Category Archives: Bodily Fluids

Forensic Tests for Semen: What you should know

By Maher Noureddine, Ph.D., President: ForensiGen, LLC

Second in the series: Bodily Fluids and Forensics

First, the biological facts about semen:

Seminal fluid is a complex mixture of secretions from at least four male urogenital glands.  The seminal vesicle gland contributes approximately 60% to this mixture, the prostate gland contributes approximately 30%, and the combined contribution of the epididymis and bulbourethral glands account for the remaining 10%. Dave Corriher & Jennifer Pietila, Human Biology Presentation (UNC-Asheville, 2008).

An average male ejaculate measures around 3.5 milliliters.  Each milliliter can contain between 10 and 50 million sperm cells.  This number can vary with the age of the male, and can be negatively impacted by medical conditions, genetic background, diet, and other habits such as smoking and illicit drug use. Some males in the population have a condition known as oligospermia, which defines an abnormally low sperm count. Aspermia refers to another condition where the affected male produces no sperm. Deficient sperm production may be affected by factors such as radiation and other environmental toxins, undescended testis, varicocele, trauma, drug effects or other factors. Randine Lewis, Ph.D., Male Factor Infertility, ACU-Denver Medical Article #RL-05 (2003).

Vasectomy, which is a surgical sterilization option, renders the male incapable of producing sperm somewhere between two and four months following that procedure. Pamela J. Schwingl & Harry Guess, Safety and effectiveness of vasectomy, 73 Fertility and Sterility 5, 925 (2000). Vasectomized, oligospermic, and aspermiac males can still produce normal amounts of seminal fluid containing both prostate gland and seminal vesicle secretions which are detectable by forensic laboratory tests as described below. M. Hochmeister et al, Evaluation of Prostate-Specific Antigen (PSA) Membrane Tests for Forensic Identification of Semen, 44 Journal of Forensic Sciences 1057 (1999).

Confirmatory Tests for semen:

1- The Christmas Tree Stain: The most reliable confirmation for the presence of semen is the positive visual identification of sperm cells (or spermatozoa) using the Christmas tree stain. Click here to read the NC State Crime Lab’s procedures for semen analysis.

Two main reagents are used consecutively to produce this distinctive stain: Picroindigocarmine stains the neck and tail portions of the sperm in green and blue, while Nuclear Fast Red (also known as Kernechtrot) gives the sperm heads a red color and the tips of the heads, an area known as acrosomal cap, a pink color.  Although this color pattern seems quite unique and may render sperm cells easily distinguishable under a microscope, sperm cells tend to deteriorate quickly after ejaculation. Jean-Paschal Allery et al, Technical Note: Cytological Detection of Spermatozoa: Comparison of Three Staining Methods. ASTM International (2001).

The sperm tails are most susceptible to damage and will break down first.  Therefore, the analyst must be trained to make visual distinctions between sperm heads and other types of cells in the mix, particularly mucosal or epithelial cells whose nuclei will also stain red. Once ejected from the body, sperm survival will depend on the surrounding environment and type of surface. It has been shown that intact sperm (sperm that retains the cap and tail sections) can be recovered from a vaginal cavity for a period of time following intercourse.  That time will depend on many physiologic factors. Intact sperm can also be recovered from surfaces and fabrics if the semen dried up quickly before natural breakdown occurs.

2- RSID-Semen Strip Test:  The RSID-Semen test provides sensitivity as well as specificity to human semen. B.C.M. Pang & B.K.K. Cheung, Identification of human semenogelin in membrane strip test as an alternative method for the detection of semen, 169 Forensic Science International 29-30 (2007). Similar in format to a pregnancy test strip, the RSID-semen test identifies the presence of the seminal vesicle-specific antigen, or semenogelin. Id. at 28; Jennifer Old et al,  Developmental Validation Studies of RSID-Semen A Lateral Flow Immunochromatographic Strip Test for the Forensic Detection of Seminal Fluid, Independent Forensics, Rev. D 3, 3 (2010). This antigen is unique to semen, and therefore, there is no cross reactivity with other bodily fluids in males and females or with semen from other mammals. Id. at 3, 31. This test can also identify semen even if the stain was stored under less favorable conditions which have been shown to affect other tests such as the Acid Phosphatase test. Id. at 10-11.

