Laser Microdissection

1
Laser Microdissection

• Advanced LMD Forensic Applications
• Patrick Wojtkiewicz, Ph.D.
• North Louisiana Crime Lab
• [phone]
• pwojtkie_at_NLCL.org

2
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

3
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)!

4
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

5
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

6
Sperm Paint

• Antibodies to.
• ESP (equatorial segment)
• SP-10 (acrosomal protein)
• CaBYR-A (tail)
• Procedure
• Add antibodies to smear
• Overnight _at_ 4ºC
• Wash with H2O
• Examine

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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

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Validation Issues for Fluorescent Sperm
Identification

• Analysis is commonly done in labs and procedure
  is simple fluorescent microscopy component is
  new
• Two issues
• A new way of looking at sperm
• Confidence in specificity of identification
• LMD component is not required for training
• Purpose of procedure (LMD or ID only)
• Plenty of practice material available

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Validation Issues for Fluorescent Sperm
Identification

• Develop lab protocol for procedure and use (eg,
  all samples, confirmations, screening)
• Effects of time after intercourse on staining (?)
• Examine slides with defined ratios of sperm to
  epithelium (sensitivity). At what point can you
  feel confident that if sperm were present they
  would be seen.
• Internal validation ? Usage (1 2 months for ID
  only LMD component must be done first)

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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.

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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
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Buccal Cells from Swab
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Validation Concerns for X-Y Chromosome Paint

• Somewhere between novel and internal validation
  and needs greater time investment.
• Likely not to be a routine procedure
• Procedure development problems
• No forensic developed kit
• Reproducibility (routineness)
• Effect on downstream analyses
• Applicable to very limited types of samples

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Validation Issues for X-Y Chromosome Paint

• Start with LMD (very limited usefulness w/o LMD)
• Procedure development and standardization.
• Post stain analyses (using DNA)
• Chromosome identification
• Cell ratios (sensitivity)
• Application to evidence sample types
• Day to day reproducibility
• Better probes or better formulation?
• More intensive analyst training
• LCN training

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Concern About Y STR Results
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Extreme Cell Mixtures
Female Male Ratio of loci in male profile of loci in female profile
991 7 12
955 9 13
7525 15 15
5050 13 11
2575 13 12
595 9 12
199 10 2
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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

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Case 1 Fingernail Scrapings
Item/Locus Victim Fingernail Scrapings Fingernail 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
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Case 2 Vaginal Swabs
VICTIM 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
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Case 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
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CODIS Searches

• In these cases, and other non-probative samples
  not shown, the male profiles were searched
  locally against approximately 1500 samples. In
  each case the partial profiles obtained by LMD
  only hit one sample, the correct one.

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Acknowledgements

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