HISTOPATHOLOGY Flashcards
The process of applying dyes on the sections to see and study the architectural pattern of the tissue and the physical characteristics of the cells.
STAINING
acidic in character
* have greater affinity for basic dyes
Nucleus
basic constituent of the cell
CYTOPLASM
Tissues constituents are demonstrated in sections by direct interaction with a dye or staining solution, producing coloration of the active tissue component.
HISTOLOGICAL STAINING
Various constituents of tissues are studied through chemical reactions that will permit microscopic localization of specific tissue substances.
HISTOCHEMICAL STAINING (HISTOCHEMISTRY)
Combination of immunologic and histochemical techniques that allow phenotypic markers to be detected and demonstrated under the microscope, using a wide range of polyclonal or monoclonal, fluorescent labeled or enzyme-labeled antibodies to detect and demonstrate tissue antigents.
IMMUNOHISTOCHEMICAL STAINING
process of giving color to the sections by using aqueous or alcoholic dye solutions.
direct staining
action of the dye is intensified by adding another agent or mordant.
indirect staining
It combines with a dye to form a colored “lake”, which in turn combines with the tissue to form a “tissue-mordant-dye-complex” that is rendered insoluble in ordinary aqueous and alcoholic solvents.
It is an integral part of the staining reaction itself, without which no staining could possibly occur.
mordant
Three ways to use mordant:
applied to the tissue before the stain
may be included as part of the staining technique
may be added to the dye solution itself.
Accelerates or hastens the speed of the staining reaction by increasing the staining power and selectivity of the dye.
ACCENTUATOR
A process whereby tissue elements are stained in a definite sequence and the staining solution is applied for specific periods of time or until the desired intensity of coloring of the different tissue elements is attained.
progressive staining
method of staining where the dye is not washed or decolorized
progressive
the tissue is first overstained to obliterate the cellular details, and the excess stain is removed or decolorized
regressive
Selective removal of excess stain from the tissue during regressive staining in order that a specific substance may be stained distinctly from the surrounding tissues.
DIFFERENTIATION (DECOLORIZATION)
This is usually done by washing the section in a simple solution (e.g.
water or alcohol), or by the use of acids and oxidizing agents.
DIFFERENTIAL STAINING
entails the use of specific dyes which differentiate particular substances.
Metachromatic staining technique
Uses more than one chemical stain to better differentiate between various microorganisms or structures/cellular components of a single organism.
Differential Staining
_____ is necessary for most metachromatic staining techniques, and metachromasia is usually lost if the section is dehydrated in alcohol after staining.
water
The property to stain biological materials with a color that is different from that of the stain itself.
METACHROMASIA
application of a different color or stain to provide contrast and background to the staining of the structural components to be demonstrated.
COUNTERSTAINING
process where specific tissue elements are demonstrated, not by stains, but by colorless solution of metallic salts
metallic impregnation
metallic impregnation produces ______ on surface of tissue or bacteria.
opaque, usually black deposit
the selective staining of living cell constituents, demonstrating cytoplasmic structures by phagocytosis of the dye particle (cytoplasmic phagocytosis).
vital staining
what is resistant to vital stains
nucleus
if nuclear structures are present during vital stain, it indicates _____.
death of the cell
done by injecting the dye into any part of the animal body (either intravenous, intraperitoneal or subcutaneous), producing specific coloration of certain cells, particularly those of the reticulo-endothelial system.
intravital staining
common dyes in intravital staining
lithium
carmine
india ink
Used to stain living cells immediately after removal from the living body.
supravital
Paraffin wax is poorly permeable to most staining solutions and should therefore be
removed from the section prior to staining.
usually done by immersing the paraffin section in a solvent (xylene) two times, at 1-2 minutes duration each, for sections up to 10 micra thick.
xylene 1
xylene 2
With the use of xylene, _____ is removed.
absolute alcohol 100%
h&e is a ____.
progressive or regressive?
regressing
cornerstone of tissue- based diagnosis
H&E
Eosin Methylene Blue(EMB) method
produces a sharp nuclear stain and reveals with marked differentiation the various structures in the tissues, which should be fixed in Zenker’s
fluid.
malory’s phloxine methylene blue stain
an alternative to iron hematoxylin nuclear stain, producing a strong and precise nuclear stain that is resistant to decolorization
produces color blue
celesteine blue hemalum
produces black clor
heidenhain’s iron
Four staining methods are commonly employed for frozen sections
Hematoxylin-Eosin method
Thionine method
Polychrome Methylene Blue method
Alcoholic Pinacyanol method
Coating the slide with dilute (thin) celloidin solutions permits firm attachment of paraffin ribbons.
