Recently Mari et al. , “exhuming” an old legend, claimed that the Grand Duke Francesco I and his wife Bianca Cappello were poisoned with arsenic by the Grand Duke’s brother, the Cardinal Ferdinand. (Mari F, Polettini A, Lippi D, Bertol E, The mysterious death of Francesco I de’ Medici and Bianca Cappello: an arsenic murder? British Medical Journal 2006; 333: 1299-1301.)
Two comments on this problem, published in the same journal (Fornaciari G, The mystery of beard hairs, bmj.com 29 Dec 2006 ; Ottini L, Who is who, that is the question, bmj.com 5 Jan 2007),  well reveal the weakness of this “romantic” hypothesis.

The mystery of beard hairs
Gino Fornaciari
Division of Paleopathology, Department of Oncology, Transplants and Advanced Tecnologies in Medicine
University of Pisa

In my quality of scientific director of the Medici Project (1,2) I feel bound to comment this article, which is important for various historical aspects.
In brief, the key of the entire study seems to be “a few beard hairs with one small fragment of skin tissue still attached”, which made it possible to establish a “high degree of similarity with the DNA” of three different pieces of “dry, thick, and crumbly material … collected within the broken terracotta jars” in the crypt of the church of Santa Maria a Bonistallo near Florence. On this basis, the Authors claim that it is “highly probable that these soft tissues were among those extracted from the body of Francesco I at autopsy”. These results are by no means possible, simply because Francesco I appeared, at re-exhumation carried out in December 2004, totally skeletonised and disarticulated in the small zinc coffin used to re-bury the skeletal remains of the Grand Duke after exhumation in the 40’s, and the skull showed no traces of soft tissues, skin or beard!
Moreover, the Authors do not provide any information about the method adopted for identification of the ancient DNA (microsatellites, mithocondrial DNA?) or about the molecular size of the DNA fragments, nor do they explain whether the DNA extractions were performed in a laboratory designed for ancient DNA manipulation (where modern human samples are not processed!). On the contrary the published data is consistent with contamination by modern DNA and, consequently, the hypothesis of arsenic poisoning is also to be rejected.
Furthermore, owing to the very frequent use of arsenical mixtures in embalming and visceral processing by the contemporary surgeons (3), it is impossible to establish whether the high arsenic concentrations in the specimens were obtained in vita or after death.
Finally, some “minor” but not less important questions should be asked regarding the findings in the crypt of the church of Santa Maria a Bonistallo: is there an archaeological report on the excavations and on the stratigraphic position of the “broken terracotta jars”? Were the fragments examined – and dated – by a post-medieval archaeologist? This information is very important because the two small crucifixes, typical of 18th and 19th century, are clearly more recent than October 1587, date of the alleged murder.
In conclusion, the article “The mysterious death of Francesco I de’ Medici and Bianca Cappello: an arsenic murder?” quite appropriately finishes with a question mark!

References:
1. Fornaciari G, Brier B, Fornaciari A. Secrets of the Medici. Archaeology, 2005; 58: 16-19.
2. Fornaciari G, Vitiello A, Giusiani S, Giuffra V, Fornaciari A. The “Medici Project”: First Results of the Explorations of the Medici Tombs in Florence (15th-18th centuries), Paleopath Newsl, 2006; 133:15-22.
3. Marinozzi S, Fornaciari G. Le mummie e l’arte medica: per una storia dell’imbalsamazione artificiale dei corpi umani nell’evo Moderno. Roma: Medicina nei Secoli, Supplemento, n.1, 2005.

Who is who, that is the question
Laura Ottini
Department of Experimental Medicine
University of Rome “La Sapienza”

I’ve read with interest the article of Mari et al. It deals with the cause of death of Francesco I de’ Medici and Bianca Cappello, the grand-ducal couple of Florence died in 1587. In their very intriguing paper the Authors affirm that “acute poisoning with arsenic was the cause of death of Francesco I de’ Medici and Bianca Cappello, in contrast to previous theories that attributed their deaths to malaria”.
Indeed, they accurately report the historical fonts that led them to re-advance the hypothesis of arsenic poisoning and extensively describe the methodologies and calculations applied to obtain the arsenic concentrations in soft biological tissues (collected from the remains of the broken terracotta jars containing, according to historical data, the viscera of Francesco I de’ Medici and his wife), and in a fragment of femur and in a beard hair of Francesco I (collected from the grave of the Grand Duke).
By contrast, the Authors very shortly describe the DNA profiling of the ancient biological materials analysed. They briefly say that the DNA profiling of two of the samples collected within the terracotta jars has “extremely high degree of similarity” with the DNA of the small skin fragment found attached to the beard hair of Francesco I and the DNA profiling of a third sample “revealed only its female origin”.
DNA profiling represents an extremely important and fundamental element in order to assign the ancient human remains the correct identity and to answer the question who were the victims of the hypothesised arsenic murder (the starting point to develop all the plot!). Surprisingly, in their article the Authors do not report the methodologies applied to obtain DNA profiling (i.e. by evaluating microsatellite loci or mitochondrial DNA regions) and the kind of analysis used to calculate the degree of similarity.
Moreover, the Authors do not describe conditions (i.e. physically isolated work area) and methods applied to perform DNA extraction from ancient biological materials (i.e soft tissue and bone) and to test the quality (i.e. PCR products size) and quantity (i.e. by real time PCR) of the ancient DNA recovered. They simply say that DNA was extracted from a “small” skin fragment attached to the beard hair of Francesco I. Why DNA extraction was not performed from the fragment of femur? This could have allowed for the possibility to repeat the results by using DNA deriving from a distinct sample, a fragment of bone whose surface can be also treated to reduce the presence of contaminants (i.e. human DNA, bacterial DNA, etc). Indeed, the results were not duplicated with different DNA preparations nor validated with independent replication in different laboratories.
Overall, the Authors do not provide any information, nor cite any references, about methods and criteria adopted for ensure and validate the authenticity of the results obtained from ancient DNA analyses. Considering that obtaining authentic DNA sequences from ancient human remains presents extreme technical difficulties due to the small amounts of DNA, quite often degraded, along with the exceptional risk of contamination (1), how can they exclude to have obtained results from DNA contaminants?
As principal investigator of the research unit devoted to molecular paleopathology investigations within the project “Paleogenetics and molecular paleopathology of the Medici family: infectious diseases and tumours in the XVI-XVIII centuries”, founded by the Italian Ministry of University and Research (2), I am aware that ancient DNA studies represent a powerful tool that can be used to obtain insights into the past (3) and that these studies are quite appealing for medical readers. However, a number of problems exist in this field that are not often properly taken into account and a rigorous scientific approach should be used in order to obtain reliable results (4).
Finally, I believe that questions exist as to how reliable the conclusions of Mari’s paper are. As the Authors appropriately say “it is highly probable that these soft tissues were among those extracted from the body of Francesco I” but this is not enough to rewrite the historical reconstruction of the death of Francesco I de’ Medici and his wife, Bianca Cappello.

References:
1. Cooper A, Poinar HN. Ancient DNA: do it right or not at all. Science 2000; 289:1139
2. Progetti di Ricerca di Interesse Nazionale (PRIN), grant 2005, prot. N° 2005067073_005
3. Ottini L, Lupi R, Falchetti M, Fornaciari G, Mariani-Costantini R, Angeletti LR. Molecular paleopathology: a novel perspective for biomedical history. Med Secoli 2005; 17(1):181-91.
4. Gilbert MTP, Bandelt H-J, Hofreiter M, Barnes I. Assessing ancient DNA studies. Trends Ecol Evol. 2005; 20:541-544