An Analysis of Body Fluids in Forensic Science

Introduction:

Forensic science is the application of scientific techniques to mainly criminal cases which are being investigated by the court of law for delivering justice to the victim. It is also known as ‘Criminalistics’ and is a crucial body of research that plays a key role in finding strong evidence for/against the accused. The branch of forensic science which deals with the identification and examination of body fluids is known as ‘Forensic Serology’. This particular branch is one of the most important fields of research in a criminal investigation but is often overlooked until the very last moment in a lot of cases.

What are Body Fluids?

 What is meant by ‘body fluids’ if someone may ask, we often assume it means practically ‘any’ fluid that is secreted or is present within the human body. In a general thought, we all will say the same but scientifically we can say the answer to this is half right, and hence it is important to know what is ‘half wrong’. The fact that there is a difference between the terms “bodily fluids” and “body fluids” may come as a surprise to many but that is how it is. The term “bodily fluids” refers to the samples taken directly from the human body and don’t need to be identified and analysed as we already know the person they are from and what exactly the fluid sample is. The term “body fluids”, on the other hand, refer to the samples taken from surfaces outside the human body such as crime scenes and here it is very necessary to identify and analyse the sample as to which body fluid it exactly is and from whom. This process is called Body fluid identification or BFID for short.

Body Fluid Identification is done both at the crime scene as well as in laboratories. To understand the dynamics of these, problems faced as well as the advantages of both these approaches to BFID we first need to understand which body fluids are generally tested, which of them have what kind of tests and where etcetera.

Body Fluids Subject to Analysis

Body fluids that are subject to analysis in forensic science are blood (normal and menstrual), semen, saliva, and vaginal material. All other fluids are considered “bodily fluids” as defined before and do not have any registered tests based on them as they generally are considered less favourable samples. Examples of such fluids are Urine etc. These are only taken into testing if all other “body fluids” are falling short or non-existent in a case. This BFID can also be broken down into another name and process which is called DNA profiling. DNA profiling is the analysis of DNA samples to know the species as well as the individual it belongs to. It is indeed valid to ask the question that if we all humans possess a similar DNA pattern then how is it possible to conduct this procedure? The answer to this is rather simple. All humans indeed have similar DNA patterns but every single individual inherits unique genetic material from their parents. As a result, some regions of DNA strands differ greatly between all humans and these are also known as polymorphic regions. This difference helps to identify a particular individual from another.

Types of Identification Tests for Body Fluids

Every single fluid has a different process of identification and examination. These processes are broadly classified into two types i.e. Presumptive and Confirmative tests.

Presumptive tests are also known as field tests and screening tests. These just show the possibility of a body fluid being present but do not confirm the presence of the specific fluid or its nature. For instance, it may tell that the sample is possible of semen but cannot confirm it or tell whether it is from a human or animal. These tests are very easy and quick to perform as well as they can be used to find samples that are not visible through the naked eye.

These are not very expensive to carry out and also are convenient enough to be carried out in a large area. Hence these tests can be conducted on the crime scene itself.

Confirmatory tests, on the other hand, confirm the exact identity of the substance. These can be one single test or a series of different tests put together. These tests are time-consuming and can cost quite a lot as compared to presumptive tests. However, these are accurate and reliable and also not overly sensitive, unlike presumptive tests.

Presumptive and Confirmatory Tests of Body Fluids

Blood (Presumptive Tests)

1. Kastle Meyer Test: This test was named after the American chemist ‘Joseph Hoeing Kastle’ who discovered this particular test. It is also commonly known as the Phenolphthalein test. In this test phenolphthalein is reacted with the sample and after some time hydrogen peroxide is added. If the sample turns pink rapidly it is said to have tested presumptive positive for blood. This works on the principle that Phenolphthalein reacts with hydrogen peroxide in the presence of haemoglobin to turn from colourless to pink[1]. The reason it is a presumptive test is that it has known to have given fake positive results from samples like saliva, pus etc.
2. Alternative Light Sources: As the name suggests, certain types of lights with different wavelengths are used to search for bloodstains. The stains tend to glow or darken depending upon the wavelength of the light used. This increases the amount of evidence to be gathered which an average scientist would have normally missed due to whatever reason.
3. Luminol Test: It gets its name from a chemical compound called “Luminol” (C8H7N3O2)[2] is applied to surfaces and the surface is seen under UV light. This particular chemical when reacted with another chemical called hematin which is formed and found in ageing stains of blood turns the spot with the stain glowy. The older the stain is, the more it glows.

