ABSTRACT: The purpose of this study was to become acquainted with methods commonly used for the detection and identification of human semen. Among these methods were the use of ALS (alternate light source) to enhance and detect semen, a presumptive test for the detection of semen known as the Kaye’s test, and confirmatory tests by microscopic analysis. Comparisons of different the ALS were done and it was concluded that the ALS was more efficient in enhancing semen than UV light was. The results from the Kaye’s test deemed the test to be efficient and specific for the detection of semen. The microscopic analysis test was also effective in identifying semen.
KEYWORDS: Forensics, serology, semen, seminal fluid, semen dilutions, Kaye’s test, acid phosphatase, presumptive tests, confirmatory tests
INTRODUCTION:
What is semen? Semen, also called seminal fluid, is a fluid that is emitted from the male reproductive tract. Seminal fluid contains cells responsible in the fertilization of female eggs, known as sperm cells. (1) Sperm cells play a crucial role in sexual assault cases, either by corroborating a person’s story or by providing a DNA profile. Sperm cells weren’t always used as evidence, though. The first scientist to ever describe the morphology of a spermatozoon was Van Leeuwenhoek in 1677. Leeuwenhoek found the bodies of spermatozoon to be oval shaped with a thin tail that was six times as long as the body (2). Although the spermatozoan was discovered in 1677, it took some time for it to be accepted that the cells found were unique to seminal fluid. Because of this sperm cells were not sought as evidence of semen presence in the case of sexual assault. This was also due to having no suitable methods for identifying seminal fluid and the fact that examinations of victims were done by matrons, rather than by physicians or scientists (1). One of the earliest tests, performed by Mathieu Orfila, were based on the appearance of the stains, changes in color and consistency, and odor given off by stains that were moistened. These stains were compared to other biological fluids, such as: nasal mucus, saliva, and vaginal secretions. Testing of seminal fluids have come a long way since then.
Some of the newer methods for screening and testing for seminal fluids are from ALS (Alternate Light Sources), the Acid Phosphatase Test, and P30 cards. These are methods used by current forensics and are very efficient and accurate. Alternate Light Sources help screen for semen by reacting with molecules called Flavins within the seminal fluid, causing the stain to fluoresce. (3) This method is only a presumptive method due to the fact that many other samples, other than seminal fluid, will fluoresce because of the flavins contained within them. Though this is not specific to seminal fluid, it does a great job of enhancing stains that may be semen.
The Acid Phosphatase Test, also known as the Kaye’s test, another presumptive test, is very commonly used in forensics lab for the detection of semen. The male prostate gland produces an enzyme, acid phosphatase, which is secreted into semen (4). The mechanism behind the acid phosphatase is that in the presence of -Napthyl Acid Phosphatase and diazo dye, acid phosphatase breaks down the substrate, creating a free phenyl phosphate. The free phenyl phosphate reacts with the diazonium salt, producing a dark purple color indicating a positive result. Although acid phosphatase can be found in other biological fluids, seminal fluid has a higher concentration of acid phosphatase than any other biological fluid, thus making the Acid Phosphatase test a suitable presumptive test for semen.
Another presumptive test that is frequently used is the P30 Test. This test is for the detection of prostate specific antigen. This antigen can be found in very small amounts in other biological fluids but is found in higher concentrations in semen, deeming it suitable as a presumptive test (3). According to the Journal of Forensic and Legal Medicine, the P30 test is highly sensitive for both fresh and frozen diluted samples of semen samples and specific for the protein it tests for (5).
A confirmatory test used to identify semen by cell morphology is the K-PICS test, also known as Christmas tree staining. Using the K-PICS reagent, the suspected seminal stain is stained to enhance visualization of the sperm cells. The tails are stained green and the heads are stained red, giving the test the name of Christmas tree staining. The morphological properties of sperm cells are unique to sperm and allows for positive identification. All of the microscopic identification for this test are done under phase contrast and brightfield microscopy.
The positive identification of semen is so crucial in sexual assault cases. It helps to corroborate a person’s story or to provide a DNA profile to link to a person. None of the tests we have today would have come to fruition had it not been for studies analyzing the effectiveness, sensitivity, and selectivity of screening, presumptive, and confirmatory tests. The following study was done to become familiarized with these methods and relate them to previous studies.