Paraffin-embedded Skin Tissue Research Articles

C3d Immunohistochemical Staining on Paraffin-Embedded Tissue for Diagnosis of Pemphigus.

Pemphigus is an autoimmune blister disease that causes blisters on the skin and mucosal surfaces. Direct immunofluorescence (DIF) testing is critical for the clinical diagnosis of pemphigus. However, it is limited to fresh tissue specimens and fluorescence microscopy. To assess the value of C3d immunohistochemistry (IHC) on paraffin-embedded skin tissue for the diagnosis of pemphigus by comparing C3d-IHC results to DIF and enzyme-linked immunosorbent assay testing in pemphigus and other blister-related skin diseases. C3d-IHC assays were retrospectively performed on paraffin-embedded skin tissue sections from 115 patients (63 with pemphigus and 52 controls). Both the case group and the control group underwent the same protocol, and cases with C3d position in the peripheral spinous layer were considered as positive samples. C3d-IHC and DIF testing had similar performance for pemphigus diagnosis, with a sensitivity of 71.0% (95% CI, 51.8%-85.1%) and 77.4% (95% CI, 58.5%-89.7%), specificity of 96.4% (95% CI, 79.8%-99.8%) and 100% (95% CI, 85.0%-100%), positive predictive value of 95.7% (95% CI, 76.0%-99.8%) and 100% (95% CI, 82.8%-100%), and a negative predictive value of 75.0% (95% CI, 57.5%-87.3%) and 80.0% (95% CI, 62.5%-90.9%), respectively. Our study indicated that C3d-IHC results for paraffin-fixed tissues were not significantly different from DIF results for the diagnosis of pemphigus. The C3d-IHC assay has the potential for routine diagnosis of pemphigus, especially in the absence of fresh-frozen tissue.

Bullous pemphigoid diagnosis: the role of routine formalin-fixed paraffin-embedded skin tissue immunochemistry

The gold standard for diagnosing bullous pemphigoid (BP) is the detection of linear deposition of IgG and/or C3 at the dermoepidermal junction using direct immunofluorescence (DIF). Because DIF has several disadvantages, primarily the requirement for frozen specimens, we assessed the diagnostic value of immunohistochemical (IHC) staining for BP detection. Eighty-eight patients with bullous lesions were included in this study. IHC staining for C3d, C4d, and IgG was performed on 88 samples, which included specimens from patients with DIF-confirmed BP (n = 43), clinicopathologically suspected BP with negative DIF results (n = 9), and other bullous diseases (n = 36). Diagnosis based on positive results for C3d, C4d, or IgG in IHC staining detected 86% of DIF-confirmed BP cases. The sensitivity of IHC staining for the detection of DIF-confirmed BP cases and clinicopathologically suspected BP cases was similar to that of DIF (80.8% vs. 84.3%), but the specificity was higher (83.3% vs. 75.0%). Five of the nine clinicopathologically suspected BP cases were diagnosed using IHC staining. Thus, IHC staining of routine biopsy material could be an alternative method for diagnosing BP. IHC staining has considerable diagnostic potential, especially in cases with a high suspicion of BP, but negative or suboptimal DIF results.Please check and confirm the author names and initials are correct. Author 2: Given name: [Chul Hwan] Family name: [Kim], Author 3: Given name: [Yoo Jin] Family name: [Lee].Checked it.

Transcriptomic Evaluation of Juvenile Localized Scleroderma Skin With Histologic and Clinical Correlation.

