Basic Microscopy

Basic Microscopy
• Fixation —
• Preserves tissues in situ as close to the lifelike state as possible —
• Ideally, fixation will be carried out as soon as possible after removal of the
tissues, and the fixative will kill the tissue quickly, thus preventing autolysis
• Five major groups of fixatives
• Dehydration —
• Fixed tissue is too fragile to be sectioned and must be embedded first in a
nonaqueous supporting medium (e.g., paraffin) — The tissue must first be
dehydrated through a series of ethanol solutions
Basic Microscopy
• Clearing
• Nonpolar solvents (e.g., xylene, toluene) are used as clearing agents; makes the tissue more translucent
• Embedding —
• Paraffin wax is the usual embedding medium;
• Plastic resin may be used, allows for thinner sections (required for electron microscopy [EM])
• Embedding is important because the tissues must be aligned, or oriented, properly in the block of paraffin
• Sectioning —
• Embedded in paraffin, which is similar in density to tissue, tissue can be sectioned at anywhere from 3 to 10
µm (routine sections are usually cut at 6 to 8 µm)
• Staining —
• Allows for differentiation of the nuclear and cytoplasmic components of cells as well as the intercellular
structure of the tissue based on chromogenic properties
• Cover-slipping —
• Thin piece of plastic or glass to protect the tissue from being scratched, to provide better optical quality for
viewing under the microscope, and to preserve the tissue section for years
Basic Microscopy - Fixatives
• Aldehydes ◆
• Formalin
• Aqueous solution of formaldehyde gas that penetrates tissue well but relatively slowly;
the standard solution is 10% neutral buffered formalin
• A buffer prevents acidity that would promote autolysis and cause
precipitation of formolheme pigment in the tissues
• Appears as golden brown (can be confused with haemosiderin)
• Tissue is fixed by cross-linkages formed in the proteins, particularly between
lysine residues
• This cross-linkage does not harm the structure of proteins greatly, preserving
antigenicity, and is therefore good for immunoperoxidase
(immunohistochemistry) techniques
Basic Microscopy - Fixatives
• Glutaraldehyde
• The standard solution is a 2% buffered glutaraldehyde and must be cold,
buffered, and not more than 3 months old
• Fixes tissue quickly and therefore is ideal for EM
• Causes deformation of α-helix structure in proteins and therefore is not good
for immunoperoxidase staining
• Penetrates poorly but gives best overall cytoplasmic and nuclear detail
• Tissue must be as fresh as possible and preferably sectioned within the
glutaraldehyde at a thickness of no more than 1 mm to enhance fixation
Basic Microscopy - Fixatives
• Mercurials
• B-5 and Zenker
• Contain mercuric chloride and must be disposed of carefully
• Penetrate poorly and cause tissue hardness but are fast and give excellent
nuclear detail
• Best application is for fixation of hematopoietic and reticuloendothelial
tissues
Basic Microscopy - Fixatives
• Alcohols
• Methyl alcohol (methanol) and ethyl alcohol (ethanol)
• Protein denaturants
• Not used routinely for tissue because they dehydrate, resulting in tissues’
becoming brittle and hard
• Good for cytologic smears because they act quickly and give good nuclear
detail
Basic Microscopy - Fixatives
• Oxidizing agents
• Permanganate fixatives (potassium permanganate), dichromate fixatives
(potassium dichromate), and osmium tetroxide cross-link proteins
• Cause extensive denaturation
• Some of these have specialized applications but are used infrequently —
• Picrates
• Bouin solution has an unknown mechanism of action
• It does almost as well as mercurials with nuclear detail but does not cause as much
hardness
• Picric acid is an explosion hazard in dry form
• Recommended for fixation of tissues from testis, gastrointestinal tract, and
endocrine organs
Basic Microscopy – Factors affecting fixation
Buffering ◆
• Fixation is optimal at a neutral pH, in the range of 6 to 8 ◆
• Hypoxia of tissues lowers the pH, so there must be buffering capacity in the fixative to
prevent excessive acidity; acidity causes formation of formalin-heme pigment that
appears as black, polarizable deposits in tissue
• Common buffers include phosphate, bicarbonate, cacodylate, and veronal
• Penetration
• Fixative solutions penetrate at different rates, depending on the diffusibility of each individual fixative
◆
• In order of decreasing speed of penetration: formaldehyde, acetic acid, mercuric chloride, methyl
alcohol, osmium tetroxide, and picric acid ◆
• Because fixation begins at the periphery, thick sections sometimes remain unfixed in the center,
