Polarization Optical Studies of Hyperkeratosis, Parakeratosis and Dyskeratosis* Daphne Anderson Roe, M.D., M.R.C.P

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POLARIZATION OPTICAL STUDIES OF HYPERKERATOSIS, PARAKERATOSIS AND DYSKERATOSIS* DAPHNE ANDERSON ROE, M.D., M.R.C.P.

Previous polarization optical studies of adultlife, the epidermis reaches its final state; at this and embryonic human epidermis have beenstage the eornified layer shows intense double devoted to the elucidation of the morphology ofrefraction. It is of interest that in the chick its fibrous structure. In unstained verticalembryo, the long axis of the cells of the periderm sections of human abdominal skin, the mostand of the embryonic horny layer is parallel to intense double refraction occurs in the stratumthe skin surface and the early keratin fibres are corneum and stratum lucidum. In these layersalso oriented in the same plane. birefringent material is oriented parallel to the There is only one polarization optical study of surface plane; below this region the birefringentpathologic epidermis. Nieuwmeijer examined the fibres are oriented perpendicularly to the surfacetonofibrils in bullous dermatoses by this method (1). and found differences between the tonofibrillar Similar observations have been made by othersystems in pemphigus and dermatitis herpeti- authors (2, 3), among them Mercer, who hasformis (6). In pemphigus, in the areas of acan- compared the appearance of the epidermis undertholysis, the tonofibrils were greatly decreased, the polarization and electron microscopes. Hewhereas in dermatitis herpetiformis the tonofibrils noted that the fibrils (tonofibrils) in the lowerwere pushed aside by the fluid in the bullae. layers of the epidermis were weakly birefringent. Recently, I used polarization optical methods At the level of the appearance of keratohyalinto investigate the anomaly of keratin formation there was a sudden rise in birefringenee associatedin psoriasis (7). It was found that the birefringent with a change in orientation of the fibres from atonofibrillar system persisted throughout the vertical to a horizontal direction. psoriatic epidermis and in the parakeratotic scale, In tissue cultures of stripped epidermis,but the mature keratin fibres failed to develop Matoltsy has shown that in 3 to 5 days' oldinto a coherent horny layer. These changes were explants the originally vertically oriented bire-reflected in the psoriatic nail (8) where immature fringent material becomes reoriented in a planekeratinization was evident from the extensive parallel to the surface (4). On the 7th day, aparakeratosis combined with persistence of a low definite keratogenous zone became visible, whiledegree of double refraction in much of the fibrous all the birefringent epidermal fibres were stillstructure of the nail plate and in the adherent oriented parallel to the skin surface. The signifi-hyponychium. These abnormalities were as- cance of these findings will be discussed later. sociated with alterations in the non-fibrous com- Studies of the development of the birefringentponents of the nail and epidermis, manifested fibrillar system of the human epidermis have yethistochemically by cytoplasmie metachromasia to be carried out, but similar observations of thein the parakeratotie cells and by an increased chick embryo have been made (5) and it has beenuptake of the Gram stain in the affected areas. shown that around the 16th day of embryonic In the present work the defects in the fibrous life the periderm or epitrichium reveals a verystructure of the epidermis were investigated in weak birefringenee. However, moderate birefrin-conditions characterized by the formation of gence appears during the next 2 days of embry-anomalous horny layers. onic life when the true cornified layer is formed. Between the 18th and 21st days of embryonic METHOD AND MATERIALS *Fromthe Department of Pathology, Memorial For the polarization optical investigations we Hospital, Wilmington, Delaware. used a Reichert monocular microscope fitted with This study was supported by a grant from thepolaroid discs as substage polarizer and tube Permanent Science Fund of the Americananalyzer. Photographic records were made with a Academy of Arts and Sciences. Presented at the Twentieth Annual Meeting ofRetina IIC camera, used in conjunction with a The Society for Investigative Dermatology, Inc.,Kodak photomicrographic unit. The sign of bire- Atlantic City, N. J., June 6, 1959. fringence was determined by means of a Zeiss first 257 258 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY order red retardation plate, together with a capalso stained with hematoxylin and eosin, buffered analyzer (9). As in our previous studies (7, 8),thionin (10) and by a modified Gram-Weigert birefringenee referred to the optic axis of themethod (11). keratin and pre-keratin fibres (tonofibrils) in the Previous routine staining technics had estab- histologic material under examination. With thislished in the specimens histologic characteristics equipment, unstained skin sections were ex-associated with hyperkeratosis, parakeratosis or amined. Biopsy specimens were fixed in 10 perdyskeratosis. The sections were classified as fol- cent formalin, embedded in paraffin and sectionedlows: to 3 m thickness. The sections were then depar- 1. Simple hyperkeratosis: callus. affinized, cleared in xylene and mounted in syn- 2. Hyperkeratosis with acanthosis: lichen thetic resin. From each specimen sections weresimplex chronicus.

