5110BMBMOL Lecture 3 2025 - Further Histological Techniques

Page 1: Lecture Overview

• Course Title: 5110BMBMOL - Cellular Pathology

• Lecture Topic: Advanced Histology Techniques

• Instructor: Dr. Nick Bryan

• Contact: n.bryan@ljmu.ac.uk

• Important Sections: liver, portal plate, interlobular regions

Page 2: Overview of Module Material

• Fundamental understanding of histology and specimen generation

• Awareness of basic stains and their identification in tissue sections

• Goals:

  • Explore quantification of histology

  • Familiarize with various cells and tissue structures in histological sections

  • Contextualize histology in diagnostic/scientific workflows (focus on biomaterials)

  • Discuss immunohistochemistry for added specificity in histology

Page 3: Lecture Objectives

• Review covered materials

• Discuss quantification of histological findings

• Look into immunohistochemistry and its value in histological analysis

Page 4: What are Biomaterials?

• Definition: Exogenous materials restoring function to damaged tissue.

• Examples include:

  • Vascular Graft

  • Pacemaker

  • Stitches/Suture

  • Stent

  • Heart Valve

  • Prosthetic Knee

Page 5: Histological Analysis Scenario

• Implant example (hip, knee, vascular graft) removed due to loosening

• Key questions for histological investigation:

  • Presence of delivered cells and infiltration by external cells

  • Integration with surrounding tissues

  • Encapsulation or fibrotic development

  • Implant condition (degradation/resorption profile)

  • Foreign body response and inflammatory cell recruitment

  • Localized necrosis assessment

  • Vascularization status (macro/microscopic blood supply)

Page 6: Key Cells Affecting Biocompatibility

• Neutrophils:

  • First responders, produce damaging molecules while coordinating healing

• Macrophages:

  • Clear debris and guide tissue healing but may cause damage if uncontrolled

• Lymphocytes:

  • Chronic inflammation, remove foreign molecules; roles in both destruction and healing

• Fibroblasts:

  • Secrete extracellular matrix (ECM) to stabilize implants

• Mast Cells:

  • Direct inflammation response through cytokine secretion

• Tissue-specific cells:

  • Include osteoblasts, chondrocytes, endothelial cells, adipocytes, neural and glial cells, with regenerative roles in device-specific applications.

Page 7: Lecture Objectives (Repeating)

• Mention of reviewed topics and future discussion points (similar to Page 3).

Page 8: ISO 10993 Standards

• Standards for evaluating medical device biocompatibility before clinical trials

• Emphasis on histopathologic data from animal models

• Pathologic Index:

  • A scoring system (0-4) based on sample criteria presence/absence:

    • Neutrophils

    • Macrophages

    • Fibroblasts

    • Encapsulation

    • Necrosis

    • Degradation

    • Neovascularization

Page 9: Statistical Analysis of Pathological Indexing

• Presented data on different features and their significance across materials

• Samples compared with particular focus on cell types and conditions.

Page 10: Quantifying Histology

• Pros:

  • Quick pathologic indexing lends to perioperative histology

  • Provides statistically testable values in a qualitative field

• Cons:

  • Interpretation is subjective and reliant on skilled histologists

  • Inter-histologist variability may introduce subjectivity

Page 11: Napthol ASD Chloroacetate Esterase Stain

• Used to identify leukocytes through specific esterase enzymes reactions

• Enzyme activity produces a colored product at the site

• Example of staining monocyte lineage cells

Page 12: Problem in Cell Counting

• Identical positive stained cells in different sample sizes do not imply similar behavior

Page 13: Solution for Cell Counting

• Perform a nuclear stain (e.g., haematoxylin) to count total cells

• Cells of interest can be expressed as a percentage of the total count.

Page 14: Reiteration of Objectives

• Same as earlier objectives, focusing on revision and quantification of findings.

Page 15: Immunohistochemistry (IHC)

• Use of antibodies to visualize specific molecules within, on, or outside a cell.

Page 16: Antibody Structure

• Overview of antibody parts:

  • Fab Region (antigen binding)

  • Fc Region (crystallisable fragment)

  • Heavy and Light Chains

  • Variable Domain as Antigen Binding Site

• Antibodies are called Immunoglobulins (Ig).

