Application of Laser Microdissection (LMD) to Expedite Forensic Sexual Assault Casework

Technology Transfer Workshop

Application of Laser Microdissection (LMD) to Expedite Forensic Sexual

Assault Casework

Kelli Raley, MSFSNorth Louisiana Criminalistics Laboratory

Shreveport, LA

NFSTC Laser Microdissection Workshop, 2007

Technology Transfer Workshop

LMD Microscopy Research and Experience: Highlights

• Why LMD for North Louisiana Crime Lab (NLCL) Casework?

• Elimination of Manual Extraction

• Reproducibility/Sensitivity

• Troubleshooting

• Single Amplification/Optimization

• Absence of Sperm

• LMD: Streamlined, Novel Process

Technology Transfer Workshop

Leica™ LMD Microscope

Technology Transfer Workshop

Why Laser Microdissection?

~45% of all NLCL DNA cases involve sexual offense

soNeed to eliminate bottleneck in

DNA analysis

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Disadvantages of LMD

• Initial cost

• Novel validation

• ~$4.25 each for PEN slides

• PEN foil contains pores ≈ sperm

• Except for PEN slides, no other consumables for Leica

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Advantages of LMD

• Eliminate traditional extraction

• Absolute separation of sperm and epithelial DNA

• Effect of traditional PCR challenges minimized

• Decrease analysis time for a sexual assault sample

Technology Transfer Workshop

Elimination of Traditional Extraction

1. Direct amplification after LMD?

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Elimination of Traditional Extraction1. Direct amplification after LMD?

2. Pre-amplification Lysis– Constraints

• Can’t adversely affect PCR• Limited volume (10µL)

Lyse-N-Go™ PCR Reagent (LNG) 25µL reaction vs. 50µL

Recombinant Proteinase K

Technology Transfer WorkshopComparison of Pre-

Amplification Treatments•150 sperm•25µL volume•Profiler Plus•30 PCR cycles

Technology Transfer Workshop

Pre-amplification Lysis: ProK/DTT• Cut directly into water

• Recombinant ProK

• Lysis incubation in TC

• PCR reaction components to same tube

• Amplify, analyze

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Additional Experiments: Sensitivity

• PCR cycles

• amplification reaction volume with reduced volume PCR (RVPCR)

– 15µL, 10µL

• PCR with fewer sperm for lowest detection limit

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ProK/DTT 150 sperm, 30 cycles

25µL

15µL

10µL

Avg PH ~880 RFUs

Avg PH ~948 RFUs

Avg PH ~2540 RFUs

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Reproducibility•Profiler Plus

•30 PCR cycles

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ProK/DTT 100 sperm, 30 cycles

25µL

10µL

Avg PH ~157 RFUs

Avg PH ~2631 RFUs

drop-out

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ProK/DTT 50 sperm, 30 cycles

drop-out

25µL

10µL

Avg PH ~174 RFUs

Avg PH ~556 RFUs

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• Elimination of traditional extraction possible

• Absolute separation of sperm and epithelial DNA possible

LMD together with Pre-amplification Lysis:

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Profiler Plus

Exciting! 50 sperm, 30 cycles, 10µL

Technology Transfer Workshop50 epi nuclei, 30 cycles, 10µL

drop-out

Profiler Plus

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25 epi nuclei, 30 cycles, 10µL

drop-out

Profiler Plus

Technology Transfer WorkshopEarly NLCL Research for LMD:

Summary• Replace DNA extraction and purification with

novel pre-amplification lysis

• Physical and complete separation of sperm and epithelial DNA

• RVPCR, 30 cycles, reproducible• Sensitivity: 50-150 sperm

• http://www.promega.com/geneticidproc/ussymp16proc/abstracts/langley.pdf

Technology Transfer Workshop

From Research to Validation . . .

• Troubleshooting

• Identifiler for single amplification

• How many sperm?

• No sperm observed

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Troubleshooting

• Static– humidity

– cuttings into 25µL vs 50µL

• Electropherogram Conundrum– “dye-saturated” e-grams,

• Contamination?• Non-specific binding?

– Profiler Plus vs. Profiler vs. Identifiler

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From Research to Validation . . .

• Troubleshooting

• Identifiler for single amplification

• How many sperm?

