Histology: Tissue Processing

Introduction to Histology

• Histology, also known as microscopic anatomy, is the study of body tissues at the molecular level and how they organize to form organs.

Tissue Processing

• Fixation involves preserving tissue with a formalin to prevent decay.
• Dehydration/Decalcification depends on the tissue type; bone uses decalcification, while soft tissue goes straight to dehydration to remove water using alcohol.
• Clearing replaces alcohol with a clearing agent like xylene to make the tissue transparent.
• Infiltration embeds the tissue in paraffin wax at 52-60°C for easy sectioning.
• Embedding solidifies the infiltrated tissue for easier sectioning.
• Trimming removes excess paraffin wax for clearer tissue visibility and precise sectioning.
• Sectioning cuts the tissue into 3-5 micron thick slices using a microtome.
• Staining applies dyes like hematoxylin and eosin to enhance tissue details.
• Mounting places the tissue on a glass slide with a cover slip for preservation and examination.
• Labeling prevents confusion with the tissue samples.

Historical Background

17th Century

• Marcello Malpighi, the "Father of Histology," discovered capillary blood vessels and the histological structure of kidneys and spleen.

Marie François Xavier Bichat [1700s]

• Marie François Xavier Bichat [1700s], the "Father of Modern Histology" and "Father of Descriptive Anatomy," worked on 21 tissues via gross dissection, disregarding microscope use.

19th Century

• August Franz Josef Karl Meyer [1819]: Coined the term histology from Greek words and book "On Histology."
• Richard Owen [1844]: Suggested using "histos" for biological tissues, similar to its usage for fabrics.

Histology Terminologies

Cell Arrangement

• Simple: single-layered (monolayer).
• Stratified: multiple layers.
• Transitional: stratified cells that transition from squamous to cuboidal or dome-shaped.
• Pseudostratified: appears stratified but is actually simple.

Normal Size of Cells

• Large: cell diameter is bigger.
• Small: cell diameter is smaller.

Normal Shape of Cells

• Squamous: flattened or "squashed" shape (wider than tall).
• Cuboidal: equal height and width like a cube.
• Columnar: height distinctly greater than width.
• Spherical: ball-like shape.
• Ovoid: egg-like shape.
• Fusiform (Spindle): elongated shape and tapering at both ends.
• Polyhedral: multiple flattened surfaces (pentagon, hexagon, etc.).
• Polarized: distinct orientation with one end different from the other.

Nucleus Properties

• Large: bigger as compared to other.
• Small: smaller in size than other.
• Euchromatic: dispersed, lightly stained.
• Heterochromatic: condensed, darkly stained.
• Central: nucleus is at the center.
• Peripheral: off-center without touching.
• Paracentral (Eccentric): nucleus off-center and touches.
• Extruded: nucleus separates.
• Nucleoli Prominent: spherical body within.
• Mitotic: shows dark, condensed chromosomal particles.
• Simple: uninucleate (round, oval, etc.).
• Segmented: distinct lobes.

Cytoplasmic Feature

• Basophilia: staining due to affinity to the basic dye.
• Acidophilia: staining due to affinity to the acidic dye.
• Granulated: stained vesicles.
• Vacuolated: cytoplasm with empty holes.
• Abundant: substantial amount (volume).
• Scant: slight amount of cytoplasm.

Other Essential Details

• Nuclear-Cytoplasmic (N:C) Ratio
• Special Staining Properties (e.g., PAS- or SBB-positive)
• Specialized Apical Structure (e.g., cilia, microvilli, stereocilia)
• Presence of Keratin (Keratinized VS Nonkeratinized)
• Cytoplasmic Inclusions
• Location (e.g., borders a lumen, surrounded by ECM)

Microscopic Description

• A <size>, <shape> cell with <nuclear feature> nucleus and <cytoplasmic feature> cytoplasm.

Cell Differentiation

• Cell Differentiation is a process wherein a cell specializes a particular cytoplasmic activity, eventually changing its shape and function.
• Stem Cell is an undifferentiated cell that undergoes cell differentiation and is capable of cell renewal and transform into any type of cell.
• Cell : the basic, living, anatomical, and physiological unit of life.

