WO2004070345A2

WO2004070345A2 - Age determination on death

Info

Publication number: WO2004070345A2
Application number: PCT/GB2004/000125
Authority: WO
Inventors: Peter Zioupos
Original assignee: Cranfield University
Current assignee: Cranfield University
Priority date: 2003-02-07
Filing date: 2004-01-15
Publication date: 2004-08-19
Anticipated expiration: 2005-08-07
Also published as: WO2004070345A3; GB0302825D0

Abstract

The invention relates to a method and means for determining the 'age at death' of an individual using only a sample of bone from said individual. The method involves determining a plurality of properties of the said bone and then using these properties to determine 'age at death'.

Description

AGE DETERMINATION ON DEATH

The invention relates to a method and means for determining the age of an individual at death.

One of the most essential aspects in any identification process where there is extensive damage to a corpse or where human remains are rendered unrecognisable by advanced decomposition is the "age at death". This is one of four important attributes that a forensic anthropologist or pathologist has to determine. The other three are sex, stature and ethnic background. An accurate estimation of "age at death" is more important in forensic anthropology than in archaeological cases because the required accuracy is higher and there may be possible legal consequences of correct versus incorrect determinations.

For the avoidance of doubt the cause of death may be due to natural circumstances, homicide, suicide, mass disaster or even war crimes including massacres caused by religious or ethnic cleansing.

Identification of the age of an individual at death becomes increasingly difficult after the individual has reached skeletal maturity. This is because aging is orchestrated by the complicated interplay of genetic, environmental and cultural factors, meaning that not everyone ages the same.

However, until skeletal maturity is reached, at the age of 30 to 35, if presented with a complete corpse, one can use a number of methods to determine the "age at death". For example, a relatively precise biological assessment, i.e. plus or minus 3 years, can be obtained by examining the chronology of dental eruption in respect of deciduous or permanent teeth. Additionally, degenerative changes occurring in teeth can also give an indication of age. These degenerative changes include tooth wearing and tooth loss. Additionally or alternatively, the fusion of the epiphyses is a further anatomical means for determining the "age at death".

At the time of birth a human has about 405 bones, over twice of that of the human adult who has 206 bones. The reduction during ontogenetic development occurs as a result of multiple bones fusing to produce a single bone by a process known as epiphyseal union. During ontogenetic development the fusion of bone epiphyses to metaphyses occurs at regular intervals and is therefore a useful indicator when quantifying the age of a skeletally immature individual at death. Several bones can be examined, for example the iliac crest or the ileum bone of the os coax, the medial epiphyses of the clavicle, the tibia and the femur. Advantageously, there are some gender variations with female epiphyses fusion occurring earlier than males. Moreover, in the aging assessment the fusion of a particular bone is usually classified into stages: nonunion, one-quarter united, one-half united, three-fourths united, and fully fused or full union. These are all useful indicators in determining the age of death of an individual with an immature skeleton. A further approach for determining "age at death" involves histomorphological features of the bone matrix based on the size of the mid-femur, the level of porosity and the relative number of primary and secondary osteonal systems. But, again, these methods are increasingly inaccurate after maturity.

The only reported analytical technique available for determining the "age at death" of a mature individual is based on the determination and exploitation of changes in the L and D forms of aspartic acid. The L converts to the D form in the non-collagenous organic matrix of bone and other tissues during the life of an individual (Nature: 1976, 262: 279 - 281). This technique has been shown to provide estimates with an accuracy of + 10 years. Whilst it is a step in the right direction the margin of error can render it ineffective. Moreover, it requires elusion methods, HPLC methods and is affected by many preparatory factors like pH, size of bone crushed particles, washing of bone tissue, the amount of bone tissue, the concentration and amount of the used acidic solutions, and the manner of agitation.

It can therefore be seen that there is a need to provide a new method and means for determining the age of an individual at death and in particular the age of a mature individual, or adult, at death. Further, this method needs to be sufficiently flexible that it can be used in instances where the corpse has been subjected to substantial damage and there may be only a small fragment of bone tissue to work with. Bone is living, growing, specialised tissue. It can be regarded as highly heterogeneous in the sense that its structure and composition varies according to skeletal size, physiological function, age and sex of the individual. Nevertheless, the basic components of bone tissue remain consistent with age the difference being in the relative properties of the tissue.

