SCIJUS-00489; No of Pages 8

Science and Justice xxx (2014) xxxxxx

Contents lists available at ScienceDirect

Science and Justice

journal homepage: www.elsevier.com/locate/scijus

Review

Chemical Differentiation of Osseous, Dental, and Non-skeletal Materials

in Forensic Anthropology using Elemental Analysis
Heather A. Zimmerman a, Cayli J. Meizel-Lambert a,b, John J. Schultz a,b,, Michael E. Sigman b,c
a
b
c

Department of Anthropology, University of Central Florida, Orlando, FL 32816, United States

National Center for Forensic Science, University of Central Florida, Orlando, FL 32816, United States
Department of Chemistry, University of Central Florida, Orlando, FL 32816, United States

a r t i c l e

i n f o

Article history:
Received 4 June 2014
Received in revised form 9 October 2014
Accepted 7 November 2014
Available online xxxx
Keywords:
Forensic anthropology
Chemical differentiation
Elemental analysis
Osseous and non-osseous materials

a b s t r a c t
Forensic anthropologists are generally able to identify skeletal materials (bone and tooth) using gross anatomical
features; however, highly fragmented or taphonomically altered materials may be problematic to identify.
Several chemical analysis techniques have been shown to be reliable laboratory methods that can be used to
determine if questionable fragments are osseous, dental, or non-skeletal in nature. The purpose of this review
is to provide a detailed background of chemical analysis techniques focusing on elemental compositions that
have been assessed for use in differentiating osseous, dental, and non-skeletal materials. More recently, chemical
analysis studies have also focused on using the elemental composition of osseous/dental materials to evaluate
species and provide individual discrimination, but have generally been successful only in small, closed groups,
limiting their use forensically. Despite signicant advances incorporating a variety of instruments, including
handheld devices, further research is necessary to address issues in standardization, error rates, and sample
size/diversity.
2014 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

Contents
1.
2.
3.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . .
Anatomy, Chemical Composition, and Variability of Bones and Teeth
Chemical Analysis of Bone in Forensic Anthropology . . . . . . . .
3.1.
Bone versus Non-Bone . . . . . . . . . . . . . . . . . .
3.2.
Human versus Nonhuman and Species Differentiation . . .
3.3.
Individual Identication . . . . . . . . . . . . . . . . .
3.4.
Commercial Cremation Analysis . . . . . . . . . . . . .
4.
Discussion . . . . . . . . . . . . . . . . . . . . . . . . . .
5.
Future Directions and Conclusions . . . . . . . . . . . . . . . .
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . .
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction
While identication of osseous and dental materials by the forensic
anthropologist is generally established on gross anatomical features, it
may be problematic to identify highly fragmented or taphonomically altered materials as osseous or dental in origin and/or to distinguish between human and non-human. Multiple chemical analysis techniques
focusing on the elemental composition of materials have been shown
Corresponding author at: Department of Anthropology, University of Central Florida
Orlando, FL 32816-1361.
E-mail address: john.schultz@ucf.edu (J.J. Schultz).

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to be reliable laboratory methods that can be used to determine if questionable fragments are osseous, dental, or non-skeletal in nature [13].
Once the forensic anthropologist determines that questionable material
is osseous or dental in nature, the next step in the analysis is to determine if osseous/dental material is human or non-human. Traditionally,
methods such as bone histology [412], protein analysis [13,14], and
DNA [15,16] may be used to determine if questionable materials are
human or non-human. Further, in the case of DNA, this method is routinely used for individual determination of commingled remains when
sorting through fragmentary material. More recently, studies suggest
that elemental analysis combined with multivariate statistical analysis
[3,1719] may be useful in the discrimination, or the initial sorting

http://dx.doi.org/10.1016/j.scijus.2014.11.003
1355-0306/ 2014 Forensic Science Society. Published by Elsevier Ireland Ltd. All rights reserved.

Please cite this article as: H.A. Zimmerman, et al., Chemical Differentiation of Osseous, Dental, and Non-skeletal Materials in Forensic
Anthropology using Elemental Analysis, Sci. Justice (2014), http://dx.doi.org/10.1016/j.scijus.2014.11.003

H.A. Zimmerman et al. / Science and Justice xxx (2014) xxxxxx

prior to DNA analysis, of multiple individuals when presented with

highly fragmentary and taphonomically modied material.
The purpose of this review is to provide a detailed background of the
chemical analysis techniques introduced in forensic anthropology to
differentiate materials as osseous, dental, or non-skeletal using the elemental composition of these materials. In addition, there are numerous
studies evaluating species and individual discrimination focusing on the
elemental composition of the samples. These studies use a variety of
chemical methods combined with multivariate statistical analyses,
and, as such, will be discussed separately. However, before discussing
how these methods are used to distinguish osseous and dental tissues
from non-skeletal materials it is rst important to provide an overview
of the anatomy, chemical composition, and variability of bones and
teeth.

