Histopath Flashcards
METHODS OF TISSUE EXAMINATION:
___
- Examined in its living state
- Allows examination of protoplasmic activities (mitosis, phagocytosis, pinocytosis, motility)
- Tissues are not permanent
Methods of FRESH TISSUE EXAMINATION:
○ Teasing or Dissociation
- Tissue is immersed in a watch glass with NSS
- Dissected or separated
- Examined under the microscope (phase contrast or bright field); Stained with methylene blue
○ ___ (Crushing): Small tissues (1mm or less) placed in a slide; Tissues are Compressed with another slide or coverslip. Supravital stains can be added
○ ___ = Cellular materials are spread lightly over a slide & useful for cytology
- ___: material is added using an applicator stick or loop in a direct or zigzag fashion
- ___: material is placed on a clean slide and spread into a moderately thick film by teasing with an applicator stick. Advantage: maintains intercellular relationships
- ___: use of 2 slides in preparing the smear; for thick secretions
- ___: (AKA: Impression smear) Freshly cut piece of tissue is brought into contact and pressed on to the surface of a clean slide; sllide is sterile & polished edges
- ___: Rapid Diagnosis
* Demonstration of lipids, nervous tissue elements, enzymes Apparatus: Cryostat, Cold microtome, Freezing microtome
FRESH TISSUES
Squash Preparation
Smear Preparation
Streaking
Spreading
Pull-Apart
Touch Preparation
Frozen Section
HISTOPATHOLOGY TECHNIQUES
1. ____: first and most important step in histopathology. Identify properly all the specimens received without the need of writing the patient’s name to the accompanying specimen tag. Entering the details of the specimen in a log book. In numbering, the specimen number is preceded by either S(__), A(__) or C(__). The year is also indicated. After numbering, the pathologist will describe the gross description of the specimen. The MT w down the description at the back of the request. Use __ in writing the description of the tissue specimen. Specimen size for Processing: ____ and ___ thick; for EM size is ___
NUMBERING (Accessioning)
surgical, autopsy, cytology
pencil
2x3cm; 3-5mm
1mm³
- ___: process of preserving cells adn tissue constituents in a condition identical to that existing during life. Prevents ___.
2 Important Goals of Fixation:
i. Preserve the morphological and chemical integrity of the cell
ii. Harden and preserve tissue for further handling
Methods of Fixation:
1. Heat Fixation
2. Perfusion - fixation via blood flow (embalming)
3. Immersion
Two Mechanisms of Action:
___: the fixative becomes part of the tissue by formation of crosslinks/complexes; stabilizes the tissue proteins; Examples: formalin, mercury, osmium tetroxide
___: fixative NOT incorporated into the tissue; alteration of the tissue composition; stabilizes the tissue by removing water. Examples: alcoholic fixatives
GENERAL EFFECTS OF FIXATIVES
Harden Soft and Friable Tissues for easy handling
Make cells resistant to damage and distortion Inhibits bacterial decomposition
Increase optical differentiation of cells
Act as mordants or accentuators
Reduce risk of infections
FIXATION
autolysis
additive
non-additive
FACTORS INVOLVED IN FIXATION
1. pH: ____
2. Temperature:
Traditionally at: ___
Autotech: ___
EM and Histochem at: ___
Rapid Fixation: __
For tissues w/ TB: __
- Thickness (Dimension of tissue):
EM: __
LM: __
*Tissues should not be more than 4 or 5 mm thick except in ___
Brain Tissue: suspended in __ for 2-3 weeks
Large Solid Tissues: uterus (open or sliced thinly)
6-8
room temp
40°C
0-4°C
60°C
100°C
1mm³/1-2mm²
2x3cm/2cm²
edematous lung tissue (10-20mm/1-2cm)
10% whole buffered formalin
- Osmolality: Slightly Hypertonic Solution around ___
- Concentration:
○ 10% Formalin
○ 3% Glutaraldehyde
○ ___: ideal for immunoelectron microscopy - Time Duration: Primary Fixation (__) in buffered formalin EM: fixation of ___ then placed in a holding buffer
PRACTICAL CONSIDERATIONS:
1. Speed: (<1hr): prevents autolysis & putrefaction
2. Rate of Penetration: Formalin __
3. Volume:___ times that of the tissue: (ideal ratio) ___
○ For expensive fixatives: ___
○ Museum Preparations: ___
4. Duration: depends on the tissue structure
○ Fibrous tissues: longer fixation time
○ Small or Loosely Textured Tissues: shorter fixation time
○ Can be hastened by: ___
OTHER CONSIDERATIONS DURING FIXATION:
* If autopsy materials are not able to be fixed as soon as possible these should be placed in
○ Mortuary Refrigerator kept at a temperature of __
o Undergo Arterial Embalming
Brain: __
Hollow Organs: __
Air Filled Lungs: __
Eyes: __
Hard Tissues (cervix, fibroids, hyperkeratotic skin, nails): washed out in running water and immersed in Tissue Softeners (ex. ___)
400-450 mOsm
0.25% Glutaraldehyde
2-6 hrs; 3hrs
1mm/hr
10-20
20:1!!
osmium tetroxide (5-10x)
at least 50x that of tissue
heating, agitation, microwave and vacuum
4°C
fix before grossing
pack in cotton soaked in fixative
tend to float, wrap in gauze
fix before grossing; inject with formol alc
Lendrum’s & Perenyi’s
FIXATIVE TYPES ACCDG TO COMPOSITION
A. ___: 1 component only
Ex. Aldehydes, Metallic Fixatives, Heat
B. ___: 2 or more components
FIXATIVE TYPES ACCDG TO ACTION:
A. ___: fixatives that permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of tissues
B. ___: preserves a specific part or element
1. ___: nucleus and chromatin material perservation; added with glacial acteic acid (increases affinity of nuclear chromatin); pH 4.6 or less
2. ___: cytoplams & organelle preservation; NO glacial acetic acid (it promotes swelling of membrane)l pH 4.6 or more
3. ___: specific chemical components
SIMPLE FIXATIVES
COMPOUND FIXATIVES
Microanatomical
Cytological
Nuclear
Cytoplasmic
Histochemical
10% Formol saline
10% neutral buffered saline
Heidenhain’s Susa
Formol Sublimate
Zenker’s
Bouin’s
Brasil’s
Microanatomical fixatives
Flemming’s
Carnoy’s
Bouin’s
Newcomer’s
Heidenhain’s Susa
nuclear fixatives
Flemming’s without HOAC
Kelly’s
Formalin with post chroming
Regaud’s
Orth’s
Cytoplasmic
10% Formol saline
Absolute ethanol
Acetone
Newcomer’s
Histochemical
ALDEHYDE FIXATIVES
A. ___ -gas produced from the oxidation of methanol
__ Formalin: concentrated stock solution
__ Formalin: routine concentration
○ Usually Buffered with PO4 buffer to pH 7 (less prone to formation of formalin pigments - brown pigment when acidic)
○ added with ___ (retards decomposition of formic acid; but no longer suitable for EM)
○ usual fixation time: ___
Advantages: Cheap, Readily Available, Easy to Prepare; Relatively Stable; Compatible with many Stains
Disadvantages: Fumes Irritating to Nose and Eyes; Solution is Irritating to Skin (allergic dermatitis)
- Prolonged Storage of 10% Formalin leads to formation of __; Remedy: ___
- Brown or Black Crystalline Precipitates (Acid Formaldehyde Hematein) on blood containing tissues (such as spleen) due to action of formic acid with blood
○ Remedy (Removal of Formalin Pigments)
■ ___: (70% ethanol & 28% ammonia water)
■ ___: (Hydrogen Peroxide & 28% ammonia water
■ Picric Acid Method (Saturated Alcoholic Picric Acid)
■ 1% KOH in 80% Alcohol
Formalin (Formaldehyde)
37-40%
10%
10% methanol
12-24 hrs
white precipitate (paraformaldehyde)
filtration or addition of 10% methanol
Kardasewitsch’s Method
Lillie’s Method
ALDEHYDE FIXATIVES
B. ___: Microanatomical Fixative
> Diluted with NaCl
> Fixation of CNS tissues and General Post Mortem Tissues for Histochemical Examination > Ideal for Silver Impregnation techniques
C. ___ (pH 7.0)
> Best Fixative for iron containing pigments and elastic fibers; BEST/RECOMMENDED ROUTINE FIXATIVE
> Inert to Lipids
> Longer to Prepare
D. ___
> contains mercury/mercuric chloride
> Excellent for stains like Sliver Reticulin Mtds
> No washing Out
> Fixes Lipids
10% Formol Saline
10% Neutral Buffered Formalin or PO4 Buffered Formalin
Formal Corrosive (Formol Sublimate)
ALDEHYDE FIXATIVES
E. ___
> has 95% ETOH with Picric acid and Glacial Acetic Acid
> Good for glycogen, microincineration techniques, sputum spx
> Fixes sputum
F. ___
> Chemical Composition: 2 formalin residues linked by 3 carbon chains
> Acts similarly to formalin
> 2.5% (Small Tx Fragments), 4% (Large Tx)
> Recommended for enzyme histochem, EM
>Widely used primary fixative for TEM
> Better preservation of cellular and fluid proteins More pleasant and less irritating but expensive
> Specimen Vial should be refrigerated
G. ___
> polymer of formalin in white powder form; 4%
> Use: thin and ultrathin sections for plastic embedding; EM
Gendre’s/Alcoholic Formalin/Alcoholic Bouin
Glutaraldehyde
Paraformaldehyde
METALLIC FIXATIVES
A. ___: most common metallic fixative
> Concentration: __; included in compound fixatives
> May produce black granular deposits except ___. Removal of black granular deposits can be done by __
o Addition of saturated iodine solution of 96% Alcohol & 5% sodium thiosulfate
> Penetrates and Hardens Tx rapidly
> Routine Fixative of Choice for Preservation of Cell Detail in Tx Photography
> Fixation of ___ and ___
Mercuric Chloride
5.7%
Heidenhain’s Susa; dezenkerization
hematopoietic and reticuloendothelial tissues
METALLIC FIXATIVES
1. ___: contains Glacial Acetic Acid
> Recommended for Small Pieces of Liver, Spleen, CT Fibers and Nuclei
> Recommended for Trichrome Staining
- ___: (AKA_) Contains Potassium Dichromate and Formalin
> Excellent Microanatomical Fixative of Pituitary Gland, BM and Blood containing Organs > Preserves Cytoplasmic Granules
> Produces BROWN pigments (remove using picric acid or NaOH) - ___: has TCA, Glacial HoAc, Formalin
> recommended for skin tumor biopsies - ___: has Anhydrous Na acetate
> for one marrow biopies, lymph nodes
Zenker’s
Zenker Formol; Helly’s/Kelly’s solution
Heidenhain’s Susa
B5 Fixative
B. Chromate Fixatives
Chromate Fixatives:
○ Concentration: ___ aqueous solutions
○ strong oxidizing agents and should not be combined with reducing agents
○ preserves ___
- ___: 3% aqueous solution
> preserves lipids and mitochondria at pH 4.5-5.2 cytoplasm, chromatin and chromosomes fixed - ___: (AKA: ___)
> chromatin, mitochondria, mitotic figures, Golgi bodies, RBC and colloid containing tissues - ___:
> for early degenerative processes and tissue necrosis demonstration of RICKETTSIA!!
> preserves myelin!
C. Lead Fixatives
4% aqueous solution and recommended for ____
Fixes Mucin also
1-2%
carbohydrates
Potassium Dichromate
Regaud’s; Mueller’s
Orth’s Fluid
~
acid mucoplysaccharides
___
Usually used in strong or saturated solutions; explosive when dry!
Chemically: ___
Major Disadvantage: yellowing of tissues
○ Remedy: Saturated Solution of Lithium Carbonate in 70% Alcohol then washed with water. The tissue is then placed in 70% ethanol followed by 5% Sodium Thiosulfate and washed with water
Uses:
> Excellent for Glycogen
> Can be used as a stain (yellow) and small tissue fragments can be seen
1. ___: recommended for embryos and pituitary biopsies
> excellent for preserving soft and delicate structures
> Preferred fixative for tissues to be stained by Masson’s trichrome stain
> NOT for kidney fixation
2. ___:
> has TCA
> better and less messy than Bouin’s > fixes glycogen
3. ___:
> for GI tract samples and for endocrine tissues
> less lysis than Bouin’s
> decalcifying property
PICRIC ACID FIXATIVES:
2,4,6 trinitrophenol
Bouin’s
Brasil’s Alcoholic Picroformol Fixative
Hollande’s Solution
____:
○ Usually incorporated in compound fixatives
○ Solidifies at 17°C
○ Precipitates Nucleoproteins, Chromatin Materials
○ Not for Cytoplasmic Fixation
___:
○ Rapidly denatures and Precipitates Proteins
○ Acts as a ___
○ Excellent for Glycogen!!!
○ Preserves Nuclear Stains but dissolves ___
1. ___
➤ for dry and wet smears (blood and bone marrow smears)
➤ Disadvantage: toxic when ingested
2. ___
➤ fixes touch preparations, Wright/Giemsa staining
3. ___
➤ simple fixative
➤ doesn’t fix glycogen
➤ useful for recovering DNA fragments for PCR
4. ___
➤ most rapid fixative
➤ Fixation time: ___
➤ contains absolute alcohol, chloroform and glacial acetic acid
➤ fixes and dehydrates at the same time
➤ recommended for chromosomes, lymph glands and urgent biopsies!!
➤ fixes Nissl granules (tigroid substance in rER) and Cytoplasmic Granules!!
5.
➤ recommended for mucopolysaccharides and nuclear proteins
➤ acts as both a nuclear and biochemical fixative
GLACIAL ACETIC ACID FIXATIVES
ALCOHOL FIXATIVES
fixative and dehydrating agent
fats and lipids
Methanol (100%)/Wood alcohol
Isopropyl Alcohol (95%)
Ethanol (70-100%)
Carnoy’s Fixative
1-3 hrs
Newcomer’s
___
Pale yellow powder that dissolves in water (6% at 20C)
Uses:
A. fix and stain unconjugated fats (stain fats black)
B. fixative for EM (secondary fixative after aldehyde)
Other Uses: Preserves mitochondria and Golgi Bodies
Disadvantage: expensive, inhibits hematoxylin; leads to corneal blindess!!!