Blood (Confirmatory Tests)

1. ABAcard HemaTrace Test: ABAcard is a testing device that is used to identify the possible presence of haemoglobin (a component found in human blood). This test is an antigen-antibody reaction.[3] The antibodies are present on the HemaTrace card and if the antigen (the heme group of the haemoglobin molecule) is present, then an antibody-antigen reaction occurs on the test site (T) of the card.[4] A positive result for human haemoglobin is evident by the formation of a pink dye band.
2. Takayama Test: This test is named after a Japanese chemist named ‘Takayama’ who also discovered the same in 1912.  It is also called the Hemochromogen crystal test. In this test when the Takayama solution is applied to a sample/stain of blood it reacts with the iron present in the heme group present in haemoglobin and forms a crystal salmon pink in colour confirming the presence of blood. This test, however, is unable to determine the species of origin of the sample. One drawback of this test is that it needs the sample in a large amount. A minimum of 0.1 milligrams of haemoglobin is needed to conduct this test.
3. Rapid Stain Identification (RSID) Test: In this test, a particular group of unique antibodies are used to detect the presence of a component called Glycophorin A which is found in RBC membranes in blood. As a result, the strips used to apply the antibodies to the sample show a colour change and confirm the presence of blood.
   Rapid Stain Identification tests are available for a lot of body fluids and just like the name suggests give fast and accurate results. However, even if the name of the test remains the same for all body fluids the procedure conducted and components used to identify every fluid are different.

Semen (Presumptive Tests)

1. Prostate-Specific Antigen Test: The male prostate gland produces a particular antigen called Prostate Specific Antigen or PSA for short. This antigen helps in conveying the possibility of the presence of semen. The reason it is just a presumptive test is that it can also give fake positive results as bodily fluids like breast milk and sweat sometimes test positive in this test.
2. Alternative Light Sources: Similar to as we saw in the presumptive blood test, this test also exists for checking the possibility of the presence of semen on the crime scene. Different lights of different wavelengths will give different coloured glows of the semen stain/sample. A lot of external factors like the nature of the surface and other biological molecules can interfere and make it extremely inaccurate but it can still be used for getting a general idea.
3. Walker Test: This test is also known as the Brentamine spot test and Acid phosphatase test. The male prostate produces an acidic enzyme called ‘Acid phosphatase’ (AP). The item is blotted with a damp sheet of filter paper, then sprayed with the substrate and the colour reagent; the appearance of coloured regions indicate the potential location of semen stains which can be then subjected to further testing.[5] In simple words, this test works based on the reaction AP in the presence of brentamine fast blue and Alpha-Naphthyl acid phosphate gives a purple colour in less than a minute.[6]

Semen (Confirmatory Tests)

1. Christmas Tree Stain Test: In this test, a combined effort of two different reagents which are Picroindigocarmine and Kernechtrot are used for visual recognition of sperm cells. The former component stains the tail of the cells in a blue-green colour whereas the latter stains the head of the cell reddish-pink and hence, due to these colours it got its name as Christmas tree stain test. This test has to be conducted very quickly due to the fragile and sensitive nature of sperm cells.
2. Rapid Stain Identification (RSID) Test: This test is considered the sure shot way to identify and examine the semen sample. In this test, an antigen called semonogelin which is unique to human semen is targeted to be identified. Due to its ‘unique’ nature, there is no confusion with other bodily fluids of males or females or even semen samples from other species. The advantage of this test is that it can work perfectly on a sample that has been stored in poor conditions and hence is a very reliable test amongst forensic scientists.

Saliva (Presumptive Tests)       

1.  Iodine Test: The suspected sample is incubated in the presence of saline water at normal body temperature.[7] Since saliva contains an enzyme called ‘alpha amylase’ which is responsible for breaking down starch when we eat food, this enzyme is targeted for identification. Iodine solution is added after some time to check for a deep blue colour. If the sample gives the colour then it is said that there is the possibility of the presence of saliva since it broke down starch and proved the existence of alpha-amylase. However, since this is just presumptive there are indeed some drawbacks. The alpha-amylase enzyme is also present in some other body fluids but at comparatively lower concentrations[8]. Hence, this affects the accuracy of the test and it is not specific for saliva.
2. Alternative Light Sources: Even Saliva, just like blood and semen as seen before will glow under UV light and other lights of different wavelengths.

