Light Microscopy
Tissue Processing Overview
- •
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
- •
- •
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
- •
- •
Clearing
- •
Ethanol is not miscible with paraffin, so nonpolar solvents (e.g., xylene, toluene) are used as clearing agents; this also makes the tissue more translucent
- •
- •
Embedding
- •
Paraffin is the usual embedding medium; however, tissues are sometimes embedded in a plastic resin, allowing for thinner sections (required for electron microscopy [EM])
- •
This embedding process 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
- •
- •
Cover-slipping
- •
The stained section on the slide is covered with a 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
- •
Fixation
- •
There are five major groups of fixatives, classified according to mechanism of action: aldehydes, mercurials, alcohols, oxidizing agents, and picrates
- •
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 formol-heme pigment in the tissues
- •
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 techniques
- •
- •
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
- •
- •
- •
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
- •
- •
- •
Alcohols
- •
Methyl alcohol (methanol) and ethyl alcohol (ethanol)
- •
Protein denaturants
- •
Not used routinely for tissue because they dehydrate, resulting in the tissues becoming brittle and hard
- •
Good for cytologic smears because they act quickly and give good nuclear detail
- •
- •
- •
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
- •
- •
- •
Factors affecting fixation
- •
Buffering
- •
Penetration
- •
Volume
- •
Temperature
- •
Concentration
- •
Time interval
- •
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
- •
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)
- •
Should be at least a 10:1 ratio of fixative to tissue
- •
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
- •
Formalin is best at 10%; glutaraldehyde is generally made up at 0.25% to 4%
- •
Formalin should have 6 to 8 hours to act before the remainder of the processing is begun
- •
- •
- •
Decalcification
- •
Tissue calcium deposits are extremely firm and do not section properly with paraffin embedding because of the difference in densities between calcium and paraffin
- •
Strong mineral acids such as nitric and hydrochloric acids are used with dense cortical bone because they remove large quantities of calcium at a rapid rate
- •
These strong acids also damage cellular morphology and thus are not recommended for delicate tissues such as bone marrow
- •
Organic acids such as acetic and formic acid are better suited to bone marrow because they are not as harsh; however, they act more slowly on dense cortical bone
- •
Formic acid in a 10% concentration is the best all-around decalcifier
- •
- •
- •
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
- •
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°C to −70°C); freezing makes the tissue solid enough to section with a microtome
Histologic 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 to red (acidophilic)
- •
Connective Tissue
- •
Elastin stain
- •
Elastin van Gieson (EVG) stain highlights elastic fibers in connective tissue
- •
EVG stain is useful in demonstrating pathologic changes in elastic fibers, such as reduplication, breaks or splitting that may result from episodes of vasculitis, or connective tissue disorders such as Marfan syndrome ( Figure 1.1 )
Figure 1.1
Elastin/Alcian blue stain.
Aortic cystic medial degeneration in Marfan syndrome. Elastin stain highlights fragmentation of elastic fibers (brown-black) and pooling of mucopolysaccharides (blue) within the media.
- •
Elastic fibers are blue to black; collagen appears red; and the remaining connective tissue is yellow
- •
- •
Masson trichrome stain
- •
Helpful in differentiating between collagen fibers (blue staining) and smooth muscle (bright red staining) ( Figure 1.2 )
Figure 1.2
Masson trichrome stain.
Cirrhosis of the liver characterized by bridging fibrosis (blue) and regenerative nodule formation (red).
- •
- •
Reticulin stain
- •
A silver impregnation technique stains reticulin fibers in tissue section black
- •
Particularly helpful in assessing for alteration in the normal reticular fiber pattern, such as can be seen in some liver diseases or marrow fibrosis
- •
- •
Jones silver stain
- •
A silver impregnation procedure that highlights basement membrane material; used mainly in kidney biopsies ( Figure 1.3A )
Figure 1.3
Membranous glomerulopathy.
A, Jones silver stain highlighting basement membrane “spikes” (arrow) along glomerular capillary loops corresponding to basement membrane material surrounding intramembranous immune complexes. B, Direct immunofluorescence showing diffuse, granular staining of the glomerular capillary basement membranes with goat antihuman immunoglobulin G. This technique requires fresh-frozen tissue sections. C, Electron microscopy showing intramembranous electron-dense immune complexes within the glomerular capillary basement membranes.
Courtesy of Pamela Gibson, MD, University of Vermont/Fletcher Allen Health Care, Department of Pathology, Burlington, VT.
- •
Fats and Lipids
- •
Oil red O stain
- •
Demonstrates neutral lipids in frozen tissue
- •
- •
Sudan black stain
- •
Demonstrates neutral lipids in tissue sections
- •
Mainly used in hematologic preparations such as peripheral blood or bone marrow aspirations for demonstration of primary granules of myeloid lineage
- •
Carbohydrates and Mucoproteins
- •
Congo red stain
- •
Amyloid is a fibrillar protein with a β pleated sheet structure
- •
Amyloid deposits in tissue exhibit a deep red or salmon color, whereas elastic tissue remains pale pink ( Figure 1.4A )
Figure 1.4
Alzheimer disease.