- ___
> most common chrome-osmium acetic acid fixative
> excellent fixative for nuclear structures
> needs lesser amount of fixative - ___
> for cytoplasmic structures especially mitochondria
____
○ Precipitates proteins
○ Another purpose: Weak Decalcifying Agent also
○ Has a softening effect on dense tissues
___ (also a dehydrating agent)
○ Used at Ice Cold Temperature from ___
○ For Diffusible Enzymes such as Phosphatases and Lipases
○ For Fixing Brain Tissues: ___
OSMIUM TETROXIDE/Osmic acid
Flemming’s
Flemming’s without Acetic Acid
~
TCA Fixatives/Trichloroacetic Acid
ACETONE FIXATIVES
-5°C to -4°C
rabies
Fixatives for Enzyme Histochemistry
○ ___
○ ___
Fixatives for EM:
- osmium tetroxide
- glutaraldehyde
- paraformaldehyde
○ Optimum temperature for Fixation: ___
Fixative for Electron Histochemistry and Electron Immunocytochemistry:
1. Best: ___
2. Addition of Acrolein with glutaraldehyde or formaldehyde
Other Fixatives combined with Rapid Microwave Techniques:
> __: mixture of methanol and PEG; cost effective alternative to formalin Able to recover RNA, DNA, proteins for molecular analysis
○ 4% formalin or formol saline
○ Acetone or formalin for cryostat sections
4°C
Karnovsky’s Paraformaldehyde Glutaraldehyde
UMFIX
___
* Thermal Coagulation of Tissue Proteins
* For Frozen Tissue Sections and Preparation of Bacteriologic Smears
Microwave Technique:
- Physical agent similar to heat(oven)
- Increases movement of molecules and accelerate fixation, staining, decalcification, EM, and immunohistochemistry (antigen retrieval)
- Optimum temperature:___
- Can penetrate tissue with thickness of ___
HEAT FIXATION
45-55°C
10-15mm
FACTORS AFFECTING FIXATION
* Retarded by:
- Size and Thickness
- Presence of Mucus (remedy: __)
- Presence of Fat
- Presence of Blood (remedy: __)
- Cold Temperature
*Enhanced by:
- Thinner and small size of tissues
- Agitation - reduce overall processing time by ___
- Heat
Remedy:
wash with NSS
wash with NSS
30%
TERMS RELATED TO FIXATION
* ___: Placing an already fixed tissue into another fixative to:
- Facilitate and improve the demonstration of substances
- Special Staining
- Ensure further and complete hardening and preservation
- ___: secondary fixation using 2.5-3% Potassium Dichromate for 1 day to act as a ___
- Also known as ___
- ___: removal to excess fixative in order to improve staining and remove artifacts
- Tap Water - chromates, formalin, osmium tetroxide
- 50-70% Alcohol - excess alcohol/picric acid
- Alcoholic Iodine - excess mercury
TRANSPORT MEDIUM: ___ - for unfixed tissues (renal,skin, oral mucosa biopsies); refrigerated; not a fixative
Secondary Fixation
Post-Chromatization; mordant
post-modanting
Washing Out
Michel’s Solution
____
- Removal of Calcium or lime salts from bones or calcified tissues FOLLOWING __
- More concentrated acid solutions decalcify more rapidly but may destroy tissue - Ratio: ___
- Heat and Agitation hastens decalcification
- __ the tissue in the decalcifying fluid hastens decalcification
- Duration: ___
Types of Decalcifying Agents
1. Acids
2. Chelating Agents
3. Ion Exchange Resins
4. Electrophoresis
DECALCIFICATION
fixation
20:1
Suspending
1-2 days
___
○ Widely Used for Routine decalcification
A. ___
- most common/routine
- Rapid Decalcifying Agent
- Acid can be removed by ___
- Imparts a ___ due to formation of (Remedy: add 5% sodium thiosulfate or urea crystals to the concentrated solution)
1. ___:
> contains formalin
> rapid; for urgent biopsies!!
2. ___:
> decalcifies and softens tissues at the same time
> no maceration occurs because of chromic acid and alcohol
> Complete decalcification cannot be determined by chemical test
3. ___:
- most rapid decalcifying agent
- For urgent Works
B. ___
- Inferior as compared to HNO3 because it is slower and produces more distortion - - - Produces good nuclear staining at 1% conc.
–> ___
> contains 36% NaCl
> Recommended for Teeth and Small Pieces of Bone
C. ___; best decalcifying agent
- Moderate Acting Decalcifying Agent but slow
- Recommended for routine decalcification of Postmortem Research Tissues
- Add’n of __ hastens decalcification
- Both a Fixative and Decalcifying Agent
- For Small Pieces of Bone and Teeth
- Suitable for Immunohistochemical Staining
–> Formic Acid Sodium Citrate Solution
○ has better nuclear staining as compared to nitric acid
○ recommended for autopsy materials, BM, cartilage and tissues for research
ACID DECALCIFYING AGENTS
Nitric Acid (5-10%)
70% alcohol
yellow color
Formol Nitric Acid
Perenyi’s Fluid
Phloroglucin Nitric Acid
HCI
Von Ebner’s
Formic Acid
citrate
ACID DECALCIFYING AGENTS
D. ___
- Weak Decalcifying Agent and Fixative
- Permits Good Nuclear Staining
- Slow
- Suitable only for small bone spicules!!!
E. ___
- Weak Decalcifying Agent and only for Minute!! Pieces of Bone!!
F. ___
- ΑΚΑ __
- Both a fixative and a decalcifying agent
- For minute bone spicules
- Chromic Acid is CARCINOGENIC !!
TCA
Sulfurous Acid
Chromic Acid
Flemming’s fluid
___
- Combine with Calcium Salts and other salts to form complexes and to facilitate removal of calcium. Commonly used is __
- Duration: 1-3 weeks for small Specimens
○ 6-8 weeks or longer for dense bones
- pH is adjusted to 7-7.4
- Excellent for immunohistochemical or enzyme staining and for electron microscopy
___ (Ammonium form of Polystyrene)
- Hastens decalcification by using formic acid containing decalcifying solutions. (Increases solubility)
- Not recommended for fluids that contain mineral acids such as nitric acid or HCl – — Formic acid decalcifying agent added is ___ the volume of the tissue
- Complete Decalcification can be measured using __ or __
CHELATING AGENTS
EDTA (Versene)
ION EXCHANGE RESIN
20-30 times
Physical or Xray Method
___
- (+) Charged Calcium ions move towards the Shorter time of calcium removal
using an electric charge
- Suited for small bone fragments!!
- Makes use of ___
- Other Techniques: ___ - faster
MEASURING THE EXTENT OF DECALCIFICATION:
1. ____
> bending or touching the tissue, using a needle
> prone to produce artifacts and may destroy cellular details
2. ____
> very expensive but most ideal
> not for mercuric chloride fixed tissues bc of radio opacity
3. ____ (AKA:__)
> simple, reliable and convenient
> detection of calcium in acid solutions by precipitation of Calcium Hydroxide or Calcium Oxalate
> Performed on the discarded fluid
○ ___ → complete decalcification
○ ___ → incomplete decalcification
ELECTROPHORESIS
88% formic acid
MICROWAVE OVEN DECALCIFICATION
Physical or Mechanical Test
Xray Method
Chemical Method/Calcium Oxalate
clear solution
Cloudiness or precipitation
POST DECALCIFICATION:
Post-Decalcification: decalcified tissues are neutralized by:
○ immersing in saturated Li2CO3 or 5-10% NaHCO3
○ rinsing in running tap water
○ storing in formol saline containing 15% sucrose or PBS with 15-20% sucrose at 4°C
____
Purpose: added to hard tissue to facilitate cutting and processing; can be added to dehydrating agents
1. Perenyi’s: ___
2. Lendrum’s: ___
3. Molliflex: Effect on Tissue: ___
4. 2% HCl
5. 1% HCl in 70% alcohol
Tissue Softeners
decalcificaltion & tissue softener
4% phenol
swolleb and soapy
- ___
- A step in tissue processing in which the intercellular and extracellular water from the tissue are removed following fixation and before wax infiltration
- ___: Solvents utilized in the removal of water usually alcohols!!
- Dehydration usually occurs by placing it in ___
Initial Concentration:
○ For Routine Tissues: ___
○ For Delicate Tissues: ___
Amount of Dehydrating Agent: at least, ___
Commonly Used Dehydrating Agents: Alcohol, Acetone, Dioxane, Cellosolve, Triethyl phosphate, Tetrahydrofuran
DEHYDRATION
Dehydrating Agents/Dehydrants
ascending grades
70%
30%
10x
ALCOHOLS
A. ___
- Boiling Point: 78.3°C
- Recommended routine tissue dehydration
- Considered to be the __
- Clear, colorless, flammable fluid
- Fast-acting, mixes with water and many inorganic solvents, penetrates tissue easily, not poisonous and expensive
B. ___
> It is primarily used for blood and tissue films, and for smear preparations.
> toxic when ingested
C. ___
> BP: ___
> It is utilized in plant and animal microtechniques
> Slow but acting but there is less shrinkage
> Odorous
D. ___ (82.8C)
> Universal Solvent
> Dehydrates and Clears
E. ___ (128C)
> miscible with 90% alcohol, toluene, xylene
> dissolves paraffin
F. ___ (82.3C)
> excellent substitute for ethanol
> not for preparing staining solutions
> best clearing agent for microwave technique!!
Notes to Remember when Using Alcohol
1. The strength of initial alcohol required in each concentration will depend upon the size, and nature of the tissue and fixative used.
○ Small and Delicate Tissue: Lower Concentration and Shorter Intervals
○ Concentrated Alcohols produce shrinkage, make tissues hard and brittle
2. Length of storage. The tissue may be stored in 70-80% alcohol (not for longer periods of time).
○ Less than 70% concentration may ___
○ Longer storage using 70-80% may interfere the staining properties of the tissue
3. Temperature: Hastens at ___
4. ____: insures complete dehydration; accelerates dehydration by removal of water from the dehydrant; ___ color: indicates presence of water
Ethyl alcohol (ethanol)
best dehydrating agent
Methanol
Butanol
117.7°C
Tertiary Butanol
Pentanol
Isopropanol
macerate the tissue
37°C
Anhydrous Copper Sulfate; blue
___
- Clear, colorless liquid, highly miscible but flammable
- Cheap, Rapid Acting
- For most urgent biopsies (dehydrates in /2 to 2 hours) but can cause considerable shrinkage and brittleness
- Lipids are removed
- Not recommended
___ (AKA ___)
- Serves as both a dehydrating and clearing agent (universal solvent)
- Miscible with paraffin, alcohol and xylene
- Produces less tissue shrinkage
- Tissues can be stored here for a longer period
- Disadvantages: EXPENSIVE, tissues tend to ribbon poorly, extremely dangerous; highly toxic
- Two Methods for Dioxane Dehydration
○ ___: Uses Pure Dioxane and Paraffin
○ ___: Tissue is wrapped in gauze and suspended in a bottle containing dioxane and anhydrous calcium oxide/quicklime
ACETONE
DIOXANE; Diethylene Dioxide
Graupner’s
Weiseberger’s
____ AKA __
- Rapid Dehydrating Agent
- Tissues can be stored for long periods of time without distortion
- DANGEROUS: combustible at ___ and toxic. Reproductive, Fetal, Urinary and Blood Systems are vulnerable to its toxic side effects
- If really necessary, use ethylene based glycol ethers for dehydration
___
- Removes water very readily and produces very little distortion and hardening
- Used to dehydrate sections and smears following certain stains
- Produce minimum shrinkage
___
- Both a Dehydrating and Clearing Agent
- Dissolve many substances including fats
- May be used for demixing, clearing and dehydrating paraffin section
- Most staining procedures give improved results with tetrahydofuran
- TOXIC if Ingested or Inhaled
- Has an OFFENSIVE ODOR!!
- Can cause ___ → blindness
- Should be used in a well ventilated room
___: dehydrate and clear at the same time
- Ex. tertiary butanol, THF, Dioxane
Dehydrating Agents for EM
○ Ethanol - for ___
○ Propylene Oxide: ___ (Transition fluid: to facilitate resin infiltration)
○ Acetonitrile: substitute for propylene oxide
CELLOSOLVE; Ethylene Glycol Monoethyl Ether
110-120°F
TRI-ETHYL PHOSPHATE
TETRAHYDROFURAN
conjunctival irritation
UNIVERSAL SOLVENTS
TEM
toxic/carcinogenic
- ___ AKA ___
- Removal of the dehydrating agent and replacement of a fluid (“clearing agent) that is miscible with both the dehydrating agent and impregnating medium
- The use of clearing agents makes the tissues “TRANSLUSCENT”
ROUTINE TISSUE PROCESSING:
Clearing Agents:
* Removal of ___
* Most are flammable liquids
* Clearing Agents have low boiling points
Duration of Clearing: over clearing may cause tissue __
Commonly Used Clearing Agents:
Xylene
Toulene
Benzene
Chloroform
Cedarwood Oil
Aniline Oil
Clove Oil
Carbon Tetrachloride
CLEARING; Dealcoholization
alcohol
brittleness
- CLEARING
___: AKA ___
- Colorless Clearing Agent; Most Commonly Used in Routine Procedures
- Rapid Clearing Time: ___
- Can be used for celloidin sections
- For tissue sections with a thickness of less than ___
- Not for Nervous Tissues and Lymph Nodes!! (chloroform should be used)
- Xylene turns __ when tissues are incompletely dehydrated
XYLENE; xylol
15-30 mins up to 1 hr
5mm
milky
- CLEARING
_____
- Substitute for Xylene or benzene
- Tissues don’t become excessively hard and brittle even if left in toluene for 1 day
- Not Carcinogenic
- Toxic upon prolonged exposure
____
- Recommended for Urgent Biopsies and for Routine Purposes (15-60 mins)
- Doesn’t make tissues hard and brittle; minimum shrinkage
- Highly Toxic (can cause ____)
___
- Slower in action than xylol but causes less brittleness
- Can be used for tissue blocks (up to 1cm)
- Recommended for big tissues, tough tissues, brain, lymph nodes
- Tissues do not ___
- Tissues tend to float in chloroform
- Toxic to ____
___
- Recommended for CNS tissues and cytology such as smooth muscles and skin
- Requires 2 changes of clearing agent
- No tissue distortion even if left in oil __
- Very Slow (2-3 days)
- Very Expensive
- Tends to become milky too upon prolonged storage
____
- Not for routine purposes
- Recommended for embryos, insects and very delicate tissues
TOLUENE (TOLUOL)
BENZENE; aplastic anemia; carcinogenic
CHLOROFORM
do not become translucent
liver
CEDARWOOD OIL
indefinitely
ANILINE OIL
- CLEARING
___
- Causes minimum shrinkage
- Becomes ___
- Recommended for skin and smooth muscle!!!