Saliva (Confirmatory Tests)

Phadebas Amylase Test: Phadebas is an artificial biochemical substrate that has certain microspheres made of starch also which are chemically bonded to a bluish dye. As a result, when this substrate is applied to the sample it gives blue colour proving that the salivary amylase present in the saliva digested the starch in the substrate and return confirming the presence of saliva.

Case Law Examples

In People v. Cumblee[9], where the body of Kathy Twarowski was found in a Geo tracker covered in blood, her pants unzipped and open along with a large splattering of blood on the rear seat (driver side) it was seen that the plaintiff who was a former police officer murdered her due to relationship problems. While investigating the case, the authorities performed a luminol test as well as a phenolphthalein test at the defendant’s residence as it was just within 1 mile of the site of the murder. Both of the tests came out to be positive. However, the court after the defendant’s objection confirmed that as both luminol and phenolphthalein tests are presumptive tests, the authorities were told to conduct confirmative tests after. The confirmative tests came negative. As a result, due to lack of additional confirmatory evidence that blood was present in the defendant’s home did not render the test results inadmissible in this case and the defendant was denied a Frye hearing.

In People v. Hernandez[10], the complainant/deponent reported that at approximately 4:45 a.m she felt a hand between her legs and when she woke up the defendant was standing at the end of the bed retracting his hand.

She further stated that she was 15 years old and did not give permission or consent to the defendant to touch her in any way, shape or form. When the sexual assault examination was conducted and the complainant’s clothes were sent to the laboratory it was found that PSA (Prostate-Specific Antigen) on the complainant’s underwear and vulvar swabs, sperm cell fraction on underwear and presumptive presence of amylase on a vulvar swab.

Conclusion

Body Fluid Identification has helped and saved thousands of innocent victims around the world. By being the strongest form of evidence in cases of murders, rapes and many more this field of forensic science restored the honour of and saved lives of thousands. Over the last few years, many advancements have improved the ways of analysis which is very important for finding out, even more, superior and convenient techniques. Techniques such as Biospectroscopy which includes methods like Raman and Fluorescence spectroscopy that provide quick, accurate and less or non-destructive identification & examination of body fluids are leading the ways to a better and safer future.

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References:

[1][1] Ellen Genovesi, Laura Blinderman & Patrick Natale, Blood Detection using the Kastle Meyer test, March 19, 2021 , https://bio.libretexts.org/Learning_Objects/Laboratory_Experiments/General_Biology_Labs/Book%3A_Unfolding_the_Mystery_of_Life_-_Biology_Lab_Manual_for_Non-Science_Majors_(Genovesi_Blinderman__Natale)/10%3A_Protein_Gel_Electrophoresis/10.1%3A_Blood_detection_using_the_Kastle-Meyer_test

[2] Luminol, https://pubchem.ncbi.nlm.nih.gov/compound/luminol

[3] Bureau of Criminal Apprehension (BCA), 2^nd line under ‘HemaTrace’, https://dps.mn.gov/divisions/bca/bca-divisions/forensic-science/Pages/dna-serology.aspx

[4] Id at BCA, line 2,3,4,5

[5] 2^nd Edition, G. Sensabaugh, Encyclopedia of Forensic and Legal Medicine, 2016

[6] Blood and Bodily Fluids, under presumptive tests for Semen, Acid Phosphatase Test, 2^nd point 5^th line onwards, https://forensicresources.org/forensic-disciplines/blood-bodily-fluids/

[7] Sangeetha Balakrishnan, Body Fluids in Forensic Analysis, March 3^rd, 2017 , https://www.slideshare.net/mobile/SangeethaBalakrishna2/body-fluids-72772131

[8] Id under heading ‘Saliva’

[9] People v. Cumblee, No. 2-93-0923, Appellate court of Illinois, 1995

[10]People v. Hernandez, 28397 NY Slip Op, 2018