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What is haemotaphonomy?
Haemotaphonomy or hemotaphonomy (from the Greek haima for blood,
taphos for burial, and nomos for law) is the science that deals with
the cytomorphology of the blood cells in bloodstains. This term was
proposed by the author in a paper published in 1992 in Forensic Science
International.
Because the
object of study of haemotaphonomy is the cytomorphology of the blood cells in
bloodstains, its subjects of study are
bloodstained specimens. The method of study
of haemotaphonomy is the analysis of images in
chiaroscuro obtained via scanning electron microscopy. Beyond
the basic-science point of view, applied haemotaphonomy seeks the
use of bloodstains as evidence, and must not be confused with bloodstain pattern
analysis.
Historical antecedents
and genesis of haemotaphonomy
The most abundant blood corpuscles are the erythrocytes. These cells were yet
observed during the last half of the 17th century by early optical
microscopists, such as Giovanni Alfonso Borelli, Jan Swammerdam, Marcello
Malpighi and Anton van Leeuwenhoek. The occurrence of (at least
cytomorphological) preservation of anucleate, mammalian
erythrocytes in
bloodstains has been reported even in Olduwan palaeolithic tools from
Sterkfontein Cave (South Africa), assigned to be ca. 2 Ma old. These corpuscles have also been
identified in prehistoric immovable items, such as an early Holocene building at
Çayönü Tepesi (Turkey), containing anucleate red blood cells, human
immunoglobulin G (IgG) and both human and non-human haemoglobin (Hb) on a stone
slab.
The
microscopic study of bloodstains began in the early 19th century
with a forensic bias. Bonaventura-Mateu Orfila is credited as the first to attempt the use of a
microscope to assess bloodstains. Throughout the criminalistic literature of the
19th and 20th centuries, this microscopic examination was only intended for
confirming the presence of blood or, at most, for discriminating taxonomically
between mammalian and non-mammalian blood. The only microscopes available for
the study of bloodstains were light microscopes until the scanning electron
microscopes (SEMs) went onto the
market. Unlike the transmission electron microscope (TEM), which was conceived
for the study of (biological) sections at high magnification, the SEM was
conceived for the study of (either biological or inorganic) surfaces at high
magnification. For this reason, SEMs are very appropriate
instruments for cytomorphological studies. The advent of the first commercial SEM (the Cambridge Instrument
Company’s Stereoscan) took place in the 1960s. The rise of molecular
biology techniques as applied to bloodstains during the same decade
led to the virtual abandonment of the use of microscopy in the
forensic assessment of bloodstains, a little-used method
in itself. Thus, this previous lack of interest in the
morphology of erythrocytes in bloodstains paved the way for the appearance of a
new science from early 1990s: the science of
haemotaphonomy.
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Taphoerythrocytes.
Three-month-old
smear on stone. Author's blood.
See more electron micrographs in Forensic Science International
vol. 55, pp. 139-159, 1992.
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Vertebrate blood and mammalian RBC morphologies in the body and in the smear
Vertebrate blood (i.e., "blood" sensu stricto) is a cell suspension into a fluid
medium (the plasma). In this histological tissue, three types of cells are
present: erythrocytes (red blood cells, RBCs), leukocytes (white blood cells,
WBCs) and thrombocytes (platelets, in mammals).
Unlike the other vertebrates,
mammals have anucleate RBCs (erythroplastids or akaryocytes). As an exception,
the slender salamanders (family Plethodontidae, order Caudata, class Amphibia)
have some proportion of anucleate RBCs, with Batrachoseps attenuatus
(Eschscholtz, 1833) possessing nearly 95% erythroplastids. Also, the pearlside
teleostean fish Maurolicus mülleri (Gmelin, 1789) (family
Sternoptychidae, order Stomiiformes, class Actinopterygii) has anucleate
erythrocytes.
Due to the lack of nucleus, the typical mammalian RBCs are shaped as biconcave
discs (discocytes). This does not apply to the family Camelidae, where RBCs are
oval (ovalocytes). Other physiological shapes – which are minor or pathologic
–
are: echinocytes (burr or berry cells), dacryocytes (tear drop cells),
schizocytes (helmet cells), keratocytes (horn cells), drepanocytes (sickle
cells), and many others.
The presence of all kind of residues on implements agrees with the criminalistic
well-known Locard’s Principle of Exchange ('every contact leaves traces').
