Department of Forensic Science, Virginia Commonwealth University Separation of Complex Biological Mixtures Using Human Leukocyte Antigen Probes and Fluorescence Activated Cell Sorting Lee Dean, Ye Jin Kwon, Sara Seashols, Jamie Sturgill, Tracey Dawson Cruz, Christopher J. Ehrhardt* Department of Forensic Science, Virginia Commonwealth University

Introduction Forensic Mixture Analysis How can we analyze biological evidence with two or more contributors? -Differential Extraction -Acoustic Separation -Laser Capture Microdissection

Introduction Mixtures containing one cell type Blood or Epithelial cell mixtures Each cell has the same size, physical characteristics A B White Blood Cells

Introduction Biochemistry of cells varies widely between individuals DNA, proteomics, immunochemistry of cell surface Use intrinsic chemical differences between contributors to physically separate cells from the mixture prior to profiling

Methods Molecular target for differentiating cells Human Leukocyte Antigen (HLA) Major Histocompatibility Complex Expressed on all nucleated cells Highly polymorphic Allelic frequencies amenable to forensic differentiation

Fluorescent Microscope Epithelial Blood BF Anti-MHC I Fluorescent Microscope Anti MHC Class I (FITC) 40X Magnification

Asian or Pacific Islander Percentage chance of allele expressed in an individual Top 30 expressed alleles Allele White Black Hispanic Asian or Pacific Islander A*02:01 45.6% C*04:01 29.0% 37.1% A*11:01 38.4% C*07:01 27.7% 25.4% A*24:02 33.7% A*01:01 27.4% C*06:02 23.0% 24.9% C*07:02 33.3% A*03:01 23.8% 22.3% 24.2% C*01:02 21.5% A*23:01 20.7% 20.8% A*33:03 23.3% 21.2% C*02:02 19.0% C*03:04 14.4% C*08:01 21.6% B*44:02 20.2% 18.7% 14.3% 19.9% B*07:02 18.1% 13.2% B*08:01 B*53:01 B*35:01 12.8% B*40:01 15.2% C*05:01 17.2% 15.8% 12.3% 14.0% 16.8% C*16:01 15.7% 11.9% B*58:01 13.3% B*15:03 13.9% 11.4% B*46:01 12.7% 15.3% 13.5% 11.0% B*51:01 12.4%

Methods Sample: labelled cells HLA alleles detected on cell with fluorescently labelled antibodies Physically separated with Fluorescence Activated Cell Sorting (FACS) High throughput, sensitive technique Optical properties (fluor, FSC, SSC) Laser Source + - + - - - - - - - - - Negative Sort Positive Sort

Methods 1st Experiment: Two-person Whole Blood Mixture White Blood Cell Mixture WBC A B HLA typing for A02 allele ACK lysis & Purification HLA A02 Antibody-FITC 1:1 Whole Blood A+B Fluorescence Activated Cell Sorting

Unstained Mixture (Negative Control) Results Unstained Mixture (Negative Control) Stained Mixture Contributor B Reference HLA-A02 - Contributor A Reference HLA-A02 +

Results Two-person Whole Blood Mixture D3S13 D16S TH01 TPOX CSF1 D7S8 Amel Mixture 15, 17 10,11,12 6,7,8,9 8, 11 7,11,12,14 8,10,13 X, Y Contrib A (HLA+) 11,12 7, 8 7, 11 8, 10 Contrib B (HLA-) 17, 17 10, 11 6, 9 12, 14 8, 13 X, X Positive Sort 15, 17 11,12 7, 8 8, 11 7, 11 8, 10 X, X Negative Sort 17, 17 10, 11 6, 9 8, 11 12, 14 8, 13 X, X

Results Two-person Whole Blood Mixture CSF1PO TH01 Positive Sort Negative Sort Positive Sort Negative Sort

Results 2nd Experiment: Four-person Whole Blood Mixture D3S13 D16S TH01 TPOX CSF1 D7S8 Amel Mixture 14,15,16,17 10,11,12 5,6,7,8,9,9.3 8,9,11 7,10,11,12,14 8,9,10,11,13 X, Y Target Indiv (HLA+) 15, 17 11,12 7, 8 8,11 7, 11 8,10 Positive Sort 15, (16), 17 11,12 7, 8 8,11 7, 11 8, 10 X, Y Negative Sort 14,15,16,17 10,11,12 5,6,8,9,9.3 8,9,11 10,11,12,14 8,9,10,11,13 X, X

Results Four-person blood mixture: Minor contributions from non-target individuals in the positive sort

Results Four-person blood mixture: Composition of non-target population TH01 TH01 Unstained Mixture Negative Sort; HLA A02-

Conclusions & Future Directions Fluorescently-labelled HLA antibody probes chemically differentiate individual contributors in a blood mixture When coupled to FACS, individual contributors can be physically separated from the mixture prior to DNA profiling ‘Multidimensional Sorts’- series of probes to isolate different contributors in successive fashion Epithelial cell mixtures

Acknowledgements VCU Forensic Science VCU Medical Campus Cristina Stanciu Elizabeth Lapatovich Alicia Zimmerman Donald Jessup Ashley Coleman Ahmad Fakourhy Kevin Martin Jordan Cox Hope Parker Lucy Carmarena Whitney Simpson Matthew Goldstein VCU Medical Campus Daniel Conrad Julie Farnsworth