[M] Week 10: Principles of Microtomy; Paraffin and Frozen Sections, Staining (H&E), Adhesives and Mounting - Part 1 Flashcards
- This is done after embedding.
- the means by which tissue is sectioned and attached to the surface of a glass slide for further microscopic examination.
- Most is performed on paraffin wax-embedded tissue blocks.
Microtomy
What is the instrument used to cut sections for microtomy?
Microtome
- This has an advancing mechanism which moves the object, the paraffin block, for a predetermined distance until it is in contact with the cutting tool, the knife or blade.
- The specimen moves vertically past this cutting surface and a tissue section is produced.
Microtome
Enumerate the Types of MICROTOME
- Rotary Microtome
- Base Sledge Microtome
- Rotary Rocking Microtome
- Freezing Microtome
- Sliding Microtome
- Ultra Microtome
MICROTOME
- This is often referred to as the “Minot” after its inventor.
- The basic mechanism requires the rotation of a fine advance hand-wheel by 360° degrees, moving the specimen vertically past the cutting surface and returning it to the starting position.
Rotary microtome
MICROTOME
The rotary microtome may be
1. completely manipulated by the operator
2. when two motors drive the fine and the coarse advance hand-wheel.
3. with one motor to advance either the fine or coarse handwheel
- Manual
- Fully Automated
- Semi-automated
MICROTOME
Rotary Microtome advantages include the ability to cut thin ____ sections and its easy adaption to all types of tissue sectioning, including those that are hard, fragile, or fatty.
2–3 μm
MICROTOME
- Here the specimen is held stationary and the knife slides across the top of it during sectioning.
- Used primarily for large blocks, hard tissues or whole mounts
Base sledge microtome
MICROTOME
Base sledge microtome are especially useful in what pathology
neurological and ophthalmic pathology
What are the disadvantage base sledge microtome
3 μm sections are difficult to produce
MICROTOME
Commonly used in cryostats, the retracting action moves the tissue block away from the knife on the upstroke, producing a flat face to the tissue block.
Rotary rocking microtome
MICROTOME
- Freezes the specimen to harden it so it can be sliced without ripping and destroying it.
- Utilizes carbon dioxide (CO2) method.
Freezing microtome
MICROTOME
- The knife or blade is stationary and the specimen slides under it during sectioning.
- This microtome was developed for use with celloidin-embedded tissue blocks used primarily for research.
- Extremely dangerous because of the blade.
Sliding microtome
MICROTOME
- Used exclusively for electron microscopy
- A microscope is present in this type of microtome. This microscope is used to view the tissue sections.
Ultra-microtome
MICROTOME
TOF
Knives were developed to fit on ur ass but not to specific types of microtome and to cope with different degrees of hardness of tissues and embedding media.
False
pag nag true ka talaga….
MICROTOME KNIVES
Most steel knives have been replaced with disposable blades, although exceptions include the?
tool-edge knive for resin and steel knives for some cryostats.
- used for routine microtomy and cryotomy.
- It provides a sharp cutting edge which can produce almost flawless 2–4 μm sections.
Disposable Stainless-Steel Blades
Disposable blade holders are incorporated into the?
microtome or an adaptor may be purchased.
The blades may be purchased in dispensers, with or without a polytetrafluoroethylene (PTFE) coating which allows ribbons to be sectioned with ease
MICROTOME KNIVES
used in electron microscopy and with plastic resin-embedded blocks
GLASS AND DIAMOND KNIVES
What are the instrument used for paraffin section cutting
- Floatation (water) bath
- Slide drying oven or hot plate
- Fine pointed or curved forceps
- Scalpel
- Slide rack
- Clean slides
- Teasing needle
- Ice tray
- Chemical-resistant pencil or pen
PARAFFIN SECTION CUTTING
A THERMOSTATICALLY CONTROLLED water bath is used for floating out tissue ribbons after sectioning
Floatatation (water) bath
FLOATATION (WATER) BATH
- The temperature of the water in the bath should be maintained at below ____ the melting point of the paraffin wax to be sectioned.
- Care should be taken to prevent ____ ____ from being trapped under the section and this can be accomplished by using distilled water in the bath.
- Floatation bath can be ____ or ___
- 10°C
- Water bubbles
- circular or rectangular
FLOATATION (WATER) BATH
This is arrded to the water to reduce the surface tension allowing the section to flatten out with ease.
