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Blood is a fluid medium that is found within the cardiovascular system-which comprises of the heart and blood vessels (Jackson and Jackson 2008). It consists of 55% blood plasma and 45% cellular material (Jackson and Jackson 2008). Blood plasma consists of dissolved materials such as antibodies, hormones, waste products and nutrients, whereas the cellular material consists of erythrocytes (red blood cells), leucocytes (white blood cells) and thrombocytes (platelets) (Jackson and Jackson 2008).
Blood is transported through the body by the pumping action of the heart. It has numerous functions including (Jackson and Jackson 2008):
Acting as an internal transport system-including the removal of waste products for excretion and moving nutrients for metabolism.
Maintaining body temperature.
Defending against infection.
Protecting the body from effects of injury.
Blood is one of main sources of DNA found at crime scenes, and is crucially important in establishing a link between a suspect and a victim of a crime (Jackson and Jackson, 2008). To detect the presence of blood at a crime scene, a presumptive test is used. These can, however, only detect whether a substance is blood and cannot distinguish between human and animal blood-a serological test is needed to do this.
The Erythrocytes (red blood cells) are the most common type of blood cell and contain haemoglobin (Jackson and Jackson 2008). They contain haemoglobin- a protein containing iron. Haemoglobin is responsible for the carriage of oxygen, and it is this property that presumptive blood tests are based on. Most of the presumptive tests rely on the ability of haemoglobin to catalyse the oxidation of a reagent, normally hydrogen peroxide (H2O2 (aq)) (Jackson and Jackson 2008). The result of oxidation normally produces a colour change in the presumptive test.
Tiny amounts of blood present as a scene can be detecting using a colour change test. Some old and dried stains look similar in appearance to blood - which can lead to a scenes of crime officer conducting a presumptive test. Other substances that could have contaminated suspected blood or other substances on their own at a scene could lead to a presumptive blood test incorrectly showing a positive result for blood. This is known as a false positive.
Once a stain has been determined as blood, then two processes must be completed. The first is to interpret any bloodstain patterns, so that a reconstruction of events can be established (Langford et al 2005). Secondly, bloodstains must then be recovered for further analysis (Langford et al 2005). Recovery of bloodstains varies according to whether the stain is wet or dry. Once recovered, the blood can then be sent to a Forensic Science Service laboratory, where it will be initially tested to ascertain whether it is human or animal blood. To do this a serological test will be conducted, which involves identifying the presence of proteins specific to humans and analysing for DNA sequences specific to humans (Jackson and Jackson 2008). The blood will then be used for DNA profiling, which will hopefully establish whether the blood belongs to the suspect or the victim.
I.II Aims and Objectives
The aim of my project is to create a definitive list of false positives for four different presumptive blood tests. Within this aim I have six objectives to complete:
To compare the false positives of four different presumptive tests.
To test substances that are known false positives- as reported by other authors.
To test unknown substances allied to those already known.
To record the time taken for a substance to react with a presumptive test.
To photograph the results of from each substance.
To create a definitive list of false positives for each presumptive test.
I am going to compare four different presumptive tests, as some tests are more practical to use in some situations than others. Consequently, analysing more than one test will allow a wider range of results.
I shall also be testing known false positives as reported by other authors, as it is important to show how the presumptive tests react. Unknown substances allied to those that are known will then be tested to see whether similar substances react alike. This will then allow me to establish whether an unknown substance has reacted or not, as I can compare the reaction times and colour changes from both the known and unknown substances.
It is important to record the time taken for a substance to react with a presumptive test, as blood should show a result straight away. An unobvious result that takes time to develop could indicate that the substance being tested is a false positive. Photographing results will allow me to document the differences in the colour change in each reaction with each substance.
I feel that it is important to create a definitive list of false positives as it can reduce the risk of using valuable resources at a crime scene. For example, if a scenes of crime officer is informed that a possible blood stain has been contaminated with horseradish (a known false positive), then they can use a presumptive test for blood that is not known to produce a false positive with horseradish. If the result is positive for blood, then serological tests for blood can be carried out.
