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tooth area ratio in upper canines by peri-apical X-rays
Legal Medicine 16 (2014) 337–343
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Accuracy and reliability of pulp/tooth area ratio in upper canines by peri-apical X-rays A.C. Azevedo a, E. Michel-Crosato a, M.G.H. Biazevic a, I. Galic´ b, V. Merelli c,d, S. De Luca d,⇑, R. Cameriere d a
Departamento de Odontologia Social da Faculdade de Odontologia da Universidade de São Paulo (FOUSP), São Paulo, Brazil Department of Dental Anthropology, School of Dental Medicine, University of Zagreb, Zagreb, Croatia c Laboratorio di Antropologia e Odontologia Forense (LABANOF), Institute of Legal Medicine, University of Milan, Milan, Italy d AgEstimation Project, Institute of Legal Medicine, University of Macerata, Macerata, Italy b
a r t i c l e
i n f o
Article history: Received 19 November 2013 Received in revised form 5 June 2014 Accepted 4 July 2014 Available online 25 July 2014 Keywords: Human identification Age estimation Pulp/tooth area Upper canines Reliability Italy
a b s t r a c t Due to the real need for careful staff training in age assessment, in order to improve capacity, consistency and competence, new research on the reliability and repeatability of methods frequently used in age assessment are required. The aim of this study was twofold: first, to test the accuracy of this method for age estimation; second, to obtain data on the reliability of this technique. A sample of 81 peri-apical radiographs of upper canines (44 men and 37 women), aged between 19 and 74 years, was used; the teeth were taken from the osteological collection of Sassari (Sardinia, Italy). Three blinded observers used the technique in order to perform the age estimation. The mean real age of the 81 observations was 37.21 (CI95% 34.37 40.05), and estimated ages ranged from 36.65 to 38.99 (CI95%-Ex1 35.42; 41.28; CI95%-Ex2 33.89; 39.41; CI95%-Ex3 35.92; 42.06). The module differences found by the three observers were 3.43, 4.24 and 4.45, respectively for Ex1 Ex2, Ex1 Ex3 and Ex2 Ex3. The module differences observed among real and observed ages were 2.55 (CI95% 1.90; 3.20), 2.22 (CI95% 1.65; 2.78) and 4.39 (CI95% 3.80; 5.75), respectively for Ex1, Ex2 and Ex3. No differences were observed among measurements. This technique can be reproduced and repeated after proper training, since it was found high reliability and accuracy. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Age estimation is a continually evolving discipline that finds its main applications in clinical fields (diagnosis, prognosis and auxological and dental treatment) and forensic human identification (age estimation of living persons, bodies, skeletal remains). In the last two decades, due to the global increase in migratory movements, there has been a growing demand for age estimates for immigrants from countries that are not members of the European Union (EU). In many cases, these non-EU citizens have no documents giving their chronological age [1–3]. When examining living sub-adults or the remains of infants, children and adolescents, the most suitable age indicators are the various stages of dental mineralization and eruption, length of long bone diaphyses, appearance of ossification centers and fusion of epiphyses [4,5]. Hands, especially the carpal bones, have been used as age indicators in several studies [6–8].
⇑ Corresponding author. Address: C/Gran Canaria, 19, 2°1a, Sabadell, Barcelona, Spain. Tel.: +34 671231425. E-mail address: [email protected] (S. De Luca). http://dx.doi.org/10.1016/j.legalmed.2014.07.002 1344-6223/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
There are several methods available to assess age in adults, depending on whether they are living subjects or not, although problems still exist concerning the standardization of techniques. Examples are the large age ranges of age-phasing methods; observer subjectivity; bias and age mimicry when appropriate reference samples are not used; and improper procedures and statistical parameters used to derive age estimates [5,9–11]. In living subjects, the number of available methods is greatly reduced because the processes of skeletal and dental maturation are complete and the methods usually employed for human remains are too invasive [5]. In this case, age can be estimated by external physical examination, hormone dosage for women and/or some dental methods. Different techniques and methods have been proposed, as of different stages of tooth formation and they often involve X-rays analysis [12–19]. Of the many possibilities, analysis of the apposition of secondary dentine is of particular interest [5]. After tooth eruption, it is well known that the size of the pulp cavity gradually decreases with age, because of the deposition of secondary dentine in the pulp cavity wall. These agerelated changes can be observed and measured from dental radiographs, establishing a valuable tool for age estimation in adults [20–22].
