Fixation + Addtl. Discussion Flashcards
This step includes entering the details of the specimen in a log book
NUMBERING
Gross description is always done by?
PATHOLOGIST
Most important and most crucial step in tissue processing. It has to be carried out adequately.
FIXATION
Step in tissue processing that is define as the killing, penetration and hardening of tissues
FIXATION
May also be defined as the alteration of tissues by stabilizing protein so that tissues become resistant to further changes
FIXATION
Primary goal of Fixation:
To preserve the morphological and chemical integrity of the cells and tissues as close to the original as possible
Secondary goal of Fixation:
To harden tissues;
To facilitate easy cutting;
To protect the tissue from trauma of further handling
2 Fixation Methods:
PHYSICAL and CHEMICAL
Types of Physical Method:
Heat Fixation and Microwave Technique
Cryo-preservation (freeze drying)/ freeze substitution
- Mostly done in microbiology to fix bacterial
smears - Not usually carried out in Histopath (rarely used)
- For rapid diagnosis
- For frozen tissue section
Heat Fixation
Fixatives under Chromate Fixatives: (CROP)
Chromic acid (1-2%)
Regaud’s/ Moller’s fluid
ORTH’S FLUID
Potassium Dichromate (3%)
- physical technique that is widely used but not a common method.
- it increases movement of molecules thereby accelerating fixation, staining, decalcification, immunohistochemistry and EM
Microwave Technique
Neurochemical substances in brain that can be used by microwave techniue
Acetylcholine
A method of fixation where the specimen is immersed in chemical fixative
CHEMICAL METHOD
Reagent used to fix tissue is called?
Fixative
Factors involved in fixation/ practical considerations
- Temperature
- Thickness/ Size
- pH
- Osmolality
- Concentration
- Time and Duration of Fixation
- Volume
Temp. required for EM and histochemistry
0-4 degree Celsius with Autotechnicon
Fixation is traditionally carried out at what temperature?
Room temperature
Required temp. in Autotechnicon?
40 degree Celsius
TRUE or FALSE. Autotechnicon is done usually in laboratory and it uses constant agitation?
TRUE
TRUE or FALSE. Formalin @ 60 deg C may be use to fix tissues with TB
FALSE
TRUE or FALSE. Formalin at 60 deg C is for rapid fixation for urgent biopsy
TRUE
Temperature for formalin required in order to fix tissues with tuberculosis
Formalin @ 100 deg C
3 Fixatives for Smear (SME)
- Methanol
- Ether/ Ethanol
- Schaudinns Fluid
Required thickness of tissue spx for electron microscopy (EM)
1-2 mm^2
Required thickness of tissue spx for light microscopy (LM)
2 cm^2 wide and not more than 4mm thick
TRUE or FALSE. The thickness of tissues spx for LM should be more than 0.5 cm
FALSE; should be no more than 0.4 cm - 0.5 cm (4-5 mm) for LM
TRUE or FALSE. Many laboratories use tissue processors that work at 45 deg C for regular tissue processing?
FALSE; 40 deg C
TRUE or FALSE. Refrigeration is used to speed up decomposition if the tissue needs to be photographed and cannot be fixed immediately
FALSE; refrigeration is used to SLOW DOWN
TRUE or FALSE. Brain cells can deteriorate very quickly
TRUE
TRUE or FALSE. Nucleic acids react with fixatives to any extent at room temperatures and chemical reactions including those involved in fixation are more slower at higher temperatures
FALSE; nucleic acids does not react; rapid at higher temperatures
TRUE or FALSE. An increase in temperature can increase the rate of fixation but can also increase the rate of autolysis
TRUE
Required thickness for processing lung specimen
1-2 cm
TRUE or FALSE. Tissues should not be more than 4-5mm except in processing lung specimen
TRUE
TRUE or FALSE. Uterus and other large solid tissues must be opened/ slice thinly to improve penetration of fixatives
TRUE
TRUE or FALSE. Fecal matter and stomach contents can inhibit the penetration of fixatives and must NOT removed before the fixation process
FALSE; it should be remove before fixation process
Brain is usually suspended whole in what fixative for how many weeks to ensure fixation and some hardening prior to sectioning
10% buffered formalin for 2-3 weeks
TRUE or FALSE. Most tissues can be cut and trimmed without prior fixation such as the brain
FALSE; most tissues EXCEPT brain is generally soft when unfixed so it must be fixed before sectioning
TRUE or FALSE. An incompletely fixed tissue may lead to improper and incomplete clearing and impregnation, and may later prove to be a hindrance to normal sectioning and staining of specimen
TRUE; FIXATION IS MOST IMPORTANT STEP IN TISSUE PROCESSING
TRUE or FALSE. Penetration into a thin section will occur more rapidly than for a thick section
TRUE
TRUE or FALSE. Formalin penetrates tissues slowly so specimens may need to be opened, incised, or sliced and left to fix for an adequate period of time prior to processing
TRUE
TRUE or FALSE. Larger tissues require more fixatives and longer fixation time
TRUE
TRUE or FALSE. To maintain an adequate fixation time of 4- hours, the recommended size of the tissue is 2 cm^2 and no more than 4mm thick
TRUE
Fixation is best carried out close to neutral pH, in what range?
