Immunohistology

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Surender Juneja - One of the best experts on this subject based on the ideXlab platform.

Anthony S.-y. Leong - One of the best experts on this subject based on the ideXlab platform.

  • Standardization in Immunohistology.
    Methods of Molecular Biology, 2011
    Co-Authors: Anthony S.-y. Leong, Trishe Y.-m. Leong
    Abstract:

    The rapid acceptance of Immunohistology as an invaluable adjunct to morphologic diagnosis has been possible because of the development of new and more sensitive antibodies and detection systems that allow its application to formalin-fixed, paraffin-embedded tissue (FFPT). More importantly, antigen-retrieval techniques have resulted in some degree of consistency allowing Immunohistology to be used reliably as a diagnostic tool. The advent of prognostic and predictive biomarkers, and the desire for individualized therapy has resulted in mounting pressure to employ the immunohistological assay in a quantitative manner. While it was not a major issue when the technique was employed in a qualitative manner, the numerous variables in the preanalytical and analytical phases of the test procedure that influence the immunoexpression of proteins in FFPT become critical to standardization. Tissue fixation is pivotal to antigen preservation but exposure to fixative prior to accessioning by the laboratory is not controlled. Antigen retrieval, crucial in the analytical phase, continues to be employed in an empirical manner with the actual mechanism of action remaining elusive. There is great variation in reagents, methodology, and duration of tissue processing and immunostaining procedure, and the detection systems employed are not standardized between laboratories. While many of these variables are offset by the application of antigen retrieval, which enables the detection of a wide range of antigens in FFPT, the method itself is not standardized. This myriad of variables makes it inappropriate to provide meaningful comparisons of results obtained in different laboratories and even in the same laboratory, as in current practice, each specimen experiences different preanalytical variables. Furthermore, variables in interpretation exist and cutoff thresholds for positivity differ. Failure to recognize false-positive and false-negative stains leads to further errors of quantitative measurement. Many of the problems relating to the technology and interpretation of immunostaining originate from failure to recognize that this procedure is different from other histological stains and involves many more steps that cannot be monitored until the end result is attained. While several remedial measures can be suggested to address some of these problems, accurate and reproducible quantitative assessment of immunostains presently remains elusive as important variables that impact on antigen preservation in the paraffin-embedded biopsy -cannot be standardized.

  • Immunohistology--past, present, and future.
    Advances in Anatomic Pathology, 2010
    Co-Authors: Trishe Y.-m. Leong, Kumarasen Cooper, Anthony S.-y. Leong
    Abstract:

    The rapid development of immunohistochemistry, a morphology-based technique, has come about through refinements in detection systems and an increasing range of sensitive and specific antibodies that have allowed application of the technique to formalin-fixed, paraffin-embedded tissues. The introduction of heat-induced antigen retrieval has been a significant milestone to compliment these developments so that the immunohistochemistry is firmly entrenched as an indispensable adjunct to morphologic diagnosis. Although this ancillary stain was initially used in a qualitative manner, problems surrounding the many variables that influence antigen preservation in formalin-fixed, paraffin-embedded tissues were not a major issue and laboratories strived to optimize their staining protocols to the material they accessioned and processed. The advent of personalized medicine and targeted cancer treatment has imposed the need to quantitate the stain reaction product and has resulted in calls to standardize the process of immunostaining. A closer examination of the variables that influence the ability to show antigens in formalin-fixed, paraffin-embedded tissues revealed many important variables, particularly in the preanalytical phase of the assay, that are beyond the control of the accessioning laboratory. Although analytical factors have the potential to be standardized, the actions of many pivotal procedures including fixation and antigen retrieval are not completely understood. Postanalytical processes including threshold and cut-off values require consensus and standardization and it is clear that some of these goals can be achieved through the direction of national and international organizations associated with cancer diagnosis and treatment. With the ability to serve as a surrogate marker of many genetic abnormalities, immunohistochemistry enters a new era and the need to better understand some of the mechanisms fundamental to the technique become more pressing and the development of true quantitative assays is imperative. There is also an increasing appreciation that the technique highlights patterns of staining that reflect exquisite localization to organelles and tissue structures that are not appreciable in routine stains, adding a further dimension to morphologic diagnosis.

  • Immunohistology and Other Diagnostic Techniques
    A Pattern Approach to Lymph Node Diagnosis, 2010
    Co-Authors: Anthony S.-y. Leong
    Abstract:

    While morphologic examination of H&E-stained sections remains the cornerstone of lymph node diagnosis, a wide variety of ancillary investigations have served to better define the malignant lymphomas and leukemias. Importantly, they have contributed to a conceptual understanding of the lymphomas, particularly those of B cell lineage. Among the more common techniques that are useful in diagnosis are Immunohistology, flow cytometry, cytogenetics, and molecular analysis, with histochemistry, electron microscopy, and bacterial and viral cultures providing useful diagnostic information in more specific situations. Gene expression profiling and proteomics are still research tools but have enhanced our knowledge and facilitated the introduction of new immunohistochemical markers.

