Age diagnosis based on incremental lines in dental cementum: a critical reflection

Age estimation based on the counting of incremental lines in dental cementum is a method frequently used for the estimation of the age at death for humans in bioarchaeology, and increasingly, forensic anthropology. Assessment of applicability, precision, and method reproducibility continue to be the focus of research in this area, and are occasionally accompanied by significant controversy. Differences in methodological techniques for data collection (e.g. number of sections, factor of magnification for counting or interpreting "outliers") are presented. Potential influences on method reliability are discussed, especially for their applicability in forensic contexts.     

Incremental lines in root cementum of human teeth —A reliable age marker?

In root cementum of teeth, alternating dark and light lines become visible in cross-sections under the light microscope. These lines bear an apparent resemblance to the annual rings of trees. Numerous studies have been done to correlate the number of cementum lines with the dental age by examining a great number of teeth of known age. Our study used a different approach. If lines in root cementum develop in an annual rhythm and are thus comparable to annual rings of trees, the same or at least a very similar number of these structures should be found in all areas of the root cementum of the same tooth. We counted cementum lines in the buccal, lingual, distal and mesial region of different sections, all from the middle third of the same root. This was repeated in eight teeth. To our surprise, we had immense difficulty in counting reproducible line numbers in the same cementum area at repeated counts. Nevertheless, the same tooth was found to differ markedly in the number of lines in different sections as well as in different regions of the same sections. These differences cannot be ascribed to variations caused by difficulties with reproducible line counting. Therefore, we are more than skeptical about the reliability of counting lines in root cementum as a method for determining the age of human teeth.     

Tooth cementum annulation for age estimation: results from a large known-age validation study

Recent research indicates that tooth-cementum annulations (TCA) may be used more reliably than other morphological or histological traits of the adult skeleton to estimate age. Until now, however, confidence intervals for age estimated by this method have not been available for paleodemographic and forensic applications. The present study addresses this problem. Based on a large known-age sample, age estimates by TCA were conducted in a blind study involving 363 teeth. Tooth-root cross sections were made using a refined preparation technique. Improved digital graphic procedures and enhancement strategies were used to produce digital images with a specially adapted software package. This resulted in high concordance between the TCA age estimates and chronological age. Assessment of the method's accuracy, as expressed by 95% confidence intervals, showed that error bounds for age estimates do not exceed 2.5 years. Sex differences, intraindividual correlations, and the effects of periodontal disease were studied. None of these indicators had a quantitative effect on the number of TCA bands when the proposed methodological standard was followed. We conclude that the TCA technique is a reliable method for estimating a subject's age from cementum annulations.     

Cementum annulations,age estimation,and demographic dynamics in Mid-Holocene foragers of North India

One of the principal problems facing palaeodemography is age estimation in adult skeletons and the centrist tendency that affects many age estimation methods by artificially increasing the proportion of individuals in the 30–45-year age category. Several recent publications have indicated that cementum annulations are significantly correlated with known age of extraction or death. This study addresses the question of how demographic dynamics are altered for an archaeological sample when cementum-based age estimates are used as opposed to those obtained via conventional macroscopic methods. Age pyramids were constructed and demographic profiles were compared for the early Holocene skeletal population from Damdama (India). The results demonstrate that the use of cementum annulations for age estimation in only a subset of the skeletal sample has a significant impact on the demographic profile with regard to specific parameters such as mean age at death and life expectancy at birth. This confirms the importance of using cementum annulations to refine age estimates in archaeological samples, which, when combined with a fertility-centred approach to demography, can provide new insights into population dynamics in the past.     

Cementum annulus counts provide a means for age determination in Macaca mulatta (primates, anthropoidea)

14 teeth of 8 rhesus macaques (Macaca mulatta) of known age were analyzed to assess the usefulness of cementum annuli counts as a means of estimating chronological age. Methods used were histological examination of stained thin sections by light microscopy, and examination of polished and etched epoxy-embedded sections by scanning electron microscopy. In 11 of 14 cases, the known chronological ages of the individuals fell within the predicted age ranges based on cementum annuli counts; in 2 other cases, it fell within half a year of the ranges. Cementum annulus counts can provide valuable information about the age of primates living in semitropical environments. This is the most accurate method for aging skeletally adult primates that has yet been tested on animals of known age.     

