Paraffin-embedded Skin Samples Research Articles

The evaluation of Congo red staining combined with fluorescence microscopy in the diagnosis of primary cutaneous amyloidosis.

Primary cutaneous amyloidosis (PCA) is a chronic pruritic skin disease. The apple-green birefringence of Congo red-stained amyloid under a polarized light microscope (CR-PLM) remains the gold standard in the diagnosis of PCA. However, there are some limitations to this approach. In this study, eighty-two paraffin-embedded biopsy skin samples were collected from patients with a clinical diagnosis of PCA. The sections were respectively stained with hematoxylin-eosin (HE), crystal violet (CV), and Congo red (CR) and observed under a light microscope. CR-stained sections were also observed under a polarized light microscope (CR-PLM) or an ultraviolet (UV)-emitted fluorescence microscope (CR-UFM). Further, 35 cases clinically diagnosed with psoriasis, lichen planus, and prurigo nodularis were selected as the negative control group. The positive rate of amyloid protein detected by CR-UFM (81.71%) was significantly higher than that detected by CR-PLM (70.73%, p = 0.004), CR staining (56.10%, p < 0.001), CV staining (30.49%, p < 0.001), or HE staining (28.05%, p < 0.001). In the control group, 34 (97.14%) cases were negative for amyloid deposits in CR staining, CR-PLM, and CR-UFM sections. The relative number of positive dermal papillae observed by CR-UFM (0.35 ± 0.27) was much more than that observed by CR-PLM (0.15 ± 0.17, p<0.001), CR staining (0.12 ± 0.16, p < 0.001), CV staining (0.07 ± 0.12, p < 0.001), or HE staining (0.05 ± 0.12, p < 0.001). The intensity of fluorescence by CR-UFM was significantly greater than that of the appl-green birefringence by CR-PLM (p < 0.001). Moreover, the amyloid was easily distinguished from the surrounding tissues using the CR-UFM method. In conclusion, the CR-UFM method was superior to CR-PLM, CR staining, CV staining, and HE staining in diagnosing PCA.

A case of indeterminate cell histiocytosis with ETV3-NCOA2 translocation.

Indeterminate cell histiocytosis (ICH) is a rare histiocytic disorder characterized by a proliferation of CD1a+ and CD207/langerin- cells. Recent molecular analyses have identified ETV3-NCOA2 translocation as a possible aetiopathogenesis of ICH. Herein, we describe the first Japanese case of ICH with ETV3-NCOA2 translocation. A 79-year-old Japanese man presented with a 1-year history of pruritic erythematous papules and nodules on his trunk and extremities. Histological examination revealed a dense and diffuse sheets-like infiltration of medium-sized histiocyte-like cells from the epidermis to the deep dermis. Immunohistochemically, the atypical cells were positive for CD1a but negative for CD207/langerin. Fluorescence insitu hybridization using NCOA2 break-apart probes confirmed a chromosomal break occurring on NCOA2 monoallele in the tumor cells. Furthermore, ETV3 exon 4-NCOA2 exon 14 translocation was identified in formalin-fixed paraffin-embedded skin samples using reverse transcription polymerase chain reaction and subsequent direct DNA sequencing. He also presented with interspersed eczematous plaques on his trunk and reactive dermatopathic lymphoadenopathy without any infiltration of ICH. He was treated with topical corticosteroids and narrowband UVB phototherapy. Four months later, his ICH skin eruptions, eczematous plaques, and lymphoadenopathy gradually regressed. Our case supports the notion that the detection of ETV3-NCOA2 translocation can be useful for diagnosis of ICH.

A Droplet Digital Polymerase Chain Reaction-Based Tool to Aid in Melanoma Diagnosis.

