Body Fluid ID by Mass Spectrometry
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1 Body Fluid ID by Mass Spectrometry Kevin Legg, PhD & Phillip Danielson, PhD This research was supported in part by DNA R&D Grants 2006-DN-BX-K001, 2009-DN-BX-K165, 2011-CD-BX-0205, and 2012-DN-BX-K035 awarded by the National Institute of Justice, Office of Justice Programs, US Department of Justice. Points of view in this presentation are those of the authors and do not necessarily represent the official position or policies of the US Department of Justice.
2 Forensic Body Fluid Identification Challenges Faced By Serologists Presumptive (Antibody, Chemical Rxn, Enzyme Activity) and/or time consuming assays (Microscopy) Chemical Rxn Kastle-Meyer Enzyme Activity Acid Phosphatase Antibody Test Kits C T S A positive result is not in itself evidence of the presence of human blood but it indicates the probability of its presence
3 Next Gen Serology: Mass Spectrometry Based Proteomics Project Objectives: 1. Identify Protein Biomarkers for body fluid identification 2. Develop a rapid multi-fluid/multiplex mass spectrometry assay Advantages: Confirmatory detection by mass spectrometry Universal sample preparation & multi-fluid multiplexing Proteomics Method Development: Discovery Phase Identify Candidate Biomarkers Validation Phase Evaluate Biomarker Specificity Assess Robustness, Reliability and Performance Limits
4 Project History Biomarker Discovery 2006-DN-BX-K001 Identify candidate biomarkers Biomarker Verification 2009-DN-BX-K165 Verify biomarker specificity Method Validation 2012-DN-BX-K035 Developmental validation Pre-Doctoral Fellowship 2011-CD-BX-0205 Pilot study using validation platform
5 Technology Implementation Sexual Assault Kit Screening Screen and prioritize sexual assault samples from rape kits Human Blood Confirmation Collaboration with Agilent Technologies and the Brasilia Civil police
6 Backlog Sexual Assault Screening
7 Sexual Assault Screening: Improving the Workflow Batch processing MS analysis Automated reporting Mass spectrometry advantages: Confirmatory stain characterization Batch analyses robotics compatible Automated data processing and reporting
8 Sexual Assault Screening: 15 minutes/sample SCIEX QTRAP 6500 Seminal Fluid Targets Acid Phosphatase PSA Semenogelin Saliva Targets Α-amylase Submaxillary protein 3B Statherin MRM mode
9 Sexual Assault Screening: ~300 Sample Tested QTRAP 6500 Casework Lubricant contamination Rectal/Buccal Surfaces (e.g. Condoms) Mixtures (e.g. Blood, Saliva) Specificity Analyze non-target fluids Buccal swabs Blood Male/Female Urine Nasal QTRAP 6500 & Antibody Tests Sensitivity Semen dilutions on swabs 1:2 to 1:16,389 Post Coital Post-intercourse vaginal swabs. Interval from 12 hour to 8 days post coitus.
10 Sexual Assault Screening: Casework & Specificity Results Casework Lubricant contamination Rectal/Buccal Surfaces (e.g. condoms, sheets) Mixtures (e.g. blood, saliva) Specificity Analyze non-target fluids Buccal swabs Blood Male/Female Urine Nasal All target proteins successfully identified in each sample. No targeted proteins detected in any non semen/saliva sample. * Α-amylase is present in buccal/nasal swabs. * Seminal fluid markers will appear in male urine
11 Sexual Assault Screening: Sensitivity Results Semen diluted & added to swab Antibody tests assayed P30/Abacus RSID Semen 1:4096 More Sensitive & More Reliable VS. Semenogelin Peptide GSISIQTEEQIHGK 1: indicates semen ID
12 Sexual Assault Screening: Post Coital Results Seminal fluid markers (Semenogelin) confirmed up to 8 days post-intercourse Semenogelin Peptide DVSQSSISFQIEK Semenogelin Peptide GSISIQTEEQIHGK
13 Backlog Developing a Blood ID Assay With the Brasilia Police
14 Developing a Blood ID Assay With the Brasilia Police Instituto De Criminalistica Laboratory serves the Brasilia Federal District Quadrupole Time-of-Flight method Chemical: Kastle Meyer Antibody: OBTI & FOB Requirements Human specific Process ~100 samples/week
15 Developing a Blood ID Assay With the Brasilia Police Developed, trained, & tested (actual case samples) in 1 week 15 Minute/sample Emphasis on high abundance targets More robust for ID in compromised or trace samples 5 blood protein biomarkers selected Protein Target Accession Number Protein Function Alpha 1 Antitrypsin P01009 Protease Inhibitor. Cellular Protection. Apolipoprotein A1 P02647 Component of high-density lipoprotein (HDL). Lipid transportation. Complement C3 P01024 Innate immunity. Complement system activation. Hemoglobin Beta P68871 Oxygen transportation. Hemopexin P02790 Heme scavenger.
16 Species Specificity BLAST search the Swissprot database for all proteins containing Hemoglobin β peptide GTFATLSELHCDK Human Bonobo Chimp Hemoglobin β peptide is not human specific.
17 Species Specificity BLAST search the Swissprot database for all proteins containing peptide Complement C3 peptide AAVYHHFISDGVR Human Complement C3 peptide is human specific.
18 Casework Sample Analyses 30 suspected bloodstains selected from casework Most of them related to homicides or attempted murders Presumptive tests results were compared to Protein-based Human Blood ID by Mass Spectrometry SOME OF THE SAMPLES AFTER STAIN EXTRACTION FROM SWAB OR GAUZE
19 Case Sample Results Presumptive Confirmatory
20 Details: Sample 18 & 23 Attempted Murder
21 Details: Sample 18 & 23 Attempted Murder S18: Sample collected from the floor S23: blood splattered plastic bottle
22 Details: Sample 18 & 23 Attempted Murder S18: Sample collected from the floor S18 S18 Presumptive Blood Tests EVIDENCE KASTLE-MEYER OBTI FOB S18 NEG NEG NEG S23 POS POS POS Mass Spectrometry PEPTIDES FOUND RELATED TO Hemo beta Apo A-1 A1AT Hemopexin POS NEG POS POS POS POS POS NEG
23 Details: Sample 18 Attempted Murder Based only on presumptive test results, sample 18 would have not been sent to DNA analysis Sample 18 was sent to DNA analysis but did not yield a DNA profile Hemoglobin, A1AT, Hemopexin identified via mass spec. Proteins likely remain viable longer than DNA Post coital serology ID may yield results when STR/YSTR testing fails
24 Summary MS method outperformed Antibody-based kits for both seminal fluid and blood detection. Sensitivity Specificity Casework Post Coital samples With added benefits of: Reliable detection and confirmatory stain ID Multiplexing and multi-fluid analysis Batch processing and Automation potential 96-well plate formats & robotics ready
25 Acknowledgements NMS Labs/Center for Forensic Sci. Dr. Christian Westring Dr. Barry Logan Heather McKiernan University of Denver Danielson Laboratory Instituto De Criminalistica (Brasilia, Brazil) Luciano Arantes Eduardo Ramalho Rodrigo Santos Agilent Technologies Andre Santos Tom Gluodenis National Institute of Justice Minh Nguyen and Chad Ernst
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