The Blood on the Shroud of Turin: Species Unknown

Kelly P. Kearse

The Shroud of Turin is an approximately 14 x 3.5 feet linen cloth of unknown origins bearing the
faint frontal and dorsal images of a man with reddish areas corresponding to wounds at the head,
hands, side, feet and back. Previous studies have shown that the reddish areas contain various blood
components including hemoglobin, heme breakdown products (bilirubin), albumin, and
immunoglobulin (1-5). For over thirty years, it has been accepted by many that the blood on the
Shroud is of human (primate) origin, but new findings that indicate the validity of previous data
must be reassessed. As discussed, cross-reactivity precludes a definitive conclusion that the
bloodstains on the Shroud are of human, even primate, origin.

Initial Characterization of the Bloodstains

The first endeavor to scientifically evaluate the nature of the bloodstains on the Shroud began in
1973 by members of a “commission of experts” led by Frache et al. Hampered by ineffective
solubilization methods, their results were negative and resulted in the conclusion that “the negative
answer to the investigations conducted does not permit an absolute judgement of the hematic nature
of the material under examination.” (5,6). In other words, the findings were inconclusive. In 1978,
the bloodstains would be evaluated again as part of the STURP (Shroud of Turin Research Project)
examination, involving over forty scientists of various disciplines; samples were also collected
independently by Bollone and colleagues at this time. To date, this remains the most thorough
investigation of the cloth and is the main depository of the data related to the characterization of the
bloodstains. John Heller and Alan Adler were the two primary investigators of the STURP team that
focused on the characterization of the bloodstains. It was concluded from such studies that the
bloodstains were composed of real blood components and not pigment or dyes (5,7). Most recently,
spectroscopic analysis has shown the bloodstains consist of methemoglobin, the deoxygenated form
of hemoglobin expected for aged blood, corroborating previous findings on the hemoglobin species
present (8,9).

Determination of Species of Origin of the Blood

To determine what type(s) of blood might be present on the Shroud, Heller and Adler performed
immunological tests using antibodies to detect specific blood proteins in Shroud samples,
specifically albumin and immunoglobulin. Albumin constitutes ~50% of total blood protein and is
important for maintaining osmotic pressure within the circulatory system; endogenous
immunoglobulin (antibody) is synthesized by B lymphocytes and plays an important role in immune
system function.
 These so-called serological (or immunochemical) experiments are done by using antibodies
generated against a protein from one species (for example, human albumin) that has been made in a
different species (for example, rabbit), (Figure 1). The production of antibodies is based on the
premise that human albumin proteins are different enough from those endogenously present in the
rabbit that the rabbit’s immune system will respond (see Figure 1). Following multiple injections to

Figure 1. Production of antibodies against human albumin proteins. See text for details.

boost the immune response, the antisera is harvested and may or may not be further purified before
use. The antisera used by Heller and Adler during the early 1980s are referred to as “polyclonal”,
because they represent a mixture of antibodies produced by many (“poly”) different cells (“clones”).

While certain proteins are different enough among various species to elicit an immune response
(such as human and rabbit), they are also alike enough among other species such that the antibody
produced against proteins of one species may recognize those endogenously present in another. This
is referred to as cross-reactivity (10,11). Cross-reactivity of anti-human antisera is most commonly
seen among similar species (humans and other primates, for example), but is by no means limited to
it. For example, in the screening of fifty anti-human antibodies directed against molecules on human
blood cells, it was found that almost half cross-reacted with canine cells (12). Anti-human
antibodies have been reported to show (strong) reactivity against certain hamster molecules (13), and
to cross react with other seemingly unrelated species, such as mouse, rat, tree shrew, and goat (14-
17).

