Refresher Flashcards
Most rapid fixative and is especially recommended for fixing lymph glands, urgent biopsies, and chromosomes
Carnoy’s fluid
The most common metallic fixative
Mercuric chloride fixatives
The most common decalcifying agent
Nitric acid
The most rapid decalcifying agent
Phloroglucin Nitric acid
Best and fast acting dehydrating agent
Ethanol
The most common clearing agent
Xylene
Most common embedding medium
Paraffin wax
Substitute for Xylene or Benzene
Toluene
The most common staining method for MICROANATOMICAL STUDY
H&E staining technique
The process by which the tissues are fixed in a physical and partly also in a chemical state
Fixation (Preservation)
Best results in fixation are obtained through what type of solutions?
Slightly hypertonic solution (400-450 mOsm)
The concentration of formaldehyde used as fixative is approximately ___ weight in volume
37-40% weight in volume or
35-40% gas by weight
Fixative that is classified as both a nuclear and histochemical fixative
Newcomer’s fluid
Paraformaldehyde deposits are formed when formalin is stored for a long time in ___ temperature, but this can be remedied by using ____.
Cold, 10%Methanol
Fixative used for sputum cytology
Alcoholic formalin (Gendre’s fixative)
A fixative that is made up of 2 formaldehyde residues linked by 3 carbon chains
Glutaraldehyde
(recommended for electron microscopy and histochemistry)
Frequently used concentration of Mercuric chloride fixatives
4-7% or 5-7%
The routine fixative for preservation of cell detail in tissue photography
Mercuric chloride
An excellent microanatomic fixative for pituitary gland, bone marrow, and blood-containing organs
Zenker-Formol (Helly’s fluid)
A mercuric chloride fixative that is commonly used for bone marrow biopsies
B-5 fixative
A fixative that is recommended for the demonstration of acid mucopolysaccharides and conective tissue mucin
Lead fixative
A fixative recommended for the demonstration of chromaffin tissues, adrenal medulla and mitochondria
Chromate
The recommended fixative for demonstration of mitochondria, chromatin, golgi bodies and mitotic figures
Regaud’s fluid (Moeller’s fluid)
The recommended fixative for study of early degenerative processes and tissue necrosis and demonstration of Rickettsia
Orth’s fluid
The recommended fixative for fixation of embryos and pituitary biopsies
Bouin’s fluid
The fixative that is used as a diluent for the Wright’s stain and excellent for fixing dry and wet smears
Methanol
Fixatives that are used in fixing brain tissues (Negri bodies) for rabies diagnosis
Acetone (1st choice), Carnoy’s fluid (2nd choice)
Recommended fixative for cytoplasmic structures such as mitochondria
Flemming’s fluid w/o acetic acid
Recommended fixative for nuclear structures such as chromosomes
Flemming’s fluid w/ acetic acid
Fixatives used for electron microscopy
Glutaraldehyde,
Osmium tetroxide/Osmic acid,
Acrolein mixture w/ glutaraldehyde-formaldehyde,
Karnovsky’s paraformaldehyde-glutaraldehyde mixture
The reagent that is used for washing out excess mercuric fixative
Alcoholic iodine
Used to wash chromate, formalin, and osmic acid
Tap water
Used to wash picric acid
50-70% Alcohol
Possible cause of too hard or brittle tissue blocks
Prolonged fixation
Failure to arrest early cellular autolysis is due to
Due to failure to fix tissue immediately or due to insufficient fixative
Too brittle and too hard blocks is caused by
Prolonged fixation
Soft and feather-like tissues is due to
Due to incomplete fixation
Removal of fixative soluble substances is caused by
Wrong choice of fixative
Presence of artifact pigments on sections is due to
Incomplete washing of fixative
Shrinkage and swelling of cells in tissue blocks is due to
Due to overfixation
Enzyme inactivation and loss is due to
Wrong choice of fixative
It is the best general tissue fixative
10% Neutral Buffered Formalin (Phosphate buffered formalin)
Best fixative for CNS, general post-mortem tissues, and silver impregnation
10% Formol-Saline
Best fixative for frozen sections and elastic fibers
10% Neutral Buffered Formalin
A specialized fixative used in frozen section which serves to localize antigens and hydrolytic enzymes. Used for preservation if lipids,
Formol-Calcium (Baker’s Formol Calcium)
Formalin usage by-products
Paraformaldehyde and Formic acid
Fixatives recommended for renal biopsies
Mercuric chloride fixative
A fixative that is used for the preservation of the tumor biopsies of the skin
Heidenhain’s Susa
This group of fixatives produces excessive yellow staining of tissues
Chromate fixatives
Air-filled lungs may float on fixative. What must be done to avoid this?
