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Forensic palynology relates to the application of the study of pollen and spores to legal matters, often used to establish links between objects, people and places based on the analysis and identification of pollen.

Pollen and Spores
Pollen refers to a powder containing the male gametophytes of seed-producing plants, produced and dispersed in order to pollinate and reproduce. Pollen grains are dispersed in a variety of ways. Anemophilous plants produce pollen to be dispersed by wind. The extent of travel can depend on a variety of factors including the strength and direction of the wind, the weight and shape of the grain, and atmospheric conditions, though the pollen will typically fall within around 2km of the parent plant. Pollen produced by hydrogamous plants is transported by water, whereas zoogamous plants use animals such as bees, birds and rodents to disperse their pollen. Finally, autogamous plants are self-pollinating, so the pollen they produce does not need to travel. If the pollen reaches a suitable plant, it can germinate (ultimately producing a plant from a seed). Pollen grains typically have a fairly hard coat to protect them as they pass from one plant to another, making them relatively resilient to destruction. Spores, on the other hand, are units of asexual reproduction.

Pollen Analysis
Pollens and spores are extremely small in size, produced in vast amounts, dispersed by various mechanisms and are fairly resistant to destruction. All of these features make them especially ideal for use in a forensic investigation. The morphology of pollen and similar substances is fairly complex, meaning that with the appropriate equipment, expertise and reference materials, it is possible to distinguish between and identify pollen grains. Pollen can particularly differ in shape, size, wall structure, and general appearance.

If possible, the palynologist should ideally visit the crime scene to collect samples, conduct a vegetation survey, and take any photographs as needed. Being familiar with the layout of the scene and the plants present can be of great use when establishing the source of pollen collected. Any evidential samples will be collected (such as from objects or people), but in addition to this control samples will be collected. This provides samples with which to compare any evidential samples.

Analysis of pollen is often carried out using transmitting light microscopy, which simply refers to any type of microscopy in which a light source is transmitted through the sample, allowing the specimen to be viewed through a lens. In addition to this, scanning electron microscopy (SEM) may be utilised. This technique may be used alongside systems such as QEMSCAN (Quantitative Evaluation of Minerals by Scanning Electron Microscopy), which allows for the automated analysis of minerals and other substances. The palynologist will study, analyse and compare pollen grains using their own expertise but also pollen reference collections if available.

Forensic Applications
Palynological samples can be recovered from a wide range of sources, including people, such as on their clothes, in their hair or even in their nasal passage, vehicle tyres, air filters in cars, on objects and in mud. Because of the dispersal mechanisms of some plants, pollen can be readily picked up and transferred. A person can easily inadvertently pick up pollen from a crime scene, whether it be in mud on their shoes or on their clothes from directly brushing against a plant in the area.

With this in mind, a primary use of palynology in a forensic investigation is to establish a link between two places, objects or people. For instance, it may be possible to link a suspect to an object, a vehicle to a crime scene, or even link two separate incident scenes. If a suspect was present at a particular crime scene at which pollen can be found (for instance a field or garden), they may have picked up pollen on their clothing or in their hair. Because pollen is so resilient, it can often stick to other objects even after that object has been washed. If the pollen recovered from the suspect matches pollen collected from the crime scene, this could suggest that the suspect was in fact present at that scene. However it must be considered that although the presence of pollen may establish a link, the lack of pollen does not necessarily prove that there is not a link.

Similarly, palynology may be able to determine the location of a crime scene if it is not known. For instance, a body that is believed to have been moved may carry pollen grains that can be analysed and traced to a likely location. This may particularly be suspected if the body carries large amounts of a particular pollen that is not found at the location in which the body was found.

The study of pollen can also be used to determine the travel history of an item. In some cases it may be necessary to ascertain where an item has originated from, especially illicit drugs, money, antiques and even food. By analysing pollen recovered from suspect items, it may be possible to trace that item to a particular country if the pollen grains identified are sufficiently distinctive. Although this application of palynology may not necessarily be able to establish an exact location, it may be least be possible to rule out certain geographical locations and point the investigation in the right direction.

It may even be possible to estimate the time of year at which a crime took place. In the investigation of a somewhat older crime scene, pollen collected may actually be released at a different time of year, indicating the crime occurred during this period.

Of course despite the links palynology may establish, further evidence may be needed to support any conclusions reached. Pollen recovered from a suspect that happens to match that of a crime scene may simply suggest that the individual had visited that area at some point recently, not necessarily prove that they have committed a crime.

Although forensic palynology has been utilised for decades, it is unfortunately not widely accepted as a reliable forensic technique, instead frequently seen as a last result failing more ‘standard’ investigative techniques. In addition to this, there are very few people properly trained to analyse palynological samples, thus samples are often collected and handled by untrained staff, inevitably leading to issues of sample preservation and contamination.

Case Study
The first documented use of the analysis of pollen and spores to a forensic investigation was in Austria in 1959. Whilst on a trip down the Danube River, an Austrian man disappeared. His body could not be found. A friend and business partner of the victim soon fell under suspicion, and was arrested and charged with murder. Unfortunately without a body, and of course the suspect proclaiming his innocence, there was not much of a case against this man.

During a search of the man’s cabin, a pair of muddy boots were recovered, providing the authorities with a new avenue of investigation. Palynologist Wilhelm Klaus of the University of Vienna was called upon to provide his expert opinion regarding a mud samples recovered from these shoes. Klaus was able to identify a number of modern pollens in the mud, including spruce, willow and alder pollen, along with a significantly older fossil pollen. Only a small area north of Vienna was consistent with this combination of types of pollen. The defendant was confronted with this new piece of information, at which point he finally cracked and confessed to the murder. He led police to the clandestine grave which was, interestingly, in the region selected by Wilhelm Klaus.

 

Bryant, V. M. Mildenhall, D. C. 1998. Forensic palynology: a new way to catch crooks. Association of Stratigraphic Palynologists Foundation Contribution Series 33, pp. 145-155.

Mildenhall, D. C. Wiltshire, P. E. J. Bryant. V. M. Forensic palynology: Why do it and how it works. For Sci Int. 163 (2006), pp. 163-172.

Mildenhall, D. C. Forensic palynology in New Zealand. Review of Paleobotany and Palynology 64. pp. 227-234.

Mildenhall, D. C. Civil and criminal investigations. The use of spores and pollen. SIAK Journal. 4 (2008), pp. 35-52.

Firearm investigation is a specialty of forensic science focusing on the examination of firearms and related subjects. Closely linked to this is ballistics, which relates to the flight path of projectiles, often associated with forensic science during the investigation of firearms. This area of study examines the path of a bullet from when it leaves the firearm up until it strikes the target. During investigations in which the use of firearms is suspected, a number of artefacts may be collected for examination, including firearms, cartridge cases, bullets, live ammunition, trace materials, and any material damaged by a projectile.

The study of firearms and firearm ballistics is often divided in internal, external and terminal ballistics. Internal ballistics refers to the processes inside the firearm, the minute space of time between the shooter pulling the trigger and the bullet exiting the muzzle of the gun. Following this, external ballistics deals with the bullet’s flight between leaving the firearm and striking a target. Finally, terminal ballistics, also known as impact ballistics, refers to the study of the projectile striking a target.

Firearm Mechanism
Although there is a wide range of types of firearm, the basic theory behind how a projectile is fired is fairly generic – the weapon aims to convert chemical energy into kinetic energy in order to expel a projectile from the firearm.

A round is first loaded and locked into the breach. This round consists of an outer cartridge case, a bullet, some form of propellant, and a percussion cap. The firing pin is generally mechanically restrained and, when the firearm is cocked, the firing pin spring is compressed. As the trigger is pulled, the hammer-mounted firing pin is forced forward to strike through a small hole in the breech face, hitting the primer cup. This contains a mixture of sensitive chemicals which rapidly burn, producing sufficient hot gases to ignite the propellant. As the gunpowder is ignited an expansion of gas occurs which, confined in a small space, eventually forces the bullet down the barrel of the firearm. Following discharge, a number of events regarding the used cartridge case may occur depending on the type of firearm. If the firearm is self-loading, a bolt will move back and pull the cartridge case out of the chamber, leading to it being ejected from the weapon. However in weapons such as revolvers, the cartridge will remain in the firearm until the shooter removes it.

The manner in which a firearm is loaded after each shot will vary depending on the type of weapon. Some firearms are bolt action, meaning a bolt ejects the spent cartridge and, when pushing forward, picks up a new cartridge and places it in the chamber, cocking the trigger during this process. Manual action firearms require manual reloading by means of a mechanical device such as lever or pump action. In recoil operated or blow back weapons, pressure generated by the ignited propellant drives back the bolt. Gas operated firearms include a gas port and, until the bullet has passed this point in the barrel, the bolt is locked. An amount of gas leaks into this port and unlocks the bolt, allowing it to move backwards. This ejects the spent cartridge case and loads another. Finally, in a revolving cylinder weapon, pressure on the trigger causes a cylinder containing the cartridges to rotate, positioning a new cartridge so that it may be fired.

Shotguns are available as either single-barrel of double-barrel. Single-barrel weapons can be further classified as single-shot, bolt-action, pump-action, lever-action or self-loading. Double-barrel shotguns encountered may contain a hinge at the barrel, allowing the shooter to open the weapon to reload cartridges. Both of these types of weapon are subject to having their barrel decreased by criminals, producing the “sawn-off” shotgun. The end of many shotgun barrels is designed to incorporate a feature known as the choke, which is a decrease in barrel diameter. This aims to focus the shot of pellets to ensure that they do not spread too much when fired. It is likely that different shotguns will express a difference degree of choke.

The ammunition of shotguns can differ quite considerably from one another. Modern cartridges consist of a metal base containing the primer and either plastic or paper sides with wads between the shot and propellant. Examination of wads, which leave the muzzle along with the shot fired, may indicate both the bore of the shotgun and even the manufacturer. Certain wads, namely those composed of plastic, may hold indentations which indicate the size of the pellet fired from the weapon.

Rifles may be single-shot, lever-action, pump-action, bolt-action or self-loading.

The mechanism of an air weapon is quite different. A piston is forced down a cylinder, often by a compressed spring. This creates a burst of high-pressure air which forces the projectile down the barrel and out of the weapon. Following each shot, the user manually compresses the spring in order to fire another shot. This action compresses the spring again, which is held along with the piston until the trigger is pulled to discharge the firearm. There are other forms of air weapon, such as pneumatic air guns, which do not pressurise the air at discharge but instead use pre-pressurised air.

Bullet Flight
Tracing the flight path of a bullet can provide important details during a forensic investigation, namely from what direction the projectile was fired. This is often vital in reconstructing the series of events throughout an incident. Establishing the path of a bullet may not be straightforward, as numerous factors must be taken into consideration. Air resistance and gravity affect the bullet’s flight path, causing it to project in a downward arc rather than a straight line. Environmental conditions such as strong winds could additionally slightly alter a bullet’s flight. How a bullet’s flight will be affected will depend on the initial velocity of a bullet, as those with a higher velocity will be less influenced, as well as the shape of the bullet. Numerous factors can result in abnormal bullet flight characteristics.
Tumbling is a phenomenon relating to flight imbalance, caused when a bullet is subjected to defective rifling or has been damaged. Another problem is ‘yaw’, an effect referring to a bullet’s deviation from a linear path. This can be caused by defective rifling, poor loading or a badly cast bullet. The choice and loading of bullet can also cause problems. If the user of the firearm loads the weapon with a bullet of incorrect calibre, the bullet may not achieve a spin rate that will ensure stability, resulting in excessive bore friction which ultimately leads to a low muzzle velocity. Incorrect bullet choice can also lead to an excessive bore velocity, causing the bullet to slide over bullet rifling and so resulting in a low spin rate.

Types of Firearm
In the UK, the Firearms Act 1968 describes a firearm as “a lethal barrelled weapon of any description, from which any shot, bullet or other missile can be discharged”. It includes 4 main classes of controlled weapons: firearms, shotguns, prohibited weapons and air weapons.

S1 – Firearms
This section covers a range of guns, including bolt action and straight pull rifles.

S2 – Shotguns
Shotguns are classed as smooth bore guns with a barrel at least 24 inches in length with a maximum diameter of 2 inches. It has no magazine or a non-detachable magazine which cannot hold more than two cartridges, and it is not a revolver gun. Anything other than this will be classed as an S5 weapon.

S5 – Prohibited Weapons
This section includes any firearm disguised as another object, rockets or ammunition not previously categorised, military ammunition and weapons, and machine guns to name a few. In 1997, an amendment was made to this act adding expanding ammunition, such as hollow point bullets, to S5.

