Virchows Arch (2004) 445:647–650 DOI 10.1007/s00428-004-1094-1
CASE REPORT
R. Ciranni · G. Fornaciari
Juvenile cirrhosis in a 16th century Italian mummy. Current technologies in pathology and ancient human tissues Received: 7 April 2004 / Accepted: 9 July 2004 / Published online: 16 September 2004 Springer-Verlag 2004
Abstract The natural headless mummy of a young man from the Basilica of Saint Domenico Maggiore in Naples (16th century) showed at autopsy a well-preserved fibrous liver with a nodular surface, suggesting a case of cirrhosis. Stereo and light microscope study confirmed this diagnosis. To identify the possible etiology of this cirrhosis, additional techniques currently used in pathology were performed. Hemochromatosis and alpha1-antitrypsin deficiency were investigated without results. Investigation regarding Wilson’s disease gave positive results, since the use of rhodamine staining, which is specific to detect the presence of copper in tissues, resulted in red-brown grains at light microscopy. The positive rhodamine test was invalidated by atomic absorption spectroscopy (AAS), which revealed normal copper levels in the tissues. These negative results and the clear and diffuse macronodularity of the liver suggest a case of post-necrotic cirrhosis.
for the excellent state of preservation of the bodies but also for the historical importance of the individuals [3]. Therefore, the study of various diseases affecting these people, their dietary habits and lifestyles make it extremely interesting to compare the paleopathological with the historical data. The analysis of particular pathological conditions and a better understanding of the pathocenosis (the whole of pathologies characterizing a human society) can provide important and useful information on the environment and social habits of the upper classes of those times [4]. In regard to the case in question, autopsy of mummy NaSD 29 showed clear features of cirrhosis. Our paleopathological target was to confirm the macroscopic diagnosis using standard histology and to investigate some of the possible causes of the disease. The conditions investigated were: hemochromatosis, a1-antitrypsin deficiency (a1-AT) and Wilson’s disease.
Keywords Paleopathology · Liver · Cirrhosis · Histology · Histochemistry · AAS
Materials and methods Introduction The Abbey of Saint Domenico Maggiore dates back to the beginning of the 14th century and is one of the most important churches in Naples. In the monumental Sacristy, on a suspended 5-m-high gateway, 37 wooden coffins contain the well-preserved bodies of ten Aragonese kings, princes and other Neapolitan nobles who died between the 15th and 17th centuries [11]. These mummies are unique in Italy, not only for their antiquity and R. Ciranni ()) · G. Fornaciari Department of Oncology, Transplants and Advanced Technologies in Medicine, Division of Pathology, Section of History of Medicine, University of Pisa, Via Roma 56, 56126 Pisa, Italy e-mail:
[email protected] Tel.: +39-50-992894 Fax: +39-50-992706
NaSD 29 is a well-preserved headless natural mummy of an adult male aged 24€2 years [10] and approximately 175 cm tall (Fig. 1). Autopsy, carried out by anterior opening of the thorax and abdomen, allowed the macroscopic examination of the internal organs. Fragments of tissues (0.80.80.8 cm) belonging to different organs were collected and rehydrated using Sandison’s method [8] and were submitted to routine histological processes. Thin sections were fixed on clean slides previously treated with an 8% solution of KCr (SO4)2 to avoid dispersion. The slides were stained with hematoxylin-eosin to evaluate the preservation of the tissue, and Masson’s trichrome was applied for the connective tissue. Hemochromatosis, a1-AT deficiency and Wilson’s disease were investigated using histochemistry. Hemochromatosis was detected by Perls’ Prussian blue method using a 2% K4Fe(CN) solution in HCl 2% for 10 min. The reaction with iron ions was evidenced by blue grains at light microscope. For a1-AT deficiency diagnosis, the periodic acid of Schiff’s (PAS) method was applied using a 1% periodic acid solution for 5 min and the Schiff reagent for 5–10 min. In a1-AT deficiency, the liver is characterized by the presence of PAS-positive dark red grains, which are diastase resistant. Wilson’s disease was detected staining selected sections of liver tissue with a solution of rhodanine 0.2% in ethanol, which is specific to detect copper, resulting in red-brown grains at light mi-
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Fig. 1 The headless mummified body of NaSD 29
croscopy. Lung tissue sections from the same individual and liver tissue from the NaSD 14 mummy were used as negative controls. Atomic absorption spectroscopy (AAS) [9] was performed to detect copper concentration in six different samples of tissue: three were from different organs of NASD 29 (left and right lobes of the liver and pectoral muscle); two of the samples belonged to the liver of other mummies of the same group (NASD 1 and NASD 14); the latter, used as negative control, was from modern liver biopsy. Dry tissue (500 mg) enveloped in muffle and diluted in 1:5 distilled water was placed in an open oven at 250C for 30 min. After this time, temperature was adjusted at 450C, and the oven was closed 30 min later. The material was maintained at 450C overnight. At the end of the procedure, the residual concentration (g/ml) of copper was evaluated for each sample.