Alcohol or a small drop of detergent
DRYING OVEN OR HOT PLATE
Drying ovens incorporate aircon which keep the warm air circulating around the slides. This removes the excess paraffin
False
Fan lang
DRYING OVEN OR HOT PLATE
TOF
The temperature setting should be approximately that of the melting point of the paraffin wax
True
DRYING OVEN OR HOT PLATE
What happen if the oven is too hot?
may be distortion to the cells causing dark pyknotic nuclei, nuclear bubbling and loss of nuclear detail.
DRYING OVEN OR HOT PLATE
Special care must be taken when drying delicate tissues such as lymph nodes or tissues from the central nervous system; what temperature is needed?
37oC for 24 hours is required to prevent splitting or cracking of the sections.
- These or teasing needles are helpful in removing folds, creases and bubbles which may form during floating out of the section on the water bath.
o Applicator stick may also be used. - They are also helpful for manipulating the section as it passes across the edge of the blade.
BRUSH AND FORCEPS
SLIDES
- For normal routine work, ____ slides are universally used.
- Although slides are available in a variety of thicknesses, ____ thickness are preferred because they do not break easily. Most slide racks are made to accommodate this slide size.
- 76 × 25 mm
- 1.0 to 1.2 mm
SLIDES
TOF
* Smaller slides are available for use with specialty tissues such as eyes or brains.
* Unique identification numbers or codes, patient name or other information should be etched, embossed or written on each slide. Automated instruments which imprint the patient’s information on the glass slide are available. (Chemical-resistant pens and pencils are routinely used to label the slide.)
- False (larger)
- True
- Slides which are ____ or ____ resist detachment of the tissue from the slide during staining.
- ____ may be used for specialized techniques (e.g., decalcification, special stains,
immunohistochemistry, etc.).
- POSITIVELY CHARGED (ELECTRONICALLY) or pretreated with an adhesive (chemically coated)
- Colored, frost-ended slides
Providing clean slides are used and sections are adequately dried, the problem of sections detaching from the slide during staining should not occur. Occasions when sections may
detach from the slide are?
- Exposure to strong alkali solutions during staining
- Cryostat sections for immunofluorescence, immunohistochemistry or intraoperative consultation
- Central Nervous System (CNS) tissues
- Sections which are submitted to extreme temperatures
- Tissues containing blood and mucus
- Decalcified tissues
Adhesives
Adhesives may alleviate the problem of?
tissue loss
Adhesives
what are the protein adhesives? what are their disadvantages
- Albumin
- Gelatin
- Starch
Disadvantage: prone to bacterial growth or heavy staining but close monitoring will prevent these problems
What are the adhesives?
- Poly-L-Lysine (PLL)
- Silanized 3 aminopropyltriethoxysilane (APES/APS)
- Charged or Plus Slides
SECTION ADHESIVES
- This is bought as a 0.1% solution which is further diluted for use 1:10 with distilled water.
○ Slides are coated with the diluted solution and allowed to dry. - The effectiveness of the coating to adhere the tissue to the slide will diminish within a few days.
Poly-L-Lysine (PLL)
SECTION ADHESIVES
- Slides are dipped in a 2% solution of APES in acetone, drained, dipped in acetone and drained again.
- The process is complete when the slides are dipped in distilled water and placed upright in a rack to dry.
- These slides are useful for cytology and specimens which may be bloody or contain proteinaceous material.
Silanized 3-aminopropyltriethoxysilane (APES/APS)
- Laboratories often use slides which have been manufactured with a ____ ____.
- Placing a positive charge on the slides is accomplished by coating the slide with a ____ ____ in which a chemical reaction occurs, leaving the amino groups linked by covalent bonds to the silicon atoms of the glass.
- These slides are superior in their ____ and ____ during staining or pretreatments such as enzyme and antigen retrieval.
- Permanent positive charge
- Basic polymer
- resistance to cell and tissue loss
PRACTICAL MICROTOMY
TOF
Expertise to become competent microtomist is obtained by practical experience under the guidance of a skilled tumor, gaining the confidence and coordination to manipulate the microtome and the \ sections produced.
FALSE
not tumor, MENTOR
adenoma kaba gurl
- Setup of the microtome. Maintenance by closely following proper care with daily, weekly, quarterly and annual preventive maintenance.
