Interesting Results with RSID-Saliva Negative Cases new pp version

Let’s Get Naked?
Mignon Dunbar
San Francisco Police Department • Forensic Services Division
October 2014

Overview
• Background
• About RSID™ -Saliva test
• Case examples
• Conclusions
• Survey
• Survey conclusions
• Suggestions

Why is this important?
• Analysts may rely on presumptive biological
screening results to determine the next step in
analysis
– Presumptive saliva positive => organic extraction
– Presumptive saliva negative => no further processing
• Only using presumptive screening results to
determine the next step in the analysis may
prematurely halt testing
– Presumptive saliva testing

RSID™-Saliva
(Rapid Stain IDentification)
Independent Forensics*
•Presumptive test for saliva
•Designed to detect a-
amylase, enzyme found in
human saliva
•Antigen-antibody reaction
•Sensitivity is < 1µL of
human saliva
•No correlation between
band intensity and amount
of amylase present
Test line
Control
line
*Old, J. et al. Developmental Validation of RSID™-Saliva: A
Lateral Flow Immunochromatographic Strip Test for the Forensic
Detection of Saliva. Journal of Forensic Science, July 2009, Vol.
54, No. 4

Case #1
• Victim reported being sexually assaulted by two men from 1/24-1/26
• Suspect 1
– Bit victim on right and left sides of neck
– Oral copulation of assailant by victim – ejaculation on victim’s face
– Vaginal penetration by penis (condom used)
• Suspect 2
– Oral copulation of assailant by victim – ejaculation occurred in victim’s mouth
• Victim had LOC and LOM
• Victim showered after assault, and washed face with a wash cloth
• Victim had consensual sex with boyfriend after assault (on 1/26)
• At RTC on 1/26, nurse observed bite marks and bruising on right and left side of
neck
• Oral swabs and bite mark swabs collected

Case #1
• Screening results:
– Oral swabs
• No sperm observed
• Nucleated and non-nucleated cells observed
– Bite mark on left side of neck:
• Presumptive saliva test = Positive
• NC = 2-10 cells observed
• NNC = 2-10 cells observed
– Bite mark on right side of neck:
• Presumptive saliva test = Negative
• NC = 0 cells observed

Case #1: How to Proceed?
Left Neck Bite Mark Swabs
• Positive presumptive saliva
test and bite mark observed
• Proceeded to DNA
• Approx 27ng of DNA
detected
• Mixture of at least 2:
– Victim and boyfriend included
Right Neck Bite Mark Swabs
• Negative presumptive saliva
test and bite mark observed
• Proceeded to DNA
• Approx 3ng of DNA
detected
• Mixture of at least 2:
– Victim and boyfriend included

Case #1: Thoughts
• DNA foreign to victim detected in sample that
tested negative using a presumptive saliva test
• Potential to miss probative information if not
proceeding to DNA after negative presumptive
saliva results
• SART nurse observations/notes and victim’s
reporting assisted in determining how to proceed

Case #2
• Victim reported being sexually assaulted by 2 suspects on 6/5
• Suspect 1
– Vaginal penetration by finger
– Unsure if object or penis was used for vaginal penetration
• Suspect 2
– Licked the right side of the victim’s neck
– Vaginal penetration by penis
– Anal penetration by penis
• No LOC or LOM
• Victim did not shower
• At RTC on 6/5, nurse collected vaginal swabs, cervical swabs, rectal swabs, anal
swabs, and right neck swabs (based on history)
• Victim had consensual sex on 6/3

Case #2
• Screening results:
– Vaginal swabs
• Sperm = 2-10 cells
observed
• Nucleated and non-
nucleated cells observed
– Cervical swabs
• Sperm = 2-10 cells
observed
– Rectal swabs
– Anal swabs
– Right neck swabs
• Presumptive saliva test =
Negative
• NNC = cells observed in
many fields

• Vaginal and cervical swabs were extracted
– Mixture of at least 2
– Unknown Male #1 detected
• Neck swabs extracted based on results from
previous case experience
– Approx 4ng DNA detected
– Mixture of at least 3
– Unknown Male #2 detected
– Deduced profile uploaded to CODIS
– The only sample with Unknown Male #2’s profile

Case #3
• On 9/15 victim was sexually assaulted by one suspect
• Possible digital penetration
• Victim remembers that the suspect had his hands in her pants and he was
touching the “front” of her vagina
• Victim was unsure if licking or kissing or any other acts occurred during assault due
to LOC and LOM
• Victim showered after assault
• At RTC on 9/16, nurse collected vaginal swabs, perianal swabs, right and left neck
swabs, and abdomen swabs
• No Wood’s Lamp positive areas were noted
• Victim had consensual sex 4 days prior to assault

Case #3
Screening results
– External vaginal swabs
• Nucleated and non-nucleated cells
observed
– Internal vaginal swabs
• Nucleated cells observed
– Perianal swabs
• Nucleated cells observed
– Left neck swabs
– Abdomen swabs
• NC = 0 cells observed

