SpermX methodology
Researchers have developed a new method that is up to six times more efficient at extracting male sperm fraction DNA profiles from sexual assault kits—particularly when analyzing low level sperm samples.
The new SpermX method is based on a novel nanotechnology-derived polymer membrane that effectively traps sperm cells during the extraction and digestion process.
The InnoGenomics Technologies team first published their validation study in Forensic Science International: Genetics earlier this year. Tuesday, at the Green Mountain DNA Conference in Vermont, they debuted supporting results from an interlaboratory study with University of Central Florida (UCF) and the Center for Forensic Science Research and Education (CFSRE).
Digestion differences
The standard differential extraction process performed in most crime laboratories involves differential lysis of the cells collected on a vaginal swab, exploiting the differential stability of the cell membranes. But, this method performed manually is time-consuming, analyst-dependent and prone to loss of DNA evidence.
There have been moderately successful attempts to automate the process recently. However, in all the approaches, the evidentiary swab is removed after epithelial cell digestion, thereby leaving some of the undigested sperm cells in the removed swab and decreasing the recovery of sperm DNA.
This is where SpermX carves a niche. With this method, epithelial cells are digested prior to capture of sperm cells in the nanofiber membrane and the swab is left in the tube after epithelial digestion for the sperm digestion, increasing sperm recovery. The process can (but doesn’t have to) be fully automated, with walkaway analysis of up to 95 rape kit swabs at one time.
Validation paper
The FSI: Genetics study compares the standard differential extraction process with the SpermX process utilizing evidence-typical buccal, semen and vaginal swabs from five males and three females. An emphasis was placed on swab sets 17 to 20, which contained lower sperm amounts with higher female-to-male ratios than other samples in the study.
While the average total female/non-sperm DNA in the epithelial fraction for SpermX and the differential extraction method was comparable, the study results showed a difference for male DNA in the sperm fraction. Overall, SpermX recovered an average of 3x more male DNA from the low-sperm samples than the standard differential extraction process.
Most importantly, the SpermX method recovered more male DNA in the final STR profiles from each of the low sperm swab sets—at 3.1-, 3.9-, 6.6- and 22.6-fold more. Specifically, both techniques detected all alleles from the male DNA for swab sets 17 and 18; but for swab sets 19 and 20, SpermX detected 13 and 15 male alleles, respectively, that differential extraction missed.
“These data demonstrate that the [SpermX] method outperforms the [differential extraction] method when dealing with very low sperm amounts,” the authors write. “This success can be attributed to the capability of the SpermX membrane to capture DNA from a small amount of sperm cells and the swab being kept in the device for sperm digestion.”
Speaking of, the average percentage of sperm DNA left on the swab for the differential extraction method was approximately 81%, in contrast to .5% to 15% for the SpermX method (as established in a previous study).
Interlaboratory study
After publication of the paper, InnoGenomics ran an interlaboratory mixture study among their lab, a forensic research lab at the University of Central Florida headed by Jack Ballantyne and Erin Hanson, and one at CFSRE with Tina Nichols and Thomas Walsh.
Each laboratory processed eight swabs in tripicate with different female-to-male ratios using the SpermX platform and standard differential extraction. According to the results, all three labs had higher male DNA yields with SpermX relative to the theoretical amount of DNA placed on the swabs. On average, SpermX recovered from 1.5 to 10 times more DNA in the sperm fraction in swab mixtures with a “typical” level of sperm. But again, just as in the validation paper, the labs reported significantly higher yields for mixtures with low-sperm—from 20 to 1,533 times more recovered DNA. The last two low-sperm swab sets used in the study contained approximately 100 cells and only 25 cells.
As part of the study, Tim Kalafut from Sam Houston State University provided metrics from STRmix analyses. The software reported more male in the STR sperm fraction profiles across all three laboratories, ranging from 2 to 36-fold. At InnoGenomics, SpermX resulted in higher likelihood ratios (LRs) than differential extraction for all four low sperm mixtures, while UCF reported higher LRs for all eight mixtures. At CFSRE, SpermX performed better for all mixtures but two, which still has comparable likelihood ratios. Overall, all three laboratories experienced “significantly more male in the profile, which rendered much better statistics for inclusion.”
Additionally, differential extraction exhibited a higher average percentage of female carryover for all swab sets for all three labs. In general, a larger amount of female carryover results in more complex mixture interpretations and/or loss of male allelic information.
Finally, across all three labs, SpermX also boasted a better recovery of unshared male alleles. With a total of 32 unshared male alleles present, UCF reported SpermX recovered all of the male alleles in 5 of 8 mixtures with an average of 92%, while differential extraction did not recover all of the unshared male alleles in any of the mixtures and resulted in an average of 33%.
Differential extraction performed better at CFSRE, where the method recovered all of the unshared male alleles in 4 of 8 mixtures, to SpermX’s 6 of 8. But, SpermX recovered more male alleles for the four swabs with low sperm counts. In the Innogenomics lab, both methods recovered all of the alleles in two low-sperm samples. In the other two, SpermX recovered 1.8 times more and 18 times more for an average increase of 10%.
“Overall, for the differential extraction set we had very little male recovery in the sperm fractions. For SpermX, the A sample had the male as the major and in the rest it was minor, but still easily detectible. We’re really quite impressed with the difference between the methods based on this data,” said UCF’s Ballantyne and Hanson.
Impact on sexual assault kits
The results the SpermX technique showed specifically on low sperm samples is a positive development for the processing of sexual assault kits. Oftentimes, victims of sexual assault and rape do not go to the police right away. In the time that passes, they may do things that reduce the likelihood of obtaining an adequate amount of DNA evidence of the assault—such as showering.
Currently, the percentage of sexual assault cases that do not have enough DNA for comparison to a known profile or for CODIS upload ranges from 30 to 50%.
With SpermX extracting DNA from mixtures with only 25 cells, the number of cases normally reported as “insufficient DNA” for analysis could drastically decrease.
“SpermX can be especially beneficial for sexual assault samples that typically have lower sperm and lower profiling success rates such as oral and rectal swabs and dried secretions,” said InnoGenomics CEO Sudhir Sinha. “With the societal benefit of each additional profile added to the CODIS database estimated to be nearly $20,000, even a modest increase in profile success rate from sexual assault samples would have a significant benefit to society.”