J . Forens. Sci. Soc. (1976), 16, 155

An Investigation into the Possible Sources of Adventitious ABH Substances in Bloodstain Grouping S. S. KIND* and B. G. LANG Home OJce Forensic Science Laboratory, Broadway West, Gosforth, Newcastle upon Tyne, England, N E 3 5HL.

"False Positives" in A B O bloodstain grouping may result from interference from homologous (i.e. human) sources or from adventitious substances such as moulds, bacteria or from other organic and inorganic origins. An ad hoc study is reported in which several possible sources of adventitious substances have been examined insofar as they affect ABO bloodstain grouping by the Absorption-Elution method after ammoniacal extraction. ( A paper presented at the Seventh International Meeting of Forensic Sciences; Zurich, Switzerland, September 1975). Introduction Blood in the circulation is normally sterile with no adventitious substances although a number of cases of acquired group B reactivity has been reported in diseases of the gut, and in carcinoma and leukaemia (reviewed in the forensic aspects by Jenkins et al., 1972). Blood typing of liquid blood samples obtained by aseptic technique thus gives no difficulty. However, the forensic serologist has no control over the condition and cleanliness of garments and other articles submitted to him, and must be aware of the possibility of contamination with ABH reactivity from non-blood sources. ABH blood group activity is found in the body fluids and secretions of cattle, sheep, goats and pigs (Neimann-Sorensen et al., 1954; Joysey, 1959; Cohen, 1962; Tucker, 1962 ; Lang, 1970). Some part of the human group B mosaic is universal in the blood of cattle, pigs, rabbits, guinea pigs, opossums, hamsters, some monkeys (New World) and the gorilla. (Friedenreich and With, 1933; Owen, 1954; Joysey, 1959; Ueno et al., 1959; Moor-Jankowski et al., 1964). This occurrence of ABH substances in non human blood normally does not pose a problem to the forensic scientist because he can apply a simple species identification test, although one must be careful to exclude the possibility of mixed human and non-human blood. The Forsmann antigen (which gives A bloodgroup reactivity) is present in the tissues, body fluids and/or on the erythrocytes of a much wider range of organisms than is normally covered by the species identification test (Schmidt, 1955; Jenkins, 1963 both cited by Prokop and Uhlenbruck, 1969). I n a n investigation of 282 Gram-negative bacterial colonies Springer showed either A, B or H activity in 96 colonies and mixtures of reactivities in a further 41 colonies (Springer et al., 1962). The coating of human and chicken erythrocytes with Gram-negative bacterial group B and H activity has been demonstrated in vitro and to a limited extent in vivo (Springer and Hahn, 1962; Springer and Horton, 1964). Blood group activity has also been reported in some plants, invertebrates and even viruses. Taxus cuspidata and Sassafras albidum show H activity (Springer, 1956; Springer et al., 1965), and the snails Helix pomatia and Helix hortensis have B activity (Prokop et al., 1965). Emulsified toxacara worms inhibit anti A and

* Present address: Home Office Central Research Establishment, Aldermaston, Reading, Berkshire, England RG7 4PN.

anti B (Heiner and Kevy, 1956; Shrand, 1964) and Ascaris lumbricoides shows A activity (Soulsby and Coombs, 1959). B activity has been demonstrated in a myxovirus (Isaacson and Holden, 1962). I n situations of active growth no doubt microorganisms are capable of confering or degrading blood group activity by enzyme action. This is borne out by a recent paper (Pereira, 1973) on the difficulties of grouping decomposing human muscle tissue. There were examples of both loss and addition of antigenic activity and even cases of substitution. The occurrence of A and B activity in house dust is mentioned briefly by Dodd (1968). We report here a n ad hoc study on a series of materials which could possibly occur as contaminants in blood stain grouping. These are house dust, soil, wood and microorganisms.