- Not for routine purposes
___
- Similar to chloroform but cheaper
- Causes tissue hardening
- Highly Toxic upon prolonged exposure
___
- Slow-acting
- For double embedding techniques
OTHER CLEARING AGENTS: *
___: for skin and smooth muscle
___: for skin
___: artificial oil; for delicate tissues
___: for smooth muscle; foul odor
___: for friable tissues
___: for delicate materials like eyes
___: for smooth muscles
___: excellent clearing agent
CLOVE OIL
adulterated
CARBON TETRACHLORIDE
METHYL BENZOATE AND METHYL SALICYLATE
Oil of Bergamot
Oil of Origanum
Oil of Wintergreen
Carbon Disulfide
Carbol Xylene
Terpineol
Phenol
High Test Aviation Lead Free Gasoline
5 AND 6. WAX IMPREGNATION AND EMBEDDING
___ (AKA ___)
- Replacing the clearing agent with the infiltrating medium to fill all tissue cavities
- Gives a firm consistency and easy handling and cutting of thin tissue sections
Embedding (AKA ___)
- impregnated tissue is placed into a precisely arranged position in a mold containing an embedding medium and allowed to solidify.
___
- Simplest, Most Common and Best Embedding Medium
- Serial Sections are cut easily without distortion
- Very rapid (Prepared within ___)
- Many Staining Procedures are Permitted
- Melting Point for Routine Work: ___
○ Do not Overheat!
○ Overheated Paraffin Tempt: __ - tissues causes brittleness and shrinkage and hardening
- Not recommended for __-
- Methods of Paraffin Wax Impregnation
○ ___ - four Changes of Paraffin of Wax at 15 minute intervals ○
○ ___ - Use of Automatic Tissue Processor (Ex. Autotechnicon, Elliot Bench Type Processor) Two Types: ___ (or “dip and dunk”) machines specimens are transferred from container to container to be processed; ___ (or “enclosed”) type specimens are held in a single process chamber or retort and fluids are pumped in and out as required
Impregnation; infiltration
Casting
PARAFFIN
24 hrs
56°C
>60°C
fatty tissues
Manual Processing
Automatic Processing
Tissue-transfer
Fluid-transfer
Number of Stations: ___
- 10 (1L ccntainers) + 2 wax baths
- Wax Bath Thermostat: ___ higher than Melting Point of Wax
- Set at Histopathologic Techniques Performed: fix, dehydrate, clear, impregnate
- Steps in an Autotechnicon
* Stations 1 and 2: __
* Stations 3-6: __
* Stations 7-8: __
* Stations 9-10: __
* Stations 11-12: __
Main Advantage: ___
* Vertical Oscillation/Mechanical Raising and Lowering of tissue basket
___
- Wax impregnation under negative atmospheric pressure inside an embedding oven ○ Hastens removal of air bubbles and clearing agent
○ Promotes rapid penetration of impregnating medium
- Fastest Method
12 stations
3°C
10% Formalin
Increasing grades of 70-95% Ethanol
Acetone
Chloroform or Xylene
Liquid Paraffin
constant agitation
Vacuum Embedding
Use of __
- For Urgent Biopsies, Delicate Tissues, CNS tissue; must be pure
○ Fresh Wax must be ___ using __
○ Reusing of Paraffin Wax: Can be only used ___
→ Water in the Wax must be removed by heating the wax at 100-105°C
- To give extra hardness to paraffin wax, paraffin can be mixed with using: ___
Paraffin Wax
filtered; coarse filter
2
10-20% beeswax or Ceresin
PARAFFIN WAX SUBSTITUTES
1. ___: Mixture of Paraffin and Synthetic Plastic Polymer (___); MP: ___
2. ___: similar to paraplast (MP: 56-58°C)
3. ___: semisynthetic; embedding of
4. ___: contains rubber
5. ___: MP: 46-48°C; but harder than paraffin
- Water insoluble but soluble in ___
- Sectioning: Use a ___ (for hard blocks)
6. ___: Polyethylene Glycols (MP 38-42C or 45-56C)
- Most Common: ___
- miscible and soluble in with water (hygroscopic)
- No need for ___
- Used for enzyme histochemistry
- Disadvantages: Hygroscopic; dissolves during fishing out in water bath; Do not overheat: it tends to crumble during sectioning
Carbowax infiltrated tissue sections should be fished out in:
○ ___: Composed of: Diethylene glycol
- Distilled Water
- 40% formalin
○ ___: Composed of: equal parts of 0.02% gelatin and 0.02% potassium dichromate
Paraplast; DMSO - bullet waxes; 56-57°C
Embeddol
Bioloid
Tissue Mat
Ester Wax; 95% ethanol; sliding microtome
Water Soluble Waxes
Carbowax
dehydration and clearing
Pearse
Blank and McCarthy
____ AKA ___
- Purified form of nitrocellulose
- For Specimens with large hollow cavities which tend to collapse, hard and dense tissues
- In 2%, 4% or 8% dissolved in equal parts of ether and alcohol
- Not requires heat during processing (less distortion and shrinkage
- Very Slow (Days to Weeks)
- Methods
* Wet Celloidin: for ____; use __ for storage
* Dry Celloidin: for processing for ___; uses ___ for storage; Composition: equal part of ___ and ___
* Nitrocellulose Method (Low Viscosity Nitrocellulose)
○ Soluble in equal concentration of ___
○ Preferred since it produces a harder tissue block and thinner sections are possible Add Plasticizer (__/__) to prevent tissue cracking
○ Explosive; tissue block becomes rubbery
> ___
- Rarely Used
- For Histochemical and Enzyme Studies and frozen sections
- Water soluble
○ Does not require ____
- Tx for processing should be <2-3 mm
- Add ___ to prevent molds
- Impregnating Medium Volume should be at least __ the volume of tissue
CELLOIDIN; Collodion
bone, brain sections and teeth; 70% alcohol
whole eye section; Gilson’s mixture; chloroform and cedarwood oi
ether and alcohol
castor oil/oleum ricini
GELATIN
clearing and dehydration
1% phenol
25x
____
- Done after Wax Impregnation
- Orientation: tissue is arranged in a precise position in the mold
- Generally the surface of the section to be cut should be parallel to the bottom of mold
◘ Tubular Structures (arteries, fallopian tubes) = cut in X-section of the lumen
◘ Skin, intestine, gall bladder and other epithelial biopsies = cut in a plane at right angles to the surface
◘ Muscle Biopsies = transverse and longitudinal planes
MOLDS USED
○ ___: 2 L Shaped Strips of Heavy Brass or Metal; size of the mould is adjustable
○ ___: made of grids and compartments; Advantage: multiple embedding
○ ___: special stainless steel base mold fitted with a plastic embedding ring (ex. Tissue Tek)
○ ___: Peel Away(3sizes: 22x22mm; 22x30mm; 22x40mm), Ice Tray, Paper Boat
○ ___: fragmentary biopsies
○ __ tissue processing and embedding system:
- Composed of 3 pieces: Perforated Bottom, Frame
- Center Piece; and a perforating lid
- These 3 pieces snap into one capsule
- Advantage: Fixation, Dehydration, Clearing and Embedding can be done using this system
EMBEDDING
Leuckhart’s Embedding Mold
Compound Embedding Unit
Plastic Embedding Rings and Base Molds
Disposable Molds
Watch Glasses
TIMS
OTHER EMBEDDING METHODS
_____ - For hard tissues and large sections of whole organs
_____ - 1st infiltration with celloidin and subsequently embedded with paraffin
_____
- Uses:
○ Embedding for ___ ; To allow tissue sectioning of 80nm thickness
○ ___: Renal Biopsies (Tissue Section of 2um); Hematopoietic Tissues
○ For Extremely Hard tissues
- Classification Accdg to Chemical Composition:
→ ___: widest application (light microscopy and electron microscopy)
- Ability to produce sections with thickness of 30-40nm
- Embedding of Choice for TEM
♣ Bisphenol A/___: Most stable but slow
♣ Glycerol/___
♣ Cyclohexene Dioxide/___: fastest; Highly toxic
- Polyester: for EM, seldom used
- Acrylic Plastics: for Light Microscopy
→ Preferred for High Resolution Light Microscopy;
→ Examples: Acrylates - (___ - ideal embedding medium for undecalcified bone)
→ ___- (Polyglycol Methacrylate/Glycol Methacrylates) - for TEM
Celloidin or Nitrocellulose Method
Double Embedding Method
Electron Microscopy
High Resolution Light Microscopy
Epoxy
Araldite
EPON
Spurr
Methyl Methacrylates
Methacrylates
5 Kinds of Microtomes
1. ___ (__ - most popular type of rocking microtome) (1881)
- simplest microtome
- Invented by ___
- Can Cut 10-12 um tissue sections
- For large paraffin embedded blocks
- Not for serial sections
- ___ (1885-1886)
- routinely used microtome
- Invented by ___.
- Cutting of ___ embedded tissues (Excellent serial sections) - ___ (1789/1798)
- For celloidin sections and hard rough tissue blocks
- Invented by: ___
- 2 Types:
→ ___ - preferred: Knife held rigidly in clamps; uses long knives; for brain tissues; most commonly used in neuropathology and opthalmology
→ Standard Sliding: more dangerous (knife is moving) - ___ (1848)
- For Frozen sections
- Invented by ___
CO2 is used as a propellant
→ Cryostat or ____
- for rush frozen section; now commonly used
- refrigerated (-5 to -30C) ave: -20°C
- Inside is a ___; Cryostat should be turned on at all times;
* 1hr-it takes for the knife to come to operating tempt.
> Soft tissue paper moistened with __: used to clean knife
* Anti-roll plate: to stop the natural tendency of frozen section to curl upwards on sectioning. Usually made of plexi glass or plastic material - ____
- Primarily for EM
- Cutting sections as thin as 0.5um (50nm)
- Tissues are usually embedded in Plastic
- Special Knife: Diamond Knife
Other Microtomes:
Vibratome -for enzyme histochem
Saw Microtome very hard material (ex. Undecalcified bone); Tx embedded in resins
Rocking Microtome/Cambridge Microtome
Trefall, Paldwell
Rotary Microtome
Minot
paraffin
Sliding Microtome
Adams
Base Sledge/Sledge Type
Freezing Microtome
Queckett
Cold Microtome; rotary microtome
alcohol
Ultrathin Microtome
- AND 8. BLOCKING AND TRIMMING
____: A step that goes hand in hand with Embedding if individual mode is used because blocks are produced after solidification. If a big mold tray is used, use a sharp knife to separate one tissue from another
____: Remove Excess Wax;
- To form a ___
- At least __ of wax should surround the TX Block
- Trimmed Tissue Block: partially expose the tissue - ___: Embedded tissue is trimmed and cut into uniformly thin slices using a microtome. Tissue blocks should be cold prior to sectioning
MICROTOMY
3 Essential parts:
- Block Holder: AKA: __
- Knife Carrier and Knife
- Pawl, Ratchet Feed Wheel and Adjustment Screws
BLOCKING
TRIMMING
truncated pyramid
2mm
SECTIONING
chuck
MICROTOME KNIVES
- Mostly made of stainless steel
- 3 Basic Types:
○ ___ (Length: ___) - shortest
→ Less Concave Side = for ___
→ More Concave Side = for ___
→ Use: for base sledge, rotary or rocking microtome
○ ___ (Length: ___)
→ both sides are concave
→ Cutting Paraffin Sections.
→ Use: ___
↪ Heiffor Knife - used for rocking microtomes with a fixed handle
○ ___ (Length: ___)
→ for frozen sections or very hard tissues
→ Use: Base Sledge or Sliding Microtome
Plane Concave; 25mm
celloidin sections
paraffin sections
Biconcave Knife; 120mm
rotary microtome
Plane Wedge; 100mm
Other Types of Knives:
○ ___: more commonly used; coated with polytetrafluoroethylene (allows easy ribboning of tissues)
○ ___: For ultrathin microtome; Glass Knives: “Ralph Knives”
Bevel = ___; found on the tapered edge of a knite
○ ___ = angle formed between the cutting edge; __ degrees
→ ___ - maintains the bevel angle
○ ___ = ___ degrees (optimum)
→ There is maximum penetration of tissue and less distortion
○ ___ = ___ degrees; Angle formed between the surface of the block and the
cutting edge of the knife.
○ ___ - angle of the upper surface of facet and surface of tissue block; High rake angles are for soft tissues
Wedge Angle-angle between the sides of knife
Disposable Blades
Glass and Diamond Knives
cutting facet
Bevel Angle; 27-32°
knife back
Cutting Angle; 15°
Clearance Angle; 0-15°
Rake angle
HONING & STROPPING
___ – Knife Hard Sharpening
- Process of Removing Nicks (irregularities of knife)
- Movement: From __ to __
- Number of Strokes per surface: __ strokes
Hones/Honing Stone/Oil Stone
- Dimension: __
- Knife Sharpeners (Mechanical Honing)
- Clean the honed knife with xylene
Types of Hones
○ ___ - best result
○ ___ - polishing effect
○ ___- for badly nicked knives
○ ___ (8x3x1 inches): excellent substitute for stone
Lubricants: (soapy water, mineral oil, clove oil, xylene or liquid paraffin)
Honing
heel to toe
10-20
8 x 3 in
Belgium Yellow
Arkansas
Fine Carborundum
Plate Glass
___ = polishing
- Removal of burrs (irregularities of the knife formed)
- Movement: From __ to __
- Use Paddle Strop made of ___
- Dimensions: ___
- Strops should be oiled on the back (DON’T USE MINERAL OIL); 40-120 double strokes
Types of Sections:
○ Paraffin Sections = ___ um sections
○ Celloidin Sections = ___ um sections
○ Renal Biopsy Sections = ___ um sections
→ Semi-thin Sections using __ embedding medium.