This is also recommended for sections that will be subjected to strong alkaline or acid solutions and for tissues that contain glycogen for demonstration.
collodionization
Stains may be effectively removed from the skin by
prompt topical application of 0.5% acid alcohol, followed by rinsing with tap water.
first Sudan dye to be introduced into histochemistry.
It is also fat soluble, and is good as a fat stain for Central Nervous System tissues, giving a less deep and lighter orange
sudan III
It is recommended for staining triglycerides
(neutral lipids), giving them a deep and intense red stain.
sudan iv
It imparts a black color on intracellular lipids,
and is recommended for paraffin sections especially
sudan black
stains phospholipids as well as neutral
fats.
does not stain crystalline cholesterol
SBB
possesses two secondary amino groups per molecule, making it a slightly basic dye which may cause non-specific staining.
Because of this molecular structure, has a much greater affinity for phospholipid than other lysochromes
SB
not real dyes in the usual sense of the word, because they do not have auxochrome groups.
They give color to lipids, simply because they are more soluble in the lipid medium of the tissues than in their medium of 70% alcohol.
lysochromes (oil soluble)
color imparted by sudan IV or scharlach r
red
causes blood cells to exhibit four major staining properties that allow the cell types to be distinguished
wrights
affinity for the oxidation products of methylene blue called azures = reddish purple
azurophilia
is used for demonstration of neuroglia in frozen sections.
victoria blue
It is recommended for staining of Nissl granules or chromophilic bodies.
* nuclei = blue
* used to differentiate different types of granules
* often used as a preliminary stain for electron
microscopy
toluidine blue
used with osmic acid to fix and stain blood and glandular tissues.
rhodamine b
is a nuclear stain
* produces red nuclei, and is used primarily as
a counterstain.
* May also be used to give a yellow color to
collagen.
SAFRANIN (or Safranin O)
utilized for the manufacture of paints, but may be used for microanatomical color contrast of specimens and for demonstration of the blood and lymph vessels by injection (intravital staining).
prussian blue
stains glycogen, mucin, mucoprotein, glycoprotein, basement membranes, capsules and blood vessels, as well as fungi and intracellular carbohydrates. Cells that secrete mucus are also strongly stained.
* Aldehydes = reddish purple
PAS
produces a cytoplasmic stain that demonstrates various tissue components more dramatically
Eosin-Phloxine B Solution
when combined with picric acid, it is extensively used in neuropathological studies;
picrocarmine
2 kinds of cochineal dyes
- best’s
2.picrocarmine
A vegetable dye extracted from certain lichens which are normally colorless.
When treated with: ✓ ammonia
✓ exposed to air
*produces colors.
orcein
“Coal Tar Dyes”
synthetic dyes
e.g. cochineal dyes, logwood dyes and vegetable extracts
natural dyes
most permanent and simplest method to demonstrate mitochondria by light microscopy
Regaud’s Hematoxylin for Mitochondria
Usually performed in patients with AIDS to rule out Pneumocystis carinii/ jiroveci
Broncho alveolar Lavage (BAL)
“the poor man’s CB”
formalin fixation
Pleural and peritoneal fluids fixative is
brasil’s fluid
specimen that uses these fixatives
1.95% ethanol
2.cough directly on a container
with Brasil’s fluid
Sputum and bronchopulmonary specimens
processing of sediments, blood clot, or grossly visible tissue fragments from cytological specimens into paraffin blocks that can be cut and stained from the same methods used for histopathology.
cell blocking
By far the best method to collect cells from body fluids (ex. Urine, pleural or peritoneal fluid).
* For those that have no access to a cytospin, centrifugation of the preparation and sampling of the centrifuged sediment is the usual method of cell concentration.
cytospin prep
Examples are peritoneal, pericardial, and pleural fluids.
These fluids are prone to jelly like clot formation, to prevent it, one must add
300 units of heparin/ 100mL of aspirates.
bronchial aspirates
It makes use of an automatic tissue processing machine (ex. Autotechnicon) which fixes, dehydrates, clears, and infiltrates tissues, thereby decreasing the time and labor needed during the processing of tissues.
automatic processing
It makes use of 12 individual processing steps, when ten 1 – liter capacity glass beakers and two thermostatically controlled wax baths with a safety device cut – out switch.
Elliott Bench – Type Processor.
Transfer tissue blocks contained in baskets through a series
of reagents housed in stationary containers.
carousel type
These processors allow maximum flexibility in the
choice of reagents and schedules.
These machines have a rapid turn-around time for day or night processing.
