AMERICAN JOURNAL OF PHYSICAL ANTHROPOLOGY 152:566–568 (2013)
Technical Note: A Histological Technique for Detecting
the Cryptic Preservation of Erythrocytes and Soft Tissue
in Ancient Human Skeletonized Remains
Teddi J. Setzer,1* Inger Birgitta Sundell,1 Susan K. Dibbley,1 and Clifford Les2
1
Department of Anthropology, Wayne State University, Detroit, MI 48201
2
Henry Ford Bone and Joint Center, Detroit, MI 48202
KEY WORDS bone marrow; ancient blood; skeletonized remains; paleohistology;
decomposition
ABSTRACT Bone samples from a Middle Bronze Age
(ca., 1600–1300 BC) site were prepared for histological analy-
sis. Preliminary results suggested that components of bone
marrow remained preserved. To verify these findings and
optimize the sample preparation procedure, we conducted
experiments varying the type of acid used to decalcify the
bones for histology preparation, as well as the exposure time
to the demineralizing agents and thickness of sections taken
It is presumed that blood and other soft tissues are
usually destroyed during the decomposition of an orga-
nism (Zimmerman, 1973; Custer and Hayhoe, 1974;
Schultz, 2001, 2011). However, recent studies utilizing
bone tissue provide results that challenge this assump-
tion. For example, using scanning and transmission elec-
tron microscopy (SEM and TEM), McNamara et al.
(2006) observed bone marrow preservation in 10 Ma fos-
silized amphibians. Schweitzer et al. (2005, 2007)
detected possible erythrocytes and vessels when examin-
ing demineralized bone fragments from the Triassic
through contemporary time periods. Similar histological
observations have been made with human bone samples.
Stout and Teitelbaum (1976) reported finding structures
consistent with erythrocytes in an undecalcified, stained,
10 lm section of a vertebral fragment. SEM analysis of
skeletal remains by Maat (1991) provided evidence of
erythrocyte preservation within marrow cavities. More
recently, blood and brain tissue have been observed
adhering to skeletonized remains (Charlier et al., 2008;
Prats-Mu~ noz et al., 2012). McNamara et al. (2006) origi-
nally attributed the marrow preservation they observed
to site- and sample-specific (i.e., preserved skin provid-
ing a protective barrier from decomposition processes)
conditions. However, upon consideration of corroborative
findings, McNamara et al. (2012) concluded that the
preservation of soft tissue is not uncommon and can
occur under a variety of environmental conditions.
Although early histological studies of bone from
archaeological sites involved decalcifying the sample,
paleohistologists moved away from this practice because
the process can degrade or damage ancient bone speci-
mens (Schultz, 2001, 2011). Instead, researchers began
preparing ground sections of undecalcified bone. This
method did not compromise the architecture of the bone,
but it obstructed the detection of cellular components,
which are more readily examined using stained, decalci-
Ó 2013 WILEY PERIODICALS, INC.
from the samples for slide preparation. Subsequent examina-
tion of the slides revealed the presence of well-preserved
erythrocytes and other cellular structures consistent with
bone marrow. Our results demonstrate that the traditional
methods used to prepare bone samples for histology may be
adjusted to improve the quality of the soft tissue architecture
and cellular morphology for histological observation. Am J
Phys Anthropol 152:566–568, 2013. VC 2013 Wiley Periodicals, Inc.
fied bone tissue. While some (de Boer et al., 2012) argue
that staining can enhance visualization in paleopatho-
logical studies, others (Schultz, 2011) note that diage-
netic and taphonomic processes can prevent the stains
from working properly. The study of undecalcified,
unstained bone remains a conventional method for
examining ancient specimens (Hollund et al., 2011;
Schultz, 2011).
A bioarchaeological study (Setzer, 2010) that revealed
a high rate of porotic hyperostosis in a population
prompted a histological investigation. We observed struc-
tures suggestive of erythrocyte and bone marrow preser-
vation during microscopic analysis. To verify the
presence and improve retrieval of soft tissue cells, we
experimented with histological methods involving decal-
cified bone samples.
MATERIALS
Serra ‘e sa Caudeba is a Bronze Age complex of two
tombs on the Campidano Plain of Sardinia, Italy. Bones
from this site were excavated from a soil matrix in the
early 1980s. Modern soil samples from this region are
composed of 41.7% sand, 22.1% silt, 36.2% clay, with a
pH 5 7 and soil organic content of 1.6% with 2.7% soil
organic matter, including high levels of the endemic
*Correspondence to: Teddi J. Setzer, Department of Anthropology,
Wayne State University, 656 West Kirby Street, 3054 Faculty-
Administration Building, Detroit, Michigan 48202, USA.
E-mail: tsetzer@wayne.edu
Received 5 March 2013; accepted 30 August 2013
DOI: 10.1002/ajpa.22375
Published online 3 October 2013 in Wiley Online Library
(wileyonlinelibrary.com).
 BLOOD AND SOFT TISSUE DETECTION IN ANCIENT BONE 567

Fig. 1. Preserved bone marrow (indicated with arrow) was

found within the medullary cavity of Sample 5.

