British Journal of Dermatology (1976) 94? 339-
Brief Communication A rapid tnethod for identifying nucleated horn cells Rupert Hallam Department of Dermatology, Hallamshire Hospital, and Department of Human Biology and Anatomy, University of Sheffield, Sheffield 10
F.C.TRING
Fluorescent microscopy has been widely used in the biological field but only occasionally as an adjunct to the usual methods applied to skin biopsies (Jarrett, Bligh & Hardy, 1956)- Similarly, although cytological examination is almost routinely carried out in several medical specialities, it is not regularly performed in examinations of skin.
FIGURE I. Micrograph of parakeratotic stratum corneum from psoriatic plaque stained with diamidinodiphenylamine dihydrochloride for approx. i min. (Magnification x iSo.)
Goldschmidt & Thew (1972) published an analysis of cytological abnormalities which they found in horn cells taken from a large series of patients with orthokeratotic and parakeratotic disease (i.e. psoriasis, seborrhoeic dermatitis and various types of dermatitis-eczema). A modified Giemsa stain technique was used to identify nuclei in horn cells obtained by several sampling methods. Significant differences between psoriasis and most other dermatoses in the frequency of halo and nucleated horn cells were reported. However, the method had a serious disadvantage for routine and extensive use because of the long time necessary to obtain reliable results with a quixotic stain. Tring & Murgatroyd (1976) have recently described the convenience and extremely rapid action of a fluorescent stain, diamidinodiphenylamine dihydrochloride. This compound was one of several aromatic diamidines deliberately incorporated into synthetic media to suppress contaminants of H. pertussis cultures (Lacey, 1951; Nicholson & Turner, 1954). A fortuitous observation that diamidmo339
34°
Brief communication
diphenylamine dihydrochloride selectively stained DNA prompted its use as a stain to identify nuclei and DNA residues in horn cells. Adhesive slides using double coated cellophane tape can be used to strip samples of horn cells. The cell coated slide is immersed in a Coplin jar containing a 0-2" „ solution of diamidinodiphenylamine dihydrochloride for l-i min. After washing under tap water for 2-3 min the sample is examined under a fluorescent microscope equipped with suitable filters to provide ultraviolet excitation. Nuclei and nuclear particles brilliantly fluoresce (Fig. i) and can be rapidly identified. This method is currently being used to evaluate various topical applications and their ability to modify parakeratosis. Preliminary experiments also indicate that the method can be used to identify r.apidly certain cutaneous fungal infections. REFERENCES GoLDSCHMlDT, H. & THEW, M.A. (1972) Exfoliative cytology of psoriasis and other common dermatoses. Archives of Dermatology, 106, 476. JAHRETT,A., BLIGH,A.& HARDY, J.A. (1956) FluoTescent micToscopy of ihc human skin. British Journal of Dermatology, 6 8 , I I I .
LACEY, B.W. (1951) Selective media for H. pertussis and H. parapertussis. Journal of General Microbiology, 5, VI. NICHOLSON, D.E. & TURNER, G.C. (1954) A selective medium for the primary isolation of W. pertussis and H. parapertussis. Journal of General Microbiology, 11, 91, TRING, F.C. & MURGATROYD, L.B. (1976) A new fluorescent stain for cell nuclei. Stain Technology (in press).
Brief Communication A rapid tnethod for identifying nucleated horn cells Rupert Hallam Department of Dermatology, Hallamshire Hospital, and Department of Human Biology and Anatomy, University of Sheffield, Sheffield 10
F.C.TRING
Fluorescent microscopy has been widely used in the biological field but only occasionally as an adjunct to the usual methods applied to skin biopsies (Jarrett, Bligh & Hardy, 1956)- Similarly, although cytological examination is almost routinely carried out in several medical specialities, it is not regularly performed in examinations of skin.
FIGURE I. Micrograph of parakeratotic stratum corneum from psoriatic plaque stained with diamidinodiphenylamine dihydrochloride for approx. i min. (Magnification x iSo.)
Goldschmidt & Thew (1972) published an analysis of cytological abnormalities which they found in horn cells taken from a large series of patients with orthokeratotic and parakeratotic disease (i.e. psoriasis, seborrhoeic dermatitis and various types of dermatitis-eczema). A modified Giemsa stain technique was used to identify nuclei in horn cells obtained by several sampling methods. Significant differences between psoriasis and most other dermatoses in the frequency of halo and nucleated horn cells were reported. However, the method had a serious disadvantage for routine and extensive use because of the long time necessary to obtain reliable results with a quixotic stain. Tring & Murgatroyd (1976) have recently described the convenience and extremely rapid action of a fluorescent stain, diamidinodiphenylamine dihydrochloride. This compound was one of several aromatic diamidines deliberately incorporated into synthetic media to suppress contaminants of H. pertussis cultures (Lacey, 1951; Nicholson & Turner, 1954). A fortuitous observation that diamidmo339
34°
Brief communication
diphenylamine dihydrochloride selectively stained DNA prompted its use as a stain to identify nuclei and DNA residues in horn cells. Adhesive slides using double coated cellophane tape can be used to strip samples of horn cells. The cell coated slide is immersed in a Coplin jar containing a 0-2" „ solution of diamidinodiphenylamine dihydrochloride for l-i min. After washing under tap water for 2-3 min the sample is examined under a fluorescent microscope equipped with suitable filters to provide ultraviolet excitation. Nuclei and nuclear particles brilliantly fluoresce (Fig. i) and can be rapidly identified. This method is currently being used to evaluate various topical applications and their ability to modify parakeratosis. Preliminary experiments also indicate that the method can be used to identify r.apidly certain cutaneous fungal infections. REFERENCES GoLDSCHMlDT, H. & THEW, M.A. (1972) Exfoliative cytology of psoriasis and other common dermatoses. Archives of Dermatology, 106, 476. JAHRETT,A., BLIGH,A.& HARDY, J.A. (1956) FluoTescent micToscopy of ihc human skin. British Journal of Dermatology, 6 8 , I I I .
LACEY, B.W. (1951) Selective media for H. pertussis and H. parapertussis. Journal of General Microbiology, 5, VI. NICHOLSON, D.E. & TURNER, G.C. (1954) A selective medium for the primary isolation of W. pertussis and H. parapertussis. Journal of General Microbiology, 11, 91, TRING, F.C. & MURGATROYD, L.B. (1976) A new fluorescent stain for cell nuclei. Stain Technology (in press).