Presumptive tests for semen:

1- Visual and Alternate Light Tests:  If the area to be examined and analyzed for semen is larger than an individual swab, forensic scientists resort to visual identification first. Clothing, undergarments, and bedding can be quickly surveyed for potential semen stains using the naked eye. Dried semen stains are often off-white to faint yellow in color. Semen can also be visualized using blue light, ultraviolet light (also known as Wood’s Lamp), or a modern light source such as CrimeScope that is properly configured with optimum wavelength filters. Under those specialized lights, semen will fluorescence due to the presence of molecules such as Flavin and Choline-conjugated proteins. The color of this fluorescence will vary from blue to yellow, depending on the light equipment used. There are many molecules (natural and artificial) that will fluoresce in a similar way as semen, and therefore, this detection technique is highly presumptive. Furthermore, not all semen stains will fluoresce under such specialized lights.  Exposure of the sample to factors such as heat, humidity, oxidizing agents, and microorganisms such as bacteria and mold can affect this fluorescent activity.  Semen fluorescence can also be masked by certain types of fabrics and fabric treatments. Hilton J. Kobux, D.Phil., Edmund Silenieks, and Jordana Scharnberg, B.Sc., Improving the Effectiveness of Fluorescence for the Detection of Semen Stains on Fabrics, 47 Journal of Forensic Sciences 4 (2002); S. Marshall, A. Bennett, and Dr. H. Fraval, Locating Semen on Live Skin Using Visible Fluorescence, Rofin Australia Pty Ltd. (2001).

2- Acid Phosphatase test:  The male prostate gland produces and secrets into semen a high amount of the enzyme acid phosphatase (AP).  Using a standard chemical reaction, a forensic laboratory can analyze a given stain for the presence of this enzyme. In the presence of Alpha-Naphthyl acid phosphate and Brentamine Fast Blue, AP will produce a dark purple color in less than a minute (test is also known as the Brentamine spot test). The shade of this purple color will depend on the activity of the enzyme, which can be negatively impacted by the age of the stain and the storage conditions. The test for AP remains highly presumptive due to the fact that vaginal secretions and other bodily fluids contain detectable levels of this enzyme.

Non-semen AP enzyme reactivity is markedly slower when using the above mentioned spot test, owing to the fact that not all AP enzymes in the body are equal in their activity level. AP activity has been detected in dried samples years after the stain was deposited.  However, moisture and heat will result in the breakdown of AP in a matter of days. Richard Li, Forensic Biology: Identification and DNA Analysis of Biological Evidence (2008). Analyses of post-coital vaginal swabs show that AP activity will markedly decrease after 24 hours and diminish after 48 hours. Jean-Pascal Allery et al , Rapid detection of sperm: Comparison of two methods, 10 Journal of Clinical Forensic Medicine 5, 6 (2003).

3- Prostate Specific Antigen:  Another presumptive test for semen is the detection of prostate specific antigen (PSA) or the P30 molecule.  Forensic labs utilize a test known as ABAcard or P30 test to screen for PSA. (This test was previously used by the SBI lab, but is no longer used). PSA is produced in high amounts by the male prostate gland. Manfred Hochmeister etal, Evaluation of Prostate-Specific Antigen (PSA) Membrane Test Assays for the Forensic Identification of Seminal Fluid, 44 J Forensic Sciences 1057 (1999). However, this antigen can also be found in very small amounts in fecal material and sweat.  Studies have shown PSA can also exist in female urine and breast milk.  A recent study identified that the majority of women have a glandular structure surrounding the urethra that is similar to the male prostate gland.  This structure was shown to produce PSA in detectable amounts. Stefan Schmidt et al, Prostate-Specific Antigen in Female Urine: A Prospective Study involving 217 Women, 57 Urology 717-720 (2001). While the PSA test remains a strong test for the presence of male semen, caution is always urged when interpreting positive PSA results which are not confirmed by the actual presence of sperm. Dale L. Laux, M.S. and Sarah E. Custis, Forensic Detection of Semen III. Detection of PSA Using Membrane Based Tests: Sensitivity Issues with Regards to the Presence of PSA in Other Body Fluids at 6.