FIG. 1. Pseudo-epitheliomatous hyperplasia associated with stasis dermatitis. The normal pattern of epidermal birefringenee is exaggerated with a widened keratogenous zone and increased tonofibrils. Magnification X400.

FIG. 2. Chronic radiation dermatitis showing moderate hypcrkeratosis and orientation of birefringent tonofibrils parallel to the surface. Magnification X400. HYPERKERATOSIS, PARAKERATOSIS AND DYSKERATOSIS 259

FIG.3

FIG. 4 Fias. 3 and 4. Keratotie basal celled carcinoma showing horn cyst and orientation of tonofibrils parallel to the surface. Magnification X200, X400.

3. Hyperkeratosis with epidermal hyperplasia: 8. Parakeratosis, hyperkeratosis and dyskera- a) Pseudo-epitheliomatous hyperplasia in stasistosis: senile keratosis. dermatitis b) Verrucous nevus pigmentosus. 0. Dyskeratosis: Darier's disease. 4. Hyperkeratosis with epidermal atrophy: 10.Dyskeratosisand hyperkeratosis involv- chronic radiation dermatitis. ing epidermal and mucosal surfaces: leukoplakia. 5. Hyperkeratosis, parakeratosis and aean- thosis: Verruca plantaris. RESULTS 6. Hyperkeratosis with dyskeratosis: cornu cutaneum. In simple hyperkeratosis, the normal pattern of 7. Parakeratosis: a) psoriasis, h) seborrheic der-epidermal birefringence was observed, though matitis, c) keratotic basal cell carcinoma. the stratum corneum containing highly hire- 260 THEJOURNAL OF INVESTTGATIVE DERMATOLOGY

I - a. a

I' h Fro. 5. Early leukoplakia showing orientation of cells and doubly refractile fibres parallel to the surface. Section stained buffered thionin and viewed under polarized light. Magnification X400.

.1 7-4 t*t_t4 1 • Ad5

Fio. 6. Plantar wart with a central area occupied by compressed cornifled cells displaying intense birefringence. Magnification X300. fringent mature keratin fibres was increased in In chronic radiation dermatitis, when hy- thickness. perkeratosis was associated with epidermal Where the hyper/ceratosis was associated withatrophy, there was a paucity of tonofibrils and acanthosis, especially in lesions with pseudo-these were oriented parallel to the skin sur- epitheliomatous hyperplasia, as in a case offace (Fig. 2). A similar orientation of the tono- chronic stasis dermatitis, this normal patternfibrils was seen in an example of keratotic basal was exaggerated. In particular, the keratogenouscell carcinoma with epidermal atrophy and in zone was broadened and very sharply defined;leukoplakia involving the muco-cutaneous junc- the hirefringent tonofibrils were increased intion of the lip (Figs. 3, 4, 5)* number, hut their orientation was identical with *Ithas been shown that in sections of the those in the normal epidermis (Fig. 1). huceal mucosa and in sections of the cow's nose HYPERKERATOSTS, PARAKERATOSIS AND DYSKERATOSIS 261

