1
Q
What has been understood about microscopical hair examinations sine 1873?
A
- it has been understood since 1873 that microscopical hair examinations ALONE do not result in individualisation (cannot be confident hair comes from individual)
- it cannot be said that any questioned hair comes from a specific individual based upon microscopy alone - link DNA and there is an increased chance of doing this
2
Q
What are the six reasons as to why hair is a great form of trace evidence?
A
- it is found on all humans and other mammals so good chance left behind as TE
- it is constantly being produced and shed in their immediate environments
- it is easily overlooked by criminals involved in nefarious activities (they remember fingerprints etc.
- it is highly stable resisting both physical and chemical degradation (persists for a long time, after 1000 of years potentially)
- it is readily transferred from one person/object to another person/object
- hairs from different individuals can be distinguished from each other and with DNA testing (can do nuclear DNA testing if has follicle)
3
Q
What mistake to forensic labs make when examining hair?
A
- stop after DNA testing but there is more to hair than this
- can provide investigative leads or help with the reconstruction of events in contention
- and about where perpetrator has moved in past (microscopic and elemental analysis)
4
Q
What are hairs composed of?
A
- hairs are composed primarily of protein (keratins)
- remarkably stable tissues - chemically and physically (can persist almost unchanged at ultrastructural level as keratin is extremely robust)
5
Q
Why is it not practical to differentiate hairs using chemical techniques?
What must be used instead?
A
- all hairs have the same chemistry
- elemental analysis and spectroscopic techniques
6
Q
What can be done when looking at isotope ratio of elements in hair?
A
- looking at isotope ratios of elements in the hair using isotope ratio mass spec
- can be used to identify locations where an individual may have travelled based on changes in drinking water isotopes in different locations
7
Q
What are three types of hairs in humans?
What is type used in forensic analysis and why?
A
1 - lanugo - hairs are formed in uterus and are fine and unpigmented
- shed before or shortly after birth so rarely a relevant form of TE unless talking about unborn/newborn children
2 - vellus - fine short unpigmented/lightly coloured hairs present on almost all skin surfaces (forehead, nose, ears, bald scalp)
- not hands/palms of feet
3 - terminal - typical hairs macroscopically visible on children and adults
- primary: head, eyelash and eyebrow
- secondary: pubic, underarm and beard
- forensic analysis of hairs generally restricted to the terminal ones (these are what have value in court):
- need a reasonable chunk of hair to be able to analyse it - vellus too short
- most commonly recovered are head and pubic
8
Q
What are three main histological layers of hair?
What are two other components of hair?
A
- cuticle (outermost layer):
- largely responsible for chemical resistance of hair
- useful and has probative value
- cortex (main bulk of hair):
- largely responsible for mechanical properties of hair (flexibility, strength, floppibility)
- contains most of pigment granules giving hair a colour
- medulla - innermost layer of hair shaft
- not very well studied/understood
- not present in all hairs (so can use this to discriminate between two samples)
- has slight pigmentation but bulk of pigmentation from cortex
- cell membrane complex (CMC) - bonds all cells together
- layer of cells found at interface between cuticle and cortex
- has slightly different mechanical properties
- rarely considered in forensic perspective
- follicle - where hair grows from and changes size and shape throughout the hair cycle
- where most likely to have any recoverable DNA
- follicle not always present
9
Q
What is the hair cycle?
What can be said about this cycle between individuals?
A
- anagen - active growing phase of hair extending progressively from the follicle root outwards from skin (85-90% of human head hairs)
- catagen - transition phase when growth slows and eventually stops
- hard to judge this phase
- telogen - resting phase when minimal force is required to remove hair and natural shedding likely to occur (exigent) (10-15% of human head hairs)
- every individual hair progresses through this cycle at a rate which depends on hair type and individuals
10
Q
What does hair shedding depend on?
A
- 30-100 hairs shed every day depending on huge range of environmental factors (how much brush hair, if we wear hat)
11
Q
What is average that hair grows a month?
A
- around 1cm a month depending on ancestral groups and age
- Asian - fastest
- European - middle
- African - slowest
12
Q
What happens when hair turns grey?
What is this use in TE?
A
- it doesn’t turn grey the pigment stops being produced giving the appearance of white/grey
- tip is pigmented but near scalp not
- different points on same strand of hair may differ in colour
- this gives us some discriminative value
13
Q
What are two methods of collection?
