Sexual Assault Kit Screening Using the QIAcube HT & Investigator Quantiplex HYres!

Transcription

1 Sexual Assault Kit Screening Using the QIAcube HT & Investigator Quantiplex HYres! Joshua Stewart, MSFS! QIAGEN North American Users Meeting! Houston, TX! July 30, 2014!

2 QIAGEN would like to thank our speaker, Joshua Stewart, MSFS, for his presentation. Disclaimer: QIAGEN is not affiliated with the Marshall University Forensic Science Center. The views expressed herein are those of the speaker, and do not necessarily express the views of QIAGEN. For up-to-date licensing information and product-specific disclaimers for QIAGEN products, see the respective QIAGEN kit handbook or user manual. QIAGEN kit handbooks and user manuals are available at or can be requested from QIAGEN Technical Services or your local distributor.

3 Disclaimer This project was supported by Award No IJ-CX-K11 awarded by the National Institute of Justice, Office of Justice Programs, U.S. Department of Justice. The opinions, findings, and conclusions or recommendations expressed in this publication are those of the author(s) and do not necessarily reflect the views of the Department of Justice.!

4 Sexual Assault Evidence Vaginal swabs! Anal swabs! Oral swabs! Dried secretions! Head hair! Pubic hair! Debris! Fingernail clippings! Fingernail scrapings! Underwear! Other clothing! Vaginal smears! Anal smears! Oral smears!

5 Screening Evidence! Deciding which evidence will be tested! Filter out items not likely to yield useful results! Tools! Serology! Medical and police reports! Lab policies!

6 Traditional Serology! Intimate sample! Suspected semen! AP & P30 POS or INC NEG Microscopy (X-mas stain) P30 & Micro NEG Hospital slides included? Yes P30 or Micro POS or INC No Report as negative case NEG POS Screen is positive: begin DNA testing

7 Cons of serology as screening tool! Experience is a factor! Occasionally subjective interpretation! Low-tech! Difficult to automate! False negative potential! Time commitment to microscopic search for sperm!

8 Screen for Male DNA ( Y-screen )! PCR-based method! Consistent interpretation! Can be automated! Process:! Sample portion of evidence! Extract DNA (include DTT)! qpcr for simultaneous autosomal and male DNA quantification! Screen samples based on presence/absence of male DNA! Autosomal-to-male ratio can be useful!

9 Y-screen Workflow Option 1! Evidence item! Cut small portion! Cut larger portion! Y-screen! Negative! Positive! Stop! Begin DNA testing!

10 Y-screen Workflow Option 2! Evidence item! Cut large portion! 1X PBS pre-treatment! Remove ~10% of cellular suspension! Remove ~90% of cellular suspension! Y-screen! Negative! Positive! Stop! Begin DNA testing!

11 Automation Options! QIAcube HT! Nucleic acid purification! 96-well format! Medium to high throughput! Silica membrane! Vacuum manifold!

12 Automation Options! Extraction Consumables! Digestion: S Block! Purification: QIAquick 96!

13 Automation Options! QIAgility! Automated pipetting! Quantification setup*! Prepare standards! Normalization! Amplification setup! CE setup! *Use in Y-screen process!

14 Y Quantification! Investigator Quantiplex HYres! Quantitative PCR! Simultaneous autosomal and male quant! Multi-copy targets! Highly conserved copy number! Multiple options for detection! Rotor-Gene Q! 7500 Real-Time PCR System! 1 hour program!

15 Validation in Progress (Workflow Option 2)! Pre-treatment! 400µL 1X PBS to cutting; agitate! Remove 40µL (10%) for Y-screen! Digestion! Buffer ATL, Pro K, 1M DTT, 56 C for 90 min.! Buffer AL, 72 C for 10 min.! Purification! QIAcube HT! QIAquick 96 plates! QIAGEN wash buffers! Heated elution buffer! qpcr setup! QIAgility! Investigator Quantiplex Hyres! 1 replicate of quantification standards! qpcr detection! 7500 Real-Time PCR System!

16 PBS Pre-Treatment Optimization! Created mock evidence swab; placed swab head into tube! Add 400μL 1X PBS! Tested variable factors:! Incubation time! 1, 2, 4 and ~18 hours! Incubation temperature! RT, 37 C, 56 C! Agitation method! Orbital shaker at 900RPM! Nutating mixer!

