Technology Transfer Workshop Application of Laser Microdissection (LMD) to Expedite Forensic Sexual Assault Casework Kelli Raley, MSFS North 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 so Need to eliminate bottleneck in DNA analysis

Technology Transfer Workshop 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

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

Technology Transfer Workshop Elimination of Traditional Extraction 1.Direct amplification after LMD?

Technology Transfer Workshop Elimination of Traditional Extraction 1.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 Workshop Comparison 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

Technology Transfer Workshop Additional Experiments: Sensitivity PCR cycles amplification reaction volume with reduced volume PCR ( RVPCR) –15µL, 10µL PCR with fewer sperm for lowest detection limit

Technology Transfer Workshop ProK/DTT 150 sperm, 30 cycles 25µL 15µL 10µL Avg PH ~880 RFUs Avg PH ~948 RFUs Avg PH ~2540 RFUs

Technology Transfer Workshop Reproducibility Profiler Plus 30 PCR cycles

Technology Transfer Workshop ProK/DTT 100 sperm, 30 cycles 25µL 10µL Avg PH ~157 RFUs Avg PH ~2631 RFUs drop-out

Technology Transfer Workshop ProK/DTT 50 sperm, 30 cycles drop-out 25µL 10µL Avg PH ~174 RFUs Avg PH ~556 RFUs

Technology Transfer Workshop Elimination of traditional extraction possible Absolute separation of sperm and epithelial DNA possible LMD together with Pre-amplification Lysis:

Technology Transfer Workshop Profiler Plus Exciting! 50 sperm, 30 cycles, 10µL

Technology Transfer Workshop 50 epi nuclei, 30 cycles, 10µL drop-out Profiler Plus

Technology Transfer Workshop 25 epi nuclei, 30 cycles, 10µL drop-out Profiler Plus

Technology Transfer Workshop Early 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: sperm p16proc/abstracts/langley.pdf

Technology Transfer Workshop From Research to Validation... Troubleshooting Identifiler for single amplification How many sperm? No sperm observed

Technology Transfer Workshop Troubleshooting Static –humidity –cuttings into 25µL vs 50µL Electropherogram Conundrum –“dye-saturated” e-grams, Contamination? Non-specific binding? –Profiler Plus vs. Profiler vs. Identifiler

Technology Transfer Workshop Profiler Plus Exciting! 50 sperm, 30 cycles, 10µL

Technology Transfer Workshop Electropherogram Conundrum

Technology Transfer Workshop From Research to Validation... Troubleshooting Identifiler for single amplification How many sperm? No sperm observed

Technology Transfer Workshop Identifiler Optimization TE -4 vs DepC H 2 O –Vary Tris, pH 8.0 in TE -4 : normal, 1/2x, 1/4x, 1/5x PCR cycling –28+6, 20+14, cycles –31 cycles MgCl 2 –Vary extra Mg 2+ added –0.5mM – 1.5mM still optimal for RVPCR

Technology Transfer Workshop drop out 24/29 50 sperm, Identifiler, 30 cycles

Technology Transfer Workshop 29/29 alleles Identifiler Optimization

Technology Transfer Workshop Electropherogram Conundrum

Technology Transfer Workshop 1/4x Tris in TE -4 Solved?

Technology Transfer Workshop 1/5x Tris in TE -4 Don’t let TE -4 sit around! Electropherogram Conundrum Solved? Lesson..

Technology Transfer Workshop From Research to Validation... Troubleshooting Identifiler for single amplification How many sperm? No sperm observed

Technology Transfer Workshop 29/29 alleles 50 sperm, Identifiler

Technology Transfer Workshop 25 sperm, Identifiler 23/29

Technology Transfer Workshop 27/29 25 sperm, Identifiler

Technology Transfer Workshop 15 sperm, Identifiler 20/29

Technology Transfer Workshop 15 sperm, Identifiler 25/29

Technology Transfer Workshop From Research to Validation... Troubleshooting Identifiler for single amplification How many sperm? No sperm observed

Technology Transfer Workshop No sperm observed Cut spot from PEN slide Organic extraction –Qiagen EZ1? YSTR and STR panels

Technology Transfer Workshop SGM Plus results, foil cut-out 1:20 male:female epithelia

Technology Transfer Workshop YSTR 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

Technology Transfer Workshop Novel Process The NLCL DNA section envisions a new way of processing and storing sexual assault samples

Technology Transfer Workshop Novel Process Prepare slide Examine using microscope (+) sperm identification: proceed with LMD, lysis, & RVPCR (-) sperm identification: excise entire spot, proceed with traditional extraction –ample extract for YSTR and STR testing

Technology Transfer Workshop Cellular Extraction Epithelial Digestion Cell Dissection Pre-amp Digestion Purification Quantification Amplification Supernatant: AP, P30 Pellet: sperm ID (~25-30uL) Sperm Epi Cut 50 sperm ~ 15 min same tube Novel Process

Technology Transfer Workshop 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.....

Technology Transfer Workshop Current Considerations Shorten lysis time of LMD harvested cells Mixture Studies –Epithelial nuclei, still necessary? Non-probative samples Considerations for casework implementation

Technology Transfer Workshop Case#2

Technology Transfer Workshop Case#2

Technology Transfer Workshop Case#3

Technology Transfer Workshop Case#3

Technology Transfer Workshop Case#4

Technology Transfer Workshop Case#4

Technology Transfer Workshop Case#1

Technology Transfer Workshop Case#1

Technology Transfer Workshop Case#1

Technology Transfer Workshop 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

Technology Transfer Workshop Problem: LCN e-grams Stochastic effects High stutter Solution: LCN e-grams Lack of mixture, interpretation easier Set software filter for stutter expectations?

Technology Transfer Workshop Promega’s Profiles in DNA Sept. ‘06 “Debunking Some Urban Legends Surrounding Validation within the Forensic DNA Community”, John Butler, NIST Member of SWGDAM Validation Subcommittee –2004 Revised Validation Guidelines esindna_902_03.pdfhttp://

Technology Transfer Workshop 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! dation.htm

Technology Transfer Workshop Acknowledgements Pat Woijkiewicz, Ph.D. NLCL DNA staff Christine Sanders Rosalind 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 (318)