• No sperm observed

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Identifiler Optimization

• TE-4 vs DepC H2O

– Vary Tris, pH 8.0 in TE-4: normal, 1/2x, 1/4x, 1/5x

• PCR cycling

– 28+6, 20+14, 20+10 cycles

– 31 cycles

• MgCl2– Vary extra Mg2+ added

– 0.5mM – 1.5mM still optimal for RVPCR

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drop out

24/29

50 sperm, Identifiler, 30 cycles

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29/29 alleles

Identifiler Optimization

Technology Transfer Workshop

From Research to Validation . . .

• Troubleshooting

• Identifiler for single amplification

• How many sperm?

• No sperm observed

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29/29 alleles

50 sperm, Identifiler

Technology Transfer Workshop25 sperm, Identifiler

23/29

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27/29

25 sperm, Identifiler

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15 sperm, Identifiler

20/29

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15 sperm, Identifiler

25/29

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From Research to Validation . . .

• Troubleshooting

• Identifiler for single amplification

• How many sperm?

• No sperm observed

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No sperm observed

• Cut spot from PEN slide

• Organic extraction

– Qiagen EZ1?

• YSTR and STR panels

Technology Transfer WorkshopSGM Plus results, foil cut-out1:20 male:female epithelia

Technology Transfer WorkshopYSTR results, foil cut-out 1:20 male:female epithelia

Technology Transfer Workshop

Advantages of LMD

• Eliminate traditional extraction

• Absolute separation of sperm and epithelial DNA

• Effect of traditional PCR challenges minimized

• Decrease analysis time for a sexual assault sample through novel process

Technology Transfer Workshop

Advantages of LMD

Effect of traditional PCR challenges minimized

– Simplify mixtures, simplifying interpretation

– Difficult statistical interpretations eliminated

– Less tendency for contaminants/inhibitors?

– Increase PCR cycles to enhance LCN sperm analysis

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Novel Process

The NLCL DNA section envisions a new way of processing and storing

sexual assault samples

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Sexual Assault Sample Processing Time - LMD

1. Presumptive testing (AP, PSA), slide preparation

2. Examine for sperm, cut nuclear material

3. Drying of TE-4

4. Pre-amp (in TC)

5. PCR and gel

2 hours (1hr shake)

15 min -1 hour

45 min

overnight (3.5 hrs)

1 day

Technology Transfer Workshop

Improves and streamlines the analysis of sexual assault

evidence

AND

Frees up analyst time!

LMD Coupled With Pre-amplification Lysis . . . . .

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Current Considerations

• Shorten lysis time of LMD harvested cells

• Mixture Studies

– Epithelial nuclei, still necessary?

• Non-probative samples

• Considerations for casework implementation

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Case#2

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Case#2

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Case#3

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Case#3

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Case#4

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Case#4

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Case#1

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Case#1

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Case#1

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Problem: Drop-Out Samples• Due to static charge?

• Loss due to tube release from holder?

• Downstream analysis human error?

Solution: Drop-Out Samples• Cut at least 2 tubes each of sample sperm

• Concordance necessary or is single amp enough?

Technology Transfer Workshop

Reproducibility•Profiler Plus

•30 PCR cycles

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Problem: LCN e-grams

• Stochastic effects

• High stutter

Solution: LCN e-grams

• Lack of mixture, interpretation easier

• Set software filter for stutter expectations?

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Promega’s Profiles in DNASept. ‘06

• “Debunking Some Urban Legends Surrounding Validation within the Forensic DNA Community”, John Butler, NIST

• Member of SWGDAM Validation Subcommittee

– 2004 Revised Validation Guidelines

• http://www.promega.com/profiles/902/profilesindna_902_03.pdf

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John Butler’s Suggestions:

• Establish “concordance” with any new technique

• Demonstrate reproducibility of the technique over time– Ongoing monitoring and performance

checks• Test a total of ~50 samples

– Not 50 samples per experiment!• http://www.cstl.nist.gov/biotech/strbase/

validation.htm

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Acknowledgements

• Pat Woijkiewicz, Ph.D.

• NLCL DNA staff

• Christine SandersRosalind Franklin University of Medicine and Science

• Andy Lee

Leica™ Microsystems

Technology Transfer Workshop

Contact Information

Kelli Raley

North Louisiana Criminalistics Laboratory

1115 Brooks St

Shreveport, LA 71101

(318)-227-2889

kraley@nlcl.org