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The main aim of this work is twofold: first, to test the accuracy of this method [23] in a sample of peri-apical radiographs of canines, in order to assess the suitability of this method in estimating chronological age; second, to obtain data on the reliability of the technique in blind trials. 2. Materials and methods Peri-apical X-rays of 81 upper canines belonging to 81 subjects (44 men, 37 women), aged between 19 and 74 years, were analyzed. The Table 1 shows the sex and age distribution of the sample. The teeth were taken from the osteological collection of Sassari (Sardinia, Italy). It is part of the Frassetto collections and it was preserved in the Museum of Anthropology, Department of Experimental and Evolutionary Biology, University of Bologna (Italy). The collection contains 606 well-preserved skeletons, 337 males and 269 females, of individuals who died in the first half of the 20th century and that were exhumed from municipal cemeteries. The cemetery registers provide the sex, age, and date of death of most individuals and in many cases their date of birth and occupation. In particular, the age-at-death is documented for 253 females and 312 males [24]. Canines were chosen for a number of reasons: they are often present in old age, they are less likely than other anterior teeth to suffer attrition or abrasion as a result of particular work, and they are the single-root teeth with the largest pulp area and thus the easiest to evaluate and perform uniform measures. In addition, canines without endodontic treatment or prosthetic fittings and with no dental fillings or significant pathologies (such as wear, caries and calculus) were chosen. The pulp cavity of the upper canine is similar in size and shape to that of the lower canine [25]. However, it is not uncommon to find two roots or at least two canals in the lower canine. Since the presence of two canals cannot be easily detected by X-rays, only the upper canine was selected for this study. All radiographs were digitized and coded to ensure that observers were blind to the gender, name and age of subjects. Each file was therefore numbered consecutively from 1 to 81, being part of a blind set-up. Three independent observers examined all the canines: two forensic odontologists and one forensic physician. When assessing the pulp/tooth ratio, they had only the instructions provided in the original text of Cameriere et al. [23]. As they only knew the tooth number and gender, after the measurements, they were able to calculate the subject’s age independently and objectively. The results of the above studies were analyzed by another examiner, who had the information with regard the sample and the real ages. The accuracy of the method was defined as its proximity with the real age, and a high reliability would be reached if this method would be reproducible with no significant differences by the three observers [26]. The nomenclature chosen to classify the canines is that proposed by the International Dental Federation (IDF). Protocols to collect radiographs for human subjects were approved by the Ethics Committee for Research Involving Human Subjects of the University of Bologna (Italy), and the study was conducted in Table 1 Age distribution of the Italian sample according to sex. Age (years)
Males
Females
N
%
19–30 31–40 41–50 51–60 61–74 Total
15 10 12 4 3 44
14 11 8 3 1 37
29 21 20 7 4 81
35.80 25.92 24.70 8.64 4.94 100.00
accordance with the ethical standards laid down by the Declaration of Helsinki (Finland). Peri-apical digital X-rays were taken and the conventional paralleling technique was used. This is the most widely accepted method of taking peri-apical radiograph owing to the dimensional accuracy of the image and better reproducibility. It involves placement of intra-oral sensor parallel to the long axis of the tooth with the central X-ray directed perpendicular to the tooth. A NOMADÒ hand-held dental X-ray device (Aribex, Orem-UT, USA) combined with a digital sensor (DSX, Anthos, Rome, Italy), linked to a portable computer, are used to place the sensor parallel to the tooth’s long axis. All radiographs were taken with a Rinn-type digital sensor holder, with 0.05 s exposure time at 65 kV. Due to technological advances with the optical plate and scintillator, all size sensors have increased sensitivity, which allows researchers to decrease the radiation dose. The teeth were X-rayed both isolated and in situ, depending on the state of conservation of each skeleton and on the possibility of extracting them without damage. Readings were taken in a horizontal plane at angles of 0° to 180° in 15° – increments. 0° was directly behind the NOMADÒ and 180° was directly in front of the cone in direct line with the radiation beam. The operator occupied what the NOMADÒ promotional material called the ‘‘zone of significant occupancy’’ from 0° to 35°. 