2. Technology Transfer Workshop Forensic LMD Research Studies at Rosalind Franklin University of Medicine and Science North Chicago, IL Christine T. Sanders geneskr@alumni.ucsd.edu

3. Technology Transfer Workshop 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

4. Technology Transfer Workshop Separation Methods Preferential Lysis (differential extraction) Flow Cytometry Microchip separation Membrane Filtration Magnetic Antibodies Y- chromosome (non-physical separation)  Laser Microdissection

5. Technology Transfer Workshop 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

6. Technology Transfer Workshop Technical Obstacles Optimizing LMD cutting parameters LMD microscope slides Environmental conditions of instrument “hanging chads” Static Collection buffer Etc…

7. Technology Transfer Workshop Histological Staining for LMD Not Stained E-Cells Sperm 63x 40x  Histological staining study considerations: –Visually discriminate sperm and epithelial cells –Effect on downstream analysis

8. Technology Transfer Workshop 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

9. Technology Transfer Workshop Acridine Orange Not Stained Christmas Tree Hematoxylin/ Eosin

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

11. Technology Transfer Workshop 62% 43%  Stained specimens exhibited RFU values significantly lower than unstained specimens (P < 0.01)

12. Technology Transfer Workshop 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 Part I: Histology Study Summary Results prompted second phase of histology study (Part II)

13. Technology Transfer Workshop 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

14. Technology Transfer Workshop RFU Differences: Stained vs. Unstained LMD Cells

15. Technology Transfer Workshop 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)

16. Technology Transfer Workshop PSA-FITC/PI stain on a PEN slide

17. Technology Transfer Workshop 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 DNA isolation study considerations: –Low manipulation, Small volume, Purity DNA Isolation Study

18. Technology Transfer Workshop Detection of Loci Using Three Isolation Methods

19. Technology Transfer Workshop Total PCR Product Detected *

20. Technology Transfer Workshop 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.

21. Technology Transfer Workshop 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

22. Technology Transfer Workshop Sample% Yield by real-time qPCR % Yield by STR RFUs 300 sperm (n=5)22.9 + 4.8 %Off scale 150 sperm (n=5)13.3 + 2.3 %Off std curve 80 sperm (n=5)16.3 + 5.4 %25.1 + 6.6% 40 sperm (n=5)18.5 + 3.8 %22.0 + 5.6% 20 sperm (n=5)12.2 + 4.7 %20.1 + 7.8% 10 sperm (n=5)14.8 + 0.8 %23.6 + 6.6% 5 sperm (n=4)17.8 + 2.9 %Off std curve AB Pos control 0.0313 -1ng 13.0 + 2.0 % (n=6) 27.5 + 4.7% (n=5) Yield of DNA Extraction (sperm)

23. Technology Transfer Workshop Sample% Yield by real-time qPCR 150 epithelial (n=5)37.5 + 2.8 % 80 epithelial (n=5)45.4 + 6.8 % 40 epithelial (n=5)37.5 + 4.2 % 20 epithelial (n=5)40.5 + 7.0 % 10 epithelial (n=5)32.9 + 5.3 % 5 epithelial (n=5)41.4 + 8.6 % 2 epithelial (n=5)40.7 + 11.0 % Yield of DNA Extraction (e-cells)

24. Technology Transfer Workshop 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.

25. Technology Transfer Workshop 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

26. Technology Transfer Workshop * * * * * ** * * * * ** * * * AmpFlST R Profiler Plus DNA mixture: sperm cells + female epithelial cells (*)

27. Technology Transfer Workshop 300 Sperm cells separated by LMD

28. Technology Transfer Workshop “150 sperm cells” from a mixture Standard PCR Extended PCR

29. Technology Transfer Workshop “75 sperm cells” from a mixture Standard PCR Extended PCR

30. Technology Transfer Workshop 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

31. Technology Transfer Workshop 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%

32. Technology Transfer Workshop 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

33. Technology Transfer Workshop 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)

34. Technology Transfer Workshop “80 sperm cells” at 34 PCR cycles

35. Technology Transfer Workshop “40 sperm cells” at 34 PCR cycles

36. Technology Transfer Workshop “20 sperm cells” at 34 cycles

37. Technology Transfer Workshop “10 Sperm Cells” at 34 PCR Cycles

38. Technology Transfer Workshop 80 40 20 10 5 LMD Collected Cells from a Mixture (34 cycles)

39. Technology Transfer Workshop Percent of Profiler Plus Profile Detected from LMD Collected Cells (34 cycles) N=5 * * *

40. Technology Transfer Workshop Percent of Profiler Plus Profile Detected from LMD Collected Cells (34 & 38 Cycles)

41. Technology Transfer Workshop 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. 16

42. Technology Transfer Workshop 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.

43. Technology Transfer Workshop Comparative Study AmpFlSTR Profiler Plus for 34 cycles

44. Technology Transfer Workshop LMD PL Plots of Sperm Fractions LMD vs. PL 1:5 Cell Mixture Ratio

45. Technology Transfer Workshop LMD PL Plots of Sperm Fractions LMD vs. PL 1:160 Cell Mixture Ratio

46. Technology Transfer Workshop LMD vs. PL : Detection of Male Donor Genotype

47. Technology Transfer Workshop LMD vs. PL : Female Carryover

48. Technology Transfer Workshop Comparison of PCR Product Quantity : LMD vs. PL of Sperm Fraction * * * *

49. Technology Transfer Workshop Preferential Lysis Method LMD Method Work Flow Chart: Comparing Methods

50. Technology Transfer Workshop 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

51. Technology Transfer Workshop 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

52. Technology Transfer Workshop

53. A Closer Look at Case B LocusPL - Sperm Fraction Male Exemplar 10 Sperm LMD (LCN) 30 Sperm *LMD (LCN) D315, 16, 1716, 1717 *** VWA15, 16, 1815, 181515, 18 FGA19, 22, 2419, 24 AMELX, Y Y D810, 13, 1410, 131310, 13 D2130, 30.2, 3130.2, 313130.2, 31 D1815, 16, 1715, 171715, 17 D58, 12, 138, 1213**8, 12 D1312, 14 1212, 14 D78, 9, 109, 109 *** Carryover from victim shown in red; Shared alleles underlined

54. Technology Transfer Workshop Case Studies Summary LMD’s performance –Case “A” - similar result to PL –Case “B” & “C” - improved results –Case “D” - worse results This study may not be the best case study comparison test of LMD to the PL method due to other variations in the analysis (i.e. LCN vs. STD PCR, and “mini-lysis”) Larger case study population required in a forensic lab setting to make final evaluation of LMD

55. Technology Transfer Workshop Research Team RFUMS –Christine T. Sanders –Daniel A. Peterson –Emily Reisenbigler LAPD –Nick Sanchez University of Central Florida –Jack Ballantyne Northern Illinois Regional Crime Lab –Kenneth Pfoser

56. Technology Transfer Workshop