Vanessa Nelson
Texas Dept. Public Safety - Weslaco
ERASE SPERM ISOLATION KIT: AN
INTERNAL VALIDATION STUDY
CURRENT METHOD: TNE DIFFERENTIAL
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Uses centrifugation to physically separate sperm
and epithelial cells
Time consuming: 2 hour incubation followed by ~1
hour of washing followed by 2 hour to overnight
incubation
Washing of sperm pellets can be difficult
Often, mixtures of male and female DNA obtained
in sperm fraction due to skill of analyst performing
washes or not enough sperm present
ERASE SPERM ISOLATION KIT
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Uses selective degradation process.
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The epithelial cells are digested using ProK.
The sperm cells are separated by centrifugation
Remaining epi DNA is removed from the sperm pellet by use
of an enzyme that degrades soluble DNA.
SHORT process: 1 hour incubation for digestion of epi
cells, 30 minutes of total incubation for sperm pellet
clean-up
Complete differential extraction in half a day
NO WASHING of the sperm pellet!!!
Cleaner profiles in the sperm cell fraction
VALIDATION OUTLINE
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Accuracy
NIST-traceability
Reproducibility
Sensitivity
Time-point study
Contamination
Concordance
Casework-type samples were contained within the
Accuracy, Concordance, and Time-point studies
NOTES ON MATERIALS & METHODS
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Our lab used the Tecan Evo Freedom 150
Combination robot to set up the Quantifiler plates,
normalize samples, and set up the amplification
plates
Quantification: Quantifiler Human on an SDS 7500
instrument
Amplification: Identifiler Plus (1.5 ng target) on a
9700 thermal cycler
CE: 3130xl at a default injection of 5 seconds (2
and 10 second injections were used as needed
and are indicated on the slides)
ACCURACY
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CTS proficiency samples were used from tests
10-582 and 11-582
Sperm cell fraction: full single-source male
profiles obtained match manufacturer results
for both samples
Epi cell fraction: full mixture profiles obtained
for both samples that match manufacturer
results. Majority of profile is female.
ACCURACY DATA
NIST-TRACEABILITY
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A sample was prepared by overlaying a 1:10
semen dilution from a known donor onto an inhouse NIST-CRM blood stain.
A single-source male profile was obtained in
the sperm fraction matching the known donor
A mixture profile in which the major component
matched the known profile of the NIST-CRM
and the minor component matched the sperm
donor was obtained in the epi fraction
NIST DATA
REPRODUCIBILITY
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6 vaginal swabs taken 24-hours post-coitus by a volunteer
donor were used in this study
2 different analysts (on 2 different days) extracted 3 swabs
each to compare reproducibility of Erase extraction
method.
SENSITIVITY
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Serial dilutions were made of semen from a
known donor into 1xPBS from neat to
1:1,310,720
The entire volume of each dilution (100ul) was
added by pipette onto buccal swabs from a
known female donor.
The entire swab was extracted for each dilution
Dilution
Factor
Sperm Loci with
at 400x male
alleles
Loci with
female
alleles
Dilution
Factor
Sperm Loci
at
with
400x male
alleles
Loci with
female
alleles
Neat
TNTC
16
0
1:5
TNTC
16
16
1:40,960
6 total 3
16
1:10
TNTC
16
0
1:81,920
3 total 0
16
1:20
TNTC
16
0
1:40
TNTC
16
0
1:163,840
1 total 0
9
1:80
TNTC
16
3
1:160
TNTC
16
10
1:327,680
None
0
11
1:320
20-30/f 16
12
1:655,360
None
0
16
1:640
10-16/f 16
16
1:1,280
1-5/f
16
11
1:1,310,720 None
0
16
1:2,560
0-5/f
16
16
1:5,120
0-3/f
6
16
1:10,240
20 total 13
16
1:20,480
6 total
16
0
Sensitivity Study: comparison
of sperm cell fractions
Profile from the sperm cell
fraction of the 1:10,240
dilution.
A profile could be deduced
from this sample that would
be suitable for entry into
NDIS
TIME POINT STUDY
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Vaginal swabs collected from a volunteer donor
24-, 48-, 72-, and 96-hours post coitus were
extracted using Erase
Time-point: comparison of sperm cell
fractions
24-hour post-coitus
(male donor profile
with female minor)
48-hour post-coitus
(male donor profile
with female minor)
72-hour post-coitus
(male donor profile
with female minor)
96-hour post-coitus
(female donor profile
with male minor)
TIME-POINT STUDY
CONTAMINATION STUDY
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Used reagent blanks from all studies in the
validation
Injected them at 5 and 10 seconds on the CE
No DNA profile detected in any of the 32
reagent blanks from the validation study
CONCORDANCE: TNE VERSUS ERASE
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Post-coital vaginal swabs from PTC were used
along with swabs of a condom and a semen stain
from a pair of panties (condom and panties
collected by a volunteer)
An entire swab/identical size stain from panty was
used for each extraction method.
