Additional comments on blood residue and analysis in archaeology.

Leach, Jeff D. ; Mauldin, Raymond P.

In the March Antiquity, Eisele et al. (1995) presented immunological results from prehistoric sites, and from simulated archaeological contexts, which suggest that residues will not survive on prehistoric stone tools. Over the last several years, we have submitted over 200 archaeological items to a commercial laboratory for immunological residue analysis. In an effort to evaluate the reliability of these archaeological results, we initiated an actualistic test using experimentally generated artefacts coated with blood of known animals. Here, we present results for n of these experimental samples. Our results also suggest that archaeologists should view immunological results with caution.

The 54 experimental samples reflect a variety of artefact types and materials encountered archaeologically. Using 10 species of animals collected from recent road kills and commercial butchers, we coated freshly knapped artefacts with blood by cutting and scrapping a specimen for several minutes. After allowing the artefacts to dry, we removed visible hairs from several of the specimens by wiping them with a cotton cloth or lightly rubbing them with sand. However, in almost all cases blood was still visible macroscopically, and it is our opinion that this procedure did not remove the blood from any specimens. Several `blank' lithic tools and sediment samples, along with sediment and fire-cracked rock (FCR) samples from an experimental hearth used to cook a rabbit, were also submitted for analysis.

All experimental samples were sent to a commercial laboratory that uses a gel immunoelectrophoresis technique, crossover immunoelectrophoresis (CIEP). Complete details of this technique, and its application to archaeological materials, are presented by Newman & Julig (1989). The experimental samples were tested against a series of polyclonal antiserum, including those for bovine, chicken, deer, dog, elk, guinea-pig, human, pronghorn, rabbit, rat, turkey, beans, corn, mesquite, pinyon, squash and yucca.

Results, reported by the laboratory (Table 1) fall into five groups: * the laboratory correctly identified the blood of the animal, or did not report any blood on a blank sample., * the laboratory correctly identified the blood of the primary animal, but also noted the blood of other animals., * the animal was correctly identified, but not as the primary blood on the item., the laboratory reported the blood of the wrong animal; * no blood was detected though blood was present.

Table 1. Experimental animals and results.

 artefact experimental laboratory
 type residue results

1 lithic tool human human
2 lithic tool elk deer, elk, human
3 lithic tool elk deer, elk
4 lithic tool elk deer, elk, human
5 lithic tool elk human, deer, elk
6 lithic tool squirrel negative
7 lithic tool squirrel negative
8 lithic tool mouse deer, elk
9 lithic tool mouse human
10 lithic tool coyote negative
11 lithic tool coyote negative
12 lithic tool bobcat negative
13 lithic tool bobcat negative
14 lithic tool bobcat negative
15 lithic tool deer deer, elk
16 lithic tool deer deer, elk, human
17 lithic tool deer deer, elk
18 lithic tool blank negative
19 lithic tool blank negative
20 lithic tool lank human
21 lithic tool blank negative
22 lithic tool blank negative
23 lithic tool blank negative
24 lithic tool blank negative
25 lithic tool cow bovine, elk
26 lithic tool cow bovine
27 lithic tool turkey negative
28 lithic tool turkey chicken
29 lithic tool turkey chicken
30 lithic tool rabbit human, rabbit
31 lithic tool rabbit rabbit
32 lithic tool rabbit rabbit
33 lithic tool rabbit rabbit
34 lithic tool rabbit rabbit
35 lithic tool rabbit rabbit
36 lithic tool rabbit rabbit, mouse
37 lithic tool rabbit turkey
38 lithic tool rabbit rabbit
39 lithic tool rabbit rabbit
40 lithic tool rabbit rabbit
41 lithic tool rabbit rabbit
42 lithic tool rabbit rabbit
43 lithic tool rabbit rabbit
44 lithic tool(1) rabbit negative
45 lithic tool(2) rabbit negative
46 lithic tool(2) rabbit negative
47 lithic tool(2) rabbit negative
48 lithic tool(2) rabbit negative
49 sediment blank negative
50 sediment(3) rabbit negative
51 sediment(3) rabbit negative
52 sediment(3) rabbit negative
53 fire-cracked rabbit negative
 rock(3)
54 fire-cracked rabbit negative
 rock(3)

1 Artefact heated to 250 [degrees]C for 30 minutes.
2 The samples are resubmitted after a 5-month period.
3 Sample recovered from an experimental hearth.

Of the five groups, we consider groups 1 and 2 to be successful: blood was correctly identified as either the only (Group 1) or the strongest (Group 2) reaction. Group 3,4 and 5 results are problematic. Group 1 results were obtained from thirteen samples and Group 2 results from seven samples. Group 3 results were obtained from five samples, Group,4 results from four samples, and Group 5 results from the remaining 25 samples. The results of these experiments are disheartening as the laboratory correctly identified only 20 of the 54 samples (37%).

Many of the misidentified samples are cross-reactions. As discussed by Nolin et al.(1994; see also Newman & Julig 1989), cross-reactions between closely related species (e.g. deer, elk, moose) within a Family (e.g. Cervidae) are not unexpected with the use of commercially purchased polyclonal sera. However, several of our results represent cross-reactions beyond the Family level: an artefact coated with elk blood (Sample 5) reacted strongest with anti-human serum and an artefact coated with mouse blood (Sample 8) strongly with anti-deer and anti-elk serum (see also Child & Pollard 1992). All of the samples exposed to heat failed to produce reactions to any antiserum, supporting the suggestion by Cattaneo et al. (1994) that excessive heat may render any residue immunologically undetectable. Yet, even eliminating those heated cases from TABLE 1, the overall success, rate is still below 50%.

Referees for Antiquity had many questions to ask about the immunological method used to analyse our experimental samples. For example, Had some of the antisera lost their original activity? Was the method standardized and controlled for the material being tested?' Such questions must be addressed by the commercial laboratory that conducted the analysis. As archaeologists submitting either experimental or prehistoric samples for analysis, we can only assume that appropriate laboratory procedures are in place. Clearly, the results presented in TABLE 1 indicate that additional experimental work is needed, including a focus on laboratory procedures and appropriate immunological techniques. (since this paper was submitted, we have sent additional samples for analysis, with results that show the same patterns.)

While laboratory procedures can be questioned, and inter-laboratory procedures vary, the discrepancies revealed by our experiment may reflect more than laboratory or procedural problems. As Sensabaugh et al. (1971; see also Eisele et al. 1995) have pointed out, the biological activity of proteins may be rapidly lost soon after death of the organism. In our study, the time-lapse between coating of the samples with blood and subsequent testing was generally 1-2 months. During this time period the artefacts were sealed in plastic bags and refrigerated. Eisele et al. (1995; see also Cattaneo et al. 1993; Gurfinkel & Franklin 1988) demonstrate that degradation of blood proteins may occur over this short length of time. As such, any dependence on immunological results, especially at time-scales commonly used by archaeologists, may be problematic.

Acknowledgements. This research is funded by the Legacy Resource Program, Department of the Army. Ms Sue Green-Leach and three Antiquity reviewers provided useful comments on a previous version of this paper.

References

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