Juvenile localized scleroderma (LS) is an autoimmune disease of the skin whose pathogenesis is not well understood due to the rarity of the disease. This study was undertaken to determine the skin transcriptome in skin biopsy tissue from children with juvenile LS compared to pediatric healthy controls, with identification of significant molecular targets using RNA sequencing (RNA-Seq). In this study, differentially expressed genes (DEGs) were assessed for correlations with histopathologic and clinical features in children with juvenile LS, and were used to group the children into distinct genetic clusters based on immunophenotype. RNA-Seq was performed on sections of paraffin-embedded skin tissue obtained from 28 children with juvenile LS and 10 pediatric healthy controls. RNA-Seq was carried out using an Illumina HTS TruSeq RNA Access library prep kit, with data aligned using STAR and data analysis using a DESeq2 platform. A standardized histologic scoring system was used to score skin sections for the severity of inflammation and levels of collagen deposition. Histologic scoring was completed by 2 pathologists who were blinded with regard to the status of each sample. Spearman's rank correlation coefficients were used to assess significant correlations between DEG expression profiles and skin histologic findings in patients with juvenile LS. We identified 589 significant DEGs in children with juvenile LS as compared to healthy controls. Hierarchical clustering was used to demonstrate 3 distinct juvenile LS immunophenotype clusters. The histologic scores of skin inflammation (based on numbers and categories of inflammatory cell infiltrates) were significantly correlated with the expression levels of HLA-DPB1, HLA-DQA2, HLA-DRA, and STAT1 genes (rs > 0.5, P < 0.01). Collagen thickness correlated with the expression levels of collagen organization genes as well as with genes found to be correlated with the severity of inflammation, including genes for major histocompatibility complex (MHC) class I, MHC class II, and interferon-γ signaling. Among children with juvenile LS, 3 distinct genetic signatures, or clusters, were identified. In one cluster, inflammation-related pathways were up-regulated, corresponding to the histologic skin inflammation score. In the second cluster, fibrosis-related pathways were up-regulated. In the third cluster, gene expression in the skin corresponded to the patterns seen in healthy controls. Up-regulation of HLA class II genes was observed within the first cluster (characterized by predominant inflammation), a feature that has also been observed in the peripheral blood of patients with morphea and in the skin of patients with systemic sclerosis.

Genetic Signatures From RNA Sequencing of Pediatric Localized Scleroderma Skin.

The purpose of this study was to explore the skin transcriptional profile in pediatric localized scleroderma (LS) to provide a better understanding of the altered immune and fibrotic pathways promoting disease. LS is a progressive disease of the skin and underlying tissue that causes significant functional disability and disfigurement, especially in developing children. RNA sequencing (RNAseq) technology allows for improved understanding of relevant cellular expression through transcriptome analysis of phases during LS disease progression (more active/inflammatory vs. inactive/fibrotic) and also permits the use of RNA extracted from existing paraffin-embedded skin tissue, which is important in pediatrics. A strong correlation was observed between the comparison of genes expressed between fresh (RNAlater) and paraffinized skin in healthy and LS subjects, supporting the use of paraffinized tissue. LS gene signatures compared to healthy controls showed a distinct expression of an inflammatory response gene signature (IRGS) composed of IFNγ-, IFNα-, and TNFα-associated genes. GSEA© enrichment analysis showed that the IRGS, including interferon-inducible chemokines such as CXCL9, CXCL10, CXCL11, and IFNγ itself, was more highly expressed in LS patients with more inflammatory lesions. The use of paraffinized skin for sequencing was proven to be an effective substitute for fresh skin by comparing gene expression profiles. The prevalence of the IFNγ signature in the lesion biopsies of active LS patients indicates that these genes reflect clinical activity parameters and may be the promoters of early, inflammatory disease.

Diagnostic value of a nested polymerase chain reaction for diagnosing cutaneous sporotrichosis from paraffin-embedded skin tissue.