A simple,low-cost method to age mammals? An alternative to cementum annuli analysis

One of the most common and ubiquitous methods to age mammals is by counting the cementum annuli in molars, premolars, incisors, or canines. Despite the ubiquity and perceived simplicity of the method, cementum annuli analysis can be time-consuming, expensive, inaccurate, and imprecise, and require specialized equipment. Using beavers (Castor canadensis) as a test species, we developed a straightforward method to age mammals that requires little specialized equipment. The method consists of: (1) digitizing longitudinally sectioned teeth and measuring the proportion of tooth surface area comprised of cementum (“proportion cementum”), (2) evaluating the relationship between proportion cementum and specimen age (determined from either known-age samples or cementum annuli analysis), and (3) using the modeled relationship to estimate the age of other individuals based solely on proportion cementum. The relationship between proportion cementum and age was strongly correlated (R² = .97–.98 depending on observer), similar between observers, and similar between known-age specimens and those aged via cementum annuli analysis. Using this proportion cementum method, two independent observers accurately predicted the age of 80%–84% of specimens within 0.5 year and 96%–98% within 1 year. We suggest this aging method will likely work with most mammal species given the relatively consistent deposition of cementum throughout mammals' lives and has promise to be a simple and quick alternative to cementum annuli analysis regardless of whether one develops proportion cementum models using known-age specimens or those aged via alternative methods.     

Basics in paleodemography: a comparison of age indicators applied to the early medieval skeletal sample of Lauchheim

Recent advances in the methods of skeletal age estimation have rekindled interest in their applicability to paleodemography. The current study contributes to the discussion by applying several long established as well as recently developed or refined aging methods to a subsample of 121 adult skeletons from the early medieval cemetery of Lauchheim. The skeletal remains were analyzed by 13 independent observers using a variety of aging techniques (complex method and other multimethod approaches, Transition Analysis, cranial suture closure, auricular surface method, osteon density method, tooth root translucency measurement, and tooth cementum annulation counting). The age ranges and mean age estimations were compared and results indicate that all methods showed smaller age ranges for the younger individuals, but broader age ranges for the older age groups.     

Age estimation of the White rhinoceros (Ceratotherium simum)

Age estimation criteria for the southern White rhinoceros ( Ceratotherium simum simum ) are presented both for free-ranging live animals and for cranial material. These are based on: (i) size appearance and horn development of live animals; (ii) stages of tooth eruption; (iii) tooth wear classes; (iv) attrition in height of the first molar tooth; (v) counts of cementum lines visible in tooth sections. Selected measurements are presented for live animals, skulls and horns.
For live animals, eight size classes are distinguished, seven of these covering immature animals up to ten years of age. Sixteen tooth wear classes are established, based on eruption and surface wear of maxillary dentition. Chronological ages were assigned from individually known animals followed in the field, and from skulls from animals for which exact records of age were available, or which could be assigned to an age category from appearance at death. Cementum line counts corresponded approximately with age in years, despite difficulties in interpreting lines. Some variability was observed, possibly related to nutritional conditions.
The maximum cementum line count obtained indicates a longevity of at least 40 years. Full body weight and socio-sexual maturity are attained by males between 10 and 15 years of age, while females first give birth between six and eight years of age. Sequences and times of tooth eruption are similar to those reported for the Black rhinoceros ( Diceros bicornis ).
Comparative cranial and body measurements are presented for the northern subspecies ( Ceratotherium simum cottoni ).     

Counting pollen grains using readily available, free image processing and analysis software

Background and Aims

Although many methods exist for quantifying the number of pollen grains in a sample, there are few standard methods that are user-friendly, inexpensive and reliable. The present contribution describes a new method of counting pollen using readily available, free image processing and analysis software.

Methods

Pollen was collected from anthers of two species, Carduus acanthoides and C. nutans (Asteraceae), then illuminated on slides and digitally photographed through a stereomicroscope. Using ImageJ (NIH), these digital images were processed to remove noise and sharpen individual pollen grains, then analysed to obtain a reliable total count of the number of grains present in the image. A macro was developed to analyse multiple images together. To assess the accuracy and consistency of pollen counting by ImageJ analysis, counts were compared with those made by the human eye.