A, Congo red–positive core of Alzheimer disease plaque. B, Apple-green birefringence of amyloid core under polarized light. C, Bielschowsky stain highlighting Alzheimer disease plaque (arrow) and neurofibrillary tangle within neuronal cell bodies (arrowhead) .
- •
When viewed under polarized light, amyloid deposits exhibit apple-green birefringence ( Figure 1.4B )
- •
The amyloid fibril–Congo red complex demonstrates green birefringence owing to the parallel alignment of dye molecules along the β pleated sheet
- •
The thickness of the section is critical (8 to 10 μm)
- •
- •
Mucicarmine stain
- •
Demonstrates epithelial mucin in tissue sections
- •
Also highlights mucin-rich capsule of Cryptococcus species
- •
- •
Periodic acid–Schiff (PAS) stain
- •
Glycogen, neutral mucosubstances, basement membranes, and fungal walls exhibit a positive PAS (bright rose)
- •
PAS with diastase digestion : diastase and amylase act on glycogen to depolymerize it into smaller sugar units that are then washed out of the section
- •
Digestion removes glycogen but retains staining of other substances attached to sugars (i.e., mucopolysaccharides)
- •
- •
Alcian blue stain
- •
May be used to distinguish various glandular epithelia of the gastrointestinal tract and in the diagnosis of Barrett epithelium
- •
pH 1.0: acid sulfated mucin positive (colonic-like)
- •
pH 2.5: acid sulfated mucin (colonic-like) and acid nonsulfated mucin (small intestine–like) positive
- •
Neutral mucins (gastric-like) negative at pH 1.0 and 2.5
- •
Pigments and Minerals
- •
Ferric iron (Prussian blue), bilirubin (bile stain), calcium (von Kossa), copper (rhodanine), and melanin (Fontana-Masson) are the most common pigments and minerals demonstrated in surgical pathology specimens
Nerves and Fibers
- •
Bielschowsky stain
- •
A silver impregnation procedure that demonstrates the presence of neurofibrillary tangles and senile plaques in Alzheimer disease ( Figure 1.4C )
- •
Axons stain black
- •
- •
Luxol fast blue stain
- •
Demonstrates myelin in tissue sections
- •
Loss of staining indicates myelin breakdown secondary to axonal degeneration
- •
Gray matter and demyelinated white matter should be almost colorless and contrast with the blue-stained myelinated white matter ( Figure 1.5 )
Figure 1.5
Luxol fast blue stain.
Demyelination in multiple sclerosis (colorless regions).
- •
Hematopoietic and Nuclear Elements
- •
Toluidine blue stain
- •
Demonstrates mast cells in tissue
- •
- •
Giemsa, Wright, and May-Grünwald stains
- •
For cellular details, including hematopoietic (peripheral blood or bone marrow) and cytology preparations
- •
- •
Leder stain (chloracetate esterase)
- •
Identification of cytoplasmic granules of granulocytes and myeloid precursors
- •
Microorganisms: Bacteria, Fungi, Parasites
- •
Brown and Brenn Gram stain
- •
Demonstration of gram-negative (red) and gram-positive (blue) bacteria in tissue
- •
- •
Giemsa stain
- •
Demonstration of bacteria, rickettsia, and Toxoplasma gondii in tissue sections
- •
- •
Grocott methenamine silver (GMS) stain
- •
Demonstration of fungi or Pneumocystis organisms (fungi may also be demonstrated by PAS-amylase stain) ( Figure 1.6 )
Figure 1.6
Aspergillus organisms in the lung stained by Grocott methenamine silver stain.
- •
- •
Warthin-Starry and Steiner stains
- •
Silver impregnation technique for spirochetes (e.g., Borrelia burgdorferi, Treponema pallidum ) in tissue sections
- •
Note: all bacteria are nonselectively blackened by silver impregnation methods such as the Warthin-Starry and Steiner stains
- •
These methods are more sensitive for small gram-negative bacteria (e.g., Legionella species, Helicobacter pylori , and Bartonella species) than tissue Gram stain
- •
- •
Ziehl-Neelsen method for acid-fast bacteria (AFB)
- •
Detect the presence of acid-fast mycobacteria (bright red) in tissue sections (background light blue) ( Figure 1.7 )
Figure 1.7
Ziehl-Neelsen stain for acid-fast bacilli.
Abundant Mycobacterium avian intracellulare organisms (red) within macrophages in the lung.
- •
Fite method should be used to demonstrate Mycobacterium leprae or Nocardia species, both of which are weakly acid fast
- •
Selected References
Bancroft J.D., Gamble M.: Theory and Practice of Histochemical Techniques.5th ed.2001.ElsevierPhiladelphia
Carson F.L.: Histotechnology: A Self-Instructional Text.2nd ed.1997.American Society for Clinical Pathology (ASCP) PressChicago
Wolff A.C., Hammond M.E., Schwartz J.N., et. al.: American Society of Clinical Oncology/College of American Pathologists guideline recommendations for human epidermal growth factor receptor 2 testing in breast cancer. Arch Pathol Lab Med 2007; 131: pp. 18-43.