Heller and Adler were well-aware of the phenomenon of cross-reactivity and performed
experiments to help evaluate the extent to which it occurred for the antibodies that were used in the
evaluation of Shroud blood (5,18). In hindsight, taking into account advances in genetic/protein
sequencing that allow efficient comparison of the homology of molecules across many different
species, together with the major technological increases in antibody production and reactivity, the
question is raised: were their tests sufficient to rule out/rule in various species in determining the
blood origin? As will be explained below, the answer is no. There are certain species that appear
relatively unrelated to humans/primates, for example, mouse, rat, cats, and others, whose proteins
are similar enough that they would show a positive reaction in such tests. This is highly significant
as these are the key experiments on which the conclusion that the blood on the Shroud is of human
(primate) origin is based.
The extent of cross-reactivity is much greater than previously realized
Before testing any Shroud samples, Heller and Adler first examined the reactivity of the anti-
human albumin antibody against blood from the following species: humans, chimpanzee, baboon,
cow, pig, and horse (5,18). They found a positive reaction against human blood (expected), and also
saw a positive result against blood from both chimpanzee and baboon (not unexpected). The
reactivities against blood from cow, pig and horse were all negative. Thus, before beginning any
experiments on samples from the Shroud, Heller and Adler knew that they could rule out a reaction
from cow, pig, and horse blood. They also knew that their conclusions would be limited to primate
at best based on their cross-reactivity data. What remains at issue here, though, is if they had
included blood from other species in such tests, for example, mouse, rat, goat, sheep, cat, camel,
(and others) would they have also gotten a positive result? The answer is yes, at least for several of
them. Recent studies have demonstrated that albumin from species that are not that similar in
overall appearance contain enough homology at the genetic/protein level that antibodies raised
against one species may strongly react with another (19-22). For example, antibodies raised against
human albumin protein can very effectively recognize albumin protein from rats. Figure 2 (top)
shows a screenshot from the Cell Applications Inc. website, a recognized global leader in isolation
and purification of primary cells, and their culture in optimized, cell specific media (22). These data
clearly show that an “anti-human” albumin antibody strongly cross reacts with rat albumin.
Screenshots are also provided from two additional independent antibody companies, R&D Systems
(Figure 2, bottom left) and CellSignal.Com (Figure 2, bottom right) that similarly demonstrate cross-
reactivity of rat (and mouse) albumin with anti-human albumin antibodies. I have also personally
communicated with one of the authors on the study listed in reference 19, who confirmed that anti-
human albumin antibody cross reacts with albumin expressed in rats (23). Had Heller and Adler
been aware of such findings, they would have widened their control panels to include many other
species, or, most likely curtailed their studies until experimental strategies could be revised.
Unfortunately, Heller and Adler never formally published their species data in a scientific journal as
their work was relatively incomplete and the amount of sample was limited. In fact, such studies are
among the very last experiments that they performed on Shroud samples. Reporting of such results
is limited to mentions in two books (5,18), one of which is the last interview Heller and Adler ever
gave together (18), and two journal articles (3,8), always as a work in progress, without any visible
data.

In hindsight, it is not intuitively obvious that an antibody generated against human albumin would
recognize albumin from rats (or cats, see below) but not albumin from cows or pigs, the latter two of
which were included by Heller and Adler as negative controls. Certainly, such possibilities were not
previously considered by myself because of their overall dissimilarity at the physiological level.
Upon further consideration, this possibility is somewhat surprising, though not entirely so. Given
the extensive advancements that have been made with genome sequencing technology, computer
modeling of conserved protein domains, and antibody generation and production within the past
twenty to thirty years, much new information has been learned regarding the similarity (often
unpredicted) of various proteins among different species (20-22). Curiously, Adler mentioned in
one symposium proceeding that dog reactivity was evaluated (24), although this was not referenced
in other sources which are more detailed (3,5,18). Canine albumin is even more similar to human
albumin than rat albumin (almost 80% for canine/human versus 74% for rat/human at the protein
level), and is known to exhibit cross-reactivity with anti-human albumin antibodies (21,25,26).
Within the past few years, human albumin has been transfused into dogs as an experimental protocol
for treatment of protein disorders in canines (27,28). In his control cross-serology studies, it is
almost certain that Adler utilized lyophilized/reconstituted dog albumin, which can maintain some
tertiary structure and thus not expose all available antibody-binding sites (which is most likely

Figure 2. Cross-reactivity of anti-human albumin antibodies with rat and mouse albumin.
Screen shots from three independent antibody companies illustrating the cross reactivity of human
and rat albumin. See text for details. Top: Screen shot from cellapplicaions.com. These data
clearly demonstrate the reaction of anti-human albumin antibody with albumin from rat cells.
Bottom left: Screen shot from R&D Systems showing reactivity of anti-human albumin antibody with
albumin from human, mouse, and rat. Bottom right: Screen shot from cellsignal.com showing the
reactivity of anti-human albumin antibody with albumin from human (HepG2) and mouse and rat
cell lines. HeLa is a human cell line that does not express albumin, included as a negative control
The full spec sheets may be found at https://www.cellapplications.com/anti-albumin-monoclonal-
albumin-antibody (2018),(top); https://www.rndsystems.com/search?keywords=albumin (2018),
(bottom left); and https://www.cellsignal.com/products/primary-antibodies/albumin-antibody/4929
(2018), (bottom right).
Figure 3. Reactivity of “anti-human” albumin antibodies with albumin from various species.
Cross-reactivity of antibodies made against human albumin with albumin expressed in other,
relatively unrelated species. See text for details.

not the case for Shroud samples). Surprisingly, cat albumin (not included as a control in Shroud
studies), shows the highest homology to human albumin (approximately 82% identical) and has also
been shown to cross-react in serological tests (21,22). Cross-reactivity with albumin from disparate
species (for example, wallaby) has been noted as well (29), and others may follow (such as ground
squirrel, guinea pig, woodchuck, flying squirrel, wolf). Such relationships are not intuitively
obvious, but in retrospection are reasonable when one considers the revealed similarities of
individual molecules at the genetic/protein level.