Key Results and Conclusions

Image analysis produced pollen counts in 60 s or less per image, considerably faster than counting with the human eye (5–68 min). In addition, counts produced with the ImageJ procedure were similar to those obtained by eye. Because count parameters are adjustable, this image analysis protocol may be used for many other plant species. Thus, the method provides a quick, inexpensive and reliable solution to counting pollen from digital images, not only reducing the chance of error but also substantially lowering labour requirements.     

Age estimation based on blood DNA methylation levels in brown bears

Age is an essential trait for understanding the ecology and management of wildlife. A conventional method of estimating age in wild animals is counting annuli formed in the cementum of teeth. This method has been used in bears despite some disadvantages, such as high invasiveness and the requirement for experienced observers. In this study, we established a novel age estimation method based on DNA methylation levels using blood collected from 49 brown bears of known ages living in both captivity and the wild. We performed bisulfite pyrosequencing and obtained methylation levels at 39 cytosine-phosphate-guanine (CpG) sites adjacent to 12 genes. The methylation levels of CpGs adjacent to four genes showed a significant correlation with age. The best model was based on DNA methylation levels at just four CpG sites adjacent to a single gene, SLC12A5, and it had high accuracy with a mean absolute error of 1.3 years and median absolute error of 1.0 year after leave-one-out cross-validation. This model represents the first epigenetic method of age estimation in brown bears, which provides benefits over tooth-based methods, including high accuracy, less invasiveness, and a simple procedure. Our model has the potential for application to other bear species, which will greatly improve ecological research, conservation, and management.     

Evaluation of Preparation, Staining and Microscopic Techniques for Counting Incremental Lines in Cementum of Human Teeth

Apposition of cementum occurs in phases resulting in two types of layers with different optical and staining properties that can be observed by light microscopy. Narrow, dark staining incremental lines are separated by wider bands of pale staining cementum. The distance from one line to the next represents a yearly increment deposit of cementum in many mammals, and counting these lines has been used routinely to estimate the age of the animals. Incremental lines in cementum have also been observed in sections of human teeth, and the object of the present investigation was to examine a number of methods for preparing and staining them for counting. Longitudinal and transverse sections, either ground or decalcified, were cut from formalin fixed human dental roots, paraffin embedded or frozen, and stained using several techniques. The cementum was investigated using conventional light, fluorescence, polarized light, confocal laser scanning, interference contrast, phase contrast, and scanning electron microscopy. Incremental lines in the cementum could be observed in ground sections and, following decalcification, in both frozen and paraffin embedded sections. Toluidine blue, cresyl violet, hematoxylin, or periodic acid Schiff (PAS) stained incremental lines allowing differentiation by conventional light microscopy. Contrast was best using fluorescence microscopy and excitation by green light since the stained cemental bands, but not the incremental lines, fluoresced after staining with cresyl violet, PAS or hematoxylin and eosin. The results with other microscopic techniques were unsatisfactory. Since incremental lines are not destroyed by acids and stain differently than the remaining cementum, it is likely that they possess an organic structure which differs from the cementum. Incremental lines in human dental cementum could be observed best using decalcified sections stained with cresyl violet excited by green light.     

EVALUATION OF AN EFFECTIVE METHOD TO ESTIMATE AGE OF CAPE FUR SEALS USING GROUND TOOTH SECTIONS

Teeth of known-age Cape fur seals Arctocephalus pusillus pusillus were used to validate age estimated from ground sections. In the canines, dentine growth layer groups (GLGs) reflected age accurately but no reliable readings could be obtained from GLGs in the cementum. Upper canines were the most suitable for age estimation. By contrast, in the postcanines where the cementum is thicker, only GLGs in the cementum could be used for age determination, but not with the same accuracy as for dentine in the canines. Therefore, it is recommended that GLGs in the dentine be used to determine age in the Cape fur seal. However, pulp cavities in canines closed at about 13 yr and consequently GLGs in the cementum of the postcanines should be used where the pulp cavities of canines are closed. Accurate estimation of age is not possible from the dentine of older animals.     

