Immunoperoxidase technique in diagnosis of oral pemphigus vulgaris: An alternative method to immunofluorescence

Short communications & case reports Immunoperoxidase technique in diagnosis of oral pemphigus vulgaris: An alternative method to immunofluorescence Janice P. Handlers, D.D.S.,* Raymond J. Melrose, D.D.S.,* Albert M. Abrams, D.D.S., iU.S.,* and Clive R. Taylor, M.A., M.B., B.Chir., D.Phil.,** Los Angeles, Cal$ SCHOOL OF DENTISTRY, UNIVERSITY COUNTY/UNIVERSITY OF SOUTHERN

OF SOUTHERN CALIFORNIA, AND CALIFORNIA MEDICAL CENTER

LOS ANGELES

Five cases of oral pemphigus vulgaris, diagnosed on the basis of clinical, histopathologic, and immunopathologic criteria, were selected. Sections were stained with the peroxidase antiperoxidase (PAP) technique of immunocytochemical staining. The findings correlated well with those reported by direct immunofluorescence demonstrating the same intercellular substance (ICS) pattern of staining. Because this staining technique is applied to routinely processed and paraffinized tissue, it may be of particular value to the oral pathologist who often receives formalin-fixed material before the need for immunopathologic studies is recognized. In addition, interpretation does not require specialized microscopy and tissue sections can be stored and retrieved for retrospective study.

P

emphigus vulgaris is a potentially fatal disease involving the skin and oral mucosa. The oral mucosa is often the first site of involvement in more than 50 percent of patients.lm5The interval between onset of oral lesions and cutaneous lesions averages 4.5 months, although the disease may remain localized without extraoral manifestations for years. Although many casesof pemphigus vulgaris may be diagnosed by light microscopy alone, the importance of both direct and indirect immunofluorescence in the diagnosis of pemphigus vulgaris has been well established.6*g-‘2These methods, however, have several inherent disadvantages.* Both methods

Funded by Biomedical Research Support Grant S07RR05303-19 (4-l-80 to 3-31-81) and by the University of Southern California School of Dentistry. *Section of Pathology, School of Dentistry, University of Southern California. **Department of Pathology, Los Angeles County/University of Southern California Medical Center. 0030-4220/82/080207+06$00.60/0@

1982

The C.V.

Mosby Co.

require specialized microscopy for the examination of stained preparations. Preparations stained by means of fluorescein-labeled antibody are not permanent. Immunofluorescence procedures are generally performed on cryostat sections with resulting poor morphology. In addition, assessmentof morphology by routine staining is not possible, as the light wavelengths and optics used for examination of flourescein-labeled preparations are not compatible with the usual histologic stains. The immunofluorescence methods generally require fresh or specially processedtissue which limits the application of the method for the diagnostic pathologist, who is usually in receipt of tissues that have been formalin fixed (and often paraffin embedded) when the need for immunologic studies becomes apparent. This may necessitate subjecting the patient to additional biopSY. Circulating pemphigus antibodies are demonstrated in indirect serum immunofluorescence and can be demonstrated in all forms of pemphigus where 207

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bazole) and hydrogen peroxide. (See Fig. 1 for illustration of this procedure.) This technique offers several advantages. Sections can be visualized by light microscopy as well as stored and retrieved for retrospective study. Morphology can be evaluated by traditional criteria, as hematoxylin is the counter stain of choice. In addition, the method can be applied to electron microscopic examination. MATERIALS