Detecting copy number variations (CNVs) at certain loci can aid in the diagnosis of histologically ambiguous melanocytic neoplasms. Droplet digital polymerase chain reaction (ddPCR) is a rapid, automated, and inexpensive method for CNV detection in other cancers, but not yet melanoma. To evaluate the performance of a 4-gene ddPCR panel that simultaneously tests for ras responsive binding element protein 1 (RREB1) gain; cyclin-dependent kinase inhibitor 2A (CDKN2A) loss; MYC proto-oncogene, bHLH transcription factor (MYC) gain; and MYB proto-oncogene, transcription factor (MYB) loss in melanocytic neoplasms. One hundred sixty-four formalin-fixed, paraffin-embedded skin samples were used to develop the assay, of which 65 were used to evaluate its performance. Chromosomal microarray analysis (CMA) data were used as the gold standard. ddPCR demonstrated high concordance with CMA in detecting RREB1 gain (sensitivity, 86.7%; specificity, 88.9%), CDKN2A loss (sensitivity, 80%; specificity, 100%), MYC gain (sensitivity, 70%; specificity, 100%), and MYB loss (sensitivity, 71.4%; specificity, 100%). When one CNV was required to designate the test as positive, the 4-gene ddPCR panel distinguished nevi from melanomas with a sensitivity of 78.4% and a specificity of 71.4%. For reference, CMA had a sensitivity of 86.2% and a specificity of 78.6%. Our data also revealed interesting relationships with histology, namely (1) a positive correlation between RREB1 ddPCR copy number and degree of tumor progression; (2) a statistically significant correlation between MYC gain and nodular growth; and (3) a statistically significant correlation between MYB loss and a sheetlike pattern of growth. With further validation, ddPCR may aid both in our understanding of melanomagenesis and in the diagnosis of challenging melanocytic neoplasms.

Comparative Study of Colorimetric In Situ Hybridization and Quantitative Real-Time Polymerase Chain Reaction for Diagnosis of Infection by Leishmania infantum in Dogs in Formalin-Fixed and Paraffin-Embedded Skin.

The zoonotic visceral leishmaniasis is caused by the protozoan Leishmania infantum and dogs are reservoirs for this parasite. For the diagnosis of Leishmania at the species level in dogs in formalin-fixed, paraffin-embedded skin (FFPES) samples, colorimetric in situ hybridization (CISH) and quantitative real-time polymerase chain reaction (qPCR) are options, but their sensitivities are not well established. Therefore, the aim of this study was to determine the sensitivity of these two techniques in FFPES for the diagnosis of the L. infantum infection in dogs using culture as the reference standard. The FFPES of 48 dogs with cutaneous infection by L. infantum confirmed by culture and by multilocus enzyme electrophoresis were examined by CISH and qPCR using specific probes for L. infantum. The sensitivities of qPCR, CISH and their combination were, respectively, 77.0%, 58.0% and 83.3%. The sensitivities of qPCR in dogs with and without clinical signs were, respectively, 74.2% and 82.4%. The sensitivities of CISH in dogs with and without clinical signs were, respectively, 61.3% and 52.9%. The CISH and qPCR showed satisfactory sensitivities for the diagnosis of L. infantum in the FFPES of dogs, even in dogs without clinical signs, and their combination increases the sensitivity for this diagnosis.

Granulomatous mural folliculitis in 16 domestic goats: Infection with malignant catarrhal fever viruses and colocalization with ovine herpesvirus-2 using in situ hybridization.

Granulomatous mural folliculitis (GMF) is an uncommon reaction pattern occasionally observed in nonadapted ruminant hosts infected with malignant catarrhal fever viruses. This report characterizes GMF and concurrent cutaneous lesions in 16 goats with crusting dermatitis using histochemistry including hematoxylin and eosin, periodic acid-Schiff, and Grocott's methenamine silver, and immunohistochemistry for CD3, CD20, ionized calcium binding adaptor molecule 1, and cytokeratin AE1/3. Infiltrates in all 16 GMF cases consisted of macrophages and fewer T lymphocytes, and variably included eosinophils, multinucleated histiocytic giant cells, and/or neutrophils. Formalin-fixed paraffin-embedded skin and fresh skin samples from caprine GMF cases were tested using pan-herpesvirus nested conventional polymerase chain reaction (PCR) and partial sequencing, ovine herpesvirus-2 (OvHV-2) real-time PCR, and OvHV-2 colorimetric in situ hybridization (ISH). Five of 16 goats with GMF (31%) were PCR positive for malignant catarrhal fever viruses, including caprine herpesvirus 3 in 1 goat and OvHV-2 in 4 goats. Three goats also had positive intranuclear OvHV-2 hybridization signal in follicular keratinocytes, among other cell types, localized to areas of GMF. Herpesviruses were not detected in the formalin-fixed paraffin-embedded skin of 9 goats without GMF. This case series describes relatively frequent detections of malignant catarrhal fever viruses in the skin of goats with GMF, including the first report of caprine herpesvirus 3, and localizes OvHV-2 infected follicular keratinocytes within areas of GMF.