Bone is made up of two components, firstly an Organic Matrix that comprises type I collagen fibrils (90% of the whole matrix) and non-collagenous components such as proteins, proteoglycans, phospholipids, glycoproteins and phosphoproteins (the remaining 10% of the matrix). The second component is known as the mineral substance, calcium phosphate hydroxyapatite. The collagen fibrils are the result of the assemblage of filamentous molecules. Bundles of microfibrils then form larger fibrils that, in turn, form larger fibres. Thus there is an orderly packing of collagen within bone structure. The non- collagenous component seems to play an important role in the degree of calcification of the bone.

In Figure 1 there is shown a cross section of bone under an electron microscope. The structure, essentially, comprises area of primary bone (I) and areas of secondary osteons (O). The primary bone (I) represents older tissue

^• and has a greater degree of calcification, so that it appears lighter. In contrast, the newer bone, which is less calcified, appears darker. In the experiments described herein we have used long bones, i.e. long tubular segments of bone such as those found in the limbs, ribs and clavicles, and in particular bone from the femur.

Other sorts of bones include short bones, i.e. those short irregular prismatic structures such as those found in the vertebrae, sternum, carpus and tarsus or flat bones, i.e. the bilaminar plates found in the skull, scapula and pelvis.

The method of the invention can be applied to any of these bone types in any species.

In the long bones osteons are orientated parallel to the axis of the bone, with an inclination of about 5 to 15°. They comprise harvesian canals which generally contain blood vessels, nerve fibres and a variety of other cell types.

Primary osteons are formed early in life and their number diminishes with increasing age. Secondary osteons form throughout life, increasing more with aging, and in bone physiology play a crucial role because they repair in-vivo cracks and allow for internal remodelling of the bone structure. Primary osteons tend to be on average well mineralised. Secondary osteons start with low mineral content and gradually mineralise more and more but not to the level of primary osteons because they are produced much later in life.

Other types of bone include Woven bone which can be formed rapidly in a disorganised manner, like in the callous formation when bone breaks. Its collagen fibres and mineral crystallites are randomly orientated in space. Woven bone has an intermediate level of mineralisation, but with more and more time in the body it mineralises considerably.

Another type of bone is Lamellar bone which is formed in a circumferential manner around the cortex of bones earlier in life. It is densely mineralised and it becomes more so with time in the body.

With increasing age the bone matrix experiences changes which are constitutional, compositional, micromechanical and/or physiochemical in nature. These changes affect the material properties of bone and we have investigated them with a view to determining whether they have any predictive power with a view to using them to determine the age of an individual at death. Our experiments have led us to conclude that these changes can be quantified and, when correctly processed, used to predict, with remarkable accuracy, i.e. + one year, the age of an individual from which the bone originated.

In the methodology to be described herein we have used long bones from males and females who are beyond the level of skeletal maturity. However, it can equally be successfully applied to individuals younger than this and for bones of the body other than the femur. STATEMENTS OF INVENTION

According to a first aspect of the invention there is provided a method for determining the "age at death" of an individual which method comprises obtaining a sample of bone from said individual and determining a plurality of properties thereof, then using these properties to determine said "age at death".

In a preferred embodiment of the invention said properties comprise at least two of the following properties: relative content of calcium to phosphorous; hardness; density; porosity; weight fraction measurements or degradation parameters (from Differential Scanning Calorimetry).

In yet a further preferred embodiment the invention involves the measurement of at least one or more of the following parameters: Apparent density; Real density;

Porosity; Mineral Fraction; Water Fraction or Organic Fraction wherein:

Apparent density = Ap.D. = wet weight in air / volume.

Real density = R.D. = wet weight in air / (wet weight in air - wet weight in water). Porosity = 1- (Apparent density / Real density).

Mineral Fraction = (dry mineralised weight - dry demineralised weight) / wet weight in air.

Water Fraction = (wet weight in air - dry mineralised weight in air) / wet weight in air. Organic fraction = dry demineralised weight / wet weight in air.

