Volume 62, Number 2Printed in the U.S.A.

INTERNATIONAL JOURNAL OF LEPROSY

An Evaluation of the S-100 Stain in the HistologicalDiagnosis of Tuberculoid Leprosy and Other

Granulomatous Dermatoses'Navjeevan Singh, V. K. Arora, M. Ramam,

S. K. Tickoo, and A. Bhatia 2

Leprosy is the most frequently biopsieddermatologic condition in our hospital andcontributes approximately 60% of all skinbiopsies (290 of 467 in 1991). The majorityof leprosy patients attending our outpatientdepartment have tuberculoid leprosy (TL)(BT leprosy in the Ridley-Jopling classifi-cation). A search for Mycobacterium lepraein tissue sections stained with the Fite stain(') is frequently futile. In such a situationwe base the diagnosis of TL on a combi-nation of physical signs (' 2 ) and histologicalfindings ( 9 ). We feel that in an endemic areaa diagnosis of leprosy should be entertainedin all cases with a compatible clinical pre-sentation and a histologic picture of com-pact epithelioid cell dermal granulomas,even in the absence of demonstrable .11. lep-rae.

As with other workers (8,11) there havebeen instances when we have had to reviseour initial clinical and histopathological di-agnosis of tuberculoid leprosy. In our ex-perience some of the lesions simulating TLinclude granulomatous secondary syphilis,post kala-azar dermal leishmaniasis, sar-coidosis, granuloma annulare, and lupusvulgaris.

Recent reports in the literature ( 2 ' 3 ) haveaddressed the utility of the immunoperox-idase staining of S-100 proteins in skin bi-opsies to aid in the diagnosis of tuberculoid

' Received for publication on 6 July 1993; acceptedfor publication in revised form on 1 March 1994.

N. Singh, M.D., Lecturer, Department of Pathol-ogy, University College of Medical Sciences and GTBHospital, Delhi 110095, India. V. K. Arora, M.D.,Reader; S. K. Tickoo, M.D., Lecturer; A. Bhatia, M.D.,Professor, Department of Pathology, University Col-lege of Medical Sciences, Delhi 110095, India. NI. Ra-mam, M.D., Assistant Professor, Department of Der-matology, All India Institute of Medical Sciences, NewDelhi 110029, India.

leprosy. The positive staining of Schwanncells allows easy recognition of intact der-mal nerve twigs or their fragments. It hasbeen proposed that the presence of intactnerve twigs, or fragmented remnants ofnerves within the granulomas, should favora diagnosis of TL ( 3 ); the nerves, being pro-tected sites for the location of leprosy ba-cilli, are the seat of the granuloma forma-tion.

We therefore decided to test the appli-cability of these findings, using the S- 100stain, in differentiating TL from the othergranulomatous conditions that we com-monly encounter.

MATERIALS AND METHODSTwelve consecutive skin biopsies from

patients with clinical diagnoses of BT lep-rosy, based on the findings of hypopig-mented, anesthetic lesions, characteristic ofthe disease in this part of the spectrum (asdefined by Ridley and Jopling), were takenfor the study. The essential histologic pic-ture in all of these cases was that of a dermalgranulomatous inflammation with destruc-tion of the appendages. M. leprae could notbe found using the Fite stain.

For comparison, all the "nonleprosy"granulomatous dermatoses (28 cases) en-countered in the 3-year period between Jan-uary 1989 and December 1991 were takenfor study (The Table). The two major groupswith histology closely resembling that of tu-berculoid leprosy were granulomatous sec-ondary syphilis (SS) and lupus vulgaris (LV).The remainder was comprised of infre-quently occurring granulomatous condi-tions with tissue reactions varying fromepithelioid cell granulomas (lichen scrofu-losorum) to histiocytic granulomas (histoidleprosy), mixed cell granuloma (fungal) andpalisading granulomas (granuloma annu-

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264^ International Journal of Leprosy^ 1994

THE TABLE.^Forty biopsies showing S-100 staining patterns.

Staining pattern

3 4

Major groups

Tuberculoid leprosy (12) 0 0 5h

Secondary syphilis (9) 2 0 7 0Lupus vulgaris (8)^ I

Other grantilomatous dermatoses

7 0 0

Granuloma annulare (2) 0 1 0Deep mycoses (2) 0 0Tuberculosis verrucosa cubs (1) 0 0 0Lichen scrofulosorum (1) 0 0 0Nodular vasculitis (I) 0 0Donovanosis (I) 0 0 0Post kala-azar dermal leishmaniasis (I) 0 0Mycetoma (I) 0 0 1 0Histoid leprosy (1) 0 0 0

" Specificity 82%.'' Specificity 100%.

lare). In all of the cases studied the clinicaland histologic criteria were sufficiently char-acteristic of the diagnosis.

Formalin-fixed, routinely processed, par-affin-embedded tissue was cut at 4 to 6 pin.

