The identification of the nature of biological traces is based on two types of tests, both of which are capable of invalidating subsequent analysis steps:
Presumptive tests: rapid, simple to perform and inexpensive but not very sensitive and non-specific tests, capable of generating false positives by cross-reacting with various exogenous substances. They provide an initial indication of the presumed nature of the biological trace under examination.
Confirmatory tests: highly specific and sensitive but more expensive tests. They do not cross-react and therefore allow identification of the nature of the biological trace.
Blood presumptive tests
COMBUR Test: The test is based on the colour change of an indicator in the presence of an organic hydroperoxide. This colour change is mediated by a peroxidase reaction and is catalysed by haemoglobin and myoglobin. Therefore, the buffer reacts by colouring green in the presence of peroxidase activity of haemoglobin.
Positive faults: plant peroxidases, rust, various oxidants, etc.
HemaStix: Strips contain: Diisopropylbenzene, Dihydroperoxide, Tetramethylbenzidine. The test exploits the peroxidase activity of haemoglobin which cuts oxygen molecules from the hydrogen peroxide molecule catalysing the reaction from the colourless reduced form to the coloured oxidised form of tetramethylbenzidine. Colour change ranges from orange to green and high blood concentrations lead to an intense blue colour.
Disadvantage: interferes with the binding of magnetic beads to DNA during the subsequent analytical step.
Luminol: Luminol is used to determine and detect traces of blood, even washed or removed. Using the natural property of luminescence, it becomes luminescent by reacting with hydrogen peroxide. Iron, present in the haemoglobin of blood, acts as a catalyst in luminescence. The colour of the reacting Luminol is blue and lasts about 30 seconds and requires near darkness to be detected. The mode of use of the test is to use an aqueous solution consisting of a mixture of sodium carbonate, sodium perborate and luminol, appropriately mixed together just before use; the resulting solution is then sprayed onto the surface being tested.
False positives: Cu, bleach, blood of animals, plants and certain types of soil.
Important drawback: it is a toxic substance, so it must be used with appropriate protection!
BLUESTAR® FORENSE has a stronger and more durable luminescence that does not require total darkness to be visible. Good quality images can be obtained with a normal camera and film. With a little practice, BLUESTAR FORENSE® makes it impossible to get confused between blood and false positives as the luminescence differs in colour, intensity and duration. BLUESTAR FORENSE® does not alter DNA and allows both subsequent DNA typing and ABO typing. BLUESTAR® FORENSE thus works on fresh blood as well as very old or altered bloodstains, pure or diluted. Commercial formulation similar to Luminol that is more stable and easier to use. The solution does not have to be prepared shortly before deposition, does not require total darkness and the chemiluminescence lasts longer. In the presence of a presumed blood trace, the intensity of chemiluminescence is higher; it is lower in the presence of a false positive.
HEXAGON OBTI: Presumptive test used to check whether the blood trace is of human origin. Human haemoglobin (hHb) in the sample reacts with a reagent consisting of red-coloured particles and monoclonal anti-hHb antibodies. The immune complex migrates into the test zone where it is captured by an immobilised secondary antibody directed against hHB. In this case there will be a line indicating a positive result (T). In order to confirm the positivity of the test, the control strip, on which other antibodies contained in the biological fluid but not specific for the desired target are immobilised, must also be positive.
The test detects whole blood up to a dilution of 1: 1,000,000. Minus 500 red blood cells are required for a positive result. A positive result is not evidence of the presence of Hb, but indicates a high probability.
Hexagon Obti does not react with: chickens, cattle, ducks, horses, goats, sheep, etc. Works well with degraded and dated material, but produces false negatives.
Advantages: Very high sensitivity; Specific for human blood; Very low risk of false positives; Fast and practical.
Blood confirmatory tests
RSID Blood Test: Monoclonal antibodies directed against glycophorin A, an integral membrane protein present in the plasma membrane of erythrocytes. Advantages: high sensitivity, specificity and speed. Specific for human blood, does not react with blood from other animals (e.g. primates etc.).
Glycophorin A: integral membrane protein present in the plasma membrane of erythrocytes. Does not react with other body fluids and blood of other animals.
Sensitivity: less than 1 µl.
Saliva confirmatory tests
RSID Salvia Test: Antigen-antibody reaction, specific for human salivary antigen (amyA).
Sensitivity: ˂ 1 µl saliva. False positives: maternal milk
Approximately 2/3 of cases involving forensic genetics concern sexual offences. Semen traces can be characterised by Acid Phosphatase, Prostate Specific Antigen (PSA or P30), Semenogelin, and Sperm Cell Detection.
Sperm confirmatory tests
RSID Semen Test: Antigen-antibody reaction: specific for semenogelin, a protein secreted in the seminal vesicles.
Specificity: does not cross-react with other biological fluids and with biological fluids of other animals (chimpanzee, gorilla, dog, mouse etc.).
Sensitivity: <1 µl seminal fluid.
Urine confirmatory tests
RSID Urine Test: Antibody directed against Tamm-Horsfall protein, secreted by tubular cells. Does not cross-react with blood, saliva, seminal fluid/sperm, vaginal fluid and menstrual blood. Sensitivity: ~ 10 µl (highly variable); Not species-specific.
About The Author
Ms. Chiara Lucanto completed her Master Degree in Forensic Biology at University of Calabria – Italy. She is a Certified Specialist of “Forensic Biology Survey” and also a researcher at the Bio Forensics Research Center.