Is it Possible to Obtain Immunofluorescence Data in Formalin-Fixed Paraffin-Embedded Skin Samples for the Diagnosis of Pemphigus Vulgaris and Bullous Pemphigoid.

The gold-standard method for assessment of autoimmune bullous disease is direct/indirect immunofluorescence (IF) examination applied to fresh frozen tissue. Since the sensitivity of IF is greatly reduced in formalin-fixed paraffin-embedded (FFPE) tissues, IF cannot be relied upon in these samples. However, immunohistochemistry with the C4d antibody is a promising marker used as a surrogate for immune complex deposition, in nephropathology practice, and the paraffin IF method is also used as a `salvage` technique when fresh frozen tissue is not available or lacks glomeruli. We aimed to investigate whether it is possible to obtain immunofluorescence data from FFPE tissues diagnosed with bullous pemphigoid (BP) and pemphigus vulgaris (PV) and its relationship with inflammatory parameters in the skin. Eighty-nine in-house cases with both IgG and C3 positivity by routine immunofluorescence examination were included in the study. Inflammation parameters were evaluated in hematoxylin-eosin sections. Immunofluorescence study with IgG protease digestion and C4d immunohistochemistry were performed. Results of 83 biopsies were obtained by paraffin immunofluorescence with IgG. There were positive reactions in 28 (34%) of these 83 biopsies. Five of the 28 positive results belonged to BP (18%), and 23 were PV (82%). Ten positive results were on lesional skin (36%), and 18 (64%) were on non-lesional skin. In the immunohistochemical study with C4d, 84 biopsy results were obtained. There were positive reactions in 34 (40.4%) of 84 biopsies. Of the 34 positive results, 12 belonged to BP (35.3%) and 22 to PV (64.7%). Again, 22 (64.7%) of 34 positive results belonged to lesional skin, and 12 (35.3%) belonged to non-lesional skin. When both techniques were used together, 44 (54%) of 81 biopsies yielded positive results for at least one of the two studies, while in 37 (46%), both tests showed negative results. The sensitivity of both IgG and C4d was less than in the literature, especially in BP-diagnosed biopsies. Positive samples were mostly PV. In conclusion, obtaining immunofluorescence data in FFPE samples is possible and is independent of the related skin being lesional or not, however, negative results should not be relied upon.

Increased epigenetic age acceleration in the hidradenitis suppurativa skin.

Epigenetic (or DNA methylation) age is calculated based on methylation of certain cytosine-guanine (CpG) repeats, and it can accurately estimate one's chronologic age. Importantly, epigenetic age acceleration (EAA) is highly predictive of age-associated morbidity and all-cause mortality. Hidradenitis suppurativa (HS) is a chronic inflammatory skin disease with significant systemic disease burden. Here, we performed a pilot study to calculate EAA from formalin-fixed paraffin-embedded skin samples using Illumina Infinium MethylationEpic BeadChip arrays. Our results demonstrated no significant difference in intrinsic EAA among HS compared to controls (- 1.00years, p-value = 0.52), significant increases in both extrinsic EAA (13.72years, p-value < 0.001) and PhenoAge acceleration (7.72years, p-value = 0.003), and asignificant decrease in GrimAge acceleration (- 5.14years, p-value < 0.001). Our findings suggest that the acceleration of epigenetic age in the HS skin may be associated with extrinsic immune-related changes and can potentially serve as a biomarker of the present and/or future disease burden in HS patients.