Air Weapons
These spring, pre-charged pneumatic or carbon dioxide weapons are also controlled by this act. They use high-pressure air, often pressurised by a piston forced by a compressed spring, to push the projectile down and out of the barrel of the firearm.

All of the above categories can be further divided into a wide range of weapons, some of which are detailed below.

Semi-Automatic: Once cocked, this weapon will load itself from the magazine, which can hold dozens of. This allows for fast reload and so a high rate of fire.

Revolver: A revolver is a type of pistol which holds the ammo in a rotating drum, holding between 5 and 7 shots. After each shot, the cylinder is rotated and the next shot is aligned with the barrel. Its relatively short barrel means it is fairly inaccurate and it has a slow reload time.

Rifle: Essentially a long rifled barrel firearm primarily designed for relatively long range use in warfare or hunting. Rifles are available as single shot, self-loading, manual action, bolt action or automatic, though most commonly encountered are self-loading. Another form of rifle is the shorter barrelled assault rifle, commonly used in the military.

Submachine Gun: A fast-loading weapon with a high rate of fire, available as single shot or fully automatic. The inaccurate SMG is a magazine-fed weapon which can hold up to around 100 rounds, designed for continuous fire.

Machine Gun: This has a very high rate of fire with a fast reload time and great power. They are generally only used by the military.

Shotgun: A smooth bored, powerful weapon with a short range and low accuracy. It can fire a number of types of ammunition, including solid slugs or pellets. Although there are numerous types of shotgun, single or double barrel are most commonly encountered. Single barrel shotguns can be single shot, self-loading or manual action. The barrel is traditionally long but is sometimes shortened to produce a ‘sawn-off’ shotgun to aid concealment. The end of the barrel may be slightly tapered in order to reduce the shot from spreading as it leaves the muzzle of the firearm. This effect is known as the choke.

Bullets
One of the primary methods of categorising bullets is based on their calibre. Calibre refers to the diameter of the bullet, which can be expressed in various terms, including millimetres (metric system), inches (imperial system) or 100ths of an inch (American system). For example, a 9mm bullet may also be referred to as a 0.35 inch bullet or .35 calibre. However it must be taken into consideration that the calibre of a bullet does not necessarily prove the calibre of the weapon used to fire it, as on occasion a cartridge too large or two small may be used.

Bullets are generally composed of metal, although substances such as plastic or rubber may be used. The core of a bullet most commonly consists of lead, as it is very dense and yet easily moulded so as to produce a gas-tight seal in the barrel upon being fired. However this also means that it will easily become deformed on impact, therefore it is commonly combined with other materials, often copper, tin or antimony, to ensure it will withstand pressure.

The use of a lead bullet can also lead to a problem known as “leading”, in which friction between the bullet and barrel results in the transfer of metal to the barrel, ultimately causing less accuracy. Therefore copper is frequently used to jacket the bullet to reduce the lead being deformed and improve the effects of rifling. For this reason lead is often combined with other metals such as copper, tin or antimony to strengthen the projectile. Lubrication may also be applied to the surface of the bullet. However these techniques can only reduce the problem of leading, therefore bullets are commonly enclosed in some form of ‘jacket’.

A full metal jacket involves a metal such as copper being used to cover the entire outer surface of the bullet. Fully jacketed bullets often display high accuracy and penetration. Some bullets are semi-jacketed, with a partial copper covering with a hollow or blunt lead tip. In this case the nose of the bullet is exposed and this type of bullet can severely deform on impact into a ‘mushroom shape’, producing greater damage. Bullets may also be unjacketed, though these projectiles have a low muzzle velocity and do not penetrate deeply.

Shotgun cartridges differ significantly from bullets. They are generally loaded with pellets or shot, which are small, round metallic balls. Shotguns can also fire single, larger projectiles as well. The shotgun cartridge may also contain a number of wads, which are designed to provide a form of gas seal in the bore. These wads may be found at the scene or even embedded in the target, and can be chemically analysed to provide details of the propellant and primer.

Cartridges
The examination of cartridges, precisely-fitting metal cases containing the bullet, propellant and primer, can provide vital information to an investigation. The composition of most cartridge cases is brass, an alloy of zinc and copper, ideal due to its low density, though other metals may be used. Similar to calibre, chambering refers to the shape and size of a cartridge. The primer, held in the primer/percussion cap at the base of the cartridge case, consists of a small amount of explosive, a fuel and an oxidiser. Modern primers generally consist of lead styphnate, barium nitrate and antimony sulphide, though the substances used may vary. The propellant, often referred to as “gunpowder”, is an explosive mixture designed to ignite and produce enough hot gas to force a projectile from the firearm.

In some instances cartridge cases may be found at the scene of a shooting, though this often depends on the type of gun used. As previously stated, self-loading handguns will eject the cases from the weapon, whereas revolvers retain the cartridges until the shooter manually removes them. However the absence of cartridge cases is not an accurate sign of the type of firearm used, as the perpetrator may have picked up the cases from the scene before leaving.

Initially, any cartridge cases retrieved from the scene should be accurately measured in all dimensions to aid future comparison. The examination of any cartridge cases found can provide clues as to the firearm used during the shooting. For example, cartridges designed for use in a revolver have projecting base rims, whereas those designed for self-loading firearms do not.

Cartridge cases also bear more distinguishing features that can be used to identify them. The headstamp is an indentation produced at the base of many cartridges during the manufacturing process. These markings can then be used to trace a cartridge back to the manufacturer and determine the make and type of the ammunition. Various sources are available to help identify headstamps. When the firing pin strikes the cartridge case, a characteristic indentation is caused that can be used to link cartridges to specific firearms, similar to the comparison of rifling marks (discussed below). Other markings that should be looked for include ejector, extractor and breech face marks. Firearms often have different firing pin, extractor and ejector designs, therefore the examination and comparison of marks produced by these implements can aid in establishing the firearm used. It should be noted that it is possible for cartridge cases to be reloaded with a new bullet and fresh propellant and primer and reused, in which case the cartridge may bear numerous additional markings. Furthermore, cartridge cases recovered from the shooting scene should be examined for fingerprints and similar forensic evidence.

Rifling
During the manufacturing process of firearm barrels, a series of spiralling lands and grooves is produced along the inside of the barrel, known as rifling. Land refers to the raised portion of these spiralling indentations, whereas the grooves are the lower portions between these lands. Rifling, which can be one of numerous types, will be cut with either a left or right hand twist, determining whether the bullet will spin clockwise or anticlockwise. As the bullet passes through the barrel, these markings cause the projectile to spin, increasing stability and accuracy whilst leaving characteristic marks on the bullet itself. The grooves present on the surfaces of bullets are unique to the barrel that caused them, making rifling patterns ideal in matching bullets to specific firearms. Bullets are often viewed side by side using a comparison microscope, allowing rifling patterns to be contrasted and any similarities noted. The type and number of spiral grooves, their measurements, and whether they rotate clockwise or anticlockwise can help narrow down the search for the weapon used. Measuring the calibre of a bullet and the angle of grooves can allow for the calculation of twists per metre and thus provide further details of the rifling of a particular firearm.

Serial Number Restoration
During the manufacturing process, legally produced firearms are stamped with a uniquely identifying serial number, usually on the barrel or action. These numbers are stamped into the firearm, a process which also impresses the digits below the surface of the metal. Even though criminals may attempt to erase these serial numbers to avoid the weapon being traced, it may be possible to restore these serial numbers to a state in which they are legible. Serial numbers are often erased by filing or grinding, which will not necessarily remove the digits below the surface. Alternatively the perpetrator may attempt to change the serial number. Various techniques and reagents have been used to successfully restore these original numbers.

Fry’s Reagent is a substance composed of hydrochloric acid, copper(II) chloride, ethanol and water, commonly used on iron and steel. Other reagents are available for use on other types of metal. Initially the metal is polished to smooth the surface, a process which in itself may partially restore some of the digits. Following this, the etching reagent is then applied using a cotton swab, removing scratches and markings covering the numbers. This process can be repeated until the entire serial number is restored. However chemical etching methods can be very time consuming and are obviously damaging to the evidence.

An alternative method of restoring serial numbers on iron or steel is the Magnaflux method. As in the chemical etching method, the surface to be treated is first smoothed. A magnet is then attached behind the area and a mixture of iron filings mixed in a light oil is added to the surface. These minute metallic pieces will hopefully arrange themselves to visualise each digit. This technique is particularly beneficial due to its non-destructive nature, however it is not effective on all types of metal.

Similar to serial numbers are proof marks, markings imprinted on a firearm specific to the manufacturer or testing facility. These unique imprints are applied to a weapon before it is released and after any significant repair work is conducted on the firearm.

Gunshot Residues
When a firearm is discharged, a cloud of gases and fine particles is released composed of gunshot residues (GSR), sometimes referred to as firearms discharge particles (FDRs) or cartridge discharge residues (CDRs). The mixture will often contain both organic and inorganic particulates, the organic matter consisting of unburned and partially combusted propellant and inorganic matter produced by hot gases acting on the bullet. When released, these fine particles will settle on any nearby surfaces and are easily carried away from the scene. The presence of such residues can provide strong links between suspects or objects and the scene of a shooting, therefore various methods of detecting gunshot and other residues have been developed.

Gunshot residues are initially collected using swabbing, washing an item with dilute acid, film lifts or adhesive tape. Once collected, these resides can be analysed and compared both physically and chemically. Initially colour tests such as the paraffin test were previously used, though this lacked sensitivity and specificity so is no longer utilised. Further methods such as the Greiss test were developed to detect gunshot residues, however it was soon established that this technique was not sensitive enough and positive results could be caused by any nitrites.

Scanning electron microscopy with energy dispersive X-ray spectroscopy has been successfully utilised in visualising and detecting minute particles associated with firearms. This technique allows for the morphology of the particles to be observed and their chemical compositions established.

Neutron activation analysis (NAA) is a technique primarily used for determining concentrations of elements and has been used in the analysis of residues from firearms. However the use of this technique is very expensive and requires access to a nuclear reactor which is not readily available to all organisations. Flameless atomic absorption spectroscopy (FAAS) largely replaced the use of NAA due to it having various advantages and costing a more reasonable price.

Distance Determination
When a weapon is discharged, various gunshot residues will be ejected onto any nearby surfaces. The examination of gunshot residue has been utilised in establishing the distance from which a firearm was discharged. For example, the closer a firearm is to the target, in theory the more concentrated the gunshot residue pattern will be, whereas shots fired from a greater distance will produce a more widespread pattern. Research has also been conducted into the study of the chemical composition of gunshot residues in determining firing distance. Analytical techniques have been used to analyse the elemental composition of gunshot residue produced during the discharge of a firearm at varying distances. Attempts have been made to produce a mathematical model by which firing distance can be determined based on the elements and their relative amounts present in the residue. However it should be taken into consideration that the use of gunshot residues in establishing firing distance can only give an estimated distance at best.

Shooting Scene Investigation
A number of points will be aimed to be established in regards to the scene of a shooting, namely the number of shots fired, the direction from which the projectiles originated, and the type of ammunition and firearm used. A thorough examination of a shooting scene is often vital to the accurate reconstruction of events. The exact location of spent bullets and cartridge cases and even the firearm itself should be thoroughly documented before the evidence is collected. Similarly, the location of any bullet damage should also be photographed and, if possible, collected. A post-mortem examination of victims may be required to retrieve bullets and fragments.

Suspects of shootings incidents may claim that the firearm was unintentionally discharged, either accidentally by the individual or through malfunction of the weapon. Various tests can be conducted on the suspect firearm to help establish the details of the shooting. Trigger pressure relates to the force required to pull a trigger and fire a weapon. In some cases firearms with light trigger pulls may result in accidental discharge, so by calculating the trigger pressure it may be possible to determine the likelihood that the trigger was accidentally pulled. Some firearms allow the user to select either normal trigger pull or light trigger pull (hair trigger); therefore it is also important to discover whether the firearm has this feature and which setting was selected. Firearms are often fitted with numerous safety mechanisms. The examination of the firearm should include the investigation of these mechanisms to conclude whether any of the safety features were malfunctioning. An investigation known as a jarring test may also be performed, in which the firearm is subjected to a series of impacts involving various surfaces and distances to determine whether the action could have resulted in the firearm being discharged.

Projectile-Surface Interactions
The effects caused when a bullet strikes the target will vary depending on numerous factors, particularly the material of the target.

Wood – When a bullet penetrates a wooden object, the entry hole will generally be neat but the exit hole will often be jagged and splintered. The type of wood will cause variations in the bullet hole, as hard wood does not usually display as much splintering.

