Rapid Lump Examination (RLE) – eine neue Technik in der mikroskopisch‐kontrollierten Chirurgie?

Background:“Micrographic surgery“ spares tissue and results in fewer recurrences. Various techniques have been described using paraffin‐embedded and cryostat sections or even optical sections from ex vivo confocal laser scanning microscopy. The presented technique is the rapid direct microscopy of the surface of a specimen (lump) for a pathological examination (RLE). Methods: Fresh surgical tissue was stained first without sectioning and then was examined directly under microscope. A 95 sec staining protocol for basal cell carcinoma (BCC) was established. 129 specimens were examined using a digital microscope and 78 specimens using a stereo microscope. Results: RLE had a high diagnostic accuracy compared to paraffin‐embedded H&E‐stained sections. Sensitivity and specificity of RLE was 91 % and 90 % for the digital and 90 % and 94 % for the stereo microscope. In addition we developed a 7 min RLE‐immunohistology protocol using the BerEp4‐antibody. Discussion: RLE is a simple and accurate technique for fresh tissue examination. Here, the technique has been established for BCC but the principle may also be transferred to histological bedside diagnosis of other tumors. The technique does not alter or destroy tissue, so that after RLE was done subsequent conventional histology is still possible. RLE might yield a time‐ and cost‐saving diagnosis in micrographic surgery.     

The utility of elastic Verhoeff‐Van Gieson staining in dermatopathology

Elastic fibers are important components of the skin and are responsible for skin elasticity. Genetic defects are well‐known in numerous hereditary elastic tissue disorders and skin biopsies are often the first step in the evaluation of those disorders. Verhoeff‐Van Gieson elastic staining is a simple method that is used for visualizing elastic fibers. With the development of modern immunohistochemical methods, the value of routine histochemical staining is sometimes underestimated. Histochemical stains are less expensive, easy to perform and help to resolve numerous diagnostic quandaries in dermatopathology. This article focuses on the value of elastic tissue staining in dermatopathology, with a focus on primary elastic tissue disorders, alopecia, inflammatory skin disorders and neoplastic proliferations.     

Characterization and quantification of wound‐induced hair follicle neogenesis using in vivo confocal scanning laser microscopy

Background: In vivo confocal scanning laser microscopy (CSLM) is a recently developed non‐invasive technique for visualizing microscopic structures with the skin. CSLM has been used to characterize proliferative and inflammatory skin diseases, neoplastic skin lesions and pigmented lesions. Objective: Here, we assessed the ability of CSLM to evaluate the formation of neogenic hair follicles after a full‐thickness wound in mice. Methods: Full‐thickness wounds were made on the dorsal skin of 3‐week‐old mice. After scab detachment (SD), the number, width, length, space and volume of neogenic hair follicles were analyzed using CSLM. The results were compared with those from conventional methods, including staining for alkaline phosphatase (AP) and keratin 17 (K17) as well as histology. Results: Quantification of neogenic hair follicles using CSLM compared favorably with the results from direct measurements on isolated epidermal tissue after immunostaining for K17, a marker for the epithelial portion of new hair follicles. CSLM detected 89% of K17‐stained follicles. CSLM more accurately quantified the number of new follicles compared with AP staining, which detects the dermal portion of the new follicle. The width and length measurement from CSLM and histology were very close and correlated with each other. The minimum length of a neogenic hair follicle that could be detected by CSLM was 21 μm. The space between neogenic hair follicles was decreased in histological sections compared with CSLM. Conclusion: CSLM is an accurate and valuable method for counting and measuring neogenic hair follicles non‐invasively. CSLM produces images similar to histology in mice. Measurements of microstructures using CSLM more accurately reflect actual sizes as this technique avoids fixation artifacts. In vivo visualization of developing follicles with CSLM allows the detection of serial changes in hair follicle formation, thus conserving the numbers of mice required for studies and improving the detection of temporal changes in developing hair follicles.     

