Sexual assault samples are particularly challenging to analyze because they often contain spermatozoa among a larger proportion of cells originating from the victim (who could be either a woman or a man) than from the offender, in a generally unfavorable ratio for a direct spermatozoa-derived STR amplification. The gender of the victim has no importance as long as the only sperm found on the samples originates from the alleged offender. When large quantities of the victim’s cells (epithelial cells, macrophages, lymphocytes, neutrophils…) are present on such samples, a direct lysis most often results in the preferential autosomal amplification of the victim’s DNA (the major female or male contributor) and thus masks the alleged offender’s DNA profile. This typically occurs when the minor contributor represents less than 5% to 10% of the total amount of DNA extracted from such a sample. This limit has been reported in other studies [1,2,3,4] and is also the lower detection limit observed in our laboratory (internal validation studies), when using NGM SElect amplification.
In absence of biological material separation, when the alleged aggressor is a male and the victim is a female, the analysis of Y chromosome-specific STRs (Y-STRs) may give access to a part of the male contributor’s genetic information [5,6,7,8]. Y-STRs are male-specific STRs and therefore their amplification should not be affected by the presence of female DNA [9, 10], although some alleles have been reported to be more difficult to call with extremely unbalanced mixture samples, such as 1:32,000 (male:female) (Promega, PowerPlex Y23 product sheet). However, the discrimination of individual with Y-STRs is much less efficient than with autosomal STRs, as all the males from a given paternal lineage share the same inherited Y-STR haplotype (unless a mutation has occurred). Y-STR profiles can be used locally or can be submitted to Y-STR national databases, wherever available, for comparison with Y-STR profiles of known individuals, or possibly to provide links between cases. Rapidly mutating Y-STRs (RM-YSTR) amplification offers a chance to differentiate related males [11,12,13,14,15,16], as they amplify another set of Y-STRs known to have a higher mutation rate (10−2 and higher).
Another approach to differentiate a minor contributor in unbalanced mixture samples, in the absence of biological material separation, lies in the use of DIP-STR markers [17,18,19,20]. DIP (deletion-insertion polymorphism)-STR are sequences found in the human genome where DIP polymorphisms (long or short, depending on the polymorphism present) and STRs are close to one another and can be amplified together using DIP-specific primers. When the minor contributor’s DNA possesses a private DIP allele, the corresponding DIP-STR haplotype can be accessed even in severely unbalanced mixtures (up to 1:1000), and this independently of the gender of the major/minor contributors.
Although quite elegant, both RM-YSTRs and DIP-STRs approaches do require reference material from all the contributors possibly at the source of the DNA being analyzed to allow direct comparisons. Another possible limitation of either Y-STRs, RM-YSTRs, or DIP-STRs-amplifications of rape samples, in the absence of separated spermatozoa fraction, is the impossibility to give information on the biological material at the origin of the observed profile. Indeed, the amplified (Y-, RM-Y-, or DIP-) STR markers observed may come from any types of male cells present in the mixture and not only spermatozoa. In a difficult scenario (i.e., presence of several male fluids (semen, blood, skin cells, and/or saliva)), it could be impossible to determine whether the Y-STR profile observed originates from spermatozoa, or from any other male cells present in the sample. For the cases where the analysis of the sperm fractions results in single source male DNA profiles, it seems reasonable to conclude that these profiles originated indeed from sperm. In other multiple contributors’ admixed samples, attributing an observed profile to a given biological fluid may be impossible to achieve.
To circumvent these issues, a separation of the spermatozoa from other cell types needs to be done prior to cell lysis and DNA amplification. This separation results in two distinct fractions, one containing the spermatozoa originating from the alleged offender (the sperm fraction), and the other, the non-spermatozoa biological material originating mainly from the victim (the non-sperm fraction). Several approaches, for the separation of spermatozoa from other cell types, have been developed over the years to process sexual assault samples (differential lysis [21,22,23,24], sieve-based filtration [25, 26], laser micro-dissection [27,28,29,30,31,32], micro-fluidic devices [33, 34], flow cytometry [35,36,37], acoustic trapping device , capillary zone electrophoresis , DEP-array sorting [40,41,42,43], antibody-based separation [44,45,46,47]). They can result in the autosomal STR profiles of the alleged offenders. These profiles can then be submitted to national, or international, DNA databases and be compared with the DNA profiles of known individuals and/or possibly to provide links between cases.
The processing of samples using the above methods within a forensic framework might be hampered by the following drawbacks:
The separation devices may only be at a prototype stage and not yet available on the market (i.e., micro-fluidic devices).
Contamination issues with degraded material and cell-free DNA present in the original sample may impair the quality of the obtained profiles (i.e., sieve-based separation, flow cytometry).
An automated approach may not be available (e.g., differential lysis, laser micro-dissection).
The equipment may be expensive, require a dedicated operator, and/or have low throughput (laser micro-dissection, flow cytometry, and DEP-array are such examples).
Erase Sperm Isolation Kit (PTC laboratories, Columbia, MO, USA) is a commercial method, based on differential lysis, routinely used in our laboratory. It relies on a two-step lysis procedure, first lysing all the non-spermatozoa cells, then increasing the lysis strength with the addition of DTT, to break the disulfide bounds found on the spermatozoa’s head and release their DNA (Erase Sperm Isolation Kit manual). A centrifugation step takes place, following the first lysis, to pellet down the spermatozoa and remove the supernatant (containing the non-spermatozoa cells free DNA). The spermatozoa pellet is subsequently treated with a deoxyribonuclease in order to digest the remaining free DNA originating from the lysed non-spermatozoa cells. This procedure normally ensures that only spermatozoa-derived DNA is found in the sperm fraction extract after the second lysis. Despite the deoxyribonuclease step prior to spermatozoa’s lysis, some challenging situations may occur when the amount of the victim’s cells are much more abundant than the alleged aggressor’s spermatozoa, leaving significant quantities of non-lysed free-DNA from the victim’s cells in the supernatant, on top of the low numbers of pelleted spermatozoa. When such sperm fractions are amplified, the remaining victim’s free DNA still present may mask, or compete with, the amplification of the alleged offender’s DNA, resulting in difficult to interpret DNA profiles or, in some extremely unbalanced samples, resulting in the amplification of the victim’s DNA only.
The MACSprep Forensic Sperm MicroBead Kit (Miltenyi Biotec, Bergisch Gladbach, Germany, Europe) separation technique specifically retains spermatozoa within a magnetic column following the recognition and binding of spermatozoa by specific antibodies coupled to magnetic beads (MACSprep Forensic Sperm MicroBead Kit manual). While retained within the column, the spermatozoa can be washed, thus removing non-spermatozoa cells and possible free DNA originating from the victim’s lysed cells. This should result, once the spermatozoa have been flushed out of the column (as the sperm fraction) and lysed, in DNA profiles originating from spermatozoa only.
The expected purity achievable with the MACSprep’s separated samples, and its apparent ease of use, led us to compare this separation method with our current separation method (Erase). Both separation kits were compared in terms of recovered material and purity of the sperm fractions, specificity, the ease of use, and hands-on time needed per sample, to seek the possible advantages of one of those two methods over the other.