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Laser Microdissection

Laser Microdissection. Novel Processing Plan for LMD Analysis of Sexual Assault Evidence Patrick Wojtkiewicz, Ph.D. North Louisiana Crime Lab (318) 227-2889 pwojtkie@NLCL.org. Goal – Comprehensive Process.

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Laser Microdissection

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  1. Laser Microdissection Novel Processing Plan for LMD Analysis of Sexual Assault Evidence Patrick Wojtkiewicz, Ph.D. North Louisiana Crime Lab (318) 227-2889 pwojtkie@NLCL.org

  2. Goal – Comprehensive Process • Improve Processing of Sexual Assault Evidence Using the Laser Microdissection Microscope • Expedite Processing of Sexual Assault Evidence • Enhance Capabilities for Analysis of Difficult Samples • Provide New Capabilities of Analyzing Mixtures

  3. Types of Sexual Assault Evidence • Vaginal swabs/washings • Bite/neck/breast swabs • Oral/rectal swabs • Suspected semen stains • Urogenital stain swabs • Fingernail samples Almost always have the potential for mixed DNA. Usually these are male/female mixtures.

  4. Potential DNA mixtures Vaginal swabs/washings Bite/neck/breast swabs Oral/rectal swabs Suspected semen stains Urogenital stain swabs Fingernail swabs Cell mixtureMethod Sp-Ep, ?Diff Ep-Ep, Sp-Ep?Std Sp-EpDiff Sp-Ep, Ep-Ep?Diff Sp-Ep, Ep-Ep?Diff Ep-Ep, ?Std Sexual Assault Evidence

  5. AP, P30 Pellet (10%) E Pellet (10%) S E Sperm Epi Differential Extraction Cellular Extraction Epithelial Digestion Sperm Wash Sperm Digestion Purification Quantification Amplification

  6. Differential Extraction • Used for semen containing samples • Strengths • Separates sperm and epithelial DNA • Microscopic identification of cells • Internal QC check • Weaknesses • Cellular extraction may not get all cells off specimen • Consumes part of sample for microscopy and quantification • User intensive and complex • Cannot always get a clean separation • Cannot separate non-sperm cell mixtures (vasectomy)

  7. Presumptive Tests? DNA Standard Extraction Cellular Extraction EpithelialDigestion Sperm Wash Sperm Digestion Purification Quantification Amplification

  8. Standard Extraction • Used for non-sperm containing samples • Strengths • Can digest sample directly with good DNA recovery • Simple procedure • Weaknesses • Cellular extraction not often done to identify cells • Cannot identify mixtures prior to data analysis • Unable to resolve some mixtures • Presumptive testing may require re-sampling

  9. Laser Microdissection Process Cellular Extraction Epithelial Digestion Cell Dissection Pre-ampDigestion Purification Quantification Amplification AP, P30 Pellet (100%) Male Female

  10. Laser Microdissection Microscopy • For all types of sexual assault samples • Strengths • Clean separation sperm and epithelial DNA • Microscopic identification of cells increases sensitivity & minimizes handling • Quantification is done by cell counting • Fluorescent attachment provides new capabilities • Weaknesses • Requires cellular extraction to prepare slide • Requires nucleated cell identification • Cannot separate same-sex epithelial cell mixtures

  11. Analysis of Sexual Assault Evidence • Brightfield microscopy • Faster analysis • Microscopy, cell separation, & purification performed in a single step • Eliminate DNA quantification • Better DNA separation • Fluorescent microscopy • Fluorescent labeling of spermatozoa • Easier & faster identification • More confidence in negative samples • Fluorescent labeling of X and Y chromosomes • Can separate different-sex epithelial mixtures! Can combine both phases into a single, comprehensive process!

  12.   LMD Analytical Process • Cellular Extraction • Examination • Cell Dissection • Pre-ampDigestion • Amplification Presumptive tests AP, P30 Spot 100% of cell pellet Microscopy – Identify cells Dissect appropriate cells directly in PCR tubes Pre-amp treatment, amplification, & data analysis

  13. Microscopy – Identify Cells • Stain or DIC • Epithelia • Spermatozoa DNA • Sperm paint Spermatozoa • Chromosome paint • Epithelia • WBC DNA DNA 4. Std/Diff Extraction

  14. Novel Process • Prepare slide • Examine using brightfield optics • Proceed with LMD & PCR • Use fluorescent capabilities • Fluorescent optics (sperm identification) • Proceed with LMD & PCR • Excise entire smear or • Chromosome Paint • Extract DNA from slide

  15. Sexual Assault Evidence Potential DNA mixtures Vaginal swabs/washings Bite/neck/breast swabs Oral/rectal swabs Suspected semen stains Urogenital stain swabs Fingernail swabs • Cell mixtureMethod • Sp-Ep, ?DIC/SP/CP • Ep-Ep, Sp-Ep" • Sp-Ep" • Sp-Ep, Ep-Ep" • Sp-Ep, Ep-Ep" • Ep-Ep, ?DIC/CP

  16. Procedure Timing • Prepare slide • Examine (brightfield optics) • Fluorescent optics (Sperm Paint) • Chromosome Paint • Pre-amp, PCR, Gel 1-2 hrs. 30-60 min. 30 min. +ovn+ex 30 min.+ovn+1 hr.+ex 1 day

  17. Goal – Comprehensive Process • Improve Processing of Sexual Assault Evidence Using the Laser Microdissection Microscope • Expedite Processing of Sexual Assault Evidence • Enhance Capabilities for Analysis of Difficult Samples • Provide New Capabilities of Analyzing Mixtures

  18. Acknowledgements • Jennifer Valentine • Kelli Raley • North Louisiana Crime Lab • LSUSHSC

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