2.1. Measurements Following Cameriere et al. [23,27], the radiographic images of the canines were processed with a computer-aided drafting program (ADOBEÒ Photoshop CS4, San Jose-CA, USA). Figs. 1 and 2 show the outlines as mentioned in the technique. A minimum of 20 points from each tooth outline and 10 points for each pulp outline were identified and connected with the line tool, also on the Draw Toolbox, and the area of both tooth and pulp was ascertained. All measurements were made without prior information about personal data of the subjects. Age was estimated by applying the equation proposed by Cameriere et al. [27] for the upper canine: Upper canine: Age = 99.937–532.775 (RAu)where RAu represents the pulp/tooth area ratio in upper canines. 2.2. Statistical analysis Descriptive statistics were performed with media, standard deviation (SD) and confidence intervals (CI); minimum and maximum values are also shown, and boxplot graphics were built. The non-parametric Kolmogorov–Smirnov test (K–S test) was used to verify sample normality. Descriptive measures were applied to determine the accuracy of the measurements, and the Bland– Altman Plot was chosen to estimate inter-observer reliability. The latter plots graphically portrayed the mean difference between the various measurements and the size of the original measurement, and thus enabled researchers to make visual judgments of the variability between observers. The data were also subjected to statistical analysis with the non-parametric Pitman’s test to determine any correlation between measurements. Also, intraclass correlation (ICC) was calculated to verify the accuracy and reliability of the measurements. The statistical program used was STATA 12.0 (College Station-TX, USA, at 5% level of significance [28]. 3. Results The Kolmogorov–Smirnov test showed that the chronological age cohort distribution was always normal for each estimated age. Considering the Kolmogorov–Smirnov test results (normal
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Fig. 1. Radiographic image of right upper canine (13), after processing and measuring tooth area with line tool: red, tooth area. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Radiographic image of right upper canine (13), after processing and measuring pulp area with line tool: blue, pulp area. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
distribution), chronological age distributions are represented in mean values, standard deviations (SD) and minimum and maximum observed values. All data are shown in Table 2 and Fig. 3 for with their minimum and maximum results; furthermore, Table 2 also shows the normality values.
Table 2 shows that the real mean age of the 81 observations was 37.21 (CI95% 34.37; 40.05), whereas estimated ages ranged from 36.65 to 38.99 (CI95%-Ex1 35.42; 41.28; CI95%-Ex2 33.89; 39.41; CI95%-Ex3 35.92; 42.06). Table 3 shows the accuracy and the reliability of the measurements among the observers and also in
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Table 2 Descriptive statistics with real age and estimated ages as per each investigator.
*
Variable
Obs*
Mean
Std. Dev.
ICC (95%)
Min
Max
Normality (p)
Age Ex1 Ex2 Ex3
81 81 81 81
37.21 38.35 36.65 38.99
12.86 13.24 12.46 13.90
34.37;40.05 35.42;41.28 33.89;39.41 35.92;42.06
19.00 17.40 17.96 17.07
74.00 73.05 71.58 78.47
0.083 0.229 0.093 0.063
Obs: number of observed teeth.
Fig. 3. Box-plot with real age and estimated ages as per each observer.
Table 3 Reliability and accuracy data of the measurements.
* ** ***
Variable
Obs*
Ex1 Ex2 Ex1 Ex3 Ex2 Ex3 Age Ex1 Age Ex2 Age Ex3
81 81 81 81 81 81
Diff ** 1.70 0.64 2.34 1.14 0.56 1.78
ICC
Diff module
ICC
0.88; 2.52 1.87; 0.59 3.56; 1.13 1.97; 0.31 0; 18; 1.30 3.17; 0.39
3.43 4.24 4.45 2.55 2.22 4.39
2.95; 3.43; 3.57; 1.90; 1.65; 3.80;
3.91 5.04 5.33 3.20 2.78 5.75
Pitmans Test (p)
ICC*** (p)
0.215 (0.054) 0.120 (0.294) 0.267 (0.052) 0.104 (0.360) 0.120 (0.284) 0.170 (0.140)
0.95 0.91 0.92 0.95 0.96 0.96
(<0.001) (<0.001) (<0.001) (<0.001) (<0.001) (<0.001)
Obs: number of observed teeth. Diff: differences between the observer’s measurements. ICC = intra-class correlation.
comparison with real ages. The module differences found by the three observers were 3.43, 4.24 and 4.45, respectively for Ex1 Ex2 (CI95% 2.95; 3.91), Ex1 Ex3 (CI95% 3.43; 5.04) and Ex2 Ex3 (CI95% 3.57; 5.33). The module differences observed among the real ages and those calculated by the observers were 2.55 (CI95% 1.90; 3.20), 2.22 (CI95% 1.65; 2.78) and 4.39 (CI95% 3.80; 5.75), respectively for Ex1, Ex2 and Ex3. No differences were observed among measurements. Fig. 4 shows the differences among observers, and Fig. 5 shows the differences among real ages and the ages as calculated by the observers. The results clearly indicate similar variations, showing high reproducibility due to positive and high correlations.
