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ABO tissue antigens of egyptian mummies
113
Science International 43 (19891113- 124 Elsevier Seientific Publishers Ireland Ltd.
Forenuic
ABO TISSUE
ANTIGENS
OF EGYPTIAN
MUMMIES
K. CRAINIC’, M. DURIGON”and R. ORION aU.E.R. De Médecine Légak, Droit Mkdàcal et Déontologie Médicale, Université René Descartes, 2, ploce Ma.zaa, F-75012 Paris, *Service d’tlnutomie Pathologique et de Médecine Légale, Hôpital Raymond Poincaré, F-92380 Garches and ‘Biochimie, CNRS UA-622, Faculté de Pharmacie, F-92296 Chatenuy-Malabry /Francel
(Received August 23rd. 19881 (Revision received September 30th, 19881 (Accepted November 22nd, 19881
Summary ABO groups were investigated on skin (and musclel, bone and hair specimens from 14 Egyptian mummies dating f’rom the Roman period. Samples were tested by the AE (absorptionelutionl, MA (mixed agglutinationl and HIF (histo-immunofluorescencel methods. in order to evaluate the reliability of each method. For half of the mummies (71the results were concordant on all samples (3-9 samples for each mummyl with al1 employed methods, suggesting an unequivocal blood group conclusion. For the other seven mummies there were discordant results with the different methods and interpretation of the results was thus inconclusive. HIF seems to be the most reliable method as specific blood group substances are identified on specific histologie structures. Failure to detect tissular ABO antigens was mainly due to excessive resin impregnation. Key worda: ABO bloed groups; Mummies; Absorption-elution; Mixed agglutination; Immunofluoresence
Introduction
The miraculeus mummification techniques, practised by ancient Egyptians, rendered immortal not only their souls, but also their biological characters, enabling US to decrypt, today, the morphology [l], biochemistry [2], pathology [3,4] and even the genomic structure (DNA) [5] of people that lived several thousand years ago. Among these characters, blood groups were intensively investigated as they enable the comprehension of anthropologic or genétic drifts (for bibliography see Ref. 6) but, as stated by Allison et al. [7] “with many divergent and confusing results, that have led to the formation of some misleading theories”. We started therefore the present study in order to re-evaluate the reliability of some of the methods used in ABO phenotyping on ancient material. 0379-0738/89/$03.50
0 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
114
The aim of our work was not an extended, statistical study, but rather a comparison of the results obtained by three different methods on several tissues from each of the mummies under study. Material and Methods The study was performed on 14 Egyptian mummies from the necropolis of the oasis of Douch, a military fortress of the Nubian desert ascribed to the Roman period (111th century B.C.-IVth century C.E.). Some of the bodies buried there have an amazing peculiarity: a golden coating of their faces, hands and legs. We undertook a nearly exhaustive sampling on this material, including skin and muscle samples from al1 parts of the body, from the scalp to the heel (under the headline “skin” are included results on skin and muscle samples, as it is often difficult to differentiate them on dehydrated specimens), bone samples (mainly vertebrae, but also ribs, phalanx, cubitus, femur, tibia and tarsusl and hair samples (scalp, eye-lash, beard and pubic hairl. The number of samples for each mummy varied between three and nine. Absorption-elution
test IAE)
Skin, muscle and bone samples were fragmented (either by aid of scissors or in a mortar) and tested by the method of Kind [8]: anti-A and anti-B sera were purchased from Ortho Diagnostic Laboratories (titer 1/2561, the anti-H reagent was the lectin 1 from Alex europaeus (titer 1/1281. By trial and error we established the optimal amounts of material as follows: 30 -50 mg per tube for skin and muscle samples, 100 mg per tube for bone samples. Hair specimens were tested by the same method [9] whenever the available material was sufficient to obtain, after mechanica1 crushing, approximately 200 mg hair powder per tube. However, for most hair specimens we used a micro-elution method [lO] which permits one to type ABO on a single hair. The AE method was equally applied on two nail samples [11,12] from two different mummies. Mixed agglutinution
test (MA)
Skin and muscle samples were examined by the method of Coombs et al. [13], modified for mummified tissues [14]. Hair specimens were examined by the method of Lincoln and Dodd [15], adapted by US to a micro-method for a single hair. The antisera used were the same as those for the absorption-elution test. Histo-immunojluorescence
test 1HIF)
Skin and muscle specimens were rehydrated in Sandison’s solution [16,17], for periods ranging from some hours to 2-3 days (the length of this step depends as wel1 on the size of the sample as on its feature). The samples
