Shroud of Turin Blog

- A special posting by Kelly Kearse -

The idea has been proposed that the bloodstains on the Shroud of Turin are the result of application of the blood meals of the medicinal leech, Hirudo Medicinalis, using a felt-tipped pouch. The identity of this illusory forger remains unknown, but has been suggested to be an overzealous medieval monk. For the purpose of discussion, we’ll call him Brother Hirudo. While many may view this idea so preposterous that it warrants no further consideration, this suggestion will be examined below with the focus on maintaining an objective evaluation of the specifics of this proposal. When a hypothesis is put forth, it is predictable that others will raise questions regarding the scientific merit of such ideas. This is standard operating procedure for scientific inquiry. Inflammatory rhetoric and insinuations regarding personal character should not be a part of the equation; it is destructive and takes the focus off of the science. A brief discussion of the procedure of blood clotting is introduced, followed by several specific, key questions regarding the basis of this hypothesis.

Blood clotting

There are four major components important in the clotting of blood: i) platelets, ii) red and white blood cells, and iii) a group of molecules collectively termed clotting factors, which include iv) fibrin, a molecule that forms a meshwork or web, joining all of the above together into a blood plug. When a tear in a blood vessel occurs, platelets first become activated and begin to adhere to the walls of the opening. Unless the tear is very small, platelets by themselves are not sufficient to stop blood flow. Various clotting factors are stimulated to reinforce the platelets, the main one being fibrinogen, which is converted to fibrin, creating a fibrous web that functions as a type of glue. Other cells, such as red and white blood cells may become trapped within the web and help fortify the clot.

The medicinal leech (Hirudo Medicinalis) begins feeding on human blood by

attaching itself to the skin and piercing the outer layer with a set of three blades, arranged at an angle to each other. Leeches contain a natural anticoagulant, or blood thinner, termed hirudin that interferes with the conversion of fibrinogen to fibrin (discussed above) that precludes the clotting of ingested blood. As the leech feeds, the watery portion of blood, the serum, is excluded to maximize the intake of red blood cells. Digestion of blood meals is extremely slow. Leeches may take up to several months to digest imbibed blood. Morphological preservation of erythrocytes ingested by leeches has been observed for up to 18 months.

1. Could the anatomical precision of bloodstains be accurately portrayed using leech-ingested blood applied with a felt stylus?

On average, the human body contains approximately 5 liters or 5000 ml of blood. A person may lose up to 10-15% of total blood volume (500-750 mls) without experiencing any major symptoms. (For those who may not regularly use the metric system, 16 oz. is equivalent to approximately 470 mls or 1 pint). A single leech may consume as much as 15 mls of blood in a feeding. Thus, Brother Hirudo would not have needed to expend tremendous effort to collect a sufficient volume of blood for the task. Moreover, there is no reason to assume that blood collection had to be restricted to a single event.

It has been suggested that the image on the Shroud of Turin shows no obvious, unequivocal evidence of wounds, which would be consistent with the requirement for application of bloodstains. However, various medical specialists would disagree (Barbet, 1963; Bucklin, 1997; Zugibe, 2005; Svensson, 2012; Svensson and Heimburger, 2012), asserting the presence of a major post-mortem wound on the right side of the body and puncture wounds located in the left wrist and middle of the right foot. The forensic accuracy of the bloodstained patterns on the Shroud has also been noted by numerous medical doctors and specialists spanning multiple decades, many of which are listed in the introduction of Y. Clement’s 2012 article “Concerning the authenticity of the Shroud of Turin: please don’t forget the evidence of the bloodstains!!!” For instance, Barbet noted that the blood flow follows a furrow between two extensor muscles of the forearm; others have discussed the gravitational flow of blood from the elbow and off the foot.

Is it reasonable to assume that someone like Brother Hirudo would have the knowledge to include such precise detail when creating the bloodstains on the Shroud? Relatedly, is it feasible that such clearly marked edges of the bloodstains could be achieved by delivering leech-sourced blood through a felt applicator, together with the use of a set of templates? Even the most open-minded scientific reviewer might struggle here.

The bordering area surrounding many of the bloodstains exhibits a halo of sorts, which is only visible under ultraviolet light (Miller and Pellicori, 1981; Jumper, et al., 1984). The medical doctor G. Lavoie, a specialist in internal and occupational medicine, has noted that such halos demonstrate the blood marks of the Shroud had exuded serum (Lavoie, 1998). Moreover, these data are consistent with the previous detection of blood serum proteins on Shroud bloodstained fibers by both chemical and immunological methods. It is unclear how application of leech-ingested blood might be considered here, as a paucity of blood serum would be expected in such mixtures since exclusion of serum occurs in the initial phase of leech feeding (see above). Considering that Brother Hirudo may have been visionary, foreseeing the use of uv analysis in the future, he might have set aside sufficient serum to decorate such wounds, using an additional set of specialized templates.