Overall, I hope that this project will aid the work of a scenes of crime officer to choose the correct presumptive test to use in different situations-minimising time spent and resources used.
Chapter II. Literature Review
II.I Background Information
The scientific analysis of blood was initially mentioned in 13th century Chinese texts, but it was Karl Landsteiner who discovered the modern science of blood typing, which categorises different types of blood into the "ABO blood typing system" (White 2010). In 1901 it was reported that blood could be determined in two week old serum stains on linen, and by 1902 the four blood types - A, B, O and AB had been discovered (White 2010). This system is based on types of antigen on the red blood cell's membrane. An antigen is a "protein molecule capable of binding on to an antibody (Erzinçlioglu 2004). The ABO system uses two antigens which are known as A and B; and the four blood groups are determined according to this system (Erzinçlioglu 2004). People that have the blood group A have the A antigen, those that are group B have the B antigen; those in the AB category have both antigens and those who belong to the O group have neither antigen (Erzinçlioglu 2004). A person's blood contains the opposite group of corresponding antibodies, so people with blood group A have b antibodies, people with blood group B have a antibodies, those with blood group AB have neither a or b antibodies and those with blood group O have both a and b antibodies (Erzinçlioglu 2004). If the wrong antibodies are introduced into the wrong blood group then death can be a result due to the red cells clumping together.
The first suspect to have been convicted largely on the basis of DNA analysis of blood samples was found guilty at Leicester Crown Court on 22nd January 1988 (White 2010). This case marks an important milestone, and DNA technology has become commonplace in forensic laboratories and is now instrumental in establishing both guilt and innocence in court cases (White 2010).
II.II Physical Properties of Blood
Blood constitutes about 7.7% of the body weight of a person (White 2010). This equates to 5-6 litres in males and 4-5 litres in females (Tortora and Anagnostakos 1987). Viscosity is resistance to flow, which in fluids is compared to water which has a viscosity of 1. (Bevel and Gardner 2002). Blood viscosity usually ranges between 4.4 and 4.7 (Tortora and Anagnostakos 1987). Blood also has a higher specific gravity (density) than water, which is the weight of a substance relative to the weight of an equal volume of water (James and Nordby 2005).
Blood is a fluid that circulates throughout the body by way of the heart, arteries, veins and capillaries-known as the circulatory system (James and Nordby 2005). A primary function of blood is to transport oxygen, electrolytes, nourishment, hormones, vitamins and antibodies to tissues and to transport waste products from tissues to the excretory organs (James and Nordby 2005).
Tortora and Anagnostakos (1987) (in Bevel and Gardner 2002) say that when 4-6 litres of blood is present in the circulatory system, it is distributed as follows:
Figure 1- Blood Distribution in the Circulatory System (Tortora and Anagnostakos 1987)
As a medium, blood is composed of 55% plasma and 45% cells (White 2010). A single drop or large volume of blood is held together by strong cohesive molecular forces that produce a surface tension (James and Nordby 2005). Surface tension is defined as "the force that pulls the surface molecules of a liquid toward its interior, decreasing the surface area and causing the liquid to resist penetration" (James and Nordby 2005).
Bevel and Gardner (2002) state that plasma is the pale yellow fluid component of blood, which is broken down by volume into 91% water, 8% protein, 1% organic acids and 1% salts. Fibrinogen is one of the proteins, and this plays an important role in the clotting of blood (Bevel and Gardner 2002). Blood serum is blood plasma minus its protein content (Jackson and Jackson 2008). The cellular component of blood consists of erythrocytes (red blood cells), leukocytes (white blood cells) and thrombocytes (platelets) (Bevel and Gardner 2002). Red blood cells are heavier than plasma, which can be seen in bodies as lividity-which is where red cells settle to the lowest extremity of a body after death (Chmiel and Walitza 1980).
Figure 2- A red blood cell, platelet and white blood cell (University of Eastern Kentucky 2010).
