Presentation on theme: "Laser Microdissection"— Presentation transcript:
1Laser Microdissection Novel Processing Plan for LMD Analysis of Sexual Assault EvidencePatrick Wojtkiewicz, Ph.D.North Louisiana Crime Lab(318)
2Goal – Comprehensive Process Improve Processing of Sexual Assault Evidence Using the Laser Microdissection MicroscopeExpedite Processing of Sexual Assault EvidenceEnhance Capabilities for Analysis of Difficult SamplesProvide New Capabilities of Analyzing Mixtures
3Types of Sexual Assault Evidence Vaginal swabs/washingsBite/neck/breast swabsOral/rectal swabsSuspected semen stainsUrogenital stain swabsFingernail samplesAlmost always have the potential for mixed DNA.Usually these are male/female mixtures.
6Differential Extraction Used for semen containing samplesStrengthsSeparates sperm and epithelial DNAMicroscopic identification of cellsInternal QC checkWeaknessesCellular extraction may not get all cells off specimenConsumes part of sample for microscopy and quantificationUser intensive and complexCannot always get a clean separationCannot separate non-sperm cell mixtures (vasectomy)
8Standard Extraction Used for non-sperm containing samples Strengths Can digest sample directly with good DNA recoverySimple procedureWeaknessesCellular extraction not often done to identify cellsCannot identify mixtures prior to data analysisUnable to resolve some mixturesPresumptive testing may require re-sampling
10Laser Microdissection Microscopy For all types of sexual assault samplesStrengthsClean separation sperm and epithelial DNAMicroscopic identification of cells increases sensitivity & minimizes handlingQuantification is done by cell countingFluorescent attachment provides new capabilitiesWeaknessesRequires cellular extraction to prepare slideRequires nucleated cell identificationCannot separate same-sex epithelial cell mixtures
11Analysis of Sexual Assault Evidence Brightfield microscopyFaster analysisMicroscopy, cell separation, & purification performed in a single stepEliminate DNA quantificationBetter DNA separationFluorescent microscopyFluorescent labeling of spermatozoaEasier & faster identificationMore confidence in negative samplesFluorescent labeling of X and Y chromosomesCan separate different-sex epithelial mixtures!Can combine both phases into a single, comprehensive process!
12LMD Analytical Process Cellular ExtractionExaminationCell DissectionPre-amp DigestionAmplificationPresumptive tests AP, P30Spot 100% of cell pelletMicroscopy – Identify cellsDissect appropriate cells directly in PCR tubesPre-amp treatment, amplification, & data analysis
14Novel Process Prepare slide Examine using brightfield optics Proceed with LMD & PCRUse fluorescent capabilitiesFluorescent optics (sperm identification)Excise entire smear orChromosome PaintExtract DNA from slide
17Validation-Comprehensive Process Sample PreparationValidation of individual componentsBetween component interferenceThe whole integrated processIs there anything lost by using this process?Can be validated incrementally
18Acknowledgements Jennifer Valentine Kelli Raley North Louisiana Crime LabLSUSHSC