4
Blood
4.1 Blood Cells and Blood Typing
Blood consists of a variety of different cell types, collectively known as blood cells, suspended in a watery fluid called serum. Traditionally, the study of blood is referred to as haematology, whilst the study of serum (and in particular the immune factors within it) is called serology. Typically, the intrusion of forensic science has complicated things and the term ‘forensic serology’ encompass not only the study of serum but also blood cells, saliva, and semen for forensic purposes.
Mature human blood cells divide into those that possess a nucleus and those that do not: both types can provide forensic information. Those lacking a nucleus are the red blood cells (RBC) (also known as erythrocytes) and the platelets. The red blood cells possess the pigment molecule haemoglobin that gives blood its red colour and is responsible for the transport of oxygen and carbon dioxide. The platelets are smaller than the red blood cells and they exhibit amoeboid‐like movement. That is, they move by sending out cell processes in a similar manner to the single‐celled organism ‘amoeba’. Consequently, they often look star‐shaped when viewed using a microscope. Platelets are responsible for the clotting mechanism and the production of chemical growth factors that maintain the integrity of the blood vessels. The cells containing nuclei are the white blood cells (WBC) or leucocytes. There are many sorts of white blood cell, but all of them are capable of amoeboid‐like movement and they are responsible for the body's immune defence capabilities. In addition to a nucleus, all WBC contain mitochondria. Therefore, unlike the red blood cells, WBC contain both nuclear and mitochondrial deoxyribonucleic acid (DNA).
All cells within the body carry upon their outer surface an array of molecules called ‘antigens’. These antigens serve a bit like a passport and identify the cell as a legitimate ‘citizen of the body’. A group of proteins, called ‘antibodies’ that are secreted by certain WBC and serve as ‘immigration control’, recognises the antigens. Any cell not possessing the correct antigens on its surface is deemed to be a foreigner and an immune response is mounted to destroy it. The nature of the antigens found on the surface of red blood cells is of medical importance, because unless one uses the correct blood type during a blood transfusion the recipient's body rejects it and this has fatal consequences. The most common antigens found on red blood cells are those that comprise the ABO system. The ABO system works as follows: an individual's red blood cells may possess either only class A antigens (type A), only class B antigens (type B), both classes A and B antigens (type AB), or neither class A or class B antigens (type O). Persons who are type A tolerate their own class A antigens, but produce antibodies against B antigens that bind to the surface of the red blood cells and form bridges between them. This causes the red blood cells to agglutinate (clump together) – a reaction that occurs quickly and can be observed using a microscope. In a similar manner, type B persons produce antibodies against class A antigens, type AB persons produce antibodies against neither class A or class B antigens, and type O persons produce antibodies against both class A and class B antigens. These interactions are summarised in Table 4.1.
Table 4.1 Summary of ABO blood group interactions.
| Blood Type | ||||
| Characteristic | A | B | AB | O |
| Ag on RBC | A | B | Both A and B | Neither A nor B |
| Ab in Plasma | Anti‐B | Anti‐A | Neither anti‐A nor anti‐B | Both anti‐A and anti‐B |
Abbreviations: Ag = antigens, Ab = antibodies, RBC = red blood cells.
Many other groups of antigens occur on red blood cell membranes. The best known are those responsible for the Rhesus (Rh) Factor (also known as antigen‐D and agglutinogen‐D). The Rhesus factor is so‐called because the antigens responsible for it were first described in rhesus monkeys. Persons possessing these antigens are Rhesus positive [Rh(+)], whilst those who do not are Rhesus negative [Rh(−)]. Eighty‐three percent of the UK population are Rh(+) and most belong to either blood types O (44%) or A (42%). Although we talk of a person being blood types A, B, AB, or O and being Rh(+) or Rh(−), all of these characteristics can themselves be divided up into many more sub‐combinations (e.g. O1 and O2). Certain ethnic groups tend to have higher proportions of particular blood groups. For example, type AB is more common in the Japanese (10%) than in Europeans (UK, 4%). A more precise indication of ethnic origin is possible if there is evidence of rare inherited disease traits or particular antigens. For example, sickle cell anaemia occurs almost exclusively among black people of African descent. By contrast, the Kell antigen occurs predominantly in Caucasian persons (Reid and Lomas‐Francis 1996). In addition to blood type and the Rhesus factor, many of the enzymes and other proteins found in red blood cells are also polymorphic – that is, they exist in more than one form. Consequently, even though an individual may have a common blood type such as type O, only a small number of the population might share the combination of other variables. If eight serological variables are used, the chances of two unrelated persons sharing the same profile is between 0.01 and 0.001 (i.e. between 1 in 100 and 1 in a 1000). This estimate is the match probability (Pm). This means that blood typing can provide a means of identification – although it is not as accurate as DNA profiling, which provides match probabilities of up to 10−10. Matching a person's blood profile to traces found at a crime scene might be incriminating, but on its own would be insufficient to prove guilt. It is, however, effective at excluding suspects and therefore allowing the police to concentrate their resources elsewhere. Blood typing suffers from several further drawbacks as a forensic tool. First, by comparison with DNA profiling, it requires relatively large amounts of sample and is therefore of limited use where only small specks of blood are available. Serological markers degrade quickly and consequently, once decay sets in, the information retrievable declines. In addition, enzymes released by bacteria growing on the dead body interfere with the analyses. Interference can be a problem where the bloodstain contains both the assailant and victim's blood (for example, after a fight). Interference also occurs if one of the parties recently received a blood transfusion (in which case their blood profile may be temporarily altered), or following a rape in which semen is left inside the victim's body and therefore will be diluted by his or her serological profile. In addition, taking a blood sample is an invasive medical procedure and a suspect may legitimately refuse to cooperate.