115
were processed by classica1 histologie techniques to obtain paraffin embedded sections. We applied the indirect immunofluorescence method [18,19] to deparaffined sections, using monoclonal anti-A and anti-B sera, titer 1/128 (Celltech, Berkshirel and goat anti-mouse IgG FITC labeled antibodies (Cappel Lab., Cochranville). The H antigen was determined with the anti-H UEA-1 lectin (VZex europaeus) revealed by a rabbit anti-UEA FITC labeled antibody (both purchased from E.Y. Lab. Inc., San Mateol. Stained sections were mounted in glycerol containing pphenylenediamine for nuclear counterstaining [20] and were examined on a Leitz epifluorescence microscope. Results Results are summarized in Tables 1 and 2. Seven out of 14 mummies gave concordant results with the three techniques (Table 11 and thus could be ascribed to one of the four ABO groups. The other seven mummies, on the contrary, show discordances among the different methods, for the same mummy (Table 21 and the ABO typing was thus inconclusive. We have to outline here that the various tissues from different mummies, and even from the same mummy, had differing degrees of conservation: some had kept excellent histologie features, while others were severely impaired. For that reason, samples were tested always in duplicate and tests were repeated on different fragments, in order to verify the reliahility of the results. A major problem in ABO typing was the presence of resin: even after prolonged prewashings in saline, test RBC were constantly haemolysed by residual resin in dark, black specimens. Some interference was also observed in the HIF method, where residual resin gave a strong yellow autofluorescence of the preparation. Blood group substances are probably wel1 preserved in al1 the explored tissues from mummies in Table 1, as could be seen by the strong agglutination of test RBC (+ + + and + + 1. Usually, haemagglutination reactions were more intense in the AE than in the MA test and stronger in skin (musclel than in bone specimens. Hair samples were usually very thick and permitted one to type the ABO groups on a single hair. The only one available specimen of beard (mummy no. 81 showed strong B and H(O) antigens, by both the AE and the MA methods. Fascinating results were observed by the HIF method, as it permits the visual localization of antigenic structures. The most common pattern was a fine green fluorescente of the vascular endothelium of the capillary vessels, either linear or speckled (Fig. lal, analogous to what is usually observed on fresh histologie preparations [21- 231. Sometimes blood group substances could be localized on the endothelium of large vessels (Fig. lb) and some images suggested even the presence of intravascular RBC. Epithelial cells of the epidermis were usually negative, except for one blood group 0 mummy who was strongly labeled with the anti-H reagent on the cel1 membranes of al1 layers of the epidermis (thumbl (Fig. ld). This aspect is characteristic for
1
82.54.12.1
82.54.12.2
82.54.12.3
82.54.12.5
82.56.12.2
82.65.12.2
8
9
10
12
15
17
“Intensity of reactions
81.8
Skin Hair Skin Bone Hair Skin Bone Hair
Bone Skin Bone Hair
Hair Skin
Skin Muscle Nail Skin
Sample
2 4
No. of samples
OF ABO PHENOTYPING
H(+
H(+ + +) H(+ +) H(+ +) Bi+ + +) Hl+ + +) B(+ +)H(+) At+ +)B(+ +) H(+ +) A(+)B(+)H(+) Af+ +)H(+ +) At+ +)H(+ +) At+ + +) H(+ + +) At+ +)H(+) Al+ + +)H(+ + At+ +)H(+ + +) At+ + +) H(+) At+ +)H(+ +) B(+ + +) Bl+ +) B(+ + +) Hl+ + +) +)
B(+)H(+
A(+)H(+) B(+ +) B(+)H(+
+)
Ai+
+)
Hl+
+)
At+
Al+)
+ +)H(+
At+
+ +)
+ +) +)
+)
+)
A(+)B(+ +) H(+ + +)
B(+)
H(+ H(+
Histoimmunofluorescente*
At+ +)H(+) A(+)H(+) A(+)H(+)
+ +)H(+
At+
Bl+ +) H(+) At+ +)B(+ +)H(+)
B(+ + +) H(+ + +)
+ +)
Mtied agglutinution”
Absorption-elution”
OF MUMMIES
was scored from ( + ) (weak) to + + + (streng).
Identification no.
RESULTS
1
No. /Lab)
CONCLUSIVE
TABLE
B
A
A
A
AB
B
0
Bloed group
2
82.54.11.1
82.54.12.4
82.54.12.6
82.54.12.7
82.64.21.2
7
11
13
14
16
of reactions was scored from
Skin Bone Hair Nail
A(+ + +) B(+ + +) A(+ + +) B(+ + +) A(+)B(+)H(+) Haemolysis A(+ +) H(+ + +) Haemolysis Haemolyis H(+ + +) A(+)B(+)H(+ +) H(+ +) B(+ + +) H(+) A(+ + +) B(+ + +) A(+)B(+)H(+) A(+ +)B(+ +) B(+ +) A(+ + +) H(+ + +) Af+) B(+) Haemolysis At+) B(+) H(+) Af+)
Absorption-ehtiona
OF MUMMIES
( + ) (weak) to + + + (streng).
2 1 2 1 1 1 3 7 1 3 1
Hair Skin Bone Hair Skin Bone Hair Skin Hair Skin Bone
81.20.51.3
4
‘Intensity
1
Bone
Hair Skin Bone Hair
1
Skin
81.18.31.1
No. of
2
Sample
OF ABO PHENOTYPING samples
Identification
RESULTS
no.
No. Lab)
INCONCLUSIVE
TABLE
+ +) + +) +) + +)H(+)
A(+
At+)
A(+
+)B(+
+ +)B(+
+)
+ +)H(+
+)H(+
A(+)B(+)H(+) A(+ +)B(+ +)
H(+ H(+ H(+ B(+
H(+ + +) Haemolysis
H(+ +) Haemolysis
H(+ + +)
Mixed agglutinution”
+)
+ +)
+ +)
A(+
+ +)
+) H(+ +)
B(+)H(+
H(+
Non-specific fhorescence (resin) H(+ +)
-
H(+
Histoimmunofluorescencea
A? AB?
B? AB?
O? B? AB?
O? AB?
O?
A? O?
O? AB?