A smear may be regarded as the result of a causal relationship,
in which a physical contact (the cause) produces a trace (the effect). On
the other hand, experimental palaeontology (actuopalaeontology) and experimental
archaeology are both based upon the Lyell’s Principle of Actualism ('the present
is the key to the past'). A short-time preservation of specimens is a sine-qua-non precondition to do feasible a (bioarchaeological
or forensic) longer one. Also, in forensic analysis the presence of
erythrocytes in a smear is considered a blood confirmation. Therefore, the study
of the different erythrocyte and plasma-matrix morphologies exhibited in
bloodstains represents a field related to disciplines such as forensics,
prehistoric archaeology, and palaeoanthropology. Mammals have
been chosen as the target taxon because this zoological class has long been
assumed to be the major animal biomass source for prehistoric man.
The largest part of the smear-origin RBC shapes share
morphology with those described in haematology. But two time-independent RBC
shapes are due specifically to blood drying phenomena. So they
can be considered as
genuine RBC morphologies characteristic of (at least mammalian) bloodstains,
and therefore not found
under physiological conditions. These shapes are:
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hecatocytes (moon-like shapes related to erythrocyte-plasma
interaction when drying), and
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janocytes (negative replicas related to imprinting by dried
plasma matrix).
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- Janocytes.
About-eight-month-old smear
on urban asphalt. Human-suspected blood. See more
electron micrographs in Microscopy and Analysis vol. 40, pp. 19-21
(UK) & vol. 28, pp. 21-23 (EU), 1994.
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Influence of the smear substrate and ageing in
erythrocyte morphology in bloodstains
From the haemotaphonomical point of view, the concrete type of bloodstain
substrate (stone, metal, paper...), whether of similar physical properties, do
not seems to play a dramatic role in RBC morphology. The most significant
bloodstain-substrate physical properties would be grouped into three categories:
(i) those related with the degree of thickness homogeneity of the smear by the
forming of thicker/thinner subareas (topography), (ii) those related with the
mechanical seizing and/or breaking of the cell fraction by surface microcrystals
while smearing, and with the adherence of the whole blood to substrate while
drying and/or ageing (roughness and texture), and (iii) related with the
infiltration of blood (mainly plasma) into the substrate (permeability, its
related absorbency, and permeability-influencing fissuration). On the other
hand, the high RBC preservation exhibited in the samples when examined under an
SEM points out that dried blood tissue is
homologous or, at least, analogous to a mummified one. Therefore, it is not
expected that the ageing time span be determinant per se of the degree of
bloodstain preservation.
Beyond science: the aesthetic background of
haemotaphonomy
Knowing the stylistic parallels between science and other human enterprises,
such as literature and the visual arts, contributes to the recognition of its
cultural framework and, by extension, to a better understanding of
science-society relationships. Literarily, haemotaphonomy is parallel in style
to the aesthetics of both tremendism and decadentism. Visually, haemotaphonomy
is stylistically parallel to the aesthetics of both Churrigueresque
architectural decoration and pictorial tenebrism. Haemotaphonomy can also be
regarded as literary realism and visual naturalism. Furthermore, because
tremendism, decadentism, churriguerism and tenebrism converge into the Baroque
culture, from the point of view of its aesthetics haemotaphonomy is a
baroquistic science.
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Author's articles on
haemotaphonomy
(full-text pdf available upon request)
Keywords:
Scanning electron microscopy, blood smears, erythrocytes, organic residues,
prehistoric archaeology, actualistic palaeoanthropology, forensic biology.
The ‘foundational minutes’
of haemotaphonomy
Mammal red blood cells (RBC) in bloodstains have been
previously detected by light microscopy on stone tools from as early as
100,000±25,000 year ago. In order to evaluate the degree of morphological
preservation of erythrocytes in bloodstains, an accidental human blood smear
on white chert and several experimental bloodstains on hard substrate (the
same stone
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white chert; another type of stone
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graywacke; a non-stone support
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stainless steel) where stored in a room, in non-sterile and fluctuating
conditions, for lenghths of time ranging from 8 to 18 months. Afterwards,
the specimens were coated with gold and examined by a Cambridge Stereoscan
120 scanning electron microscope. Results revealed a high preservation of
RBC integrity, with the maintenance of several discocytary shapes, a low
tendency to echinocytosys and a frequent appearance of a moon-like
erythrocytary shape in the thinner areas of the bloodstains.
The first studies on haemotaphonomy
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SEM characterization of blood stains on
stone tools. The Microscope vol. 40 (2), pp. 111–113
[Editorial ‘Errata’
on scale bars in vol. 40 (3), p. vi],
1992.