Table 2
Common Trace Elements found in Bone Tissue and Tooth Enamel.
Bone Tissue and Tooth Enamel

Bone Tissue

Tooth Enamel

Calcium (Ca)
Chlorine (Cl)
Chromium (Cr)
Cobalt (Co)
Copper (Cu)
Iodine (I)
Iron (Fe)
Manganese (Mn)
Molybdenum (Mo)
Nickel (Ni)
Phosphorus (P)
Potassium (K)
Tin (Sn)
Vanadium (V)
Zinc (Zn)

Carbon (C)
Fluorine (F)
Hydrogen (H)
Lead (Pb)
Magnesium (Mg)
Nitrogen (N)
Oxygen (O)
Selenium (Se)
Silicon (Si)
Sodium (Na)
Sulfur (S)

Arsenic (AS)
Barium (Ba)
Cadmium (Cd)
Cerium (Ce)
Lanthanum (La)
Neodymium (Nd)
Niobium (Nb)
Rubidium (Rb)
Strontium (Sr)
Titanium (Ti)

2. Anatomy, Chemical Composition, and Variability of Bones

and Teeth

(Compiled using [21], [22])

Dental and osseous tissues consist of both organic and inorganic

components (Table 1). The organic component of bone consists of the
bone cells (osteoblasts, osteoclasts, and osteocytes) and the osteoid,
the organic portion of the bone matrix. The osteoid consists of collagen,
proteoglycans, and glycoproteins, all of which are secreted by osteoblasts, and makes up approximately one-third of the bony matrix.
The organic component of bone, particularly collagen, provides
bone with great tensile strength and exibility, as well as the ability
to resist stretching and twisting [20]. The inorganic component of
bone, accounting for the remaining two-thirds of the bony matrix,
consists of hydroxyapatites, or mineral calcium phosphates, with the
empirical formula, Ca5(PO4)3OH. The hydroxyapatite gives bone its incredible strength, allowing it to resist compression [20].
Teeth also have a chemical composition similar to bone (Table 1). An
acellular material called enamel composes the crown. Enamel is the
hardest substance in the body, with a chemical composition of 96% inorganic material (mostly hydroxyapatite), 1% organic material, and 3%
water [20]. The enamel protects the underlying dentin, which is a living,
porous, bone-like material that forms the bulk of the tooth. Dentin is
harder than bone but softer than enamel, with a chemical composition
of 70% inorganic material (mostly hydroxyapatite), and 30% inorganic
material and water. A substance called cementum covers the outer surface of the root, attaching the tooth to the periodontal ligament and anchoring the tooth in the bony alveolus. The layer of cementum is thin
and composed of 55% organic material and 45% inorganic material
(mostly hydroxyapatite) [20].
Once laid down, the trace element component of the inorganic portions of bone can change due to ionic substitution of other elements
(Table 2). Different portions of the calcium phosphate matrix can be
replaced by various trace elements [23]. Calcium ions are reported
to be replaced by: Li, Be, Na, Mg, Al, Si, K, V, Cr, Mn, Fe, Cu, Zn, Ga, Sr,
Y, Zr, Nb, Cd, Sn, Ba, Au, Hg, Pb, Bi, Ra, Ac, Th, U, and Pu. The phosphate group (PO4), may be substituted by citrate, phosphate esters,
diphosphonates, pyrophosphates, and amino acids in vitro. Finally, the

hydroxyl group (OH) can be replaced by F or Cl [23]. Bone turnover

and external conditions can also cause these elements to lter back
out of the bone matrix [24].
As a result of these substitutions and the resulting uctuations of the
calcium-phosphorus ratios within individual bones, presence and quantities of trace elements can vary signicantly between individuals. Factors inuencing trace elemental disbursement include bone location
and type, diet, growth environment, and taphonomic modications.
One of the strongest inuential factors related to elemental distribution
within the bone matrix is bone type as trace elements are not evenly
distributed within individuals or even individual bones [25]. For example, Zn, V, Ni, Cr, Pb, Mn, Co, and Sn are found in higher concentrations in
bone epiphyseal regions while Ca, Sr, Na, and K are found more often in
the central portion of the diaphysis [21].
Diet and growth environment also have a signicant impact on the
distribution of trace elements within bone. Research is being conducted
to establish past dietary habits by using stable isotope analysis to detect
specic trace element ratios associated with particular dietary patterns
[2632]. Environmental contaminants, such as F or Pb in the water supply, will also be incorporated into the bone matrix [33]. Research has
also shown increases in Fe, Mn, and Cu is possible due to leaching
from the burial environment [34].
Similar conditions impact chemical alterations to dental materials.
Dental materials higher hydroxyapatite concentration results in a
more homogenous composition [35] which makes them less susceptible
to change [36]. Similar to osseous materials, trace elemental concentration in dental materials is most effected by location. Concentrations of
heavy metals increase when progressing from the outer enamel to the
inner dentin root, while leeched materials exhibit decreased concentrations towards the inner portion of the tooth [34]. Finally teeth in different
portions of the dental arc will reect different life periods as a result of
dental formation patterns. As a result, while trace elements can be
deposited in bone during ones lifetime, due to factors such as diet or

Table 1
Bone and tooth organic and inorganic components [20].