Materials and Methods The ammoniacal extract method of absorption-elution bloodstain grouping (Kind and Cleevely, 1969) has been used. I n this method the bloodstains are extracted into a volume of 5% v/v of 0.880 ammonia in deionised water for a minimum of 30 minutes before drying down onto triple cavity microscope slides. These are then tested by a normal absorption-elution thecnique usually in duplicate for A, B and H activity. For tests with dust, soil, wood and microbiological colonies several concentrations and amounts of extract were used. Dust samples Thirty household dusts (mainly from vacuum cleaners) were collected by members of the laboratory staff. Twenty of the dust samples were part of a collection made two years previously. Preliminary tests with 5 dust samples were carried out with two sets of extracts both 1% w/v in 5% v/v of 0.880 ammonia solution in deionised water. Series (a) 6mg dust in 0.6ml of ammonia (10mg/ml). Series (b) 50mg dust in 5ml of ammonia (10mg/ml). The remaining 25 dust samples were tested using the series (a) 1% w/v extract. I n addition 15 series (a) extracts were tested at 2% w/v (i.e. double the amount of dust). Each extract was dispensed onto three triple-cavity microscope slides to give dried extracts from 0.72mg, 0.48mg and 0.25mg of dust for slide 1, 2 and 3 for the 1 % extracts. Each quantity of extract was tested with anti A, anti B and anti H (Ulex europaeus) against A,, B and 0 test cells. Soil samples Twenty four soil samples were collected by laboratory staff mainly from suburban gardens. These samples were dried in a 56°C incubator; large pieces of grit, stones and obvious vegetable matter like roots and stalks were picked out and aggregates were broken down by slightly grinding with a pestle and mortar. Two series of extracts were prepared both a t 2% w/v. Series (a) 12mg of soil in 0.6ml of ammonia (20mg/ml) Series (b) lOOmg of soil in 5ml of ammonia (20mg/ml) Slides with dried extracts were prepared as for dust, but the three amounts of extract were from 1.65mg, 1almg and 0.5mg of soil respectively on slides 1, 2 and 3. Of the twenty-four soils tested 10 were tested by both extract series (a) and series (b) and the remaining fourteen by series (b) only. Wood samples Forty-two woods were selected in alphabetical order of genera and species from the reference wood collection (see Appendix 1). A minimum of 600mg of wood was planed off the end grain with a block plane producing a satisfactory quantity of friable shavings and dust. The extracts were prepared by soaking 500mg of wood dust and shavings in 5ml of ammonia at 5OC overnight. After centrifugation the wood free extract was doubling diluted in approximately 0.18ml amounts (with a marked Pasteur pipette) from neat to 1/16 i.e., five 156

different concentrations in doubling steps. Each concentration was pipetted out ( 3 0 ~ 1per cavity) onto triple cavity slides producing five slides for each sample of wood, and these were tested with anti A, anti B and anti H. The reason for diluting the 10% w/v extract is to avoid the possibility of negative results due to antigen excess (see Kind and Cleevely, 1969). The amount of dried extract on the slides 1-5 is extracted from the following quantities of wood in milligrams: 6, 3, 1.5, 0.75 and 0-375. Micro~r~qanism samples Twenty-four blood agar plates (Oxoid) were issued to the laboratory staff for exposure (15 minutes) in the various departments (5 plates) or in their homes (19 plates). These were incubated for periods ranging from 4-9 days a t temperatures varying from 25O-33OC over this period. The resultant colonies were sampled by lifting with a heat sterilised section lifter and spreading onto the surface of Whatman No. 1 filter paper circles. These were air dried at room temperature. A total of 257 colonies was sampled in this way with the exception of colonies 1-15 which were sampled by cutting lmm plugs from the agar plate with a Pasteur pipette of that internal bore. Colonies 16-257 were extracted after periods of storage dry at room temperature ranging from 21-48 days from sampling by cutting 3mm squares of Whatman No. 1 filter paper smeared with colony. These and the plugs from colonies 1-15 were extracted in 10 drops (approx. 0.3ml) of 5% ammonia for a minimum of hour. These extracts were distributed on two 3 cavity slides in the ratio 2 :1. With large fungal type colonies it often proved difficult to sample the crust composed of mycelium free from the partially digested blood agar. In these cases the smeared blood agar was tested with the suffix/l and the crusty mycelium with the normal number e.g. 2211 and 22. Blood agar plates The blood agar plates themselves were examined and no significant amounts of ABH activity under the present conditions of test were found.

+

Results Dust The result of grouping the dust extracts are shown in Table 1. From the table it can be seen that 22 dusts out of 30 reacted of which 11 were single reactions, 7 were double reactions and 4 had all three reactions. A, B and H reactivity was obtained in 13, 12 and 12 samples respectively. Five of the samples were subjected to further tests at different concentrations of dust material i.e., lOmg/ml and 20mg/ml. Table 2 shows that the changing of the concentrations resulted both in the apparent disappearance of antigens originally present and the appearance of antigens originally absent. I n order to clarify these anomalies a third series of extracts Series (c) was prepared for dusts SSK and KJK producing dried extract from the weights of dust indicatcd in Tablc 3. It is clcar from Tablc 3 that different results arise TABLE 1 SAMPLES OF HOUSE DUST SHOWING ABH REACTIVITY Reaction A

B H

No. of Samples Showing Sin,ele Combined 5 AH 2 AB 4 BH ABH

11 -

22 dusts react out of 30

1 3 3 4

11 -

TABLE 2 COMPARISON O F ABH ACTIVITY O F DUST EXTRACTS PREPARED FROM DIFFERENT AMOUNTS O F DUST Amount of Dust Volume of Ammonia Concentration Dusts SSK KJK SD JG GBS

