Paper Chase: DNA Analysis and the Shroud of Turin: Development of a Shroud CODIS

imageSTERA has just published another paper by Kelly Kearse, DNA Analysis and the Shroud of Turin: Development of a Shroud CODIS. This is Kelly’s fourth paper at shroud.com. This paper will certainly shape discussions about future research on the shroud.

Barrie Schwortz, in introducing the paper this morning on shroud.com, writes:

once again, he has taken a very technical subject and made it interesting and understandable for everyone,”

Based so far on an early morning first read (the coffee is still brewing), I agree. More importantly, what do you think?

ABSTRACT: 

Since its development in the mid 1980s, DNA analysis has become a standard procedure utilized by law enforcement and legal systems in the forensic examination of human remains, and to help establish or exclude a connection to a crime scene. The recent progression of gene amplification and enrichment strategies, together with next generation sequencing techniques, have made the analysis of ancient and degraded DNA samples much more feasible than previously imagined. Human DNA has been isolated from the Shroud of Turin, although the results remain rather limited and controversial. Indeed, it is unknown if such DNA truly originates from blood cells present on the cloth or is the result of contamination from exogenous sources. Here, the potential and limitations of modern molecular biology techniques in the analysis of the Shroud of Turin are reviewed, including the evaluation of both nuclear and mitochondrial DNA.

The High Price Of Success at STERA

clip_image001Barrie Schwortz writes on the Late Breaking News page of shroud.com:

As I said at the beginning of this update, a "perfect storm" of events recently led to massive international media coverage of the Shroud, the most in many years. The effect on STERA, Inc. and Shroud.com was immediate and I think the above graphic chart says it all. We had nearly 150,000 unique visitors in March (nearly triple our norm) and 90,000 in April. Consequently, our bandwidth tripled in March to 87GB and was double the norm in April at 50GB. Although we receive very reasonable rates from our service provider and a gracious allowance of free bandwidth (which we only rarely exceed), we dramatically exceeded our allowance over March and April. This put an unexpected burden on our operating expenses and we have to ask our viewers for assistance. We are hoping to raise at least $3000.00 to help offset these recent expenses and you can help by making a safe, online tax deductible contribution to STERA, Inc. using our 256 bit encrypted Secure Contribution Form. Please help us manage the high price of success.

Again: Secure Contribution Form

Blood Clotting and the Strange Case of Brother Hirudo

– A special posting by Kelly Kearse –

 

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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.


Other essays and postings by Kelly Kearse:

 

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

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

Distinguishing human blood from that of other species:

Too much monkey business?

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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.

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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.

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


Whose DNA is it, anyway?

Immunoglobulin, the T cell receptor, and the Shroud of Turin

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Below is a brief synopsis of the paper “DNA on the Shroud of Turin: Distinguishing endogenous versus exogenous DNA” recently published on shroud.com. The full- length manuscript is available at http://www.shroud.com/pdfs/kearse2.pdf.

In the 1990s, Garza-Valdes reported in the book “The DNA of God” the cloning and sequencing of human gene segments from blood remnants on the Shroud. The presence of human DNA on the Shroud is sometimes viewed as corroborative evidence that the bloodstains are composed of real blood. Mature red blood cells in the human lack a nucleus and do not contain DNA; any DNA present from bloodstains must come from the white blood cells, which include B and T lymphocytes, or B and T cells for short. It is the DNA present within these two cell types that could prove useful in verifying that (certain) DNA on the Shroud is truly from blood cells and not from contaminating DNA, originating from other sources (see below).

Contamination of handled objects: Touch DNA

A major issue that persists in DNA analysis of objects that have been handled by numerous individuals is contamination. The average person sheds roughly 400,000 skin cells per day, a portion of which contains DNA that may be transferred to handled objects, referred to as touch DNA. DNA may be transferred by direct contact with the cloth, or by touching an object, which then comes in contact with the cloth (or with threads removed from it). A determined skeptic might argue that contaminating DNA is responsible for the previous results, a charge that is somewhat difficult to counter.