- Water bath and microtome should be ergonomically positioned to reduce stress and tension on the neck and shoulders.
- Blade should be sharp and defect free
Practical microtomy
Sectioning
Trimming the tissue blocks-paraffin block may be faced or “rough cut” by
setting the micrometer at ____ or by advancing the block using the coarse feed mechanism. ‘moth-holes’ artifact may be formed with aggressive trimming as well as damage to tissue by gouging or scoring.
15-30mm
Sectioning
Cutting sections-blocks should be arranged in ____ ____ on a cooling device, cooling both tissue and paraffin giving them a similar consistency. Routine surgical materials should be cut at ____. Ribbons of sections are the most convenient way of handling sections
- numerical order
- 3-4microns
Sectioning
- Floating out sections-must be smooth with the ____ of the ribbon making contact with the water first.
- Folds may be removed by simply teasing with the forceps. (TOF)
- Approximately ____ should be long enough for a ribbon to flatten.
- Circular structures like eyes are difficult to flatten, can be placed on slides pre-flooded with ____
- trailing end
- True
- 30 seconds
- 50% alcohol
- Further flatten tissues
- Remove paraffin before staining
- Use of drying ovens in automated stainers or hot plate
- Hot plate may cause localized overheating of slides
Drying sections
Cutting hard sections
- Less problematic since the introduction of ____ ____
- Cutting difficulties is more likely ____ or ____
- Prolonged soaking of the block, or exposing the block surface to running tap water for ____ minutes, often overcome many of the problems associated with cutting hard tissues.
- Slight reduction in knife slant or application of ____ ____ on the surface of the block may yield results
- Disposable Blades
- Poor fixation or overprocessing
- 30 min
- Softening agents
This is a discussion of the methods used to produce sections which preserve cellular morphology without the use of dehydrating and clearing solutions and heat.
FROZEN AND RELATED SECTIONS
- Frozen sections are used for?
- Moh’s Procedures are used for?
- rapid intraoperative diagnoses
- surgical margins and sentinel node evaluation
USE OF FROZEN SECTIONS
- Rapid production of sections for intra-operative diagnosis
- Diagnostic and research enzyme histochemistry for labile enzymes
- Immunofluorescent methodology
- Immunohistochemistry techniques when heat and fixation may inactivate or destroy the antigens
- Diagnostic and research non enzyme histochemistry, (e.g., lipids and some carbohydrates)
- Silver demonstration methods particularly in neuropathology
- This is a refrigerated cabinet in which a modified microtome is housed.
- All the controls for the microtome are operated outside the cabinet.
- The first machine were introduced in 1954 and developments in design have improved both sectioning and laboratory safety.
Cryostat
The fresh tissue should be frozen as rapidly as possible without creating freeze artifacts. Suitable techniques include?
- Liquefied nitrogen (−190°C).
- Isopentane (2-methylbutane) cooled by liquid nitrogen (−150°C).
- Dry ice (−70°C).
- Carbon dioxide gas (−70°C).
- Aerosol sprays (−50°C).
- Internal freezing shelf or bar
FREEZING OF FRESH UNFIXED TISSUE
TOF
The best frozen sections are obtained when the tissue is frozen quickly.
True
Method of choice for freezing of fresh unfixed tissue
isopentane cooled by liquid nitrogen
FREEZING OF FRESH UNFIXED TISSUE
The problem with using liquid nitrogen alone is the formation of ____ around the tissue which act as an insulator and inhibit rapid, even cooling of the tissue. This can produce ____ ____ in the tissue making diagnostic interpretation difficult, especially in muscle biopsies.
- nitrogen vapor bubbles
- Freeze artifact
FREEZING OF FRESH UNFIXED TISSUE
- may be used for freezing
tissue blocks - Tissues can be successfully frozen directly in the cryostat, using the?
- Solid carbon dioxide (dry ice)
- freeze (cryo) bar
CRYOSTAT SECTIONING
TOF
The temperature of the microtome and the cryostat chamber should not be suitable for the tissue type.
False
SHOULD BE boang kaba
CRYOSTAT SECTIONING
- Tissues containing large amounts of water will section best?
- harder tissues and those that contain fat require?
- warmer temperature
- colder temperature
CRYOSTAT SECTIONING
- Most unfixed material will section well between ____
-
Most fixed tissues will section best within the range of ____ depending on the hardness of the
tissue.