○ Ultra-thin Sections (EM)
→ Recommended Section: 80nm (Silver or Straw Colored Sections)
○ Frozen Sections (Cryostat) = ___ um if microtome used is Rotary
Stropping
toe to heel
horse leather/cordovan
3-4 in x 18 in
4-6
10-15
2
plastic
4
FROZEN SECTION:
- Rapid diagnosis
- For Enzyme Histochemistry
- Demonstration of Soluble Substances
- Immunofluorescent and Immunocytochemical Staining
- Specialized Silver Stains
Two Methods:
___: Use of Conventional Freezing Microtome (Carbon Dioxide)
- Tissue Block: 3-5mm
- ___ = point in which sections may be cut at 10um; frozen tissue starts to thaw (warmed with the finger) and becomes visible to the naked eye
- ___ - used to remove the ribbon that sticks to the knife blade
___: Use of – ; Optimum Temperature = -18 to -20C (Average: -20C)
Cold Knife
Dew Line
Use of Camel Hairbrush
Cryostat
Methods of Freezing:
○ ___ (-190C)= most rapid; Disadvantage: frozen tissue cracks producing freeze artifacts
○ ___ (-150C) = liquid at Room Temp
○ ___ (-50C) (Cryokwik) = getting popular; not for muscle; made of fluorinated hydrocarbons
○ Carbon Dioxide (-50C)
○ ___ -high thermal conductivity (better that liquid nitrogen)
FROZEN SECTIONS:
- Clearing Agents Used: ___
- No need for dealcoholization
→ function of clearing agent: increase refractive index
___ = use of synthetic water soluble glycols and resins embedding medium
Liquid Nitrogen
Isopentane
Aerosol Sprays
Freon 2.2
glycerin and sum syrup
mounting
Tissue Tek Tissue Embedding Center
- Paraffin Reservoir
-Tissue Tank
- Warm Plate
- Cold Plate
Flotation Water Bath
- Temp: __C below MP of Wax
→ Overheating can lead to artefact formation: “__” artifact on tissues
- Colored Enamel Black
- Diameter = __inch; Height = ________inch;_____L capacity
- Filled within 1/2-1cm from the top
Drying Oven
- Temp: __C Higher than MP of wax
- Forceps and Squirrel Hair Brush
- Clean Slides = 76x25mm 1-1.2mm thick; Frosted; Non-frosted
10°
parched earth
11in; 4in; 2L
5°
_____
* Place the ribbon of sections into the water bath for it to flatten
* 30 Seconds: time it takes for ribbons to flatten
* Do not let the ribbon stay on the water bath for more than 1-2 mins
* Separate the ribbon of sections into individual sections using forceps
*Start fishing out each section using a slide (lightly smeared with adhesive)
* Immerse the slide vertically
* The __ in a ribbon are never picked up on slides
* Fish-out a section
* Gently lift the slide vertically
* Slide is then stood to drain at and angle of 60-85 degrees for 2-5mins
* dry the section using either of the following methods
→ __ 45-55C for 30-45 mins
→ leaving it in a incubator overnight = best for ___
→ wax oven 56-60C for 2 hours
→ in a blower type electric slide dryer for 20-30mins 50-55C
→ above a ___ until the wax melts - urgent method
Difficulties during Sectioning
Mushy tissue block that leads to sections to crumble and feather Tearing of tissue from block
Sections fail to form ribbons
Sections roll up on cutting that they adhere; wrinkled or jammed
Sections adhere to knife or other parts of microtome
“___” -horizontal or parallel lines across the sections
___ -thick/thin areas
Floaters and Contaminants
FISHING OUT
first one or two sections
hot plate
nervous tissue
Bunsen Burner
Chatters
Venetian Blinds
TISSUE ADHESIVES
1. ___: most common
→ Adhesive added on slide (thin)
→ Components:
- Egg White
- ___: is also added to increase viscosity and prevent complete drying.
- ___: preservative is added to prevent growth of molds
- Disadvantage: Background staining may be detected due to its uptake of dyes.
- ___ - Provides firmer attachment than albumin
→ Concentration: ___
→ has to be gently heated before use to melt the gelatin.
→ Added to the ___- Disadvantage: Stains with many dyes.
- ___ - Advantage: Not staining to any appreciable extent with commonly used in stains of histochemical reagents.
- ___ – Use as a general-purpose section adhesive.
- Widely used as an adhesive in ___
- No production of background staining.
Albumin (Mayer’s Egg Albumin)
glycerol
thymol crystals
Gelatin
1%
waterbath
Cellulose in the form of 1% Methyl cellulose
Poly-L-lysine
immunohistochem
TISSUE ADHESIVES
5. ___ - commercial syrup = 1:10 dilution
-Had strong adhesive properties
- Advantages: Little tendency to staining with most dyes; not affected by the use of mild alkaline solutions
- Disadvantages: Blackening in some silver impregnation techniques, in some reticulin methods, and red staining in methyl green pyronin technique
6. ___- Greatest adhesion.
- ___- made of epoxy resins
» Diluted 1:10 with acetone
» Little affected by most fluids in any treatment of sections
Sodium Silicate
Resins
Araldite
SPECIAL PROCESSING TECHNIQUES
For Histochemical Evaluation (Enzyme Studies)
○ ___
Preservation by rapid freezing (quenching) at ___
Desiccation by transferring the frozen tissue in a vacuum at -40C (Sublimation)
2mm tissue thickness
Expensive and Time Consuming!!!
Four Stages to Freeze Drying:
→ __ -stops the chemical reaction and diffusion in tissue
→ __ - most time consuming, introduction of heat to cause sublimation of heat; transfer of water vapor from ice crystals through dry portion of tissue; removal of water vapor from surface of specimen
→ Fixation and Embedding
→ __ - most impt is formalin
○ ___
similar to freeze drying except the vacuum procedure
* Makes use of Rossman’s Formula or in 1% Acetone and Dehydrated in Absolute Alcohol
* Routine Paraffin Wax Impregnation and Embedding is done
Freeze Drying
160°C
Quenching
Drying
Vapor Fixation
Freeze Substitution
- ___
- Application of dyes on tissue sections to study the architectural patterns and physical characteristics of cells
- Different tissues and cells have varying affinities for most dyes and stains
AFFINITY:
→ Nucleus: stained by basic stains
→ Cytoplasm: stained by acidic stains
- For H and E:
○ Deparaffinize sections in xylene, then rehydrate sections (Absolute and 95% alcohol) prior to actual staining
○ Removal of pigments/artefacts should be done during the __ stage prior to staining.
STAINING
hydration
GROUPS OF TISSUE STAINING
1. ___
- Direct interaction with a dye or staining solution
- Active tissue component is colored
- This include: micro-anatomical stains, bacterial stains, specific tissue stains (e.g. muscles, connective tissue and neurologic stains)
- ___
- Study of the tissue constituents through their chemical reactions
- Microscopic Localization of Specific Tissue Substances
- The process whereby various constituents of tissues
Examples:
→ Perl’s prussian blue reaction for __
→ Periodic Acid Schiff staining for __
♦ Enzyme histochemistry
- Enzymes act as a ___ for most biological reactions
- Enzymes can be found inside organelles or found free and soluble in cytoplasm and body fluids
- Tissues Required:
→ ____
→ ____
- Enzyme Substrate: Active reagent
- Enzyme: present in tissue
Histologic Staining
Histochemical Staining
iron
glycogen
biochemical catalyst
frozen sections
tissues fixed in 4% formalin/formol saline
Types of Enzymes:
→ Oxidases, Peroxidases, Dehydrogenases, Diaphorases, Hydrolases, Lyases
- The final opacity or coloration is produced from the substrate rather than the tissue.
- Controls are needed
- ___
- Combination of Immunologic and Histochemical Techniques
- Detection of ___ that are detected by antibodies
- Ex. Monoclonal, Polyconal, Fluorescent Labeled or Enzyme Labeled Antibodies
Immunohistochemical Staining
phenotypic markers
METHODS OF STAINING
1. ___: Uses aqueous or alcoholic dye solutions (e.g. methylene blue, cosin) to produce a color
2. ___: Uses a mordant or another agent to intensify the action of the dye used
What is a Mordant?
- Serves as a ___ between the tissue and the dye
- Dye + Mordant = insoluble tissue mordant dye complex
- E.g. Potassium alum with hematoxylin in Ehrlich’s hematoxylin. Iron in Weigert’s hematoxylin
___
- Not essential and does not participate to the chemical reaction of the tissue and dye
- Hastens the staining reaction by increasing the staining power & selectivity of the dye
- E.g. Potassium hydroxide in Loeffler’s methylene blue, Phenol in Carbol thionine and Carbol fuchsin
___
- Tissue elements are stained in a definite sequence
- The staining with specific periods of time or until desired color is attained
- __ is Applied
- The distinction of tissue detail relies solely on the selective affinity of the dye for various cellular elements
___
- overstaining is done
- Excess stain is removed or decolorized from unwanted parts of the tissue and until the desired color is obtained
___
- Selective Removal of Excess Stain so that a specific substance may stain distinctly from the surrounding tissue
- Usually done by washing the section in simple solution (e.g. water or alcohol) or use of acids and oxidizing agents
- Ex. Acid Alcohol
Direct staining
Indirect staining
link/bridge
ACCENTUATOR
PROGRESSIVE STAINING
no decolorizer
REGRESSIVE STAINING
DECOLORIZER/DIFFERENTIATION
___
- Staining with a color that is different from that of the stain itself
- Usually employed in staining cartilage, connective tissue, epithelial mucins, amyloid and mast cell granules.
- All are cationic/basic or Basic Dyes belonging to thizine and triphenylmethane groups
→ Ex. Methyl violet, Bismarck Brown, Methylene Blue, Toluidine Blue, Cresyl Blue
→ ___ is important in metachromatic staining; ___ tends to lose the metachromatic stain
___
- For contrast and background
- Stain with a different color that of the primary stain
- Common Counterstains
____:
→ Red: Eosin Y, Eosin B, Phloxine B
→ Yellow: Picric Acid, Orange G, Rose Bengal
→ Green: Light Green SF, Lissamine Green
____:
Red: Neutral Red, Safranin, O Carmine, Hematoxylin
Blue: Methylene Blue, Toluidine Blue, Celestine Blue
___
- Demonstration of tissue elements using solutions of metallic salts that are deposited on the tissue surface
- It is not absorbed by the tissues, could be a precipitate or a reduction product on certain tissues; E.g. Gold chloride, Silver nitrate
- Precaution: ___ are potentially explosive; Avoid Using Metallic Instruments; Do not throw in the sink
→ Contact with Sodium Azide can cause explosion
METACHROMATIC STAINING.
water; alcohol
COUNTER STAINING
Cytoplasmic Stains
Nuclear Stains
METALLIC IMPREGNATION
Ammoniacal Silver
STAINS: NATURAL DYE
♦ ___
- Extracted from insects (cochineal bug: coccus cacti)
- With alum
→ Carmine dye
→ Chromatin and nuclear stain for fresh and smear preparation
- With picric acid
→ Picrocarmine (Neuropathological stain)
- With aluminum chloride
→ ___: Demonstration of glycogen
♦ ___
- Vegetable dye
- Extracted from lichens
- Used for staining __
- Colorless, treated with ammonia, exposed to air to produce a blue or violet color
- Also the source of ____ - used as a pH indicator
Cochineal dyes
Best’s carmine
Orcein
elastic fibers
litmus paper
___ - coloring substances
Two Categories:
1. ____
- Derived from plants and animals
- E.g. Hematoxylin, Cochineal dyes, Orcein, Saffron (dried stigmata of ___)
♦ ___
- Natural Dye obtained from hematoxylin campechianum (___)
- Extracted with hot water and precipitated from the aqueous solution using urea.
- Hematoxylin in itself is not a stain
- __ - Active coloring, anionic; it is inadequate in itself without a mordant
→ Obtained by ___ = oxidation of hematoxylin
- Used in combination with a __ such as alum, iron, chromium and copper salts
Ripening:
○ ___: Expose the substance to sunlight and air
- A slow process, 3-4 months
- Hematoxylin Stains ripened by Natural Ripening: Ehrlich’s and Delafield’s
○ ___: Chemical oxidation
- Hydrogen peroxide, mercuric oxide, potassium permanganate,
- Sodium perborate, sodium iodate
- Hematoxylin Stains ripened by Artificial Ripening: Mayer’s, Harris
STAINS
NATURAL DYES
crocus sativus
Hematoxylin
Mexican tree
hematein
Ripening
mordant
Natural ripening
Artificial ripening
___:
- The selective staining of living cell constituents
- Demonstrates cytoplasmic structures
→ By engulfment of the dye particle
→ By staining of pre-existing cellular components
- ___is resistant to vital stains
→ Staining of Nucleus indicates: __
Two Types of Vital Staining:
1. ___: by injecting the dye into any part of the animal body
e.g. lithium, carmine and India ink
2. ___: used immediately after removal of cells from the living body
-e.g. Neutral red, Janus green, Trypan blue, Nile blue, Thionine and Toluidine Blue Best Vital Dye:
VITAL STAINS
Nucleus
cell death
Intravital staining
Supravital staining
COMMON STAINING SOLUTIONS
A. ___ (pH 2.5-2.9)
- Most commonly used for histologic studies
- Combined with Mordants (such as Alum, Iron) to form the dye mordant tissue complex
- Filter the stain prior to use (to remove the metallic sheen formed especially in Harris Hematoxylin)
1. ___: routinely used in H and E staining
- Mordant Used: ___
- Initially, it Stains the nuclei reddish
- Forms Blue Lakes after blueing (scott’s tap water, ammonia water)
- Blueing Agent (pH 8) for 2 minutes
- For Progressive and regressive staining
Examples of Hematoxylin
○ ___: Routinely Used in nuclear staining
- Ripened with ___
- Cytology: Nuclear Stain in Pap’s
- Staining of Sex Chromosomes
- Addition of ___ gives a precise nuclear staining (Add this in the working solution)
- Staining Method: ___
Hematoxylin
Aluminum hematoxylin
Alum
Harris Hematoxylin
mercuric oxide
glacial acetic acid
regressive
___ ΑΚΑ ___
- Derived from hydrocarbon benzene
- ___
○ Substances with definite atomic groupings that are capable of producing visible color but color isn’t permanent
○ ___: simple benzene compounds that contain chromophores
- ___
○ Substances that are added to a chromogen, which alters the property of the chromogen by altering its shade, enabling it to form salts with another compound and enables it to retain its color in the tissue - ___- impart color that is permanent;
→ Composed of ___ and ___
SYNTHETIC DYES; Coal tar dyes/Aniline dyes
Chromophore
Chromogen
Auxochrome
Dyes
chromophore and auxochrome
3 GROUPS OF SYNTHETIC DYES:
1. ____
* The coloring substance is found in the acid component
* Basic cell structures have an affinity for acid dye ions and are called __
* Ex. eosin, picric acid
- ____
* The coloring substance is found in the basic component that combines with the acid radical
* Acidic structures have an affinity for basic dyes and are called __
Ex. methylene blue - ___
* Formed by combining aqueous solutions of acid and basic dyes
* Stains the cytoplasm and nucleus simultaneously and differentially
* Insoluble to barely soluble in __
* Soluble in __
* Ex. Romanowsky dyes (Giemsa, Wright’s)
Acid dyes
acidophilic
Basic dyes
basophilic
Neutral dyes
water
alcohol
EXAMPLES OF HEMATOXYLIN:
○ ___: Excellent nuclear stain; stains mucins, recommended for bone and cartilage!!
- Not for frozen sections
- shelf life of hematoxylin
- ___ added to slow oxidation and to prolong
- Staining Method: ___
○ ___: Naturally ripened; similar longevity to Ehrlich’s
○ ___: Chemically ripened with sodium iodine; Primarily a regressive stain
○ ___: Artificially Ripened with alcoholic iodine
○ ___: Artificially ripened with potassium iodide; For frozen sections
Ehrlich’s Hematoxylin
glycerin
regressive
Delafield’s Hematoxylin
Mayer’s Hematoxylin
Cole’s Hematoxylin
Carazzi’s Hematoxylin
- ___
- Uses Iron salts as both a mordant and a ripening agent
- Staining Method: __
- Examples:
○ ___: Mordant/Oxidizer: Feric ammonium chloride
- Standard iron hematoxylin
- Used in demonstrating muscle fibers and CT!!