The gold standard for diagnosis of cutaneous sporotrichosis involves the isolation of the fungus, Sporothrix, by a culture test. Generally, the sampling for the culture test is performed at the same time as skin biopsy under local anaesthesia. However, the culture test may occasionally return a false negative result. The aim of our study was to investigate the diagnostic value of a molecular method for diagnosing cutaneous sporotrichosis from formalin-fixed and paraffin-embedded (FFPE) tissues. Over a 30-year period, we collected 52 cases of cutaneous sporotrichosis from biopsied specimens that had been positively diagnosed by a culture test. A nested PCR specific for Sporothrix detection was applied using FFPE tissue as template. The results were compared with control samples from 79 patients diagnosed with other cutaneous diseases according to histopathological, clinical findings and a cutler test. Of the 52 patients who were tested positive on the culture test, all cutaneous diseases were detected by PCR. Of the 59 patients in the control group, 58 tested negative by PCR. Under our conditions, the calculated sensitivity of this method was 100%, the specificity was 98.7% and the kappa coefficient was 0.984 (95% CI: 0.953-1.000). The specific PCR assay used appears to be a useful tool for the prompt and accurate diagnosis of sporotrichosis. Using this method, it would be possible to diagnose cutaneous sporotrichosis for patients who were suspected of cutaneous sporotrichosis but tested negative on culturing, and for pathologically suspected cutaneous sporotrichosis patients for whom the culture test was not undertaken.

Combined Use of PCR-Based TCRG and TCRB Clonality Tests on Paraffin-Embedded Skin Tissue in the Differential Diagnosis of Mycosis Fungoides and Inflammatory Dermatoses

The distinction between mycosis fungoides (MF) and inflammatory dermatoses (ID) by clinicopathologic criteria can be challenging. There is limited information regarding the performance characteristics and utility of TCRG and TCRB clonality assays in diagnosis of MF and ID from paraffin-embedded tissue sections. In this study, PCR tests were performed with both TCRG and TCRB BIOMED-2 clonality methods followed by capillary electrophoresis and Genescan analysis using DNA samples from 35 MF and 96 ID patients with 69 and 133 paraffin-embedded specimens, respectively. Performance characteristics were determined for each test individually and in combination. TCRG and TCRB tests demonstrated identical sensitivity (64%) and specificity (84%) when analyzed as individual assays. The positive predictive value, negative predictive value, and change of posttest MF probability over a range of MF pretest probabilities were obtained. These data were used to construct an algorithm for sequential use of TCRG and TCRB. As single tests, commercially available BIOMED-2 PCR-based TCRG and TCRB clonality tests on paraffin-embedded tissue have no significant difference in terms of sensitivity and specificity. Combined use of the two tests in patients with intermediate pretest probabilities as proposed in the algorithm could improve test utility.

Abnormal overexpression of mastocytes in skin biopsies of fibromyalgia patients

Formalin-fixed, paraffin-embedded skin tissue sections were collected from a matched cohort of 63 fibromyalgia syndrome (FMS) patients and 49 volunteers from the general population with both alpha1-antitrypsin (AAT) normal and deficiency variants. These tissues were examined for the expression of the broad-spectrum inhibitor AAT, the serine proteinases elastase and tryptase, the proinflammatory cytokines MCP-1 and TNFα, the endothelium biomarker VEGF, and the inflammation/nociception-related receptor PAR(2). The most relevant finding of the study was a significantly increased number of mast cells (MCs) in the papillary dermis of all FMS patients (greater than or equal to five to 14 per microscopic high power field) compared to zero to one in controls (p < 0.001). MCs strongly stained with tryptase, AAT and PAR(2) antibodies, exhibited a spindle-like shape and were uniformly distributed around blood vessels and appendages. MCP-1 and VEGF expressed weak/moderate positivity in most samples, with a higher expression in controls than in FMS patients (p < 0.001 and 0.051, respectively). No differences in elastase and TNFα were found between both groups. Moreover, no histological differences were found between samples from AAT deficiency and normal AAT phenotypes. Our results indicate that FMS is a MC-associated condition. MCs are present in skin and mucosal surfaces throughout the human body, and are easily stimulated by a number of physical, psychological, and chemical triggers to degranulate, releasing several proinflammatory products which are able to generate nervous peripheral stimuli causing CNS hypersensitivity, local, and systemic symptoms. Our findings open new avenues of research on FMS mechanisms and will benefit the diagnosis of patients and the development of therapeutics.