Inpsoriasis, as previously described (7), the prekeratin fibrils persisted throughout the epidermis and in the parakeratotie scale. The birefringent fibres were oriented perpendicularly or at an angle to the surface plane. Mature keratin fibres occurred in isolated groups, oriented parallel to the surface. This pattern is appar- ently specific for psoriasis; in other pathologic material no comparable distribution was found. Thus in seborrheic dermatitis, with marked parakeratosis, the normal orientation of the doubly refractilc epidermal fibres was retained, though in parakeratotic areas the bircfringcnce was less intense than in the normal horny layer. Dyskeratosis, defined as a faulty keratinization of individual cpidermal cells (12), is reflected £ under the polarization microscope. In Darier's disease, the villous epidermal projections showed normal tonofibrils. The corps ronds contained bircfringent fibres of low intensity, oriented parallel to the long axis of the cells. In the abnormal horny layer, the grains appeared to be

FIG. 7. Plantar wart showing peripheral area with alternating columns of highly bircfringent and poorly birefringcnt fibres, following the areas of fully cornified and parakeratotic cells and oriented in the long axis of these cells. A well-defined keratogenous zone can be seen. Magnification X200.

The plantar wart showed a peculiar pattern of bircfringence. The central area, occupied by a mass of compressed cornified cells displayed intense birefringence with orientation of the component fibres parallel to the surface. In the periphery, the alternating columns of parakcratotic and fully cornified cells showed varying degrees of double refraction. In effect, the orthokcratotic areas revealed a more intense bircfringence than the parakeratotie zones. The orientation of the keratin fibres followed the direction of the long axis of the cells within the modified horny layer, and thus varied with the papillomatous elevations and depressions. The keratogcnous zone was clearly demarcated and the tonofibrils somewhat increased in number, in a manner similar to that seen in pseudo- epitheliomatous hypcrplasia (Figs. 6, 7, 8). epidermis (which histologically resemble one another with respect to their differentiated epi- FIG. 8. Plantar wart demonstrating details of thelium), the tonofibrils are oriented parallel tothe keratogenous zone and tonofibrils. Magnifica- the surface. tion X400. 262 THE JOURNAL OF INVESTIGATIVE DERMATOLOGY