compromising both histology and antigenicity of the cells (important for immunohistochemistry [IHC])
• It is important to section the tissues thinly (2 to 3 mm)
Basic Microscopy – Factors affecting fixation
• Volume
• Should be at least a 10:1 ratio of fixative to tissue —
• Temperature
• Increasing the temperature, as with all chemical reactions, increases the speed of fixation
• Hot formalin fixes tissues faster, and this is often the first step on an automated tissue processor —
• Concentration ◆
• Formalin is best at 10%; glutaraldehyde is generally made up at 0.25% to 4% —
• Time interval
• Formalin should have 6 to 8 hours to act before the remainder of the processing is begun ●
• Decalcification —
• paraffin wax and calcium have different densities between calcium
• Strong mineral acids such as nitric and hydrochloric acids are used to remove large quantities of calcium at a
rapid rate
• Organic acids (acetic and formic acid) are better. However, they act more slowly on dense cortical bone
• Formic acid in a 10% concentration is best
 Basic Microscopy – Factors affecting
fixation- Please Note
• Prolonged fixation can affect immunohistochemical results owing to alcohol precipitation of
antigen at the cell surface;
• to optimize antigenicity of the tissue for IHC, the American Society of Clinical Oncology/ College of
American Pathologists (ASCO/CAP) guidelines recommend fixation of tissue destined for IHC in neutral
buffered formalin for a minimum of 6 hours and a maximum of 48 hours (see Wolff et al, 2007)
• Urate crystals are water soluble and require a nonaqueous fixative such as absolute alcohol
(makes diagnosis of gout challenging)
• If tissue is needed for immunofluorescence (e.g., kidney or skin biopsies) or enzyme profiles
(e.g., muscle biopsies), the specimen must be frozen without fixative; enzymes are rapidly
inactivated by even brief exposure to fixation
• For rapid intraoperative analysis of tissue specimens, tissue can be frozen, and frozen
sections can be cut with a special freezing microtome (“cryostat”); the pieces of tissue to be
studied are snap-frozen in a cold liquid or cold environment (−20° to −70°C); freezing makes
the tissue solid enough to section with a microtome
Basic Microscopy – Histological stains
• The staining process makes use of a variety of dyes that have been chosen for their
ability to stain various cellular components of tissue ●
• Hematoxylin and eosin (H&E) stain — The most common histologic stain used for
routine surgical pathology —
• Hematoxylin, because it is a basic dye, has an affinity for the nucleic acids of the cell nucleus —
• Hematoxylin does not directly stain tissues but needs a “mordant” or link to the tissues; this is
provided by a metal cation such as iron, aluminum, or tungsten —
• The hematoxylin-metal complex acts as a basic dye, and any component that is stained is
considered to be basophilic (i.e., contains the acid groups that bind the positively charged basic
dye), appearing blue in tissue section — The variety of hematoxylin stains available for use is
based partially on choice of metal ion used, which can vary the intensity or hue —
• Conversely, eosin is an acid aniline dye with an affinity for cytoplasmic components of the cell
• Eosin stains the more basic proteins within cells (cytoplasm) and in extracellular spaces
(collagen) pink (eosinophilic) to red (acidophilic)
Pattern Recognition
• Inflammatory Reaction Patterns
• Spongiotic
• Interface
• Psoriasiform
• Vesicular and bullous
 Skin
Inflammatory Conditions Pattern Recognition
Spongiotic dermatitis
• Due to the presence of spongiosis or intercellular edema
• stretches apart keratinocytes and
• sometimes results in the formation of intraepidermal vesicles.
• Exocytosis of inflammatory cell may be seen
• Eosinophilic
• Neutrophilic
• Involve skin appendages
• Follicular
• No specific and occurs in a number of conditions
Spongiotic Dermatitis
• Conventional
• Allergic dermatitis
• Polymorphous light eruption
• Drug eruption
• Transient acantholytic dermatitis
• Eosinophilic
• Pemphigus
• Bullous pemphigoid
• Neutrophilic
• Phototoxic dermatitis
• Follicular
• Atopic dermatitis
• Eosinophilic folliculitis
Interface Dermatitis
• morphologic alteration at the junction or interface between the epidermis and
dermis.
• Specific features
• vacuolization (vacuoles or discrete clear spaces) either within basilar keratinocytes or
within the basement membrane zone.
• dyskeratotic keratinocytes (which are probably apoptotic cells),
• disruption keratinocytic maturation
• clefts resulting from coalescence of vacuoles.
