Tissue Processing
Tissue Biology/Histology
Pathology: branch of medical science that
= Study of the morphological features of normal cells and tissues mainly involves the study and diagnosis of
using a light microscope. disease through the examination or
= Understanding how the body functions. surgically removed organs, tissues (biopsy
= Can’t recognize abnormal until understand normal. samples), bodily fluids, and in some cases
the whole body (autopsy).
Tissue: groups of cells that have a similar structure (morphology) and
function. Types: blood, supporting/connective, nervous, muscle, epithelia.
Tissue Fixation
▪ Unfixed tissue will degrade by autolysis.
▪ Fixative required to maintain structure.
▪ Either denatures proteins (e.g. alcohol based) or cross-links proteins (e.g.
formaldehyde). Formalin (37% formaldehyde) commonest fixative.
Processing & Embedding
1. Dehydration: tissue blocks passed through graded alcohols.
2. Clearing: solvent that is miscible with alcohol an paraffin wax.
3. Infiltration with wax: provides rigidity and support to allow sections to
be cut.
▪ Impregnated tissue embedded and encased with more molten wax.
Sectioning→ 0.004mm thick section are cut using a microtome. Sections are
floated onto a water bath and then picked onto a glass slide.
Staining→ tissue elements are colourless. To reveal structural detail using a
light microscope, staining is required.
Tissue Staining
• Basic Dyes: have positive charge so bind to negatively charged tissue
components. E.g. haematoxylin.
• Acidic Dyes: have negative charge so bind to positively charged tissue
components. E.g. eosin
• Basophilia is the binding of basic dye to tissue component. It is blue, its
components include: nuclear chromatin, cytoplasmic RNA, certain extracellular matrix proteins e.g.
cartilage.
• Acidophilia is the binding of acidic dye to tissue. It is pink. Its
components include: cytoplasmic proteins including cytoskeleton
intracellular membranes, most extracellular protein fibres e.g. collagen.
Microscopy
= Light microscope (x1000)
= Electron microscope (x100,000)
= Fluorescence microscope (antibodies or probes
with fluorescent tag)
,Layer of The Skin
-The view of the skin as being a covering to prevent fluid loss and injury
persisted.
Functions:
• It is the largest organ.
• Thickest on the soles of the feet and thinnest on eyelids.
• Resists bacterial and fungal invasion.
• It is rich in antigen presenting cells (Langerhans cells) for immune
response upon breach.
• Pigmentation from melanin provides protection from UV.
• Skin is able to facilitate synthesis of vitamin D3.
• The skin helps thermoregulate via blood circulation in extremities (hands and feet) and sweat.
• The skin contains receptors for touch, pressure, pain and temperature.
Specialised Appendages;
Layers: - Nails
Epidermis→ continuously proliferating stratified squamous epithelium which - Hair follicles
produces a non-living layer of keratin. - Eccrine (sweat)
Dermis→ consists of fibrous and fibroadipose tissue which supports the epidermis glands
physically and metabolically. - Sebaceous glands
Subcutis→ consists of adipose tissue and supporting fibrous bands. - Apocrine glands
Histology Structure of the Epidermis
▪ Keratinising stratified squamous epithelium constitutes the epithelium of skin=
epidermis.
▪ Adapted to withstand constant abrasion and desiccation due to tough non-living
surface layer composed of protein keratin wrapped in plasma membrane.
▪ The epidermis is avascular, nourished by diffusion from the dermis.
▪ Keratinocytes are the major constituent, constituting 90% of the epidermis.
▪ Other cell types in the epidermis; melanocytes, Langerhans cells, Merkel cells,
inflammatory cells.
Dermoepidermal Junction:
→ Area of tissue that joins the epidermis and the dermal layers.
→ It is highly corrugated and has many downward, ridge like extensions of epidermis called epidermal, or rete
ridges.
→ These project between altering, upward projections of dermis is known as dermal papillae. It is responsible
for the exchange of nutrients from the epidermis to the dermis.
→ Rete ridges are finger-like projections in the junction increase the surface area of the epidermis that is
exposed to the blood vessels and the needed nutrients.
Distinct Layers/Strata:
▪ Basal Layer (Stratum Basale): deepest, single layer of closely
packed basophilic cuboidal to columnar epithelial cells
(keratinocytes) resting on basement membrane.
▪ Prickle Cell Layer (Stratum Spinosum):several cells thick,
polyhedral cells that become progressively flatter towards the
surface.
▪ Granular Layer (Stratum Granulosum): 3 to 5 layers of flattened
cells, contain numerous basophilic granules (keratohyalin).
▪ Keratin Layer (Stratum Corneum): dead anucleate cornified cells, keratin replaces cytoplasm.
▪ Stratum Lucidum: absent in thin skin, but present in thick skin. It comprises of a few layers of tightly
packed squamous cells that lack organelles and nuclei. This is translucent and represents a transition from
the stratum granulosum to the corneum.
