A method of distinguishing between amalgam and graphite in tissue Edmund Peters, D.D.S., M.Sc.,* and David G. Gardner, D.D.S., M.S.D.,** Johannesburg, South Africa, and Houston, Texas

UNIVERSITY OF THE WITWATERSRAND. SOUTH AFRICAN INSTITUTE FOR MEDICAL RESEARCH, AND UNIVERSITY OF TEXAS HEALTH SCIENCE CENTER AT HOUSTON

The clinical and histologic appearance of amalgam and graphite tattoos are similar; both appear as dark particles, although amalgam, after degradation, displays a characteristic binding to blood vessels, nerves, basement membrane, and fibers. Graphite particles also consistently exhibit a light yellow peripheral birefringence under strong polarized light. Amalgam particles may exhibit a similar birefringence, but often it is weak or absent. When histologic sections containing particles exhibiting birefringence are treated with 10% ammonium sulfide solution, graphite is unaltered, whereas the birefringence of amalgam particles, if present, is changed to orange, or the particles undergo dissolution. (ORAL SURC. ORAL MED. ORAL PATHOL. 62173-76, 1986)

T he clinical and histologic appearance of amalgam tattoo and lesions caused by the implantation of graphite in the form of pencil lead are similar. This study compares the appearance of graphite and amalgam particles in tissue and describes a method of distinguishing between them.

MATERIALS AND METHODS Experimental investigation

Crushed particles of freshly prepared amalgam (Dispersalloy, Johnson & Johnson Dental Products Co., East Windsor, NJ) and of graphite from lead pencil were implanted into two 150 gm male Wistar rats. These procedures were performed after admin- istration of general anesthetics. The materials were placed subcutaneously over each haunch and into the buccal mucosa by injection with a 2 l-gauge spinal tap needle. For subsequent identification, the amal- gam was always placed on the right side of the animals, the graphite on the left. The rats were killed after 21 days and the area around each of the twelve implants was harvested.

The specimens were fixed in buffered formalin, proeessed for routine histologic examination, and stained with hematoxylin and eosin. They were then studied by conventional and polarized light micros-

*Lecturer, Department of Oral Pathology, University of the

Witwatersrand. **Professor and Chairman, Department of Pathology and Radiol- ogy, University of Texas Health Science Center at Houston.

Dental Branch.

copy. An attempt was made to distinguish between the two materials by assessing the difference in their peripheral birefringence, since birefringence is a feature of graphite particles from lead pencil in tissue sections.‘-3 The sections were then reexamined by polarizing microscopy after reacting the sections with 10% ammonium sulfide solution for l- and 8-hour intervals.

Retrospective study of biopsy specimens

Ten recent cases diagnosed as amalgam tattoo in the files of the Oral Pathology Diagnostic Service of the University of Western Ontario and the Universi- ty of the Witwatersrand were similarly studied.

RESULTS Experimental investigation

Histologic examination of sections of the amalgam and graphite specimens revealed similar, irregular- shaped, black-brown particles of varying sizes within the connective tissue. No selective binding of the particles to basement membrane, blood vessels, fibers, or nerves, as often occurs in clinical amalgam tattoos, was noted in either case. There was no difference between the histologic appearance of the material implanted subcutaneously and that implanted in the buccal mucosa.

Both materials exhibited a light yellow peripheral birefringence when examined under strong polarized light (Figs. 1 and 2). The particles of each material remained indistinguishable by conventional light

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74 Peters and Gardner Oral Slug. July, 1986

Fig. 1. A, Graphite particles implanted into the subcutis of a rat for 21 days. They appear as black, irregular-shaped particles with no selective binding to connective tissue structures. (Hematoxylin and eosin stain. Magnification, x 117.) B, Same section under strong polarized light. Particles exhibit light yellow peripheral birefringence.

Fig. 2. Amalgam particles implanted into the subcutis of a rat for 21 days under conventional (A) and polarized (B) light. Histologic appearance is similar to that in Fig. 1, A and B. Amalgam cannot be distinguished from graphite. (Hematoxylin and eosin stain. Magnification, X 117.)

microscopy after the sections were treated with ammonium sulfide. However, the amalgam particles exhibited a strong peripheral orange birefringence under polarized light, whereas the graphite particles continued to exhibit light yellow birefringence. This change in the amalgam sections was apparent after both the l- and &hour periods. Using this criterion, another oral pathologist was able to identify reliably the amalgam and graphite particles.

Retrospective study of biopsy specimens

the selective attachment of fine dark granules to basement membranes, nerves, or connective tissue fibers; birefringence in these sections was an uncom- mon feature. Two cases without this selective stain- ing which exhibited a strong peripheral birefringence were positively established as amalgam tattoos when an alteration in the color of the birefringence from light yellow to orange was observed after a reaction with ammonium sulfide. The best results were noted after the 8-hour interval. A further unexpected finding in all the cases of amalgam tattoo was the

Seven of the ten cases previously diagnosed as dissolution of many of the particles to form semi- amalgam tattoo could easily be confirmed as such by translucent, stippled structures. This latter finding

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Method of distinguishing between amalgam and graphite 75

Fig. 3. Clinical case of amalgam tattoo after reaction with ammonium sulfide for 1 hour. Arrows indicate semitranslucent remnants of amalgam particles. (Hematoxylin and eosin stain. Magnification, ~357.)

was partially evident after 1 hour and became a marked feature after 8 hours (Fig. 3).

