A Laboratory-based Analytical Method to Determine Age at Death

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					                                                          REVIEW REPORT

 A laboratory based analytical method to determine ‘age at death’ for forensic purposes using human
                                               bone.

                                                Principal Investigator: P. Zioupos1
                      1
                          Dept of Materials & Applied Science, Cranfield University, DCMT, Shrivenham, UK
                                                     Co-investigator: S. Black2
                           2
                               Faculty of Life sciences, Dept of Anatomy, University of Dundee, Scotland, UK
                                                 Academic partner: J.G. Clement3
                           3
                               School of Dental Science, The University of Melbourne, Melbourne, Australia



1. Background/Context
Age is one of the four important attributes that a forensic anthropologist is called to answer in the case of a body or
body parts of unknown origin, together with sex, stature and ethnic background. Identification is a difficult process in
situations where human remains are rendered unrecognisable by advanced decomposition, or are completely
skeletonized, or there is fragmentation of the body. Estimating the ‘age at time of death’ becomes most challenging
once adulthood has been reached.
Ageing brings about a ‘constellation of changes’ and is orchestrated by the complicated interplay of genetic,
environmental, and cultural factors, meaning that not everyone ages like anyone. However some basic biological
processes that all humans undergo are reflected in the skeleton, from the prenatal months of development through
infancy, childhood and finally adulthood, giving experts at least a fighting chance to begin tackling the problem of age
determination. The real challenge, in fact, becomes one where researchers are required to pinpoint processes that change
with age in a well defined and constrained manner and thereby deduce the age of the individual by tracking down the
evolutionary stage of these processes during ontogeny.
Several qualitative methods exist that provide a relative biological assessment of age, like the chronology of dental
eruption, or the fusion of the epiphyses at different anatomical sites in the human skeleton, but the reliability of
estimation declines with increasing age and beyond the age of 30-35 most ageing methods are termed as unreliable [1].
During the sub-adult years of development the eruption of deciduous teeth, and permanent teeth occurs at fairly regular
intervals, therefore, age estimation of sub-adults can be based dental eruption quite accurately (±3yrs). After adulthood
is reached an age estimate can be obtained by examination of the fusion of bone epiphyses to metaphyses, and several
bones can be examined for such age determination, i.e. the iliac crest, the medial epiphysis of the clavicle, the tibia, and
the femur. Based upon the predicable changes of growth, and development in childhood and early adulthood, age
determination by the estimation of the time of fusion of the epiphysis can consistently provide age assessment only until
the 30s, while is rendered ineffective past the 40s. Comparative examination of 4 methods (i) single rooted teeth, (ii) the
sternal end of the fourth rib, (iii) the symphyseal face of the pubis or, (iv) the degree of femoral cortical remodelling [2]
does not also provide estimates more accurate than ±10yrs. It is fair to say that although an approximate age of the
individual can be determined by these qualitative methods, the actual age cannot be established.
Several attempts have been made over the past years to establish more quantitative estimates of age at death in a
consistent way and even semi-automated way (not operator depended) by assessing histomorphological features of
bones [3-8]. These are based on the fact that after adulthood is reached bone intracortical porosity starts increasing and
bone starts remodelling. In remodelling older bone lamellar matrix is substituted by new osteons, which in turn at a later
date are themselves also substituted by other osteons that follow at a later stage. Efforts were also made [3] to measure
the area and perimeter of the femoral cortex, and finally an effort was undertaken to measure the number and the areas
of occupied by Haversian canals within the cortex [8]. Results showed that although the number of Haversian canals
measured from the entire bone cortex changes with age, because of the spread of values at any given age, the method
was of limited value in age determination and the errors exceeded ±8yrs for over half of the cases in these studies.
The only strictly quantitative analytical technique available today, analyses the variation of optical isomers of aspartic
acid present in the organic proteins of the skeleton. This method was introduced in a paper published in Nature [9],
which underlines the importance of analytical methods for age determination, not only in forensics but in all disciplines
where human remains need to be characterised e.g. archaeology. The aspartic acid method does not achieve in the best
of cases accuracy better than ±5yrs for bone tissue and no better than ±3yrs for perfectly preserved teeth. However, the
method is very lab- and protocol- depended [10], it is complex, slow and has inherent inaccuracies for females. The
reason for this the racemization process, it is based on, is a truly time and temperature depended process. The method
needs therefore, skeletal tissue, which is metabolically isolated and maintained at constant temperature throughout (like
in teeth). Inaccuracies in females arise from the fact that their bone matrix is highly metabolically active especially after
the menopause (>55r) when osteoporosis ensues.