Water (H2O)
Organic Components
Main Component(s)
Function(s)
Inorganic Components
Main Component(s)
Function(s)

Bone

Tooth

10-20% in living bone, but b1% in dry bone

~ 33% (dry bone)
Bone Cells (osteoblasts, osteoclasts, osteocytes), Osteoid (collagen,
proteoglycans, glycoproteins)
gives bone its exibility and great tensile strength; allows bone to resist stretch
and twisting
~ 66% (dry bone)
Hydroxyapatite (Ca5(PO4)3OH (mineral calcium phosphates))
give bone its incredible strength; allowing bone to resist compression

~ 3% in living tissue, b1% in dry tissue

~ 1-55% (dry tissue) depending on tooth element
Protein (amelogenins and enamelins in enamel; odontoblasts
in dentin)
Framework for the development of enamel/dentin
~ 45-96% (dry tissue) depending on tooth element
Hydroxyapatite (Ca5(PO4)3OH (mineral calcium phosphates))
makes teeth strong and resistant to degradation; protects
living portion of tooth

Please cite this article as: H.A. Zimmerman, et al., Chemical Differentiation of Osseous, Dental, and Non-skeletal Materials in Forensic
Anthropology using Elemental Analysis, Sci. Justice (2014), http://dx.doi.org/10.1016/j.scijus.2014.11.003

H.A. Zimmerman et al. / Science and Justice xxx (2014) xxxxxx

environment, and not in parts of the teeth that are mineralized, it may
be possible to separate out individuals based on differences in trace
elements.
3. Chemical Analysis of Bone in Forensic Anthropology
The use of chemical analysis in forensic anthropology revolves
around specic questions that researchers strive to answer about a set
of potential remains. The main questions in forensic anthropology
include:
1. How can we differentiate bone from non-bone?
This is usually only a problem when the remains are highly fragmented and/or taphonomically modied; researchers rely on microscopic and elemental analysis to answer this question when necessary.
2. How can we differentiate human from nonhuman bone?
Several microscopic and analytical techniques have been proposed
for differentiating human and nonhuman remains, but histological and
biological analyses are the most common.
3. Is it possible to determine identity or discriminate between individuals?
In instances of commingling and fragmentation in a medicolegal
context, DNA is traditionally used for individuation.
In some cases, these questions may be easy to answer, as the morphology of whole bones or large fragments of bone will quickly indicate
that the material is bone, and whether it is or is not human. However, if
the fragments are too small to show details consistent with human bone
or even detail consistent with bone in general, it may be necessary for
the forensic anthropologist to refer to a laboratory method for help
with identication of the material. For this purpose, researchers have
developed microscopic, biological, and analytical methods.
3.1. Bone versus Non-Bone
A limited number of techniques have been proposed for distinguishing osseous and dental tissues from non-skeletal materials.
The common technique is to determine the elemental composition of
the material and compare the Ca/P ratio to those that have been determined by researchers [1,2]. Research has shown that dry bone typically
has a Ca/P atomic ratio within the range of 1.24 to 1.56 and a weight percent ratio between 1.61 and 2.02 when a calibrated analytical method
based on peak height is used [1,37]; however, additional research has
shown that archaeological bone may have an atomic ratio as high as
1.99 and a weight percent ratio as high as 2.58 [1]. This is important
to note since in some cases the bone being examined by a forensic
anthropologist may be from an archaeological, rather than a forensic
context [1].
One of the early applications of analytical chemistry techniques to
the eld of forensic anthropology consisted of Ubelaker and colleagues
[1] sorting fragmentary osseous and dental tissue from other materials
of similar chemical composition. The researchers developed a method
using scanning electron microscopy/energy dispersive X-ray spectroscopy (SEM/EDS). This technique, which is capable of detecting elements
carbon through uranium, is commonly used to determine the structure
and elemental composition of unknown/unidentied materials from
the X-ray spectrum that is produced during the EDS portion of the
analysis [1].
The seminal article by Ubelaker et al. [1] proposed the use of SEM/
EDS to sort bone/teeth from other materials, which allowed the
researchers to make presumptive identication of materials that
were later corroborated with DNA testing. In this method, samples
underwent elemental analysis, producing a spectrum, and this spectrum was then compared against a spectral library developed by the
FBI called SLICE, or Spectral Library for Identication and Classication