Glass – The effects of a bullet on glass will vary significantly depending on the type of glass. Some forms of glass will simply shatter, whereas on other occasions the projectile will pass straight through it, leaving bullet holes. Laminated glass, such as that used in car windshields, is manufactured with a layer of plastic sandwiched in the middle. Therefore, if penetrated by a bullet, although the glass will break, it will often be held together in a sheet. Tempered glass will break into many small, dull pieces. Some forms of glass, namely used in banks or by the military, are bullet-resistant and will not generally shatter when fired at.

The velocity of the projectile will also produce varying effects. High velocity rounds often result in small, tidy holes with little glass fracturing, whereas low velocity bullets will cause more damage to the glass. Studying the radiating cracks formed around the bullet hole can also help determine the order of bullet impacts. If the radiating line meets a pre-existing fracture, it will stop, indicating it was produced after the pre-existing crack.

Metal – Depending on the type of metal the surface is composed off, the bullet may ricochet off the target, dent or become lodged in the material, or produce a bullet hole. If the bullet does penetrate the metal, the entry hole will generally be fairly neat. Any exit holes will project outward slightly in a funnel-like manner.

Fabric – Bullets tend to pass straight through fabrics, producing a bullet wipe ring and, if close enough, depositing gunshot residues around the bullet hole. Different types of fibre will be affected differently by the interaction with a bullet. Natural fabrics tend to tear, resulting in a frayed bullet hole edge, whereas synthetic fibres may melt slightly.

Firearm Examination
Before the examination of any firearm can be conducted, it must first be established that the weapon is not loaded and it is safe to handle. This check should be carried out by an appropriately trained individual. During the initial examination of a firearm, a number of details should be noted. This includes the state of the weapon when received, its type, the make and model, the serial number and any other unique engravings and anything else that the investigator deems important. It is vital to document whether the weapon was locked or cocked, the position of the safety catch, and how many cartridges remain in the firearm’s cylinder or magazine.

In some instances it may be necessary to load and even test-fire a weapon into water or a gelatine block to obtain test bullets and cartridge cases, often for purposes of comparison with items of evidence. Again, this should only be carried out by a competent person in a safe environment, and at times it may be necessary to use a remote firing device rather than using the firearm by hand. When dealing with a self-loading weapon that ejects spent cartridge cases, the direction and distance travelled by ejected cartridges should be documented. Test-firing a suspect weapon should only occur once other forensic tests have been completed (fingerprinting, swabbing, etc). It may also be necessary to measure the trigger pressure of the weapon. Following this, the weapon should once again be checked to make sure it is not loaded before being returned to storage.

One of the primary focuses of the investigation of a firearm is often to establish whether or not the weapon was responsible for firing the shots in question. Various pieces of evidence recovered from the shooting scene can be used to establish this, including spent ammunition, cartridge cases, gunshot residues and the characteristics of wounds or other bullet damage. This evidence can help narrow down the search for the weapon used. For example, if scattered shotgun pellets are found at a scene, it is likely that investigators are looking for some form of shotgun. However it must be taken into account that weapons can be modified to fire a variety of ammunition, so the investigator should always keep an open mind.

Databases
Databases have been produced to store images of bullets and cartridge cases, allowing comparisons and matches to be made. In the UK, the Forensic Science Service and the Association of Chief Police Officers set up the National Firearms Forensic Intelligence Database, a system allowing information on weapons and firearms to be stored and analysed. This was later replaced by the National Ballistics Intelligence Service database. The FBI sponsors the database Drugfire, an automation nation system combining images input by users with a reference library.

 

Firearms Act 1968

Jackson, A. R. W, Jackson, J. M., 2011. Forensic Science. Essex: Pearson Education Limited.

White, P. C., 2004. Crime Scene to Court: The Essentials of Forensic Science. Cambridge: The Royal Society of Chemistry.

1 – Almeida, A A. Magaljaes, T. Santos, A. Sousa, A V. Vieira, D N. Firing Distance Estimation through the Analysis of the Gunshot Residue Deposit Pattern Around the Bullet Entrance Hole by Inductively Coupled Plasma Mass Spectrometry – An Experimental Study. American Journal of Forensic Medicine and Pathology. 2007, 28(1), 24-30.

Fire investigation involves the examination of all fire-related incidents once firefighters have extinguished the fire. The practice is similar to the examination of crime scenes in that the scene must be preserved and evidence collected and analysed, but with numerous additional difficulties and dangers. The investigation will include closely surveying the damaged scene to establish the origin of the fire and eventually establishing the cause.

However in order to effectively examine and evaluate a fire scene, it is imperative that the investigator has a detailed knowledge of the chemistry and behaviour of fire and its effects.

Nature & Chemistry of Fire
Fire occurs due to the exothermic reaction of combustion (burning), producing heat and light. In order for a fire to occur, three vital components must be present: a fuel source, an oxidant (O₂) and a sufficient amount of energy in the form of heat. Together these make up the fire triangle. A fourth factor can also be described – a self-sustaining chemical chain reaction – to produce the fire tetrahedron. The absence of any of these conditions will result in a fire not starting or extinguishing through smothering (oxygen removal), cooling (heat removal) or starving (fuel removal).

Solid and liquid materials do not actually combust, but the process of heating causes them to produce vapours which can burn. This is the process of pyrolysis. Through this pyrolysis products will be formed, flammable and volatile substances of low molecular weight caused through the decomposition of materials by fire.

The colour of flames can vary depending on the materials involved in the combustion. The colour of a flame is basically determined by the wavelength of light emitted, which varies depending on the material. For example, red/yellow/orange flames are commonly encountered when carbon is present. Inorganic substances can produce more obvious colour differences, such as copper which will cause a green flame.

Heat produced by a fire can spread in one of three ways; convection, conduction and radiation. Convection is the transfer of heat through air circulation, and only occurs in liquids and gases. An example of convection is the heat from a fire rising and heating the ceiling of a room. Conduction is the transfer of heat through a medium by direct contact, such as a fire heating a metal beam which transfers the heat elsewhere. Radiation is the emission of heat as infrared radiation without a medium, such as a fire heating and igniting a nearby sofa.

Ignition
Ignition will occur when all required conditions to start a fire occur, producing either a smouldering or flaming fire. This will often be induced by the addition of heat to a fuel in air, which can be caused by various sources such as exothermic chemical reactions, friction, solar radiation and electricity.

The temperature required for ignition to occur varies depending on the fuel. The flash point is the minimum temperature at which fuel favour is momentarily ignited in air by an external ignition source. However this will not necessarily sustain combustion and produce a fire. The flame or fire point is the minimum temperature at which enough vapour is produced to allow continued combustion. This is usually a few degrees higher than the flash point. Both the flash and flame point of a substance can be determined by placing a small amount of sample in an airtight container, gradually increasing its temperature whilst periodically adding an ignition source, and then measuring the point at which the flash and flame point is reached.

The spontaneous ignition temperature, also known as the auto-ignition point, is the lowest temperature at which a substance will ignite without any external ignition source. This is measured by heating a sample, studying the central temperature of the material and documenting the temperature at which ignition spontaneously occurs.

The flash point, flame point and spontaneous ignition temperature are the lowest temperatures at which a material has ignited when heated experimentally, though these actual temperatures can vary and so should only be used as a guideline. Different fuels also have individual lower and upper flammability limits, the lowest and highest concentrations of flammable gas required for combustion. If the concentration falls outside of this flammability range, combustion will not generally occur. Substances such as hydrogen have wide flammability ranges, making them particularly dangerous.

Smouldering
Not all types of fire produce flames. Smouldering is a form of flameless combustion which occurs at the surface of the material in cellulosic substances that can form a solid char. The presence of a smouldering fire is characterised by extremely localised burning and the production of thick, tarry smoke. The surface temperature can be linked to the colour of the smouldering. For example, dark red surfaces suggest a temperature of 500-600^oC, whereas a white surface indicates temperatures in excess of 1400^oC. The rate of propagation is dependent on the material burning and the amount of oxygen available. Only low concentrations of oxygen are required for smouldering combustion, but if sufficient oxygen is supplied, smouldering fires can then produce flames. Cigarettes are a common cause of smouldering fires when left in contact with upholstered furniture, for example.

Spontaneous Combustion
Spontaneous combustion refers to the sudden ignition of a material without an external ignition source such as a flame or spark. The phenomenon occurs as a result of exothermic chemical reactions occurring within the material, releasing heat. In cases where the material is piled together, the heat cannot dissipate effectively and so the temperature within the material rises. The rise in temperature causes chemical reactions to accelerate, producing even more heat. The temperature can rise until the flame point of the material is reached, causing ignition. Spontaneous combustion tends to be characterised by the apparent source of the fire being the centre of the material, as heat is dissipated more readily from the surface, thus resulting in the centre reaching the highest temperature. Rags soaked with oil, sawdust or piles of hay have been known to spontaneously combust.

Fire Scene Investigation
The primary purposes of a fire investigation is to establish the origin (seat) of the fire, determine the likely cause, and thus conclude whether the incident was accidental, natural or deliberate. It is vital to establish the cause to ensure similar events do not occur (in the case of natural or accidental) or to allow a legal investigation to be conducted (in the case of deliberate fires).

Safety
The initial concern with regards to a fire incident scene is safety. Such a scene has an increased risk factor with possible hazards including heated materials, structural collapse, damaged electricity and gas mains, debris, asbestos, dangerous combustion products and other toxic substances. A dynamic risk assessment should be conducted, the scene must be declared safe and all individuals entering the scene should wear appropriate protective clothing such as hardhats, fire-resistant overalls, steel-capped boots, thick gloves and, in some cases, a face mask. Supplies of gas and electricity should be switched off before the investigation begins.

Witnesses
Information regarding a fire can be obtained from witnesses. Witnesses may be able to provide details of the premises prior to the fire in addition to details of the fire itself, such as suspicious activity or apparent fire spread and smoke colour. Onlookers may even have taken photographs or video recordings of the incident on their mobile phones or cameras. The owner of the building/area may be able to detail the contents and layout of the building as well as any other potentially pertinent facts. However it should always be taken into consideration that civilian witnesses may be unreliable and could even have been involved in the fire incident. Emergency service personnel, such as police and fire fighters, are considerably more reliable as witnesses. Fire fighters in particular may be able to provide useful information on the possible origin of the fire and any unusual conditions. Fire fighters should also be interviewed to identify any disturbances made to the scene during fire-fighting efforts.

Ideally eyewitnesses should be interviewed by an objective individual with experience in interviewing in such a way that the information they provide is not influenced.

Scene Examination
A fire incident should be treated as a crime scene in that the area should be strictly controlled by a cordon to preserve evidence and allow access to authorised personnel only, with the scene and evidence being fully documented. A plan of the premises should be produced where possible to include the locations of objects, though it must be taken into consideration that disturbance may have been caused during fire-fighting efforts.

The investigation should ideally begin with an external examination of the scene. This allows for the identification of entry point, signs of forced entry, indications as to the origin and cause of the fire, artefacts, and any possible safety concerns. All doors and windows should be examined to establish whether or not they were locked during the fire. Once again, fire-fighters may have forcefully entered the building or smashed windows to provide ventilation, and damage caused by the fire itself may appear similar to signs of forced entry. The external examination will also allow for the search for items relevant to the incident, such as tools used to break into the building, ladders or containers of flammable substances. It may also be important to note weather conditions, as temperature and wind conditions can affect a fire in terms of fire propagation and direction.

The interior examination of the scene is then conducted, usually with the production of the layout of the scene detailing the location of items and any bodies. The investigator will generally begin with the area of least damage, allowing investigators to backtrack to the seat of the fire, which will typically be found in a more damaged region.

Establishing the Origin
A vital aspect of the forensic fire investigation is to establish the point of origin of the fire, also known as the seat of fire. There are numerous indicators that can be used to determine the possible origin. The region in which a fire started will generally burn for a longer amount of time, thus will be an area with the worst damage. Fires tend to burn upwards, therefore the seat of the fire is likely to be found at a lower point of burn damage. However this is not always reliable as fires can spread downwards, particularly in the presence of certain fuel sources.

Fire effects on certain materials can indicate the direction of fire. As fire burns upwards and outwards, V-shaped smoke/burn patterns may be found on surfaces adjacent to the fire, with the end of the V pointing towards the point of ignition. However ventilation can affect the path or shape of V-shaped patterns. Smoke deposits of object surfaces can suggest the direction from which the fire originated, and glass and plastics tend to melt in the direction of fire, thus distortion of such materials can act as directional indicators.

