technology transfer workshop laser microdissection advanced lmd forensic applications patrick...
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Technology Transfer Workshop
Laser Microdissection
Advanced LMD Forensic Applications
Patrick Wojtkiewicz, Ph.D.
North Louisiana Crime Lab
(318) 227-2889
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 WorkshopValidation Issues for Fluorescent Sperm Identification
• Analysis is commonly done in labs and procedure is simple; fluorescent microscopy component is new
• Two issues1. A new way of looking at sperm2. Confidence in specificity of identification
• LMD component is not required for training
• Purpose of procedure (LMD or ID only)
• Plenty of practice material available
Technology Transfer WorkshopValidation 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)
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
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
Technology Transfer Workshop
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
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 Workshop
Case #3 Bitemark SwabsITEM/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 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.
Technology Transfer Workshop
Acknowledgements
• Jennifer Valentine
• Kelli Raley
• North Louisiana Crime Lab
• LSUSHSC