Technology Transfer Workshop
Forensic LMD Research Studies at
Rosalind Franklin University of
Medicine and Science
North Chicago, IL
Christine T. Sanders
geneskr@alumni.ucsd.edu
Technology Transfer Workshop
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
63x
Histological
40x
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 (H&E)
• 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
Spermatozoa
Buccal Cell s
Sample
1
2
3
UNSTN
+/+/+/-
H&E
+
+
+
1
2
3
+/+/-
+
+
+
UNSTN = not stained.
H&E = hematoxylin/eosin.
CTS = nuclear fast red/picroindigocarmine.
MG = methyl green.
WRT = Wright's stain.
AO = acridine orange.
Histological Stain
CTS
MG
+
-++
++
-++
++
++
--
WRT
--
AO
+
+
+
----
+/+
- - : cannot ID or highly
challenging
- : poor
+ / - : satisfactory
+ : good
+ + : excellent
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Percent RFUs Normalized
to Unstained Samples
RFU Levels of Stained Cells

100%
H&E
CTS
80%
60%
40%
20%
0%
62% 43%
1
Stained specimens exhibited RFU values
significantly lower than unstained specimens (P < 0.01)
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Part I: Histology Study Summary
• Methyl green, Wright’s 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
– H&E best choice
Results prompted second phase of histology study (Part II)
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Part II: Histology Study
Stains Tested:
• H&E 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.
• H&E 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 8C97C
• 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 (n=5)
22.9 + 4.8 %
Off scale
150 sperm (n=5)
13.3 + 2.3 %
Off std curve
80 sperm (n=5)
16.3 + 5.4 %
25.1 + 6.6%
40 sperm (n=5)
18.5 + 3.8 %
22.0 + 5.6%
20 sperm (n=5)
12.2 + 4.7 %
20.1 + 7.8%
10 sperm (n=5)
14.8 + 0.8 %
23.6 + 6.6%
5 sperm (n=4)
17.8 + 2.9 %
Off std curve
AB Pos control
0.0313 -1ng
13.0 + 2.0 %
(n=6)
27.5 + 4.7%
(n=5)
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Yield of DNA Extraction (e-cells)
Sample
% Yield by real-time qPCR
150 epithelial (n=5)
37.5 + 2.8 %
80 epithelial (n=5)
45.4 + 6.8 %
40 epithelial (n=5)
37.5 + 4.2 %
20 epithelial (n=5)
40.5 + 7.0 %
10 epithelial (n=5)
32.9 + 5.3 %
5 epithelial (n=5)
41.4 + 8.6 %
2 epithelial (n=5)
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 (*)
*
* *
*
**
AmpFlST
R Profiler
Plus
*
*
**
*
*
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300 Sperm
cells
separated
by LMD
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“150 sperm cells” from a mixture
Standard PCR
Extended 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” 71+12% alleles
– “150 sperm” 96+3% 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”
79.3+2.9%
– “150 sperm” 81.8+4.3%
– “300 sperm” 82.0+1.4%
• Under “extended cycles” PCR (34 cycles)
– “75 sperm”
67.0+13.2%
– “150 sperm” 85.2+6.7%
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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
guide’s 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)
*
*
*
N=5
N=5
N=5
N=5
N=5
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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?
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
Mixtures of sperm cells and female epithelial cells
1:5, 1:10, 1:20, 1:40, 1:80 & 1:160 (SP:EC)
9µl smeared on PEN slide for LMD
25µl used for Preferential Lysis
Collected 20-30 sperm cells by LMD
with Lyse-N-Go Extraction
Differential Extraction
with Organic Purification
PCR mastermix added directly
to collection/extraction tube
qPCR DNA Quanification
Maximum volume of extract
input into PCR
AmpFlSTR Profiler Plus for 34 cycles
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Plots of Sperm Fractions LMD vs. PL
1:5 Cell Mixture Ratio
LMD
PL
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Plots of Sperm Fractions LMD vs. PL
1:160 Cell Mixture Ratio
LMD
PL
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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
Sexual Assault Kit Swab:
Elute Cells & Prepare Smear
LMD
Method
Microscopic ID
Microscopic ID
LMD ~5 cells/min.
Preferential Lysis Separation
3- 4 hrs.
DNA Isolation
Lyse-N-Go 20 min.
DNA Isolation
Chelex: 70 min. or
Organic: 2 hrs.
PCR
Capillary Electrophoresis
Data Analysis (single source genotype)
DNA Quantification 4 hrs.
PCR
Capillary Electrophoresis
Data Analysis
Technology Transfer Workshop
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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Locus
Case A sp
LMD
D3S1358
vWA
FGA
AMEL
D8S1179
D21S11
D18S51
D5S818
D13S317
D7S820
16, 17
17
21, 23
X, Y
12, 14
28, 31
18
9, 12
11, 12
8, 12
Locus
Case C sp
LMD**
D3S1358
vWA
FGA
AMEL
D8S1179
D21S11
D18S51
D5S818
D13S317
D7S820
15
16, 18
24, 25
X, Y
13, 15
28, 31.2
14, 16
11, 12
10, 13
11, 12
Case A sp
original PL
results
16, 17
17
21, 23
X, Y
12, 14
28, 31
18
9, 12
11, 12
8, 12
Case B sp
LMD**
17
15, 18
19, 24
X, Y
10, 13
30.2, 31
15, 17
8, 12
12, 14
9
Case B sp
original PL
results
15, 16, 17
15, 16, 18
19, 22, 24
X, Y
10, 13, 14
30, 30.2, 31
15, 16, 17
8, 12, 13
12, 14
8, 9, 10
Case C sp
original PL
results
15
16, 18
Case C
victim*
Case D sp
LMD**
16
16
15
18
24
X
12
32.2
13
10
13
10
X, Y
13, 15
28
X
10, 14
30, 31.2
11, 12
10, 11
9, 10
* Partial profile of victim assumed from the epithelial cell fraction of vaginal swab
** LMD performed after “mini-PL”
sp = sperm fraction
Case B
suspect
Case B
victim
16, 17
15, 18
19, 24
X, Y
10, 13
30.2, 31
15, 17
8, 12
12, 14
9, 10
15, 16
15, 16
22, 25
X
14, 17
30, 31.2
16
13
12
8, 12
Case D sp
original PL
results
15, 16
16, 18
24
X, Y
12, 13
28, 32.2
10, 13
12, 13
Case D
victim*
15
17, 18
X
12, 13
28, 30
8, 11
8
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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
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
17 ***
9 ***
Carryover from victim shown in red; Shared alleles underlined
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Case Studies Summary
• LMD’s 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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