Age determination of the African buffalo, Syncerus coffer (Sparrman) in Zimbabwe

Age determination methods using teeth are described for African buffalo in Matusadona National Park, Kariba, Zimbabwe. Tooth eruption stages and age classes for sub-adult animals were established by reference to a seasonal breeding peak and known dates of death. Teeth in adult animals were allocated age classes based on wear using measurements of crown height and by reference to previously published material. Layering in the dental cement indicated the deposition of one cementum line per annum and line counts were used to assign chronological ages to individual animals. Regressions of wear on age were confirmed by independent counts of cementum lines using a predictive wear model.     

Quantitative analysis of disseminated tumor cells in the bone marrow by automated fluorescence image analysis

Accurate quantification of disseminated tumor cells in hematological samples is of fundamental importance in clinical oncology. However, even highly standardized protocols allow only a rough estimation of the total analyzed cell number, as sample processing may have adverse effects on the number of cells available for analysis. The fluorescence-based microscopic scanning system (MetaCyte) detects, counts, captures, and relocates immunolabeled tumor cells in hematopoietic samples. We report on a cell-counting approach that has been implemented into the scanning system to precisely quantify the number of cells per slide. The cell-counting function, which was designed to determine the number of all nucleated (DAPI-stained) cells on the slide, allows an accurate counting of the tumor cells and the total number of cells analyzed in the given microscopic sample. The reliability of the cell-counting approach was demonstrated by the analysis of DAPI-stained images with 18-1,363 nucleated cells. A good correlation (r(2) = 0.965) between the manually and automatically gained results was observed. The counting accuracy could even be optimized after implementing a correction factor. To prove or disprove an interslide variation, routine bone marrow cytospin preparations from neuroblastoma patients were immunostained for GD2/FITC and counterstained with DAPI. Automatic cell counting of cytospin preparations from the same patients showed significant differences in the total cell number (up to 67% cell loss during preparation, with a maximum interslide difference of 4.7 x 10(5) mononuclear cells). We conclude that determination of the tumor cell content in hematopoietic samples is only reliable when it is performed together with accurate cell counting.     

Incremental lines of dental cementum in biological age estimation

The aim of this study is to evaluate the use of counting incremental lines of dental root cementum for biological age determination, and to compare it with alternative methods. Two samples were taken: 51 teeth from 49 individuals of known age obtained at the Stomatological Clinic, Vilnius University, as well as the canine teeth from the remains of 48 individuals from the mass graves of Tuskulenai in Vilnius (inhumed 1944-47). In the latter sample, the chronological age of 43 individuals was known through personal identification. Undecalcified teeth were sectioned with the Leica SP 1600 microtome diamond saw, and incremental line count as a blind test was made on sections of 35 to 100 microns thickness. Incremental line count was possible in 82-86 percent of cases. The results of three independent counts showed that intra-observer bias has no significant impact. Biological age was estimated by adding incremental line number to the average age of tooth eruption. It was found that mean absolute error was 6.46 years for the 1st sample, 6.27 years for the 2nd sample, and in some cases exceeded 10 years. For the 2nd sample, the results were compared to those of other methods such as endocranial suture ossification, pubic symphysis morphology and the "combined" method of Nemeskéri. All four methods yield a similar correlation in regard to an individual's chronological age. The highest correlation was found for the combined method, and the lowest one for public symphysis morphology. All correlations had a similar standard error. Thus our assessment is less enthusiastic than in some past studies; it is suggested that the incremental lines rather have a similar use as other methods.     

COUNTS OF GROWTH LAYER GROUPS IN CEMENTUM AND DENTINE IN RINGED SEALS (PHOCA HISPIDA)

We compared counts of growth layer groups (GLGs) in the dentine of un-decalcified, unstained cross-sections and in the cementum of decalcified, stained longitudinal sections of canine teeth from 144 ringed seals ( Phoca hispida ). Although there was a statistically significant correlation until approximately 10 GLGs, about 75% of paired readings at ≤ 10 cementum GLGs disagreed. After 10 GLGs, the number of GLGs in the cementum usually was greater. The maximum GLG count in cementum was 33, compared to a maximum in dentine of only 19. Interobserver differences in median counts were not statistically significant using cementum or dentine counts. Regression analysis revealed that for cementum in female seals, readers differed at higher counts (P < 0.05), and for dentine, there was a constant difference of about 0.6 GLGs ( P < 0.05) for male seals and 1.1 GLGs ( P < 0.05) for female seals. Counting GLGs in the cementum of decalcified and stained longitudinal sections provided higher counts and more agreement between readers, and it was the better of the methods examined for ageing ringed seals.     

