MTAP (Gross Exam- Fixation) Flashcards
A properly completed histopathology requisition form contain:
- patient’s name
- age
- sex
- relevant clinical data
- surgical findings
- nature of operation
- name of tissue submitted
The specimen placed on the cutting board in an anatomic position and the following information must be recorded:
Quick recall ^_^
- Types of specimen
- Structure include
- Dimensions (length and width)
- Weight
- Shape
- Color
- Consistency
- Surgical margin
In recording the suture margin of the specimen what must be included?
- Sutures
- Surgical inking
These should be measured by aggregate pieces in volume
Endometrial and prostatic tissue
Measurements are usually given in _________ unless the specimen is very small in which ______ can be used
centimeters; millimeters
Most specimens from solid tissues are cut in the form of pieces measuring ___________ on the slides and _______ in thickness
10-15 mm; 2-3 mm
Discrete areas of calcification or ossication should be taken out and should be decalcified in?
3% nitric acid
Small fragments of tissue must be wrapped in?
thin paper
Ovaries filtrated with hard tissues have white portions which are _____ and discolorations that could be _______
tumors; cancers
Decalcification in nitric acid takes about
24 hrs to 3 days
Its aim is to have good output in biopsy
Fixation
As soon as cells or tissues are removed from the body they begin to die and undergo?
post mortem changes (decomposition)
A self-destructive process where cells release autolytic enzymes, leading to the breakdown of cell components.
Autolysis
Action from cells
Autolysis
Occurs when bacteria invade the tissue and cause decomposition; unlike autolysis, it is driven by bacterial action
Putrefaction
Action from bacteria
Putrefaction
To stop the decomposition what fixative is commonly used?
10% buffered formalin
List the main aims of fixation
A quick recall lang hehehe ^_^
- Prevent autolysis and putrefaction.
- Penetrate evenly and rapidly (from the periphery inward).
- Harden tissues to maintain structure.
- Enhance optical differentiation of cells and tissues.
- Avoid causing shrinkage or swelling (especially if fixation exceeds 48 hours).
- Not react with receptor sites, ensuring staining procedures are not interfered with.
- Be cost-effective, considering the large volumes required (e.g., a 20:1 ratio).
Most commonly used fixative because it penetrates tissues effectively, prevents autolysis/putrefaction, and is relatively inexpensive compared to other fixatives.
10% buffered formalin
Fixative to specimen ratio
20:1 (10:1 sa other books daw)
What factors are crucial for obtaining satisfactory fixation results? CUP
- Correct choice of fixative
- Use of fresh tissue
- Proper penetration of the fixative
What is a key limitation regarding the penetration of fixatives?
They will NOT penetrate a piece of tissue thicker than 1 cm, so larger specimens must be sliced (ex. solid organs should be cut into slices not thicker than 5 mm).
These are cut into slices as necessary but not thicker than 5 mm
Solid organ
It is fatty and not fully miscible in formaldehyde and require to be cut twice before soaking in 10% buffered formalin
Breast tissue
It is either opened or filled with fixative or pack lightly with wool soaked in fixative.
Hollow organ
It requires dissection, injection of fixative along the vessels or bronchi in case of lung so that it reaches all parts of the organs.
Large specimen
At the molecular level, _______ have the property of coagulating proteins in the tissue, through the formation of crosslinks between protein molecules thereby keeping their relation to each other
fixative
Most of these act by denaturing or precipitating proteins which then form a sponge or meshwork, tending to hold the other constituents
fixatives
Methods of fixation:
- Heat fixation
- Perfusion fixation
- Immersion fixation (most common)
- Vapor method
- Phase partition method
last 4 (PIVP) are quite expensive
Method of fixation requires special equipment to deliver the fixative.
Perfusion fixation
Method of fixation that uses the vapors of the fixative to fix the tissue
Vapor method
Method of fixation often used in microbiology
Heat fixation
Method of fixation where the tissue is submerged in the fixative
Immersion fixation
What is the simplest form of physical fixation?
Heat fixation
It refers to using an elevated temperature to fix tissue or cells.
Heat fixation
How does microwave fixation speed up the fixation process?
Microwave heating accelerates fixation by increasing temperature, improving penetration, and reducing fixation time from hours to minutes (from 12 hrs to less than 20 mins)
Speeds fixation and can reduce times for fixation of some gross specimens and histological sections from more than 12 hrs to less than 20 mins.
Microwave fixation
Factors that speed up fixation: AIA
- agitation in fixations
- Increase temperature
- add in osmolality or osmolarity of the solution to penetrate easily
Useful technique for studying soluble materials and small molecules; tissues are cut into thin sections, immersed in liquid nitrogen, and the water is removed in a vacuum chamber at -40°C. Used for cryostat.
Freeze-Drying
Similar to freeze-drying but involves replacing water in frozen tissue with an organic solvent at low temperatures. The tissue can be post-fixed with formaldehyde afterward.
Freeze-substitution
Utilizes organic or non-organic solutions to maintain adequate morphological preservation.