There are roughly 4.8 to 5.4 million red blood cells per cubic millimetre of blood (Tortora and Anagnostakos 1987). They are bioconcaved discs in shape. The main role of the red blood cells is "to transport oxygen from the lungs via the arterial system and return carbon dioxide to the lungs for expiration via the venous system" (James and Nordby 2005). Red blood cells contain haemoglobin which is a red pigment that gives blood its colour (Bevel and Gardner 2002). Haemoglobin is composed of globin, which is made up of four folded polypeptide chains, and four haem groups that join with iron (University of Eastern Kentucky 2010).
Figure 3- Haemoglobin, containing four haem groups (University of Limerick, 2010).
As the oxygen content increases in the blood, the bright red pigment of the haemoglobin also increases (Bevel and Gardner 2002). A red blood cell does not contain a nucleus.
Red blood cells are expressed as a percentage of the "packed (red) cell volume (PCV)", also known as the haematocrit (Wonder 2001). Nelson and Rodak (1983) state that the haematocrit in humans is variable between individuals.
Possible people with range of haematocrit
Chronic alcoholics or drug abusers, steroid abusers, women after traumatic child birth or illegal abortion, malnourished homeless, elderly.
Normal range for nontraumatic venipuncture (blood drawn in a clinic or hospital) samples.
Dehydrated individuals, people in shock, those living at high altitude, impending and active heart attack victims, newborn babies, people suffering from hypothermia, and people after extreme exercise.
Table 1- Table to show the range of haematocrit ratios (Wonder 2001).
White blood cells act to fight infections, destroy old cellular material and to destroy other invading microbes (Bevel and Gardner 2002). White blood cells can be further subdivided into phagocytes - which are responsible for the capture and ingestion and foreign substances, and lymphocytes- which are responsible for the production of antibodies (Jackson and Jackson 2008). They make up less than 1% of the cellular component of blood, which equates to 5000 to 9000 white blood cells per cubic millimetre (Tortora and Anagnostakos 1987). The nuclei of white blood cells are the source of DNA in the blood (James and Nordby 2005).
The other part of the cellular component of blood is the platelets. Like red blood cells, platelets also lack a nucleus (Bevel and Gardner 2002). Bevel and Gardner (2002) say that there are generally about 250,000 to 400,000 platelets per cubic millimetre of blood. Platelets are major components of the clotting mechanism of blood, and this is their primary function (James and Nordby 2005). Platelets have irregular shapes and are normally quite small, however when they encounter a damaged blood vessel they increase their size and their shapes changes (Bevel and Gardner 2002). They also become sticky and adhere to surrounding fibres in the vessel wall, which results in the accumulation of platelets called the "platelet plug" (Bevel and Gardner 2002).
II.III Blood at a Crime Scene
Blood is normally found at a crime scene due to a person sustaining an injury either by accident or on purpose. When a breach in the circulatory system occurs- due to an injury- the body reacts in different ways to control the loss of blood (Bevel and Gardner 2002). Initially the vascular spasm occurs, which is which the "smooth vessels in the blood vessel wall contract to decrease the size of the vessel", which reduces the flow of blood through it (Bevel and Gardner 2002). Tortora and Anagnostakos (1987) say that this reduces blood loss for up to 30 minutes following injury, which gives time for the other blood loss mechanisms to engage. The platelet plug then follows which reduces, if not stops, the blood loss (Bevel and Gardner 2002). The final step is coagulation, or clotting. This is what is normally seen at crime scenes, where the clotted mass of fibrin fibres and blood cells is surrounded by blood serum (Bevel and Gardner 2002).
There are three types of bleeding that can occur from damage to blood vessels (BUPA 2009):
Arterial bleeding usually is spurting bright red blood, due to the blood having come from the heart and lungs-so it is oxygen rich (Walter et al 2004). The pumping action of the heart adds rhythmic surges to move blood vessels away from the heart (Wonder 2001). It is the most serious type of bleeding, and the most difficult to control due to the blood in the arteries being under pressure from the heart (Walter et al 2004). Arterial wounds results in "volume stains" (Wonder 2001). Loss from the carotid artery or the aorta can rapidly lead to death (Wonder 2001). Examples of arterial injuries, and how they may occur are listed in Table 2.