arouo”
Blood
118
the thick areas of the epidermis [24] and it was most surprising to observe an identical staining on the mummified tissue as compared to the fresh one (Fig. lfl. Specific staining was observed once on a sweat duet and in another case we could observe a faint positivity on a sweat gland (Fig. IC). The positive reaction on a fresh sweat duet is shown for comparison (Fig. lel. Besides this specific staining, which may decide, even alone, an ABO blood group attribution, we could observe also another non-specific, equally characteristic pattern - a cloudy positivity spread through the tissue, unrelated to any histologie structure. Probably this aspect is due to bacterial proliferation and was observed also on al1 specimens of a recent collective disaster where corpses had been preserved at 15- UlOC, for 10 days, before being sampled. This may confirm the hypothesis [25] that Egyptian corpses were not processed immediately after their death, so that bacterial cultures could meanwhile develop. The subsequent mummification procedures might afterwards fix those colonies, which are easily identified on histologie preparations with classica1 colorations. Bone specimens proved to be useful for complementary investigations, as they confirm, in half of the mummies, the ABO blood group. Nevertheless, in the other half of the samples, phenotyping on bone specimens was difficult, either because of the haemolysis of the RBC, or of probably non-specific absorptions. On the contrary, nail samples might be useful tissue for blood grouping and we regret that we had only two nail samples available to check this hypothesis. However, it seems reasonable that nails preserve the blood group substances wel1 as they are subjected neither to postmortem degradation, nor to environmental influences. For the seven mummies which gave concordant results for at least three different tissues, each of them explored by three different methods, a reasonably good estimate of the ABO blood group can be made. As can be seen in the results in Table 1, one mummy was group 0, three group A, two group B and one group AB. For nearly al1 samples we found the H specificity, besides.A or B specificities. The results on the other seven mummies (Table 21 are much more difficult to interpret, as divergent results were observed on different tissues. For two of them (nos. 4 and 71 specimens were severely damaged and constantly yielded RBC haemolysis by AE and MA methods. Tissue sections examined in HIF showed a sparked, widespread fluorescente due to resin impregnation. The only exploitable samples for those two mummies were the hair and both reacted as blood group 0 hair. When looking at our results, a striking aspect would be the low incidence of the blood group 0. In fact, statistic analysis on ancient material yields two types of errors: either an excess of blood group 0 (in experiments performed with only anti-A and anti-B sera, negative results were reported as group 01, or an excess of blood group AB (due to non-specific reactionsl. The results illustrated in Table 2 suggest this last situation: by only the AE method, five out of seven mummies might be attributed to group AB, whereas by the HIF
Fig. 1. HIF of paraffin-embedded skin and muscle samples of mummies. (al Mummy no. 9 (group AB) tested with anti-B serum. Green fluorescent staining of the vascular network lining the muscular fibres (arrows). The epidermal zone is negative, except a faint staining of the cornean layer (double arrows1.t x 1001. (bl Mummy no. 15 (group Al reacted with anti-A serum. Fluorescent staining of the vascular endothelium of a large vesse1.f x 250). (cl Mummy no. 9 (group AB) stained with anti-A. Sweat glands show green positivity.(x 2501. (dl Epidermis of a thumb (mummy no. 1, group 01 reacted with anti-H. Strong fluorescence of the cell membrane of all layers of the epidermis4 x 2501. (el Skin sample of a recent cadaver of a group A, Le(a - b + 1 individual stained with anti-A. All cells of the sweat duct are positive (arrow), as are the small capillary vessels (double arrowsl( x 2501. (fl Palmar epidermis of a recent cadaver of group 0, Le(a- b+) individual reacted with anti-H. The membrane of all epidermis cells is positive tarrows) with the exception of the basement membrane which is negative. Capillaries (double arrows) and red blood cells are also positive.( x 2501.
121
method three of them are 0 (nes. 2, 11 and 131, one A (no. 16) and one group B (no. 141. In the discordant cases it seems reasonable to attribute the HIF method as being the most specific test for tissular ABO phenotyping, even if additional A and/or B specificities were observed by the AE and MA methods. We have to suppose that, most probably, these last reactions are the expression of non-specific absorptions, as is frequently mentioned for archeologica1 material. Based on this assumption, the 14 mummies tested in this study would be 6 group 0 (if mummy no. 4 is grouped accordingly to the MA test), 4 group A, 3 group B and one group AB. This distribution fits wel1 with the statistical data on Egyptian populations [26]. Discussion The remarkable preservation of blood group antigens through centuries and even millenia is mainly due to their glycosidic structure and it has repeatedly been demonstrated that the glucidic structures of ancient material are nearly identical to those of fresh tissue [27,28]. After half a century of research work on phenotyping mummies, the question is no longer whether or not ABO blood groups may be identified, but rather which method is the best. The absorption-inhibition test, largely utilized during the first period [29,30] was subjected to severe criticism, due to its lack of sensitivity and rate of errors [31-341. Subsequently other different methods were applied for ancient material, like AE [35], MA [14,36,37], absorption of tissue antigens on group 0 RBC [38,39], induction of antibodies [7], purification of blood group substances [40-421 and multiple absorption [43]. The most utilized materials were bones and muscle specimens, and in one case the spleen [39]. In our study, each mummy was explored on the basis of a plurivalent material, including skin tand musclel, bone and hair samples and each sample was investigated according to a multi-method protocol. We hoped to increase, in this way, the chances of finding a good preserved tissular antigen and simultaneously to minimize the errors due to each one of the employed methods. In fact, the lack of controls, when testing this kind of material, always placed doubt on the results, the most current errors being due to destroyed antigens or to additional reactions due to bacterial or fungal contamination of the sample [31,32,44]. The HIF method has been shown to be specific and of high sensitivity for the identification of ABH antigens as wel1 on fresh cryostat sections [21-231 as on fixed, paraffin-embedded tissues [45- 471: specific staining was identified on vascular endothelium, digestive mucosae and skin in large series of donors of known erythrocyte ABO and secretory salivary phenotype. The same specificity and sensitivity proved to be confirmed on ancient material, based on the following arguments: 61 ABH antigens were identified, in mummified tissue, on the same specific structures as those described on fresh tissues; (iil specific staining of a minute histologie structure, like a cap-