A
- forceps: for individual hairs forceps can be used to isolate and collect
- be careful with damaging hairs as too much pressure can ruin hair and this is bad as we are interested in the morphology of hair
- tape lift: most efficient when collecting hairs from large surfaces
- solves problems with forceps but is less precise
14
Q
What must be done during collection of known samples?
A
- need to collect a representative sample due to inherent variation
- ENFSI recommendations suggest collecting 20 hairs from 5 head regions and package them separately in order to give you enough variation
- collect through combination of plucking (high pressure) and combing (low pressure)
- some more likely to get follicle and some shape is going to be different and some will be at different growth phases
15
Q
Define the seven step analytical workflow for hairs?
A
- gross examination, recovery, and collection
- preliminary evaluation of physical characteristics
- microscopic techniques
(a lot of labs stop here – but there is a lot more you can get from hair)
- DNA
- SEM (Very occasionally TEM)
- spectroscopic techniques - IR & raman
- chromatographic techniques & mass spectrometry
16
Q
What microscopic techniques can be used in forensic investigation of hair?
A
- main ones: stereoscopic, comparison, polarised, reflected light and brightfield
- if something more useful to collect: fluorescence and SEM
- very unlikely - thermal
17
Q
What are four morphological characteristics of hair observed at macroscopic level?
What is used to observe these?
A
1 - macroscopic colour - colourless, blonde, red, brown, black (may use coloured backgrounds to provide contrast so can assess colour)
2 - length - measured in absolute units e.g. mm
3 - general contour and curliness - straight, wavy, curly, kinked
4 - approximate diameter - thin, medium, thick
- stereoscopic microscope (can use at scene to aid recovery)
18
Q
What microscope is used after stereoscopic one?
What must be done when comparing questioned and known hairs?
What 9 features are evaluated in this step? and why?
A
- hairs mounted in appropriate medium for examination with compound light microscope with polarising filters (these allow for more info)
- questioned and known hairs should be in same media to provide equivalent contrast
- to provide more discriminatory value between samples:
1 - colour (colourless, blonde, red, brown, black)
2 - cross sectional shape (round, oval, flattening
3 - biological damage (insect bites, fungal, bacterial activity)
4 - cosmetic treatments (bleached, dyed)
5 - shaft irregularities (buckling, twisting)
6 - adhering material (blood, nots, particles, residues)
7 - thickness range (um)
8 - general damage (split, frayed, broken, crushed, burnt (burnt allows more discriminatory value)
9 - non-root morphologies (rounded, cut, broken)
19
Q
What are 6 microscopic features of cuticle that could be analysed?
A
1 - colour e.g. colourless, or unnatural colour indicative of dyeing
2 - inner margin e.g. distinct/indistinct, smooth, cracked, ragged, serrated, flattened
3 - pigment granules – presence or absence
4 - thickness – thin, medium, thick
5 - damage e.g. lifted, cracked, or looped
6 - scale protrusion e.g. indistinct, minimal, prominent etc
20
Q
What are 3 microscopic features of cortex that could be analysed?
A
1 -pigment granules - density, size, shape, aggregates, distribution - uniform, clumped, granular)
2 - texture e.g. fine, medium, or coarse
3 - Cortical Fusi or Ovoid Bodies
21
Q
What is most important feature when examining medulla?
A
- presence or lack of presence
22
Q
What are 3 microscopic features of root that could be analysed?
A
- growth stage
- follicular material
- abnormalities
23
Q
What is done after known and unknown has been studied in same conditions?
What is important to note about this next step?
After one examiner done, what happens?
When is this the last step in hair analysis?
A
- hairs determined to be similar to each other should then be examined using a comparison microscope with transmitted light
- all instrumental parameters and preparation conditions should be exactly the same for comparison (mounting media has to be same to be able to do direct comparison)
- must be peer reviewed by a second examiner observing the hairs directly using a microscope
- carried out ‘blind’ to initial conclusions to independently verify what it is and what it looks like
- last step unless there is a unique feature that will give more discriminative value that more detail required
- SEM/Fluorescence or Elemental Analysis
- or root requires evaluation to explore possibility of DNA testing.
24
Q
What type of hairs exhibit more variation between people?
What does this mean for their examination?