Mycobacterium leprae and Mycobacterium lepromatosis Infection: a Report of Six Multibacillary Cases of Leprosy in the Dominican Republic.

The causative agents of leprosy are Mycobacterium leprae and M. lepromatosis. Mycobacterium lepromatosis was found in 2008 to cause diffuse lepromatous leprosy in Mexican patients. This study aimed to identify M. leprae and M. lepromatosis in paraffin-embedded skin samples from Caribbean patients with leprosy. A total of six skin samples were obtained from the Dominican Republic. All cases presented the multibacillary form; five were nodular lepromatous leprosy, and one was borderline lepromatous leprosy. All patients received multidrug therapy. Molecular identification was achieved using the M. leprae-specific repetitive element for M. leprae and the hemN gene for M. lepromatosis. Mycobacterium leprae was identified in two lepromatous leprosy cases, and one borderline lepromatous leprosy case; M. lepromatosis was found in one nodular lepromatous leprosy case. Both Mycobacterium species were present in two nodular lepromatous leprosy cases. This is the first report of M. lepromatosis in the Dominican Republic.

Accumulation of Cytotoxic Skin Resident Memory T Cells and Increased Expression of IL-15 in Lesional Skin of Polymorphic Light Eruption.

Patients with polymorphic light eruption (PLE) develop lesions upon the first exposure to sun in spring/summer, but lesions usually subside during season due to the natural (or medical) photohardening. However, these lesions tend to reappear the following year and continue to do so in most patients, suggesting the presence of a disease memory. To study the potential role of skin resident memory T cells (Trm), we investigated the functional phenotype of Trm and the expression of IL-15 in PLE. IL-15 is known to drive Trm proliferation and survival. Multiplex immunofluorescence was used to quantify the expression of CD3, CD4, CD8, CD69, CD103, CD49a, CD11b, CD11c, CD68, granzyme B (GzmB), interferon-gamma (IFN-γ), and IL-15 in formalin-fixed, paraffin-embedded lesional skin samples from PLE patients and healthy skin from control subjects. Unlike the constitutive T cell population in healthy skin, a massive infiltration of T cells in the dermis and epidermis was observed in PLE, and the majority of these belonged to CD8+ T cells which express Trm markers (CD69, CD103, CD49a) and produced cytotoxic effector molecules GzmB and IFN-γ. Higher numbers of CD3+ T cells and CD11b+CD68+ macrophages produced IL-15 in the dermis as compared to healthy skin. The dominant accumulation of cytotoxic Trm cells and increased expression of IL-15 in lesional skin of PLE patients strongly indicates the potential role of skin Trm cells in the disease manifestation and recurrence.

Comparison of immunohistochemical and qPCR methods from granulomatous dermatitis lesions for detection of leishmania in dogs living in endemic areas: a preliminary study

BackgroundIn canine leishmaniosis (CanL) endemic areas, pathologists often receive skin biopsies for testing with histopathologic findings suggestive—but not conclusive for a definitive diagnosis—of CanL lesions. I the absence of data on the infective status of animals, the diagnosis can therefore be challenging. The aim of this retrospective study was to evaluate the ability of immunohistochemistry (IHC) and quantitative PCR (qPCR) methods to detect Leishmania infection in skin biopsies with a histopathologic diagnosis of lymphoplasmacytic/histiocytic and/or granulomatous dermatitis and to correlate the pattern, depth and severity of the histopathologic lesions with the parasite load detected by qPCR and IHC.MethodsThirty formalin-fixed, paraffin-embedded skin samples were evaluated by hematoxylin–eosin (H&E) staining, IHC, conventional PCR (cPCR) and qPCR. The severity, pattern and depth of the dermal inflammation and parasite load were graded.ResultsLeishmania was detected by H&E staining in 8/30 sections (26.66%) and by IHC in 14/30 samples (46.66%). Parasite DNA was detected in 14/30 samples (46.66%) by cPCR and in 21/30 samples (70%) by qPCR, with an extremely variable parasite load (1.32–62.700 copies). The level of agreement was fair between H&E staining and cPCR (κ = 0.32), and moderate between H&E staining and IHC (κ = 0.58). The level of agreement between IHC and cPCR was good (κ = 0.65); between IHC and qPCR, moderate (κ = 0.41); and between cPCR and qPCR, fair (κ = 0.28). A significant association was found between the severity of dermal inflammation and the parasitic skin load by IHC, although with weak linear correlation.ConclusionsOur study underlines the difficulty of obtaining a definitive diagnosis of CanL cutaneous lesions, even with the most accurate diagnostic tests currently available. Based on our results, no single test is suitable on its own for the diagnosis of cutaneous lesions caused by Leishmania. However, in the presence of a moderate/severe lymphoplasmacytic/histiocytic and/or granulomatous dermatitis, we suggest performing IHC, as in our study this technique proved to be the method with the highest discriminatory power to estimate the role of the parasite in skin lesions. In mild lesions, IHC loses its discriminatory power and should be effectively combined with techniques such as qPCR.Graphical