AND METHODS

Five cases of pemphigus vulgaris, diagnosed on the basis of clinical, histopathologic, and immunopathologic criteria, were selected from the files of the 0 University of Southern California School of Dentistry and the Los Angeles County/USC Medical Cen0 A 0 PEROXIDASE RABBIT ter. Care was taken that the biopsy specimens were TERTIARY not from the gingiva in light of the report by ANTI-PEROXIDASE t ANTISERUM COMPLEX Nisengard and co-workers6 that these may give rise to false positive results of intercellular antibodies by immunofluorescent study. At the time of initial evaluation, the two cases studied for circulating antibodies reacting with intercellular substance (ICS) were positive. Direct immunofluorescence of lesional tissue for IgG showed ICS deposition in all three of the cases tested. Fig. 1. Diagram of the peroxidase antiperoxidase (PAP) Paraffin sections, fixed in ten percent buffered technique of immunocytochemical staining. The human neutral formalin, were cut at 6 microns. These were tissue antigen X is bound to rabbit specific antihuman X. mounted by hot water bath and allowed to dry in air. Excess swine antirabbit IgG is then used to link the primary antibody to the peroxidase rabbit antiperoxidase The slides were then incubated for 2 to 3 hours at 55 complex. The sites of peroxidase localization are visualized to 60°C. in order to facilitate adherence. Sections of by use of the chromatogenic hydrogen donor (amino tonsillar tissue were also processed as a positive ethylcarbazole) and hydrogen peroxide. The product is control. seen as a red granular deposit. Slides were stained with the PAP method using the DAK0 PAP (K505) kit for IgA, IgG, and IgM. Only slight modifications were made in the procethe disease is active. However, negative results have dural instructions as supplied by DAKO. The slides been obtained in patients with inactive disease or in were first deparaffinized and rehydrated as follows: early stages of disease, with only a few lesions.6*‘3 xylene, 5 minutes; absolute alcohol, 3 minutes two Also, indirect immunofluorescent staining requires times; 95 percent alcohol, 3 minutes two times; and careful attention to tissue antigen, patient’s sera, quick rinse in tap water. conjugate characteristics, and interpretations of The slides were then treated with a hydrogen results.6 Many of the above problems can be avoided peroxide solution to destroy endogenous peroxidase with the use of the peroxidase antiperoxidase (PAP) activity that might be present. A 30-minute H,O,technique applied to formalin-fixed, paraffinized methanol bath composed of 50 ml. of three percent tissue. H,O, in 200 ml. of absolute methanol was used in The PAP technique of immunocytochemical stainlieu of the direct application suggested by DAKO. ing was developed by Sternberger and co-workers.7 A This helped to reduce tissue disruption and loss of more recent review of the technique has been preadherence. This was followed by a 5-minute bath in pared by Taylor.8 Unlabeled antibodies are used to 0.05M TRIS buffer, pH 7.6. link horseradish peroxidase to a tissue antigen. The The slides were then incubated with normal swine peroxidase can then be visualized by use of chroserum to suppress nonspecific binding of immunomatogenic hydrogen donor (that is, aminoethylcarglobulins. Next, the rabbit antihuman IgG, IgA, and

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Fig. 2. There are intraepithelial clefts with the basal cells remaining attached to the lamina propria. The immunoperoxidase PAP technique demonstratesthe IgG antibodies bound to the intercellular substanceas a granular deposit. (Original magnification, X100.)

Fig. 3. Higher-power view of the intercellular substancepattern of staining in an area where characteristic features of pemphigus vulgaris by light microscopy are not present. (Original magnification, X250.)

IgM antisera were applied to separate sections from each case and allowed to incubate for 20 minutes. The slides were then rinsed gently and placed in a TRIS buffer wash for 5 minutes (in lieu of the 20 minutes suggested by DAKO). The swine antirabbit immunoglobulin (“link”

antibody) was applied to bind any primary rztbbit antibody remaining on the section. A 20-mi nute incubation was allowed. The sections were a.gain washed for 5 minutes and the PAP reagent was applied. The DAK0 antiperoxidase is also a ri tbbit immunoglobulin which will bind to the free bin(ding

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4. This demonstrates the granular nature of the final reaction product. (Original magnification, X400.)

Fig.

Fig.

5. Positive staining with rabbit anti-IgM. (Original magnification, X250.)

side of any link antibody remaining on the section. Incubation was done for 20 minutes and the slides were again washed in TRIS buffer for 5 minutes. The aminoethylcarbazole substrate was then applied for 40 minutes. the slides were gently rinsed and then counterstained with Mayers hematoxylin. The coverslips were mounted with liquid glycerol gelatin. Tonsillar tissue was used for positive control tissue. Negative controls were accomplished by omit-

ting the primary antibody and substituting normal swine serum. Staining of a nonspecific mucositis case with no clinical or histopathologic features suggesting pemphigus was also done as a negative tissue control. RESULTS

The immunoperoxidase staining of the five cases showed the same ICS pattern observed with direct immunofluorescence (Figs. 2 to 4). (Negative con-

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Fig. 6. A, Demonstration of positive staining of IgG antibodies to the intercellular substance.B, The same tissue section in which the primary antiserum was deleted and normal swine serum used in its place producing a negative control. (Original magnification, X250.)

trols stained negative [Fig. 6, A and B].) All five casesdemonstrated ICS deposition with IgG. Three of the five cases showed positive staining with IgM (Fig. 5) and one other case was equivocal. One case showed questionable positivity with IgA. DISCUSSION

The findings on immunoperoxidase study correlate well with those reported by direct immunofluorescence.6*9* lo*‘* However, it was interesting that three of the five casesshowed definitive staining with

IgM. Only four of the fifty-eight cases studied by Laskaris,‘* using direct immunofluorescence, showed positivity with IgM. We suspectthat IgM is probably always present as a small portion of the pemphigus antibody but that immunofluorescence is not sensitive enough to detect it. Becauseof its larger molecular size, IgM may be able to resist deterioration by formalin fixation enabling the more sensitive immunoperoxidase staining technique to elucidate its presence. The immunoperoxidase technique proved to be a