122
illary vessel, permits an ABH typing on a 5-Pm section, whereas much more material wil1 be required for AE and/or MA; (iiil the visualization of different patterns for specific and non-specific staining (e.g. related or not to a characteristic histologie structure) permits one to discriminate specific results from non-specific ones. Therefore, whenever possible, this method has to be included as the most sensible and the most specific one for ABO blood group identification. Several samples, taken from different parts of the body, have to be examined, as preservation of tissues may vary greatly from one specimen to another [ll. In our study, the best preserved tissues originated from the face, neck and hands, probably because they are less impregnated with resin. We might suppose that the exposed regions of the body were treated in a special manner and therefore are the most suitable for ABO tissue typing. AE and MA constitute valuable additive methods in ABO blood group determination, both on soft tissues (skin and musclel, and on hard ones (bene, nail and hair). Results observed by these two methods depend mainly on the quality of the sample and very black, resin-impregnated samples are unacceptable. More confidence may be attributed to hair samples results since, theoretically, they are usually wel1 preserved structures. Nevertheless, we have always to keep in mind the high percentage of non-specific reactions repeatedly observed on hair samples [10,48,49]. Conclusions We have to conclude that group-specific antigens are wel1 preserved on archeological material but false positive and false negative reactions are equally frequent. The most reliable results are those obtained by several methods, in different samples, when they are concordant. The basic requirement for a successful blood group determination remains the quality of the sample (mainly the degree of resin impregnationl. Acknowledgements The authors want to thank Mr. Prof. P.F. Ceccaldi and Mrs. Prof. C. Roubet for having initiated this study and having supplied the samples. The skillful technical assistance of Mrs. S. Sanna and Mrs. C. Doucet is gratefully acknowledged. References B.A. Barraco, Paleobiochemistry. In A. and E. Cockburn teds.), Mummies, Diseaaes aud Ancient Cultures, Cambridge University Press, 1980, pp. 312-326. S.M. Borgognini-Tarli and C. Paoli, Biochemieal and immunologieal investigations on early Egyptian remains. J. Hum, Evol, 1 (1972)281- 287. A.T. Sandison, Diseases in ancient Egypt. In A. and E. Cockburn (eds.1, Mummies, Diseases and Ancient Cultures, Cambridge University Press, 1980, pp. 29-44. D. Brothwell and A.T. Sandison (eds.), Diseases in Antiquity. Springfield, 111:Thomas, 1967.
123 5 6
S. Pääbo, Molecular cloning of ancient Egyptian mummy DNA. Nuture, (Lond.1 314 (1985) 644-645. S.M. Borgognini-Tarli, Paleoserology - genera1 bibliography. J. Hum. Evol, 8 (1979) 735740.
7
8
9 10 11 12 13
M.J. Allison, A.A. Hossaini, N. Castro, J. Munizaga and A. Pezzia, ABO blood groups in Peruvian mummies. 1. An evaluation of techniques. Am. J. Physiol Anthropol, 44 (1976) 55-62. S.S. Kind, Absorption-elution
grouping of blood stains on fabrics. Nuture (Lond.1 187 (19601 789-790. S. Yada, M. Okane and Y. Sano, Blood grouping of human hairs derived from various parts of the body by means of elution technique. Acts Crìm. J~R, 32 (1966116- 19. K. Crainic and M. Durigon, Determination du groupe ABO sur un seul cheveu par microélution. Abstracts XXXVII Congr. Intern. Langue Franc. Méd Légale, Paris, 1983, pp. 59. R. Outteridge, Determination of the ABO group from fingernails. Med. Sci Luw, 3 (19631 275-276. S. Yada, M. Okane and Y. Sano, A simple method 32 (1966) 96 -98.
for grouping fingernails.
Acts
Grim. Jpn.,
R.R.A. Coombs, D. Bedford and L.M. Rouillard, A and B blood group antigens on human epidermal cells demonstrated by mixed agglutination. Lancet, 270 (19561 461- 463. 14 C.M. Otten and L.L. Flory, Blood typing of chilean mummy tissue: a new approach. Am. J. PhysioL Anthropol, 21 (19631283-285. 15 P. Lincoln and B. Dodd, Mixed agglutination as a method for the determination of A, B and H blood group substances in hair. Med. Sci. Law, 8 (19681 38-40. 16 A.T. Sandison, The histological examination of mummified material. Stain TechnoL, 30 (1955) 277 - 283. 17 T.A. Reyman and A.M. Dowd, Processing of mummified tissue. In A. and E. Cockburn (eds.1, Mummies, Diseases and Ancient Cultures, Cambridge University Press, 1980, pp. 258 - 266. 18 D. Barres, V. Phat, M. Durigon, R. Oriol, K. Crainic and A. Hadengue, Blood group typing on different tissues by histoimmunofluorescence. 9th Meet. Int. Ass. Forens& Sci, Bergen, 1981. 19 R. Oriol, J. LePendu and R. Mollicone, Genetics of ABO, H, Lewis, X and related antigens. Vox Sang., 51 (19861 161- 171. 20 R. Oriol and R. Mantilla-Jimenez, Fluorescent staining of nuclei and amyloid substance. Two useful properties of pphenylenediamine. J. ImmunoL Methods, 62 (1983) 185- 192. 21 L.E. Glynn and E.J. Holborow, Distribution of blood group substances in human tissue. Br. Med. BulL, 15 (19591 151- 153. 22 A.E. Szulman, The histologieal distribution of blood group substances A and B in man. J. Exp. Med., 111 (19601785-800. 23 A.E. Szulman, The histological distribution of blood group substances in man as disclosed by immunofluorescence. 11. The H antigen and its relation to A and B antigens. J. Exp. Meo!., 115 (19621977-996. 24 R. Oriol, ABH and related tissue antigens. Biochem. Sec. Trans., 15 (19871596-599. 25 A. Cockburn, R. Barraco, W. Peck and T. Reyman, Autopsy of an Egyptian mummy. Science, 187 (1975) 1155-1160. 26 S.M. Borgognini-Tarli and G. Paoli, Les groupes sanguins du système ABO à partir des tissus d’anciens égyptiens. BuZL Mém. Sec. Anthropol, (Paris), 8 (1981) 297-305. 27 R.A. Barraco, Preservation of proteins in mummified tissues. Am. J PhysioL AnthropoL, 48 11978) 487 - 491. 28 P. Michelin-Lausarot, C. Ambrosino, F. Fauro, A. Conti and E. Rabino-Massa, Preservation and amino acid composition of Egyptian mummy structure proteins. J. Hum. EvoL, 1 (1972) 489 - 499. 29 W.C. Boyd and L.G. Boyd, Blood grouping tests on 300 mummies. J. ImmunoL, 32 (19371307 - 319.
