Technology Transfer Workshop
Laser Microdissection
Novel Processing Plan for LMD Analysis of Sexual Assault Evidence
Patrick Wojtkiewicz, Ph.D.
North Louisiana Crime Lab
(318) 227-2889
Technology Transfer Workshop
Goal – Comprehensive Process
• Improve Processing of Sexual Assault Evidence Using the Laser Microdissection Microscope
1. Expedite Processing of Sexual Assault Evidence
2. Enhance Capabilities for Analysis of Difficult Samples
3. Provide New Capabilities of Analyzing Mixtures
Technology Transfer Workshop
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.
Technology Transfer Workshop
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
Technology Transfer Workshop
Differential Extraction
Cellular Extraction
Epithelial Digestion
Sperm Wash
Sperm Digestion
Purification
Quantification
Amplification
AP, P30Pellet (10%)
Sperm Epi
Pellet (10%)
E
S E
Technology Transfer Workshop
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)
Technology Transfer Workshop
Standard Extraction
Cellular Extraction
Epithelial Digestion
Sperm Wash
Sperm Digestion
Purification
Quantification
AmplificationDNA
PresumptiveTests?
Technology Transfer Workshop
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
Technology Transfer Workshop
Laser Microdissection Process
Cellular Extraction
Epithelial Digestion
Cell Dissection
Pre-amp Digestion
Purification
Quantification
Amplification
AP, P30Pellet (100%)
Male Female
Technology Transfer Workshop
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
Technology Transfer Workshop
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 Can combine both phases both phases into a single, into a single, comprehensive comprehensive process!process!
Technology Transfer Workshop
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
Technology Transfer Workshop
Microscopy – Identify Cells
1. Stain or DICEpitheliaSpermatozoa
2. Sperm paintSpermatozoa
3. Chromosome paintEpitheliaWBC
4. Std/Diff Extraction
DNADNA
DNADNA
DNADNA
Technology Transfer Workshop
Novel Process
1. Prepare slide
2. Examine using brightfield optics+ Proceed with LMD & PCR– Use fluorescent capabilities
3. Fluorescent optics (sperm identification)+ Proceed with LMD & PCR Excise entire smear or
4. Chromosome Paint
5. Extract DNA from slide
Technology Transfer Workshop
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
Technology Transfer Workshop
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. +ovn+ex
30 min.+ovn+1 hr.+ex
1 day
Technology Transfer Workshop
Goal – Comprehensive Process
• Improve Processing of Sexual Assault Evidence Using the Laser Microdissection Microscope
1. Expedite Processing of Sexual Assault Evidence
2. Enhance Capabilities for Analysis of Difficult Samples
3. Provide New Capabilities of Analyzing Mixtures
Technology Transfer Workshop
Acknowledgements
• Jennifer Valentine
• Kelli Raley
• North Louisiana Crime Lab
• LSUSHSC