Kamla-Raj 2014

Anthropologist, 17(3): 929-936 (2014)

Anthropological Age Estimation with Bone Histomorphometry

from the Human Clavicle
+
U-Young Lee1, Go-Un Jung2, Seung-Gyu Choi3 and Yi-Suk Kim2*
1

Department of Anatomy, Catholic Institute for Applied Anatomy, College of Medicine,

The Catholic University of Korea, Seoul, Republic of Korea 137-701
2
Department of Anatomy, Ewha Womans University School of Medicine, Seoul,
Republic of Korea 158 -710
3
Division of Forensic Medicine, National Forensic Service, Seoul, Republic of Korea 158-707
KEYWORDS Bone Histology. Compact Bone. Osteon. Grinding Method. Forensic Anthropology
ABSTRACT The purpose of present study was to apply histomorphometry to estimate the age at death using the
clavicular osteonal remodeling rate and to develop an age-predicting equation. 46 clavicular samples consisting 28
males and 18 females were obtained from Korean cadavers. Two sequential 1-mm-thick sections, were prepared
using a diamond wheel, and a 100 um-thick section was then made by manual grinding with polarizing filter optical
microscopy. Three histomorphometric variables, including relative cortical area (RCA), osteon population density
(OPD), and mean osteon area (OA), were measured. The mean values are indicated because no significant difference
in any variable was noted by a paired t test between the two sequential sections. Analysis of covariance demonstrated
that stratification of the statistical results of OPD and OA based on sex was unnecessary, except for RCA.
Regression analysis indicated that the highest coefficient value was OPD (R 2 = 0.583), which was selected as a
significant value with RCA for an age-predicting equation (R2 = 0.628) by the multiple regression analysis stepwise
method. In conclusion, a histomorphometric method using the clavicle is reliable for age estimation in physical
and forensic anthropological fields.

INTRODUCTION
In forensic anthropology, biological profiles
including age, sex, and populations group
should be assessed to identify skeletal remains
(Trotter and Gleser 1958; Han et al. 2009). To do
this, it is crucial to first distinguish human bones
from animal bones (Mughal et al. 2013; Formicola 1993). If the skeletal remains are confirmed to
be human, procedures for assessing the biological profiles depend on individual skeletal measurements (Balseven-Odabasi et al. 2013; Absolonova et al. 2013; Buikstra and Ubelaker 1994).
Among the biological profiles, the age at death
provides significant information about the cause
of death, irrespective of the elapsed time after
death. Therefore, it is vital to determine the age
at death of unidentified remains, especially in
forensics. Until now, two major methods have
Address for correspondence:
Dr. Yi-Suk Kim
Department of Anatomy,
Ewha Womans University School of Medicine
911-1, Mok-5-dong,
Yangcheon-gu, Seoul 158-710,
Republic of Korea
Fax: 82-2-2650-5716,
E-mail: [email protected]

been used for age estimation: the conventional

method of visual inspection and a histomorphometric method (Kerley 1965). In visual inspection, the examiners study a specific articulation
surface with the naked eye and measure the level of aging-related degeneration. However, the
outcome is likely to produce a wide range and a
large standard error of estimate. In addition, if
the articulation surface is severely damaged, visual inspection might be difficult to satisfy the
scientific demands.
In contrast, the histomorphometric method
is a quantitative determination based on the measurable morphology of the osteon (Frost 1987).
Microscopic examination of a cross-section of
the bone is used to measure the histomorphometric features of the osteon and the measured
variables are applied to confirm the regression
relationship between bone and age at death. The
histomorphometric method reduces subjective
judgments and the level of experience needed
for age estimation, and this method is available
even for small fragmentary.
Currently, histomorphometric age estimation
is widely performed for a number of population
groups and various types of bone elements (Frost
1958; Thompson 1983). The osteonal remodeling process that contributes to bone variation

930

U-YOUNG LEE, GO-UN JUNG, SEUNG-GYU CHOI ET AL.