Mammalian red blood cells (RBC) in bloodstains have been
previously detected by light microscopy on stone tools circa 100,000
years old. To observe and characterize the bloodstain-original RBC, a modern
replica of ancient blood residues was examined by scanning electron microscopy
(SEM). To simulate a Stone Age process a collared peccary (Tayassu tajacu)
cadaver was skinned using a paleolithic-like white chert knife, which was then
smeared with blood plus serous liquid. After drying in the open air for one
week, the tool was stored at unsterile and fluctuant room conditions. After six
months, a detached fragment of the bloodstain on a SEM stub was coated
with gold and examined at an accelerating voltage of 15 kV using a Cambridge
STEREOSCAN 120 scanning electron microscope. Results reveal protuberant
moon-like shapes which are interpreted to be characteristic of RBC.
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Application of SEM to the study of red blood
cells in forensic bloodstains. Microscopy and
Analysis [early series]
vol. 40,
pp. 19–21 (UK)
& vol. 28, pp.
21–23
(EU), 1994.
Although in forensic analysis the presence of red blood cells in a smear is
considered to be a confirmation of blood, to date morphological researches using
electron microscopy dealing with superficial preservation or imprints
characterisation of erythrocytes in bloodstains have not been carried out.
Short-time preservation of specimens is a "sine qua non" precondition to
bioarchaeological or palaeobiological preservation.
So a part of author’s researches on ancient
blood residues has been focused on developing a methodology to study forensic
suspected bloodstains – considering as 'forensic' all modern blood smears of
unknown origin– covering substrates from hard to (absorbent) soft ones.
In this article dealing with
'urban' forensic hemotaphonomy, field and scanning electron microscopy
procedures, as well as showing examples of achieved results on either soft
(paper handkerchief) and hard (urban asphalt on a crossing-walk) smear
substrates are reported.
The
presence of morphologically complete mammalian erythrocytes (red blood
cells, RBC) from bloodstains has been previously evidenced in prehistoric
implements. While the presence of ancient non-human blood on a prehistoric
tool is an evidence of the real use of this on an animal resource, the
presence of RBC in a smear is evidence of blood. In a simulation of a
prehistoric predation human operative chain, mammalian bloodstains on
palaeolithic-like chert implements were obtained from two specimens
belonging to the order Artiodactyla: collared peccary (Tayassu tajacu,
family Tayassuidae) and Dorcas gazelle (Gazella dorcas, family
Bovidae). After one year, the unburied peccary blood smear and the buried
gazelle smear were coated with gold and then examined by a scanning electron
microscope. Results revealed the presence of preserved RBC with several
shapes like those found in haematological studies, as well as curved plasma
fractures and negative imprints, two bloodstain-characteristic morphologies
which are interpreted as due, respectively, to erythrocyte-plasma
interaction when drying and to imprinting by dried plasma matrix.
Mammalian erythrocytes or red blood cells (RBC) have been
previously reported as forming part of residues on prehistoric implements.
On the basis of the Principle of Actualism, several thick smears of human
blood were obtained on chert.
After increasing lengths of storage time span (1‑36 months), the bloodstains
were micromorphologically studied via scanning electron microscopy. Results
revealed, in all the smears, the presence of an erythrocyte acme‑zone with
packed RBC, as well as negative replicas and moon‑like shapes. Morphologies
were found to be time‑independent, and furthermore those erythrocyte
acme‑zones with packed RBC to be thick‑bloodstain characteristic.
The
systematisation of erythrocyte morphologies in bloodstains
Mammalian
erythrocytes or red blood cells (RBC) -whose presence in a
smear is a blood evidence- have been previously reported as forming part of
residues on prehistoric implements assigned to be as far as around two
million years old. On the basis of the Principle of Actualism, bloodstains
from human individuals were obtained on obsidian, limestone and chert, and
then stored in a unsterile room under microclimatically unmanipulated
fluctuating conditions, for lengths of time ranging from 7 years, 6 months
to 10 years, 2 months. Afterwards, the bloodstains were doubly coated with
carbon and gold and then examined by a JEOL JSM-6400 scanning electron
microscope (SEM). Results revealed a high preservation of erythrocyte
integrity, with several shapes as those found under physiological conditions
and a significant presence of moon-like shapes plus a minor one of negative
replicas, two morphologies that are interpreted as specifically related to
blood drying phenomena. These results agree with several previously reported
SEM analyses of younger mammalian bloodstains on chert and materials other
than obsidian and limestone, and lead to consider the moon-like shapes
(hecatocytes, a term ex novo) and negative replicas (janocytes, another term
ex novo) as the genuine RBC morphologies characteristic of (at least
mammalian) bloodstains.