The three gene segments that have been isolated and analyzed from the Shroud are: the betaglobin gene (a subunit of hemoglobin), and the amelogenin-X and amelogenin-Y genes (present on the X and Y chromosomes, respectively). Such genes are not exclusive to blood cells, but are present in essentially all cells throughout the body, including skin cells. Thus, analysis and sequencing of such gene segments is unable to distinguish if they originated from DNA from bloodstains present on the cloth, or from contaminating DNA from exogenous sources, i.e. skin cells.

Same technique: Different Target Genes

Previous Shroud DNA studies utilized the polymerase chain reaction (PCR) technique that works by repeated cycling of DNA replication to exponentially amplify DNA. Such methods allow even small gene fragments to be very rapidly and effectively analyzed: a billion DNA copies can be created from a single DNA strand in just three hours time.

Unlike past Shroud studies, which focused on genes that appear identical in all cell types, the suggested experimental approach uses PCR techniques to specifically target lymphocyte receptor genes. B and T lymphocytes are unique in that unlike any other cell type in the body, the DNA encoding their cell surface receptors undergo rearrangement and splicing: immunoglobulin, or antibody for B cells, and the T cell receptor for T cells. As a normal process of B and T cell development, receptor genes separated by large distances on the chromosome are brought close together and edited to create a final product. Although other cell types, including skin cells, contain such genes, they remain in the unrearranged (germline) configuration throughout their lifetime: DNA rearrangement is specific for lymphocytes. Detection of rearranged lymphocyte receptor genes is an established and routine laboratory

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procedure, commonly used in the diagnosis of various leukemias and lymphomas. Multiple B cell and T cell receptor genes exist that could be suitable for DNA analysis. Moreover, as many of the genes are present on different chromosomes, this might increase the chances for detection if certain DNA regions are more fragmented than others. Samples taken from several sites on the Shroud, particularly those below the fibers on the surface, would yield the most definitive conclusion. Such an approach may help establish that DNA in the blood areas of the Shroud of Turin originated from white blood cells (lymphocytes) present on the cloth.

Positive for AB is not the same as AB positive

Kelly Kearse writes:

Blood typing and the Shroud:
Positive for AB is not the same as AB positive

 

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The bloodstains on the Shroud are reported to be type AB, as determined forward typing methods. Although semantically similar, being positive for AB blood is not physiologically equivalent to being AB positive. Here’s the difference: blood typing nomenclature typically lists the blood type first (A, B, AB, or O), followed by expression of a molecule termed the Rh factor. The ABO and Rh molecules are not genetically linked and are encoded on separate chromosomes. ABO and Rh are listed together in blood typing because both are important in blood transfusions. (Individuals that are Rh negative can mount an immune response to red blood cells that are Rh positive). You either express the Rh factor or you don’t, which is why the designation positive or negative is used.

A person that is AB positive (AB +) would contain red blood cells that express AB antigens as well as Rh molecules. Relatedly, a person that is AB negative (AB -) would contain red blood cells that express AB antigens, but not Rh molecules. Technically, (semantically), both individuals are positive for AB, but only the first is truly AB positive. Although the blood type of the Shroud is frequently reported as AB positive (or AB negative), to the best of my knowledge, expression of the Rh factor on the bloodstains on the Shroud has never been evaluated. In a personal communication with Baima Bollone last year (through Emanuela Marinelli), he replied that the Rh factor was too degraded for study at the protein level. The positive (and negative) designations that are at times assigned to the Shroud bloodstains are most likely semantic in nature (due to nonstandard use of the words “positive” or “negative”), and not data-driven. It is correct to say that the blood on the Shroud is positive for AB, which is most precisely stated as “types as AB”. It is not accurate to say that the blood on the Shroud is AB positive. This implies additional information about the expression of the Rh molecule, which is unknown

MUST READ: Cloning the man on the Shroud of Turin

 

Cloning the man on the Shroud of Turin:

The Media’s Hyperbole with the Double Helix

by Kelly P. Kearseimage

The subject of a recent blog post about a comic book series that is now into its fifth issue, Punk Rock Jesus, involves a rather popular storyline regarding the Shroud: using DNA extracted from bloodstained threads to clone Jesus. Search on amazon.com and you will find over twenty fiction novels based on this premise; include those available exclusively as e-books and you can add about ten more. There has also been an Outer Limits television series episode, and a feature film released in 2010, “I’m not Jesus, Mommy”, centered on this idea.