2. Is the presence of hydroxyproline in blood samples sufficient evidence of leech involvement?

Mass spectrometry is among the most powerful methods that exist for the identification and characterization of small amounts of substances present within a sample. To this end, pyrolysis (heating) coupled with mass spectrometry was performed on samples from the Shroud, having the primary goal of the sensitive detection of impurities (e.g. painting materials and sebum), (R. Rogers, 2008). A blood-spotted ( “Zina”) sample taken from the heel area was shown to emit hydroxyproline following treatment with low-temperature. These results helped to define an upper limit on the highest temperature the blood on the cloth was exposed to, as related to suggestions the Shroud was at one time boiled in oil (Rogers, 2008); also towards Rogers’ refutation of photons of particular wavelength playing a role in image formation (Rogers, 2008).

A tenet of the leech hypothesis is that hydroxyproline is not a regular constituent in human blood, that there is scarcely any worth speaking of. Moreover, as hydroxyproline is known to be present in connective tissue (collagen) of animals, including leeches, this can account for the hydroxyproline signal at m/e 131.

While true that hydroxyproline does not represent a principle component of human blood, its scarcity may be overhyped here. Hydroxyproline can be detected in normal human blood serum using simple immunological techniques that do not approach the sensitivity of mass spectrometry (ELISA kits, see kamiyabiomedical.com and mybiosource.com for examples). Using HPLC (high pressure liquid chromatography) methods, serum levels of hydroxyproline may be evaluated in patients as a measure of liver and renal function (E. Kucharz, Rom J Intern Med Oct-Dec; 32: 271, 1994; Inoue, et al. Analyst Apr. 120: 1141, 1995; Inoue, et al., Biol. Pharm. Bull. Feb 19: 153, 1996). The clinical significance of a hydroxyproline-containing protein in human plasma was reported by Carwile LeRoy and Sjorerdsma as early as 1965 (J. Clin. Investigation 44: 914, 1965). It is not a given that the presence of hydroxyproline is indicative of contamination by animal protein, i.e. leeches. Perhaps it is an oversimplification, but has it also been considered that the sample taken from the heel area could contain trace components (hydroxyproline) of abraded skin? Or that the sample might have been contaminated by exposed skin during its collection and handling? Analysis of multiple blood areas would help determine if this finding were unique to this particular area of the cloth, and further validate the detection of hydroxyproline in Shroud bloodstains.

3. What are the colorometric and chemical properties of leech-ingested human blood?

One of the main issues that is raised regarding the involvement of someone such as Brother Hirudo, is what is known regarding the properties of leech-ingested blood?

Other than trying to imagine how someone might have gotten around the problem of using normal blood subject to clotting, what empirical evidence is there that the appearance of the bloodstains is telling of leeches? This raises some interesting points as to what may be known regarding the properties of leech-ingested blood that is put to further use. For example, when expelled, does such blood eventually clot upon drying? If so, what are the kinetics and what is the appearance of such bloodstains? Have any spectrophotometric studies ever been performed to compare normal vs. leech-ingested blood to evaluate the oxidation state of hemoglobin that is present? Such information could help establish a preliminary basis for further consideration of this novel idea.

Concluding Remarks

I do not have a satisfactory explanation for why the blood on the Shroud of Turin has a red appearance. I would like to know. I am not convinced that bilirubin is the answer. I am not sure I completely understand the proposed effect(s) Saponaria treatment might have. I am willing to consider the involvement of other possibilities that involve some type of conversion of chemical bonds, by natural or even supernatural means. Leeches? It’s a creative idea, I’ll admit, but I need a lot more to go there. When I was a teenager, we used to wade out to up above our waist to use a pitchfork to remove lily pads that had overgrown on our neighborhood lake in the summer. The average leech count on each laborer was easily in the mid-thirties, upper torso to bottom toe, dorsal and ventral. I guess that’s why our dads sent us out to perform the task while they “held down the fort.” Of course, Brother Hirudo would have anticipated as much.

