A holding reply to Thibault Heimburger MD re the longevity of blood stains on the Shroud of Turin

This is an instant response to a post that has appeared on The Other Site, in which I am taken to task for underestimating the ability of blood to resist biodegradation. Stone age tools  have been mentioned, with Thibault Heimburger reminding me that they still have recognizable species-specific blood proteins, as detected by immunological procedures.

As I said, I am a biochemist (long since retired), not an immunologist. But while impressed by the sensitivity of immunological procedures, especially those that cleverly use fluorescent tags to detect antigen-antibody reactions (fluorescence can be picked up with great sensitivity), I also have some old-fashioned scientific reservations based on qualitative v quantitative response. OK, so one can pick up a signal of undegraded protein – or at any rate the portion thereof that is responsible for immunological recognition – but how much in microgram terms, and what proportion of the protein that was there originally?  One can/could go on making one’s immunological assays more and more sensitive, but if one was able to detect just 0.1% of the original blood protein that has survived aeons of biodegradation, how valid is it to say that proteins are amazingly resilient to  centuries, millennia even of  biodegradation?

One also has to remember that the blood on a sheet of linen that is stored with  intermittent exposure to moist air, with its fungal and bacterial spores, is a somewhat different situation from stone tools that have been unearthed  in caves and other relatively protected situations.

A Google search unearthed this abstract from a PhD thesis that gives a pointer to the kind of geological and other factors that may contribute to the preservation of blood. The key term is probably calcification, or more generally mineralisation, which I doubt has been a factor where the Shroud is concerned. Here’s the abstract in full (my bold and italics):

Author Ms Peta Jane Jones

Thesis Title Geoarchaeological investigation into the preservation of archaeological blood residues, Sterkfontein, South Africa, with an application of a systems based methodology School, Centre or Institute School of Social Science Institution The University of Queensland Publication date 2007-10

Thesis type PhD Thesis

Supervisor Fairbairn, Andrew
Weisler, Marshall Subjects 430000 History and Archaeology Formatted abstract

Two million year old blood residues were preserved on stone tools from the
Sterkfontein cave site, South Africa. Sterkfontein is renown for its archaeological
record that comprises Oldowan and Acheulean stone tool assemblages and significant
hominid fossils from the Australopithecus and Homo species. Sterkfontein is part of a
karst landscape; karsts are cyclic, dynamic systems, which exhibit a variety of
features as a result of active and interacting processes governed by intrinsic and
extrinsic factors. The cycles of cave-forming, precipitation, calcification, dissolution, collapse and in-fill produce crenulated, pock-marked and often dangerous landscapes underlain by a variety of air-filled voids, avens and similar speleothemic features, which are hosts to delicate crystal structures and complicated water systems. The study documented in this thesis found Sterkfontein presented a strong and durable system-type, which was responsible for the survival of blood residues in the  taphonomic interval for millennia.This geoarchaeological study investigated the taphonomic diagenetic processes of the microenvironment to understand the conditions facilitating blood residue preservation.
Previous investigations offered insight into the biostratinomic processes responsible
for blood residue preservation, which include the amount of tool use, thickness of
blood residue, sequestering of blood residues into microcracks, ultra violet ray exposure and drying prior to deposition.

The majority of the Sterkfontein deposits contain brecciated infills comprising
approximately equivalent fractions of clay sediments and calcium carbonate (calcite),
therefore the physico-chemical nature of clay sediment, clay minerals and calcite were
studied through literature review. It was determined that protein adsorption by clay is
influenced by physico-chemical characteristics that include, cation exchange reaction,
molecular weight of proteins, valency of cations, hydrogen bonding, an increase in
calcite and magnesium cations and a pH range of 5-9. Calcite in solution has a
positive charge and may create a weak ionic bond with the negatively charged clay
surface, which may produce a protective shield for the blood residues. The
crystallised form of calcite can help protect the clay and blood from degradative processes through the tight packing of crystals and continual growth from solution and further sequestration of the proteins away from the active soil environment
. This  precipitation of calcite enhances the structural stability of finely grained deposits, in particular clay sediment, which results in a more impervious matrix. In addition the presence of calcium in clay sediment encourages flocculation of particles and  therefore holds water more strongly, thus protecting organic material.
Rock samples were taken from the depositional members 1, 2, 3, 4 and 5 of the
Sterkfontein Formation and from stratigraphically uncorrelated deposits within the
Lincoln Cave and Jacovec Cavern. These samples were studied macroscopically
before thin section production. The microscopic analysis of these sections utilised
petrographic techniques to identify the major constituents and to determine structure,
roundness, shape and deterioration of constituents. Thin section appearance and the
relationships between constituents were also studied enabling the interpretation of
formation processes. From the samples studied it was determined that the cultural and
non-cultural deposits entered the cave via gravity drop, hydrological events, including
stochastic events indicative of sheet flooding and/or talus cones or slopes. The cultural
deposits were made part of a finely intermixed matrix that was cemented by calcite
precipitation shortly after deposition, consequently preserving the deposits and archaeological material.