Results Autopsy revealed well-preserved thoracic and abdominal organs, dried and partially collapsed on the posterior wall. The mediastinum and the diaphragm were clearly identified as well as the bronchial tree and lungs. The pericardial sac contained the heart, wrinkled (74 cm) and flattened. The liver—reduced in size (189cm) but of typical shape, with sickle-like ligament and easily recognizable hepatic lobes—showed a granular surface with many single or clumped 0.5- to 1.5-cm nodules (Fig. 2a). The kidneys were typically shaped (73 cm) and appeared flattened up to 0.5 cm. Histology, using hematoxylin-eosin and Masson’s trichromic staining, showed excellent preservation of the
Fig. 2 The NaSD 29 liver appears reduced in size but of typical shape. The sickle-like ligament, the hepatic lobes and the nodular surface (arrow). Stereoscopic image (4) showing some of the hepatic nodules (a)
tissues, making it possible to identify some pathological conditions, such as pulmonary apical fibrosis and severe anthracosis. Liver histology evidenced coarse bands of fibrous tissue dissecting and surrounding areas of homogeneous, brown and granular material representing residual hepatic parenchyma, which resulted in a sort of pseudo-lobular organization (Fig. 3). Some nodular formations, with multi-layered fibrous connective bands, surrounding regions of degenerated parenchyma, were well evident (Fig. 3a). Therefore, the macroscopic and microscopic picture clearly revealed a picture of micro-macronodular cirrhosis. The histochemical results, specific for iron, were positive in the liver and in lungs, where light microscopy revealed massive deposits of the metal. PAS staining was negative for a1-AT hepatic accumulation, owing to the absence of the typical grains. Positive results were also obtained using differential rhodamine staining, specific for copper: this result, suggestive of Wilson’s disease, was not confirmed by AAS, which revealed normal copper levels in the tissues.