Between pH of 6-8
TRUE or FALSE. Acidity favors formation of formalin-heme pigment that appears black, polarizable deposits in tissue
TRUE
Common buffers includes…
Phosphate
Bicarbonate
Cacodylate
Veronal
Commercial formalin is buffered with phosphate at a pH of…?
pH of 7
What will happen if the cells are fixed in a hypertonic solution?
Cell Shrinkage
Effect of hypotonic solution?
Cells may swell
Preferred osmolality for fixation
Slightly hypertonic (400-450 mOsm)
What component is commonly added to osmium tetroxide fixatives for electron microscopy?
Sucrose
Osmolality classification used in practice and may be use as holding solutions for tissues to be transported to frozen sections or kidney biopsies for special processing
Isotonic solution
TRUE or FALSE. Concentration of fixative should be adjusted down to the lowest level possible
TRUE
Concentration of fixative used for formaldehyde?
10% solution
Concentration of fixative used for glutaraldehyde and used for EM
3% solution
Ideal concentration of fixative for immuno-electron microscopy
0.25% glutaraldehyde
Ideal time to perform fixation
20-30 minutes following interruption blood supply
TRUE or FALSE. In order to maintain tissue morphology, samples SHOULD BE FIXED IMMEDIATELY after removal or death to prevent autolysis or putrefaction.
TRUE
TRUE or FALSE. More cellular organelles will be lost, more nuclear shrinkage and artefactual clumping will occur and tissue spx can be irreversibly damaged when the fixation is delayed
TRUE
It refers to the period the tissue is exposed to formalin
Fixation time
Fixation is retarded by:
Large and thick tissues
Presence of mucus
Presence of blood
Presence of fat
Cold temperature
(can make the fixation time LONGER/ PROLONG)
TRUE or FALSE. Cold temperature can inactivate enzymes
TRUE
TRUE or FALSE. Cold temperature can inactivate enzymes
TRUE
Fixation is accelerated by:
Smaller and thin tissues
Agitation — continuous stirring
Heat (37-56 degrees C)
(can make fixation time SHORTER/FASTER)
Required temperature for heat in tissues
37-56 deg C only
TRUE or FALSE. Beyond 56 deg C heat is acceptable for tissues spx
FALSE; 37-56 deg C heat only, beyond 56 deg C can be damaging to tissues
Fixative volume for maximum effective fixation
20x the volume of the specimen
Ratio of fixative to tissue
20:1
Formalin diffuses into the tissue at the rate of:
1mm per hour
Fixatives used for EM: (KAPOG)
Karnovsky’s
Acrolein
Paraformaldehyde
Osmium tetroxide
Glutaraldehyde
Volume required if osmium tetroxide is used for EM
5-10 times the volume of specimen
Volume required for museum preparations
Should not be less than 50-100 times the volume of the specimen
TRUE or FALSE. Agitation will also enhance fixation of the specimen
TRUE
Most common error in histotechnology is…
Insufficient ratio of tissue volume to fixative
Human brain must undergo in what method in fixation process
INTRAVASCULAR PERFUSION
This is used to wash out blood in human brain during intravascular perfusion
RINGER’S LACTATE
TRUE or FALSE. Eyes should be dissected before they are fixed
FALSE; it SHOULD NOT be dissected; tissues may wrinkle; inject formol alcohol before immersing the organ to fixative
If the autopsy materials is not possible to fix immediately after death, the body must be placed where and at what temperature?
Mortuary ref (4 deg C)
Hard tissues (cervix, uterine, fibroid, etc.) must undergo in what method?
LENDRUM’S METHOD
What method washes with running water overnight then immerse specimen in 4% aqueous phenol for 1-3 days.
Method of Lendrum’s Method
Lendrum’s method is done before, during or after fixation?
After fixation
Hollow organs (stomach, intestine) should be…
Packed with cotton soak in fixative;
The spx can be sliced and it must be completely open before immersing it in adequate fixing solution
What specific organ may float on fixative?
Air filled lungs
How to prevent air filled lungs floating on a fixative?
May cover it on several layers of gauze to keep it at the bottom of the container
Effects of fixatives in general:
Harden tissues
Makes cells resistant to damage
Increase optical differentiation of cells
Acts as mordants or accentuators to facilitate easy staining
(Problems in Fixation) What artefacts that may appear in tissue spx when the washing was incomplete?
Presence of artefacts —> white paraformaldehyde, precipitate artefacts
PROBLEMS IN FIXATION
- Incomplete washing may lead to: presence of artefacts on tissues
- Overfixation will render the tissue brittle and hard, shrinkage and swelling
- Loss of substance soluble in fixing agent, loss or inactivation of enzyme may result from wrong choice of fixative.
Cause of failure to arrest early autolysis of cells
Failure to fix immediately (the tissue was probably allowed to dry before fixing); Insufficient fixative
Cause of removal of substances soluble in fixing agent
Wrong choice of fixative
Presence of artefact pigments on tissue sections
Incomplete washing of fixative
Cause of issues are soft and feather-like in consistency
Incomplete fixation
Cause of loss or inactivation of enzymes needed for study
Wrong choice of fixative
Cause of shrinkage and swelling of cells and tissue structure
Overfixation
Cause of tissues block are brittle and hard
Prolonged fixation
Well-known artifact that may be produced under acid conditions
Formalin pigment
What substance should use to eliminate the pigment or may be used to reduce the fixation of the specimen?
