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

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. LMD Microscopy Research and Experience: Highlights.

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

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  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 • PEN slides are Leica™ product • 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. Exciting! 50 sperm, 30 cycles, 10µL Profiler Plus

  24. Electropherogram Conundrum

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

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

  27. 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

  28. Identifiler Optimization 29/29 alleles

  29. Electropherogram Conundrum

  30. Solved? 1/4x Tris in TE-4

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

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

  33. 50 sperm, Identifiler 29/29 alleles

  34. 25 sperm, Identifiler 23/29

  35. 15 sperm, Identifiler 20/29

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

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

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

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

  40. 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

  41. 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

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

  43. 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

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

  45. 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

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

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

  48. Pat Woijkiewicz, Ph.D. NLCL DNA staff Christine Sanders Rosalind Franklin University of Medicine and Science Andy Lee Leica™ Microsystems Acknowledgements

  49. Contact Information Kelli Raley North Louisiana Criminalistics Laboratory 1115 Brooks St Shreveport, LA 71101 (318)-227-2889 kraley@nlcl.org

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