Comparison between MACSprep™ forensic sperm microbead kit and Erase Sperm Isolation kit for the enrichment of sperm fractions recovered from sexual assault samples

Abstract

Sexual assault samples often contain mixtures of cells coming from at least two donors. Ideally, one would need to separate the cells into two cellular fractions: one consisting of the alleged aggressor's spermatozoa (the sperm fraction) and the other containing the victim's epithelial cells (the non-sperm fraction). This separation increases the probability of obtaining the alleged offender's autosomal DNA profile. However, spermatozoa are often collected along with an excess of biological material originating from the victim, and with unfavorable male:female biological material ratios, the absence of separation could result in the PCR amplification of the victim's DNA profile only. Several approaches are available to enrich/purify the spermatozoa present on sexual assault samples. In this paper, we compare a new method, the MACSprep™ Forensic Sperm MicroBead Kit (MACSprep, based on microbeads conjugated with antibodies bound to spermatozoa and their retention within a magnetic column) with the Erase Sperm Isolation Kit (Erase, a standard differential lysis separation procedure combined with a specific removal of free DNA) routinely used in our lab. The performance of both kits was tested using sets of vaginal and buccal swabs loaded with different dilutions of sperm, or azoospermic semen, representing a total of 120 independent samples. For the samples containing undiluted sperm, an average recovery of 58% was observed for the MACSprep's sperm fractions and 43% for Erase's. Significantly better recovery of azoospermic semen was observed in MACSprep's non-sperm fractions (~ 85%) compared to Erase (~ 28%). Erase performed significantly better than MACSprep in terms of recovery for diluted sperm samples (1:10 to 1:800 sperm dilutions) in the presence of vaginal cells, while the purities of the achieved sperm fractions were in favor of MACSprep for the highest sperm dilutions tested. Similar trends were observed with buccal swabs loaded with 1:200 sperm dilutions. Increased sperm dilutions on vaginal swabs resulted in higher variability in the male material recovered, whatever the separation method used. Both methods were easy to perform and resulted in male DNA extracts ready to use in less than 2 h. Both kits showed their specificities in terms of recovery efficiency and purity of the sperm fractions. Ideally, additional experiments should be performed in different laboratories, using workflow and chemistries different than ours, to better define the peculiarities observed with MACSprep for high dilutions. Improving the recovery of MACSprep for diluted samples, in addition to its better purity observed in the experiments performed, could make it a method of choice for laboratory workflow, despite MACSprep's current price per sample being about twice the price of Erase's.

Keywords: DNA extraction; Differential lysis; Forensic genetics; STR profile; Sexual assault samples; Spermatozoa separation.

Fig. 1

A Male material recovered from sperm-loaded swabs using either MACSprep or Erase separation kits. Twenty microliters of undiluted sperm was loaded on swabs and recovered using either one of the separation kits. The material in the different fractions (sperm fraction (SF), non-sperm fraction (NSF), and flow-through (FT)) was quantified and compared to references (20 µl of the same sperm solution in Eppendorf, lysed and quantified). A, B, and C stand for triplicate. (B) Male material recovered from azoospermic semen-loaded swabs using either MACSprep or Erase separation kits. Twenty microliters of azoospermic semen was loaded on swabs and recovered using either one of the separation kits. The material in the different fractions (sperm fraction (SF), non-sperm fraction (NSF), and flow-through (FT)) was quantified and compared to references (20 µl of the same azoospermic semen solution in Eppendorf, lysed and quantified). A, B, and C stand for triplicate. Asterisk indicates a significant difference in quantities of recovered material between both methods (p < 0.05)

Fig. 2

Purity of the different sperm fractions. Vaginal swabs, loaded with either 20 µl of 1:10 PBS-diluted sperm or 20 µl of 1:200 PBS-diluted sperm, were processed with both separation kits in triplicates. Human and male materials were quantified for each sample. The purity of the sperm fraction was calculated as the male DNA contribution to the total human DNA quantified. The closer to 100%, the purer the fraction is, regarding male material. Error bars are from the triplicate processed for each kit by each technician

Fig. 3

Influence of biological mixture on male material recovery. Vaginal swabs, buccal swabs, and new, unused swabs were loaded with 20 µl of 1:200 PBS-diluted sperm. Triplicates of each conditions were separated using both MACSprep and Erase in parallel. The recovered material was then compared to references (20 µl of the same sperm dilution in an Eppendorf, directly lysed and quantified). Based on the results shown in Fig. 1A, MACSprep flow-through was not quantified. Asterisks indicate significant difference in male material recoveries in the sperm fractions (p < 0.05)

Fig. 4

Purity of the sperm fractions in the presence of vaginal or buccal cells. The purity of each of the sperm fraction was calculated for each of the vaginal or buccal swabs loaded with 20 µl of 1:200 PBS-diluted sperm. Error bars are from triplicate processed for each kit

Fig. 5

Sensitivity of the kits. Both separation kits were tested on vaginal samples loaded with 20 µl of increasing dilutions of sperm (1:10, 1:100, 1:200, 1:400. 1:800, and 1:1600) in duplicates. The male material in the sperm fractions was quantified and compared to references. Asterisks indicate significant differences in male materiel recovery (p < 0.05)

Fig. 6

Purity of the sperm fractions with increasing sperm dilutions. Vaginal swabs loaded with 20 µl of the indicated sperm dilutions were separated using both kits in duplicates (A and B). The resulting sperm fractions were extracted and quantified. The purity of the sperm fractions is shown for each sample