4.1.1 Locating Bloodstains and Presumptive Tests
The standard procedure at a crime scene involving an assault or homicide would be to first identify the location of suspect stains, then confirm whether there was blood and then determine whether the stains were of human or animal origin. Bloodstains are not always obvious because of the manner in which they are formed or because the assailant cleans up the crime scene after committing an assault. However, once blood spills, it is extremely difficult to remove all the traces. Any attempt at cleaning up inevitably means using either the kitchen or bathroom as a ‘base’ – so it is good place to start looking for blood. In the cleaning process, blood can flow beneath tiles or linoleum or between the boards of wooden flooring. Similarly, when grasping a water tap with a bloody hand, the blood may flow into the screw mechanism. Consequently, blood is detectable in even an apparently spotlessly clean room if one knows where to look. To identify the presence of this hidden or ‘latent’ blood and to confirm whether suspect stains really are blood, an investigator uses an indicator substance. There are many of these, but most work on the basis of haemoglobin catalysing the oxidation of an oxidant (e.g. hydrogen peroxide), and the products of oxidation then interacting with other chemicals to bring about a colour change. These tests can be highly sensitive, but a positive reaction only suggests the presence of blood and is not proof. They are therefore called ‘presumptive tests’. In addition, all of them suffer from interference from common environmental chemicals that break down the oxidant or interact directly with the test reagent, thereby inducing false positive reactions. There are discrepancies in the literature regarding the extent to which presumptive tests produce false positives and the chemicals responsible. Various vegetables (e.g. potatoes, horseradish, and red onions) and inorganic substances (e.g. ferric sulphate) are the most commonly cited sources of interference. In addition, in order to be sure the test is working, one should always perform a negative control (e.g. a spot of distilled water) and a positive control (e.g. a spot of dried animal blood) immediately before assessing an unknown stain. Without a negative and a positive control, the results are questionable.
4.1.1.1 Luminol
Luminol was first used to detect bloodstains in 1937 and it remains one of the most effective presumptive tests. It has the added advantage of not interfering with subsequent DNA analysis, so it can be sprayed directly onto the suspect stains (Tobe et al. 2007). Several of the other presumptive tests (e.g. Kastle‐Meyer test) destroy DNA and therefore when employed, a sample of suspect stain is scratched onto filter paper and the test performed on that. To perform the luminol test, luminol (5‐amino‐2,3,‐dihydro‐1,4‐phthalazinedione) is mixed with sodium carbonate to form an alkaline solution, whilst a separate solution of either sodium perborate or hydrogen peroxide is prepared to act as the oxidant. Immediately before use, the two solutions are mixed together and then sprayed onto the suspect stains. If haemoglobin is present, the oxidant breaks down and highly reactive free oxygen radicals form. The oxygen radicals bring about the conversion of luminol to 3‐aminophthalate in a chemiluminescent reaction. This manifests as a faint bluish glow best seen in dim light (Figure 4.1).
Figure 4.1 Blue luminescence following treatment with luminol indicates the (presumptive) presence of (presumptive) blood. The smeared appearance is because of an attempt to clean up the bloodstains. Confirmatory tests are required to prove that the stains were due to blood and that the blood was from a human.
Source: Reproduced from James et al. (2005), © Taylor & Francis.
Luminol is so sensitive that it can detect the presence of blood on machine‐washed clothing. However, after washing, the stains become diffuse and bear little resemblance to the original patterns. Consequently, caution is necessary when interpreting such stains. Luminol can also demonstrate the presence of blood in soil for up to a year after the blood was shed. However, it may be necessary first to scrape off the top layer of soil before spraying. After blood spills onto soil, a person or animal subsequently walking over the stained area, even months afterwards, may leave tracks that stain positive with luminol. In the absence of other evidence, it would not be possible to know whether the tracks were associated with the spillage of blood or a subsequent artefact (Adair et al. 2006). The luminescence does not last long and the requirement for dim light conditions presents practical difficulties in some circumstances. A residual light amplifier enhances the low signals given off by the luminol reaction. For example, the SCENEview BV800 is a hand‐held device that facilitates the identification of luminescent spots and records them as still or video images. Another problem that can arise is autoluminescence owing to degradation or contamination. For example, bleach containing sodium hypochlorite causes false‐positive reactions. Bleach is often used to remove bloodstains on clothing, sinks, and floors. Pre‐treatment of areas suspected to contain bloodstains with 8 M urea prevents false‐positives caused by sodium hypochlorite and increases the intensity of the luminol reaction (Stoica et al. 2016). Spraying luminol solution produces luminescent spots that are of a similar size to the mist‐like bloodstains caused by shooting injuries or severe beatings. Consequently, where such stains might be present, it is best not to spray luminol.
A luminol‐based product called BlueStar Forensic® has similar sensitivity to luminol. However, it is easier to mix and use at the crime scene, remains stable for several days after mixing, and produces a luminescence that does not require complete darkness to become visible. In addition, it does not alter the size and shape of bloodstains on fabric (Grafit et al. 2014) and does not interfere with subsequent DNA analysis.
4.1.1.2 Kastle‐Meyer Test
The Kastle‐Meyer test is used on suspect stains that are clearly visible to the naked eye. A sample of the stain is scraped onto filter paper and treated with a drop of ethanol (to improve the sensitivity of the test). A drop of phenolphthalein (a colour indicator) is then added, followed by a drop of hydrogen peroxide solution. If the phenolphthalein changes colour before the hydrogen peroxide is added, the test is negative. The hydrogen peroxide interacts with the haem molecule of haemoglobin and is broken down into water plus free oxygen radicals – these radicals then interact with the phenolphthalein, resulting in the solution changing from colourless to pink. The Kastle‐Meyer reaction is slightly less sensitive than the luminol test and must not be performed directly on stains, since it interferes with subsequent DNA extraction. However, it is quick, simple, and cheap, and the colour change is immediately apparent.
4.1.1.3 Infra‐Red Imaging
Infra‐red imaging shows potential for demonstrating the presence of latent bloodstains without the use of chemicals. The area or object is viewed using a camera or video recorder capable of detecting wavelengths in the 760–1500 nm region. The technique is still in the development stages and compromised if the background material has the same infrared absorbance and reflectance characteristics (e.g. nylon), but otherwise exhibits high sensitivity (DeJong et al. 2015).
