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Histological Variables at Multiple Locations and the Effect on Age Estimation

Left or right sixth ribs from five Caucasian male postmortem human subjects (8292 years, SD = 6.24
Three seriated sections were taken from each of three locations measured from the vertebral end of the
rib: posterior (25%), middle (50%), and anterior (75%) (Figure 1). Nine sections were made per individual.
All slides were imaged at 100x magnification and observed on the microscope using bright field and
polarized light at 200x magnification.
Two histological variables were quantified and analyzed (Table 1). Osteon Population Density (OPD) was
collected live from the microscope and Cortical Area (Ct.Ar) was measured manually from still images
(Figure 2).
Serial section analysis
OPD was normally distributed, Ct.Ar was normalized with a ln transformation.
Repeated measures ANOVAs determined that neither mean OPD (F (2, 28) = 3.347,p= 0.05) nor Ct.Ar (F
(2, 28) = 3.347,p= 0.324) differed significantly between sections.
Location analysis
OPD was normally distributed, but Ct.Ar could not be normalized and thus was analyzed using non-
parametric statistics.
Victoria M. Dominguez; Angela L. Harden; Amanda M. Agnew
Skeletal Biology Research Laboratory, The Ohio State University
Current histological methods for estimating adult age-at-death using the rib were developed exclusively
from cross-sections of the midshaft 1, 2. However, in forensic practice, it is not uncommon for histological
samples to come from fragmented or previously segmented ribs, leading to uncertainty of sampling
The potential for error increases when sampling location on the rib is uncertain and utilizing a section
beyond the midshaft (either anterior or posterior) may result in erroneous age estimates.
As histology is a two-dimensional approach to three-dimensional structures, it is not surprising that
strong similarities exist across adjacent sections. This suggests it may be possible to get accurate age
estimates using only one slide.
OPD and Ct.Ar differ across the three locations considered in the rib, supporting the importance of using
the midshaft portion of the rib when utilizing current methods.
OPD does not vary significantly between the anterior and middle locations, suggesting that when
sampling locations are uncertain, a more anterior section should be used, as it is more consistent with
the histomorphology of the midshaft.
Future studies should explore OPD and Ct.Ar in a more diverse sex and age sample to determine if the
pattern observed here is consistent throughout the population and the significance of sampling location
on age estimation.
1. Stout SD, Pain RR, “Brief communication: histological age estimation using rib and clavicle,American Journal of Physical Anthropology 87 (1992): 111115.
2. Cho H, Stout SD, Madsen RW, Streeter MA, “Population-specific histological age-estimating method: a model for known African-American and European-American skeletal remains,Journal
Forensic Science 47 (2002): 1218.
3. Frost HM, “Tetracyline-based histological analysis of bone remodeling,Calcified Tissue Research 3 (1969): 211237.
The authors would like to thank the donors for their generous gifts. Thank you to Nicole
Crowe for preparation and imaging of the slides used in this project. Lastly, thank you to
all the members of the Skeletal Biology Research Laboratory and the Injury Biomechanics
Research Center at OSU.
Table 1. Histological Variables
Figure 2. Diagram of Ct.Ar measurement
Table 2. Paired Samples Test
Paired t-tests reveal that OPD
for the anterior and middle
locations are not significantly
different from each other, but
that the posterior location is
significantly different from both
(Table 2).
Wilcoxon Signed Rank tests showed that Ct.Ar in the anterior location is significantly different from both
the middle and posterior locations, but that the middle and posterior locations do not demonstrate
significant differences between themselves.
Frost 3recommended reading a minimum of 50 mm2of cortical bone when assessing skeletal remodeling,
resulting in the common practice of reading two to three serial sections in the ribs and averaging the
results to account for local variation. Problems arise when there is not enough bone to create or
insufficient time to properly analyze multiple serial sections.
This study aims to determine the importance of serial sections and the midshaft distinction for age
assessment by analyzing histological variables of multiple sections at multiple sampling locations along
the length of the rib.
Figure 1. Exemplar cross-sectional images from the posterior (25%), middle (50%), and anterior (75%)
locations, imaged in bright field illumination with a 10x objective (orientation = cutaneous cortex is on top,
superior is to the left). Schematic of seriated sections from each location at a thickness of 120 μm each.
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Full-text available
Histological methods for estimating age at death using osteon population densities for the rib, clavicle, and rib and clavicle combined are presented. Predicting formulas were generated from a sample of 40 individuals of known age, sex, and race. Independent samples of 12 ribs and 7 clavicles were used to test the formulas. Mean differences between known and predicted ages were 1.1 years, 2.6 years, and 3.4 years for the clavicle, rib and clavicle combined, and rib formulas respectively. An analysis of variance found no significant differences among the means for predicted and known ages. Since the formula based upon rib and clavicle combined has the higher standard error and r2, and includes data from different bones, it should provide better overall accuracy and reliability, and is recommended whenever both bones are available.
Previously developed histological age-estimating methods have been based on samples lacking interpopulation variability. A comparison of age-associated rib histomorphometrics between an European-American sample and an African-American sample indicates that ethnicity can have a significant effect on osteon population density (OPD), osteon cross-sectional area (On.Ar), and relative cortical area (Ct.Ar/Tt.Ar). Based upon these findings, new histological age-predicting formulae are presented that are recommended when estimating age for African-American or European-American skeletal remains. A general formula that is applicable to remains of unknown ethnicity is also provided.