○ ___: Mordant/Oxidizer: ferric chloride
- For nuclear and cytoplasmic inclusions; cytological stains
- Result: Gray-Black
○ ___: - for frozen sections
○ ___: - elastic fibers (black)
- ___
- Mordant: 1% aqueous phosphotungstic acid
- Oxidizer: Potassium Permanganate
→ ____: Natural ripening achieved with light and air; Progressive stain; For CNS, general tissue - ___ for demonstration of granules of endocrine cells of alimentary tract; argyrophil cells
- ___ for the study of spermatogenesis
Iron Hematoxylin
regressive
Weigert’s Hematoxylin
Heidenhain’s Hematoxylin
Loyez Hematoxylin
Verhoeff Hematoxylin
Tungsten Hematoxylin
Phosphotungstic Acid Hematoxylin
Lead Hematoxylins
Copper Hematoxylin
B. ___: A red acid (xanthene)dye (pH4-4.5)
- Routinely used as a counterstain after hematoxylin and before methylene blue
- Stains connective tissues and cytoplasm differentially
3 forms:
○ ___: most commonly used; Soluble in water; yellow fluorescence
○ ___: deeper red color
○ ___: soluble in alcohol
Eosin
Yellow (Eosin Y)
Eosin B, Erythrosin B
Eosin S, Ethyl eosin
Other Stains:
Acid Fuchsin-Picric Acid (__ Stain)
○ For demonstration of connective tissues
___ (Masson Stain)
○ Basic acridine fluorochrome
○ Discriminates dead and living cells
○ DNA- __ fluorescence
○ RNA- __ fluorescence
___
○ Demonstration of calcium salt deposits and Phosphatase activities
Alcian Blue
○ Water soluble phthalocyanin dye
○ For connective tissue and epithelial mucin
Aniline Blue
○ Cytoplasmic stain
○ For counterstaining of epithelial sections
Van Gieson’s
Acridine orange
green
red
Acridine Red 3B
Other Stains:
___
○ Plasma stain
○ For staining acid-fast organisms, for mitochondria, for differentiation of smooth muscles (with the use of picric acid)
Basic Fuchsin
A. Carbol-Fuchsin
B. Coleman’s Feulgen Reagent
C. Schiff’s Reagent
D. Mallory’s Fuchsin Stain
E. Aldehyde Fuchsin (Gomori’s stain)
Benzidine
○ For staining __
Bismarck Brown
○ Used as counterstain for Gram’s technique, for acid fast, for Papanicolau method
○ Used for staining ___ organisms
Carmine
○ Used as chromatin stain for fresh materials in smear preparations Combined with aluminum chloride to stain glycogen (___ solution)
Celestine Blue
○ Used for routine staining of fixed sections
○ Resistant to strong acid dyes
○ Good nuclear stain
___
○ Best known as an indicator
○ Stains elastic tissues, amyloid, myelin
___
○ A nuclear or chromatin stain
○ Stains amyloid in frozen sections, platelets in blood
○ Gentian violet (crystal violet, methyl violet, dexterin)
___
○ Used for staining blood to differentiate WBCs
Basic Fuchsin
hemoglobin
diphtheria
Best Carmine
Congo Red
Crystal Violet
Giemsa Stain
Other stains:
___
○ Stain used for metallic impregnation
○ Made up of gold chloride and mercuric chloride
___
○ Stains amyloid, cellulose, starch, carotenes, glycogen
○ Used to remove mercuric fixative artifact pigments
○ Used as a reagent to alter crystal and methyl violet which may be retained by certain bacteria and fungi
○ ___: Stains microorganisms and fibrin in tissue sections
○ ___: Used as test for glycogen, amyloid and corpora amylacea
Janus Green B
For demonstrating mitochondria during supravital staining
___
○ Counterstain for ascaris eggs, RBCs, bacterial spores
○ Used as a decolorizer and counterstain
___
○ Common basic nuclear stain used with eosin
○ Stains plasma cells, cytological examination of sputum for malignant cells, evaluation and differentiation of bacteria, diagnosis of diphtheria, vital staining of nervous tissues
___
○ Basic stain
○ For demonstration of cell granules and vacuoles of phagocytic cells
___
○ Stains clastic fibers
○ Recommended for dermatological studies
○ Demonstrates the finest and most delicate fibers in the skin
Osmium Tetroxide
○ Used to stain fats
Gold Sublimate
Iodine
Gram’s iodine
Lugol’s iodine
Malachite Green
Methylene Blue
Neutral Red
Orcein
Other stains:
____
○ Counterstain for acid fuchsin, connective tissues (in Van Gieson’s stain), cytoplasmic stain in contrast to basic dyes. counterstain for crystal violet
____
○ Colored salt of ferric ferrocyanide
○ Used for the manufacture of paints
○ Used as contrast stain, intravital staining of the circulatory system
____
○ Used with osmic acid to fix and stain blood and glandular tissues
____
○ Used for identification of spirochetes, reticulum, fiber stains
____
○ Used as nuclear stain in fixed tissues, stains Nissl granules or chromophilic bodies
____
○ Demonstrates neuroglia in frozen sections
Picric Acid
Prussian Blue
Rhodamine B
Silver Nitrate
Toluidine Blue
Victoria Blue
STAINS FOR FATS:
- Fats are either simple lipids or complex lipids (phospholipids and glycolipids), or derived lipids
- ____ property of tissues to be stained with fat or oil
soluble dyes
OIL SOLUBLE DYES (LYSOCHROMES):
- Not real dyes
- Lack ___
- Gives color to lipids because they are more soluble in lipid medium of the tissues than in 70% alcohol
- Sudan Black B, Sudan III, Sudan IV
- ___: most sensitive of the oil soluble dyes
- Stains: phosphoipids, neutral fats (triglycerides)
- Color:black/blue-black - ___ (AKA ___)
- Has no secondary amino group
- Most commonly used
- Stains neutral fats (triglycerides)
- Add benzoic acid to intensify the fat staining
- Color: red - ___: first Sudan dye
- Stains Fat in CNS tissues
- Less deep, light orange stain
OTHER STAINS FOR FAT
Oil Red O = stains neutral fats and
__ = stain for unsaturated fats in frozen section
__ = differentiates two lipid classes
> Red Oxazone: dissolves neutral lipids
> Blue Oxazine: basic and reacts with phospholipids and free fatty acids
Sudanophilia
auxochrome
Sudan Black B
Sudan IV; Scharlach R
Sudan III
Osmic Acid
Nile Blue Sulfate Method
Solvents Used for Staining
1. Water: Distilled, unless otherwise specified
2. Alcohol: Ethyl alcohol or Methyl alcohol
○ Absolute methyl alc or Acetone-free if for use on blood stains
3. ___: 10ml of aniline per 1⁄2 to 1L of hot dist. water
4. ___: Used in aqueous solution of 0.5 to 5%
Aniline Water
Phenol
CARBOHYDRATE STAINS:
Carbohydrates - main sources of energy
- Forms: Monosaccharides, Polysaccharides
- __ Stored form of CHO in humans and animals
→ stored in liver
___ - polysaccharides bound to other substances; Found in in epithelial and intestinal glands
- Forms the matrix of connective tissues; secreted by goblet cells,
respiratory lining cells, certain glands
- Classified as:
→ Mucopolysaccharides: Acid Mucopolysaccharides
- Primarily composed of hyaluronic acid, heparin sulfate and chondroitin sulfate
- Metachromatic Substances
- PAS (-)
→ Mucoproteins (mucoids and glycoproteins): Neutral Mucopolysaccharides
- Contains hexoses
- Do not stain with Alcian Blue
glycogen
Mucins
Stains for Electron Microscopy: (heavy metals)
- ____ (superior stain)
- ____ (1st general stain)
- ____ (1° or 2° stain)
Precautions of Staining:
To remove stains on skin: add decolorizer
uranyl acetate
phosphotungstic acid
lena
___
- Identification of specific or selective tissue or cellular antigens
found on tissues
- Used for detection of organisms in cytologic preparations (eg. Fluids, sputum samples, FNA) Makes use of antigen-antibody reactions
→ Antigen: found in cells
→ Antibodies: ___ most commonly used antibody class
Types:
→ ___ Antibodies: produced by different cells; react with various epitopes; Most common source: ___
Other animal sources: goat, pig, sheep, horse, guinea pig
→ ___ Antibodies: product of an individual clone of plasma cell; Source: ___
- Storage: -80C in aliquots
Labels:
○ Enzyme Labels
- Use of HRP (horse radish peroxidase) most widely used; and AP/ALP (Alkaline Phosphatase)
- Chromogens such as Diaminobenzidine (dark brown) and AEC (brick red); Other chromogens: nitroblue tetrazolium, fast red TR salt
→ ___- used for most applications and provides strong and permanent stains. Other chromogens such as AP Red is employed for skin sections where brown DAP can be masked by melanin pigment; Two chromogens such as DAB and AP can be used in the same section at the same time to allow visualization 2 antigens in one section. This is called double staining
→ Optimum Incubation time for linking antibodies with peroxidase conjugates is ____ at room tempt
→ Produces a stable colored reaction end product suitable for ___
Fluorochrome Labels:
- Use of ___
- Need of ___ microscope
Other Labels: Colloidal Metals such as gold (+) pink under light microscope, Radiolabels
___ - plant or animal proteins that can bind to tissue CHO. Can also be labelled like Ab.
IMMUNOHISTOCHEMISTRY
IgG
Polyclonal; rabbits
Monoclonal; mice
DAB
30-60 mins
LIGHT MICROSCOPY
fluorescein
fluorescence
Lectins
Specimens for Immunohistochemistry
- Cryostat Frozen Sections and fixed in a few seconds in __ or __
- Formalin-fixed or paraffin-embedded tissues can be used
→ ___ - widely used fixative
→ Other fixatives: 4% paraformaldehyde; Bouin’s, Zamboni’s (EM Immunocytochemistry)
- Prior to processing, unmasking of Ag/Ag retrieval is a prerequisite!!! especially for paraffin embedded tissues
→ Tissue sections are dewaxed, rinsed in alcohol and washed in water
Methods to Retrieve Antigens (Ag unmasking) from Routinely Processed Tissue:
1. Proteolytic Enzyme Retrieval (use of pepsin and bromelin, trypsin, protease)
2. Microwave Antigen Retrieval (Heat induced Epitope Retrieval)
3. Pressure Cooking Ag Retrieval
4. Autoclave Heating
5. Waterbath Heating (90C; better retrieval at 95-98C)
6. Steamer Heating
7. Decloaker Heating
8. Combination of Microwave and Enzyme digestion
Counterstain: traditional nuclear stains such as ___
Other counterstains: methyl green, nuclear fast red (source: Fisher Scientific)
Absolute Methanol or Acetone
`
10% Neutral Buffered Formalin
hematoxylin
IMMUNOHISTOCHEMISTRY:
Adhesives - ____
Use of Inactivators and Blocking Agents - prior to staining
○ ___ - ex. 3% Hydrogen Peroxide- methanol (for HRP); levamisole (for ALP) inhibit endogenous enzymes to prevent non- specific binding
○ ___ - ex. Animal serum; used to block residual sites on the tissue section may bind to secondary antibodies leading to false positive results. 10-30 minutes at room temperature
Mounting Medium - must be __ pH resins, with 50% glycerol (short term)
Key Steps in Processing Paraffin Embedded Tissues for IHC
___
Controls:
1. ___: A section known and proven to have the antigen in question
2. ___: Omit the primary antibody from the staining schedule; Replace the specific primary antibody by an Ig that is directed against unrelated antigen
3. ___: “built-in” control; Contains the target antigen but not in the tissue elements under investigation
APES diluted in acetone
Inactivators
Blocking Agents
neutral
Tissue Preparation > Deparaffinization > Inactivation using 3% Hydrogen Peroxide > Antigen Retrieval > Blocking > Application of Antibody > Staining > Mounting
Positive control
Negative control
Internal tissue control
Methods:
- Traditional Direct Technique: Primary Ab is labelled
- EPOS - ___ = direct labelling system; use of primary Ab conjugated with labeled long chain dextran polymer
- ___ - more sensitive than direct; increased signal amplification; Use of labeled secondary Ab - ___ - Secondary Ab conjugated to an enzyme labelled polymer chain
Other Methods:
- Unlabeled Antibody enzyme complex techniques
- Immunogold Silver Staining - silver is added for enhancement
- Streptavidin Biotin Techniques
Extended Polymer One-Step Staining
Two step indirect technique
Polymer Chain Two-step indirect technique
TUMOR MARKERS (ANTIGENS)
I .Epithelial Tumor Markers
1. ___: marker of epithelial cells
2. ___: Helpful in determining the site of tumor, (+) for breast adenocarcinomas, lungs and kidneys
3. ___: An oncofetal antigen present carcinomas of GI tract, pancreas, lung, breast, ovary, uterus, cerix Differentiates adenocarcinoma (+) from mesothelioma (-)
4. ___: Distinguishes lung adenoCA from mesothelioma; Present in thyroid gland neoplasms
5. ___: (+) Prostate adenoCA, pancreatic and salivary tumors
6. ___: Normally found in breast epithelial cells but not myoepithelial cells; Prognosis and treatment of breast CA, identification of metastatic breast CA
Keratin
EMA - epithelial memvrane antigen
CEA (Carcinoembryonic Antigen)
TTF-1 (Thyroid transcription factor-1)
PSA (Prostate Specific Antigen)
ER/PR (Estrogen/Progesterone Receptor)
Intermediate Filament Markers:
1. ___: A Sensitive Maker for muscle diferentation Identify tumors derived from smooth, skeletal and cardiac muscles
2. ___: Normally present in mesenchymal cells and neoplastic cells such as sarcomas, lymphomas, leukemias, seminomas, neural tumors;
- Melanomas and schwannomas! always stain __
3. ___: Normally expressed by smooth, skeletal, cardiac muscle
- Highly Specific for ___
4. ___: Normally expressed by CNS glial cells especially glial cells/astrocytes; Diagnosis of astrocytoma
5. ___: Expressed by cells of neural origin; (+) in neuroblastomas, ganglioneuromonas, neuromas, chemodectomas
6. ___: calcium binding protein; Expressed by CNS glial cells, Schwann cells, melanocytes, histiocytes, chrondrocytes, skeletal and cardiac muscles, myoepithelial cells, epithelial cells of the breast, salivary and sweat glands
Neuroendocrine Markers
1. NSE (Neuron-specific enolase)
⚫ (+) for tumors with neural or neuroendocrine differentiation
2. Chromogranin
Found in neural secretory granules of endocrine tissues
(+) neuroendocrine differentiation
Keratin (+) and Chromogranin (+) = neuroendocrine carcinomas
3. Synaptophysin
Present in presynaptic vesicles of neurons, normal neurons and neuroendocrine cells
Actin (thin filament)
Vimentin (mesenchymal cells); positive
Desmin; myogenic tumors
GFAP (Glial Fibrillary Acidic Protein)
NF (Neurofilament)
S100 Protein
Neuroendocrine Markers
1. NSE (Neuron-specific enolase)
2. Chromogranin
3. Synaptophysin
IV. Germ cell tumor markers
1. ___: synthesize by placental cytotrophoblast/syncytiotrophoblast in late pregnancy; (+) germ cell tumors, choriocarcinomas
2. ___: synthesized by normal liver; (+) hepatocellular CA
3. ___: produced by placental syncytiotrophoblast in late pregnancy
human chorionic gonadotropin (HCG)
alpha-fetoprotein (AFP)
placental-like alkaline antigen (PLAP)
V. Mesenchymal Tumor Markers
___: melanocytes are derived from the neural crest (+) S100 protein; melanosome (+) HMB45
___: (+) LCA (leukocyte common antigen or CD45; best screening marker for lymphoma
Melanoma
Lymphoma
11 and 12. MOUNTING AND LABELLING
- Last 2 Steps in Histopathology
___
- Use of a medium and a coverslip to facilitate the handling, storage, protection of the tissue section
- Mounting Medium is used
→ syrupy fluid to prevent movement of the coverslip;
→ protects and prevents scratches to the tissue section
→ Coverslips Used:
○ Coverslip # 1.5 (180nm thick): ideal range for photomicrography
○ Coverslip # 1 (150nm thick): routinely used
Sizes: 18x18 mm, 22x22 mm, 20x20 mm, 24x24mm;
Methods of Mounting of Sections:
○ Slide lowered on to coverslip and quickly inverted
○ Coverslip lowered on to slide
A Mounting Medium should have a refractive index close to that of glass ____
Mounting
1.518
Kinds of Mounting Medium:
- ___: used to mount water miscible preparations
○ Composition:
→ gelatin, glycerin jelly or gum Arabic: solidify the medium
→ ___: to prevent drying and cracking
→ ___: to increase refractive index
→ Preservative: ex merthiolate
- ___: For preparations that are dehydrated and cleared in xylene or toluene
Aqueous Mounting Media
1. Water: cheapest
2. ___ (Semi-permanent) sections are kept preserve for years if sealed with paraffin
- Standard Mounting Medium (as in Fat Stains)
- Refractive Index: 1.46
- Ringing is required (sealing of edges)
3. Farrant’s Medium (___ Medium)
→ Refractive Index: 1.44
4. Apathy’s Medium (Methylene Blue Stained Nerve Preparations)
5. ___ = Mounting Frozen Sections from Water
6. Levulose (Fructose)
Aqueous
glycerol
sugar
Resinous
Glycerin
Gum Arabic
Brun’s Fluid
Resinous Mounting Media:
1. ___ =transparent and colorless but darkens and oxidizes with ages; for whole mounts and thick sections; acidic pH
→ Natural Resin coming from ___ (Albus balsamea)
→ Refractive Index: ___
→ Diluents: ___
2. ___ = small tissue sections; dries rapidly
3. ___= synthetic resin mixture dissolved in xylene
4. Clarite (1.544) = synthetic resin; preferred over DPX
5. Permount, HSR, Clearmount
____
- Sealing of Margins of the Coverslip to prevent escape of fluid and evaporation; prevents sticking of slides
- Kinds of Ringing Media
→ Kronig Cement (2 Parts Paraffin mixed with 4-9 parts Powdered Colophonium Resin)
→ Durofix/Du Noyer’s (Cellulose Adhesives)
____
- Process of Indicating the year and Specimen Number on one end of the prepared slide for proper identification
EXFOLIATIVE CYTOLOGY
.