4.1.1.4 Vibrational Spectroscopy
Infra‐red spectroscopy and Raman spectroscopy are both examples of vibrational spectroscopy. They are non‐destructive techniques and provide both confirmatory and quantitative information on the substance being analysed. The two techniques complement one another and provide different information about the chemical(s) studied. They are particularly useful for the analysis of trace evidence such as fibres, glass, and paint fragments. If a database of spectra is available, then the unknown substance can be identified relatively quickly. It is also possible to confirm whether there is a match between evidence found on a suspect and that at a victim/crime scene. Vibrational spectroscopy is likely to play an increasing role in many more aspects of forensic science (Muro et al. 2014).
4.1.1.4.1 Infra‐Red Spectroscopy
Infra‐red spectroscopy involves determining the absorption of light by the test substance across the infra‐red wavelength spectrum. When the energy of a particular wavelength matches the energy required to cause a chemical bond to bend and stretch (vibrate), a photon of light is absorbed. The different parts of the infra‐red spectrum, usually divided into near‐, mid‐ and far‐infra‐red, cause different effects and the absorption spectra can identify a substance by providing information on its component chemical bonds and functional groups. Infra‐red hyperspectral imaging combines photography with spectroscopy and may be a useful means of localising blood stains that are otherwise difficult to visualise – for example, those on dark backgrounds (Schuler et al. 2012).
4.1.1.4.2 Raman Spectroscopy
Raman spectroscopy is used in analytical chemistry to determine the chemical composition of substances. Like infra‐red spectroscopy, Raman spectroscopy can provide a confirmatory test for the presence of blood. There are even claims that Raman spectroscopy can distinguish between blood from different ethnic groups (Mistek et al. 2016). Raman spectroscopy works by shining a monochromatic (single wavelength) light at an object and measuring the wavelength spectrum of the light emitted (scattered). When photons interact with a molecule, they transfer some of their energy to it. This results in a wavelength shift when the photons are scattered. Because all molecules have their own unique vibrational energy levels, they induce unique wavelength shifts to the scattered light. The resultant spectra of scattered light are measured using a detector and provide a non‐destructive means of identifying chemical composition. Following the development of hand‐held Raman spectrometers, one can use the technique in the field.
4.1.1.4.3 Hyperspectral Imaging
A normal digital camera records an image in which each pixel contains three spectral channels (red, green, and blue), but a hyperspectral imaging system records hundreds of spectral channels from each pixel (and hence a lot more information). This technology has been used for several years in the food industry for identifying contaminants and for environmental monitoring. Some machines are about the same size as a large digital SLR (single lens reflex camera) and are therefore easily portable to the crime scene. The applicability of the technique for the detection of blood stains is still in its infancy and will depend upon identifying unique spectral characteristics of blood and ensuring there is no interference from the substrate. Preliminary reports suggest that it has a lot of potential and may be able to not only identify blood stains but also age them and distinguish between the blood of two or more individuals and the blood of humans and other animals. It also has potential for identifying other biological stains and drugs at crime scenes. Because hyperspectral imaging is non‐invasive, it would not interfere with subsequent forensic tests.
4.2 Distinguishing Human and Animal Blood
Even if blood is detectable, one cannot assume that it is human blood. Household pets are just as likely to fight, scratch and otherwise injure themselves as any human and therefore leave their bloodstains on upholstery and clothing. Similarly, when handling raw meat in the kitchen, one often leaves bloodstains upon surfaces and clothing. Sometimes it is important to identify animal blood, for example, when investigating crimes such as badger baiting or when a pet was injured or killed during the course of a break‐in or homicide. In the past, a common means of determining whether blood comes from a human or an animal was the Ouchterlony Double Diffusion Technique. This precipitin test involves reacting antigens (proteins) in the blood sample with anti‐human antibodies – these are available commercially and raised in rabbits. The blood samples and the antisera (anti‐human antibodies) are placed in wells punched into an agar gel that is spread over a glass dish or slide. The samples move towards one another through the agar by diffusion or the process can be speeded up using an electric current. If a white line – called the precipitin line – forms at the point at which the two samples meet, this indicates an interaction between the antigens in the blood and antibodies in the rabbit antisera and therefore the blood is the human. If no precipitin line forms and there is a suspicion that the blood belongs to an animal, then the procedure is repeated using antibodies raised against the appropriate animal sera. A development of this is the ABAcard® HemaTrace® method that works on a similar immunological basis in which haemoglobin in the stain interacts with anti‐human haemoglobin antibodies. However, the procedure has been simplified and speeded up so that a result is possible within as little as 10 minutes. It is therefore suitable for use at crime scenes. The sample (150 μl) is applied to a test well on a strip mounted on a plastic card. A second well in the card allows the operator to observe whether a line develops that is indicative of a positive reaction. The technique is higher primate specific but not human specific and therefore care is required in cases of suspected trafficking of primate bushmeat.
DNA‐based techniques are now available to differentiate between human and non‐human blood and tissue samples. For example, Matsuda et al. (2005) describe a highly specific polymerase chain reaction (PCR)‐based protocol that utilises primers for the human mitochondrial cytochrome b gene. They state that following the agarose electrophoresis, human DNA produces a single band, whilst blood from other vertebrates fails to produce any bands at all. Raman spectroscopy can detect blood and initial studies indicate that it can also discriminate between human and animal blood (McLaughlin et al. 2014; Mistek and Lednev 2015). Whether or not the technique would also work with the old dry spots of blood that are typical of forensic casework is currently uncertain.
4.3 Bloodstain Pattern Analysis
Bloodstain pattern analysis is a specialised branch of forensic science, in which the investigator deduces evidence from the shape and distribution of bloodstains. From careful analysis, one can determine whether or not a crime was committed and if so, how the conflict developed and how the wounds were inflicted. The earliest record of bloodstains being used in court proceedings relate to the trial of the unfortunate Richard Hunne in 1514. He was being held in Lollard's Tower in London on five charges of heresy, but before he could be examined, he was found hanged in his prison cell. This obviously upset the authorities, because he was promptly charged with 13 more counts of heresy and suicide. (Committing suicide was a serious offence and the victim's body was not allowed burial in consecrated ground. Suicide remained a criminal offence in England and Wales until 1961.) However, bloodstains were found in his cell and after reviewing these and medical evidence, it was concluded that ‘whereby it appearth plainly to us all, that the neck of Hunne was broken, and the great Plenty of Blood was shed before he was hang'd. Wherefore all we find by God and all our Consciences, that Richard Hunne was murder'd. Also we acquit the said Richard Hunne of his own Death’ (Forbes 1985).