Histomorphologic correlation with routine histology and optical coherence tomography

BACKGROUND: Optical coherence tomography (OCT) is a new and promising diagnostic technique for investigation of skin tumours. We describe a method that makes evaluation and definition of specific morphologic structures of skin tumours via OCT more accurate. MATERIALS AND METHOD: We investigated three patients with basal cell carcinoma and three patients with melanocytic nevi. Three-dimensional (3D) images were obtained from these skin tumours via OCT according to previously applied marks, which were tattooed with special histological marking dye after excision of the tumours. Corresponding to these marks, we investigated serial histological sections (haematoxylin&eosin staining). RESULTS: We could prove similar morphological structures both in OCT and histology. Due to tissue deformation, the compared measurements of structures like cell nests or epidermal thickness were slightly deviated. However, by this method we could prove similar tissue formations in OCT and histology. CONCLUSIONS: Due to the deformation by histological processing and slightly different sectioning levels, the comparison of histological pictures and OCT images seems difficult. Nevertheless, in two cases it was possible to demonstrate the same morphological structures with OCT imaging and histological investigation. Our method could play an important role for further evaluation of OCT images. We estimate better evaluation of OCT imaging using a 3D reconstruction method.     

Mikrographisch kontrollierte Chirurgie (3D‐Histologie) beim Melanom

Frederic E. Mohs was the first to apply micrographic surgery in cutaneous surgery. As a result in the English literature, micrographic surgery (3D‐histology) is usually called Mohs micrographic surgery (MMS). Techniques of 3D‐histology have been applied in various skin tumours. The method is most suitable for tumours with continuous spread. Excisions with 3D‐histology can be performed with smaller initial safety margins. Thus less normal tissue is excised and often better cosmetic results can be obtained. Techniques of 3D‐histology such as the Mohs technique (MMS) and the “Tuebingen Cake” technique are compared with routine histopathology. In addition, the current status of 3D‐histology in cutaneous melanoma is assessed. Prospective randomized studies on 3D‐histology in cutaneous melanoma are not available. Nevertheless, 3D‐histology should usually be employed for lentigo maligna melanoma. In functionally and cosmetically important areas (face, hand, foot) 3D‐histology may allow reduced safety margins in other histological types of cutaneous melanoma.     

Subtle intracorneal findings in inflammatory disorders: hyphae or not?

BACKGROUND: Spongiotic and lichenoid dermatitides are frequently stained with periodic acid-Schiff (PAS) stains to check for the presence of dermatophytes. PAS+ structures without a septate morphology are often seen with lichenoid dermatitides, however, their nature has not been previously characterized. METHODS: Fifteen consecutive biopsies of lichenoid and five spongiotic dermatitides were stained with hematoxylin and eosin (H&E), PAS, and antibodies to CD1a. RESULTS: Twelve of 15 lichenoid and none of the five spongiotic dermatitis showed PAS+ structures in the stratum corneum. Distinct septation or branching was not identified in these PAS+ structures. Eleven of 15 from the lichenoid group, but none from the spongiotic group, showed CD1a+ structures in the stratum corneum. This staining pattern suggests that the intracorneal structures represent the dendritic processes of Langerhans' cells (LCs) within the stratum corneum. CONCLUSIONS: PAS+ and CD1a+ structures are present in the stratum corneum of lichenoid, but not in spongiotic, dermatitis. This study morphologically confirms extension of LC dendrites into the stratum corneum in lichenoid but not in spongiotic dermatitides.     

Broad histopathologic patterns of non‐glabrous skin and glabrous skin from patients with a new variant of endemic pemphigus foliaceus‐part 1