Phenol-formalin
May be found in surgical specimens particularly in liver biopsies, associated with an intense eosinophilic staining at the center of the tissue stained in H&E stained sections
Crush artifact
Can be used to vapor-fix freeze-dried tissues
Paraformaldehyde and Osmium tetroxide
TRUE or FALSE. Fixation does not prevents degeneration, decomposition, putrefaction, and distortion of tissues after removal from the body
FALSE; fixation PREVENTS
TRUE or FALSE. Fixation in 10% buffered formalin will initially cause slight swelling of tissue specimens
TRUE
TRUE or FALSE. During processing, the spx may shrink and lose 20%-30% of its volume
TRUE
TRUE or FALSE. The particular fixative employed will also influence the degree to which individual elements will stain with various histochemical and immuno-histochemical reagents
TRUE
TRUE or FALSE. Leaving a tissue spx in air for a prolonged period of time will cause it to dry out, and will result in distortion of its morphological appearance
TRUE
BENEFITS OF FIXATION
- Allows thin sectioning of tissue by hardening it
- Prevents autolysis and inactivates infectious agents (except prion diseases)
- Improves cell avidity for special stains
Factors to be considered when choosing the right fixative
- The need for immediate examination: urgency of the case (urgent biopsy = use a rapid fixative in action)
- Type of specimen to be processed
- The tissue structure to be studied.
- Structure technique to be applied (the fixative must be compatible with the stain to be used)
Types of fixatives as to MECHANISM OF ACTION
Additive
Non-additive
Fixatives that when used is ABSORBED BY THE TISSUE; stabilizes tissue proteins
Additive Fixatives
Examples of Additive Fixation
Formaldehyde
Mercuric Chloride
Chromium Trioxide
Picric Acid
Glutaraldehyde
Osmium Tetroxide
Zinc Sulfate or Chloride
Fixing agent that is not incorporated into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attach to H-bonds of certain groups within the protein molecule.
Non-additive Fixation
Fixative is not absorbed by the tissue; alteration of tissue components
Non-additive Fixation
Example of Non-additive Fixatives
Alcohol
Acetone
Acts by cross-linking proteins
Aldehyde and Oxidizing Agents
Protein-denaturing agents
Alcohol based fixatives
Acts by forming insoluble metallic precipitates
Metallic fixatives
- Cheap
- Stable
- Safe to handle
- Must kill the cell quickly
- Must inhibit bacterial decomposition and autolysis
- Produce minimum shrinkage of tissue
- Permit rapid and even penetration of tissues
- Must harden tissues
- Must be isotonic (some are hypotonic solutions)
- Must make cellular component insoluble to hypotonic solutions and render them insensitive to subsequent processing
- Must permit subsequent application of many staining procedures to facilitate easier and more profitable examination
Characteristics of a good fixative
Non-additive fixative that alters the tissue component
Acetone
Types of Fixative According to Action: (MCH)
Cytological
Microanatomical
Histochemical
Use to preserve PART OF THE CELLS (nucleus or cytoplasm)
Cytological
Preserves parts of cytoplasm;
pH greater than 4.6;
DO NOT CONTAIN GLACIAL ACETIC ACID because HAc destroys the mitochondria and golgi bodies of the cytoplasm
Cytoplasmic Fixatives
Fixatives that (should) contain glacial acetic acid;
pH of < 4.6;
Preserves nuclear structures like nuclear chromatin and chromosomes
Nuclear Fixatives
Examples of Nuclear Fixatives: (BF(e)NCH)
Bouin’s Fluid
Flemming’s Fluid
Newcomer’s Fluid
Carnoy’s Fluid
Heidenhain’s susa
Fixatives for Cytoplasmic: (HORFF)
Helly’s/ Kelly’s/ Zenker Formol
Orth’s Fluid
Regaud’s Fluid (Moller’s)
Flemming’s without HAc
Formalin with post-chroming
Fixes Rickettsia org. and other bacteria
Orth’s fluid
Fixative that reacts with viruses, and causes the loss of their infective power
Mercuric Chloride
TRUE or FALSE. Glacial acetic acid destroys mitochondria and golgi bodies of the cytoplasm
TRUE
Gives the best quantitative results using frozen tissues as the standard
Ethanol and Acetone
Allows the GENERAL MICROSCOPIC STUDY of tissue structures WITHOUT ALTERING THE STRUCTURAL PATTERN normal intercellular relationship of tissues
Microanatomical Fixatives
Fixatives for Microanatomical:
10% Formol saline
10% Neutral buffered formalin
Heidenhain’s susa
Formol sublimate/ formol corrosive
Zenker’s formol (helly’s)
Zenker’s solution
Bouin’s
Brasil’s
Used to preserve chemical components of tissues like enzymes
Histochemical
Fixatives for Histochemical: (FAcNAe)
10% Formol saline
Acetone
Newcomer’s fluid
Absolute ethyl alcohol
Fixative used for the detection of rabies
Acetone
Types of Aldehyde Fixatives: (FGG)
Formaldehyde/ Formalin
Glutaraldehyde
Glyoxal
Types of Metallic Fixatives: (MCL)
Mercuric Chloride
Chromate Fixatives
Lead Fixatives
Types of Picric Acid:
Bouin’s solution
Brasil’s alcoholic picformol
Holllande’s solution
Types of Alcohol Fixatives (MINCER)
Methyl alcohol
Isopropyl
Newcomer’s
Carnoy’s
Ethyl alcohol
Rossmann’ solution
Others: Clarke’s solution, Methacarn
Used for routine; m
