Chemical enhancements for detection of bloodstains

Chemical enhancement for the detection of bloodstains Abstract The evaluation of any forensic technique is dependent upon the detection, observation, measurement and validation of data through experimentation. The credibility of forensic science has suffered in the past due to bad science. It continues to suffer until this day as there hasn't been much change in methodology, application, practice and research. The work carried out by the criminal justice community during investigations in crime scenes has unfortunately been on the basis of a hit-and-trial method. Forensic sciences and especially bloodstain analysis need to move more towards a scientific foundation and standardization to have a better foothold in the scientific community. This article will review the components of blood, basic principles of blood detection like peroxidase reaction with hemoglobin and biochemical reactions behind the major serological tests for blood stains. The idea is to understand the effects that dilution and substrates have on the observed sensitivity of the tests. The limitations of these tests will also be reviewed and discussed. The intention of this research review is to present a careful and balanced evaluation of detection methods for bloodstains, and making appropriate suggestions for applying the research in case work, as dependent upon type of blood used in the research. Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains Table of contents 1. Introduction 2. Composition of bloodstains 2.1. Red blood cells 2.2. White blood cells and platelets 2.3. Blood plasma 3. Reaction Mechanism of Chemiluminescence in Blood 4. Methods of chemical enhancement of bloodstain detection 4.1. Confimatory Tests for Laboratory 4.2. Presumptive Tests Used in crime scenes 4.2.1 Luminol 4.2.2. Bluestar® Forensic 4.2.3. Fluorescein 4.2.4 Phenolphthalein/Kastle-Meyer Test 4.2.5. Lecuo malachite green 4.2.6. Leuco crystal violet 4.2.7. DAB 4.2.8. Alternative Light Source 5. Substrates and Environments 5.1. Blood detection in Soil 5.2. Blood detection in layers of paint 5.3. Blood detection on porous substrates 5.4. Blood detection on nonporous substrates 5.5. Blood detection on dark surfaces 5.6. Blood detection in arson crime scene 5.7. Fingerprints in Blood 5.8. Foot impressions in blood 5.9. Effects of presumptive tests on aged bloodstain 6. Comparison of presumptive tests of bloodstain Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains 7. Discussion and Conclusion References Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains 1. Introduction Bloodstains are one of the most common substances found at the crime scene. They are critical information for any forensic case. Its identification and enhancement with different chemicals helps a lot in analyzing the nature of the blood and its interaction with its surroundings. Usually blood is not detectable at a crime scene for a variety of reasons, which may include; time, weather, and attempts by the perpetrator to clean the crime scene. In such cases, it is important to use forensic chemical enhancements for the detection of blood to discover the location of latent stains. This information is critical in determining subsequent confirmation of blood in the laboratory with the help of forensic DNA analysis [3,33]. Blood can be detected by chemical and biological tests. This article investigates chemical enhancement and subsequent detection of bloodstains with different techniques. Chemical enhancement and detection tests are dependent on the presence of hemoglobin in blood and thus it will give positive results for both animal and human blood [12]. Most chemical enhancement serological tests performed at the crime scene are presumptive tests, while tests performed in the laboratory for further forensic analysis are confirmatory tests. They include confirmation of the nature of concentrated or aged bloodstain, and the detection of non-visible bloodstains on different substrates like fabric and paint or enhancement of weak bloodstains with the help of alternative light source and IR photography [20]. This article compares and contrasts the relative sensitivities, specificity of laboratory and commercial preparations of various chemical enhancement reagents. This comprehensive study also examines the relative sensitivity of each reagent in regards to differing substrates, ranging from smooth fabrics to Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains carpet, tile and hardwood, porous and nonporous surfaces, and the effect of colors and dyes on the substrate in altering the observed sensitivity of the tests. Results indicate that observed sensitivity varies depending on commercially prepared versus homemade preparations, and, as seen in other studies, that sensitivity is drastically impacted by the substrate that the latent bloodstain is present upon [12,46,50]. Bloodstain enhancement is particularly important in instances where footprints, fingermarks, hand prints are made in blood. The chemical enhancement methods are useful as they can enhance the stain which in turn can be visualized and analyzed properly. 