A prospective, controlled epidemiologic survey performed in El Bagre, Colombia revealed a new variant of endemic pemphigus disease, occurring in a gold mining region. The disease resembled Senear‐Usher syndrome, and occurred in an endemic fashion. The aim of this study is to describe the most frequent histopathologic patterns in non‐glabrous skin and in glabrous skin observed in these patients, and their clinical correlation. The study was performed on non‐glabrous skin biopsies of 30 patients from the dominantly clinical affected areas (either on the chest, arms or face). Simultaneously, biopsies from the palms were obtained in 10 randomly chosen patients of the 30 total patients. The specimens were examined following hematoxylin and eosin (H&E) staining. The most common blisters observed were subcorneal, although in some cases intraspinous and subepidermal blisters were visualized. Our results showed a very heterogeneous pattern of histopathologic patterns in non‐glabrous skin, which seemed to correlate with the clinical features. The most common pattern was typical pemphigus foliaceus‐like, with some lupus erythematosus‐like features. A non‐specific, chronic dermatitis pattern prevailed in the clinically controlled patients taking daily corticosteroids. In the patients who have had the most severe and relapsing pemphigus, early sclerodermatous changes and scleredermoid alterations prevailed in their reticular dermis. In addition to the scleredermoid alterations, the reticular dermis showed a paucity of appendageal structures. On the contrary, in the palms, a similar pattern was seen in all cases, including thickening of the stratum corneum, hypergranulosis, edema in the papillary and reticular dermis and a dermal perivascular lymphocytic infiltrate. The direct immunofluorescence of the glabrous vs. the non‐glabrous skin also showed some differences. We conclude that the histopathologic features of this new variant of endemic pemphigus are complex, therefore, classical histopathologic features previously described for superficial, endemic pemphigus cannot be used alone to diagnose this disease. Howard MS, Yepes MM, Maldonado‐Estrada JG, Villa‐Robles E, Jaramillo A, Botero JH, Patiño PJ, Hashimoto T, Abreu‐Velez AM. Broad histopathologic patterns of non‐glabrous skin and glabrous skin from patients with a new variant of endemic pemphigus foliaceus‐part 1.     

‘Hints’ in the horn: diagnostic clues in the stratum corneum

The stratum corneum or horny layer is the uppermost layer of the epidermis, and is mainly responsible for the skin's barrier function. In spite of its complexity at the ultrastructural and molecular level, the features accessible to visualization on conventional histology are relatively limited. Nevertheless, knowledge of subtle clues that one may observe in the stratum corneum can prove useful in a wide range of situations in dermatopathology. We herein review a selection of common and rare entities in which the horny layer may reveal significantly important hints for the diagnosis. These clues include parakeratosis and its different patterns (focal, confluent, alternating, associated with spongiosis, epidermal hyperplasia or lichenoid changes), subcorneal acantholysis, infectious organisms in the stratum corneum (including fungal, bacterial and parasitic), thickening or thinning of the stratum corneum and the presence of different kinds of pigment. Even when normal, the horny layer may prove to be useful when seen in association with severe epidermal damage, a combination of features testifying to the acute nature of the underlying pathological process.     

Effects of a topical aqueous oxygen emulsion on collagen deposition and angiogenesis in a porcine deep partial‐thickness wound model

A porcine deep partial‐thickness wound model was used to evaluate the effects of a newly developed topical aqueous oxygen emulsion (TOE) on wound repair. The wounds were treated with TOE, which contains super‐saturated oxygen or vehicle control. Semiquantitative immunofluorescent staining was performed to examine protein production for type I and type III collagen and vascular endothelial growth factor (VEGF). Immunofluorescent staining revealed higher protein levels of type I and type III collagen and VEGF in the TOE treatment group. Histological analysis also revealed improved angiogenesis and granulation tissue formation with topical TOE treatment and was consistent with the protein expression. In addition, the histology examination demonstrated faster epithelialization in wounds treated with TOE. The study suggests that sustained high levels of oxygen released by TOE may promote the process of wound repair through increasing collagen deposition and angiogenesis as well as stimulating epithelialization.     

Improved cutaneous wound healing after intraperitoneal injection of alpha‐melanocyte‐stimulating hormone

