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 Quantification is done by cell counting Fluorescent attachment provides new capabilitie

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)!

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

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

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

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

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

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)

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.

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

Buccal Cells from Swab

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

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

Concern About Y STR Results

# of loci in male profile # of loci in female profile Extreme Cell Mixtures Female: Male Ratio # of loci in male profile # of loci in female profile 99:1 7 12 95:5 9 13 75:25 15 50:50 11 25:75 5:95 1:99 10 2

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

Case #1 Fingernail Scrapings Item/Locus Victim Fingernail Scrapings Vaginal Swab Suspect Known 30 Female Cells 15 Male Cells 25 Male Cells (Non-sperm) D3S1358 14,15 15 15,16 vWA 16,17 18 16,18 FGA 23,24 ND 19,25 AMEL X Y XY D8S1179 13,14 16 13,(15),16,(17) 13,16 D21S11 29,31.2 30,31 D18S51 14,17 14.2,16 D5S818 8,12 14 12,14 D13S317 12 (8),11,12 11,12 D7S820 8 8,9 D16S539 10 9 THO1 TPOX 10,11 CSF1PO 11 D2S1338 18,23 18,25 D19S433 14.2 14.2,16.2

Case #2 Vaginal Swabs VICTIM VAGINAL SWAB SUSPECT KNOWN 20 MALE CELLS ACTUAL EVID D3S1358 14,16 17,18 14,16,17,18 vWA 14,17 14,17,18 FGA 19,21 ND 19,21,23,25 23,25 AMEL X XY D8S1179 11,13 12,13 11,12,13 D21S11 30 29 29,30 D18S51 13 D5S818 11,12 12 D13S317 8,12 11 8,11,12 D7S820 D16S539 11,(12) THO1 6,9.3 7,(9.3) 6,7,9.3 6,7 TPOX 8,11 CSF1PO 9,10 9,10,11 D2S1338 17,23 25 17,18,23,25 18,25 D19S433 14 13,14

Case #3 Bitemark Swabs ITEM/LOCUS VICTIM VAGINAL SWAB BITEMARK SWAB SUSPECT KNOWN 30 FEMALE CELLS 7 MALE CELLS ACTUAL D3S1358 14,15 14 15,16 14,15,16 vWA 16,17 16 16,17,18 16,18 FGA 23,24 24 19 ND 19,25 AMEL X XY D8S1179 13,14 13,14,16 13,16 D21S11 29,31.2 29,30,31 30,31 D18S51 14,17 14.2,16 D5S818 8,12 8 12,14 D13S317 12 11 11,12 D7S820 8,9 D16S539 10 9 THO1 TPOX 8,10,11 10,11 CSF1PO D2S1338 18,23 25 18,25 D19S433 16.2 12,14,14.2,16.2 14.2,16.2

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.

Acknowledgements Jennifer Valentine Kelli Raley North Louisiana Crime Lab LSUSHSC