Most common fixative reagent
Soluble in water to the extent of 37-40% solution volume
AKA 100% formalin
Formaldehyde/ Formalin
Fixative recommended for mailing specimens (tolerant fixative) and for colored tissue photography
Formalin
Diluted form of the concentrated solution;
Routine tissue fixative;
Most commonly used fixative
10% formalin
Advantages of Formalin:
Cheap
Easy to prepare
Readily available
Can preserve fats
Stable
Disadvantages of Formalin:
Fumes are irritating
May cause dermatitis on prolonged contact
May form brown pigment on blood containing tissues like spleen
Methods in removal of Formalin pigments:
Kardasewitsch method
Lillie’s method
Picric Acid method
1% KOH in 80% alcohol
Component of Kardasewitsch method:
70% ethanol
28% ammonia water
Components of Lillie’s method:
Hydrogen Peroxide
28% ammonia water
Acetone
Components of Picric Acid method:
Saturated Alcoholic Picric Acid
Most widely used fixative for routine histology buffered to pH 7 with phosphate buffer
10% neutral buffered formalin (phosphate buffer)
It is the considered the fixative of choice for many other procedures that require paraffin embedding, including immunohistochemistry and interphase Fluorescent In-Situ Hybridization (FISH)
10% neutral buffered formalin
Fixative which is for post-mortem tissue and CNS tissues
Classified as histochemical fixative
10% formol saline
Remedy for precipitation of white paraformaldehyde
May add 10% methanol or filter it
Prolonged storage of formaldehyde = ?
Precipitation of white paraformaldehyde
TRUE or FALSE. Overnight fixation is generally indicated for 10mm thick slices of tissues
TRUE
TRUE or FALSE. Variations in time and conditions of fixation cause the majority of problems in histochemistry
TRUE
TRUE or FALSE. Formalin cannot preserve fats, mucin, and glycogen
FALSE; Formalin can preserve
TRUE or FALSE. Formalin can preserve proteins but it does not precipitate proteins
TRUE
TRUE or FALSE. Formalin does not make tissues brittle and therefore, it is the recommended for nervous tissue preservation
TRUE
TRUE or FALSE. Formalin is a soft fixative and does not harden some cytoplasmic structures adequately enough for paraffin embedding
TRUE
Prolonged fixation may produce:
- Bleaching of the specimen and loss of natural tissue colors
- Dispersal of fat grom the tissue into the fluid
- Dissolution or loss of glycogen and urate crystals
Recommended for fixing tissues with iron pigments and elastic fibers
10% Neutral Buffered Formalin or Phosphate Buffered Formalin
AKA FORMOL SUBLIMATE
Formol Corrosive
Mercuric chloride + formaldehyde; recommended for lipids, neutral fats, and phospholipids
Formol corrosive/ Formol sublimate
AKA Gendre’s solution
Alcoholic Formalin
Composition of Alcoholic Formalin:
95% ETOH
Picric Acid
Glacial Acetic Acid
Fixative to use to fix sputum specimen and for microincineration techniques
Alcoholic Formalin
When small tissues are burn into ashes to identify mineral elements from the ashes
Microincineration Techniques
Fixative used for enzyme histochemistry and electron microscopy
Glutaraldehyde
Formula of 10% formal-saline:
40% formaldehyde: 100ml
Distilled water: 900ml
Sodium dihydrogen phosphate monohydrate: 4gm
Disodium hydrogen phosphate anhydrous: 6.5gm
Formula of 10% neutral -buffered formalin
Distilled water
40% formaldehyde
Sodium dihydrogen phosphate, anhydrous
Sodium dihydrogen phosphate
Fixative that considered as alternatives to mercuric chloride formulations;
Can give improves results with immunohistochemistry
Zinc Formalin
Fixatives under Glutaraldehyde that are also for EM
Karnovsky’s paraformaldehyde-glutaraldehyde and ACROLEIN
Required solution of glutaraldehyde for small tissue fragments
2.5% solution
Required solution of glutaraldehyde for large tissues less than 4mm thick
4% solution
TRUE or FALSE. Glutaraldehyde can cause rapid and irreversible changes but it can fixes quickly and well suited for EM, it fixes well at 4 deg C and gives best overall cytoplasmic and nuclear detail
TRUE
TRUE or FALSE. Glutaraldehyde does not cause dermititis
TRUE
TRUE or FALSE. Glutaraldehyde is more expensive and less stable
TRUE
Supplied as 40% aqueous solution;
Commercially available;
Suited for urgent biopsies
Glyoxal
Fast acting fixative;
Can fix tissues rapidly;
Smallest aldehyde fixative;
Suited in urgent biopsies
Glyoxal
Surgical specimens are fixed within how many hours using glyoxal fixativesurgical specimens are fixed within how many hours using glyoxal fixative
4-6 hours
Small biopsy specimens are fixed within how many minutes using glyoxal fixative
45 minutes
Most common metallic fixative and may form black mercury deposits
Mercuric Chloride
Remedy for black mercury deposits formed by Mercuric Chloride
Wash tissue with alcoholic iodine (treating the section with 0.5% iodine solution in 70% ethanol for 5-10 minutes)
Mercuric Chloride is excellent for:
Trichrome staining
TRUE or FALSE. Mercuric chloride precipitates all proteins; has greater affinity to acid dyes and preferred in lieu of formaldehyde for cytoplasmic staining.
