Diagnosis of thalassemia in ancient bones • 75 



Discussion 



Literature cited 



The results described above demonstrate that the immu- 

 nochemical technique employed in this work is sufficiently 

 sensitive and reliable to allow quantitative titration of hemo- 

 globin and its constituent polypeptide chains in bone ex- 

 tracts, even some thousands of years after burial of the indi- 

 vidual. It is important that, as expected, the results indicate 

 that bones with higher erythropoietic activity contain greater 

 amounts of titratable hemoglobin or hemoglobin fragments 

 than those characterized by reduced erythropoiesis. More- 

 over it is clear from data in Table 2 that, within the accuracy 

 of the methodology, no significant decrease of hemoglobin 

 content is observed with the archeological age of the speci- 

 men, within a range of 15-2000 years. Previous results ( As- 

 cenzi et al. 1985) indicated a loss of immunochemically 

 titratable hemoglobin with time of burial, but it may be that 

 characteristics of the soil, humidity, or other interferences 

 may be more important than age after death in determining 

 the final content of detectable protein. It should moreover be 

 emphasized that some uncertainties about the quantitative 

 estimate of hemoglobin in bone extracts remain, owing to 

 the interferences by false positive immunochemical reac- 

 tions. 



As an example, it may be mentioned that two samples of 

 lumbar vertebrae of adult humans from different burial sites 

 (both approximately 2000 years old) were tested against an 

 affmity chromatography purified antialpha chain Ig fraction 

 of the rabbit antiserum as well as against the antialpha de- 

 pleted fraction of the same serum. The hemoglobin content in 

 the two tests was comparable (1-3 |xg/IOO g), while the 

 nonhemoglobin reactivity was zero in one specimen and 

 almost eight times higher than that of hemoglobin in the 

 other. 



Up to now. the immunochemical technique has been ap- 

 plied only to morphologically normal bones, but it may be 

 possible to determine the content of hemoglobin remnants 

 even in bones whose morphology indicates hyperactive 

 erythropoiesis. As reported above, differential diagnosis of 

 chronic anemias with increased but insufficient compensa- 

 tory erythropoiesis is usually impossible on the mere anatom- 

 ical evidence of porotic hyperostosis. In this respect, the 

 immunochemical technique employed here represents a pos- 

 sible tool for an unequivocal diagnosis of alpha and beta 

 thalassemias (and possibly other hemoglobinopathies) in 

 very old skeletal remains. Further work along these lines is in 

 progress. 



Asccnzi. A. 1976. Physiological Relationship and Pathological In- 

 terferences Between Bone Tissue and Marrow. In G.H. Bourne, 

 cd.. The Biodwmislry and Physiology of Bone. 403-444. New 

 York; Academic Press. 



1979. A Problem in Paleopathology: The Origin of Thalas- 

 semia in Italy. Virchows Archiv A Pathological Anatomy and 

 Histology. 384:121-130. 



Ascenzi, A.. M. Brunori. G. Cilro. and R. Zito. 1985. Immu- 

 nological Detection of Hemoglobin in Bones of Ancient Roman 

 Times and of Iron and Eneolithic Ages. Proceedings oj the Na- 

 tional Academy of Sciences. 82:7170-7172. 



Bucci, E., andC. Frontieelli. 1965. A New Method for the Prepara- 

 tion of Alpha and Beta Chains of Human Hemoglobin, yourna/ of 

 Biological Chemistry. 240:PC 551 . 



Caffey, J. 1951. Cooley's Erythroblastic Anemia. Some Skeletal 

 Findings in Adolescent and Young Adults. American Journal of 

 Roentgenology and Radium Therapy. 65:547-560. 



Chalevelakis. C, J.B. Clegg. and D.J. Weatherall. 1975. Im- 

 balanced Globin Chain Synthesis in Heterozygous p-Thalas- 

 semic Bone Marrow. Proceedings of the National Academy of 

 Sciences. 72:3853-3857. 



Cooley, T B . . and P. Lee . 1 925 . Series of Cases of Splenomegaly in 

 Children with Anemia and Peculiar Bone Changes. Transactions 

 of the American Pediatric Society. 37:29-30. 



Moseley. J.E. 1963. Bone Changes in Hematologic Disorders 

 (Roentgen Aspects). Orlando, Fla.: Grune and Stratton. 



1965. The Palcopathologic Riddle of '■Symmetrical Os- 

 teoporosis. " American Journal of Roentgenology, Radium Thera- 

 py and Nuclear Medicine, 95:135-142. 



Summary of aiidif.nce discussion: Before one can attempt to 

 correlate porotic hyperostosis with the chemical findings of globin 

 pattems common to thalassemia, one must develop laboratory 

 methods not only for alpha and beta chains but also delta and gam- 

 ma. The work presented in this study used trabecular bone becau.se 

 of its hematopoietic marrow and therefore because hemoglobin was 

 present there in vivo. Unfortunately during interment its porosity 

 also invites diagenetic changes which might alter protein structure. 

 Use of ribs as source material might minimize the diagenetic haz- 

 ard. Dental pulp is even more sheltered from the environment. 

 Conceivably the problem of antibodies against alpha and beta 

 chains also cross-reacting with gamma chains might be prevented 

 by raising antibodies in appropriate animals against an antigen com- 

 posed only of a peptide, preferably one with a known amino acid 

 sequence; even better would be production of a monoclonal anti- 

 body in the usual manner. Considering the small quantity of protein 

 present in fossil material, the use of radioimmunoassay methodol- 

 ogy would appear to be desirable. 



Zagreb Patfopathotogy Symp. 1988 



