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