Aim of Research.
The determination of ABH blood groups in human skeletal remains from the City of Oxford and its environs. An examination of skeletal remains to determine the presence of pathology, abnormality, anomaly, age and sex. Special note will be made of the presence and incidence of osteoarthitic changes.
Methods of Research.
Serological techniques would be those used by Boyd & Boyd, (1931; 1933; 1934; 1937; 1939), Candela (1936; 1937; 1939; 1939a; 1940), Berti & Parenti (1964), Kellerman, Lengyel & Nemeskeri (1965; 1967), and Borgognini. The techniques used by Kellerman (1971; 1972;) and Borgognini (1967;1969), have achieved positive results in approximately 88% of cases. ABH antigens have been found to be present in bone 2000 BC at death. In some cases the use of radiographic techniques to confirm pathology, abnormality, age and to assist analysis of developmental criteria (e.g., Harris Lines).
Some 400 to 500 complete and incomplete skeletons from the City of Oxford and its environs. Covering a period from sub-Roman to late medieval times. Most material is from the priories of Blackfriars, Greyfriars, All Saints Church (High Street), St Frideswides at Osney, St Budoc’s, the Roman site at the University Museum, Queensford Farm at Dorchester, Appleford, and Abingdon. The material is obtainable from the Oxfordshire Archaeological Committee. Portions of the skeletons for serological analysis will be mainly proximal portions of femora and/or vertebrae.
Results of Research.
Possible determination of the ABH system distribution, this having value historically, serologically and in terms also of population genetics and human biology. This palaeoserological and palaeopathological work will help provide information concerning disease and arthritis incidence in earlier populations. Indicate the presence of anomalies and abnormalities in earlier populations, this being of value genetically and medically. Palaeoserological results will have also some value in the field of forensic medicine.
Work to Date.
Analysis for Oxford Archaeology Unit of some 35 skeletons from All Saints, High Street, 1973 excavation. Evidence to date indicates high incidence of arthritic changes, plus an age of death in the 30’s to 40’s. Specimens are from infancy to late 40’s. No serological work possible as yet. Various non-metrical evidence apparent including metopism, lambdoid ossicles, sacralisation of lumber 5th.
Overall Analysis of Skeletal Material (Lengyel & Nemeskeri).
Allows for decomposition, and permits a general biochemical and anatomical analysis. Combined chemico-analytical, serological, histological method. Analysis of post-mortem decomposition and fossilisation processes (Lengyel, 1967).
Two steps. (1) fresh bones, and (2) fossil bones. Method (a) is determination of water content by drying to 105 degrees centigrade. Then incineration and removal of chemically bound water. Method (b) is the determination of organic substances:
Method (b) determines total nitrogen content; non-protein nitrogen; the water soluble organic fraction; the protein-polysaccaharide complex; collagen; and other resistant proteins after extraction of collagen. Standard tests are available. In addition determination of citrate for sex determination; carbonate content; and organic phosphorus.
Method (c): determines calcium, inorganic phosphorus, magnesium, and sulphur.
Determination of Blood Groups.
Determination of blood groups are achieved by modified fluorescent antibody technique by microscope; and also by the Boyd-Candela precipitation test which is macroscopic.
Histological analysis comprises staining with Haematoxylin-Eosin, Schwart’s Method, and Azan. The histochemical analysis includes metachromatic staining, and the peroxide reaction.
Boyd and Boyd Method.
Evidence of presence of group specific antigen may be presumed if antiserum of suitable strength is inhibited in its activity with red cells of appropriate group. Prolonged contact with tissue sample is required. There are four naturally occurring human anti-A sera and four naturally occurring anti-B sera. Use on each specimen with specimen quantity permitting. Dilution of resulting titres of absorbed and unabsorbed sera to convenient level for titration. Titrations are fresh 2% suspensions of A2 and B cells.
Candela’s Technique (also as modified by Smith/Glemser).
Uses different dilution ranges. From each dilution tube take 0.5cc serum to which is added 0.05cc of 1% suspension of cells. Agitation followed by final microscope readings after 30 minutes. This micro-method means there are more test cells relative to the serum. This method is better for ground up bone.
Candela’s Technique modified by Smith (Glemser) uses 0.2 gm of prepared tissue placed in a 75 x 10mm tube. Then 0.4 ml of antiserum added to each sample. Tubes placed in a refrigerator for 24 hours then moderate centrifuging for 2 to 3 minutes. Super-natant is removed to a clean tube and any remaining bone particles removed. Tritration according to Boorman & Dodd (1961). A buffered saline of 1% sodium azide is used as preservative. The result is the observation of consistent inhibition over a series of tests. See Smith (1959; 1960; and Glemser, 1963).
The Borgognini Method for the ABH System
There are three methodological improvements to the Boyd and Candela method (Berty, 1964). There is (1) colorimetric reading of the degree of haemoglobin which ensures greater sensitivity; (2) the use of aqueous extracts instead of bone powder as the absorbing medium in haemoagglutination-inhibition test. This reduces or eliminates non-specific absorption; (3) the systematic use of anti-H sera for group O determination.
