Will PAXgene substitute formalin? A morphological and molecular comparative study using a new fixative system

Formalin fixation and paraffin embedding present the standard procedures for conserving clinical tissues for histological analysis. However, molecular analysis is impaired by the cross linking properties of formalin. The PAXgene tissue system (PreAnalytix, Switzerland) is a new formalin-free tissue collection device.AimsIn this study we aimed to evaluate this new tissue preservation technique in comparison with formalin fixation and fresh frozen tissue samples.Methods12 melanoma biopsy samples were divided and fixed simultaneously with formalin, PAXgene or fresh frozen in liquid nitrogen and analysed with regard to morphology, immunohistochemistry, DNA and RNA content and quality. Markers of melanocytic differentiation and tumour cell proliferation were used.ResultsMorphology was well preserved in PAXPE samples. However, 5 out of 11 immunohistochemical markers showed significantly lower overall staining and staining intensity with PAXPE tissues in comparison with formalin-fixed, paraffin-embedded (FFPE). Increasing membrane permeability through adding a detergent did proportionally increase staining intensity in PAXPE samples. Amplification of different mRNA amplicons showed a direct relationship with the size of the amplicon with greater template integrity observed in PAXPE samples. Sequencing and mutational analysis of DNA samples were comparable for all the different fixation methods, while the level of DNA fragmentation seemed to be lower in PAXPE compared with FFPE tissues.ConclusionsThe switch from formalin to PAXgene fixation would require a re-evaluation of immunohistochemical markers and staining procedures originally developed for FFPE tissues. Our data demonstrate that PAXPE fixation offers some advantages concerning molecular analysis. However, these advantages would not justify substituting formalin fixation in any routine pathology laboratory.

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Concordance between comprehensive cancer genome profiling in plasma and matched tumor specimens.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e23146 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e23146-e23146

Author(s):

Roopika Menon ◽

Judith N Müller ◽

Markus Falk ◽

Sotirios Lakis ◽

Erika Mariotti ◽

...

Keyword(s):

Lung Cancer ◽

Cancer Patients ◽

Mutational Analysis ◽

Massively Parallel Sequencing ◽

Point Mutations ◽

Liquid Biopsies ◽

Genomic Alterations ◽

Pathology Laboratory ◽

Tissue Samples ◽

Routine Diagnostics

e23146 Background: The importance and clinical use of liquid biopsies to identify somatic, targetable alterations in the plasma of cancer patients is steadily increasing. However, their concordance with alterations identified in cancer specimens tested in routine diagnostics is not being monitored. In a cohort of non-squamous cell lung cancer patients, our aim was to systematically compare alterations found by a massively parallel sequencing liquid biopsy assay covering 39 cancer related genes (NEOliquid) with alterations identified by routine diagnostics in a certified central pathology laboratory. Methods: Routine Diagnostics: DNA mutational analysis was performed using cobas or Sanger-Sequencing. Rearrangements were identified using immunohistochemistry or FISH. NEOliquid assay: Cell-free DNA from plasma was subjected to hybrid-capture based next-generation sequencing to detect point mutations, small insertions and deletions, copy number alterations and genomic translocations in 39 clinically relevant genes in a single assay. To evaluate and compare the performance of liquid biopsies, we applied the NEOliquid tests on blood samples of 82 non-squamous NSCLC patients and correlated them with results of routine diagnostics of matched tissue samples. Results: Routine diagnostics for lung cancer related oncogenes (EGFR, ALK, ROS1, KRAS, BRAF) identified a total of 50 somatic alterations of which 37 were point mutations, 11 InDels and 2 gene fusions. NEOliquid analysis of the corresponding plasma sample revealed a total of 34 alterations, including 21 targetable driver alterations. Overall, the concordance of mutation calls between routine diagnostic testing and NEOliquid was 97.6%, with a sensitivity of > 70% and a specificity of 100%. Importantly, NEOliquid identified additional clinically relevant genomic alterations, not covered by routine testing. Conclusions: We have shown high concordance of genomic alterations identified from liquid biopsies and tumor tissue specimens. The excellent specificity of actionable alterations identified by NEOliquid offers analytical performance for routine diagnostic use.

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Collection and Handling of Thoracic Small Biopsy and Cytology Specimens for Ancillary Studies: Guideline From the College of American Pathologists in Collaboration With the American College of Chest Physicians, Association for Molecular Pathology, American Society of Cytopathology, American Thoracic Society, Pulmonary Pathology Society, Papanicolaou Society of Cytopathology, Society of Interventional Radiology, and Society of Thoracic Radiology

Archives of Pathology & Laboratory Medicine ◽

10.5858/arpa.2020-0119-cp ◽

2020 ◽

Vol 144 (8) ◽

pp. 933-958 ◽

Cited By ~ 5

Author(s):

Sinchita Roy-Chowdhuri ◽

Sanja Dacic ◽

Mohiedean Ghofrani ◽

Peter B. Illei ◽

Lester J. Layfield ◽

...