2. Composition of bloodstains Bloodstains originate from droplets of whole blood that are dried out. Whole blood contains blood cells, proteins and amino-acids; suspended in a liquid called blood plasma. There are three major types of blood cells and cell fragments: red blood cells, white blood cells and platelets. The subsequent paragraphs help us understand the dynamics and biochemical properties of blood which are then utilized in chemical enhancement of blood [58]. 2.1. Red blood cells Red blood cells have the highest concentration in blood. It makes up 97% of the blood's dry content. It does not have a nucleus and thus it does not contain DNA. Hemoglobin, the oxygen carrying protein, is the main component of blood Hemoglobin can occur in different forms, called hemoglobin derivatives. It is about 45% of total volume of blood. It also contains an iron containing protein which binds to 97% of the oxygen in the body [58]. 2.2. White blood cells and platelets Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains The amount of white blood cells in blood is less compared to red blood cells (1:700) [58]. It is produced in the bone marrow and its nucleus contain DNA and RNA which is used for DNA analysis in blood [58]. 2.3. Blood plasma Blood plasma consists of many serum proteins along with clotting factors, water, electrolytes and various other biochemicals. Albumin and immunoglobins are important components as they are speciespecific and serve as the basis for many biological based tests for blood identification. Some of the serum proteins have genetic variation which is useful bio-marker in DNA analysis [1]. 3. Reaction Mechanism of Chemiluminescence in Blood Blood is not a naturally fluorescent substance, nor are most of the blood print enhancement techniques [2,28]. Chemiluminescene is described as emission of light as a form of energy as a result of a chemical reaction. The light emitted in such reactions differs in intensity, lifetime and wavelength, with the spectrum of wavelength varying from near infra red through visible light to near ultraviolet [3]. The intensity of emission from the chemical reaction is usually dependent upon the rate of reaction and efficiency of the production of intermediate species which when form final products, release energy in the form of light [3]. Heme protein is specifically the hemoglobin protein in red blood cells. Hemoglobin, is a hemoprotein, which in the presence of H2O2 acts as peroxidase [1]. These peroxidase when exposed to an environment of chemical enhancement reagents cause chemiluminescence which helps in the detection of blood at the crime scene. For example, chemical enhancement reagents like luminol and fluorescein Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains act on the heme group in a substrate, releasing free oxygen radicals, which in turn yields a visible response. In the case of luminol, 3-aminophthalhydrazide is oxidized to 3-aminophthalate, releasing energy in the form of visible light [3]. Whereas in the reaction of fluorescein with blood, fluorescin is oxidized to fluorescein, which fluoresces when under a 450 nm alternate light source [15-18] . These are the tests that will be analyzed in this review of chemical enhancement of bloodstains. Though the visible colour change is indicative of the presence of blood but it is important to realize that that there are numerous other oxidizers like enzymes and metals which can produce the same result with any chemical enhancement reagent. This is one of the primary reason that these tests are taken as the presumptive tests for blood. Some of the reagents which are used for chemical enhancement of blood are; Luminol, Bluestar® Forensic, Leuco-malachite green, Leuco-crystal violet, Amido Black, and DAB. 4. Methods of chemical enhancement of bloodstain detection Biological specimens when found in the different forms are exposed to identification tests as the basic step of forensic examination. It is important to know the presence or absence of bloodstains when investigating criminal cases. Biological tests like antigen-antibody tests are more specific in nature, and they can be specie-specific. Though they take longer time to perform but they are more confirmatory tests in nature. At the crime scene, chemical tests are usually preferred over biological tests as they are much faster and they can provide preliminary basis for further testing required for identification. Bloodstain analysts make such detection on the basis of these three tests; crystal, catalytic and chromatographic Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains tests for blood. Chromatographic techniques like HPLC and crystal methods like pyridine are more confirmatory tests in nature while a catalytic test like luminol is more presumptive in nature [1]. 4.1. Confirmatory tests for laboratory Forensic DNA analysis for different biological evidence require context. Presumptive tests conducted at the crime scene need to verified further with the help of confirmatory tests. Confirmatory tests are used to identify the biological sample after it has been screened with a presumptive test., which is then typed for subsequent analysis. A positive confirmatory test is needed to identify rigorously blood [1]. Crystal and chromatographic tests conducted in the laboratory are confirmatory tests for blood. In crystal tests, blood reacts with specific salt reagents to form heme crystals, which is indicative of blood. This method might be used in a laboratory rather than at a crime scene, as many different evidence are in need to be collected in limited time. Pyridine is such reagent which forms crystals on contact with the bloodstain [46]. 