Explorer. The spectral library, SLICE, was demonstrated in the past by

researchers as useful in identifying materials in a number of forensic
contexts [38,39], however, the availability of the database following
2008 cannot be conrmed (by means of an internet search). A number
of considerations of this method include: (1) the method is applied as a
presumptive test to identify materials as osseous or non-osseous; (2) it
is largely based on the relative proportion of Ca/P found in the bone,
which prevents some non-bone materials from being completely discriminated from bone and teeth, such as synthetic hydroxyapatite, mineral apatite, a specic species of octocoral, and ivory; (3) it requires
signicant sample preparation; and (4) it can be destructive to the sample, as samples must be relatively small (approximately no larger than
the size of a dime) or fragments must be removed from a larger sample
in order for the specimen to t in the instrument for analysis. Additionally, Ubelaker et al. [1] recommend grinding all samples into powders
for analysis in order to optimize the homogeneity of each sample; however, this increases the destructive nature of the technique.
Christensen et al. [2] recently proposed an alternative to Ubelakers
[1] analytical technique for fragmentary bone discrimination.
Christensen and colleagues [2] used X-ray uorescence (XRF) to analyze
the same samples from the SEM/EDS research by Ubelaker et al. [1], in
order to determine if XRF was an appropriate method for distinguishing
bone/teeth from non-skeletal materials of similar chemical composition. X-ray uorescence can be derived from all elements in the periodic
table using most bench top instruments [40]; however, the low-Z elements can be difcult to detect in portable or handheld versions
(pXRF or HHXRF) [2,41]. Low-Z elements are those occurring below
Ca [41]. The results from XRF are provided in spectral form, with emissions from each element observed at known energies and varying in intensity depending on the sample.
The study by Christensen et al. [2] involved the analysis of a variety
of materials, including human and non-human osseous and dental tissues; additional biological hard tissues that include shell, coral, horn,
and beak; and non-biological materials including glass, plastic, metal,
wood, and minerals. The materials were examined in a variety of states,
such as burned/charred, weathered, antiquity, and exposure to erosive
chemicals [2]. This analysis technique, like that of Ubelaker et al. [1], is
largely based on the relative proportions of Ca/P found in the sample,
which prevented some non-bone categories of specimens from being
distinguished from osseous and dental tissue conclusively. These
categories of specimens were: mineral apatite, a specic octocoral,
and brachiopod shells. There are some advantages associated with
XRF, including: (1) it requires minimal sample preparation and
(2) is non-destructive to the sample (as long as the sample preparation is nondestructive), making it an appealing method for forensic
anthropologists [2].
More recently, Zimmerman and colleagues [3] published a study
regarding the utility of handheld X-ray uorescence (HHXRF) in
distinguishing human and nonhuman bone/teeth from non-skeletal
material using multivariate statistical analysis. The study examined
a variety of materials (Table 3), which included several examples
found in previous studies on the topic [1,2]. The study involved testing
the reliability of the instrument rst, by examining prepared samples of
synthetic hydroxyapatite and calcium carbonate at known molar ratios.
By plotting the peak intensity ratios of Ca/P detected by the instrument
against the known molar ratios in the sample, the researchers were able
to determine if the HHXRF was capable of detecting Ca and P reliably
due to direct proportionality of the instrumental response to the
molar ratios [3].
In the second phase of the study, the researchers examined the
accuracy of the technique [3]. Data collection involved collecting eight
spectra from each specimen in four distinct locations, followed by compiling the data for statistical analysis. Before statistical analysis, the
researchers removed background noise and the dominating Ca peak
from each specimen; the spectra were also identied as being either
Ca-Dominated or Non-Ca Dominated and only those spectra

Please cite this article as: H.A. Zimmerman, et al., Chemical Differentiation of Osseous, Dental, and Non-skeletal Materials in Forensic
Anthropology using Elemental Analysis, Sci. Justice (2014), http://dx.doi.org/10.1016/j.scijus.2014.11.003

H.A. Zimmerman et al. / Science and Justice xxx (2014) xxxxxx

Table 3
Summary of materials analyzed in Zimmerman et al., [3] using HHXRF.
Human Elements

Nonhuman Species/Element

Other Biological Materials

Non-biological Materials

Taphonomically Modied Materials

Parietal*
Zygomatic*
Humerus
Femur
Rib
Fibula
Foot phalanx
Metacarpal
Molar
Premolar
Canine

Pig (femur)
Turtle (femur)
Turtle shell (shell)
Dog* (femur)
Deer (femur)
Deer (antler)
Gator (femur)
Bird (femur)
Armadillo (femur)
Raccoon (femur)
Turkey (tarsometatarsus)

Scallop
Clam
Sand dollar
Octocoral
Coral
Ivory*
Starsh
Spur

Twig
Bark
Root
Seeds
Float glass
Beer bottle
Limestone
Synthetic
hydroxyapatite
Rock apatite

Human bula (burned)

Human molars (burned)
Human femur (fetal, archaeological)
Human metacarpal (weathered)
Plastic (burned)
Wood (burned)

Rock phosphate

* Examined by Ubelaker et al. [1]

Examined by Christensen et al. [2]

considered Ca-Dominated were included in subsequent statistical

analyses. The remaining spectra were subjected to principal component
analysis (PCA), followed by scatterplots analysis and quadratic discriminant analysis (QDA), all using the PCA scores as variables. The PCA
scores plots showed distinct groups of bone/teeth and non-skeletal
materials with minimal overlap, attributed to materials known to
have a similar chemical composition to bone (rock apatite, synthetic hydroxyapatite, ivory and octocoral). Using QDA, the researchers obtained
high correct classication rates of 94% for bone and non-bone identication. There are several advantages to HHXRF spectrometry in forensic
anthropology, including: (1) it requires minimal sample preparation,
(2) is non-destructive to the sample, (3) rapid data collection and analysis is possible, and (4) samples of varying sizes can be examined without difculty.
Meizel-Lambert and colleagues [19] are currently conducting
research in response to previous chemical analysis studies [13] and
have presented preliminary results. This study aims to assess differentiation of osseous, dental and non-skeletal materials, as well as
potentially human and non-human osseous/dental materials, using
scanning electron microscopy energy dispersive x-ray spectrometry
(SEM/EDX) and statistical analysis. Data was collected as trace element
weight percentages and processed using PCA, linear discriminant analysis (LDA), and QDA. Preliminary results suggest high potential for discrimination of osseous and non-osseous materials [19].
3.2. Human versus Nonhuman and Species Differentiation
Following the identication of questionable material as consistent
with bone/tooth, the next step is determining whether the skeletal material is human or nonhuman. Differentiating human and nonhuman
skeletal material is important because material identied as nonhuman
may be excluded from subsequent analyses, if it is determined not to be
of forensic signicance. While some forensic cases may involve nonhuman remains that are of forensic signicance, for example pet remains
associated with a missing child, many nonhuman remains found can
be sorted out as unrelated material. However, there are a large number
of factors that inuence the discrimination of human and non-human
osseous materials. Primarily, this is a result of their congruent base compositions - hydroxyapatite. Though previous studies have not statistically assessed differentiation between human and non-human
osseous materials, determined Ca/P ratios indicate that the hydroxyapatite foundations of human and non-human osseous materials are
highly similar [1,2]. Additionally, there is a strong overlap between
the trace elements exhibited in each of the samples due to similarities in diet and environment between represented species. A limited
number of analytical chemistry techniques have been proposed for
use in differentiating human and nonhuman bone and teeth, and
these include: near-infrared (NIR) Raman spectroscopy [42], Fourier
transform (FT) Raman spectroscopy [43,44], NIR-FT Raman spectroscopy [45], laser induced breakdown spectroscopy (LIBS)[46], and