Just how realistic is this scenario? What would be required to accomplish the cloning of a person under such circumstances? Would a clone be an exact duplicate of the Turin Shroud man? These and related issues are discussed below.

What exactly is cloning?

Cloning is the creation of an identical genetic copy of a living organism. Several types of cloning exist, but the most germane to the discussion of the Shroud is reproductive cloning using a method known as somatic cell nuclear transfer (SCNT). Somatic cells are cells other than sex cells (sperm or egg), which under normal circumstances do not provide DNA in the generation of an organism. Development of the SCNT technique began in the early 1950s using frogs, and was further refined and eventually popularized in the mainstream media years later with the success of Dolly, a cloned sheep, in 1996. The basics of this method are shown in the figure below. The nucleus of adult cell (a skin cell, for example) is isolated and transplanted into an egg cell (oocyte), which has had its own nucleus removed. The egg cell is then implanted into a surrogate mother, who also receives various hormones to simulate the normal course of pregnancy. Since the only source of nuclear DNA in the developing embryo is from the adult cell, the resulting offspring will be genetically identical to the organism from which it was taken.

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In the creation of Dolly, the scientists used a very clever strategy to monitor their success: the skin cell containing the DNA to be transferred was taken from a type of sheep that was purely white-faced; the host egg cell into which this nucleus was transplanted was from a black-faced animal. If truly a clone, the offspring would have to be purely white-faced (which was also verified by DNA analysis). Cloning Dolly required significant effort; success was achieved only after 276 previous attempts by the same group resulted in failure.

Send in the clones

Since the creation of Dolly, other types of animals have been cloned using this method, including mice, rats, cats, dogs, goats, deer, cows, mules, and horses. To date, however, reproductive cloning has not been successful in primates. Although cloning of a Rhesus monkey was reported in 2007 (by embryo splitting), this is not equivalent to reproductive cloning by SCNT using DNA from adult cells in the creation of an exact genetic copy. Refinement of this method for use in primate cells has been especially hampered by the fact that removal of the nucleus from the egg cell disrupts important host proteins that are essential for subsequent division and development. It is certainly possible that current limitations to reproductive cloning in primates will be overcome in the future as techniques continue to be developed and refined. Reports of cloned human embryos have periodically surfaced in the media, but all have been subsequently found to be bogus.

Cloning and the Shroud

Apart from the existing technical roadblocks in the reproductive cloning of primates, if such a system were currently in place, cloning the man on the Shroud using DNA isolated from bloodstains still lies well within the realm of science fiction. Multiple problems exist with this scenario. First and foremost, to clone an organism, you need a full complement of nuclear DNA. The DNA on the Shroud is badly fragmented; while certain regions on particular chromosomes may be intact (for example, portions of the betaglobin and amelogenin X and Y genes sequenced by Garza-Valdes and coworkers), it is extremely unlikely that sufficient DNA is present to represent the entire genome. As mentioned above, even with technically pristine DNA present in a freshly isolated nucleus, successful transfer and development often requires numerous attempts together with a generous amount of luck.