Keyword(s):

Systematic Review ◽

Mutational Analysis ◽

Expert Panel ◽

Diagnostic Testing ◽

Clinical Pathology ◽

Pathology Laboratory ◽

Tissue Samples ◽

Core Needle ◽

Fine Needle ◽

Ancillary Studies

Context.— The need for appropriate specimen use for ancillary testing has become more commonplace in the practice of pathology. This, coupled with improvements in technology, often provides less invasive methods of testing, but presents new challenges to appropriate specimen collection and handling of these small specimens, including thoracic small biopsy and cytology samples. Objective.— To develop a clinical practice guideline including recommendations on how to obtain, handle, and process thoracic small biopsy and cytology tissue specimens for diagnostic testing and ancillary studies. Methods.— The College of American Pathologists convened an expert panel to perform a systematic review of the literature and develop recommendations. Core needle biopsy, touch preparation, fine-needle aspiration, and effusion specimens with thoracic diseases including malignancy, granulomatous process/sarcoidosis, and infection (eg, tuberculosis) were deemed within scope. Ancillary studies included immunohistochemistry and immunocytochemistry, fluorescence in situ hybridization, mutational analysis, flow cytometry, cytogenetics, and microbiologic studies routinely performed in the clinical pathology laboratory. The use of rapid on-site evaluation was also covered. Results.— Sixteen guideline statements were developed to assist clinicians and pathologists in collecting and processing thoracic small biopsy and cytology tissue samples. Conclusions.— Based on the systematic review and expert panel consensus, thoracic small specimens can be handled and processed to perform downstream testing (eg, molecular markers, immunohistochemical biomarkers), core needle and fine-needle techniques can provide appropriate cytologic and histologic specimens for ancillary studies, and rapid on-site cytologic evaluation remains helpful in appropriate triage, handling, and processing of specimens.

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Z Probe, an Efficient Tool for Characterizing Long Non-Coding RNA in FFPE Tissues

10.20944/preprints201806.0096.v1 ◽

2018 ◽

Author(s):

Manish Tripathi ◽

Chidi Zacheaus ◽

Kyle Doxtater ◽

Fatemeh Keramatnia ◽

Lani Gao ◽

...

Keyword(s):

Tissue Samples ◽

Protein Coding ◽

Dna And Rna ◽

Non Coding Rna ◽

Formalin Fixed Paraffin ◽

Lncrna Malat1 ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

Single Transcript ◽

Long Non Coding Rna

Formalin-Fixed Paraffin Embedded (FFPE) tissues are a valuable resource in studying different markers and mechanistic molecules (protein, DNA and RNA) in order to understand the etiology of different cancers as well as many other diseases. Degradation and modification of RNA is the major challenge in utilizing FFPE tissue samples in medical research. Recently, non-protein coding transcripts long non-coding RNAs (lncRNAs), have gained significant attention due to their important biological actions and potential involvement in cancer. There is no validated method except qRTPCR or RNAseq to evaluate and study lncRNA expression. We have standardized and are reporting a sensitive Z probe based in situ hybridization method to identify, localize and quantitate lncRNA in FFPE tissues. This assay is sensitive to single transcript and localizes lncRNA in individual cells within tumor. We have characterized a tumor suppressor lncRNA-NRON (non coding repressor of NFAT), which is scarcely expressed, a moderately expressed oncogeneic lncRNA UCA1 (urothelial cancer associated 1), and a highly studied and expressed lncRNA MALAT1 (metastasis associated lung adenocarcinoma transcript1) in different cancers. High MALAT1 staining was found in colorectal, breast and pancreatic cancer. MALAT1 expression increased with the progression of the stage in colorectal cancer and invasiveness in breast cancer.