AH ABH H A H

ABH ABH AH A H

AH ABH A A

TABLE 3 ABH GROUPING O F T W O DIFFERENT SAMPLES O F HOUSE DUST EXTRACTED INTO AMMONIA Extract from Dust in mp

SSK Dust Reactivitv

K J K Dust Reactivitv

L=complete haemolysis Bracketed results indicate weak reactions prefix indicates particularly strong reactions A

+

from various weights of any one dust sample. I n addition since the dust samples are heterogeneous in their constitution any random sampling variation in preparing the extracts will also affect the result both quantitatively and qualitatively. Without making a n extensive investigation we determined the blood groups of as many individuals in each household as was conveniently possible and compared them with the reactivity in the house dust. Quite a good correlation is found (Table 4). Soils Of the 24 soils tested only 3 gave any reaction when the extracts were prepared from lOOmg of soil in 5ml of 5% ammonia i.e., 20mg/ml (Table 5 ) . These were all soils which showed some colour in the extract, doubtless caused TABLE 4 BLOOD GROUPS O F T H E OCCUPANTS O F T H E VARIOUS HOUSEHOLDS TOGETHER W I T H T H E REACTIVITY O F T H E DUST COLLECTED FROM T H E HOUSEHOLD Occupants 0 A, B '41 0 0 A2 0 A 0

Reactivity

;(Bg)

no reaction ABH BH

TABLE 5 A COMPARISON O F REACTIONS OBTAINED FROM TWO SERIES O F EXTRACTS O F SOIL IN AMMONIA Series (a) 12mg of soil in 0.6ml of ammonia (20mglml) Reactivitv Single in combination A 1 1 ABH 1 B H I J

1

Series (b) lOOmg of soil in 5ml of ammonia (20mglml) Reactivity Single in combinatzon I ABH I A 2 }BHl B H 1 2 J

by higher humus content. The remaining soils were low in organic content. When only 12mg of soil was extracted in 0.6ml of ammonia only one soil reacted despite the two series of extracts being 20mglml and both having identical amounts of material per cavity. Once again this is a n indication of the heterogeneity of the soil samples. With random sampling variation more positive results are produced by the larger sample in this case. woods Twenty-four out of the 42 woods selected alphabetically showed significant blood group reaction as indicated in Table 6. No A reactivity was found. Microbiological colonies A total of 257 colonies was tested and 80 showed reactions (Table 7). No colony showed combined ABH reactivity but every other reactivity and combination was demonstrated at the level tested. Of the 19 modified blood agar smears tested separately from the crusty colonies producing the liquified agar, TABLE 6 ABH REACTIVITY O F AMMONIACAL EXTRACTS O F 42 DIFFERENT WOODS SELECTED ALPHABETICALLY ACCORDING T O GENERIC NAME Reaction

Single

A

-

combined

-

TABLE 7 ABH REACTIONS FROM AMMONIACAL EXTRACTS O F MICROBIAL COLONIES DEVEI.OPED O N BLOOD AGAR SEEDED BY ATMOSPHERIC CONTAMINATION IN VARIOUS LOCATIONS Reaction A B H

No. of Colonies Showing Single 47 14 11 -

Combined AB 4 AH 1 BH 3 -

72

8

-

-

80 Colonies reacted out of 257

13 showed some reactivity not present in the original agar (Table 8). Only two of the colonies themselves showed reactivity--one H the other A. The modified agar from these showed AH and A respectively. 159

