Tissue Microarray Analysis Applied to Bone Diagenesis

Abstract

Taphonomic processes affecting bone post mortem are important in forensic, archaeological and palaeontological investigations. In this study, the application of tissue microarray (TMA) analysis to a sample of femoral bone specimens from 20 exhumed individuals of known period of burial and age at death is described. TMA allows multiplexing of subsamples, permitting standardized comparative analysis of adjacent sections in 3-D and of representative cross-sections of a large number of specimens. Standard hematoxylin and eosin, periodic acid-Schiff and silver methenamine, and picrosirius red staining, and CD31 and CD34 immunohistochemistry were applied to TMA sections. Osteocyte and osteocyte lacuna counts, percent bone matrix loss, and fungal spheroid element counts could be measured and collagen fibre bundles observed in all specimens. Decalcification with 7% nitric acid proceeded more rapidly than with 0.5 M EDTA and may offer better preservation of histological and cellular structure. No endothelial cells could be detected using CD31 and CD34 immunohistochemistry. Correlation between osteocytes per lacuna and age at death may reflect reported age-related responses to microdamage. Methodological limitations and caveats, and results of the TMA analysis of post mortem diagenesis in bone are discussed, and implications for DNA survival and recovery considered.

Figure 1

(a) Upper: Tissue microarray TMA-NA containing eleven exhumed and five control bone subsamples in duplicate, with a positive control (liver) for immunohistochemical analysis. Lower: Tissue microarray TMA-EA containing nine exhumed and four control bone subsamples, with a positive control (liver) for immunohistochemical analysis; (b) specimen 012 in TMA-NA showing osteocyte hot-spot location, ×400; (c) specimen 030 in TMA-EA with (d) same field illustrating identification of bone matrix loss in ImageJ, ×100; (e) specimen 030 in TMA-EA stained with PAS, ×100; (f) specimen 030 in TMA-EA stained with SM, ×100; (g) specimen 005 in TMA-NA immunohistochemically stained with anti-CD31, ×100; (h) specimen 005 in TMA-NA immunohistochemically stained with anti-CD34, ×100; (i) specimen 005 in TMA-NA stained with Picrosirius red, ×100; (j) control 019 in TMA-NA stained with Picrosirius red, ×100.

Figure 2

Charts showing plots and coefficients of correlation (R²) for selected variables: (a) Osteoctye count v. lacuna count (TMA-NA), (b) osteocyte count v. lacuna count (TMA-EA), (c) period of burial v. decalcification time (TMA-EA), (d) length v. decalcification time (TMA-EA), (e) osteocytes per lacuna v. age at death (TMA-NA), (f) osteocytes per lacuna v. age at death (TMA-EA).