PART 1 - HP - PT Flashcards
Type of Tissue Sections and its SIZE (micra)
✓Paraffin section?
✓Paraffin section- 4-6 micra
Type of Tissue Sections and its SIZE (micra)
✓Celloidin section?
✓Celloidin section- 10-15 micra
Type of Tissue Sections and its SIZE (micra)
✓Frozen section?
✓Frozen section- 4 micra
practical consideration of FIXATION?
Speed
Penetration (1 mm/hr) —- ideal thickness (2-3 mm)
Volume (20x)
Duration of fixation - shortened by heat, vacuum, agitation, microwave
Fixative for minute specimens
PICRIC ACID FIXATIVES (ex. Bouins) – imparts color YELLOW
A specialized fixative used in (FROZEN SECTION which serves to localize ANTIGENS and hydrolyze ENZYMES and used for preservation of LIPIDS
Formol Calcium (Bakers)
Formalin Usage by-products
PARA FORMALDEHYDE
FORMIC ACID
Fixative NOT for H&E
osmium tetroxide (inhibit action of hematoxylin ex. Ehrlich’s)
It is promoted by slow freezing
FORMATION OF ICE CRYSTAL ARTIFACTS
knife for celloidin?
PLANE CONCAVE KNIFE (less Concave)
gold standard stain for Glomerular Basement Membrane
jones methenamine silver stain
OTHERS:
Azocarmine
Periodic acid schiff
AlciaN blue
“JAPAn”
importance of 5% sodium thiosulfate in Dezenkerization?
to remove excess iodine, removed by tap water
MJ disadvantage of OSMIC ACID as fixative?
very expensive
adhesive for cytology
APES / 3-aminopropyl-thriethoxysilane
The routine fixative of choice for preservation of cell detail in tissue photography
Recommended for renal tissues, fibrin, connective tissues, and muscles
5-7 % MERCURIC CHLORIDE
fixative of choice for demonstration of URATE CRYSTALS?
95-100% ethanol
The fixative of choice for VERY MINUTE/SMALL SPECIMENS (fragmentary biopsies) and DELICATE TISSUES such as embryos
picric acid (bouin’s)
Used both as a FIXATIVE and as a STAIN FOR FATS
OSMIUM TETROXIDE / OSMIC ACID
Formalin fixes tissue by
STABILZATION OF PROTEINS (cross-linkages)
It is made up of 2 formaldehyde residues, linked by three carbon chains.
Most commonly used FIXATIVE FOR ELECTRON MICROSCOPE (EM) & HISTOCHEMISTRY
GLUTARALDEHYDE
Cause of Failure to arrest early cellular autolysis
Due to the failure to fix IMMEDIATELY or INSUFFICIENT fixative
Cause of too brittle and too hard blocks/ OVERHARDENED TISSUE
Due to PROLONGED fixation
cause of Soft and feather-like tissues
Due to Incomplete fixation
cause of Presence of artefact pigments on sections
INCOMPLETE washing of fixative
cause of Shrinkage and swelling of cells in tissue blocks
Due to OVER-FIXATION
Cause of enzyme inactivation and loss and Removal of fixative soluble substances
WRONG choice of fixative
The process of removing intracellular and extracellular WATER FROM THE TISSUE following fixation and prior to wax impregnation
DEHYDRATION
Excellent DEHYDRATING and CLEARING agent miscible to water, melted paraffin, alcohol and xylol
DIOXANE (DIETHYLENE DIOXIDE)
CELLUSOLVE (ETHYLENEGLYCOL MONOETHYL)
Procedure whereby calcium or lime salts are removed from tissues
DECALCIFICATION
Most rapid decalcifying agent
PHLOROGLUCIN NITRIC ACID
Routine/ most common decalcifying agent
NITRIC ACID
FORMIC ACID (Best for CELLULAR PRESERVATION)
Yellow color imparted by nitrous acid is removed through
- Neutralization with 5% SODIUM SULFATE and running tap water for 12 hours
- Addition of 0.1 UREA to pure concentrated nitric acid
DECAL AGENT & TSE SOFTENER
PERENYI’S FLUID
Von Ebner’s fluid Formula
36% Sat. Aq. NacCl,
Conc. HCL
Distillef water
The process whereby alcohol or a dehydrating agent is removed from the tissue and replaced by a fluid (clearing agent) that will dissolve the wax with which the tissue must be impregnated
CLEARING