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tissue and cancellous bone. Am. J. PhysioL
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H.E. Ezra-Cohn and S.F Cook, Blood typing compact human bone tissue. Nature (Land.), 191 (1961) 1267- 1268. M.P. Gray, A method for reducing non specific reactions in the typing of human skeletal material. Am. J. PhysioL AnthropoL, 16 (1958) 135-139. F.P. Thieme and C.M. Otten, The unreliability of blood typing aged hone. Am. J. AnthroPOL, 15 (1957) 387-397. M. Smith, Blood groups of the ancient dead. SC~Q~CQ,131 (1960) 699-702. F.R. Carnese and M. Palatnik, Estudios paleoserologicos di restos momificados de aborigenes argentinos. Sungre, 17 (1972) 201- 210. L. Harsanyi and G. Gerencser, Nachweis von Gruppensubstanzen in Geweben mit der Mixed cel1 agglutination Methods. Dtsch. Zschr. GQS. GQT. MQ~, 63 (1968) 162- 165. L.K. Lippold, The mixed cel1 agglutination method for typing of mummified human tissue. Am. J. PhysioL AnthropoL, 34 (1971) 377-383. R.C. Connolly, R.G. Harrison and A. Abdalla, Kinship of Smenkhkare and Tutankhamen affirmed by serological micromethod. Nature (Land.), 224 (1969) 325-326. G.D. Hart, 1. Kvas and M. Soots, Blood group testing of ancient material with particular reference to the mummy Nakht. Transfusion, 18 (1978) 474-477. G. Paoli, Determinazione del gruppo sanguino del sistema ABO in scheleti egiziani di età dinastica. Atti Sec. Toscana Sci Nut., SB, 77 (1970) 88- 100. G. Paoli, Further biochemieal and immunologieal investigations on early Egyptian remains. J. Hum. EvoL, 1 (19781 45’7-466. S. Micle, E. Kobilansky, M. Nathan, B. Arensburg and H. Nathan, ABO typing of ancient skeletons from Israel. Am. J. PhysioL Anthropol, 47 (1977) 89-91. R. Heglar, Paleoserology techniques applied to skeletal identification. J. Forensic Sci, 17 (1972) 358-363. M. Pereira, ABO grouping of decomposed human tissue. J. Forensic Sci Sec., 13 (1973) 3336.
45 46
41
48 49
R. Oriol, J.E. Cooper, D.R. Davies and P.W.N. Keeling, ABH antigens in vascular endothelium and some epithelial tissues of baboons. Lab. Znvest., 50 (1984) 514-519. R. Mollicone, J. Le Pendu, J. Bara and R. Oriol, Heterogeneity of the ABH antigenic determinants expressed in human pyloric and duodenal mucosae. Glycoconjugate J., 3 (1986) 187 - 202. R. Mollicone, A.M. Dalix, A. Jacobsson, B.E. Samuelsson, G. Gerard, K. Crainic, T. Caillard, J. Le Pendu and R. Oriol, Red Cel1 H deficient salivary ABH secretor phenotype of Reunion island. Genetic control of the expression of H antigen in the skin. Glycoconjbgate J., 5 (1988) 499-512. B. Brinkmann, J. Lemke and R. Lengfeld, The reliahility of determination of ABO blood groups in hairs. Ad. KriminoL, 164 (1979) 167-171. 1. Oepen and H. Noever, Zur ABO-Blutgruppenprägung des menschlichen Haares. Z. Rechtsmed., 85 (1980) 205- 209.
Science International 43 (19891113- 124 Elsevier Seientific Publishers Ireland Ltd.
Forenuic
ABO TISSUE
ANTIGENS
OF EGYPTIAN
MUMMIES
K. CRAINIC’, M. DURIGON”and R. ORION aU.E.R. De Médecine Légak, Droit Mkdàcal et Déontologie Médicale, Université René Descartes, 2, ploce Ma.zaa, F-75012 Paris, *Service d’tlnutomie Pathologique et de Médecine Légale, Hôpital Raymond Poincaré, F-92380 Garches and ‘Biochimie, CNRS UA-622, Faculté de Pharmacie, F-92296 Chatenuy-Malabry /Francel
(Received August 23rd. 19881 (Revision received September 30th, 19881 (Accepted November 22nd, 19881
Summary ABO groups were investigated on skin (and musclel, bone and hair specimens from 14 Egyptian mummies dating f’rom the Roman period. Samples were tested by the AE (absorptionelutionl, MA (mixed agglutinationl and HIF (histo-immunofluorescencel methods. in order to evaluate the reliability of each method. For half of the mummies (71the results were concordant on all samples (3-9 samples for each mummyl with al1 employed methods, suggesting an unequivocal blood group conclusion. For the other seven mummies there were discordant results with the different methods and interpretation of the results was thus inconclusive. HIF seems to be the most reliable method as specific blood group substances are identified on specific histologie structures. Failure to detect tissular ABO antigens was mainly due to excessive resin impregnation. Key worda: ABO bloed groups; Mummies; Absorption-elution; Mixed agglutination; Immunofluoresence
Introduction
The miraculeus mummification techniques, practised by ancient Egyptians, rendered immortal not only their souls, but also their biological characters, enabling US to decrypt, today, the morphology [l], biochemistry [2], pathology [3,4] and even the genomic structure (DNA) [5] of people that lived several thousand years ago. Among these characters, blood groups were intensively investigated as they enable the comprehension of anthropologic or genétic drifts (for bibliography see Ref. 6) but, as stated by Allison et al. [7] “with many divergent and confusing results, that have led to the formation of some misleading theories”. We started therefore the present study in order to re-evaluate the reliability of some of the methods used in ABO phenotyping on ancient material. 0379-0738/89/$03.50
0 1989 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland
114
The aim of our work was not an extended, statistical study, but rather a comparison of the results obtained by three different methods on several tissues from each of the mummies under study. Material and Methods The study was performed on 14 Egyptian mummies from the necropolis of the oasis of Douch, a military fortress of the Nubian desert ascribed to the Roman period (111th century B.C.-IVth century C.E.). Some of the bodies buried there have an amazing peculiarity: a golden coating of their faces, hands and legs. We undertook a nearly exhaustive sampling on this material, including skin and muscle samples from al1 parts of the body, from the scalp to the heel (under the headline “skin” are included results on skin and muscle samples, as it is often difficult to differentiate them on dehydrated specimens), bone samples (mainly vertebrae, but also ribs, phalanx, cubitus, femur, tibia and tarsusl and hair samples (scalp, eye-lash, beard and pubic hairl. The number of samples for each mummy varied between three and nine. Absorption-elution