Sections were stained by hematoxylin andeosin (H&E) and a commercially available

FIG. 1. Nerve twig within a granuloma (pattern I)

detected by S-100 protein in a case of lupus vulgaris

(PAP x400).

kit (DAKOPATTS, K524) for demonstra-tion of S-100a antigen by the peroxidase-an tiperoxidase (PAP) method ( 4 ). This iden-tifies nerves in tissue sections. Statisticalanalysis of the three major groups was doneusing Fisher's exact test.

RESULTSFibrillar structures staining positively for

S-100 protein were identified as nerve twigs.The smallest twigs were approximately 30-pm in size. Four patterns of nerve twig dis-tribution were seen: 1) nerve twigs withingranulomas (Fig. 1), 2) nerve twigs betweengranulomas, 3) nerve twigs within and be-tween granulomas (Fig. 2), and 4) unde-tectable nerve twigs in an adequate biopsywhich included some subcutaneous tissue.In all 12 of our cases of tuberculoid leprosy(The Table) nerve twigs were detectable ei-ther within the granulomas (7) or not at all(5). The complete absence of nerve twigs(pattern 4) in an adequate biopsy is the mostreliable criterion for the diagnosis of TL (p< 0.05). In the seven cases of TL with nervetwigs present within granulomas, nerve twigsinvariably were absent between them (pat-tern 1). A pattern similar to this also wasobserved in two cases of SS and one of LV.In LV and SS the dominant patterns were2 and 3, respectively.

In the remaining cases nerve twig distri-bution patterns were either 2 or 3. However,the numbers were insignificant for charac-

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FIG. 2. Nerve twigs both within (open arrowhead)and between (arrow) granulomas (pattern 3) in a caseof granulomatous secondary syphilis (PAP x400).

FIG. 3. Numerous macrophages packed with pos-itively staining M. leprae (arrow) (PAP x400).

terizing any pattern of distribution. An ad-ditional finding was the positive staining of/11. leprae (Fig. 3) in nodular leprosy as wellas the granules of mycetoma (Fig. 4) in onecase each.

DISCUSSIONAn etiological diagnosis of epithelioid

granulomas of the skin is often difficult be-cause it is seldom possible to demonstratethe inciting agent. Also, different etiologicalagents produce a confusingly similar mor-phological picture. Demonstrable evidenceof nerve involvement by the granulomatousinfiltrate has been considered an importantfeature of tuberculoid leprosy, its signifi-cance being almost the same as the findingof acid-fast bacilli in a nerve ( 10). The dif-ficulty in recognizing small nerve twigs onroutine H&E sections has led to the use ofthe S-100 stain as an aid to the histologicaldifferentiation of granulomatous skin in-flammations.

It has been advocated that the finding ofnerve twigs within a granuloma should sug-gest a diagnosis of leprosy ( 2- 3 ). This study

FIG. 4. Positively staining mycetoma granule (ar-row) (PAP x400).

266^ International Journal of Leprosy^ 1994

suggests that such an hypothesis might bemisleading because nerves within granulo-mas also were found in other conditions,notably lupus vulgaris (1 of 7) and granu-lomatous secondary syphilis (2 of 9). More-over, even with use of the S-100 stain wewere unable to distinguish between the mor-phology of intact small nerve twigs and frag-ments of destroyed nerves in the tissue sec-tions, the smallest structures recognizableas nerves consisting of at least 2 to 3 posi-tively staining, fibrillar Schwann cells of ap-proximately 30 pm in size.

It is evident from The Table that two pat-terns of staining, the presence of nerve twigswithin granulomas (pattern 1) or the com-plete absence of recognizable nerve twigs(pattern 4) in the biopsy, were seen in TL.Although in the majority of the other gran-ulomatous lesions studied the patterns var-ied, a striking common histological featurewas perineural lymphocytic infiltrate,sometimes infiltrating the perineurium.Careful neurological testing in nonleprosygranulomatous dermatoses is likely to re-veal clinical evidence of nerve damage asdescribed in cutaneous leishmaniasis ( 5 ) andnecrobiosis lipoidica ( 7 ).

In conclusion, our findings in granulo-matous dermatoses suggest that, contrary tothe existing view, it is the complete absenceof nerve twigs (pattern 4) in an adequateskin biopsy that is the most reliable crite-rion for the diagnosis of TL. The finding ofnerve twigs within but not between granu-lomas (pattern 1) is highly suggestive of lep-rosy. The other two patterns, nerve twigsboth within and between (pattern 3) or onlybetween granulomas (pattern 2) should sug-gest diagnoses other than TL. An unex-pected finding of this study is the positivestaining of the granules of mycetoma. Also,we substantiate a previous report of thestrongly positive staining of M. leprae (6 ) innodular leprosy.

