Formalin, an aqueous solution of formaldehyde, has been the gold standard for fixation of histological samples for over a century. Despite its considerable advantages, growing evidence points to objective toxicity, particularly highlighting its carcinogenicity and mutagenic effects. In 2016, the European Union proposed a ban, but a temporary permission was granted in consideration of its fundamental role in the medical-diagnostic field. In the present study, we tested an innovative fixative, glyoxal acid-free (GAF) (a glyoxal solution deprived of acids), which allows optimal tissue fixation at structural and molecular level combined with the absence of toxicity and carcinogenic activity. An open-label, non-inferiority, multicentric trial was performed comparing fixation of histological specimens with GAF fixative vs standard phosphate-buffered formalin (PBF), evaluating the morphological preservation and the diagnostic value with four binary score questions answered by both the central pathology reviewer and local center reviewers. The mean of total score in the GAF vs PBF fixative groups was 3.7 ± 0.5 vs 3.9 ± 0.3 for the central reviewer and 3.8 ± 0.5 vs 4.0 ± 0.1 for the local pathologist reviewers, respectively. In terms of median value, similar results were observed between the two fixative groups, with a median value of 4.0. Data collected indicate the non-inferiority of GAF as compared to PBF for all organs tested. The present clinical performance study, performed following the international standard for performance evaluation of in vitro diagnostic medical devices, highlights the capability of GAF to ensure both structural preservation and diagnostic value of the preparations.
- MeSH
- Tissue Fixation * methods MeSH
- Fixatives * chemistry MeSH
- Formaldehyde * chemistry MeSH
- Glyoxal * MeSH
- Humans MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Equivalence Trial MeSH
- Multicenter Study MeSH
- Comparative Study MeSH
Pomocí molekulárně genetických metod lze prokázat infekční organismy virového, bakteriálního a houbového původu, stejně jako protozoa a parazitické červy. Molekulární metody detekují specifické úseky v sekvencích nukleových kyselin infekčních agens, a není tedy nutné zachování viability hledaných mikroorganismů. Proto je možné použít tyto metody i pro přímý průkaz infekčního agens z fixované tkáně, nejčastějšího dostupného materiálu v patologii. Tento krátký přehledový článek vychází z více než dvacetiletého fungování molekulárně mikrobiologického úseku v rámci patologie a naším cílem je přiblížit možnosti molekulárně genetické detekce infekčních organismů pro patologickou diagnostiku.
Using molecular methods, infectious organisms of viral, bacterial and fungal origin, as well as protozoa and helminths, can be detected. Molecular methods detect specific segments in the nucleic acid sequences of infectious agents and therefore do not require the maintenance of viability of the microorganisms of interest. Therefore, these methods can also be used for direct detection of infectious agents from fixed tissue, the most commonly available material in pathology. This short review article is based on more than 20 years of molecular microbiology within pathology and our aim is to present the possibilities of molecular detection of infectious organisms for pathological diagnosis.
- MeSH
- Molecular Diagnostic Techniques * classification methods MeSH
- Formaldehyde MeSH
- In Situ Hybridization methods MeSH
- Communicable Diseases * diagnosis etiology pathology MeSH
- Humans MeSH
- Microbiota genetics MeSH
- Nucleic Acids analysis isolation & purification MeSH
- Polymerase Chain Reaction classification methods MeSH
- High-Throughput Nucleotide Sequencing methods MeSH
- Check Tag
- Humans MeSH
- Publication type
- Review MeSH
With the onset of the COVID-19 pandemic, a problem arose with classic body donation programmes for obtaining cadavers for anatomical dissections, science and research. The question has emerged whether bodies of individuals who died of COVID-19 or were infected by SARS-CoV-2 could be admitted to Departments of Anatomy. To determine the risk of SARS-CoV-2 transmission to employees or students, the presence and stability of SARS-CoV-2 RNA in cadavers after fixation agents' application and subsequent post-fixation baths over time were examined. The presence of viral RNA in swabs from selected tissues was assessed by the standardized routine RNA isolation protocol and subsequent real-time PCR analysis. To support the results obtained from the tissue swabs, samples of RNA were exposed in vitro to short and long-term exposure to the components of the injection and fixation solutions used for the bodies' conservation. Substantial removal of SARS-CoV-2 RNA was observed in post-mortem tissue following perfusion with 3.5% phenol, 2.2% formaldehyde, 11.8% glycerol and 55% ethanol, and subsequent post-fixation in an ethanol bath. In vitro experiments showed significant effects of formaldehyde on SARS-CoV-2 RNA, while phenol and ethanol showed only negligible effects. We conclude that cadavers subjected to fixation protocols as described here should not pose a considerable risk of SARS-CoV-2 infection while being handled by students and staff and are, therefore, suitable for routine anatomical dissections and teaching.