A clearing agent to use in processing tissues for paraffin embedding should be
- Miscible with alcohol and remove DEHYDRATING AGENT/DEHYDRANT
- Miscible with and easily removed by melted PARAFFIN WAX and/or MOUNTING MEDIUM
The most rapid clearing agent
XYLENE - EXCELLENT I TRUE CLEARING AGENT
Cause of The XYLENE becomes TURBID or MILKY when the tissue or section is added to it
INCOMPLETE DEHYDRATION
it also becomes milky when PROLONGED STORAGE
Cedarwood oil
Clearing time of xylene
30-60 MINS/
30 MINS - 2 HRS
Newly opened/urgent biopsies - 15-30 mins
agent best for NERVOUS TISSUE, LYMPH NODES, EMBRYO, granulation tissue, and fetal and other delicate, highly cellular specimens
CHLOROFORM
Carcinogenic or may damage bone marrow (Aplastic anemia)
ΒΕΝΖΕΠΕ
Can be used as a substitute for xylene/benzene as a clearing agent
TOLUENE
The process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will fill all natural cavities, spaces, and interstices of the tissues
IMPREGNATION/INFILTRATION
how many times should paraffin wax should be
USE?
REUSED?
USE twice
REUSED once
The process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium, which is then allowed to solidify
Embedding
Embedding medium for electron microscopy
PLASTIC / RESIN (EPOXY RESINS)
made up of esters of acrylic or methacrylic acid and are used extensively for light microscopy.
ACRYLIC PLASTICS / RESINS
The process in which tissues are first infiltrated with CELLOIDIN and subsequently embedded in PARAFFIN mass
double embedding
The excess wax is cut off from the block to expose the tissue surface in preparation for actual cutting
trimming
Tissues are cut into uniformly thin slices with the aid of machine to facilitate the studies under the microscope
Sectioning/section cutting
This method is normally utilized when a rapid diagnosis of the tissue in question is required, and is essentially recommended when lipids and nervous tissue elements are to be demonstrated.
FROZEN SECTION
Commonly used freezing agent
liquid nitrogen
Advantages of FREEZE- DRYING
- Minimum tissue shrinkage
- Allows tissues to be processed in a fresh state
- Less displacement
Advantages of FREEZE- SUBSTITUTION
MORE ECONOMICAL/CHEAPER
SUITABLE FOR ROUTINE PURPOSES
type of microtome used for cutting celloidin embedded sections
SLIDING MICROTOME (ADAMS - 1789)
A TYPE OF SLIDING MICROTOME favored in laboratories where very hard tissue or blocks are sectioned
Base-Sledge Microtome
A TYPE OF SLIDING MICROTOME THAT IS MOST DANGEROUS because of its movable exposed knife (moved backward and forward)
Standard Sliding Microtome
for cutting paraffin embedded sections
MOST POPULAR and MOST COMMON type used for routine and research studies
microtome inside the CRYOSTAT
ROTARY MICROTOME (MINOT 1885-1886)
THE SIMPLEST type of microtome
for cutting serial sections of large blocks of paraffin embedded tissues
ROCKING MICROTOME/CAMBRIDGE
aka Cambridge PALDWELL TREFALL (1881)
typeof microtomr for cutting unembedded frozen sections
FREEZING microtome (QUECKETT - 1848)
for cutting specimens into extremely thin slices (0.5u) for electron microscopy work
ULTRATHIN MICROTOME (BY GERMAN’S in 1800s)
COLD ULTRAMICROTOMY by who?
HUMBERTO HERNANDEZ HORAN
MICROTOME KNIFE
One side of the knife is flat/straight while the other is concave
Base-sledge/Rocking/Rotary microtome
MEASUREMENT?
PLANE
(25mm in length)
MICROTOME KNIFE
with both sides concave (ROUTINE PARAFFIN SECTIONS)
Recommended for ROTARY MICROTOME
MEASUREMENT?
BICONCAVE
(120mm in length)
MICROTOME KNIFE
have both sides straight (FROZEN SECTIONS and extremely HARD AND TOUGH specimens)
Base-sledge/Sliding microtome
MEASUREMENT?