differs among population groups and bone types

(Thompson and Guness-Hey 1981; Stout and
Lueck 1995). In addition, age-estimating research
based on diverse population groups with different materials has been expanded, especially in
Korean populations. In 2007 and 2009, the sternal end of the 4th rib and anterior cortex of the
femur were tested for use to as materials (Kim et
al. 2007; Han et al. 2009). However, previous research has not examined the extent and range
for applying additional bone materials that can
be assessed in diverse forensic situations. Moreover very few studies have focused on the clavicle, except in Caucasian populations (Stout and
Paine 1992)
The clavicle has several clear benefits and
was selected as the target material in this study.
First, the size determines the relatively high ratio
of compact bone to spongy bone, so there is
little possibility of damage to the clavicle by a
fracture. Second, the bone is likely to be found
intact rather than in fragments. Third, the clavicle is not a weight-bearing bone, so few factors,
including daily lifestyle, can affect the bone remodeling rate over time. Finally, the clavicle does
not influence other anthropological tests (Stout
and Paine 1992); the bone is not an essential
elements for tests that determine utile biological
profiles such as statue and sex.
The purpose of this study is to provide a
new regression equation using various histomorphometric features of the clavicle for age estimation that can be applied in different forensic
situations. Additionally, this study also aimed
to show a positive application of the equation,
which is founded on histomorphometric methods. In future studies that utilize diverse bone
materials for age estimation, histomorphometric
age estimation methods can be applied in the
forensic field.
METHODOLOGY
Materials
Right clavicle samples were obtained from
46 dissected cadavers with a known cause of
death, age, and sex (28 males and 18 females).
The mean age was 47.5 15.8 for men and 47.5
15.8 for women (Table 1). The causes of death
from chronic disease, primary bone disease, or
other serious conditions that could affect the
condition of the bone were excluded. Samples (3

cm long) were cut from the sternal end of the

right clavicle and then two sequential slides were
made by manual grinding on graded silicon carbide abrasive paper. The bone unit, including
features of the osteon, was observed, measured,
and calculated using a microscope and an image
analysis program.
Table 1: Age distribution of specimens
Age range
(years)

Pooled sexes
n (%)

20-29
30-39
40-49
50-59
60-69
Over 70

7
8
11
9
5
6

(15.2)
(17.4)
(23.9)
(19.6)
(10.9)
(13.0)

Total

46 (100)

Male
n (%)
3
5
8
6
3
3

(10.7)
(17.9)
(28.6)
(21.4)
(10.7)
(10.7)

28 (100)

Female
n (%)
4
3
3
3
2
3

(22.2)
(16.7)
(16.7)
(16.7)
(11.1)
(16.7)

18 (100)

Chemical Treatments
The remaining soft tissues from the muscle,
fascia, and periosteum adhering to the clavicle,
were excised carefully without damaging the subperiosteal space. The remnants could produce
measuring error, so pretreatment was performed
at each of the following steps as needed. To prevent morphologic change from protein denaturation, the sample was fixed in 10% formalina
mixture of formalin and distilled waterover seven days to ensure complete permeation. Fixed
samples were removed from formalin and soaked
in cold running water to remove formalin. After
deep rinsing, the samples were transferred to
another mixture of chloroform and methanol for
2 days to eliminate the remaining fat inside the
bone marrow, without obstructing the examiners field of vision during observation with the
microscope. Finally, the samples were bleached
in a 2% H2O2 solution for 1 day to maintain sterility.
Serial Sectioning
A 1-mm thick clavicular sample was crosssectioned using a diamond wheel (IsoMet 5000
Precision Saw, Buehler Instruments, Lake Bluff,
IL USA). Then, two sequential thin sections (approximately 100-m thick) were obtained by manual grinding on graded silicon carbide abrasive
papers. The 4-inch wheel operated at a speed of
300 rpm, and the samples were fully locked in the
clamp. Despite this high speed, the samples were