According to a further aspect of the invention there is therefore provided a method for determining the "age at death" of an individual comprising taking a sample of bone from a corpse of said individual and subjecting said bone to laboratory analysis to determine any one or more of the following parameters:

a) the relative content of calcium to phosphorous; b) hardness; c) Organic Fraction, i.e. dry demineralised weight / wet weight in air; d) Water fraction, i.e. (wet weight in air - dry mineralised weight in air) / wet weight in air; e) Mineral Fraction, i.e. (dry mineralised weight - dry demineralised weight) / wet weight in air; or f) collagen degradation or a DSC value

and then feeding this information into any one or more of the following equations.

1 ) Age = 55.6 + 161 (Organic Fraction) - 0.253 (Delta H Demin.)

2) Age = -81.9 + 2.99 ( Ogf in Osteon) - 0.164 (Delta H Demin.)

3) Age = -86.0 + 2.63 (10gfin Matrix) - 0.134 (Delta H Demin.) 4) Age = - 88.4 + 1.52 (10gf in Osteon) + 1.36(10gf in Matrix) - 0.146(Delta H Demin.)

5) Age = - 23.6 + 2.36(1 Ogf in Osteon) - 27.8(Ca/P% in Matrix) +

14.1 (Organic Fraction. )

6) Age = 564 - 733(Organic Fraction) - 723(Mineral Fraction) -0.066(1 Ogf in Osteon) + 2.90(1 Ogf in Matrix)

7) Age = 105 - 9.3 (1 Ogf in Osteon) + 8.26 (lOgf in Matrix) -0.414 (Delta H Demin.)

8) Age = -192 + 150 (Ap.D./R.D.) + 2.92 (lOgf in Osteon) -0.281 (Delta H Demin.)

9) Age = -264 - 2481 (Water Fraction) + 6.Q(10gf in Osteon) - 6.76(10gf in Matrix)

In a preferred embodiment of the invention a) the relative content of calcium to phosphorous and/or b) hardness is determined for osteons and/or interstitial (Matrix) tissue. Most preferably equations 5 + 6 are used to estimate the "age at death" of a sample believed, or suspected, to be of male origin and equations 7, 8 or 9 are used to estimate the "age at death" of a sample believed, or suspected, to be of female origin.

In yet a further preferred embodiment of the invention said methodology employs the use of a standard wherein bone from an individual whose age is known is analysed in parallel with a sample of bone whose "age at death" is to be determined.

More preferably a plurality of standards are used, and most ideally bone from at least one relatively young individual is used and bone from at least one relatively old individual is used. Advantageously, bone from an individual of 30 years of age is used as one standard and bone from an individual of 70 years of age is used as a second standard.

The use of standards is particularly preferred where samples from a number of different species are analysed and most preferably the standards used are selected (in terms of their ages and species) on the basis of the species whose bone is thought to be under investigation.

In yet a further aspect of the invention there is provided means for performing the above methodology wherein said means comprises a resin block in which there is embedded a sample of bone whose age is to be determined and at least one other standard as herein described.

By using the aforementioned parameters, and in particular at least two thereof, it is possible to predict the "age at death" with some accuracy as indicated below where R² + p are the levels of significance between predicted and actual age values based on a validation test sample of 12 individuals: 7 male, 5 female.

1 ) Age = 55.6 + 161 (Organic Fraction) - 0.253 (Delta H Demin.)

(p=0.043) (p=0.025) (p=0.070) R² = 55.6% Readjusted) = 45.7% p = 0.026

2) Age = -81.9 + 2.99 (1 Ogf in Osteon) - 0.164 (Delta H Demin.)

(p=0.001) (p=0.000) (p=0.004)

R²=94.8% R²(adjusted)=93.7% p=0.000

3) Age = -86.0 + 2.63 (10gfin Matrix) - 0.134 (Delta H Demin.) (p=0.001) (p=0.000) (p=0.0012)

R²=94.9% R²(adjusted)=93.8% p=0.000

4) Age = -88.4+1.52 (10gf in Osteon)+ 1.36(1 Ogf in Matrix)-0.146(Delta H

Demin)

(p=0.000) (p=0.072) (p=0.068) (p=0.004)

R²=96.7% R²(adjusted)=95.4% p=0.000 5) Age = -23.6+2.36(1 Ogf in Osteon)-27.8(Ca/P% in Matrix)+ 4Λ (0rganic Fraction.)

