methods

Lecture 3 - Advanced Histology Techniques

Instructor Information

  • Presenter: Dr. Nick Bryan

  • Room: 10.02B

  • Email: n.bryan@ljmu.ac.uk

Overview of the Material so Far

By this point in the module, students should have:

  • A fundamental understanding of histology as a field and the processes involved in generating histology specimens.

  • Familiarity with some basic stains, how they are performed in the laboratory, and their identification in tissue sections.

This lecture builds upon the previous knowledge by:

  • Exploring methods for quantifying histology.

  • Familiarizing students with the appearances of different cells and tissue structures in histological sections.

  • Contextualizing histology in diagnostic workflows, particularly focusing on biomaterials.

  • Discussing how specificity can be added to histology using a technique known as immunohistochemistry.

Objectives

By the end of this lecture, participants will have:

  • Revised previously covered topics.

  • Discussed how histological findings are quantified.

  • Examined the immunohistochemistry technique and its value in histological interrogation.

What are Biomaterials?

Biomaterials are defined as "exogenous materials which restore function to damaged tissue." Examples of biomaterials include:

  • Vascular grafts

  • Pacemakers

  • Stitches/sutures

  • Stents

  • Heart valves

  • Prosthetic knees

Scenario Consideration

A clinical scenario is presented: An implant (e.g., hip, knee, vascular graft) seeded with cells is removed from a patient due to becoming loose. Histological examination can reveal:

  • The status of implanted cells (e.g., presence or absence).

  • Infiltration by external cells.

  • Integration with surrounding tissue, checking for encapsulation or fibrosis.

  • The extent of degeneration or resorption of the implant.

  • The extent of a foreign body response by inflammatory cells.

  • Potential localized necrosis in adjacent tissues.

  • Vascularization of the implant (both macroscopic and microscopic blood supply).

Important Cells for Biocompatibility

Several crucial cells impact the biocompatibility of implanted materials:

  • Neutrophils:

    • First responders in immunological defense, produce damaging molecules but are vital for coordinating tissue healing.

  • Macrophages:

    • Clear debris at the implant site and secrete factors that direct tissue healing. Uncontrolled activation can harm the implant and surrounding tissue.

  • Lymphocytes:

    • Involved in chronic inflammation, they are responsible for the removal of persistent foreign molecules and play roles in both tissue healing and material destruction.

  • Fibroblasts:

    • Known as matrix-secreting cells, they produce extracellular matrix (ECM) to stabilize the implant.

  • Mast Cells:

    • Tissue-resident cells directing inflammation through the secretion of cytokines and inflammatory mediators.

  • Tissue-specific Cells:

    • Such as osteoblasts (bone), chondrocytes (cartilage), endothelial cells (blood vessels), adipocytes (fat), and neural/glial cells (nervous tissue) that have regenerative roles in device-specific applications.

ISO 10993 Standards

  • ISO 10993: A set of standards for evaluating medical device biocompatibility prior to clinical trials.

  • Histopathologic Data: Plays a heavy role, typically presented in the form of a 'Pathologic Index' where several criteria are scored, often on a numerically scaled system (often 0-4) based on presence or absence:

    • Neutrophils

    • Macrophages

    • Fibroblasts

    • Encapsulation

    • Necrosis

    • Degradation

    • Neovascularization

Material

Neutrophils

Macrophages

Fibroblasts

Encapsulation

Necrosis

Degradation

Neovascularization

Material A

1

1

1

1

0

0

3

Material B

3

3

4

4

1

3

4

Material C

1

2

2

1

0

1

2

Quantifying Histology

Pros

  • Pathologic Indexing:

    • Quick process suitable for perioperative histology.

  • Statistical Validity:

    • Adds statistically testable values to a qualitative field.

Cons

  • Interpretation Dependency:

    • Results heavily dependent on skilled histologists' interpretations, with potential variances in conclusions.

  • Subjectivity Issues:

    • Inter-histologist differences can introduce biases in interpretation.

Immunohistochemistry (IHC)

Overview

Immunohistochemistry employs antibodies to visualize specific molecules located in or on cells.

Antibody Structure

  • S = Short segment, indicating the various regions of the antibody including:

    • Fab Region: Antigen binding fragment.

    • Fc Region: Crystallizable fragment involved in immune responses.

    • Heavy and Light Chains: Comprise the antibody structure.

    • Variable Domain: The antigen binding site.

Antibody Functionality

  • Antibodies or immunoglobulins play significant roles in identifying cells and substances throughout molecular biology due to their specific binding to antigens.

  • They are part of the humoral immune system, targeting immune responses to minimize cytotoxic damage to tissues.

Producing IHC Antibodies

  1. In Animals:

    • Inoculate an animal with the target antigen, leading to the generation of antibodies against the antigen.

    • The produced mixture is termed polyclonal antibodies because it reacts with multiple epitopes on the antigen.

  2. In Cell Lines:

    • Hybridoma cells engineered from B cells and myeloma cells produce monoclonal antibodies that target one specific epitope of an antigen.

Visualization Techniques

  • Fluorescence: Antibodies can be conjugated to fluorophores for visualization under fluorescence microscopy.

  • Colorimetric Methods: Enzymes like horseradish peroxidase can also be used in combination with substrates to produce a color change at the antibody binding site.

Antigen Retrieval

  • During formalin fixation, methylene bridges can mask antigenic sites, necessitating retrieval methods:

    • Heat-Induced Antigen Retrieval (HIAR)

    • Enzymatic Antigen Retrieval

Fluorescence Properties

  • Each fluorophore is characterized by its excitation and emission wavelengths, critical for visualizing multiple targets without color overlap.

Important Terminologies and Concepts

  • CD Markers: These are surface proteins on cells recognized by antibodies, with a diverse range of specificity.

  • Isotype Importance: Different antibody isotypes have unique characteristics critical for reliable interactions in assays.

Immunohistochemistry Summary

  • The lecture emphasized the dual potential of IHC for both specificity and quantification in histology.

  • Targeted histological tools and antibody applications enhance the precision of diagnosis and understanding in tissue response and disease progression.

Concluding Remarks

  • Histology can be quantified, although conventional methods may pose subjectivity issues.

  • Modern quantification often uses image analysis.

  • Antibodies incorporated into histological analyses greatly enhance specificity, using techniques such as immunohistochemistry alongside various staining methods to elucidate cellular functions and pathology.

Examples of Specific Stains

  • Von Kossa: Bone

  • Alizarin Red: Bone

  • Alcian Blue: Cartilage

  • Oil Red O: Adipose Tissue