TABLE 1. Sample preparation techniques

Fig. 2. Material from the medullary cavity of Sample 9 con-
Section tained erythrocytes (a) and nucleated cells (b) consistent with
Demineralization Concentration thickness hemopoietic tissue preservation (12 h demineralization in 1%
agent (%) pH Time (mm) citric acid pH 3.5, H&E stained, 10 lm, 10003).

EDTA 10 7.4 Eight days 5

to two
weeks
HCl 1 2 24 h 5 and
HCl 1 2 12 h 5 and
Citric acid 1 3.5 12 h 5 and
EDTA 1 8 12 h 5 and
species of the Fusarium and Tricoderma genera (Balmas
et al., 2010). The area receives an average of 741 mm of
rainfall per year, with temperatures ranging from 7.1 C
to 23.8 C (Balmas et al., 2010).
Archaeologists, who have macroscopically examined
these skeletal remains, described them as highly frag-
mented and of poor to average research quality (Atzeni
et al., 2012). Human skeletal remains from this collec-
tion were sampled for further analysis (Setzer, 2010).
Three long bone fragments, one from an infant (Sample
2), an adolescent (Sample 5), and an adult (Sample 9),
were subsampled for histological analysis.
METHODS
The first specimen examined in this study, Sample 2,
was selected randomly. During sample preparation,
material from the medullary cavity was collected in a
conical tube. The matter was washed with distilled
water, followed by three alcohol baths, and the resulting
button was embedded in paraffin and cut with a micro-
tome. Five micrometer sections of the button were
mounted on glass slides and stained with hematoxylin
and eosin (H&E), a common, frequently-used stain for
the general histology during routine tissue preparation,
or Giemsa, which is used for bone marrow samples, and
examined by light microscopy. When cells suggestive of
blood and bone marrow preservation were observed,
additional methods were used with multiple specimens
to confirm the results.
To verify the presence of bone marrow, we examined
material found within the medullary cavity of Samples 5
and 9 (Fig. 1). The fine-grained nature of the substance
precluded paraffin embedding without pretreatment,
which could potentially degrade the sample. Instead, the
10 material was directly embedded in optimum cutting tem-
10 perature medium, frozen, and cut into sections using a
10 cryotome. Once cut, the samples were sprayed with an
10 alcohol fixative for slide preparation and subsequent
storage at room temperature. The mounted sections
were stained with H&E and examined by microscopy.
Demineralization time, acid, and thickness of sections
were varied to find a method that would improve the
preservation and recovery of soft tissues from archaeo-
logical bone for light microscopy (Table 1). After demin-
eralization, the tissue was rinsed in 0.1 mol=L
phosphate buffer (PBS, pH 7.4), and incubated in PBS
for 6 h before fixation in 10% formalin. Fixed tissues
were embedded in paraffin blocks. About 5- and 10-mm-
thick sections were cut with a microtome and placed on
glass slides. The sections were deparaffinized using
xylene and rehydrated through serial incubations in
graded ethanol. After rinsing with distilled water, the
samples were incubated with 0.1 mol=L PBS (pH 7.4).
Sections were stained using H&E and examined by light
microscopy.
RESULTS
We noted hemalum-stained (e.g., cell nuclei) and eosin-
ophilic (e.g., protein, cytoplasm, erythrocytes) structures
consistent with bone marrow. Red, disc-shaped, biconcave
cells within a size range of 5–8 lm, typical of erythro-
cytes, as well as nucleated cells were detected in all three
specimens. These structures were observed in both the
material isolated from the medullary cavity and the
demineralized bone sections. Samples that were partially
demineralized, in this case for 12 h, and sectioned at 10
lm produced slides that were both qualitatively and
quantitatively superior. A 20-fold excess in the number of
erythrocytes was observed in one case in which the sam-
ples were demineralized using citric acid (Fig. 2).
DISCUSSION
Although erythrocytes and erythrocyte-like structures
in ancient skeletal material have been reported (Stout

