Forensic laboratory personnel are well aware that the entire structure of DNA profiling begins with the identification of a questioned stain from an article of evidence or from the analysis of an evidence swab. There are both criminalistic and laboratory procedural reasons to identify the source of the biological material that will be processed for DNA, as coming from blood, saliva, semen or sperm. As fully half of all forensic biology laboratory analysis involves sexual assault evidence, the identification of semen and sperm are particularly important. Recall that sperm is a specialized cell with distinctive morphology that is also the source of the overwhelming majority of DNA-containing cells in human ejaculate.
Once seminal fluid has been identified on sexual assault evidence, the DNA analyst must attempt to determine not only whether sperm are present, but which item of evidence or swab has the most sperm, in order to identify the sample most likely to provide a DNA profile. Here again, the forensic methods involved are unchanged for forty years, and current forensic identification of sperm uses a generalized cell staining method coupled with brightfield microscopy. In theory, this should be sufficient to identify sperm, in practice sperm isolated from sexual assault evidence has lost many, if not all, of its distinctive sub-cellular organelles upon which morphological identification depends. Hence, DNA analysts spend many hours searching for sperm using a less than optimal microscopic technique.
In order to provide a more scientifically and procedurally robust sperm searching technique, Independent Forensics has developed a fluorescent monoclonal antibody-based kit, SPERM HY-LITER™, for the microscopic identification of sperm from sexual assault evidence. SPERM HY-LITER™ is designed to provide positive identification of sperm using a unique monoclonal antibody that has been chemically tagged with an Alexa 488 fluorophore. The kit incorporates a second fluorescent dye, 4',6-diamidino-2-phenylindole (DAPI) that will stain all cell nuclei; this is a fluorescent analogue of the KPIC stain currently used in most DNA forensic laboratories. By combining both fluorescent dyes, SPERM HY-LITER™ provides several visually confirmatory steps for the identification of sperm. Sperm can be visualized in the fluorescein channel (the fluorescent spectra of Alexa 488 falls conveniently within the emission maximum for fluorescein); all cell nuclei can be seen in the DAPI channel; and using specialized dual filter ‘cubes,’ epithelial nuclei and sperm can be visualized simultaneously.
The monoclonal antibody used in SPERM HY-LITER™ provides an unprecedented degree of specificity that allows the identification of human sperm from previously unsearchable samples. Furthermore, while the signal-to-noise advantage of fluorescent microscopy (and the very low background seen with the fully optimized SPERM HYLITER™ kit) increases the sensitivity of sperm detection by orders of magnitude compared to current brightfield microscopic techniques. The incorporation of both DAPI and Alexa dyes was designed for image processing software such that sperm recognition could be essentially automated. By using computer-aided image analysis software, SPERM HY-LITER™ stained preparations can first be scanned for "features",i.e., fluorescent signals above background – and second, these features can then be analyzed further for the color (or hue) of the observed fluorescence. Only those features that have both DAPI (from the DNA) and the Alexa 488 (from the monoclonal antibody) fluorescence would be scored by the software as sperm.
SPERM HY-LITER™ provides all required solutions for slide staining in pre-calibrated dropper bottles – two slightly different version allow staining of smear slides (often included in sexual assault evidence kits, "rape kits") or of extracts made from evidence swabs or identified stains. The addition of phase contrast to the method, although not required, gives less experienced crime laboratory personnel the ability to visualize cells, nuclei, and sperm in one image.
As an illustration of the specificity and sensitivity of the SPERM HY-LITER™ method, we show a mixture of sperm from a variety of animal species, with and without human sperm, stained with SPERM HY-LITER™ (Figure 1).
Fig. 1: A mixture of canine, feline, bovine, equine, caprine, ovine, porcine, murine (left panels) and a mixture of canine, feline, bovine, equine, caprine, ovine, porcine, murine, and human sperm (right panels) were stained with SPERM HY-LITER™ and visualized using phase contrast, DAPI, fluorescein filters (top, middle, and bottom, respectively). Note that only the mixture containing human sperm is labeled in the fluorescein channel thus demonstrating species specificity of SPERM HY-LITER™ staining. Photomicrographs taken on a Leica DM2500 microscope fitted with A4 and L5 filters. Final magnification: 400X
The sensitivity and cell type specificity of SPERM HY-LITER™ is demonstrated from images provided by a crime laboratory case work validation study of SPERM HY-LITER™ (Figure 2). Here a smear slide made by a sexual assault nurse examiner from a vaginal swab collected from a sexual assault victim was stained using SPERM HY-LITER™. These types of slides are notoriously difficult for crime laboratory personnel to analyze for the presence of sperm, as the cell density, collection method, and storage conditions all conspire to destroy sperm cell morphology and inhibit KPIC staining, making standard sperm identification methods all but impossible. The series of images demonstrate the complexity of the original slides (see phase contrast image), the ability to detect sperm in the preparation (see combined phase and FITC image), as well as confirmatory steps in the process where both epithelial and sperm cells can be simultaneously identified (see combined dual cube and phase contrast image). SPERM HY-LITER™ stains sperm in all layers of the preparation.
Fig. 2: SPERM HY-LITER™ staining of a sexual assault smear slide performed by forensic DNA crime laboratory. Smear slide was stained according to SPERM HY-LITER™ supplied protocol. Photomicrographs taken with PAX-IT 2 camera using DAPI, FITC and dual DAPI/FITC cubes. Note air bubble at top of image.
The job of the forensic analyst often involves screening many items of evidence in a case. Although current forensic laboratory protocols vary, screening for sperm is usually performed with 40X objectives (400X final magnification). Here again, SPERM HY-LITER™ provides an advantage over current methods as stained preparation can be easily visualized using 10X and 20X objectives (100X and 200X final magnification) greatly increasing the field of view and therefore decreasing the time needed to scan stained slides. In fact, the signal from SPERM HY-LITER™ stained slides is such that sperm can scanned using appropriately configured fluorescent-capable stereomicroscopes (Figure 3)! The stereomicroscope can be fitted with either a traditional mercury light source or newer fiber-optic metal arc lamps, and accept the same filter cubes as traditional fluorescent compound microscopes. Given the field of view and working distance of these instruments (and therefore the speed and ease of slide manipulation on the stereo microscope), this approach promises to dramatically change the way in which crime laboratories search for sperm from sexual assault evidence.