Laser Microdissection Separation of Pure Spermatozoa from Epithelial Cells for Short Tandem Repeat Analysis^*

Abstract

Short tandem repeat (STR) analysis is a valuable tool in identifying the source of biological stains, particularly from the investigation of sexual assault crimes. Difficulties in analysis arise primarily in the interpretation of mixed genotypes when cell separation of the sexual assailant's sperm from the victim's cells is incomplete. The forensic community continues to seek improvements in cell separation methods from mixtures for DNA typing. The feasibility of applying laser microdissection (LMD) technology to precisely separate sexual assault cell mixtures by visual inspection coupled with laser dissection was assessed through three experiments. First, various histological stains were evaluated for use with LMD and DNA analysis. Second, different DNA isolation methods were evaluated on LMD-collected cells. Finally, STR analysis was performed on LMD-separated sperm cells from mixtures of semen and female buccal epithelial cells. The results indicated (a) hematoxylin/eosin staining performed best in its ability to differentiate sperm and epithelial cells while exhibiting the least negative effect on further downstream analysis; (b) both QIAamp and Lyse-N-Go methods were useful for recovery of DNA from LMD-collected sperm cells; and (c) LMD separation provided clear STR profiles of the male donor with the absence of any additional alleles from the female donor. This report describes an efficient, low-manipulation LMD method for the efficient separation of spermatozoa from two-donor sperm/epithelial cell mixtures.

Avoiding Errors and Pitfalls in Evidence Sampling for Forensic Genetics

“…The different cell types can be recognized by morphological characteristics, various chemical staining or fluorescence labeling techniques. These methods allow to establish a clear DNA profile from few cells present in a mixture samples that otherwise had not be detected while swabbed by the major component and not detectable in the profile ( Elliott et al, 2003;Anslinger et al, 2005;Anoruo et al, 2007 ;Sanders et al, 2006). With laser micro dissection techniques ( Anslinger et al, 2007;Vandewoestyne et al, 2009), it has been shown that cells derived from a male contributor can be analyzed separately from those derived from a female contributor after morphological or fluorescent labeling identification.…”

A Novel On‐Chip Method for Differential Extraction of Sperm in Forensic Cases

“…However, it has been reported that this cell separation process results in losses of 60–90% of the male DNA 10, 11, 12, 13. Although there have been multiple attempts for alternative methods to differentially extract sperm using acoustic trapping,14 antibody‐based capture,15 laser microdissection,16, 17, 18 nuclease‐based approaches,19 and magnetic bead‐based separation,20, 21 these methods have not been broadly available in practical applications due to the complexity and low separation yield for sperm. As a result, they are not widely in use in the community.…”

Automatic detection of spermatozoa for laser capture microdissection

“…The use of laser capture microdissection (LCM) has been evaluated for the isolation of spermatozoa from microscope slides containing both spermatozoa and vaginal cells [4][5][6]. Elliot et al showed that LCM outperforms differential extraction for the recovery of DNA from sperm cells, especially in cases where only a few sperm cells are present [7].…”

“…Standard methods to identify spermatozoa are based on non-specific staining techniques such as hematoxylin/eosin (HE) and nuclear fast red/ picroindigocarmine (CTS or "Christmas tree stain") [10], which rely on sperm morphology for identification [4] and cannot be used for automated sperm detection. Moreover HE staining reduces the yield of DNA [4,5,11] and CTS staining results in DNA damage, probably due to the picric acid component [4].…”