(p=0.802) (p=0.065) (p=0.465) (p=0.813)

R²= 90.5% R²(adjusted)=80.9% p=0.049

6) Age = 564-733 (Organic Fraction)-723(Mineral Fraction)-0.066(1 Ogf in Osteon)+2.90(10gfin Matrix)

(p=0.401 ) (p=0.353) (p=0.346) (p=0.949) (p=0.062)

R²=98.6% R²(adjusted)=95.8% p=0.028

7) Age = 105 - 9.3(10gf in Osteon)+8.26(1 Ogf in Matήx)-0A 4(Delta H Demin.)

(p=0.066) (p=0.140) (p=0.185) (p=0.329)

R²=99.5 R²(adjusted)=98.2 p=0.086

8) Age = -192 + 150 (Ap.D./R.D.)+2.92(10gf in Osteon)-0.281 (Delta H Demin.)

(p=0.887) (p=0.826) (p=0.900) (p=878)

R²=94.8 R²(Adjusted)=79.3% p=0.287

9) Age = -264-2481 (Water Fraction) + 6.8(1 Ogf in Osteon)-6.76(1 Ogf in Matrix)

(p=0.016) (p=0.070) (0.027) (0.038)

R²=100.0 R²(adjusted)=99.9 p=0.019 MATERIALS AND METHODS

All the experiments achieved during this project were performed from bone received from the North London Tissue Bank. The bone received was from 7 males and 7 females of various ages, who had died from causes that did not affect the experimental requirements of the bones, and had not been hospitalised for any length of time. The bones were transported in plastic bags packed with ice; subsequently they were thoroughly washed with running water and finally were stored in plastic bags at -20°C. During sample preparation and according to the requirements of several of the experiments performed, a number of samples were either stored in airtight containers at -20°C with saline solution, or in class container at +39°C, in a GALLENKAMP Hot Box Oven Size 1.

The bone tissue used was freely donated.

DENSITY AND POROSITY AND WEIGHT FRACTION MEASUREMENTS Wet Weight in Water (WWW) Initially the diameter and height of all the samples was measured (in mm) by the means of a Digital Caliper MITUTOYO ABSOLUTE DIGIMATIC, and therefore their volume (in mm³) was calculated by multiplying the two together. During the measuring processes, several measurement of the height and diameter were taken from each sample in order to establish the lower and higher values which was finally averaged. The digital calliper measured up to two decimal places and the error values for this instrument was determined to be approximately 0.01mm. Nevertheless, the measurements were taken up to three decimal places, due to the calculation processes.

Following the measurements the samples were centrifuged immersed in deionised water for 3 min at lOOOrpm in a MISTRAL 1000 unit. Centrifugation was performed in order to ascertain the water uniformity in the pores of the samples. Subsequently the wet weight in water (in grams) of the samples was measured under water by using a metal tray provided for measurement of density of solid materials by the Archimedes principle. All measurements were performed by the means of a high precision METLER TOLEDO College B154 balance at room temperature. The precision balance measurements were displayed with four decimal places, and with an error of approximately 0.0001 to 0.0002 g.

Wet Weight in Air (WWA)

After that the samples were centrifuged again for 3 min at lOOOrpm, but this time wrapped in a wet cloth in order to remove excess amounts of water from the major pores found in the bone matrix of the samples. Thereafter the samples were again wet weighted, but this time in air, and not immersed in water, at room temperature, therefore producing the wet weight in air (in grams). Dry Mineralised Weight (DMW)

The dry mineralised weight (in grams) of the samples were also measured in air, at ambient room temperature and humidity, after the samples have been left to dry for 5 days and 7 days, at +39°C in the hotbox oven. Great care was undertaken to eliminate the factor of humidity from the measuring process.