Types of Stem Cell

Based on Residency

• Embryonic Stem Cell (ESC): derived from embryo; inner cell mass of a blastocyst (early-stage embryo).
• Adult Stem Cell or Somatic Stem Cell or Tissue-Specific Stem Cell (TSSC): derived from a postnatal individual; found throughout the body among differentiated cells in a tissue or organ after development.

Based on Differentiation Potency

• Totipotent Stem Cell (TSC) or Omnipotent Stem Cell (OSC): capable of differentiating into all possible cell types under embryonic and extraembryonic cell lineages.
• Pluripotent Stem Cell (PSC): capable of developing into nearly all type of cell which makes up the body of an individual.
• Multipotent Stem Cell (MSC) or Progenitor Cell: capable of differentiating into numerous but closely related cells.
• Unipotent Stem Cell (USC) or Precursor Cell: committed cells capable of producing only one cell type and is also capable of self-renewal.

Embryonic Development

• Embryonic development starts with fertilization where a sperm and egg joins to form a zygote, which undergoes cleavage, dividing into a solid ball of cells called the morula.
• In the next phase, the morula becomes a hollow blastocyst that attaches to the uterus.
• Gastrulation follows forming three layers: the ectoderm (skin and brain), mesoderm (muscles, bones, heart), and endoderm (lungs, stomach, organs).
• Next neurulation and organogenesis: the brain, spinal cord, and organs begin forming.
• Lastly, by eight weeks, the embryo becomes a fetus, continuing to grow until birth.

Organogenesis

• Organogenesis is the process of organ formation during embryonic development.

Ectoderm-derived

• Nervous system, cornea and lens of eye, epidermis of skin, and epithelial lining of mouth and rectum

Mesoderm-derived

• Skeletal system, Circulatory system, Lymphatic system, Muscular system, Excretory system, Reproductive system, Dermis of skin and Lining of body cavity

Endoderm-derived

• Epithelial lining of digestive tract, respiratory tract, reproductive tract, urinary tract, Liver, Pancreas, Thyroid gland, Parathyroid glands and Thymus

Specialized Cells General Cellular Activity

• Muscle and other Contractile Cells: movement
• Epithelial Cells: form adhesive and tight junctions between cells
• Fibroblasts, Bone Cells, Cartilage: synthesize and secrete components of extracellular matrix (ECM)
• Neurons and Sensory Cells: convert physical and chemical stimuli into action potentials (AP)
• Cells of Digestive Glands: synthesis and secretion of degradative enzymes
• Cells of Mucous Glands: synthesis of glycoproteins
• Cells of Adrenal Gland & Gonads: synthesis and secretion of steroids
• Renal Cells and Salivary Ducts: ion transport
• Macrophages & Polymorphonuclear Leukocytes (PMNs): intrecellular digestion
• Fat Cells (Adipocytes): lipid storage

Histological Development in Cytology

16th Century

• Hans Jansen and Zacharias Jansen (1590): Dutch opticians who developed the first compound microscope by aligning several lenses in a tube.

17th Century

• Robert Hooke (1665): coined the term "cell", Made improvements to the compound microscope by adding the specimen stage, illuminator, coarse adjustment and fine adjustment and "Micrographia": introduced cell and microscopic wonders.
• Anton van Leeuwenhoek (1674): "Father of Microscopy" and first person to use microscope in studying biologic specimen.

19th Century

• Matthias Jakob Schleiden (1838): German botanist and microscopist who proposed the first tenet of cell theory "the cell is the basic unit of life".
• Theodor Schwann (1839): German zoologist who discovered the second tenet of cell theory "all living organisms are made of cells".
• Rudolf Virchow (1858): German pathologist who introduced the third tenet of cell theory "Omnis cellula e cellula" or "All cells [come] from cells."

Cell Anatomy and Physiology

Main Parts of the Cell

• Plasma Membrane (Cell Membrane)
• Cytoplasm
• Nucleus

Definition of Plasma Membrane

• It is a lipid bilayer forming cell flexible outer surface.
• composed of lipids, membrane proteins, and glycocalyx.
• Function of Plasma Membrane: physical barrier electrochemical gradient, communication
• Properties of Plasma Membrane: selective permeability, fluidity and asymmetry.

