Bodily Fluids Analysis – The Forensics Library (2023)

The detection and identification of body fluids at a crime scene can provide essential information as to the events which may have occurred and the people involved. The presence of body fluids, their location and DNA profile obtained from them can supply police agencies with a wealth of information to aid the investigation.

Unfortunately, body fluid analysis can be a lengthy process. Visualisation techniques may be employed at an incident scene to initially locate potential body fluids, for instance alternative light sources (ALS). An ALS utilises different wavelengths of light to visualise otherwise invisible or ambiguous stains, with some even having the ability to show stains that have been wiped clean or covered by paint. However the use of alternative light sources will not typically allow for differentiation between different body fluids. Once potential biofluids are located in-situ, presumptive tests may be used to indicate the possible identity of the stain, and following sample collection confirmatory analysis and DNA analysis will often be conducted. Unfortunately there is no single method for the analysis of all body fluids, and it may be necessary to employ multiple techniques depending on the type of biofluid.

Blood
Undoubtedly the most common body fluid of interest encountered at crime scenes, blood is primarily composed of water, along with blood cells, proteins, minerals, hormones, glucose and many metabolites. Due to the obvious importance of blood in a forensic context, a range of tests for identifying blood has been developed. Initially presumptive tests may be used at a crime scene to establish whether a suspected stain is in fact blood. Luminol, perhaps the most well-known presumptive test, results in a distinctive blue luminescence in the presence of blood, based on the oxidation of luminol by haemoglobin. Similarly, Fluorescein is based on the heme-accelerated oxidation of fluorescin to fluorescein, though requires an alternate light source to visualise the change. Another well-known presumptive test for blood is Phenolphthalein, also known as the Kastle-Meyer test, which causes an alkaline solution to take on a pink colouring in the presence of blood. Leucomalachite green, or LMG, is another popular presumptive test, resulting in a distinctive green colour change in the presence of heme in blood. In addition to these, there are also a number of commercially available immunological tests for blood, including Heme Select, ABAcard and HemaTrace. The number of presumptive tests available for the detection of blood is seemingly endless, and different techniques may be employed throughout different countries, and even between different police forces. However the tests are, as the name suggests, only presumptive, and further analysis may be necessary to confirm the identity of a suspected bodily fluid.

The Rapid Stain Identification (RSID) test is an antibody-based method that can be used to confirm the presence of blood based on the detection of glycophorin A. Furthermore, enzyme-linked immunosorbet assays (ELISA) can be used to both identify blood and differentiate between different blood groups. Certain crystal tests may also be used to confirm the presence of blood, such as the Teichman test, which is based on the formation of hematin, or the Takayama test, which uses the formation of hemochromogen crystals. Additional analytical techniques can also be applied to blood identification, such as microscopy for the identification of blood cells, or spectroscopic methods such as UV-vis and fluorescence spectroscopy.

Saliva
Saliva can be very variable in terms of composition, particularly due to recent food consumption or even the time of day saliva is produced. Typically it is made up of almost entirely water, along with electrolytes, epithelial cells, mucus, proteins, enzymes and various exogenous and endogenous metabolites. Although perhaps less common, saliva may also be encountered during a criminal investigation and, as with blood, may be initially visualised using alternative light sources.

Unfortunately there are significantly fewer techniques for the identification of saliva. Perhaps the most widely used type of presumptive test is based on the presence of amylase, an enzyme found in various body fluids but present in greater amounts in saliva. The starch-iodine test will result in a blue colour change when in contact with saliva, caused by the presence of starch produced from the breakdown of salivary amylase. Similarly, the Amylose Azure test produces a blue colour change following hydrolysis in amylase. Another commonly used test for saliva is the Phadebas test, also directed towards the detection of salivary amylase. A Rapid Stain Identification (RSID) test has also been developed for the detection of saliva, specifically designed to focus on the presence of salivary amylase.

Urine
Primarily composed of water along with urea, organic salts, proteins, hormones and a huge range of metabolites, urine may be encountered in a forensic context in cases of abuse or sexual assault, for instance. Urine is a difficult body fluid to detect and, although it will typically fluoresce under alternate light sources, the fact that it is generally very dilute makes it problematic to establish a sufficiently sensitive and reliable test.