                                                           IGR ‘Age at death’ - Page 1 of 6
In contrast to previous histomorphometric studies of bone, which are only based in phenomenological changes in the
bone cortex, the principal investigator of the present project and his colleagues [11-15] have been over a number of
years concentrating their attention on age related changes of some more robust features of the bone matrix such as the
material characterisation of the various bone phases at the macroscale or the microscale. In an effort to try and evaluate
the factors affecting the biomechanical bone properties as function of age a number of physical characteristics were
measured in situ or in homogenised (bone powder) form: the porosity, the mineral content, the calcium to phosphorus
ratios, the dry density, the condition of collagen (thermal shrinkage and content in mature x-links), the elasticity of
osteonal and interstitial lamellae and the numerical and surface-density of the in vivo fatigue microcracks and other
similar microstructural features. It was observed that meaningful relationships can be established that can predict some
of these age related factors as function of others, i.e. the elastic modulus and hardness of secondary osteonal and
interstitial bone (fig. 1), was examined through the thickness of the cortex of human femoral bone from 9 male subjects
of ages 35-95 by nano-indentation. By combining results on the area fraction occupied by secondary osteons, the
nanoproperties of these osteons, with the chronological age, one could predict the modulus of whole bone in bending
with an R2=0.88 (fig. 2). It became apparent in those applications that inclusion of ‘age’ as an independent additional
variable was improving significantly the accuracy of predictions. It was, therefore, only reasonable to assume that
similarly successful inferences can be obtained if one reverses the analysis where then age is the ‘unknown’ variable.
This approach utilises parameters measured at either macroscopic (M) or microscopic (μ) level. These are: wet apparent
density-M, dry density-M, porosity-M, organic fraction-M, mineral content-M, collagen degradation peak shrinkage
temperature-M, total amount of energy required for collagen degradation-M, calcium to phosphorus ratios-μ, osteonal
and matrix microhardness-μ, ash content-M and image analysis of cortical sections-μ for porosity. This method
produced successful age estimates on a cohort of 12 donors of age 53-85 yr (7 male, 5 female), where the age of the
individual could be approximated within ±3yrs (mean error = 0.4 ± st.dev=1.9 yrs) R2=0.93 (Fig. 3).

                                                                              16.5                                                                      90
           (1)                                                                             (2)                                                                      (3)
                                            predicted bending modulus (GPa)




                                                                              16.0                                          35
                                                                                                                                                                  age vs all                          81 85
                                                                                                                                                        80                                          79




                                                                                                                                 predicated age (yrs)
                                                                              15.5                                     48                                                                      74
                                                                                                                                                                                          70   75
                                                                              15.0                                                                      70
                                                                                                                  56
                                                                                                                                                                                         68
                                                                                                                                                                                        67
                                                                              14.5                                50
                                                                                                      70                                                                           64
                                                                                                             42
                                                                                                           6052
                                                                                                                                                        60
                                                                              14.0
                                                                                                                                                                         55
                                                                                                                                                                        54
                                                                                           92                                                                           53
                                                                              13.5              46
                                                                                                                                                        50
                                                                              13.0

                                                                              12.5                                                                      40
                                                                                                                                                             40    50         60         70         80        90
                                                                                     12.5 13.0 13.5 14.0 14.5 15.0 15.5 16.0
                                                                                        measured bending modulus (GPa)                                                  actual age (yrs)

These results show that an examination of a combination of the various constitutional, compositional, micromechanical
and physicochemical changes that occur in bone matrix with age can be used to derive accurately the ‘age at death’
using solely analytical and laboratory based (and thus non-expert witness depended) methods. The challenge was to
further develop, validate and assess the (1) experimental methodology; (2) address the biological/tissue related issues;
and (3) the legal issues so as to offer accurate age determination that can be applied in a wide range of criminal and civil
investigations.