- -15 and - 23°C
- -7 to -12°C
CRYOSTAT SECTIONING
Small blocks of undecalcified cancellous bones can be sectioned but care must be taken to remove any ____ prior to freezing.
cortical bone fragments
Staining
the most widely used histological stain. It is simple to use, easy to automate and demonstrates different tissue structures clearly
Hematoxylin and eosin (H&E)
HEMATOXYLIN AND EOSIN (H & E) STAIN
- stains the cell nuclei blue-black, showing clear intranuclear detail
- stains cell cytoplasm and most connective tissue fibers in varying shades and intensities of pink, orange and red.
- Hematoxylin
- Eosin
- individual manually dips the slides in the container of the stain.
- he slide in the slide
rack is programmed and the results will automatically be displayed on the monitor.
- Manual Method
- Automatic Slide stainer
jusko po baka di niyo pa to masagutan
fluorescent, xanthene dye which binds to salts with eosinophilic compounds containing positive charges
Eosin
Staining
- It is the most suitable stain to combine with an alum hematoxylin to demonstrate the general histological architecture of a tissue.
- Has the ability, with correct differentiation, to distinguish between the cytoplasm of different types of cell, connective tissue fibers and matrices, by staining these
differing shades of red and pink.
Eosin
EOSIN
- eosin yellowish, water-soluble
- eosin S, alcohol-soluble
- eosin bluish, erythrosin B
- Eosin Y
- Ethyl Eosin
- Eosin B
EOSIN
- the most widely used and is soluble in water and alcohol.
- As a cytoplasmic stain, it is usually used as a 0.5 or 1.0% solution in distilled water, with a crystal of thymol added to inhibit the growth of fungi.
- The addition of a little acetic acid (0.5 ml to 1000 ml stain) is said to sharpen the staining.
EOSIN Y
- extracted from the heartwood (‘logwood’) of the tree Hematoxylon campechianum.
- It is extracted from the logwood with hot water and then precipitated out from the aqueous solution using urea
Hematoxylin
Hematoxylin itself is not a stain, but ____, the major oxidization product, is a natural dye responsible for the color properties.
Hematein
Hematein can be produced from hematoxylin in two
ways:
- Natural oxidation or ‘ripening’
- Chemical oxidation
Hematoxylin
- by exposure to light and air.
- This slow process can take up to 3–4 months, but the resultant solution retains its staining ability for a long time.
- Ehrlich’s and Delafield’s hematoxylin solutions are examples of naturally ripened hematoxylins.
Natural oxidation or ‘ripening’
- Chemical oxidizing agents convert the hematoxylin to hematein rapidly,
- Sodium iodate used in Mayer’s hematoxylin
- mercuric oxide used in Harris’s hematoxylin
Chemical oxidation
Hematoxylin
Hematein is anionic, having a ____ for tissue, and is inadequate as nuclear stain without the presence of mordant
Poor Affinity
Hematoxylin
confers a net positive charge to
the dye-mordant complex, enables it to bind to anionic tissue sites, such as nuclear chromatin
Mordant/metal cation
The type of mordant used influences the type of tissue components stained and their final color.
Hematoxylin
most useful mordants for hematoxylin
Aluminum, iron and tungsten salts
Hematoxylin
demonstration of argyrophil cells.
Solutions using lead
Hematoxylin
Hematoxylin solutions can be arbitrarily classified according
to which mordant is used:
- Aluminum hematoxylin
- Iron hematoxylin
- Tungsten hematoxylin
- Molybdenum hematoxylin
- Lead hematoxylin
- Hematoxylin without mordant
Hematoxylin
These are the most frequently used hematoxylins in the H&E and produce good nuclear staining.
ALUMINUM (ALUM) HEMATOXYLIN
Hematoxylin
What is the usual mordant for aluminum hematoxylin
- aluminum potassium sulfate (potash alum)
- aluminum ammonium sulfate (ammonium alum).
Hematoxylin
process of converting the color of nuclei from red to blue-black when section is washed in a weak alkali solution like tap water or alkaline solutions such as saturated lithium carbonate, 0.05% ammonia in distilled water (ammonia water) or Scott’s tap water substitute
Blueing
The alum hematoxylins can be used either regressively or progressively.
- section is over-stained and then differentiated in acid alcohol, followed by blueing.