In more recent models the tissue basket is enclosed within an integrated fume hood during agitation and transfer cycles thus overcoming the disadvantages of earlier styles.
carousel
Tissue cassettes are loaded in a retort chamber where they remained throughout the procedure.
Agitation is achieved by tidal action.
Self-contained vacuum processor
2 basic types of automatic processing
Tissue transfer processors
Fluid transfer processors
Vertical oscillation – provides the agitation for rapid contact between the tissue and the reagents is used by
carousel type
Recommended for the demonstration of lipids and nervous tissue elements.
frozen section
Almost any microtome can be utilized for the purpose, provided means are made available for freezing and maintaining the specimen and the knife at low temperatures.
Usually utilizes the carbon dioxide technique.
cold knife procedure
cold knife temp
-40° to -60°C
This method makes use of the Cryostat, an apparatus used in fresh tissue microtomy, consisting of an insulated microtome housed in an electrically driven refrigerated chamber.
- It is maintained at temperatures near -20°C, where microtome, knife, specimen, and atmosphere are kept at the same temperature.
cryostat procedure
type of choice in cryostat
rotary microtome
-18° to - 20°C.
Most rapid of the commonly available freezing agents.
liq nitrogen
for brain, lymph nodes, liver, spleen, uterine curettings, soft cellular tumors; temp
-5° to -15°C
Frozen tissue is fixed in Rossman’s formula or in 1% acetone and dehydrated in absolute alcohol.
utilizes chemical fixative
freeze substitution
Use of antibodies as histological tools for identifying patterns of antigen distribution within a tissue organism.
immunohistochemistry
Products of an individual clone of plasma cells.
uses mice
monoclonal ab
Useful in differentiating lung adenocarcinomas from
mesotheliomas
* Positive: Thyroid, lung, and neuroendocrine tumors
TTF – 1 (Thyroid Transcription Factor – 1)
Useful in diagnosis of prostatic adenocarcinoma
PSA (Prostate Specific Antigen)
Melanomas and Schwannomas always stain positive
in
vimentin
Used as a marker for germ cell tumors,
particularly germinomas.
plap
cervical mucus exhibits a “palm leaf” pattern due to formation of salt crystals
* Signifies a high persistent estrogen effect
* One of the basis of the diagnosis of early
pregnancy
ferning
Fern or palm leaf crystal
increase estrogen & decrease progesterone
* ovulation or fertile period
squamous epithelial cells that show the cytopathic effects of HPV
* cell with an atypical “wrinkled prune” nucleus surrounded by a perinuclear halo
koilocytosis??
auses Strawberry Cervix
T. vaginalis
having a “honeycomb appearance” when
view on end
Endocervical glandular cells
45-50 um
* cells having pale, pink-staining cytoplasm and dark
pyknotic nuclei
* with true acidophilia (under estrogen influence)
* display the most maturity, have been affected by
estrogen.
Mature superficial/superficial cells
the routine staining procedure used in a cytopathology laboratory.
PAPANICOLAOU STAIN
fluids are obtained by paracentesis, done on the middle of the abdomen below the naval (at the linea alba)
PERITONEAL CAVITY
fluids are obtained by thoracentesis done commonly on the left lateral thoracic wall
PLEURAL CAVITY
for patients with hysterectomy
vag scrape
for localization of vaginal adenosis
four quad vag scrape
wet gangrene
result of venous occlusion, wherein bacterial infection supervenes in ischemic injury to the tissue, causing putrefactive changes
refers to the massive death or necrosis of tissue, caused by combination of ischemia (coagulation) and superimposed bacterial infection (liquefaction)
gangrenous necrosis
exhibits “tombstone formation”
* found in myocardium (heart), lungs, kidneys and spleen.
coag necrosis
Refers to the purplish discoloration or lividity of the skin in the dependent portions of the body, due to stasis and eventual settling down of blood into the dependent vessels
LIVOR MORTIS
Currant jelly clot
Chicken fat appearance
* Assumes the shapes of
blood vessels (cylindrical)
* Rubbery consistency
postmortem clot
Refers to the rigidity or stiffening of the
muscles, occurring about 6 to12 hours after death
rigor mortis
lesion and surrounding tissue is firm to the touch
induration
the simplest, least invasive test and uses smallest needle to simple remove cells from the area of abnormality.
* This type of needle biopsy uses a thin, hollow needle to draw cells from your body.
fine needle biopsy
Removes not only cells, but also a small amount of the surrounding tissu
core needle biopsy
takes out even more surrounding tissue.
Some of the abnormal tissue is taken
out but not all. The doctor will slice into the lesion and remove only a portion of it.
incisional biopsy
tissue is scooped or spooned to remove tissue or growths from body cavities such as the endometrium or cervical canal.
currettings
