Search Results (610)

Background: Stage II/IIIA colorectal cancer (CRC) patients have a relatively high 5-year overall survival rate after surgical resection alone, but more than 50% of patients receive adjuvant chemotherapy. This study investigates a technology that applies artificial intelligence (AI) to conventional histopathology images to identify patients at risk of recurrence. Methods: HistoNav, a novel AI deep-learning algorithm based on Vision Transformer, Graph Neural Networks and Convolutional Neural Networks was designed to analyse H&E-stained formalin-fixed paraffin-embedded (FFPE) samples (n = 2095) to stratify Stage II/IIIA CRC patients into low-, intermediate-, and high-risk groups. Results: Data analysis revealed a 5-year disease-specific survival (DSS) of 93.2%, 84.3%, and 69.3% for low-, intermediate-, and high-risk groups, respectively. The hazard ratio for the high versus low-risk group (HR = 4.611, 95% CI: 2.776–7.662; p < 0.000001) was statistically significant, demonstrating HistoNav’s potential to stratify patients based on recurrence risk. Conclusions: HistoNav can effectively identify CRC patients with good prognosis using the digital images of H&E-stained resection samples and will support clinical decisions around the use of adjuvant chemotherapy. Full article

Background/Objectives: Fungal skin infections are commonly assessed using clinical examination and conventional histopathology, including hematoxylin-eosin (HE), periodic acid–Schiff (PAS), and Grocott methenamine silver (GMS) staining. However, these methods provide limited spatially resolved biochemical information. This proof-of-concept study investigated whether Fourier transform infrared (FTIR) chemical imaging can identify biochemical patterns associated with histologically defined fungal infection-associated tissue compartments in human skin. Methods: Archived formalin-fixed, paraffin-embedded skin samples with histological evidence of fungal infection were investigated. The study group comprised 19 patients, of whom 12 fulfilled the histological and technical eligibility criteria for quantitative FTIR analysis. These 12 independent biological cases yielded 46 histologically defined regions of interest (ROIs), comprising 16 fungal infection-associated ROIs, 14 keratosis/keratinised tissue ROIs, and 16 vital epidermis ROIs. ROI assignment was guided by corresponding HE-, PAS-, and GMS-stained sections. Results: Fungal infection-associated tissue compartments showed partially distinct spectral characteristics compared with vital epidermis and keratinised tissue. The most prominent exploratory differences occurred within the 900–1300 cm⁻¹ fingerprint region. Case-level statistical analysis showed significant differences between fungal infection-associated tissue and vital epidermis at approximately 1185 and 1240 cm⁻¹ after false discovery rate correction, whereas substantial overlap with keratinised tissue remained. Case-level PCA retained tissue-associated spectral structure after biological aggregation, although fungal infection-associated and keratinised tissue showed partial overlap. Unsupervised clustering further demonstrated spatially coherent spectral compartments corresponding to histologically identifiable tissue structures. Conclusions: FTIR chemical imaging may complement conventional histopathology by providing label-free, spatially resolved biochemical information on fungal infection-associated tissue compartments. Because fungal elements are embedded within surrounding keratinised and epithelial tissue, the observed spectral characteristics should be interpreted as exploratory infection-associated tissue signatures rather than fungal-specific diagnostic biomarkers. Larger independent studies with case-wise validation are required before diagnostic application can be considered. Full article

Conventional histopathological diagnostics rely on morphological assessment of stained tissue sections, requiring extensive sample preparation and subjective expert interpretation. Mid-infrared (MIR) imaging offers a complementary approach by providing spatially resolved, label-free access to the intrinsic biochemical composition of tissue without exogenous contrast agents. This work introduces a preprocessing and analysis pipeline for multiphotometric MIR data, applied to formalin-fixed, paraffin-embedded tissue sections from two patients with histopathologically confirmed head and neck squamous cell carcinoma. Combining differential scattering correction, sub-pixel channel registration, and automated tissue segmentation with unsupervised K-Means clustering, the pipeline achieves label-free discrimination of biochemically distinct tissue compartments. K-Means clustering identified four distinct clusters, of which three corresponded to tissue compartments with protein-to-lipid ratios tentatively consistent with epithelial, tumor-associated, and stromal compartments. The resulting cluster maps showed partial spatial correspondence with mIF reference stainings targeting epithelial and stromal markers, supporting the potential of this approach for label-free tissue characterization in digital pathology. Full article

