44, 45 - Histological Diagnosis I Flashcards
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review
Why are histochemical stains used in pathology?
Which stains highlight salient cell features?
Highlight salient features of cells to aid in diagnosis
* Alcian blue, Mucicarmine
Which stains help define tissue architecture?
- Highlight tissue architecture
- Trichrome, Periodic Acid Schiff, H&E
Which stains help identify intracellular deposits
Help identify intracellular deposits:
Iron stain, Periodic Acid Schiff (PAS)
Which stains help define tissue architecture in pathological processes?
elastin and reticulin
Which stains help identify extracellular deposits?
Which stains helps visualize infectious microorganisms?
- Highlight infectious organisms that may be difficult to visualize using an H and E stain
- Grocott-Methenamine silver (GMS), Mucicarmine, Periodic Acid Schiff (PAS), Warthin Starry
How is IHC used in pathology?
- Determining cell of origin when this is not clear from
morphology alone - e.g. CK7, vimentin, desmin, chromogranin,
synaptophysin, CD45 - Prognostic markers – help oncologists determine
prognosis - e.g.Ki-67
- Predictive markers - predict response to targeted
therapy - ER, PR, Her-2
What are the priniciples of IHC?
Immunohistochemistry (IHC) involves the use of antibodies to identify specific antigens within
tissues. In IHC, the primary antibody has specificity for the antigen of interest (Figure 12).
After incubation and binding of the primary antibody, a secondary antibody is applied. The
secondary antibody has specificity for the Fc region of the primary antibody. The secondary
antibody is also bound to a “label”. This label can be fluorescent or enzymatic. In the case of
fluorescently labeled antibodies, staining is viewed using a microscope that emits wavelengths of
light that excite the fluorochrome (epifluorescence, inverted fluorescence and confocal
microscopes). With enzyme-linked antibodies, the tissue sections are exposed to the enzyme’s
substrate, and a colored reaction product precipitate, called a chromogen, is formed. This
chromagen can be seen by light microscopy. Many types of enzyme-linked antibody/substrate
pairs are used in pathology, but most commonly, horseradish peroxidase/diaminobenzidene is
used.
What are the patterns of staining with IHC?
IHC staining patterns are based on where the antigen of interest is located within the cell.
Nuclear staining is seen for transcription factors and other proteins that would normally be located in the nucleus. One example is Ki-67, a proliferation marker (Figure 13, top left). In the virtual microscopy collection, there is a slide with tonsillar tissue stained for Ki-67. Note the different rates of proliferation (i.e. number of cells that are positive for Ki-67) of the lymphocytes in the different parts of this lymphoid tissue.
Other antigens are cytoplasmic such as CK7, vimentin, desmin, and chromogranin (Figure 13, top right).
Finally, some antigens are expressed on the cell surface, such as CD45 (a.k.a. leukocyte common antigen). This marker is found on the surface of many types of hematopoietic cells (Figure 13, bottom panel).
compare and contrast IHC to IF
IHC is routinely employed by Pathologists to detect specific molecules within cells for diagnostic purposes, primarily because samples can be viewed with a light microscope. However, the advantage of IF over IHC is that it is more easily applied for detecting multiple molecular markers in a single tissue section (Figure 14). In addition, due to the precision of the fluorescent signal, co-localization of two molecules can be seen in the same subcellular compartment.
Sections of adult human kidney were labeled by IHC (A) or IF (B) using antibodies to cadherins and collagen IV. Three different tissue sections labeled by IHC were counterstained with hematoxylin. The chromogen precipitate is brown, and nuclei are blue. For IF, the same section was labeled with the E-cadherin antibody localized with the fluorochrome Alexa 488 (green) and collagen IV antibody detected with the fluorochrome Alexa 555 (red). Nuclei were stained blue. The merged images show the distribution of E-cadherin
and collagen IV in the same section.
What is in situ hybridization? What is FISH?
- Labeled probe hybridizing to nucleic acid
- Probe label can be enzymatic or fluorescent
- Can detect mRNA or DNA
- Fluorescence In situ hybridization used in
cytogenetics to detect specific regions in chromosomes - ISH used to detect mRNA in cells
In situ hybridization (ISH) is a technique used to detect nucleic acids. An anti-sense oligonucleotide probe for either DNA or mRNA is coupled with an enzyme or fluorescent
molecule. Under special conditions, the labeled probe hybridizes with the complementary nucleic acid sequence within the tissue of interest. Fluorescence in situ hybridization (FISH), a type of ISH, can be used to detect specific genes, chromosomal regions of interest (i.e. DNA), or messenger RNA (Figure 15). FISH is useful for detecting two mRNAs within the same section and for precise labeling of chromosomes. In the case of FISH, the fluorescent signal is detected using an epifluorescent or confocal microscope
How can ISH be used to detect chromagens?