One case involved a palatal lesion from a 55 year-old woman. The lesion exhibited no selective binding of connective tissue structures. Moreover, the particles exhibited the constant peripheral bire- fringence characteristic of graphite. Because no physical alteration or change in the color of the birefringence occurred after treatment with ammo- nium sulfide, the lesion was rediagnosed as graphite tattoo (Fig 4).

DISCUSSION

Graphite is a crystalline form of carbon that occurs naturally and can also be made synthetically by the carbonization of oi1.4 It has been used in pencils since 1564.’ Traumatic implantation of graphite in the oral cavity is probably more common than is apparent from the literature. The clinical and histologic similarities of these lesions to amalgam tattoos may result in their lack of recognition. They are mentioned in some oral pathology textbooks,5,6 but no articles on the subject appear to have been published. A clinically similar cutaneous lesion caused by the implantation of pencil lead has been reported by Mehregan and Faghri,7 and there are scattered reports concerning traumatic implantation of graphite in other anatomic sites.‘,3.8.9 Graphite particles in tissue are irregularly shaped, dark brown, and opaque; they invariably display a light yellow peripheral birefringence under strong polarized light. Unlike silver particles of amalgam, they do not exhibit selective binding to vessels, nerves, collagen fibers, or basement membranes.

This selective binding is considered reliable evi-

dence for the diagnosis of amalgam tattoo in the oral cavity. It occurs after tin and mercury are lost from the implanted amalgam particles; the resultant disso- ciated granules, which contain silver and sulfur, subsequently attach to the various connective tissue elements,‘O much like a reticulin stain. The lack of selective binding in the experimental sections was probably the result of the relatively short period of implantation and the consequent lack of degrada- tion.

This study illustrates that a focal area of dark particles that are neither melanin nor hemosiderin cannot be assumed to be amalgam when the charac- teristic selective binding of the particles is absent. A graphite tattoo resulting from pencil injury should also be considered. However, the peripheral birefrin- gence of graphite particles is not reliable for distin- guishing between the two materials because amal- gam may also be birefringent, particularly if it has been implanted recently. In the latter case, the characteristic selective binding of the particles to connective tissue structures is often not present. Reaction with ammonium sulfide will confirm the presence of amalgam by altering the color of the birefringence or by changing the dark particles to granular semitranslucent structures. Graphite is unchanged by this procedure.

This work was performed in part while we were associ- ated with the University of Western Ontario, London, Ontario, Canada. We thank J. Crooks for her technical assistance.

REFERENCES

I. Johnson FB: Identification of graphite in tissue sections. Arch Path01 Lab Med 104: 491-492, 1980.

76 Peters and Gardner Oral Surg. July, 1986

Fig. 4. A, Palatal lesion from a 59-year-old woman. There are black, irregular-shaped particles present within the connective tissue. They exhibit no selective binding to connective tissue structures. (Hematoxylin and eosin stain. Magnification, ~74.) B, Higher power magnification of an area from Fig. 4, A. C, Particles exhibit light yellow peripheral birefringence under polarized light. Color of the birefringence was not altered by overnight treatment with ammonium sulfide, indicating that the particles were graphite. (Hematoxylin and eosin stain, treated with ammonium sulfide. Magnification, ~228.)

2. Johnson FB: Crystals in pathologic specimens. Pathol Annu 7: 321-344, 1972.

3. Johnson FB: Case for diagnosis. Milit Med 142: 93, 221. 1977.

4. Town JD: Pseudoasbestos bodies and asteroid giant cells in a patient with graphite pneumoconiosis. Can Med Assoc J 98: 100-104, 1968.

5. McCarthy PL, Shklar G: Diseases of the oral mucosa, ed. 2, Philadelphia, 1980, Lea & Febiger, p. 374.

6. Eversole LR: Clinical outline of oral pathology: Diagnosis and Treatment, ed. 2. Philadelphia, 1984, Lea & Febiger, p. 44.

7. Mehregan AH, Faghri B: Implantation dermatoses. Acta Derm Venereol (Stockh) 54: 61-64, 1974.

8. Foy P, Sharr M: Cerebral abscesses in children after pencil- tip injuries. Lancet 2: 662-663, 1980.

9. Sinha SN: Foreign body (lead pencil) in the maxillary sinus. J Laryngol Otol 82: 473-476, 1968.

IO. Elcy BM: Tissue reactions to implanted dental amalgam, including assessment by energy dispersive x-ray microanaly- sib. J Pathol 138: 251-272. 1982.

Keprinr reqwsts lo.

Dr. E. Peters Dcpartmcnl of Oral Pathology University of the Witwatersrand I Jan Smuts Avenue Johannesburg 2001 1 South Africa