Structural damage to the building can also be used to locate the seat of the fire. In some instances buildings may collapse in such a way that the area first weakened by the fire is clear, suggesting this is where fire damage first occurred and thus is the origin. Similarly, windows and ceiling structures are likely to fail in areas close to the seat of the fire first. However this is by no means an accurate method of locating the seat of the fire, as the collapse and damage of a building is affected by numerous factors, not just the fire itself.

It may be possible to determine the area in which a fire began based on the operation of smoke and fire alarms. There may be some form of record of which alarm was triggered first, suggesting the fire is likely to have started in that room. The order in which alarms were triggered can be used further to establish the path of propagation of the fire. However such information is not available for all premises.

The investigator may be required to excavate the scene and systematically remove debris in order to identify the possible origin. Once debris and other evidence can be collected the scene can be lightly cleaned to expose fire burn patterns. However, depending on the extent of fire damage, the seat of fire may have been destroyed, particularly if the fire has been burning for a significant length of time.

The growth of the fire, whether fast or slow, and its heat can be suggested by fire damage at the scene. Spalling of plaster suggests a rapid increase in temperature, though the quality of the plaster and fire-fighting efforts can distort the usefulness of this. Intense charring is indicative of a slow, smouldering fire acting as the source. Fire damage to glass can also suggest the heat of the fire. The rapid increase in temperature can cause clear breaks in the glass, whereas a very slow build-up of heat tends to cause the glass to soften rather than break. Examining the extent to which wooden structures have been charred can provide insight into the fire, as exposed wood chars at a rate related to the exposure time and amount of radiant heat.

There may be multiple seats of fire, which in some cases can indicate arson if the arsonist has started fires in numerous places. However burning wallpaper, curtains or debris can also produce apparently distinct ignition points. Due to the range of factors affecting the origin of a fire, it may not be possible to specify the exact point of ignition of a fire. Therefore investigators generally define a confidence perimeter or radius of error. This is an extended section somewhere within which is the seat of the fire, with the most probable origin placed in the centre of the circle. Generally, the radius of this circle will decrease as the investigator becomes more confident in establishing the origin.

Establishing the Cause
Determining the cause of the fire is often greatly aided by locating the seat of fire, at which point investigators can identify characteristics or artefacts associated with ignition. The investigator will aim to establish whether the cause of the fire was accidental, natural, deliberate or undetermined. Accidental fires generally involve no malicious human contact, with examples including the malfunction of an electrical appliance or an unattended candle. Natural fires include “acts of God”, such as lightning strikes. Deliberate fires are those ignited purposely by individuals, often with malicious intent, in an act known as arson. Finally, if the cause of the fire cannot be ascertained due to lack of evidence, it may be classed as undetermined.

Evidence directly linked to the fire may be found at the point of origin, such as fuel sources, incendiary devices, electrical appliances or pools of accelerant. In addition to examining the artefacts present at the scene, the lifestyle of individuals living or working in the building should be taken into consideration. For example, factors such as whether individuals were smokers, used candles or kept large amounts of possible fuel packages such as newspapers and magazines may be relevant.

Arson
There are numerous indications of the deliberate ignition of a fire, also known as arson. Cases of arson are of particular importance to the forensic investigator, and such incidents may arise for a variety of reasons, such as insurance fraud, terrorism, in attempts to harm a person or their property, mental health problems, or to conceal a previous crime.

A particularly significant indication of arson is the lack of evidence suggesting an accidental or natural fire, though it is possible that the cause of even an innocent fire has been destroyed and cannot be ascertained. Signs of forced entry into the premises can suggest arson, displayed through broken windows, forced doors, tools found at the scene or disabled intruder alarms.

Flammable liquids are commonly used by arsonists to accelerate a fire, particularly patrol, diesel, kerosene and turpentine. The use of accelerants is suggested by extremely localised burning patterns with clear demarcation between burnt and unburnt areas, multiple seats of fire or trailing marks, and the detection of hydrocarbon vapours using sniffer dogs or hydrocarbon detectors. Flammable liquid containers may also be found at the scene. However it must be taken into account that flammable liquids may be present for innocent purposes, therefore it is necessary to determine whether such accelerants were stored on the premises prior to the fire. Other fuel packages may also be used, such as newspaper, which may be suspiciously piled up and ignited. If an incendiary device was used to ignite the fire, evidence of the device may be found amongst the debris. Furthermore if numerous devices were used, they could be found intact if they failed to detonate.

Investigators should attempt to ascertain the contents of the building prior to the fire. The removal of items from the premises, such as business stock or objects of sentimental or monetary value, is a strong indication of arson, commonly linked to cases of insurance fraud. The owner of the premises should be investigated and any possible financial or business problems searched for, which would provide further evidence in the form of a motive.

Fires are occasionally started to conceal a previously committed offence. However if the fire was ignited to conceal a murder, it is extremely unlikely that the victim’s body will actually be completely destroyed, as this would require temperatures of hundreds of degrees Celsius for 2-3 hours.

In some cases the arsonist may make attempts to shield the cause of the fire or attempt to make it appear to be natural or accidental. For example, they may start to fire close to an appliance or pile newspapers near a potential ignition source. Arsonists may block windows to shield the fire until it has developed, or conversely prop doors open to provide ventilation. They may also place objects to hinder entrance to the building and fire-fighting efforts.

In cases of suspected arson, it may prove beneficial to observe or photograph any onlookers. Arsonists have been known to return to the scene to watch the fire and the investigation. Certain indicators at a fire scene may not only suggest arson, but can also provide an insight into the possible motives of the individual responsible. People connected to the premises should be interviewed and investigated to search for any possible motives for arson.

Electrical Fires
When an electrical current passes through any material resistance will be encountered, producing some heat. Electrical wiring is usually produced and installed in such a way that any heat produced is relatively low and will be dissipated. However there are some occasions in which the heat produced can reach sufficient temperatures to cause ignition. Electricity is a common cause of accidental fires, often through the occurrence of an electrical arc.

An electrical arc occurs when two conductors come into contact following the insulation in the cable being damaged. This damage can occur for various reasons, particularly overheating, overloading, mechanical damage or manufacturing defects. If the cable becomes too hot, perhaps due to coiling of wires, heat will be unable to dissipate and the insulation may melt, allowing conductors to touch. Overloading occurs when more power is drawn through the cable than it is designed to handle, such as if too many plugs are inserted into one socket. This can also occur through the fitting of incorrect fuses or cable sizes. This will also cause insulation melting. Mechanical damage can occur through direct damage or continuous movement, weakening the cable at a certain point and thus allowing contact between the conductors. Similarly, damage may be the result of defects in the manufacturing process. Arcs are characterised by beading on the cable caused by the wire melting. It should be taken into account that although electrical arcs can lead to fires, fires can equally cause arcs.

If the suspected cause of the fire is an electrical appliance, the equipment must be thoroughly investigated, with a record being kept of details such as the brand, model and serial number. The expert must first conclude whether the appliance was turned on or off, whether it had a power supply, and whether the power supply was active or if the fuse had blown. Unfortunately an appliance which has caused a fire will most likely have suffered a great deal of damage and so confirming the cause of the fire may be extremely difficult or even impossible. It may be necessary to consult an expert for advice.

Upholstery Fires
The spread of a fire, the extent to which it grows and the pyrolysis products formed partly depends on the types of fuel available. In compartment fires in homes and other buildings, there will often be large amounts of upholstered furniture present, including beds, mattresses, sofas, armchairs and futons, all of which are a potential source of fuel. Upholstered furniture generally consists of a frame, filling material such as foam, and an outer covering fabric.

Various problems have been encountered with upholstered furniture in fires, particularly the flammability of materials used in their manufacture and the toxicity of materials used. In the 1970s-1980s a type of foam filling was used which produced toxic fumes when burned. The Furniture and Furnishings (Fire Safety) Regulations 1988 applied various fire resistance standards to upholstered furniture such as sofas, beds and armchairs. Following this legislation, modern upholstered furniture must include labels with fire resistance information. Furthermore, modern furniture is often produced using flame retardant textiles. For example, nitrogen and chlorine inhibit the burning rate of textiles and so are often used to treat fabrics. Other substances are added to increase the amount of charring and so create a heat barrier to prevent the fire from spreading further.

Flashover
Flashover is a phenomenon known to occur in compartment fires following a series of events, eventually resulting in the compartment’s full involvement in the fire.

Radiation-induced flashover is one particular form of this. As a fire burns in the room and the fire plume cannot escape, a layer of hot gases produced by the fire rise and form at the ceiling, increasing the temperature of the upper portion of the room. Flameover may occur, which is the fast horizontal spread of flames. As temperature increases, the rate of heat radiation increases. Temperatures at this point can reach around 600^oC, with radiant heat flowing down to floor level. Soon flames across the ceiling can reach between 750 and 850^oC. At this point all available combustible materials in the room can reach their auto-ignition temperature and burst into flames. This process is known as radiation-induced flashover. Furthermore, if a compartment is breached through the opening of a window or door or due to structural collapse, the influx of oxygen can result in the occurrence of an explosion known as ventilation-induced flashover.

However flashover will not occur if there is insufficient fuel, inadequate heat production, too little ventilation or too great a flow of heat out of the compartment.

Outdoor Fires
When investigating an outdoor fire, there are various differences from compartment fires that must be taken into consideration. A fire burning on a flat, open surface will move outwards towards any available fuel whilst producing hot gases above the fire. Assuming the fire is surrounded by a similar fuel source and there is no wind to take into account, the fire will most likely spread in a circular pattern. A fire on a sloped surface will most likely spread in an uphill direction, provided there is a fuel source, producing a fan-shaped spread.

Evidence Collection & Analysis
In the collection of evidence during the investigation of a fire scene, the same rigorous preservation and anti-contamination methods used in crime scene investigation should be employed.

In cases of suspected arson, samples are collected from the incident scene for the analysis of accelerants. The use of accelerants is not always apparent, therefore investigators may need to use detection dogs or hydrocarbon sniffers to detect these volatile substances. Hydrocarbon sniffers are vapour detectors used to discover the presence of fuel and solvent vapours associated with flammable liquids. Early devices implemented treated paper or crystals which changed colour when exposed to hydrocarbons, whereas more modern devices are essentially portable gas chromatographs or flame ionisation detectors. However these devices can only ever act as a preliminary test for accelerants, as similar substances can also be produced through the thermal decomposition of various natural and synthetic materials that may be found at the scene.

Once likely regions have been located, fire test samples are collected from the suspected point of ignition. In addition to this, a control sample should also be obtained, which consists of the same material as that of the fire sample but collected from an area uncontaminated by the suspected fuel, and a negative control sample. When collecting samples of possible accelerants, surface samples may be collected however, in some instances, charring of floors may be too severe. In this case samples can be collected from grooves between or beneath floorboards or even from soil below the floorboards.

All samples containing potentially volatile substances should be stored in airtight containers such as metal containers, glass jars or impervious plastic bags. All samples should be stored and submitted separately. The analysis of volatile samples is generally conducted using a technique known as headspace analysis. A common method used in the employment of headspace analysis uses a piece of activated charcoal or a similar adsorbent material which is stored in an airtight container with the volatile sample. Volatile compounds are drawn into this material either passively or dynamically and later desorbed for analysis.

Gas chromatography is the technique most commonly utilised in the analysis of fire debris. This allows for volatile substances, whether from bulk or trace samples, to be separated, displayed in the form of a chromatogram, and identified. The technique is also able to isolate and identify mixtures of various compounds. The use of gas chromatography not only permits samples to be identified, but can also allow for numerous samples to be compared to establish whether or not they are the same substance.

 

Daeid, N N, 2005. Fire Investigation. Florida: CRC Press.

Fairgrieve, S I, 2008. Forensic Cremation: Recovery & Analysis. Florida: Taylor & Francis Group.

TC Forensics. [online] Available at: [http://www.tcforensic.com.au

The majority of cells making up the human body are diploid cells carrying identical DNA, with the exception of haploid gametes (egg and sperm) and red blood cells (which have no nucleus). Several types of biological evidence are commonly used in forensic science for the purpose of DNA analysis, including blood, saliva, semen, skin, urine and hair, though some are more useful than others. The use of biological evidence in DNA and genetic analysis varies, with areas of study including blood typing, gender determination based on chromosome analysis (karyotyping), DNA profiling and, more recently, forensic DNA phenotyping. Since the advent of DNA profiling in the 1980s, it has been successfully utilised in criminal cases, disaster victim identification and paternity testing to name a few. However despite their merits, DNA fingerprints are not ideally used as the sole piece of evidence in a case, and in certain countries, such as the United Kingdom, DNA fingerprints must be presented in conjunction with other evidence.