Chemical fixation
Chemical fixatives can be considered as members of three major categories:
3Cs
● Coagulant
● Cross-linking
● Compound fixatives
It is prepared by mixing 40% Formaldehyde gas in 100 w/v of distilled water.
Formalin
- It forms cross-links between amino acids of proteins, thereby making them insoluble.
- It fixes 4 mm thick tissue in 8 hours.
Formalin
Made up of two formaldehyde residues, linked by a three-carbon chain.
Glutaraldehyde
Causes rapid and irreversible changes, fixes quickly, and is well suited for electron microscopy
Glutaraldehyde
This is satisfactory for electron microscopy
A standard fixative (2% buffered glutaraldehyde) followed by secondary fixation in osmium tetroxide
Glutaraldehyde in fixing small tissue fragment and neddle biopsies:
2.5% solution fixed in 2-4 hours at room temperature
Glutaraldehyde in fixing larger tissues less than 4mm thick:
4% solution fixed in 6-8 hours up to 24 hours
Osmium Teroxide is a pale yellow powder which dissolves in water up to ________ at ____ to form a strong oxidizing solution.
up to 6% at 20°C
Causes the complete denaturation of protein and is used in electron microscopy both as a fixative and a heavy metal stain.
Osmium Tetroxide
A secondary fixative by reacting with lipids.
Osmium Tetroxide
Penetrates slower than glutaraldehyde (0.5 mm/hr) and the tissue takes on a progressively blackened appearance
Osmium Tetroxide
Most common chrome-osmium acetic acid, for nuclear preparation of such sections.
Flemming’s Solution
Flemming’s Solution formula & fixation time:
Formula:
● Aqueous chromic acid 15 ml
● 1% Aqueous osmium tetroxide 4 ml
● 2% Glacial acetic acid 1 ml
Fixation time: 24 - 48 bouts/hours(?).
Chromic and osmic acid, for cytoplasmic structures, particularly the mitochondria
Flemming’s Solution without Acetic Acid
Flemming’s Solution without Acetic Acid fixation time:
24-48 hours
Why is acetic acid removed in Flemming’s Solution without Acetic Acid?
To improve the cytoplasmic detail of the cell.
Used in a 3% aqueous solution, it fixes but does not precipitate cytoplasmic structures. It also preserves lipids and mitochondria.
Potassium Dichromate
Potassium dichromate 2.5gm 100 ml, Sodium sulfate (optional) 1 gm.
➔ Fixation time: 36-72 hours.
Orth’s Fluid
3% Potassium dichromate - 80 ml.
➔ Fixation time: 12-48 hours
Regaud’s (Muller’s) Fluid
Most common metallic fixative, frequently used in saturated aqueous solutions of 5-7%.
Mercuric Chloride
Formula:
● Mercuric chloride- 5 gm
● Potassium dichromate- 2.5 gm
● Distilled water- 100 ml
● Acetic acid, glacial- 5 ml
➔ Fixation time: 12-24 hours.
Zenker’s Solution
Widely used as a secondary fixative reacting with a number of amino acid residues.
Mercuric Chloride
Due to the presence of a buffer, the pH of the solution remains at neutral or near neutral. It also prevents Formalin pigment formation.
10% Neutral Buffered Formalin (NBF)
Staining of the nucleus, cytoplasm, and connective tissue are good with _________
Helly’s fluid
Fixation time of Lillie’s B-5 Fixative
4-8 hours
By adding __________ to Zenker’s solutions, the beneficial effects of both fixatives are combined, minimizing their disadvantages.
formalin
Formula:
● Mercuric chloride- 5 gm
● Potassium dichromate- 2.5 gm
● Distilled water- 100 ml
➔ Fixation time: 4-24 hours.
Zenker-Formol (Helly’s) Solution
Rapidly penetrate tissues and permit excellent staining of nuclei and connective tissues.
Zenker’s Fluid (Mercuric Chloride Fixative)
Lillie’s B-5 Fixative working solution (prepare immediately before use):
● B-5 stock solution - 20 ml
● 40% formaldehyde - 2 ml
Fixed tissue should be washed overnight in running tap water before processing.
Disadvantage: Causes hardening of tissues.
Zenker’s Fluid (Mercuric Chloride Fixative)
These are used to preserve the anatomy of the tissue.
Microanatomical Fixatives
Used to fix intracellular structures.
Cytological Fixatives
Causes short fixation time. Fixed tissue should be transferred to 70% alcohol.
Formula:
● Saturated aqueous solution of picric acid - 75 ml
● Formalin (~40% aqueous solution of formaldehyde) - 25 ml
● Glacial acetic acid - 5 ml
➔ Fixation time: 6 hours
Bouin’s Fluid
Formula:
● Distilled water - 950 ml
● Potassium dichromate - 25 g
● Mercuric chloride - 50 g
● Glacial acetic acid - 50 g
➔ Fixation time: 4-24 hours
Zenker’s Fluid (Mercuric Chloride Fixative)
Lillie’s B-5 Stock solution:
● Mercuric chloride - 12 g
● Sodium acetate anhydrous - 2.5 g
● Distilled water - 200 ml
Explosive hazard in dry form.