4. Discussion High accuracy and high reliability were verified using Pitman’s and intra-class correlation tests and Bland Altman graphics have shown these results. Other techniques for age estimation also found high agreement between observers [29]. As some age estimation methods may not be as reliable as they are claimed to be, some authors recommend that caution should be taken not to rely too much on the results of one single examiner and stress the importance of obtaining a second opinion [30,31]. As Cunha et al. [5] noted, the best methods are sometimes not those with the best standard error, but those which have been
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Fig. 4. Box-plot of the observers’ differences.
Fig. 5. Box-plot of the difference among real age and the observers’ evaluations.
tested by many researchers on many different populations. Our investigation observed a high reliability among the observers, confirming that the method can be applied by professionals, after appropriate training. In addition, it is commonly believed that almost any physician is qualified to ‘‘read’’ X-rays and that any dentist can evaluate dental radiographs. However, the more highly qualified is a professional, the more weight that expert’s opinion will carry. Most juries will appreciate that diagnostic images must be interpreted and explained to them by a competent expert, familiar with both medical and technical factors applicable to the evidence. A physical anthropologist, for instance, may qualify as an expert to testify on radiographs of the skeleton, but will probably be required to
explain or document special training or experience in the use of this tool [32,33]. Other studies [23,27,34,35] have already discussed the importance and precision of the pulp/tooth ratio in canines by peri-apical X-rays for age assessment in adults. However, since then, very few published studies have validated the previous results of the technique. In particular, very little research has been carried out in order to determine intra- and inter-observer variations in analysis of the apposition of secondary dentine using the pulp/tooth area ratio in canines [36]. One study tested the accuracy of Camerierés method in medieval samples of unknown age at death, comparing the dental age estimation with other archaeological measurements [36]. Another one has performed 3D age estimations based on the
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pulp-tooth volume ratios of mono-radicular teeth, and more studies are necessary to reach precise results [37], on both ancient and today’s samples. As regards the first aim of this study, the final results showed that the pulp/tooth area ratio gradually decreases with age from 19 to 74 years, indicating age related narrowing of the pulp cavity from the cervical pulp chamber to the terminal part of the root canal. Nevertheless, real ages compared with estimated ones were found to be similar among the observers, demonstrating the high accuracy of measures. The mean differences between real and estimated ages were low, with slight over-estimation of age. The mean difference between real and estimated ages was about three years. This corroborated some previous results on two-dimensional measurements as age-related variables [38–42]. The coronal third of the root pulp is the most stable region in canines, with minimal morphological diversity and few influences due to various types of dental wear and other post-development changes, such as caries, especially if slowly progressing, or the use of restorative materials [43]. All these clinical factors have been reported to affect or be associated with post eruptive changes in dentine, resulting in enhanced mineralization [43]. The stability of this part of the root pulp should lead to few measurement errors and little inter observer variability. Regarding the reliability of Cameriere’s method, the measurements performed by our three observers did not differ (no statistical differences), showing good inter-observer agreement. This proves that the method is highly reproducible and may also be applied after proper training. The inter-observer evaluations revealed no statistically significant differences in the measurements of any of the observers, probably because they had had lengthy experience with the technique, and were familiar with age estimation techniques in particular. This is an important feature, considering the need for extensive international comparisons between the results provided by several observers from many different populations [5,44]. The present work also showed no significant influence of sex and age groups on age estimation, which was similar to the findings of previous studies [27,34–36]. However, several adaptations of current research set-ups could improve future results. Manual correction of the automatic selection of the layer PULP CHAMBER was most clearly required for the apical quarter of the tooth root. On these smallest tooth and pulp contours, the computerized distinction of tooth parts based on the grayscale threshold became less reliable, so that necessary checking and correcting procedures took up most of the time needed for image processing. In addition, manual operations may have a negative influence on the precision of pulp and tooth measures. More studies are necessary in order to test the method in other population groups in order to verify its accuracy, after training in order to reach a high degree of inter-observer concordance. 5. Conclusions Cameriere’s method is useful for age estimation and it presented high accuracy and reliability. Novelty statement Since 2004, Roberto Cameriere et al. have published several papers on a method of age estimation using the pulp/tooth area ratio to quantify the apposition of secondary dentine. A decrease in the area of the pulp cavity by secondary dentine formation is known to occur with age. The researchers reported extremely high regression coefficients (R2 = 0.94) when using labio-lingual X-ray images of upper and lower canines. The reliability of this method has recently been
