Issue 26
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Category		Title		Author
Newsletter		Taking a look into skin		Ronald Marks

Conventional microscopy which for the most part inspects vertical sections, does not do justice to this delicate structure. Fixation, dehydration, embedding and mechanical sectioning shatter and distort the stratum corneum and anyway do not allow a view of the structure in the dimension in which it functions - the horizontal dimension.

Fig. 1: Diagram to show cyanoacrylate glue being pressed onto skin by glass microscope slide and below the final skin surface biopsy specimen with transparent cyanoacrylate glue and stratum corneum specimen.

Our interest and good fortune met when we came across the skin surface biopsy technique (1). This technique relies on the use of rapidly bonding cyanoacrylate adhesives to remove a thin layer of SC. Several of these adhesives including methyl, ethyl and octyl cyanoacrylate have all been used for this purpose. They rapidly polymerise with slight pressure and moisture and form a very strong optically transparent bond. In practice a drop of the adhesive is placed on a glass microscope slide which is then pressed against the skin site to be sampled. After some 20-seconds the slide is "rolled off" the skin taking with it an intact layer of SC some 2 or 3 cells thick with it (Figure 1). The SC is undisturbed and is precisely the same on the microscope slide as it is in vivo.

Polymerised cyanoacrylate adhesive has very similar optical properties to glass so that is it easy to inspect the SC specimen by light microscopy. Taking skin surface biopsies is virtually painless - the worst discomfort arises from trapping hair in the adhesive and then yanking inadvertently these out. The adhesives appear non toxic and anyway are completely removed with the taking of the specimen. Indeed various cyanoacrylates have been used as tissue cements for some years and recently there has been a resurgence of interest in octyl cyanoacrylate to seal small lesions in what is sometimes termed needless suture(2). As already mentioned cyanoacrylates are not toxic, but problems can arise from the rapid bonding that occurs when the adhesive contacts skin. The classic problem is that the fingers become stuck together - although this is easily solved by dunking the fingers in a beaker of acetone which instantly dissolves the adhesive. Particular care must be taken when sampling facial skin. To prevent the adhesive running into the eyes, the procedure should only be performed with the patient sitting up.

Fig. 2: Photomicrograph of skin surface biopsy on glass slide and arm from which it has been taken.

Skin surface morphology (3)

Skin surface biopsies (SSB) from the arms and legs and from some sites on the trunk have a characteristic geometric pattern with the surface arranged in a series of rhomboids (Figure 2). When the skin is put on the stretch, the rhomboids become narrower and if the change in width of these geometric figures is measured it can be used to assess compliance of the stratum corneum. If the degree of extension is known, this can be used as a simple test of mechanical function.

There are striking regional differences in the skin surface pattern (3). The palms and soles, for example, show the dermatoglyphic ridges which make up the characteristically unique 'finger prints'. At the peaks of the ridges there are the openings of the eccrine ducts which are quite easy to see. In fact it is quite difficult to obtain skin surface biopsies from the palms and soles because the bonding strength of the palms and soles is often equal to or even greater than the bonding strength of the adhesive. Gently hydrating the region first makes the obtaining of an SSB from the palm or sole somewhat easier and with a little patience and perseverance it is usually possible to successfully obtain a specimen.

Fig. 3: Photomicrograph of skin surface biopsy from back of hand showing typical rhomboidal pattern (unstained x25).

Facial skin has interesting surface morphology which differs markedly from limb and trunk skin by not having the same rhomboidal patterning. In male beard areas there is prominence of the hair follicles with curved ridges arranged around these in marked contrast to the delicate hair follicle openings seen on the forehead and cheek.