A NewMycobacteriumSpecies Causing Diffuse Lepromatous Leprosy

Mycobacterium leprae causes leprosy. M leprae strains collected worldwide have been genetically clonal, which poorly explains the varying severity and clinical features of the disease. We discovered a new Mycobacterium species from 2 patients who died of diffuse lepromatous leprosy (DLL). The Mycobacterium was purified from heavily infected, freshly frozen autopsy liver tissue followed by DNA extraction in 1 case. Paraffin-embedded skin tissue was used for DNA extraction in another case. Six genes of the organism were amplified by polymerase chain reaction, sequenced on cloning or from amplicons, and analyzed. Significant genetic differences with M leprae were found, including a 2.1% divergence of the 16S ribosomal RNA (rRNA) gene, a highly conserved marker of bacterial evolution, and 6% to 14% mismatches among 5 less conserved genes. Phylogenetic analyses of the genes of 16S rRNA, rpoB, and hsp65 indicated that the 2 most related organisms evolved from a common ancestor that had branched from other mycobacteria. These results and the unique clinicopathologic features of DLL led us to propose Mycobacterium lepromatosis sp nov. This species may account for some of the clinical and geographic variability of leprosy. This finding may have implications for the research and diagnosis of leprosy.

Detection of a T Cell Clone in the Peripheral Blood at Diagnosis and in the Skin in Complete Clinical Remission after PUVA and Interferon-α Combination Therapy Is Not a Negative Prognostic Factor in Early Mycosis Fungoides.

In the skin of 50–75% patients with early mycosis fungoides (MF) a dominant T-cell receptor (TCR) γ gene rearrangement can be detected. An identical T-cell clone is also detectable in the peripheral blood (PB) in 15–40% of these patients, as well as in 39–61% of skin biopsies showing histological remission after therapy. At the present time, it is still unclear if molecular analyses might be helpful to recognize an unfavourable subset of patients less responsive to skin-directed therapy or with a higher risk of disease recurrence. 89 patients (56 men, 33 women) with a median age of 60 years (range, 17–80), with a histologically confirmed diagnosis of early MF, were treated with a combination protocol of PUVA and low dose IFN-α for 14 months and then closely followed up. Only patients with a dominant clone in the affected skin at diagnosis, and in whom peripheral blood samples at diagnosis and a second tissue biopsy at the end of treatment were available, were selected for the present study. Twenty-four patients (10 men, 14 women; median age 62.5 yrs, range 17–77; 6 in stage IA, 15 IB, 3 IIA) met the inclusion criteria. PCR amplification for TCR γ gene was performed on DNA extracted from formalin-fixed and paraffin-embedded skin tissue samples and from frozen PB lymphocytes, as previously reported. Amplification products were visualised by 10% polyacrylamide gel electrophoresis at the same time, on the same gel, thereby allowing precise comparison of the dominant clonal populations. In one patient the identity of T-cell clones in the skin at diagnosis and at the end of therapy was assessed by sequencing analysis. After a mean time of 4 months (range 1–11), all 24 patients responded to the combination therapy, obtaining a clinical complete remission (CCR), even if three of them showed histological persistence of disease. During the follow-up, 11 patients had a disease recurrence (median time 70 months range 27–108). PCR analysis of TCR γ gene in the PB showed a circulating T-cell clone identical to the one detected in the skin in 9 cases (37.5%) at diagnosis. An identical T-cell clone was observed in the skin at the end of therapy in 17 cases (71%). Disease-free survival curves plotted by Kaplan-Meier method showed that patients with or without a peripheral T cell clone did not behave differently, that is, half of them would have experienced a relapse after a similar period of time in any case. The same behaviour was observed in patients showing different molecular responses after therapy. Only in one third of patients a molecular cure for the disease could be obtained by combination IFN-α and PUVA therapy, since a high rate of persistence of monoclonal PCR signals following CR was observed (71% of CR cases with a dominant clone at diagnosis); surprisingly, such a molecular outcome seems not to protect patients from subsequent disease relapses. On the other hand, our data seems to indicate that the combination of a skin-directed therapy like PUVA in addition to the systemic immunoregulatory effects of IFN-α may abolish the negative influence of a circulating clone.

