1. Laser Microdissection
Novel Processing Plan for LMD Analysis of Sexual Assault Evidence
Patrick Wojtkiewicz, Ph.D.
North Louisiana Crime Lab
(318)

2. 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

3. 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.

4. 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

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

6. 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)

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

8. 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

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

10. 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

11. 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!

12. 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

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

14. 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

15. 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

16. 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

17. 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

18. Acknowledgements Jennifer Valentine Kelli Raley
North Louisiana Crime Lab
LSUSHSC