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


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


Leica™ LMD Microscope


Why Laser Microdissection?

~45% of all NLCL DNA cases involve sexual offense

soNeed to eliminate bottleneck in

DNA analysis


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


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


Elimination of Traditional Extraction

1. Direct amplification after LMD?


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


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

• Recombinant ProK

• Lysis incubation in TC

• PCR reaction components to same tube

• Amplify, analyze


Additional Experiments: Sensitivity

• PCR cycles

• amplification reaction volume with reduced volume PCR (RVPCR)

– 15µL, 10µL

• PCR with fewer sperm for lowest detection limit


ProK/DTT 150 sperm, 30 cycles

25µL

15µL

10µL

Avg PH ~880 RFUs

Avg PH ~948 RFUs

Avg PH ~2540 RFUs


Reproducibility•Profiler Plus

•30 PCR cycles



25µL

10µL

Avg PH ~157 RFUs

Avg PH ~2631 RFUs

drop-out



drop-out

25µL

10µL

Avg PH ~174 RFUs

Avg PH ~556 RFUs


• Elimination of traditional extraction possible

• Absolute separation of sperm and epithelial DNA possible

LMD together with Pre-amplification Lysis:


Profiler Plus

Exciting! 50 sperm, 30 cycles, 10µL

Technology Transfer Workshop50 epi nuclei, 30 cycles, 10µL

drop-out

Profiler Plus


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


From Research to Validation . . .

• Troubleshooting

• Identifiler for single amplification

• How many sperm?

• No sperm observed


Troubleshooting

• Static– humidity

– cuttings into 25µL vs 50µL

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

• Contamination?• Non-specific binding?

– Profiler Plus vs. Profiler vs. Identifiler



• Troubleshooting


• How many sperm?



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


drop out

24/29

50 sperm, Identifiler, 30 cycles


29/29 alleles

Identifiler Optimization



• Troubleshooting


• How many sperm?



29/29 alleles

50 sperm, Identifiler

Technology Transfer Workshop25 sperm, Identifiler

23/29


27/29




20/29



25/29



• Troubleshooting


• How many sperm?



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


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


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


Novel Process

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

sexual assault samples


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


Improves and streamlines the analysis of sexual assault

evidence

AND

Frees up analyst time!

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


Current Considerations

• Shorten lysis time of LMD harvested cells

• Mixture Studies

– Epithelial nuclei, still necessary?

• Non-probative samples

• Considerations for casework implementation


Case#2


Case#3


Case#4


Case#1


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?


Reproducibility•Profiler Plus

•30 PCR cycles


Problem: LCN e-grams

• Stochastic effects

• High stutter

Solution: LCN e-grams

• Lack of mixture, interpretation easier

• Set software filter for stutter expectations?


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


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


Acknowledgements

• Pat Woijkiewicz, Ph.D.

• NLCL DNA staff

• Christine SandersRosalind Franklin University of Medicine and Science

• Andy Lee

Leica™ Microsystems


Contact Information

Kelli Raley

North Louisiana Criminalistics Laboratory

1115 Brooks St

Shreveport, LA 71101

(318)-227-2889

[email protected]

application of laser microdissection (lmd) to expedite forensic sexual assault casework

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