Histopathological and Clinical Analysis of Skin Rashes in Children With Multisystem Inflammatory Syndrome Associated With COVID-19.

A new entity, which occurs a few weeks after SARS-CoV-2 infection and resembling incomplete Kawasaki disease or toxic shock syndrome, has been defined and named multisystem inflammatory syndrome (MIS-C) associated with COVID-19 in children. The aim of our study was to describe histopathological characteristics of skin lesions of MIS-C patients to reveal whether there is a relationship between histopathological features and clinical manifestations. Seventeen who had skin involvement of 57 patients who were diagnosed with MIS-C between December 2020 and February 2021 were included in this prospective study. Demographic information, laboratory findings, and patients' managements were recorded. Skin biopsies were taken simultaneously of each patient. Formalin-fixed, paraffin-embedded skin samples were examined microscopically. The rate of skin rash was 30% in patients with MIS-C and was predominantly the maculopapular type. The anatomical distribution of the rash was evaluated as localized in 10 and generalized in 7 patients. In patients with myocarditis, C-reactive protein and fibrinogen were found to be significantly higher, and lymphocyte and albumin values were found to be low. Herpes-like inclusions were found in the microscopic examination of 2 patients with a history of zona zoster in themselves or in their mother. There was a significant difference between keratinocyte necrosis and some clinical parameters. Localized skin lesions appear to be associated with a more severe inflammatory.

Immunoexpression of Adhesion Molecules During Human Fetal Hair Development

SummaryBackground: Hair follicles are produced in a cyclical manner and the machinery involved in the reproduction of these follicles is present since the fetal stage. Although extensive research has been done on the human hair follicle, very little is known about the importance of adhesion molecules in its development. Methods: We analyzed here, the immunoexpression of beta-1 integrin, p-cadherin, e- cadherin, and beta-catenin in hair follicles from 26 formalin-fixed and paraffin-embedded skin samples from human embryos and fetus between 12-23 weeks of gestational age. Findings: The adhesion molecules beta-1 integrin and e-cadherin/p-cadherin were expressed from 12 weeks and seemed to play a role in regulating epidermis invagination. Beta-catenin immuno stainingwas negative in all cases; down regulation of this protein may be necessary for fetal hair development and thus facilitating hair follicle down growth. Conclusion: Adhesion molecules are essential for hair follicle down growth and proliferation; integrins and cadherins play a major role in this process. More studies are needed to describe hair follicle development.

Diagnostic Value of Genotypic Analysis in Primary Cutaneous Lymphomas using Standardized BIOMED-2 Polymerase Chain Reaction Protocols: Experience in Daily Clinical Practice.

