Inadequacies in Serological ABO Typing of Ancient Artifacts: The Shroud of Turin as a Case Example

Results

The Shroud of Turin is an approximately fourteen- foot-long cloth with the faint image of a man containing bloodstains consistent with scourging and crucifixion. Carbon-14 dating tests performed in 1988 assigned the Shroud a proposed date 1260-1390 [14] although such results have recently been questioned, both in terms of data homogeneity and whole cloth representation [15, 16, 17]. The bloodstains contain authentic blood components including hemoglobin, albumin, and immunoglobulin, although the species of origin remains to be demonstrated [18, 19, 20]. In the early 80’s, Baima Bollone reported a designation of AB in a Shroud-specialty journal, resulting from experiments using the mixed agglutinin (forward typing) method [21]. In this technique, multivalent antibodies specific for a particular blood group antigen are added to the sample; unbound antibodies are removed by washing, and fresh RBCs of the same type as the antibody are added. For a positive result, a type of clumping, or agglutination or RBCs is observed (Figure 1, left-hand side). In aged archaeological samples, this method is typically problematic, in typing of fresh blood (left), antibodies bind to red blood cell antigens and after washing, new red blood cells of the identical type are added, forming a type of sandwich (agglutinin), which is visible under a microscope. With aged blood, antibodies may recognize similar antigens present on bacteria (shown) and fungi, resulting in a false positive result (Figure 1, right-hand side), (Table 2), a limitation acknowledged by the original researchers, [21] (Table 2). Note that antigens, antibodies, and red blood cells are not drawn to scale in this figure.

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As a control in these studies, white fibers adjacent to the bloodstain were included, which tested negative [21], indicating that contamination was not widespread. It could be argued, however, that contamination would be greatest in the most nutrient rich areas of the cloth, i.e., the bloodstains. Regarding this issue, a microscopic examination of bloodstained threads taken from the Shroud showed heavy contamination by both bacteria and fungi; threads chosen from other areas, less so [22].

In a follow up study, a different method (immuno chemistry) involved peroxidase-labeled antibodies. These studies yielded similar results as before, type AB [22]; however, this experimental design suffers from the same limitations as the original studies, false positives resulting from contamination cannot be excluded [9].

To cross-check the forward typing results, a reverse typing method (Lattes technique) was used to evaluate the presence or absence of anti-A and anti-B antibodies (Table 1). Unfortunately, this technique is even more problematic with aged samples than forward typing, as it is extremely unlikely that antibodies remain functionally stable over time [9]. Typically, reverse typing is never used in studies of ancient material because the most likely result is AB, regardless of the original (true) blood type (Table 3). Not unexpectedly, this was the reported result [21]. Note that even if the designation of AB for Shroud fibers was correct, this method cannot distinguish between a true AB result and a negative finding with another blood type due to antibody instability and loss of function (Figure 2). With any blood type other than AB, verification is possible, but since the serum of AB individuals does not contain anti-A or anti-B antibodies to begin with (Table 1), the contention is exceptionally circular. Therefore, these findings are not useful for cross-checking or confirmation of blood typing of older samples [9].

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Figure 2: Mixed Agglutinin Method for Forward Typing of Fresh Vs Aged Samples. In (A), fresh and aged blood samples that are type A are evaluated using the Lattes method. A visible reaction is not observed with aged blood as antibodies have become nonfunctional over time (compare left and middle); note that this result is the same as a person having AB blood (right). (B) Similar to (A), except that an example is shown using red blood cells of the B type. Note that antigens, antibodies, and red blood cells are not drawn to scale in this figure.

Discussion

Serological blood typing of ancient artifacts is particularly challenging when little to no background information exists regarding the genotype of the sample in question. With articles such as skeletal remains or relatively well-preserved

mummies, environmental or familial information may be available to help corroborate blood type findings. For objects like the Shroud, where bloodstains are relatively solitary in terms of typing evidence, reliability on the data is essential for confidence in the results.

As each artifact is accompanied by unique and special circumstances of preservation, storage, and exposure to environmental conditions, the limitations of techniques must be determined on an individual basis. Thus, what may work for one object does not guarantee success with another. Ideally, having results from both serological and molecular biology studies on the same object to cross-check and verify one other is ideal, although this is not always possible. As use of serological methods for blood typing of older materials is frequently unreliable, the strictest caution must be used in the interpretation of the results, especially those for which contamination has already been established. In consideration of the current serological data that exists, the blood type designation for the Shroud bloodstains is best described as inconclusive and should be considered type unknown pending further testing.

Conclusions

The current perspective has evaluated the existing blood typing data for the bloodstains on the Shroud of Turin. As discussed, unreliability in serological evaluation of aged materials limits the conclusions that may be drawn from such results. After modern review and evaluation, the designation for the Shroud of Turin bloodstains should be listed as inconclusive, type unknown.