Right neck swab
• Proceeded to
DNA
• Approx 4ng of
DNA detected
• Mixture of at
least 2
Left neck swab
• Proceeded to
DNA
• Approx 4ng of
DNA detected
• Mixture of at
least 2
• Proceeded to
DNA
• Approx 4ng of
DNA detected
• Mixture of at
least 2
Abdomen swab
• Same Unknown Male detected on neck and abdomen
• Deduced profile uploaded to CODIS
• Negative screening report previously issued

Conclusions
• Important to read incident reports/SART nurse
observations
• Solely relying on presumptive screening test results
may result in stopping testing, which may result in not
obtaining probative information
• A negative presumptive test for saliva does not mean
there is no foreign DNA present
• Showering after the assault does not mean there is no
foreign DNA present*
*Sweet, D. and Shutler, G. Analysis of salivary DNA evidence from a bite mark on a body
submerged in water. Journal of Forensic Sciences, 1999, 44(5):1069-1072

Conclusions
• 28 samples with RSID™ -Saliva negative results extracted
– Results: ND or consistent with victim
• 18 samples
– Results: DNA foreign to victim
• 10 samples (3 profiles uploaded to CODIS)
– 4 samples with no nucleated cells observed
• Where is the DNA coming from?
– Amylase enzyme dissipated or below detection limit, epithelial
cells from mouth contact still present
– Nucleated cells from body parts, other than mouth
– Cell free DNA* aka Naked DNA
*Quinones, I. and Daniel, B. Cell free DNA as a component of forensic evidence recovered
from touched surfaces. Forensic Science International: Genetics 6 (2012) 26-30
*Vandewoestyne, M. et al. Presence and potential of cell free DNA in different types of forensic
samples. Forensic Science International: Genetics 7(2013) 316-320

Survey
• Survey Monkey
– Case scenarios
– Biological screening results
– Questions
• Association of Forensic Quality Assurance
Managers (AFQAM) forum
• Friends DNA
• Anonymous survey
• Total participants: 72-85 people

Case #1
• Case scenario
– Victim sexually assaulted by two suspects
– Suspect 1 bit victim on right and left sides of neck
– Victim had loss of consciousness and loss of memory
– Victim showered after assault
– Victim had consensual sex with boyfriend after
– The nurse collected bite mark swabs
• Biological screening results
– Bite mark on left side of neck
• Presumptive saliva test = Positive
– Bite mark on right side of neck

Case #1: Survey
• Given the previous case scenario and screening results,
how would you proceed regarding the bite marks swabs?
Total Responses = 85
28.24%
0.00%
60.00%
11.76%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
–Take the left neck bite mark
swabs through DNA
–Take the right neck bite mark
swabs through DNA
–Take both left and right neck
bite mark swabs through DNA
–Take neither one of the bite
mark swabs through DNA
PercentageofResponses
Answer Choices
Case#1: Percentage of Responses vs. Answer Choices

Case #1: Survey
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
40.00%
45.00%
50.00%
The victim’s
account of
the incident
The SART
nurse’s
observations
and/or what
he/she
collected
The
presumptive
saliva test
results
The
microscopic
analysis
results
Your
laboratory
protocols, if
any
Influence
Case #1: Rankings of Decision on How to Proceed Regarding Right Neck Bite Mark Swabs
(Start by taking both left and right bite mark swabs through DNA)
1 = Insignificant–
2–
3–
4–
5 = Vital–
N/A–

Case #1: Survey
11.59%
26.09%
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
–Take the right neck bite mark swabs through DNA –Stop testing
Answer Choices
Case#1: How to Proceed after taking only left neck bite mark through DNA
If the victim’s boyfriend’s DNA was detected on the left neck bite mark, how
would you then proceed?

Case #1: Survey
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
The victim’s
account of the
incident
The SART
nurse’s
observations
and/or what
he/she
collected
The
presumptive
saliva test
results
The
microscopic
analysis
results
Your
laboratory
protocols, if
any
Influence
(Start by taking only the left bite mark through DNA)
2–
3–
4–
5 = Vital–
N/A–

Case #1: Survey
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
100.00%
The victim’s
account of
the incident
The SART
nurse’s
observations
and/or what
he/she
collected
The
presumptive
saliva test
results
The
microscopic
analysis
results
Your
laboratory
protocols, if
any
PecentageofRespponses
Influence
(Taking neither the left nor the right bite mark swabs through DNA)
2–
3–
4–
5 = Vital–
N/A–

Case #2
• Case scenario
− Victim reported being sexually assaulted by two
suspects
− Suspect 1 licked the right side of the victim’s neck +
other acts
− No loss of consciousness and loss of memory
− Victim did not shower
− Nurse collected vaginal swabs, cervical swabs, rectal
swabs, anal swabs, and right neck swabs (based on
history)
− Right neck swabs