BIOMED-2 Concerted Action BMH4-CT98-3936 (BIOMED-2) PCR protocols are an important diagnostic tool in the evaluation of cutaneous lymphomas. The aim of this study was to assess the diagnostic value of the genotyping results obtained by these techniques in daily clinical practice. A total of 360 paraffin-embedded skin samples were retrospectively reviewed from 114 cutaneous T-cell lymphomas and 35 cutaneous B-cell lymphomas. A total of 249 biopsies from 180 patients with benign lymphoid infiltrates served as controls. T-cell receptor and immunoglobulin gene rearrangements were assessed using the BIOMED-2 method. A combined T-cell receptor gamma and beta assay approach reliably distinguished cutaneous T-cell lymphomas from benign skin T-cell infiltrates (sensitivity 89.4%; specificity 81.5%). Analysis of complete immunoglobulin heavy chain rearrangements also differentiated cutaneous B-cell lymphomas from benign B-cell infiltrates (sensitivity 85.7%; specificity 82.4%). In conclusion, the full BIOMED-2 protocol is a useful aid combined with clinical, histological and immunophenotypical findings for assessment of lymphoid clonality in skin lymphoid proliferations.

PO 8505 LEISHMANIASIS IN ANGOLA – AN EMERGING DISEASE?

BackgroundPoverty, lack of resources, inadequate treatments and control programmes exacerbate the impact of infectious diseases in the developing world. Leishmaniasis is a vector-borne disease that is among the ten major neglected tropical diseases. Although endemic in more than 90 countries, the ones most affected, representing over 90% of new cases, are Bangladesh, Brazil, Ethiopia, India, Kenya, Nepal, and Sudan. In Africa south of the equator, the impact of leishmaniasis is much lower. In several countries, like Angola, little is known about this infectious neglected disease. In the 1970s, a group of Portuguese researchers described three cases of cutaneous leishmaniasis in children from Huambo district and in the 1990s visceral leishmaniasis was diagnosed in an African patient. More recently a canine survey in Luanda revealed two Leishmania-infected dogs.After some suspected cases of human cutaneous leishmaniasis in Huambo region in 2017, the Angola health authorities and the Instituto de Higiene e Medicina Tropical (IHMT), Lisbon, Portugal, established a collaboration to analyse samples from some suspected cases.MethodsThree paraffin-embedded human skin samples from dermatological lesions were sent to IHMT for molecular analysis. After DNA extraction, PCR was performed by using four protocols with different molecular markers.ResultsOne PCR protocol using a nested approach was positive in two of the samples. Sequencing analysis confirmed Leishmania sp. DNA.ConclusionThis was the first time that suspected human cutaneous samples were screened for leishmaniasis by molecular methods with detection of Leishmania sp. DNA. These preliminary studies highlight the need for higher awareness of health professionals for leishmaniasis clinical forms, to recognise risk factors and the epidemiological features of leishmaniasis in the Huambo province. It would be relevant to perform further epidemiological studies to confirm if this vector-borne disease could be emergent in this country.

Characterization of inflammatory infiltrate of ulcerative dermatitis in C57BL/6NCrl-Tg(HMGA1P6)1Pg mice.

Ulcerative dermatitis (UD) is an idiopathic, spontaneous and progressive disease typically affecting C57BL/6 aged mice with an unknown aetiopathogenesis. For this study, we evaluated 25 cases of UD in C57BL/6NCrl-Tg(HMGA1P6)1Pg mice. Formalin-fixed, paraffin-embedded skin samples were submitted to morphological investigations. Immunohistochemical analysis was performed to characterize and quantify inflammatory cells using CD3, CD45/B220, CD4, CD8 and IL-17 antibodies. Mast cell-bound IgE was investigated by immunofluorescence, whereas serum and cryopreserved skin samples were collected for molecular analysis. Student's t-test (two-tailed) was performed to assess significant differences between the two groups. Affected skin showed extensive areas of ulceration and diffuse, severe and mixed inflammatory infiltrates. No relevant changes were observed in control mice. Immunohistochemical analysis showed a predominant CD3 + CD4 + leukocyte population with fewer CD45/B220 and IL-17 immunolabelled cells and mast cell-bound IgE. Increases in TNFα, IL-1β and Il-6 mRNA expression were observed in the skin of affected animals compared to controls. Serum TNFα and IL-6 did not vary between affected and control mice. Inflammatory infiltrates and cytokine expression were consistent with both Th2/IgE and Th17 differentiation, a typical pattern of a type I hypersensitivity reaction. Overall, our data suggest an allergic-based aetiopathogenesis of UD in C57BL/6NCrl-Tg(HMGA1P6)1Pg mice.