Background: Fibroblast Growth Factor Receptor 3 (FGFR3) mutations are common and clinically relevant alterations in bladder cancer, with implications for diagnosis, monitoring, and patient selection for targeted therapies. Tissue-based testing is often limited by sample availability, invasiveness, and the need for repeated sampling. This study aimed to develop and analytically validate a sensitive double drop-off droplet digital polymerase chain reaction (ddPCR) assay for simultaneous detection of four FGFR3 hotspot mutations in tissue and liquid biopsy samples. Methods: The assay targeted four FGFR3 mutations (S249C, R248C, Y373C, G370C) using reference probes that generate a constant fluorescence signal and wild-type-specific drop-off probes that lose binding when a mutation is present, thereby distinguishing wild-type double-positive droplets from mutant droplets with reduced drop-off fluorescence. Analytical validation was performed using synthetic mutant oligonucleotides, wild-type genomic DNA, and cell-free DNA (cfDNA) from healthy donors (HDs). Specificity, limit of blank (LOB), limit of detection (LOD), and assay precision were evaluated. Performance was compared with next-generation sequencing (NGS) in formalin-fixed paraffin-embedded (FFPE) tissue DNA. FGFR3 mutations were also assessed in matched plasma and urinary cfDNA from bladder cancer patients. Results: The assay demonstrated clear cluster separation, no cross-reactivity, and reliable detection of all mutations down to 0.2% mutant allele frequency (MAF). Strong agreement was observed with a mutation-specific singleplex ddPCR assay for S249C. Concordance with NGS in tissue DNA was 74.2%, with ddPCR identifying additional low-abundance mutations not reported by NGS. FGFR3 mutations were detected in plasma and urinary cfDNA, with complete concordance between matched plasma and urine samples. Conclusions: This ddPCR assay provides a rapid, sensitive, and cost-effective method for detecting clinically relevant FGFR3 mutations and may complement sequencing-based approaches for molecular monitoring using tissue, plasma, and urine specimens. Full article

Background/Objectives: Breast cancer is the most frequent malignant neoplasm in women and presents marked biological heterogeneity. The androgen receptor (AR) has emerged as a biomarker with important prognostic and therapeutic implications, its effect varying according to the molecular subtype. The objective of this study was to analyze AR expression in breast carcinoma samples and its relationship with the different molecular subtypes and clinicopathological variables. Methods: An observational, descriptive, cross-sectional, and prospective study was conducted based on the immunohistochemical analysis of 215 formalin-fixed, paraffin-embedded breast carcinoma samples. AR expression was digitally evaluated as the percentage of positive tumor cells after incubation with an anti-AR monoclonal antibody. Results: A high frequency of AR expression was demonstrated in the cohort, with a median of 53.3%. There were statistically significant differences between molecular subtypes (p < 0.001), detecting greater expression in luminal tumors and markedly low levels in triple-negative breast cancer (TNBC) (median 0.41%). A significant negative correlation was evidenced between AR expression and the Ki-67 proliferation index (ρ = −0.272; p < 0.001), both in the overall sample and in the TNBC subgroup. Conclusions: The androgen receptor is associated with specific molecular subtypes and lower tumor proliferation, suggesting a less aggressive phenotype and supporting its role as a biological biomarker and potential therapeutic target in the management of breast cancer. Full article

