Presentation on theme: "Technology Transfer Workshop Laser Microdissection Advanced LMD Forensic Applications Patrick Wojtkiewicz, Ph.D. North Louisiana Crime Lab (318) 227-2889."— Presentation transcript:
Technology Transfer Workshop Laser Microdissection Advanced LMD Forensic Applications Patrick Wojtkiewicz, Ph.D. North Louisiana Crime Lab (318) 227-2889 pwojtkie@NLCL.org
Technology Transfer Workshop 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
Technology Transfer Workshop 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)!
Technology Transfer Workshop 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
Technology Transfer Workshop 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
Technology Transfer Workshop Sperm Paint Antibodies to:. –ESP (equatorial segment) –SP-10 (acrosomal protein) * –CaBYR-A (tail) Procedure –Add antibodies to smear –Overnight @ 4ºC –Wash with H 2 O –Examine
Technology Transfer Workshop 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
Technology Transfer Workshop 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.
Technology Transfer Workshop 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
Technology Transfer Workshop Buccal Cells from Swab
Technology Transfer Workshop Diploid cells Microdissection
Technology Transfer Workshop Concern About Y STR Results
Technology Transfer Workshop Extreme Cell Mixtures Female: Male Ratio # of loci in male profile # of loci in female profile 99:1712 95:5913 75:2515 50:501311 25:751312 5:95912 1:99102
Technology Transfer Workshop 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
Technology Transfer Workshop Case #1 Fingernail Scrapings Item/LocusVictimFingernail Scrapings Vaginal SwabSuspect Known30 Female Cells 15 Male Cells25 Male Cells (Non-sperm) Known D3S135814,15 1515,16 vWA16,17 1816,18 FGA23,24 ND19,25 AMELXXYXY D8S117913,14 1613,(15),16,(17)13,16 D21S1129,31.2 ND30,31 D18S5114,17 ND14.2,16 D5S8188,12 1412,14 D13S31712 ND(8),11,1211,12 D7S8208,12888,9 D16S53910 ND99 THO19998,9 TPOX88ND10,11 CSF1PO11,12 1111,12 D2S133818,23 ND18,25 D19S43312,14 14.214.2,16.2
Technology Transfer Workshop 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.
Technology Transfer Workshop Acknowledgements Jennifer Valentine Kelli Raley North Louisiana Crime Lab LSUSHSC