SEM/EDX [19]. A summary of the techniques used to discriminate

human and nonhuman skeletal material (to be discussed in this section) can be found in Table 4.
Several studies have suggested that the elemental composition of
bones and teeth differ at the species level [25,4750]. Most of the analytical techniques proposed for distinguishing human and nonhuman
skeletal material in forensic anthropology have involved the use of
Raman spectroscopy [4245]; however, limited research has also been
proposed involving laser induced breakdown spectroscopy [46]. These
analytical techniques are advantageous in that they are nondestructive
or minimally destructive and require less sample preparation than histological and protein techniques, yet still provide species-specic information. However, these techniques are limited in that they are not
standardized or routinely used methods in forensic anthropology.
Several researchers have published work demonstrating the utility
of different types of Raman spectroscopy in discriminating bone and
tooth specimens at the species level [4245]. Raman spectroscopy is
an analytical technique that involves the use of a laser, but is nearly
non-destructive [42], an added benet for forensic anthropology.
There are several forms of Raman spectroscopy, including Fourier transform or FT Raman spectroscopy, dispersive near-infrared Raman, and
micro-Raman spectroscopy, to name a few [51]. In addition to being a
nondestructive technique, Raman spectroscopy also requires limited
sample preparation and allows for rapid analysis [42].
Shimoyama and colleagues [45] demonstrated the usefulness of
near-infrared (NIR) Fourier transform (FT) Raman spectroscopy combined with chemometrics in discriminating between hard ivory, soft
ivory (both from African elephant) and mammoth tusks. In their
research, the researchers collected spectra from 10 hard ivory, 10 soft
ivory, and 5 mammoth ivory samples using a NIR FT-Raman spectrometer. The spectra were normalized to correct for differences in intensity
and principal components analysis (PCA) was performed on the spectral
data. The scores of the principal components were plotted and used to
determine discrimination between the samples [45]. The researchers
determined that all three types of ivory were distinct from one another
in their scores plots, even when a leave-one-out classication method
was used to test the ruggedness of the method [45].
The combination of FT-Raman and chemometrics has also been
shown by Brody et al. [43] to be useful in discriminating ivory, bone
and tooth samples from elephant, sperm whale, mammoth, bovine,
walrus, ovine, hippopotamus, and porcine samples. Ten spectra
were collected from each specimen, individually normalized to the
dominant peak in each sepctrum, and then analyzed using PCA [43].
Scatterplots of the scores from the rst two principal components
showed overlap between several of the specimens, but results of
the stepwise discriminant analysis, showed 90% correct classication
or higher for all groups except the Asian elephant samples and the
mammoth samples, both of which overlapped with the African elephant samples [43]. A follow-up study conducted by Edwards et al.
[44] using infrared FT-Raman spectroscopy and the same method

Please cite this article as: H.A. Zimmerman, et al., Chemical Differentiation of Osseous, Dental, and Non-skeletal Materials in Forensic
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H.A. Zimmerman et al. / Science and Justice xxx (2014) xxxxxx

Table 4
Summary: Advantages and disadvantages of methods used in discriminating skeletal and non-skeletal materials and human and non-human skeletal materials.
Method

Advantages

Disadvantages

Raman spectroscopy (Near-infrared

(NIR), Fourier transform (FT))

Nondestructive
Little to no sample preparation
Potentially species-specic
discrimination
High correct classication rates
Relatively nondestructive
Minimal sample preparation

Advanced statistical analysis may

require training
Only forensic application included
small sample analyzing fresh bone
Not standardized
Small sample
Incomplete results
Not standardized
Not standardized

Laser induced breakdown spectroscopy

(LIBS)
X-ray uorescence
Handheld X-ray uorescence

Scanning electron microscopy/energy

dispersive X-ray spectrometry
(SEM/EDX)

Nondestructive
Minimal sample preparation
Nondestructive
Minimal sample preparation
High correct classication rates
High correct classication rates