In situ microbiology of skin

The SSB technique is ideal for the study of the in situ microbiology of skin (4). Not only are SC invaders easily revealed, but the density of the infection and their exact positioning within the SC can also be studied. When the SSB is stained with periodic acid schiff reagent an excellent view may be obtained of ringworm fungi, pityriasis versicolor, candida species and the erythrasma micro-organisms (Figure 3, Figure 4 and Figure 5). The taking of the SSB and its subsequent staining is easier to perform and the micro-organisms are easier to see than the usual skin scrapings and potassium hydroxide 'clearing'. SSB is my preferred diagnostic technique for suspected ringworm. It is also possible to culture the fungus by 'reversing' one of the SSB's into the Sabouraud culture medium. If for some reason a higher magnification is needed with a more detailed view of the relationship between the micro-organism and the SC then scanning electron microscopy (SEM) can be employed. A small portion of the SSB is cut to size and stuck to an SEM stub before it is 'coated' with gold and then viewed in the SEM.

Fig. 4: Photomicrograph of skin surface biopsy stained by periodic acid schiff reagent showing mycelium (red) from ringworm (x50).

Histochemical applications

The SSB technique can be used to trace the presence of particular substances or chemical activities (5). Amongst the simplest of these is visualisation of melanin particles with silver stain. This is of major use clinically when there is some doubt as to the nature of brown/black pigmentation of the skin. Silver staining will show abundant black melanin particles while staining with potassium ferricyanide (Prussian Blue reaction) will show bluish clumps with blood pigments.

Sebum can be demonstrated using lipid stains and it is possible to produce a rough estimate of the rate of sebum secretion using this technique. At the start of the investigation the forehead is washed and wiped clean with a lipid solvent - isopropyl alcohol swabs are quite suitable and convenient for the purpose. Then an SSB is taken from one side of the forehead and 30 minutes later another is taken from an adjoining site. At one and two hours other SSBs are taken from other adjoining sites across the forehead. All the SSBs are then stained together for the same length of time with a lipid stain such as Sudan red. The density of the lipid staining material and the area of staining is an indication of the amount of sebum secreted during the interval between the initial cleaning and the taking of the SSB.

Sweat can also be detected by treating the SSB with one of the reagents that reveals its presence. The SSB specimen is taken at a defined time after cleaning and drying the site and then stained by the starch - iodine method or with orthophthalaldehyde or one other of the sweat revealing substances. This can be useful to check for the adequacy of therapeutic manoeuvres to stop excess sweating such as sympathectomy or the injection of Botulirum Toxin intracutaneously.

Staining the SSBs with haematoxylin and eosin shows up nuclei in parakeratotic SC. This can have diagnostic importance in distinguishing psoriasis from chronic eczema - the latter not having aggregates of polymorphs within the SC. It can also be useful in confirming a diagnosis of solar keratosis as the presence of abnormal nuclei is characteristic of epidermal dysplasia.

Fig. 5: Photomicrograph of skin surface biopsy stained with periodic acid schiff reagent to show pseudo mycelium and clusters of spores from lesion of pityriasis versicolour (x50).

A variety of enzyme histochemical tests have been used on SSBs, most of these having a research application. For example, it has been possible to study the metabolism of various dermatophyte fungi present in SSB samples using enzyme histochemical reactions such as succinic dehydrogenase and lactic dehydrogenase. This is not quite such an esoteric exercise as it may sound as the mode of action of antifungal agents can be checked in this way.

Studies of percorneal penetration

The skin surface biopsy technique is ideal for the study of the penetration of drugs into the skin (6, 7, 8). It is often convenient to use a radiolabelled drug so that an estimate of the concentration of the drug in an SSB sample can be made by solubilising part of the SSB and "counting" the resulting fluid in a scintillation counter. The same area of SSB is assessed from each SSB so that comparisons can be made. Other analytic techniques also can be employed including the radioimmune assay method and HPLC. SSBs are taken at increasing depths right through the SC at different times in adjoining sites. It is thus possible to build a profile of the concentrations throughout the SC at various time points. By taking a full thickness skin biopsy at the end of the study a complete profile of the penetration can be constructed.

We have investigated the penetration of many drugs into the skin in this way including corticosteroids, antifungal imidazoles and nonsteroidal anti-inflammatory reagents.

