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Analytica Chimica Acta Nov 2023Paraformaldehyde (PFA) fixation is necessary for histochemical staining, and formalin-fixed and paraffin-embedded (FFPE) tissue archives are the largest repository of...
BACKGROUND
Paraformaldehyde (PFA) fixation is necessary for histochemical staining, and formalin-fixed and paraffin-embedded (FFPE) tissue archives are the largest repository of clinically annotated specimens. Single-cell gene expression workflows have recently been developed for PFA-fixed and FFPE tissue specimens. However, for tissues where intact cells are hard to recover, including tissues containing highly interconnected neurons, single-nuclear transcriptomics is beneficial. Moreover, since RNA is very unstable, the effects of standard pathological practice on the transcriptome of samples obtained from such archived specimens like FFPE samples are largely anecdotal.
RESULTS
We evaluated the effects of polyformaldehyde (PFA) fixation and paraffin-embedding on transcriptional profiles of the mouse hippocampus obtained by RNA sequencing (RNA-seq). The transcriptomic signatures of nuclei isolated from fresh PFA-fixed and fresh FFPE tissues were comparable to those of cryopreserved samples. However, more differentially expressed genes were obtained for brains after PFA fixation for more than 3 days than in fresh PFA-fixed samples, especially genes involved in spliceosome and synaptic-related pathways. Importantly, the real cell states were destroyed, with oligodendrocyte precursor cells depleted in the 1day fixed hippocampus. After fixation for 3 days, the proportions of neuronal cells and oligodendrocytes decreased and microglia increased; however, relative frequencies remained constant for longer fixation durations. The storage time of FFPE samples had a negligible effect on the cell composition.
SIGNIFICANCE
This represents the first work to investigate the effects of fixation and storage time of brains on its nuclear transcriptome signatures in detail. The fixation time had more influences on the nuclear transcriptomic profiles than FFPE retention time, and the cliff-like effects appeared to occur over a fixed period of 1-3 days. These findings are expected to guide sample preparation for single-nucleus RNA-seq of FFPE samples, particularly in transcriptomic studies focused on brain diseases.
Topics: Formaldehyde; Paraffin Embedding; Animals; Mice; Tissue Fixation; Gene Expression Profiling; Cell Nucleus; Brain; Transcriptome; Polymers; Mice, Inbred C57BL; Hippocampus; Male; Fixatives
PubMed: 38783731
DOI: 10.1016/j.aca.2023.341861 -
Frontiers in Oncology 2023Triple-negative breast cancer (TNBC) presents significant challenges to female health owing to the lack of therapeutic targets and its poor prognosis. In recent years,...
Triple-negative breast cancer (TNBC) presents significant challenges to female health owing to the lack of therapeutic targets and its poor prognosis. In recent years, in the field of molecular pathology, there has been a growing focus on the role of intra-tumoral microbial communities and metabolic alterations in tumor cells. However, the precise mechanism through which microbiota and their metabolites influence TNBC remains unclear and warrants further investigation. In this study, we analyzed the microbial community composition in various subtypes of breast cancer through 16S rRNA MiSeq sequencing of formalin-fixed, paraffin-embedded (FFPE) tissue samples. Notably, , a microbe associated with cancer response, exhibited a significantly higher abundance in TNBC. Similarly, mass spectrometry-based metabolomic analysis revealed substantial differences in specific metabolites, such as nutriacholic, pregnanetriol, and cortol. Furthermore, we observed significant correlations between the intra-tumoral microbiome, clinicopathological characteristics, and human epidermal growth factor receptor-2 expression(HER2). Three microbial taxa (, and ) were associated with tumor-infiltrating lymphocytes(TILs), which are indicative of antitumor immunity. This study creatively utilized FFPE tissue samples to assess intra-tumoral microbial communities and their related metabolic correlations, presenting avenues for the identification of novel diagnostic biomarkers, the development of therapeutic strategies, and the early clinical diagnosis of TNBC.