A branch of General Cytology which deals wit the microscopic study of that have been desquamated from the from the epithelial surfaces.
Recommended for:
detection of malignant cells or cancerous conditions in the body; detection of asymptomatic or precancerous cervical lesions in women; assessment of female hormonal status in case of sterility and endocrine
disorders;
determination of genetic sex;
detection of the presence of infectious agents
Specimens for Examination
-peritoneal, pericardial and pleural fluids
- CSF
- nipple discharge
-bronchial brushings/washings
- sputum
PREPARATION OF SMEARS
→ smears should be from fresh material
- gastric washings
-urine sediment
Barr Body
- prostatic secretions / fluid
- cervicovaginal (paps) smear
→ see requisition form (patient’s ID: name, age; date and type of specimen requested
Canada Balsam
Canadian Fir tree
1.524
xylene, toluene
DPX(1.52)
XAM
Ringing
Labelling
____
A branch of General Cytology which deals wit the microscopic study of that have been desquamated from the from the epithelial surfaces.
Recommended for:
- detection of malignant cells or cancerous conditions in the body;
- detection of asymptomatic or precancerous cervical lesions in women;
- assessment of female hormonal status in case of sterility and endocrine disorders;
- determination of genetic sex; Barr Body (inactivated X chromosome)
- detection of the presence of infectious agents
PREPARATION OF SMEARS:
→ smears should be from fresh material
→ see requisition form (patient’s ID: name, age; date and type of specimen requested
→ label the slide
Methods of Smear Preparation:
1. ___
2. ___
3. ___
4. ___
SPECIMENS THAT REQUIRE ADHESIVE AGENT
⚫ Urinary Sediment
*
Bronchial Lavage Specimen
* Specimens that utilizes proteolytic enzymes during processing (eg. Trypsin, conc. Sputum and enzymatic lavage sample from the GIT)
CHARACTERISTICS OF A GOOD ADHESIVE
1. must be permeable to both fixative and the stain
2. must not retain the stain
GOOD ADHESIVES FOR CYTOLOGIC METHOD
Pooled human sera or plasma
⚫
Celloidin Ether-Alcohol
*
Leuconostoc culture
APES (3-aminopropyltriethoxysilane)
FIXATION
* WHY FIX?
⚫ exfoliated cells decompose rapidly which may destroy cellular and nuclear details, in turn will give inadequate results for diagnosis.
Fixation Time:
COMMON FIXATIVES
1. equal parts of 95% EtOh and ether
2. 95% EtOH
3. Carnoy’s fluid
equal parts of tertiary butyl alcohol and 1 part 95% EtOH 5. SCHAUDINN’S FLUID sat. aq. HgCl, absolute HoAc 6. MeOH
7. Saccomano Preservative: (50% alcohol and carbowax) 8. Spray Fixatives
PAPANICOLAU SMEAR AND STAIN
Credited to:
⚫ Screening test for:
Uses
-
PAPS STAIN
Advantage:
Determination of etiology of certain infections (STD) Determination of ovarian function (Hormonal Cytology)
One test to assess in infertility
Evaluation of response of malignancy to post radiation or chemotherapy Barr Body Determination
Medico Legal Examination of Sexual Assault
-transparent blue staining of cytoplasm is observed
- excellent nuclear staining
color range is predictable and of great value in identification of cells
Disadvantage:
procedure is lengthy and complicated
does not give accurate acidophilic index
Stains for Pap’s:
1. Harris Hematoxylin:
2. OG 6 Stain: strong affinity for,
Orange Green 6, 0.5 solution in 95% ROH Phosphotungstic acid
3. EA 50: strong affinity for,
; stain keratin
light green SF, yellowish 0.1% solution in 95% ROH
Bismarck brown 0.5 in 95% ROH
Eosin Y, 0.5% in 95% ROH
EXFOLIATIVE CYTOLOGY
spreading
spreading
pull apart
touch or impression smear
SPECIMENS THAT REQUIRE ADHESIVE AGENT
- Urinary Sediment
- Bronchial Lavage Specimen
- Specimens that utilizes proteolytic enzymes during processing (eg. Trypsin, conc. Sputum and enzymatic lavage sample from the GIT)
CHARACTERISTICS OF A GOOD ADHESIVE
1. must be __ to both fixative and the stain
2. must not __ the stain
GOOD ADHESIVES FOR CYTOLOGIC METHOD: (4)
FIXATION:
-WHY FIX?
- exfoliated cells decompose rapidly which may destroy cellular and nuclear details, in turn will give inadequate results for diagnosis.
Fixation Time: ___
COMMON FIXATIVES
1. equal parts of 95% EtOh and ether - BEST
2. 95% EtOH
3. Carnoy’s fluid
4. equal parts of tertiary butyl alcohol and 1 part 95% EtOH
5. SCHAUDINN’S FLUID sat. aq. HgCl, absolute HoAc
6. MeOH - for blood smears
7. Saccomano Preservative: (50% alcohol and carbowax)
8. Spray Fixatives
permeable
retain
- Pooled human sera or plasma
- Celloidin Ether-Alcohol
- Leuconostoc culture
- APES (3-aminopropyltriethoxysilane)
at least 15 mins
PAPANICOLAU SMEAR AND STAIN
Credited to: ___
Screening test for: ___
Uses: (STD) Determination, ovarian function, Hormonal Cytology, infertility, response of malignancy, Barr Body Determination, Sexual Assault
PAPS STAIN
Advantage:
- transparent blue staining of cytoplasm is observed
- excellent nuclear staining
- color range is predictable and of great value in identification of cells
Disadvantage:
- procedure is lengthy and complicated
- does not give accurate acidophilic index
Stains for Pap’s:
1. ___: nuclear stain
2. ___: strong affinity for ___; stain keratin
→ Orange Green 6, 0.5 solution in 95% ROH
→ Phosphotungstic acid
3. ___: strong affinity for ___ (lower part of vagina)
→ light green SF, yellowish 0.1% solution in 95% ROH
→ Bismarck brown 0.5 in 95% ROH
→ Eosin Y, 0.5% in 95% ROH
→ Phosphotungstic acid
→ Lithium Carbonate. Sat. aq. Solution
- EA 50 is comparable to EA 36
- EA 65 differs from EA 50 or EA 36 only with respect to the concentration of the ___
Results:
Nucleus: vesicular nucleus - ___
pyknotic nucleus - __
Cytoplasm:
→ OG-6: Orange with a hint of green
→ EA 36 to 50: Olive Green with a hint of brown and red
bacteria - __
mycelia - __
Trichomonas vaginalis - pale greenish blue blob of cytoplasm PAP
George Nicolas Papanicolau
Cervical Cancer
Harris Hematoxylin
OG 6 Stain; mature cells
EA 50; immature cells
light green stock solution
blue
dark blue to black
dark blue
violet
GYNECOLOGIC SPECIMENS:
ANATOMIC SITES FOR Cytologic Samples:
1. ____ Ideal for studying the evaluation of inflammatory conditions, classification of the normal flora and rarely detection of malignant vaginal lesions
2. ____ Most common for cancer screening; Use of Ayre’s spatula (can reach the T-zone); T-zone = where most malignancy arise
- Histology: Stratified Squamous Non-Keratinizing
3. ____ For detection of endocervical lesions, intrauterine lesions
- Histology: Simple Columnar Epithelium
Cytologic Collection and Preparation:
- Materials Used for Conventional Paps Smear
Endocervical Brush - for endocervical canal
___ - for patients with hysterectomy
Lateral Vaginal Scrape - for ___
___ - for localization of vaginal adenosis
___ - for detection of herpetic lesions or carcinoma
Equipment for Vaginal, Endocervical and Endometrial Aspirations
- Glass Pipet and Rubber Bulb - ___; 6-8 inches x 4 inches
- ___ - swab smear
- Laryngeal Cannula attached to a 10cc syringe - endocervixal/endometrial aspiration
- Antiseptic Used: Zephiran (QUATS)
Methods for Pap’s Smear
Conventional Pap’s Smear
* Liquid Based Pap’s (Sure Path and Thin Prep) - use of spatula or brush/broom to collect sample. Placed in a vial containing preservative. A thin layer of cells is places on a slide
Upper (Proximal) Third of the Vaginal Wall
Ectocervix
Endocervix
Vaginal Scrape
hormonal cytology
Four Quadrant Vaginal Scrape
Vulvar Scrape
vaginal aspiration
Ayre’s Spatula
Methods for Pap’s Smear
○ Conventional Pap’s Smear
○ ___ (Sure Path and Thin Prep) - use of spatula or brush/broom to collect sample. Placed in a vial containing preservative. A thin layer of cells is places on a slide
___: Based on the specific response of the vaginal epithelium to steroid hormones
- ___: produced by proliferating granulose theca cells of the ovarian follicles; Acts upon the ___
- ___: produced by corpus luteum formed after ovulation; Acts upon the ___
SPECIMEN COLLECTION:
* Vaginal smears may be taken regularly and often.
* Hormonal changes are best mirrored in the __ of the vagina.
* They can also be taken from the lateral walls because their more accessible and less likely to be contaminated by cellular debris or discharge.
Liquid Based Pap’s
HORMONAL CYTOLOGY
ESTROGEN; superifical cells
PROGESTERONE; intermediate cells
upper third
CELLS IN VAGINAL SMEARS:
1. ___:
- Large (30-60u)
- Polyhedral flat cells
- Cytoplasm: may be acidophilic or basophilic
- Presence of small dark pyknotic nuclei (less than 6u)
- ___:
- medium large (20-30u); polyhedral or elongated
- Cytoplasm: basophilic with vacuoles
-Vesicular nuclei (6-9u)
→ ___: intermediate cells with a strong tendency to fold and curl their edges. Expression of the combined estrogen-progesterone effect found in the latter half of menstrual cycle, during pregnancy, menopause may also be found as a result of abnormal androgen stimulation, either endogenous or exogenous
→ ___: Round or oval to oval shaped cell; has a translucent basophilic cytoplasm; cytoplasm stains deep blue or blue green + cell membrane; Appearance: ___ - ___:
- Round to oval cells
- Smaller than intermediate (15-25 u)
- Thick
- Appearance: fried egg/sunny side up
- Have strong basophile cytoplasm and vesicular nucei (6-9 u)
- Found from 2 weeks of age to puberty, after childbirth, abortion or miscarriages and after menopause.
→ ___: small (13-20u); Round, slightly oval cells, with relatively large nucleus that occupying half or more of the cell volume Cytoplasm: strongly basophilic; Found in vaginal smears only before pregnancy and after menopause
CYTOHORMONAL SMEAR
Used to evaluate the hormonal status based on the distribution of the cells (superficials, intermediates, parabasals) CHMI: Cytohormonal Maturation Index (% per 100 cells)
CHMI = parabasals: intermediates: superficials
Results should be correlated with age, and LMP of patient
Examples:
0/10/90: ⚫0/70/30:. 100/0/0:
OTHER NORMAL CELLS THAT MAY BE SEEN IN A PAP SMEAR ENDOMETRIAL CELLS
Superficial Cells
Intermediate Cells
Navicular Cells
Pregnancy Cells; double wall
Parabasal Cells
Basal Cells
___:
- Used to evaluate the hormonal status based on the distribution of the cells (superficials, intermediates, parabasals)
- CHMI: Cytohormonal Maturation Index (% per 100 cells)
- CHMI = ___:____:___
Results should be correlated with age, and LMP of patient
Examples:
0/10/90: shift to the right; estrogen effect; menopausal who are taking estrogen
0/70/30: shift to the mid zone; progesterone; pregnancy
100/0/0: shift to the left; menopausel pre-menarche (no mens yet)
CYTOHORMONAL SMEAR
parabasals: intermediates: superficials
OTHER NORMAL CELLS THAT MAY BE SEEN IN A PAP SMEAR
____:
- Found during menstruation period (in groups) and 1-4 days after the cessation of the period (single)
- ___: seen in tight clusters of small, oval dark cells!!; Glandular cells: slightly larger!!.