SUMMARYForty biopsies of granulomatous derma-

toses, 12 of which were tuberculoid leprosy(TL), were studied for patterns of nerve twigdistribution using an immunoperoxidasetechnique for S-100 protein. Four distinctpatterns of nerve twigs were identified: 1)within granulomas, 2) between granulomas,

3) within and between granulomas, and 4)undetectable nerve twigs in an adequate bi-opsy. Pattern 4 was seen exclusively in TL(p < 0.05). The other patterns occurred innonleprosy dermatoses as well, suggestingthat pattern 4 is the best indicator towarda diagnosis of TL. The granules of myce-toma and illycohaelerium leprae also stainedpositively with the S-100 stain.

RESUMENUtilizando un têcnica de inmunodetecciOn con Pe-

roxidasa para la proteina S-100, se estudiaron 40 biop-

sias de dermatosis granulomatosas (12 de las cuales

fueron de lepra tuberculoide, TL) para establecer el

patrOn de distribucffin de las ramificaciones nerviosas.

Sc identi ficaron 4 patrones distintos de distribuciOn de

las ramificaciones nerviosas: (1) dentro de los granu-

lomas, (2) cntre los granulomas, (3) dentro y cntre los

granulomas, y (4) ausencia de ramificaciones nerviosas

aim cuando la biopsia foe adecuada. El patron 4 se

observO exclusivamente en los pacientes TL (p menor

de 0.05). Ya que los otros patrones se observaron tarn-bi(ffi en dermatosis no leprosas, se sugiere que el patron

4 es el mejor indicador para establecer un diagnOstico

de TL. Los griinulos de micetoma y Mycobacterium

leprae tambiên se tifieron positivamente con la tincion

para S-100.

RESUME

Quarante biopsies de dermatoses granulomateuses,

parmi lesquelles 12 de lepre tuberculoide (LT), ont etc

tudié.es quant n leurs modes de distribution en filets

des nerfs par une technique d'immunoperoxydase pour

la proteine S-100. Quatre types distinets de filets ner-veux ont etc identifies: 1) dans les granulomes, 2) entre

les granulomes, 3) dans et entre les granulomes, et 4)

des filets nerveux indêtectables dans une biopsie ade-quate. Le type 4 êtait vu exclusivement dans la lêpre

tuberculoide (p < 0.05). Les autres types survenaientCgalement dans les dermatoses non-l'épreuses, suggê-

rant que le type 4 est le meilleur indicateur pour undiagnostic de 10re tuberculoide. Les granules de my-

cêtomes et de Mycobacterium leprae prenaient &gale-

ment la coloration au S-100.

REFERENCES1. FITE, G. L., CAMBRE, P. J. and TURNER, M. H.

Procedure for demonstrating lepra bacilli in par-

affin sections. Arch. Pathol. 43 (1947) 624-625.2. FLEURY, R. N. and BACCHI, C. E. S-100 protein

and immunoperoxidase technique as an aid in the

histopathologic diagnosis of leprosy. Int. J. Lepr.

55 (1987) 338-344.

3. Joe, C. K., DRAIN, V., DEMING, A. T., HASTINGS,R. C. and GERBER, M. A. Role of S-100 protein

as a marker for Schwann cells in the diagnosis of

6") , ")^

Singh, et al.: Evaluation of S-100 Stain^267

tuberculoid leprosy. (Letter) Int. J. Lepr. 58 (1990)392-393.

4. KAHN, H. J., MARKS, A., THOM, H. and BAUMAI,R. Role of antibody to S-100 protein in diagnosticpathology. Am. J. Clin. Pathol. 79 (1983) 341-347.

5. KUBBA, R., EL-HASSAN, A. M., AL-GINDAN, Y.,OMER, A. H., BUSRA, M. and KurrY, M. K. Pe-ripheral nerve involvement in cutaneous leish-maniasis (Old World). Int. J. Dermatol. 26 (1987)527-531.

6. LEFFORD, M. J. S-I00 protein marker. (Letter) Int.J. Lepr. 59 (1991) 122.

7. MANN, R. J. and HARMAN, R. M. Cutaneous an-esthesia in necrobiosis lipoidica. Br. J. Dermatol.110 (1984) 323-325.

8. PAVITHRAN, K. Chromoblastomycosis masquer-ading as tuberculoid leprosy. (Letter) Int. J. Lepr.60 (1992) 657-658.

9. RIDLEY, D. S. Histological classification and im-munological spectrum of leprosy. Bull. WHO 51(1974) 451-465.

10. RIDLEY, D. S. Skin Biopsy in Leprosy. 2nd edn.Basle: CIBA-GEIGY, 1985, p. 30.

11. SINGH, K., DAWAR, R. and RAMESH, V. Lympho-cytic infiltration of skin of Jessner-Kanof mas-querading as borderline leprosy. Indian J. Lepr.57 (1985) 804-806.

12. WORLD HEALTH ORGANIZATION. A Guide to Lep-rosy Control. 2nd edn. Geneva: World Health Or-ganization, 1998.