Cover the lungs with gauze
Used to decontaminate prions-infected tissue
Immersion of specimen in:
Formalin (48hrs)
Formic acid (1hr)
Formalin(48hrs)
A procedure whereby calcium or lime salts are removed from tissues
Decalcification
List of decalcifying agents (in general)
- Acids (routinely used)
- Chelating agents (EDTA)
- Ion-exchange resin
- Electrophoresis
Substances which combine with calcium ions and other salts to form weakly dissociated complexes and facilitate removal of calcium salt
Chelating agents (EDTA)
-Versene, Sequestrene
EDTA-decalcified tissue rinsing process
- Wash in running tap water
- Overnight immersion in formol saline or phosphate buffered formalin
The most common decalcifying agent used
Nitric acid
How to prevent discoloration caused by Nitric acid
- By neutralizing the tissue with 5% sodium sulfate and washing in running tap water for at least 12 hours or by
- Addition of 0.1% urea to pure concentrated nitric acid.
A decalcifying agent that can also be used as a tissue softener
Perenyi’s fluid
The most rapid decalcifying agent and is recommended for urgent works
Phloroglucin nitric acid
The weakest decalcifying agent and is used only for minute pieces of bone
Sulfurous acid
Von Ebner’s fluid is recommended for teeth and small pieces of bones. Its components include:
- 36% Saturated Aqueous Sodium Chloride
- Concentrated Hydrochloric acid
- Distilled water
Methods to determine the extent of decalcification
- Physical method
- Radiological
- Chemical method
A method to detect the extent of decalcification which cannot be used for mercuric chloride-fixed tissues
Radiological (X-ray)
The process of removing intracellular and extracellular water from the tissue
Dehydration/Dessication
It is the best dehydrating agent
Ethanol
A dehydrating agent used as a substitute for ethanol
Isopropanol
A dehydrating agent utilized for plant and animal microtechniques
Butanol
A toxic dehydrating agent usually for blood and tissue films
Methanol
A dehydrating agent that is limited inly to small pieces of tissue due to its extreme volatility and flammability
Acetone
The reason why a clearing agent turns milky when the tissue is transferred to it
Incomplete dehydration
The process whereby alcohol is removed from the tissue and replaced by a fluid that will dissolve the wax with which the tissue must be impregnated.
Clearing/Dealcoholization
The most commonly used and is considered as an excellent and true clearing agent
Xylene
A clearing agent that is carcinogenic and may cause aplastic anemia
Benzene
A clearing agent that becomes milky on prolonged storage and its quality is not always good and uniform
Cedarwood oil
The clearing agent recommended for clearing embryos, insects and very delicate specimens
Aniline oil
A slow-acting clearing agent that can be used when double-embedding techniques are required
Methyl salicylate/ Methyl benzoate
Properties are very similar to chloroform but is cheaper
CCL4 (Carbon tetrachloride)
The process whereby the clearing agent is completely removed from the tissue and replaced by a medium that will fill all natural cavities, spaces and interstices of the tissues to provide solid and firm consistency.
Inflitration/Impregnation
The process by which the impregnated tissue is placed into a precisely arranged position in a mold containing a medium, which is then allowed to solidify.