A
Head hairs typically exhibit more variation between people than any other type of hair, thus have the most discriminative value
25
When is SEM carried out?
What can be done with SEM?
What is it combined with?
How often is SEM used in hair analysis?
- when additional features of hair examined under light microscopes further analysis in higher resolution
- directly examine surface of hair to highlight scales or physical damage within hair
- combined with secondary electron, backscatter electron and EDX modes to provide elemental data about hair or residues on surface of hair
- only occasionally used
26
Can TEM be used in analysis of hair?
- no current examples of usage of TEM in forensic cases of hair
- takes a lot of time, expensive, doesn’t really add anything new for hair analysis
27
What use do IR and raman have in hair analysis?
- to distinguish hair treatments and chemical damage along the length of a hair to give us more discriminatory value
- can also distinguish between untreated hair and chemically damaged hair
- possible to distinguish between dye colours and brands but requires chemometrics and strong training
28
What is a mass spectrometer?
What is it used for?
- a device for producing ions from a compound in order to obtain molecular weight and structural information about what is there
- used for the detection and analysis of minute amounts of materials
29
What is electrospray ionisation (ESI)?
How is this used in analysis?
- sample of interest is taken from probe or GC column, flowed through capillary and potential is applied between edge of capillary and sample in order to create a charge with the flow of gas
- as solution flows through capillary a potential is applied across cone as it is emerging
- this gives charge to all droplets as they are emerging
- going towards Rayleigh limit droplets get smaller and move towards surface
- charged droplets are drawn into mass analyser in order to analyse what is going on in there
- instead of just putting sample of interest into tube and analysing it - can use flow of charge ions to extract further information
30
what is laser desorption electrospray ionisation (LDESI)?
What is this particularly useful for?
- instead of firing at surface with out electrospray in order to ionise it, we use a mid-infrared laser to ablate the sample which creates a cloud of neutral molecules and chips them off from surface
- everything is neutral which is not useful as not going to be detected by mass spectrometer
- cloud is hit with electrospray from above to cause ionisation
- collected in MS for analysis
- particularly useful for anything with animal tissue, plant tissue and live-cell imaging
31
What is the ionisation process?
What are four ionisation approaches (analytes, sample, probes, mass, type, advantage, disadvantage)
- process of conversion to a charged species
- electron
- small molecule volatile
- GC or probe
- <1000
- hard (destroys everything)
- structure
- not useful for protein analysis
- chemical
- small molecule volatile
- GC or probe
- <1000
- soft (doesn't give right down to structural ionisation)
- good for picking out molecular ion M+H
- electrospray
- start to pick out non-volatiles peptides (lots of TE from human body made from proteins and peptides)
- LC (electrospray has to be attached to some kind of column to create ions)
- <200,000
- soft
- provides us with multiply charged ions
- MALDI
- non-volatiles peptides
- works by mixing surface of sample with matrix (we charge matrix and then matrix passes charge to sample)
- <500,000
- soft
- highest mass and a lot of biological molecules are large so this is good
- identification of furs of domestic dog, racoon dog, rabbit and domestic cat by hair analysis using MALDI-ToF mass spectrometry
32
What is relevance of mass analyser?
What are the three most useful types (resolution, mass range, mass accuracy, advantages and disadvantages)
- after ionisation need to do something to measure what is there
- choosing the right analyser is vital to define what fragments can be identified and at what resolution to give a degree of discrimination often offset against cost, ease of use and size of instrumentation
- quadrupole - 4 poles where charge switched between poles and path of energy moves through them and this allows us to discriminate different ions of different masses by separating by the speed at which they moved
- 500 - 2000 resolution
- 2 - 2000 mass range
- 0.1 amu mass accuracy
- reproducibility, low cost
- low resolution, low mass accuracy
- time of flight - where you use the speed of different ions of different sizes and how they change as they flow down a flight tube with smallest fragments arriving first
- 500 - 12,000 resolution
- 50 - 1,000,000 mass range
- 0.0001 amu mass accuracy
- highest mass range, limit of detection
- large
- magnetic sector - applying a magnetic sector
- 800 - 50,000 resolution
- 2 - 15,000 mass range
- 0.0001 amu mass accuracy
- highest resolution and mass accuracy
- expensive, large
33
What are 6 examples of modern instrumentation?