The analysis of the finely intermixed matrix identified constituents comprising mainly
clay and calcite, dolomite, quartz, with small amounts of organic material. The
constituents displayed slight to moderate degradation. Evidence for multiple episodes
of calcification and decalcification was displayed macroscopically with calcite bands  viewed across thin sections and a variety of calcite crystal forms observed  microscopically. From thin section analysis the constituents had not been extensively reworked, demonstrating that sediment had not travelled far prior to deposition. Some samples displayed layering of sediment indicating microstratigraphy suggestive of  quieter, less disturbed episodes of deposition.
In addition, a modified application of systems theory was used to integrate site
information, including archaeology, geology and taphonomy into a methodologicaland interpretive framework based on event type and designated time period. The
major influencing subsystems of the formation processes for the Sterkfontein
preserving system included hydrology, calcium carbonate, clay burial matrix,
archaeology, stone tools and residues, clay, geology, sediments, hominids and
dissolution/exposure/decalcification. The subsystems hydrology/archaeology and
archaeology/clay burial matrix were observed to interact more in the preserving  system than other subsystems. The results of this systems application facilitate predictive modelling of where blood residues are most likely to be preserved.
Based on this approach Sterkfontein was
defined as a stable and organised system that maintained and regulated diagenetic
processes and burial conditions of the finely grained clay/calcite matrix.


I shall try to respond more fully at a later date. At present I am preparing a post that divides the Shroud’s history into two key phases, possibly, indeed probably pre and post the 1532  Chambery fire. Based on the Lirey badge, aka Cluny medal, I shall set out a  rationale for thinking that the blood stains, or at any rate the more striking ones, were post 1532, together with a prediction that the blood stains will yield a later radiocarbon date than the linen, probably by at least some 150-200 years.

Late addition: in response to some knocking copy from one of the usual suspects on The Other Site, here’s an article from 1995 Science  News that I am reproducing in full. Some of it is undoubtedly dated, I’m sure, but the principle still holds – that an immunological test is something that is done blind – one has nothing but the result – positive or negative – and if positive, might well be a false positive. Sadly, there is rarely a means of independently corroborating the result, given the tiny amounts of material that are involved – amounts that get smaller with each increase in assay sensitivity EXCEPT by patiently assembling a varied database and checking  scrupulously for internal consistency, through collaborative multi-lab trials to check on each others’ analytical precision and accuracy. Archaeo-immunology, if one may describe it as such is no place for glib sound bites or for scoring cheap debating points.

There are many passages I was minded to emphasise, but I have contented myself with putting just one in bold face. Apols for the erratic spacing (as happens often when one cuts and pastes using MS Office as an interface).

Blood from Stones: Tests for prehistoric blood cast doubt on earlier results


Copyright ©1995 by Science Service

Forensic experts aren’t the only scientists who mine bloodstains for clues. For more than a

decade, archaeologists have been borrowing crime-lab techniques to hunt for ancient blood on scraps of stone.

Using antibodies to detect blood and the species it came from, some researchers have

seemingly obtained astonishing results. Margaret E. Newman of the University of Calgary in

Alberta and her colleagues reported finding buffalo blood on stone knives at a 5,600-year-old  butchering spot in Canada. Thomas H. Loy’s team picked up human blood in paint dating to  20,000 years ago on a cave wall in Australia. And at an Iraqi site, Loy says, he detected  180,000-year-old blood spilled by a man whittling wood.

It seems that dirt stuck in the grooves of a stone scraper or a dark spot on a rock slab can

reveal such secrets as what creatures early peoples sacrificed and when they turned from

hunting to farming.  But just as discoveries of ancient DNA have met with skepticism, researchers’ zeal for  archaeological blood tests, known as residue analysis, has begun to fizzle. In a recent spate of  papers, scientists question not only one another’s findings, but whether it’s even possible for  traces of buried blood to survive thousands of years.

“People are getting very capricious and puzzling and different results,” says Christopher

Chippindale, editor of Antiquity, a journal on whose pages the debate is unfolding. “There’s

something in the biochemistry that is giving false positives. That really puts quite a question

mark on the various studies.”

Loy, now at the University of Queensland in Australia, leads the field in archaeological

blood claims, having reported ancient blood on more than 1,000 tools since 1983. Initially, he  identified prehistoric hemoglobin, a protein in blood, by crystallizing it. That test has come  under heavy criticism, but Loy stands by his results.

When he and others began using immunological tests, they seemed to move to firmer

ground. These tests, which detect blood proteins, date back more than 40 years.

(Archaeological DNA tests, used since the 1980s, decode genetic material.) To devise a test for,  say, deer blood, scientists inject fresh deer blood into a rabbit, which makes millions of antibodies  to the blood. The antibodies in rabbit serum, called antiserum, can then be used to  search for deer blood.

To test a stone tool for traces of such blood, a researcher would generally wash the tool,

then pour the washing extract onto a solid to which the blood proteins stick—a plastic membrane, for example. At that point, he or she rinses the solid with deer antiserum, then with a  second antibody that sticks to the antiserum. Because this second antibody is tagged with a  fluorescent molecule or some other marker, it flags any deer blood in the sample.