The
dissemination of knowledge on haemotaphonomy
Mammals are
the only vertebrates that have anucleate red blood cells (RBC's). In this
zoological class, RBC's typically have the shape of biconcave discs. The
cytomorphological study of RBCs in bloodstains is an issue with implications
in fields such as forensic biology or prehistoric archaeology. Using scanning
electron microscopy, the author has pioneered a new approach to the study of
bloodstains, which has led moreover to a general terminology and systematics
for smear-origin mammalian RBC's. This paper summarises the results of more
than 10 years of research in this field, by presenting the main morphological
features of mammalian erythrocytes, when in smears.
The technical advances in the examination and imaging of
bloodstains
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SEM examination of human erythrocytes in
uncoated bloodstains on stone: use of conventional as environmental-like SEM
in a soft biological tissue (and hard inorganic material).
Journal of Microscopy
vol. 218 (2), pp. 94–103, 2005.
Although nowadays the so-called
environmental scanning electron microscopes (ESEMs) allow the observation of
the samples without metal or carbon coating, many conventional scanning
electron microscopes (SEMs) are still in use. On the other hand, the
presence of erythrocytes (red blood cells, RBCs) in a smear is considered a
blood confirmation. Such a presence has been previously reported even in
Lower Stone Age implements. In previous works, I have reported several
studies dealing with cytomorphology of RBCs in bloodstains using scanning
electron microscopy with standard specimen preparation procedures, i.e. via
coating the samples before SEM analysis. In order to explore the potential
of conventional SEM as environmental-like SEM in haemotaphonomical studies,
two alkaline (limestone) and two acid (flint) rock fragments were smeared
with human blood from a male and a female. The bloodstains obtained in this
way were then air dried indoors and stored into a non-hermetic plastic box.
Afterwards, the smears and their rock substrates were examined directly
without coating, via secondary electrons, using a JEOL JSM-6400 scanning
electron microscope. Satisfactory results reveal the capability of a
conventional SEM to work in secondary-electron mode as an environmental-like
SEM on these kinds of biological and inorganic materials, and probably in
many other biological and non-biological samples.
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Using an SEM as an ESEM to study minute
human bloodstains on stainless steel.
Microscopy and
Analysis [new series] vol. 20 (6),
pp. 15–17
(UK), pp. 5–7
(EU), pp. 23–25
(US) & pp. 11–13
(AP), 2006.
Because many conventional,
high-vacuum scanning electron microscopes (SEMs) are still in use, their
full potential should always be explored. With this aim in mind, two
uncoated stainless-steel blood lancets, used for finger puncturing in a
study carried out 20 months earlier, were examined for possible blood smears
in an SEM using secondary electron imaging at an accelerating voltage of 0.5
kV. Minute bloodstains and some of their erythrocytes were found. As was
previously revealed in uncoated bloodstains on stone, this study clearly
demonstrated that a conventional SEM can be used in secondaryelectron mode
just like an environmental SEM to examine these biological materials on a
stainless-steel substrate.
The imaging of most biological
samples via conventional scanning electron microscope (SEM) in
secondary-electron mode involves routinely some kind of specimen
preparation. Conventional SEMs are still used when a low-vacuum or
variable-pressure SEM (usually known as ‘environmental’, or ESEM) is not
available. But that preparatory approach may be undesirable in certain
cases, for instance in museum specimens, forensic evidences or clinical
samples. This report details a simple, low-cost, and sample-saving
bioimaging protocol without specimen preparation, by using removable plastic
conducting carbon cement, and then working under ex-profeso SEM conditions,
i.e., by using an SEM in secondary-electron mode just like an ESEM. The
successful use to image up to high magnifications human erythrocytes in
bloodstains on an extensively bloodsmeared, high-carbon steel surgical blade
is reported as an example of the potential of this procedure.