4.2. Presumptive tests used in crime scenes Presumptive tests are generally quick and sensitive but not necessarily specific at all times. Presumptive tests for blood are catalytic tests which are based on the fact that heme of hemoglobin possesses catalytic activity and it can act as a peroxidase i.e. it speeds up oxidation reaction with hydrogen peroxide [36]. False positives are possible for such tests and thus a confirmatory test is needed to establish a level of confidence in the biological sample collected from the crime scene. 4.2.1 Luminol Luminol (C8H7N3O2) is one of the presumptive tests that is used mostly at the crime scenes by Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains investigators. It has been around for a while now and thus a lot of study has been done on the chemical structure of luminol, different formulation and its effectiveness in enhancing blood on different substrates. It follows the principle of hemoglobin and its derivatives to enhance oxidation in presence of an alkaline medium [21]. It exhibits chemiluminescence, with a striking blue glow, when mixed with an oxidizing agent like hydrogen peroxide [37]. Luminol is a yellow crystalline solid at room temperature. It is sensitive to light, combustible, incompatible with strong oxidizing agents, strong acids, strong bases and strong reducing agents [2]. Luminol solutions are sensitive to light and they are stable for only 8-12 hours [21]. It can be used locate traces of blood at crimes even if it has been cleaned or removed [34,35]. DeHaan and Adair [9,22] has found it to be useful for porous surfaces . The reaction mechanism is based on the iron present in hemoglobin and its catalysis with the help of hydrogen peroxide in Luminol. Half-life of luminol`s luminescence is between 20-40 seconds [2, 35] which means that the light emission period is very short and more luminol needs to be sprayed subsequently. In Luminol, 3- aminophthalhydrazide is oxidized to 3-aminophthalate, releasing energy in the form of blue light [35]. 4.2.2. Bluestar® Forensic Blood enhancement reagent, Bluestar®, is a modified form of the luminol molecule which produce a very bright chemiluminescence-based blue-light emission [4,10,19]. Bluestar® tablets (all tablet kit) are usually diluted in 125 mL of deionised water to make a solution of Bluestar® [10]. Bluestar® chemiluminescence intensity is not as great as that of Luminol but the life time of the intensity is greater than that of Luminol [4,10,19]. Blum [10] study shows that the dilution factor of blood was correlated with the intensity of light produced during the reaction. It allows detection of diluted bloodstains up to 1:1000. Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains 4.2.3. Fluorescein Fluorescein is based on the chemical reaction of peroxidase-reduction principle [18]. The reduced colourless form of Fluorscin with hydrogen peroxide is immediately oxidized to Fluorescein, when it interacts with hemoglobin [18]. The reaction product fluoresces when it is excited under light of wavelength between 425- 485 nm, with the help of yellow or orange filter [16-18]. Cheeseman et al., [15-18] have been conducting research on the effectiveness of Fluorescein for quiet some time. It can be problematic to use Fluorescein on non-porous and vertical surface as it can distort the bloodstain pattern [15]. Cheeseman suggested the use of a commercial thickener, Keltrol RD, or xanthan gum, an exo-cellular heteropolysaccharide, which can reduce the issue of run down pattern of bloodstain [15]. In its reduced (colorless) state, fluorescin was found to have a very short shelf life [18]. Cheeseman's study also found that the fluorescin reaction occurs for several minutes before the bloodstain pattern begins to degrade which allows sufficient time for the photographers to document the bloodstain [1518]. 4.2.4 Phenolphthalein / Kastle Meyer Test Phenolphthalein is used as an indicator for different acid-base reaction tests, and it also produces a colour change from colourless to pink when it comes in contact with blood. Cox conducted the study to evaluate the sensitivity of phenolphthalein [46]. Grodsky, Wright, and Kirk [35] presented a comparative study of phenolphthalein with other presumptive tests. They used blood solutions to test the sensitivity of the reagents rather than dry bloodstains. They prepared the solution in stock and working solution to conserve the lifetime of the solution due to keep it in its reduced form. Test solutions of blood were made in dilutions of 1:50, 1:100, 1:500, 1:1000, 1:5000, 1:10 000, 1:50 000, Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains 1:100 000, 1:500 000, 1:1 000 000, and 1:2 000 000 parts of blood in distilled water [46]. The findings by Cox [46] were that the sensitivity is up to 1:1 000 000 for blood solutions but it wasn't as specific as tetramethylbenzidine and the orthotolidine tests [46]. 4.2.5. Leuco malachite green Leuco malachite green (LMG) produces a bright green colour when reacts positively with blood. Cox [46] in his study for specificity of different presumptive tests for blood described it as the least sensitive test. It was found to be about 1:5000. He used the following formula for it; Leucomalachite green (Aldrich Co.), 0.1 g ; Glacial acetic acid, 66 mL; distilled water, 33 mL; and hydrogen peroxide [46]. The sensitivity was found to be far less than reported by Grodsky, Wright and Kirk [35], whereas it was found to be more specific than tetramethylbenzidine and the orthotolidine tests [46]. 