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Fig. 3 Coarse bands of fibrous tissue dissecting and surrounding the hepatic parenchyma, resulting in a sort of pseudo-lobular organization (hematoxylin-eosin staining 10). a Nodular formations, with multi-layered fibrous connective scars, surrounding degenerate parenchyma (Masson’s trichrome staining 10)
Discussion Cirrhosis is currently a very common pathological condition in Western countries. Even though it is well documented in the ancient medical texts [1], the paleopathological diagnoses are rare. Overview of the literature has shown some cases of mummies with cirrhotic livers, clearly caused by parasitic infections. In the Egyptian Nakht case (first half of the12th century B.C.), for example, schistosomal ova were found in the liver, suggesting cirrhosis caused by hepatic schistosomiasis [5]. Another parasitic cirrhosis, due to clonorchiasis and Schistosoma japonicum, was also described in a Jiangling
male body, a very well-preserved Chinese mummy dated back to 167 B.C. [7]. The only case of cirrhosis not dependent on parasites has been described recently by Zimmerman in a frozen body belonging to a young Eskimo girl (800–1000 A.D.), who died of pulmonary edema and severe emphysema [12]. This condition was a complication of genetic a1-AT deficiency, as histologically evidenced by the author using PAS and diastase methods. Alcoholism, at present the most frequent cause (70– 80%) of cirrhosis, was initially attributed to NaSD 29. However, several decades of alcohol abuse are necessary to generate cirrhosis [2], and this strongly contrasts with the young age (24€2 years) of the man. Histochemistry was strongly positive for iron in both the liver and lung of the mummy. These results could be consistent with a diagnosis of hemochromatosis, but the significance of its massive presence in the lung remains unclear. Forensic medicine provides a simple explanation for iron accumulation, for colliquative post-mortem events: hemoglobin present in the erythrocytes supports severe degradation, releasing high levels of iron throughout the whole body, but particularly in the lungs and liver, through their rich venous and capillary net [6]. During macroscopic examination, our attention, supported by the finding of pulmonary fibrosis and by the young age of the individual, was firstly focused on a1-AT deficiency. Since the test results for a1-AT deficiency were negative, this hypothesis was abandoned. Lung fibrosis was considered an independent condition, probably depending on apical tuberculosis, very common in the past. Because of its rarity, Wilson’s cirrhosis was the last hypothesis we considered. At present, the incidence of Wilson’s disease is very low (0.005%) [2]. The positive results obtained with rhodamine method made this diagnosis seem possible, but the elemental analysis using AAS revealed normal levels of copper. Therefore, the positive rhodamine reaction could be referred to the normal copper concentration in a mummified and shrunken organ such as cirrhotic liver. In conclusion, despite its very typical macroscopic and histological picture and the different modern tests, the real etiology of this juvenile cirrhosis is still unclear, but macroscopic and histological picture and, in particular, presence of large nodules (macronodules) is peculiar of post-necrotic cirrhosis. Acknowledgements We would like to thank Mrs. Debora Barraco and Mr. Marcello Gambini (Santa Chiara Hospital) for their precious technical support.
References 1. Brothwell DR, Sandison AT(1967) Disease in antiquity. Charles C. Thomas Publisher, Springfield 2. Cotran RS, Kumar V, Robbins SL (1994) Pathologic basis of disease. W.B. Saunders Company, Philadelphia
650 3. Fornaciari G (1998) Italian mummies. In: Cockburn A, Cockburn E, Reyman TA (eds) Mummies, disease and ancient culture. Cambridge University Press, 2nd edn. Cambridge, pp 266–281 4. Grmek MD (1983) Les maladies l’aube de la civilisation Occidentale. Payot, Paris 5. Hart GD, Cockburn TA, Millet NB, Scott JW (1977) Editorial: autopsy of an Egyptian mummy. Can Med Assoc J 117:461 6. Janssen W (1984) Forensic histopatology. Springer, Berlin Heidelberg New York 7. Peng L, Wu Z (1998) China: the Mawangtui-type cadavers in China. In: Cockburn A, Cockburn E, Reyman TA (eds) Mummies, disease and ancient culture, 2nd edn. Cambridge University Press, Cambridge, pp 328–335
8. Sandison AT (1955) The histological examination of mummified material. Stain Technology 30:277–283 9. Szpunar CB, Lambert JB, Buikstra JE (1978) Analysis of excavated bone by atomic absorption. Am J Phys Anthropol 48:199–202 10. Todd TW (1920) Age changes in the pubic bone: I. The male white pubis. Am J Phys Anthropol 3:285–334 11. Valle R, Minichini B (eds) (1854) Descrizione storica, artistica e letteraria della chiesa di San Domenico Maggiore. Napoli 12. Zimmerman MR (1998) Alaskan and Aleutian mummies. In: Cockburn A, Cockburn E, Reyman TA (eds) Mummies, disease and ancient culture, 2nd edn. Cambridge University Press, Cambridge, pp 138–153
