Forensic LMD Research Studies at Rosalind Franklin University of Medicine and Science North Chicago, IL

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Forensic LMD Research Studies at Rosalind
Franklin University of Medicine and ScienceNorth
Chicago, IL

• Christine T. Sanders
• geneskr_at_alumni.ucsd.edu

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How did this start?

• 2003 - Sanders investigates the applicability of
  LMD for forensic use as a thesis topic
• Jan 2004 - Sanders and Peterson at RFUMS write
  NIJ grant proposal to investigate LMD technology
  for separation of sperm from mixtures.
• Feb 2004 - AAFS presentation of preliminary data
• June 2004 - Awarded two year grant
  2004-DN-BX-K215
• Several presentations made throughout the grant
  period
• Published paper in July 2006, JFS
• July/Aug. 2007 - NFSTC Technology Transfer
  Workshop
• Second manuscript in progress

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Separation Methods

• Preferential Lysis (differential extraction)
• Flow Cytometry
• Microchip separation
• Membrane Filtration
• Magnetic Antibodies
• Y- chromosome (non-physical separation)
• Laser Microdissection

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Studies for LMD Development

• Histological staining study
• DNA isolation study
• Yield Evaluation qPCR
• Mixture separation study
• Low Copy Number study (LCN)
• Comparative study
• Case Study

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Technical Obstacles

• Optimizing LMD cutting parameters
• LMD microscope slides
• Environmental conditions of instrument
• hanging chads
• Static
• Collection buffer
• Etc

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Histological Staining for LMD
Not Stained
E-Cells
Sperm
40x
63x

• Histological staining study considerations
• Visually discriminate sperm and epithelial cells
• Effect on downstream analysis

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Histological Staining Part 1

• Stains tested
• Hematoxylin / Eosin (HE)
• Christmas Tree stain (nuclear fast red /
  picroindigocarmine)
• Acridine Orange
• Wright Stain (azure blue / eosin)
• Methyl Green
• Evaluation
• Stains were evaluated for ability to ID sperm
• LMD collected cells were isolated with Qiagen
  QIAamp extraction
• STR/Profiler Plus analysis performed

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Acridine Orange
Not Stained
Christmas Tree
Hematoxylin/ Eosin
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Microscopic ID scores of sperm epithelial cells

UNSTN not stained. HE hematoxylin/eosin.
CTS nuclear fast red/picroindigocarmine. MG
methyl green. WRT Wright's stain. AO
acridine orange.
- - cannot ID or highly challenging - poor
/ - satisfactory good excellent
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62
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• Stained specimens exhibited RFU values
  significantly lower than unstained specimens (P lt
  0.01)

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Part I Histology Study Summary

• Methyl green, Wrights stain not suitable for LMD
• Cells stained with Acridine orange resulted in no
  amplified product
• Christmas tree stain Hematoxylin/Eosin
• Good stains for sperm ID.
• Significantly lower RFU values than unstained
  control
• However, genotyping could still be obtained with
  300 sperm cells or 150 E-cells
• HE best choice

Results prompted second phase of histology study
(Part II)
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Part II Histology Study

• Stains Tested
• HE Modified - shorter exposure times
• Nuclear Fast Red - nuclear dye
• SYBR 14/Propidium Iodine - fluorescent duel stain
• PSA/PI - fluorescein-conjugated Pisum sativum
  agglutinin (FITC-PSA) propidium iodide
• Evaluation
• Stains were evaluated for ability to ID sperm
• LMD collected cells were isolated with Lyse-N-Go
  extraction
• STR/Profiler Plus analysis performed

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RFU Differences Stained vs. Unstained LMD Cells
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Part II Histology Study Summary

• SYBR 14/Propidium Iodine
• No STR results could be obtained - not suitable
  for DNA analysis. PI tests indicate SYBR 14 as
  the problem agent.
• HE Modified and Nuclear Fast Red
• Good stains for sperm ID.
• No significant difference in RFUs obtained from
  that of unstained controls
• PSA-FITC/PI
• Good duel stain but staining consistency
  difficult to control between samples. STR
  genotypes can be obtained from PSA-FITC stained
  cells. (more optimization required)

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PSA-FITC/PI stain on a PEN slide
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DNA Isolation Study