212 Handlers et al. viable alternative to the use of direct immunofluorescence. It may be of particular value to the oral pathologist, who is more likely to be dealing with patients whose diseasei‘sconfined to the oral mucosa and/or is minimal or inactive. It has been suggested that the detection of circulating pemphigus antibodies in these casesmay be diflicult.6, 12.13 Another advantage to the oral pathologist is that oral biopsy specimens are often small and distorted. Intact oral blisters are often not available for study and the characteristic features of acantholysis and intraepithelial clefts may not be present. The immunoperoxidase staining technique makes possible the detection of the bound antibodies, even in these areas (Fig. 3), permitting the diagnosis of pemphigus vulgaris to be made in the absenceof characteristic microscopic features. The PAP technique may also be applied to formalin-fixed tissue, prevents the need for repeat biopsy, does not require specialized microscopy, and can be stored and retrieved for retrospective studies. We realize that this article deals only with the diagnosis of pemphigus vulgaris and further study of a larger number of casesis necessary.However, this is only one of the many areas in which immunoperoxidase staining could play a significant role in both diagnosis and research of oral disease. The authors would like to thank Mr. Ray Russell and Ms. Cathy Cooper for their technical assistance. REFERENCES 1. Lever, W. F.: Pemphigus and Pemphigoid, Springfield, Ill., 1965, Charles C Thomas, Publisher, pp. 16-39. 2. Ryan, J. G.: Pemphigus: A 20-Year Survey of Experience with 70 Cases, Arch. Dermatol. 104: 14-20, 1971. 3. Rosenberg, F. R., Sanders, S., and Nelson, C. T.: Pemphigus: A 20-Year Review of 107 Patients Treated With Corticosteroids, Arch Dermatol. 112: 962-970, 1976.

Oral Surg. August, 1982 4. Pisanti, S., Sharov, Y., Kaufman, E., and Posner, L. N.: Pemphigus Vulgaris: Incidence in Jews of Different Ethnic Groups, According to Age, Sex, and Initial Lesions, ORAL SURG. 38: 382-387, 1974. 5. Zegarelli, D. J., and Zegarelli, E. V.: Intraoral Pemphigus Vulgaris, ORAL SURG. 44: 384-393, 1977. 6. Nisengard, R. J., Jablonska, S., Beutner, E. H., Shu, S., Chorzelski. T. P.. Jarzabek. M.. and Blaszczvk. M.: Diaenostic Importance of 1mmunoAuo;escence in &al Bullous-Disease and Lupus Erythematous, ORAL SURG. 40: 365-375, 1975. 7. Sternberger, L. A., Hardy, P. H., Cuculis, J. J., and Meyer, H. G.: The Unlabeled Antibody-Enzyme Method of Immunohistochemistry. Preparation of Properties of Soluble AntigenAntibody Complex (Horseradish Peroxidase-Antihorseradish Peroxidase) and Its Use in Identification of Spirochetes, J. Histochem. Cytochem. 18: 315-333, 1970. 8. Taylor, C. R.: Immunoperoxidase Techniques: Practical and Theoretical Aspects, Arch. Pathol. Lab. Med. 102: 113- 12 1, 1978. 9. Hasler, J. F.: The Role of Immunofluorescence in the Diagnosis of Oral Vesiculobullous Disorders, ORAL SURG. 33: 362-374, 1972. 10. Gilmore, H. K.: Early Detection of Pemphigus Vulgaris, ORAL SURG. 46: 641-644,

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1 I. Laskaris, G., Papanicolaaou, S., and Angelopoulos, A.: Immunofluorescent Study of Cytologic Smears in Oral Pemphigus Vulgaris: A Simple Diagnostic Technique, ORAL SURG 51: 531-534,

1981.

12. Laskaris, G.: Oral Pemphigus Vulgaris: An Immunofluorescent Study of Fifty-Eight Cases, ORAL SURG. 51: 626-631, 1981. 13. Jablonska S., Chorzelski, T. P., Beutner, E. H., Michel, B., Cormane, R. t-l., Holubar, K., Bean, S. F., Blaszczyk, M., Ploem, J., and Saiki, N. K.: Utilization of Immunofluorescence in the Diagnosis of Bullous Disease, Lupus Erythematosus, and Certain Other Dermatoses, Int. J. Dermatol 14: 83-100, 1975.

Reprint requests to: Dr. Janice P. Handlers Department of Pathology School of Dentistry University of Southern California 925 W. 34th St. Los Angeles, Calif 90007