• Classification
• according to the density and pattern of the inflammatory cell infiltrate in the papillary
dermis as
• (1) the vacuolar or cell-poor type, based on perivascular or patchy infiltrates in the papillary dermis or
• (2) the lichenoid or cell-rich type, which shows a dense bandlike infiltrate that fills the papillary
Interface Dermatitis
• Vacuolar interface dermatitis
• Erythema multiforme
• Fixed drug eruption
• Viral xanthems
• HIV interface dermatitis
• Connective tissue disease
• Lupus erythematosus
• Dermatomyositis
• Graft-versus-host reaction
• Vitiligo
• Pigmented purpuric dermatitis
• Lichenoid interface dermatitis
• Lichen planus and variants
• Lichenoid drug eruption
• Lichenoid keratosis
• Lichen striatus
• Porokeratosis
• Histologic regression of many tumors
Psoriasiform Dermatitis
• Characterised by pattern of epidermal hyperplasia
• elongation of the epidermal rete ridges
• epidermal surface is unaffected, that is, remains essentially flat-topped.
• The epidermal alteration further described as either regular or irregular.
• Regular psoriasiform hyperplasia,
• elongated epidermal rete ridges of fairly uniform length and thickness and
• typical of psoriasis in a well-developed stage.
• Often accompanied other alterations (notable in psoriasis):
• broad zones of parakeratosis and absence of the granular layer,
• exocytosis of neutrophils (Munro micro abscess),
• Pallor of keratinocytes (intracellular edema),
• Supra papillary thinning and prominent dilated and tortuous papillary dermal microvessels,
• papillary dermal edema.
• Irregular psoriasiform epidermal hyperplasia
• chronic eczematous dermatitis, lichen simplex chronicus, or mycosis fungoides
Psoriasiform Dermatitis
• Psoriasis and Reiter syndrome
• Subacute to chronic eczematous dermatitis, Seborrheic dermatitis
• Lichen simplex chronicus
• Pityriasis rubra pilaris, Parapsoriasis
• Mycosis fungoides, Psoriasiform drug eruption
• Erythroderma
• Candidiasis, Secondary syphilis, scabies
• Inflammatory linear verrucous epidermal nevus (ILVEN)
• Lamellar ichthyosis
• Clear cell acanthoma
• Pellagra
• Acrodermatitis enteropathica
• Migratory necrolytic erythema
Vesicular and bullous dermatitis
• Characterised by the formation of tissue clefts or spaces
• may or may not be accompanied by cellular inflammatory infilitrates.
• In general, these disorders are classified according to whether the level of cleavage is
• (1) intraepidermal or (2) subepidermal
• Intraepidermal blisters
• subcorneal or intragranular cleavage, cleavage through the superficial layer, suprabasal layer
• Subepidermal
• blisters may be further delineated as cleavage
• through the lamina lucida of the basement membrane zone or through the superficial dermis.
• predominantly inflammatory or noninflammatory. If inflammatory, the composition of the
infiltrate and possibly immunofluorescence and serological studies will further aid classification.
• Mechanism of vesicle/blister
• spongiotic, acantholytic
Vesicular and bullous dermatitis
• Intracorneal and subcorneal vesicles and pustules
• Impetigo
• Staphylococcal “scalded skin”syndrome
• Superficial fungal infection
• Pemphigus foliaceus
• Pemphigus erythematosus
• Subcorneal pustular dermatosis
• Infantile acropustulosis
• Intraepidermal vesicles and pustules
• Spongiotic vesicles
• Viral vesicles
• Palmoplantar pustulosis
• Friction blister
• Epidermolysis bullosa
Vesicular and bullous dermatitis
• Subepidermal blisters with little inflammation
• Epidermolysis bullosa
• Porphyria Pseudoporphyria Bullous pemphigoid (cell-poor type)
• Burns Toxic epidermal necrolysis
• Subepidermal blisters with lymphocytes
• Erythema multiforme Fixed drug eruption Lichen planus pemphigoides Polymorphous light eruption Bullous mycosis
fungoides Bullous fungal infections
• Subepidermal blisters with eosinophils
• Bullous pemphigoid, Epidermolysis bullosa acquisita, Pemphigoid gestationis, Arthropod bites Drug reactions
• Subepidermal blisters with neutrophils
• Dermatitis herpetiformis, Linear IgA bullous dermatosis, Pustular vasculitis, Bullous lupus erythematosus Sweet
syndrome, Erysipelas Bullous urticaria
• Subepidermal blisters with mast cells
• Bullous mastocytosis
• Miscellaneous blistering diseases
• Drug-overdose-related bullae, PUVA-induced
Overlapping patterns
• Spongiotic psoriasiform dermatitis
• Spongiotic interface dermatitis
• Spongiotic psoriasiform interface dermatitis
• Psoriasiform interface dermatitis
Dermal Inflammatory Reactions
• Absent vascular injury
• pattern, depth, density, and composition of the inflammatory cell infiltrate, as
well as whether it is granulomatous or not.