Fluorescence Microscopy
- •
Tissue is exposed to short-wavelength ultraviolet (UV) light (2500 to 4000 angstroms) through a mercury or halogen lamp; the energy is absorbed by molecules that then release the energy as visible light (4000 to 8000 angstroms)
- •
In immunofluorescence techniques, antibodies are labeled with a fluorescent dye such as fluorescein isothiocyanate (FITC)
- •
Direct immunofluorescence
- •
Fluorescein-labeled antihuman globulin primary antibodies are applied to frozen, unfixed tissue sections to locate and combine with antibodies, complement, or antigens deposited in tissue
- •
- •
Indirect immunofluorescence
- •
Unlabeled primary antibody is applied to the tissue section, followed by application of an FITC-labeled antibody that is directed against a portion of the unlabeled primary antibody
- •
More sensitive and more expensive
- •
Primary application in surgical pathology is detection of autoimmune diseases involving the skin and kidney ( Table 1.1 )
TABLE 1.1
Immunofluorescence Patterns and Disease Associations
Disease
Antibodies
Pattern
Histologic Manifestation
Skin
Pemphigus vulgaris
Antidesmosomal
Intercellular chicken-wire IgG in epidermis
Suprabasal vesiculation
Bullous pemphigoid
Antiepithelial BM; anti-hemidesmosome (collagen XVII [BP180])
Linear IgG along BM; in salt-split skin, reactivity along roof
Subepithelial vesiculation
Epidermolysis bullosa acquisita (EBA)
EBA Ag
Linear IgG along BM; in salt-split skin, reactivity along floor
Subepithelial vesiculation
Dermatitis herpetiformis
Antigluten
Granular IgA, especially in tips of dermal papillae
Subepithelial vesiculation
Kidney
Anti–glomerular basement membrane (anti-GBM) disease
Anti-GBM COL4-A3 antigen
Linear GBM staining for IgG, corresponding granular staining for C3
Crescentic GN
Membranous glomerulopathy
Subepithelial deposits secondary to in situ immune complex formation (antigen unknown; associated with lupus nephritis, hepatitis B, penicillamine, gold, malignancy)
Diffuse, granular GBM staining for IgG and C3
Diffusely thickened glomerular capillary loops with lacelike splitting and “spikes” identified on Jones silver stain
IgA nephropathy
Deposited IgA polyclonal: possible increased production in response to exposure to environmental agents (e.g., viruses, bacteria, food proteins such as gluten)
IgA ± IgG, IgM, and C3 in mesangium
Focal proliferative GN; mesangial widening
Membranoproliferative glomerulonephritis
Type I: immune complex
Type II: autoantibody to alternative complement pathway
Type I: IgG + C3; C1q + C4
Type II: C3 ± IgG; no C1q or C4
Mesangial proliferation; GBM thickening; splitting
BM , Basement membrane; GBM , glomerular basement membrane; GN , glomerulonephritis; Ig , immunoglobulin.
- •
Selected References
D’Agati V.D., Jennette J.C., Silva F.G.: Non-neoplastic kidney diseases.2005.Armed Forces Institute of PathologyWashington, DC:
Kalaaji A.N., Nicolas M.E.O.: Mayo Clinic Atlas of Immunofluorescence in Dermatology: Patterns and Target Antigens.2006.Informa HealthcareNew York, NY
Electron Microscopy
- •
The electron microscope has a magnification range of 1000 to 500,000 diameters (×) (the upper limit of light microscopy is approximately 1000 diameters), thereby allowing for analyzing the ultrastructure of a cell
- •
There are two types of EM:
- •
Transmission EM
- •
Scanning EM
- •
Two-dimensional (2D) black-and-white image is produced
- •
Tissue either transmits electrons (producing “lucent” or clear areas in the image) or deflects electrons (producing electron “dense” or dark areas in the image)
- •
Useful in the diagnosis of nonneoplastic diseases of the kidney
- •
Three-dimensional (3D) black-and-white image results as an electron beam sweeps the surface of the specimen and releases secondary electrons
- •
Lower resolution than transmission EM and used primarily in the research setting to study cell surface membrane changes
- •
- •
Application in surgical pathology: EM is a useful diagnostic technique to supplement morphologic, immunohistochemical, cytogenetic, and molecular analysis of tissues
- •
Immunoperoxidase techniques have largely replaced EM for tumor diagnosis in surgical pathology
- •
EM is useful in
- •
Renal, skin, myocardial, nerve, and muscle biopsies
- •
Undifferentiated or poorly differentiated neoplasms
- •
Diagnosis of lysosomal storage disorders
- •
Ciliary dysmorphology
- •
Visualization of infectious agents
- •
- •
Technical Overview
- •
