British Journal of Dermatology (1981) 105, 645-651.

Direct immunofluorescence on cytological smearsin oral pemphigus

A.E.ACOSTA,* J.HIETANENf AND L.IVANYI*

*Department of Oral Medicine and f Department of Pathology, Institute of Dental Surgery, London

Accepted for publication 12 February t98i

SUMMARY

Direct immunofluorescence was performed on washed oral epithelial smears from thirteenpatients with pemphigus vulgaris, thirteen patients with other oral diseases and from tensubjects with clinically healthy oral mucosa.

The intercellular deposition of IgG was observed on cytological smears from oral lesions in allpatients with active pemphigus. In contrast, smears from pemphigus patients in remission, frompatients with other oral diseases and from healthy controls, did not show any fiuorescence.Therefore, direct immunofiuorescence on cytological smears may be of value in the diagnosis ofpemphigus.

Pemphigus vulgaris is an autoimmune bullous disease involving the skin and mucousmembranes. In this condition, scrum antibodies which react with an intercellular substance ofstratified squamous epithelium can be detected by direct and indirect immunofiuorescence.

Recently, intercellular deposition of IgG was demonstrated on cytological smears from oralpemphigus lesions (Decherd et ai, 1972). However, it was also reported that smears from otheroral lesions (mainly aphthous ulcers) showed intercellular fluorescence similar to pemphigus(Skeete, 1977)-

The aims of this investigation were to evaluate the specificity of immunoglobulin deposits onsmears from pemphigus by direct immunofluorescence.

MATERIALS AND METHODS

PatientsEpithelial cells were obtained from the oral mucosa of thirteen patients with pemphigus vulgaris(seven males and six females) aged 48-82 years. Ten of these patients had active oral lesions andthree were in remission. The diagnosis was confirmed by histology and by direct immunofiuor-escence on biopsy specimens. Furthermore, indirect immunofluorescence was performed inorder to evaluate the serum intercellular (IC) antibody titre of these patients.

OO07-0963/81/1200-0645S02.00C) 1981 British Association of Dermatologists

645

646 A.E.Acosta,J.Hietanen andL.IvanyiControl epithelial smears were taken from seven patients with recurrent oral ulceration

(aphthous ulcers), two patients with mucous membrane pemphigoid, three patients with erosivelichen planus, one patient with erythema multiforme and ten subjects with clinically healthyoral mucosa. The control patients were matched for age (46-84 years) and sex (thirteen malesand ten females).

Collection and processing of epithelial cellsBefore collecting the epithelial cells, the oral mucosa around the lesions was dried, the roof of thelesions, if present, was removed, and the base of the lesions was gently scraped with a woodentongue depressor. The epithelial cells from healthy oral mucosa were scraped in the same way.

The cytological smears were prepared by two techniques;(1) The scraped epithelial cells were spread directly on glass slides, air dried, rinsed for i min

in PBS and used for direct immunofluorescence. Some slides were stained with haematoxylinand eosin to identify the acantholytic cells. The presence of acantholytic cells on unstainedsmears was also confirmed by phase contrast microscopy. For scanning electron microscopy, thescraped cells were spread directly on Agralon (ICI) polyester film.

(2) The cells were collected in 5 ml of tissue culture medium (TC-199, Wellcome Reagents)and washed twice by centrifugation (2000 r.p.m. for 10 min). Immediately after washing, thecells were counted and cytocentrifuged on to glass slides at a concentration of 4 x io"* per slide.

FIGURE I. Topography of unwashed epithelial cell from healthy oral mucosa by SEM ( x 1800).

Direct immunofluorescence 647For scanning electron microscopy, the cells were cytocentrifuged on Agralon polyester film bythe same method.

Scanning electron microscopyThis was done in order to assess the effect of washing procedure on the topography of theepithelial cells. Cells were fixed in 3 "« glutaraldehyde in 0-085 M cacodylate buffer pH 7-4 for 90min, rinsed three times with 5",, sucrose in cacodylate buffer, dehydrated and immersed inacetone and critical point dried in a Polaron critical point drying apparatus (E.3000, PolaronEquipment Limited). Selected areas were mounted and coated with gold using argon in aPolaron SEM unit (E.5000, Polaron Equipment Limited). The cells were studied in a JEOLJSM-35 scanning electron microscope at 25 kV using a tilt angle of 60' .