Since 1964 Borgognini (1967; 1969) introduced 4 modifications to the Berti & Parenti (1964) technique. There followed (1) systematic decalcification of bone powder with disodic and trisodic EDTA solutions at pH 7.0; (2) the use of an electric stirrer to standardise readings; (3) the systematic purification of aqueous extracts from bone powder by gel filtration, though columns. This uses the macromolecular portion only and removes degradation products. Finally (4) occasional removal of lipids from the powder with solvents such as acetone, chloroform or ether.
The Kellerman Method for the ABH System.
Requires 4 to 5 gm of spongiosa ground in a mortar. Extraction of bone powder mixed with 10ml of 5% Na2/Na3 EDTA at pH 6.5-7.0, for 12 hours on a magnetic stirrer at room temperature. Centrifugation for 10 mintes at 5000 rpm and then decantation of the supernatant.
Centrifugation again for 20 minutes at 25,000 rpm. This gives a volume of extract of 6 to 8 ml. Decantation with partial purification gel filtration through Sephadex G.25 in coarse columns of 1.5 x 30 cm. Aqua dest with 0.02% NaN3. Bacterioatatium used as eluting medium. Bring back eluate to original volume, then concentrate by ultrafiltration.
Blood typing is with the Ouchterlyontest and immunophoresis. Using immunophoresis anti-sera is diluted to 1:4. With the Ouchterlyonytest the human anti sera (anti-A; anti-B; and anti-H) is from Ulex europeans. Preparation of anti-sera is using the Borgognini method (1967). All sera are titred at 1:1024 to 1:2048. Test anti-sera with human saliva.
Summary of other researches.
Borgognini (1968) tested ancient bones dated to 2000 BC. !20 tests of 33 skeletons typed 88% or 29 skeletons. Thus the results were 12 x A; 9 x B; 1 x AB; and 7 x O groups. Four noy diagnosed.
Kellerman (1971) tested 169 femora out of which 149 (88.2%) showed a reaction. This meant that the ABH blood group distribution for the period 15th to 17th centuries gave the result: 149 = 36.2 x A; 22.8 x B; 30.9 x H; and 3.5 x AB. From 891 modern specimen samples the results were: A x 50.3; B x 8.8; H x 37.4; AB x 3.5.
Kellerman (1972) tested 151 femora from the 12th to 14th centuries. !26 or 83.4% showed a positive reaction. Specimens numbering 126 = A x 53.9%; B x 16.7%; H x 23.8%; AB x 5.6%.
Appendix 1. Diagram of Methodology.
Appendix 2. Diagram of Factors of Decomposition.
References Cited and Sources Consulted. (some lost over passage of time).
Borgognini. (1969). Proc.XVIII. Int.Cong.Anth & Ethno Sci.
Boyd & Boyd. (1931). J. of. Immun. 26 (6).
Boyd & Boyd. (1933). Science. 78 (578-).
Boyd & Boyd. (1934). Proc. Sci. Exp. Biol. Med. 81 (671-).
Boyd & Boyd. (1937). Blood Grouping in Forensic Medicine. J. Immun.
Boyd & Boyd. (1939). Blood Group Reactions in Preserved Bone and Muscle. Am.J.Phys.Anth. 21 (429-).
Candela. (1936). Blood Group Reactions in Human Skeletons. Am.J.Phys.Anth. 23 (429-).
Candela. (1937). Blood Group Determination upon Skeletal Material. Am.J.Phys.Anth. 23 (71-).
Candela. (1939). Blood Group Determination upon 30 Aleut Mummies. Am.J.Phys.Anth. 24 (36-).
Candela. (1939a). Blood Group Tests on Stains, Mummified Tissues, Bone. Am.J.Phys.Anth. 25.
Candela. (1940). Reliability of Blood Group Tests on Human Bones. Am.J.Phys.Anth.
Garlick, J. D. (1969). Buried Bone – N content and Blood Group Activity. In: Science in Archaelogy ed Brothwell, D.
Glemser (nee Smith). 1963. Science in Archaeology. 1st edition, 1963.
Kellerman. (1971). Humangenetik. (14).
Kellerman. (1972). Humangentik. (14).
Lengyel & Nemeskeri (1963).Application of Biochemical Methods to Biological Reconstruction. Z.Morph.Anthrop. 54 (1).
Lengyel & Nemeskeri. (1965). Investigation of Chemical Composition of Aged Bones. Int.Conf.Gerontogy.
Lengyel. (1967). Biochemical Aspects of Early Skeletons. In: Skeletal Biology. Brothwell, D. ed.
Smith, M. (1959). Blood Grouping of the Remains of Swedenborg. Nature. 184 (867-).
Smith, M. (1960). Blood Groups of the Ancient Dead. Science. 131.
The programme was unable to obtain facilities or a grant to carry out the research. Reasons given in a department of Oxford University were that it was unlikely that blood groups or other genetic material could be obtained from bone or teeth. This despite active interest and cooperation from the Pathology Department, Nuffield Orthopaedic Centre, and Faculty of Medicine, Oxford, and offer of preliminary help from H. M. Forensic Science Department, Harwell, Berkshire. As well as the availability of skeletal material from Oxfordshire Archaeological Services.