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

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.

Modified differential lysis for sexual assault samples using a combined enzymatic and alkaline approach.

Sexual assault sample processing, despite recent funding and research efforts, remains time-consuming, labourious, and inefficient. These limitations, combined with the prevalence of sexual assaults, have prompted the need to develop a cheaper, quicker, and more robust method for separating victim and perpetrator contributions within sexual assault evidence so that analysts can keep pace with submissions and cases can be resolved in a timely manner. Thus, this study examined the use of a combined enzymatic and alkaline approach for differential cell lysis-with the goal of developing a quick, cheap, and more efficient DNA isolation method. Quantification results for this assay revealed that (72.0 ± 18.3)%, (15.8 ± 14.2)%, and (29.5 ± 23.7)% of total DNA were retained in sperm fractions for neat semen, neat vaginal, and semen-vaginal mixture eluates, respectively. Short tandem repeat (STR) analysis of mixture samples processed with this technique exhibited sperm fraction DNA profiles with mean male-to-female ratios of 1.74:1, which was a 3.01 ± 2.30-fold improvement in male-to-female ratios and led to the recovery of 5.90 ± 7.80 unshared male contributor alleles in sperm fractions that were otherwise undetected in unseparated controls. Overall, this study presented a modified differential lysis approach using prepGEM™ and sodium hydroxide treatments that can accomplish cell elution and fractional lysis within 25 min. Future studies should investigate alternative "non-sperm" cell lysis methods to enhance lysis efficiency and minimize the potential for inhibition, as well as the optimization and automation of this technique. Key points: Traditional sexual assault sample processing methods are time-consuming and inefficient.This modified differential lysis method produces lysates with sufficient DNA yield and quality.A combined technique using enzymatic and alkaline lysis can accomplish fractional separation.Lysis with prepGEM and NaOH absent purification is compatible with downstream processes.

Human Papillomavirus (HPV) Infection and Its Impact on Male Infertility.

Nowadays, the striking numbers of infertile couples that turn to assisted reproductive technologies (ART) drive the research toward a more comprehensive understanding of the underlying causes. Male factors contribute to the inability to conceive in half of the cases, and it has been suggested that sexually transmitted infections could have a role in the onset of spermatozoa impairments. Since the impact of HPV infection on sperm quality and sperm DNA integrity is debated, we wanted to analyze its impact on conventional seminal parameters and the sperm DNA fragmentation index (DFI). Therefore, 117 semen samples of patients undergoing IVF were evaluated for the following characteristics: HPV DNA detection and sperm DNA fragmentation, concentration, motility, and morphology. The results showed a higher rate of HPV-negative patients (59.8% vs. 40.2%) and no HPV-related effect on DFI, sperm concentration, total sperm number, and total motility. Only progressive motility and morphology were found as significantly influenced by HPV positivity. Moreover, we observed a statistically significant difference in DFI when comparing high-risk HPV (HR-HPV) and low-risk HPV (LR-HPV) genotypes. Our data suggest that the presence of any HPV type, whatever the exact localization of the virions, can impair some sperm parameters, while HR-HPVs specifically affect the integrity of spermatozoa DNA.

Alternative direct-to-amplification sperm cell lysis techniques for sexual assault sample processing.

The prevalence of sexual assault cases and increasingly sensitive DNA analysis methods have resulted in sexual assault kit backlogs in the United States. Although traditional DNA extraction and purification utilizing detergents, proteinase K, and DTT have been the primary technique for lysing sperm cell fractions from these samples, it is labor-intensive and inefficient regarding time and sperm DNA recovery - hindering the ability of forensic analysts to keep pace with evidence submissions. Thus, this study examined seven alternative sperm cell lysis techniques to develop a method that could efficiently lyse sperm and consistently generate high-quality profiles while also reducing time, labor, and cost requirements. Microscopic examination of lysates indicated only Casework Direct and alkaline techniques could lyse all spermatozoa within samples, while quantification results demonstrated all methods performed comparably to the control method of forensicGEM™ Sperm (p > 0.06). Amplification with 0.25 ng DNA revealed that unpurified lysates from Casework Direct, alkaline, and NP-40 techniques produced DNA profiles with acceptable mean STR peak heights and interlocus balance, both of which were similar to or better than the control. Overall, this study demonstrated the ability of Casework Direct, alkaline, and NP-40 methods to efficiently lyse spermatozoa and provide high-quality STR profiles despite the absence of a purification step. Ultimately, based on the data reported herein, alkaline lysis is the recommended alternative sperm lysis approach given its ability to generate high-quality profiles, save time, and decrease the cost per reaction when compared to traditional sperm cell lysis methods.

Differential Fractionation of Erythrocytes Infected by Plasmodium berghei.

The study of host/pathogen interactions at the cellular level during Plasmodium intra-erythrocytic cycle requires differential extraction techniques aiming to analyze the different compartments of the infected cell. Various protocols have been proposed in the literature to study specific compartments and/or membranes in the infected erythrocyte. The task remains delicate despite the use of enzymes or detergents theoretically capable of degrading specific membranes inside the infected cell. The remit of this protocol is to propose a method to isolate the erythrocyte cytosol and ghosts from the other compartments of the infected cell via a percoll gradient. Also, the lysis of the erythrocyte membrane is done using equinatoxin II, which has proven to be more effective at erythrocyte lysis regardless of the cell infection status, compared to the commonly used streptolysin. The parasitophorous vacuole (PV) content is collected after saponin lysis, before recovering membrane and parasite cytosol proteins by Triton X-100 lysis. The lysates thus obtained are analyzed by Western blot to assess the accuracy of the various extraction steps. This protocol allows the separation of the host compartment from the parasite compartments (PV and parasite), leading to potential studies of host proteins as well as parasite proteins exported to the host cell.

Improved resolution of mixed STR profiles using a fully automated differential cell lysis/DNA extraction method.

Sexual assault evidence often contains sperm cells, which are typically separated from nonsperm cells using manual differential lysis procedures. The goal of this study was to evaluate the automated QIAGEN QIAcube for this purpose and to compare it to manual QIAGEN and manual organic differential methods using DNA yields and STR profile data for assessment. DNA yields were determined by qPCR, followed by multiplex STR amplification, CE analysis, and mixture interpretation. The automated method was capable of effective cell separation, producing DNA yields sufficient for STR amplification. Further, sperm fraction human:male DNA ratios from the QIAcube samples were consistently closer to the desired 1:1 and STR profiles were less likely to result in mixtures, with 6-8× fewer female alleles detected (median 1.5 alleles). Ultimately, using the QIAcube for automated differential processing of semen-containing mixtures reduces the need for downstream mixture interpretation and improves STR profile quality with substantially less hands-on time.

The Discussion of semen stains examination in the field of forensic applications / 中国法医学杂志

Objective To investigate the significance of a comprehensive test on the semen stain in the field of forensic evidence;Methods PSA tests performed on samples obtained supernatant and sediment STR genotyping by differential lysis method;Results Through STR testing,there are inconsistencies with the PSA test results in which 13 cases of false positive and false negative in 1 case.The supernatant was detected in two cases of male component,coeducational 9 cases;Conclusion PSA test strips there are some false positive and false negative rate,test isolated by the difference lysis supernatant can not only increase the success rate of the fine on the semen stain can also as a supplement to bring more valuable information for the semen stain.