Skin wound healing is a complex process involving many types of cells and molecules and often results in scar tissue formation in adult mammals. However, scarless healing occurs in foetal skin and minimal scars may occur after cutaneous healing in the adult with reduced inflammation. Alpha‐melanocyte‐stimulating hormone (α‐MSH) is widely distributed within the central nervous system and in other body regions, such as the skin, and has strong anti‐inflammatory activity. The aim in the present experiments was to learn whether intraperitoneal (i.p) injection of α‐MSH just before skin wounds antagonize inflammation and improves skin wound healing in adult mice. C57BL/6 young adult mice received an i.p. injection of 1 mg/kg of α‐MSH and, 30 min later, two circular through‐and‐through holes (6.5 mm diameter) were made in their dorsal skin under anaesthesia. Control mice were wounded after vehicle injection. The wound healing process was analysed macroscopically and microscopically at 3, 7, 40 and 60 days. Skin samples were fixed in formalin, embedded in paraffin, sectioned at 5 μm, stained with H&E or toluidine blue for cell analysis or Gomori's trichrome for extracellular matrix (ECM) analysis. Other samples were fixed in DMSO+methanol, embedded in paraplast and incubated with anti‐CD45, antismooth muscle actin, anticollagen‐I and anticollagen‐III for immunofluorescence analysis. Alpha‐MSH significantly reduced the number of leucocytes, mast cells and fibroblasts at 3 and 7 days after injury. On days 40 and 60, α‐MSH reduced scar area and improved the organization of the collagen fibres indicating that it may direct the healing into a more‐regenerative/less‐scarring pathway.     

Clinical evidence of benefits of a dietary supplement containing probiotic and carotenoids on ultraviolet‐induced skin damage

Background Lactobacillus johnsonii (La1) has been reported to protect skin immune system homeostasis following ultraviolet (UV) exposure. Objectives To assess the effects of a dietary supplement (DS) combining La1 and nutritional doses of carotenoids on early UV‐induced skin damage. Methods Three clinical trials (CT1, CT2, CT3) were performed using different UV sources: nonextreme UV with a high UVA irradiance (UV‐DL, CT1), extreme simulated solar radiation (UV‐SSR, CT2) and natural sunlight (CT3). All three clinical trials were carried out in healthy women over 18 years of age with skin type II–IV. In CT1, early markers of UV‐induced skin damage were assessed using histology and immunohistochemistry. In CT2, the minimal erythemal dose (MED) was determined by clinical evaluation and by chromametry. Chromametry was also used to evaluate skin colour. Dermatologists’ and subjects’ assessments were compiled in CT3. Results A 10‐week DS intake prevented the UV‐DL‐induced decrease in Langerhans cell density and the increase in factor XIIIa+ type I dermal dendrocytes while it reduced dermal inflammatory cells. Clinical and instrumental MED rose by 20% and 19%, respectively, and skin colour was intensified, as shown by the increase in the ΔE* parameter. The efficacy of DS was confirmed by dermatologists and subjects under real conditions of use. Conclusions Nutritional supplementation combining a specific probiotic (La1) and nutritional doses of carotenoids reduced early UV‐induced skin damage caused by simulated or natural sun exposure in a large panel of subjects (n = 139). This latter result might suggest that DS intake could have a beneficial influence on the long‐term effects of UV exposure and more specifically on skin photoageing.     

Histology‐guided protein digestion/extraction from formalin‐fixed and paraffin‐embedded pressure ulcer biopsies

Herein we present a simple, reproducible and versatile approach for in situ protein digestion and identification on formalin‐fixed and paraffin‐embedded (FFPE) tissues. This adaptation is based on the use of an enzyme delivery platform (hydrogel discs) that can be positioned on the surface of a tissue section. By simultaneous deposition of multiple hydrogels over select regions of interest within the same tissue section, multiple peptide extracts can be obtained from discrete histological areas. After enzymatic digestion, the hydrogel extracts are submitted for LC‐MS/MS analysis followed by database inquiry for protein identification. Further, imaging mass spectrometry (IMS) is used to reveal the spatial distribution of the identified peptides within a serial tissue section. Optimization was achieved using cutaneous tissue from surgically excised pressure ulcers that were subdivided into two prime regions of interest: the wound bed and the adjacent dermal area. The robust display of tryptic peptides within these spectral analyses of histologically defined tissue regions suggests that LC‐MS/MS in combination with IMS can serve as useful exploratory tools.     