鲫耳石重量与年龄的关系及其在年龄鉴定中的作用

耳石重量在年龄组间重叠较少,大小相近的个体,年龄大的,即生长慢的耳石重量比年龄小的,即生长快的大,不同龄组之间耳石重量有显著差异（P＜0．05）,按年龄组在耳石重量与相应的体长作图,可初步判断观测年龄的可靠性,分析耳石重量频率分布能分离出体长相近,年龄不同的个体,其结构与耳石年轮观测的基本一致,耳石重量与年龄呈显著线性正相关（P＜0．05）,用耳石重量与年龄关系估算的年龄从耳石上直接读取的年龄无显著差异（P＞0．05）,文中对耳石重量直接用于确定鱼类年龄的可能性作了分析和探讨。     

Pulmonary regional ventilation. C15O2 single breath method. New technical improvements

After a single inhalation of C15O2 labelled air, from residual volume to total lung capacity, followed by a few seconds apnea, it is possible to record in four or six lung zones the regional activity changes on a time basis. A first fast rise in activity is seen during the inhalation phase, corresponding to the ventilation in the region of interest (ROI). During the apnea, a slower decreasing component is observed in the different ROI, proportional to the regional blood flow, as almost instantaneously C15O2 is converted to labelled water. After the pioneer work of West & Dollery (1962) very few papers appeared on the subject despite technical improvements as, for instance, coincidence counting of radioactive events. We thus decided to improve the reproducibility and accuracy of the method which were of such a poor quality that no individual measurements were possible in the past. Were of critical importance: a careful positioning of the patients between the counters, a faster electronics improving true coincidence counting. In six healthy volunteers (age 22-35) the values obtained for regional ventilation with the C15O2 single breath were compared to the values given by the inhalation of a poorly soluble gas (Nitrogen13). The correlation between the two sets of measurements is high (r = 0.975). The slope of the regression line is 0.859, ordinate at origin is 2.35. This reveals a systematic error. A mathematical model was therefore developed to take into account the "washout" occurring during the constitution of the ventilation peak, and to correct its value.(ABSTRACT TRUNCATED AT 250 WORDS)     

The assessment of the subjective second by using the "individual minute" test]

Correlation of the two characteristics of the "individual minute" test with age and sex of the subjects was examined in 151 persons. Close correlation was observed between the first (counting in the course of 60 s) and the second (counting time up to 60 s) modes of testing, but not between the two modes of testing and age and sex. Results are discussed from the viewpoint of the existence of the common system of time interval estimation the method of their quantum organization being used in different investigation modes.     

Estimating red deer Cervus elaphus populations: an analysis of variation and cost-effectiveness of counting methods

• 1 Different counting methods are currently used to estimate red deer populations in the open range in Scotland, but there are few data available to compare variation in estimates, or relative cost‐effectiveness.
• 2 While it is impossible to determine the accuracy of counts (as real numbers are unknown), variation within and between different methods can be measured by repeat counts of the same area within as short a period as possible.
• 3 This study aimed to quantify the variation observed from repeat counts using each of four methods (ground, helicopter, infrared helicopter and dung‐counting methods) at one of three study sites in late winters 2003, 2004 and 2005. Additional data from digital camera images of groups from counts in other areas of Scotland were also used to assess the accuracy of visual counts.
• 4 Coefficients of variation (CVs) within any method of between 5% and 16% were recorded, consistent with previous comparisons for red deer open range counts in Scotland. CVs were lowest for ground and helicopter counts. The infrequency of optimal conditions was likely to limit the applicability of infrared counts in Scotland.
• 5 In terms of cost‐effectiveness, helicopter counting was the least labour‐intensive, with costs of other techniques depending on the availability of existing manpower as an overhead cost.
• 6 It is concluded that helicopter counts are most likely to minimize errors while maximizing cost‐efficiency. Accuracy can be improved by the use of digital photography for counting larger deer groups. Estimates are likely to be improved further by increasing the frequency of counts and using the same methods, counters and routes for repeat counts.