of Sperm and Epithelial Cells
Mark Timken, PhD*, and Martin R. Buoncristiani, MSc, California
Deptartment of Justice Richmond DNA Lab, 1001 West Cutting
Boulevard, Suite 110, Richmond, CA 94706
After attending this presentation, attendees will be introduced to a
new method based on the principle of dielectrophoresis (DEP) for
separating sperm cells from epithelial cells (e-cells). This introduction
will include a presentation of the general principles of DEP for cell
separation, a description of a specific chip-based DEP system for
separating sperm and e-cells, and results to evaluate the efficacy of the
DEP separation system relative to the differential chemical lysis method
that is currently used in most forensic DNA labs for sexual assault cases.
This presentation will impact the forensic community by providing
information about a new approach for handling biological evidence in
sexual assault cases.
A critical step in the successful DNA analysis of most sexual assault
cases is the effective separation of male sperm and female epithelial
cells, which are typically collected on a vaginal swab at the hospital soon
after the event. In the differential extraction procedure currently used by
most forensic DNA analysts, the swab containing both cell types is rehydrated,
cells are collected and a two-step differential lysis is
performed. In the first step, the epithelial cell fraction is removed by a
mild chemical lysis (detergent and proteinase), leaving the majority of
the sperm heads intact. Cell separation relies on the more robust nature
of the sperm head membranes, in particular, on the use of a chemical
agent (e.g., DTT) in the second step to reduce disulfide bonds to assist
in digesting the sperm membranes. Although the preferential lysis
extraction is, by and large, effective, it suffers from several drawbacks.
For example, some sperm DNA is lost to the epithelial fraction in the
first lysis step, as well as in subsequent wash steps, and the procedure is
labor intensive and not particularly amenable either to automation or to
the microfluidic devices that are being examined for forensic
applications.
To address these issues, we have been investigating the use of
dielectrophoresis (DEP) for separating sperm and epithelial cells. DEP,
a phenomenon first described by Pohl in 1978, is the movement of cells
in the presence of a non-uniform electric field. Such an electric field can
induce an electric dipole on a cell. The resulting interaction of this
dipole with the non-uniform electric field can lead to a net force on the
cell, inducing cell movement. DEP is particularly useful for selectively
separating and sorting cell types because the strength and direction of the
induced forces are very sensitive to cellular structure and composition.
Experimental conditions (e.g., applied field strengths, applied field
frequencies) can be chosen to fine-tune the DEP forces. Consequently,
DEP can be used to separate cell types based on differences in size or in
membrane or cytoplasm composition. In this presentation, we describe
the use of a DEP system (the Silicon BiosystemsTM SlideRunnerTM DEP
system) to separate sperm and epithelial cells in a chip-based format.
Results (e.g., purity of the separated fractions, yield, sensitivity, and
reproducibility) will be presented to describe the efficacy of the DEP
separation for mixtures of sperm and female (buccal) epithelial cells.
Dielectrophoresis, Differential, Extraction
B53 Comparison of Commercial Blood
Test Kits for Use in Crime Scenes
Lin Kiak Lee, BSc*, Kiak Eng Lee, BSc, Yuen San Vicky Chow, MSc,
Christopher K.C. Syn, PhD, Chin Chin Lim, MBA, Wai Fun
Tan-Siew, MSC, and Ming Kiong Michael Tay, PhD, Health Sciences
Authority, 11 Outram Road, Singapore, 1690878, SINGAPORE
After attending this presentation, attendees will learn the about the
similarities and differences between the two commercial test kits
“HEXAGON OBTI” and “ABAcard HemaTrace” in terns of design,
sensitivity, response time, and cost for forensic identification of human
(higher primate) blood.
This presentation will impact the forensic community by
demonstrating how the identification of human blood at the crime scene
using a fast and reliable technique is important for investigative
purposes, for collection of useful stains for subsequent DNA profiling,
and bloodstain pattern analysis. Two commercially available test kits
based on the immunochromatographic technique were compared and
reported.
To date, validation and implementation studies have been
conducted for each of the two test kits but they have not been compared
with each other. The objective of this study is to evaluate and compare
the “HEXAGON OBTI” and “ABAcard HemaTrace” test kits in terms
of their specificity, sensitivity, cost and more importantly, their ease of
use at crime scenes. Human bloodstains, human body fluids (saliva,
urine, semen) and animal bloodstains (sheep, cow, fish, pig, chicken,
dog, goose, goat, cat, macaw, buffalo and eight kinds of higher primates)
were tested according to the manufacturer’s instructions.
These findings indicated that the sensitivities, specificity and cost
of the two commercial test kits were comparable. Both kits were
specific to human and higher primate blood and could detect up to two
nanoliters of blood. The key differences between them were in their
response time and the design of their component parts. The
“HEXAGON OBTI” test card exhibited a much faster response time
(almost immediately), compared to the one to two minutes lag time
required for the “ABAcard HemaTrace” test card, for the different
concentrations of blood. In terms of design, the “HEXAGON OBTI” kit
design was more user-friendly than the “ABAcard HemaTrace” kit. In
the former, the cotton swab for collection was designed as an integral
367 * Presenting Author