Ultrapath XVIII 2016, Lisboa


Precongress Course

The Skin: Layers of information below the Surface


Dr. rer. nat. Ingrid Haußer-Siller

Institute of Pathology (IPH), University Clinic Heidelberg, Germany

Electron Microscopy Core Facility (EMCF), Heidelberg University, Germany

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What can a skin biopsy tell us?

We get improved comprehension of cellular processes, development, differentiation and get insight in pathogenetic pathways, enabling eventually diagnostic EM.

To date, EM diagnostics in dermatology is asked for mainly in rare diseases, that is genodermatoses: heritable bullous diseases/epidermolyses; heritable keratinization disorders; heritable connective tissue disorders, as well for some special entities (e.g. CADASIL, lysosomal storage diseases, some rare histiocytoses, deposition disorders/argyrosis, gadolinium deposits in nephrogenic systemic fibrosis), not to forget virus diagnostics by negative staining approaches. We will concentrate on some basic aspects and discuss examples:


TEM Preparation of skin biopsies

Prerequisites for the evaluation of morphological phenotypes are standardized fixation protocols, yielding standardized artefacts; awareness of individual variability; and comparison of multiple matched specimens.

A standard protocol for conventional transmission electron microscopy (TEM) of skin comprises chemical fixation with glutaraldehyde and osmium tetroxide followed by embedding in epoxy resin. Semithin survey sections to preselect area of interest are stained with methylene blue or toluidin blue, ultrathin section are treated with uranylacetate and lead citrate (for connective tissue components: pretreatment of ultrathin sections with tannic acid).


Morphological aspects of skin development and terminal differentiation

Skin/epidermis and extra-embryonic membranes are continuous and derive from the ectoderm. The epidermis develops from embryonic epidermis (<60d, basal keratinocytes and periderm) to embryonic-fetal transition (ca. 60d, third intermediate cell layer) to a four-layer epithelium at the end of the first trimester (ca. 90d, second intermediate layer). Keratinisation or terminal differentiation, consists in a gene-controlled well-defined sequence of interacting structural proteins and enzymes. It starts at ca. 20 WMA around the orifices of hair follicles, and at ca. 24 WMA the interfollicular epidermis is continuously keratinized. The overall postnatal epidermis is stratified in basal layer (with the dermoepidermal junction as border to dermal connective tissue), spinous layer, granular layer and horny layer. Main cell types are keratinocytes with a cytoskeleton of keratin tonofilaments (keratins are expressed).

Important pecial structural components of keratinization (granular layer) are keratohyalin granules and lamellar bodies.


Diagnostic EM of heritable keratinisation disorders

Ichthyoses and related keratinization display a broad clinical heterogeneity. Some entities show distinct ultrastructural markers having contributed in many cases to the identification of candidate genes, and enabling diagnostic classification. In most cases light microscopy is not informative (enough). Examples are autosomal dominant ichthyosis vulgaris (with lack of granular layer/”crumbly” keratohyalin, FLG mutations); some autosomal-recessive lamellar ichthyoses ( TGM1-deficient type with cholesterol clefts within horny lamellae); EM type III with granular layer vesicle complexes and membranes, mostly NIPAL4 mutations; ichthyosis prematurity syndrome with trilamellar deposis in granular layer and horny cells, FATP4 mutations; Harlekin ichthyosis with lamellar body defect, ABCA12 mutations; PNPLA1 lamellar ichthyosis with clefts in horny cells and corresponding membraneous material deposits in granular cells); epidermolytic ichthyosis, dominant and recessive (suprabasal keratin filament aggregations and clumping; mutations in K1/10).


Morphology of normal dermal connective tissue compared to aberrations of dermal connective tissue components in some conditions and heritable connective tissue disorders


The extracellular matrix of the dermis consists of an intricate system of components and macromolecules typical of all connective tissues. Therefore, “skin is a window” for evaluating abnormalities caused by internal and external factors modulating matrix metabolism. Ground substance material is rarely feasible by conventional TEM because of the preparation procedure. Main components to be investigated are the two fibrous components, collagen fibrils and elastic fibres. Collagen fibrils, conferring tensile strength, can be best evaluated within the reticular dermis, where they are present with uniform diameters, circular cross sections and characteristic banding of longitudinal sections in compact bundles. Elastic material is distributed throughout the dermis in elastic fibres with a homogeneous elastin component with elastic property, within a network of elastic microfibrils (built of numerous proteins). For evaluation of the dermal architecture and ultrastructural morphology, several aspects have to be kept in mind: The width of dermal connective tissue varies with age; a thin dermis of a newborn maybe mixed up with the typical architecture of vascular Ehlers-Danlos Syndrome (EDS). In papillary dermis, collagen bundles are loosely packed and display considerable variability of fibrils’ diameter (as in vascular EDS).

In principle, collagen fibrils show only a limited repertoire of ultrastructural changes which have to be combined with clinical symptoms to a pattern defining or typical for a heritable connective tissue disorder. General differential aberrations are characteristic for EDS types I-IV, but qualitatively similar collagen fibril changes are seen adjacent to aberrant elastic fibres in pseudoxanthoma elasticum and in restricted areas not prone to normal turnover and physiological exchange as around blood vessels, smooth muscle cells and in lymphedema. Unique collagen fibril aberrations are caused by ADAMTS2 mutation in dermatosparaxis (EDS VIIC).

Elastic fibres show signs of degradation in papillary dermis due to light/UV exposure (actinic elastosis) and are increased in aged skin (overlap with vascular EDS!). Focal deposits within the elastin component is seen in PXE heterozygotes, in patients with spontaneous dissections and in the lamina elastica of arterioles in aged skin. Rarefied elastic material is a hallmark of cutis laxa, with rare small elastic fibres with otherwise normal aspect, or fibres with decreased deposition of elastin component or fibres with separate deposition of elastin and elastic microfibrils. A series of genes involved in general metabolic pathways also cause rare variants of cutis laxa syndromes.

Taken together, clinical history, body localisation of biopsy and area of interest have to be thoroughly compiled to get overall comprehensive morphological results.






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