Histological age-at-death estimation in white South Africans using stereology

  • D. BothaEmail author
  • M. Steyn
  • N. Lynnerup
Original Article


Various methods are available for estimating age from skeletal remains, amongst them the use of histomorphometry. It is generally argued that age estimation standards are population specific, but this in itself creates problems as the reference samples used are often not large enough and/or lack substantial representation of all age cohorts. Traditional age methods have been shown to suffer from problems such as age mimicry. This paper aims at establishing histological age-at-death standards for the white South African population by supplementing the available sample (lacking an adequate number of young adults) with another sample of European descent to avoid over-estimation of age in younger individuals caused by age mimicry. Bone microstructures related to the number of osteons and fragments, osteon size and Haversian canal size that change with advancing age were used for the development of regression formulae. A histomorphometric assessment of the anterior cortex of the femur was done using stereology for the estimation of age at death. All sections were analysed using the optical fractionator and nucleator probes. A sample of 94 bone sections (n = 50 male, n = 44 females) of white South African individuals were used. A sample of Danish individuals (n = 14 males, n = 16 females) was combined with the South African sample to create a normal age distribution for the reference sample. Single and multiple regression equations were developed after randomly selecting a hold-out sample (n = 14) for validation. Osteon size (average length, surface area and volume) showed the highest correlation with age, followed by the number of osteons and fragments per grid area. Haversian canal size showed inconsistent changes with advancing age. Using the regression equations, predicted ages were obtained for the 14 individuals. RMSE values ranged between 14 and 17 years, which we deemed acceptable.


Femur Age estimation Stereology Linear regression Age mimicry 



The authors would like to thank the curators of the Pretoria Bone Collection and Raymond A. Dart Skeletal Collection for granting access to the remains, Prof. Paul Manger for use of the stereology system and Dr. A. Bhagwandin for assisting with the production of the stereology images.

Funding information

This project received financial support from the National Research Foundation.

Compliance with ethical standards

Conflict of interest

The authors declare they have no conflict of interest.

Statement of informed consent

Ethical approval for the project was obtained through the University of the Witwatersrand.