Embedding/Blocking/Casting
Processes done by the automatic tissue processor (Elliot bench-type tissue processor)
Fixation,
Dehydration,
Clearing,
Impregnation
Manual paraffin wax impregnation requires at least ____ changes of wax with _____minutes interval.
4 changes, 15 minutes
The most rapid impregnation technique is ___; nit is impregnation under _____.
Vacuum method, negative atmospheric pressure
Substitutes for Paraffin wax:
- Paraplast
- Embeddol
- Bioloid
- Ester wax
- Carbowax
Melting point of paraffin wax
54-58 deg Celsius
Recommended infiltrating agent when histochemical and enzymatic studies are considered
-Gelatin
-Carbowax
Embedding medium that is used for Electron microscopy
Plastic/Resin (Epoxy)
A semisynthetic wax that is used for embedding eyes
Bioloid
Double-embedding method is the process in which tissues are first infiltrated with ____ and subsequently embedded in_____.
Celloidin; Paraffin
A process whereby tissues are cut into uniformly thin slices with the aid of microtome to facilitate the studies under the microscope.
Sectioning/Cutting
The thickness of paraffin section is about
4-6 micrometer
(Celloidin= 10-15 um)
The removal of gross nicks and irregularities from the knife is called
Honing
Available in a wide variety of fineness; Used only for badly nicked knives
Carborundum hone
A stone of medium fineness
Arkansas stone
The finest hone
Yellow Belgian/Belgium
In plane concave knife, one side of the knife is flat/straight while the other is concave. The straight side is used for cutting ____ while the concave side is used for cutting____
Celloidin sections,
Paraffin sections
A knife that is used for cutting paraffin sections
Biconcave
A knife with straight sides that is used for cutting frozen sections and extremely hard and tough specimens
Plane wedge
The temperature of water bath is kept at
45-50 deg Celsius (6-10 deg C below the melting point of wax)
Temperature of paraffin oven (infiltration)
2-5 deg Celsius above the melting point of wax
A microtome that is used for fluorescent antibody staining techniques or histochemical enzyme studies
Cryostat/Cold microtome
The process by which a tissue is arranged in a precise position in the mold during embedding, on the microtome before cutting, and on the slide before staining
Orientation
Base sledge is a type of
-Sliding microtome
Cryostat chamber is set at what temperature
5-30 deg Celsius/
18-20 deg Celsius
(common is 20degC)
The process whereby various constituents of tissues are studies thru chemical reactions that will permit microscopic localization of a specific tissue substance
Histochemical staining/Histochemistry
Natural dyes
-Hematoxylin
-Cochineal
-Orcein
-Saffron
Sometimes known as “coal tar dyes” since they were originally manufactured from substances that have been taken from Coal tar.
Synthetic dye
Staining of tissues by simple dyes
Direct/Simple staining
Continuous staining until desired intensity is achieved
Progressive staining
Overstaining and then decolorizing to reach desired intensity
Regressive staining
Used to stain tissues immediately after removal from the body
Supravital staining
Injecting the dye into living cells
Intravital staining
Selective staining of the living calls and shows cytoplasmic activities
Vital staining
What is the best vital stain?
Neutral Red
The most common counterstain used in routine hemoxylin stain
Eosin Y
Most commonly used in metallic impregnation. Used for the demonstration of CNS tissues and reticulin
Silver nitrate
Most important stain derived from the core of heartwood tree H. campechianum
Hematoxylin
From vegetables sources and lichens
Orcein
A substance which when taken up by the tissue, helps make the dye in return serving as a link or bridge to make the staining reaction possible.
Mordant
Most reliable and specific stain for DNA. Stains DNA red-purple.
Feulgen stain
Stains RNA red and DNA green.