- Nucleus: small and moderately dark
- Cytoplasm: basophilic and maybe vacuolated
- If seen in a post-menopausal woman indicate a possible endometrial carcinoma or endometrial hyperplasia
____:
- Slightly cylindrical appearance; columnar epithelial cells occurs in groups and strips of three or more cells
- Nuclei: basally oriented; vacuolated
- cytoplasm: deeply basophilic the that of the parabasal cells; mucin filled cytoplasm
- Denotes: satisfactory specimen collection
Characteristic “___” appearance
___ AKA ____:
- Gram + slender rod bacteria (cultured in tomato juice)
- Predominant organism of the vaginal normal flora: establishes the low pH that inhibits the growth of pathogens
- Stains ___ to ___
- Energy is obtained by the fermentation of glycogen derived from
disintegrating epithelial cells
- Numerous in the luteal phase and during pregnancy
___: normally increased just before, during and shortly after menstruation
___: seen during traumatic collection, menstruation; PAPS smear should be done 10days after her LMP
___: long thin filamentous bacilli; increased in high vaginal pH; indirectly tells possibility of infection
___: usual contaminant; ovoid bodies,
ENDOMETRIAL CELLS
Endometrial stromal cells
ENDOCERVICAL CELLS; honeycomb
DODERLEIN BACILLI; Lactobacillus acidophilus
pale blue to lavender
NEUTROPHILS
RBCs
Leptothrix sp.
Talcum
ABNORMAL CELLULAR COMPONENTS OF A PAPS SMEAR
___: budding yeast; seen among diabetics, taking oral contraceptives,
immunocompromised patients, prolonged steroid therapy
___: STD; pear-shaped organism; granular cytoplasm; eccentric dark nucleus
___: tiny pleomorphic coccobacilli that clings to cytoplasm of epithelial cells; Presence of ___
___: squamous epithelial cells that show cytopathic effects of HPV
→ Nucleus appears like a “wrinkled prune” that is
surrounded by a perinuclear halo
→ Presence of koilocyte is diagnosed as Low Grade Squamous Intraepithelial Lesion
___: cytopathic effect of cell is characterized as macrosomia, multinucleation, nuclear molding and ground glass chromatin pattern
Cancerous Lesions (Carcinomas)
Dysplastic Cells
Candida albicans
Trichomonas vaginalis
Gardnerella vaginalis; clue cells
Koilocytes
HSV II
FERNING (ARBORIZATION):
Mucus on drying exhibits a ____
Due to salt crystal formation (high NaCl concentration) under the influence of ___
(+) Ferning: High Persistent Estrogen Effect in the Absence of progesterone
Basis for the Diagnosis of ___
CRITERIA FOR CYTOLOGIC DIAGNOSIS OF NORMAL PREGNANCY
- Marked Progesterone Effect
- At least 50% of intermediate cells in clusters
- Presence of Typical Pregnancy Cells (3-7 cells)
- Less than 30% Superficial Cells
- Presence of Doderlein Bacilli
fern/palm leaf pattern
estrogen
Early Pregnancy
QUANTITATION IN VAGINAL CYTOLOGY
___ - percentage of cells that stain pink-orange to red with Pap’s and red in Shorr method
___ - “karyo-pyknotic index”; percentage of cells having shrunken, dark, small (less than 6μ) structures nuclei.
___ - percentage proportion of cells from the three layers of the vaginal epithelium.
MANNER OF REPORTING PAPS SMEARS
Two Systems:
Bethesda System
1. Class System (No Longer Used; Last used December 1988)
- Based on: presence of malignant cells
- No classes for non-cancerous entities
- Not reproducible
- Obsolete
Class System:
Class I: Negative for Malignant cells
Class II: Atypical cells present, but __
Class III: ___
Class IV: Strongly Suggestive for Malignant Cells
Class V: ___
- Bethesda System: developed at National Cancer Institute in December 1988
- Report Format
Specimen Adequacy:
○ Satisfactory
○ Limited
○ Unsatisfactory
General Categorization:
○ Negative for Intraepithelial lesion or malignant cell
○ Epithelial cell abnormality
○ Benign Cellular Changes
Descriptive Diagnosis:!!!
○ Infections/Radiation effects
○ ASCUS?
○ LGSIL?
○ HGSIL?
○ Squamous Cell Carcinoma
○ Glandular cell abnormality
○ Atypical glandular cells
○ Adenocarcinoma
○ Others
~ 3rd reworking of the Pap Smear Classification system
ACIDOPHILIC INDEX (A.I.)
PYKONTIC INDEX (P.I)
MATURATION INDEX (M.I.)
Negative for Malignancy
Suspicious for Malignant Cells
Conclusive for malignant Cells
Atypical squamous cells of unknown significance
Low grade squamous intraepithelial lesion
High grade squamous intraepithelial lesion
NON GYNELOGIC SPECIMENS:
For Fluid Specimens, 3 preparations can be made:
a. Smears (__)
b. Cell Block - formalin (fixative)
c. Cytospin-Centrifuge 1000rpm for 1minute; best for ___
____
○ Can be prepared from a wide variety of cytology specimens
○ Neutral Buffered Formalin - universal fixative
→ Other fixatives - Formalin/95% ethanol rinse, Nathan Alcohol formalin substitute (less toxic), methanol (the automated Cellient cell block technique)
Methods of Preparation
1. ___
- Centrifuge the fluid, then obtain sediment and add 10% formalin
- Disadvantage: Easily Washed Out
2. ___ - most popular
- Centrifuge, then obtain the sediment, add 1ml of plasma and add thrombin
- This will form a gel like substance; place in a filter paper and add 10
- Advantage: Minimizes washing out, but expensive
3. ___
-centrifuge, obtain sediment and add carbowax; place in a paraffin block
- advantage: no dehydration step!!!; but expensive
2 smears usually
body fluids
Cell BLOCK
Direct Filtration
Plasma Thrombin
Carbowax Method
Other methods of cell blocking:
○ ___ - express the contents of the aspirate onto a glass slide. Allow to dry/clot. Scrape the material and wrap in tissue paper. Process as routine tissue.
○ BBC Cell Block Fixative Method
○ Collodion Bag Cell Block
○ Shandon Cytoblock
○ Cellient Automated Cell Block System
○ Histogel
Fluid Specimens:
1. ____: usually used to rule out malignancies or infectious agents such as pneumocystis (in pneumonia px)
- Specimens: Bronchoalveolar Lavage/ Bronchial Washings and Bronchial Brushings; sputum
- Manner of Collection of Sputum: ___
- Presence of ___ indicates satisfactory sputum collection
- Fixatives that can be used: Sputum (Saccomano Fluid); Bronchial brushing (Spray Fixative or 95% Ethanol)
- ___ should be made for each patient
- ____: quite difficult to prepare; specimens should be sent to laboratory immediately; no delay beyond 30mins
- ____ is required for gastric washing - ____: used for detection of presence of malignant cells; Jelly like clots can be prevented by the addition of 300 units of ___ per 100ml aspirate
- ____: discharges from the nipple except during lactation and post lactation periods are abnormal; and may be due to benign breast lesions such as ducta ectasia and papilloma or endocrine problems.
- Smear Preparation using ___ technique; fix using spray fixative or 95% isopropanol - ____: Specimens: Voided Urine
- ___ - for women
- Washings from bladder or renal pelvis: for urine cytology (dont use 1st morning urine) - ____: used for malignancies
- include CSF, pleural fluid, ascetic fluid, pericardial fluids
- specimens should be submitted fresh ; formalin or alcohol should not be used; anticoagulants such as heparin may be used;
- CSF specimens = _ minimum amount.
Clot and Scrape method
Respiratory Tract Specimens
deep cough early in the morning
alveolar macrophage
2 Smears
Gastric Secretions and Aspirates
8hour fasting
Peritoneal, Pleural and Pericardial Fluids
Heparin
Breast Secretion
Pull up
Urinary Tract Specimens
Catheterized Specimen
Body Cavity Effusions
1ml
GENERAL PATHOLOGY
Review of Basic Parts of Cell
FOUR MAIN TISSUE TYPES:
A. ___ -derived from the 3 germ layers
B. ___- derived from mesoderm
C. ___- derived from mesoderm
D. ___- derived from ectoderm
___
- Covers and lines surfaces
- Cells are close to each other
→ membranes or sheets of cells, Glands (endocrine or excocrine)
- Cells are attached to a basement membrane
- Apical Surface: free surface of epithelial tissues
→ Apical Specializations Present:
1. ___ - motile
2. ___- non-motile (for absorption); seen in epididymis and vas deferens
3. ___ - non-motile; “brush border”
- avascular
EPITHELIAL
CONNECTIVE
MUSCULAR
NERVOUS
Epithelial Tissues
cilia
stereocilia
microvilli
CLASSIFICATION OF EPITHELIALTISSUES:
○ cell shape (columnar, cuboidal, squamous)
○ arrangement (simple, stratified, pseudostratified)
○ Examples:
◘ ___ - ex. Bowman’s capsule, endothelium, Loop of Henle, lung alveoli Simple
◘ ___ - ducts of glands, walls of thyroid follicles, kidney tubules
◘ ___ - gall bladder (non-ciliated), uterine tube (ciliated)
◘ ___ - skin (keratinized), vagina, esophagus, cervix
◘ ___ - ducts of sweat glands
◘ ___- male urethra
◘ ___- urinary bladder
◘ ___ - trachea (ciliated), epididymis
Glands:
○ exocrine
→ tubular:
♦ simple tubular: ex. sweat glands, pyloric glands
♦ compound tubular: ex. Brunner glands
→ acinar:
♦ simple: ex Littre glands
♦ compound: ex. submandibular glands
○ endocrine
methods of secretion:
1. ___ - no loss of cytoplasm ex. goblet cells and sweat glands
2. ___ - cytoplasmic loss ex. mamary glands
3. ___ - complete breakdown of the cell; ex. Sebaceous glands
Simple Squamous
Cuboidal
Simple Columnar
Stratified Squamous
Stratified Cuboidal
Stratifed Columnar
Transitional
Pseudotrafied columnar
merocrine
apocrine
holocrine
_____
- most abundant tissue
- vary in appearance adn functions
- main functions: BIND, PROTECT AND SUPPORT
COMPONENTS:
Cells: fixed or wandering
○ fixed - fibroblast and adipocytes
○ wandering - WBC, RBC & macrophage
Fibers: produced by fibroblasts
○ collagen - most abundant
○ elastic - stretch and recoil
○ reticular - “argyrophilic” silver stains
CONNECTIVE TISSUES
Muscular Tissue
- highly specialized
- Function: Movement
3 Types:
○ ____:
a. Skeletal Muscle - cylindrical, elongated, non-branching, multi-nucleated (periphery), vlountary muscle, undergoes mitosis and regeneration
b. Cardiac Muscle - branching, 1-2 nuclei, involuntary, no mitosis, permanent cells, has intercalated disks
○ ___
- non-striated, spindle, tapered, 1 nucleus, involuntary, mitosis, regenerates
○ ___
* FUNCTION: RAPID COMMINICATION
Cell Types:
➤ ___: receives stimuli; conducts waves of excitability; permanent cells
➤ ___: nerve glue; more in number than above; tumor in this cell is called __
→ Function: support, protection, insulation;
Striated Muscle
Visceral/Smooth Muscle
Nervous Tissue
Neurons
Neuroglia; glioma
INTRODUCTION TO PATHOLOGY
Etymology: pathos - pain; logos - study
Also called ___.
* Bridges discipline of basic science and clinical practice
* Study of the structural and functional changes in cells, tissues, and organs that underlie disease What can we learn from pathology?
* Etiology, Disease Process, Morphologic Changes, Clinical
DIVISIONS OF PATHOLOGY (3)
CELLULAR GROWTH AND DIFFERENTIATION:
- Organs of the body possess tissue reserves which could be brought into action when needed
- These reserve tissues are maintained by intermittent blood flow
- Aside from these, cells can grow and differ if subjected to excess strain
3 CLASSES OF CELLS
a. ___: frequent division to replace lost cells
b. ___: definite pattern of replication with cells lost by wear and tear being replaced by the mitotic activity of others.
c. ___: non-replicating (ie. cardiac, neurons)
pathobiology
A. GROSS AND MICROSCOPIC PATHOLOGY
B. ANATOMIC PATHOLOGY
C. CLINICAL PATHOLOGY
LABILE CELLS
STABLE CELLS
PERMANENT CELLS
~ Abnormalities can occur during cell growth and differentiation and these are an important part of many disease processes
ABNORMALITIES OF CELL GROWTH:
___:
➤ incomplete/defective tissue development; seen commonly in kidneys, gonads and adrenals
___:
➤ non appearance of organs
___:
➤ failure of an organ to reach or achieve its full mature or adult size
___:
➤ failure of an organ to form an opening
Cell Adaptation:
- Cells may undergo various adaptations in certain physiologic and pathologic conditions.
- Controlled by complex mechanisms
Aplasia
Agenesia
Hypoplasia
Atresia
___: Shrinkage in the size of the cell by loss of cell substance
- Characteristic feature: Autophagic Granules (intracytoplasmic vacuoles containing debris from degraded organelles)
Classified as:
→ __- due to decreased work load (e.g., decreased size of uterus following child birth), reduction of lymphoid tissue
→ __- primarily due to denervation of muscle, diminished blood supply, nutritional deficiency, old age, disuse, some are idiopathic
Atrophy
Physiologic
Pathologic
___: increase in the size which results in an increase in the size of the organs just LARGER ones
- NO NEW cells (no mitosis)
→ Synthesis of more structural components
→ Can be physiologic or pathologic
- Cause by increased functional demand or specific hormonal stimulation
- Can be seen in skeletal and heart muscles
- Can be reverted if the cause is removed
→ Physiologic: exercise
→ Pathologic: myocardial hypertrophy
Hypertrophy
___: Increase in the number of cells in an organ or tissue, leading to increased organ or tissue size
- There is mitosis /cell division
- Occurs if the cellular population is capable of synthesizing DNA, permitting mitotic division
Hyperplasia
___: Change of one cell type to another; Reversible
- Adaptation to abnormal localized environmental changes or to new
functional demands.