2. Key advances and Supporting Methodology
Following the kick-of meeting a set of more specific objectives were set to achieve the previous aims. It was decided
that various confounding factors that may affect the predictive power of the method had to be examined/analysed.
1. The introduction of nanoindentation measurements to analyse the previous set of human samples, to complement the
other physical measurements, as it was felt that nanohardness offers both modulus and hardness measurements and
potentially other variables to quantify plasticity, creep indentation properties etc.
2. Artificially modify bone samples by embalming fluids and/or histological preservation fluids. Subsequently apply the
full set of techniques employed in this method in order to find out the likely effect of these preservations treatments on
the predictive power of the method.
3. Examine the effect of the state of hydration of bone samples in the application of the differential scanning
calorimetry (DSC) results.
4. Examine the effect of the hydration level at a burial site on the hardness properties and mineral/organic content of
samples from a single individual.
5. Examine the effect of the pH of the burial site over a period of a year (in 3-month intervals) on the full set of
properties measured by all techniques used by this method.
6. Apply the full set of techniques on a new set of freshly retrieved samples from donors in AUS supplied to us by our
colleagues in the Forensic institute in Melbourne to corroborate the evidence collected in the British cohort.


                                                                                         IGR ‘Age at death’ - Page 2 of 6
2.1 Nanoindentation
Nanoindentations were performed on the first cohort of samples
(7M+5F) on selected areas of histological significance, by using
the CSM NHT system at 10mN and 100mN max loads and
standard loading protocols (load/hold/unload), each indentation
lasting ~180s.
Stepwise regression of the produced data gave a number of
significant relationships. To keep the number of variables few
and meaningful we show one which utilises Real density values-
M, Apparent density-M, optical porosity-M, indentation
modulus−μ and hardness−μ of the interstitial matrix areas at
10mN, and the enthalpy of the DSC test on demineralised bone
matrix-M :
Age = 199 - 133 Real Density + 52.7 App. Density + 106 Opt-
                                                                                                          Fig. 4
Porosity + 2.96 Em10 + 0.0550 UHm10 - 0.632 dem-DeltaH,
R2= 0.97, and residuals within of ±3yrs of actual age values.
2.2 Influence of fixation technique on the material properties of compact bone
A series tests were planned on control human and bovine specimens in order to establish whether the fixation technique
of the bone affected the material properties of the bone on which the age estimation techniques are based. The effect of
heat on the differentially-fixated bone was examined through ash tests and DSC experiments on both mineralised and
demineralised compact bone. Material and apparent densities were also calculated alongside the other techniques. A
section of bovine femoral shaft and a section of human femoral shaft (left proximal femur from a 55 year old male
individual) were each sectioned using a bandsaw into three slices approximately 1 cm thick. One slice from each was
subjected to a different method of fixation for 14 days: a) native – kept in Ringer’s solution in a deep freezer (-20°C); b)
70% ethanol – as used in histological sectioning; c) 100% embalming fluid (Plasdoform-based Metasyn arterial
chemical, Dodge Company Ltd). Following 14 days of treatment the slices were rinsed and cut using a circular saw into
three thinner slices (approximately 5 mm thick). Four discs were drilled from each slice using a diamond-encrusted