4.3.1 Types of Bloodstain
An adult human contains about 5 l of blood; loss of approximately 30% blood volume (~1.5 l) usually results in loss of consciousness or incapacitation, whilst the loss of 40% (~2 l) blood volume can be fatal. Bleeding takes place both internally and externally, so the amount of blood surrounding a body may not reflect the amount lost. Arterial blood (with the exception of that going from the heart to the lungs) is bright red in colour owing to its high oxygen content, whilst venous blood is darker in coloration as it is deoxygenated. However, once shed, blood darkens, and begins to clot within about three minutes and it is impossible to tell whether it originated from an artery or vein from its colour. There are currently (2019) no reliable tests to determine the age of dry bloodstains, although studies on the extent of DNA decomposition have shown some potential.
Many texts classify bloodstains as low‐, medium‐ and high‐velocity spatter, based upon their size distribution and hence the force necessary for their production. Briefly and crudely, these causes are dripping, beating, and shooting, i.e. the higher the velocity, the smaller the stains. However, there is a move away from this approach, because it is difficult to be so prescriptive. For example, a severe beating and certain gunshot injuries can generate similar bloodstain size distributions. However, these two causes are often categorised as medium‐ and high‐velocity respectively. Other mechanisms, such as sneezing or coughing, can produce stains within the same size category as medium‐ to high velocity. Perhaps the best classification system is that set out in detail by James et al. (2005), in which they recognise three main categories: passive, spatter, and altered, that are subdivided according to the mechanism most likely to have produced them.
4.3.1.1 Passive Stains
Passive stains are subdivided into:
- Drops: These stains occur when blood drips passively under the influence of gravity (Figure 4.2). They may occur singly, in clusters or provide a trail and thereby indicate movement and its direction. However, direction may be difficult to determine if the victim moves slowly and therefore the drops fall at 90°, resulting in the stains being more or less oval. For example, if you cut yourself, you leave a drip trail of drop stains from where the accident occurred to the nearest washbasin.
- Flows: These occur when blood flows passively to produce small rivers or streams of blood (Figure 4.3). These are useful for determining orientation at the time of attack or movement after death. For example, if a person is stabbed in the chest whilst standing, a trickle of blood will head vertically down the body. If, however, the victim was lying on the floor, the flow veers to whichever side of the body angles to the floor. Similarly, if someone drags a bleeding victim, one finds multiple flow paths, although this is most obvious when the skin is uncovered, since clothing diffuses and complicates flow patterns.
- Transfers: These occur when wet blood transfers from one object to another (Figure 4.4). For example, a bloody knife wiped onto clothing, a bloody hand leaving prints on doorknobs or weapons, and a person stepping in blood, leaving behind impressions of their footwear.
- Large Volumes: A large volume of blood sometimes forms a pool around a dead body and seeps underneath it (Figure 4.5). Considering the volume of blood in relation to the extent of the injuries provides an indication of the time the body was resting in that position. Persons dying of pulmonary TB and a variety of other diseases may cough up or vomit large amounts of blood shortly before or at the time of their death. Consequently, their body might be found in a pool of blood. This can appear suspicious and it is a not unusual scenario in the deaths of vagrants and those who have become cut off from society.
Figure 4.2 Factors affecting the spatter pattern of passively falling droplets of blood. (a) The droplet falls 5 cm onto a smooth hard surface and forms a circular‐shaped stain. (b) The droplet falls from a greater height, 90 cm, and forms a sunburst‐shaped bloodstain. (c) A second droplet falls upon a bloodstain that is still wet and causes the formation of satellite spatter. (d) The droplet falls 5 cm onto a textured surface (calico) and promptly disintegrates to form satellite spatter. Square scale = 20 mm.
Figure 4.3 The flow of blood vertically down the chest and abdomen from a neck wound indicates that this person was standing upright when they were injured.
Source: Reproduced from James et al. (2005), © Taylor & Francis.
Figure 4.4 Transfer bloodstain from a bloody finger on a tap. Transfer stains like this are often made during clean‐up attempts.
Figure 4.5 This man was shot in the head. Because his body is on an incline, the blood is flowing away and collecting in the gutter. The amount of blood and the flow distribution indicates that the body was not moved after death.
Source: Reproduced with permission from Arlo Bailey BSc.
4.3.1.2 Spatter Stains
James et al. (2005) subdivide spatter stains into three subcategories based upon their cause: secondary mechanisms, impact mechanisms, and projection mechanisms.
- Secondary mechanisms are those that are secondary to the cause of the initial wound. For example, if blood drips onto an existing wet stain, it will distort the original stain's shape and form a patchwork of surrounding spatter (Figure 4.2c).
- Impact mechanisms, as the name suggests, indicates the means by which the wounds were caused. Although separate categories, such as gunshot, beating (sharp/blunt implements) and industrial tools are recognised, it is not always easy to ‘work backwards’ and establish the cause solely from the bloodstain evidence. For example, a variety of factors affects the blood spatter resulting from gunshot wounds. These include the type of firearm, the type of ammunition, the distance of the victim from the firearm, the presence of clothing and the location of the wound. Gunshot wounds often produce back spatter (i.e. towards the firearm) and forward spatter (i.e. in the direction of the projectile leaving the body) (Figure 4.6a and b). Both may include small fragments of flesh and bone. Suicidal gunshot injuries are distinguishable from homicidal ones by the presence of both blood spatter and gunshot residues on one of the hands of the victim and/or within the cuffs of long‐sleeved clothing. The absence of both raises suspicions that the gun was placed in the victim's hand after they were shot. However, Karger et al. (2002) state that when calves were shot in the head with a 9‐mm Luger, there were always cases in which backspatter did not happen. Betz et al. (1995) also found that backspatter was sometimes absent in human suicides. As the speed of impact increases, the dimensions of the blood droplets decreases. High velocity gunshot wounds cause the formation of a mist‐like array of tiny stains 1 mm in diameter or less (Figure 4.6b).