  • Quantitative Immunohistology: tissue section thickness, another glitch in the path to standardization.
    Applied Immunohistochemistry & Molecular Morphology, 2009
    Co-Authors: Anthony S.-y. Leong
    Abstract:

    Immunohistology was introduced in anatomical pathology as a means of labeling cells to allow their identification. Through the labeling of proteins, enzymes, andother cellular products it became possible to specifically recognize cells that otherwise could not be distinguished on the basis of morphology alone. Immunohistochemistry served as a specific ‘‘special stain’’ that provided largely qualitative information, which, at best, was semiquantitative. Nonetheless, this qualitative function served the diagnostic role of Immunohistology well and the technique has rapidly become entrenched as an important adjunct to morphologic diagnosis

  • Variables That Influence Outcomes in Immunohistology
    The Australian Journal of Medical Science, 2007
    Co-Authors: Trishe Y.-m. Leong, Anthony S.-y. Leong
    Abstract:

    Despite entering its fourth decade of existence, Immunohistology as a diagnostic tool continues to experience poor reproducibility. This problem is highlighted as the technique is employed as a quantitative assessment of therapeutic and prognostic markers in a number of neoplasms. While qualitative applications continue to increase with the range of sensitive antibodies available, numerous variables that influence the immunoexpression of proteins in formalin fixed tissue exist in the pre-analytical and analytical phases of the test procedure. Pre-analytical variables are currently beyond the control of the laboratory. Tissue fixation is critical but exposure to fixative prior to accessioning in the laboratory cannot be controlled. Antigen retrieval, another pivotal procedure in Immunohistology, continues to be employed in an empirical manner with the actual mechanism of action remaining elusive. There is great variation in reagents and duration of tissue processing and the immunostaining procedure is not standardised between laboratories. Variables in interpretation exist and cut-off thresholds for positivity are variable. Failure to recognise false positive and false negative stains leads to errors of qualitative interpretation. Many of the problems related to the technology and interpretation of immunostaining originate from failure to recognise that this procedure is different from other histological stains. Accurate and reproducible quantitative assessment of immunostains remains elusive as important variables that impact on antigen preservation in the paraffin embedded biopsy currently cannot be controlled.

Michel Piotin - One of the best experts on this subject based on the ideXlab platform.

  • Acute ischemic stroke thrombi have an outer shell that impairs fibrinolysis
    Neurology, 2019
    Co-Authors: Lucas Meglio, Jean-philippe Desilles, Véronique Ollivier, Mialitiana Solo Nomenjanahary Msci, Sara Di Meglio, Catherine Deschildre, Stéphane Loyau, Jean-marc Olivot, Raphaël Blanc, Michel Piotin
    Abstract:

    OBJECTIVES: Thrombi responsible for large vessel occlusion (LVO) in the setting of acute ischemic stroke (AIS) are characterized by a low recanalization rate after IV thrombolysis. To test whether AIS thrombi have inherent common features that limit their susceptibility to thrombolysis, we analyzed the composition and ultrastructural organization of AIS thrombi causing LVO. METHODS: A total of 199 endovascular thrombectomy-retrieved thrombi were analyzed by Immunohistology and scanning electron microscopy (SEM) and subjected to ex vivo thrombolysis assay. The relationship between thrombus organization and thrombolysis resistance was further investigated in vitro using thrombus produced by recalcification of citrated whole blood. RESULTS: SEM and Immunohistology analyses revealed that, although AIS thrombus composition and organization was highly heterogeneous, AIS thrombi shared a common remarkable structural feature in the form of an outer shell made of densely compacted thrombus components including fibrin, von Willebrand factor, and aggregated platelets. In vitro thrombosis experiments using human blood indicated that platelets were essential to the formation of the thrombus outer shell. Finally, in both AIS and in vitro thrombi, the thrombus outer shell showed a decreased susceptibility to tissue plasminogen activator-mediated thrombolysis as compared to the thrombus inner core. INTERPRETATION: Irrespective of their etiology and despite their heterogeneity, intracranial thrombi causing LVO have a core shell structure that influences their susceptibility to thrombolysis.

Grant Stotts - One of the best experts on this subject based on the ideXlab platform.