DNA Structure and Function
It is vital to understand the structure and function of DNA and how this relates to DNA analysis in forensic science. DNA, deoxyribonucleic acid, is a molecule arranged into a double-helix, its structure first described by James Watson and Francis Crick in 1953. It is composed of nucleotide trisphosphate molecules, referred to as the ‘building blocks’ of DNA. These molecules consist of a trisphosphate group, a deoxyribose sugar and one of four nitrogenous bases. The four bases involved in a DNA molecule are adenine and guanine (purines) and thymine and cytosine (pyrimidines). These bases bond to the deoxyribose sugar and one of the other bases to form base pairs, with adenine and thymine bonding through two hydrogen bonds, and guanine and cytosine bonding with three hydrogen bonds.

DNA is essentially the molecule that holds all genetic information and ‘instructions’ for an organism. The human genome is composed of over 3 billion base pairs of information organised into 23 chromosomes. Genes are the regions of DNA that encode and regulate protein synthesis, though this involves just 1.5% of the entire genome. A significant amount of the human genome, approximately 75%, consists of extragenic DNA, which contains regions that do not actually contain known gene sequences. About 50% of extragenic DNA is made up of something called repetitive DNA, which is of particular use in forensic DNA analysis. Repetitive DNA is further sub-divided into tandem repeats (including satellite DNA, microsatellites and minisatellites) and interspersed repeats (SINE, LINE, LTR and Transposon). Tandem repeat DNA and the variation between them (polymorphisms) is the focus of many DNA profiling techniques. It is due to the number and location of these polymorphisms that every individual has unique DNA which produces a distinctive band pattern when analysed.

It is through the extensive study of the genome that DNA fingerprinting has been produced as a useful and reliable technique in forensic science.

Sources of DNA Evidence & DNA Extraction
In terms of forensic DNA analysis, there is a variety of possible sources of DNA evidence. The more useful sources include blood, semen, vaginal fluid, nasal secretions and hair with roots. It is theoretically possible to obtain DNA from evidence such as urine, faeces and dead skin cells, though this is often classed as a poor source due to the lack of intact cells and high levels of contaminants preventing successful analysis. Such samples will be collected depending on the type of sample (see crime scenes page for more details of evidence collection and preservation).
Prior to analysis, it will be necessary to extract DNA from the sample. This is generally achieved through the following simplified steps.

• The sample cells are lysed (broken down) in a buffer solution.
• Denatured proteins and fats are pelleted through centrifugation.
• The cleared lysate is then passed through a column, often containing a positively charged medium that binds to the DNA.
• Contaminating proteins, fats and salts are then removed through several washes.
• The DNA is recovered in a buffer solution/water.
• The amount of DNA is often then quantified using spectrophotometric techniques.
• Various methods of extraction have been devised for different types of sample.

DNA samples for comparison are generally collected from suspects using buccal swabs, in which a sterile swab is scraped along the inside of the cheek to collect epithelial cells to use in producing a DNA fingerprint. Sir Alec Jeffreys is often described as the father of DNA fingerprinting.

Single Nucleotide Polymorphisms
The use of DNA analysis in forensic science is based on a variety of techniques focusing on polymorphisms, which essentially refers to variation in sequences. Different sequences are studied in different techniques, including single nucleotide polymorphisms, minisatellites (variable number tandem repeats), microsatellites (short tandem repeats) and mitochondrial DNA, each different with regards to length and repetition.
Single Nucleotide Polymorphisms (SNPs) are the simplest and most common type of genetic variation, composing around 90% of genetic variation in humans. They occur during meiosis when DNA is replicated, with each SNP representing a difference in a single nucleotide. For example, a SNP may replace cytosine with a thymine in a stretch of DNA, and this will not change the length of the DNA. There are about 10 million SNPs in the human genome, with one found at every 100-300 base pair. These can act as biological markers.

In the analysis of SNPs, which can consist of up the four alleles, the specific base present in the SNP must be established. This is achieved using DNA sequencing techniques. However DNA sequencing is not generally used in forensic science except in the analysis of mitochondrial DNA.

Dideoxy Method
Also known as the Sanger Method, this form of DNA sequencing was developed by Fred Sanger in 1975. A labelled primer is utilised to initiate the synthesis of DNA. Four dideoxy nucleotides are added and randomly arrest synthesis. Fragments are produced which are subsequently separated using electrophoresis or, in more modern automated systems, capillary electrophoresis. Specialised software is then used to convert the band patterns into a DNA sequence. Initially unstable, hazardous radioactive tags were used, soon replaced by fluorescence dyes. However the varying mobility of these different coloured dyes meant that the order of bands did not necessarily accurately represent the order of the nucleotide sequence. The dyes would also sometimes behave unpredictably, making them unreliable and not particularly ideal for use in forensic cases. If conditions were unsuitable for the DNA template or the dideoxy nucleotides, the reaction would be unsuccessful. The computer software itself was problematic, with peaks often overlapping, making it difficult to establish the correct order of nucleotides.

Minisatellites or Variable Number Tandem Repeats (VNTRs)
Variable Number Tandem Repeats (VNTRs) are highly polymorphic sequences in the exon region of DNA repeated at various points along the chromosome, 4-40 times. They are about 20-100 base pairs in length, though their specific lengths are not strictly defined. These tandem repeats are inherited from both parents, therefore no one will have the same VNTRs as either of their parents. The number of repeats at the locus affects its position after electrophoresis and the length of the DNA after the chromosome has been cut with a restriction enzyme. Minisatellites were the first type of polymorphism to be used in a criminal investigation in the case of British murderer and rapist Colin Pitchfork.

However VNTRs are not generally used in forensics, as this technique is often a more expensive process and takes longer due to VNTRs having a greater length than, say, STRs.

Microsatellites or Short Tandem Repeats (STR)
Short Tandem Repeats (STRs) are regions of the genome composed of approximately 1-5 bases and repeated up to 17 times. STR markers will either be simple (identical length repeats), compound (two or more adjacent repeats) or complex (several different length repeats). They are found on 22 autosomal chromosomes as well as both X and Y sex chromosomes, though those on the Y chromosome differ less due to lack of recombination. Only a select number of STR markers are used in forensic DNA profiling (10 in the UK and 13 in the US).They express a high degree of polymorphism, making them of particular use to the forensic scientist. The variability in STRs is caused by the inaccuracy of DNA polymerase in copying the region. As STR regions are non-coding, there is no selective pressure against the high mutation rate, resulting in high variation between different people.

Though there have been thousands of short tandem repeats found in the human genome, only a small number are utilised in forensic DNA analysis. STRs used in forensic science tend to be tetra- and penta-nucleotide repeats, as they are both robust, suffer less environmental degradation, and provide a high degree of error free data. STR loci are ideal for use in forensic science for a number of reasons. They represent discrete alleles that are distinguishable from one another, they show a great power of discrimination, only a small amount of sample is required due to the short length of STRs, PCR amplification is robust and multiple PCR can be used, and there are low levels of artefact formation during amplification. An early use of microsatellites is in the identification of Auschwitz camp doctor Josef Mengele.

Restriction Fragment Length Polymorphisms (RFLPs)
Restriction Fragment Length Polymorphisms (RFLPs) were used in the first technique developed to analyse variable lengths of DNA fragments produced through DNA digestion. It exploits variations in DNA sequences due to the differing locations of restriction enzyme sites. The method uses restriction endonucleases to ‘digest’ the DNA by cutting it at specific sequence patterns. The resulting restriction fragments are then separated using gel electrophoresis and transferred to a membrane using the Southern Blot technique. After the separated DNA fragments are transferred, probe hybridisation is used to detect the fragments.

However DNA analysis with RFLP required relatively large amounts of DNA and degraded samples could not be analysed with accuracy. More effective, faster and cheaper DNA profiling techniques have seen been developed, so RFLP is generally no longer used in forensic science.

Polymerase Chain Reaction (PCR)
The amount of DNA evidence obtained during the investigation of a crime is often very small, thus for successful DNA profiling some form of amplification is ideal. Polymerase Chain Reaction (PCR) is a technique which allows for the exponential amplification of DNA fragments to lengths of approximately 10,000 base pairs. This means that, theoretically, a single copy of a DNA fragment could be amplified to millions of copies in just a few hours. PCR is particularly beneficial in the amplification of minute amounts or degraded samples.

A successful PCR reaction requires a number of vital primary components. Oligonucleotide primers which are complementary to the DNA target and mark the target to be amplified, with two primers being used. The base sequence of one primer binds to one side of the target whilst the other primer binds to the other side of the target, with the DNA between the primers being amplified. Fluorescent tags are often added to the primers to visualise amplified DNA in electrophoresis. DNA polymerase enzyme allows the DNA strand to be copied by adding nucleotides to the 3’ end of the primers. Other components required include a reaction buffer with MgCl to ensure ideal conditions for the functioning of the DNA polymerase enzyme, deoxyribonucleotides to build the DNA molecule, and template DNA. Modern PCR uses thermostable DNA polymerases. Most commonly used is the Taq polymerase, which has largely replaced the previously used E.coli-derived polymerase. This was isolated from Thermus aquaticus, which is an organism capable of surviving in temperatures over 70oC. However Taq polymerase lacks the ability to proof read. VENT polymerase is from Thermococcus litoralis, which can survive in temperatures over 100oC.

The PCR cycle consists of three primary steps: denaturation, annealing and extension. The process is generally conducted in a small, plastic centrifuge tube with the temperature carefully controlled using a thermal cycler.

• Denaturation: The sample is heated to 94-95oC for about 30 seconds. This separates the double-stranded DNA by breaking hydrogen bonds, allowing primers access.
• Annealing: The samples is kept at 50-65oC, depending on the primer sequence, to allow hydrogen bonds to form between the primers and the complementary DNA sequence.
• Extension: Also known as the elongation stage. The sample is heated to 72oC for a duration depending on the length of the DNA strand to be amplified and the speed of the polymerase enzyme (Taq polymerase) which builds up the strand. Deoxynucleotide triphosphates are added to the 3’ end of the primer.

Each PCR cycle can take only 5 minutes. This procedure can then be repeated as necessary until the original sequence has been amplified a sufficient amount of time, with the amount being doubled with each cycle. Following PCR, the products are separated using electrophoresis.

Unfortunately PCR is not suitable in the analysis of longer strands of DNA, and so cannot be used with earlier techniques such as RFLP. It must be taken into consideration that certain compounds can inhibit PCR reactions, often substances associated with the stages of extracting and purifying the DNA. Such substances include proteinase K (which degrades the polymerase enzyme), ionic detergents and gel loading dyes. Similarly, certain substances present in blood can inhibit PCR, such as haemoglobin and heparin.

Various alterations have been made to improve the PCR method. Multiplex Polymerase Chain Reaction involves the amplification of numerous DNA sequences in a single reaction through the use of primers that produce non-overlapping allele sizes, allowing numerous regions of a sample to be tested simultaneously.

PCR Errors
Various factors can contribute to errors and inaccuracies in data produced by the polymerase chain reaction technique. PCR is often carried out using DNA polymerases such as Taq DNA polymerase, which does not have the ability to ‘proof read’, resulting in errors in amplification. The greater the amplification, the more likely it is that such errors will occur. Mispriming is also a potential problem, with products being formed from non-target sites. Excessive primer dimers may be formed, which are by-products of PCR produced when one primer is annealed to another causing primer extension. This may all result in unexpected variability in PCR success across a series of samples or previously successful conditions failing.

Electrophoresis
As mentioned, during DNA analysis the individual fragments of DNA can be separated using electrophoresis to produce the distinct ‘DNA fingerprint’. Electrophoresis is essentially a method of separating molecules by their size through the application of an electric field, causing molecules to migrate at a rate and distance dependent on their size. In gel electrophoresis, a porous gel matrix is used, often consisting of agarose gel for simple work or polyacrylamide gel for more specific procedures. The gel is often floating in a buffer solution to ensure the pH level is maintained and the applied electric current is conducted. Samples to be analysed are placed in small wells at the top of the gel using pipettes. A control sample and a standard/marker sample will often be run simultaneously. As the electric current is applied, the negatively charged DNA fragments begin moving through the gel towards the positively charged anode. The gel essentially acts as a type of molecular sieve, allowing smaller molecules to travel faster than larger fragments. Following electrophoresis, it may be necessary to visualise these bands using radioactive or fluorescent probes or dyes. Electrophoresis not only separates DNA but also allows for the fragments to be measured, often expressed in base pairs. Measuring the length of these fragments can ultimately allow the number of repeats to be determined and thus the genotype at that locus.