Background/Objectives: Colorectal cancer (CRC) is molecularly heterogeneous, and the KRAS (Kirsten rat sarcoma viral oncogene homolog) genotype both governs eligibility for anti-EGFR (epidermal growth factor receptor) therapy and carries prognostic weight. Central Asian data are scarce: no Kazakhstani series has described the KRAS variant spectrum, linked it to survival, or reported mutation detection rates across specimen types and PCR platforms. Methods: We studied 332 patients with morphologically confirmed stage II–IV colorectal adenocarcinoma. KRAS status was determined in formalin-fixed paraffin-embedded (FFPE) tumor tissue by allele-specific real-time PCR (RT-PCR) covering six selected codon 12 and 13 variants. Overall survival (OS) was estimated by the Kaplan–Meier method for the whole cohort and with stage stratification. Separately, mutation detection rates were recorded in three non-overlapping groups of different patients: plasma RT-PCR on the Idylla platform (n = 30), plasma nanoplate digital PCR (dPCR) on QIAcuity One (n = 120), and a routine tissue RT-PCR series (546 evaluable of 550). Because these groups differed in patients, specimen type, and mutation panel, this comparison describes observed detection rates only and supports no inference about analytical sensitivity, specificity, or concordance. Results: KRAS was mutated in 149/332 tumors (44.9%); codon 12 supplied 80.5% of variants, led by G12D (32.2%), G12V (24.8%) and G13D (19.5%). Median OS did not differ between mutant and wild-type tumors (39.0 vs. 36.6 months; p = 0.40). Variant-level differences were directionally consistent, but none was significant, and all were exploratory and unadjusted for multiplicity. Observed detection rates were 40.3% for tissue RT-PCR (220/546), 13.3% for plasma RT-PCR (4/30; continuity-corrected p = 0.006 vs. tissue), and 50.8% for plasma dPCR (61/120; continuity-corrected p = 0.044, Pearson p = 0.034 vs. tissue). BRAF V600E was detected by plasma dPCR in 11/120 cases (9.1%). Conclusions: This first Kazakhstani series places KRAS frequency within the internationally reported range and shows that variant-level reporting reveals prognostic structure that a binary call conceals. The higher detection rate seen with plasma dPCR is hypothesis-generating, not evidence of platform superiority, and motivates a prospective paired-sample study with harmonized mutation panels. Full article

Background/Objectives: B-cell receptor (BCR) signalling is implicated in Burkitt lymphoma (BL) lymphomagenesis, although its activation may differ across biological and epidemiological settings. While surface IgM is the predominant immunoglobulin isotype of BCR in BL, cases expressing IgA transcripts or lacking detectable surface immunoglobulin (sIg) expression have been reported, suggesting a more complex pattern of sIg expression than previously recognized. This study aimed to evaluate sIg heavy-chain expression by immunohistochemistry in a relatively large series of BL cases from endemic and sporadic settings and to investigate the mutational landscape of surface immunoglobulin-undetectable (sIg-UND) cases. Methods: sIg heavy-chain expression was assessed by immunohistochemistry in 55 formalin-fixed paraffin-embedded BL samples. Targeted next-generation sequencing was performed on four sIg-UND cases using an Illumina capture-based custom panel covering 74 lymphoma-related genes. KRAS and NRAS hotspot mutations were additionally assessed by real-time PCR. Results: Overall, 41/55 cases showed IgM expression, including 31 IgM-positive and 10 IgM+/UND cases, whereas 8/55 cases showed IgA expression (IgA+/UND). IgA-expressing cases were significantly enriched at mucosal sites, particularly the oral cavity and gastrointestinal tract, consistent with the relevance of these anatomical sites to mucosal IgA production. Six cases lacked detectable sIg expression, showing negativity for all tested immunoglobulin heavy chains in more than 90% of neoplastic cells. Four sIg-UND cases were available for sequencing. These cases harbored mutations affecting genes commonly altered in BL, together with alterations in genes less commonly represented in recurrent BL series, such as MEF2B, CREBBP, PRDM1, PIM1, ARID3A, HIST1H1B, and HIST1H1C. Conclusions: Our study provides a systematic immunohistochemical characterization of surface immunoglobulin heavy-chain expression in a relatively large series of endemic and sporadic BL. We identified a subset of IgA-expressing BL cases enriched at mucosal sites, whereas rarer cases lacked detectable surface immunoglobulin expression. The mutational profile of sIg-UND cases highlights alterations affecting genes involved in epigenetic regulation, chromatin organization, and B-cell differentiation, which may contribute to the biological heterogeneity of these cases. No KRAS or NRAS hotspot mutations were detected in the four sequenced sIg-UND cases, as assessed by targeted NGS and real-time PCR. Further functional studies are needed to clarify the biological and clinical significance of sIg-UND BL cases and to determine the functional relevance of the identified alterations. Full article