Neue und etablierte Indikationen der UV‐B‐311‐nm‐Phototherapie

Phototherapy with ultraviolet (UV) irradiation of wavelengths between 280 and 320 nm (UV‐B) is a safe and effective treatment for a variety of inflammatory skin diseases. In addition to standard broad band UVB, narrow band phototherapy with fluorescent bulbs emitting near monochromatic UV between 310 – 315 nm has become an important treatment for diseases such as psoriasis, atopic dermatitis or vitiligo. Other diseases respond favorably to narrow band UV‐B phototherapy, the number of potential indications for such phototherapy is continuously growing. The differential effects of narrow band UV‐B phototherapy in comparison to other UV phototherapies, as well as new and established indications for this treatment modality are reviewed.     

New non‐invasive method for evaluation of the stratum corneum structure in diseases with abnormal keratinization by immunofluorescence microscopy of desmoglein 1 distribution in tape‐stripped samples

The corneodesmosomes in the stratum corneum are critical for the maintenance of stratum corneum integrity. To evaluate the normal and diseased keratinization states in the epidermis, we studied the distribution of desmoglein 1 (DSG1), a major component of corneodesmosomes, in samples of the stratum corneum obtained by tape stripping, a non‐invasive method. Samples were collected from lesional skin of four patients with psoriasis and three with lichen planus, and from non‐lesional skin of three volunteers. Upper stratum corneum cells were obtained by tape stripping and skin biopsies were obtained from adjacent sites. Tape‐stripped samples were examined by immunofluorescence microscopy using anti‐DSG1 monoclonal antibody, in combination with histopathology of skin biopsies. In normal human stratum corneum, which shows basket‐woven orthokeratosis, DSG1‐containing fluorescent dots were distributed on the lateral cell–cell contact areas of plasma membrane, but not on the dorsal/ventral plasma membrane, and formed a well‐ordered hexagonal network structure. In psoriatic stratum corneum, fluorescent dots were distributed throughout the cell membrane at ventral aspects of corneocytes as well as at the lateral cell–cell contacts. In lichen planus, fluorescent dots were distributed homogeneously and/or heterogeneously on the ventral surface in some cells. Adjacent cells lacked DSG1 at the lateral cell–cell contacts, but were instead separated by distinctive black‐gap lines. These results suggest that the intercellular adhesion by DSG1 may depend on the lateral plasma membrane in normal human stratum corneum, on the dorsal/ventral plasma membrane in lichen planus, and on both lateral and dorsal/ventral plasma membranes in psoriatic stratum corneum. Tape stripping and DSG1 immunofluorescence visualizes adhesion features of corneocytes and has considerable potential for evaluation of abnormal keratinization and the process of healing in response to treatment.     

Glycyrrhizic acid prevents ultraviolet‐B‐induced photodamage: a role for mitogen‐activated protein kinases,nuclear factor kappa B and mitochondrial apoptotic pathway

Glycyrrhizic acid (GA), a natural triterpene, has received attention as an agent that has protective effects against chronic diseases including ultraviolet UV‐B‐induced skin photodamage. However, the mechanism of its protective effect remains elusive. Here, we used an immortalized human keratinocyte cell line (HaCaT) and a small animal model (BALB/c mice), to investigate the protective effects of GA against UV‐B‐induced oxidative damage, and additionally, delineated the molecular mechanisms involved in the UV‐B‐mediated inflammatory and apoptotic response. In the HaCaT cells, GA inhibited the UV‐B‐mediated increase in intracellular reactive oxygen species (ROS) and down‐regulated the release of pro‐inflammatory cytokines interleukin (IL)‐1α, ‐1β and ‐6, tumor necrosis factor (TNF)‐α and prostaglandin E2 (PGE2). GA inhibited UV‐B‐mediated activation of p38 and JNK MAP kinases, COX‐2 expression and nuclear translocation of NF‐κB. Furthermore, GA inhibited UV‐B‐mediated apoptosis by attenuating translocation of Bax from the cytosol to mitochondria, thus preserving mitochondrial integrity. GA‐treated HaCaT cells also exhibited elevated antiapoptotic Bcl‐2 protein, concomitant with reduced caspase‐3 cleavage and decreased PARP‐1 protein. In BALB/c mice, topical application of GA on dorsal skin exposed to UV‐B irradiation protected against epidermal hyperplasia, lymphocyte infiltration and expression of several inflammatory proteins, p38, JNK, COX‐2, NF‐κB and ICAM‐1. Based on the above findings, we conclude that GA protects against UV‐B‐mediated photodamage by inhibiting the signalling cascades triggered by oxidative stress, including MAPK/NF‐κB activation, as well as apoptosis. Thus, GA has strong potential to be used as a therapeutic/cosmeceutical agent against photodamage.     