Beatings with a blunt instrument, whether by fists, feet, or an iron bar, can produce a wide variety of bloodstain patterns. Where the beating is prolonged and brutal, blows are inflicted upon already open bleeding wounds. This spatters large amounts of blood over the surrounding area. In extreme cases, mists of small droplets form similar to those caused by gunshots. With so much blood being spilt, anything getting in the way of the projected blood, such as the assailant or an item of furniture, results in a clean region or ‘void’ on the surrounding vertical surfaces (e.g. wall or door). Such voids are useful for determining the position of people during the course of an assault. The spattering of large amounts of blood means that it is highly unlikely that the assailant would leave the scene without substantial staining to their body, clothes, and footwear. It can also indicate whether doors were open of closed at the time of the assault. For example, if a door was open, projected blood might be found on the hinges or parts of the door frame that would be covered if the door was shut.
- Projection mechanisms are divided into spurt patterns, sneezed, coughed or breathed blood, and cast off stains.
Figure 4.6 Blood spatter from gunshot wounds: (a) backspatter stains on the right hand of a man who committed suicide using a shotgun.
Source: Reproduced from James et al. (2005);
(b) forward spatter stains (misting) after close range shot with high velocity firearm.
Source: Reproduced with permission from Arlo Bailey BSc.
Arterial blood flows under high pressure and will spurt out over a considerable distance should the vessel be cut (Figure 4.7). Furthermore, it will continue to spurt out in bursts owing to the beating of the heart, thereby producing a characteristic undulating ‘arterial spurt stain’. By contrast, when a vein is cut, the blood usually seeps out rather than spurts, because of the comparatively lower blood pressure. However, restricting the terminology to arteries could give rise to interpretation errors, since in certain circumstances pulse‐type stains result when veins are severed (Brodbeck 2007). This is particularly the case in venous insufficiency syndrome, in which blood pools in the lower legs and often involves the formation of varicose veins. Varicose veins result from the valves within the vein becoming leaky – this disrupts the normal flow of the blood allowing it to pool and therefore cause localised swelling. Varicose veins that are close to the skin surface are vulnerable to being knocked and ruptured. This results in pulse stains that are commonly found on the floor and lower vertical surfaces. Although not a common cause of death, elderly sufferers of varicose veins may stagger around after breaking open a vein, spreading large amounts of blood and giving the impression of a physical assault.
Figure 4.7 This stain was caused when the victim slit their wrist. It exhibits the typical undulating pattern caused by arterial bleeding. Note the accompanying spray of smaller spatter stains.
Source: Reproduced from James et al. (2005), © Taylor & Francis.
Coughing and sneezing generates a stream of fast moving air that carries a mixture of spatter consisting of small droplets just visible to the naked eye and an aerosol of microscopic droplets <100 μm in diameter. Wounds to the chest, mouth, or nose will result in the coughing and sneezing of blood droplets of a similar range of sizes. Stains formed from these droplets are often contaminated with strings of mucus, thereby indicating their probable origin. Droplets >100 μm lack the mass and kinetic energy to travel far, but aerosolized droplets (<100 μm) can remain airborne for long periods and the distance travelled will depend, in part, on the local air currents. Small droplets may also be projected when a person is breathing face down through a pool of blood (e.g. after a beating or being shot in the head). The absence of such stains would indicate that the person was possibly dead by the time their head hit the floor. Bloodstains containing tiny air bubbles are an indicator that the blood was expired.
Cast off stains result from blood thrown from a moving weapon or limb. For example, pulling a knife outwards and upwards from a body projects a series of bloodstains from the blade. The second and any further blows tend to be the ones causing the most blood splatter. Axes and similar hefty weapons tend to be used with a downward chopping action and the returning upswing leaves a cast off pattern of blood on the ceiling. Weapons that pick up a lot of blood produce cast off stains on both their outward and return stroke. Typically, in an arc of bloodstains, the stains become longer, more elliptical at the end of the stroke, and more circular as the blow centres above the assailant's head. If there is only one victim and cast off bloodstains present in more than one room, it indicates that the attack was prolonged and there was a chase. However, if there are cast off bloodstains in only one room, but passive or transfer bloodstains in other rooms, it is possible that the attack took place in one room after which the victim staggered or was dragged elsewhere.
4.3.1.3 Altered Stains
After its initial formation, any bloodstain is alterable. During an assault, bloodstains can fall upon one another and a bleeding person might fall across a spatter pattern. Changes also occur when blood dries out and clots, when it diffuses through clothing, or as the result of cleaning‐up operations or the movement of insects through the bloodstains. Although blood tends to remain fluid within the body after death, clots will form at wound sites and shed blood also clots. Consequently, a severely wounded body dragged away some hours after the assault took place leaves large clots of blood among the drag pattern (Figure 4.8). The absence of such clots suggests movement of the body shortly after death. Similarly, attempts at cleaning up bloodstains result in smears and smudges, as does movement of a bleeding body across the floor. The pattern of a blood smear indicates the direction in which a person or bloodstained object was dragged: the smear usually begins as a series of drops and these then become ragged along one edge, indicating the direction of travel. The initial spots might be disrupted by the passage of the head (especially if it has long hair) through them – for example, if the victim is wounded in the chest and dragged by the feet. However, the direction of travel will remain obvious. A ‘thinking murderer’ wishing to move their victim's body will drag it by its feet, because this reduces the risk of transferring blood onto their clothes. Holding a dead body under its armpits and then dragging it results in a smear from one or both heels passing through the initial bloodstains.
Figure 4.8 Smeared bloodstain pattern formed by dragging a bloody body across the floor. The presence of large blood clots among the smear pattern indicates the victim was not moved for some time after they were wounded.
Source: Reproduced from James et al. (2005), © Taylor & Francis.
