Laser Microdissection

1
Laser Microdissection

• Novel Processing Plan for LMD Analysis of Sexual
  Assault Evidence
• Patrick Wojtkiewicz, Ph.D.
• North Louisiana Crime Lab
• [phone]
• pwojtkie_at_NLCL.org

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.
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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
• Sp-Ep, Ep-Ep ?Diff
• Ep-Ep, ? Std

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Differential Extraction
Cellular Extraction Epithelial Digestion Sperm
Wash Sperm Digestion Purification Quantification A
mplification
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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)

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Standard Extraction
Cellular Extraction Epithelial Digestion Sperm
Wash Sperm Digestion Purification Quantification A
mplification
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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

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

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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!
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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
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Microscopy Identify Cells

• Stain or DIC
• Epithelia
• Spermatozoa

?DNA

• Sperm paint
• Spermatozoa

• Chromosome paint
• Epithelia
• WBC

?DNA
?DNA
4. Std/Diff Extraction
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Novel Process

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

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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 "
• Sp-Ep, Ep-Ep "
• Ep-Ep, ? DIC/CP

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Procedure Timing

1. Prepare slide
2. Examine (brightfield optics)
3. Fluorescent optics (Sperm Paint)
4. Chromosome Paint
5. Pre-amp, PCR, Gel

1-2 hrs. 30-60 min. 30 min. ovnex 30 min.ovn1
hr.ex 1 day
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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

18
Acknowledgements

• Jennifer Valentine
• Kelli Raley
• North Louisiana Crime Lab
• LSUSHSC