Some developed presumptive tests for urine are based on the presence of urea, an organic compound found in urine in high concentrations. This type of presumptive test is often based on the enzyme urease, which breaks down into urea and releases ammonia and carbon dioxide in the process. The DMAC test is perhaps one of the most common methods for detecting urine based on the presence of urea, producing a pink or magenta colour if positive. Creatinine is another compound found in high concentrations in urine, thus a number of tests have attempted to utilise this for urine identification tests. The Jaffe test utilises picric acid which, in the presence of creatinine, forms a red compound known as creatinine picrate. The colour change is proportional to the concentration of creatinine present. Similarly, the Salkowski test produces a blue colour in the presence of urine via a reaction between sodium nitroprusside and creatinine when heated. Finally, the immunological RSID test has also been developed for the detection of urine, based on the presence of a urinary protein known as the Tamm-Horsfall protein

Semen
Semen is a viscous fluid secreted by the testes, containing a variety of constituents including sugars, enzymes, lipids, and of course spermatozoa. In a forensic context, the presence of semen is of particular importance during the investigation of sexual assaults, so understandably a wide range of tests have been developed for its presumptive and confirmatory identification following visualisation by ALS.

The tests developed for the identification of semen are largely based on the presence of certain enzymes. Perhaps the most common presumptive tests aims to detect acid phosphatase, an enzyme produced in large amounts by the prostate, resulting in an observable colour change if positive. Unfortunately this test can also react with vaginal acid phosphatase, resulting in false positives. Other tests have been developed based on the detection of other enzymes, such as the leucine aminopeptidase test, however these are not commonly utilised.

Confirmation that a sample is semen can typically be achieved using simple microscopy to identify the presence of sperm cells, usually using a technique known as the Christmas tree stain, which allows the heads of the sperm cells to be visualised. Of course in some situations this test is useless, for instance if the donor has had a vasectomy or if they are azoospermic (in which the semen contains no sperm cells). If this is the case, additional tests are available to help confirm the identity of the sample. Numerous commercially available test kits are based on the detection of prostate-specific antigen (PSA), however understandably it is possible for male urine to give false positive results. Another immunological method is the rapid station identification test for semen, which is based on the detection of semenogelin, a primary protein in human semen.

Vaginal Fluid
Vaginal secretions may consist of a mixture of fluid secreted through the vaginal walls, cervical mucus and residual urine. Although this particular bodily fluid may is not commonly encountered at most crime scenes, it can be of vital importance in the investigation of sexual assault cases. Unfortunately the development of a means of identifying vaginal secretions has proven difficult, particularly as the chemical composition of this bodily fluid can change throughout the menstrual cycle.

Perhaps the most common method is based upon the detection of glycogenated epithelial cells using a periodic acid Schiff reagent, resulting in the cells being stained a magenta colour. The intensity of this colour relates to the concentration of the cells present. However a major downfall of this particular method is that post-menopausal or pre-pubescent females may not show glycogenated cells in their vaginal secretions, rendering the test useless.

Some older methods were based on the presence of certain enzymes, such as vaginal peptidase and lactate dehydrogenase isoenzymes, however these tests are not widely utilised. Research has also aimed to develop methods of identifying vaginal secretions based on levels of specific bacteria and based on RNA profiling, however in short no reliable method has so far been produced.

A vast number of techniques have been developed for the identification of body fluids in a forensic context, ranging from presumptive tests for a quick, in-situ estimation of the likely identity to laboratory-based confirmation tests. However there are various problems associated with existing techniques. The primary concern with all presumptive tests is the lack of specificity, as the tests will often react with a range of different substances and so false positives are an issue. Furthermore some of the tests are not particularly sensitive and so small amounts of a body fluid may result in false negatives. Finally, presumptive and confirmatory tests are typically destructive to the sample, obviously not ideal if only a small quantity of evidence is available in the first place.

As a result of this on-going research is aiming to develop non-destructive, sensitive and specific body fluid identification techniques.

Virkler, K. Lednev, I. K. Analysis of body fluids for forensic purposes: From laboratory testing to non-destructive rapid confirmatory identification at a crime scene. For Sci Int. 188, 1-17 (2009).