The emerging role of viruses in the etiology of human neoplastic diseases has reinforced interest in defining the elusive causative agent in laryngeal papilloma. A rational therapeutic approach might be anticipated should such an agent be identified. Most of the evidence found in the literature at this time points to a viral etiology. The purpose of this study was to examine the ultrastructure of papillomas of children and adults for viral particles, and to identify virus within the cells of the tumor by immunofluorescent techniques. METIIODS AND MATERIALTissues from seven different papilloma specimens obtained from three children and one adult were examined in this study. Two children and the adult had specimens obtained on two separate occasions, All specimens were obtained by surgical excision at direct laryngoscopy.Five serum samples were also examined in an attempt to find circulating antibodies against the tumorgenic virus. Four of the serum samples were from the above patients and one was from a patient who was considered cured of his disease, and from whom no tissue was obtained for study. This patient had been treated surgically on repeated occasions with complete regression of his disease over the past three and one-half years. This patient was included in the study since it was felt there was an excellent chance of finding circulating antibody in his serum.Tissue specimens were examined by light and electron microscopy and by immunofluorescent techniques. Light microscopy was performed after standard paraffin block sectioning technique and staining with hematoxylin and eosin.Electron microscopy was performed after overnight fixation in 3% distilled glutaraldehyde buffered to pH 7.4 with phosphate or cacodylate buffer. Postfixation was for 90 minutes in osmium tetroxide, dehydration in ethanol and embedding in araldite.In the immunofluorescent study, frozen sections of tissues were examined by both single and double plating techniques. Single plating (direct) technique was done with fluorescein-tagged antihuman globulin obtained from rabbits. This was also used for the over-stain in the double plating technique. This serum contained antibodies against human IgG, IgA, and IgM. Portions of normal human pharyngeal mucosa were used as negative controls.The double plating technique (indirect) was done in three ways, with different sera used in the intermediate step: 1) Patient's autologous serum; 2) Homologous and autologous serum pooled from the five samples; and 3) Antihuman globulin serum (Coomb's ) which was not fluorescent tagged. The third method was done to induce blocking of fluorescent staining, and help confirm specificity.Staining was done in the following manner: frozen sections on clean glass slides were washed twice for five minutes each in phosphate buffered pH 7.2 saline at room temperature, then immersed in cold (4 0 C) acetone for 30 seconds, then washed twice more with buffered saline. In the single plating technique, slides were incubated with fluoresFrom the Department of Otolaryngology,...

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