Our stratum corneum (SC) provides vital protection against the potentially injurious
external environment. It acts as a membrane controlling the flow of water, xenobiotics,
gases and radiation in and out of the body's internal environment.
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.
1a: 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. |
 |
| Fig.
3: Photomicrograph of skin surface biopsy from
back of hand showing typical rhomboidal pattern (unstained x25). |
 |
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.
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.
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.
| 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). |
 |
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.