test IAE)
Skin, muscle and bone samples were fragmented (either by aid of scissors or in a mortar) and tested by the method of Kind [8]: anti-A and anti-B sera were purchased from Ortho Diagnostic Laboratories (titer 1/2561, the anti-H reagent was the lectin 1 from Alex europaeus (titer 1/1281. By trial and error we established the optimal amounts of material as follows: 30 -50 mg per tube for skin and muscle samples, 100 mg per tube for bone samples. Hair specimens were tested by the same method [9] whenever the available material was sufficient to obtain, after mechanica1 crushing, approximately 200 mg hair powder per tube. However, for most hair specimens we used a micro-elution method [lO] which permits one to type ABO on a single hair. The AE method was equally applied on two nail samples [11,12] from two different mummies. Mixed agglutinution
test (MA)
Skin and muscle samples were examined by the method of Coombs et al. [13], modified for mummified tissues [14]. Hair specimens were examined by the method of Lincoln and Dodd [15], adapted by US to a micro-method for a single hair. The antisera used were the same as those for the absorption-elution test. Histo-immunojluorescence
test 1HIF)
Skin and muscle specimens were rehydrated in Sandison’s solution [16,17], for periods ranging from some hours to 2-3 days (the length of this step depends as wel1 on the size of the sample as on its feature). The samples
115
were processed by classica1 histologie techniques to obtain paraffin embedded sections. We applied the indirect immunofluorescence method [18,19] to deparaffined sections, using monoclonal anti-A and anti-B sera, titer 1/128 (Celltech, Berkshirel and goat anti-mouse IgG FITC labeled antibodies (Cappel Lab., Cochranville). The H antigen was determined with the anti-H UEA-1 lectin (VZex europaeus) revealed by a rabbit anti-UEA FITC labeled antibody (both purchased from E.Y. Lab. Inc., San Mateol. Stained sections were mounted in glycerol containing pphenylenediamine for nuclear counterstaining [20] and were examined on a Leitz epifluorescence microscope. Results Results are summarized in Tables 1 and 2. Seven out of 14 mummies gave concordant results with the three techniques (Table 11 and thus could be ascribed to one of the four ABO groups. The other seven mummies, on the contrary, show discordances among the different methods, for the same mummy (Table 21 and the ABO typing was thus inconclusive. We have to outline here that the various tissues from different mummies, and even from the same mummy, had differing degrees of conservation: some had kept excellent histologie features, while others were severely impaired. For that reason, samples were tested always in duplicate and tests were repeated on different fragments, in order to verify the reliahility of the results. A major problem in ABO typing was the presence of resin: even after prolonged prewashings in saline, test RBC were constantly haemolysed by residual resin in dark, black specimens. Some interference was also observed in the HIF method, where residual resin gave a strong yellow autofluorescence of the preparation. Blood group substances are probably wel1 preserved in al1 the explored tissues from mummies in Table 1, as could be seen by the strong agglutination of test RBC (+ + + and + + 1. Usually, haemagglutination reactions were more intense in the AE than in the MA test and stronger in skin (musclel than in bone specimens. Hair samples were usually very thick and permitted one to type the ABO groups on a single hair. The only one available specimen of beard (mummy no. 81 showed strong B and H(O) antigens, by both the AE and the MA methods. Fascinating results were observed by the HIF method, as it permits the visual localization of antigenic structures. The most common pattern was a fine green fluorescente of the vascular endothelium of the capillary vessels, either linear or speckled (Fig. lal, analogous to what is usually observed on fresh histologie preparations [21- 231. Sometimes blood group substances could be localized on the endothelium of large vessels (Fig. lb) and some images suggested even the presence of intravascular RBC. Epithelial cells of the epidermis were usually negative, except for one blood group 0 mummy who was strongly labeled with the anti-H reagent on the cel1 membranes of al1 layers of the epidermis (thumbl (Fig. ld). This aspect is characteristic for
1
82.54.12.1
82.54.12.2
82.54.12.3
82.54.12.5
82.56.12.2
82.65.12.2
8
9
10
12
15
17
“Intensity of reactions
81.8
Skin Hair Skin Bone Hair Skin Bone Hair
Bone Skin Bone Hair
Hair Skin
Skin Muscle Nail Skin
Sample
2 4
No. of samples
OF ABO PHENOTYPING
H(+
H(+ + +) H(+ +) H(+ +) Bi+ + +) Hl+ + +) B(+ +)H(+) At+ +)B(+ +) H(+ +) A(+)B(+)H(+) Af+ +)H(+ +) At+ +)H(+ +) At+ + +) H(+ + +) At+ +)H(+) Al+ + +)H(+ + At+ +)H(+ + +) At+ + +) H(+) At+ +)H(+ +) B(+ + +) Bl+ +) B(+ + +) Hl+ + +) +)
B(+)H(+
A(+)H(+) B(+ +) B(+)H(+
+)
Ai+
+)
Hl+
+)
At+
Al+)
+ +)H(+
At+
+ +)
+ +) +)
+)
+)
A(+)B(+ +) H(+ + +)
B(+)
H(+ H(+
Histoimmunofluorescente*
At+ +)H(+) A(+)H(+) A(+)H(+)
+ +)H(+
At+
Bl+ +) H(+) At+ +)B(+ +)H(+)
B(+ + +) H(+ + +)
+ +)
Mtied agglutinution”
Absorption-elution”
OF MUMMIES
was scored from ( + ) (weak) to + + + (streng).