  1. 1.
    Garvin HM, Passalqua NV, Uhl NM, Gipson DR, Overbury RS, Cabo LL (2012) Developments on forensic anthropology: age-at-death estimation. In: Dirkmaat DC (ed) A companion to forensic anthropology. Blackwell Publishing, UK, pp 202–238CrossRefGoogle Scholar
  2. 2.
    Bocquet-Appel JP, Masset C (1982) Farewell to paleodemography. J Hum Evol 11:321–333CrossRefGoogle Scholar
  3. 3.
    Konigsberg LW, Frankenberg SR (1992) Estimation of age structure in anthropological demography. Am J Phys Anthropol 89:235–256CrossRefGoogle Scholar
  4. 4.
    Boldsen JL, Milner GR, Konigsberg JW, Wood JW (2002) Transition analysis: a new method for estimating age from skeletons. In: Hoppa RD, Vaupel JW (eds) Paleodemography: age distributions from skeletal samples. Cambridge University Press, pp 73–106Google Scholar
  5. 5.
    Cho H, Stout SD, Madsen RW, Streeter MA (2002) Population-specific histological age-estimating method: a model for known African-American and European-American skeletal remains. J Forensic Sci 47:12–18CrossRefGoogle Scholar
  6. 6.
    Cool SM, Hendrikz JK, Wood WB (1995) Microscopic age changes in the human occipital bone. J Forensic Sci 40:789–796CrossRefGoogle Scholar
  7. 7.
    Singh IJ, Gunberg DL (1970) Estimation of age at death in human males from quantitative histology of bone fragments. Am J Phys Anthropol 33:373–382CrossRefGoogle Scholar
  8. 8.
    Drusini A, Businaro F (1990) Skeletal age determination by mandibular histomorphometry. Int J Anthropol 5:235–243CrossRefGoogle Scholar
  9. 9.
    Stout SD, Porro MA, Perotti B (1996) Brief communication: a test and correction of the clavicle method of Stout and Paine for histological age estimation of skeletal remains. Am J Phys Anthropol 100:139–142CrossRefGoogle Scholar
  10. 10.
    Lee U-Y, Jung G-U, Choi S-G, Kim Y-S (2014) Anthropological age estimation with bone histomorphometry from the human clavicle. Anthropologist 17:929–936CrossRefGoogle Scholar
  11. 11.
    Kimura K (1992) Estimation of age at death from second metacarpals. Z Morph Anthropol 79:169–181Google Scholar
  12. 12.
    Stout SD, Dietze WH, İşcan MY, Loth SR (1994) Estimation of age at death using cortical histomorphometry of the sternal end of the fourth rib. J Forensic Sci 39:778–784CrossRefGoogle Scholar
  13. 13.
    Crowder C, Heinrich J, Stout SD (2012) Rib histomorphometry for adult age estimation. In: Bell LS (ed) Forensic microscopy for skeletal tissues: methods and protocols. Springer Science and Business Media, New York, pp 109–127CrossRefGoogle Scholar
  14. 14.
    Thompson DD (1979) The core technique in the determination of age at death of skeletons. J Forensic Sci 24:902–915CrossRefGoogle Scholar
  15. 15.
    Yoshino M, Imaizumi K, Miyasaka S, Seta S (1994) Histological estimation of age at death using microradiographs of humeral compact bone. Forensic Sci Int 64:191–198CrossRefGoogle Scholar
  16. 16.
    Kerley ER (1965) The microscopic determination of age in human bone. Am J Phys Anthropol 23:149–164CrossRefGoogle Scholar
  17. 17.
    Narasaki S (1990) Estimation of age at death by femoral osteon remodelling: application of Thompson’s core technique to modern Japanese. J Anthropol Soc Nippon 98:29–38CrossRefGoogle Scholar
  18. 18.
    Ericksen MF (1991) Histological estimation of age at death using the anterior cortex of the femur. Am J Phys Anthropol 84:171–179CrossRefGoogle Scholar
  19. 19.
    Watanabe Y, Konishi M, Shimada M, Ohara H, Iwamoto S (1998) Estimation of age from the femur of Japanese cadavers. Forensic Sci Int 98:55–65CrossRefGoogle Scholar
  20. 20.
    Keough N, L’Abbé EN, Steyn M (2009) The evaluation of age-related histomorphometric variables in a cadaver sample of lower socioeconomic status: implications for estimating age at death. Forensic Sci Int 191:114.e1–114.e6CrossRefGoogle Scholar
  21. 21.
    Hauser R, Barres D, Durigon M, Derobert L (1980) Identification par l’histomorphometrie du femur et du tibia. Acta Med Leg Soc 30:91–97Google Scholar
  22. 22.
    Kerley ER, Ubelaker DH (1978) Revisions in the microscopic method of estimating age at death in human cortical bone. Am J Phys Anthropol 49:545–546CrossRefGoogle Scholar