PubMed: 37901331
DOI: 10.3389/fonc.2023.1143163 -
Epigenomes May 2024Nucleosomes are non-uniformly distributed across eukaryotic genomes, with stretches of 'open' chromatin strongly associated with transcriptionally active promoters and... (Review)
Review
Nucleosomes are non-uniformly distributed across eukaryotic genomes, with stretches of 'open' chromatin strongly associated with transcriptionally active promoters and enhancers. Understanding chromatin accessibility patterns in normal tissue and how they are altered in pathologies can provide critical insights to development and disease. With the advent of high-throughput sequencing, a variety of strategies have been devised to identify open regions across the genome, including DNase-seq, MNase-seq, FAIRE-seq, ATAC-seq, and NicE-seq. However, the broad application of such methods to FFPE (formalin-fixed paraffin-embedded) tissues has been curtailed by the major technical challenges imposed by highly fixed and often damaged genomic material. Here, we review the most common approaches for mapping open chromatin regions, recent optimizations to overcome the challenges of working with FFPE tissue, and a brief overview of a typical data pipeline with analysis considerations.
PubMed: 38804369
DOI: 10.3390/epigenomes8020020 -
Genome Biology Oct 2023Technologies to study localized host-pathogen interactions are urgently needed. Here, we present a spatial transcriptomics approach to simultaneously capture host and...
Technologies to study localized host-pathogen interactions are urgently needed. Here, we present a spatial transcriptomics approach to simultaneously capture host and pathogen transcriptome-wide spatial gene expression information from human formalin-fixed paraffin-embedded (FFPE) tissue sections at a near single-cell resolution. We demonstrate this methodology in lung samples from COVID-19 patients and validate our spatial detection of SARS-CoV-2 against RNAScope and in situ sequencing. Host-pathogen colocalization analysis identified putative modulators of SARS-CoV-2 infection in human lung cells. Our approach provides new insights into host response to pathogen infection through the simultaneous, unbiased detection of two transcriptomes in FFPE samples.
Topics: Humans; Transcriptome; Tissue Fixation; Formaldehyde; COVID-19; SARS-CoV-2
PubMed: 37858234
DOI: 10.1186/s13059-023-03080-y -
Journal of Colloid and Interface Science Dec 2023Phase change slurries (PCS) have emerged as a promising class of oil-in-water emulsions for energy applications, but stability remains an issue. Pickering phase change...
HYPOTHESIS
Phase change slurries (PCS) have emerged as a promising class of oil-in-water emulsions for energy applications, but stability remains an issue. Pickering phase change slurries (PPCS) stabilized solely by nanoparticles could offer enhanced stability. We hypothesize that stability in PPCS can be achieved by tuning environmental variables of salinity and temperature.
EXPERIMENTS
A paraffin-based PPCS stabilized using fumed silica nanoparticles was developed and assessed under varying NaCl concentrations (up to 150 mM) and temperatures (up to 70 °C). Extended-DLVO modeling, confocal, and cryogenic electron microscopy analyzed the silica-paraffin interactions. Rheological experiments examined the impact of effective volume fraction, thermal expansion, and salinity on the viscosity and shear stability of PPCS. The stability of the resulting formulation was assessed under high pressure and temperature conditions.
FINDINGS
Increased salinity did not change the packing density of the silica at the oil-water interface (82% ± 6%) but did increase the adsorbed layer thickness and network formation, enhancing the formulation's resistance to shear-induced instability. A critical volume fraction of 0.51 ± 0.01 was identified, beyond which viscosity increased significantly. The resulting formulations remained stable under high pressures and temperatures, regardless of salinity. These findings offer insights into the variables affecting PPCS properties, assisting in designing stable PPCS formulations for diverse applications.
PubMed: 37597365
DOI: 10.1016/j.jcis.2023.07.187 -
American Journal of Transplantation :... Mar 2024Immunocompromised patients are at risk of chronic hepatitis E (HEV) infection. Recurrent T cell and borderline rejections in a pediatric patient with high HEV copy...