Dry Demineralised Weight (DDW)

Subsequently some samples were selected and demineralised with the use of Ethylenediaminetetraacetic acid 0.5M, pH=7.4 (EDTA), by continuously stirring the samples for a period of 13 days and by changing the solution 7 times. After the demineralisation process was finished, the samples were thoroughly washed to remove EDTA, and were dried out, for a period of 5 days. Following drying, the samples were weighed (in grams), at ambient temperature and humidity, so determining the dry demineralised weight, which basically is the weight of the organic material of the bone matrix.

Calculations

The following calculation processes were used in order to determine, Apparent Density (ApD), Real Density (RD), Porosity (P), Mineral Fraction (MF), Water Fraction (WF), and finally Organic Fraction (OF). Due to the fact that weight measurements were measured up to four decimal places, in addition to the volume measurements, which were obtained up to three decimal places, in all the calculation precision used was up to four decimal places. (2) Apparent Density = Ap.D. = WWA / Volume

(3) Real Density = R.D. = WWA / (WWA - WWW)

(4) Porosity = P = 1 - (ApD / RD)

(5) Mineral Fraction = MF = (DMW - DDW) / WWA (6) Water fraction = WF = (WWA - DMW in air) / WWA

(7) Organic Fraction = OF = DDW / WWA

Microhardness Measurements

Hardness of a solid material is defined as the resistance it exhibits towards penetration by another solid. Microhardness testing is accomplished by indenting the material under investigation with a specially designed microscope mounted with a very small indenter. In this case, the type of the indenter used was a Vickers™. This is a pyramidal diamond indenter with an apical angle of 136°. Although the microhardness values obtained were automatically calculated by the testing device, INDENTEC HWDM-7, Vickers microhardness number (HV) is given by the following equation:

HV = 2P sin (θ/2) / d² Where: P = applied load in Kg d = mean length of di and d₂ in mm, the two diagonal of the indentation

(see Figure 3.4) θ = 136°

For the purposes of this experiment, the samples under investigation were embedded in resin METPREP Polyester Castins Resin KLEER-SET TYPE SSS, in a BUEHLER VACUUM IMPREGNATION EQUIPMENT I, and left to dry. Following drying the samples were metallurgically polished to mirror finish, in a METESERV rotary pregrinder, by the use of 800 and 1200 Grinding paper and finally with a polish cloth, MASTERTEX, with adhesive backing under

MICROPOLISH ALUM 3B 6OZ. Great care was undertaken to assure a good quality polished area. Indentation values were obtained for secondary osteons, and interstitial lamellae from five locations in each sample, in the following fashion, north, south, east, west and from the centre. The indentation loads used in each location were 10gf, 50gf and 100gf. The experimentation processes are illustrated in Figure 2. However, it is to be noted that, as per the formulae above, it is sufficient to measure hardness using only one load in either Osteon or Matrix tissue.

But in these experiments, in total, 30 hardness values were obtained from each sample, and in general, 360 indentations were performed. Different weights were used for selection purposes due to the fact that different weights produce different indentation diagonals, and therefore the easiness of measuring under the microscope varies according to weight. The error observed during these processes was found to be approximately + 1-2 Vickers hardness units. During experimentation, great care was undertaken to avoid confounding factors such as, levelling the sample thus allowing the indenter to penetrate in right angles, keeping the loading mechanism free of any vibrations, and discarding asymmetric or problematic readings. Differential Scanning Calorimetry Measurements

Differential Scanning Calorimetry (DSC) subjects a material and a reference to the same heat treatment, with the purpose of measuring the amount of energy necessary for the material to maintain the same temperature as that of the reference. In this project, DSC was used to determine the thermodynamic parameters of the denaturation of bone collagen, in the mineralised and demineralised state.