TRUE
It is recommended for renal tissue, fibrin, connective tissue and muscle
MERCURIC CHLORIDE
Contains mercuric chloride and glacial HAc;
Recommended for fixing liver, spleen, connective tissue fibers, and nuclei
Zenker’s Fluid
AKA Helly’s Fluid
Zenker’s formol
Contains potassium dichromate and 40% formaldehyde;
Preserves pituitary glands, bone marrow, and other blood containing organs
Zenker’s formol
Mercuric deposits may be removed by immersing tissues in ALCOHOLIC IODINE prior to staining, through a process known as …
De-zenkerization
Done by oxidation with iodine to form mercuric iodide
De-zenkerization (iodine - sodium thiosulfate - water)
Excellent fixative for bone marrow, extramedullary hematopoiesis and intercalated discs of cardiac muscle
Zenker’s Formol or HELLEY’S
Fixative combined with anhydrous acetate;
For preserving bone marrow
Lillie’s B5 fixative
Composition of B-5 Fixative
4% aqueous formaldehyde with 0.22M chloride and 0.22M acetic acid
TRUE or FALSE. Mercuric chloride ensures rapid structural stabilization and also facilitates bright staining by many of the dyes used in microtechnique
TRUE
Estimate fixation time for Lillie’s B5 fixative
4-8 hours
Fixatives under Mercuric Chloride: (BHZZ)
B5
Heidenhain’s susa
Zenker’s Fluid
Zenker’s Formol
Others: Schaudinn’s Ohlmacher’s, Carnoy-Lebrun solution
Used for acid mucopolysaccharides and tissue mucin
Lead Fixatives
Chromate fixative that preserves carbohydrates and precipitates all proteins
Chromic acid (1-2%)
Chromate fixative that preserve lipids and mitochondria
Potassium dichromate (3%)
AKA Muller’s fluid
Regaud’s
Chromic fixative for mitochondria, mitotic figures, golgi bodies, RBC, and containing colloid tissues
Regaud’s (Moller’s)
- chromate fixative for rickettsia and other bacteria
- also for tissue necrosis, and the early degenerative process.
Orth’s fluid
pH value used in potassium dichromate to fix mitochondria
pH 4.5-5.2
Agent can act as a fixative, stain, and decalcifying agent
PICRIC ACID
Excellent for glycogen demonstration and Brilliant staining with trichome method
PICRIC ACID
Major drawback of Picric Acid
Imparts a YELLOW color when used as a fixative
Remedy for drawback (yellow impart) of Picric Acid:
Saturated solution of Lithium carbonate in 70% alcohol then wash with water
The tissue is then placed in 70% ethanol followed by sodium thiosulfate and washed with water
TRUE or FALSE. Orth’s fluid preserves myelin better than buttered formalin
TRUE
TRUE or FALSE. Regaud’s and Orth’s fluid can preserve fats
FALSE; Regaud’s and Orth’s fluid does not preserve fats
Good fixative for connective tissue, preserves glycogen well, and extracts lipids to give superior results in immunostaining of biogenic and polypeptide hormones
Picric Acid Fixatives
TRUE or FALSE. Picric Acid is explosive in dry form
TRUE; should be stored in moist distilled water or saturated alcohol
This stain use combinations of anionic dyes with phosphotungstic or phosphomolybdic acid to impart contrasting colors to cytoplasm, collagen fibers and other components of tissues
Trichrome stains
- Fixative used for the fixation of embryo, pituitary biopsies, and endometrial curetting
- Usually an excellent fixative for preserving soft and delicate structures
Bouin’s solution
- This fixative is NOT for kidneys, lipids and mucus
- It abolishes Feulgen’s reaction
Bouin’s Fluid
Demonstrates RNAs and DNAs
Feulgen reaction
Excess picric should be washed from tissues prior to staining with…?
70% ethanol
A fixative known to be excellent for glycogen
Brasil’s Alcoholic Picformol
Fixative for GIT biopsies and endocrine tissues
Hollande’s solution
TRUE or FALSE. One disadvantage of using Bouin’s solution is that it causes RBC hemolysis and reduces the amount the demonstrable ferric iron in tissue
TRUE
TRUE or FALSE. Hollande’s solution produces LESS lysis than Bouin’s solution
TRUE
Fixative that is considered as Picric Acid and Alcohol
Gender’s fluid
It is the preferred fixative for tissues to be stained by Masson’s trichrome for collagen, elastic or connective tissue;
It does not need “washing out”
Gender’s fluid
Fixative that is better and less messy than Bouin’s solution
Brasil’s Alcoholic Picroformol Fixative
The major effects of Acetic Acid are…
Precipitate DNA and for preservation of nuclei
A compound fixative, recommended for nucleoproteins
Glacial Acetic Acid
Glacial acetic acid solidifies at what temperature?
