View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector PRIMARY FLUORESCENCE OF NORMAL AND PATHOLOGIC KERATIN * KARLSTEINER, M.D. WITH THE TECHNICAL AID OF ETHEL StJBIN, BA. The present study was undertaken in order to(fig. 1), the keratogenous zone, the intracorneal elucidate certain difficulties encountered in asweat ducts, the collagen and particularly the previously published study of the secondaryelastic fibers (fig. 2), the eponychium, and the fluorescence of normal and pathologic keratin-secretory sweat granules. The sweat granules izations (10). These difficulties concerned differ-fluoresce strongly, all other structures moder- ences in intensity of fluorescence and variabilitiesately but distinctly. of orthochromatic and metachromatic staining, In comparison with sections stained with acri- particularly with acridine orange. A comparisondine orange, the unstained specimens reveal a of primary and secondary fluorescence of identi-conspicuous lack of fluorescence of nuclear struc- cal or similar keratin promised to offer sometures, especially nucleoli,of keratohyaline, explanation for certain of these problems. trichohyaline, Huxley's layer of the inner root The literature on primary fluorescence dis-sheath, and of the nail plate. Collagen shows closed only a few fundamental reports on normalweak fluorescence only, and the epidermal cyto- skin (1, 6, 2, 7, 4, 5), some of them of relativelyplasm almost no fluorescence at all. old date. No systematic study of primary fluo- Parakeratoses and Hyperkeratoses. These types rescence in skin diseases could be found, particu-of keratinization often display strong fluorescence larly not in disease with pathologic cornification.(fig. 3). On the other hand, although always It was, therefore, decided to review the primarypresent, the fluorescence of the horny layers is fluorescence of normal keratinization first andsometimes weak and even less than in normal then that of pathologic keratin structures. skin (fig. 4). Horn pearls and onion-shaped in- clusions of benign as well as of malignant lesions MATERIAL AND METHOD (seborrheic keratoses, kerato-acanthomas, spi- The material consisted of the biopsy specimensnous cell epitheliomas, etc., fig. 3, 5, 6) generally used in the previous study (10), i.e., 10 specimensshow strong fluorescence. Parakeratotic layers of normal skin and 80 specimens from 21 diseaseare sometimes more conspicuously fluorescent states. In addition, sections of a keratosis follicu-than hyperkeratoses, in tissue sections as well as laris Darier, and embedded and sectioned scales of psoriasis and callous were examined. The prepa- in scales (fig. 7, 8, 9). However, when hyijer- ration of all of these specimens was the same askeratoses have a loose fibrous structure they are already reported (10) except that no stains werealso strongly fluorescent (fig. 10), while densely applied. Likewise, the same "Large Fluorescencepacked parakeratoses usually reveal weak fluo- Equipment" (Zeiss) was used. The basic principles of fluorescence microscopyrescence only. Keratogenous zones fluoresce have been fully discussed by de Lerma (3) andstrongly (fig. 7, 11). There is a lack of fluorescence Price and Schwartz (8). of nuclear structures, including parakeratotic RESULTS pyknoses (fig. 7), and of keratohyaline (fig. 3, 8). Dyskerato.ses. The primary fluorescence of Normal Skin. Primary fluorescence is shownthese lesions is completely different from that of by the keratin fibers of the stratum corneumnormal skin and of hyper- and parakeratotic *Fromthe Medical Service, Section of Derma-lesions. Benign, as well as malignant dyskera- tology, Veterans Administration Hospital, Brook- lyn, New York, and the Department of Medicine,toses generally show moderate, or at least weak, Division of Dermatology and Syphilology, Statebut always distinct fluorescence of nucleolar University of New York, Downstate Medicaland/or nuclear structures and of cell membranes Center at New York City. This study was supported by a grant from the(fig. 5, 6, 9, 10, 11, 12). Often, not only the National Institutes of Health, U. S. Public Healthdyskeratotic cells but the entire epidermis shows Service, Grant RG-4961. Received for publication July 14, 1961. this abnormal fluorescence (fig. 11). However, in 357 358 THEJOURNAL OF INVESTIGATIVE IIIERMATOLOGY All figures are about 120% enlarged from the Fm. 4. Scale from callus. X 32 magnifications of the original films as given in the captions. FIG. 1. Keratin Fibers of the Stratum Corneum. Normal sole. X 104. Fm. 5. Kerato-Acanthoma. Dyskeratotie cells and horny inclusions. X 26. FIG. 2. Normal hairs, elastic fibers and colla- gen. X 32. FIG. 3. Seborrheic keratosis. ilyperkeratosis FIG. 6. Spinous cell epithelioma. Dyskeratoses and horn inclusions. X 26. and horn pearls. X 32. FLUORESCENCE OF KERATIN 359 comes from the stratum corneum and other kera- tinization products. In this connection, it is pertinent that keratohyaline and trichohyaline which show very strong and brilliant ortho. chromatic secondary fluorescence with acridine orange, do not exhibit any primary fluorescence. Otherwise, a comparison with secondary fluo- FIG. 7. Psoriasis. Parakeratosis and microab- scess. X 32. FIG. 9. Keratosis follicularis Darier. Dyskera. toses. X 128. .ra S..