Field cancerization describes molecular alterations occurring in histologically normal tissues surrounding tumors that may contribute to cancer initiation and progression. In prostate cancer (PCa), the molecular characteristics of peritumoral tissue (PTT) remain incompletely understood. Because microRNAs (miRNAs) play key roles in gene regulation, tumor progression, and microenvironmental remodeling, we investigated miRNA expression patterns and regulatory networks across benign tissue (BT), PTT, and tumor tissue (TT). Small RNA sequencing was performed on matched formalin-fixed paraffin-embedded samples from 40 patients with PCa. Differential expression analysis was conducted using DESeq2, adjusting for age and Gleason grade, while functional enrichment analysis and weighted gene co-expression network analysis (WGCNA) were used to identify dysregulated pathways and conserved miRNA modules. PTT exhibited a molecular profile intermediate between BT and TT, consistent with a field cancerization effect. Compared with BT, 102 miRNAs were differentially expressed in TT and 57 in PTT, with 39 miRNAs (68% of the PTT-associated miRNAs) overlapping the tumor signature. Shared dysregulated pathways included PI3K–Akt, p53, and HIF-1 signaling; whereas, PTT showed additional enrichment in pathways related to epigenetic regulation (Polycomb Repressive Complex) and cellular stress responses (mitophagy, protein processing in ER) exclusively through up-regulated miRNAs; no pathways were uniquely enriched from down-regulated miRNAs in PTT. WGCNA identified conserved miRNA modules enriched for members of the let-7, miR-200, miR-103/107, and miR-106a~363 families, which have established roles in epithelial–mesenchymal transition, tumor progression, and microenvironmental remodeling. Collectively, these findings demonstrate that histologically benign peritumoral tissues harbor tumor-associated miRNA programs and regulatory networks that closely resemble those observed in prostate tumors, providing molecular evidence of field cancerization in PCa and identifying potential miRNA-mediated mechanisms relevant to disease progression and biomarker development. Full article

Background: Germline and somatic variants in DNA Damage Repair (DDR) genes are found in approximately one-fourth of patients with metastatic prostate cancer (PC). However, their precise prognostic role and predictive impact on standard therapies remain controversial. Methods: This retrospective, single-center study evaluated the prevalence of germline/somatic DDR aberrations in 287 eligible patients with metastatic prostate cancer (mPC), treated between 2017 and 2022. Clinical characteristics and treatment outcomes (PFS and OS) for chemotherapy (taxanes) or next-generation hormonal therapies (NHT) were compared between DDR-mutated (DDRmut) and wild-type (DDRwt) cohorts. Results: Sixty-three patients (21.9%) were DDRmut, with BRCA2 (12.5%), ATM (3.1%), and BRCA1 (1.39%) being the most common alterations. A family history of breast, ovarian, or prostate cancer strongly predicted DDRmut status (47.0% vs. 14.0%, p = 0.0001). Tissue samples remained evaluable for sequencing up to 180 months from collection. Overall baseline characteristics were similar between cohorts, and BRCA1/2- and ATM-mutated patients treated with first-line taxanes for mCRPC presented with non significantly highermedian OS compared to DDRwt patients (70 vs. 36 months; p = 0.30). On the contrary, the DDRmut subgroup showed a trend toward shorter PFS (12 vs. 18 months; p = 0.04) when treated with first-line NHT. No significant differences were observed with third-line Cabazitaxel. Conclusions: Formalin-fixed paraffin-embedded (FFPE) prostate tissue is highly reliable for DDR, possibly integrating novel liquid biopsy approaches for DDR evaluation. In a real-world setting, BRCA1/2 and ATM variants identify a distinct molecular subgroup that derives preferential survival benefit from first-line taxanes over standard hormonal intensification. Full article

Leprosy presents a broad clinical–immunological spectrum, whose heterogeneity challenges early diagnosis and disease stratification. Metabolomic approaches have emerged as promising tools for identifying potential biomarkers associated with the disease’s pathophysiology. This study aimed to investigate metabolic profiles associated with the different clinical forms of leprosy using untargeted metabolomics in formalin-fixed paraffin-embedded (FFPE) tissue samples. A retrospective cross-sectional study was conducted with 55 patients classified according to the Ridley–Jopling spectrum. Metabolites were extracted from FFPE skin biopsies and analyzed by liquid chromatography–mass spectrometry (LC-MS). From 908 metabolites initially detected, 27 were retained after frequency filtering. Six metabolites ultimately met the criteria of one-way analysis of variance (ANOVA, p < 0.05) and fold-change (FC ≥ 2.0) for differential expression, while N-stearoyl tryptophan was identified as an additional candidate metabolite based on its contribution to multivariate group discrimination. These included 11-hydroperoxy-H4-neuroprostane, which showed a specific association with bacterial load, and the Gly-Pro-Lys tripeptide, which correlated with markers of infection progression. Metabolomics applied to FFPE samples proved feasible for discriminating the clinical spectrum of leprosy and annotating signatures associated with immune response. This approach represents an innovative strategy for exploratory biomarker discovery using archived histopathological samples in translational research. Full article