• DNA isolation study considerations
• Low manipulation, Small volume, Purity

• Lyse-N-Go commercial lysis buffer (low
  manipulation)
• Series of heating and cooling incubations 8C-97C
• Microlysis commercial lysis buffer (low
  manipulation)
• Series of 65C and 96C incubations
• Qiagen QIAamp commercial kit
• DNA binding membrane columns
• Chelex
• DTT (dithiothreitol) added to all three protocols

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Detection of Loci Using Three Isolation Methods
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Total PCR Product Detected

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DNA Isolation Study Summary

• MicroLYSIS method was not suitable for LMD with
  forensic STR analysis
• QIAamp performed best for collection of stained
  epithelial cells
• Both Lyse-N-Go and QIAamp performed well for
  isolating DNA from LMD collected sperm cells
• Lyse-N-Go provides a low manipulation method and
  inexpensive
• QIAamp provides a cleaner DNA extract but higher
  manipulation required and higher cost.

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DNA Yield Study

• LMD process may provide an estimate of DNA
  quantity.
• Efficiency of DNA extraction process must be
  considered
• Two methods of DNA quantification performed from
  LMD collected cells extracted with the Qiagen
  QIAamp protocol
• Real-Time qPCR
• Relative amounts of STR PCR product to a standard
  curve

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Yield of DNA Extraction (sperm)
Sample Yield by real-time qPCR Yield by STR RFUs
300 sperm (n5) 22.9 4.8 Off scale
150 sperm (n5) 13.3 2.3 Off std curve
80 sperm (n5) 16.3 5.4 25.1 6.6
40 sperm (n5) 18.5 3.8 22.0 5.6
20 sperm (n5) 12.2 4.7 20.1 7.8
10 sperm (n5) 14.8 0.8 23.6 6.6
5 sperm (n4) 17.8 2.9 Off std curve
AB Pos control 0.0313 -1ng 13.0 2.0 (n6) 27.5 4.7 (n5)
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Yield of DNA Extraction (e-cells)
Sample Yield by real-time qPCR
150 epithelial (n5) 37.5 2.8
80 epithelial (n5) 45.4 6.8
40 epithelial (n5) 37.5 4.2
20 epithelial (n5) 40.5 7.0
10 epithelial (n5) 32.9 5.3
5 epithelial (n5) 41.4 8.6
2 epithelial (n5) 40.7 11.0
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DNA Yield Study Summary

• Extraction efficiency surprisingly low but
  consistent
• Cells can easily be counted during LMD
  collection, starting DNA material calculated, and
  then final DNA quantity can be estimated
  factoring in extraction efficiency prior to PCR
• Laborious and sample consuming DNA quantification
  step can be eliminated when using LMD.

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Mixture Study

• Mixtures were prepared with the equivalent of
  half a female oral cotton swab 1µl of semen.
• Collection amounts of 75, 150 and 300 sperm cells
  were recovered from the mixtures.
• PCR amplification was performed using standard 28
  cycles
• Extended PCR was performed by amplifying half of
  the PCR product an additional 6 cycles

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DNA mixture sperm cells female epithelial
cells ()












AmpFlSTR Profiler Plus
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300 Sperm cells separated by LMD
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150 sperm cells from a mixture
Extended PCR
Standard PCR
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75 sperm cells from a mixture
Standard PCR
Extended PCR
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Detection of Profiler Plus Alleles

• Under standard PCR conditions (28 cycles)
• 75 sperm 7112 alleles
• 150 sperm 963 alleles
• 300 sperm 100 alleles
• Under extended cycles PCR (34 cycles)
• 75 sperm 100 alleles
• 150 sperm 100 alleles

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Allelic Balance

• Heterozygote peak height ratio Height of the
  lower peak divided by the height of the higher
  peak, expressed as a percentage
• Under standard PCR conditions (28 cycles)
• 75 sperm 79.32.9
• 150 sperm 81.84.3
• 300 sperm 82.01.4
• Under extended cycles PCR (34 cycles)
• 75 sperm 67.013.2
• 150 sperm 85.26.7

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Summary Mixture Study

• LMD separation of sperm cells from a epithelial
  cell mixture results in a single semen donor
  genotype
• The lower limit of detection using ABI user
  guides PCR protocol (standard conditions) is
  75-150 sperm cells.
• Extended cycle analysis can extend the lower
  limit of detection

