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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. Validation Issues for Fluorescent Sperm Identification • Analysis is commonly done in labs and procedure is simple; fluorescent microscopy component is new • Two issues • A new way of looking at sperm • Confidence in specificity of identification • LMD component is not required for training • Purpose of procedure (LMD or ID only) • Plenty of practice material available

  9. Validation Issues for Fluorescent Sperm Identification • Develop lab protocol for procedure and use (eg, all samples, confirmations, screening) • Effects of time after intercourse on staining (?) • Examine slides with defined ratios of sperm to epithelium (sensitivity). At what point can you feel confident that if sperm were present they would be seen. • Internal validation  Usage (1 – 2 months for ID only; LMD component must be done first)

  10. 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.

  11. 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

  12. Buccal Cells from Swab

  13. Validation Concerns for X-Y Chromosome Paint • Somewhere between novel and internal validation and needs greater time investment. • Likely not to be a routine procedure • Procedure development problems • No forensic developed kit • Reproducibility (routineness) • Effect on downstream analyses • Applicable to very limited types of samples

  14. Validation Issues for X-Y Chromosome Paint • Start with LMD (very limited usefulness w/o LMD) • Procedure development and standardization. • Post stain analyses (using DNA) • Chromosome identification • Cell ratios (sensitivity) • Application to evidence sample types • Day to day reproducibility • Better probes or better formulation? • More intensive analyst training • LCN training

  15. Concern About Y STR Results

  16. Extreme Cell Mixtures

  17. 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

  18. Case #1 Fingernail Scrapings

  19. Case #2 Vaginal Swabs

  20. Case #3 Bitemark Swabs

  21. CODIS Searches In these cases, and other non-probative samples not shown, the male profiles were searched locally against approximately 1500 samples. In each case the partial profiles obtained by LMD only hit one sample, the correct one.

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

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