Earlier techniques used flat-bed gel electrophoresis, though the faster and automated capillary electrophoresis is now used more often. In this technique, abbreviated to CE, the gel is held in a fine capillary tube through which the fluorescently-labelled DNA passes through much as in gel electrophoresis, often with an added DNA size standard. Positioned along the capillary is a laser beam which causes the DNA to fluoresce as it passes. This can then be detected and fed directly to a computer system in the form of an electropherogram. Unfortunately capillary electrophoresis is not able to separate more than one sample at a time, though a genetic analyser can be used to separate a series of samples one after the other.

Blotting Techniques
Following gel electrophoresis, probes are generally used to detect specific molecules. However because probes cannot be directly applied to the gel, blotting methods were initially utilised. There are a number of common blotting techniques: Southern blot, Western Blot, Northern Blot, Eastern Blot and Southwestern Blot.

• Southern Blot – Used in DNA analysis. DNA is extracted, separated with electrophoresis and transferred to the membrane. Labelled probes are added, which hybridise to specific sequences and identify them.
• Western Blot – Used to detect proteins. SDS-PAGE used to separate proteins, which are transferred to membrane. Specific antibodies are then added, followed by a substrate to visualise bands.
• Northern Blot – Used to study gene expression. Similar to the Western blot, except RNA is being analysed.
• Eastern Blot – Used to study proteins. Considered to be an extension of the Western blot.
• Southwestern Blot – Combines features of the Southern and Western blot. Used for the rapid characterisation of DNA binding proteins and their sites.

Low Copy Number (LCN) DNA Analysis
Low Copy Number DNA Analysis, referred to as LCN, is a technique developed by the UK’s Forensic Science Service in an attempt to increase the sensitivity of DNA profiling methods. Samples containing small amounts for badly degraded DNA often leads to problems such as poor quality fingerprints or even completely negative results. This technique reduced these issues.

Developed in 1999,LCN is essentially an extension of the Second Generation Multiplex Plus (SGM+) technique, and is generally used when the previous technique has failed or has produced weak results. Improved sensitivity is achieved through an increased number of PCR cycles, with standard techniques generally using 28 cycles but LCN using 34. This could ultimately allow for DNA profiles to be successfully obtained from minute amounts of sample and even from single cells (see below).

However the increased sensitivity of this DNA profiling technique brings about amplified concerns over issues of ease of contamination and amplification of these contaminants, mixed profiles being produced and wrongful accusations. With techniques such as LCN, it is now more important than ever that investigators wear suitable protective clothing and follow strict anti-contamination procedures, and controls are used in analyses.

In 2007, LCN came under great scrutiny through the case of R vs Hoey, which led to the use of this technique being temporarily banned until a thorough review could be conducted. Sean Hoey was tried for involvement in the Omagh bombing in 1998, charged with numerous counts of murder in addition to terrorism and explosives charges. The DNA evidence used against him was based on the relatively new LCN technique which, at the time, was only being used in British courts. However due to the lack of data and known error rates regarding the technique, serious concerns were raised. Hoey was subsequently found innocent.

Single Cell DNA Fingerprinting
Closely linked to LCN analysis is single cell DNA profiling. Dr Ian Findlay and his colleagues at the Australian Genome Research Facility first reported the successful development of a DNA fingerprint from a single cell in 1997. 226 buccal cells from four individuals were isolated using micromanipulation, amplified using the UK Forensic Science Service’s Second Generation Multiplex (SGM) assay to increase sensitivity. Six STR markers and amelogenin for sex-typing were amplified. The results from single-cell analysis were compared with known DNA profiles. Whereas a full and correct profile was only obtained in 50% of the single-cell tests, 91% of tests exhibited some form of result.

Previous DNA fingerprinting methods often required hundreds of cells in order to obtain a profile. The single cell is obtained by swabbing the material and identifying the cell to be analysed using microscopy prior to analysis. This technique is particularly fast, taking a matter of hours, and has a specificity of about 1 in ten billion.

Single-cell DNA profiling is particularly useful in rape cases, as DNA in sperm cells is highly conserved due to it being so compacted in the protein head. There is also potential for the technique in use in documents. Human DNA can be placed in documents such as Government bonds, wills and security documents, to track their flow. However the main issue with this particular use is that close relatives may handle the documents, particularly when dealing with documents such as wills, and so the technique may not be appropriate. Single-cell DNA analysis is also ideal in IVF procedures, in which single cells can be analysed for genetic defects.

However there are some major concerns with this method. As an increased number of PCR cycles are required to amplify the DNA, this brings about problems of allele drop-in, where additional alleles are added to the sample. Allele drop-out is a similar problem with increasing amplification samples. When starting with a single cell or small amount of sample, any contaminants already present in the sample will be amplified. Similarly, any PCR inhibitors present will be more concentrated, causing amplification problems. One of the primary concerns is the ease of contamination by cells from other individuals. This could result in samples being contaminated and rendered useless or, worse still in the case of forensics, innocent individuals being wrongly accused. More work is required to validate the technique, particularly to use it in forensic science.

The work in single-cell DNA analysis led to the Forensic Science Service in the UK developing low-copy number DNA analysis.

Mitochondrial DNA (mtDNA)
Mitochondrial DNA is a circular molecule of DNA 16,569 base pairs in size, first referred to as the Anderson sequence, obtained from the mitochondrion organelle found within cells. This organelle is involved in the production of cell energy. There can be anywhere between 100 and 1000 mitochondria within a cell, each one containing numerous copies of the mitochondrial genome. This sequence is entirely functional and highly conserved, so there is very little variation between individuals. However there is a 1000 base pair long non-coding D-loop, known as the control region, which contains two hypervariable regions referred to as HV1 and HV2. The variations within these regions are generally single nucleotide polymorphisms (SNPs). SNPs do not alter the length of the mtDNA, and it is these regions that are focused on in the forensic analysis of mitochondrial DNA. Mitochondrial DNA is often subject to a relatively high rate of mutation due to its lack of DNA reparation, causing variation between individuals. The variation within this small portion is itself not especially significant, with HV1 and HV2 differing by 1-3% between non-related individuals.

In the analysis of mitochondrial DNA, the DNA is extracted and the HV1 and HV2 regions amplified using PCR. The base pair sequence of these regions is then established through DNA sequencing (see DNA sequencing section). This is then compared with the Cambridge Reference Sequence and differences noted. Other samples can then also be analysed an comparisons made to establish potential similarities. Mitochondrial DNA is generally used when other methods such as STR analysis have failed. This is often in the case of badly degraded bodies, in cases of disaster or accidents where an individual is too badly damaged to identify, and sometimes in taxonomy to determine species using the cytochrome b gene.

The most significant advantage of the use of mitochondrial DNA is the possibility of analysing even highly degraded samples. If a specimen is severely decomposed to the point that it is not possible to successfully extract a DNA profile using nuclear DNA, it may be possible through mitochondrial DNA. Additionally, only a very small sample size is required.

However the use of mtDNA does have its disadvantages. As mitochondrial DNA is only maternally inherited, this cannot form a full DNA fingerprint of the individual, thus this technique is only beneficial if the DNA profiles of maternal relatives are available, such as the individuals mother or biological siblings. Because of this, mtDNA is significantly less discriminatory than, for example, Short Tandem Repeats. Detecting sequence differences is also relatively time consuming and expensive.

Forensic DNA Phenotyping
Attempts have been made to utilise DNA analysis in the identification of phenotypic characteristics such as skin colour, hair colour and eye colour in a study known as forensic DNA phenotyping (FDP) or phenotypic profiling. This is generally achieved using single nucleotide polymorphisms (SNPs) rather than STRs. SNPs have a lower mutation rate and so are more likely to become fixed in a population, thus they are often found to be population-specific. It may be possible to estimate ethnic origin based on the presence of rare SNPs or STRs linked to particular population groups, though this theory becomes problematic with individuals of mixed ancestry.

Most work on phenotype SNPs has focused on pigmentation, and SNPs in a number of pigmentation genes have been associated with various human hair, skin and eye colour phenotypes.

Advances are already being made in this area of study. The Forensic Science Service has developed an SNP typing assay involving mutations in the human melanocortin 1 receptor gene (MC1R), which is associated with red hair. Specific alleles along this gene are associated with red hair, thus an individual inheriting this allele from each parent results in a high likelihood of that individual having red hair. Establishing that an individual is likely to have red hair is limited in forensic science, as red hair is not particularly common, though it is more common to certain populations.

Some research has been carried out into the genetics of eye colour, namely relating to the OCA2 gene on chromosome 15, which is also involved in the pigmentation of both skin and hair. IrisPlex is a recent test developed which allows for the accurate prediction of blue and brown eye colour.

Studies have been conducted examining the frequency of specific short tandem repeat alleles in groups of varying geographic ancestry. This research has resulted in the possibility of likely ancestry being established due to certain alleles being more likely in specific groups. However this particular use of DNA analysis is not infallible and can only be used as an estimation. The Forensic Science Service has developed an ethnic inference test, which provides the likely origin of a DNA sample from a number of groups (white European, Afro-Caribbean, Middle Eastern, South-East Asian and Indian Sub-continental).

Some countries and states are implementing specific legislation relating to the use of phenotypic DNA analysis. Many jurisdiction presently only allow the analysis of non-coding DNA, though the Netherlands currently allows forensic phenotyping under certain regulations. The phenotypic use of SNPs have also been used in non-forensic analysis, such as in determining the ethnicity, hair and skin colour of ancient remains.

However there are a number of problems and concerns with this approach. It is unlikely that a few chosen SNPs will provide a foolproof ‘picture’ of the sample’s source due to the complexity of multigenic traits as well as external factors such as environment and aging. Even if the technique was perfected for use in forensic science, phenotypes such as hair or eye colour can easily be masked through dyes and coloured contact lenses, limiting its forensic use. There are also privacy concerns relating to the possible traits determined, though it has conversely been argued that visible traits such as hair and eye colour cannot be considered private. Though should further advances be made to the extent that genes were located for traits such as aggression and predispositions to certain diseases, more serious concerns would be raised over the sensitivity of such information.

Researchers anticipate that further advances could allow for additional details to be ascertained, such as the likelihood that the individual smokes, along with the possibility for genes for the likes of handedness, aggression and homosexuality.

Y Chromosome Analysis
Much of modern DNA profiling is based on the analysis of short tandem repeats found on autosomes. However one particular branch of DNA analysis focuses on the amelogenin marker, the only marker on the sex chromosome, useful in the analysis of the Y chromosome. The Y chromosome, generally found only in males, is a small chromosome which, unlike other genes, is only altered through the infrequent occurrence of mutation. However similar to mitochondrial DNA (which is maternally inherited), the combination of alleles in this instance is theoretically identical between father and son, assuming mutation does not occur. Furthermore, Y chromosome analysis discrimination is comparatively low. Y chromosome analysis is particularly useful in cases of sexual assault and rape in which mixed DNA profiles may be encountered. Numerous systems have been developed to analyse some of the STRs present on this chromosome, such as Applied Biosystems’ Yfiler.

DNA Databases
Numerous countries have produced computerised databases containing DNA profiles to aid in the comparison of DNA fingerprints and the identification of suspects and victims. The first Government DNA database was established in the United Kingdom in April 1995, known as the National DNA Database (NDNAD). As of 2011, there were over 5.5 million profiles of individuals in the system. Similarly, the FBI in the US formed their own DNA database, the Combined DNA Index System (CODIS), in 1994, though it was not implemented in all states until 1998. Staff members involved in the handling and analysis of evidence will often also submit their DNA profiles to the database in the case of accidental contamination. There is the possibility for DNA databases to be shared between countries, however some countries focus on different loci in DNA fingerprinting.

DNA databases are not only used to make direct matches between the DNA fingerprints of one person, but to also conduct familial searching. This involves the search for genetic near-matches between a victim/suspect and a member of their family whose DNA profile is stored. This technique is based on the principle that related individuals are likely to express similarities in their DNA profiles. The first prominent use of familial searching was in the case of British serial killer Joseph Kappen, who was seized after his son’s DNA profile was obtained and then linked to him.

The production of DNA databases has allowed for the faster apprehension of suspects through comparing new crime scene samples to those already stored in the database, providing links between criminals and other crimes. It has also been widely used in cold cases, in some instances proving the guilt of an individual decades after they committed the crime. Conversely, wrongly imprisoned individuals have been exonerated through the advent of new DNA analysis techniques and databases. There is also the potential benefit of identifying bodies that have been too badly damaged or decomposed to identify, provided the individual’s DNA profile is stored.