ISH can also be used to detect mRNA using oligonucleotides coupled with an enzyme that when provided with its substrate, produces a chromagen precipitate, as described above (Figure 16). The chromogen can be detected using light microscopy (Figure 16). ISH and FISH are particularly useful when it is necessary to detect a secreted protein, such as kappa or lambda light chains. Pathologists use kappa and lambda light chain expression as a marker of clonality in plasma cell disorders. Because kappa and lambda light chains are secreted, staining for these proteins using IHC is often diffuse, making it difficult to determine
which cells are truly secreting the labeled protein. However, kappa and lambda mRNA are confined to the cell of interest, allowing for identification of the specific cells transcribing genes for these proteins
What stain is this? What is it used for?
Highlight salient cell features
What stain is this? What is it used for?
Highlight salient cell features
What stain is this? What is it used for?
Help define tissue architecture – Liver Cirrhosis
What stain is this? What is it used for?
Help define tissue architecture – Liver Cirrhosis
What stain is this? What is it used for?
Help define tissue architecture in pathologic processes
What stain is this? What is it used for?
Help define tissue architecture in pathologic processes
What stain is this? What is it used for?
Identify intracellular deposits
What stain is this? What is it used for?
Identify intracellular deposits
What stain is this? What is it used for?
Identify extracellular deposits
What stain is this? What is it used for?
Identify extracellular deposits
What stain is this? What is it used for?
Visualize infectious microorganisms
What stain is this? What is it used for?
Visualize infectious microorganisms
What stain is this? What is it used for?
What is alcian blue stain used for? How does it work?
Alcian blue: This stain is used by pathologist to identify mucoid (mucus-like) substances within cells. For example, this stain can be used to identify mucin (family of glycoproteins with high sialic acid or sulfated polysaccharides) containing goblet cells as part of intestinal metaplasia at the gastroesophageal junction (Barrett’s esophagus) (Figure 1). Using this stain, acid mucins
(those seen in intestinal glands) and cartilage are blue, while nuclei appear red, and cell cytoplasm appears pink.
What is congo red stain used for? How does it work?
Congo red: This stain is used to detect amyloid protein. However, it is unable to distinguish different types of amyloid proteins (e.g. Lambda light chains in AL amyloid vs. acute phase reactant proteins in AA amyloid). Using this stain, amyloid appears red-pink and takes on an
apple-green color upon exposure to polarized light (Figure 2) while nuclei are blue. In the virtual microscopy slide collection, a section of soft tissue is stained with Congo red. Please note the abundant, amorphous, red-pink material throughout the section.
What is elastin stain used for? How does it work?
Elastic Stain (Verhoeff-vanGieson): Clinically, this stain can be used to help identify arteries
and veins in the setting of vasculitis (inflammation of the vessels). This stain highlights elastic
fibers, staining them a blue-black to black color. Nuclei are stained blue/black while collagen is
red and other tissues are generally yellow. In the figure below (Figure 3), panels “A” and “B”
show low and high power views, respectively, of an artery involved by giant cell arteritis. In “C”,
the artery is stained with an elastic stain which helps delineate the elastin fibers within the wall of
the vessel (labeled “E”). These fibers are partially disrupted by the vasculitic process (red
arrow). In the virtual microscopy slide collection, please examine slide 220, an artery stained
with an elastin stain. In this slide, the elastic fibers stain blue while the background tissue stains
blue-yellow. Please note the wavy inner elastic lamina layer (closest to the lumen of the vessel).
What is iron stain used for? How does it work?
Iron (colloidal iron): This stain highlights iron deposition in tissues. Clinically, this can be used
to evaluate iron stores in the bone marrow or to highlight excess iron deposition in the liver, seen
in a condition called hemochromatosis. When this stain is used, ferric iron appears blue, cell
nuclei appear red, and cell cytoplasm appears pink. In the virtual microscopy slide collection,
there is a slide with a section of liver stained with an iron stain. Please note the extensive
collections of iron within the hepatocytes highlighted in blue by the iron stain.
What is GMS (Grocott methenamine-silver nitrate) stain used for? How does it work?
Grocott methenamine-silver nitrate (GMS): This stain is used to highlight infectious
organisms within tissues. For example, fungi and Pneumocystis jerovecii appear black in a GMS
stain while the background tissue appears green due to a counter stain (Figure 4). In the virtual
microscopy slides, a portion of thick skin (either from the palm of the hand or the sole of the
foot) has been stained using a GMS stain. Please note that in the most superficial, acellular
layers of the epidermis, there are numerous organisms highlighted in black. These are likely
fungal elements as part of a fungal infection of the skin.
What is Masson’s Trichrome stain used for? How does it work?
Masson’s Trichrome stain: The Masson’s trichrome stain is used in pathology to highlight areas
of fibrosis, for example, fibrosis seen in the liver in cirrhosis (Figure 6). Using this stain, tissues
usually stain one of three colors: collagen stains blue (mature, type I collagen stains dark blue
while immature collagen, types 3 and 4, stains light blue); mucin stains green or blue; nuclei
stain black; and keratin and the cytoplasm of cells stain red. In the virtual microscopy collection,
please focus on the section of colon stained using a trichrome stain and note how different
elements of the colon wall stain differently using this stain.
