Innovative techniques for the localization of seminal stains and the identification of spermatozoa
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Semen is a commonly found bodily fluid in sexual assault cases and with an ever increasing Sexual Assault Evidence Collection Kit (SAECK) backlog, the ability to localize seminal stains and identify spermatozoa in an accurate and efficient manner is crucial. Items of evidence are first visualized under white light followed by an Alternate Light Source (ALS) if no stains are readily visible. When potential semen stains are localized, preliminary chemical testing can be performed to guide the analyst to conduct further testing or to disregard a certain stain. Preliminary testing for semen often employs the Acid Phosphatase (AP) test which can be applied directly to an item if a stain is visible, or using a mapping technique if no stains are identified under white light or ALS. Preliminarily positive stains then undergo confirmatory testing for semen during which at least one spermatozoon must be identified to make a definitive statement that semen is present in a given stain. STK® Sperm Tracker™ is a new product that is able to perform AP mapping while in contact with an item, allowing easier localization to occur over a larger area. STK® Sperm Tracker™ yielded similar results to the AP Spot reagent when used for mapping purposes, however, traditional AP mapping was slightly more sensitive when a 10-minute reaction window was used. Rapid SpermBlue® stain and Goldcyto-SB pre-stained slides are commonly used in fertility testing as quick methods to differentially stain spermatozoa and epithelial cells. These two methods are similar to the hematoxylin and eosin (H&E) stain in that they provide a monochromatic range of coloration with darker blue nuclei compared to cytoplasm. Rapid SpermBlue® and Goldcyto-SB pre-stained slides were compared to H&E and Kenerchtrot-Picroindigocarmine (KPIC) stains in their ability to stain aged samples, samples exposed to decomposition fluids, and compromised samples in which wet evidence was sealed in plastic and left at an elevated temperature. All methods stained the compromised samples as effectively as freshly prepared samples. Simulated sexual assault swabs made with overwhelming ratios of epithelial cells to spermatozoa were prepared and the times to prepare and search slides were recorded. While Goldcyto-SB pre-stained slides were the fastest to prepare, they were also the slowest to search due to a larger surface area than is utilized by the other methods. An attempt was made to optimize a protocol in which a thiol-specific probe could be utilized as a sperm-specific dye based on the number of disulfide bonds within spermatozoa. Spermatozoa protamines are known to contain several cysteine residues which can be taken advantage of with the fluorescent probe, N-(7-dimethylamino-4-methyl-3-coumarinyl)-maleimide (DACM). Spermatozoa heads and tails fluoresced brightly with DACM and weaker fluorescence was observed on epithelial cells. To allow easier penetration of the fluorescent probe, cells were lysed with Sodium Dodecyl Sulfate (SDS) and disulfide bonds broken by the reducing agent, Tris-(2-carboxyethyl)phosphine (TCEP). DACM was able to penetrate the spermatozoa head and bind to thiols even before the addition of SDS and TCEP, though their presence did increase fluorescence. While there is a stark difference between the appearance of spermatozoa and epithelial cells using this method, proteomic work will be necessary before the method can be altered to be spermatozoa specific.