The main fixative used for EM is glutaraldehyde, which penetrates tissues more slowly than formalin; cubes of tissue 1 mm or smaller are needed
- •
Processing post fixation with osmium tetroxide, which binds to lipids in membranes for better visualization; dehydration with graded alcohols; infiltration with propylene oxide and epoxy resin; embedding in epoxy resin
- •
1-μm sections (semithin) are cut and stained with toluidine blue to verify that the area of interest has been selected for EM
- •
100-nm sections (ultrathin) are cut and collected on copper grids
- •
Tissues are stained with heavy metals (uranyl acetate and lead citrate)
- •
Electron dense : darker in color as a result of heavy impregnation with heavy metal
- •
Electron lucent : lighter in color
Ultrastructure of a Cell
Nucleus
- •
Nuclear membrane
- •
Nuclear pore
- •
Nucleolus
- •
Dense, rounded basophilic structure that consists of 80% to 90% protein
- •
Produces most of the ribosomal RNA (rRNA)
- •
Mitotically or metabolically active cells have multiple nucleoli
- •
- •
Chromatin
- •
Heterochromatin: stainable, condensed regions of chromosomes seen as intensely basophilic nuclear material in light microscopy
- •
Euchromatin: nonstainable, extended portions of the chromosomes that consist of genetically active DNA
- •
Cytoplasm
- •
Plasma membrane
- •
Appears as two electron-dense (dark) layers with an intervening electron-lucent (light) layer
- •
- •
Basement membrane = basal lamina (lamina densa + lamina lucida) + lamina reticularis + anchoring fibrils + microfibrils
- •
Lamina densa
- •
Electron-dense membrane made up of type IV collagen fibers coated by a heparan sulfate proteoglycan
- •
Approximately 30 to 70 nm thick with an underlying network of reticular collagen (type III) fibrils, which average 30 nm in diameter and 0.1 to 2 μm in thickness
- •
- •
- •
Mitochondria
- •
The energy-producing component of the cell; these membrane-bound organelles undergo oxidative reactions to produce energy
- •
Energy generation occurs on the cristae, which are composed of the inner mitochondrial membrane
- •
Most cells contain shelflike mitochondrial cristae
- •
Steroid-producing cells (i.e., adrenal cortex) contain tubular cristae
- •
Mitochondrial crystals are always pathologic
- •
Hürthle cell change occurs when the cytoplasm of a cell becomes packed with mitochondria
- •
- •
Ribosomes
- •
Sites of protein synthesis
- •
Usually responsible for the basophilic staining of the cytoplasm on H&E-stained sections
- •
- •
Endoplasmic reticulum
- •
Membrane-bound channels responsible for the transport and processing of secretory products of the cell
- •
Granular or rough endoplasmic reticulum is abundant in cells that actively produce secretory products (e.g., plasma cells producing immunoglobulin [Ig] and pancreatic acinar cells producing digestive enzymes); the granular appearance is due to attached ribosomes
- •
Smooth endoplasmic reticulum is abundant in cells that synthesize steroids (i.e., adrenal cortex, Sertoli-Leydig cells) and in tumors derived from these types of cells
- •
- •
Golgi apparatus
- •
Concentrates and packages proteins into secretory vesicles for transport to the cell surface
- •
Prominent in cells that secrete proteins
- •
Single Membrane–Bound Structures
- •
Cytoplasmic granules are classified based on size and morphology ( Table 1.2 )
TABLE 1.2
Cytoplasmic Granules
Type
Size
Morphology
Product
Cell Type/Tumor
Mucigen
0.7–1.8 μm
Electron lucent
Glycoprotein
Mucin secreting
Serous, zymogen
0.5–1.5 μm
Electron dense
Proenzyme/enzyme
Example: acinar cells of pancreas
Neuroendocrine
100–300 nm
Dense core
Example: biogenic amines
Neuroendocrine cells
- •
Lysosomes
- •
Contain enzymes that assist in digesting material to be disposed of in the cell
- •
Endogenous and exogenous pigments can be collected in lysosomes; can be large and filled with undigested cellular components in lysosomal storage disorders
- •
- •
Dense core granules: seen in cells and tumors with neuroendocrine differentiation ( Figure 1.8 )
Figure 1.8
Electron microscopy.
Neuroendocrine granules in small cell carcinoma of the lung.
- •
Melanosomes and premelanosomes are specific single membrane–bound structures
- •
Weibel-Palade bodies are specific for endothelial cells
- •
Birbeck granules are seen in Langerhans cell histiocytosis ( Figure 1.9 )
Figure 1.9
Electron microscopy.
Birbeck granules (arrow) in Langerhans cell histiocytosis.
Photo courtesy of Janet Schwarz, Senior Research Technician, Microscopy Imaging Center, University of Vermont, Burlington, VT.