Neck, front throat
Stab wound, Gunshot, Decapitation
Under collar bone
Gunshot, Stab wound
Slit wrists, Bone Break, Stab wound
Gunshot, Stab wound
Bone Break, Crushing
Upper arm muscle
Table 2- Areas and actions that may involve arterial damage (Wonder 2001)
External venous bleeding is normally as a result of wounding, as veins are closer to the skin than arteries (Walter et al 2004). It results in the steady flow of dark red (almost brown) blood, and is darker than arterial blood as it has released oxygen to the tissues in the body and is flowing back to the heart and lungs for more oxygen (Walter et al 2004).
Capillary wounding is common in minor wounds as capillaries are very small vessels that are under very little pressure with a low volume of blood (Walter et al 2004). Capillary bleeding results in the oozing of either bright or dark red blood, which will normally stop on its own (Walter et al 2004).
As well as the three main types of bleeding, there is a further category which is traumatic bleeding. There are different types of wounds which can cause traumatic bleeding, and these can be categorised as follows:
Abrasion- also known as a graze, where an object brushes on the skin but does not break it.
Hematoma- where blood vessels are damaged, causing blood to collect under the skin.
Laceration- where a blunt impact to soft tissue causes a deep wound.
Incision- where a precise cut is made into the skin.
Puncture Wound- where an object penetrates the skin and deeper layers.
Contusion- also known as a bruise, where a blunt trauma causes damage under the skin, but does not break it.
Crushing injuries- where a great amount of force is applied over a period of time, causing initially internal bleeding.
Ballistic trauma- where a projectile weapon has entered and exited the area of the body, causing a wound between the two.
Scenes of Crime officers attend many types of crime scene where blood is present. These include:
Burglary- When an offender breaks a glass window or door to gain entry to a premises, they risk cutting their hands/arms. This leads to blood being left on fragments of glass in the window and on the floor.
Assault/Wounding- Open wounds are normally the result of an attack on a victim. Blood can be left at an assault scene on the weapon that was used in the assault, on the ground, on the offender and on the victim. If the victim is bleeding heavily then blood will be left whenever the victim comes into contact with another surface.
Manslaughter/Attempted Murder/Murder- Blood left at these scenes is not only important for swabbing purposes, but the pattern in which the blood is left can determine the order of events at a major scene.
Road Traffic Crash- Blood at this scene can be found in the victims car and, if involved, the offenders car. This is important as it can place people in their respective cars- allowing investigators to work out the positions of people at the time of the incident.
The collection, packaging and preservation of blood evidence at a crime scene should not take place until the scenes of crime officer has documented the bloodstain patterns (Lee, Palmbach and Miller 2001). Whenever biological fluids are encountered at a crime scene, protective clothing, gloves and masks should be worn due to the biohazard nature of blood (Lee, Palmbach and Miller 2001).
To recover dry blood, an area near the blood that is unstained should be swabbed using a sterile swab, as a control sample (Derbyshire Constabulary 2008). Then, the stain should be swabbed using a sterile swab that has been moistened using sterile water (Derbyshire Constabulary 2008). The remains of the stain should then be dry swabbed using a sterile swab (Derbyshire Constabulary 2008). The swabs should be returned to their tubes immediately and stored frozen as soon as possible (Derbyshire Constabulary 2008). A "batch control" of both the water and swabs should always be made, and should be exhibited separately to the swabbed stain and background control (Derbyshire Constabulary 2008). Items that have areas of dried blood on them should be packaged in paper bags which are sealed securely and clearly marked as biohazard. Blood and bloodstained evidence should never be packaged in airtight containers (Lee, Palmbach and Miller 2001).
To recover wet blood, a control swab of the surrounding area of the stain should be taken using a sterile swab (Derbyshire Constabulary 2008). The wet stain should then be swabbed using a dry, sterile swab (Derbyshire Constabulary 2008). The swabs should be returned to their tubes immediately, and should be stored frozen as soon as possible (Derbyshire Constabulary 2008). Again, a "batch control" of the swab should be exhibited separately (Derbyshire Constabulary 2008). If a removable item has an area of wet blood on it, then the entire object should be exhibited and left to dry in a drying room at the police station.