4.3.2 Interpreting Bloodstains
In the initial stages of an investigation, the characteristics of all suspicious stains at a crime scene are noted. This will include the exact position of every stain, along with its size and shape and the nature of the material on which it was formed (clothing, plastic, wood, etc.): these records are made using photography and a written report, so that every spot that is to form part of the evidence is given an identifying number for future reference. From the distribution and shape of the stains, one can determine how the blood was shed and hence how a wound was inflicted. For example, large (4 mm or more in diameter) circular drops of blood on the floor indicate that the blood was travelling slowly and that the victim was stationary or hardly moving. This is indicative of blood dripping passively from a wound or a weapon. The shape of these ‘passive stains’ indicates how far it had fallen – blood falling vertically onto the floor from 1 to 50 cm tends to form circular drops with slightly frayed edges, whilst blood falling from a greater height forms a sunburst pattern. However, above a certain height, it is impossible to determine how far a blood droplet has fallen, because once it reaches terminal velocity, it cannot impact with any greater force. Larger droplets develop a greater terminal velocity than smaller ones. The distance a droplet must fall to reach terminal velocity should be determined by experimentation, since it is affected by local factors such as air currents. However, the shape of a bloodstain is much more strongly affected by the nature of the surface it impacts upon. For example, blood falling onto a textured surface such as concrete distorts more than blood falling onto a smooth surface such as glass. It is therefore important to verify any conclusions drawn from a bloodstain pattern by experimentation.
If more than one person is present when a violent crime is committed, it may be possible to distinguish their roles from the types of bloodstains upon them. For example, the person pulling the trigger or wielding the murder weapon may have only blood spatter stains on their clothes, whilst the one shifting the heavily bleeding corpse may acquire only transfer bloodstains. The absence of bloodstains on a suspect is not necessarily an indication of innocence, since they may have had time to wash and change their clothes. Alternatively, they may have removed their clothes before committing the crime or they were sufficiently far away from any flying blood not to be hit by it, or shielded from it in some way.
4.3.2.1 Determining the Area of Haemorrhage
Most texts use the terms ‘area of origin’ or ‘point of origin’ to refer to the site from which bloodstains originated, but I find ‘area of haemorrhage’ more apposite, because it clearly indicates ‘blood loss began here’. Its determination is notoriously difficult and relies upon knowledge of physics and a lot of experience. Therefore, in complicated cases, analysts may arrive at different interpretations of the crime scene.
There are five principle methods of determining the area of haemorrhage and hence the position of a person at the time of the injury: observation, trigonometry, stringing, graphics, and computer programs.
4.3.2.1.1 Observation
Simple observation of the crime scene cannot provide the numerical data that most forensic investigations require, but it can sometimes provide an initial indication of the approximate area of haemorrhage/origin without the need for time‐consuming analysis. For example, if a pattern of aspirated bloodstains is found 1.8 m up a wall, this would indicate that the victim must have been standing upright and must have been close to the wall, since such small droplets travel only very short distances.
4.3.2.1.2 Trigonometry
When a drop of blood falls, it is held together by surface tension and behaves as though it has an elastic skin. Surface tension results from cohesive forces between molecules at the surface of a liquid and manifests in the drop pulling into its smallest possible area. Consequently, a drop of blood will not fall unless it is subjected to a force greater than its surface tension and once in flight the drop will tend to stay together in a sphere (rather than break up or form a teardrop shape) until it comes into contact with another object. This assumption of a spherical shape is crucial to the behaviour of a blood droplet whilst in flight and following contact with a solid object.
If a blood droplet hits a smooth clean flat surface at 90° (i.e. ‘head on’), it forms an oval shape with about the same diameter as the droplet and with a number of spines dependent upon the velocity with which it impacts and the nature of the surface it hits. If, however, the droplet strikes the surface at an angle, then it forms an ellipse, the length of which depends on both the diameter of the original droplet and the angle with which it hit the surface. Basically, as the angle of impact decreases, the length of ellipse increases, i.e. a droplet impacting at an angle of 15° produces a longer ellipse than one impacting at 45° (Figure 4.9). The ellipses are seldom perfectly symmetrical and the narrower end and/or where tails form, indicates the direction in which the blood droplet was travelling and hence the direction of the force that was propelling it. Because the shape of the ellipse is dependent upon the angle of impact (Figure 4.10), one can calculate this angle using basic trigonometry.
Figure 4.9 (a)–(c) The influence of angle of impact on the shape of bloodstain. For a blood droplet falling onto a smooth hard surface, as the angle of impact decreases, the length of the ellipse increases.
Figure 4.10 Diagrammatic representation of how the length of an elliptical bloodstain is related to its impact angle.
Angle θ = angle of impact (θ = Greek symbol ‘theta’)
Therefore
If bloodstain A is 4 mm wide and 7 mm long, then the calculation would be
However, it is impossible to determine the point of haemorrhage from this one calculation, so one needs measurements from at least one other stain. Assume therefore that a second bloodstain B lies to one side of bloodstain A and has the dimensions 5 mm wide and 10 mm long. The calculations would be:
Provided both stains result from the same event, one can calculate their area of convergence and thereby estimate the distance of the stains from the area of haemorrhage. This is done by drawing lines (sometimes called ‘strings’) through the centre of the long axis of the stains. The point where the two lines meet is the area of convergence (Figure 4.11a). The distances between the centres of the bloodstains and the area of convergence are then measured. Let us assume that in our case the distance between bloodstain A and the area of convergence is 0.70 m and for bloodstain B it is 0.882 m (bigger droplets tend to travel further than smaller ones).
Figure 4.11 (a) and (b) Diagrammatic representation of how the point of convergence and area of origin can be determined using trigonometry. For details, see the text.