Filaments and Tubules
- •
Filaments are classified based on size ( Table 1.3 )
TABLE 1.3
Filaments and Tubules
Component
Diameter
Location
Microfilaments (actin, nonmuscle myosin)
6–8 nm
Cytoskeleton of all cells
Intermediate filaments
10 nm
Cytokeratin
>19 proteins 40–68 kD
Epithelial cells
Glial fibrillary acid protein
55 kD
Astrocytes
Neurofilament
68, 160, 200 kD
Neural tissue
Vimentin
57 kD
Mesenchymal tissues
Desmin
53 kD
Muscle
Microtubules
25 nm
Neural derivatives (e.g., neuroblastoma)
kD , kilodaltons; nm, nanometers; 50 kD = ∼4 nm.
- •
Microtubules are seen in association with the mitotic spindle and in cells or tumors of neural origin (e.g., neuroblastoma)
Cell Surface
- •
Cell processes are seen in cells that are capable of movement; some tumors, such as schwannomas and meningiomas, demonstrate interdigitating processes
- •
Villi are prominent and regular in cells or tumors of glandular origin ( Figure 1.10 )
Figure 1.10
Electron microscopy.
Short villi lining an intracytoplasmic lumen in adenocarcinoma of the breast.
- •
Terminal web and rootlets in villi are seen in foregut derivatives (e.g., colon)
- •
Junctions are seen in virtually all cells except those of hematopoietic origin
- •
Basal lamina is seen surrounding all endodermal and ectodermal derivatives; cells with muscle differentiation also may have a basal lamina, which may be incomplete
Extracellular Matrix
- •
Collagen shows a regular structure amyloid
- •
Fibrils measuring approximately 10 nm in diameter, with an electron-lucent core
- •
Fibrils are straight, nonbranching, and arranged randomly
- •
Selected References
Ghadially F.N.: Diagnostic Electron Microscopy of Tumors.1986.Butterworth-HeinemannBoston
Ghadially F.N.: Diagnostic Ultrastructural Pathology.2nd ed.1998.Butterworth-HeinemannBoston
Ghadially F.N.: Ultrastructure of the Cell and Matrix.4th ed.1997.Butterworth-HeinemannBoston
Immunohistochemistry
Introduction
IHC combines anatomic, immunologic, and biochemical techniques to identify specific tissue components using a specific antigen-antibody reaction labeled with a visible reporter molecule. This binding is then visualized through the use of various enzymes that are coupled to the antibodies being used. The enzyme acts on a chromogenic substrate to cause deposition of a colored material at the site of antibody-antigen bindings. Hence IHC permits the visualization and localization of specific cellular components within a cell or tissue while importantly preserving the overall morphology and structure of the tissue section. Key improvements in protein conjugation, antigen preservation and antigen retrieval methods, and enhanced immunodetection systems have enshrined IHC as a major adjunctive investigative tool for both surgical and cytopathology. IHC is not only critical for the accurate diagnosis of malignancies but also plays a pivotal role in prognostic evaluation (e.g., estrogen and progesterone receptors in breast cancer) and treatment strategies (e.g., c-kit protein for gastrointestinal stromal tumors and HER-2-neu in certain breast cancers).
Technical Overview
- •
Formalin cross-links proteins in tissues; success of immunohistochemical staining depends on the availability of an antigen after fixation
- •
Various techniques may unmask antigens, such as digestion by enzymes (e.g., trypsin) or antigen retrieval using heat, metallic mordants, or alkaline buffers
- •
Commonly used enzymes include peroxidase, alkaline phosphatase, and glucose oxidase
- •
Most commonly used chromogen substrates produce brown 3,3’-Diaminobenzidine (DAB), or red 3-Amino-9-Ethylcarbazole (AEC) reaction products
- •
- •
Definition of terms
- •
Polyclonal antibody: conventional antiserum produced by multiple plasma cells of an animal that had been injected with an antigen; a polyclonal antibody may have multiple determinants (binding sites)
- •
Monoclonal antibody: produced by fusion of a malignant cell with a plasma cell producing antibody to a specific epitope; antibodies may be grown in tissue culture
- •
- •
Antibodies for the detection of cellular components
- •
Intermediate filaments (see Table 1.3 )
- •
Other cellular and tissue components: (e.g., α 1 -antitrypsin, myeloperoxidase, synaptophysin and chromogranin, myoglobin)
- •
Leukocyte antigens and Ig components commonly used in paraffin-embedded tissues
- •
- •
T-cell
- •
CD1a: thymocyte; also marks Langerhans cells
- •
CD3: Pan–T-cell marker that shows cytoplasmic and membrane staining
- •
CD5: Pan–T-cell marker also expressed by some B-cell lymphomas
- •
CD43: Pan–T-cell marker also expressed by some B-cell lymphomas
- •
CD45RO (UCHL-1), CD4, CD8: T-cell markers
- •
- •
B-cell
- •
CD20: Pan–B-cell marker
- •
Ig heavy and light chains: used for demonstration of clonality in B-cell neoplasms
- •
- •
Myeloid
- •
CD15 (Leu-M1): pan-myeloid antigen that also marks Reed-Sternberg cells of Hodgkin lymphoma
- •
- •
Monocyte and histiocyte
- •
CD163, CD68
- •
- •
Natural killer cell
- •
CD57 (Leu-7)
- •
CD56 (neural cell adhesion molecules, NCAM, Leu-19)
- •
- •
Megakaryocyte
- •
CD41
- •
Factor VIII–von Willebrand factor (vWF)
- •
Ulex europaeus agglutinin-1 (UEA-1)
- •
- •
Hormones and hormone receptors
- •
Presence may have prognostic significance
- •
Estrogen and progesterone receptors in breast carcinomas
- •
Androgen receptors
- •
- •
Infectious agents
- •
Oncogenes and oncogene products
- •
May correlate with prognosis
- •
bcl-1, bcl-2, bcl-6 in lymphoid neoplasms
- •
HER-2-neu and C-erbB2 in breast carcinomas ( Figure 1.11 )
Figure 1.11
Immunohistochemistry for HER-2-neu in a breast adenocarcinoma showing (3+) membranous staining.
