Advanced Concepts in Fixation: 1. Effects of Fixation on Immunohistochemistry, Reversibility of Fixation and Recovery of Proteins, Nucleic Acids, and other Molecules from Fixed and Processed Tissues. 2. Developmental Methods of Fixation

Eltoum, Isam A.; Fredenburgh, Jerry; Grizzle, William E.

doi:10.1179/his.2001.24.3.201

Cited by 58 publications

(20 citation statements)

References 47 publications

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“…Formalin fixation is a time‐dependent process in which increased fixation time results in continued formaldehyde groups binding to proteins to a point of equilibrium. Studies have shown that formalin fixation, especially if prolonged, results in decreased antigenicity, limiting the use of formalin‐fixed tissues for diagnostic IHC (Goto et al., 2000 and Eltoum et al., 2001).…”

Section: Discussionmentioning

confidence: 99%

Pathological, Immunological and Molecular Diagnosis of Rabies in Clinically Suspected Animals of Different Species Using Four Detection Techniques in Jordan

Faizee

Hailat

Ababneh

et al. 2011

Transboundary and Emerging Diseases

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A total of 29 brain tissue samples (BTS) were examined for rabies infection by different diagnostic techniques. None of the examined brain tissues were presented as a whole intact brain. Twenty-seven brain tissue samples from various animal species - dog (13 cases), cat (one case), fox (one case), pig (one case), cow (three cases), sheep (two cases), goat (one case), camel (one case), horse (one case) and donkey (three cases) - were provided by the Vaccine and Sera Department/Al-Bashir Central Hospital in Amman/Jordan from July 2009 up to May 2010. All these samples were frozen at -20°C, for a period of time and then fixed in 10% formalin after being tested for rabies virus by fluorescence antibody test (FAT). The results showed that 21 (77.77%) of 27 BTS were positive for rabies by FAT. Seventeen samples (58.62%) of 29 were positive by histopathology, 2 (6.90%) were positive by histopathology, immunohistochemistry (IHC) and of those which were fixed for 24h only, and 21 (72.42%) were positive using RT-PCR assay. Five of 29 BTS had no pathological lesions, 17 had Negri bodies and the remaining had non-suppurative encephalitis and necrosis. Thirteen BTS that were diagnosed positive by FAT were also positive by RT-PCR and histopathology, but negative by IHC. Four BTS that were positive by FAT were negative by histopathology, IHC and RT-PCR. Also, 3 BTS (cases 19, 22, and 25) that were negative by FAT were positive by RT-PCR and negative by IHC. One of these was negative, while two were positive by histopathology. Therefore, definitive diagnosis of rabies under these conditions in Jordan needs one or more other diagnostic tests in addition to FAT. Also, freezing and prolonged formalin fixation of BTS is not suitable for the detection of rabies virus antigen using IHC.

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Section: Discussionmentioning

confidence: 99%

Pathological, Immunological and Molecular Diagnosis of Rabies in Clinically Suspected Animals of Different Species Using Four Detection Techniques in Jordan

Faizee

Hailat

Ababneh

et al. 2011

Transboundary and Emerging Diseases

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“…The major advancement of the MaCIA is its extended shelflife, at least for 14 weeks, that was achieved through formalinfixation of HCT-8 cells. Formalin is routinely used to preserve tissues and cells and it protects protein from denaturation (Eltoum et al, 2001). Therefore, receptor molecules on formalin-fixed HCT-8 cells, remained active and enabled viable Salmonella binding without diminishing MaCIA's performance.…”

Section: Discussionmentioning

confidence: 99%

“…In this study, we took an innovative approach and developed a shelf-stable Mammalian Cell-based ImmunoAssay (MaCIA) platform for the detection of live pathogenic bacteria. Shelflife of MaCIA was prolonged by fixing the mammalian cells in formalin (4% formaldehyde) which is a common practice in histology and tissue imaging to preserve the cells by preventing protein degradation (Eltoum et al, 2001). Furthermore, instead of measuring cytotoxicity, we took advantage of the adhesion ability of enteric pathogens to the intestinal cells followed by antibody-based assay for specific detection of the adhered target pathogens.…”

Section: Introductionmentioning

confidence: 99%

Mammalian Cell-Based Immunoassay for Detection of Viable Bacterial Pathogens

Bai

Tenguria

et al. 2020

Front. Microbiol.