The samples under investigation were initially thinned down manually by the use of grinding paper, under continuous irrigation, to approximately 1 to 2mm in height, and left to dry for 5 days. Following drying, the mineralised samples under investigation, were, firstly weighted in a METLER AJ50 precision balance, secondly embedded in PYRIS 1 aluminium crucibles, and finally their

Temperature/Heat Flow spectra were obtained by a PERKIN ELMER PYRIS 1 , DISC unit. An identical methodology was used to examine the previously demineralised samples, although the aluminium crucibles used were from the METLER HANDLING SET ME-119091 , and the DSC unit used was METLER M3 DISC UNIT. In both the mineralised and demineralised samples, the values of Peak temperature of collagen degradation and the total energy needed were measure. The samples were heated from 30°C up to 300°C at a heating rate of 5.00°C per min. The extrapolation of the data was performed automatically from the software used by both DSC units. An explanation of the extrapolation process is shown below in Figure 4.

It is to be noted that, as per the formulae above, it is sufficient to measure the DSC value of only the demineralised samples.

ENERGY DISPERSIVE ANALYSIS MEASUREMENTS

Mineral characterisation of the samples, their Calcium to Phosphorous ratio, was accomplished by Energy Dispersive Analysis through X-rays (EDAX). EDAX is specialised detector mounted upon a Scanning Electron Microscope (SEM) unit.

The previously embedded in resin samples (used in Vickers Microhardness) were carbon coated, in a FISONS INSTRUMENTS CARBON COATER, and were analysed with a PRINCENTON GAMMA TECH PGT IMIX MICROANALYSER EDAX detector. The detector was mounted on a JEOL JSM-840A SEM unit. In general, four calcium to phosphorus, ratio values were obtained from each sample. Two values were obtained from secondary osteons, and two from interstitial matrix lamellae. The variables obtained were the percentage values of the normal weights of calcium and phosphorus. The ratio values were calculated by dividing the calcium values with those of the phosphorus. The accelerating voltage used was 10.00keV, with a takeoff angle of 40°, for the period of 200 seconds, in each location measured. The ratio values were calculated automatically from the spectra produced, by the PGT software used by the detector. Again, it is to be noted that, as per the formula above, it is sufficient to measure the Calcium to Phosphorous ratio for Matrix tissue.

CONCLUSIONS

Age Determination for Mixed Samples

The above analysis was carried out in respect of each sample of bone tissue and as a result a vast amount of data was collected. We then sifted through this data and discovered that when age was correlated with organic fraction and DSC values, i.e. Delta H of the demineralised samples, it produced a regression formula with an R² value of 55.6% and p = 0.026.

Formula 1

Age = 55.6 + 161 (Organic Fraction) - 0.253(Delta H Demin.) (p=0.043) (p=0.025) (p=0.070) R² = 55.6% Readjusted) = 45.7% p = 0.026

Following the same approach when age was examined in correlation with the Vickers Microhardness results for the weight of 10gf in the osteons and the Delta H demineralised samples a highly significant Formula 2 was produced in relation to age with an R² value of 94.8% and p = 0.000.

Formula 2

Age = -81.9 + 2.99 (10gfin osteons) - 0.164 (Delta H demin.) (p = 0.001) (p=0.000) (p=0.004) R² = 94.8% Readjusted) = 93.7% p=0.000

In Formula 3 age was correlated with the Vickers microhardness results for the 10gf weight in the matrix, and the Delta H of the demineralised samples producing a slightly higher significant regression formula than Formula 2 with R² values of 94.9%, p=0.000 and residuals ranging from approximately -4 to +4 years. Again, all of the coefficients were found to be highly significant.

Formula 3

Age = -86.0 + 2.63 (10gfin Matrix) - 0.134 (Delta H demin.) (p=0.001) (p=0.000) (p=0.0012)

R² = 94.9% Readjusted) = 93.8% p=0.000

The most successful relationship of all was when age was correlated with, the Vickers microhardness results for weights of 10gf in the osteons and in the matrix, in addition to Delta H demineralisation. The statistical results of this regression Formula 4 showed a surprisingly R² value of 96.7% and p=0.000/ Nevertheless, the level of significance of the coefficients was not highly significant in the cases of the Vickers microhardness results rather than formulas, but the table of residuals showed a fluctuation between approximately -3 to +3 years.