A detailed discussion of the Gram staining of some of these lesions and its possible significance is published in another paper (13). Cytoplasmic Gram positivity was associated not only with parakeratotic cells, but also with areas of dyskeratosis, especially where the dyskeratotic cells -.1— -- were exfoliated from the epidermal tissue. Under I —% - V -a - 41 oil immersion it could be seen that this cytoplasmic uptake of the Gram stain was localized to I — c'Z .o.:. - granules; no evidence was obtained that Gram 4t - positivity was related to the fibrous structure of p..••—:.- — 2 the cell. — — --..- DISCUSSION From these observations, certain pathological patterns of epidermal birefringenee emerge. Where the whole epidermis shows simple hy- pertrophy, there is an exaggeration of the normal pattern with two significant features; an increase in the number of tonofibrils and a wide, well- defined keratogenous zone. Preliminary measure- ments with a micrometer eye-piece suggest that the width of the keratogenous zone and the abun- dance of tonofibrils are related to the actual FIG. 9.Darier'sdisease showing dyskeratoticamount of hyperkeratosis. When hyperkeratosis cells of low intensity birefringenee. Magnifica-occurs in the absence of these features, it is only a tion X200. relative and not an absolute hyperkeratosis. In this ease the horny layer forms a greater propor- more fully keratinized, judged from their greatertion of the epidermis, but is not thickened and birefringence. In the horny layer there were alsoeven may be decreased in width. amorphous masses of keratin which displayed a Two other characteristic patterns have been patchy double refraction with scattered groupsconsistently observed. One is the peculiar struc- of horny cells showing the normal birefringenccture of the psoriatie epidermis which has already of the stratum corneum (Fig. 9). Certain similarbeen described in detail. The other is the "de- features were observed in senile keratosis. Iso-differentiation" of the epidermal fibrous system lated cells within the characteristic epidermalin chronic epidermal atrophy and in certain clefts resembled the corps ronds under the polari- premalignant dermatoses, as manifested by the zation microscope. In the horny layer, penetrat-orientation of the tonofibrils parallel to the ing into the prickle cell layer, amorphous keratinsurface. It is of interest to recall that this pattern masses revealed a stippled or patchy birefrin-is identical with that found in skin explants gence. The pattern of birefringenee of the epi-(4), in the epidermis of chick embryos (5) and dermal fibres was irregular, with a few tonofibrilsin normal keratinizing mucous membranes. oriented at various angles to the skin surface. In our study we found that the orientation of Although the parakeratotie scale showed low hire-the keratin and prekeratin fibres was directly fringenee of the component fibrous structure, there were areas in the lesion where maturerelated to the long axis of their parent cells. The factors which orient the keratin and triehohyalin fibres could be observed, layered in the form of fibres in the hair follicle were studied by Mereer. cones (Fig. 10 a, b). In the cornu cutaneum, well-defined andHe observed that the cells in which these fila- packed mature keratin fibres of high intensityments formed were slowly distorted into spindle birefringence could be recognized. At the base ofshapes as synthesis proceeded; nevertheless, the the tumor, these fibres were looser; between themincrease in length of the cells was not sufficient to there were dyskeratotie cells, similar to those inaccount for the drawing out of the filaments. the clefts of the senile keratosis (Fig. 11). Mercer has assumed that, because the filaments FIG. 10 a and b. Senile keratosis showing amorphous keratin masses, the irregular pattern of the ani- sotropie epidermal fibres and isolated dyskeratotic cells. Magnification X200, X400.

FIG. 11. Cornu cutaneum with packed mature keratin fibres of high intensity birefringence and isolated dyskeratotic cells, displaying a lower degree of birefringencc. Magnification X200. 263 264 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY

FIG. 12 a and b. Intra-epidermal squamous celled carcinoma with loss of tonofibrils in the affected areas. Magnification X200, X400. grow parallel to the long axis of the cell, the slightelectron microscopic studies (17) of the hair flow of cell contcnts oriented the initial filamentsfollicle. This author has shown that triehohyalin and that later growth maintained the samegranules in the root sheath cells do not elongate orientation. Furthermore, he believed that lateralinto fibres, but that the nonfibrous trichohyalin association between adjacent fibrils was a sig-is deposited around individual tonofilaments. nificant factor in this process (14). Similarly, keratohyalin is precipitated on the Such lateral association of fibrils has beentonofibrils in the course of epidermal keratiniza- demonstrated, not only in electron micrographstion within the granular layer (18). of the human epidermis (15), but also in electron Despite these conflicting viewpoints, it would micrographs of the protein tonofibrin, which hasappear reasonable to assume that the orientation been extracted from the epidermis. This protein isof tonofibrils may be related to changes in cell believed to form the substance of the tonofila-shape and perhaps to alterations in the viscosity ments (16). Some of these interpretations mustof the nonfibrous cell contents. remain tentative in the light of Charles' recent From Weiss's experiments (19), two important HYPERKERATOSIS, PARAKERATOSIS AND DYSKERATOSIS 265 points should be mentioned. First, the alignment The pattern in psoriasis has been found to be of fibres may influence the shape of cells; sec-specific for this disease. A peculiar development ondly, cell and fibre orientation may be a resultof the tonofibrils was demonstrated in chronic of differential adhesion. We are now sufficientlyradiation atrophy and with certain premalignant familiar with the methods of adhesion of prickleand malignant conditions; the prekeratin fibres cells to realize the significance of the latterwere oriented parallel to the surface, as in em- observation. According to Selby (15), thebryonic skin and in skin explants. The doubly familiar intercellular bridges or desmosomesref ractile fibres in the dyskeratotic cells in surround the cells of the stratum germinativumDarier's disease have been compared with those and there is some electron dense material betweenfound in parakcratotic cells. the cell membranes within the desmosomes. Yet, Various factors which contribute to the for- in the stratum granulosum the desmosomes liemation and orientation of keratin fibres in the closer to each other along the cell periphery andnormal and pathologic epidermis were discussed. the material within the desmosomes is more conspicuous. Selby suggested that the close ADDENDUM approximation of desmosomes is a result of an Quite recently the author has had the oppor- overall decrease in cell volume. This may indeedtunity to study sections of an intra-cpidermal be an important factor in the altered orientationsquamous cell carcinoma, revealing the malignant of tonofibrils within this layer. dyskeratotic cells characteristic of Bowen's At present, we can only form hypothesesdisease. Under polarized light, in these sections concerning the abnormal orientation and lack ofthe anisotropic tonofibrils had disappeared in the maturation of keratin fibres in certain derma-affected areas except peripherally, where a few toses. Anomalous cell orientation would seemremained oriented parallel to the surface. (see to be of foremost importance in the peculiarFig: 12 a, b). This finding coincides with ob- alignment of tonofibrils seen with epidermalservations made by von Albertini (21) in his "dc-differentiation" as in pre-malignant dcrma-electron microscope studies of epidermal car- toses. Abnormal cell adhesion may be the primarycinoma. He showed that tonofibrils were factor in producing the patterns of epidermalmarkedly increased with epidermal hypcrplasia birefringence in dyskeratoscs and particularly inbut that with the loss of cytoplasmic differen- Daricr's disease. Indeed it has been shown thattiation involved in malignant change, there was in Daricr's disease the formation of corps rondsfirst a malformation and later, complete lack of occurs because cpidcrmal cells arc unable to formformation of tonofibrils. pricklcs (intercellular bridges) on some or all sections of their cell walls (18). In psoriasis, we REFERENCES have certain experimental corroboration for our 1. MATOLTSY, A. G.: The Chemistry of Kera- tinization. The Biology of Hair Growth. p. theory that abnormal cell adhesion and incom- 59. ed, Montagna, W. and Ellis, R. A. New plete synthesis or deposition of kcratohyalin York. Academic Press Inc. 1958. together lead to the persistence of tonofibrils and 2. MONTAGNA, W.: The Structure and Function of Skin. p. 44—45. New York. Academic Press the inhibition of mature keratin fibre formation Inc. 1956. (18). 3. MERcRR, E. H.: The Electron Microscopy of Keratinized Tissues. The Biology of Hair Growth. p. 107—108. ed. Montagna, W. and SUMMARY Ellis, R. A. New York. Academic Press Inc. Unstained skin sections were studied under 1958. 4. MATOLTSY, A. G. AND SINE5I, S. J.: A study the polarization microscope, with relation to the of the mechanism of keratinization of human orientation, maturation and degree of bircfrin- cpidermal cells. Anat. 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In Press. len Glykoproteine und Mukopolysaecharide 14. MEEcEE, E. H.: Electron Microscopy and bei Psoriasis. Dermat. Wschr., 139: 321— Biosynthesis of Fibers. The Biology of Hair 324. 1959. Growth. p. 128. ed. Moritagna, W. & Ellis,21. VON ALBERTINI, A.: Electron microscopic R. A. New York. Academic Press Inc. 1958. study of epidermal carcinoma induced by 15. SELBV, C. C.: An electron microscope study methyl eholanthrene in the mouse. J. Nat. of thin sections of human skin. II. Super- Cancer. Inst., 13: 1473—1479, 1953.