4.2.6. Leuco crystal violet Leuco crystal violet (LCV) is one of the many reagents used for blood enhancement in different crime scenes. It is a completely reduced form of crystal violet, which makes it colourless in nature [45]. The main reason many analysts find it useful as it is that it is vividly visible under light [45]. It gives a vivid violet colour on its reaction with blood. Bodziak in his study discusses LCV as a better blood enhancement reagent for processing the floor areas of crime scene [45]. His study showed that it can be applied quickly bloody impression, which provides improved visual under ambient light [45]. Leuco crystal violet is usually used for enhancing bloody footwear impressions. 4.2.7. DAB DAB is described as being usable for both crime scenes and laboratories [28]. It is applied on a two Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains stage process on a blood impression. An application of fixative 5-sulfosalicylic acid in distilled water, with subsequent application of a phosphate buffer, and 3,3’-diaminobenzidine (DAB) and distilled water [28]. DAB developed prints are very bright and dark brown in colour. It was also observed by Sahs et al. that samples applied with DAB show no indication of fading, even after several months [28]. Blood prints were found to luminate readily, usually within 450-585 nm excitation, with most consistent clarity in the 450 nm range [28]. When mixed with MDB, it was found to give better results in terms of luminescence. Omniprint 1000 was used for the experiment by Sahs [28]. 4.2.8. Alternative Light Source Alternative light source (ALS) is one of the simplest and non-invasive ways to detect latent blood at a crime scene. Ultraviolet light, Polilight, Luma‐lite, Spectrum 9000, and IR are some of the light sources used or have been used at the crime scene. UV has been described to be particularly helpful when the bloodstain on a dark surface [8,21]. Klasey and Levine found UV light to be a better light source in reducing any background interference [30]. UV light is not advised to be used in crime scenes since certain UV wavelengths can destroy DNA evidence in blood which is required for further analysis. It has been found that exposure to 255nm light for more than 30 sec can cause enough damage that there will be no detection of DNA during PCR amplification and quantitation [6,21]. An alternative light source can also be a versatile light source like Polilight which contains a wide range of wavelengths and it can be also be used to reveal stains covered by paints [6,8]. In a study conducted by Vandenberg, Polilight was described as a relatively safe, simple, noninvasive, and Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains nondestructive technique suitable for use in forensic casework [6]. They found that 415 nm was the best absorption band for blood to be detected under Polilight. This observation was applicable for bloodstain on various materials, with the exception of green polar fleece [6]. The efficiency of detection of blood with the help of alternate light source described to be dependent on the absorbency of substrate, humidity and background colour of the substrate [6,21]. 5. Substrates and Environments 5.1. Blood detection in Soil Blood detection using chemiluminescent reagents is different for indoor and outdoor environments. Adair et al., conducted experiments on blood detection in soil by using Luminol and Bluestar® [23-26]. The experiments were conducted over the period of six years and data were collected during that period of time. Luminol formula used for the experiment was similar to Grodsky formula [23]. The purpose of the experiment was defined by the authors as to effectively test for the detection of blood while controlling false positives at the same time. It was noted that the luminescence of Luminol with blood reduced with the passage of time. Photographs of the reactions were taken successfully with the help of digital cameras such as the Nikon D50, D100, D2H, Fuji Finepix S7000, and the Fuji Finepix S20pro. Exposures were from 10-30sec. at a focus of f 3.5- f 4.0. Sony “night shot” 8mm video camera was found to be unsuitable for the process as no successful photographs were taken [23-26]. For confirmation, samples of soil from the reaction area were also tested with phenolphthalein. The soil samples were found to be indicative of blood [23-26]. 