Histologic pitfalls in the Mohs technique

The success of the Mohs procedure depends on the reliability of each step in the technique. Pitfalls in histologic preparation of the tissue specimens may occur during debulking, excising, orienting, creating the map, sectioning, inking, tissue flattening and freezing, cutting, slide fixation, staining, and mapping the tumor. Challenges are also present in interpreting the slides. Diagnostic pitfalls include floaters, inflammatory conditions resembling tumor, and perineural invasion. The technique requires time, teaching, and a sufficient quantity of cases from which to learn, as well as attention to the pitfalls that occur while processing tissue specimens and interpreting and mapping the histology.     

Activation of human vascular endothelium in melanoma metastases induces ICAM‐1 and E‐selectin expression and results in increased infiltration with effector lymphocytes

Lymphocytic infiltration into melanoma tissue is an important prerequisite for effective antitumoral immunity. However, analysis of human metastatic melanoma has shown that leucocyte adhesion receptor expression on melanoma blood vessels is very low or absent, thereby impairing the entry of cytotoxic lymphocytes into tumor tissue. We hypothesized that adhesion molecules can be induced on melanoma vasculature allowing better infiltration of cytotoxic lymphocytes. Quantitative real‐time PCR and immunofluorescence staining indicated that the adhesion molecules ICAM‐1 (CD54) and E‐selectin (CD62E) can be significantly induced by intralesional application of TNF alpha in tissue from human melanoma metastases either in vitro or in vivo when grafted onto immunodeficient NSG (NOD.Cg‐PrkdcscidIl2rgtm1Wjl/SzJ) mice that preserved human vessels. Furthermore, activated human autologous CD3⁺ lymphocytes were injected intravenously into mice bearing melanoma xenografts treated with TNF‐α or PBS in addition to the leucocyte chemoattractant TARC (CCL17). Significantly increased numbers of CD8⁺ cells were detected in TNF‐α–treated melanoma metastases compared with PBS‐treated controls. In addition, tumor cell apoptosis was enhanced and melanoma cell proliferation reduced as shown by TUNEL assay and KI‐67 staining. We conclude that adhesion molecules can be induced on human melanoma vasculature resulting in significantly improved homing of activated autologous cytotoxic T cells to melanoma tissue and inhibition of melanoma cell proliferation. These observations should be considered when designing protocols for immunotherapy of malignant melanoma.     

Evaluation of coexistence of the Human Herpesvirus type 8 (HHV‐8) infection and pemphigus

Background Human Herpesvirus type 8 (HHV‐8) is a new member of the herpes virus family, first found in the tissue of acquired immune deficiency syndrome (AIDS)‐related Kaposi’s sarcoma. Environmental factors including viral infection may play a role in the onset and/or development of pemphigus. Some studies based on polymerase chain reaction findings suggest an association between HHV‐8 and pemphigus. The aim of this study is investigation of the association of pemphigus with HHV‐8 and the relationship between inflammatory and acantholytic cells with HHV‐8 infection. Methods Tissue‐extracted DNA from 41 paraffin‐embedded skin tissues from patients first presenting with pemphigus was tested using nested PCR for the presence of HHV‐8. A total of 37 cases had pemphigus vulgaris (PV) and 4 patients had pemphigus foliaceus (PF). For our control group, normal skin of 21 cosmetic surgery patients were included. Furthermore, microscopic slides with H&E staining were evaluated for the number of inflammatory and acantholytic cells per microscopic field. Results Skin lesions from 13 of 37 patients (35.1%) with PV and 2 of 4 cases (50%) with PF were positive for HHV‐8 DNA. All specimens in our control group were negative for this virus. Conclusion HHV‐8 infection might be a contributing factor in the initiation or development of pemphigus.