  • 18f fluorodeoxyglucose pet ct imaging as a marker of carotid plaque inflammation comparison to Immunohistology and relationship to acuity of events
    International Journal of Cardiology, 2018
    Co-Authors: Myra S Cocker, Robert R. Hammond, Robert A. Dekemp, Cheemun Lum, George A. Wells, Jordan Bernick, Andrew Hill, Sudhir Nagpal, David J Spence, Grant Stotts
    Abstract:

    Abstract Background [18F]-fluorodeoxyglucose (18FDG) uptake imaged with positron emission tomography (PET) and computed tomography (CT) may serve as a biomarker of plaque inflammation. This study evaluated the relationship between carotid plaque 18FDG uptake and a) intraplaque expression of macrophage and macrophage-like cellular CD68 Immunohistology; b) intraplaque inflammatory burden using leukocyte-sensitive CD45 Immunohistology; c) symptomatic patient presentation; d) time from last cerebrovascular event. Methods 54 patients scheduled for carotid endarterectomy underwent 18FDG PET/CT imaging. Maximum 18FDG uptake (SUV max ) and tissue-to-blood ratio (TBR max ) was measured for carotid plaques. Quantitative immunohistological analysis of macrophage-like cell expression (CD68) and leukocyte content (CD45) was performed . Results 18FDG uptake was related to CD68 macrophage expression (TBRmax: r = 0.51, p  90 days (SUVmax:3.15 ± 0.87 vs. 2.52 ± 0.45, p = 0.015). Conclusions In this CAIN cohort, 18FDG uptake imaged with PET/CT serves a surrogate marker of intraplaque inflammatory macrophage, macrophage-like cell and leukocyte burden. 18FDG uptake is greater in plaque associated with patient symptoms and those with recent cerebrovascular events. Future studies are needed to relate 18FDG uptake and disease progression.

  • [18F]-Fluorodeoxyglucose PET/CT imaging as a marker of carotid plaque inflammation: Comparison to Immunohistology and relationship to acuity of events
    International Journal of Cardiology, 2018
    Co-Authors: Myra S Cocker, J. David Spence, Robert R. Hammond, Robert A. Dekemp, Cheemun Lum, George A. Wells, Jordan Bernick, Andrew Hill, Sudhir Nagpal, Grant Stotts
    Abstract:

    Abstract Background [18F]-fluorodeoxyglucose (18FDG) uptake imaged with positron emission tomography (PET) and computed tomography (CT) may serve as a biomarker of plaque inflammation. This study evaluated the relationship between carotid plaque 18FDG uptake and a) intraplaque expression of macrophage and macrophage-like cellular CD68 Immunohistology; b) intraplaque inflammatory burden using leukocyte-sensitive CD45 Immunohistology; c) symptomatic patient presentation; d) time from last cerebrovascular event. Methods 54 patients scheduled for carotid endarterectomy underwent 18FDG PET/CT imaging. Maximum 18FDG uptake (SUV max ) and tissue-to-blood ratio (TBR max ) was measured for carotid plaques. Quantitative immunohistological analysis of macrophage-like cell expression (CD68) and leukocyte content (CD45) was performed . Results 18FDG uptake was related to CD68 macrophage expression (TBRmax: r = 0.51, p  90 days (SUVmax:3.15 ± 0.87 vs. 2.52 ± 0.45, p = 0.015). Conclusions In this CAIN cohort, 18FDG uptake imaged with PET/CT serves a surrogate marker of intraplaque inflammatory macrophage, macrophage-like cell and leukocyte burden. 18FDG uptake is greater in plaque associated with patient symptoms and those with recent cerebrovascular events. Future studies are needed to relate 18FDG uptake and disease progression.

Lucas Meglio - One of the best experts on this subject based on the ideXlab platform.

  • Acute ischemic stroke thrombi have an outer shell that impairs fibrinolysis
    Neurology, 2019
    Co-Authors: Lucas Meglio, Jean-philippe Desilles, Véronique Ollivier, Mialitiana Solo Nomenjanahary Msci, Sara Di Meglio, Catherine Deschildre, Stéphane Loyau, Jean-marc Olivot, Raphaël Blanc, Michel Piotin
    Abstract:

    OBJECTIVES: Thrombi responsible for large vessel occlusion (LVO) in the setting of acute ischemic stroke (AIS) are characterized by a low recanalization rate after IV thrombolysis. To test whether AIS thrombi have inherent common features that limit their susceptibility to thrombolysis, we analyzed the composition and ultrastructural organization of AIS thrombi causing LVO. METHODS: A total of 199 endovascular thrombectomy-retrieved thrombi were analyzed by Immunohistology and scanning electron microscopy (SEM) and subjected to ex vivo thrombolysis assay. The relationship between thrombus organization and thrombolysis resistance was further investigated in vitro using thrombus produced by recalcification of citrated whole blood. RESULTS: SEM and Immunohistology analyses revealed that, although AIS thrombus composition and organization was highly heterogeneous, AIS thrombi shared a common remarkable structural feature in the form of an outer shell made of densely compacted thrombus components including fibrin, von Willebrand factor, and aggregated platelets. In vitro thrombosis experiments using human blood indicated that platelets were essential to the formation of the thrombus outer shell. Finally, in both AIS and in vitro thrombi, the thrombus outer shell showed a decreased susceptibility to tissue plasminogen activator-mediated thrombolysis as compared to the thrombus inner core. INTERPRETATION: Irrespective of their etiology and despite their heterogeneity, intracranial thrombi causing LVO have a core shell structure that influences their susceptibility to thrombolysis.