Immunocompromised patients are at risk of chronic hepatitis E (HEV) infection. Recurrent T cell and borderline rejections in a pediatric patient with high HEV copy numbers led us to study HEV infection within renal transplants. To investigate the frequency of renal HEV infection in transplanted patients, 15 samples from patients with contemporaneous diagnoses of HEV infection were identified at our center. Ten samples had sufficient residual paraffin tissue for immunofluorescence (IF) and RNA-fluorescence-in-situ-hybridization (RNA-FISH). The biopsy of the pediatric index patient was additionally sufficient for tissue polymerase chain reaction and electron microscopy. HEV RNA was detected in paraffin tissue of the index patient by tissue polymerase chain reaction. Subsequently, HEV infection was localized in tubular epithelial cells by IF, RNA-FISH, and electron microscopy. One additional biopsy from an adult was positive for HEV by RNA-FISH and IF. Focal IF positivity for HEV peptide was observed in 7 additional allografts. Ribavirin therapy was not successful in the pediatric index patient; after relapse, ribavirin is still administered. In the second patient, successful elimination of HEV was achieved after short-course ribavirin therapy. HEV infection is an important differential diagnosis for T cell rejection within transplanted kidneys. Immunostaining of HEV peptide does not necessarily prove acute infection. RNA-FISH seems to be a reliable method to localize HEV.
Topics: Adult; Humans; Child; Hepatitis E; Hepatitis E virus; Ribavirin; Antiviral Agents; Paraffin; RNA, Viral; Kidney; Peptides
PubMed: 38072120
DOI: 10.1016/j.ajt.2023.11.016 -
Journal of Immunoassay & Immunochemistry May 2024The data referring to the value of direct immunofluorescence on formalin-fixed, paraffin-embedded tissue (IF-Paraffin) in the diagnosis of renal diseases is... (Comparative Study)
Comparative Study
BACKGROUND
The data referring to the value of direct immunofluorescence on formalin-fixed, paraffin-embedded tissue (IF-Paraffin) in the diagnosis of renal diseases is controversial. The aim of this study was to investigate whether renal biopsies evaluated by routine immunofluorescence on frozen tissue (IF-Frozen) would yield adequate findings to confirm diagnoses when the IF-Paraffin technique was applied.
METHODS
To show immunoglobulins, complement components, and light chains, 55 native renal biopsies were subjected to IF-Paraffin and IF-Frozen staining techniques. The intensity of the staining was compared, and the sensitivity and specificity were calculated.
RESULTS
The IF-Paraffin technique showed a sensitivity of 89%, 81%, 86%, 30%, 71%, 60%, and 77% for IgG, IgM, IgA, C1q, C3, κ, and λ, respectively, whereas specificity was 91%, 100%, 100%, 96%, 94%, 98%, and 100%. It showed diagnostic findings in 87% of cases. Compared to cases that had both IF-Paraffin and IF-Frozen staining techniques, 43 of 55 showed either equal intensity for the diagnostic immunoglobulin/complement or a little difference.
CONCLUSIONS
Direct immunofluorescence on formalin-fixed, paraffin-embedded sections cannot replace immunofluorescence on frozen sections in the assessment of renal biopsies, but may be a "salvage technique" when frozen tissue is insufficient or unavailable and must be interpreted with great caution.
Topics: Humans; Paraffin Embedding; Formaldehyde; Biopsy; Kidney; Fluorescent Antibody Technique, Direct; Male; Frozen Sections; Female; Middle Aged; Aged; Adult
PubMed: 38263688
DOI: 10.1080/15321819.2024.2306324 -
Actas Dermo-sifiliograficas Jun 2024Paraffin-embedded margin-controlled Mohs micrographic surgery (PMMS) includes various procedures such as slow Mohs or deferred Mohs technique, the Muffin and Tübingen... (Review)
Review
Paraffin-embedded margin-controlled Mohs micrographic surgery (PMMS) includes various procedures such as slow Mohs or deferred Mohs technique, the Muffin and Tübingen techniques, and staged margin excision, or the spaghetti technique. PMMS is a variation of conventional Mohs micrographic surgery (MMS) that allows histopathological examination with delayed margin control. PMMS requires minimum training and may be adopted by any hospital. The setback is that PMMS can require procedures across multiple days. PMMS lowers the rate of recurrence of basal cell carcinoma vs wide local excision in high-risk basal cell carcinoma, and improves the rates of recurrence and survival in lentigo maligna. PMMS can be very useful in high-risk squamous cell carcinoma treatment. Finally, it is a promising technique to treat infrequent skin neoplasms, such as dermatofibrosarcoma protuberans, or extramammary Paget's disease, among others. In this article, we present a literature narrative review on PMMS, describing techniques and indications, and highlighting long-term outcomes.