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Accuracy and reliability of pulp/tooth area ratio in upper canines by peri-apical X-rays A.C. Azevedo a, E. Michel-Crosato a, M.G.H. Biazevic a, I. Galic´ b, V. Merelli c,d, S. De Luca d,⇑, R. Cameriere d a
Departamento de Odontologia Social da Faculdade de Odontologia da Universidade de São Paulo (FOUSP), São Paulo, Brazil Department of Dental Anthropology, School of Dental Medicine, University of Zagreb, Zagreb, Croatia c Laboratorio di Antropologia e Odontologia Forense (LABANOF), Institute of Legal Medicine, University of Milan, Milan, Italy d AgEstimation Project, Institute of Legal Medicine, University of Macerata, Macerata, Italy b
a r t i c l e
i n f o
Article history: Received 19 November 2013 Received in revised form 5 June 2014 Accepted 4 July 2014 Available online 25 July 2014 Keywords: Human identification Age estimation Pulp/tooth area Upper canines Reliability Italy
a b s t r a c t Due to the real need for careful staff training in age assessment, in order to improve capacity, consistency and competence, new research on the reliability and repeatability of methods frequently used in age assessment are required. The aim of this study was twofold: first, to test the accuracy of this method for age estimation; second, to obtain data on the reliability of this technique. A sample of 81 peri-apical radiographs of upper canines (44 men and 37 women), aged between 19 and 74 years, was used; the teeth were taken from the osteological collection of Sassari (Sardinia, Italy). Three blinded observers used the technique in order to perform the age estimation. The mean real age of the 81 observations was 37.21 (CI95% 34.37 40.05), and estimated ages ranged from 36.65 to 38.99 (CI95%-Ex1 35.42; 41.28; CI95%-Ex2 33.89; 39.41; CI95%-Ex3 35.92; 42.06). The module differences found by the three observers were 3.43, 4.24 and 4.45, respectively for Ex1 Ex2, Ex1 Ex3 and Ex2 Ex3. The module differences observed among real and observed ages were 2.55 (CI95% 1.90; 3.20), 2.22 (CI95% 1.65; 2.78) and 4.39 (CI95% 3.80; 5.75), respectively for Ex1, Ex2 and Ex3. No differences were observed among measurements. This technique can be reproduced and repeated after proper training, since it was found high reliability and accuracy. Ó 2014 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Age estimation is a continually evolving discipline that finds its main applications in clinical fields (diagnosis, prognosis and auxological and dental treatment) and forensic human identification (age estimation of living persons, bodies, skeletal remains). In the last two decades, due to the global increase in migratory movements, there has been a growing demand for age estimates for immigrants from countries that are not members of the European Union (EU). In many cases, these non-EU citizens have no documents giving their chronological age [1–3]. When examining living sub-adults or the remains of infants, children and adolescents, the most suitable age indicators are the various stages of dental mineralization and eruption, length of long bone diaphyses, appearance of ossification centers and fusion of epiphyses [4,5]. Hands, especially the carpal bones, have been used as age indicators in several studies [6–8].
⇑ Corresponding author. Address: C/Gran Canaria, 19, 2°1a, Sabadell, Barcelona, Spain. Tel.: +34 671231425. E-mail address: [email protected] (S. De Luca). http://dx.doi.org/10.1016/j.legalmed.2014.07.002 1344-6223/Ó 2014 Elsevier Ireland Ltd. All rights reserved.
There are several methods available to assess age in adults, depending on whether they are living subjects or not, although problems still exist concerning the standardization of techniques. Examples are the large age ranges of age-phasing methods; observer subjectivity; bias and age mimicry when appropriate reference samples are not used; and improper procedures and statistical parameters used to derive age estimates [5,9–11]. In living subjects, the number of available methods is greatly reduced because the processes of skeletal and dental maturation are complete and the methods usually employed for human remains are too invasive [5]. In this case, age can be estimated by external physical examination, hormone dosage for women and/or some dental methods. Different techniques and methods have been proposed, as of different stages of tooth formation and they often involve X-rays analysis [12–19]. Of the many possibilities, analysis of the apposition of secondary dentine is of particular interest [5]. After tooth eruption, it is well known that the size of the pulp cavity gradually decreases with age, because of the deposition of secondary dentine in the pulp cavity wall. These agerelated changes can be observed and measured from dental radiographs, establishing a valuable tool for age estimation in adults [20–22].