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LCN Study of Mixtures

• Prepared mixtures of human female oral swabs
  (epithelial cells) and male semen (sperm cells).
  Equivalent to 1 swab 1µl of semen
• Collected 80, 40, 20, 10 and 5 sperm cells by
  laser microdissection
• Profiler Plus PCR amplification was performed
  using 34 38 cycles. (6 10 cycles)

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80 sperm cells at 34 PCR cycles
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40 sperm cells at 34 PCR cycles
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20 sperm cells at 34 cycles
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10 Sperm Cells at 34 PCR Cycles
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LMD Collected Cells from a Mixture (34 cycles)
80
40
20
10
5
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Percent of Profiler Plus Profile Detected from
LMD Collected Cells (34 cycles)



N5
N5
N5
N5
N5
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Percent of Profiler Plus Profile Detected from
LMD Collected Cells (34 38 Cycles)
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Epithelial Cell Carryover Outlier?
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STR plots from LMD collected sperm cells with
epithelial cell DNA carryover. Female donor
alleles (indicated by asterisks) were detected in
the 40 and 80 sperm cell collections from one of
the slide smears in this study.
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LCN Mixture Study Summary

• Minute numbers of sperm cells can be separated
  and recovered by LMD from epithelial cell
  mixtures.
• STR genotyping can be obtained with as little as
  5 sperm cells captured by LMD using increased PCR
  cycles.

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Comparative Study
AmpFlSTR Profiler Plus for 34 cycles
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Plots of Sperm Fractions LMD vs. PL 15 Cell
Mixture Ratio
LMD
PL
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Plots of Sperm Fractions LMD vs. PL 1160 Cell
Mixture Ratio
PL
LMD
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LMD vs. PL Detection of Male Donor Genotype
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LMD vs. PL Female Carryover
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Comparison of PCR Product Quantity LMD vs. PL
of Sperm Fraction




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Work Flow Chart Comparing Methods
Preferential Lysis Method
LMD Method
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Comparative Study Summary

• LMD provides improved separation and detection of
  sperm from epithelial cell DNA over the
  preferential lysis method at higher e-cell to
  sperm-cell ratios.
• LMD Does not require a DNA quantification step
• Single sample processing more rapid using LMD

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Case Studies using Low Copy

• 4 case studies
• Non-probative or adjudicated cases
• Originating crime lab performed organic
  differential extraction and attempted typing of
  13 core loci
• Case A - public masturbation (tissue paper
  recovered from the scene)
• Cases B, C D - sexual assaults (vaginal
  swabs obtained)
• Case A - ample sperm available
• 30 sperm collected by LMD followed by LCN
  analysis
• Case B, C D - Difficulty locating sperm
  and limited sample available.
• modified the LMD protocol to include a
  mini-lysis
• 18-30 sperm collected by LMD followed by LCN
  analysis

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A Closer Look at Case B
Locus PL - Sperm Fraction Male Exemplar 10 Sperm LMD (LCN) 30 Sperm LMD (LCN)
D3 15, 16, 17 16, 17 17
VWA 15, 16, 18 15, 18 15 15, 18
FGA 19, 22, 24 19, 24 19, 24 19, 24
AMEL X , Y X, Y Y X, Y
D8 10, 13, 14 10, 13 13 10, 13
D21 30, 30.2, 31 30.2, 31 31 30.2, 31
D18 15, 16, 17 15, 17 17 15, 17
D5 8, 12, 13 8, 12 13 8, 12
D13 12, 14 12, 14 12 12, 14
D7 8, 9, 10 9, 10 9
Carryover from victim shown in red Shared
alleles underlined
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Case Studies Summary

• LMDs performance
• Case A - similar result to PL
• Case B C - improved results
• Case D - worse results
• This study may not be the best case study
  comparison test of LMD to the PL method due to
  other variations in the analysis (i.e. LCN vs.
  STD PCR, and mini-lysis)
• Larger case study population required in a
  forensic lab setting to make final evaluation of
  LMD

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Research Team

• RFUMS
• Christine T. Sanders
• Daniel A. Peterson
• Emily Reisenbigler
• LAPD
• Nick Sanchez
• University of Central Florida
• Jack Ballantyne
• Northern Illinois Regional Crime Lab
• Kenneth Pfoser

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