Additional notes on pre-ejaculation fluid:

Pre-ejaculation fluid originates from a male anatomic structure known as the bulbourethral gland (also known as the Cowper’s gland) and functions as a natural lubricant during intercourse. In the absence of full male ejaculation, what is the forensic significance of this fluid? It is widely accepted that pre-ejaculation fluid can contain traces of acid phosphatase and prostate specific antigen; although no evidence for the semen specific antigen semenogelin has been found to date. There is still debate on whether sperm is expected to be present in pre-ejaculation fluid. Most scientists agree that the presence of sperm will depend on the individual male, and that pre-ejaculate sperm can be attributed to previous full ejaculation in that male. Stephen R. Killick et al, Sperm content of pre-ejaculatory fluid, 14 Human Fertility 1, 48-52 (2011); Zvi Zukerman et al, Short Communication: Does Preejaculatory Penile Secretion Originating from Cowper’s Gland Contain Sperm? 20 Journal of Assisted Reproduction and Genetics 4, 157-159 (2003).

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Forensic Tests for Saliva: What you should know

By Maher Noureddine, Ph.D.

First in the series: Bodily Fluids and Forensics

You may encounter cases in which the prosecution claims that SBI tests establish the presence of human saliva on the victim as part of its proof that an assault occurred. This type of evidence is often seen in sexual assault cases.  In cases like this it is important to understand how the SBI or law enforcement tests for saliva and how the problem of false positives limits those tests.

The non-invasive nature of saliva sample collection has earned that bodily fluid significant medical attention. Saliva tests can reveal certain disease markers, viral infections, and the presence of therapeutic as well as illicit drugs in the body. Saliva is rich in the enzyme alpha-amylase (a.k.a. α-amylase, salivary amylase, or Ptyalin), an enzyme that breaks down complex carbohydrates into smaller sugar molecules. The biological function of this enzyme was first described by Erhard Friedrich Leuchs in 1831.

Almost 100 years later (1928), the German investigator B. Mueller conceived the use of alpha-amylase as a forensic target to validate the presence of saliva on a given surface. This idea gained popularity, and for the next 50 years several investigations endeavored to refine and enhance the methods to test for alpha-amylase. However, some important caveats continue to limit this test as a presumptive or screening test for human saliva, even to this day. Alpha-amylase is an enzyme that has changed very little throughout the process of evolution: alpha-amylases from bacteria, fungi, or chimps are very similar in structure and function to that of the human alpha-amylase. In humans, there are at least four variants (or versions) of alpha-amylase, two of which are found in saliva, and the other two are secreted in the pancreas. Those variants are almost indistinguishable at the enzymatic activity level. Because the presumptive test for saliva detects the enzymatic activity of alpha-amylase, and not alpha-amylase molecule itself, that test will yield a positive result if any alpha-amylase enzyme is present, regardless of the organism it came from.

Crime labs, including the SBI lab (See p. 13 of their protocol here) use a reagent (chemical) called Phadebas to conduct this presumptive test for alpha-amylase. This test is relatively cheap, quick, and highly sensitive to any alpha-amylase enzymatic activity. Myers, J. and Adkins W., Comparison of modern techniques for saliva screening, 53 J Forensic Sci 4 (2008).  However, it is important to keep in mind that this test alone cannot confirm the presence of human saliva because this presumptive test will give a positive result if the alpha-amylase enzyme from any organism is present.

Laboratory tests for saliva remained presumptive until the late 1980s, when a group of researchers in Japan succeeded in developing a monoclonal antibody that is specific for the alpha-amylase variant that is present in human saliva in particular. Ito, K. et al., Preparation of human salivary alpha-amaylase specific monoclonal antibody, 97 J Biochem 5 (1985). Therefore, instead of testing for enzymatic activity, now we can detect the alpha-amylase molecule itself, and specifically, the alpha-amylase from human saliva. This ushered the development of test kits that are now being used in forensic laboratories around the world to screen for human saliva (known to many as Lateral Flow Immunochromatographic Strip Test or Rapid Stain Identification (RSID) Saliva kits).  The SBI lab used a combination of the presumptive Phadebas test and the RSID test to “confirm” the presence of human saliva (See p. 14 of their old protocol here).  The SBI lab protocols have been updated recently and reflect important changes in interpretation language.  Under current protocols (see p. 2, available here), the SBI lab acknowledges that the RSID test for saliva is a presumptive test.