Identification of Mycobacterium leprae and Mycobacterium lepromatosis in Formalin-Fixed and Paraffin-Embedded Skin Samples from Mexico.

BackgroundThe causative agents of leprosy are the well-known Mycobacterium leprae and the newly discovered Mycobacterium lepromatosis. This agent was found in 2008, and it was found to be the cause of diffuse lepromatous leprosy in two Mexican patients.ObjectiveThe objective of this work was to determine if M. leprae and M. lepromatosis were present in formalin-fixed and paraffin-embedded skin samples from cases from different regions in Mexico.MethodsA total of 41 skin samples were obtained from 11 states of Mexico. All patients' samples were diagnosed by clinical and histopathological analyses. Total DNA was isolated using a Qiagen-DNeasy blood and tissue kit and molecular identification was achieved by two semi-nested polymerase chain reactions.ResultsThe 41 patient included 33 samples from men and 8 samples from women; 29 samples were polymerase chain reaction (PCR)-positive to Mycobacterium and 12 samples were PCR-negative. From those 29 samples, 13 were PCR-positive to M. leprae, 8 to M. lepromatosis and 8 were positive to both species. The histopathological diagnosis included; Nodular lepromatous leprosy (NLL); Diffuse lepromatous leprosy (DLL); and Borderline leprosy (BL). The 29 PCR-positive samples were classified as follow: 14 NLL, 4 DLL, and 11 BL. In the 12 samples negative to Mycobacterium, 7 showed the NLL, 2 DLL and 3 BL.ConclusionThese findings add evidence to the M. leprae and M. lepromatous distribution, clinical forms and participation of dual infections in Mexico.

Reduced immunohistochemical expression of CCN3 in vitiligo.

Defective adhesion seems to be involved in the chronic loss of melanocytes observed in vitiligo. Recent findings showed an association of genetic variants of an adhesion gene with vitiligo and reduced immunohistochemical expression of some adhesion molecules in vitiligo skin. To compare CCN3 immunohistochemical expression in lesional and non-lesional epidermis of individuals with vitiligo. A total of 66 skin specimens from 33 volunteers with vitiligo were analyzed by immunohistochemistry using anti-CCN3 antibodies. Absence of topical or systemic treatment for vitiligo over the previous 30 days and availability of an area of non-lesional skin for biopsy at least 15 cm away from any vitiliginous macules were the main inclusion criteria. A significant reduction of CCN3 expression was observed in lesional skin as compared to non-lesional skin (P = 0.001). Paraffin embedded skin samples do not allow investigation by molecular biology methods. Not all samples allowed analysis due to the lamina preparation technique. Complete clinical data was not available for all patients. Our results support the hypothesis of impaired cell adhesion in vitiligo suggested by genetic studies. The pattern of immunohistochemical expression suggests that vitiligo might be an epithelial disease and not just a melanocyte disorder.

Analysis of Bacterial and Fungal Nucleic Acid in Canine Sterile Granulomatous and Pyogranulomatous Dermatitis and Panniculitis.

Next generation sequencing (NGS) studies are revealing a diverse microbiota on the skin of dogs. The skin microbiota of canine sterile granulomatous and pyogranulomatous dermatitis (SGPD) has yet to be investigated using NGS techniques. NGS targeting the 16S rRNA and ITS-1 region of bacterial and fungal DNA, respectively, were used to investigate if bacterial and fungal DNA were associated with skin lesions in cases of canine SGPD. The study included 20 formalin-fixed paraffin-embedded (FFPE) skin samples and 12 fresh samples from SGPD-affected dogs, and 10 FFPE and 10 fresh samples from healthy dogs. DNA was extracted from deep dermis and panniculus, and microbial DNA was amplified using primers targeting the bacterial 16S rRNA V1-V3 and fungal ITS-1 regions. The amplified DNA was utilized for NGS on an Illumina MiSeq instrument. The sequences were processed using QIIME. No differences in fungal or bacterial alpha diversity were observed between the SGPD and control samples. Beta diversity analysis demonstrated differences in the bacterial communities between SGPD and control, but not in the fungal communities. Compared to controls, the family Erysipelotrichaceae and genus Staphylococcus were significantly more abundant in the SGPD FFPE samples, and genus Corynebacterium were more abundant in fresh samples. The bacteria found to be more abundant in SGPD are common inhabitants of skin surfaces, and likely secondary contaminants in SGPD cases. This study provides additional evidence that SGPD lesions are likely sterile.