Anaglyphy is a stereoscopic
technique based on the superimposition of two images of the same view, taken
from slightly different angles as well as in two different colours. This
superimposition produces a depth effect when viewed through glasses having
one red and one green, blue or cyan lens acting as a colour filter. The
nearly-flat surface of a chip of grey chert was thin-smeared with peripheral
human blood. Two months later, the specimen was fixed to a SEM stub as
horizontal as possible, coated with gold, and examined via secondary
electrons by a scanning electron microscope. After obtaining SEM
micrographs, anaglyphs were digitally generated. The best results were
obtained using pairs of SEM micrographs acquired at 10° differing angle and
at SEM-stage tilts that were symmetric from the horizontal plane. The relief
effect was more accentuated at low magnification. The most ergonomic colour
combination for viewing bloodstain anaglyphs was red-cyan. Digital anaglyphy
provides a simple and feasible method to improve the relief effect of SEM
micrographs of bloodstains. Moreover, as a first consequence of this
improvement, the results obtained in this research reveal that the outermost
erythrocyte layer of a thin bloodstain – coinciding, in general, with the
smear surface – can be much more uneven than that previously suggested by
customary SEM micrographs.
When dealing with microscopic
still images of some kinds of samples, the out-of-focus problem represents a
particularly serious limiting factor for the subsequent generation of fully
sharp 3D animations. In order to produce fully-focused 3D animations of
strongly uneven surface microareas, a vertical stack of six digital
secondary-electron SEM micrographs of a human bloodstain microarea was
acquired. Afterwards, single combined images were generated using a
macrophotography and light microscope image post-processing software.
Subsequently, 3D animations of texture and topography were obtained in
different formats using a combination of software tools. Finally, a 3D-like
animation of a texture–topography composite was obtained in different
formats using another combination of software tools. By one hand, results
indicate that the use of image post-processing software not concerned
primarily with electron micrographs allows to obtain, in an easy way,
fully-focused images of strongly uneven surface microareas of bloodstains
from small series of partially out-of-focus digital SEM micrographs. On the
other hand, results also indicate that such small series of electron
micrographs can be utilized for generating 3D and 3D-like animations that
can subsequently be converted into different formats, by using certain
user-friendly software facilities not originally designed for use in SEM,
that are easily available from Internet. Although the focus of this study
was on bloodstains, the methods used in it well probably are also of
relevance for studying the surface microstructures of other organic or
inorganic materials whose sharp displaying is difficult of obtaining from a
single SEM micrograph.
Although in the
scientific-research literature the micrographs from scanning electron
microscopes (SEMs) are usually displayed in greyscale, the potential of
colour resources provided by the SEM-coupled image-acquiring systems and,
subsidiarily, by image-manipulation free softwares deserves be explored as a
tool for colouring SEM micrographs of bloodstains. After acquiring greyscale
SEM micrographs of a (dark red to the naked eye) human blood smear on grey
chert, they were manually obtained in red tone using both the SEM-coupled
image-acquiring system and an image-manipulation free software, as well as
they were automatically generated in thermal tone using the SEM-coupled
system. Red images obtained by the SEM-coupled system demonstrated lower
visual-discrimination capability than the other coloured images, whereas
those in red generated by the free software rendered better magnitude of
scopic information than the red images generated by the SEM-coupled system.
Thermal-tone images, although were further from the real sample colour than
the red ones, not only increased their realistic appearance over the
greyscale images, but also yielded the best visual-discrimination capability
among all the coloured SEM micrographs, and fairly enhanced the relief
effect of the SEM micrographs over both the greyscale and the red images.
The application of digital colour by means of the facilities provided by an
SEM-coupled image-acquiring system or, when required, by an
image-manipulation free software provides a user-friendly, quick and
inexpensive way of obtaining coloured SEM micrographs of bloodstains,
avoiding to do sophisticated, time-consuming colouring procedures. Although
this work was focused on bloodstains, well probably other monochromatic or
quasi-monochromatic samples are also susceptible of increasing their
realistic appearance by colouring them using the simple methods utilized in
this study.
The unraveling of the
cultural framework of haemotaphonomy
This study intends to provide
insight into the aesthetics of the science of haemotaphonomy (HTN), by
identifying its stylistic parallels with literature and the visual arts. The
object of study of HTN is the cytomorphology of the blood cells in
bloodstains. Its subjects of study are bloodstained specimens, while its
method of study is the analysis of images in chiaroscuro obtained via
scanning electron microscopy. Literarily, HTN is stylistically parallel to
the aesthetics of both tremendism and decadentism. Visually, HTN is
stylistically parallel to the aesthetics of both Churrigueresque
architectural decoration and pictorial tenebrism. In accordance with the
results of this study, HTN can also be stylistically regarded as literary
realism and visual naturalism. Furthermore, because tremendism, decadentism,
churriguerism and tenebrism converge into the Baroque culture, it must be
concluded that, aesthetically, HTN is a baroquistic science.
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