5.2. Blood detection in layers of paint At times, perpetrators use paint to hide blood evidence, which makes enhancement and detection of Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains blood a difficult task. Vandenberg et al., [6] conducted an experiment for detection of blood under painted coloured walls. They used a polilight for their experiment. The found that the darker-coloured green paint, was better at blocking out the appearance of a bloodstain. Bloodstains on wood, when painted with the water-based paint, a trend of decreasing visibility was observed. It was most visible in white paint and least visible for green-coloured paint [6]. Adair et al., also used different alternate light sources to detect blood under paint [22]. They used Omniprint™ 1000, Polilight™ PL-10, and SPEX™ models MCS-400 and CS16-500 Krimesite™ reflective ultraviolet viewer for their experiment. Their research showed that SPEX ALS units provided the best results when set at 445nm while using a deep yellow filter even under four coats of paint [22]. Quickenden and Creamer [39-43] have shown that there are a number of substances that produce luminescence with hemoglobin when luminol is sprayed, including enamel paint (Duluxs). Quickenden and Creamer [39-43] also showed that acrylic paint (Taubmans), matte-finish paint (Duluxs) and flat oil-based paint produced no chemiluminescence on treatment with luminol. This was found to be different from the research conducted by Bily and Maldonado [38] where they were able to detect blood under eight layers of paint with the help of luminol. Howard and Messan [44] compared alternate light source (ALS), Infrared photography, Bluestar® Forensic, Luminol and fluorescein for detection of blood under painted surface. Their results showed that Infrared was the best way to visualize blood patterns. For chemical reagent, they suggested Bluestar® with high intensity of chemiluminescence [44]. Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains 5.3. Blood detection on porous surface The substrate surface texture upon which stain is located plays an important role in chemical enhancement and successful detection of blood. An absorbent material consists of substrates with irregular porous surfaces such as wood-finish panelling, walls, and interstitial spaces between tiles or wood objects which, due to the grooves or cracks onto the surface. These materials show superficial absorbent properties and they are able to have blood remains, even after thorough scrubbing of the surface, for a long time [2, 34]. In this way, they are often able to retain significant amounts of blood, maintaining it in relatively undegraded form even for a long period of time [2,7], thus giving intense reaction with presumptive tests for blood. Porous surfaces not only helps in prevention of degradation by environmental biological agents such as bacterial hydrolytic enzymes but it also protects blood from physical or chemical environmental agents such as solar rays, moisture, water, or cleaning attempts after the crime has been committed [2,7,9, 22, 34]. Luminol [22] , Bluestar® [10] and Fluorescein [15-18] have been found to be suitable chemical enhancement reagents for blood for porous surfaces. 5.4. Blood detection on non-porous surface Non-porous surfaces are non-absorbent substrates such as non-textured linoleum, vinyl, tile, glass, metal etc. They present difficulty in both reagent application and in the quality of chemiluminescence [2]. They don't have the capacity to retain and store blood and, moreover, cannot prevent degradation of blood by physical and chemical agents. These surfaces can be cleaned completely with mild washing by water and soap. This results in negative response of presumptive tests of blood. The surface can also complicate analysis as it can lead to the bloodstain pattern running, due to the limited retention of the Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains resulting solution by the smooth surface. This can lead to complete loss of the bloodstain pattern [34]. It is recommended that minimum amount of reagent should be used when applying reagent on nonporous surfaces [15-18]. Luminol on such surfaces can be used with along with nebulizer [2,34], whereas Cheeseman suggested that Fluorescein should be used along with a second application of commercial thickener, Keltrol RD, or xanthan gum, an exo-cellular hetero-polysaccharide, which can reduce the issue of run down pattern of bloodstain on non-porous surfaces [15]. Bergeron found methanol and titanium oxide to be a better alternate solution for the detection of blood on non-porous surface [51]. DeHaan [9] reported that Amido black is very sensitive and works well on non-porous surfaces. 5.5. Blood detection on dark surfaces Hungarian red, merbromin, and DFO have been used to detect blood on darker surface but they require the simultaneous use of an ALS [51]. Leucomalachite green, amido black, and ninhydrin chemically react with components in blood to form a dark-colored dye complex, which is not useful when blood needs to be detected on a darker surface [9]. Luminol was unsuccessful on such surfaces as well due to its limited luminosity. Bergeron [51] in his study explored titanium Oxide with methanol for detection of bloodstain on darker surface. The study described it as safe and effective solution for latent prints on dark surfaces with prominent ridge detail. Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains Klasey and Levine [30] studied non-invasive way to detect bloodstain on darker surface. They studied UV light as an alternate light source. In their study, they concluded, UV light to be useful in finding blood on darker substrates. DeForrest et al.[52], used photography for imaging bloodstains. They found IR photography to be a better alternate solution in detection and preservation of blood sample. With IR photography, they used background corrections on digital images. The combination of digital photographs at several wavelengths helped in enhancing the visualization of blood on dark coloured substrates [52]. 