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

2. 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

3. 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)!

4. 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

5. 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

6. 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

7. 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

8. Technology Transfer Workshop Validation Issues for Fluorescent Sperm Identification Analysis is commonly done in labs and procedure is simple; fluorescent microscopy component is new Two issues 1.A new way of looking at sperm 2.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. Technology Transfer Workshop 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. 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.

11. 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

12. Technology Transfer Workshop Buccal Cells from Swab

13. Technology Transfer Workshop 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. Technology Transfer Workshop 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. Technology Transfer Workshop Concern About Y STR Results

16. 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

17. 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

18. 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

19. Technology Transfer Workshop Case #2 Vaginal Swabs VICTIMVAGINAL SWAB SUSPECT KNOWN20 MALE CELLSACTUAL EVIDKNOWN D3S135814,1617,1814,16,17,1817,18 vWA14,1717,1814,17,1817,18 FGA19,21ND19,21,23,2523,25 AMELXXY D8S117911,1312,1311,12,1312,13 D21S11302929,3029 D18S5114,17ND13 D5S81811,121211,1212 D13S3178,12118,11,1211 D7S8208,12ND8,12 D16S5391211,(12)11,12 THO16,9.37,(9.3)6,7,9.36,7 TPOX8,11118,11 CSF1PO9,10119,10,1111 D2S133817,232517,18,23,2518,25 D19S4331413,14

20. Technology Transfer Workshop Case #3 Bitemark Swabs ITEM/LOCUSVICTIMVAGINAL SWABBITEMARK SWAB SUSPECT KNOWN30 FEMALE CELLS7 MALE CELLSACTUALKNOWN D3S135814,151415,1614,15,1615,16 vWA16,17 1616,17,1816,18 FGA23,242419ND19,25 AMELXXNDXY D8S117913,14 ND13,14,1613,16 D21S1129,31.2ND 29,30,3130,31 D18S5114,17ND 14.2,16 D5S8188,128ND12,14 D13S31712ND11ND11,12 D7S8208,12 ND 8,9 D16S53910 999 THO1998,9 TPOX88ND8,10,1110,11 CSF1PO11,12ND11,12ND11,12 D2S133818,23ND25ND18,25 D19S43312,14 16.212,14,14.2,16.214.2,16.2

21. Technology Transfer Workshop 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. Technology Transfer Workshop Acknowledgements Jennifer Valentine Kelli Raley North Louisiana Crime Lab LSUSHSC