Laser Microdissection Novel Processing Plan for LMD Analysis of Sexual Assault Evidence Patrick Wojtkiewicz, Ph.D. North Louisiana Crime Lab (318) 227-2889 pwojtkie@NLCL.org

Goal – Comprehensive Process Improve Processing of Sexual Assault Evidence Using the Laser Microdissection Microscope Expedite Processing of Sexual Assault Evidence Enhance Capabilities for Analysis of Difficult Samples Provide New Capabilities of Analyzing Mixtures

Types of Sexual Assault Evidence Vaginal swabs/washings Bite/neck/breast swabs Oral/rectal swabs Suspected semen stains Urogenital stain swabs Fingernail samples Almost always have the potential for mixed DNA. Usually these are male/female mixtures.

Sexual Assault Evidence Potential DNA mixtures Vaginal swabs/washings Bite/neck/breast swabs Oral/rectal swabs Suspected semen stains Urogenital stain swabs Fingernail swabs Cell mixture Method Sp-Ep, ? Diff Ep-Ep, Sp-Ep ?Std Sp-Ep Diff Sp-Ep, Ep-Ep ?Diff Ep-Ep, ? Std

Differential Extraction Cellular Extraction Epithelial Digestion Sperm Wash Sperm Digestion Purification Quantification Amplification AP, P30 Pellet (10%) Sperm Epi E S E

Differential Extraction Used for semen containing samples Strengths Separates sperm and epithelial DNA Microscopic identification of cells Internal QC check Weaknesses Cellular extraction may not get all cells off specimen Consumes part of sample for microscopy and quantification User intensive and complex Cannot always get a clean separation Cannot separate non-sperm cell mixtures (vasectomy)

Standard Extraction Cellular Extraction Epithelial Digestion DNA Presumptive Tests? Cellular Extraction Epithelial Digestion Sperm Wash Sperm Digestion Purification Quantification Amplification

Standard Extraction Used for non-sperm containing samples Strengths Can digest sample directly with good DNA recovery Simple procedure Weaknesses Cellular extraction not often done to identify cells Cannot identify mixtures prior to data analysis Unable to resolve some mixtures Presumptive testing may require re-sampling

Laser Microdissection Process Cellular Extraction Epithelial Digestion Cell Dissection Pre-amp Digestion Purification Quantification Amplification AP, P30 Pellet (100%) Male Female

Laser Microdissection Microscopy For all types of sexual assault samples Strengths Clean separation sperm and epithelial DNA Microscopic identification of cells increases sensitivity & minimizes handling Quantification is done by cell counting Fluorescent attachment provides new capabilities Weaknesses Requires cellular extraction to prepare slide Requires nucleated cell identification Cannot separate same-sex epithelial cell mixtures

Analysis of Sexual Assault Evidence Brightfield microscopy Faster analysis Microscopy, cell separation, & purification performed in a single step Eliminate DNA quantification Better DNA separation Fluorescent microscopy Fluorescent labeling of spermatozoa Easier & faster identification More confidence in negative samples Fluorescent labeling of X and Y chromosomes Can separate different-sex epithelial mixtures! Can combine both phases into a single, comprehensive process!

LMD Analytical Process Cellular Extraction Examination Cell Dissection Pre-amp Digestion Amplification Presumptive tests AP, P30 Spot 100% of cell pellet Microscopy – Identify cells Dissect appropriate cells directly in PCR tubes Pre-amp treatment, amplification, & data analysis

Microscopy – Identify Cells Stain or DIC Epithelia Spermatozoa DNA Sperm paint Spermatozoa Chromosome paint Epithelia WBC DNA DNA 4. Std/Diff Extraction

Novel Process Prepare slide Examine using brightfield optics Proceed with LMD & PCR Use fluorescent capabilities Fluorescent optics (sperm identification) Excise entire smear or Chromosome Paint Extract DNA from slide

Sexual Assault Evidence Potential DNA mixtures Vaginal swabs/washings Bite/neck/breast swabs Oral/rectal swabs Suspected semen stains Urogenital stain swabs Fingernail swabs Cell mixture Method Sp-Ep, ? DIC/SP/CP Ep-Ep, Sp-Ep " Sp-Ep " Sp-Ep, Ep-Ep " Ep-Ep, ? DIC/CP

Procedure Timing Prepare slide Examine (brightfield optics) Fluorescent optics (Sperm Paint) Chromosome Paint Pre-amp, PCR, Gel 1-2 hrs. 30-60 min. 30 min. +ovn+ex 30 min.+ovn+1 hr.+ex 1 day

Validation-Comprehensive Process Sample Preparation Validation of individual components Between component interference The whole integrated process Is there anything lost by using this process? Can be validated incrementally

Acknowledgements Jennifer Valentine Kelli Raley North Louisiana Crime Lab LSUSHSC