  23. 23.
    Stout SD, Paine RR (1992) Histological age estimation using rib and clavicle. Am J Phys Anthropol 87:111–115CrossRefGoogle Scholar
  24. 24.
    Robling AG, Stout SD (2008) Histomorphometry of human cortical bone: applications to age estimation. In: Katzenberg MA, Saunders S (eds) Biological anthropology of the human skeleton. Wiley, Hoboken, pp 149–182CrossRefGoogle Scholar
  25. 25.
    Crowder C, Heinrich J, Dominquez V (2009) Histological age estimation. In: Blau S, Ubelaker D (eds) Handbook of forensic anthropology and archaeology. Left Coast Press, Walnut Creek, pp 222–235Google Scholar
  26. 26.
    Pfeiffer S, Heinrich J, Beresheim A, Alblas M (2016) Cortical bone histomorphometry of known-age skeletons from the Kirsten Collection, Stellenbosch University, South Africa. Am J Phys Anthropol 160:137–147CrossRefGoogle Scholar
  27. 27.
    Black J, Mattson R, Korostoff E (1974) Haversian osteons: size, distribution, internal structure and orientation. J Biomed Mater Res 8:299–319CrossRefGoogle Scholar
  28. 28.
    Pfeiffer S, Crowder C, Harrington L, Brown M (2006) Secondary osteon and Haversian canal dimensions as behavioral indicators. Am J Phys Anthropol 131:460–468CrossRefGoogle Scholar
  29. 29.
    Howard CV, Reed M (2005) Unbiased stereology: three-dimensional measurements in microscopy, 2nd edn. New York, BIOS Scientific PublishersGoogle Scholar
  30. 30.
    Botha D, Lynnerup N, Steyn M (submitted) Inter-population variation of histomorphometric variables used in the estimation of age-at-death. Int J Leg MedGoogle Scholar
  31. 31.
    L’Abbé EN, Loots M, Meiring JH (2005) The Pretoria bone collection: a modern South African skeletal sample. HOMO – J Comp Hum Biol 56:197–205CrossRefGoogle Scholar
  32. 32.
    Maat GJR, Van den Bos RPM, Aarents MJ (2002) Manual for the preparation of ground sections for the microscopy of bone tissue. Barge’s Anthropologica Nr. 7. Leiden University Medical Centre: Leiden, NetherlandsGoogle Scholar
  33. 33.
    Villa C, Lynnerup N (2009) A stereological analysis of the cross-sectional variability of the femoral osteon population. Am J Phys Anthropol 142:491–496CrossRefGoogle Scholar
  34. 34.
    West MJ (2012) Introduction to stereology. Cold Spring Harb Protoc 2012:843–851Google Scholar
  35. 35.
    Botha D, Bhagwandin A, Lynnerup N, Steyn M (2018) The use of stereological methods in the histomorphometric assessment of bone for age-at-death estimation. Forensic Sci Int 290:353.e1–353.e7CrossRefGoogle Scholar
  36. 36.
    West MJ, Slomianka L, Gundersen HJG (1991) Unbiased stereological estimation of the total number of neurons in the subdivisions of the rat hippocampus using the optical fractionator. Anat Rec 231:482–497CrossRefGoogle Scholar
  37. 37.
    Gundersen HJG (1977) Notes on the estimation of the numerical density of arbitrary profiles: the edge effect. J Microsc 111:219–223CrossRefGoogle Scholar
  38. 38.
    Gundersen HJG, Bagger P, Bendtsen S et al (1988) The new stereological tools: disector, fractionator, nucleator and point sampled intercepts and their use in pathological research and diagnosis. APMIS 96:857–881CrossRefGoogle Scholar
  39. 39.
    Seber GAF, Lee AJ (2003) Linear regression analysis, 2nd edn. Hoboken, WileyCrossRefGoogle Scholar
  40. 40.
    Rankin G, Stokes M (1998) Statistical analysis of reliability studies. Clin Rehab 12:187–199CrossRefGoogle Scholar
  41. 41.
    Maat GJR, Maes A, Aarents MJ, Nagelkerke NJD (2006) Histological age prediction from the femur in a contemporary Dutch sample: the decrease of nonremodeled bone in the anterior cortex. J Forensic Sci 51:230–237CrossRefGoogle Scholar
  42. 42.
    Aykroyd RG, Lucy D, Pollard AM, Solheim T (1997) Technical note: regression analysis in adult age estimation. Am J Phys Anthropol 104:259–265CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Human Variation and Identification Research Unit, School of Anatomical SciencesUniversity of the WitwatersrandJohannesburgSouth Africa
  2. 2.Department of Forensic PathologyUniversity of CopenhagenCopenhagenDenmark

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