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Body fluid examination

Vivek Sahajpal, SFSL Shimla, limitations of the tests used in identifying and examining different types of body fluids.
So bodily fluids, body fluids and their stains are of biological evidence for forensic, serological and DNA analysis and may be useful in solving crimes. The most common biological fluids in forensic analysis are blood, seminal fluid and saliva. Sometimes urine stains, fecal material are also related to assault crimes as well. The identification of bodily fluids can be carried out using presumptive and confirmatory tests to identify the type of bodily fluid in question. So initially, we go for the presumptive assays, but they have certain issues. They are not specific and are used just for the purpose of screening. They are definitely rapid, sensitive and simple.
Positive reaction indicates possibility of presence of a bodily fluid in the questioned sample, but it does not confirm it. After this, we need to go for the confirmatory tests. So confirmatory tests, they are basically more specific for the bodily fluid in question. They provide higher certainty than the presumptive assays. Once the stain has been identified, the evidence is tested to determine whether it is blood of human origin or not. Non-human samples which are unrelated to the investigation can be excluded. Further investigation proceeds after this. So, the next level is the grouping where we try to identify the ABO type of blood groups, depending on based on the ABO blood system.
So as far as blood is concerned, we have two type of assays to identify whether it is blood, these are basically the presumptive test, and then we have the confirmatory assays. So the presumptive test, they are basically based on the peroxidase activity, which is an enzyme which is present inside the blood. So there are three or four common tests which are used for identification of blood, which is basically the presumptive test. One is the Kastle Meyer test or the phenolphthalein test. Then is the Benzidine test, which is based on the tetramethyl benzidine and the Leucomalachite Green test, which is based on the leucomalachite green.
So basically what happens is the peroxide enzyme present inside the blood, it oxidized the H2O 02 to release nascent oxygen, which interacts with phenolphthalein to give a pink colour, interacts with tetramethyl benzidine to give a bluish colour. Similarly interacts with the leucomalachite green to give a greenish colour. So if these colours are formed, it is a preliminary indication that the sample is possibly a blood sample. The next presumptive test, which we can use for identification of blood are the luminol test and the fluorescent test, which are basically based on the fluorescence. And in this case, the oxidized fluorescent emits intense yellowish green fluorescent light, which indicates the presence of a blood stain.
After the preliminary test, we go for the confirmatory tests to identify whether a particular stain is blood or not, in a confirmatory way. So we use the Takayama test and the Teichmann test. In case of the Takayama test, the blood stain is treated with pyridine and glucose, and there is formation of crystals. Similarly, in the Teichmann test, the blood stains are treated with glacial acetic acid and salt and subsequently heated. So that leads to the formation of hematin chloride, which has a prismatic, brown coloured crystal. And this crystal is specific to blood samples, so this is basically a confirmatory test for the presence of blood.
Another important biological fluid, which is found in case of, specifically in case of sexual assault cases, is semen. So identification of semen is again based on the preliminary test and confirmatory test. So preliminary tests, they are basically based on uses of different light sources. The seminal stain, it can be visualized under a UV light or under an argon laser light where it gives a particular colour and fluorescence. The next level of testing is using the colour test, and the commonly used test is the acid phosphatase test. So acid phosphatase test is basically based on the acid phosphatase enzyme, which is present in the case of semen samples. So a formation of purple blue colour indicates the presence of semen.
However, this is not a confirmatory test. In order for confirmation, we go for the next level of testing. Once a biological stain or a fluid or a sample has been indicated to be semen, then we go for the confirmatory test. In case of semen samples, the confirmatory test is a microscopic test. Serological examination has its limitation. First of all, the biggest limitation of serological examination is that it consumes a significant amount of the biological evidence. And many times what happens is that significant amount of evidence or a stain is consumed, and very little amount of evidence is left for the further DNA analysis. The second drawback of serological technique is that these are not universal techniques.
For every type of fluid, you require a new type of test or a different type of test. So in case of semen, a test is different. In case of blood, the test is different. As such, a number of tests need to be performed for these type of biological evidences. So it is challenging to identify biological fluids at crime scene because these tests are not easy to perform at scene of crime. The preliminary test, like the benzidine test or the luminol test, can be done at a crime scene. But confirmatory tests, they cannot be done at the crime scene. It’s difficult to do them. Further, it is very much time consuming because you have to perform end number of tests.
You have to go for preliminary tests, then you have to go for confirmatory tests and then you have to go to different tests for different type of body fluids. So this is also very, very time consuming. The next major limitation of the serological test is the false positive results, specifically in case of blood samples and in case of semen samples. We know that the test, which are used for identification of blood. They are based on the peroxidase activity, so any sample which has peroxidase activity can give a false positive result. For example, luminol can give a false positive result with household bleach, some metals and some vegetables.
Similarly, horseradish, potatoes and some lichens, they can also give false positive results for the benzidine test, and these tests, therefore should be confirmed with the precipitant test. Similarly, the acid phosphatase test, which is used for identification of semen, can also sometimes give false positive result. For example, the vaginal secretions, certain contraceptive creams, they show activity, which gives false positive results as far as the acid phosphatase test is concerned. The acid phosphatase enzyme is present in vaginal secretions also, but in a lower concentration. But sometimes during testing, you may find that it gives a very strong result. So it appears that the sample is a semen sample, but actually it is not.
So hence, it needs to be confirmed with the microscopy, whether semen is present or not.

In this video, Dr Sahajpal introduces you to the commonly examined body fluids. He will also explain the different tests conducted for identification of blood and semen, and the limitations of these tests.

As you watch the video, recall if you have come across serological reports pertaining to identification of body fluids in your casework. Did they mention the tests used for identification of the body fluids? If yes, were those tests presumptive or confirmatory? Share your comments below.

*References for images

Certain images in this video are sourced from ‘ Li, R., Norman, S., & Schober, J. : Forensic biology, CRC Press (2015)’.

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Decoding Forensics for Legal Professionals

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