TABLE 8 ABH REACTIONS FROM AMMONIACAL EXTRACTS O F BLOOD AGAR MODIFIED BY MICROBIOLOGICAL ACTION

Reaction A B

Blood A ~ a r Single ~Abined 8 AB 2 AH 3

Colonies Single 1 -

13 modified agars reacted out of 19

Discussion Blood stains may occur on wood, or mixed with house dust, or on soil or contaminated with bacteria. Our findings demonstrate clearly the necessity for forensic scientists to be on guard against possible adventitious contamination. Generally speaking the amount of source materials in this study were more, sometimes substantially more than we would use in blood group determinations on stains. When blood stains occur on any substratum then meticulous control testing must be carried out. Bacterial contaminated samples (e.g. caused by slow drying) are unreliable for grouping purposes. References COHEN,C., 1962, Editor, Blood Groups in Infrahuman Species, Ann. N.Y. Acad. Sci., 97, 1, 1-328. DODD,BARBARA E., 1968, in Clinical Aspects of Immunology. Ed. by R. P. G. Gel1 and R. R. A. Coombs, Blackwell Scientific Publ. FRIEDENREICH, V. and WITH,S., 1933, Zschr. Immunit. forsch., 78, 152. HEINER, D. C. and KEVY,S. V., 1956, New Engl. J. Med., 254, 129. P. and HOLDEN, D. M., 1962, Virology, 17, 494. ISAACSON, JENKINS, C. R., 1963, Advances Immunol., 3, 351. J., LINCOLN, P. J. and DODD,BARBARA E., 1972, JENKINS,G. C., BROWN, J. Forens. Sci., Soc. 12, 597. JOYSEY, VALERIE C., 1959, Brit. Med. Bull., 15, 2, 158-164. KIND,S. S. and CLEEVELY, ROSALYN M., 1969, J. Forens. Sci. Soc., 9, 131-1 34. LANG,B. G., 1970, Vox. Sang., 18, 257-264. MOOR-JANKOWSKI, J., WIENER, A. S. and ROGERS, C. M., 1964. Nature, 202,663 NEIMANN-SBRENSEN, A., RENDEL, J. and STONE,W. H., 1954, J. Immun., 73, 407-414. OWEN,R. D., 1954, J. Immun., 73, 29. PEREIRA, MARGARET, 1973, J. Forens. Sci. Soc., 13, 33. A. and SCHLESINGER, D., 1965, S. Afr. J. Forens. Med., PROKOP, O., RACKWITZ, 12. 3. 108. P R O ~ O0 ~. ,and UHLENBRUCK, G., 1969, Human blood and Serum Groups. Maclaren and Sons, London. H., 1955, Fortschritte der Serologie, Stein Kopff Darmstadt. SCHMIDT, SHRAND, H., 1964, Lancet, 1, 1367. E. J. L., and COOMBS, R. R. A., 1959, Parasitology, 49, 505. SOULSBY, SPRINGER, G. F., 1956, J. Immun., 76, 1, 399. J., 1962. Proc. Congress int Soc. SPRINGER, G. F., and ANSELL-HAHN, NORMA, Blood Transf. Tokyo 1960, 219-221. SPRINGER, G. F. and HORTON, R. E., 1964, J. Gen. Physiol., 47, 1229. G. F., TAKAHASHI, T., DESAI,P. R. and KOLECKI,B. J., 1965, SPRINGER, Biochem., 4, 10, 2099. P. and READLER, BARBARA L., 1962, Ann. N.T. SPRINGER, G. F., WILLIAMSON, Acad. Sci., 97, 1,104. TUCKER, ELIZABETH M., 1962, Vox. San.~., - . 7,- 239-241. UENO,S., MATSUZAWA, S., KITAMURA, S. and MISHIMA, H., 1959, J. Immun., 82, 385.

Appendix 1 Woo& tested for Blood Group Acliuity 1. Acacia melanoxylon 2. Acanthopanax ricinifolius 3. Acerplatanoides 4. Acer pseudoplatanus 5. Acer saccharum 6. Acer Saccharum 7. Adina cordifolia 8. Aesculus hippocastanum 9. Afromorsia sp. 10. Afzelia sp. 1 1. Agathis alba 12. Alnus glutinosa 13. Amoora sp. 14. Anisoptera sp. 15. Araucaria augustifolia 16. Astronium sp. 17. Aucoumea Klaineana 18. Backhousia bancroftii 19. Brachystegia sp. 20. Calophyllum brasilense 2 1. Calophyllum sp. 22. Carapa guranensis 23. Cedrus libani 24. Eucryphia cordifolia 25. Fagus sylvatica 26. Fagus sylvatica 27. Grevillea robusta 28. Gossweilerodendron balsamiferum 29. Gonystylus warburgianus 30. Gonystylus sp. 3 1. Fraxinus excelsior 32. Fagus sp. 33. Guarea cedrata 34. Guarea thompsonii 35. Hura crepitans 36. Juglans negis 3 7. Khaya ivorensis 38. Koompassia malaccensis 39. Larix decidua 40. Laurelia aromatics 4 1. Liquidamber styrac$ua 42. Lophira alata var. procera

Australian Blackwood SEN Norway Maple Sycamore Birds Eye Maple Rock Maple Kanluang Horse Chestnut Kokrodus Afzelia Borneo Kauri Alder Tasua Mersawa Parana Pine Zebra Wood Gaboon Johnstone River Hardwood Okwen .Jacareuba Bintangor Crabwood Cedar ULMO European Beech French Beech Silky Oak AGBA Melawis Ramin European Ash Japanese Beech Guarea Black Guarea Hura European Walnut African Mahogany Kempas European Larch Chilian Laurel American Red Gum (Sap Gum) Ekki