Washing out fluids off of clothes in a crime scene

The purpose of this study was to investigate the retention of blood stains on cotton fabric after treatment and washing with four different types of detergent and two different types of pre-wash treatment. By using various types of detergent alone, then the same detergents with cleaning enhancers such as pre-wash treatments, we were able to compare and contrast the effects of each detergent and each detergent with pre-wash treatment on pure blood stains on cotton. The techniques used for this experiment included staining the cotton cloths with pure blood, performing Phenolphthalein Tetramethylbenzidine, or P-TMB and Luminol tests, and washing the cloths using a standard washer and dryer. Our results using the P-TMB test yielded more inconclusive results more than positive or negative results. The Luminol testing yielded positive results across the board. While the P-TMB test may not be the most reliable presumptive test for the presence of blood, it appears that the Luminol presumptive test for blood may play a vital role in discovering the presence of blood on washed clothing.

Keywords: Phenolphthalein Tetramethylbenzidine, Luminol, detergent, blood

Introduction

Most people do not ever expect to get caught up in a crime. Some of them are the perpetrators, some of them are the victims, and some become accessories. In any case, an altercation can happen, whether physical or otherwise. At times, the altercation can result in the unfortunate loss of life. Blood, among various bodily fluids, can accompany an altercation and the loss of too much can lead to death. Where there is blood, there must be a cleanup job as well. Many know that blood is not an easy stain to remove or hide. Blood has many stark components that make the identification of the body fluid much easier through new technologies such as presumptive and confirmatory testing.

The most common type of body fluid found at most crime scenes is blood. Blood evidence is associated with a crime that can provide essential information that may help solve a case, collaborate witness testimony, define a scene of a crime, link a suspect and scene, or point the investigation in a new direction (Tobe et al., 2007). Investigators may be presented with washed clothing that is suspected to contain bloodstains from criminal acts such as assault, homicide, or sexual assault. Suspects or victims may wash clothing following the bloodshed, thereby destroying any blood evidence and complicate the reconstruction process. Diluted bloodstains resulting from machine washing may not be visible. In such cases, the use of chemical reagents may be the only viable option for developing latent bloodstains. Presumptive tests for blood have been developed over the years and progressing into more advanced and more accurate methods. The most important component pertaining to this study is the forensic presumptive application of utilizing hemoglobin in blood as the unique characteristic that shows the results. This component of blood isn't commonly found in our everyday environment, hence why most tests rely on the peroxidase activity that hemoglobin exhibits (Tobe et al., 2007). Hemoglobin is a protein that gives red blood cells their red hue and helps them to carry oxygen. Hemoglobin is involved in the transport of O2 and CO2 in the body. Hemoglobin contains a catalytic properties that has peroxidase like activity called heme (Holland, 1974) . Peroxidases catalyze the oxidation by peroxide of a wide variety of substrates, many of which give colored oxidation products.
One of the presumptive tests for the identification of blood include the Phenolphthalein Tetramethylbenzidine, or P-TMB test. The P-TMB test is used as a presumptive, or probable test to identify if the stain that is being tested could possibly be blood or not. In 1901, Kastle and Shedd found that biological material could cause the oxidation of phenolphthalein to phenolphthalein in slightly alkaline solutions (Culliford, 1972).The phenolphthalein test uses a basic environment and can be reversed. When in a basic environment phenolphthalein is oxidized to phenolphthalein which turns bright pink. When in a basic environment, phenolphthalein reverts to phenolphthalein when ph is acidic environment. Saliva, pus, malt extract, vegetable extracts, and heavy metals such as cobalt, manganese, lead, and iron give false positive reactions (Gaensslen 1983). In 1974, a study reported on the synthesis of 3,3’, 5,5 tetramethylbenzidine and suggested its possible use in the detection of blood (Garner 1976). Beforehand the presumptive used to test for blood stains was the benzidine color test, but due to the carcinogenic nature of benzidine, it was replaced with phenolphthalein (Higaki 1976). In their study, TMB was investigated by its physical properties and its carcinogenic activity. All tumors found in rats given TMB either were benign tumors at the sight of injection (Garner 1976). TMB had been found to give blood tests approximately four times as sensitive as the benzidine test (Holland 1974). The TMB test uses an acidic environment that gives the product a bright yellow-green to green-blue color product. In this study TMB and phenolphthalein are used together, which is called a combined P-TMB test which utilizes both of their selective and specific properties. TMB is more sensitive than phenolphthalein alone, meaning fewer false positive results are indicated. However, TMB is less specific than phenolphthalein.
In 1942, a forensic scientist Walter Spech, recommended the Luminol test for use in blood detection in crime scenes and can be used for presumptive purposes (White 2011). Pathologists Proescher and Moody’s research showed that the older the stain, the longer and more pronounced the luminescence (Barni 2007). This is forensically important, because perpetrators often wash away visible signs of blood at the scene, in an effort to remove all possible evidence of the crime. The major advantage of Luminol is its high sensitivity. It can detect nanogram traces of blood that are invisible to the naked eye, which makes it up to 20 times more sensitive than any other blood detection test (Proescher 1939). Luminol is an chemiluminescent compound that produces a blue glow without ALS and an barrier filter. The reaction pathway of Luminol is the breakdown of sodium perborate. The free radicals in the hydroxyl ions catalyze the oxidation of 3-aminophthalhydrazide to 3-aminophthalate which creates a domino effect that excites the electrons moving them to the upper shell, as the electrons settle down they move the ground state and releasing the energy that emits light, which is chemiluminescent; however, this reaction only lasts a few seconds (Merényi 1990). Chemiluminescence occurs when a molecule goes to an excited state during a chemical reaction all the way to the upper shell. As it goes down to the ground state, energy in the form of light is emitted (Laux 1998).
This study was conducted in order to test the results of various detergents on dried bloodstain patterns on 100% pure cotton fabric. This was to simulate a conventional crime scene in a sense. Many clothes are 100% cotton or made with a cotton blend. Blood is a substance that has biomarkers that are very easy to identify in the lab as well as through the five senses. Because of its apparent biomarkers, it leaves behind many clues and is very hard to get rid of, giving way to the ease in use of presumptive tests to discover the presence of blood.
During the study, it was apparent that none of the bloodstains had been visibly altered to an effect where we could not see them with our own eyes. The bloodstain patterns, although no longer bright red, now grayish appearing, remained visible on the cotton fabric after washing. None of the detergents alone or with a pre-treatment altered the bloodstain patterns or had any visual effects upon the stains other than the discoloration of the stains resulting from the combination of detergents, treatments, and wash and dry cycles.
The PTMB results mostly resulted in negative outcomes, giving only three positive results. Shout with all detergents did the best job of inhibiting the PTMB test. This is due to the composition of Shout, which includes citric acid, sodium hydroxide and sodium borate. These components are defined as “ph adjusters.” Ph adjusters may change the alkaline level of the blood stain and affect the sensitivity of the PTMB test. During the soaking pre-treatment of the bloodstained washcloths in Shout, it was visibly evident that the Shout solution began lifting the bloodstain from the washcloths immediately. ECOS Natural Laundry Detergent did the second best job of inhibiting the PTMB test. It is a neutral detergent consisting of three coconut based surfactants which may have obscured the PTMB test by itself as well as with the combined with pretreats. All of the detergents have surfactants in them that aid in stain removal. Surfactants have a hydrophobic end that binds to the stain to lift it (Salager 2002). While the blood stained washcloths had residual stain marks on them, surfactants may have lifted off enough of the blood to leave too small of a concentration to yield a positive PTMB result, but not enough to yield a negative Luminol result. There were immediate strong and positive Luminol results on each washcloth tested. The entire stain fluorescenced in all of the washcloths, including the negative control. This could be due to the blood dispersal and possible residue left in the washer after each wash cycle. Errors in in this study was the negative control was washed in the middle of the procedure instead of the beginning, so residual blood may have settled in the negative control fabric, leading to the scattered fluorescence of the negative control washcloth. Most of the PTMB tests may have been negative due to unsubstantial amount of stain swabbed off the washcloths, although they were vigorously rubbed with a clean, wet cotton swab before testing.
This study could have been furthered by performing a confirmatory test, such as the Takayama Test, in order to confirm whether or not the luminescence was a direct result of blood on the washcloths or residue left behind from the detergents and/or the pre-treatments. Future studies could be done to improve and enhance presumptive blood tests to make them more sensitive and closer to acting in the fashion of confirmatory tests.
In any case, this study has concluded that no detergent or pre-wash treatment is able to inhibit the presence of blood to multiple presumptive tests such as the Phenolphthalein Tetramethylbenzidine Test or to Luminol Testing. This finding is crucial to both criminals and crime investigators.

