Comparative study of presumptive and confirmatory tests for detection of blood on serial dilutions and washed stains

Background and aims: Detection of blood from blood stains is the first crucial step for forensic analysis, such as DNA profiling. After committing the crime, the criminal tries to destroy evidence such as blood stains by washing their clothes or other circumstances found at the crime scene make the blood or blood stains diluted due to washing by water or detergent. This study aimed to identify the most sensitive presumptive test among phenolphthalein, tetramethylbenzidine (TMB), benzidine, leuco-malachite green (LMG), and luminol for the washed and serially diluted blood and bloodstains and confirmatory test of blood among Takayama, Teichmann, and Wagenaar. Materials and methods: In this study, serially diluted blood, stains of the serially diluted blood, and blood-stained clothes were prepared and subjected to different kinds of washings. Blood was detected using reagents of phenolphthalein, TMB, benzidine, LMG, and luminol with variable protocols reported in various literatures. The samples were further tested for the confirmatory tests of blood using Takayama, Teichmann, and Wagenaar tests. Results: It is observed that in presumptive testing of serially diluted bloodstains, luminol shows positive results in all (1:10 to 1:100,00,000) followed by TMB (1:10000). TMB in acetate buffer solution works better for liquid blood; however, solution of TMB in glacial acetic acid works better for stains. LMG solution with zinc has higher sensitivity as compared to a solution without zinc. Conclusion: Luminol is the most sensitive presumptive test for detecting blood on washed and diluted bloodstains, and sensitivity decreases with an increase in wash cycles. Among confirmatory tests, the Takayama test is more promising than Teichmann and Wagenaar test.