Leaflets

• Inner or Protoplasmic Face (P Face): rich in phosphatidylserine and phosphatidylamine, crucial for cell structure and apoptosis
• Outer or Extracellular Face (E Face): rich in phosphatidylcholine and sphingomyelin for cell signaling
• Lipid Composition: 75% Phospolipids, 20% Cholesterol and 5% Glycolipid

Lipid Composition of Lipid Bilayer

• Glycerophospholipid: Phosphatidylethanolamine, Phosphatidylserine, Phosphatidylinositol and Phosphatidylcholine
• Sphingolipid: Sphingomyelins and Glycosphingolipids
• Phospholipids: fats that make up cell membranes with hydrophilic head and hydrophobic tails
• Phospholipids Movement: Lateral Diffusion, Flexion, Rotation and Flip-Flop
• 20% Cholesterol is weakly amphipathic
• 5% Glycolipid is lipid conjugated to a carbohydrate group
• Three Types of 5% Glycolipid are Sphingolipids, Glycerol glycolipids and Glycosylphosphatidylinositols (GPIs
• Membrane Fluidity: ability of membrane lipids and proteins to rotate and move

Membrane Protein

Classification of Membrane Proteins

• Peripheral Proteins: not firmly embedded; attached to the polar heads of membrane lipids or to integral proteins at either face of the lipid bilayer
• Integral Proteins: extend into or through the lipid bilayer and are firmly embedded

Functions of Membrane Proteins

• Ion channel (Integral): creates a pore so that the specific ion can flow to get across membrane..
• Carrier (Integral): known as transporters as it facilitates substance movement across the membrane by undergoing changes in shape.
• Receptor (Integral): detects and responds to chemical signals.
• Enzyme (Integral and Peripheral): catalyzes reaction either inside or outside depending where it faces.
• Cell Identity Marker (Glycoprotein): enable cell recognition
• Linker (Integral and Peripheral): provides structural stability by connecting cells to its surroundings (cytoskeleton).

Cytoplasm

• Definition: consists of all the cellular contents between the plasma membrane and the nucleus
• Components of the Cytoplasm: Cytoplasmic Matrix and Organelles

Non-Membranous Organelles

• Cytosol makes up 55% of the total cell volume and varies in composition and consistency (amorphous)., viscid, translucent and colloidal mixture and site of chemical reaction & organellar functions
• Composition: 70% (75-90%) water and 30% dissolved components (e.g., ions, glucose, amino acids, fatty acids, proteins, ATP, and waste products
• Proteasome are tiny barrel-shaped structures with four stacked rings of proteases (proteolytic enzymes) around the central core and discards cells that unnecessary proteins
• Cytoskeleton maintains shape and stabilizes the cell
• Centrosome is microtubule-organizing center

Membranous Organelles

• Lysosome is dense vesicles in golgi complex
• Peroxisomes contain oxidases and catalases to break down hydrogen peroxide and superoxide
• Rough Endoplasmic Reticulum is continuous with nuclear envelope
• Smooth Endoplasmic Reticulum is extension of rough endoplasmic reticulum
• Mitochondrion produces adenine triphosphate (ATP) through cell respiration
• Golgi Apparatus is made up of 3-20 cisterns
• Inclusions involve accumulations of metabolically inactive materials

Nucleus

• Definition: the largest organelle and the "command or control center" of the cell
• Parts of Nucleus are the Nuclear Membrane, Nuclear Pores and Nucleoplasm

Epithelial Tissue

• Definition: a sheet of cells with lipid-rich membranes
• Function: covering exposed surfaces, lining internal passageways & chambers forming glands
• Characteristics: Cellularity, polarity, attachment and avascularity

Functional Classification of Epithelium

• Surface Epithelium: covering and lining
• Glandular Epithelium: epithelial tissue that produce secretions through glandular cells

Surface Epithelium - Morphological Classification

• Based on arrangement or the number of cell layers
• Simple: monolayered
• Stratified: multi-layered
• Pseudostratified: looks layered but not
• Based on number of layers
• Squamous: flat cells; "wider than tall"
• Cuboidal: box-like; cube-like; "as tall as they are wide"
• Columnar: column-like; "taller than wide"
• Transitional: cells change shape; from squamous to cuboidal and vice versa