References
Tobe, S. S., Watson, N., & Daeid, N. N. (2007). Evaluation of Six Presumptive Tests for Blood, Their Specificity, Sensitivity, and Effect on High Molecular‐Weight DNA. Journal of forensic sciences, 52(1), 102-109.
White, J. H., Lester, D., Gentile, M., & Rosenbleeth, J. (2011). The utilization of forensic science and criminal profiling for capturing serial killers. Forensic science international, 209(1), 160-165.
Barni, F., Lewis, S. W., Berti, A., Miskelly, G. M., & Lago, G. (2007). Forensic application of the luminol reaction as a presumptive test for latent blood detection. Talanta, 72(3), 896-913.
Laux, D. L. (1998). The detection of blood using luminol. Interpretation of Bloodstain Evidence at Crime Scenes, 153.
Merényi, G., Lind, J., & Eriksen, T. E. (1990). Luminol chemiluminescence: chemistry, excitation, emitter. Journal of Bioluminescence and Chemiluminescence, 5(1), 53-56.
Culliford, B. J. (1972). The examination and typing of bloodstains in the crime laboratory (No. 7). National Institute of Law Enforcement and Criminal Justice.
Higaki, R. S., & Philp, W. M. S. (1976). A study of the sensitivity, stability and specificity of phenolphthalein as an indicator test for blood. Canadian Society of Forensic Science Journal, 9(3), 97-102.
Holland, V. R., Saunders, B. C., Rose, F. L., & Walpole, A. L. (1974). A safer substitute for benzidine in the detection of blood. Tetrahedron, 30(18), 3299-3302.
Garner, D. D., Cano, K. M., Peimer, R. S., & Yeshion, T. E. (1976). An evaluation of tetramethylbenzidine as a presumptive test for blood. J Forensic Sci, 21(4), 816-21.
Proescher, F., & Moody, A. M. (1939). Detection of blood by means of chemiluminescence. J Lab Clin Med, 24(11), 1183-9.
Gaensslen, R. E. (1983). Sourcebook in forensic serology, immunology, and biochemistry (pp. 112-15). US Department of Justice, National Institute of Justice.
Laundry Detergent | Earth Friendly Products. (n.d.). Retrieved December 3, 2015, from http://www.ecos.com/ecosliquid.html#spec
Shout® Trigger. (n.d.). Retrieved December 3, 2015, from http://www.shoutitout.com/en-US/Products/Pages/shout-trigger.aspx
Salager, J. L. (2002). Surfactants types and uses. Fire p booket-E300-attaching aid in surfactant science and engineering in English. Merida Venezuela, 2, 3.