Formula 4

Age = -88.4 + 1.52 (10gf in Osteon) +1.36(1 Ogf in Matrix) -0.146(Delta H Demin) (p=0.000) (p=0.072) (p=0.068) (p=0.004)

R²=96.7% R²(adjusted)=95.4% p=0.000

The actual age of the individual at the time of death against the predicted values of age at the time of death can be determined by using Formula 4. Formula 4 suggests that the combined knowledge and usage of parameters, such as hardness of bone measured in the osteons and in the matrix and the Delta H energy of the demineralised samples of bones can make possible accurate predictions of age in mature adult bone.

Age Determination for the Male Samples

A similar approach as that followed for the mixed sample group was followed for the male and female samples as well. With the unknown as age, several combinations of several parameters were tested in order to establish the most successful formula for age determination at death, at least knowing the sex of individual.

Initially for males, a combination of Vickers Micro hardness results with weight 10gf in the osteons, with calcium and phosphorous ratio in the matrix, and the organic fraction, produced Formula 5 with R² values of 90.5% and p=0.049.

Nevertheless, the coefficient level of significant was found not to be significant, and residuals ranged from approximately -5 to +4 years. Formula 5

Age = - 23.6 + 2.36(1 Ogf in Osteon) - 27.8(Ca/P% in Matrix) + UA (Organic Fraction.)

(p=0.802) (p=0.065) (p=0.465) (p=8.13) R²=90.5% R²(adjusted)=80.9% p=0.049

In a second attempt to determine age at death for males, a combination of Organic fraction, mineral fraction, and Vickers Micro hardness results with 10gf weight in the osteons and in the matrix, produced the most successful formual 6 with R² values of 98.6 and p=0.028. On the other hand, the coefficients level of significant was found not to be significant, as on Formula 5. Interestingly residuals ranged approximately between -1 to +2 years. The data for this formula is shown in Figure 5.

Formula 6

Age = 564 - 733(Organic Fraction) - 723(Mineral Fraction) - 0.066(1 Ogf in Osteon) + 2.90 (10gfin Matrix)

(p=0.401) (p=0.353) (p=0.346) (p=0.949) (p=0.062)

R²=98.6% R²(adjusted)=95.8% p=0.028

Age Determination for the Female Samples

In female samples, the Vickers micro hardness measurements with 10gf weights in the osteons and in the Matrix, were combined with Delta H measurements in the demineralised samples. This Formula 7 was proven to be more successful than the previous one with an R²=of 99.5, but still the significance level of the formula but as well of the coefficient was still less successful. The residuals ranged approximately between -0.305 - 0.26 years.

Formula 7

Age = 105 - 9.3 (10gf in Osteon) + 8.26 (10gf in Matrix) - 0.414 (Delta H Demin.)

(p=0.066) (p=0.140) (p=0.185) (p=0.329)

R²=99.5 R²(adjusted)=98.2 p=0.086 Also in the female samples, a combination of Apparent density over Real density, with the results from Delta H in the demineralised samples, and Vickers micro hardness measurements with 10gf weights in the osteons, produced a formula 8 with R² values of 94.8% but the significance level of the formula as well for the coefficients was less successful. The residuals ranged from -1.2 to 0.02 years.

Formula 8

Age = -192 + 150 (Ap.D./R.D.) + 2.92 (10gfin Osteon) -0.281 (Delta H Demin.) (p=0.887) (p=0.826) (p=0.900) (p=878) R²=94.8 R²(Adjusted)=79.3% p=0.287

Astonishingly enough Formula 9 proved to be the most successful of all in female samples with an R² value of 100.0% and p=0.019. In this formula, the water fraction was combined with the Vickers micro hardness measurements with 10gf weights in the osteons and in the Matrix. In addition all the significant levels of the coefficient were under acceptable limits (p<0.05) except that of the Water Fraction (p=0.070), and the residuals showed not significant deviation from the actual ages of the females examined. The data for this formula is shown in Figure 5.

Formula 9

Age = -264 - 2481( Water Fraction) + 6.8(10gfin Osteon) - 6.76(10gfin Matrix) (p=0.016) (p=0.070) (0.027) (0.038) R²=100.0 R²(adjusted)=99.9 p=0.019

Claims

1. A method for determining the "age at death" of an individual which method comprises obtaining a sample of bone from said individual and determining a plurality of properties thereof, then using these properties to determine said "age at death".

