1. 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

2. 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

3. Leica™ LMD Microscope

4. Why Laser Microdissection? ~45% of all NLCL DNA cases involve sexual offense so Need to eliminate bottleneck in DNA analysis

5. 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

6. 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

7. Elimination of Traditional Extraction • Direct amplification after LMD?

8. Elimination of Traditional Extraction • Direct amplification after LMD? • 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

9. Comparison of Pre-Amplification Treatments • 150 sperm • 25µL volume • Profiler Plus • 30 PCR cycles

10. Pre-amplification Lysis: ProK/DTT • Cut directly into water • Recombinant ProK • Lysis incubation in TC • PCR reaction components to same tube • Amplify, analyze

11. Additional Experiments: Sensitivity • PCR cycles • amplification reaction volume with reduced volume PCR (RVPCR) • 15µL, 10µL • PCR with fewer sperm for lowest detection limit

12. 25µL 15µL 10µL ProK/DTT150 sperm, 30 cycles Avg PH ~880 RFUs Avg PH ~948 RFUs Avg PH ~2540 RFUs

13. Reproducibility • Profiler Plus • 30 PCR cycles

14. drop-out ProK/DTT100 sperm, 30 cycles 25µL Avg PH ~157 RFUs 10µL Avg PH ~2631 RFUs

15. drop-out ProK/DTT50 sperm, 30 cycles 25µL Avg PH ~174 RFUs 10µL Avg PH ~556 RFUs

16. LMD together with Pre-amplification Lysis: • Elimination of traditional extraction possible • Absolute separation of sperm and epithelial DNA possible

17. Exciting! 50 sperm, 30 cycles, 10µL Profiler Plus

18. drop-out 50 epi nuclei, 30 cycles, 10µL Profiler Plus

19. drop-out 25 epi nuclei, 30 cycles, 10µL Profiler Plus

20. 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: 50-150 sperm • http://www.promega.com/geneticidproc/ussymp16proc/abstracts/langley.pdf

21. From Research to Validation . . . • Troubleshooting • Identifiler for single amplification • How many sperm? • No sperm observed

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

23. From Research to Validation . . . • Troubleshooting • Identifiler for single amplification • How many sperm? • No sperm observed

24. 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

25. 50 sperm, Identifiler, 30 cycles drop out 24/29

26. Identifiler Optimization 29/29 alleles

27. From Research to Validation . . . • Troubleshooting • Identifiler for single amplification • How many sperm? • No sperm observed

28. 50 sperm, Identifiler 29/29 alleles

29. 25 sperm, Identifiler 23/29

30. 25 sperm, Identifiler 27/29

31. 15 sperm, Identifiler 20/29

32. 15 sperm, Identifiler 25/29

33. From Research to Validation . . . • Troubleshooting • Identifiler for single amplification • How many sperm? • No sperm observed

34. No sperm observed • Cut spot from PEN slide • Organic extraction • Qiagen EZ1? • YSTR and STR panels

35. SGM Plus results, foil cut-out1:20 male:female epithelia

36. YSTR results, foil cut-out 1:20 male:female epithelia

37. 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

38. 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

39. Novel Process The NLCL DNA section envisions a new way of processing and storing sexual assault samples

40. Sexual Assault Sample Processing Time - LMD • Presumptive testing (AP, PSA), slide preparation • Examine for sperm, cut nuclear material • Drying of TE-4 • Pre-amp (in TC) • PCR and gel 2 hours (1hr shake) 15 min -1 hour 45 min overnight (3.5 hrs) 1 day

41. LMD Coupled With Pre-amplification Lysis . . . . . Improves and streamlines the analysis of sexual assault evidence AND Frees up analyst time!

42. Current Considerations • Shorten lysis time of LMD harvested cells • Mixture Studies • Epithelial nuclei, still necessary? • Non-probative samples • Considerations for casework implementation

43. Case#2

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49. Case#1

50. Case#1