Identification no.
RESULTS
1
No. /Lab)
CONCLUSIVE
TABLE
B
A
A
A
AB
B
0
Bloed group
2
82.54.11.1
82.54.12.4
82.54.12.6
82.54.12.7
82.64.21.2
7
11
13
14
16
of reactions was scored from
Skin Bone Hair Nail
A(+ + +) B(+ + +) A(+ + +) B(+ + +) A(+)B(+)H(+) Haemolysis A(+ +) H(+ + +) Haemolysis Haemolyis H(+ + +) A(+)B(+)H(+ +) H(+ +) B(+ + +) H(+) A(+ + +) B(+ + +) A(+)B(+)H(+) A(+ +)B(+ +) B(+ +) A(+ + +) H(+ + +) Af+) B(+) Haemolysis At+) B(+) H(+) Af+)
Absorption-ehtiona
OF MUMMIES
( + ) (weak) to + + + (streng).
2 1 2 1 1 1 3 7 1 3 1
Hair Skin Bone Hair Skin Bone Hair Skin Hair Skin Bone
81.20.51.3
4
‘Intensity
1
Bone
Hair Skin Bone Hair
1
Skin
81.18.31.1
No. of
2
Sample
OF ABO PHENOTYPING samples
Identification
RESULTS
no.
No. Lab)
INCONCLUSIVE
TABLE
+ +) + +) +) + +)H(+)
A(+
At+)
A(+
+)B(+
+ +)B(+
+)
+ +)H(+
+)H(+
A(+)B(+)H(+) A(+ +)B(+ +)
H(+ H(+ H(+ B(+
H(+ + +) Haemolysis
H(+ +) Haemolysis
H(+ + +)
Mixed agglutinution”
+)
+ +)
+ +)
A(+
+ +)
+) H(+ +)
B(+)H(+
H(+
Non-specific fhorescence (resin) H(+ +)
-
H(+
Histoimmunofluorescencea
A? AB?
B? AB?
O? B? AB?
O? AB?
O?
A? O?
O? AB?
arouo”
Blood
118
the thick areas of the epidermis [24] and it was most surprising to observe an identical staining on the mummified tissue as compared to the fresh one (Fig. lfl. Specific staining was observed once on a sweat duet and in another case we could observe a faint positivity on a sweat gland (Fig. IC). The positive reaction on a fresh sweat duet is shown for comparison (Fig. lel. Besides this specific staining, which may decide, even alone, an ABO blood group attribution, we could observe also another non-specific, equally characteristic pattern - a cloudy positivity spread through the tissue, unrelated to any histologie structure. Probably this aspect is due to bacterial proliferation and was observed also on al1 specimens of a recent collective disaster where corpses had been preserved at 15- UlOC, for 10 days, before being sampled. This may confirm the hypothesis [25] that Egyptian corpses were not processed immediately after their death, so that bacterial cultures could meanwhile develop. The subsequent mummification procedures might afterwards fix those colonies, which are easily identified on histologie preparations with classica1 colorations. Bone specimens proved to be useful for complementary investigations, as they confirm, in half of the mummies, the ABO blood group. Nevertheless, in the other half of the samples, phenotyping on bone specimens was difficult, either because of the haemolysis of the RBC, or of probably non-specific absorptions. On the contrary, nail samples might be useful tissue for blood grouping and we regret that we had only two nail samples available to check this hypothesis. However, it seems reasonable that nails preserve the blood group substances wel1 as they are subjected neither to postmortem degradation, nor to environmental influences. For the seven mummies which gave concordant results for at least three different tissues, each of them explored by three different methods, a reasonably good estimate of the ABO blood group can be made. As can be seen in the results in Table 1, one mummy was group 0, three group A, two group B and one group AB. For nearly al1 samples we found the H specificity, besides.A or B specificities. The results on the other seven mummies (Table 21 are much more difficult to interpret, as divergent results were observed on different tissues. For two of them (nos. 4 and 71 specimens were severely damaged and constantly yielded RBC haemolysis by AE and MA methods. Tissue sections examined in HIF showed a sparked, widespread fluorescente due to resin impregnation. The only exploitable samples for those two mummies were the hair and both reacted as blood group 0 hair. When looking at our results, a striking aspect would be the low incidence of the blood group 0. In fact, statistic analysis on ancient material yields two types of errors: either an excess of blood group 0 (in experiments performed with only anti-A and anti-B sera, negative results were reported as group 01, or an excess of blood group AB (due to non-specific reactionsl. The results illustrated in Table 2 suggest this last situation: by only the AE method, five out of seven mummies might be attributed to group AB, whereas by the HIF
Fig. 1. HIF of paraffin-embedded skin and muscle samples of mummies. (al Mummy no. 9 (group AB) tested with anti-B serum. Green fluorescent staining of the vascular network lining the muscular fibres (arrows). The epidermal zone is negative, except a faint staining of the cornean layer (double arrows1.t x 1001. (bl Mummy no. 15 (group Al reacted with anti-A serum. Fluorescent staining of the vascular endothelium of a large vesse1.f x 250). (cl Mummy no. 9 (group AB) stained with anti-A. Sweat glands show green positivity.(x 2501. (dl Epidermis of a thumb (mummy no. 1, group 01 reacted with anti-H. Strong fluorescence of the cell membrane of all layers of the epidermis4 x 2501. (el Skin sample of a recent cadaver of a group A, Le(a - b + 1 individual stained with anti-A. All cells of the sweat duct are positive (arrow), as are the small capillary vessels (double arrowsl( x 2501. (fl Palmar epidermis of a recent cadaver of group 0, Le(a- b+) individual reacted with anti-H. The membrane of all epidermis cells is positive tarrows) with the exception of the basement membrane which is negative. Capillaries (double arrows) and red blood cells are also positive.( x 2501.