TruSeq-Based Gene Expression Analysis of Formalin-Fixed Paraffin-Embedded (FFPE) Cutaneous T-Cell Lymphoma Samples: Subgroup Analysis Results and Elucidation of Biases from FFPE Sample Processing on the TruSeq Platform.

Cutaneous T-cell lymphomas (CTCLs) are a heterogeneous group of malignancies with courses ranging from indolent to potentially lethal. We recently studied in a 157 patient cohort gene expression profiles generated by the TruSeq targeted RNA gene expression sequencing. We observed that the sequencing library quality and depth from formalin-fixed paraffin-embedded (FFPE) skin samples were significantly lower when biopsies were obtained prior to 2009. We also observed that the fresh CTCL samples clustered together, even though they included stage I–IV disease. In this study, we compared TruSeq gene expression patterns in older (≤2008) vs. more recent (≥2009) FFPE samples to determine whether these clustering analyses and earlier described differentially expressed gene findings are robust when analyzed based on the year of biopsy. We also explored biases found in FFPE samples when subjected to the TruSeq analysis of gene expression. Our results showed that ≤2008 and ≥2009 samples clustered equally well to the full data set and, importantly, both analyses produced nearly identical trends and findings. Specifically, both analyses enriched nearly identical DEGs when comparing benign vs. (1) stage I–IV and (2) stage IV (alone) CTCL samples. Results obtained using either ≤2008 or ≥2009 samples were strongly correlated. Furthermore, by using subgroup analyses, we were able to identify additional novel differentially expressed genes (DEGs), which did not reach statistical significance in the prior full data set analysis. Those included CTCL-upregulated BCL11A, SELL, IRF1, SMAD1, CASP1, BIRC5, and MAX and CTCL-downregulated MDM4, SERPINB3, and THBS4 genes. With respect to sample biases, no matter if we performed subgroup analyses or full data set analysis, fresh samples tightly clustered together. While principal component analysis revealed that fresh samples were spatially closer together, indicating some preprocessing batch effect, they remained in the proximity to other normal/benign and FFPE CTCL samples and were not clustering as outliers by themselves. Notably, this did not affect the determination of DEGs when analyzing ≥2009 samples (fresh and FFPE biopsies) vs. ≥2009 FFPE samples alone.

Multi-elemental imaging of paraffin-embedded human samples by laser-induced breakdown spectroscopy

Chemical elements play central roles for physiological homeostasis in human cells, and their dysregulation might lead to a certain number of pathologies. Novel imaging techniques that improve the work of pathologists for tissue analysis and diagnostics are continuously sought. We report the use of Laser-Induced Breakdown Spectroscopy (LIBS) to perform multi-elemental images of human paraffin-embedded skin samples on the entire biopsy scale in a complementary and compatible way with microscope histopathological examination. A specific instrumental configuration is proposed in order to detect most of the elements of medical interest (i.e. P, Al, Mg, Na, Zn, Si, Fe, and Cu). As an example of medical application, we selected and analysed skin biopsies, including healthy skin tissue, cutaneous metastasis of melanoma, Merkel-cell carcinoma and squamous cell carcinoma. Clear distinctions in the distribution of chemical elements are observed from the different samples investigated. This study demonstrates the high complementarity of LIBS elemental imaging with conventional histopathology, opening new opportunities for any medical application involving metals.