The washing procedure did not affect the topography of oral epithelial cells (Figs t and 2).These cells showed surface details with no alteration of microridges.

Direct immunofluorescenceImmunofiuorescent staining on unwashed and washed epithelial cell smears was carried out,according to the method of Coons & Kaplan (1950) with slight modifications. Briefly, the smearswere washed for 30 min in phosphate buffered saline (PBS pH 74) and overlaid with EITCconjugated sheep antihuman immunoglobulins (Wellcome Reagents; anti IgG, F/P ratio 4-5dilution 1130; anti IgA F/P ratio 4-5 dilution i: 10; anti IgM F/P ratio 31 dilution 1:15). Afterincubation at 37 C for 30 min, smears were washed in PBS three times for 10 min each andmounted in buffered glycerol pH 8-6. All slides were examined immediately under a LeitzSM-Lux microscope equipped with a 50 W mercury lamp and the following filter system: heatfilter KG-1, red suppressor filter LP-1, exciter filter K-490, barrier filter K515 and dichromaticmirror TK-510.

FIGURE 2. Topography of washed oral epithelial cell from healthy oral mucosa by SEM ( x 1500).

648 A.E.Acosta, J.Hietanen and L.Ivanyi

FIGURE 3. IgG deposition on unwashed epithelial ceils from pemphigus lesion by immunofluorescence( X 700).

FIGURE 4. Immunofluorescence of acantholytic cells from pemphigus lesion with IgG after washing( X 1120).

Direct immunofluorescence 649

Indirect immunofluorescenceSera from thirteen patients with pemphigus were tested by indirect immunofiuorescence(Coons & Kaplan, 1950) for the IC antibody titre using human skin as substrate.

RESULTS

Comparison of immunofluorescent patterns in unwashed and washed epithelial smearsDirect immunofiuorescence in unwashed epithelial celis from pemphigus lesions showed adeposition of IgG around some individual acantholytic cells and an intercellular fiuorescence onsheets of cells. However, many cells were in clumps and exhibited confluent fiuorescence on thecell membranes (Fig. 3). This deposition of IgG on the cell membranes was also observed in themajority of smears from various oral lesions and in several smears from healthy oral mucosa. Afew smears from patients with recurrent oral ulcers and erosive lichen planus showedintercellular fiuorescence in some sheets of the cells.

In contrast, when washed and cytocentrifuged epithelial cell preparations were used, the cellswere free of the cell membrane fluorescence and were evenly distributed on the slides with onlyfew clumps present. Smears from pemphigus lesions showed the presence of individualacantholytic cells with a ring-shaped deposition of IgG around them (Fig. 4). Sheets of cellswere also seen showing an intercellular fluorescent pattern (Fig. 5). Therefore, only washed andcytocentrifuged epithelial cells were used in the next experiment.

Deposition of immunoglobulins in oral epithelial smearsTen patients with active oral pemphigus and three in remission were investigated (Table i). All'active' patients had serum IC antibody with titres ranging from 1140 to i: 160. Intercellularantibody was not present in sera from two patients in remission, whilst one patient with recently

. Intercellular IgG deposition of washed sheet of cells from pemphigus lesion ( x 1120).

650 A.E.Acosta, y.Hietanen and L.IvanyiTABLE I. Deposition of immunoglobulins in smears from oral pemphigus lesions

Direct immunofluorescence

Patient Clinical status IC*-antibody titre Presence of acantholytic cells IgG IgA IgM

1

2

3456

78

91 0

I I

12

13

ActiveActiveActiveActiveActiveActiveAaiveActiveActiveActive

RemissionRemissionRemission

:i6o1:160

1:160

:i6o:i6o: 160

:80:8O:40

1:401:10

NegativeNegative

*IC = intercellular.

healed lesions had a serum antibody titre of i : 10. Acantholytic cells were only seen in lesionsfrom active pemphigus.

Direct immunofiuorescence on epithelial cells from 'active' lesions showed IgG deposition inall ten patients. Smears from two patients (Nos 6 and 9) also showed intercellular IgAdeposition. IgM positive fluorescence was not detected in any of the patients tested. Oralepithelial smears from three patients in remission did not show any IgG, IgA or IgM deposition.Finally, smears from thirteen patients with various oral diseases and from ten subjects withhealthy oral mucosa showed no positive fluorescence for IgG, IgA or IgM (Table 2).