17 PBS Pre-Treatment Optimization! Variable Incubation Times Variable Incubation Temperatures Male [DNA] (ng/ul) hour 2 hours 4 hours Overnight Male [DNA] (ng/ul) Room Temp 37C 56C Male [DNA] (ng/ul) Variable Agitation Conditions Chosen conditions:! Room temperature! Overnight (15-18 hrs.)! Nutating mixer! Nutator Thermal Mixer

18 Precision! Assessed precision of HYres setup on the QIAgility and qpcr on the 7500! Previously-extracted DNA sample quantified 24 times!! Human [DNA]! Male [DNA]! Average! ! ! St Dev! ! ! % CV! 5%! 6%! Min! ! ! Max! ! ! Range! ! !

19 Precision/Sensitivity! Assessed precision of HYres/QIAgility/7500 at decreasing concentrations.! 3X dilution series! Each dilution quantified 4 times.! Dilution! Average male [DNA]! St Dev! % CV! 1! ! ! 3%! 2! ! ! 6%! 3! ! ! 3%! 4! ! ! 4%! 5! ! ! 8%! 6! ! ! 19%! 7! ! ! 45%! 8! ! ! 45%! 9! ! ! 96%!

20 Precision! Assessed precision of entire Y-screen system! 44 replicates of 1:10 semen dilution plated in checkerboard pattern! First Run! Average male ! [DNA]! St Dev! ! % CV! 26%! Minimum! ! Maximum! ! Range! ! Second Run! Average male ! [DNA]! St Dev! ! % CV! 20%! Minimum! ! Maximum! ! Range! !

21 Sensitivity Comparison! Compared sensitivity of Y-screen to traditional semen serology.! 3X dilution series of semen mixed with female saliva; spotted 45μL onto 2 swabs; air dried! Performed AP, p30, Y-screen, differential extraction & STR amp!

22 Sensitivity Comparison! Dilution! (3X series)! Traditional! Serology! Y-screen! (Threshold of 0)! Male Profile Obtained?! A! POS! POS! Yes (full)! B! POS! POS! Yes (full)! C! POS! POS! Yes (full)! D! POS! POS! Yes (full)! E! POS! POS! Yes (full)! F! POS! POS! Yes (1 replicate full, 1 replicate partial)! G! NEG! NEG! Yes (partial)! H! NEG! NEG! No! Yellow: at least 1 replicate = 0!

23 Contamination Assessment! 2 plates in checkerboard pattern! 44 blanks, 44 positive samples (1:10 semen dilution) per plate! 16/88 negatives quantified >0ng/μL! Quantification negative control also >0ng/μL! Plate 1! ! *! *! *! ! ! ! Quant neg = ! Plate 2! ! *! *! *! *! ! ! ! ! Quant neg = !

24 Cost analysis per sample*! Traditional Semen Serology! AP! $0.19! ABAcard p30! $5.28! Xmas microscopy! $1.10! TOTAL! $6.57! Y-screen! QIAGEN-assembled kit will soon be available! Said to include reagents and consumables for whole process (digestion qpcr)! PROJECTED cost per sample is $3.80! *Reagents and consumables only!

25 Current Status! HYres/QIAgility/7500 has good precision down to 0.008ng/uL! Work with QIAGEN to improve sensitivity, precision of extraction! Continue to monitor contamination; identify step(s)!

26 Y-screen: Future Questions to Address! How much to sample?! E.g. if 4 vaginal swabs submitted:! Cut and screen all 4 separately?! Combine cuttings and screen together?! What is being retained?! Differential or non-differential?! Use serology to determine?! Use medical report to determine?! Make a reasonable assumption?! Contingency plan for male victim and/or female assailant cases.!

27 Y-screen: Future Questions to Address! Can a threshold be set?! How should the report be phrased?! Male DNA was indicated! Male DNA was identified! Do replicate quantifications need to be performed on low samples?! Can autosomal-to-male ratio be used to send a sample to Y-STR?!

28 Acknowledgements! QIAGEN! Mark Guilliano! Mary Jones Dukes! Danielle Kobi! Colleagues at MUFSC DNA Lab!

29 QUESTIONS?!! Contact Information:! Joshua Stewart, MSFS! Forensic DNA Analyst III! DNA Training Officer! MUFSC! (304) ! forensics.marshall.edu!