- •
- •
p53 tumor suppressor gene: mutations are seen in a variety of malignant tumors
Ground Rules for Quality Application of Immunohistochemistry in Surgical Pathology
- •
Technique
- •
It is imperative that the pathologist work closely with the immunohistotechnologist to optimize, validate, and interpret the IHC assay for any particular antibody reagent
- •
Adequate fixation of tissue or specimen in 10% buffered formalin is essential to high-quality IHC; it is probably better to overfix (because modern antigen retrieval systems can unmask epitopes) rather than underfix (because inadvertent alcohol fixation during tissue processing precipitates and masks epitopes)
- •
It is best to use a polymer-based detection system, which has the advantage of being avidin-biotin free, thereby avoiding false immunoreactivity with endogenous biotin
- •
Appropriate antigen retrieval systems should be optimized for each antibody (noting that different antibodies require unique systems, and some require none)
- •
- •
Antibody choice
- •
A generic screening panel of antibodies should be chosen initially, followed algorithmically by a specific panel to further characterize a neoplasm
- •
Avoid using a single antibody in isolation (because this may result in a potentially erroneous diagnosis), and always use more than one antibody to target a specific antigen
- •
The choice of a panel of antibodies to target a specific antigen should always be made in the context of the morphology and clinical presentation of any neoplasm; avoid use of the “buckshot” approach in hope that an IHC assay returns a positive reaction
- •
Avoid preordering an IHC panel of antibodies before previewing the morphology; remember that IHC is an ancillary or adjunctive technique to the quality practice of surgical pathology and not vice versa
- •
- •
Interpretation
- •
Interpretation of IHC should always be made in the context of the known subcellular localization or distribution of the targeted antigen (e.g., membranous, cytoplasmic, nuclear, or perinuclear “Golgi pattern” of immunoreactivity) ( Figures 1.12 and 1.13 )
Figure 1.12
Immunohistochemistry for HepPar-1 highlighting strong cytoplasmic staining of normal hepatic parenchyma.
Figure 1.13
Immunohistochemistry for TTF-1.
A, Nuclear immunoreactivity in normal thyroid parenchyma. B, Nuclear immunoreactivity in pulmonary adenocarcinoma.
- •
- •
Controls
- •
Finally, the importance of adequate incorporation of appropriate tissue and reagent (both positive and negative) controls in every run of IHC cannot be overemphasized; this is ultimately the highest form of quality control of the IHC assay and should be reviewed daily to avoid false-positive and false-negative interpretation
- •
A Practical Tabular Approach to Using Immunohistochemistry for Common Diagnostic Problems
- •
Because a complete technical overview of IHC and comprehensive listing of available antibodies is beyond the scope of this chapter, our goal is to provide a practical approach to IHC application in surgical pathology; the following tables are presented as guidelines to assist with the choice of an antibody panel when confronted with certain differential diagnoses ( Tables 1.4 through 1.36 )
TABLE 1.4
Immunohistochemistry Approach to Undifferentiated Tumors
Pan-CK
EMA
S-100
SALL4
LCA
CD138
Carcinoma
+
+
−
−/v
−
−
Melanoma
−/v
−
+
−
−
−
Germ cell
v
−
−
+
−
−
Lymphoma
−
−
−
−
+
−
Anaplastic plasmacytoma/myeloma
−
+
−
−
−/+
+
EMA , Epithelial membrane antigen; LCA , leukocyte common antigen; Pan-CK , pan-cytokeratin; SALL4 , sal-like4; v , variable; +, positive; −, negative; −/+, rarely positive.