Often at crime scenes, stains that are composed of unknown substances can easily be confused with blood. Identifying whether a substance is blood allows further analysis to confirm species, and the individual (Spalding 2006).
II.IV Presumptive Tests for Blood
James and Nordby (2005) say that a presumptive test is one which allows the scenes of crime officer to make a qualified conclusion that blood is present in the tested sample, when positive. They also say that when a test is negative, stains that need no further consideration are eliminated. Presumptive tests may be recognised as those that produce a visible colour reaction or those that result in the release of light (James and Nordby 2005). Both of these rely on the catalytic properties of blood to drive the reaction (James and Nordby 2005). Lee, Palmbach and Miller (2001) write this as a chemical reaction:
AH2 + H2O2 ƒ A + 2H2O
Oxidisable chemical Hydrogen peroxide Haeme Oxidised
James and Nordby (2005) state that catalytic tests involve the "chemical oxidation of a chromogenic substance by an oxidising agent catalyzed by the presence of blood". They also say that the catalyst of the reaction is the peroxidise-like activity of the haeme group of haemoglobin.
Cox (2004) describes the attributes that a good presumptive test for blood should be sensitive, specific, quick, simple and safe. In order for presumptive tests for blood to function properly, they must detect a component of blood (Tobe, Watson and Daéid 2007). Most presumptive tests therefore act on the peroxidise activity of haemoglobin. This component is not found in the everyday environment, but other substances found in items such as fruit and vegetables perform a similar function (Tobe, Watson and Daéid 2007).
A very popular presumptive method is the phenolphthalein test, which is also known as the Kastle- Meyer test (Virkler and Lednev 2009). Lee, Palmbach and Miller (2001) say that the Kastle-Meyer test was introduced in 1901 by Kastle. Phenolphthalein will cause an alkaline solution to turn pink after it has been oxidised by peroxide when blood is present (Spalding 2006). The reagent consists of reduced phenolphthalein in alkaline solution, which is oxidised by peroxide in the presence of haemoglobin (James and Nordby 2005). The test result is normally immediate, and a positive result a minute or more after the test is performed is usually not considered as reliable (James and Nordby 2005). It has a sensitivity of 1:100,000 (Lee, Palmbach and Miller 2001).
James and Nordby (2005) say that Adler and Adler in 1904 investigated the reduced or colourless form (leuco) of the dye malachite green, which is also referred to as McPhail's reagent. This test involves the Leuco base of malachite green (Lillie 1969). Leucomalachite Green oxidation is catalyzed by haeme to produce a green colour (James and Nordby 2005). The reaction is usually carried out in an acid medium with hydrogen peroxide as the oxidiser (James and Nordby 2005). It has a sensitivity of 1: 20,000 (Lee, Palmbach and Miller 2001).
"Bluestar is a luminol preparation developed by Professor Loic Blum in France that is extremely sensitive and stable and produces a very bright, long lasting chemiluminescence" (James and Nordby 2005). The extreme sensitivity of Bluestar Forensic allows detections of bloodstains down to 1:10,000 dilutions (Bluestar Forensic 2004). It does not require total darkness to be visible, and works well on either fresh blood or old bloodstains (Bluestar Forensic 2004). Bluestar works by mixing the Bluestar Forensic solution with Bluestar Forensic tablets, which is then left to dissolve. This is sprayed onto the area of suspected blood. A positive result will cause a bluish luminescence (Bluestar Forensic 2004).
The Hemastix test, created by Miles Laboratories in 1992, is particularly useful when solutions can be hazardous, or inconvenient (James and Nordby 2005). The test consists of a plastic strip with a "reagent treated filter tab at one end" (James and Nordby 2005). The tab contains TMB, diisopropylbenzene, dihydroperoxide, buffering materials and non reactants (James and Nordby 2005). A bloodstain is tested by moistening a swab with distilled water, sampling the stain, and touching the swab onto the reagent tab on the strip (James and Nordby 2005). The tab is normally yellow, and turns form orange to green or blue when positive.
Quality control testing is necessary and should be completed with known blood samples on every new batch of test reagents to verify that the reagents are working as expected (Lee, Palmbach and Miller 2001).