However despite the advantages of such databanks, there has been significant controversy relating to the subject. Standard practice in many locations is to take a DNA sample of an individual when arrested, but this raises concerns over whether their DNA should be retained if they are then found innocent. There are also worries over innocent people being identified as matches or partial matches to DNA found at a crime scene even if they were not involved in the crime but had innocently attended the scene at some other point in time. This particular problem is becoming increasingly concerning as DNA fingerprinting techniques become more sensitive. There are also privacy concerns over the availability of sensitive genetic information, such as susceptibility to certain diseases and familial relationships. A more administrative disadvantage of such databases relates to the need for a facility that is both large enough to store such data but also has adequate security, a combination that can prove extremely expensive.

DNA Profile Interpretation
The primary purpose of forensic DNA profiling is to obtain a DNA ‘fingerprint’ from a biological sample and compare this to profiles obtained from DNA from a crime scene, an individual or profiles stored on a database. The process of modern DNA profiling includes statistically determining the chance that two people will share the same profile by establishing how common a genotype or collection of genotypes is within a population. The more loci studied and the greater heterozygosity of these loci, the smaller the chance two people will share a profile. However such figures can only ever be estimations and do not take certain factors into consideration, such as biological relatives. DNA evidence tends to be presented in terms of a random match probability, rather than definitively stating whether two profiles match or not.

Jeffreys, A J. Thein, S L. Wilson, V. Individual-Specific Fingerprints of Human DNA. Nature. 1985, 316)6023), 76-79.

Watson, J D. Crick, F H. A Structure for DNA. Nature. 1953, 171, 737-738.

Findlay, I. Taylor, A. Quirke, P. et al. DNA Fingerprinting from Single Cells. Nature. 1997, 389(6651), 555-556.

Jackson, A. R. W, Jackson, J. M., 2011. Forensic Science. Essex: Pearson Education Limited.

White, P. C., 2004. Crime Scene to Court: The Essentials of Forensic Science. Cambridge: The Royal Society of Chemistry.

Often found at the scenes of violent crimes, the analysis of bloodstains can provide vital clues as to the occurrence of events. Though bloodstain pattern analysis (BPA) can be a subjective area of study at times and often reliant on the experience of the investigator, the idea that blood will obey certain laws of physics enables the examination of blood at an incident scene and on items of evidence to offer at least an insight into what was likely to have occurred.

The successful interpretation of bloodstain patterns may provide clues as to the nature of the offence, the possible sequence of events, any disturbance to the scene that may have occurred, and even the position of individuals and objects during the incident. It may prove beneficial in refuting or corroborating eyewitness accounts.

Types
The appearance of a bloodstain can depend on a number of factors, including the velocity at which it was travelling, distance travelled, the amount of blood, the angle of impact, and the type of target onto which it lands.

Single Drop
These bloodstains typically refer to blood drops that have fallen vertically, whether it be from an injured person or another object, and landed onto another surface. As a blood drop falls perpendicular to a surface it maintains a spherical form until impacting. The size and appearance of this stain will depend on a number of factors. The volume of a single drop of blood will vary depending on the quantity of blood present and the surface area available from which the drop is falling. As would be expected, a larger surface area would allow for larger drop of blood to form before falling. The height from which the blood falls will affect the size of the stain, with greater heights tending to result in larger bloodstains. Furthermore, the target surface itself will cause an effect, with absorbent surfaces usually producing smaller stains than non-absorbent targets. The nature of the target can alter the appearance of the stain. For instance, a rough target surface can result in increased distortion to the stain and even satellite stains, which are additional stains radiating outwards. A drop of blood falling into an existing bloodstain will result in a drip pattern.

Impact Spatter
This type of bloodstain is the result of a forceful impact between an object and wet blood, causing the blood to break into smaller droplets. A greater force will typically produce smaller droplets, with the density of blood drops decreasing moving further away from the initial blood source. The study of impact spatter may provide insight into the relative position of individuals and objects during an incident and the nature of the incident.

Cast-Off Stain
Cast-off bloodstains occur when centrifugal force causes blood drops to fall from a bloodied object in motion. Similarly, cessation cast-off patterns may result from the sudden deceleration of an object. In this instance, the blood flung from a blood-stained object, such as a weapon, may produce characteristic patterns of numerous individual blood drops forming a curved or straight line. If an object is repeatedly moved, each subsequent swing will result in less cast-off as less blood remains on the object. Bloodstains produced in this fashion can be particularly difficult to interpret as there is a great deal of possible variation in patterns produced. However depending on the nature of the motion of the bloodied object, cast-off blood will at least produce relatively linear stains.

Transfer Bloodstains
Transfer or contact stains result when a bloodied surface comes into contact with another surface, transferring blood to that secondary target. The study of this type of bloodstain can prove particularly beneficial in establishing a sequence of events at the incident scene and tracing the movement of objects or individuals. In some cases it may even be possible to establish what object the transfer stain was likely to be caused by, for instance if a particular pattern is produced that can be traced to a blood-bearing object. Similarly, such bloodstains may be left by the hands of an individual, thus opening the possibility of fingerprint evidence.

Projected Pattern/Arterial Damage Stain
This type of bloodstain results from the discharge of pressurised blood onto a target surface, for instance the ejection of blood from a punctured artery. Areas of the body in which wounding may cause arterial bloodstains include the carotid artery, the radial artery in the wrist, the femoral artery in the inner thigh, the brachial artery in the arm, temporal regions of the head, and the aorta (though damage to the aorta is less likely due to increased protection of the chest cavity). Blood is expelled from the artery as the heart continues to pump and, as the blood travels, it breaks up into smaller individual droplets. Bloodstains produced will usually represent the beating of the heart as blood is expelled in periodic spurts. The resulting bloodstains can vary depending on a variety of factors, including whether the victim was stationary or moving as blood was being ejected, where on the body the injury occurred and the extent of the wound. If a wound is smaller in size, naturally smaller blood drops will be produced, which can subsequently be expelled further from the injury site than larger blood drops.

Pool Stains
Pooling bloodstains refer to the accumulation of blood on a particular surface, generally from prolonged bleeding from a wound or accumulation of arterial blood. If a body is not present at the incident scene, depending on the quantity of blood present, it may even be possible to roughly estimate whether the victim is likely to be dead or alive based on how much blood they have lost.

Insect Stains
These are bloodstains resulting from insect activity. The presence of insects such as flies at an incident scene, particularly one involving blood, is not uncommon (see the forensic entomology page). Flies may feed on blood and tissues at the scene and then, following regurgitation or excretion, produce small circular stains known as flyspeck. This minute stain could be mistaken for alternative bloodstains, such as expirated blood. Furthermore, small additional stains may be caused by insects walking through a stain, thus spreading the blood.

Expiration Stains
Often associated with injury to the respiratory tract, this type of bloodstain is caused by blood being coughed or otherwise expelled from the mouth. The stains will often be slightly diluted in appearance due to the additional presence of saliva or mucous. When blood is expirated from the mouth, it will often produce a pattern of small, round stains that could be likened to a fine mist.

Examination of Bloodstain Patterns
Various factors must be taken into account in order to successfully interpret a bloodstain. The surface onto which the blood is found may have had an effect on the behaviour and appearance of the stain. For instance, a bloodstain pattern may appear different if landing on an absorbent surface such as fabric as oppose to tile or plastic. Studying the state of the bloodstain may be able to shed light onto how much time has passed since the blood was shed, as over time blood will naturally coagulate (the process by which liquid blood turns into a gelatinous substance through various clotting factors). Furthermore, the extent of drying or coagulation will depend on the quantity of blood present – for instance a single drop will dry significantly faster than a large pool of blood. During this process of coagulation serum stains may be formed, which occur when the serum (liquid portion of the blood) separates.

Bloodstains at an incident scene may not always be visible to the naked eye, either due to low amounts of blood present or an individual cleaning in attempts to remove signs of bloodshed. Despite the use of cleaning reagents or even attempting to cover the stains with paint, detectable traces will generally remain, which can be visualised using various chemicals or specialised light. Although blood will not fluoresce under UV light like some bodily fluids, it will significantly darken, thus enhancing its visibility. Furthermore, certain chemical reagents can be used to visualise latent bloodstains. These tests, such as luminol and phenolphthalein, generally work by reacting with a constituent of blood to produce some kind of chemiluminescence. However it should always be remembered that these chemical reagent tests are often presumptive, meaning that they can only indicate that the stain is possibly blood. In reality, other substances may react with the reagent in the same way.

A lack of a bloodstain can be just as revealing. The absence of blood in a continuous bloodstain is known as a void, and may suggest that something or someone was present in that area when the bloodstain was caused. This could indicate an object present at the time of the incident has been removed from the scene, or an individual (or even multiple individuals) were present in specific locations when blood was shed.

It can easily be incorrectly assumed that blood found at an incident scene belongs to a victim, however it must be taken into account that some bloodstains may have resulted from the perpetrator being injured at some point. Either way, the information available from the presence of bloodstains is not limited to bloodstain pattern analysis, but also DNA analysis. See the DNA analysis page for more information.

Point of Origin – Directionality and Angle of Impact
In the reconstruction of an incident scene involving bloodstains, it is often beneficial to establish the point of origin of bloodstains, based on directionality and angle of impact. The examination of certain bloodstains may allow for the determination of the direction of travel of blood as it impacted the target. Whereas a drop landing perpendicular to a surface (depending on the type of surface) will tend to produce a more circular pattern, those landing at an angle will result in an elongated stain. The tapered end of this stain will generally point in the direction in which the droplet was travelling. Small amounts of blood may break away from the parent stain entirely – these are known as satellite stains.

Although it may be possible to estimate area of origin purely through visual observation of bloodstain patterns, in some instances trigonometry may be utilised to determine a more precise point of origin. Depending on the type of bloodstain pattern, it may be possible to establish the angle at which a blood droplet hit a target, referred to as the angle of impact. By measuring the ratio of the width of the bloodstain to the length, it can be possible to calculate the angle of impact. If the angle of impact of multiple bloodstains is established, it may be possible to determine the area of convergence (the point where lines of travel from multiple stains meet) through stringing techniques and establish the area of origin.

Documentation and Collection
Documentation of bloodstain evidence will most typically be carried out using photography, including photographs of the wider scene along with close-up images of particular bloodstains. A ruler or other form of scale may be placed in the photograph in order to give perspective as to the size of a bloodstain. Sketches and even videos may also be utilised for further documentation. Collection of bloodstain evidence can be a complex matter, as the evidence will not likely be confined to a small object that can be easily removed from the scene. After rigorous documentation of the evidence, ideally the bloodstains themselves will be collected. This can involve simply removing objects from the scene or, more problematically, sections of carpet or large pieces of furniture. Evidence removed should be packaged in such a way that the stains are not altered or damaged. Collection of blood evidence for the purpose of DNA profiling will generally be conducted using a swab.

 

Jackson, A. R. W, Jackson, J. M., 2011. Forensic Science. Essex: Pearson Education Limited.

Scientific Working Group on Bloodstain Pattern Analysis. [online] Available at: [http://www.swgstain.org]

White, P. C., 2004. Crime Scene to Court: The Essentials of Forensic Science. Cambridge: The Royal Society of Chemistry.

Forensic anthropology refers to a specialised branch of physical anthropology particularly applied to medico-legal matters. When dealing with a set of human remains, a primary fact to ascertain is the identity of the individual and how they may have died, which is understandably not straightforward if all that remains of a body is the skeleton.

Through the study of bones, an array of information can be ascertained regarding the remains including, but by no means limited to, age, gender, ethnicity, cause of death, and even indications of lifestyle such as where a person might have lived. The adult human skeleton consists of some 206 individual bones, with there being even more in the skeleton of a child, whose bones have not undergone certain fusion processes yet, and many of these bones may prove useful to the anthropologist.

Bones develop from cells known as osteoblasts, first beginning as soft cartilage before the bone hardens through the introduction of various minerals, a process known as ossification. Bones can be divided into a number of classes; short, long, flat, sesamoid and irregular bones (Gunn, A, 2009). Short bones, such as the carpal bones within the wrist, tend to be as wide as they are long. Long bones are, as the name suggests, longer in length and also tend to be slightly curved, for example the femur. Flat bones, such as the ribs and breastbone, could be described as being fairly flat and plate-like. Sesamoid bones refer to small bones embedded in a tendon, often found in joints, such as in the knees and wrists. Finally, irregular bones refer to a certain class of bone that do not belong in the other categories, such as the bones composing the spine.