A line, we can call it ‘H’, is now drawn at 90° from the area of convergence, so creating two right‐angled triangles: the one formed from bloodstain A and the one formed by bloodstain B and both of them will share H as one of their sides (Figure 4.11b). This is done by drawing a line with an angle of 34.85° from bloodspot A, and another with an angle of 30.00° from bloodspot B, and where they meet on line H will be the area of haemorrhage. This is calculated as follows:
- For bloodstain A
H = Tangent 34.85° × 0.70 = 0.487 m
- For bloodstain B
H = Tangent 30.00° × 0.882 = 0.509 m
Note that the two H values are not identical. This is because in real life there is a lot of ambiguity, not least because blood droplets travel in parabolic flight paths rather than straight lines and the further a droplet travels the more it assumes a parabolic arc. In addition, when we are hit by something, our body is sent into motion and when we bleed from a serious wound, we do not (usually) lose blood one drop at a time from a single point source. Hence, the source of any two spots of blood in three‐dimensional space may not be exactly the same, even if they were caused by the same event. Consequently, some workers use the terms ‘area of convergence’ and ‘area of origin/haemorrhage’, rather than ‘point of convergence’ or ‘point of origin/haemorrhage’ because it is seldom possible to establish these sites with absolute precision. Accuracy can be increased by carefully choosing which stains to use – those with the lowest impact angles are thought to provide the greatest accuracy (Willis et al. 2001) – and whilst there are no recommended minimum numbers, it is obviously prudent to use more than two or three if many more suitable stains are available. Several workers have developed more complicated equations for the determination of the area of haemorrhage that provide a more realistic account of droplet's flight path (e.g. Knock and Davison 2007), but these remain at the experimental stage. Absolute accuracy is not always necessary, because the investigator often only needs to know the general position of a person when they shed their blood. For example, if a suspect claims that he was defending himself but the bloodstains indicate that the blood was shed from a fatal wound to the head that was at a height of about 14 cm from the ground when struck – then this is evidence that the victim was already on the floor at the time and therefore hardly a threat.
4.3.2.1.3 Stringing
After labelling and measuring all the stains to be used in the analysis, the scene is recorded using a camera. A scale is included with each photograph along with directional indicators. On a horizontal surface, these take the form of the points of the compass and an indication as to whether they are on the upper/lower surface or ceiling/floor as appropriate. On a vertical surface, an arrow indicating up/down is required and the arrow should be drawn at 90° vertically downwards (i.e. as a ‘plumb line’). Alternatively, a spirit level is used to draw ‘level line’ underneath the stain. This enables a record to be made of the stains' orientations in relation to the surface they are found on. Next, an elastic cord (or ‘string’) is placed through the mid‐line of each stain and run backwards in the direction the blood came from. The individual cords are secured at either end and where they overlap one another is the point of convergence. The distance from the leading edge of each stain to the area of convergence is then recorded. Let us assume that our stains are on a vertical surface, and in this case one should extend the level line so that it cuts across the elastic cords that are running to the area of convergence. The angle formed between the plumb line and individual cords can be recorded using a protractor and is called the ‘direction of flight angle’. In order to work out the distance from the vertical surface and hence the area of haemorrhage/origin, a stick or similar object needs to be secured so that it projects at 90° from the area of convergence. The angle of impact can be determined for each stain from its dimensions, as described above, and a cord with this angle to the surface (as determined using a protractor) run back to the stick. Where the cords cross on the stick is the area of haemorrhage (Figure 4.12). The whole exercise can take several hours, especially where there are numerous separate ‘events’ to record. Furthermore, the tendency of the cords to droop adds to the difficulties. In addition, the technique requires experienced practitioners and the cords must be removed as soon as possible to allow further investigations of the scene to be made. Thus, there is a lot of interest in developing techniques that can make measurements from digital recordings of blood spatter patterns. This provides a permanent record of the scene and enables repeated computer‐based analysis of the staining patterns. This is particularly useful should there be a subsequent dispute in court about the interpretation of the evidence.
Figure 4.12 Bloodstain pattern analysis using ‘stringing’.
Source: Reproduced from James et al. (2005), © Taylor & Francis.
4.3.2.1.4 Graphics
The angle of impact and distance to the area of convergence are calculated for each stain as described in the trigonometry method. This information is then plotted on an X‐Y graph, in which the distance to the area of convergence is the horizontal X‐axis and the Y‐axis represents the distance to the area of haemorrhage/origin. For example, let us assume that there are four bloodstains whose characteristics are:
| Distance to area of convergence (cm) | Angle of Impact | |
| Stain A | 72.4 | 50.9° |
| Stain B | 99.0 | 41.6° |
| Stain C | 128.0 | 35.8° |
| Stain D | 160.0 | 30.0° |
As can be seen from Figure 4.13, by drawing a line with the appropriate angle from each stain to the Y‐axis, the point at which they meet represents the area of haemorrhage – i.e. the distance above/below/to the side (as appropriate) of the point of convergence.
Figure 4.13 Determination of the area of haemorrhage (origin) by the graphics method. For details, see the text.
4.3.2.1.5 Computer Programs
The use of computer programs, such as Hemospat® and BackTrack®, to analyse the distribution of bloodstains, is starting to replace manual procedures such as stringing. Not only do these programs permit more rapid analysis than traditional stringing, they also provide numerical estimates of the error associated with individual measurements. This makes the recording of data more scientifically rigorous, the reconstruction of the events easier to understand, and facilitates the subsequent presentation of evidence in court.
The crime scene is recorded using a pre‐calibrated digital camera and a scale included with each photograph along with a directional indicator. Ideally, this should indicate the points of the compass and up/down if the stains are on a vertical surface. Unless calibrated reference markers (fiducial markers) are used, photographs should be taken at 90° to each stain to avoid perspective distortion. The inclusion of a calibrated chequerboard in the photographs enables rectification of angled shots by the computer to appear as though taken from overhead. The computer superimposes an ellipse over any chosen bloodstain and uses algorithms to identify its angle of impact. Then, following the analysis of several bloodstains, the computer can generate lines to identify their area of origin and illustrate this in either two or three dimensions.
Current computer programs require numerous photographs of the crime scene, because it is seldom possible to take just one or two photographs that both show the bloodstains with sufficient resolution to facilitate measurement and display them in the wider context of the room. Most current computer programs still require manual measurements of the stains and the subsequent analysis can be time‐consuming. An analytical process called HemoVision incorporates reference markers when taking the photographs and by exploiting computer vision algorithms, it speeds up both the recording process at the crime scene and the subsequent analysis (Joris et al. 2015).
