Forensic LMD Research Studies at Rosalind Franklin University of Medicine and Science North Chicago, IL

1. Forensic LMD Research Studies at Rosalind Franklin University of Medicine and ScienceNorth Chicago, IL Christine T. Sanders geneskr@alumni.ucsd.edu

2. How did this start? • 2003 - Sanders investigates the applicability of LMD for forensic use as a thesis topic • Jan 2004 - Sanders and Peterson at RFUMS write NIJ grant proposal to investigate LMD technology for separation of sperm from mixtures. • Feb 2004 - AAFS presentation of preliminary data • June 2004 - Awarded two year grant #2004-DN-BX-K215 • Several presentations made throughout the grant period • Published paper in July 2006, JFS • July/Aug. 2007 - NFSTC Technology Transfer Workshop • Second manuscript in progress

3. Separation Methods • Preferential Lysis (differential extraction) • Flow Cytometry • Microchip separation • Membrane Filtration • Magnetic Antibodies • Y- chromosome (non-physical separation) • Laser Microdissection

4. Studies for LMD Development • Histological staining study • DNA isolation study • Yield Evaluation qPCR • Mixture separation study • Low Copy Number study (LCN) • Comparative study • Case Study

5. Technical Obstacles • Optimizing LMD cutting parameters • LMD microscope slides • Environmental conditions of instrument • “hanging chads” • Static • Collection buffer • Etc…

6. Histological Staining for LMD Not Stained E-Cells Sperm 40x 63x • Histological staining study considerations: • Visually discriminate sperm and epithelial cells • Effect on downstream analysis

7. Histological Staining Part 1 • Stains tested: • Hematoxylin / Eosin (H&E) • Christmas Tree stain (nuclear fast red / picroindigocarmine) • Acridine Orange • Wright Stain (azure blue / eosin) • Methyl Green Evaluation: • Stains were evaluated for ability to ID sperm • LMD collected cells were isolated with Qiagen QIAamp extraction • STR/Profiler Plus analysis performed

8. Acridine Orange Not Stained Christmas Tree Hematoxylin/ Eosin

9. Microscopic ID scores of sperm & epithelial cells - - : cannot ID or highly challenging - : poor + / - : satisfactory + : good + + : excellent UNSTN = not stained. H&E = hematoxylin/eosin. CTS = nuclear fast red/picroindigocarmine. MG = methyl green. WRT = Wright's stain. AO = acridine orange.

10. 62% 43% • Stained specimens exhibited RFU values significantly lower than unstained specimens (P < 0.01)

11. Part I: Histology Study Summary • Methyl green, Wright’s stain not suitable for LMD • Cells stained with Acridine orange resulted in no amplified product • Christmas tree stain & Hematoxylin/Eosin: • Good stains for sperm ID. • Significantly lower RFU values than unstained control • However, genotyping could still be obtained with 300 sperm cells or 150 E-cells • H&E best choice Results prompted second phase of histology study (Part II)

12. Part II: Histology Study Stains Tested: • H&E Modified - shorter exposure times • Nuclear Fast Red - nuclear dye • SYBR 14/Propidium Iodine - fluorescent duel stain • PSA/PI - fluorescein-conjugated Pisum sativum agglutinin (FITC-PSA) & propidium iodide Evaluation: • Stains were evaluated for ability to ID sperm • LMD collected cells were isolated with Lyse-N-Go extraction • STR/Profiler Plus analysis performed

13. RFU Differences: Stained vs. Unstained LMD Cells

14. Part II: Histology Study Summary • SYBR 14/Propidium Iodine • No STR results could be obtained - not suitable for DNA analysis. PI tests indicate SYBR 14 as the problem agent. • H&E Modified and Nuclear Fast Red • Good stains for sperm ID. • No significant difference in RFUs obtained from that of unstained controls • PSA-FITC/PI • Good duel stain but staining consistency difficult to control between samples. STR genotypes can be obtained from PSA-FITC stained cells. (more optimization required)

15. PSA-FITC/PI stain on a PEN slide

16. DNA Isolation Study DNA isolation study considerations: • Low manipulation, Small volume, Purity • Lyse-N-Go : commercial lysis buffer (low manipulation) Series of heating and cooling incubations 8C-97C • Microlysis: commercial lysis buffer (low manipulation) Series of 65C and 96C incubations • Qiagen QIAamp: commercial kit DNA binding membrane columns • Chelex DTT (dithiothreitol) added to all three protocols

17. Detection of Loci Using Three Isolation Methods

18. Total PCR Product Detected *

19. DNA Isolation Study Summary • MicroLYSIS method was not suitable for LMD with forensic STR analysis • QIAamp performed best for collection of stained epithelial cells • Both Lyse-N-Go and QIAamp performed well for isolating DNA from LMD collected sperm cells • Lyse-N-Go provides a low manipulation method and inexpensive • QIAamp provides a cleaner DNA extract but higher manipulation required and higher cost.