Background/Objectives: Granulomatous inflammation in tissue biopsies presents a diagnostic challenge, particularly in differentiating between Mycobacterium tuberculosis complex (MTB) and non-tuberculous mycobacteria (NTM) in high-HIV-prevalence areas. This study evaluates the prevalence and species distribution of mycobacteria in formalin-fixed, paraffin-embedded (FFPE) tissues from patients with granulomatous inflammation and correlates these findings with demographic and clinical data. Methods: Conducted at the NHLS-Dr George Mukhari Tertiary Laboratory, the retrospective descriptive study analysed 37 FFPE samples (January 2019–December 2020) after obtaining ethical approval and collecting clinicopathological data. Histopathological assessments using H&E and Ziehl–Neelsen (ZN) stains were carried out, and mycobacterial species were identified utilising multiplex PCR with reverse hybridisation (GenoType MTBC/CM/AS assays). Results: The cohort had an average age of 38.59 years, with a predominance of females (59.5%) and a high HIV positivity rate (62.2%). The most common biopsy site was lymph nodes (32.4%). All samples exhibited necrotising granulomas, but only 8.1% displayed acid-fast bacilli (AFB) on ZN staining. Molecular testing revealed mycobacterial DNA in 75.7% of samples, identifying NTM alone in 50.0%, MTB alone in 10.7%, and both in 28.6%. M. fortuitum was the most frequently recognised species. Importantly, there was no significant association between mycobacterial detection and HIV status (p > 0.05). Conclusions: The findings demonstrate that molecular methods substantially improve mycobacterial detection rates and speciation. This underscores the diagnostic limitations of ZN staining and highlights the importance of molecular diagnostics for accurately identifying bacterial aetiology, crucial for appropriate antimicrobial therapy in immunocompromised patients. Full article

Background/Objectives: Cytological analysis of pleural and peritoneal effusions is minimally invasive but may show limited sensitivity for malignancy detection because tumor cells can be scarce or morphologically difficult to identify. Proteomics-based mass spectrometry imaging (MSI) enables direct molecular profiling of cytological specimens and may improve the distinction between malignant and benign samples. This study investigated whether MALDI-based MSI profiling of formalin-fixed paraffin-embedded (FFPE) cytology cell blocks could discriminate high-grade serous ovarian carcinoma (HGSOC) from benign effusions and identify discriminatory peptide signatures. Methods: Forty-one FFPE cytological specimens derived from pleural and peritoneal effusions were analyzed, including 18 malignant samples and 23 benign control specimens with reactive or inflammatory cytological backgrounds. MALDI-MSI analyses were performed using proteomic profiling. In a preliminary comparative experiment, two section thicknesses (3 µm and 5 µm) were evaluated to optimize ion peak intensity yield. Classification analyses using linear discriminant analysis (LDA) and support vector machine (SVM) models were performed to identify discriminatory peptide signatures. Results: Comparative analysis of average mass spectra identified multiple differentially expressed ions with significant discriminatory performance (AUROC ≥ 0.7 or ≤0.3; Wilcoxon/Kruskal–Wallis, p < 0.001). Classification analyses achieved accuracy ranged from 91% to 94% for the discrimination of malignant and benign samples. Differential proteomic profiling identified Complement C3, Perilipin-3, arachidonate 5-lipoxygenase, Leukotriene A-4 hydrolase, fibrinogen beta and gamma chains, and serotransferrin as discriminatory proteins associated with immune modulation, lipid metabolism, cytoskeletal and extracellular matrix organization, and metabolic regulation. Notably, Complement C3 was found to be overexpressed in malignant tumor cells, supporting its potential role as a marker of tumor presence and progression. Conclusions: Proteomics-based mass spectrometry imaging enabled reliable discrimination of HGSOC from benign cytological specimens and revealed cancer-associated proteins linked to relevant biological processes. These findings support MALDI-MSI as a complementary molecular approach for the classification of challenging cytological specimens in routine diagnostic pathology. Full article