17 deg C
TRUE or FALSE. Acetic acid is always incorporated into other fixatives to form a compound solution, most commonly at a concentration of approximately 5%
TRUE
Can be a fixative or decalcifying agent;
Also used for precipitation of proteins and nucleic acids
Trichloroacetic Acid (TCA)
TRUE or FALSE. TCA is a poor penetrating agent, and suitable only for small pieces of tissues or bones
TRUE
- Fixative used at ice cold temperature (-5-4 deg C);
- Can also fix and dehydrate tissue at the same time
ACETONE
Fixative recommended for preservation of water-diffusable enzymes (lipases, phosphatases) and for to fix brain tissue for diagnosis of rabies
ACETONE
Type of fixatives that rapidly denatures and precipitates proteins;
Can also act both as fixative and dehydrating agents;
Ideal for small tissue fragments
Alcohol Fixatives
TRUE or FALSE. Alcohols are protein denaturant and can cause too much brittleness and hardness, hence, not used routinely for tissues
TRUE
TRUE or FALSE. Solid specimens taken from patients with gout are usually fixed with 70% methanol for subsequent histochemical detection of sodium urate crystals
FALSE; 95% ETHANOL
Alcohol fixative that appears to give the most usable DNA fragments for PCR
ETHANOL
TRUE or FALSE. Alcohol can cause tissue shrinkage and not good for EM
TRUE
TRUE or FALSE. Absolute alcohol can be used to fix and preserve glycogen, pigments, blood, tissue films and smears
TRUE
Alcohol fixative for fixing wet and dry smears, blood smears and BM tissues;
Fixes and dehydrates at the same time
METHANOL (100%) or Methyl Alcohol
Alcohol fixative that is recommended for touch preparations, for special staining (Wright-Giemsa staining)
ISOPROPYL (95%)
For blood, tissue films and smears, and DNA (alcohol fixative);
Preserves nucleoproteins and nucleic acids, used for histochemistry especially for enzyme studies
ETHYL ALCOHOL (70-100%)
Most rapid fixative;
Contains alcohol, glacial HAc, and chloroform;
Can also used as fixative and can dehydrate at the same time
Carnoy’s fluid
Most rapid alcohol fixative;
For urgent biopsies (Chromosomes, Lymph glands), brain for diagnosis of Rabies
Carnoy’s fluid
- Fixative that is classified both as nuclear and histochemical fixative
- Can also preserve mucopolysaccharide and nuclear proteins
NEWCOMER’S FLUID
Alcohol fixative for CT mucins and umbilical cord
ROSSMANN’S
TRUE or FALSE. Lower concentration of ethanol (70-80%) will cause TBC hemolysis and inadequately preserve leukocytes
TRUE
Alcohol fixative that preserves Nissl granules and cytoplasmic granules;
Also permits good nuclear staining and differentiation
CARNOY’S
TRUE or FALSE. Carnoy’s fluid causes considerable tissue shrinkage and is suitable only for small pieces of tissues due to slow penetration. It also dissolves fat, lipids, and myelin
TRUE
- Alcoholic fixative
- Recommended for frozen sections and smears
- Can produce fair results after conventional processing if fixation time is kept very short
- Preserves nucleic acids but extracts lipids.
Clarke’s solution
Fixative for EM that is not commonly used and quite expensive;
It is also slow-acting fixative;
Excellent stain for lipids in membranous structures and vesicles
OSMIUM TETROXIDE
Tissues fspx of OSMIUM TETROXIDE
Myeline
Peripheral nerves
Processing neurological tissues
Most common chrome osmium acetic acid fixative, and excellent for nuclear structures
Flemming’s
- Known for cytoplasmic fixatives
- Preserves cytoplasmic structures particularly the mitochondria
- Composed of osmic acid and chromic acid
Flemming’s without HAc
Fixation time for Flemming’s solution
24-48 hours
Flemming’s solution has a tendency to form artifact pigments, what is the remedy of it?
Washing the fixed tissue in running tap water for 24 hours before dehydration
Fixatives for Enzyme Histochemistry:
4% formaldehyde
Formal saline
Fixative for electron histochemistry and electron immunocytochemistry
Karnovsky’s paraformaldehyde-glutaraldehyde
What substance is used in post chromatization during secondary fixation occurs
2.5-3% potassium dichromate (chromate-containing)
- Acts as a mordant for better staining
Placing an already fixed tissue to a second fixative to:
- Improve demonstration of particular substance
- Ensure complete hardening and
- For special staining
(not a mandatory step in tissue processing)
Secondary fixation
What fluids or solution that can be used during washing out process in order to remove excess fixative in tissue?
- Tap Water (often used)
- Excess chromates in tissues fixed and in Helly’s, Zenker’s and Flemming’s
- To remove excess - Alcoholic Iodine
50-70% alcohol
First step of tissue processing wherein the spx will be placed in 10% formalin for the first time?