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Reactivation of BK Polyomavirus in Urine Cytology Is Not Associated with Urothelial Cell Carcinoma

Viruses ◽

10.3390/v12121412 ◽

2020 ◽

Vol 12 (12) ◽

pp. 1412

Author(s):

Faisal Klufah ◽

Ghalib Mobaraki ◽

Axel zur Hausen ◽

Iryna V. Samarska

Keyword(s):

Cell Carcinoma ◽

Urine Cytology ◽

Urothelial Cell ◽

The Other ◽

Urothelial Cell Carcinoma ◽

Tissue Samples ◽

Bk Polyomavirus ◽

Ffpe Tissues ◽

Immunosuppressed Patients ◽

History Of

BK polyomavirus (BKPyV) has been associated with some high-grade and special urothelial cell carcinoma (UCC) subtypes in immunosuppressed patients. Here, we evaluated the relationship of BKPyV-positive urine cytology specimens (UCS) with UCC. A large single-institution database was retrospectively searched for UCS positive for decoy cells, suggesting BKPyV infection. These were tested for the presence of BKPyV by PCR and immunohistochemistry (IHC) in urine sediments and formalin-fixed paraffin-embedded (FFPE) tissue samples of UCC. Decoy cells were reported in 30 patients out of the database with 22.867 UCS. Of these 30 patients, 16 (53.3%) had no history of UCC. Six patients out of these 16 had a history of transplantation, 4 had a history of severe chronic medical conditions, and 6 had no chronic disease. The other fourteen patients were diagnosed with either in situ or invasive UCC of the urinary bladder (14/30; 46.6%) prior to the detection of decoy cells in the urine. Nine of these UCC patients received intravesical treatment (BCG or mitomycin) after the first presentation with UCC. However, the clinical data on the treatment of the other five UCC patients was lacking. IHC identified BKPyV-positivity in the urine samples of non-UCC and UCC patients, while no BKPyV positivity was found in FFPE tissues of primary UCCs and metastases. In addition, BKPyV-PCR results revealed the presence of BKPyV DNA in the urine of the UCC cases, yet none in the UCC tissues itself. These data strongly indicate that BKPyV reactivation is not restricted to immunosuppression. It can be found in UCS of the immunocompetent patients and may be related to the intravesical BCG or mitomycin treatment of the UCC patients.

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Alcoholic fixation over formalin fixation: A new, safer option for morphologic and molecular analysis of tissues

Saudi Journal of Biological Sciences ◽

10.1016/j.sjbs.2021.08.075 ◽

2021 ◽

Author(s):

Md. Asabur Rahman ◽

Nasrin Sultana ◽

Ummay Ayman ◽

Sonali Bhakta ◽

Marzia Afrose ◽

...

Keyword(s):

Molecular Analysis ◽

Formalin Fixation

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Molecular Diagnostics in Melanoma: Current Status and Perspectives

Archives of Pathology & Laboratory Medicine ◽

10.5858/2009-0623-rar1.1 ◽

2011 ◽

Vol 135 (7) ◽

pp. 860-869 ◽

Cited By ~ 3

Author(s):

Soheil S. Dadras

Keyword(s):

Molecular Diagnostics ◽

Ribonucleic Acid ◽

New Technologies ◽

Mutational Analysis ◽

Transcriptional Profiling ◽

Clinical Information ◽

Current Status ◽

Comparative Genomic ◽

Molecular Approaches ◽

Fresh Frozen

Abstract Context.—In the current “molecular” era, the advent of technology, such as array-based platforms, systems biology, and genome-wide approaches, has made it possible to examine human cancers, including melanoma, for genetic mutations, deletions, amplification, differentially regulated genes, and epigenetic changes. Advancement in current technologies is such that one can now examine ribonucleic acid (RNA), deoxyribonucleic acid (DNA), and protein directly from the patient's own tumor. Objective.—To apply these new technologies in advancing molecular diagnostics in melanoma has historically suffered from a major obstacle, namely, the scarcity of fresh frozen, morphologically defined tumor banks, annotated with clinical information. Recently, some of the new platforms have advanced to permit utilization of formalin-fixed, paraffin-embedded (FFPE) tumor specimens as starting material. Data Sources.—This article reviews the latest technologies applied to FFPE melanoma sections, narrowing its focus on the utility of transcriptional profiling, especially for melastatin; comparative genomic hybridization; BRAF and NRAS mutational analysis; and micro ribonucleic acid profiling. Conclusion.—New molecular approaches are emerging and are likely to improve the classification of melanocytic neoplasms.

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Muscle Biopsy Samples for Histochemical Processing: Alterations Induced by Storage

Veterinary Pathology ◽

10.1177/030098588802500111 ◽

1988 ◽

Vol 25 (1) ◽

pp. 77-82 ◽

Cited By ~ 6

Author(s):

K. G. Braund ◽

K. A. Amling

Keyword(s):

Skeletal Muscle ◽

Cell Morphology ◽

Type I ◽

Type Ii ◽

Tissue Samples ◽

Frozen Tissue ◽

Fresh Frozen ◽

Healthy Dogs ◽

Morphology And Morphometry ◽

Type Ii Fibers

Skeletal muscle samples from two healthy dogs were stored in ice at 0 C for up to 30 hours to examine the influence of time on cell morphology and morphometry. Cytochemical and histochemical properties of muscle to 18 hours were not markedly different from fresh frozen tissue. Samples stored to 30 hours were still satisfactory, despite a decline and unevenness in depth of staining. Morphometry from samples stored at 0 C for 6 hours or longer is not recommended, due to the statistically significant increase in diameter (from 21 to 25%) of type I and type II fibers.

