Letter to Editor Unanticipated issues in serological analysis of blood species - The Shroud of Turin as a case example Kelly P. Kearse Knoxville Catholic High School, 9245 Fox Lonas Road, Knoxville, TN 37923, United States A B S T R A C T Serological analysis of blood on objects can be challenging, particularly those for which limited background information is available. The Shroud of Turin is an aged linen cloth that shows the images of a man bearing wounds corresponding to scourging and crucifixion. The bloodstains on the Shroud are often reported as being human in origin; however, a modern revaluation of the prior experimental design indicates that blood from other species would have also tested positive. The current perspective underscores the importance of consideration of a broad spectrum of potential cross-reactivity in bloodstain analysis, particularly with objects that are of indefinite origin. A R T I C L E I N F O Keywords: Serology Antibody Shroud of Turin Blood Species Two primary methods exist for determining the species of origin of bloodstained artifacts: serological techniques and DNA testing. Whereas DNA analysis is often at the forefront of modern scientific evaluation, examination of certain aged artifacts may be hampered by DNA degradation and/or contamination by many individuals throughout its history. In such cases, serological testing can provide an alternative method, provided the antibody reactivity is well-defined. As discussed below using the Shroud of Turin as a case example, species assignment using serological methods can be challenging, particularly in situations where the cross-reactivity is unanticipated or broader than previously recognized. The Shroud of Turin is a linen cloth of unknown origin containing the faint imprint of the ventral and dorsal images of a human body, and bloodstains consistent with scourging and crucifixion. Although the Shroud has been viewed by millions of pilgrims throughout modern history, and is referred to as the “most studied artifact in existence”; very little direct forensic examination of the cloth itself has been performed. This is particularly true in relation to the bloodstains and what species of blood might be present. Previous studies have shown that authentic blood components are present within the Shroud bloodstains, including hemoglobin, bilirubin, albumin, and immunoglobin [1,2]. The blood is typically described as being human (primate) in origin, based on certain serological experi- ments performed in the early 1980s. Unlike many of the other studied characteristics of the Shroud that are readily available in the scientific literature, the blood species results are incomplete, only referred to in several books, conference proceedings, and related Shroud-specialty journals [3–6]. Although these studies were at best a work in progress, the perception exists that these results are well-established and scientifically confirmed. Indeed, the detail that the blood on the Shroud has been scientifically demonstrated to be “human” (or primate) is regularly cited as evidence towards the Shroud’s possible authenticity. From even the limited results that are available, a modern revaluation indicates that the blood on the Shroud should be reclassified as species unknown. This conclusion is based on an updated analysis of antibody (cross-)reactivity in the context of the original experimental design. The studies performed on the Shroud to determine blood species used serological techniques, involving antibodies that recognize two major blood proteins, human albumin and human immunoglobulin. A crucial requirement in the design of this type of experiment is to first demonstrate that the antibodies show limited reactivity with the same molecules present in other species. To be effective, the antibodies used in the test should react positively with blood from humans (and most likely related primates, such as chimpanzee), but negatively with blood from unrelated species, such as horse, cow, and pig (Fig. 1). This is what was found, and when subsequent experiments on Shroud samples tested positive, led to the conclusion that the blood on the Shroud is likely of human (primate) origin [3,4]. Unfortunately, the amount of Shroud samples that were available to further confirm these results were limited, and the findings were never fully completed. Related studies were performed on serum immunoglobin by separate investigators on Shroud samples, although remarkably, potential reactivity with other species was never addressed [5,6]. The Shroud of Turin is an excellent case example of the types of challenges that can arise when serological testing is used to define what type of blood may be present on an object. The conclusion is only as strong as the data is able to effectively discriminate between the types of species that might be present; in this case, primate versus a select number of other animal types. E-mail address: kelly.kearse@knoxvillecatholic.com (K.P. Kearse). http://doi.org/10.1016/j.fsir.2020.100073 Received 11 November 2019; Received in revised form 11 February 2020; Accepted 11 February 2020 Available online 14 February 2020 2665-9107/© 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc- nd/4.0/). Forensic Science International: Reports 2 (2020) 100073 Contents lists available at ScienceDirect Forensic Science International: Reports jo u r nal h o mep ag e: ww w.els evier .c om /lo cat e/fs ir