• Patterns of inflammatory infiltrates in the dermis generally are described as:
• lichenoid, perivascular,
• periadnexal,
• interstitial (infiltrating collagen bundles),
• nodular, or diffuse (occupying the entire dermis)
• The depth of involvement is important to recognize because many
dermatitides correlate with depth.
• Drug eruptions and viral exanthems often show superficial perivascular involvement only,
• whereas conditions such as lupus erythematosus,
Dermal Inflammatory Reactions
• polymorphous light eruption, and secondary syphilis are prone to
involvement of the deep dermal vascular plexus (so called superficial and
deep perivascular pattern).
• deep infiltrates also may be indicative of a systemic disease process, as in
lupus or secondary syphilis.
• Density of an infiltrate is difficult to assess except in rather subjective terms,
such as
• sparse, moderate, or dense.
• Recognizing the density of an infiltrate has relevance for particular disease processes,
such as acute urticaria (which is sparse),
• figurate erythema (which is moderately dense),
• and cutaneous lymphoid hyperplasia or lymphoma (which tends to be dense).
Dermal Inflammatory Reactions
• A granuloma is a circumscribed aggregate of macrophages.
• Cells vary from mononuclear cells with abundant pale, vacuolated, or lipidized (foamy)cytoplasm to cells with plentiful pink
cytoplasm, resembling epithelial cells and hence the term epithelioid cells (Fig. 1-6).
• Multinucleated giant cells are commonly present and are generally one of two types:
• foreign-body
• Langhans
• Granulomas often contain a variable admixture of other cell types, such as lymphocytes, plasma cells, neutrophils,
and mast cells.
• Poorly defined granulomatous infiltrates often are referred to as granulomatous inflammation.
• Classification of Granulomas
• Infectious or noninfectious
• morphological features,
• Sarcoidal or epithelioid cell granulomas,
• tuberculoid or caseating granulomas,
• foreign-body granulomas,
• suppurative granulomas,
• palisading/necrobiotic granulomas, and (6)
• elastolytic granulomas.
• In general, these reaction patterns are nonspecific
Dermal Inflammatory Reactions
• Disorders of skin appendages
• The skin appendages, principally the hair follicle and the eccrine sweat
apparatus, may show primary inflammatory involvement.
• Disorders of the hair follicle
• In general, folliculitis can be
• infectious or noninfectious
• superficial only or superficial and deep.
• The hair follicle also may show a peculiar reaction—
• follicular mucinosis—which may be associated with a number of processes, including
mycosis fungoides and inflammatory conditions such as arthropod bites and lupus
erythematosus.
Dermal Inflammatory Reactions
• Disorders of the sweat apparatus
• The eccrine duct may be involved primarily in an inflammatory reaction termed miliaria
and categorized according to depth of involvement as
• Superficial, miliaria crystallina, miliaria rubra, and miliaria profunda.
• Deep, hidradenitis refers to an inflammatory disorder involving the sweat coil as in
neutrophilic eccrine hidradenitis, which may be infectious or noninfectious.
• Panniculitis
• The primary focus of inflammation may be in the subcutaneous fat, fascia, or both.
• panniculitis has been classified as septal or lobular, in fact, in most instances the
inflammatory process is both septal and lobular, often spreading from the septae.. The
reaction pattern of adipose tissue to injury is limited.
• Initially one observes an influx of neutrophils (erythema nodosum pattern), then mononuclear cells
(lymphocytes and macrophages), and finally, reparative fibrosis
Dermal Inflammatory Reactions
• Vascular Injury present
• endothelial perturbation or activation
• frank fibrinoid necrosis
• presence of inflammation for an interpretation of vasculitis.
• Vasculopathy
• Small-vessel vasculitis
• Neutrophilic/leukocytoclastic vasculitis
• Lymphocytic vasculitis
• Granulomatous vasculitis
• Medium-Sized vessel Vasculitis