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The main aim of this work is twofold: first, to test the accuracy of this method [23] in a sample of peri-apical radiographs of canines, in order to assess the suitability of this method in estimating chronological age; second, to obtain data on the reliability of the technique in blind trials. 2. Materials and methods Peri-apical X-rays of 81 upper canines belonging to 81 subjects (44 men, 37 women), aged between 19 and 74 years, were analyzed. The Table 1 shows the sex and age distribution of the sample. The teeth were taken from the osteological collection of Sassari (Sardinia, Italy). It is part of the Frassetto collections and it was preserved in the Museum of Anthropology, Department of Experimental and Evolutionary Biology, University of Bologna (Italy). The collection contains 606 well-preserved skeletons, 337 males and 269 females, of individuals who died in the first half of the 20th century and that were exhumed from municipal cemeteries. The cemetery registers provide the sex, age, and date of death of most individuals and in many cases their date of birth and occupation. In particular, the age-at-death is documented for 253 females and 312 males [24]. Canines were chosen for a number of reasons: they are often present in old age, they are less likely than other anterior teeth to suffer attrition or abrasion as a result of particular work, and they are the single-root teeth with the largest pulp area and thus the easiest to evaluate and perform uniform measures. In addition, canines without endodontic treatment or prosthetic fittings and with no dental fillings or significant pathologies (such as wear, caries and calculus) were chosen. The pulp cavity of the upper canine is similar in size and shape to that of the lower canine [25]. However, it is not uncommon to find two roots or at least two canals in the lower canine. Since the presence of two canals cannot be easily detected by X-rays, only the upper canine was selected for this study. All radiographs were digitized and coded to ensure that observers were blind to the gender, name and age of subjects. Each file was therefore numbered consecutively from 1 to 81, being part of a blind set-up. Three independent observers examined all the canines: two forensic odontologists and one forensic physician. When assessing the pulp/tooth ratio, they had only the instructions provided in the original text of Cameriere et al. [23]. As they only knew the tooth number and gender, after the measurements, they were able to calculate the subject’s age independently and objectively. The results of the above studies were analyzed by another examiner, who had the information with regard the sample and the real ages. The accuracy of the method was defined as its proximity with the real age, and a high reliability would be reached if this method would be reproducible with no significant differences by the three observers [26]. The nomenclature chosen to classify the canines is that proposed by the International Dental Federation (IDF). Protocols to collect radiographs for human subjects were approved by the Ethics Committee for Research Involving Human Subjects of the University of Bologna (Italy), and the study was conducted in Table 1 Age distribution of the Italian sample according to sex. Age (years)
Males
Females
N
%
19–30 31–40 41–50 51–60 61–74 Total
15 10 12 4 3 44
14 11 8 3 1 37
29 21 20 7 4 81
35.80 25.92 24.70 8.64 4.94 100.00
accordance with the ethical standards laid down by the Declaration of Helsinki (Finland). Peri-apical digital X-rays were taken and the conventional paralleling technique was used. This is the most widely accepted method of taking peri-apical radiograph owing to the dimensional accuracy of the image and better reproducibility. It involves placement of intra-oral sensor parallel to the long axis of the tooth with the central X-ray directed perpendicular to the tooth. A NOMADÒ hand-held dental X-ray device (Aribex, Orem-UT, USA) combined with a digital sensor (DSX, Anthos, Rome, Italy), linked to a portable computer, are used to place the sensor parallel to the tooth’s long axis. All radiographs were taken with a Rinn-type digital sensor holder, with 0.05 s exposure time at 65 kV. Due to technological advances with the optical plate and scintillator, all size sensors have increased sensitivity, which allows researchers to decrease the radiation dose. The teeth were X-rayed both isolated and in situ, depending on the state of conservation of each skeleton and on the possibility of extracting them without damage. Readings were taken in a horizontal plane at angles of 0° to 180° in 15° – increments. 0° was directly behind the NOMADÒ and 180° was directly in front of the cone in direct line with the radiation beam. The operator occupied what the NOMADÒ promotional material called the ‘‘zone of significant occupancy’’ from 0° to 35°. 