Primary fixation
Microscopic study of NORMAL tissues
HISTOLOGY
4 Types of Tissues:
Connective tissues
Epithelial tissue
Muscle tissues
Nervous tissue
Microscopic study of ABNORMAL tissues
HISTOPATH
3 Types of Specimen that is processed in Histopathology:
Specimen of cytology
Autopsy specimen
Biopsy specimen
Tissues specimen obtained from a patient (ALIVE) for the examination in the lab
Biopsy specimen
Most common type of biopsy:
Incisional
Excisional
Needle biopsy/ Aspiration/ Fine needle aspiration biopsy (FNAB)
Type of biopsy that are not usually carried out:
Core biopsy and Shave biopsy
Type of biopsy that involves the removal of a part of a mass or organ
Incisional biopsy
Type of biopsy that involves removal of ENTIRE mass or organ;
Known as the most reliable and will give a greater chance to detect cancer
Excisional biopsy
Type of biopsy that uses needle and syringe to collect tissue spx
FNAB or Needle biopsy
Type of specimen that is obtained from a dead patient;
Also called as necropsy
Autopsy specimen
Specimens obtained for studying cell biology;
Example is PAP’s smear
Cytology specimens
Small fragments are shave from a surface usually skin
Shave biopsy
- Kind of specimen that will not undergo tissue processing
- It allows examination of protoplasmic activity (phagocytosis, motility)
- Tissues are not permanent it cannot be kept for future purposes
Fresh specimen
- Kind of specimen that usually facilitate or examine in histopath lab
- Specimen that are better and more effective
- Can keep the specimen on slides for future references
- Better and more effective
Processed tissues
- Type of cytology spx
- Example is PAPANICOLAU STAIN
- Used as a screening procedure for cervical cancer
Gynecologic specimens
- Cytology specimens such as sputum, CSF, gastric lavage, urine
- Can be used to detect urothelial malignancies
Non-gynecological specimen collected
4 method of examination are:
Teasing (Dissociation,
Squash Preparation (Crushing)
Smear Preparation
Frozen Section
Steps in Teasing/ dissociation:
- Get the watch glass, place small pieces of tissues, then add NSS
- Using a loop or needle aspirator, dissociate or separate.
- Place the separated tissues on a slide and examine it under the microscope
Stains that can be used in fresh tissue specimen
Supravital dyes/stains
Type of microscope that can be used to detect movement and mitotic division
Phase Contrast Microscope
Steps in squash preparation:
- On a slide, place the specimen
- Get another slide
- Compress the tissue in between two slides
(slide-to-slide then compress)
4 types of smear preparation
- Streaking
- Spreading
- Pull-apart
- T ouch preparation
Most recommended method of examination if dealing with cells
Smear preparation
Manner used in Streaking preparation
Zigzag manner
Advantage of Spreading preparation
Preserve intercellular relationship
Types of smear preparation that is suited for viscous specimens
Pull-apart and Streaking
Smear preparation that also called Impression smear and Abraded cytology
Touch preparation
Recommended glass slides for Touch Preparation
A. Frosted Glass Slide
B. Polished Ends Glass Slide
B. Polished Ends Glass Slide
Method of examination for rapid diagnosis and done intraoperatively;
Method used to demonstrate heat sensitive structures
Frozen section
Smear technique that allows slide to come in contact with freshly cut tissues (lymph node)
Touch Preparation/ Impression Smear/ Abdraded Cytology
Smear technique recommended for fresh sputum and bronchial aspirate, and thick mucoid secretions;
It also maintains intercellular components
Spreading
Method used for demonstration of fats/ lipids, nervous tissues elemets, and enzymes
Frozen section
Apparatus used in Frozen Section:
Freezing Microtome and Cryostat/ Cold Microtome
- Rapid diagnosis during surgery
- Diagnostic and research enzyme histochemistry
- Demonstration of soluble substances like lipids and carbohydrates
- Immunofluorescent and immunohistochemical staining
- Special staining in neuropathology
Applications of Frozen Sections:
FREEZING AGENTS:
- Liquid nitrogen
- Isopentane cooled by liquid nitrogen
- Carbon dioxide gas
- Aerosol sprays
BENEFITS OF FIXATION/ EFFECTS OF FIXATION:
- Prevents autolysis, reduce risk of infection
- Allows thin sectioning
- Acts as mordant thus facilitating staining
Results from frozen section must be released by how many minutes? (TAT)
5-15 minutes
Most common freezing agent used
Carbon dioxide
Average temp of cryostat or comb microtome
-20 deg C
First and most important step;
This is where you enter the details of patient in LOGBOOK
Numbering/ Accessioning
TRUE or FALSE. BRAIN tissues must be FIXED BEFORE GROSSING
TRUE
Before Fixation, large solid tissues should be…?
Opened or sliced thinly
Factors that can accelerate fixation:
- Heat application = 37-56 deg C only
- Size and thickness of tissue
- Agitation - continuous mixing —> allows rapid entry of fixative to tissue
Request form must include…
- Name of the patient
- Age
- Attending doctor
- Initial diagnosis
- Type of specimen
Not computerized
NUMBERING
Computerized
ACCESSIONING
Size of a tissue cassette
2.5 x 4 cm
Depth of a tissue cassette
5mm
An automatic tissue processor that can do fixation, dehydration, clearing, and infiltration;
Consists of 10 1L beakers that arranged in circular position
Autotech
Highest concertation of formalin
37 - 40% Formalin
Factors that can DECREASE the fixation time:
Increased heat
Agitation
Vacuum
Microwave
Small and thinner tissues specimen
Factors that ENHANCE fixation process:
Size and thickness
Agitation
Moderate heat (37 deg C)
TRUE or FALSE. Presence of mucus and blood in a specimen can be flush with NSS if there’s too many
TRUE
- Spx should be transferred to fixative immediately after surgery (< 1 hr.)