Advanced statistical analysis may

require training
Not standardized
Signicant sample preparation
Requires small fragments for analysis
Not standardized

proposed by Brody et al. [43], found better discrimination, with 98% of

ivory specimens from six species classifying correctly.
McLaughlin and Lednev [42] recently proposed the use of NIR
Raman spectroscopy to differentiate bones from different species. The
researchers examined chicken, turkey, cow and pig bone samples
cut from freshly cleaned bone that was recently purchased from a
meat market [42]. A total of 36 spectra were collected from each
bone fragment, with one fragment representing each species [42].
The researchers normalized the data to the dominant peak in each
spectrum and analyzed the data using partial least squares discriminant analysis (PLS-DA) [42]. Partial least squares-DA reduces the dimensionality of the spectral data, as occurs in PCA, but extends the
analysis to include the denition of specic classes and show the
loading of the components in terms of the amount and type of variation
contained in the component [42]. The results of the PLS-DA were
positive, showing discrimination between all four species, with only a
small area of overlap when a 95% condence interval ellipsoid was
applied to the results scatterplot [42].
However, regarding the application of this technique to forensic
anthropology, there are several limitations to this study, specically in
terms of the sample. The sample set chosen was small in terms of the
number of species represented. Additionally, while 36 spectra were collected from each species, the area where the spectra were collected
from was only approximately 75 square micrometers, rather than representative of the entire bone. The bones were also all fresh, which
may sometimes occur in a forensic case, but dry bone should also be
included for comparison purposes.
Another method was proposed by Vass et al. [46] that used laser induced breakdown spectroscopy (LIBS) to sort human and nonhuman
skeletal material, but the only available data is preliminary and rather
incomplete. Laser induced breakdown spectroscopy is an analytical
technique that provides information regarding the elemental composition of a sample [52]. Vass et al. [46] compared eight adult human femora with the tibiae and femora of 14 species of animals, all fully matured
at the time of analysis. Cortical bone was chosen for the site of analysis
since it is less variable than cancellous bone, and remodels in a predictable pattern throughout life [46]. Preliminary results of the spectral data
showed separation between both human and nonhuman samples and
between human bones as resulting from differences in the elemental
signatures of the bones [46]. However, more research is necessary
in this area, in addition to more specic information as to how the
researchers performed their analyses.
There are several advantages and disadvantages to using LIBS in
forensic anthropology analyses. The advantages include that it is a relatively nondestructive technique, as the laser produces a small (approximately 200-m) impact crater in which a very low amount of mass is

Discriminating
Skeletal/Non-Skeletal

Discriminating
Human/Nonhuman
[4245]

[46]

[2]
[3]

[1]

[19]

removed from the sample (a few micrograms) [52]. Other positive characteristics of LIBS include its rapid analysis time, and its requirement of
minimal to no sample processing [5254]. The main disadvantage cited
in the literature was a problem with reproducibility [55]. However, the
main limiting factor associated with LIBS in forensic anthropology is the
lack of research using the method in this eld.
3.3. Individual Identication
Recent research has also indicated that elemental analysis may be
useful in identifying individuals [17]. Castro et al. [17] uses laser ablation
to discriminate bone and tooth samples of individuals based on elemental analysis for trace metals. Laser ablation, or laser ablation-sector eldinductively coupled plasma mass spectrometry (LA-ICP-SF-MS), is a
combination of several methods [17]. Inductively coupled plasma
mass spectrometry (ICP-MS) is an analytical technique that provides
highly sensitive elemental analysis using plasma as the ionization
source, with a mass spectrometer to analyze the ions produced by the
plasma [56]. This allows for the measurement of nearly all elements
found on a periodic table and makes ICP-MS capable of trace element
analysis down to the parts-per-trillion (ppt) level.
The study by Castro et al. [17] used bone reference standards to
develop the method, which was then applied to real bone samples
from 12 individuals. The researchers found that the individuals were
best discriminated when just the femur or humerus were considered
separately, with 42.7% correct classication with all elements versus
75.2% and 63.1% for femoral and humeral bones, respectively. Individuals were also discriminated based on elemental composition of
whole teeth samples. The elements used for discrimination purposes
were: Al, Ba, Cu, Fe, Mg, Mn, Pb, Sr, and Rb. The study used PCA as a
data dimensionality reduction method, as well as a variety of statistical
methods for discrimination, including ANOVA and LDA. With correct
classication rates ranging from 43-75%, this method is not currently
useful for forensic anthropology as this indicates that commingled
skeletal material could be incorrectly associated as high as 57% of the
time. However, with further renement this technique may be applicable to forensic anthropology, particularly in the sorting process of
commingled human remains [17].
Another method using handheld XRF recently reported higher correct classication rates for discriminating between individuals when
using a small sample [18]. Gonzalez-Rodriguez and Fowler [18] examined ve individuals excavated from a medieval period cemetery in Lincoln, United Kingdom, and analyzed 23 bones from each skeleton. The
researchers analyzed each element three times, and averaged the spectra to obtain a single measurement for each bone sampled [18]. The
researchers selected specic elements relating to diet and metabolism

Please cite this article as: H.A. Zimmerman, et al., Chemical Differentiation of Osseous, Dental, and Non-skeletal Materials in Forensic
Anthropology using Elemental Analysis, Sci. Justice (2014), http://dx.doi.org/10.1016/j.scijus.2014.11.003