- the section is stained for a predetermined time, staining the nuclei adequately, but leaving the background tissue relatively unstained.
- Regressive Staining
- Progressive Staining
Hematoxylin
TOF
The times required for hematoxylin staining with satisfactory differentiation vary according to the type and age of the alum hematoxylin, the tissue type and the personal preference of the pathologist.
True
Hematoxylin
For routine H&E staining of tissues, the most commonly used are:
- Ehrlich’s
- Mayer’s
- Harris’s
- Gill’s
- Cole’s
- Delafield’s
Hematoxylin
occasionally used for urgent frozen section.
Carazzi’s hematoxylin
- strong hematoxylin solution staining nuclei intensely and crisply.
- Particularly useful for staining sections for tissues that have been exposed to acids
EHRLICH’S HEMATOXYLIN
Hematoxylin
- The stain fades more slowly than those stained with other alum hematoxylins.
- It is suitable for acid-decalcified tissues, and tissues stored
in formalin fixatives for long periods of time which become increasingly acidic during the storage period.
EHRLICH’S HEMATOXYLIN
Hematoxylin
Ehrlich’s hematoxylin is also suitable for tissues which have been fixed in acid fixatives such as?
Bouin’s
Hematoxylin
Ehrlich’s hematoxylin is not ideal for
frozen section
last step in tissue processing that results in a permanent histological
preparation suitable for microscopy, after adhesion of the sections on to the slide and appropriate staining of the tissue.
MOUNTING
A process by which the maximum degree of transparency of stained tissue sections is covered by coverslip obtained by using a mounting medium that approximate to that of dried protein between ____ and ____ which is close
to the RI of glass.
between 1.53 and 1.54
Purpose is to make the tissue transparent for evaluation
Mounting
use this slide to famillarize the mounting procedure
- Place enough amount of mounting medium on the center of tissue section.
- Place a dry, clean coverslip on the edge of the slide and release gently.
- Pressed gently on the coverslip allowing the mounting medium to spread quickly.
- Properly label the slide.
- Allow the slide to dry up.
TYPES OF MOUNTING MEDIA
- Mounting media can be?
- Most commercially available mountants are usually?
- ____ acetate may be added to mountants
- Aqueous or Resinous
- non aqueous/resinous
- Potassium
Aqueous/hydrophilic/non-aqueous mounting media are still important for special stains (Oil Red O staining), solvents dissolve the fats, few with RI higher than 1.5, most being in
the range 1.4 to 1.45
Non-Resinous or Aqueous Mounting Media (Temporary)
3 types of aqueous mounting media:
- Syrups
- Gelatin media
- Gum Arabic
Give the Refractive Index
- Apathy’s mountant (modified by Lilie and Ashburn 1943)
- Apathy’s mountant (Highman’s modification, 1946)
- RI 1.41
- RI 1.436
Match
- Water
- Glycerine Jelly
- Apathy’s Medium
- Farrant’s Medium
- Highmans’s Medium
- Fructose Syrup
- Polyvinyl Aclohol
- Glycerine-Glycerol
A. 1.47
B. 1.333
C. 1.52
D. 1.43
- B
- A
- C
- D
- C
- A
- walang sagot
- A
- adhesive, organic, non-aqueous, hydrophobic
- They may be divided into natural and synthetic resins
Resinous Mounting Media (Permanent)
a natural resin extracted from the Canadian tree, Abus Balsamea.
Canada Balsam
most commonly used synthetic resinous media
are the?
polyesterenes
such as Kirkpatrick & Lendrum’s mountant and Gurr’s distrene plasticizer xylene (DePex)
such as Kirkpatrick & Lendrum’s mountant and Gurr’s distrene plasticizer xylene (DePex)
DPX (DePeX) - Distrene 80
Match the following
- DPX (Depex) – Distrene 80
- Histomount
- Cover Bond
- Gurr’s Neutral Mounting Medium
- Histoclad
A. 1.49 - 1.50
B. 1.52
C. 1.54
D. 1.53
E. 1.51
- B
- A
- D
- E
- C
Match the following
- Permount
- Pro-Texx
- Technicon Resin
- UV-Inert
- XAM
- Eukitt
A. 1.526
B. 1.62
C. 1.52
D. 1.495
E. 1.517
F. 1.49 - 1.50 / 1.515
- A
- D
- B
- E
- C
- F