PLANE WEDGE
(100 mm in length)
uses hones/grinding stones
Purpose: To remove the gross NICKS and IRREGULARITIES on the knife
Direction: Heel to Toe (Edge first)
honing
uses paddle strap made up of horse leather
Purpose: To polish and SHARPEN DULL KNIFE. FREE OF NICKS. Removal of BURRS formed during honing
Direction: Toe to Heel (Edge last)
stropping
Used for rapid preparation of urgent tissue biopsies for intraoperative diagnosis
CRYOSTAT / COLD MICROTOME
Used in order to promote adhesion of sections to slides
ADHESIVES
used as a section adhesive in immunohistochemistry
Poly-L-lysine
adhesive that is very useful in cytology particularly for cytospin preparations of proteinaceous or bloody material
3-APES
A syrupy fluid applied between the section and the coverslip after staining, setting the section firmly, preventing the movement of the coverslip.
mounting medium (mountant)
An aqueous mounting medium was prepared 3 months ago and stored in 4º refrigerator. Mold is now observed in the medium. To prevent this in future, preparing the medium, one should:
thymol crystals - to avoid mold formation & growth of bacteria
Process of SEALING THE MARGINS OF THE COVERSLIP TO PREVENT ESCAPE OF FLUID or semi-fluid mounts and evaporation of mountant, to immobilize the coverslip, and to prevent sticking of slides upon storage.
RINGING (uses kronig cement)
Important things to do in giving slides for Pathologist
check slides have proper labels and are accounted for
KRONIG CEMENT is composed of?
2 parts paraffin wax mixed with
4.9 parts powdered Colophonium resin
(heated and filtered)
Other equipment for SECTIONING
- Water bath
- Drying oven or hot plate
- Forceps (fine pointed or curved) and squirrel hair brush
- Clean slides (76x25 mm; 1-1.2mm thickness)
- Ice tray
- Pencil
Tissue 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 thru chemical reactions that will permit microscopic localization of a specific tissue substance.
HISTOCHEMICAL STAINING
the most important Natural dye?
hematoxylin
a natural dye; a vegetable dye extracted from lichens which are normally colorless
orcein (elastic fibers becomes brown/DB
a natural dye derived from an extract from the female Cochineal Bug (Coccus cacti)
cochineal
a natural dye; a plant with orange stigmas yielding a dye.
saffron
aka “coal tar dyes”
a synthetic dye
Derived from the hydrocarbon benzene
aniline dyes
TYPE OF STAINING:
The tissues are overstained and the excess dye is then removed until the desired intensity is obtained.
REGRESSIVE STAINING
TYPE OF STAINING:
Staining is continued in a definite sequence until the desired intensity of coloring of the different tissue elements is attained. No washing/ differentiation/ decolorization
PROGRESSIVE STAINING
TYPE OF STAINING:
A process of selective removal of excess dye/stain around the tissue
DIFFERENTIATION
TYPE OF STAINING:
A tissue section is overstained in a relatively neutral solution of hematoxylin. The excess stain is removed with an acid alcohol, this is what type of staining?
REGRESSIVE STAINING
TYPE OF STAINING:
H and E staining w/ DIFFERENTIATION?
REGRESSIVE STAINING
Modified H and E staining W/O DIFFERENTIATION
PROGRESSIVE STAINING
Color of cytoplasm of cells?
PINK (PALE)
RED
L-Red
Substances capable of producing visible COLORS THAT ARE NOT PERMANENT
CHROMOPHORE
any substance that contain chromophore?
chromogen
Composition of chromogen?
benzene + chromophore —> chromogen
For a chromogen to be a dye, it must be composed of an acid and a base, and therefore have salt forming properties, ultimate retaining its color
AUXOCHROME
how to rinse EDTA decalcified tissue?
- rinse in running water
- overnight immersion in formol- saline/ phosphate-buffered saline
how many changes of Paraffin in manual processing?
4 changes w/ 15 mins interval
1 hr (complete processing time)
what are the common SUPRAVITAL DYES?
“JNNTTT”
Janus Green B
Neutral Red
Nile blue
Trypan Blue
Toulidine Blue
Thionine
best VITAL/SUPRAVITAL DYES?
NEUTRAL RED