H.A. Zimmerman et al. / Science and Justice xxx (2014) xxxxxx

for use in discrimination (Ca, Fe, K, Pb, Sr and Zn), quantitated the
amounts of these elements present in each bone using the XRF software,
and then performed PCA on the quantitated data for these elements
only [18]. Using LDA for classication, the researchers obtained 96100% correct classication when discriminating between three
individuals.
In a follow-up analysis (within the same study), the researchers
used elemental ratios for discrimination (Pb/Ca, Zn/F, and Sr/Pb). The
importance of contributing elements was determined based on the
PCA factor loadings and signicant ratios were rationalized based on
biological processes [18]. The factor loadings provide information on
which elements are contributing most to the variation in a principal
component, and in this case, the bone/element the PC is describing.
Using these elemental ratios as data for PCA and LDA, the researchers
obtained 83-96% correct classication when discriminating between
four individuals, but only 53-96% correct classication when discriminating between ve individuals [18]. While the researchers obtained
relatively high classication rates for four individuals, their results are
limited in that the analysis of ve individuals led to a signicant
decrease in discrimination for some comparisons. While this study
shows promise for individuation, a study containing a larger sample
set would be helpful in determining whether discrimination would
continue to decrease as the number of individuals increases. This
could be specically problematic since the number of individuals
being discriminated in forensic commingling events such as airplane
crashes, mass disasters (i.e., World Trade Center attack), and natural disasters (i.e., Hurricane Katrina), typically involve much larger numbers
of individuals.
3.4. Commercial Cremation Analysis
The use of elemental analysis in sorting and identifying cremains,
has become commonly accepted in forensic anthropology, with much
work being published on the topic in recent years. Most work with
elemental analysis in this area of forensic anthropology deals with classifying cremains as human or contaminated (by some other type of
material). The use of proton induced X-ray emission (PIXE) was used
by Warren and colleagues [57] determine that a questionable set of cremains did not represent osseous material.
Another type of elemental analysis used in the classication of
cremains is inductively coupled plasma-optical emission spectroscopy
(ICP-OES). In a study by Brooks and colleagues [58](with a pilot study
published previously as Bodkin et al., [59], ICP-OES combined with multivariate statistical analysis was used to determine the legitimacy of
human cremains following a crematorium disaster in Nobel, GA. A
total of 21 elements were chosen for examination in the study, with
only seven of the 21 being chosen for use in the statistical analysis.
By combining ICP-OES with multivariate statistical analysis, the
researchers were able to develop a classication method by which
the analyzed remains would be classied as (1) cremains, (2) questionable remains, or (3) concrete, based on the percentage of human
cremains detected in the sample.
The analysis by Brooks and colleagues [58] included the comparison
of human cremains to those of Canis familiaris (a specimen that visually
and chemically resembles human bone) and wood ash (a specimen that
visually resemble human bone) in order to determine if it was possible
to separate cremains up to the genus level. The method classied the
wood ash as concrete and the dog ash as cremains, allowing the
researchers to conclude that the method was unable to provide species
differentiation due to the chemical similarity between human and animal bone at the cremains level. Since their analysis was based on only
seven elements, it may be possible to differentiate species using additional elements, but further research is needed on this topic.
Brooks and colleagues [58] also examined the remains of two individuals in their study that contained high levels of metallic alloys in
their cremains indicative of projectile (bullet) residue. The researchers

concluded (through elemental analysis and background research on

the individuals) that the metallic residue was the result of projectiles
acquired during life that remained in the soft tissue of each individual
through the cremation process resulting in increased levels of specic
metals in the elemental analysis (increased levels of Pb corresponded
to lead buckshot and increased levels of Pb, Cu, and Mn corresponded
to a copper-jacketed bullet) [58].
More recently, the use of a different type of inductively coupled plasma was proposed for use in the classication of cremains by Schultz and
colleagues [60]. In their study, Schultz et al. [60] used ICP-MS to determine the probable identity of a set of remains that appeared to have
been tampered with due to their appearance (dark gray color). The
study involved comparing the problem cremation with a Standard
Reference Material (SRM) bone ash sample (required when performing
elemental analysis using ICP-MS) that was provided by NIST (National
Institute of Standards and Technology), as well as three known specimens that would act as a baseline for the unknown specimen: (1) a recent historical cremation from Europe, (2) cremains of a donated
anatomical specimen, (3) known commingled cremation. The results
of the analysis showed that all four samples had comparative Ca/P ratios
and common trace elements that were consistent with bone, indicating
that the problem cremation had not been contaminated with any foreign substances.
The elemental analysis also showed that the problem cremation
contained high levels of gadolinium (Gd), a type of rare trace element
not commonly found in bone samples, which could be matched to his/
her life history for identication purposes. Since Gd can be introduced
to the body during life as a product of radiological procedures, and
then accumulate in the bones and liver due to the inability of the body
to excrete it, the researchers concluded that the individual likely
acquired the Gd during MRI and CT imaging studies conducted before
death. This was consistent with the individuals medical history of
renal failure and the imaging conducted to diagnose and treat the condition [60]. This research provides further evidence of the value of elemental analysis in the examination of cremains, especially when rare
trace elements found in a sample can be matched to an individuals history, providing a positive identication rather than a likely or probable
identication.

4. Discussion
Forensic anthropologists use a variety of methods (Summary,
Table 4) to identify osseous and dental materials, including visual identication in the case of whole bones or large fragments, and laboratory
methods in the case of more fragmentary materials. The laboratory
techniques typically used in forensic anthropology to identify questionable remains as (1) bone, and as (2) human or nonhuman bone, include
histology [412] and protein analysis [13,14,6164]. These techniques
are destructive, time intensive and costly. Additionally, these methods
may not be able to discriminate highly fragmented or taphonomically
altered materials. Chemical analysis techniques (HHXRF, XRF, SEM/
EDS, Raman spectroscopy, LIBS) have been proposed for discriminating
bone/teeth from non-skeletal material [13,19] and human from nonhuman skeletal material [42,46]. However, their use is sporadic and
more dependent on the availability of the instrument.
Chemical analysis can be especially useful in identifying fragmentary
or questionable remains, including commercial cremations, which
cannot be identied using alternative laboratory methods due to a
high degree of fragmentation or taphonomic alteration. For example,
chemical analysis of commercial cremations has been shown to be a
valuable step in forensic anthropology when determining the composition of purported cremains [58,60]. At the same time, chemical analysis
of cremains can also be used for providing supporting evidence for identifying a descendent by detecting rare trace elements, or elevated levels
of normal minor elements, acquired during ones life [58,60].