931

HISTOMORPHOMETRIC AGE ESTIMATION OF CLAVICLE

unexposed to frictional heat because over onethird of the wheel surface was submerged from
the beginning. The paper was moistened to avoid
frictional heat throughout the manual grinding
work. Researchers followed the Frost technique
(Ahlqvist 1969) to apply strength equally during
the overall manual grinding process. Once the
sample appeared transparent, with an expected
thickness of approximately 100 m, manual grinding was stopped and the sections were immersed
in distilled water using ultrasonic cleaner for approximately 2 min to loosen any tiny debris.
Slide Preparation
Dehydration was performed at room temperature using varying concentrations of alcohol,
from 60% to 100%. Dehydration at each concentration took 3 min, except at the absolute (100%)
concentration. With 100% alcohol, extra moisture inside the sections was eliminated in 5 min.
After the dehydration procedure was complete,
the specimens were soaked in xylene for 1 min to
remove any alcohol. The sections were mounted
in Entellan (Merck, Darmstadt, Germany) and
covered with a glass cover slip.
Histomorphometric Analysis
The histomorphometric analysis performed
in this study was based on captured images from
a polarizing microscope (BX-51, Olympus, Tokyo, Japan) equipped with a polarizing filter (BXPOL). The total magnification was 100x, fitted
with a 10 10 mm2 eyepiece reticle with a grid

area of 1.0 mm2. These grids enable consistent

visual measurements. Three histomorphometric
variables including the ratio of the relative cortical area (RCA), osteon population density (OPD),
and mean osteon area (OA), were measured microscopically and counted using an image analysis program (Image J, NIH Image, Bethesda,
MD). For OA, data were obtained from 8 surface
points in all directions and read in a checkerboard pattern (Fig. 1). The measured OA of the 8
points was regarded as a representative value of
the entire cross-sectional OA for analysis in this
study.
Statistics
SPSS 13.0 was used for data analysis (SPSS,
Inc., Chicago, IL). The difference between the
two serial slides was verified by a paired sampled t-test. The statistical differences were confirmed between sexes through the analysis of
covariance (ANCOVA) when age was adjusted.
A simple linear regression analysis showed a
general association between the estimated age
at death and each variable. Finally, multivariate
linear regression analysis using the stepwise
method provided an age estimation equation
applicable to the clavicle.
RESULTS
Descriptive statistics of all histomorphometric variables for age classification and sex are
recorded in Table 2. As seen in Table 2, a nega-

Fig. 1. Measuring osteon area (OA) by using image analysis program (image J,NIH Image,
Bethesda, MD)

932

U-YOUNG LEE, GO-UN JUNG, SEUNG-GYU CHOI ET AL.

Table 2: Descriptive statistics of histomorphometric variables for the pooled and separate sexes (mean
standard deviation)
RCA (ratio)
Pooled Sexes (n = 46)
2029 years
3039 years
4049 years
5059 years
6069 years
Over 70 years
Males (n = 28)
2029 years
3039 years
4049 years
5059 years
6069 years
Over 70 years
Females (n = 18)
2029 years
3039 years
4049 years
5059 years
6069 years
Over 70 years

OPD (#D mm2)

OA (mm 2 )

64.740
62.734
62.539
55.519
44.204
44.222

24.559
10.209
9.145
7.819
7.678
18.618

21.714
22.526
23.524
26.008
26.911
33.216

1.945
3.761
3.166
3.072
3.860
4.434

0.025
0.025
0.027
0.026
0.027
0.019

0.002
0.003
0.002
0.005
0.005
0.002

84.763
66.160
67.035
56.421
47.117
61.189

27.402
5.789
5.615
6.973
9.078
1.358

22.343
23.222
24.273
26.950
29.339
30.081

2.999
3.565
2.992
1.865
1.632
3.113

0.025
0.023
0.026
0.025
0.027
0.020

0.003
0.001
0.002
0.004
0.004
0.002

49.724
57.024
50.550
53.716
39.834
27.254

2.031
14.187
3.365
10.755
2.706
1.050

21.242
21.367
21.525
24.125
23.269
36.351

0.937
3.985
3.248
4.590
3.172
3.160

0.024
0.028
0.028
0.029
0.027
0.018

0.002
0.004
0.003
0.007
0.007
0.002

Fig. 2. Bivariate plots of histomorphological variables of relative cortical area (RCA)

according to the age. The related regression results are summarized in Table 2.

tive correlation was observed between RCA and

OA with age increment (Figs. 2 and 4) and a

positive correlation with OPD was observed

using simple linear regression analysis (Fig. 3).