121
method three of them are 0 (nes. 2, 11 and 131, one A (no. 16) and one group B (no. 141. In the discordant cases it seems reasonable to attribute the HIF method as being the most specific test for tissular ABO phenotyping, even if additional A and/or B specificities were observed by the AE and MA methods. We have to suppose that, most probably, these last reactions are the expression of non-specific absorptions, as is frequently mentioned for archeologica1 material. Based on this assumption, the 14 mummies tested in this study would be 6 group 0 (if mummy no. 4 is grouped accordingly to the MA test), 4 group A, 3 group B and one group AB. This distribution fits wel1 with the statistical data on Egyptian populations [26]. Discussion The remarkable preservation of blood group antigens through centuries and even millenia is mainly due to their glycosidic structure and it has repeatedly been demonstrated that the glucidic structures of ancient material are nearly identical to those of fresh tissue [27,28]. After half a century of research work on phenotyping mummies, the question is no longer whether or not ABO blood groups may be identified, but rather which method is the best. The absorption-inhibition test, largely utilized during the first period [29,30] was subjected to severe criticism, due to its lack of sensitivity and rate of errors [31-341. Subsequently other different methods were applied for ancient material, like AE [35], MA [14,36,37], absorption of tissue antigens on group 0 RBC [38,39], induction of antibodies [7], purification of blood group substances [40-421 and multiple absorption [43]. The most utilized materials were bones and muscle specimens, and in one case the spleen [39]. In our study, each mummy was explored on the basis of a plurivalent material, including skin tand musclel, bone and hair samples and each sample was investigated according to a multi-method protocol. We hoped to increase, in this way, the chances of finding a good preserved tissular antigen and simultaneously to minimize the errors due to each one of the employed methods. In fact, the lack of controls, when testing this kind of material, always placed doubt on the results, the most current errors being due to destroyed antigens or to additional reactions due to bacterial or fungal contamination of the sample [31,32,44]. The HIF method has been shown to be specific and of high sensitivity for the identification of ABH antigens as wel1 on fresh cryostat sections [21-231 as on fixed, paraffin-embedded tissues [45- 471: specific staining was identified on vascular endothelium, digestive mucosae and skin in large series of donors of known erythrocyte ABO and secretory salivary phenotype. The same specificity and sensitivity proved to be confirmed on ancient material, based on the following arguments: 61 ABH antigens were identified, in mummified tissue, on the same specific structures as those described on fresh tissues; (iil specific staining of a minute histologie structure, like a cap-
122
illary vessel, permits an ABH typing on a 5-Pm section, whereas much more material wil1 be required for AE and/or MA; (iiil the visualization of different patterns for specific and non-specific staining (e.g. related or not to a characteristic histologie structure) permits one to discriminate specific results from non-specific ones. Therefore, whenever possible, this method has to be included as the most sensible and the most specific one for ABO blood group identification. Several samples, taken from different parts of the body, have to be examined, as preservation of tissues may vary greatly from one specimen to another [ll. In our study, the best preserved tissues originated from the face, neck and hands, probably because they are less impregnated with resin. We might suppose that the exposed regions of the body were treated in a special manner and therefore are the most suitable for ABO tissue typing. AE and MA constitute valuable additive methods in ABO blood group determination, both on soft tissues (skin and musclel, and on hard ones (bene, nail and hair). Results observed by these two methods depend mainly on the quality of the sample and very black, resin-impregnated samples are unacceptable. More confidence may be attributed to hair samples results since, theoretically, they are usually wel1 preserved structures. Nevertheless, we have always to keep in mind the high percentage of non-specific reactions repeatedly observed on hair samples [10,48,49]. Conclusions We have to conclude that group-specific antigens are wel1 preserved on archeological material but false positive and false negative reactions are equally frequent. The most reliable results are those obtained by several methods, in different samples, when they are concordant. The basic requirement for a successful blood group determination remains the quality of the sample (mainly the degree of resin impregnationl. Acknowledgements The authors want to thank Mr. Prof. P.F. Ceccaldi and Mrs. Prof. C. Roubet for having initiated this study and having supplied the samples. The skillful technical assistance of Mrs. S. Sanna and Mrs. C. Doucet is gratefully acknowledged. References B.A. Barraco, Paleobiochemistry. In A. and E. Cockburn teds.), Mummies, Diseaaes aud Ancient Cultures, Cambridge University Press, 1980, pp. 312-326. S.M. Borgognini-Tarli and C. Paoli, Biochemieal and immunologieal investigations on early Egyptian remains. J. Hum, Evol, 1 (1972)281- 287. A.T. Sandison, Diseases in ancient Egypt. In A. and E. Cockburn (eds.1, Mummies, Diseases and Ancient Cultures, Cambridge University Press, 1980, pp. 29-44. D. Brothwell and A.T. Sandison (eds.), Diseases in Antiquity. Springfield, 111:Thomas, 1967.
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