II.V False Positives
Sutton (1999) points out that a false positive is "an apparent positive test result obtained with a substance other than blood. James and Nordby (2005) say that misleading results can be attributed to
Chemical oxidants (often producing a reaction before the application of peroxide)
Plant materials (vegetable peroxidises are thermolabile)
Materials of animal origin (that contain traces of blood).
Substances that produce false positives generally take detectably longer to react and, therefore, may be eliminated through observational interpretation (Tobe, Watson and Daéid 2007).
False positives were initially noted only with copper salts (Glaister 1926).
Tobe, Watson and Daéid (2007) state that "saliva, semen, potato, tomato, tomato sauce, tomato sauce with meat, red onion, red kidney bean, horseradish, 0.1 ascorbic acid, 5% bleach, 10% cupric sulphate, 10% ferric sulphate and 10% nickel chloride" are all known false positives.
Bluestar False Positives (2008) say that Bluestar has false positives that include oil based paint, alkyd varnish, turnip, banana, leek, green bean, carrot, ginger, manganese sulphate, copper sulphate, iron sulphate and potassium permanganate.
Lee, Palmbach and Miller (2001) write that many household cleaning products contain oxidising agents that can produce false positives. Many fruit and vegetables produce false positives including apples, horseradish and broccoli (Lee, Palmbach and Miller 2001).
Bleach is a false positive that provides an (immediate and intense reaction) according to Gardner (2005). Hunt et al (1960) say that faeces often gave a false positive depending on the food that had been eaten previously.
Ponce and Pascual (1999) state that lemon juice added to a bloodstain can cause a positive result due to its acidity.
A false negative is when "there is some interference with the oxidation-reduction reaction", normally in the presence of a strong reducing agent, which results in a delay of the oxidation reaction; thus resulting in a coloured formation (Lee, Palmbach and Miller 2001). False negatives are less common but problematic as an actual blood sample may be overlooked or left at the scene (Lee, Palmbach and Miller 2001).
Many of the false positive reactions can be identified during the presumptive testing procedure if any changes observed and the exact point in the reaction of these changes is recorded and compared to that of blood (Lee, Palmbach and Miller 2001).
Chapter III. Experimental Methodology
I will be investigating known substances previously reported by other authors that show a false positive and then analysing other substances similar to those already known to see if these also produce a false positive.
III.I Project Design
Each of the substances will need to be repeated to ensure a wide enough range of consistent results. Therefore, a grid will be drawn on a piece of Perspex measuring 1.5m2, and a piece of filter paper placed in each of the grid spaces, to allow the even distribution of substances and to allow the easy identification of false positives.
New technique to detect metabolites from a single drop of blood
New technique to detect metabolites from a single drop of blood
New technique to detect metabolites from a single drop of blood
Figure 4- A diagram to illustrate an example of the project layout.
For each of the presumptive reagents tested, the filter paper in each grid space will be exposed to a substance to be tested. This will be allowed to dry for a minimum of 1 hour. Each substance will then be tested with a reagent. Each substance will be repeated three times to give a fair indication of performance. The time taken for a substance to register a positive result will be recorded. If a colour change occurs then the test will be classed as positive. If no colour change is noted within 5 minutes of the reagent being added, then the test will be classed as negative.
III.II Sample Preparation
The substances that I have chosen to analyse are known false positives as reported by other authors and then substances allied to known false positives.
Known false positives to be tested:
Bleach Solution (5%).
Unknown substances to be tested:
Bleach Solution (less than 5%).
III.III Choice of Presumptive Tests
I have chosen to use the following presumptive reagents to test substances for false positives:
III.IV Control Tests
I will test all of the presumptive tests on blank filter paper before proceeding to test with substances. This ensures that there is no reaction from the filter paper to the presumptive tests. I shall also test all of the presumptive tests with horse blood before proceeding to test with substances. This shows that the tests do recognise a sample of blood.
I have chosen to use 2.5g of each substance as I feel this is an amount that is representative of a stain at a scene.
Blood is reported to have been diluted to 1:10000 in previous tests, and as this dilution has proved the most successful, I have chosen to use this dilution.