5.6. Blood detection in arson crime scene Crime scenes as a result of fire and explosions are difficult to process. Investigators need to go through debris to identify and recognize biological substance [3]. When charred by high temperatures, biological and non biological stains usually appear similar [3]. In arson cases, where most of the evidence is burnt, sensitive and rapid screen tests for blood are required for the selection of relevant biological material for further DNA analysis [4]. In such crime scenes, stains which yield positive result for blood screening tests are chosen for further analysis. Bilous et al tested the efficiency of Luminol, Bluestar®, Fluorescein and Hemascein have been used to detect burnt bloodstain patterns [4] Fluorescein, a blood enhancement reagent, has been shown to be more successful than Bluestar® and Luminol for detection of burnt bloodstains [15-19]. Bilous et al. exposed 1:10 dilute liquid blood samples to 400-600 C of temperature for 1,3 and 5 minutes [4]. It was found that HemasceinTM produced constant fluorescence which surpassed than that of Luminol and Bluestar® after 1st minute [4]. They concluded that HemasceinTM was a better chemical reagent for burnt blood samples than Luminol and Bluestar®. Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains 5.7. Fingerprints in Blood Fingerprints are the most common and useful physical evidence for the apprehension and conviction of crime perpetrators. Chemical methods for the enhancement of residual blood fingerprints have been successfully used for years. Leucomalachite green, amido black, and ninhydrin chemically react with components in blood to form a dark-colored dye complex and have all been used successfully on lightcolored or transparent surfaces [9]. Leucomalachite green and ninhydrin have low background colours but they are unsuitable for prints found on non-porous surfaces. Amido black is very sensitive and works well on non-porous surfaces and it has been proven to provide vivid ridge detail but it is unsuitable for porous surfaces for fingerprints in blood [9]. Strongin et al., [53] analyzed fluorogenic reagents for detecting and enhancing fingerprints in blood. They found them to be a bit challenging as fluorogenic reagents are unstable for dark and multi-coloured substrates. They diffuse away, degrading detail. They developed fluorogenic compounds for detecting fingerprints in blood that have suitable sensitivity and stability, enhance and preserve print details, and will work on dark and multi-colored substrates. Their research found that oxidation products are stable, allowing one to capture images after long periods of time (at least 8 months) [53]. Fluorescence can vary with the dilution of blood and it can be improved by spraying the treated fingerprint with a solution of reagent at higher pH. Fingerprints developed using fluorescent dyes were visualized in the range of 400 – 570 nm, using alternative light sources [53], caution is required for not using UV light as it destroys DNA in blood [53]. Bergeron found methanol and titanium oxide to provide a prominent ridge detail for the detection of blood [51] while being safe for use at the same. Sears et al., [54] carried out study on detection of latent prints on both porous and nonporous surfaces. Most effective reagent for porous surfaces were Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains determined to be DFO and ninhydrin, which react with amines in the secretion from finger. On nonporous surfaces, no process was found to be as effective at developing fingerprint detail as the protein dye benzoxanthene yellow. Acid yellow 7 was determined to be a suitable replacement [54]. 5.8. Foot impressions in blood Apart from fingerprints, foot impressions and shoe prints are the most common forensic evidence. Morgan et al., [31] found ninhydrin and amido black to be a better chemical enhancement reagents for foot impressions in blood compared to leuco crystal violet [31]. With the use of amido black, the researchers found the impression to be visible up to 16 weeks in external environment. Cullen et al., studied the subsequent development of surviving foot impressions post excavation, using chemical enhancement techniques of ninhydrin, acid black 1, leucocrystal violet (LCV), and Bluestar®. The authors found that LCV and Bluestar® are most effective for enhancing and retrieving impressions, which were later examined to identify class, individual, and wear characteristics of itself [57]. Bodziak recommended the use of leuco crystal violet for the detection of foot wear impression in comparison with luminol [45]. Leuco crystal violet [4,4',4"methylidynestris(N,N dimethyl aniline)] is a reduced form of crystal violet which produces a vivid violet once it comes in contact with blood [9], a reagent for developing dark-colored prints on light-colored backgrounds. Cheeseman and DiMeo [17] successfully applied fluorescin to bloody fingerprints in a viscous medium to reduce running. Farrugia et al., investigated the optimisation of peroxidase based enhancement techniques like Luminol, LCV, LMG and Fluorescein, for footwear impressions made in blood on various types of fabric [56]. Luminol was found to be the successful reagent for the fabrics tested. Gorn et al., reported Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains a case study where they were able to recover foot wear impressions in blood which were burnt by the fire [5]. Leuco crystal violet was found to be effective at the crime scene for footwear impressions [5]. 