Methyl green pyronin
Fluorescent stain for DNA (yellow-green fluorescence) and RNA (orange-red fluorescence)
Acridine orange
Best staining procedure for reticulin fibers
Metallic/Silver impregnation
Red chromogen in immunoperoxidase reaction
Aminoethylcarbazole (AEC)
Brown chromogen in immunoperoxidase reaction
Diaminobenzidine (DAB)
Mixture of picric acid and acid fuchsin for the demonstration of connective tissues, mucin, and elastic tissue
Acid fuchsin-Picric acid (Van Gieson’s stain)
The oldest of all stains; stains amyloid, cellulose, starch, carotenes, and glycogen
Iodine
An excellent stain for elastic fibers
Orcein
Used for visualizing mitochondria
Janus Green B
Stains that are utilized for staining ferrous iron
Turnbull’s Blue reaction
Hematoxylin that is used for the demonstration of spermatogenesis
Copper hematoxylin
Special stain for hemoglobin
Benzidine
Special stain for blood
Rhodamine B
Special stain for Calcium salts
Acridine Red 3B
Special stain for acid mucopolysaccharides
Alcian blue
Special stain for neuroglia
Victoria blue
Special stain for reticulocytes
Cresyl blue
Specific stain used to identify Hemosiderin (ferric iron)
Perl’s Prussian Blue
The adhesive agent that is very useful in cytology particularly for cytospin preparations of proteinaceous or bloody material
3-APES (Aminopropyltriethoxysilane)
Composition of Mayer’s egg albumin
Egg white 50 mL
Glycerin 50 mL
Thymol crystals 100 mg
Composition of 1% Gelatin
Gelatin 1g
Distilled water 100 mL
Glycerin 15 mL
Phenol crystals 2g
Composition of Dried Albumin
Dried albumin 5g
Sodium chloride 5 g
Distilled water 100 mL
Thymol crystals
What does 3-APES mean?
Aminopropyltriethoxysilane
Composition Starch Paste
Powdered starch 1g,
Distilled water 30 mL,
Hydrochloric acid 2 drops
Thymol crystals
Readily available from outdated blood stored in blood banks, dispensed into sterile tubes of 0.5 mL each.
Plasma
Very useful cytology particularly for cytospin preparations of proteinaceous or bloody material.
3-APES
Added to the starch paste in order to prevent mold formation
Thymol crystals
Refractive index of a glass
1.518
Used for Methylene blue-stained nerve preparations and as a general purpose aqueous mountant
Apathy’s medium
A natural resin that is recommended for whole mounts and for thick sections
Canada balsam
It is incorporated in Pap staining technique for cytologic studies of immature cells
Eosin azure
The process by which a tissue is arranged in a precise position in the mold during embedding, on the microtome before cutting, and on the slide before staining.
Orientation
The process of sealing the margins of the coverslip to prevent escape of fluid or semi-fluid mounts and evaporation of mountant to immobilize the coverslip and to prevent sticking of slides upon storage
Ringing
Marker for choriocarcinoma
Human Chorionic Gonadotropin
Useful in differentiating lung adrenocarcinomas from mesotheliomas
TTF1 (Thyroid Transcription Factor 1)
Useful in diagnosis of prostatic adenocarcinoma. Positive in certain pancreatic and salivary gland tumor
PSA (Prostate Specific Antigen)
Used to identify tumors derived from smooth, skeletal and cardiac muscles
Actin
Most widely used to confirm the diagnosis of astrocytoma
GFAP (Glial Fibrillary Acidic Protein)
Best screening marker for lymphoma
LCA (Leukocyte Common Antigen/CD 45)
Provides strong evidence of neural and neuroendocrine differentiation
NSE (Neuron Specific Enolase)
Recognized as a marker for neuroendocrine differentiation
Chromogranin
Highly specific for myogenic tumors including leiomyoma and rhabdomyosarcoma
Desmin
Polyclonal antibodies used in immunohistochemical techniques are derived from
Rabbits
An excellent dehydrating and clearing agent, but is expensive, extremely dangerous and its vapor is toxic to man
Dioxane/Diethyl dioxide
It is made up of mercuric chloride stock solution to which glacial acetic acid has been added just before its use to prevent turbidity and formation of dark precipitate. It is recommended for fixing small pieces of liver, spleen, connective tissue fibers and nuclei.