- Fixative to specimen: 20:1 or 10:1
- Anatomical barriers to fixation (should be removed)
- fascia (covering of organ)
- bones
- feces
- thick tissue - Large specimens must be sectioned or inflated with fixative or opened and cleaned to allow penetration
- Lungs —> inflate/ sectioned
- Intestines/gastrointestinal tract —> opened - Fixatives diluted and contaminated fixative - should be replaced to ensure effectiveness
- Pinning of spx to corkboard or inserting a paper or gauze “wick” into tubular structures can improve fixation and reduce tissue distortion
Practical Considerations to Optimize Fixation of Tissue
May be more difficult to reverse and may also result in loss of immunohistochemical antigenicity
Prolonged fixation
Increased fixation –> decreased immunohistochemical antigenicity
2 types of COMPOSITION for fixative:
Simple fixative
Compound fixative
Composition of Saline:
Distilled water
NaCl
Formaldehyde linked by 3C’
Glutaraldehyde
TRUE or FALSE. Methyl alcohol can cause BLINDNESS
TRUE
Microanatomical fixative should never contain ________ because it inhibits hematoxylin (for staining nucleus)
Osmium tetroxide
Preserving or demonstrating chemical substances in the tissue
Histochemical fixatives
Formaldehyde waste ways:
- Recycle by distillation
- Drain disposal
- Disposal by a licensed waste howler
- Detoxification by a commercial product
Mercurial reagents/ used to dezenkerize
- Releases mercury and must not go through drain
Disposal of mercury is expensive but it can replaced by ____
Zinc formalin or Glyoxal solutions
Fixative agent cannot be used in lipid fixation
ALCOHOL
Agents used in Lipid Fixation: (FB MAP D)
- Formaldehydes
- Baker’s formol-calcium -> for phospholipids
- Mercuric chloride
- Aldehydes
- Potassium dichromate - lipid for cryostat
- Digitonin -> cholesterol for ultra-structural demonstration
Fats (Frozen Section)
- Frozen section
- Formalin
- Potassium dichromate
- Osmic acid
- Formol calcium
Agents for Protein Fixation:
- Neutral Buffered Formalin
- Formaldehyde Vapor
Agents for Carbohydrate Fixation:
- Alcoholic fixatives (glycogen fixation)
- Rossman’s fluid
- Cold absolute alcohol
BRAIN must suspend WHOLE in ____ for 2-3 weeks to ensure hardening
10% buffered formalin
TRUE or FALSE. Formaldehyde should never be neutralized because it may cause violent explosion
TRUE
REMOVAL OF PARAFORMALDEHYDE
- Use methanol to prevent its decomposition to formic acid
- Can filter paraformaldehyde
Color of formalin pigments
Brown or Black
Formalin pigments occur because of…?
Reaction of formic acid and hemoglobin (blood)
Formaldehyde (Formalin) recommended for ___
CNS
It fixes sputum since it coagulates mucus
Alcoholic formalin (Gendre’s)
All mercuric chloride fixatives cause black precipitate except…?
A. Zenker’s fluid
B. Zenker’s formol
C. Heidenhain’s Susa
D. B5
C. Heidenhain’s Susa
TRUE or FALSE. Mercuric chloride is used for tissue photography
TRUE
What to use for removal of black deposits from Mercuric chloride?
Use saturated iodine in 96% alcohol
- Contains glacial acetic acid, mercuric chloride, potassium dichromate, sodium sulfate, and distilled water
- Recommended for small pieces of liver, spleen, connective tissue fibers, nucleic components
Zenker’s fluid
- Zenker’s formol
- Contains formaldehyde, mercuric chloride, potassium dichromate
- Good for microanatomical fixative
- Pituitary gland, bone marrow, blood-containing organs (spleen and liver)
Helly’s fluid
- Recommended for tumor biopsies of the skin
Heidenhain’s Susa
Fixative used for PRESERVATION OF BONE MARROW BIOPSIES
B5 fixative
Composition of Bouin’s solution:
Picric acid saturated aqueous solution
40% Formaldehyde
Glacial acetic acid
- Process of removing the excess fixative after fixation
- To improve staining
- To remove artifacts from the tissue
Washing out
- Less concentrated alcohol fixatives = less lysis
- Used in small tissue fragments
Alcoholic fixatives used in concentration of 70-100%
In WASHING OUT:
- Excess chromates in tissues fixed and in Helly’s, Zenker’s and Flemming’s
- To remove excess formalin and cosmic acid
Tap water (washing out)
In WASHING OUT:
- To remove excess picric acid fixatives (Bouin’s solution)
50-70% alcohol (washing out)
In WASHING OUT:
- To remove excess mercuric fixatives
Alcoholic iodine (washing out)
- Tissue in a fixated
- 450 watts at 55 deg C for 1.5 minutes to 4 minutes
- May create fumes
Microwave assisted fixation
ADVANTAGE OF FIXING TISSUES IN MICROWAVE:
- Tissues is heated through the block for a very short time; potentially allowing rapid study of cellular processes.
- Can used in neurochemical substances (brain, acetylcholine)
- For rapid fixation: routine surgical specimens
- Immunohistochemistry and In-Situ hybridization
DISADVANTAGE OF FIXING TISSUES IN MICROWAVE:
- Only penetrates tissues to thickness of 10-15mm
- No significant cross-linking of protein molecules, and subsequent chemical fixation may be needed
- Viable spores and pathogens may remain in tissues processed with alcohol-based fixatives or microwave alone
Physical method that preserves tissue by rapid exposure of the spx to cold temperatures (-160 to -180 dec C)
Freeze drying/ Freeze substitution
3 types of Specimen
Biopsy
Autopsy
Cytology
