Body fluid identification is important in the field of forensic science as it can provide valuable information to an investigation. An accurate method for detecting blood at a crime scene or on evidence is beneficial to an analyst or investigator. A piece of evidence may be any house-hold object or material; therefore, a test must be able to accurately detect blood on a variety of substrates. The most common preliminary testing method for blood is based on the peroxidase-like activity of hemoglobin. Tests such as phenolphthalein (Kastle-Meyer), Ortho-Tolidine (O-Tol), and Luminol utilize this method. The sensitivity of presumptive blood tests was evaluated using a series of diluted bloodstains on six fabrics: fleece, felt, linen, denim, flannel, and terrycloth. In addition to a direct testing method, two indirect methods were tested utilizing a piece of dry filter paper or a moistened cotton swab. The last portion of this study compared commercial field kits to the laboratory-prepared reagents. This study yielded overall sensitivities for Kastle-Meyer, O-Tol, and Luminol of 1:1000, 1:5000, and 1:10000, respectively. The direct testing resulted in a slightly lower sensitivity with fleece versus the other fabrics. Fleece also resulted in slower and weaker reactions compared to thinner fabrics such as denim, linen, and terrycloth. This suggests that highly absorbent fabrics, such as fleece, can have a negative effect on the sensitivity of catalytic color tests such as Kastle-Meyer and O-Tol. The indirect testing methods utilizing a moistened swab or a dry filter paper were less sensitive compared to direct testing methods. The field kits tested in this study mimic the methods of a moistened swab technique, and the results demonstrated that the field kits were about the same sensitivity or less sensitive compared to the indirect testing methods.

Blood is considered one of the most widely tested biological matrices. The first step in blood identification involves visual examination followed by presumptive testing. Once a positive presumptive result is observed, confirmatory tests are performed to determine that a stain is human blood, thus reducing the time and resources spent on forensically irrelevant samples. When interfering agents are present, this general workflow is hindered as presumptive tests can render false-negative results. General awareness of these interfering agents can help analysts to recognize forensically relevant evidence that may have otherwise been deemed immaterial. The main objective of this study was to understand various interfering agents and their effects on presumptive blood tests such as Kastle Meyer (KM) and Orthotolidine (O-tol) reagents and confirmatory tests such as HemaTrace® and Rapid Stain Identification (RSID™) Blood. Additional experiments explored the effects on downstream DNA analysis. In the first part of the study, bloodstains in varying concentrations were exposed to ten chemical interferents over a period of time to understand how blood dilution, age of the stain, and the chemical nature of the interferent affect presumptive blood test results. Antioxidants, active oxygen, and tannins are known to interrupt the mechanism of presumptive tests. Thus, ten interfering agents (ascorbic acid, chlorogenic acid, catechin, sodium percarbonate, hydrogen peroxide, oxalic acid, proanthocyanidins, quebracho extract, chestnut extract, and theaflavin) were selected based on these characteristics. Six blood dilutions (neat, 1:10, 1:50, 1:100, 1:500, and 1:1000) were exposed to the interferents, and presumptive tests for blood were conducted on six days (day 1, 8, 22, 43, 71, and 106). The second part of the study examined bloodstains deposited on real-world samples (wines, citrus fruit juices, teas, coffee, cleaning agents, and leather products) containing chemical interferents. In addition, confirmatory testing for human blood was conducted with HemaTrace® and RSID™-Blood on day 106 using the 1:500 dilution. Finally, DNA analysis of 1:10 dilution stains was performed on day 150 to study whether downstream DNA analysis was compromised due to the presence of the interferents. The results showed that as blood concentration reduced, more false-negative results were observed when chemical interferents were present. Further, chemical interferents produced frequent atypical color changes in tests with KM and O-tol reagents, while only some atypical color changes were observed with the household products tested. Immunochromatographic assay results indicated both HemaTrace® and RSID™-Blood could detect the presence of blood when interfering agents are present, although the positive result bands with RSID™-Blood were faint and sometimes difficult to visualize. Poor DNA results from the untreated blood sample limited any interpretation of the DNA results obtained from bloodstains deposited on household products. Overall, the data indicates that valuable blood evidence may be overlooked due to faint or false-negative results when these interferents are present. Future studies should focus on how these interferents may affect downstream DNA analysis.