- MeSH
- COVID-19 * MeSH
- Ethanol MeSH
- Phenols MeSH
- Formaldehyde MeSH
- Humans MeSH
- Cadaver MeSH
- Pandemics MeSH
- RNA, Viral MeSH
- SARS-CoV-2 * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
Biomarker testing is crucial for treatment selection in advanced non-small cell lung cancer (NSCLC). However, the quantity of available tissue often presents a key constraint for patients with advanced disease, where minimally invasive tissue biopsy typically returns small samples. In Part 1 of this two-part series, we summarise evidence-based recommendations relating to small sample processing for patients with NSCLC. Generally, tissue biopsy techniques that deliver the greatest quantity and quality of tissue with the least risk to the patient should be selected. Rapid on-site evaluation can help to ensure sufficient sample quality and quantity. Sample processing should be managed according to biomarker testing requirements, because tissue fixation methodology influences downstream nucleic acid, protein and morphological analyses. Accordingly, 10% neutral buffered formalin is recommended as an appropriate fixative, and the duration of fixation is recommended not to exceed 24-48 h. Tissue sparing techniques, including the 'one biopsy per block' approach and small sample cutting protocols, can help preserve tissue. Cytological material (formalin-fixed paraffin-embedded [FFPE] cytology blocks and non-FFPE samples such as smears and touch preparations) can be an excellent source of nucleic acid, providing either primary or supplementary patient material to complete morphological and molecular diagnoses. Considerations on biomarker testing, reporting and quality assessment are discussed in Part 2.
- MeSH
- Biomarkers MeSH
- Tissue Fixation methods MeSH
- Fixatives MeSH
- Formaldehyde MeSH
- Humans MeSH
- Lung Neoplasms * diagnosis pathology MeSH
- Carcinoma, Non-Small-Cell Lung * diagnosis pathology MeSH
- Nucleic Acids * MeSH
- Paraffin Embedding MeSH
- Expert Testimony MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Review MeSH
BACKGROUND: Clinical laboratories routinely use formalin-fixed paraffin-embedded (FFPE) tissue or cell block cytology samples in oncology panel sequencing to identify mutations that can predict patient response to targeted therapy. To understand the technical error due to FFPE processing, a robustly characterized diploid cell line was used to create FFPE samples with four different pre-tissue processing formalin fixation times. A total of 96 FFPE sections were then distributed to different laboratories for targeted sequencing analysis by four oncopanels, and variants resulting from technical error were identified. RESULTS: Tissue sections that fail more frequently show low cellularity, lower than recommended library preparation DNA input, or target sequencing depth. Importantly, sections from block surfaces are more likely to show FFPE-specific errors, akin to "edge effects" seen in histology, while the inner samples display no quality degradation related to fixation time. CONCLUSIONS: To assure reliable results, we recommend avoiding the block surface portion and restricting mutation detection to genomic regions of high confidence.
Mitochondrial oxidative phosphorylation (OXPHOS) generates ATP, but OXPHOS also supports biosynthesis during proliferation. In contrast, the role of OXPHOS during quiescence, beyond ATP production, is not well understood. Using mouse models of inducible OXPHOS deficiency in all cell types or specifically in the vascular endothelium that negligibly relies on OXPHOS-derived ATP, we show that selectively during quiescence OXPHOS provides oxidative stress resistance by supporting macroautophagy/autophagy. Mechanistically, OXPHOS constitutively generates low levels of endogenous ROS that induce autophagy via attenuation of ATG4B activity, which provides protection from ROS insult. Physiologically, the OXPHOS-autophagy system (i) protects healthy tissue from toxicity of ROS-based anticancer therapy, and (ii) provides ROS resistance in the endothelium, ameliorating systemic LPS-induced inflammation as well as inflammatory bowel disease. Hence, cells acquired mitochondria during evolution to profit from oxidative metabolism, but also built in an autophagy-based ROS-induced protective mechanism to guard against oxidative stress associated with OXPHOS function during quiescence.Abbreviations: AMPK: AMP-activated protein kinase; AOX: alternative oxidase; Baf A: bafilomycin A1; CI, respiratory complexes I; DCF-DA: 2',7'-dichlordihydrofluorescein diacetate; DHE: dihydroethidium; DSS: dextran sodium sulfate; ΔΨmi: mitochondrial inner membrane potential; EdU: 5-ethynyl-2'-deoxyuridine; ETC: electron transport chain; FA: formaldehyde; HUVEC; human umbilical cord endothelial cells; IBD: inflammatory bowel disease; LC3B: microtubule associated protein 1 light chain 3 beta; LPS: lipopolysaccharide; MEFs: mouse embryonic fibroblasts; MTORC1: mechanistic target of rapamycin kinase complex 1; mtDNA: mitochondrial DNA; NAC: N-acetyl cysteine; OXPHOS: oxidative phosphorylation; PCs: proliferating cells; PE: phosphatidylethanolamine; PEITC: phenethyl isothiocyanate; QCs: quiescent cells; ROS: reactive oxygen species; PLA2: phospholipase A2, WB: western blot.