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Rapid detection of live pathogens is of paramount importance to ensure food safety. At present, nucleic acid-based polymerase chain reaction and antibody-based lateral flow assays are the primary methods of choice for rapid detection, but these are prone to interference from inhibitors, and resident microbes. Moreover, the positive results may neither assure virulence potential nor viability of the analyte. In contrast, the mammalian cell-based assay detects pathogen interaction with the host cells and is responsive to only live pathogens, but the short shelf-life of the mammalian cells is the major impediment for its widespread application. An innovative approach to prolong the shelf-life of mammalian cells by using formalin was undertaken. Formalin (4% formaldehyde)-fixed human ileocecal adenocarcinoma cell line, HCT-8 on 24-well tissue culture plates was used for the capture of viable pathogens while an antibody was used for specific detection. The specificity of the Mammalian Cell-based ImmunoAssay (MaCIA) was validated with Salmonella enterica serovar Enteritidis and Typhimurium as model pathogens and further confirmed against a panel of 15 S. Enteritidis strains, 8 S. Typhimurium, 11 other Salmonella serovars, and 14 non-Salmonella spp. The total detection time (sample-to-result) of MaCIA with artificially inoculated ground chicken, eggs, milk, and cake mix at 1–10 CFU/25 g was 16–21 h using a traditional enrichment set up but the detection time was shortened to 10–12 h using direct on-cell (MaCIA) enrichment. Formalin-fixed stable cell monolayers in MaCIA provide longer shelf-life (at least 14 weeks) for possible point-of-need deployment and multi-sample testing on a single plate.

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“…Care also should be taken to quantify the length of fixation, particularly in formalin, in which cross‐linking of nucleic acids can hinder molecular testing by ISH, amplification‐based testing, or sequencing. Length of fixation also may have an effect on IHC testing . It should be noted that cell block samples fixed in formalin might not tolerate extensive fixation times due to their markedly small size.…”

Section: Introductionmentioning

confidence: 99%

Analytic inquiry: Validation and practical considerations

Berry¹

2017

Cancer Cytopathology

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Molecular diagnostics and cytopathology ideally are complementary medical services. When used together, they provide optimal benefit to patient care with the least risk of complications. However, many cytopathology laboratories are reluctant to bring in molecular tests, in part due to inexperience with regard to molecular test validation. This article is a brief review of the basic principles of molecular test validation as it applies to cytopathology samples. Regulatory constraints, practical considerations, and issues that are particular to cytopathology samples are discussed, along with a brief review of issues that pertain specifically to next-generation sequencing. This review should serve as a general guide to validating molecular tests in the cytopathology laboratory. Cancer Cytopathol 2017;125(6 suppl):465-9.

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Advanced Concepts in Fixation: 1. Effects of Fixation on Immunohistochemistry, Reversibility of Fixation and Recovery of Proteins, Nucleic Acids, and other Molecules from Fixed and Processed Tissues. 2. Developmental Methods of Fixation

Cited by 58 publications

References 47 publications

Pathological, Immunological and Molecular Diagnosis of Rabies in Clinically Suspected Animals of Different Species Using Four Detection Techniques in Jordan

Pathological, Immunological and Molecular Diagnosis of Rabies in Clinically Suspected Animals of Different Species Using Four Detection Techniques in Jordan

Mammalian Cell-Based Immunoassay for Detection of Viable Bacterial Pathogens

Analytic inquiry: Validation and practical considerations

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