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Laser Microdissection

Laser Microdissection. Advanced LMD Forensic Applications Patrick Wojtkiewicz, Ph.D. North Louisiana Crime Lab (318) 227-2889 pwojtkie@NLCL.org. LMD Strengths. Microscopic identification of cells increases sensitivity & minimizes handling Easily separates Sperm and Epithelial DNA

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Laser Microdissection

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  1. Laser Microdissection Advanced LMD Forensic Applications Patrick Wojtkiewicz, Ph.D. North Louisiana Crime Lab (318) 227-2889 pwojtkie@NLCL.org

  2. LMD Strengths • Microscopic identification of cells increases sensitivity & minimizes handling • Easily separates Sperm and Epithelial DNA • Quantification is done by cell counting • Fluorescent attachment provides new capabilitie

  3. Fluorescent Capabilities • Adds Capabilities for Analysis of All Types of Sexual Assault Evidence • Provide Improved Capabilities in Sperm Identification • Enable New Capabilities of Identifying Male and Female Diploid Cell Mixtures • These procedures are in the development stage but have been successfully performed on actual case evidence (non-probative)!

  4. Sperm Identification Problems • Few spermatozoa • Searches are often labor intensive • Analyst uncertainty about ID • Case processing based upon sperm id • Excessive quantity of epithelial cells • Spermatozoa buried under cells • Combined with few spermatozoa • Low quality microscope • Poor staining

  5. Sperm Labeling by Fluorescent Dyes • Based upon antibodies conjugated to AlexaFluor. • Antibodies are specific to human sperm components. • Easier, faster, and confident searches • Backwards compatible (not LMD) • Adds extra step in analysis • Requires high quality microscope with fluorescence capabilities

  6. Sperm Paint • Antibodies to:. • ESP (equatorial segment) • SP-10 (acrosomal protein)* • CaBYR-A (tail) • Procedure • Add antibodies to smear • Overnight @ 4ºC • Wash with H2O • Examine

  7. Fluorescent Sperm Identification • Advantages • Simple procedure & Sequential processing of samples • Antibodies should not adversely affect DNA analysis • Samples can be examined at lower magnification • Improved capability - Fast examination & confidence in negative results. Can be done on older slides. • Previous slides are from casework-like material

  8. Problems in Diploid Cell Mixtures • Sample may not be identified as a mixture prior to analysis. • Mixture may not be apparent when there is a preponderance of DNA from one of the donors. • Interpretation issues • No current way of separating diploid cell mixtures.

  9. Chromosome Paint • Fluorescent in situ hybridization (FISH) • X and Y chromosomes • commercial kit • Interphase nuclei • New capability of analyzing male & female epithelial cell mixtures • Time & reproducibility Lymphocytes

  10. Buccal Cells from Swab

  11. Concern About Y STR Results

  12. Extreme Cell Mixtures

  13. Real Case Results • Identification and dissection of spermatozoa well established • Following results based upon diploid cell analysis • 40% of cases had at least one sample with enough diploid cells to obtain a male profile • Vaginal swab • Bitemark • Fingernail scraping

  14. Case #1 Fingernail Scrapings

  15. Case #2 Vaginal Swabs

  16. Case #3 Bitemark Swabs

  17. CODIS Searches In these cases, and other non-probative samples, the profiles were searched locally against approximately 1500 samples. In each case the partial profiles obtained by LMD only hit one sample, the correct one.

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

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