- MeSH
- Adenosine Triphosphate metabolism MeSH
- Autophagy * MeSH
- Cysteine metabolism MeSH
- Dextrans metabolism MeSH
- Respiration MeSH
- Endothelial Cells metabolism MeSH
- Fibroblasts metabolism MeSH
- Formaldehyde metabolism MeSH
- Phosphatidylethanolamines metabolism MeSH
- Inflammatory Bowel Diseases * metabolism MeSH
- Isothiocyanates MeSH
- Humans MeSH
- Lipopolysaccharides metabolism MeSH
- DNA, Mitochondrial metabolism MeSH
- Mitochondria metabolism MeSH
- Mechanistic Target of Rapamycin Complex 1 metabolism MeSH
- Mice MeSH
- AMP-Activated Protein Kinases metabolism MeSH
- Microtubule-Associated Proteins metabolism MeSH
- Reactive Oxygen Species metabolism MeSH
- Sirolimus MeSH
- Animals MeSH
- Check Tag
- Humans MeSH
- Mice MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
AIM: Activating mutations in the epidermal growth factor receptor (EGFR) are predominantly detected in pulmonary adenocarcinoma and have been reported in small cell lung cancer (SCLC) for decades. This retrospective single-center study aimed to determine the frequency and types of EGFR mutations in SCLC in Taiwan. METHODS: This study comprises a consecutive cohort of 161 patients histologically diagnosed with SCLC between January 1992 and August 2014 at the Department of Pathology in Keelung Chang Gung Memorial Hospital, Taiwan. Archived formalin-fixed paraffin-embedded sections from 71 patients were eligible for molecular analysis. EGFR mutation analysis was performed using a fully-automated IdyllaTM EGFR Mutation Test and confirmed a comparable result through Qiagen Therascreen® EGFR RGQ PCR. In addition, EGFR gene copy number was assessed in EGFR-mutated tumors by fluorescence in situ hybridization (FISH). RESULTS: Mutational status of the EGFR gene was successfully analyzed in 63 specimens by both IdyllaTM and Qiagen platforms. Both methods detected L858R point mutation in exon 21 in an 81-year-old female and a 47-year-old male non-smoker. Both tumors show no concurrent EGFR gene amplification. The overall agreement between results obtained with the IdyllaTM EGFR Mutation Test and Qiagen Therascreen® EGFR RGQ PCR was 100% Conclusions. Our results showed that EGFR mutation is a rare mutation type in a consecutive series of de novo SCLC. Furthermore, the performance of IdyllaTM EGFR Mutation Test and Qiagen Therascreen® EGFR RGQ PCR on archived paraffin sections of limited quantities is available with the high agreement of results.
- MeSH
- ErbB Receptors genetics MeSH
- Formaldehyde MeSH
- In Situ Hybridization, Fluorescence MeSH
- Humans MeSH
- Small Cell Lung Carcinoma * genetics MeSH
- Mutation MeSH
- DNA Mutational Analysis methods MeSH
- Lung Neoplasms * diagnosis MeSH
- Carcinoma, Non-Small-Cell Lung * diagnosis MeSH
- Paraffin MeSH
- Retrospective Studies MeSH
- Check Tag
- Humans MeSH
- Male MeSH
- Female MeSH
- Publication type
- Journal Article MeSH
- Case Reports MeSH
In Slovakia, the Roma population forms the second-largest ethnic minority. A large part of the Roma is semi-nomadic or lives in segregated settlements with poor living standards and limited access to health facilities. More than 40 years ago, a cross-sectional survey revealed a high prevalence of parasitic infections. There is a paucity of recent data, and hence, we designed a study to investigate the current status of intestinal parasitic infections in this population. Overall, 259 children aged 7 months to 18 years from 32 different segregated settlements provided faecal samples for microscopic examination using a sodium acetate-acetic acid-formalin concentration and the Paraprep L technique. Almost 40% of the samples yielded a positive result, with Ascaris lumbricoides (27.4%) and Giardia intestinalis (9.3%) being the most frequent helminth and intestinal protozoa species, respectively. Many children younger than 2 years were found to be infected, which suggests that community transmission is important. In view of our findings, there is a pressing need for targeted action to improve the health status of this neglected population.