Glandular Epithelium

• Structural Classification: Based on the presence of ducts
• Endocrine Gland is ductless
• Exocrine Gland - secretes products into body cavities or onto surfaces

Exocrine Gland

• Based on the Nature of Secretion
• Mucus-secreting (Mucous) Glands secretes mucus
• Serous-secreting (Serous) Glands secretes serous fluid
• Mixed Glands secretes both mucus and serous fluid
• Based on Number of Cells
• Unicellular composed of a single cell type
• Multicellular more than one cell type

Multicellular Glands

• Based on the Mode of Secretion
• Merocrine Glands release via exocytosis
• Apocrine Glands release by shedding the apical part of their cell body
• Holocrine Glands the whole cell dissolves completely
• Based on the Presence of a Branch on the Glandular Duct
• Simple: unbranched duct
• Compound: branched duct
• Based on the Shape of the Secretory Portions of the Gland
• Tubular tubular; blind-ending tube
• Acinar/ Alveolar rounded or globular; basket-like
• Tubuloacinar/ Tubuloalveolar/ Tubuloacinous both tubular and acinar

Properties of Epithelial Tissue

• Cellularity
• Types of Cellularity involvesInterdigitations and Interconnections
• Cell Junctions: Tight Junctions. Adherens Junctions, Desmosomes, Gap Junctions and Hemidesmosomes
• Layers of Basement Membrane involvesBasal Lamina and Reticular Lamina
• Types of Basal Lamina involves Lamina Rara Externa , Lamina Densa and Lamina Rara Interna
• APICAL MODIFICATION involves Cilia, MIcrovilli and Sterocilia
• BASOLATERAL MODIFICATION involves Infoldings (Basal Folds)

Definition of Connective tissues

• most diverse and abundant type of tissue

Function of Connective tissues

• structural framework (bone), protection (cartilage), storage within the body (adipocytes), repair and healing (collagen) and transportation (blood)

Characteristics of Connective tissues

• embryonic origin: derived from mesenchyme, abundant ecm with relatively few specialized cells, has variety of vascularity and innervated by nerves

Classification of Connective tissues

• Embryonic Connective Tissue
• Mesenchymal Tissue: found in the embryo and has stem cells
• Mucoid CT: found in the umbilical cord
• Mature Connective Tissue
• Connective Tissue Proper: loose CT, dense CT.
• Supportive CT: cartilage, bone
• Fluid CT: blood, lymph

General Composition of Extracellular Matrix

• Ground Substance is amorphous, transparent, gel-like medium.
• Protein Fibers are Collagen Fibers, Reticular Fibers and Elastic Fibers
• Cellular Elements are Fixed (Resident) Cells and Wandering (Visiting) Cells

Collagen Synthesis Processes

• Intracellular Phase involves preprocollagen is synthesized in the RER
• Extracellular Phase involves, tropocollagen units assemble into collagen fibrils, stabilized by cross-linking.

Medical Significance of Elastase

• Emphysema: excessive elastase activity damages alveolar walls

Nervous Tissue

• Definition: network of fine cellular processes emerging from neurons and glial cells or neuropil
• embryonic origin: ectoderm

Division of Nervous System

• Central Nervous System (CNS): composed of the brain and spinal cord
• Peripheral Nervous System (PNS): includes cranial nerves and spinal nerves
• Division of Peripheral Nervous System
• Sensory Division and Motor Division
• Autonomic NS Division

Neurons

• highly polarizer: conduct signals in one direction.
• Function: Conductivity, Extreme Longevity, Permanent Cells and High Metabolic Rate
• Main Parts of Neurons Cell Body and Neuronal Processes

Main Parts of Neurons of Parts

• Cell Body includes nucleus and cytoplasm
• Inclusive Parts: Nissl bodies, fat droplets and melanin

Nerve Fibers

• Types of Nerve Fibers involves Dendrites and Axon
• Classifications:Based on Number of Processes,Based on their Functions