TABLE 1.5
Immunophenotypic Distribution of Cytokeratins 7 and 20
Carcinoma Type a
CK7
CK20
Colorectal and Merkel cell
−
+
Hepatocellular
−
−
Salivary gland
+
−
Lung, non–small cell carcinoma
+
−
Lung, neuroendocrine carcinoma
−
−
Breast, ductal
+
−
Ovarian, serous, and endometrioid
+
−
Endometrial and endocervical
+
−
Renal cell
−
−
Prostatic
−
−
Urothelial
+
+
Pancreas
+/−
+/−
Mesothelioma
+
−
CK , Cytokeratin; +, positive; −, negative; +/−, variably positive.
a Only approximately 70% to 90% of these tumors follow the given CK7/20 immunoprofile; therefore reliance solely on this profile to determine the primary site of carcinomas is not recommended.
TABLE 1.6
Specific Antibody Reagents to Identify Primary Site of Metastatic Carcinoma
Modified from Kakar S, Gown AM, Goodman ZD, Ferrell LD. Best practices in diagnostic immunohistochemistry: hepatocellular carcinoma versus metastatic neoplasms. Arch Pathol Lab Med . 2007;131:1648–1654; Bishop JA, Teruya-Feldstein J, Westra WH, et al. p40 (ΔNp63) is superior to p63 for the diagnosis of pulmonary squamous cell carcinoma. Mod Pathol . 2012;25:405–415.
From Conner JR, Hornick JL. Metastatic carcinoma of unknown primary: diagnostic approach using immunohistochemistry. Adv Anat Pathol . 2015;22(3):149–167.
From Miettinen M, McCue PA, Sarlomo-Rikala M, et al. GATA3: a multispecific but potentially useful marker in surgical pathology: a systemic analysis of 2500 epithelial and nonepithelial tumors. Am J Surg Pathol . 2014;38(1):13–22.
Carcinoma Type
Antibody
Signal Localization
Other Tumors Identified
Breast
GCDFP-15
Cytoplasmic
Salivary, sweat gland
Breast
Mammaglobin
Cytoplasmic
Salivary, sweat gland
Breast
GATA3
Nuclear
Urothelial, Salivary glands
Colon
CDX2
Nuclear
Subset of pancreas, gastric
Hepatocellular
HepPar-1 Ag
Cytoplasmic
Hepatoid carcinomas of stomach, ovary
Hepatocellular
pCEA or CD10
Bile canaliculi
Hepatoid carcinomas
Hepatocellular
GPC-3
Membranous and cytoplasmic
Melanoma, a subset of chronic active hepatitis
Lung and thyroid except mucinous adenocarcinoma in situ (formerly mucinous BAC)
TTF-1
Nuclear
Neuroendocrine carcinoma extrapulmonary
Lung squamous cell carcinoma
Ovarian serous
p40
WT-1, p16
Nuclear
Nuclear
—
Mesothelioma (WT-1)
Prostate
NKX3.1
Nuclear
Prostate
PSA, PAP
Cytoplasmic
Squamous, urothelial, thymic
p63
Nuclear
Salivary gland, neuroendocrine, subset prostate
Thyroid
Thyroglobulin
Cytoplasmic
—
Urothelial
Uroplakin III
Membranous
—
Renal, clear
RCC
Membranous
BAC , Bronchoalveolar carcinoma; GATA3 , GATA Binding Protein 3; GCDFP-15 , gross cystic disease fluid protein-15; GPC-3 , glypican 3; NKX3.1 , NK3 Homeobox 1; PAP , prostatic acid phosphatase; pCEA , polyclonal carcinoembryonic antigen; PSA , prostate-specific antigen; RCC , renal cell carcinoma; TTF-1 , thyroid transcription factor-1; WT-1 , Wilms tumor gene protein 1.
TABLE 1.7
Immunohistochemistry Panel for Interpretation of Lung Mesothelioma and Adenocarcinoma
Modified from Marchevsky AM. Application of immunohistochemistry to the diagnosis of malignant mesothelioma. Arch Pathol Lab Med . 2008;132:397–401; Bishop JA, Sharma R, Illei PB: Naspin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum Pathol . 2010;41:20–25.
From Erber R, Warth A, Muley T, et al. BAP1 loss is a useful adjunct to distinguish malignant mesothelioma including the adenomatoid-like variant from benign adenomatoid tumors. Appl Immunohistochem Mol Morphol . 2019 Jan 11. doi: 10.1097 [Epub ahead of print].
Antibody
Epithelioid Mesothelioma (Percentage Positive)
Sarcomatoid Mesothelioma (Percentage Positive)
Adenocarcinoma (Percentage Positive)
Epithelial Marker
Naspin A
mCEA
Negative
3
Negative
—
83 (lung)
81
Ber-Ep4
10
0
80
B72.3
7
0
80
CD15 (Leu-M1)
7
0
72
MOC-31
7
0
93
TTF-1
Negative
0
72 (lung)
Mesothelial Marker
Cytokeratin 5/6
83
13
15
Calretinin
82
88
15
WT-1
77
13
4
D2-40
86–100
0
36 (weak)
Mesothelin
100
0
—
BAP1 (loss of nuclear expression)
55.4
41.7
__
BAP1 , BRCA1-associated protein 1; mCEA , monoclonal carcinoembryonic antigen; TTF-1 , thyroid transcription factor-1; WT-1 , Wilms tumor gene protein 1.