American Journal of Physical Anthropology

568 T.J. SETZER ET AL.
and Teitelbaum, 1976; Maat, 1991; McNamara et al.,
2006; Schweitzer, 2007; Charlier et al., 2008), the pre-
sumption that the soft tissues and blood cells within ske-
letonized remains are destroyed through autolysis and
subsequent taphonomic and diagenetic processes contin-
ues to shape the methodology of paleohistology. Partially
decalcified bone histology, which can be conducted in a
standard histology lab, can be used with other analytical
methods (e.g., ground bone, Raman spectroscopy, SEM)
to provide an additional line of evidence when studying
skeletonized remains.
The persistence of soft tissue within these bones could
represent a unique case of preservation that occurred at
this specific site, or it could be indicative of a more com-
mon biological mechanism that occurs during decomposi-
tion. Numerous gaps exist in both our understanding of
the environmental history of Sardinia (Massoli-Novelli,
1986) and the overall impact of the environment on
taphonomic processes (Behrensmeyer et al., 2000) mak-
ing it difficult to assess factors affecting the preservation
of soft tissues.
ACKNOWLEDGMENTS
Authors thank the Soprintendenza per i Beni Archeolo-
gici per le Province di Cagliari e Oristano, the Museo de
Villanovaforru, E. Atzeni, A. Usai, M. Perra, R. Forresu, O.
Fonzo, L. Tait, D. Sullivan, P. Hortol
a, L. Lai, R. Tykot, and
T. W. Killion.
LITERATURE CITED
Atzeni E, Usai A, Bellintani P, Fonzo O, Lai L, Tykot R, Setzer
TJ, Congiu R, Simbula S. 2012. Le tombe Nuragiche di Sa
Sedda ‘e Sa Caudela (Collinas – CA). Scavi 1982–84. Qua-
derni 23:25–50.
Balamas V, Migheli Q, Scherm B, Garau P. 2010. Multilocus
phylogenetics show high levels of endemic fusaria inhabiting
Sardinian soils (Tyrrhenian Islands). Mycologia 102:803–812.
Behrensmeyer A, Kidwell SM, Gastaldo RA. 2000. Taphonomy
and paleobiology. Paleobiology 26:103–147.
Charlier P, Georges P, Bouchet F, Huynn-Charlier I, Carlier R,
Mazel V, Richardin P, Brun L, Blondiaux J, Lorin de la
Grandmaison G. 2008. The microscopic (optical and SEM)
examination of putrefaction fluid deposits (PFD). Potential
interest in forensic anthropology. Virchows Archiv 453:377–386.
Custer RP, Hayhoe FGJ. 1974. An atlas of the blood and bone
marrow. Philadelphia: W.B. Saunders.
American Journal of Physical Anthropology
deBoer HH, Aarents MJ, Maat GJR. 2012. Staining ground sec-
tions of natural dry bone tissue for microscopy. Int J Osteoar-
chaeol 22:379–386.
Hollund HI, Jans MME, Collins MJ, Kars H, Joosten I, Kars
SM. 2011. What happened here? Bone histology as a tool in
decoding the postmortem histories of archaeological bone
from Castricum, The Netherlands. Int J Osteoarchaeol 22:
537–548.
Maat GJR. 1991. Ultrastructure of normal and pathological fos-
silized red blood cells compared with pseudopathological bio-
logical structures. Int J Osteoarchaeol 1:209–214.
Massoli-Novelli R. 1986. The geology, environment, and natural
resources of Sardinia. In: Balmuth, MS, editor. Studies in
Sardinian archaeology. Vol. II: Sardinia in the Mediterranean.
Ann Arbor: University of Michigan Press. p 3–8.
McNamara ME, Orr PJ, Alcal a L, Anadon P, Pe~
nalver E. 2012.
What controls the taphonomy of exceptionally preserved
taxa—environment or biology? A case study using frogs from
the Miocene Libros Konservat-Lagerst€ atte (Teruel, Spain).
Palaios 27:63–77.
McNamara ME, Orr PJ, Kearns SL, Alcal a L, Anad on P,
Pe~nalver-Molla E. 2006. High-fidelity organic preservation
of bone marrow in ca. 10 Ma amphibians. Geology 34:641–
644.
Prats-Mu~ noz G, Malgosa A, Armentano N, Galt es I, Esteban J,
Bombi JA, Tortosa M, Fern andez E, Jordana X, Isidro A,
Fullola JM, Petit MA,  Guerrero VM, Calvo M, Fern andez PL.
2012. A paleoneurohistological study of 3,000-year-old
mummified brain tissue from the Mediterranean Bronze Age.
Pathobiology 79:239–246.
Schultz M. 2001. Paleohistopathology of bone: a new approach
to the study of ancient diseases. Am J Phys Anthropol 116:
106–147.
Schultz M. 2011. Light microscopic analysis of macerated patho-
logically changed bones. In: Chowder, C and Stout, S editors.
Bone histology: an anthropological perspective. Boca Raton:
Taylor & Francis Group. p 253–296.
Schweitzer MH, Wittmeyer JL, Horner JR. 2007. Soft tissue
and cellular preservation in vertebrate skeletal elements from
the Cretaceous to the present. Proc R Soc B 274:183–197.
Schweitzer MH, Wittmeyer JL, Horner JR, Toporski JK. 2005.
Soft-tissue vessels and cellular preservation in Tyrannosaurus
rex. Science 25:1952–1955.
Setzer TJ. 2010. Malaria in prehistoric Sardinia (Italy): an
examination of skeletal remains from the middle Bronze Age.
Unpublished PhD, University of South Florida.
Stout SD, Teitelbaum SL. 1976. Histological analysis of unde-
calcified thin sections of archaeological bone. Am J Phys
Anthropol 44:263–269.
Zimmerman MR. 1973. Blood cells preserved in a mummy 2000
years old. Science 180:303–304.