2.1. Measurements Following Cameriere et al. [23,27], the radiographic images of the canines were processed with a computer-aided drafting program (ADOBEÒ Photoshop CS4, San Jose-CA, USA). Figs. 1 and 2 show the outlines as mentioned in the technique. A minimum of 20 points from each tooth outline and 10 points for each pulp outline were identified and connected with the line tool, also on the Draw Toolbox, and the area of both tooth and pulp was ascertained. All measurements were made without prior information about personal data of the subjects. Age was estimated by applying the equation proposed by Cameriere et al. [27] for the upper canine: Upper canine: Age = 99.937–532.775 (RAu)where RAu represents the pulp/tooth area ratio in upper canines. 2.2. Statistical analysis Descriptive statistics were performed with media, standard deviation (SD) and confidence intervals (CI); minimum and maximum values are also shown, and boxplot graphics were built. The non-parametric Kolmogorov–Smirnov test (K–S test) was used to verify sample normality. Descriptive measures were applied to determine the accuracy of the measurements, and the Bland– Altman Plot was chosen to estimate inter-observer reliability. The latter plots graphically portrayed the mean difference between the various measurements and the size of the original measurement, and thus enabled researchers to make visual judgments of the variability between observers. The data were also subjected to statistical analysis with the non-parametric Pitman’s test to determine any correlation between measurements. Also, intraclass correlation (ICC) was calculated to verify the accuracy and reliability of the measurements. The statistical program used was STATA 12.0 (College Station-TX, USA, at 5% level of significance [28]. 3. Results The Kolmogorov–Smirnov test showed that the chronological age cohort distribution was always normal for each estimated age. Considering the Kolmogorov–Smirnov test results (normal
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Fig. 1. Radiographic image of right upper canine (13), after processing and measuring tooth area with line tool: red, tooth area. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 2. Radiographic image of right upper canine (13), after processing and measuring pulp area with line tool: blue, pulp area. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
distribution), chronological age distributions are represented in mean values, standard deviations (SD) and minimum and maximum observed values. All data are shown in Table 2 and Fig. 3 for with their minimum and maximum results; furthermore, Table 2 also shows the normality values.
Table 2 shows that the real mean age of the 81 observations was 37.21 (CI95% 34.37; 40.05), whereas estimated ages ranged from 36.65 to 38.99 (CI95%-Ex1 35.42; 41.28; CI95%-Ex2 33.89; 39.41; CI95%-Ex3 35.92; 42.06). Table 3 shows the accuracy and the reliability of the measurements among the observers and also in
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Table 2 Descriptive statistics with real age and estimated ages as per each investigator.
*
Variable
Obs*
Mean
Std. Dev.
ICC (95%)
Min
Max
Normality (p)
Age Ex1 Ex2 Ex3
81 81 81 81
37.21 38.35 36.65 38.99
12.86 13.24 12.46 13.90
34.37;40.05 35.42;41.28 33.89;39.41 35.92;42.06
19.00 17.40 17.96 17.07
74.00 73.05 71.58 78.47
0.083 0.229 0.093 0.063
Obs: number of observed teeth.
Fig. 3. Box-plot with real age and estimated ages as per each observer.
Table 3 Reliability and accuracy data of the measurements.
* ** ***
Variable
Obs*
Ex1 Ex2 Ex1 Ex3 Ex2 Ex3 Age Ex1 Age Ex2 Age Ex3
81 81 81 81 81 81
Diff ** 1.70 0.64 2.34 1.14 0.56 1.78
ICC
Diff module
ICC
0.88; 2.52 1.87; 0.59 3.56; 1.13 1.97; 0.31 0; 18; 1.30 3.17; 0.39
3.43 4.24 4.45 2.55 2.22 4.39
2.95; 3.43; 3.57; 1.90; 1.65; 3.80;
3.91 5.04 5.33 3.20 2.78 5.75
Pitmans Test (p)
ICC*** (p)
0.215 (0.054) 0.120 (0.294) 0.267 (0.052) 0.104 (0.360) 0.120 (0.284) 0.170 (0.140)
0.95 0.91 0.92 0.95 0.96 0.96
(<0.001) (<0.001) (<0.001) (<0.001) (<0.001) (<0.001)
Obs: number of observed teeth. Diff: differences between the observer’s measurements. ICC = intra-class correlation.
comparison with real ages. The module differences found by the three observers were 3.43, 4.24 and 4.45, respectively for Ex1 Ex2 (CI95% 2.95; 3.91), Ex1 Ex3 (CI95% 3.43; 5.04) and Ex2 Ex3 (CI95% 3.57; 5.33). The module differences observed among the real ages and those calculated by the observers were 2.55 (CI95% 1.90; 3.20), 2.22 (CI95% 1.65; 2.78) and 4.39 (CI95% 3.80; 5.75), respectively for Ex1, Ex2 and Ex3. No differences were observed among measurements. Fig. 4 shows the differences among observers, and Fig. 5 shows the differences among real ages and the ages as calculated by the observers. The results clearly indicate similar variations, showing high reproducibility due to positive and high correlations.