- Examples:
→ Columnar to squamous: most common seen in smokers
→ squamous to columnar (___)
→ Mesenchymal tissue (e.g., formation of bone in skeletal muscle).
Metaplasia
Barrel’s esophagus
___: AKA:___
- abnormal growth and differentiation
- Variation of size, shape and orientation
- a preneoplastic lesion (a stage in the cellular evolution to cancer)
- May lead to cancer but not necessarily
Dysplasia; atypical hyperplasia
___: ΑΚΑ:___
- Cellular or tissue change from more to a less differentiated form.
- More primitive and embryonic looking
- Exhibits pleomorphism, hyperchromatism, increased N:C ratio, prominent nucleoli, abnormal mitoses, cellular dyspolarity
- IRREVERSIBLE!!!
Anaplasia; undifferentiated cell
___: limits of adaptive response are exceeded, or when the cell is exposed to an injurious agent or stress
- The affected cells may recover from the injury (reversible) or may die (irreversible).
Causes :
1. oxygen deprivation (anoxia) (ex. Ischemia, Anemia, CO poisoning,
Decreased perfusion of tissues by oxygen carrying blood, Poor oxygenation of blood)
2. physical agents
3. chemical agents
4. infectious agents
5. immunologic reactions
6. genetic defects
7. nutritional imbalances
- NOTE: Hypoxic injury can be irreversible after:
→ 3-5 minutes for ___
→ __ for myocardial cells and hepatocytes
→ Many hours for __
Cell Injury
Neurons
1-2 hours
skeletal muscle
CELL INJURY
Morphology:
I. ___
Patterns of Cell Injury:
a. Microscopic Changes
b. Ultrastructural Changes
c. Fatty changes
d. Accumulation of exogenous pigments
e. Accumulation of endogenous pigments
- ___ - yellowish fat soluble pigment and product of membrane peroxidation Also known as wear and tear pigment; Seen in elderly patients (hepatocytes and poles of nuclei of myocardial cells)
f. Pathologic Calcification
g. dystrophic
Reversible
Lipofucsin
CELL INJURY
II. ___
> leading to CELL DEATH
- enzymatic digestion of dead cellular elements, denaturation of proteins and autolysis (by lysosomal enzymes)
- Cytoplasm
→ Larger Cells “__”
→ increased eosinophilia
- Nucleus
→ Nonspecific breakdown of DNA
> pyknosis - ___
> karyolysis - ___
> karyorrhexis - __
Irreversible/Necrosis
Cloudy Swelling
shrinkage
fading
fragmentation
Cell Death:
Two Patterns of Cell Death:
A. ___ (irreversible injury)
- changes produced by enzymatic digestion of dead cellular elements
- Protein denaturation
Patterns of Necrosis In Tissues or Organs
1. ___: the outline of the dead cells are maintained and the tissue is somewhat firm.
- Cell death due to ischemia; Hallmark Appearance: ___ Appears eosinophilia like cell (in H and E); anucleated
- Ex. myocardial infarction; also involves kidney, adrenal glands, spleen;
- Note: This type of necrosis does not occur in the ___.
- ___: autolysis or heterolysis; liquified; Complete destruction of cells
- Ex. cerebral infarction - ___: “cheesy & white appearance”; usually seen in tuberculosis, tlaremia, LGV
- Microscopically it appears as an amorphous eosinophilic appearance - ___: description of focal areas of fat destruction due to release of pancreatic lipases
> Appearance: ___
> Ex. pancreatitis - ___: necrosis (secondary to ischemia) usually with superimposed; eg. necrosis of distal limbs
○ Types of Gangrene
→ ___: caused by arterial occlusion; Ex. Embolism of foot
→ ___: result of venous occlusion; Ex. Bacterial infection
Necrosis
Coagulative
tombstone; brain
Liquefactive necrosis
Caseous necrosis
Fat necrosis
“chalky white”
Gangrenous necrosis
Dry gangrene
Wet gangrene
B. ___: programmed cell death;
- vital process that helps eliminate unwanted cells
- an internally programmed series of events
- unlike Necrosis, there is ___
Chief Morphologic Features: (5)
Apoptosis
no inflammatory reaction
○ Chromatin Condensation
○ Chromatin Fragmentation
○ Cell Shrinkage
○ Cytoplasmic Bleb Formation
○ Phagocytosis of Apoptotic Cells
___: Death of an organism as a WHOLE
___: “-itis”
* A complex reaction to various injurious agents
* Consists of vascular responses, migration and activation of leukocytes, and systemic reactions
* Double Edged Sword
* A protective response
- Ultimate goal:
→ Remove initial cause of injury
→ Remove consequences of injury
* Important in tissue repair
- Destroy, dilute, wall of infectious process
- Sets in motion tissue repair
→ Regeneration
→ scarring
* Inflammation is terminated when the inciting agent is eliminated
and the mediators have degenerated.
Cardinal Signs of Inflammation:
Named by ____
Common Causes:
- Infection, trauma, injury due to thermal extremes or from ionizing radiation, chemical injury, immunologic injury, tissue death
SOMATIC DEATH
Inflammation
Celsus (1st 4) and virchow (5th)
___:
- A rapid response to an injurious agent that aims to rapidly bring mediators of inflammation to the site of injury Alterations in blood flow
Hallmark Sign: increased vascular permeability
Infiltration by: polymorphonuclear cells (neutrophils)
Cellular response of leukocytes:
- __: passage of inflammatory leukocytes between endothelial cells into adjacent interstitial tissue
- Chemotaxis
- Phagocytosis
- Opsonization
- Intracellular microbial killing
~ May progress to Chronic Inflammation if it fails to subside in the course of several weeks
___: Refers to an excess fluid in the interstitial tissue or serous cavities
___: The escape of fluid, proteins, and blood cells from the vascular system into interstitial tissue or body cavities
- Two types
1. __:
Pus: exudate rich in WBC and cellular debris
2. __:
Outcomes of Acute Inflammation:
- Resolution of tissue structure and function
- Tissue Destruction and Persistent Acute Inflammation
→ Abscess - cavity filled with pus
→ Ulcer-loss of cellular epithelium
→ Fistula - abnormal communication between organs or between an organ and a surface
→ Scar
- Conversion to Chronic Inflammation
Acute Inflammation
Emigration
Edema
Exudation
Exudate
Transudate
___:
- An inflammation of prolonged duration
- Cellular Morphology of Chronic Inflammation
1. Infiltration of mononuclear cells (macrophages, lymphocytes, plasma cells)
2. Tissue destruction
3. Attempts at healing by CT replacement (angiogenesis and fibrosis)
___:
○ A distinctive pattern of chronic inflammation
○ Characterized by formation of granulomas
○ Granuloma
→ Focal aggregation of activated macrophages which are transformed in an epithelial-like (epithelioid) cells, have an abundant pink cytoplasm, and are surrounded by numerous lymphocytes (on the rim) and plasma cells
○ Presence of multinucleated giant cells (ex. Langhan’s Giant Cell, foreign giant cell)
○ Seen in: TB, leprosy, schistosomiasis, syphilis, bartonella, henselae
___- resolution of inflammation
○ ___
→ No destruction of normal tissue
→ Offending agent is neutralized
→ Vessels return to their normal permeability state
→ Excess fluid is reabsorbed
→ Clearance of mediators and inflammatory cells
○ ___
→ Replacement of lost or necrotic tissue with a new tissue that is structurally and functionally similar to those that were destroyed
→ The intact, healthy neighboring cells surrounding the dead cells will proliferate to replace the affected cells
○ Replacement by a CT scar
Chronic Inflammation
Granulomatous Inflammation
Healing
Simple resolution
Regeneration
___
- Greek Word neo = “new”; plasma = “creation”
- Means the process of “new growth”
- A new growth is called a neoplasm, commonly known as tumor (swelling)
- ___- the study of tumors or neoplasms
- ___ - common term for all malignant tumors (Latin for crab)
Neoplasm
- Abnormal mass of tissue
- Growth exceeds and uncoordinated with that of the normal tissues
- Behave like parasites and compete with normal cells and tissues for metabolic needs
- Persists in the same excessive manner after cessation of stimuli which evoked the change
- Persistence results from heritable genetic alterations that are passed down to the progeny of the tumor cells
Characteristics of Tumors
1. ___: proliferating neoplastic cell
2. ___: support, nutrition, made up of blood vessel & connective tissue
○ ___: suffix-oma
> tumor is localized and doesn’t metastasize
○ ___: “cancer”
> invasive and destroys adjacent areas
>Anaplastic: malignant neoplasms that are undifferentiated; pleomorphic
NEOPLASIA
Oncology
Cancer
PARENCHYMA
SUPPORTIVE STROMA
Benign
Malignant
Nomenclature of Neoplasms:
Benign tumors - attach suffix-oma to the cell of origin
○ MESENCHYMAL CELLS
→ Fibroblasts - ___
→ Chondroblasts - ___
→ Osteoblasts - ___
→ Lipoblasts - ___
EPITHELIAL CELLS
○ Glandular pattern - ___(glands)
○ Finger-like or warty projections - ___
○ Large cystic mass - ___
fibroma
chondroma
osteoma
lipoma
adenoma
papilloma
cystadenoma
Malignant tumors
MESENCHYMAL TUMORS - __
- Fibrosarcoma
- Chondrosarcoma
- Osteosarcoma
- Leukemias / Lymphomas
EPITHELIAL TUMORS - ___
- Adenocarcinoma
- Squamous cell carcinoma
Mixed tumors
Teratomas
___
- Extent to which neoplastic cells are comparable to normal cells, both morphologically and functionally
___
- Tumor implants discontinuous with the primary tumor
- most reliable feature of malignancy
- Cancer cells penetrate into blood vessels, lymphatics and body cavities providing opportunity for spread
- All neoplasms metastasize EXCEPT: ___ and ___
Manner of Dissemination of Malignant Neoplasms:
- Seeding within body cavities: neoplasm penetrates into a “natural field”. Most often in the peritoneal cavity
- Lymphatic spread: MOST COMMON PATHWAY for ___
- Hematogenous spread: MOST COMMON PATHWAY for ___
> Liver and Lungs: frequently involved
sarcomas
carcinomas
Differentiation
Metastasis
GLIAL CELLS AND BASAL CELL CARCINOMA
carcinomas
sarcomas
Grading and Staging of Tumors:
____: Based on the degree of differentiation of tumor cells and the number of mitoses
- Well-differentiated tumors as a rule are less malignant than undifferentiated tumors.
- Differentiated cells resembling normal cells.
- Undifferentiated cells-younger forms (anaplastic)
___: Differentiated Cells; Undifferentiated Cells
Grade I:
Grade II:
Grade III:
Grade IV:
Value of Grading:
○ Guide for Treatment: ↓ grade - surgery; ↑ grade - radiation
○ Prognostic Guide: ↓ grade - better; ↑ grade - poorer prognosis
___
- Based on the size of the primary lesion, extent of spread to regional lymph nodes, presence or absence of metastases
- The most common systems for staging employs the TNM classification.
- A “__” score is based upon the size and/or extent of invasion. (1-4)
- The “N” score indicates the extent of __ involvement. (0-3)
- The “M” score indicates whether ___ are present. (0-1)
Grading
Broder’s Classification
diff: 100-75%; undif: 0-25%
diff: 75-50%; undif: 25-50%
diff: 50-25%; undif: 50-75%
diff: 25-0%; undif: 75-100%
Staging
T
lymph node
distant metastases
BIOPSY
* Biopsy (in Greek: bios = life and opsy = look/appearance)
* Excision and Examination of Adequate Tissue Sample coming from a living person
* Use: Diagnosis of Malignant Disease
Types of Biopsy:
* ____: Pap’s smear; desquamated cells
* ____:complete removal of tissue lesion; Most Reliable Biopsy
- Allows examination the whole tumor lesion
* ____: partial removal of tissue lesion; Preferred for large tumors that cannot be excised completely
* ____:fluid aspiration of cells; Simple, Inexpensive, No need for hospitalization; least invasive test Ex. FNAB, BM Aspiration
- ____: Incisional Biopsy; Small pieces of tumor tissue are removed by special forceps; Ex. Endoscopic Biopsy
- ____: For skin biopsies (Dx of melanoma)
- ____: obtaining a deeper skin sample that removes a short cylinder or “apple core” of tissue; full thickness skin specimens
- ____: This type of biopsy involves removing the top layers of skin by shaving it off. Shave biopsies are also performed with a local anesthetic.Incision biopsy from solid organs Wedge Shaped
- ____: Incision biopsy from solid organs; Wedge Shaped
AUTOPSY
Etymology: “auto” “opsis”
- Meaning=
Other terms:
Systematic examination of a cadaver for study or for determining the cause of death.
Prosector: Diener:
Giovanni Battista Morgagni-father of Anatomical Pathology
The first law authorizing human dissection (1231) is credited to Frederick II (1194-1250), Holy Roman Emperor.
PURPOSE OF AUTOPSY
1. Determine the etiology or cause of death of a patient
2. Determine the pathogenesis
3. Preservation of tissues of the dead person for further examinations and for further research
4. Improvement of safety standards of living
An autopsy is done as soon as possible in order to prevent various post-mortem changes that can occur in the dead bod
Exfoliative Biopsy
Excisional Biopsy
Incisional Biopsy
Needle Biopsy
Bite Biopsy
Cutaneous Biopsy
Punch Biopsy
Shave Biopsy
Wedge Biopsy
- examination of the organs of the three major cavities of the body (3)
- spinal cord is not removed and examined
- blood vessels of arms and legs are also not examined
COMPLETE AUTOPSY
- abdomen
- chest
- head
Other Related Notes:
Histopathology Reports
○ Surgical Pathology
○ Cytopathology
○ Autopsy Report
Number of Copies = __ (4 copies of death certificate = 1 for PSA)
- ___ - Signs the Request Form
- ___- Signs the Pathology Result Forms
Retention of Pathology Reports (Henry)
○ Clinical Pathology Lab Reports - ______
○ Autopsy Forensic Reports -______
○ Surgical Pathology (and BM) Reports- ______
○ Cytogenetics Reports -______
Specimens
○ Pathology Tissue Blocks- ______
○ Serum/Body Fluids - ______
○ Slides-______
Turn-Over of Results:
○ Surgical Pathology and Cytology-______
○ Frozen Sections-______
○ Autopsy Report-______
3
Attending Physician
Pathologist
3 years
indefinite
10 yrs
20 yrs
10 yrs
2 days
indefinite
2 days
5-15 mins
7 days
Average Weights of Organs (Autopsy Manual - Navy Medicine)
___= 1400g (Males); 1275g (Females)
___ = 300g (Male) 250g (Female)
___ = 375g
___ = 450g
___= 1650g
-Pancreas-110g
Kidneys-__ (Males) ___ (Females)
Thyroid - __
Adrenal - 6g
Brain
Heart
Left Lung
Right Lung
Liver
313g; 288g
40g