Page 17: Role of Antibodies in Molecular Biology

• Specificity of antibodies is crucial for identifying cells/substances.

• Help target immune responses and limit damage to native tissues.

• Use of antibody specificity in molecular biology for identification purposes.

Page 18: Production of IHC Antibodies (1)

• Method involves inoculating animals with an antigen

• Production of polyclonal antibodies against various epitopes of the antigen.

Page 19: Production Steps

1. Inoculate mammal with antigen

2. Collect blood and isolate antibodies

3. Chickens yield antibodies from both blood and eggs

4. Typically involves 3-4 inoculations over 28 days.

Page 20: Production of IHC Antibodies (2)

• Approach using hybridoma cells

  • Hybridoma cells fuse B-cell and myeloma cancer cell properties

  • Yield monoclonal antibodies targeting one epitope of the antigen.

Page 21: Production Process Overview

• Inoculation, blood extraction, B-cell isolation, fusion with myeloma cells, hybridoma growth and harvesting antibody.

Page 22: Polyclonal vs. Monoclonal Antibodies

• Polyclonal: Multiple B cell clones, detect different epitopes.

• Monoclonal: Single parent clone, detects one epitope.

Page 23: Visualization Methods in IHC

• Fluorescence: Antibodies conjugated to fluorophores viewed via fluorescence microscopy.

• Enzyme-linked methods produce color changes at antibody binding sites.

Page 24: Antigen Retrieval

• Formalin fixation can mask antigenic sites; retrieval needed for antibody binding.

• Methods:

  • Heat Induced Antigen Retrieval

  • Enzymatic Antigen Retrieval

Page 25: Antigen Retrieval Process Visual

• Steps in unmasking antigens to allow antibody binding post-fixation.

Page 26: Physics of Fluorescence

• Explanation of electron excitation in fluorophores and energy release.

Page 27: Fluorophore Properties

• Details on excitation and emission wavelengths of fluorescein, a common fluorophore in IHC.

Page 28: Using Multiple Fluorophores

• Different excitable fluorophores allow visualization of multiple targets in a single sample.

Page 29: Fluorescence Microscope Technology

• Discussion on traditional light sources in fluorescence microscopy.

Page 30: Common CD Markers

• Overview of significant CD markers and their associated cell types.

• Examples include:

  • CD31 (Endothelial Cells)

  • CD14 (Monocytes/Macrophages)

  • CD45 (White Blood Cells)

Page 31: Antibody Terminology

• Species specificity of antibodies and nomenclature explained.

Page 32: Importance of Antibody Species

• Use of secondary antibodies enhances signal strength in immunohistochemistry.

Page 33: Antibody Isotypes

• Importance of knowledge on the shape of antibodies for molecular biology applications.

Page 34: Analytical Antibody Conjugation

• Importance of knowing conjugates in order to select proper reading instruments and avoid crossovers in experiments.

Page 35: Immunohistochemistry Direct Conjugation

• Visual representation of direct conjugation process in IHC.

Page 36: Visualizing Antibodies on Cells

• Use of secondary antibodies to increase fluorescence signal.

Page 37: Multiplexing in IHC

• Using multiple antibodies to visualize different targets in the same sample.

Page 38: Multiplexing Challenges

• Ensure distinct spectral properties among antibodies to avoid color overlap.

Page 39: Counter Stains in IHC

• Nuclear Stains:

  • DAPI as a commonly used DNA intercalating dye.

• Cytoskeletal Stains:

  • Example: Phalloidin highlighting actin filaments.

Page 40: Final Notes on IHC

• Techniques can include colorimetric methods using enzymes for visible changes in samples.

Page 41: Case Study Applications

• Example of macrophages visualized on biomaterials.

Page 42: Breast Cancer Treatment Relevance

• IHC utilized to determine effective drug treatments for cancers based on receptor status.

Page 52: Summary of Lecture Insights

• Mention of standard histology stains and versatile multipurpose histological tools.

• Emphasizing the need for quantitative approaches in modern histology, including advanced image analysis and immunohistochemistry techniques.