Erase: Sperm cell fractions
TNE: Sperm cell fractions
Sample
Profile
type
Male
(#/16)
Female
(#/16)
Sample
Profile
type
Male
(#/16)
Female
(#/16)
PC165
(0-6 hr)
Single
source
16
0
PC165
(0-6 hr)
Mixture
16
16
PC225
(31-36
hr)
Single
source
16
0
PC225
(31-36
hr)
Mixture
(M/M)
5
16
(Minor) (Major)
Panties
Single
source
16
0
Panties
Single
source
16
0
Inside
condom
Single
source
16
0
Inside
condom
Single
source
16
0
Outside
condom
Single
source
16
0
Outside
condom
Mixture
(M/M)
16
2
(Major) (Minor)
Erase: Epi cell fractions
TNE: Epi cell fractions
Sample
Profile
type
Male
(#/16)
Female
(#/16)
Sample
Profile
type
Male
(#/16)
Female
(#/16)
PC165
(0-6 hr)
Single
source
0
9
PC165
(0-6 hr)
Single
source
0
16
PC225
(31-36
hr)
Single
source
0
9
PC225
(31-36
hr)
Single
source
0
16
Panties
Single
source
0
4
Panties
Mixture
(M/M)
4
16
(Minor) (Major)
Inside
condom
Mixture
5
4
Inside
condom
Mixture
16
16
Outside
condom
Single
source
0
16
Outside
condom
Single
source
0
16
WHAT HAPPENED TO THE EPI FRACTION?
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4 of the 5 samples extracted with Erase
produced partial profiles for the epi fraction
Epi fractions can be probative in some sexual
assault cases, so it is important to make sure
complete profiles obtained for this fraction
PTC revealed that sporadic loss of epi fraction
was seen in other labs
SPORADIC LOSS OF EPI FRACTION
22.4 ng/ul in 55 ul volume
40.5 ng/ul in 25 ul volume
Post-clean up with chelex beads
to remove possible inhibitors
POSSIBLE DEGRADATION?
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We presume that there was possible degradation occurring in
the epi fraction during the Erase extraction and subsequent
clean up procedures
We tried several methods of purifying our DNA samples
including Qiagen DNA mini kit, several washes with P:C:I:, and
several TE washes with Vivacon membrane filtration devices
PTC recently issued an improved extraction buffer for us to test.
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The buffer components were adjusted to provide better lysis of the
epithelial cells and prevent possible degradation of this fraction without
affecting sperm cell fraction yields.
EPITHELIAL CELL FRACTION RECOVERY
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Since most of the previous samples from the concordance
study were depleted, new samples (from the same individuals)
were obtained from PTC. Additionally, new swabs were taken
from the same condom and another cutting was taken from the
same area on the same panty.
These samples were re-extracted using Erase and the new
extraction buffer
Improved results were seen for all the epithelial cell fractions.
Any loss of DNA seen in the sperm cell fractions is believed to
be due to sample condition
Old Buffer: Epi fractions
New Buffer: Epi fractions
Sample
Profile
type
Male
(#/16)
Female
(#/16)
Sample
Profile
type
Male
(#/16)
Female
(#/16)
PC165
(0-6 hr)
Single
source
0
9
Single
source
0
16
PC225
Single
(31-36 hr) source
0
9
PC204
(13-18
hr)
PC250
(31-36
hr)
Single
source
0
16
Panties
Single
source
0
4
Panties
Mixture
3
16
Inside
condom
Mixture
5
4
Inside
condom
Mixture
10
14
Outside
condom
Single
source
0
16
Outside
condom
*condom stored at 2-8C for 6 months
Mixture
11
15
*condom stored at 2-8C for 6 months
Old Buffer: Sperm fractions
New Buffer: Sperm fractions
Sample
Profile
type
Male
(#/16)
Female
(#/16)
Sample
Profile
type
Male
(#/16)
Female
(#/16)
PC165
(0-6 hr)
Single
source
16
0
Single
source
16
0
PC225
Single
(31-36 hr) source
16
0
PC204
(13-18
hr)
PC250
(31-36
hr)
Single
source
13
0
*Swab was AP neg, 2 sperm seen
Panties
Single
source
16
0
Panties
Single
source
16
0
Inside
condom
Single
source
16
0
Inside
condom
Single
source
16
0
Outside
condom
Single
source
16
0
Outside
condom
Single
source
13
0
*condom stored at 2-8C for 6 months
CONCLUSIONS
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Erase Sperm Isolation kit is a valid method for
performing differential extractions
Erase Sperm Isolation kit gave comparable results to
the TNE differential currently in use in our lab
Erase Sperm Isolation kit is faster, easier to use, and
gave cleaner sperm cell fractions than the TNE
method
We hope to implement this method for casework in
our lab soon!
ACKNOWLEDGEMENTS
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Thanks to Nicole Hahn who did the bulk of the validation work
Thanks to Catharine Worthen for finishing up my samples for
me
Thanks to Tina Trevino for doing additional sensitivity study
work