vanessa nelson texas dept. public safety - weslaco

ERASE SPERM ISOLATION KIT: AN INTERNAL VALIDATION STUDY

Vanessa Nelson Texas Dept. Public Safety - Weslaco

CURRENT METHOD: TNE DIFFERENTIAL

• 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

• Uses selective degradation process. • 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

• 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

• 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

• 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

• A sample was prepared by overlaying a 1:10 semen dilution from a known donor onto an in-house 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

• 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

• 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 at 400x

Loci with male alleles

Loci with female alleles

Neat TNTC 16 0

1:5 TNTC 16 16

1:10 TNTC 16 0

1:20 TNTC 16 0

1:40 TNTC 16 0

1:80 TNTC 16 3

1:160 TNTC 16 10

1:320 20-30/f 16 12

1:640 10-16/f 16 16

1:1,280 1-5/f 16 11

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 0 16

Dilution Factor

Sperm at 400x

Loci with male alleles

Loci with female alleles

1:40,960 6 total 3 16

1:81,920 3 total 0 16

1:163,840 1 total 0 9

1:327,680 None 0 11

1:655,360 None 0 16

1:1,310,720 None 0 16

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

• Vaginal swabs collected from a volunteer donor 24-, 48-, 72-, and 96-hours post coitus were extracted using Erase

24-hour post-coitus (male donor profile with female minor)



96-hour post-coitus (female donor profile with male minor)

Time-point: comparison of sperm cell fractions

TIME-POINT STUDY

CONTAMINATION STUDY

• 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

• 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.

Sample Profile type

Male (#/16)

Female (#/16)

PC165 (0-6 hr)

Single source

16 0

PC225 (31-36 hr)

Single source

16 0

Panties Single source

16 0

Inside condom

Single source

16 0

Outside condom

Single source

16 0

Sample Profile type

Male (#/16)

Female (#/16)

PC165 (0-6 hr)

Mixture 16 16

PC225 (31-36 hr)

Mixture (M/M)

5 (Minor)

16 (Major)


16 0

Inside condom

Single source

16 0

Outside condom

Mixture (M/M)

16 (Major)

2 (Minor)

Erase: Sperm cell fractions TNE: Sperm cell fractions

Sample Profile type

Male (#/16)

Female (#/16)

PC165 (0-6 hr)

Single source

0 9

PC225 (31-36 hr)

Single source

0 9


0 4

Inside condom

Mixture 5 4

Outside condom

Single source

0 16

Sample Profile type

Male (#/16)

Female (#/16)

PC165 (0-6 hr)

Single source

0 16

PC225 (31-36 hr)

Single source

0 16

Panties Mixture (M/M)

4 (Minor)

16 (Major)

Inside condom

Mixture 16 16

Outside condom

Single source

0 16

Erase: Epi cell fractions TNE: Epi cell fractions

WHAT HAPPENED TO THE EPI FRACTION?

• 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

22.4 ng/ul in 55 ul volume

Post-clean up with chelex beads to remove possible inhibitors

40.5 ng/ul in 25 ul volume

SPORADIC LOSS OF EPI FRACTION

POSSIBLE DEGRADATION?

• 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. • 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

• 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

Sample Profile type

Male (#/16)

Female (#/16)

PC165 (0-6 hr)

Single source

0 9

PC225 (31-36 hr)

Single source

0 9


0 4

Inside condom

Mixture 5 4

Outside condom

Single source

0 16

Sample Profile type

Male (#/16)

Female (#/16)

PC204 (13-18 hr)

Single source

0 16

PC250 (31-36 hr)

Single source

0 16

Panties Mixture 3 16

Inside condom

Mixture 10 14

Outside condom

Mixture 11 15

Old Buffer: Epi fractions New Buffer: Epi fractions

*condom stored at 2-8C for 6 months


Sample Profile type

Male (#/16)

Female (#/16)

PC165 (0-6 hr)

Single source

16 0

PC225 (31-36 hr)

Single source

16 0


16 0

Inside condom

Single source

16 0

Outside condom

Single source

16 0

Sample Profile type

Male (#/16)

Female (#/16)

PC204 (13-18 hr)

Single source

16 0

PC250 (31-36 hr)

Single source

13 0


16 0

Inside condom

Single source

16 0

Outside condom

Single source

13 0

Old Buffer: Sperm fractions New Buffer: Sperm fractions

*Swab was AP neg, 2 sperm seen


CONCLUSIONS

• 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

• 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

vanessa nelson texas dept. public safety - weslaco

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