Topics: Mohs Surgery; Humans; Skin Neoplasms; Margins of Excision; Paraffin Embedding; Carcinoma, Basal Cell; Carcinoma, Squamous Cell; Neoplasm Recurrence, Local; Paget Disease, Extramammary; Hutchinson's Melanotic Freckle; Dermatofibrosarcoma
PubMed: 38395222
DOI: 10.1016/j.ad.2024.02.017 -
Pharmacological Research Aug 2023Small-cell lung cancer (SCLC) is generally considered a 'homogenous' disease, with little documented inter-tumor heterogeneity in treatment guidance or prognosis...
Multi-dimensional characterization of immunological profiles in small cell lung cancer uncovers clinically relevant immune subtypes with distinct prognoses and therapeutic vulnerabilities.
Small-cell lung cancer (SCLC) is generally considered a 'homogenous' disease, with little documented inter-tumor heterogeneity in treatment guidance or prognosis evaluation. The precise identification of clinically relevant molecular subtypes remains incomplete and their translation into clinical practice is limited. In this retrospective cohort study, we comprehensively characterized the immune microenvironment in SCLC by integrating transcriptional and protein profiling of formalin-fixation-and-paraffin-embedded (FFPE) samples from 29 patients. We identified two distinct disease subtypes: immune-enriched (IE-subtype) and immune-deprived (ID-subtype), displaying heterogeneity in immunological, biological, and clinical features. The IE-subtype was characterized by abundant immune infiltrate and elevated levels of interferon-alpha/gamma (IFNα/IFNγ) and inflammatory response, while the ID-subtype featured a complete lack of immune infiltration and a more proliferative phenotype. These two immune subtypes are associated with clinical benefits in SCLC patients treated with adjuvant therapy, with the IE-subtype exhibiting a more favorable response leading to improved survival and reduced disease recurrence risk. Additionally, we identified and validated a personalized prognosticator of immunophenotyping, the CCL5/CXCL9 chemokine index (CCI), using machine learning. The CCI demonstrated superior predictive abilities for prognosis and clinical benefits in SCLC patients, validated in our institute immunohistochemistry cohort and multicenter bulk transcriptomic data cohorts. In conclusion, our study provides a comprehensive and multi-dimensional characterization of the immune architecture of SCLC using clinical FFPE samples and proposes a new immune subtyping conceptual framework enabling risk stratification and the appropriate selection of individualized therapy.
Topics: Humans; Small Cell Lung Carcinoma; Retrospective Studies; Neoplasm Recurrence, Local; Prognosis; Lung Neoplasms; Tumor Microenvironment
PubMed: 37392900
DOI: 10.1016/j.phrs.2023.106844 -
STAR Protocols Sep 2023Organoids are unique tools to mimic how tumors evolve in a 3D environment. Here, we present a protocol to embed spheroids invading a 3D matrix into a paraffin mold. We...
Organoids are unique tools to mimic how tumors evolve in a 3D environment. Here, we present a protocol to embed spheroids invading a 3D matrix into a paraffin mold. We describe steps for preparing spheroids, collagen and agarose inclusion, and paraffinization. We then detail procedures for sectioning, staining, and visualization. This protocol allows histological identification of markers expressed in cells escaping the tumor. For complete details on the use and execution of this protocol, please refer to Guyon et al. (2022)..
Topics: Humans; Glioblastoma; Organoids; Collagen; Histological Techniques; Paraffin
PubMed: 37597188
DOI: 10.1016/j.xpro.2023.102521