The RSID-Saliva kits have been tested on samples from various types of surfaces such as paper, cigarette butts, plastic and glass bottles, and metal cans. The specificity of RSID-Saliva kits has been scrutinized by researchers. Although RSID-Saliva kits were found to be sensitive and specific to human saliva, positive reactions were also noted in samples containing alpha-amylases from  mammals such as  gorillas and rats. Positive reactions were also noted in other bodily fluids such as semen, blood, vaginal discharge and sweat. See Old J. et al., Developmental Validation Studies of RSID-Saliva Lateral Flow Immunochromatographic Strip test for the forensic detection of Saliva, Independent Forensics (2010); Pang, B. et al., Applicability of Two Commercially Available Kits for Forensic Identification of Saliva Stains, 53 J Forensic Sci 5 (2008). The RSID-Saliva test gives positive results from breast milk, likely because the presence of alpha-amylase aids the nursing infant in food digestion. High reactivity of this test is also observed in samples containing human feces. Not surprisingly, humans swallow copious amounts of saliva, which travels through the entire digestive system and, as a result, alpha-amylase from saliva (as well as alpha-amylase from the pancreas) is thought to reach the colon where it can mix with fecal material. You will note that the SBI lab’s procedure is to not use this test on rectal samples. Reactivity was also noticed in urine samples, a result that remains inconsistent between studies. Butterworth, P. et al., Human α-amylase and starch digestion: An interesting marriage, Starch 63 (2011). These limitations clearly demonstrate why the RSID-Saliva test is presumptive in nature.

It is important to consider these limitations when examining or reviewing tests for the presence of the assailant’s saliva based on swabs from sexual assault kits. Improper swabbing and other factors relating to personal hygiene, personal behavior, and indirect saliva transfer from mouth to surface can result in “false” positives. Since saliva can harbor mucosal cells from the perpetrator, the validation of the saliva sample can be coupled with forensic DNA analysis (STR/Y-STR) to rule in or rule out the connection between an individual and the forensic evidence. If DNA analysis not completed, it is worth questioning whether the positive test for saliva could be caused by personal hygiene practices or another innocent explanation for the presence of the salivary amylase.

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Bodily Fluids and Forensics: Introduction to the Series

By Maher Noureddine, Ph.D. and Sarah Rackley

The field of forensic investigation continues to reap tremendous benefits from advancements made in various scientific disciplines including physics, chemistry, biology and others. The detection and analysis of biological molecules have been at the forefront of this advancement, even to the level of revolutionizing forensics as we know it. Arguably, DNA takes center stage as the molecule with the most impact. There are, however, other biological molecules that have played a significant role in forensics for many decades, and will continue to do so for years to come.

In this blog series, we will highlight such molecules, the bodily fluids they lurk in, the important issues relating to their detection and analysis in forensics, and what you might want to know when confronting this type of evidence. The bodily fluids we will examine include: saliva, blood, and semen.

We will start this series on Monday by tackling salivary amylase, the biological molecule that is found in saliva and the main target for analysis when examining saliva as a forensic sample in cases such as murder, kidnapping, sexual assault, and others. Stay tuned for Dr. Noureddine’s first post in this scientific series.

Dr. Noureddine is a doctor in molecular genetics with extensive background and experience in scientific research and training in human genetics. He has a B.S. in Biology from Radford University in Virginia, an M.S. in Molecular Biology from the University of North Carolina at Greensboro, and a Ph.D. in Molecular Genetics from the University of North Carolina at Chapel Hill. He completed a postdoctoral fellowship at Duke University Medical Center (The Center for Human Genetics), where he published many articles on the genetics of Parkinson Disease and other human genetic disorders. He was also a Senior Fellow at the National Institute for Environmental Health Sciences/National Institute of Health, where he conducted research in cancer using advanced methodologies in genomics. His expertise include specialized training in complex genomics, DNA fingerprinting, SNP analysis, mitochondrial genetics, and state of the art methodologies in genetic variations in the population. He owns and operates ForensiGen, LLC, a consulting company that specializes in DNA forensics, biological evidence, and disease genetics. His profile can be found here. His email address is mnoureddine@theiacd.com.

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