933

HISTOMORPHOMETRIC AGE ESTIMATION OF CLAVICLE

No significant difference in any measurement was

noted between the two serial slides (P>0.05).
Therefore, a mean value of the two slides was
indicated and referred to in this research (Table
3). Table 4 shows the results of the analysis of
covariance with age as a covariate. No significant difference was seen in OPD and OA between
sexes when age was adjusted, with the exception
of RCA. Therefore only the value of pooled sexes was applied to the analysis. The results of the
standard regression analysis used in this study
are shown in Table 5. Among the three indicated
parameters in Table 5, OPD showed the highest
correlation between estimated age (R2= 0.583).
The OPD and RCA were selected for the agepredicting equation from the multiple regression
analysis using the stepwise method (R2= 0.628)
(Table 6). Because OA showed the lowest correlations, it was not selected for multiple regression analysis for the age estimation equation.

DISCUSSION
Conventional methods such as visual inspection of the articular surface are common estimation techniques and that have been widely
used. These methods determine the degenerative conditions and the beginning of the epiphyseal union using medical imaging analysis based
on gross morphology (Balseven-Odabas 2013;
Kellinghaus et al. 2010; Mughal et al. 2013). The
typical articulation regions used for age estimation include the auricular surface on the ilium,
the sternal end of the rib, the suture closure on
the cranium, and the symphyseal surface of the
pubis (Iscan 1984; Lovejoy et al. 1985; Meindl
1985; Brooks and Suchey 1990). There is currently absolute criteria for age estimation that
has been establish using this technique, and thus
the estimated results had a wide range with a
large standard estimation error. The histomor-

Fig. 3.Bivariate plots of histomorphological variables of osteon population density

(OPD) according to the age. The related regression results are summarized in Table 2.
Table 3: Results of paired t-test between two serial slides
Measurements

RCA1-RCA2
OPD1-OPD2
OA1-OA2

46
46
46

P > 0.05.

Mean SD
0.424 1.566
0.029 1.270
0.001 0.005

Differences
45
45
45

Significance *
0.073
0.877
0.060

934

U-YOUNG LEE, GO-UN JUNG, SEUNG-GYU CHOI ET AL.

Table 4: Results of multivariate and univariate analysis of covariance

Variable
*

RCA

OPD *
OA*

Parameter
Intercept
Age
Sex (F)
Intercept
Age
Sex (F)
Intercept
Age
Sex (F)

B
84.426
-0.427
-16.481
15.878
0.201
-1.254
0.028
-7E-005
0.001

Significance

18.573
-5.044
-5.255
11.682
7.943
-1.338
16.450
-2.205
0.632

0.000
0.000
0.000
0.000
0.000
0.188
0.000
0.033
0.531

Partial n
0.889
0.372
0.391
0.760
0.595
0.040
0.863
0.102
0.009

The parameter of each dependent variable [Sex = M] was set to zero.

Table 5: Simple regression analysis between age and histomorphometric variables

Variable
RCA
OPD
OA
*

Intercept
84.604
-24.566
84.439

Slope
-0.627
2.911
-1428.534

SEE

15.746
11.934
17.538

0.274
0.583
0.100

0.000
0.000
0.033

SEE, Standard Error of Estimate (years).

P < 0.05.

Fig. 4. Bivariate plots of histomorphological variables of osteon area (OA) according to

the age. The related regression results are summarized in Table 2.

phometric method usually measures and quantifies various features of the osteon, mainly the
secondary osteon with a clear cement line, to
provide more absolute criteria in age estimation
(Bednarek 2008). Based on this process, the his-

tomorphometric method can be used even when

the bone is discovered in fragments. Under normal circumstances, fragmented or eroded bone
increases uncertainty in forensic fields, with little success in identification, and can hinder the

935

HISTOMORPHOMETRIC AGE ESTIMATION OF CLAVICLE

Table 6: Age-predicting equations conducted from stepwise regression analysis
Equation *

Adjusted R

SEE

Age = 1.412 - 0.282(RCA) + 2.519 (OPD)

0.628

11.403

Multicollinearity did not exist among the independent variables, and the linear regression model was verified
by ANOVA at the 1% level of significance.

SEE, Standard Error of Estimate.