hole-drill bit of internal diameter 5 mm. Each disc was subjected to a different combination of techniques, in order to
maximise the amount of information obtainable from each disc: (1st) osteonal and matrix micro-hardness and nano-
hardness, optical porosity; (2nd) wet apparent density, dry density, mineral density and demineralised DSC; (3rd) ash
content; (4th) mineralised DSC. Those discs destined for hardness testing were marked with an indelible pen before
drilling so that the endosteal and periosteal aspects of the slice were known during the hardness testing. The DSC
experiments (on discs 2 and 4) were carried out as before, using a Mettler Toledo-M3 DSC machine, and heating the
bone samples from 30°C to 500°C at a constant rate of 5°C/min. These experiments were done to test whether the
fixation method applied to the bone affected the behaviour of the bone as it was heated. In conclusion, all the techniques
available to us were applied to bone samples from the same source, in order to reduce experimental variables. The bone
samples were then differentially modified so that the effect of the chemical processes on the bone samples could be
isolated in the experiments. To mention just one of the effects that we are interested in, fixation by either method
reduced the recorded hardness for the samples by on average 3-7% (this was also different in matrix or osteons), the
implication is that this was the case in the relationship mentioned above the age would have been underestimated by as
much as 4 years. Given that the effect of fixation on other factors could go either way we estimate that these effects
would introduce an inaccuracy of 3 years. We are currently studying these effects carefully.
2.3 Factors that may affect DSC results.
Differential Scanning Calorimetry (DSC) is a standard chemical technique used to characterise compounds that exhibit
thermal transitions. It provides information about how the structure of a compound changes as it is heated. For bone
samples, DSC has successfully been used to investigate the heat-induced degradation of collagen [16], which is known
to vary with age of the individual. Experiments were carried out to examine the effect of two factors: the presence or
absence of the bone mineral and the hydration level of the bone. This led to the examination of four different
experimental conditions of the bone: dry-mineralised; wet-mineralised; dry-demineralised and wet-demineralised. The
aim of these pilot tests was to determine how the DSC properties varied with the condition of the bone, in order to
enable characterisation of unknown samples from their DSC results, as well as to find the combination of conditions
that produced the most consistent, repeatable results for the later experiments.
Specimens of cortical bone were obtained from 5-6 cm long mid-shaft cortices of fresh bovine femur from one
individual animal approximately 18 months old. Twelve test specimens were obtained by drilling (drill bit with internal
diameter 5 mm) and were cut and polished to an equal size of approximately 5 mm in diameter and 2-3 mm thick. The
decision was taken not to powder the bone samples, as this has been shown to damage the collagen. Six of the twelve
test specimens were demineralised in 0.5M EDTA solution (pH 7.4) for 14 days. The solution was refreshed every two
days. Once decalcified, the samples were rinsed thoroughly in distilled water. Three of the mineralised test specimens
and three of the demineralised test specimens were then oven-dried at 36°C for 4 days in order to remove all traces of
water from the cortical bone. The remainder of the specimens were kept in distilled water in airtight containers in the
deep freeze until required for testing. As a result of this preparation, cortical bone specimens in four different states

