1. 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

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

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

4. 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

5. 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

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

7. 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%

8. 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

10. 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

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

12. 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 , , , , , , X, X
Negative Sort , , , , , , X, X

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

14. 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 , (16), , , , , , X, Y
Negative Sort ,15,16, ,11, ,6,8,9, ,9, ,11,12, ,9,10,11, X, X

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

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

17. 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

18. 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