Table 7: Comparison with previous study using clavicles

Present study
Stout and Paine (1992)
*

Bone element

Population

Clavicle
Clavicle

Korean (n = 46)
American (n = 40)

R
0.628
0.699

SEE *
11.403
-

SEE, Standard Error of Estimate.

assessment of biological profiles. Therefore, the

histomorphometric method can be applied as a
reliable and alternative method of age estimation.
Because the osteonal remodeling rate is different for each bone, histomorphometric studies have progressed based on the various bone
elements (Frost 1958; Thompson and GunnesHey 1981; Thompson 1983). Among these
bones, the clavicle is mostly often found intact
and has a low possibility for fragmentation. The
clavicle is not a weight-bearing bone, and so is
not affected by physical exercise, labor activity and life style that could influence the osteonal remodeling rate over time (Stout and
Paine 1992). Despite this, only a few studies
have incorporated the clavicle to estimate age
at death except in Caucasian populations.
Therefore, the researchers attempted to determine the reliability of the clavicle as an age indicator by confirming its regression relationship with age at after the rib and femur, especially in the Korean population.
The researchers used materials of various
ages to support our conclusion. If the age groups
were disproportionate, the research results
would be biased, distorted, and thus less convincing (Stout et al. 1994). Therefore, this study
endeavored to consider all age groups, which
allows the deduced regression equation for age
estimation to be applied in different age groups
which are indicated in Table 1.
ANCOVA was the method used to observe
the effect of covariates and age in this study.
The researchers adjusted age to determine how
sex affected variables thorough a general linear
model (Osborne et al. 2004). When age is adjusted, only RCA was significantly different between
sexes in this study (Table 4). As a result, sex

should have been considered in RCA measurement. However, RCA essentially differs depending on sex (Mughal et al. 2014), and RCA is a
ratio and not a directive value. In addition, considerable preceding research reported no significant relationship between bone composition
and sex (Kerley 1965; Ericksen 1991). Therefore,
in our final analysis, the researchers do not divide our data set into two parts (male and female) to develop an equation, although sex is
considered in RCA measurement.
Table 5 shows that the OA was fairly unrelated with estimated age. However, another study
showed contrasting results (Osborne et al. 2004).
In this study, OA was not measured at all surfaces but at the surrounding 8 parts in all directions using the protractor on cortical bone. Until now, no clear objective criteria have been
outlined to measure the average osteon area for
age estimation. Accordingly, to take an objective approach for the overall distribution of the
osteon area, the selected 8 parts were presented
as criteria in this study. However, the results
showed that OA did not adequately reflect the
remodeling rate. Thus, for our additional study,
the researchers proposed that the unremodeled
area rather than the osteon area alone should be
applied for histomorphometric age estimation.
The result of previous research regarding
age estimation with bone histology from the clavicle is shown in Table 7. Stout and Paine (1992)
used the clavicle as material and developed individual regression equations. The regression
correlation based on the sole clavicle in the Stout
and Paine study (R2=0.699) is similar to those
reported in this study (R 2=0.628). Nonetheless,
comparison of descriptive statistics with previ-

936

U-YOUNG LEE, GO-UN JUNG, SEUNG-GYU CHOI ET AL.

ous study reveals that the osteonal remodeling

rate is not in agreement with population groups.
Age estimation has to be approached using multifactorial methods. Therefore, to resolve the populational differences, applying various types of
bone elements measurement techniques, is suggested to achieve a more accurate estimate of
the age at death. Consequently, the different
bones should be collectively studied and compared with the clavicular remodeling rate to determine the age correlation between ages on histomorphometric analysis.
In conclusion, this study identified a reliable age estimation equation using repeatable
and constant histomorphometric measurements
of the clavicle which can be applied especially in
Korean adults. In addition, age estimation using
the clavicle can be applied in situations such as
and international airplane crash that involves
multinational victims. The results of this study
could be applied and expanded if more diverse
bone elements from all age groups are included
and verified for accurate forensic assessment.
ACKNOWLEDGEMENTS
This research was supported by the Basic
Science Research Program through the National
Research Foundation of Korea (NRF) funded by
the Ministry of Education, Science and Technology (2010-0002383).
REFERENCES
Absolonova K, Veleminsky P, Dobisikova M, Beran M,
Zocova J 2013. Histological estimation of age at
death from the compact bone of burned and unburned human ribs. J Forensic Sci, 58:135-145.
Ahlqvist J, Damsten O 1969. A modification of Kerleys method for the microscopic determination of
age in human bone. J Forensic Sci, 14: 205-212.
Balseven-Odavasi A, Yalcinozan E, Keten A, Akcan R,
Tuner AR, Onaa A, Canturk N, Odavasi O, Dince AH
2013. Age and sex estimation by metric measurements and fusion of byoid bone in a Turkish population. Journal of Forensic and Legal Medicine, 20:
496-501.
Bednarek J 2008. Methods of age at death estimation
based on compact bone histomorphometry. Arch
Med Sadowej Kryminol, 58: 197-204.
Brooks S, Suchey JM 1990. Skeletal age determination
based on the os pubis: A comparison of the AcsdiNemeskri and Suchey-Brooks methods. Human
Evolution, 5: 227-238.
Buikstra JE, Ubelaker DH 1994. Standards for Data
Collection from Human Skeletal Remains. Arkan-