Additionally, because numerous individuals are known to have handled the cloth, it is unclear that any DNA isolated would belong exclusively to the man on the Shroud. The average human being sheds approximately 400,000 skin cells per day, a portion of which contains DNA that may be transferred by contact, referred to as touch DNA; how long touch DNA may survive is unclear and unique to each object. The extent of contamination of the Shroud by exogenous DNA is unknown, but given the communal nature of the cloth in both its past and even more recent history, it is reasonable to speculate that DNA from numerous individuals may be present on the Shroud. If it were possible to obtain a full nuclear complement of DNA from a sample taken from the Shroud, it is likely to be a mosaic, resulting from the contribution of multiple persons. In the previously mentioned 2010 film “I’m not Jesus, Mommy”, the scientist responsible for the breakthrough, Dr. Gabriel, announces “What you are holding in your hands is the first human embryo cloned from red blood cells.” This is a miraculous feat indeed, as red blood cells in humans (and all mammals) are devoid of DNA because they lack a nucleus. In the non-Hollywood version, DNA from the Shroud would have to originate from white blood cells in the bloodstains.

A True Duplicate Copy: Ob-La-Di, Ob-La-Di or Ob-La-Da?

Ethical and moral issues aside, which are without question, hugely significant and relevant; and strictly speaking from a scientific viewpoint: if a full complement of intact, unfragmented nuclear DNA were available, and if it were purely from the bloodstains on the Shroud, and if current methods were in place for reproductive cloning in primates, would a clone be identical to the man on the Shroud? Genetically speaking, the answer is yes and no. Although a clone contains exactly the same nuclear DNA as the organism from which it originated, it is not entirely identical. There is no such thing as an exact clone. In addition to nuclear DNA, cells also contain mitochondrial DNA, which encode genes necessary for cellular function. In reproductive cloning, only the nuclear DNA is transferred to the donor egg cell. All mitochondrial DNA originates from the host egg cell, which will be expressed in the resulting organism (clone) throughout its lifetime. In normal organisms (non-clones), while nuclear DNA is inherited from both parents, mitochondrial DNA is transmitted solely from the mother.

Genes are only part of the story in the development of an organism. Environmental factors may influence which genes are turned on and which genes are switched off. Even monozygotic twins, which are truly genetically identical, do not have the same fingerprints. Twins that are raised together may appear at times indistinguishable, but each possesses unique personality traits and even physical features that are distinct characteristics. Unlike cinematic portrayals of cloning, which at times border on the irrational (e.g. Multiplicity, 1996), clones are not born as adults, equivalent in age to the individual from which they were propagated. A clone would be born as an infant and subject to unique experiences and environmental influences, which would impact the genetic blueprint. A clone would be expected, of course, to be very similar to the organism from which it came, but an identical carbon copy is not likely.

What the future holds in terms of cloning, particularly in relation to higher organisms, remains to be determined. Technology has advanced relatively rapidly compared to the full consideration of moral and ethical issues that accompany such scientific progress. Concerning the Shroud, such cloning scenarios are best categorized as science fiction rather than science fact.

Just how old is the AB blood type?

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?

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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 4th 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.clip_image006

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.

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

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Kelly P. Kearse, a card-carrying immunologist writes:

I appreciate the opportunity to address the issue that “All old blood types as AB”, particularly in reference to the study of the Shroud. The idea that “aged blood is degraded to (or reverts to) type AB” is rather misleading.

Blood typing is typically performed using two distinct methods that measure two very different things. First, there is forward typing, which measures the presence of specific molecules on the surfaces of red blood cells (RBC): the ABO molecules. Second, there is reverse typing, which measures the presence of antibodies in the serum (essentially the fluid component of blood), specifically the presence of antibodies to ABO blood group molecules. Both forward and reverse typing methods have been utilized in the study of the Shroud.

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ABO molecules and serum antibodies

The presence of ABO molecules on red blood cells and antibodies directed against these molecules in the serum are complementary within an individual. That is, a person with a particular blood type (A, B, AB, or O) doesn’t contain antibodies in his or her serum against the A or B molecules they themselves express, but will contain antibodies specific for the A or B molecules they lack.

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ABO molecules are a branched chain of carbohydrates (sugars) that share the same core structure, but are uniquely modified by a different terminal sugar to create A and B molecules. The O molecule doesn’t receive a terminal sugar, but exists as the (unmodified) core structure.