Case #2: Survey
Given the previous case scenario and screening results, if the same unknown male
was detected on both the vaginal and cervical swabs, how would you proceed
regarding the neck swabs?
– Total Responses = 75
69.33%
30.67%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
Take the neck swabs through DNA Do not take the neck swabs through DNA
Answer Choices
Case #2: Percentage of Responses vs. Answer Choices

Case #2: Survey
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
40.00%
45.00%
50.00%
The victim’s
account of
the incident
The SART
nurse’s
observations
and/or what
he/she
collected
The
presumptive
saliva test
results
The
microscopic
analysis
results
Your
laboratory
protocols, if
any
Influence
Case #2: Ranking of Decision on How to Proceed Regarding the Neck Swabs
(Taking the neck swabs through DNA)
1= Insignificant–
2–
3–
4–
5 = Vital–
N/A–

Case #2: Survey
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
The victim’s
account of
the incident
The SART
nurse’s
observations
and/or what
he/she
collected
The
presumptive
saliva test
results
The
microscopic
analysis
results
Your
laboratory
protocols, if
any
Influence
Case#2: Ranking of Decision on How to Proceed Regarding the Neck Swabs
(Not taking the neck swabs through DNA)
1= Insignificant–
2–
3–
4–
5 = Vital–
N/A–

Case #3
• Case scenario
− Victim was sexually assaulted by one suspect
− Possible digital penetration
− Victim remembers that the suspect had his hands in her pants and he was
touching the “front” of her vagina
− Victim was unsure if licking or kissing or any other acts occurred during assault
due to loss of consciousness and loss of memory
− Victim showered after assault
− Nurse collected right and left neck swabs, and abdomen swabs
– Left neck swabs
– Abdomen swabs

Case #3: Survey
• Given the previous case scenario and screening results, how would you proceed
regarding the neck and abdomen swabs?
– Total responses = 72
0.00% 2.78% 2.78%
41.67%
52.78%
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
Take one of the neck
swabs through DNA
Take both of the neck
swabs through DNA
Take the abdomen swabs
through DNA
Take all the neck swabs
and abdomen swabs
through DNA
Take neither the neck
swabs, nor the abdomen
swabs through DNA
Answer Choices
Case #3: Percentage of Responses vs. Answer Choices

Case #3: Survey
0.00%
5.00%
10.00%
15.00%
20.00%
25.00%
30.00%
35.00%
40.00%
45.00%
50.00%
The victim’s
account of
the incident
The SART
nurse’s
observations
and/or what
he/she
collected
The
presumptive
saliva test
results
The
microscopic
analysis
results
Your
laboratory
protocols, if
any
Influence
Case #3: Ranking of Decision on How to Proceed Regarding the Neck and Abdomen Swabs
(Taking all the neck and abdomen swabs through DNA)
2–
3–
4–
5 = Vital–
N/A–

Case #3: Survey
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
The victim’s
account of
the incident
The SART
nurse’s
observations
and/or what
he/she
collected
The
presumptive
saliva test
results
The
microscopic
analysis
results
Your
laboratory
protocols, if
any
Influence
Case #3: Ranking of Decision on How to Proceed Regarding the Neck and Abdomen Swabs
(Not taking the neck swabs and abdomen swabs through DNA)
2–
3–
4–
5 = Vital–
N/A–

Survey Conclusions
• Lab protocols and victim’s account were a vital influence
• 5 people commented that their lab has a policy of not
proceeding with body swabs if the victim has showered
Case 3
10 people
Case 1
10 people
No bite mark
swabs to DNA
No abdomen
and neck
swabs to DNA

Survey Conclusions
• Lab protocols, victim’s account, and SART nurse’s observations were
important
• Comments regarding using Quant Duo as a screening tool to determine
if swabs should move forward to DNA
Case 1
40 people
Case 2
52 people
Neck swabs to
DNA
Left and right
neck bite mark
swabs to DNA
Left neck bite
mark swabs to
DNA
No swabs to
DNA
6 people
6 people

Survey Conclusions
Case 3
Case 2
19 people
38 people
No neck swabs
to DNA
No abdomen
and neck
swabs to DNA
19 people
Yes, neck swabs to
DNA
Lab procedure, victim’s account and SART
nurse collection/observation was vital
No overwhelming
influence

Survey Conclusions
– Victim's account, SART nurse testing, and lab protocols had a vital
impact on how to proceed
– Analyst experience may be a factor
Case 3
30 people
Yes, abdomen
and neck
swabs to DNA
Case 1
27 people
Yes, right
and left
bite mark
swabs to
DNA
Case 2
28 people
Yes, neck
swabs to
DNA

Suggestions
• Have a discussion regarding protocol
– Update
– Clarification for analysts
• Consider performing DNA analysis on
presumptive saliva negative samples

Acknowledgements
• San Francisco Police Department Criminalistics
Laboratory
• Anonymous Survey Participants
• Association of Forensic Quality Assurance
Managers
• California Association of Criminalists

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Editor's Notes