, *The stratum basale and spinosum are sometimes collectively or singularly called the Malpighian layer.
Epidermal Cell Types
1. Melanocytes
Are melanin pigment producing cells that determine colour of skin and hair.
Not linked by desmosomes, each melanocyte establishes contact via dendritic
process.
Melanin is produced in membrane bound organelles known as melanosomes.
Two major forms of melanin eumelanin (brown to black) and pheomelanin
(yellow to red).
2. Langerhans Cells
Monocyte-derived dendritic cells that reside in the epidermis.
Phagocytic and antigen-processing and presenting cells.
Monitor and capture invading surface antigens, then migrate to regional
lymph nodes.
Induce an immune response via antigen presentation to CD4+ and CD8 + T
lymphocytes.
3. Merkel Cells
Intra-epidermal touch receptors.
Contain membrane bound vesicles in their cytoplasm.
Make synaptic junctions with sensory nerves in the upper dermis.
They are difficult to detect in routine H&E sections.
Epidermal Skin Appendages
• Eccrine (Sweat) Glands;
= Simple, coiled tubular glands consisting of secretory and narrower
excretory duct portions.
= Thermoregulatory role and maintain body temperature by evaporation.
= The tightly convoluted secretory part of a gland deep in dermis
consists of two types of cuboidal to pyramidal secretory cells-clear
cells and dark cells.
= Clear cells primarily secrete water and electrolytes.
= Dark cells secrete macromolecules.
= Smaller, intensely eosinophilic myoepithelial cells, border the
secretory cells.
= Myoepithelial cells are mainly contractile and help expel sweat into the lumen.
= The excretory duct is made of two layers of dark staining cuboidal epithelial cells has a smaller
diameter than the secretory acinus and lacks myoepithelial.
• Apocrine Glands:
= Are often confined to a few localised areas, mainly in the axilla and groin.
= The secretory component is located in deep reticular dermis or subcutis.
= A duct system carries the secretion into the upper part of the hair follicle
above the sebaceous duct.
= Apocrine gland secretions in humans have no defined function.
= The secretory gland a coiled tubular type with a widely dilated lumen.
= The secretory cells are usually low cuboidal with eosinophilic cytoplasm.
= They have a discontinuous layer of myoepithelial cells between the secretory cells and the basement
membrane.
= The duct is histologically similar to that of eccrine sweat gland.
Tissue Biology/Histology
Pathology: branch of medical science that
= Study of the morphological features of normal cells and tissues mainly involves the study and diagnosis of
using a light microscope. disease through the examination or
= Understanding how the body functions. surgically removed organs, tissues (biopsy
= Can’t recognize abnormal until understand normal. samples), bodily fluids, and in some cases
the whole body (autopsy).
Tissue: groups of cells that have a similar structure (morphology) and
function. Types: blood, supporting/connective, nervous, muscle, epithelia.
Tissue Fixation
▪ Unfixed tissue will degrade by autolysis.
▪ Fixative required to maintain structure.
▪ Either denatures proteins (e.g. alcohol based) or cross-links proteins (e.g.
formaldehyde). Formalin (37% formaldehyde) commonest fixative.
Processing & Embedding
1. Dehydration: tissue blocks passed through graded alcohols.
2. Clearing: solvent that is miscible with alcohol an paraffin wax.
3. Infiltration with wax: provides rigidity and support to allow sections to
be cut.
▪ Impregnated tissue embedded and encased with more molten wax.
Sectioning→ 0.004mm thick section are cut using a microtome. Sections are
floated onto a water bath and then picked onto a glass slide.
Staining→ tissue elements are colourless. To reveal structural detail using a
light microscope, staining is required.
Tissue Staining
• Basic Dyes: have positive charge so bind to negatively charged tissue
components. E.g. haematoxylin.
• Acidic Dyes: have negative charge so bind to positively charged tissue
components. E.g. eosin
• Basophilia is the binding of basic dye to tissue component. It is blue, its
components include: nuclear chromatin, cytoplasmic RNA, certain extracellular matrix proteins e.g.
cartilage.
• Acidophilia is the binding of acidic dye to tissue. It is pink. Its
components include: cytoplasmic proteins including cytoskeleton
intracellular membranes, most extracellular protein fibres e.g. collagen.
Microscopy
= Light microscope (x1000)
= Electron microscope (x100,000)
= Fluorescence microscope (antibodies or probes
with fluorescent tag)
,Layer of The Skin
-The view of the skin as being a covering to prevent fluid loss and injury
persisted.
Functions:
• It is the largest organ.
• Thickest on the soles of the feet and thinnest on eyelids.
• Resists bacterial and fungal invasion.
• It is rich in antigen presenting cells (Langerhans cells) for immune
response upon breach.
