Technology Transfer Workshop

Laser Microdissection

Advanced LMD Forensic Applications

Patrick Wojtkiewicz, Ph.D.

North Louisiana Crime Lab

(318) 227-2889

pwojtkie@NLCL.org

Technology Transfer Workshop

LMD Strengths

• Microscopic identification of cells increases sensitivity & minimizes handling

• Easily separates Sperm and Epithelial DNA

• Quantification is done by cell counting

• Fluorescent attachment provides new capabilitie

Technology Transfer Workshop

Fluorescent Capabilities

• Adds Capabilities for Analysis of All Types of Sexual Assault Evidence – Provide Improved Capabilities in Sperm

Identification

– Enable New Capabilities of Identifying Male and Female Diploid Cell Mixtures

• These procedures are in the development stage but have been successfully performed on actual case evidence (non-probative)!

Technology Transfer Workshop

Sperm Identification Problems• Few spermatozoa

– Searches are often labor intensive

– Analyst uncertainty about ID

– Case processing based upon sperm id

• Excessive quantity of epithelial cells– Spermatozoa buried under cells

– Combined with few spermatozoa

• Low quality microscope

• Poor staining

Technology Transfer Workshop

Sperm Labeling by Fluorescent Dyes

• Based upon antibodies conjugated to AlexaFluor.

• Antibodies are specific to human sperm components.

• Easier, faster, and confident searches

• Backwards compatible (not LMD)

• Adds extra step in analysis

• Requires high quality microscope with fluorescence capabilities

Technology Transfer Workshop

Sperm Paint

• Antibodies to:.– ESP (equatorial segment)– SP-10 (acrosomal

protein)*

– CaBYR-A (tail)

• Procedure– Add antibodies to smear– Overnight @ 4ºC– Wash with H2O– Examine

Technology Transfer Workshop

Fluorescent Sperm Identification

• Advantages– Simple procedure & Sequential processing of

samples– Antibodies should not adversely affect DNA

analysis– Samples can be examined at lower magnification

• Improved capability - Fast examination & confidence in negative results. Can be done on older slides.

• Previous slides are from casework-like material

Technology Transfer Workshop

Problems in Diploid Cell Mixtures

• Sample may not be identified as a mixture prior to analysis.

• Mixture may not be apparent when there is a preponderance of DNA from one of the donors.

• Interpretation issues

• No current way of separating diploid cell mixtures.

Technology Transfer Workshop

Chromosome Paint

• Fluorescent in situ hybridization (FISH) – X and Y chromosomes– commercial kit

• Interphase nuclei

• New capability of analyzing male & female epithelial cell mixtures

• Time & reproducibility Lymphocytes

Technology Transfer Workshop

Buccal Cells from Swab

Technology Transfer Workshop

Concern About Y STR Results

Technology Transfer Workshop

Extreme Cell Mixtures

Female: Male Ratio

# of loci in male profile

# of loci in female profile

99:1 7 12

95:5 9 13

75:25 15 15

50:50 13 11

25:75 13 12

5:95 9 12

1:99 10 2

Technology Transfer Workshop

Real Case Results

• Identification and dissection of spermatozoa well established

• Following results based upon diploid cell analysis

• 40% of cases had at least one sample with enough diploid cells to obtain a male profile– Vaginal swab

– Bitemark

– Fingernail scraping

Technology Transfer Workshop

Case #1 Fingernail ScrapingsItem/Locus Victim Fingernail

ScrapingsFingernail Scrapings

Vaginal Swab Suspect

Known 30 Female Cells 15 Male Cells 25 Male Cells (Non-sperm)

Known

D3S1358 14,15 14,15 15 15,16 15,16

vWA 16,17 16,17 18 16,18 16,18

FGA 23,24 23,24 ND 19,25 19,25

AMEL X X Y XY XY

D8S1179 13,14 13,14 16 13,(15),16,(17) 13,16

D21S11 29,31.2 29,31.2 ND 30,31 30,31

D18S51 14,17 14,17 ND 14.2,16 14.2,16

D5S818 8,12 8,12 14 12,14 12,14

D13S317 12 12 ND (8),11,12 11,12

D7S820 8,12 8 8 8,9 8,9

D16S539 10 10 ND 9 9

THO1 9 9 9 8,9 8,9

TPOX 8 8 ND 10,11 10,11

CSF1PO 11,12 11,12 11 11,12 11,12

D2S1338 18,23 18,23 ND 18,25 18,25

D19S433 12,14 12,14 14.2 14.2,16.2 14.2,16.2

Technology Transfer Workshop

Case #2 Vaginal SwabsVICTIM VAGINAL SWAB VAGINAL SWAB SUSPECT

KNOWN 20 MALE CELLS ACTUAL EVID KNOWN

D3S1358 14,16 17,18 14,16,17,18 17,18

vWA 14,17 17,18 14,17,18 17,18

FGA 19,21 ND 19,21,23,25 23,25

AMEL X XY XY XY

D8S1179 11,13 12,13 11,12,13 12,13

D21S11 30 29 29,30 29

D18S51 14,17 ND 13 13

D5S818 11,12 12 11,12 12

D13S317 8,12 11 8,11,12 11

D7S820 8,12 ND 8,12 8,12

D16S539 12 11,(12) 11,12 11,12

THO1 6,9.3 7,(9.3) 6,7,9.3 6,7

TPOX 8,11 11 8,11 8,11

CSF1PO 9,10 11 9,10,11 11

D2S1338 17,23 25 17,18,23,25 18,25

D19S433 14 13,14 13,14 13,14

Technology Transfer WorkshopCase #3 Bitemark Swabs

ITEM/LOCUS VICTIM VAGINAL SWAB BITEMARK SWAB BITEMARK SWAB SUSPECT

KNOWN 30 FEMALE CELLS 7 MALE CELLS ACTUAL KNOWN

D3S1358 14,15 14 15,16 14,15,16 15,16

vWA 16,17 16,17 16 16,17,18 16,18

FGA 23,24 24 19 ND 19,25

AMEL X X ND XY XY

D8S1179 13,14 13,14 ND 13,14,16 13,16

D21S11 29,31.2 ND ND 29,30,31 30,31

D18S51 14,17 ND ND ND 14.2,16

D5S818 8,12 8 ND 12,14 12,14

D13S317 12 ND 11 ND 11,12

D7S820 8,12 8,12 ND ND 8,9

D16S539 10 10 9 9 9

THO1 9 9 8,9 8,9 8,9

TPOX 8 8 ND 8,10,11 10,11

CSF1PO 11,12 ND 11,12 ND 11,12

D2S1338 18,23 ND 25 ND 18,25

D19S433 12,14 12,14 16.2 12,14,14.2,16.2 14.2,16.2

Technology Transfer Workshop

CODIS Searches

In these cases, and other non-

probative samples, the profiles were

searched locally against approximately

1500 samples. In each case the partial

profiles obtained by LMD only hit one

sample, the correct one.

Technology Transfer Workshop

Acknowledgements

• Jennifer Valentine

• Kelli Raley

• North Louisiana Crime Lab

• LSUSHSC