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.
Based on the results of the Alternate Light Source study, ALS(UltraLite) was superior at enhancing the biological fluids than the UV was. This could be explained by the differences in wavelengths of each device and the wavelengths within the fabric being tested. The dilutions of the biological fluids were difficult to visualize under UV light, making ALS more sensitive in detecting the Flavins in the fluids. Flavins are present in seminal fluid in far higher concentrations than other biological fluids, but their presence in other fluids allows them to fluoresce as well when being screened with UV or ALS. This lead to many of the positive results shown in table 1. It is also noted how strong of a fluorescence each sample had. All of the neat samples of the fluids tested yielded positive results on both UV and ALS, possibly due to it having a higher concentration of flavins. As more dilute samples were used, more negative and weaker positive reactions occurred, possibly due to a lower concentration of flavins within the fluids.
There are limitations to using any forms of alternate light, such as the fact that many other types of materials can fluoresce and give false positives. These materials fluoresce because of the different molecules they contain in them that will react with the light. ALS also require the use of filters to protect the eyes, while UV light can be used and visualized with the naked eye. Both of these methods successfully enhanced biological fluids and are a powerful tool when screening for semen at a crime scene, especially when performed with the acid phosphatase test and microscopic identification of semen.
For the acid phosphatase test, every dilution of seminal fluid yielded positive results. With each dilution increase, the reaction took a longer time to give a positive result and results went from dark purple to light pink. This could be explained by each dilution having less of a concentration of acid phosphatase. Seminal fluid mixed with bleach gave negative results, which could be due to the bleach killing the sperm cells within the seminal fluid. Seminal fluid mixed with blood gave a positive reaction. The only other positive result was yielded by the mixture of vaginal fluid and dilute blood. The following post-test observations were recorded: mayonnaise, neat saliva, and neat vaginal fluid all turned light pink after some time. Neat vaginal fluid, originally, should have given positive results and did after some time, probably due to the possibility of acid phosphatase being present in vaginal fluid. The reason the reaction may have taken longer could have been due to the size of the sample or the concentration of acid phosphatase within the sample. The acid phosphatase test was specific in reacting with seminal fluid, but there were still some positive results by other biological fluids. This test is useful in narrowing down the amount of samples from a crime scene possibly being seminal fluid. This test is still only a presumptive test and needs to be done in conjunction with a confirmatory test in order to conclude that a sample is seminal fluid.
The confirmatory test performed was microscopic identification. On the 1:5 blood/semen mixture, it appeared to look more like blood than semen. No semen was found in this mixture. For the 1:5 semen dilution, there were shapes found, but no spermatozoon. This could be due to the fact that the seminal fluid was diluted. The neat semen yielded the desired observations. Only one spermatozoon with a tail was observed. There was another sperm head observed with no tail in the neat semen sample. The other sperm head could have been degraded throughout time or from the K-PICS staining, which is why no tail was found.
A study was conducted comparing three staining methods. The three methods studied were the Christmas Tree, hematoxylin-eosin, and alkaline fuchsin. The study consisted of 174 consenting women and accurately knowing the date of their last sexual intercourse. 3 slides were prepared from each swab sample and were stained with the three different methods. The results from this study concluded that the Christmas Tree Stain was the most useful test in the first 72 hours (6).
The tests done in this study proved to be effective. Of the screening and presumptive tests, no test alone was enough to accurately identify semen. The components that each test reacts with are found in other biological fluids, making it difficult to use just one test, but the combination of both tests helps screen for semen more effectively and allows for more positive identification when used with a confirmatory test. None of these tests are perfect, but through them forensic scientists have been able to use their findings in sexual assault cases. It’s imperative that we continue studying and developing better techniques so that we can become quicker at identifying semen in a sexual assault case and more accurate. Some of the issues with the accurate techniques we have now are that they are time consuming and expensive. Sometimes, it’s the amount of sample used up is too much. Further studies can be done to come up with a way that are efficient and inexpensive. The science behind the identification of semen has come along way, but still has a long way to go.
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