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Influence of prolonged formalin fixation of tissue samples on the sensitivity of chromogenic in situ hybridization

Journal of Veterinary Diagnostic Investigation ◽

10.1177/1040638711425584 ◽

2011 ◽

Vol 23 (6) ◽

pp. 1212-1216 ◽

Cited By ~ 10

Author(s):

Meike M. Mostegl ◽

Barbara Richter ◽

Nora Dinhopl ◽

Herbert Weissenböck

Keyword(s):

In Situ Hybridization ◽

Nucleic Acid ◽

Signal Intensity ◽

Formalin Fixation ◽

Proteinase K ◽

Cross Linking ◽

Fixation Time ◽

Tissue Samples ◽

Chromogenic In Situ Hybridization

Chromogenic in situ hybridization (ISH) is a commonly used tool in diagnostic pathology to detect pathogens in formalin-fixed, paraffin-embedded (FFPE) tissue sections. Prolonged formalin fixation time was identified to be a limiting factor for the successful detection of nucleic acid from different pathogens, most probably due to the cross-linking activity of formalin between RNA, DNA, and proteins. Therefore, in the current study, the influence of formalin fixation time on ISH signal intensity of 2 viral ( Porcine circovirus-2 [PCV-2] and Porcine respiratory and reproductive virus [PRRSV]) and 2 protozoal agents ( Cryptosporidium serpentis and Tritrichomonas sp.) was evaluated. Tissue samples were fixed in 7% neutral buffered formaldehyde solution, and at defined intervals, pieces were embedded in paraffin wax and subjected to pathogen-specific ISH. For all 4 pathogens, the signal intensity remained comparable with the starting ISH signal for different periods of fixation (PCV-2: 6 weeks, PRRSV: 23 weeks, C. serpentis: 55 weeks, Tritrichomonas sp.: 53 weeks). Thereafter, the signal started to decline until loss of nucleic acid detection. The influence of increased proteinase K concentrations for inverting the formalin-induced cross-linking activity was examined compared with the standard protocol. With all 4 infectious agents, a 4-fold proteinase K concentration restored the ISH signals to a level comparable with 1 day of fixation. In conclusion, the influence of prolonged formalin fixation on the intensity of detected ISH signal highly depends on the analyzed infectious agent and the pretreatment protocol.

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A Pilot Study of Rare Renal Amyloidosis Based on FFPE Proteomics

Molecules ◽

10.3390/molecules26237234 ◽

2021 ◽

Vol 26 (23) ◽

pp. 7234

Author(s):

Shuang Meng ◽

Wenwen Xia ◽

Li Xia ◽

Li Zhou ◽

Jing Xu ◽

...

Keyword(s):

Pilot Study ◽

Bioinformatics Analysis ◽

Great Promise ◽

Individual Treatment ◽

Renal Amyloidosis ◽

Tissue Samples ◽

Retrospective Diagnosis ◽

Formalin Fixed Paraffin Embedded ◽

Ffpe Tissues ◽

End Stage

Renal amyloidosis typically manifests albuminuria, nephrotic-range proteinuria, and ultimately progresses to end-stage renal failure if diagnosed late. Different types of renal amyloidosis have completely different treatments and outcomes. Therefore, amyloidosis typing is essential for disease prognosis, genetic counseling and treatment. Thirty-six distinct proteins currently known to cause amyloidosis that have been described as amyloidogenic precursors, immunohistochemistry (IHC) or immunofluorescence (IF), can be challenging for amyloidosis typing especially in rare or hereditary amyloidosis in clinical practice. We made a pilot study that optimized the proteomics pre-processing procedures for trace renal amyloidosis formalin-fixed paraffin-embedded (FFPE) tissue samples, combined with statistical and bioinformatics analysis to screen out the amyloidosis-related proteins to accurately type or subtype renal amyloidosis in order to achieve individual treatment. A sensitive, specific and reliable FFPE-based proteomics analysis for trace sample manipulation was developed for amyloidosis typing. Our results not only underlined the great promise of traditional proteomics and bioinformatics analysis using FFPE tissues for amyloidosis typing, but also proved that retrospective diagnosis and analysis of previous cases laid a solid foundation for personalized treatment.

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