                                                     IGR ‘Age at death’ - Page 3 of 6
were produced: dry-mineralised; wet-mineralised; dry-demineralised and wet-demineralised. Before each test, an empty
aluminium crucible was weighed using a Mettler pan balance, and the crucible and sample weighed together. The pre-
testing weight of the sample was then calculated. The crucible was sealed and the domed lid of the crucible was pierced
with a pin to allow the release of gases during the heating process. The bovine cortical bone samples were then
subjected to thermal testing using a Mettler Toledo M3 DSC machine. The temperature inside the central furnace of the
machine was raised uniformly at a rate of 5°C per minute from 30°C to 600°C. The reference sample was an identical,
empty aluminium crucible. The sample and crucible were weighed after the heating was finished, and the weight of the
crucible subtracted from the total, giving the post-testing sample weight.
The output graphs from the Mettler DSC machine show a clear endotherm, starting at 30°C and peaking at
approximately 140°C (mean=139.47°C, s.d.=1.514). The mean temperature at onset of the melting phase was found to
be 98.16°C (s.d.= 9.65). The point at which the output graph has the steepest gradient represents the temperature at
which the greatest rate of change in heat flow between the sample and the reference occurs. The mean value for this was
found to be 123.26°C (s.d.=5.75). The mean enthalpy of the dry mineralised samples was determined by calculating the
integral of the curve representing the endotherm, as was determined to be 244.02 J/g (s.d.= 60.03) (Fig. 5).
A simple qualitative analysis of the output graphs showed
that testing the cortical bone sample in the dry, mineralised
state produced the most consistent, reproducible results.
This is intuitive as bone tested dry and in its native state is
easier to control as the process of demineralisation or
hydration/dehydration can not always be perfectly
controlled and could introduce variability. Further analysis
of the ‘ageing’ method used therefore, dry-mineralised and
dry-demineralised samples.
Fig. 5. A typical DSC output graph showing the difference
in heat flow between the sample and the reference as a
function of temperature.
2.4 Hydration level at a burial site of samples from a single individual.
This experiment used sections of bone from the mid left femur of one only 75yr old male donor to minimise inter-
individual variations. We used neutral pH soil and kept sections for 2 years at 4oC temperature in containers in native
(wrapped in gauze in Ringer’s), wet and dry soil. Multiple sections were cut to embed in resin for hardness
measurements and for ash content and density determination. Two factors were examined in analysis of variance, the
site on the bone (anterior/lateral/medial/posterior) and 3 burial conditions. There were no effects of site on the bone for
hardness and density and mineral content, regardless of the burial conditions. There were however, significant effects
on all, hardness, density and mineral content brought about by the 3 burial regimes. Hardness increased from the native
to the dry and wet soil and at the same time the organic content dropped and the relative mineral content increased.
Although the results are unambiguous and show that there was a degradation of the organic phase of bone in the soil
[17], we are planning in the future to follow this up with some XRD work to confirm what the state of crystallinity of
the mineral of the buried bone actually is. The average effects on hardness were similar to section 2.2 above (±3% ash
content; ±3-7% hardness => ±4 years in age terms).
2.5 Influence of burial conditions on the material properties of compact bone.
This experiment represents a long term study of the effect of burial condition and burial time on the material properties
of compact bone. Porcine limbs have been used as analogous to human due to: the similarities between human and pork
flesh and therefore decomposition; the consistency in pig bone material properties; and for their availability. 12 pairs of
hind legs from 12 individual farm-reared pigs were severed at the knee joint to include the tibia, fibula and tarsal bones
but were kept fully-fleshed. Each pair was divided so that one leg of each pair was designated as the control specimen
and the contra-lateral leg was designated as the experimental specimen. In the 12 control specimens the tibia and fibula
were excised; a mid-shaft section was cut using a band saw; the bone marrow was removed, and were placed in deep
freeze. Carefully labelling by corrosion-resistant stainless steel tags ensured that each control leg was compared to each
contra-lateral which was sent for burial. The 12 experimental contra-lateral legs were buried (Fig. 6) in an
Environmental Change Network site in Wytham Woods, Oxfordshire, which is a research site belonging to the
University of Oxford. Access was made possible through mediation with members of Forensic Alliance (nowadays
LGC Forensics). The site is a secure, ecologically controlled site comprising a variety of different soil types and
conditions. Three burial locations of different soil types (an acidic sand-rich soil (pH value <6); a limestone-rich
alkaline soil (pH value >7.5); a clay-rich neutral soil (pH value 6 - 7.5) were chosen by a forensic geologist curating the
site (Fig. 7). The precise organic and inorganic composition of the soil was also analysed by a forensic geologist.
Within 3 months of the initial burial the site was disturbed by scavenging badgers and as a result the experiment was
restarted with 6 months delay by placing the legs in protective large metal badger-proof cages. These samples
decomposed well and were unharmed at the times of excavation which took place at 3, 6, 9 and 12 months. Excavated
legs were pressure-washed at site to remove any adherent flesh and the bone marrow removed. The specimens were
labelled, matched to their controls and immediately frozen. The full set of physical and mechanical testing took place
from May 2006 onwards as to test all samples in one batch to avoid instrument fluctuation and calibration errors. Early

