1. Laser Microdissection
Advanced LMD Forensic Applications
Patrick Wojtkiewicz, Ph.D.
North Louisiana Crime Lab
(318)

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

7. 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

8. 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

9. 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)

10. 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.

11. 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

12. Buccal Cells from Swab

13. 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

14. 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

15. Concern About Y STR Results

16. # of loci in male profile # of loci in female profile
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
50:50 11
25:75
5:95
1:99 10 2

17. 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

18. Case #1 Fingernail Scrapings
Item/Locus
Victim
Fingernail Scrapings
Vaginal Swab
Suspect
Known
30 Female Cells
15 Male Cells
25 Male Cells
(Non-sperm)
D3S1358
14,15 15
15,16
vWA
16,17 18
16,18
FGA
23,24 ND
19,25
AMEL X Y XY
D8S1179
13,14 16
13,(15),16,(17)
13,16
D21S11
29,31.2
30,31
D18S51
14,17
14.2,16
D5S818
8,12 14
12,14
D13S317 12
(8),11,12
11,12
D7S820 8
8,9
D16S539 10 9
THO1
TPOX
10,11
CSF1PO 11
D2S1338
18,23
18,25
D19S433
14.2
14.2,16.2

19. Case #2 Vaginal Swabs VICTIM VAGINAL SWAB SUSPECT KNOWN 20 MALE CELLS
ACTUAL EVID
D3S1358
14,16
17,18
14,16,17,18
vWA
14,17
14,17,18
FGA
19,21 ND
19,21,23,25
23,25
AMEL X XY
D8S1179
11,13
12,13
11,12,13
D21S11 30 29
29,30
D18S51 13
D5S818
11,12 12
D13S317
8,12 11
8,11,12
D7S820
D16S539
11,(12)
THO1
6,9.3
7,(9.3)
6,7,9.3
6,7
TPOX
8,11
CSF1PO
9,10
9,10,11
D2S1338
17,23 25
17,18,23,25
18,25
D19S433 14
13,14

20. Case #3 Bitemark Swabs ITEM/LOCUS VICTIM VAGINAL SWAB BITEMARK SWAB
SUSPECT
KNOWN
30 FEMALE CELLS
7 MALE CELLS
ACTUAL
D3S1358
14,15 14
15,16
14,15,16
vWA
16,17 16
16,17,18
16,18
FGA
23,24 24 19 ND
19,25
AMEL X XY
D8S1179
13,14
13,14,16
13,16
D21S11
29,31.2
29,30,31
30,31
D18S51
14,17
14.2,16
D5S818
8,12 8
12,14
D13S317 12 11
11,12
D7S820
8,9
D16S539 10 9
THO1
TPOX
8,10,11
10,11
CSF1PO
D2S1338
18,23 25
18,25
D19S433
16.2
12,14,14.2,16.2
14.2,16.2

21. 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.

22. Acknowledgements Jennifer Valentine Kelli Raley
North Louisiana Crime Lab
LSUSHSC