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

pwojtkie@NLCL.org

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

• 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

• 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

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

Technology Transfer Workshop

Acknowledgements

• Jennifer Valentine

• Kelli Raley

• North Louisiana Crime Lab

• LSUSHSC