- MeSH
- Child MeSH
- Ethnicity MeSH
- Feces parasitology MeSH
- Formaldehyde MeSH
- Giardiasis epidemiology parasitology MeSH
- Helminthiasis epidemiology parasitology MeSH
- Infant MeSH
- Humans MeSH
- Minority Groups MeSH
- Adolescent MeSH
- Parasites MeSH
- Child, Preschool MeSH
- Prevalence MeSH
- Cross-Sectional Studies MeSH
- Risk Factors MeSH
- Animals MeSH
- Check Tag
- Child MeSH
- Infant MeSH
- Humans MeSH
- Adolescent MeSH
- Male MeSH
- Child, Preschool MeSH
- Female MeSH
- Animals MeSH
- Publication type
- Journal Article MeSH
- Geographicals
- Slovakia MeSH
Microsatellite instability (MSI) is present in 15-20% of primary colorectal cancers. MSI status is assessed to detect Lynch syndrome, guide adjuvant chemotherapy, determine prognosis, and use as a companion test for checkpoint blockade inhibitors. Traditionally, MSI status is determined by immunohistochemistry or molecular methods. The Idylla™ MSI Assay is a fully automated molecular method (including automated result interpretation), using seven novel MSI biomarkers (ACVR2A, BTBD7, DIDO1, MRE11, RYR3, SEC31A, SULF2) and not requiring matched normal tissue. In this real-world global study, 44 clinical centers performed Idylla™ testing on a total of 1301 archived colorectal cancer formalin-fixed, paraffin-embedded (FFPE) tissue sections and compared Idylla™ results against available results from routine diagnostic testing in those sites. MSI mutations detected with the Idylla™ MSI Assay were equally distributed over the seven biomarkers, and 84.48% of the MSI-high samples had ≥ 5 mutated biomarkers, while 98.25% of the microsatellite-stable samples had zero mutated biomarkers. The concordance level between the Idylla™ MSI Assay and immunohistochemistry was 96.39% (988/1025); 17/37 discordant samples were found to be concordant when a third method was used. Compared with routine molecular methods, the concordance level was 98.01% (789/805); third-method analysis found concordance for 8/16 discordant samples. The failure rate of the Idylla™ MSI Assay (0.23%; 3/1301) was lower than that of referenced immunohistochemistry (4.37%; 47/1075) or molecular assays (0.86%; 7/812). In conclusion, lower failure rates and high concordance levels were found between the Idylla™ MSI Assay and routine tests.
- MeSH
- Tissue Fixation * MeSH
- Fixatives MeSH
- Formaldehyde MeSH
- Immunohistochemistry * MeSH
- Colorectal Neoplasms chemistry genetics pathology MeSH
- Automation, Laboratory MeSH
- Humans MeSH
- Microsatellite Instability * MeSH
- Mutation * MeSH
- DNA Mutational Analysis * MeSH
- Biomarkers, Tumor * analysis genetics MeSH
- Predictive Value of Tests MeSH
- Reproducibility of Results MeSH
- Paraffin Embedding * MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
- Multicenter Study MeSH
- Comparative Study MeSH
Proteomics of human tissues and isolated cellular subpopulations create new opportunities for therapy and monitoring of a patients' treatment in the clinic. Important considerations in such analysis include recovery of adequate amounts of protein for analysis and reproducibility in sample collection. In this study we compared several protocols for proteomic sample preparation: i) filter-aided sample preparation (FASP), ii) in-solution digestion (ISD) and iii) a pressure-assisted digestion (PCT) method. PCT method is known for already a decade [1], however it is not widely used in proteomic research. We assessed protocols for proteome profiling of isolated immune cell subsets and formalin-fixed paraffin embedded (FFPE) tissue samples. Our results show that the ISD method has very good efficiency of protein and peptide identification from the whole proteome, while the FASP method is particularly effective in identification of membrane proteins. Pressure-assisted digestion methods generally provide lower numbers of protein/peptide identifications, but have gained in popularity due to their shorter digestion time making them considerably faster than for ISD or FASP. Furthermore, PCT does not result in substantial sample loss when applied to samples of 50 000 cells. Analysis of FFPE tissues shows comparable results. ISD method similarly yields the highest number of identifications. Furthermore, proteins isolated from FFPE samples show a significant reduction of cleavages at lysine sites due to chemical modifications with formaldehyde-such as methylation (+14 Da) being among the most common. The data we present will be helpful for making decisions about the robust preparation of clinical samples for biomarker discovery and studies on pathomechanisms of various diseases.
- MeSH
- Formaldehyde MeSH
- Humans MeSH
- Proteome * MeSH
- Proteomics * MeSH
- Reproducibility of Results MeSH
- Digestion MeSH
- Paraffin Embedding MeSH
- Check Tag
- Humans MeSH
- Publication type
- Journal Article MeSH