Because of the time taken to make a photographic record of a crime scene and blood spatter evidence, there is an interest in developing 3D laser scanners that can rapidly map a whole room and its contents in three dimensions. Computer digital pattern analysis can then identify the oval and elliptical shapes of typical bloodstains within the room and analyse them as described above (Lee and Liscio 2016). This would permit the recording of bloodstain evidence far more quickly than is currently the case. Currently, laser scanners are deployed fixed to a tripod, but in the future they could be mounted onto a small drone and flown over the crime scene. The output could then be analysed by computer to enable fully automated blood spatter analysis (Acampora et al. 2015). The use of remote sensing devices mounted on small drones could potentially allow the investigation of a crime scene, with minimal disturbance or risk to the investigators. However, a means of reducing down draft from the rotors would be required to avoid lightweight evidence such as paper and fibres blowing away.
Computer analysis software can also be particularly useful in complex situations in which two or more blood patterns overlap; for example, if blows are delivered in quick succession or if a conflict progresses back and forth over the same area. The probability that an individual stain resulted from one blow or another can be calculated through the use of statistical techniques such as fuzzy cluster analysis.
4.3.2.2 Collecting Blood‐Stained Evidence
Blood‐stained evidence required for further analysis such as DNA or as an exhibit at a future trial should be collected after the crime scene has been fully documented. The evidence should be given a unique identification number, so that its exact position in the scene can be determined. Dry evidence should not be placed in an airtight container, because if any moisture is present this can result in the growth of mould and bacteria. Unstained areas adjacent to the stain(s) should also be sampled to determine whether the DNA (or whatever is being analysed for) was already present on the substrate before the blood was spilled. For example, a chair seat or bed will contain DNA from naturally shed skin cells and if blood is spilled on top of this, then samples from the bloodstains can contain a mixed DNA profile, even though only one person shed blood.
The evidence may be collected as follows:
- Whole item
- Tape lifting
- Scraping
- Swabbing
- Elution
- Whole item: This typically relates to objects that have caused the injuries, such as a knife or brick. It may also include items such as the stained clothing and footwear of both the suspect and the victim. If there is more than one item, then each one should be placed in a separate evidence bag.
- Tape lifting: The effectiveness of this method depends upon the surface properties of the material on which the bloodstain is found. Fingerprint tape is placed over the stain and afterwards placed on vinyl acetate for storage.
- Scraping: The dry stain is scraped with a clean sterile sharp object, such as a scalpel blade, into a paper evidence bag. Plastic bags should not be used because the associated static electricity can result in the fragments being lost through dispersal and sticking to the sides of the bag. To avoid the risk of contaminating evidence, scrapings from different stains should be stored separately and each stain should be scraped with a different clean scraper.
- Swabbing: Wet stains can be collected using a sterile cotton swab, which is then allowed to dry before storage. If the stain is dry, then the swab needs to be moistened with sterile distilled water or sterile saline before gently rubbing it onto the stain. If more than one swab is used to sample a stain, then the order with which they are taken should be recorded. The swabs are allowed to dry before storage in separate evidence bags.
- Elution: A small volume of sterile saline is pipetted onto the stain to dissolve it. The liquid is then collected and stored.
4.4 Fake Blood
Where the conclusions drawn from bloodstain analysis are ambiguous or likely to be controversial, then it is always a good idea to verify them through experimentation. The ideal test material is, obviously, human blood, or failing that, equine or pig blood. However, working with real blood presents Health and Safety problems and is not necessary for demonstration or training purposes. In these latter situations, one can use fake blood. It is better to refer to ‘fake blood’ rather than ‘artificial blood’, because the latter term is used to describe substance(s) used to mimic some of the functions of real blood during blood transfusions. Fake blood for use as make‐up is available on the internet from a variety of suppliers and there are many recipes that you can use to make your own (Millington 2004). However, these do not necessarily have the same physical characteristics as real blood and therefore may not yield the same spatter patterns.
4.5 Post‐Mortem Toxicological Analysis of Blood
Most toxicological studies involve the analysis of blood samples, but the collection and analysis of post‐mortem blood presents far more difficulties than it does when the donor is still living (Skopp 2004). Once a person dies, the blood stops circulating and settles to the dependent regions, whilst chemical and microbial decay begins. Consequently, many drugs become redistributed and their concentration at a particular site may increase, decrease, or remain more or less the same as it was at the time of death. All drugs behave differently. There are also differences between their behaviour in different individuals, whilst the post‐mortem environment and time since death are also important factors. For example, drug addicts often develop a tolerance to their drug of choice, but there is no way of estimating this once they are dead. Therefore, an otherwise healthy drug addict may have much higher drug levels in their body after death than a person not addicted to that drug, but in the drug addict the levels would not have been toxic. Similarly, post‐mortem changes mean that the blood drug concentration can differ markedly in different regions and there is little consensus about how many regions should be sampled or whether the highest, lowest, or mean value of those samples should be used in any calculations. For example, if a drug is prone to redistribution (e.g. morphine), its concentration tends to be much higher in blood taken from within one of the heart's chambers (cardiac blood) or the surrounding pericardial sack than it does in the peripheral blood – which is usually taken from the left and right femoral veins. Microbial fermentation causes the formation of ethanol and could potentially lead to a false assumption of alcohol intake prior to death. Although microbial ethanol production is not usually marked until 3–10 days after death it might, presumably, occur sooner under conditions that promote microbial growth. Therefore, any findings should be considered with a level of caution appropriate to the circumstances.
4.6 Future Directions
There is still a need for a reliable means of estimating the age of bloodstains. It might be achieved through estimating the rates of DNA degradation, although a range of biological and environmental factors will affect this.
The analysis and interpretation of blood spatter patterns will probably be speeded up and, hopefully, improved with increased use of automated scanning and computer‐based techniques. If blood spatter patterns at crime scenes can be recorded reliably as computer files, it will be easier to send these to experts around the country or between countries for their opinion. Although this is already done through photographic documentation, computer technology allows one to record the scene in three dimensions and therefore gain a more accurate impression of the scene. It also becomes possible to develop 3D computer models of how the crime scenario unfolded. This evidence could also be presented to the jury when a case comes to court.