Determining the blood group – A, B, AB or O – in a FRESH sample of blood is fairly straightforward.
You can do it one of two ways, masterfully described recently by Kelly P. Kearse (a self-styled “card-carrying immunologist”) with a gift for exposition on the Other Site. Better still, as he was careful to point out, you do it both ways. One is by “forward” typing, where you add serum containing antibodies to blood group A and B separately, and look to see agglutination (“clumping”) of the red blood cells . If both anti-A and anti-B serum cause clumping, then it is blood group AB. If neither anti-A nor anti-B causes clumping then it is blood group O ; if anti-A but not anti-B causes clumping it is blood group A, and if anti-B but not anti-A causes clumping then it is blood group B.
Before considering the problems of applying this procedure to aged blood – blood that is centuries old, or millennia even – let’s first ask ourselves what one is determining when one says “blood group A” or whatever. For starters, let’s keep it immunological rather than biochemical (or try to at any rate).
A red cell membrane has immunological markers on its surface, ones that distinguish between self and non-self. It is particular surface sugars and/or sugar derivatives that are responsible for ABO incompatibilities, i.e. where one cannot give a blood transfusion of, say, blood group B into a blood Group A individual (since the anti-A in the first would attach and clump to the A markers in the second, causing agglutination and blocking of blood vessels).
Antibodies such as anti-A or anti-B have the ability to recognize the markers (“antigens”) on the red cell membrane, and having attached cause red cells to clump. So our immunological test with FRESH blood depends, one might think, on the red blood cells being in a reasonably intact state. The membrane has to be the right way round, with markers pointing to the outside rather than inside, and red cells have to be able to interact with each other, no doubt relying on Brownian motion (i.e. those frenetic “vibrations” that one sees under the microscope due to kinetic energy, in particular transient imbalances in the direction of collisions with water and other small molecules).
So will the test work on ancient degraded blood? Answer: it depends. It depends on the extent of degradation, notably the extent to which either the intactness of the marker OR the intactness of the RBC membrane or BOTH are affected, and one’s ability as a archaeo-blood typer to “work around” these alterations.
There are two phases in the degradation of blood that can be envisaged, both mentioned briefly in the Kelly Kearse (KK) write-up. The first is rupture (lysis) of the red cell membrane, tha latter being basically a phospholipid bilayer with embedded proteins and glycoproteins (proteins with attached carbohydrate chains that “stick out” into the surrounding medium).
However, ruptured membranes have a tendency to reseal, often preserving sidedness. A prime example are the stacked endoplasmic reticular and Golgi membranes : when tissue is homogenised they break up, but reseal as tiny “microsomes”, aka closed vesicles, preserving sidednesss, e.g. enzymes that are on the inner face are similarly on the inner face of the microsomes. So lysis per se, provided it is followed by resealing and retention of sidedness, should not affect an immunological test per se as far as the amount of available antigen is concerned, provided the chemical marker, e.g. glycoprotein sugar and its immediate neighbours are preserved sufficiently for the antibody to detect their chemical markers and the SHAPE of their surroundings. However, there is a caveat. The test with FRESH blood relied up intact red blood cells to produce the agglutination, generally viewed on a microscope slide. What if the RBC were too damaged by rupture and random resealing to function properly as a detection system? That is the prime question that needs to be addressed if devising protocols that reliably extend the ABO test to aged blood.
This writer is a biochemist and not an immunologist. He has to read between the lines, and supplement where necessary with internet searches to seek confirmation that he is making the right connections, and valid deductions.
Example: KK says that testing with old blood requires adding fresh blood at the end, at least in the original protocols. Why the need for fresh blood one may ask? I assume that it’s for the reason already suggested – that while the aged RBC are intact enough to preserve markers they are not suitable in the other half of their dual role as agglutination-detectors. So the reasoning went as follows: add an excess of antibody, so that a fraction attaches to the membrane markers, while the rest remains free. Then add a suitable source of blood to measure the surplus of free, and that “titre” can then be used to estimate how much of the antibody had attached to the RBC membrane. A 1957 paper confirmed my hunch that this is/was the original approach adopted with ancient blood (bone actually in which blood was the active ingredient).
But let’s be clear: this revised protocol is one where the analyst is operating entirely in the dark, using an immunological tool to probe for the presence of particular glycoprotein markers, which are in turn the phenotypical expression (outward appearance) of genetic markers that are ultimately encoded in specific DNA sequences. Seen in those terms, the ABO testing, done by a backdoor route in the case of aged blood, is a test in which the end-result – agglutination of ADDED fresh blood – is used to deduce the presence of absence of a particular kind of DNA. Thus my choice of title.
In fact KK alludes to the perceived weakness of the modified protocol. An alternative means has been found for testing antigen-antibody precipitation that does not require added blood. It uses antibodies that carry fluorescent tags or markers instead. That is obviously better than adding fresh blood to an assay system allegedly specific for aged blood, allaying the suspicions of sceptics that what one lacked, the other magically restored! But the forward assay of ABO in ancient blood is still not perhaps as robust a procedure as some might imagine. Beware then the so-called “belt and braces” approach of combining it with the so-called reverse testing because that, as we shall see, offers no real corroboration if the hypothesis is that one is dealing specifically with the AB blood group. Why not?
The reverse procedures work on the basis that blood group A individuals have antibodies to blood group B (goodness knows why!), that blood group B have antibodies to blood group A, that blood group O has antibodies to A and B, and that, importantly for the Shroud, blood group AB has neither antibodies for A nor B. So corroboration of a tentative identification of blood group AB depends on a negative finding in the reverse test – i.e. finding NO antibodies to A or B. But while a negative test might be due to the presence of AB antigens, it might also be due to a thousand other reasons, like the technician having mixed up the H2O and the H2SO4….