20. DNA Yield Study • LMD process may provide an estimate of DNA quantity. • Efficiency of DNA extraction process must be considered • Two methods of DNA quantification performed from LMD collected cells extracted with the Qiagen QIAamp® protocol • Real-Time qPCR • Relative amounts of STR PCR product to a standard curve

21. Yield of DNA Extraction (sperm)

22. Yield of DNA Extraction (e-cells)

23. DNA Yield Study: Summary • Extraction efficiency surprisingly low but consistent • Cells can easily be counted during LMD collection, starting DNA material calculated, and then final DNA quantity can be estimated factoring in extraction efficiency prior to PCR • Laborious and sample consuming DNA quantification step can be eliminated when using LMD.

24. Mixture Study • Mixtures were prepared with the equivalent of half a female oral cotton swab + 1µl of semen. • Collection amounts of 75, 150 and 300 sperm cells were recovered from the mixtures. • PCR amplification was performed using standard 28 cycles • Extended PCR was performed by amplifying half of the PCR product an additional 6 cycles

25. * * * * DNA mixture: sperm cells + female epithelial cells (*) * * * * * * * * * * * * AmpFlSTR Profiler Plus

26. 300 Sperm cells separated by LMD

27. “150 sperm cells” from a mixture Extended PCR Standard PCR

28. “75 sperm cells” from a mixture Standard PCR Extended PCR

29. Detection of Profiler Plus Alleles • Under standard PCR conditions (28 cycles) • “75 sperm” 71+12% alleles • “150 sperm” 96+3% alleles • “300 sperm” 100% alleles • Under “extended cycles” PCR (34 cycles) • “75 sperm” 100% alleles • “150 sperm” 100% alleles

30. Allelic Balance • Heterozygote peak height ratio: Height of the lower peak divided by the height of the higher peak, expressed as a percentage • Under standard PCR conditions (28 cycles) • “75 sperm” 79.3+2.9% • “150 sperm” 81.8+4.3% • “300 sperm” 82.0+1.4% • Under “extended cycles” PCR (34 cycles) • “75 sperm” 67.0+13.2% • “150 sperm” 85.2+6.7%

31. Summary: Mixture Study • LMD separation of sperm cells from a epithelial cell mixture results in a single semen donor genotype • The lower limit of detection using ABI user guide’s PCR protocol (standard conditions) is ~75-150 sperm cells. • Extended cycle analysis can extend the lower limit of detection

32. LCN Study of Mixtures • Prepared mixtures of human female oral swabs (epithelial cells) and male semen (sperm cells). Equivalent to 1 swab + 1µl of semen • Collected 80, 40, 20, 10 and 5 sperm cells by laser microdissection • Profiler Plus PCR amplification was performed using 34 & 38 cycles. (+6 & +10 cycles)

33. “80 sperm cells” at 34 PCR cycles

34. “40 sperm cells” at 34 PCR cycles

35. “20 sperm cells” at 34 cycles

36. “10 Sperm Cells” at 34 PCR Cycles

37. LMD Collected Cells from a Mixture (34 cycles) 80 40 20 10 5

38. Percent of Profiler Plus Profile Detected from LMD Collected Cells (34 cycles) * * * N=5 N=5 N=5 N=5 N=5

39. Percent of Profiler Plus Profile Detected from LMD Collected Cells (34 & 38 Cycles)

40. 16 Epithelial Cell Carryover : Outlier? STR plots from LMD collected sperm cells with epithelial cell DNA carryover. Female donor alleles (indicated by asterisks) were detected in the 40 and 80 sperm cell collections from one of the slide smears in this study.

41. LCN Mixture Study Summary • Minute numbers of sperm cells can be separated and recovered by LMD from epithelial cell mixtures. • STR genotyping can be obtained with as little as 5 sperm cells captured by LMD using increased PCR cycles.

42. Comparative Study AmpFlSTR Profiler Plus for 34 cycles

43. Plots of Sperm Fractions LMD vs. PL 1:5 Cell Mixture Ratio LMD PL

44. Plots of Sperm Fractions LMD vs. PL 1:160 Cell Mixture Ratio PL LMD

45. LMD vs. PL : Detection of Male Donor Genotype

46. LMD vs. PL : Female Carryover

47. Comparison of PCR Product Quantity : LMD vs. PL of Sperm Fraction * * * *

48. Work Flow Chart: Comparing Methods Preferential Lysis Method LMD Method

49. Comparative Study Summary • LMD provides improved separation and detection of sperm from epithelial cell DNA over the preferential lysis method at higher e-cell to sperm-cell ratios. • LMD Does not require a DNA quantification step • Single sample processing more rapid using LMD

50. Case Studies using Low Copy # • 4 case studies • Non-probative or adjudicated cases • Originating crime lab performed organic differential extraction and attempted typing of 13 core loci • Case “A” - public masturbation (tissue paper recovered from the scene) • Cases “B”, “C” & “D” - sexual assaults (vaginal swabs obtained) • Case “A” - ample sperm available • 30 sperm collected by LMD followed by LCN analysis • Case “B”, “C” & “D” - Difficulty locating sperm and limited sample available. • modified the LMD protocol to include a “mini-lysis” • 18-30 sperm collected by LMD followed by LCN analysis