                                                    IGR ‘Age at death’ - Page 4 of 6
analysis of the results shows that there no detrimental effects of either soil condition or time since burial (death) on the
physical characteristics we measure here. This was unlike the results of previous sections (2.2-2.4) and the only
difference being the presence of the flesh, which appears to have somehow protected those areas of bone material deep
inside the bone cortex. This is good news, but we would expect that with burial time beyond 12 months diffusion of
harmful chemicals and enzymolysis would eventually start breaking down the bone matrix.

      Fig. 6. A photograph of the first set of porcine legs
      buried in the acidic pit. (left)
      Fig. 7 A map of Oxford University’s Wytham woods
      with the three burial sites in acidic, neutral and alkaline
      soil. (right)




2.6 Examine a different AUS cohort
In this we applied all available techniques in a new set of freshly retrieved samples from donors in AUS supplied to us
by our colleagues in the Forensic institute in Melbourne to corroborate the evidence collected in the British cohort. This
group of samples was of a broader age range and was very well age/sex matched.
The results (predicting age with an error of ±3yrs) were in line
with those of our pilot study if only slightly worse. However,
two of these samples were inadvertently placed in 70% ethanol,
which is the standard preservation fluid for histological slides,
before delivered to us. We are currently adjusting the parameters
values using the information we derived in section 2.2 to correct
for such effects.
Project Plan Review
There were only a few small deviations from the original project plan : (1) the mid-term meeting of the project main
partners took place in the fringes of ‘17th Meeting of the International Association of Forensic Sciences, Hong Kong,
21-26 August 2005’ as opposed to a UK location; (2) there was no testing of samples from embalmed or other tissue
collections as the act by Scottish Parliament that made these samples available for research was only passed in Autumn
2006 at the end stages of the project (this was not by any means detrimental to the project, it was a kind offer made by
Professor Sue Black initially should future legislation allowed the use of such tissue); (3) Dr Carl Edwards (CE) moved
on in Sept 2004 to the Knowledge transfer unit of the Imperial college and later on to the become Managing Director of
NHS Innovations East Midlands ( www.em-nhs-hub.org ) and as such he did not have a major input in the exploitation
of the current method as originally envisaged. His role was taken over by the PDRA employed in the project although it
is fair to admit that maintaining the contact with some partners (like the FSS) suffered in the absence of CE, however,
again at no detriment to the scientific output of the project. On the other hand, there was a whole new set of additional
activities that took place (2.1-2.5) as outlined above following suggestions in the first kick-off meeting between the
partners and a major input provided by Dr M Higginson of LGC Forensics as outlined in the supporting letter attached.
Research Impact and Benefits to Society
The project outcomes will aid forensic investigation authorities and institutes, the crown prosecution service, the
security services and more widely in cases of archaeological and anthropological/demographic investigations. We had
inquiries from around the globe on certain cases and also official requests from the Dutch and Czech police authorities
to provide training to some of their experts. Although the method is no longer protected by an international patent, our
current assessment of the international stage is that we are the only ones internationally who can provide a reliable
service by using this method because (although the method is not expert based) it does still require significant expertise
and equipment to apply it, which gives us a commanding position for exploitation world-wide.

                                                      IGR ‘Age at death’ - Page 5 of 6
One major success of this project is the professional and other developments relating to our PDRA Dr Anna Williams
(AW). She is an Oxford MA and a Sheffield PhD in forensic anthropology. Having a natural sciences background had
to be completely retrained in this interdisciplinary field where the contribution of physical sciences is only now
becoming evident. AW was subsequently employed in our dept as a Lecturer and was instrumental in the setting up of
the new MSc course in forensic anthropology archaeology and the launch of the Cranfield Forensic Institute, what is
essentially a completely new area of scientific discipline for our campus. ( www.cranfield.ac.uk/forensics )
Explanation of Expenditure
The expenditure went according to plan with only small amounts of funds diverted between subsections to cover minor
events (like damage to the nanoindenter machine) or purchasing an extra freezer needed to store samples. Some funds
for travel and subsistence remained unclaimed for Dr Carl Edwards (due to his departure from Cranfield) and Professor
Sue Black (she had to travel extensively to Iraq, Bosnia, Kosovo) and we compensated for this by using our
universities’ video-conferencing facilities. PZ also participated in the EPSRC - Crime Prevention and Detection
Technologies Dissemination Event, Forensic Science, Thistle Hotel Barbican, London, 30th March 2006.
Further Research and Dissemination Activities
The project activities appeared in our Cranfield staff magazine, our research and business development web pages and
in those of various international and governmental organisations (72 Google hits), which are updated regularly.
(PZ) http://www.cranfield.ac.uk/forensics/projects/projects.html
(PZ) http://www.dcmt.cranfield.ac.uk/dmas/cmse/materials/biomechanics/research/forensic_bone
(PZ) http://www.businessgateway.cranfield.ac.uk/index.cfm?page=newsdetails&id=844
(JC) http://www.dent.unimelb.edu.au/dsweb/about_school/staff_profiles/clement_j.html

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                                                          IGR ‘Age at death’ - Page 6 of 6

				
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Description: A Laboratory-based Analytical Method to Determine Age at Death