The application of a pCR technique for the detection of immunoglobulin heavy chain gene rearrangements in fresh or paraffin-embedded skin tissue

Although detection of a clonal sequence of the heavy chain gene of immunoglobulin by the polymerase chain reaction (PCR) is frequently used to assess lymphoid infiltrates in skin biopsy specimens, there are no data on the sensitivity and specificity of this test in detecting clonal B cell populations. Having refined a PCR technique for the detection of immunoglobulin heavy chain (IgH) gene rearrangement in both fresh and formalin-fixed, paraffin-embedded skin samples, we undertook to define the role of this assay in the diagnostic setting. Thirty-one cases of cutaneous B cell lymphoma (CBCL), 19 cases of B cell pseudolymphoma (lymphocytoma cutis), 34 cases of benign lymphocytic infiltrates of the skin and one case of cutaneous T cell lymphoma (CTCL) were studied using the polymerase chain reaction assay. All biopsies were formalin-fixed, paraffin-embedded skin sections apart from 13 of the 31 CBCL specimens which were fresh skin specimens. DNA from the framework region 3 (FR3) sequence of the IgH genes was amplified to ascertain the presence of a clonal IgH gene rearrangement. The findings were correlated with histological and immunophenotyping results on all samples. The assay performed with 73% sensitivity and 100% specificity, comparable to results obtained examining fresh lymphoid tissue specimens from patients with B cell tumours. The results indicate that this technique is a useful tool in the work up of suspected CBCL and in differentiating between CBCL and mixed lymphocytic infiltrates, a clearly important distinction with regards to prognosis and treatment.

Antigen Retrieval in Immunofluorescent Testing of Bullous Pemphigoid

Direct immunofluorescence is an immunopathological technique frequently utilized for diagnosis of vesiculobullous disease such as bullous pemphigoid. Fresh-frozen tissue is required for immunofluorescent testing, making retrospective analysis difficult. In this study, we compared two methods of antigen retrieval in formalin-fixed, paraffin-embedded skin tissue from patients with bullous pemphigoid to determine if archival tissue, after use of an unmasking antigen, can be substituted for fresh-frozen tissue in the immunopathological study of skin. Paraffin-embedded tissue blocks from patients with bullous pemphigoid and patients with eosinophilic spongiotic dermatitis as the prodromal stage of bullous pemphigoid were obtained. Sections were mounted on poly-L-lysine-coated slides and the slides were deparaffinized. The methods of antigen retrieval included incubation with trypsin (0.1%) and microwave irradiation in urea (6 M). Antigen retrieval was followed by indirect immunofluorescence. Microwave irradiation was more effective in antigen retrieval than was incubation with trypsin (0.1%). Microwave irradiation in urea (6 M) produced more intense immunofluorescent staining than did trypsinization. Overall, positive basement membrane zone immunofluorescent staining was found in 60% of patients with a diagnosis of classical bullous pemphigoid and in 50% of patients with eosinophilic spongiotic dermatitis as the prodromal stage of bullous pemphigoid. Although the frozen-tissue method appeared more effective than the antigen-retrieval method in immunofluorescent testing of skin, the antigen-retrieval method can certainly be considered an option in retrospective studies. Antigen retrieval may be particularly advantageous in patients with eosinophilic spongiotic dermatitis in whom the diagnosis of bullous pemphigoid may not be suspected initially.