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Just how old is the AB blood type?

October 13, 2012 8 comments

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

February 16, 2012 64 comments

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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.

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