- solid phase micro-extraction
- hybrid instruments
- tandem mass spec
- isotope ratio and ion mobility spectrometry
- inductively coupled plasma mass spectrometry
- atom probe mass spectrometry
34
Tandem mass spec:
- what is it
- underlying principle
- other names
- even more advanced version
- what mass analysers can be used?
- two mass analysers stuck next to each other
- employ a first mass analysis stage to select precursor (parent) ion
- excite selected ion species (via collision with neutral target gas
- causes it to fragment further to give one or more daughter ions plus neutral fragments
- then employ a second mass analysis stage to determine the mass spectrum of the product ions
- mass spec/mass spec
- MS/MS
- MS^2
- MS/MS/MS where determine the mass spectrum of the granddaughter ions
- tandem in space or tandem in time
35
Tandem-in-space:
- how is it structured
- what mass analysers are used
- what is used in analysis of dri=ug concentration in hair
- mass analysis stages are physically separated from each other spatially so that they occur in different regions of the overall instrument
- instruments employ ion beam-type mass analysers such as magnetic (plus electric) sectors, ToF, quadrupole mass spectrometers or hybrid arrangements comprising two or more different beam-type analysers
- drug concentrations in hair are considerably lower than those found in other matrices (e.g., blood), so MS/MS is often used
- can look at concentration of drug in different stages within hair
- hair grows around 1cm a month – can identify timelines of use of substances of abuse
36
How can isotope mass spectroscopy be used in analysis of hair?
- providing evidence in tracking the movement of people and or chemicals e.g. substances of abuse or water we consume
- a lot of common elements have more than one natural abundance
- looking at isotope ratios of elements in the hair can be used to identify locations where an individual may have travelled based on changes in drinking water isotopes in different locations
- ratio will be reflected exactly within hair
- can find where human has been based on oxygen in their hair using mass spec
- a lot of earth isn't always the same so hard but certain areas are identifiable e.g. by a lake
- can then use as method to trace people and their movements
37
Why is hair different to many other forms of trace evidemce?
- hair is of biological origin
- hair exhibits significant amounts of morphological variation within an individual and between individuals
38
How can hair provide discriminating potential?
What can't this do?
- variation between individuals provides the discriminating potential for forensic hair comparisons
- hair from different individuals may appear different & therefore it is possible to distinguish hairs from different individuals on the basis of their macroscopic and microscopic morphology
- however cannot be individualised on the basis of these physical characteristics alone – using microscopy alone can’t determine if an unknown originates from a specific individual
39
What are three main conclusions that can be drawn from the interpretation of microscopical hair analysis alone?
- association
- inconclusive
- exclusion
40
Define association (definition, factors strengthening association and factors weakening it)
- questioned hair cannot be distinguished from known sample
- factors strengthening it:
- distinctive cosmetic treatments e.g. dye
- abormalities
factors weakening it:
- short hairs
- incomplete hairs
- colourless/highly pigmented
41
Define inconclusive (definition,
- questioned hair exhibits some similarities & some differences with a known sample, but limiting factors are complicating the comparison
e.g. hairs have been exposed to different environmental conditions – buried vs. direct sunlight
42
Define exclusion (definition, how is confidence of this improved, certainty)
- questioned hair exhibits a meaningful difference compared to a known source
- to improve this confidence, important to collect many hairs from multiple sub-areas for known sample
- hard to be certain unless from different ancestral characteristics e.g. Asian, European, or African
43
What are the nine fundamental principles of transfer and persistence of hairs?
1 - object texture
2 - hair loss
3 - secondary transfer
4 - wearing a hat
5 - washing e.g. in washing machine
6 - fingernail scrapings
7 - animal hair transfer
8 - artificial dying
9 - migration
44
Why can't it be predicted absolute number of hairs expected to be transferred or remain on an object in a real world scenario?
- transfer and persistence of hairs are complex issues with far too many variables to be able to predict the absolute number of hairs expected to be transferred or remain on an object in a real‐world scenario
45
What is vital to be maintained, stored safely and updated regularly in any forensic microscopy lab for hairs?
What should this include for human hair?
- reference collections for hairs are of vital importance
- basic lab human hair reference collection will include:
- different ancestral groups
- different locations on body
- different cosmetic treatments
- different damage types
- exhibiting different diseases
- degraded in different ways
FSCI6030 - Trace Evidence
flashcards
Decks in class (13)
# Cards