Species Determination
It is initially essential to establish whether recovered bones are of human or animal origin. Whereas the answer to the question may be obvious when a full set of skeletonised remains are present, a great deal more expertise is needed when only a few or even a single bone is found. The general shape, size and structure of the bones may be sufficient to determine likely species, and methods for distinguishing between human and non-human remains have also been established based on microscopic differences in bone structure (Urbanova, P et al, 2005). If the bones are relatively recent they may contain the proteins required to carry out serological tests to establish the species.

Sex
Establishing the sex of skeletonised human remains is not generally too difficult, as there are a number of morphological differences between the skeletons of males and females. If the remains have not reached the latter stages of decomposition, some indicators of sex may still be present in the softer tissues. For instance, the prostate gland in males and the uterus in females do not decay until later than other soft tissues. Should the bones be all that remains, perhaps the most significant indicator of sex is the pelvis. In a female the pelvis presents a U-shaped sub pubic arch, as oppose to the V-shape found in a male pelvis. As would be expected, the female’s pelvis is also generally more spacious to allow for childrearing, with a wider sub-pubic angle and sciatic notch. Furthermore, examination of a female’s pelvis may even indicate whether or not she has previously given birth, offering a further detail for identification purposes. Through examination of the skull it may also be possible to determine the likely sex of the remains, with the skull of a male tending to display a larger, squarer and more pronounced jaw, a more prominent supraorbital ridge (brow), and more rectangular eye sockets (orbits). Though not an infallible means of sex determination, the general size of bones can provide some indication as to whether the remains belong to a male or female. As the muscles in a male tend to be larger and better developed, the bones are generally larger and to an extent more robust than those of a female. However it should be noted that establishing sex based on the human skeleton is often challenging when dealing with the remains of pre-pubescent children, as certain indicators, such as the widening of the hips in a female, may not have occurred until puberty. Furthermore, the natural sex of an individual may not be consistent with the gender of that individual (for instance, a person designated female at birth may be living as a male), hindering the identification process.

Ethnicity
Establishing the ethnicity of an individual is generally carried out by studying the skull, which is typically classed as belonging to a Caucasoid (or Caucasion), Negroid or Mongoloid. The cranium itself is typically long, narrow and high in Caucasians, similar in Negroids but lower, and more rounded in individuals of Mongoloid ancestry. The size of the nasal opening may be used as an indicator for ethnicity, with the nasal cavity of a Caucasian being narrower and higher in comparison to the broader opening belonging to an individual of Negroid origin, and Monogloids sitting somewhat in between. The eye orbits can also provide clues as to the possible ethnicity, with individuals of Caucasian ancestry tending to have sloped orbits, as oppose to Negroids who generally possess more rectangular eye orbits and people of Mongoloid ancestry with rounder orbits. The mastoid process, which refers to a particular part of the skull just behind the ear, tends to appear as a wider projection in Negroids whereas this is often more pointed and narrow in Caucasian individuals. The teeth may also prove beneficial to a certain extent, with individuals of a Mongoloid ethnicity tending to have upper incisors that could be described as ‘shovel-shaped’ with a curved inner surface, as oppose to the more flat surface found in the teeth of Negroids and Caucasians. An additional observation to note relating to teeth, Caucasians tend to have smaller teeth and more overcrowding, commonly resulting in impacting third molars that must be removed. However the determination of ethnicity is certainly not straightforward, with the occurrence of ‘racial hybridity’ being a problem in establishing ethnicity of an unknown individual. This is the result of breeding between different racial groups, resulting in individuals possessing features that are typical of two or more racial groups.

Age
Once a body has decayed to such an extent that only bones remain, the estimation of age may not be as obvious as if soft tissues were present. Fortunately, there are many indicators in the skeleton that can be used to establish the likely age of the victim at the time of death. As previously mentioned, the skeleton of a younger individual is composed of significantly more than the 206 bones found in an adult. This is because as the human skeleton develops, a process known as ossification occurs in certain areas, which is essentially the fusion of bones. The appearance of certain ossification centres can estimate the age of a younger individual, though this becomes decreasingly beneficial as an individual ages. In the human skull there is a series of zigzag lines known as sutures which separate the plates of the skull. Over time these sutures harden and become less distinct. The epiphyseal fusion of bones (the fusion of the shaft of a bone to the end of the bone) can equally act as an age indicator. In addition to this, the study of tooth eruption can also be a useful indicator of the age of an individual, though from approximately the mid-twenties this is not necessarily valuable. Once an individual has reached a particular point in adulthood, generally around the mid-20s, accurately determining age becomes difficult, because tooth eruption has generally reached a stage of completion as has the fusion of bones. Depending on the age of the individual, there may be some signs of age-related conditions such as osteoporosis or arthritis, though once again there are inaccuracies to take into account as not all older people will develop these conditions and furthermore some younger people may be susceptible to them.

Age of Remains
Establishing the age of a set of remains, as in how long ago the individual died, is often a difficult task. With more recent sets of remains, there may still be some tissues present on the body to help pinpoint the age of the body. Certain soft tissues and ligaments can last for up to 5 years, so the presence of these may at least be able to narrow down time since death to the last few years.

Typically, isotope analysis can prove to be particularly beneficial in establishing the likely age of remains. Isotopes are atoms of the same element, and with the same chemical properties, but differing in the number of neutrons within the nucleus (and thus have a slightly different atomic mass). Among the most common elements to be studied in isotope analysis are carbon, nitrogen, oxygen, strontium and hydrogen. This branch of study, which can be focussed upon unstable or stable isotopes, is based on the principle that many elements within the body exist as various isotopes, many of which are taken into the body by eating, for instance. Bones and teeth are usually subjected to isotopic analysis in cases of dating skeletonised human remains, though if present hair and nails may also be used.

The isotopic analysis of stable isotopes, such as carbon-14, is beneficial in estimating time since death. Whilst an organism is living it is constantly taking in carbon, for instance through the ingestion of other organisms containing the isotope. However upon death, it ceases acquiring new ¹⁴C and so levels of ¹⁴C begin to decline. It is thus possible to establish a likely time period since death by measuring the quantities of ¹⁴C present in the remains. Despite the benefits of this technique, it is not as accurate as would be ideal, and establishing time since death using isotope analysis can only allow for an estimation. ¹⁴C dating is also only ideal for older remains (older than at least 100 years), as the technique is simply not accurate for younger remains. Due to this fact, the study of the ¹⁴C isotope is not necessarily of the greatest benefit to forensic science as other isotopes may be.

For example, the analysis of the lead-210 isotope (²¹⁰Pb) is perhaps of more use to the forensic scientist due to its shorter half-life of 22.3 years. As with the ¹⁴C isotope, ²¹⁰Pb is absorbed by the body through the ingestion of food and, upon death and thus the halt of food intake, the isotope will begin to decay. By measuring the amount of this isotope in a set of remains in comparison to the levels of the isotope seen in the body of a living person, it is possible to determine the likely time elapsed since death. Similarly, research is being conducted into the analysis of a polonium isotope (²¹⁰Po), which has a significantly shorter half-life of only 138 days and thus has the potential to pinpoint time since death with greater accuracy.

However it must be taken into account that exposure to certain conditions can affect the isotopes present in a sample and the ratios of these isotopes. Furthermore, isotopes are ubiquitous in the environment thus it is entirely possible for detected isotopes in a sample to actually be the result of contamination.

Lifestyle
As previously discussed, the study of isotopes making up a set of remains can be used to provide various forms of information, such as the likely age of the remains. Another particularly beneficial use of isotopic analysis is to determine the possible geographical origin of remains (known as the provenance) through the study of stable isotopes, often through the use of stable isotope ratio mass spectrometry (SIRMS). The study of various elements such as hydrogen, carbon, oxygen, nitrogen and strontium can theoretically be used to trace an individual across various locations throughout their life.

Different geographical locations are known to have different, often distinct isotope ratios. The varying isotope ratios in different areas occurs due to a process known as isotopic fractionation, in which certain isotopes become enriched over others. The elements studied in this instance exist in different isotopic forms – for instance, strontium exists as four stable isotopes; ⁸⁴Sr, ⁸⁶Sr, ⁸⁷Sr and ⁸⁸Sr. Because their ratios vary between different geographical locations, thus people and animals will ingest food and water from those locations, the ratio of isotopes measured in a sample can act as a kind of ‘fingerprint’, indicating the likely location in which a person had lived. Furthermore, because isotopes are taken into the body at different points throughout an individual’s life, isotope ratios can also infer certain changes in a person’s location throughout his or her life.

Stature
In order to estimate the height of an individual from the complete skeleton, direct measurements can be taken and thus height determination is usually relatively straight forward, provided the presence of tissue prior to decomposition is taken into account. When dealing with an incomplete skeleton or only certain individual bones, estimating the height of an individual may still be possible. By measuring the length of the long bones, ideally the femur, fibula and tibia in the leg, it is possible to estimate height based on stature tables. These tables take into account the race, sex and age of the individual along with the measurement of the long bone. Determination of an individual’s stature based on a skeleton or bones will only ever be an estimation, as it is not always possible to accurately take into account the changes soft tissue would have had on the individual’s height in life.

Injuries and Cause of Death
By X-raying skeletal remains, it may be possible to obtain information that could lead to both establishing a cause of death and even identifying an individual. There may be evidence of injuries obtained earlier in life that have left noticeable markings on the bones, for instance fractures and breakages or even the presence of artificial bones. Furthermore, there may be evidence of bone disease such as osteoporosis. Studying the teeth of the remains may provide important clues, particularly if the individual had any distinguishing dental features or dental work such as fillings carried out. All of this information may be compared to the medical records of known individuals to aid in confirming or disputing the identity of the skeletonised remains. Dental records in particular often prove beneficial in identifying an individual who cannot be identified by any other means, providing they have had dental work carried out and have some dental records stored somewhere.

DNA
Unsurprisingly, bone contains very little nuclear DNA, thus typical DNA profiling methods are not likely to be possible. However bone does contain mitochondrial DNA, which typically persists for longer than nuclear DNA. Mitochondrial DNA is solely inherited from the maternal bloodline, thus it does not contain any genetic information from the individual’s father. If mitochondrial DNA can be successfully extracted and analysed, it may be possible to compare it with living maternal relatives to aid in identification.

Facial Reconstruction
Provided the skull is in a reasonable state, it may be possible to reconstruct the face of an individual based on the skull by various available methods to aid investigation of an individual. The process of photosuperimposition is fairly rudimentary. A photograph of the skull is taken and a photograph of an individual in life overlaid to determine whether the features of the individual match those of the skull. This technique is not always ideal, as the photograph of the individual must be altered in size to match that of the skull, which may not always be accurately possible. A little more complex, facial reconstruction essentially involves rebuilding the likely facial features of an individual using a cast of the skull as a baseline. The technique generally utilises facial markers placed in specific locations on the skull and modelling clay, which is intricately applied to the surface of the skull to the required depths to simulate facial tissue, smoothed and coloured to resemble skin. Further additions such as the nose, ears and lips will then be constructed. Following this additional features such as prosthetic hair and eyes can be added in attempts to reconstruct the facial features of the individual. Of course when dealing with an entirely unidentified victim certain presumptions must be made, such as the colour of a person’s hair and eyes or the presence of facial hair. Even the depth of tissues on an individual’s face can only be estimated to a certain extent, as the underlying bone structure will not indicate this. The technique of facial reconstruction may also be carried out using sophisticated computer software if available. Upon completion of any forms of facial reconstruction, the image produced can be distributed as necessary to aid identification.

Decomposition
Please see the human decomposition page for more details.

 

Beard, B. L. et al. Strontium isotope composition of skeletal material can determine the birth place and geographic mobility of humans and animals. J Forensic Sci. 45(2000), pp1049-61.

Gunn, A (2009). Essential Forensic Biology. Oxford: John Wiley & Sons.

Forensic Magazine. Tracing Unidentified Skeletons Using Stable Isotopes. [online][Accessed 13 Jan 2015] Available: http://www.forensicmag.com/articles/2007/01/tracing-unidentified-skeletons-using-stable-isotopes

The University of Western Ontario Journal of Anthropology. Racial Identification in the Skull and Teeth. [online][Accessed 20 Feb 2015] Available:http://ir.lib.uwo.ca/cgi/viewcontent.cgi?article=1137&context=totem

Urbanova, P et al. Distinguishing between human and non-human bones: histometric method for forensic anthropology. Anthropologie. 43(2005), pp. 77-85.