Tests for identification of body fluids

Examination of body fluids or serology involves presumptive and confirmatory tests. It is important to note that presumptive tests broadly examine a “class characteristic” i.e. characteristics that may be common to a wide range of substances and therefore, are not specific in nature. Due to this, presumptive tests often lead to false positives.

False positives occur when test results indicate the target substance to be present even when it is absent, therefore giving wrong results. Presumptive test results are indicative of a possibility of the substance being present and cannot confirm the presence of the substance. Therefore, it is essential to conduct confirmatory tests and a serological report should not solely rely on the results of a presumptive test. It is important to note that DNA profiling is not a confirmatory test for detection of body fluids as it cannot determine the type of body fluid from which the DNA profile originates.

False positives in case of presumptive tests for blood:

• Tetramethyl Benzidine test also known as the TMB test is one of the most common presumptive tests for blood. TMB is a modification of the Benzidine test (as benzidine is carcinogenic) and both these tests work on the same principle. Both these tests are used for detecting the peroxidase activity found in blood. The haemoglobin present in blood contains iron and is responsible for the red appearance of blood. Haemoglobin in our blood has peroxidase like activity, which means that it can catalyse (hasten/assist) the breakdown of a peroxide (like Hydrogen peroxide) and release an oxygen molecule, which on combining with the test reagent (TMB or Benzidine) results in a visible colour change. Here, blue colour will appear once reagents have been added to blood. It is important to note that ‘peroxidase like activity’ is a class characteristic and substances other than blood can provide the same result as blood. TMB gives false positive results for horseradish, garlic, green grapes, red grapes as well as typing and recycled paper. Certain studies have shown that TMB gives false positive results for saliva as well. Benzidine gives false positive results for ginger, garlic, coriander, onion, tomato, cabbage, beet root, spinach, guava, banana, cucumber, sugarcane, rust, ferrous sulphate and shoe polish as well.¹
• Phenolphthalein works on the same principle as the TMB and Benzidine test. Also known as the Kastle Meyer test, the reagent here is Phenolphthalein which, in the presence of peroxidase like activity, produces pink colour. It gives false positive results with horseradish, potato, turmeric, malt extract, cobalt, manganese, iron, lead, saliva and pus.¹
• Luminol is a compound that is sprayed onto a suspected area and observed under Ultraviolet light (UV light). The compound reacts with haematin, a substance found in aged bloodstains, to produce a strong blue fluorescence that can only be observed under UV light. Luminol provides false positive results in case of copper salts, brass, bronze, and other copper containing alloys.²

False positives in case of presumptive tests for semen:

• Acid Phosphatase test is a presumptive test for detection of semen. It is also known as Walker test or Brentamine spot test. In the presence of the test reagents, Acid Phosphatase, an enzyme found in semen, produces dark purple colour. Similar reactions can be observed for substances other than semen and false positive results have been reported for certain varieties of tea as well as vegetables such as cauliflower. Vaginal secretions and other body fluids also contain small levels of this enzyme and can give false positive results.³
• Prostate specific antigen (PSA) testing is a presumptive test that detects the presence of this antigen in semen. PSA is produced in high amounts by the male prostate gland. Detectable levels of PSA are also found in breast milk, and female urine and give false positive results.⁴
• In case of the Florence test, a substance containing Choline produces dark brown crystals in the presence of reagents. These can be observed under the microscope. Choline is found in large amounts in semen but can be found at detectable levels in other animal tissues as well. False positives for Florence test can be observed for meat and dairy products, as Choline is a nutrient that is found across different animal tissues.⁵

References

1. Ashutosh Sharma, Sensitivity of phenolphthalein and benzidine tests in field of forensic sciences, International Journal of Research Culture Society, (2017)
2. Wendy J. Koen and C. Michael Bowers (ed), Presumptive and confirmatory blood testing in Forensic Science Reform, Protecting the Innocent (2017)
3. Ajay SR, Priyanka V, Hiren V, Priyanka M. Study on acid phosphatase enzyme activity in semen mixed with various body fluids, J Forensic Sci & Criminal Inves (2019)
4. SERATEC, PSA in body fluids
5. Gaensslen, R. E., Sourcebook in forensic serology, immunology, and biochemistry, Washington, DC: US Department of Justice, National Institute of Justice (1983).