5.9. Effects of chemical enhancement reagents on aged bloodstain The bloodstains deteriorate over time whether they are in indoor or sheltered environments. Morgan et al., conducted the experiment in various environment [31]. Their study showed that the intensity and detail of the impression taken with the help of reagents deteriorated over time. It was found that the indoor impressions showed least deterioration, followed by outdoor and then the impressions which were totally exposed to the environment [31]. Leuco crystal violet gave the most intense enhancement but it was also the first one to lose its intensity over time. Their experiments concluded ninhydrin was the best reagent for treating aged impression on paper substrates. For wooden and linoleum substrates, they found amido black was the best of the reagents. Garfano et al., probed effectiveness of Luminol and Fluorescein for diluted and aged bloodstain detection at crime scenes. It is a comparative study of sensitivity and selectivity of these two presumptive blood tests using a series of diluted blood (from 1:10 to 1:10.000.000) on different substrates. Luminol and Fluorescein showed sensitivity on different substrates with varied level of intensity. Luminol was most sensitive on porous substrates like wood and rubber [55], whereas Fluorescein was more sensitive with blood on iron and baked clay [55]. 6. Comparison of Presumptive Blood Tests Luminol and Fluorescein are chemicals commonly used in the detection of latent bloodstains. Both Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains classes of reagents are catalytic tests which share a common chemical pathway for visible chemiluminescence. Both methods require relative darkness for visualization. While the fluorescein method requires an additional alternate light source and filter to visualize, it is nontoxic in nature. In contrast, the luminol reaction requires no additional equipment, but dries to leave a residue that is slightly toxic [12,13]. For use of fluorescein, it is recommended to store a portion of a sample first. This is needed due to the high alkaline nature of the fluorescein technique which inhibits any humoral analysis (ie; ABO, anti-human) afterwards [15]. Bluestar® being a derivative of Luminol provides better luminescence though its not as sensitive as Luminol [10, 34,35, 36]. Luminol, fluorescein and Bluestar® are not recommended to be used for non-porous surface. In contrast, Ninhydrin, amido black, leuco crystal violet and leuco malachite green are recommended for non-porous surfaces with respect to different substrates. UV light despite being very useful for detecting blood on darker surface, destroys DNA which is not useful for crime scene investigation. Polilight as an alternate light source has been found to have various uses at the crime scene but it is advised to be used as secondary to any presumptive test for blood. 7. Discussion and Conclusion In the literature reviewed, colour change catalytic tests were found to be very sensitive, but not specific. It was concluded that a positive detection was dependent on a number of variables like background colours of the substrate; nature of substrate; the texture and porosity of the substrate; ease of preparing solutions; ease of application; the expertise of the examiner; toxicity level of the reagent and the nature of the blood impression [9,21]. Chemical enhancement being a catalytic peroxidase reaction involves false positive results for blood. Literature showed that making a distinction between chemiluminescence by bloodstain or chemiluminescence with bleach was dependent upon the Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains examiner. An experienced practitioner may be able to distinguish luminescence catalyzed by hemoglobin from that of luminescence produced from other substances, based on the intensity, duration or distribution of the luminescence [14]. A negative result is generally proof of the absence of detectable quantities of heme, however a false positive can be generated in the presence of a reducing agent. This is one of the main reasons that positive colour change alone should not be interpreted as positive proof of blood. Compounds which compete for the reduction reaction with chemical enhancement reagent are called interferences. Bleach was found to be one of the major interferences found at the crime scene. It gives a positive reaction with Bluestar® and Luminol upon interaction. However, this reaction is visually different from the reaction with a bloodstain [10]. The luminescence is not as intense as it is with blood and it does not slow down gradually. Instead, it gives off luminescence in the form of inconsistent sparks. There are a wide range of environmental, pharmaceutical, domestic and industrial substances which are able to affect blood-induced chemiluminescence [2, 39-43, 47,49 ]. This has been attributed to catalytic activity of the substance, their redox properties, and their chemical reactivity with the mixture or with iron in the bloodstains [2]. Some of the examples of such interferences are soils, detergents, bleaches, carpet, metal objects, tools, plastic panels, wood, and vegetable compounds [2, 47,49]. There was inconsistency observed in formulation of chemical reagents for enhancement of blood. There was no standardization found in the literature and there were no validation studies done on such formulation, except that of luminol [21]. Formulation of luminol was determined to have advantages and disadvantages regarding sensitivity, intensity and duration of illumination, and effect on subsequent Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains DNA analysis [14,21]. Luminol is often used in crime scenes for blood detection and enhancement. Dilbeck [19] compared the use of Bluestar® against Luminol. She compared the evaluation of photographic results, ease of preparation and the level of visualization in ambient light. It was determined that Bluestar® has distinct advantages when compared to Luminol [19]. Virkler et al., [21] in their review of properties of luminol deduced that the luminol test has been popular due to the lack of false positives and false negatives in comparison with other screening tests. Luminol was found to have four distinct formulations. Grodsky formulation for luminol was found to have higher sensitivity but it has detrimental effects on subsequent DNA analysis when compared to the Weber, Weber II, and Bluestar1 alternatives [21] . Both luminol and Bluestar® reagents have a short shelf life and must be prepared just before use. Furthermore, the crime scene must be darkened in order to see the short-lived light emissions [4]. Fluorescein, Bluestar® and Phenolphthalein were shown not to destroy DNA in blood for subsequent analysis. No validation study was found on Hungarian Red, Amido Black, Ninhydrin and other chemical reagents and their better substitutes. In a study conducted by Bilous et al. [10] for analysis of burnt bloodstains, diluted blood ranging from 1:10 to 1:10,000 were tested with Luminol, Bluestar® and Hemascein. It was found that intensity of fluorescence and luminescence decreased with increase in dilution of blood sample. The experimental results showed that there was a significant difference in the intensity of burnt blood sample and nonburnt blood sample. It was confusing as to why the samples were exposed to a temperature of 400 C600 C. It was also not specified in the experimental details if there was any consistency in temperature or if there was supposed to be any variance in luminosity with difference in temperature. The article Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains also did not account for the duration of exposure to heat properly. Even though the experiment was conducted successfully in laboratory conditions, there is a need of the evaluative study to compare and analyze burnt bloodstain results from simulated arson crime scenes [4]. It was also observed that Bluestar® and Luminol gave high intensity for the first 60 sec, followed by a gradual decline in luminescence, while Hemascein did not produce a higher intensity as that of Luminol and Bluestar® but it was constant for the entire test period. There needs to be another alternative than Hemascein for burnt bloodstain exhibits as it destroys DNA, which means the sample will not be used for further DNA analysis [11]. Though there was also a case report by Gorn et al., [5] where they reported detection of burnt latent blood impression with the help of Leuco crystal violet. In different research articles, it was observed that there was no consistency and continuity of experimental procedure. For example, the study conducted by Adair et al., [23-26] on detection of blood in soil with luminol, they didn't control any of the parameters of the experiment. It is unknown what effect higher humidity, precipitation, microbial activity and weedy vegetation would have on the effectiveness of the luminol reagent over extended periods of time. The experiment was started off with the interest in luminol in soil but in at the end of the 6th year, the researchers tested the surface with Bluestar® and reported it as a part of the research. This is problematic as it does not maintain consistency in the experiment protocol. The authors did not report the amount of luminol that was used on different soil sample grids for chemiluminescence. This is important as spraying too much chemical reagent can dilute the blood sample which is not helpful in further analysis of blood. When examining exhibits for latent bloodstain using Polilight, it is important to be mindful of the Ayesha Asghar FRSC 3900 Chemical enhancement for the detection of bloodstains background colour of the fabric and its potential fluorescence and to choose an appropriate wavelength of light in order to enhance the contrast between the stain and the background [6,8,9,11]. It is important to recognize that in most instances the history of fabric will be unknown and other fluorescent staining may be present, which can be attributed to the presence of other fluids that are known to fluoresce like urine or semen [9,11]. Ideally, the examination of evidence exhibits with the Polilight would be best performed in corroboration with other screening tests to better target an area of interest [6] Overall, the literature review of chemical enhancement of bloodstains shows that there is a need for validation studies, standardization of protocols and methodology and rigorous research. 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