TABLE 2. Direct immunofluorescence on smears from various oral lesions and healthy oral mucosa

Direct immunofluorescence

Diagnosis Number of subjects IgG IgA IgM

Recurrent oral ulceration 7 _ _ _Erosive lichen planus 3 _ _ _Mucous membrane pemphigoid 2 _ _ _Erythema multiforme i _ _ _Healthy controls 10 _ _ _

DISCUSSION

The specificity of direct immunofiuorescence for the detection of pemphigus antibodies in oralcytological smears has been a subject of controversy (Decherd et al, 1972, Skeete, 1977).

The results show that the processing of scraped oral epithelial cells before directimmunofiuorescence is of importance. Using unwashed cell preparations, an intercellular

Direct immunofluorescence 651deposition of IgG was observed not only on smears from pemphigus lesions, but also in somesheets of cells from several patients with other oral diseases. Furthermore, the deposition of IgGon the cell membranes was seen in the majority of epithelial cell samples studied. Examination ofunwashed smears by scanning electron microscopy showed that most of the cells scraped fromoral lesions were coated with a film masking the cell surface details (results not presented). Thisseems likely to be due to non-specific attachment of serum proteins to the epithelial cellmembranes. Coruh & Mason (1980) have demonstrated the intercellular and intracellulardeposition of immunoglobulins and other sertim proteins in various healthy and inflamedhuman epithelia. The most probable explanation is that they represent a leakage of serumproteins on and into epithelial cells (Coruh & Mason, 1980; Brandtzaeg, Surjan & Berdal, 1978).This has particular importance in the case of an inflamed tissue where the extravasation of serumproteins is increased. Another possible source of non-specific staining could be the mechanicaltrapping of immunoglobulins in clumps of cells (especially where the cellular borders overlap).

In contrast, washed and cytocentrifuged epithelial cells were free of the cell membranepositive fluorescence, and they were evenly distributed on the slides with only few clumpspresent. Furthermore, intercellular deposition of IgG was observed only in smears frompemphigus lesions. Interestingly, IgA localization was seen in intercellular substance areas insmears from two patients with pemphigus. Similar results were reported by other authors(Jordan et al., t974), but the role of IgA in the pathogenesis of pemphigus has not beenelucidated.

Our results confirm the specificity of IgG deposits on smears from pemphigus lesions bydirect immunofluorescence. This corroborates the initial report of Decherd et al. (1972) on thevalue of direct immunofluorescence on cell smears as an aid to the diagnosis of pemphigusvulgaris.

ACKNOWLEDGMENTS

We wish to thank Dr J.J.H.Gilkes and Dr G.M.Levene for allowing us to investigate theirpatients. We also wish to thank the National University of Mexico for a grant to one of us(E.Acosta Gio). Finally, we are grateful to the Institute of Neurology for letting us use thescanning electron microscope.

REFERENCES

BRANDTZAEG, P., SURJAN, L . & BERDAL, P. (1978} Immunoglobulin system of human tonsils. Clinical and ExperimentalImmunology, 31, 367.

COONS, A.H. & KAPLAN, M.H. (1950) Localization of aniigen in tissue cells, II. Improvements in method of detection ofantigen by means of fluorescent antibody. Journal of Experimental Medicine, 91, i.

CORUH, G . & MASON, D . Y . (1980) Serum proteins in human squamous epithelium. British Journal of Dermatology, 102,

497-DECHERD, J.W., RtmiN, M.B., GOLDSCHMIDT, H . & HEISS, H . B . (1972) Immunofluorescence of Tzanck smears in

pemphigus vulgaris. Acta dennato-venereologica (Stockholm), 52, 116.JORDAN, R.E., SCHROETER, A.L., ROGERS, R.S. & PERRY, H . (1974) Classical and alternate pathway activation of

complement in pemphigus vulgaris lesions. The Journal of Investigative Dermatology, 63, 256.SKEETE, M . V . H . (1977) Evaluation of ihe usefulness of immunofiuorescence on Tzanck smears in pemphigus as an aid to

diagnosis. Clinical and Experimental Dermatology, 2, 57.