Assessment of thyroid cartilage ossification processes by radiological methods in forensic identification

Introduction: There are different opinions about the existence of a relationship between age and the degree of ossification of the thyroid cartilage; some articles argue that this relationship is valid, while others argue that the influence of lifestyle, nutrition and genetics on the ossification processes of the thyroid cartilage is more important. In this framework, although it is considered that a combination of several methods is necessary for accurate estimation of age, it is important to clarify whether the examination of thyroid cartilage calcification using radiographic features can also be applied as a valid method among all methods. In this study, it is aimed to contribute to the thyroid cartilage ossification data pool of the Turkish population, which can be used for identification processes, based on the measurement of the throid cartilage ossification values among the Turkish population by CT method and evaluation of the changes according to age and gender through the statistical analysis of the obtained data. Method: In addition to the approval of Bezmialem Vakıf University Non-Interventional Clinical Research Ethics Committee dated 29/12/2020 and numbered 22/438; all retrospective and prospective procedures of this study followed the principles of the 1964 Declaration of Helsinki, with subsequent development versions. The sample group was formed with patients who underwent CT without contrast in the neck region, including the thyroid cartilage, between 01/01/2015 and 01/01/2021 at the Bezmialem Vakıf University Faculty of Medicine, Health Application and Research Center, Radiology Department. A total of 540 cases between the ages of 11 and 29 years who underwent non-contrast CT were identified from the records in a randomized manner in order to simulate the normal population distribution. However, randomizedly detected cases were evaluated according to their clinical data to see if they have any bias potential through either abnormal Ca-P values or any chronical disease directly related with Ca-P metabolism at the CT graphy formation time. 11 cases were found to have neoplasia diagnoses before CT graphy, however only one of them had had a 0.2-0.7 decrease in Ca level one year before the CT graphy and other Ca-P levels before CT graphy day were between normal values and two of these 11 neoplasia cases were not having Ca-P tests in their file. Except these three cases, eight neoplasia cases were having always normal Ca-P values before the CT graphy day. Among 529 remaining cases, 373 were having Ca-P tests in their file and all of them were with normal results and, moreover, acute reasons of CT referral (284 with injuries); 164 were found to have no Ca-P tests in their file but acute reasons of CT referral (154 with injuries). The potential but uncertain bias risks of these three neoplasia cases and remaining eight neoplasia cases with a lower bias risk were decided to be neglected. For the equal distribution of age groups, the cases were chosen among11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 and 29 year old age groups constituting 19 groups in total. Besides, equal distribution of both genders as 15 females and 15 males was taken into account in the determination of the groups. Computed tomography images were scanned retrospectively with the use of Synapse system and PACS (Picture Archiving and Communication System, Synaps Fujifilm, Japan) system, study cases were uploaded to Syngo.via (software version syngo.via VB30A_HF06, Siemens, Germany) workstation. All cases were opened at the Workstation and the number of ossified areas at the right and at the left side and the total ossified volume with cm3 unit at the right and at the left side for each case were calculated. The data obtained from the measurements were recorded in the 2013 Microsoft Office Excel program. The recorded values were transferred to the IBM SPSS 22.0 (Statistical Package for the Social Sciences, Armonk, NY, USA) program and the parameters of ossification onset age, ossification initiation site, number of ossified areas at the right and at the left side according to age groups, distribution of number of ossified areas at the right and at the left side by age-sex and distribution of total ossification volume at the right and at the left side by sex were analyzed. Before the data analysis, intra-observer and inter-observer compatibility tests were carried out. In data analysis, Chi-square, Mann Whitney U, Kruskal-Wallis, Post-hoc Dunn and non-parametric correlation tests were used as well as descriptive statistical applications. Intra-class Correlation Coefficient was used for interobserver evaluations. Significance level was accepted as p<0.05. Results: The earliest age of ossification was found to be 13 in women and 15 in men. In females, ossification onset was found to be most common in the posterior triangle and the upper half of the inferior horn on the right and the posterior triangle and the middle site of the posterior lamina on the left; in men, in the upper and lower halves of the inferior horn and the posterior triangular area on the right, and the posterior triangle, the upper half of the lower horn, and the middle site of the posterior lamina on the left. Accordingly, it was determined that the initial focus of ossification in women occurred at a higher level than in men, and when the symmetry status was evaluated, it was found to occur at a higher level on the left side than on the right for both genders. In general, the number of ossified regions were found to increase with age, except for the lower number of ossified regions in both sexes, more prominently in women, in the 21 and 28 age groups, and only in women in the 24 and 26 age groups –as an unexpected finding-. In addition, it was determined that the rate of increase in the number of ossified regions was higher in women until around the age of 17, while it was higher in men after this age and to increase in advancing ages. It was observed that the total number of ossification regions equalized at the age of 19, and then increased in men in direct proportion to the rate of increase. It was determined that the first ossification in the upper half of the anterior midline of the thyroid cartilage lamina was 16 years old in women, 19 years old in men while 25 years old in women and 17 years old in men in the lower half. Since the groups before the age of 29 and with one age difference were evaluated, parameters such as radiolucent window and reaching complete ossification which were related with the progression pattern according to ossified area numbers and pregnancy-maternity data which were absent in the clinical files were not evaluated. On both sides of the thyroid cartilage, a significant positive and moderately correlating relation between increasing age and measured ossification volume was determined regardless of gender (p˂0.001, r=0.622(right), r=0.589(left)); It was also determined that the ossification volume differences compared to later ages decreased as the age increased, however statistical analysis of this situation was not performed. In addition, the total volume of ossification was found to be statistically significantly higher on both sides in males than females (p˂0,001(right), p˂0,001(left)) Conclusion: In this study, contrary to the frequent data in the available literature, it was determined that ossification started as early as 13 years of age in women and 15 years in men. The facts that rate of increase in the ossifying region is higher in women until the age of 17, and afterwards in men; and the ossification volume is higher in men in the total process; and the difference in ossification volume compared to later ages decreases as age increases are remarkable findings. More importantly, although the significant positive and moderately correlating relation between age growth and increased ossification volume regardless of gender suggests that measuring ossification volume may normally provide a use for age estimation in the identification process, when the internal and external distribution differences of the groups are examined individually, it was observed that the variability between individuals is too large to support a direct and strong correlation with age. Therefore, according to the results of this study, due to the moderately correlating, not a statistically high relation achieved, it can be said that the place of thyroid cartilage ossification among age estimation methods is still preserved as an additional method of assistance for conventional age determination perfomed today. New interpretations for thyroid cartilailage ossification will be possible with advanced examinations such as 3D tests, electrical tissue resistance measurement and meta-analyses of powerful bioinformatics.