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When the statement “all old blood is degraded to AB” is evaluated in the context of ABO structure, it doesn’t make much sense, as degradation would result in aged blood being converted to type O. This statement inadvertently refers to reverse typing, which is somewhat less informative and more difficult to interpret on its own (see below).

Forward typing methods: What the data show

In forward typing of fresh blood, samples are mixed with freshly added antibodies and evaluated for their ability to clump, or agglutinate. This is the type of test typically performed at local blood drives. For fresh blood, typing tests are fast, inexpensive, and relatively foolproof. When aged blood is involved, these techniques are slightly modified since red blood cells become dehydrated and eventually rupture. Remarkably, red blood cells have been detected microscopically in ancient samples, including mummies, prehistoric rock tools, and also the Shroud. What is most likely being visualized in such samples are aged red blood cell membranes that have resealed during the preparation procedure, representing reconstituted cells, not intact red blood cells that have survived over large periods of time.

In 1983, Bollone and colleagues reported that bloodstained fibers were positive for both A and B molecules, assigning a blood type of AB. A few years later, these findings were extended using more sensitive methods, involving antibodies tagged with specific dyes, that bypass the need for adding fresh red blood cells in the final step. These data demonstrated that bloodstained fibers were positive for both A and B antigens, but not O antigens. Evaluation by electron microscopy showed equal intensities of anti-A and anti-B binding. Unstained fibers failed to react with anti-A, anti-B, or anti-O specific antibodies. Most significantly, even colorless fibers taken from the bed of the stain were negative. Collectively, these findings are in agreement with the supposition that the blood on the Shroud is type AB. In 1998, Garza-Valdes performed similar studies and reported bloodstained TS fibers were positive for B, but not O antigens; anti-A reactivity was not evaluated.

One criticism that has been raised regarding forward typing of aged samples is that because ABO molecules are carbohydrates, which are shared by multiple organisms (including bacteria, fungi, and insects), contamination may preclude accurate typing results, a concern noted by the original investigators. Thus, one might expect that “all old blood types as AB”. While true that studies have been published in which ancient materials serologically type with a relatively high incidence as AB, this is certainly not always the case. Often, reported errors in serotyping of ancient specimens result from the failure to determine any blood type, not an error in specific blood type determination. Thus, while tempting to dismiss serological typing methods of ancient materials in one broad stroke, the preservation of red blood cell antigens and the extent of contamination in aged material must be evaluated specifically for each case study.

Throughout its history, the Shroud has been handled by untold numbers of people and stored under less than pristine conditions. The extent of the bacterial and fungal biofilm present on the Shroud is unknown, but it is reasonable to assume that the surface is less than aseptic. False positives from antigens present on contaminating bacteria, fungi, or insects is the main objection to the validity of blood typing studies done on the Shroud; a logical deduction given the wavering reliability of serological studies on ancient artifacts. An underlying assumption that must accompany this criticism, however, is that contamination is stringently restricted to bloodstained areas of the cloth. Indeed, colorless fibers of the Shroud taken from the bed of the bloodstain gave no response with anti-A or anti-B antibody, indicating that any contaminating false positive antigens that may exist cannot be widespread. It might be countered that bacterial or fungal antigens would be expected to be concentrated in areas that are relatively enhanced with body fluids and cellular debris. Rationally, one would expect that such contamination would also extend to areas immediately adjacent to (e.g. at bed of) bloodstains and (at least slightly) beyond, making it somewhat difficult to reconcile these findings with the simple explanation that contamination is responsible for the results. Thus, taken at face value, it appears that the typing results are specific and cannot be attributed to (nonspecific) contamination. Isolation of ABO molecules specifically associated with human red blood cell proteins (for example, Band 3) would help distinguish these results. In studies unrelated to the Shroud, Kimura and colleagues have used this approach in accurate typing of bloodstains on fibers only 1-1.5 cm in length.

