CONFIRMATORY BODY FLUID IDENTIFICATION | DNA ANALYSIS
THE MAIN STEPS IN FORENSIC DNA ANALYSIS
THE SEARCH FOR TRACES AND THE DETECTION OF BODY FLUIDS
The analyses aimed at establishing the presence or absence of bodily fluids are the first step in a forensic medicine laboratory investigation. The goal is to establish whether a reddish stain is a bloodstain, a yellowy one is saliva, sperm or urine, or whether a fibre is natural (a hair, for example) or artificial.
At this stage of the investigation, specific tests are carried out. These tests can be divided into preliminary or orientation tests, and confirmatory tests.
The orientation screening tests are fairly sensitive and can be carried out with a remarkably small amount of material, but they are not equally specific. Other follow-up tests are therefore necessary.
Confirmation of the presence of blood, sperm, saliva, etc, is obtained with techniques that provide certainty as to the nature of the sample and the animal species from which it derives. Unfortunately, tests of this kind require a considerable amount of material and could therefore put at risk the follow-up DNA investigations, which are much more sensitive.
In general, there are visual, physical, microscopic and chemical methods for establishing the presence of blood or sperm. Apart from the visual method, these approaches are all capable of providing indicative or even certain results.
The visual method can be carried out immediately. Nowadays it is still the preliminary step and allows to guide the search for traces to be collected and submitted for subsequent laboratory tests. It is worth remembering that blood stains can acquire colours from red to brown or dark green and sperm traces may appear white to greyish or yellowy, with characteristically 'map-like' edges. Saliva or sweat traces may be completely invisible.
The suspicious areas therefore must be submitted for laboratory tests.
Sperm traces, as well as saliva, sweat, urine and other biological liquids, are visible under ultraviolet light or using light sources with specific wavelengths. These forensic lights are particularly useful when large surfaces must be explored, since the stains turn luminous and can be easily seen.
Microscope examination is particularly important in detecting sperm, since the sperm cells have unique features which are therefore revealed. The detection of spermatozoa provides confirmatory results in the case of normospermic and oligospermic ejaculations. Chemical methods to obtain confirmatory results in the case, e.g, of azoospermic ejaculations, are detection of the P-30 protein, prostatic-specific antigen (PSA) or prostatic acid phosphatase (AcP), identified through conventional electrophoresis, immunoelectrophoresis or the ELISA method.
There are various methods for detecting blood, semen or saliva. These are specialised tests, although some use extremely outdated techniques. The well-known Luminol test, for example, is based on a principle that has been known for at least 90 years. New and more sensitive RNA-based tests are now being employed.
Hair identification is based on microscopic examination of hair-like structures to check whether they are artificial structures (such as textile fibres) or human hair. This is achieved by confirming the presence of the root and the shaft - which are different in the human species compared with animal ones - and the tip.
The limited amount of biological material available often requires complex decisions. Sometimes the preliminary steps must be omitted to preserve the material for DNA analysis. The apparent scientific paradox that arises is that an individual can perfectly be identified as the donor of a trace even if the exact nature of the biological material cannot be established.
CONFIRMATORY BODY FLUID IDENTIFICATION
The confirmation of body fluid identification tests proves what part of the body the sample comes from. These tests are based on the detection of proteins or other specific factors (mRNA, for example) in a tissue.
Some of the most used tests, employ techniques that require a lot of biological material. More recent techniques, such as mRNA, are more sensitive but have not been completely validated for forensic use yet.
DNA ANALYSIS
The real revolution in personal identification occurred with the discovery of DNA polymorphisms. These are much more informative, resistant and widely usable on all biological materials than any of the old and "traditional" protein polymorphisms (AB0).
The study of genetic profiles is based on the examination of certain variable loci of the DNA molecule. Each of our somatic cells (except our mature red blood cells) contains in its nucleus around 6 billion base pairs, or nucleotides, which constitute the entire genetic profile of the individual, organized in 23 pairs of chromosomes. Most of this DNA is identical in all human beings, but some regions are highly variable.
These variable regions are used in forensics since we know their exact chromosomal location (genetic locus) and the frequency with which this variability, or polymorphism, appears in the population.
An individual's DNA profile is visible in the form of electropherogram peaks. The position of these peaks reflects the number of repeat units for each genetic marker examined. A genetic marker containing few repeat units will have a position in the left side of the electropherogram, while a fragment with more repeat units will be located further right.
THE MAIN STEPS IN FORENSIC DNA ANALYSIS
Extraction and quantification.
To be analysed, the DNA must be extracted from the stain or reference sample and purified of any proteins or substances that may be present in the trace (e.g. dust, soil, clothing fibres, etc.).
It is necessary to establish the quantity and quality of DNA in order to use the appropriate amount of DNA in the following stages (PCR).
PCR
A second revolution in forensic investigation came with the introduction of the polymerase chain reaction (PCR) technique, which allows a polymorphic DNA fragment to be multiplied millions of times.
With this technique, different polymorphic genetic loci can be simultaneously amplified millions of times to reach an extremely high identification power.
One of the worst enemies of PCR, however, is contamination, since the technique is able to identify only a very few DNA copies.
Comparison of genetic profiles
The study of DNA polymorphisms in forensics is carried out by comparing genetic profiles.
A comparison may be made, for example, between the DNA profile extracted from bloodstains found on the accused's trousers and those extracted from the victim's blood. Or, the DNA profile extracted from seminal liquid samples from a victim of rape may be compared with that of a suspect.
The result may be exclusion or non-exclusion, but not identity.
A forensic genetic scientist is asked to establish whether a certain individual can be excluded as the one who left his/her/their traces at the crime scene.
In the case of exclusion, the answer may be quite swift, since a total inconsistency between the two genetic profiles is detected.
Non-exclusion is a more complicated matter. It requires an assessment of the frequency with which the genotype of each marker appears in the reference population (Italian, Dutch, English, etc.). An examination of all markers produces a cumulative frequency value, which reveals the greater or lesser rarity of the profile.
The reciprocal of this frequency value refers to the number of individuals in the reference population who randomly share the same profile (Random Man Not Excluded - RMNE). Until a few years ago values of 1 out of 10,000 or 1 out of 100,000 were applied. These figures are rapidly giving way to much more significant values of 1 out of several billion or even more. This is because we can now use increasingly informative genetic systems, with extremely low cumulative frequencies.
From:
Cazzaniga, Cattabeni, Luvoni, Zoja.
Compendio di Medicina Legale e delle Assicurazioni.
UTET Giuridica, 2022
Book chapter by A. Piccinini (mod.).