TABLE 1.8
Immunohistochemistry Panel for Lung Adenocarcinoma and Breast Adenocarcinoma
Data from Takeda Y, Tsuta K, Shibuki Y, et al. Analysis of expression patterns of breast cancer-specific markers (mammaglobin and gross cystic disease fluid protein 15) in lung and pleural tumors. Arch Pathol Lab Med . 2008;132:239; Striebel JM, Dacic S, Yousem SA. Gross cystic disease fluid protein (GCDFP-15): expression in primary lung adenocarcinoma. Am J Surg Pathol . 2008;32:426; Bishop JA, Sharma R, Illei PB. Naspin A and thyroid transcription factor-1 expression in carcinomas of the lung, breast, pancreas, colon, kidney, thyroid, and malignant mesothelioma. Hum Pathol . 2010;41:20–25.
Immunostain
Lung Adenocarcinoma (Percentage Positive)
Breast Adenocarcinoma (Percentage Positive)
TTF-1
77
0
Naspin A
83
0
Mammaglobin
17
85
GCDFP-15
2
53
ER
4
72
ER , Estrogen receptor; GCDFP-15 , gross cystic disease fluid protein-15; TTF-1 , thyroid transcription factor-1.
TABLE 1.9
Immunohistochemistry Comparison of Spindle Cell Areas in Metaplastic Carcinoma, Phyllodes Tumor, and Fibromatosis of the Breast
Modified from Dunne B, Lee AH, Pinder SE, et al. An immunohistochemical study of metaplastic spindle cell carcinoma, phyllodes tumor and fibromatosis of the breast. Hum Pathol . 2003;34:1009–1015.
CD34
SMA
34βe12
Pan-CK
β-catenin
Desmin
p63
Metaplastic carcinoma
−
+/−
+/−
−/+
−
−/+
+
Phyllodes
+/−
+/−
−
−
−
−/+
−
Fibromatosis
−
+/−
−
−
+
−
−
Myofibroblastoma
+
+/−
−
−
−
+
−
Myoepithelial tumor
−
+/−
+/−
+
−
−/+
+/−
Pan-CK , Pan-cytokeratin; SMA , smooth muscle actin; +, positive; −, negative; +/−, often positive; −/+, rarely positive.
TABLE 1.10
Useful Antibody Panel to Demonstrate Myoepithelial and Basal Cells in Breast Lesions to Distinguish Benign (+) From Invasive (−) Carcinoma
Modified from Rabban JT, Chen YY. D2-40 expression by breast myoepithelium: potential pitfalls in distinguishing intralymphatic carcinoma from in situ carcinoma. Hum Pathol . 2008;39:175–183.
Myoepithelial/Basal Cells
Stromal Myofibroblasts
Smooth muscle heavy-chain myosin
+ (Cytoplasmic)
−/+
p63
+ (Nuclear)
−
α-SMA
+ (Cytoplasmic)
+/−
S-100
+ (Nuclear and cytoplasmic)
v
Calponin
+ (Cytoplasmic)
−/+
D2-40 a
−/+
−
SMA , Smooth muscle actin; v , variable; +, positive; −, negative; −/+, rarely positive.
a D2-40 is a useful marker to highlight lymphatic endothelium in lymphovascular invasion (LVI) by carcinoma but may in addition occasionally stain myoepithelial and basal cells—hence the use of D2-40 to demonstrate that LVI should always be accompanied by p63/SMHCM immunohistochemistry.
TABLE 1.11
Immunohistochemical Panel Approach to Differential Diagnosis of Hepatocellular Carcinoma
Modified from Kakar S, Gown AM, Goodman ZD, Ferrell LD. Best practices in diagnostic immunohistochemistry: hepatocellular carcinoma versus metastatic neoplasms. Arch Pathol Lab Med . 2007;131:1648–1654; Yan BC, Gong C, Song J, et al. Arginase-1: a new immunohistochemical marker of hepatocytes and hepatocellular neoplasms. Am J Surg Pathol . 2010;34:1147–1154.
Arginase-1
HepPar-1
CK19
MOC-31
GPC-3
pCEA
CDX-2
TTF-1
RCC
Inhibin/Melan-A/D2-40
Hepatocellular carcinoma
+
+
−
−/+
+
+
−
− a
−
−
Cholangiocarcinoma
−/+
−
+/−
+/−
−
−
−
−
−
Metastatic adenocarcinoma
−
Colon
−
−
−
−
+
−
−
−
Thyroid, lung
−
−
−
−
−
+
−
−
Tumors with polygonal cells
−
RCC
−
−
+
−
−
−
+
−
Adrenocortical carcinoma
−
−
−
−
−
+
Neuroendocrine tumors b
−
−
+
−
v
Hepatoid carcinoma (e.g., gastric, ovary)
−
+
Stay updated, free articles. Join our Telegram channel
Full access? Get Clinical Tree