What is Mucicarmine stain used for? How does it work?
Mucicarmine stain: In pathology, the mucicarmine stain is used to help identify the presence of
mucin within cells (Figure 7, right). A mucicarmine stain can also be used to highlight the thick,
polysaccharide capsule of the fungus, Cryptococcus (Figure 7, left). Using the mucicarmine
stain, both mucin and the cryptococcus polysaccharide capsule appear deep rose to red while
nuclei appear black, and background tissue appears blue or yellow. In the virtual microscopy
slide collection, there is a slide of small bowel stained with a mucicarmine stain. Please note that
the mucin droplets within the goblet cells of the small bowel epithelium are pink while the
remaining tissue is yellow, due to the counterstain.
What is Oil Red O staining used for? How does it work?
Oil red O: This stain is used by pathologists to highlight fat. This stain requires frozen tissue
sections as most fixatives dissolve lipids during tissue processing. Using this stain, fat appears
red, and nuclei appear blue (Figure 8). We do not have an example of an oil red o stain for our
virtual microscopy slide collection, but an example of this stain is pictured below.
What is Periodic acid-Schiff reaction (PAS) stain used for? How does it work?
Periodic acid-Schiff reaction (PAS): This stain reacts with complex carbohydrates, staining
them a deep red or magenta color. Structures/cells that stain magenta with this stain are
considered “PAS positive”. PAS positive structures include mucin within goblet cells of the
gastrointestinal and respiratory tracts; basement membranes and brush borders of kidneys, small,
and large intestine; cartilage; collagen (somewhat); fungi and glycogen. A PAS stain can also be
used with a diastase digestion. Diastase digests glycogen, preventing it from staining. Staining
with PAS both with and without diastase can help determine whether or not PAS positive
material is glycogen. In a disease called alpha 1 antitrypsin deficiency, PAS positive deposits
within the cytoplasm of hepatocytes (liver cells) are diastase resistant, indicating that they are not
composed of glycogen (Figure 9). In the virtual microscopy slide collection, there is a slide with a section of liver stained with PAS without diastase digestion. Please note the small, deep pink granules within many of the hepatocytes (cells of the liver). These are PAS positive deposits.
What is Reticulin stain used for? How does it work?
Reticulin stain: This stain is used to stain reticulin fibers that are composed primarily of type III
collagen. In the bone marrow, increased reticulin fibers can be associated with some bone
marrow disorders such as idiopathic myelofibrosis (Figure 10). Increased reticulin fibers can
also be seen in the liver as part of liver fibrosis or veno-occclusive disease. A reticulin stain
stains reticulin fibers black, mature collagen (type I) brown, and immature collage (types 3 and
4) black. In the virtual microscopy collection, please look at the slide containing liver tissue
stained with a reticulin stain. Note the presence of reticulin fibers (black) along the edges of the
hepatic sinusoids (the light, “empty” spaces between the cords of hepatocytes).
What is Warthin-Starry stain used for? How does it work?
Warthin-Starry: This stain is used to highlight certain infectious organisms within tissues
including spirochetes (syphilis), helicobacter (H. pylori), and Bartonella henselae (cat scratch
fever). Using this stain, the aforementioned organisms stain black while the background tissue
stains pale yellow to light brown (Figure 11). Although we do not have an example of this stain
in our virtual microscopy collection, please see figure 11, which demonstrates use of this stain
for identifying H. pylori in gastric biopsies.
What stain is this?
Congo Red
What stain is this?
Elastin
What stain is this?
GMS
What stain is this?
GMS
What stain is this?
GMS
What stain is this?
Iron
What stain is this?
Iron
What stain is this?
Iron
What stain is this?
Trichrome
What stain is this?
Trichrome
What stain is this?
Mucicarmine
What stain is this?
Mucicarmine
What stain is this?
Mucicarmine
What stain is this?
PAS without Diastase
What stain is this?
PAS without Diastase
What stain is this?
PAS without Diastase
What stain is this?
Reticulin
What stain is this?
Ki-67
What stain is this?
Ki-67
What stain is this?
CD45
What stain is this?
CD45
What stain is this?
CD45
What stain is this?
CK7
What stain is this?
CK7
What stain is this?
Vimentin
What stain is this?
Vimentin
What stain is this?
Vimentin
What stain is this?
Desmin
What stain is this?
Desmin
What stain is this?
Desmin
What stain is this?
Desmin
What stain is this?
Desmin
What stain is this?
Desmin
What stain is this?
Chromogranin
What stain is this?
Chromogranin
What stain is this?
CEA
What stain is this?
CEA
What stain is this?
ISH - Lambda
What stain is this?
ISH - Lambda