Whatever the pathway, the coloration of the bloodstains on the Shroud must have a definable, molecular basis. Further characterization of the chemical nature of the blood is central in any effort to define the basis for the resultant color. It is reasonable that more could be learned by careful examination of older (unrelated) blood samples. Others may argue that because this situation is totally unique, such comparisons will eventually become futile; even so, perhaps important knowledge could be gained before eventually is reached. Finally, any evaluation of blood coloration should be considered in the context of adhering to/binding the fibers of the cloth; this is an important variable, which should be part of the matrix. It is also one of the most challenging. The coloration of the bloodstains is an interesting scientific question, regardless of where one stands on possible mechanisms involved in image formation, or even on the proposed age of the cloth.

Guest Posting by Kelly Kearse: Distinguishing human blood from that of other species

Guest Posting by Kelly Kearse: Whose DNA is it, anyway?

Positive for AB is not the same as AB positive

MUST READ: Cloning the man on the Shroud of Turin

Just how old is the AB blood type?

MUST READ: A lot of old blood types as AB: Not Exactly

Distinguishing human blood from that of other species:

Too much monkey business?

The term “human blood” is consistently used in discussions of the bloodstains on the Shroud. Just how is human blood distinguished in the laboratory? And where exactly does the data on the Shroud stand? These questions are briefly discussed below.

Human blood versus animal blood

A scientist cannot just look at a bloodstain with the naked eye and tell that it’s human blood. For fresh blood, microscopic analyses may allow one to distinguish mammalian red blood cells from non-mammalian red blood cells due to the absence and presence of a nucleus, respectively. If sufficient numbers of (white) blood cells are present, chromosomal characterization (karyotyping) may be performed, at significantly higher magnification. Among primates, only humans contain 46 chromosomes; chimpanzees, gorillas, and orangutans contain 48. The chromosome number of other species is quite variable, for example pigs have 38, sheep have 54, dogs have 78, and cows have 60.

In aged bloodstains, such microscopic tests are not practical because blood cells become dehydrated and rupture within hours of drying. Species characterization of dried, aged bloodstains relies on serological (immunology) tests or molecular (DNA) analysis. Chemical tests cannot distinguish human from animal blood. For serological studies, human blood components (usually albumin or immunoglobulin proteins) are detected using antibodies that are generated in another species, for instance, rabbits. When utilized in the laboratory, such antibodies would not react with blood components from other animals, for example chickens or cows, because enough difference exists from their human counterpart proteins that the antibodies fail to recognize them.

A positive reaction in such tests usually results in the conclusion that human blood is present. However, this is where things get somewhat tricky. Even though such reagents are designated as “anti-human”, this only refers to the species in which they were generated. Cross-reactivity (or the lack thereof) must be independently verified. Species that are closely related to humans (i.e. non-human primates) express blood components (albumin and immunoglobulin) that are similar enough to those of humans to also react positively in such tests (see picture above). In forensic settings this is typically disregarded unless special circumstances warrant that such possibilities be considered (at a crime scene within a zoo, for example, or if someone were known to keep apes or monkeys as pets). In most situations, when it is stated that bloodstains tested positive for human blood, this underlying supposition exists. Strictly speaking, such serological tests do not distinguish human blood from the blood of other primates (monkeys or apes). If sufficient DNA is intact for molecular biology analysis, specific regions of certain genes may be targeted that have sequences unique to humans, which allow them to be effectively distinguished from those of closely related species.

Human origin of the blood on the Shroud

In the vast majority of discussions of the human nature of the bloodstains on the Shroud, the studies that are typically referenced are the experiments of Adler and colleagues and Baima Ballone and coworkers, utilizing serological tests for detection of common major blood components: albumin, immunoglobulin, ABO antigens. In fact, such studies do not distinguish human blood from the blood of other primates.

With the Shroud, primate may imply human but this is an extension beyond what the data actually show. Adler was appropriately cautious in concluding that the data only demonstrated that the blood was of primate origin, and even conducted experiments to evaluate the cross-reactivity of such “anti-human” reagents. Adler would also comment, “If you choose to think that the image you see is that of a chimp or an orangutan, you’re perfectly welcome to believe that…”

To date, the only study that directly addresses the human nature of the Shroud bloodstains is an often overlooked report by Baima Ballone et al. that evaluated the expression of additional blood components found on red blood cells, specifically the M,N, and S antigens. (Such antigens have also been studied in the blood analysis of King Tut). The conclusion was that the bloodstains on the Shroud are characterized as MNS positive. What is most significant about these studies is that unlike M and N antigens, which are shared between certain primates and humans, the S antigen is exclusive to humans only. No S counterpart exists in other species, including apes or monkeys. This point was not emphasized (or mentioned) in the report, as the significance of this relationship among primates was not fully elucidated until several years later (in non-Shroud related studies). Of the six serological analyses of blood components on the Shroud, this brief study remains the single most definitive piece of serological evidence that directly addresses the human origin of the blood on the Shroud. For a more detailed discussion see: “Empirical evidence that the blood on the Shroud of Turin is of human origin: Is the current data sufficient?” recently published on shroud.com. The full-length manuscript is available at http://www.shroud.com/pdfs/kearse1.pdf.