Please cite this article as: H.A. Zimmerman, et al., Chemical Differentiation of Osseous, Dental, and Non-skeletal Materials in Forensic
Anthropology using Elemental Analysis, Sci. Justice (2014), http://dx.doi.org/10.1016/j.scijus.2014.11.003

H.A. Zimmerman et al. / Science and Justice xxx (2014) xxxxxx

The use of nondestructive chemical analysis techniques in forensic

anthropology can be particularly useful as an initial sorting method
(human bones and teeth from non-skeletal material) when analyzing
fragmentary and questionable material. For example, mass disasters including airline crashes, the September 2001 terrorist attacks, and Hurricane Katrina, resulted in fragmentary human remains that may have
been subjected to any number of taphonomic processes that involved
burning and weathering [65,66]. In these cases, techniques using nondestructive chemical analysis methods, such as HHXRF, would be useful
as the initial step in quickly identifying questionable material as bone or
non-bone [3]. Once questionable material is identied as skeletal, it can
be further analyzed using histological or biological methods (i.e., DNA)
to determine if the bone is human, and to make an identication if
DNA is present.
There are several benets to using chemical analysis in forensic
anthropology, most notably in terms of statistical integration and identication without destruction of the material. The majority of the chemical methods assessed are highly specic, nondestructive, and require
minimal sample preparation or operator training, but most have not
provided classication rates. Of all of the methods proposed for species
differentiation and individualization, Raman spectroscopy has shown
the most potential, demonstrating species classication rates between
84.5 and 97% [4244]. However, these studies were performed using
limited samples; larger sample sets need to be assessed to determine
the validity of this method.
5. Future Directions and Conclusions
While the use of chemical analysis is now becoming a standard
when examining cremations [58,60], there have been signicant
advances in chemical differentiation of skeletal and non-skeletal materials using elemental analysis [13] in forensic anthropology.
These methods [13], if applied as a preliminary step for discriminating the origin of osseous, dental and non-skeletal materials, could assist
in determining the potential forensic signicance of unidentied materials. In turn, this would reduce time and monetary costs traditionally
expended on analysis of non-skeletal samples. However, further
research is necessary to rmly establish chemical analysis as a viable
method within forensic anthropology. There are two main directions
for the expansion of this topic: renement of methods and their incorporation into forensic investigations.
Previous studies have set the framework for developing methods
aimed at chemically differentiating skeletal from non-skeletal but
there are areas in which the eld still needs to progress. Forensic anthropologists need a rapid, non-destructive, and cost efcient method
that can identify a range of elements and concentrations indicative of
skeletal material [67,68]. It is necessary to continue to address the application of chemical differentiation technologies towards differentiation
of skeletal and non-skeletal material. Research must be undertaken to
expand the data sets to include more representative samples, including
additional human bones and teeth, non-human bones and teeth, nonskeletal materials, and taphonomically modied materials. Homogeneity within samples and within species should be assessed to determine
how this might impact classication during real-world application. Additionally, problematic methods or substances should be re-examined
to determine the underlying cause of poor classication. This may
allow for better understanding of the discrimination process and help
to determine if there are alternative analysis processes or data analysis
methods that would provide more accurate discrimination or more
accessible testing.
Additional research also needs to be contributed to differentiation of
human and non-human skeletal materials. This is the second step in determining the forensic signicance of unidentied materials [68], as
well as the more difcult task. Being able to chemically determine the
origin of skeletal materials would allow for more rapid determination
of forensic signicance and, as a result, more direct and concise forensic

investigations. Specic research queries in this area should be directed

at determining the chemical overlap between species and if there are
any antemortem conditions or postmortem events that could change
composition signicantly enough to shift classication.
Subsequently, attention needs to be directed towards incorporating
successful chemical differentiation methods into criminal investigations. Large scale blind studies, using materials frequently encountered
in forensic investigations, should be performed to assess the applicability of these methods for criminal investigations and to identify potential
issues that might arise. This would also contribute to establishing error
rates. Once a successful method has been identied protocols should be
established and introduced to the forensic community, encouraging incorporation of this approach into criminal investigations. Overall, continued research involving the chemical analysis of osseous and dental
material can lead to standardization protocols for the use of chemical
analysis in forensic anthropology.

Acknowledgements
Portions of this manuscript are derived from the thesis of the rst author (Zimmerman HA. 2013. Preliminary validation of handheld X-ray
uorescence (HHXRF) spectrometry: Distinguishing osseous and dental
tissue from non-bone materials of similar chemical composition [M.A.
thesis]. Orlando, Florida: Department of Anthropology, The University
of Central Florida). We would also like to thank the anonymous
reviewers for their valuable comments that improved this manuscript.

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Please cite this article as: H.A. Zimmerman, et al., Chemical Differentiation of Osseous, Dental, and Non-skeletal Materials in Forensic
Anthropology using Elemental Analysis, Sci. Justice (2014), http://dx.doi.org/10.1016/j.scijus.2014.11.003