Reverse typing methods: what the data show

In reverse typing of fresh blood, blood serum is mixed with freshly added red blood cells and evaluated for their ability to clump, or agglutinate. This type of test is typically done in the blood lab to corroborate forward typing results. A similar, slightly modified procedure is used when aged samples are under study.

Consistent with their forward typing results, Bollone and colleagues reported that no naturally occurring anti-A or anti-B antibodies were present in bloodstained fibers on the Shroud. Reverse typing studies on aged samples are somewhat difficult to interpret, however, because they cannot distinguish between the possibilities that (i) no anti-A or anti-B antibodies were ever present (truly type AB); or (ii) anti-A or anti-B antibodies were once present in bloodstains, but over time were denatured and degraded (type A, B, or O). While proper controls were included to demonstrate that the reverse typing method was operational and specific, these can only be applied to control fiber samples experimentally daubed with (fresh) human blood of known type. Given that the expected result for AB blood type is that no naturally occurring anti-A or anti-B should be present (even in freshly obtained samples), this issue becomes somewhat circular. To quote the late Ray Rogers, “Nothing is ever simple with the Shroud”.

Why does it matter what the blood type is?

While some may view the scientific details of blood components as somewhat irrelevant, for example blood typing, such knowledge contributes to our overall understanding of the physical characteristics of the Shroud. Scientific data from blood studies provide an important component in this investigation.

In 1985, Baima Bollone and colleagues performed similar typing experiments on samples taken from the Oviedo cloth, concluding that blood group AB molecules were present in the bloodstains. The findings were confirmed by Goldini et al., in 1993, who also reported a series of standard chemical tests, similar to those used by Heller and Adler, to verify that blood is present, including the demonstration of hematoporphyrin.

Comparative blood typing is an exclusion test, the most solid conclusions can be made when blood types are not shared between samples, ruling out that such cloths wrapped the same person. When blood types are shared, the results are consistent, but still circumstantial. Mitochondrial DNA analysis would greatly extend the investigation into the relationship of these two cloths.

Is the blood on the Shroud type AB? Probably

Ideally, forward and reverse serological typing methods cross-check and complement each other, but as is the case with certain aged samples, such as the Shroud, the validity is somewhat in question, particularly the reverse typing results. Thus, it is best concluded that the results suggest that the Shroud bloodstains are type AB as shown by forward typing methods. To dilute the significance of these results by adding that “ however, all old blood types as AB” or “all old blood is degraded to AB” unfairly oversimplifies the issue. Number one, it totally depends on which typing method is involved, and number two, this is simply not always the case. As correctly noted in a response to the earlier post, serological typing has been successfully used in the study of mummies by Robert Connelly, including King Tutankhamun with blood type A. Clearly, serological evidence is most useful when corroborated by additional experimentation, for example molecular (DNA) analysis of blood group genes. The DNA on the Shroud is badly fragmented, although the extent to which specific chromosome regions survive remains to be determined. Expression of human ABO blood groups is controlled by a single locus in exons (coding segments) 6 and 7 of chromosome 9. If molecular analysis of this region were feasible, such studies would help address previous concerns raised with serological techniques regarding the blood type.

AB negative or AB positive: which is it?

Finally, in addition to ABO, the designation “positive” or “negative” is often given following a person’s blood group (for example, A positive or O negative). The “+” or “-” refers to expression of the Rh molecule, which is distinct and separate from ABO molecules. Individuals either express Rh molecules (Rh+) or they don’t (Rh-). Although widely reported on the internet that the blood on the Shroud is AB positive (AB+) or AB negative (AB-), there is no scientific basis for these claims. In previous tests, the condition of the blood was such that analysis of the Rh factor was not feasible (personal communication with Baima Bollone through Emanuela Marinelli). Therefore, the expression of Rh antigens on bloodstained fibers of the Shroud remains to be determined. It is unknown if the blood type is AB positive or AB negative.