Concluding Remarks

Taken together, the current serological data indicate the blood on the Shroud is of primate, i.e. human origin. Could more work be done in the laboratory to strengthen the conclusion that the blood is indisputably human? The answer is yes. Should there be sufficient doubt that the blood is in fact, human, and may represent the blood of a monkey or ape? The MNS studies say no. All other data are consistent with this finding. Moreover, Adler has effectively commented on the difficulties a forger would encounter in trying to apply clotting blood [of any species] to various regions on the Shroud. Since the original blood studies were performed some thirty years ago, significant advancements have been made in the development of serological and molecular tools that could prove useful in advancing previous information. For example, within the past few years, monoclonal antibodies have been generated that effectively distinguish human blood from that of closely related species. Similar to blood typing data, additional analyses would cross-check and verify previous findings on the bloodstains of the Shroud.

Kelly Kearse writes:

While watching the Orioles go down in flames this evening, I wrote a posting about the origin of the AB blood type . . . .

He attached it and here it is:

Just how old is the AB blood type?

In response to an earlier posting on the upcoming BTST meeting, there has been much discussion about when the AB blood type first appeared in history. AB blood type has been reported in skeletal remains that are approximately 1,600-2,000 years old (Am. J. Phys. Anthrop. 47: 89-91, 1977), and in tissues of mummies from 3 B.C. to 4^th century (Forensic Science International, 43: 113-124, 1989). These studies were done using serological methods (antibodies), which recognize the ABO molecules on the surfaces of blood cells (see Figure below). As ABO antigens are also found in many organisms including bacteria, fungi, and insects, the issue of “false positives” is often mentioned when discussing blood typing of ancient samples (see “Blood on the Shroud of Turin: An Immunological Review, available on shroud.com, for a detailed discussion).

An alternative method for blood typing exists, which involves molecular biology (DNA) techniques to probe for the genes responsible for conferring ABO blood type. Because human red blood cells (RBCs) lack a nucleus, this method evaluates gene expression in white blood
cells (WBCs), or other nucleated cell types (see Figure below). In this technique, which uses the polymerase chain reaction (PCR) to amplify DNA, possible issues with false positives due to bacteria are circumvented because it is the (internal) gene responsible for encoding the surface antigens that is being evaluated. Unlike the surface antigens which may be similar in humans & bacteria, human ABO genes are easily distinguished from DNA of other organisms. While this may seem like the method of choice for analyzing ancient samples, such studies are often precluded because of DNA degradation issues. Indeed, for typing studies of aged bloodstains, more reports exist in the literature using serological (surface) analysis relative to molecular (DNA) analysis.

Within the past couple of years, I have written to numerous scientists who specialize in the fields of anthropological and molecular evolution (my research background is in cell biology & immunology) to inquire about the origins & appearance of the AB blood type. Specifically, I asked what is the first human or human-related sample that has typed as AB using molecular biology (DNA) methods; and when is the AB blood type believed to have emerged in human history? The consensus from the answers I received is represented in several specific responses quoted below, “ABO systems have not been widely analyzed in ancient remains [using molecular biology techniques], there are very few papers on the blood system.” “There certainly is controversy as to when group B (and thus AB people) emerged.” A specialist at Harvard replied, “It is CERTAINLY [his caps, not mine] well before 10,000 years ago”, “some place the origin of B @ 100,000 years ago.” “I am very sure it was long before 1,000 years ago-based on DNA divergence from the primordial group A gene”. One of the world’s foremost authorities on the molecular analysis of the ABO blood group, whose laboratory first cloned the human ABO genes, was quite adamant in his response that D’Adamo’s claims that the AB blood type is only 1,000 years old “does not fit with the current theory of the evolution of the ABO gene”…and “is not based on scientific evidence”.

In the literature, the few samples that have been analyzed (by molecular DNA techniques) are reported as O; for example, 2 Neanderthals analyzed by Lalueza-Fox in 2008, and Otzi the 5,300 year old “ice man”. In the current population, ~44% of people are type O, compared to ~5% for type AB (A and B are 35% and 17%, respectively). Thus, perhaps it is not that surprising with such a very limited amount of DNA studies reported, type O would be more prevalent. Clearly, much more work remains to be done in this particular area, especially in relation to molecular analysis of aged samples.