2. A method according to Claim 1 wherein said properties comprise at least two of the following properties: relative content of calcium to phosphorous; hardness; density; porosity; at least one Weight Fraction measurement; or collagen degradation parameters.

3. A method according to Claim 1 or Claim 2 comprising a determination of any one or more of the following properties: Apparent density; Real density; Porosity; Mineral Fraction; Water Fraction or Organic Fraction.

4. A method according to Claim 3 wherein said Apparent density = wet weight in air / volume.

5. A method according to Claim 3 wherein Real density = wet weight in air / (wet weight in air - wet weight in water).

6. A method according to Claim 3 wherein Porosity = 1 -(Apparent density / Real density).

7. A method according to Claim 3 wherein Mineral Fraction = (dry mineralised weight - dry demineralised weight) / wet weight in air.

8. A method according to Claim 3 wherein Water Fraction = (wet weight in air - dry mineralised weight in air) / wet weight in air.

9. A method according to Claim 3 wherein Organic Fraction = dry demineralised weight / wet weight in air.

10. A method for determining the "age at death" of an individual comprising taking a sample of bone from a corpse of said individual and subjecting said bone to laboratory analysis to determine any one or more of the following parameters:

(a) the relative content of calcium to phosphorous;

(b) hardness;

(c) Organic Fraction, i.e. dry demineralised weight / wet weight in air;

(d) Water Fraction, i.e. (wet weight in air - dry mineralised weight in air) / wet weight in air;

(e) Mineral Fraction, i.e. (dry mineralised weight - dry demineralised weight) / wet weight in air; or

(f) collagen degradation or a DSC value; and then feeding this information into one or more of the following equations: (1 ) Age = 55.6 + 161 (Organic Fraction) - 0.253 (Delta H Demin.)

(2) Age = -81.9 + 2.99 (lOgfin Osteon) - 0.164 (Delta H Demin.)

(3) Age = -86.0 + 2.63 (10gfin Matrix) - 0.134 (Delta H Demin.)

(4) Age = -88.4 + 1.52 (10gf in Osteon) + 1.36(1 Ogf in Matrix) - 0.146(Delta H Demin.) (5) Age = -23.6 + 2.36(1 Ogf in Osteon) - 27.8(Ca/P% in Matrix) +

14.1 (Organic Fraction. ) (6) Age = 564 - 733(Organ/c Fraction) - 723{Mineral Fraction) - 0.066(70gf in Osteon) + 2.90(1 Ogf in Matrix) (7) Age = 105 - 9.3 (10gf in Osteon) + 8.26 (10gf in Matrix) - 0.414 (Delta H Demin.)

(8) Age = - 192 + 150 (Ap.D./R.D.) + 2.92(1 Ogf in Osteon) - 0.281 (Delta H Demin.) (9) Age = -264 - 2481 (Water Fraction) + 6.8(1 Ogf in Osteon) - 6.76(1 Ogf in

Matrix).

11. A method according to Claim 10 wherein:

(a) the relative content of calcium to phosphorous and/or

(b) hardness is determined for Osteons and/or interstitial (Matrix) tissue.

12. A method according to Claim 10 wherein equations (5) and (6) are used to estimate the "age at death" of a sample believed, or suspected, to be of male origin.

13. A method according to Claim 10 wherein equations (7), (8) or (9) are used to estimate the "age at death" of a sample believed, or suspected, to be of female origin.

14. A method according to any preceding Claim wherein said methodology employs the use of a standard wherein bone from an individual whose age is known is analysed in parallel with a sample of bone whose "age at death" is to be determined.

15. A method according to Claim 14 wherein a plurality of standards are used.

16. A method according to Claim 15 wherein said standards comprise bone from at least one relatively young individual and bone from at least one relatively old individual.

17. A method according to Claim 16 wherein bone from an individual of 30 years of age + 5 years is used as one standard and bone from an individual of 70 years of age + 5 years is used as a second standard.

18. Means for performing the methodology according to any preceding Claim comprising a resin block containing a sample of bone whose age is to be determined and at least one standard according to Claims 14 to 17.