In practice, the assays are more complicated. Because closely related species have similar

blood proteins, the antiserum for, say, elk can react with blood from a deer or cow. So it’s necessary  to test each antiserum against many other species’ blood for cross-reactions and to be  aware of these reactions when testing a piece of stone.

The test itself varies from one laboratory to the next. Some people buy commercial antisera,  while others make their own. Some testing methods are a thousand times more sensitive  than others. An antiserum can be made to react with a single protein, such as albumin or  hemoglobin, or even with one region of a protein instead of the many proteins in whole blood.

A chemist for 27 years, Judith A. Eisele had these things in mind 4 years ago when she

began looking at blood residues on tools for an anthropology master’s project at the University  of Nevada at Reno. Working with biochemist Roger A. Lewis, she used a dozen antisera, from  turkey to bear, to test for blood on more than 150 flaked stone tools from the Southwest.

When only seven tools tested positive for blood and these results proved ambiguous, she

tried another experiment. She coated clean stone tools with deer blood and buried them for

several months. The results, published in the March ANTIQUITY: Tools buried in dry dirt tested  positive for blood for only 10 months. As for tools stored in damp dirt, the blood couldn’t be detected after just a single month.

Eisele’s adviser, archaeologist Donald D. Fowler, sent her master’s thesis to researchers

across the country a year ago. “The dovecotes were definitely fluttering,” says Jerold M.

Lowenstein, an immunologist at the University of California, San Francisco. Other reports

added to these doubts. Researchers from the United Kingdom buried stone tools daubed with  blood; only one tested positive for blood a year later. When scientists in Texas and New Mexico  recently sent 54 tools dipped in fresh animal bloods to a commercial laboratory, it incorrectly  identified half the samples.

Lowenstein and retired Boston physician Elinor F. Downs also reported confusing results in

the January/February JOURNAL OF ARCHAEOLOGICAL SCIENCE. They split up washings from a set of  stone tools, sending one-third to another university laboratory and keeping one-third each for  themselves. Downs used crossover immunoelectrophoresis, Lowenstein radioimmunoassay,  and the third group a dipstick clinicians use to detect hemoglobin in urine.  While the three groups agreed about tools that held fresh blood or nothing, their results for  ancient blood didn’t match. On a particular tool, for instance, one team found human blood,  another bear blood, and the third nothing at all.

So is it possible to get ancient blood from a stone? The answer depends on whom you ask.

Howard Ceri of Newman’s group at the University of Calgary argues that the immunological

techniques are valid, even on aged blood. “Look at the wealth of forensic evidence that’s out

there,” he says.  Loy says others have gotten negative results because their tests aren’t sensitive enough to  detect minuscule amounts of blood and because they don’t begin by screening for blood visually.

“They’re either archaeologists using techniques that they really don’t understand in terms

of chemistry or immunology,” he says, or they are immunologists who have never “actually

looked at a tool.” Loy tests for a single region of immunoglobin, and he is among the few who

claim to have seen red blood cells on artifacts through a microscope.

At the other extreme is Eisele, who wonders whether blood can endure in prehistoric bone,

much less on stone tools, except under freezing conditions. Those who think they’ve found

blood on artifacts, she says, more likely have picked up proteins from microbes or plants.

Lowenstein remains confident that he has detected blood on some tools. So does biochemist

Noreen Tuross of the Smithsonian Institution in Washington, D.C. In the spring Journal

of Field Archaeology, she and Tom D. Dillehay of the University of Kentucky, Lexington, reported

a strong indication of hemoglobin, possibly from a mastodon, on a tool from a site in Chile

dated to 11,000 B.C.

But Tuross speaks for many when she says, “Immunoreactivity to ancient, degraded molecules  is an area we don’t fully understand. To take modern techniques and to apply them to  ancient results is inappropriate.” Antibodies designed to find fresh, folded proteins could yield  misleading results when used on old proteins that have lost their shape and broken into fragments  or formed denser shapes, Tuross warns.

Adding to the confusion, there’s no consistency across groups on how they test, how they

deal with cross-reactions, or even how they wash possible blood from tools. And unlike

chemists, researchers who publish in archaeological journals aren’t accustomed to describing  their procedures, Eisele says.

Blind tests may help iron out these problems. One has just been set up by University of

Colorado Health Sciences Center in Denver researchers and a Golden, Colo., company, Paleo  Research Laboratories. The group sent stone tools covered with modern blood to a half dozen  research teams, which will analyze them using their usual methods and send back the results.

Lowenstein, a participant, says the study is a good first step. “I think we’re just getting

into the scientific phase of this work, and I think it’s badly needed.” But the real check will be  blind tests for ancient blood, he says.



About Colin Berry

Retired science bod, previous research interests: phototherapy of neonatal jaundice, membrane influences on microsomal UDP-glucuronyltransferase, defective bilirubin and xenobiotic conjugation and hepatic excretion, dietary fibre and resistant starch.
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