So the identification of AB blood group depends crucially on the forward test. While the reverse test should, in competent hands be done to seek confirmation, the latter is in reality of little value unless the forward test is really giving unequivocal confirmation of the presence of a particular kind of DNA, albeit measured by a highly indirect means.
So it all comes down to that forward test, and its reliability in measuring a particular DNA marker by a somewhat distant downstream immunological knock-on effect.
Is it a reliable. How confident can one be that the blood on the Shroud really is blood group AB.
Let’s first dismiss the old chestnut about “all ancient blood reverts to AB”. That makes no sense in chemical terms, as KK has pointed out, since the ‘capping’ AB markers would be the first to be lost on degradation, being at the termini of the core structure. That misconception arises presumably because of the nature of the “reverse test” where one is looking for NO response and where degradation could be just one of those 1000 reasons why one would see the desired “no response” if testing for AB.
A more serious objection to AB result in forward testing is that of contamination by bacteria, fungi etc that also carrying the kind of sugars and sugar derivatives on the peptidoglycan cell walls that we see on RBC. KK has addressed that question skilfully. The localisation of AB markers to the bloodstain, with relatively little spread to surrounding regions argues against contamination (assuming that microorganisms would tend to grow beyond the immediate pigmented area, e.g. to feed on serum that has diffused beyond the stain).
Contamination is certainly the major obstacle to uncritically accepting a blood group designation, whether AB or something else. But not so much microbial contamination (while not be lightly dismissed) as, wait for it, deliberate human contamination. Yes, in the absence of any radiocarbon-dating of blood flakes (why not?) we have no means of knowing whether the blood is 1st century AD, medieval or 20th century. There could have been blood residues initially at the time of the Shroud’s genesis, that have subsequently been “touched up” by surreptitious additions of real blood, possibly blood of a carefully chosen group that best fits with “authenticity” – and what better group to choose than AB, an overzealous custodian might have imagined, given that AB is rare worldwide but highly prevalent among Jews, or at any rate modern Jews ( shame that is considered by genetic historians to have been a late arrival – medieval era – through admixing of A and B.
There are broader objections that can be made to the “blood” on the Shroud being AB. Its permanent red colour for a start, that suggests it to be fake blood. The porphyrin chemist Alan Adler solitarily brainstormed that oxidised methaemoglobin might be stabilised by an excess of bilirubin, the latter he proposed having been massively augmented by the trauma of crucifixion. Well, he may have been an expert in porphyrins, noted for their exceptional chemical stability on account of their having cyclic system of delocalised pi-electrons reminiscent of benzene, the model par excellence for aromatic pi-electron delocalisation, but he clearly knew next to nothing about bilirubin and other linear tetrapyrroles. Bilirubin especially is notoriously susceptible to bond-breakage when exposed to light – both as regards its multiple internal hydrogen bonds as well as the C-H bonds of the central methylene bridge. Bilirubin undergoes a photoisomerisation that leads to a profound change in physical properties notably solubility in aqueous electrolytes (allowing its excretion in bile without glucuronide conjugation – as happens in phototherapy of neonatal jaundice) and via a range of photo-oxidation reactions, some involving singlet oxygen and/or other active oxygen species. Bilirubin never stabilised anything – and it’s not red either, but orange.
My main objection to the “AB blood” identification is that a indirect test that depends on a reasonable survival of biochemical integrity – i.e. glycoproteins that can be recognized by antibodies – is being used to support the authenticity of something that is at least centuries old. Yet the intactness of the RBC membrane, or even fragments thereof, has never been demonstrated. Indeed numerous test for blood “albumins” (tested with bromocresol green to give an, er, green colour) , porphyrins (atypical uv/visible spectra) etc etc” have been less than conclusive. The validity of the ABO blood systems depends to a greater or lesser extent on intactness, and in the absence of such evidence, the answers they give for aged blood are highly questionable. Indeed, there is an element, yet again, of begging the question – to assume that since it was real blood originally, enough of it has survived to give the kind of positive result that might be expected of, er, real blood. When it is a test that detects little more than one component binding to another, allegedly in a highly specific way because the test involves antigen/antibody reaction, when really there is little hard evidence for specificity when dealing with aged blood, then the entire exercise starts to look especially dubious. It is perhaps unfortunate from a methodological standpoint that the presumed blood group is AB, with both markers present or absent in the forward and reverse tests respectively. It might have been better if the blood had been either A or B, but not both, since it is harder then to deny a degree of specificity.
My own position at present (which could change in the light of new evidence) can be simply expressed. Any real blood on the Shroud of Turin that still responds to tests for “blood”, e.g. porphyrins, albumins, physiological electrolytes (Na, K, Ca, Mg,P etc) is unlikely to be medieval blood, far less 1st century. That’s not to say that real blood was not deposited on the Shroud at the time of its genesis by whatever mechanism, or whether that blood preceded the body image or not. Anything that tests as intact non-degraded albumin, or still immunologically-competent “blood group AB”, assuming those tests are not ‘false positives’ (which they may well be) is probably of relatively recent addition.
The only thing that would convince me that blood stains, if real, support authenticity is radiocarbon dating.
Given there is still alleged blood on the Shroud that can, we are told, be detached as scrapings without damaging the linen, then why not test- date some blood immediately? Why were blood flakes not included in the original C-dating protocol anyway?