Serological studies were also performed on Shroud fibers to detect endogenous immunoglobulin,
although discussion of cross-reactivity was much less detailed (nonexistent). Bollone et al. would
report positive reactivity of Shroud samples with anti-human immunoglobulin with the apparent
assumption that specificity of the “anti-human immunoglobulin” was definite and restricted. Such
results have been published solely in books, Shroud specialty journals, and symposia proceedings
(4,30,31). The STURP team would only briefly mention positive results for detection of
endogenous immunoglobin, although no information was provided regarding verification of cross-
reactivity or the lack thereof (8,18). Without any such demonstration of restricted specificity for the
“anti-human” reagents that were used for the detection of immunoglobulin, any solid conclusion that
the blood is of human origin from such data may be easily challenged. Indeed, cross-reactivity of
immunoglobulin among species is an equal or even greater concern than albumin as structural and
functional immunoglobulin domains are highly conserved (32-34).
Discussion
What does all of this mean regarding Shroud blood studies? These findings indicate that the
interpretation of the results of having immunological evidence that the blood is of primate (human)
origin has to be seriously reconsidered. One cannot validly make such a conclusion from the data
after knowing that the possibility of cross-reactivity is even broader than previously thought (Figures
2 and 3). Indeed, as stated on the Proteintech Group website, a company that has manufactured
antibodies against 12,000 targets, and also references cross-reactivity of rat albumin with anti-human
antibody in studies unrelated to the Shroud, “ Cross-reactivity can invalidate the results of an
experiment and thereby impact scientific reproducibility. Thus, testing an antibody for cross-
reactivity with closely related proteins is a critical validation experiment.” (35).

While other serological tests have been performed on a limited number of Shroud samples (ABO,
MNS antigens), most recently discussed here (36,37), such studies cannot be applied to the
determination of species of origin for blood. The MNS test would have been the most helpful,
particularly the studies on the S antigen, but is greatly lacking in detail; no data was presented for the
S antigen in the brief report (36,38). The key data on which the conclusion that the blood is of
human (primate) origin was based are the immunological tests on serum albumin and
immunoglobulin, with the albumin analysis being the stronger of the two. Is one to assume that the
results show that the blood on the Shroud is of feline origin or from another animal species? Of
course not. The appropriate conclusion is that the scientific determination of the blood origin is
currently unknown; the previous data can no longer be used to support the claim that human (or
primate) origin has been demonstrated from such serological studies on Shroud fibers, the results are
inconclusive.

Adler realized the importance of the immunological tests to help determine the species origin of
the Shroud blood. Regarding their findings, he would state, “But the most interesting thing is now
there is immunological evidence that it is primate blood” (18). What was unrealized, however, was
the extent of cross-reactivity with relatively unrelated species, such as rats, mice, feline (or other),
recently elucidated due to advances in genetic sequencing and protein modeling. Had Adler known
this, it is safe to assume that he would have never made the above conclusion and redesigned or even
postponed his experimental procedure. Such strategies are much easier to develop some forty years
later as major advances in the technology of antibody production and screening are now available
that allow investigators to more efficiently generate reagents that are even further restricted in their
reactivity among species.

Finally, it is noteworthy to mention that no immunological experiments have ever been done to
directly address the possibility that blood from any species other than primate (human) might be
present on the Shroud (36). All of the tests performed to date have exclusively examined the
reactivity of Shroud blood samples with “anti-human” reagents. The possibility that blood from
other species might (also) be present has not been considered. Although it is beyond the intent and
scope of the current article to go into detail, if blood from another species were present on the
Shroud, it would have gone undetected due to the experimental design. This is important as even if
the blood on the Shroud were eventually demonstrated to be human, it would be most definitive if
the existence of other blood types could also be ruled out.

Summary
The current paper provides evidence that calls for serious reconsideration of the interpretation of
previous immunological evidence suggesting that the blood on the Shroud is of primate (human)
origin. One cannot validly support such a conclusion realizing that the cross-reactivity is much
broader than previously thought. Cross-reactivity precludes a definitive conclusion that the blood on
the Shroud is of human, or even primate, origin.

Acknowledgements
Thank you to Drs. David Wiest and Richard Franklin for helpful discussion. As always, thank you
to my wife Kathy, for everything.

Biographical Information
Kelly P. Kearse holds a BS and MS in Biology and a Ph.D. in Immunology. Following completion
of postdoctoral fellowships at Johns Hopkins University School of Medicine in Biochemistry and the
National Cancer Institute, National Institutes of Health in Immunology, he became a Principal
Investigator in the Experimental Immunology Branch at the National Institutes of Health. After
several years, he transferred to the Department of Microbiology and Immunology at East Carolina
University School of Medicine to have the opportunity to do both research and teaching. He has
authored over forty peer-reviewed research articles in the fields of cell biology and immunology in
such journals as the Proceedings of National Academy of Sciences, the Journal of Biological
Chemistry, and the Journal of Experimental Medicine; and was the Editor in Chief of T Cell
Protocols, Development and Activation. In 2000, he semi-retired from laboratory research to
relocate to his original hometown and teach high school science, something that he had always
wanted to do. He has been a science instructor at Knoxville Catholic High School, in Knoxville, TN
for the past twenty years. Correspondence/comments may be sent to:
kelly.kearse@knoxvillecatholic.com

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