Investigation of follicles contents and comedogenicity

When an SSB is taken from the face or upper trunk of the follecular contents are also removed and this has proved of great value to investigators such as Cunliffe and to Kligman in studying the pathogenesis of acne and the results of treatment of this disease.

Some topically applied agents such as cocoa butter and isopropyl myristate irritate the follicle and cause the formation of comedones and even folliculitis or acne in some instances. It is clearly of importance to the manufacturers of topical agents to know that they do not have this comedogenic or acnegenic potential. Originally a rabbit 'ear test' was employed to detect agents with this propensity (comedogenic agents) but this test is now hardly ever used as it is considered that human based tests are better in all respects. We have employed the SSB method to examine human skin onto which preparations whose comedogenic status is unclear have been applied. The skin of the back of human volunteer subjects is used. These subjects are selected because they have a degree of clinical acne. The materials are applied under occlusion for a 4 week period alongside a known comedogenic agent as well as a negative control. At the end of the period of application SSB's are taken from the test sites and examined using low power microscopy. They are scored on an arbitrary scale according to the amount of impacted horn in the follicular lumen.

DNA analysis

A recent adaptation of the SSB technique has been to characterise the amount and type of DNA present in the stratum corneum. It was thought that epidermal nuclear DNA was completely destroyed in the granular cell layer and absent from the stratum corneum but our studies have shown that this is not so. It was possible to identify native DNA in SSBs from normal skin after PCR (polymerase chain reaction) and very much more was found as might be expected in SSBs from psoriatic skin(9). The ability to look at human DNA using this "non invasive" technique opens up a multitude of investigative possibilities and we look forward to seeing how this particular application develops.

Conclusion

The technique of SSB allows a detailed examination of the stratum corneum as it exists in vivo. Its ability to allow both detailed microscopic inspection and analysis of its contents is unique and permits many diagnostic and research applications. New ways of employing this simple non-invasive method are continually coming to light and although nearly 30 year old the SSB technique looks like it will continue to produce fresh information on the stratum corneum.

References

1. Marks, R., Dawber, R. Skin surface biopsy: An improved technique for the examination of the horny layer. Br J Dermatol 1971; 84: 117-123.
2. Maw, J., Quinn, J., Ramotar, K., Wenckebach, G., Wells, G., Octoylcyanoacrylate tissue adhesives versus suture wound repair in a contaminated wound model. Surgery 1997; 122:69-72.
3. Marks, R., Saylan, T. The surface structure of the stratum corneum. Acta Derm (Stockholm) (1972), 52: 119-125.
4. Marks, R. , Dawber, RPR. In situ microbiology of the stratum corneum. Arch Derm (1972), 105:216-221.
5. Marks, R., Histochemical applications of skin surface biopsy. Br J Dermatol 1972; 86: 20-26.
6. Marks, R., Dykes, P.J., Williams, D.L., Thorne, E.G., Lufrano, L. In vivo stratum corneum pharmacokinetics of econazole following once and twice daily application to human skin. J Dermatol Treat (1990), 1: 195-197.
7. Marks, R., Dykes, P.J. Plasma and cutaneous drug levels after topical application of Piroxicam gel: a study in healthy volunteers. Skin Pharmacol (1994), 7: 340-344.
8. Dykes, P., Hill, S., Marks, R. Pharmacokinetics of topically applied metronidazole in two different formulations. Skin Pharmacol (1997), 10: 28-33.
9. Yahya, H. Estimating the DNA content of stratum corneum by skin surface. MSc in Dermatology, UWCM 1999.

Author

Professor Ronald Marks FRCP FRCPath



Professor Ronald Marks is Emeritus Professor at the University of Wales College of Medicine. He is Clinical Professor at the University of Miami School of Medicine and is based at Skin Care Cardiff Ltd., South Wales, UK. He has authored extensively and is the author/co-author of over 35 books and 350 articles in scientific and medical journals. He has organised several international symposia and has been editor/co-editor of several dermatology journals. Just recently he has been chairman of the Scientific Committee of the Stratum Corneum III congress, which was very successfully organized (Basel, September 2001).


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