A capillary-driven microfluidic electrical screening platform was developed using silver nanoparticle (AgNP)-decorated laser-patterned villous microstructures on a glass substrate for the analysis of H3N2-infected A549 cells. The device integrated nanosecond laser patterning, AgNP conductive thin-film formation, passive capillary transport, and direct electrical readout within a single microfluidic sensing structure. Villous-like arrays were fabricated using a 1064 nm IR pulsed laser at a fluence of 4.35 J/cm², with a repetition rate of 300 kHz, pulse overlap of 96.7% and scanning speed of 500 mm/s. The fabricated structures exhibited a diameter of 60 μm, height of 80 μm and interpillar pitches ranging from 30 to 90 μm. After AgNP deposition, the surface showed a dominant Ag content of 59.2%, confirming successful formation of conductive microstructured electrodes. The 30 μm pitch structure produced the highest current response of 22 μA at 1 V and the highest ΔI_norm of 0.053 after introduction of H3N2-infected A549 samples. Wettability and capillary transport were tunable by pitch, with contact angles (CAs) decreasing from 140° to 30° and flow velocities decreasing from 0.1 mm/s to 0.03 mm/s. Formalin-fixed H3N2-infected A549 cells were electrically distinguished from non-infected A549 controls over 10¹–10⁶ PFU/μL, with detectable responses down to 10¹ PFU/μL. These results demonstrate a label-free, self-driven, and fabrication-oriented microfluidic strategy for electrical screening of virus-associated cellular samples. Full article

Malignant pleural mesothelioma (MPM) is an aggressive pleural tumor associated with asbestos exposure. Poor clinical outcome of MPM is often driven by late-stage diagnosis due to non-specific clinical presentation, similarity to pleural lesions (e.g., inflammatory changes, metastatic adenocarcinoma), and limitations of current diagnostic methods. We employed Fourier transform infrared (FTIR) spectroscopy combined with convolutional neural networks (CNNs) to analyze formalin-fixed paraffin-embedded (FFPE) pleural tissue samples from patients with MPM, metastatic adenocarcinoma, pleural inflammation, and normal (healthy) pleura. Glycan analysis of FFPE normal pleura and MPM was performed using ultra-high-performance liquid chromatography (UPLC) and mass spectrometry (MS). Our FTIR-spectral analysis uncovered a strong spectral fingerprint of MPM that was especially apparent in the region typical for C-O and C-C stretches as well as local symmetry region typical for deformation vibrations of CH₂ and C-OH groups, all appearing in carbohydrates. Our orthogonal validation of these findings through a targeted glycomics approach using UPLC confirmed that the MPM N-glycome exhibits a distinct fingerprint that distinguishes it from normal pleural tissue. Through utilization of MS for identifying the exact structures of differentially expressed N-glycan peaks, we also identified two high-mannose N-glycan structures that show a specific biomarker potential for MPM and need to be examined in future studies. Full article

This study reports atypical findings indicating that extracellular particles (EPs) isolated from leftover discarded formalin-fixed tissues (FFTs) contain atypical RNA profiles that may be highly fragmented. Using particle purification liquid chromatography (PPLC), we isolated EPs from five tissue types that had been immersed in 10% neutral buffered formalin, stored at room temperature, and collected at the point of discard. Tissues were de-identified and maintained at room temperature until EP separation and exRNA purification. Purified EPs were stored at −80 °C before physicochemical characterization and small RNA sequencing. PPLC separation profiles were broadly consistent with expected EP signatures, although subtle shifts in physicochemical properties were observed. exRNA was successfully recovered from all samples; however, RNA-Seq analysis showed that most reads were unclassified, while others mapped to microbial taxa rather than host-derived transcripts. These results suggest that EP-associated RNA in FFT may be substantially degraded and that microbial particles or nucleic acids were present in the tissues despite formalin fixation. Microbial viability was not assessed, as this was outside the scope of the study. Nonetheless, several explanations are plausible, including RNA fragmentation due to prolonged formalin exposure, microbial contamination during pre-discard storage, or inadequate or ineffective fixation that permitted microbial persistence or enrichment. Collectively, these findings indicate that leftover discarded tissues that were not flash-frozen or properly stored may be unsuitable—or at minimum require caution—when used as a source of EPs or EP-associated RNA. Investigators should consider RNA degradation/fragmentation, microbial confounders, and fixation-related artifacts when interpreting EP or exRNA data derived from FFT. Full article