• Pigmentation from melanin provides protection from UV.
• Skin is able to facilitate synthesis of vitamin D3.
• The skin helps thermoregulate via blood circulation in extremities (hands and feet) and sweat.
• The skin contains receptors for touch, pressure, pain and temperature.
Specialised Appendages;
Layers: - Nails
Epidermis→ continuously proliferating stratified squamous epithelium which - Hair follicles
produces a non-living layer of keratin. - Eccrine (sweat)
Dermis→ consists of fibrous and fibroadipose tissue which supports the epidermis glands
physically and metabolically. - Sebaceous glands
Subcutis→ consists of adipose tissue and supporting fibrous bands. - Apocrine glands
Histology Structure of the Epidermis
▪ Keratinising stratified squamous epithelium constitutes the epithelium of skin=
epidermis.
▪ Adapted to withstand constant abrasion and desiccation due to tough non-living
surface layer composed of protein keratin wrapped in plasma membrane.
▪ The epidermis is avascular, nourished by diffusion from the dermis.
▪ Keratinocytes are the major constituent, constituting 90% of the epidermis.
▪ Other cell types in the epidermis; melanocytes, Langerhans cells, Merkel cells,
inflammatory cells.
Dermoepidermal Junction:
→ Area of tissue that joins the epidermis and the dermal layers.
→ It is highly corrugated and has many downward, ridge like extensions of epidermis called epidermal, or rete
ridges.
→ These project between altering, upward projections of dermis is known as dermal papillae. It is responsible
for the exchange of nutrients from the epidermis to the dermis.
→ Rete ridges are finger-like projections in the junction increase the surface area of the epidermis that is
exposed to the blood vessels and the needed nutrients.
Distinct Layers/Strata:
▪ Basal Layer (Stratum Basale): deepest, single layer of closely
packed basophilic cuboidal to columnar epithelial cells
(keratinocytes) resting on basement membrane.
▪ Prickle Cell Layer (Stratum Spinosum):several cells thick,
polyhedral cells that become progressively flatter towards the
surface.
▪ Granular Layer (Stratum Granulosum): 3 to 5 layers of flattened
cells, contain numerous basophilic granules (keratohyalin).
▪ Keratin Layer (Stratum Corneum): dead anucleate cornified cells, keratin replaces cytoplasm.
▪ Stratum Lucidum: absent in thin skin, but present in thick skin. It comprises of a few layers of tightly
packed squamous cells that lack organelles and nuclei. This is translucent and represents a transition from
the stratum granulosum to the corneum.
, *The stratum basale and spinosum are sometimes collectively or singularly called the Malpighian layer.
Epidermal Cell Types
1. Melanocytes
Are melanin pigment producing cells that determine colour of skin and hair.
Not linked by desmosomes, each melanocyte establishes contact via dendritic
process.
Melanin is produced in membrane bound organelles known as melanosomes.
Two major forms of melanin eumelanin (brown to black) and pheomelanin
(yellow to red).
2. Langerhans Cells
Monocyte-derived dendritic cells that reside in the epidermis.
Phagocytic and antigen-processing and presenting cells.
Monitor and capture invading surface antigens, then migrate to regional
lymph nodes.
Induce an immune response via antigen presentation to CD4+ and CD8 + T
lymphocytes.
3. Merkel Cells
Intra-epidermal touch receptors.
Contain membrane bound vesicles in their cytoplasm.
Make synaptic junctions with sensory nerves in the upper dermis.
They are difficult to detect in routine H&E sections.
Epidermal Skin Appendages
• Eccrine (Sweat) Glands;
= Simple, coiled tubular glands consisting of secretory and narrower
excretory duct portions.
= Thermoregulatory role and maintain body temperature by evaporation.
= The tightly convoluted secretory part of a gland deep in dermis
consists of two types of cuboidal to pyramidal secretory cells-clear
cells and dark cells.
= Clear cells primarily secrete water and electrolytes.
= Dark cells secrete macromolecules.
= Smaller, intensely eosinophilic myoepithelial cells, border the
secretory cells.
= Myoepithelial cells are mainly contractile and help expel sweat into the lumen.
= The excretory duct is made of two layers of dark staining cuboidal epithelial cells has a smaller
diameter than the secretory acinus and lacks myoepithelial.
• Apocrine Glands:
= Are often confined to a few localised areas, mainly in the axilla and groin.
= The secretory component is located in deep reticular dermis or subcutis.
= A duct system carries the secretion into the upper part of the hair follicle
above the sebaceous duct.
= Apocrine gland secretions in humans have no defined function.
= The secretory gland a coiled tubular type with a widely dilated lumen.
= The secretory cells are usually low cuboidal with eosinophilic cytoplasm.
= They have a discontinuous layer of myoepithelial cells between the secretory cells and the basement
membrane.
= The duct is histologically similar to that of eccrine sweat gland.