Zenker’s fluid
It is not suitable for fixing kidney structures, lipid and mucus. Destroys cytoplasmic structures like mitochondria
Bouin’s fluid
It is highly explosive when dry, and therefore must be kept moist with distilled water or saturated alcohol at 0.5-1% concentration during storage.
Picric acid
It is recommended for fixing mucopolysaccharide and nuclear proteins. Acts as both Nuclear and Histochemical fixative
Newcomer’s fluid
Used for detection of herpetic lesions and carcinoma
Vulvar scrape
Used to collect samples of endocervical canal
Endocervical brush
The best staining method of choice for exfoliative cytology
Pap’s stain/Pap’s smear
An equipment used for vaginal aspiration
Glass pipette and rubber bulb
Equipment used for swab smear
Ayre’s spatula
Equipment used for endocervical and endometrial aspiration
Laryngeal cannula attached to a 10-cc syringe
Programmed cell death
Apoptosis
Accidental cell death from due to trauma
Necrosis
Organs removed one by one is a technique of whom?
Virchow
Technique characterized by in situ dissection, in part combined with en bloc removal
Technique of Rokitansky
Technique by which thoracic and cervical organs, abdominal organs and the urogenital system are removed as organ blocks
Technique of Ghon
En bloc removal technique is widely used
Modification of Ghon’s technique
Autopsy technique by which thoracic, cervical, abdominal and pelvic organs are removed en masse and subsequently dissected into organ blocks
Technique of Letulle
Which knife is used for celloidin sections?
Plane concave
What is the paraffin impregnation temperature?
2-5deg C above the melting point of the wax
Plastic resin utilized for light microscopy
Acrylic plastics
Plastic resin embedding medium recommended for EM but not for immunohistochemistry
Epoxy
Most commonly used fixative for cytological examination
95% ethanol
Who does the autopsy?
The pathologist
Factors for tumor grading
- Polarity of the cell
- N:C ratio
- Number and characteristics of mitoses
- Hyperchromaticity
Changes of intercellular structural pattern
- Increase in size
- Irregular shape
- Irregular chromatin
- Anisocytosis and anisokaryosis
- Excessive growth and overcrowding of cells
Classes of Cancer
I- Absence of abnormal or atypical cells
II- Atypical cytologic feature but not suggestive of malignancy
III- Cytologic feature suggestive but not conclusive of malignancy
IV- Cytologic feature strongly suggestive of malignancy
V- Cytologic feature conclusive of malignancy
Primary signs of death
- Respiratory failure
- Cardiovascular failure
- CNS failure (1st to occur)
The first demonstrable change of death
Algor mortis (cooling of the body)
Occurs 2-3 hours after death
Rigor mortis (stiffening of the skeletal muscle)
Post-mortem lividity/suggilations; purplish discoloration
Livor mortis
Drying and wrinkling of the anterior chamber if the eye
Dessication
Decomposition; Invasion of intestinal microorganisms
Putrefaction
Self-digestion of cells
Autolysis
Formation of chicken fat and currant jelly-like clot
Post-mortem clotting
An increase in size of tissues because of the increase in size of individual cells
Hypertrophy
An increase in size of tissue because of increase in the number of cells that make up the tissue
Hyperplasia
A retrogressive change in cellular growth pattern characterized by non-appearance of an organ.
Agenesia
Incomplete or underdevelopmemt of the organ
Hypoplasia
Incomplete or defective development of a tissue or organ
Aplasia
Failure of an organ to form an opening
Atresia
Considered to be the most common type of necrosis usually observed in the myocardium, lungs, kidneys, and the spleen
Coagulative necrosis