san Archaeological Survery Report Number 44, Fayettevill, Arkansas

Formicola V 1993. Stature reconstruction from long
bones in ancient population samples: An approach
to the problem of its reliability. Am J Phys Anthropol, 90: 351-358.
Frost HM 1958. Preparation of thin undecalcified bone
sections by rapid manual method. Stain Technol, 33:
273-277.
Frost HM 1987. Secondary osteon populations: An algorithm for determining mean bone tissue age. Year
book of Physical Anthropology, 30: 221-238.
Han SH, Kim SH, Ahn YW, Huh GY, Kwak DS, Park
DK, Lee UY, Kim YS 2009. Microscopic age estimation from the anterior cortex of the femur in
Korean adults. J Forensic Sci, 54: 519-522.
Ican MY, Loth SR, Wright RK 1984. Age estimation
from the rib by phase analysis: White males. J Forensic Sci, 29: 1094-1104.
Kellinghaus M, Schulz R, Vieth V, Schmidt S, Schmeling
A 2010. Forensic age estimation in living subjects
based on the ossification status of the medial clavicular epiphysis as revealed by thin-slice multidetector computed tomography. Int J Legal Med, 124:
149-154.
Kerley ER 1965. The microscopic determination of
age in human bone. Am J Phys Anthropol, 23: 149163.
Kim YS, Kim DI, Park DK, Lee JH, Chung NE, Lee
WT, Han SH 2007. Assessment of histomorphological features of the sternal end of the fourth rib for
age estimation in Koreans. J Forensic Sci, 52: 12371242.
Lovejoy CO, Meindl RS, Pryzbeck TR, Mensforth RP
1985. Chronological metamorphosis of the auricular surface of the ilium: A new method for the determination of adult skeletal age at death. Am J Phys
Anthropol, 68: 15-28.
Meindl RS, Lovejoy CO 1985. Ectocranial suture closure: A revised method for the determination of skeletal age at death based on the lateral-anterior sutures. Am J Phys Anthropol, 68: 57-66.
Mughal AM, Hassan N, Ahmed A 2014. Bone age assessment methods: A critical review. Pak J Med Sci,
30: 211-215.
Osborne DL, Simmons TL, Nawrocki SP 2004. Reconsidering the auricular surface as an indicator of age
at death. J Forensic Sci, 49: 905-911.
Stout SD, Dietze WH, Ican MY, Loth SR 1994. Estimation of age at death using cortical histomorphometry of the sternal end of the fourth rib. J Forensic
Sci, 39: 778-784.
Stout SD, Lueck R 1995. Bone remodeling rates and
skeletal maturation in three archaeological skeletal
populations. Am J Phys Anthropol, 98: 161-171.
Stout SD, Paine RR 1992. Brief communication: Histological age estimation using rib and clavicle. Am J
Phys Anthropol, 87: 111-115.
Thompson DD, Galvin CA 1983. Estimation of age at
death by tibial osteo remodeling in an autopsy series. Forensic Sci Int, 22: 203211.
Thompson DD, Gunness-Hey M 1981. Bone mineralosteon analysis of Yupik-Inupiaq skeletons. Am J
Phys Anthropol, 55: 1-7.
Trotter M, Gleser GC 1958. A re-evaluation of estimation of stature based on measurements of stature
taken during life and of long bones after death. Am J
Phys Anthropol, 16: 79-123.