• a FIG. 8. Lichen planus. Hyperkeratosis. X 32 these instances, the dyskeratotic cells fluoresce more strongly than the other epidermis. DISCUSSION Various normal and pathologic structur of the skin emit a more or less intensive primary FIG. 10. Leukoplakia. Dyskeratotic nuclei and fluorescence. A major part of this fluorescencecell membranes. X 128. 360 THEJOURNAL OF INVESTIGATIVE DERMATOLOGY globular keratins of dyskeratoses (fig. 9 through 12) excludes the assumption that this fluo- rescence could be a property of fibrous proteins only. The pathologic kcratin structures with primary fluorescence include all of the various types of abnormal keratinizations: hyper- and para- keratoses, ichthyosis, "superkeratinizations" (9) L.- - - and dyskeratoses. Among these pathologic structures the primary fluorescence of the dyskeratoses, both benign and malignant, is particularly interesting, not Fig. 11. Bowen's disease. Dyskeratoses. X 104only because it agrees with the presumably ke- ratinous character of these structures but also because this fluorescence emanates from the nuclei and cell membranes (fig. 11, 12), a fact which is in conspicuous contrast to the other- wise general lack of nuclear and nucleolar fluo- rescence. For this reason, the primary fluo- rescence of dyskeratoses may even be considered as having diagnostic value. There is, in general, a regular relationship between primary and secondary fluorescence of horn structures. Those keratins which exhibit orthoehromatic orange fluorescence with acridine orange, reveal a strong primary fluorescence and, vice versa, secondary acridine orange fluores- cence of metachromatic green color usually corre- sponds to weak or absent primary fluorescence. For a demonstration of these relationships the figures of this paper may be compared with the corresponding figures of the same structures in the previous paper on secondary fluorescence (e.g.figs.3 and 4 of this paper with figs. 13 and Fm. 12. Bowen's disease. Dyskeratotic nuclei8 of the previous paper). and cell membranes. X 320. However, some of the pathologic keratini- zations do not conform to this rule of "strong rescence reveals that the only other structurespriniary-orthochromatic secondary fluorescence" which do not fluoresce, if not stained, are theand "weak primary-metachromatie secondary cell nuclei and the nucleoli of the epidermis,fluorescence." Parakeratoses often show strong Huxley's layer and the nail plate. Incidentally,primary but metachromatic secondary fluores- the lack of primary fluorescence of the nucleolicence, and in some hyperkeratoses there is weak excludes ribonucleic acid from the mechanism ofor moderate but orthochromatic secondary this fluorescence. fluorescence. In these instances, the texture of Except for the various keratins, the elasticthe keratinous structure seems to be decisive for fibers also show distinctive primary fluorescence.the intensity and quality of its primary and For this reason, the primary fluorescence ofsecondary fluorescence. Loose, fibrous or flaky fibers, elastic, keratin or others, cannot be relatedkeratia is, without exception, brilliantly ortho- to a periodic molecular structure since elasticchromatic (e.g.,psoriatickeratin fibers, flakes in fibers do not have such a structure (9). kerato-acanthomas, spinous cell epitheliomas, On the other hand, the primary fluorescenceetc.), while dense, compact masses of horny of lipid sweat granules (7), inflammatory cells oflayers show dull metachromasia (e.g.callus,some cutaneous infiltrates (fig. 7, 8), and the abnormalchronic dermatitides, etc.). FLUORESCENCE OF KERATIN 361 SUMMARY 2. CORNELEET, T. AND POPPER, H.: Properties of human skin revealed by fluorescence micros- Almost all normal and pathologic keratinous copy. The normal skin; the vitamin A con- structures exhibit primary fluorescence. The in- tent of the skin. AMAArch.Derm., 46: 59— 65, 1942. tensity of this fluorescence ranges from weak and 3. DE LERMA, B.: Die Anwendung von Fluores- dull to bright and brilliant. zenzlicht in der Histochemie, in Handbuch der Histochemie, Vol. 1, Pt. 1, pp. 78—159. In contrast to secondary fluorescence, cell Stuttgart, Gustav Fischer, 1958. nuclei, nucleoli,