4. Discussion High accuracy and high reliability were verified using Pitman’s and intra-class correlation tests and Bland Altman graphics have shown these results. Other techniques for age estimation also found high agreement between observers [29]. As some age estimation methods may not be as reliable as they are claimed to be, some authors recommend that caution should be taken not to rely too much on the results of one single examiner and stress the importance of obtaining a second opinion [30,31]. As Cunha et al. [5] noted, the best methods are sometimes not those with the best standard error, but those which have been
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Fig. 4. Box-plot of the observers’ differences.
Fig. 5. Box-plot of the difference among real age and the observers’ evaluations.
tested by many researchers on many different populations. Our investigation observed a high reliability among the observers, confirming that the method can be applied by professionals, after appropriate training. In addition, it is commonly believed that almost any physician is qualified to ‘‘read’’ X-rays and that any dentist can evaluate dental radiographs. However, the more highly qualified is a professional, the more weight that expert’s opinion will carry. Most juries will appreciate that diagnostic images must be interpreted and explained to them by a competent expert, familiar with both medical and technical factors applicable to the evidence. A physical anthropologist, for instance, may qualify as an expert to testify on radiographs of the skeleton, but will probably be required to
explain or document special training or experience in the use of this tool [32,33]. Other studies [23,27,34,35] have already discussed the importance and precision of the pulp/tooth ratio in canines by peri-apical X-rays for age assessment in adults. However, since then, very few published studies have validated the previous results of the technique. In particular, very little research has been carried out in order to determine intra- and inter-observer variations in analysis of the apposition of secondary dentine using the pulp/tooth area ratio in canines [36]. One study tested the accuracy of Camerierés method in medieval samples of unknown age at death, comparing the dental age estimation with other archaeological measurements [36]. Another one has performed 3D age estimations based on the
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pulp-tooth volume ratios of mono-radicular teeth, and more studies are necessary to reach precise results [37], on both ancient and today’s samples. As regards the first aim of this study, the final results showed that the pulp/tooth area ratio gradually decreases with age from 19 to 74 years, indicating age related narrowing of the pulp cavity from the cervical pulp chamber to the terminal part of the root canal. Nevertheless, real ages compared with estimated ones were found to be similar among the observers, demonstrating the high accuracy of measures. The mean differences between real and estimated ages were low, with slight over-estimation of age. The mean difference between real and estimated ages was about three years. This corroborated some previous results on two-dimensional measurements as age-related variables [38–42]. The coronal third of the root pulp is the most stable region in canines, with minimal morphological diversity and few influences due to various types of dental wear and other post-development changes, such as caries, especially if slowly progressing, or the use of restorative materials [43]. All these clinical factors have been reported to affect or be associated with post eruptive changes in dentine, resulting in enhanced mineralization [43]. The stability of this part of the root pulp should lead to few measurement errors and little inter observer variability. Regarding the reliability of Cameriere’s method, the measurements performed by our three observers did not differ (no statistical differences), showing good inter-observer agreement. This proves that the method is highly reproducible and may also be applied after proper training. The inter-observer evaluations revealed no statistically significant differences in the measurements of any of the observers, probably because they had had lengthy experience with the technique, and were familiar with age estimation techniques in particular. This is an important feature, considering the need for extensive international comparisons between the results provided by several observers from many different populations [5,44]. The present work also showed no significant influence of sex and age groups on age estimation, which was similar to the findings of previous studies [27,34–36]. However, several adaptations of current research set-ups could improve future results. Manual correction of the automatic selection of the layer PULP CHAMBER was most clearly required for the apical quarter of the tooth root. On these smallest tooth and pulp contours, the computerized distinction of tooth parts based on the grayscale threshold became less reliable, so that necessary checking and correcting procedures took up most of the time needed for image processing. In addition, manual operations may have a negative influence on the precision of pulp and tooth measures. More studies are necessary in order to test the method in other population groups in order to verify its accuracy, after training in order to reach a high degree of inter-observer concordance. 5. Conclusions Cameriere’s method is useful for age estimation and it presented high accuracy and reliability. Novelty statement Since 2004, Roberto Cameriere et al. have published several papers on a method of age estimation using the pulp/tooth area ratio to quantify the apposition of secondary dentine. A decrease in the area of the pulp cavity by secondary dentine formation is known to occur with age. The researchers reported extremely high regression coefficients (R2 = 0.94) when using labio-lingual X-ray images of upper and lower canines. The reliability of this method has recently been
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