Picric Acid
Recommended for research specimens but may also be used for surgical and post-mortem specimens. Prevents the formation of troublesome acid formalin pigments.
10% Neutral Buffered Formalin (NBF)
It is a microanatomical fixative. Ideal for fixation of the brain and is recommended for fixation of general surgical biopsy specimens and tissues from CNS.
1-10% Formal Saline
Formula:
● 95% Ethanol saturated with picric acid - 800 ml
● 40% Formaldehyde - 150 ml
● Acetic acid glacial - 50 ml
➔ Fixation time: 4 - 18 hours
Gendre’s Solution
Formula:
● Formaldehyde (37-40%) - 10 ml
● Distilled water - 90 ml
● Mix well.
1-10% Formal Saline
Formula:
● Formaldehyde (37-40%) - 100 ml
● Distilled water - 900 ml
● NaH₂PO₄ - 4.0 g
● Na₂HPO₄ (anhydrous) - 6.5 g (for buffer)
● Mix to dissolve.
10% Neutral Buffered Formalin (NBF)
Causes even fixation and very little shrinkage due to its isotonicity.
1-10% Formal Saline
Disadvantage of 10% Neutral Buffered Formalin (NBF)
● Laborious and time-consuming.
● A lot of residue and artifacts seen during microscopic examination.
This is NOT used to fix Pap smear
Formalin
Used for Pap smear and FNAB
Alcohol fixative
Formula:
● Absolute alcohol - 60 ml
● Chloroform - 30 ml
● Glacial acetic acid - 10 ml
➔ Fixation Time: 1-3 hours
Carnoy’s Fluid
When using this fixative, the fixed tissue should be processed immediately or transferred to 80% alcohol.
Carnoy’s Fluid
Formula:
● Isopropyl alcohol - 60 ml
● Propionic acid - 30 ml
● Petroleum - 30 ml
● Ether - 10 ml
● Acetone - 10 ml
● Dioxane - 10 ml
➔ Fixation time: 12-18 hours at 3°C
Newcomer’s Fluid
Formula:
● Ethanol (absolute) - 75 ml
● Acetic acid glacial - 25 ml
➔ Fixation time: 3 - 4 hours
Clarke’s fluid
Formula:
● Chromium trioxide - 1 g
● Potassium dichromate - 3 g
● Osmium tetroxide - 1 g
● Distilled water - 250 ml
Champy’s fluid
Formula:
● Potassium dichromate 3% - 80 ml
➔ Fixation time: 12-48 hours
Regaud’s (Muller’s) Fluid
Used to demonstrate the chemical constituents of the cell.
Histochemical Fixatives
These are the reagents used for histopathology and are monitored by the government:
● Benzene
● Chloroform
● Some stains
● Acetone
Include formaldehyde (formalin) and glutaraldehyde. Good for immunohistochemistry techniques.
Aldehydes
Penetrates tissue well but is relatively slow.
Formalin
Aldehydes standard solution
10% neutral buffered formalin.
Fix tissue by an unknown mechanism. Contain mercuric chloride and include well-known fixatives like Zenker’s.
Mercurials
Factors Affecting Fixation:
➔ Temperature: Increasing temperature increases the speed of fixation.
➔ Size of the specimen.
➔ Volume ratio:
-The volume of the fixative is important.
-There should be a 10:1 ratio of fixative to tissue.
➔ Duration of Fixation.
➔ Choice of fixatives.
➔ Penetration: Depends on the diffusibility of each individual fixative.
➔ Tissue Storage.
➔ Buffer & pH:
-Best carried out close to neutral pH, in the range of 6-8.
➔ Osmolality
Penetrate relatively poorly and cause some tissue hardness but are fast and give excellent nuclear detail
Mercurials
These histochemical fixatives are used for fatty tissue
Mercurials and aldehydes
Includes methyl alcohol (methanol) and ethyl alcohol (ethanol). Very good for cytologic smears as they act quickly and give good nuclear detail.
➔ Used for: Pap Smear, FNAB.
Alcohols
Other Factors Affecting Fixation: CT
- Concentration of fixative (should be adjusted to the lowest level possible)
- Time interval (time interval from removal of th tissues to the fixation)
Compound Fixative include:
- Microanatomical Fixative (1-10% Formol Saline, 10% Neutral Buffered Formalin)
- Cytological Fixatives (Alcohol Fixative and Nuclear Fixative which under this kay ang Carnoy’s Fluid, Clarke’s Fluid, Newcomer’s Fluid, Flemming’s Solution)
- Cytoplasmic Fixative (Champy’s Fluid and Regaud’s Fluid)
- Histochemical Fixatives
Simple fixatives include:
- Formalin
- Glutaraldehyde
- Osmium Tetroxide (Flemming’s Solution, Flemming’s Solution without Acetic Acid)
- Potassium Dichromate (Regaud Fluid, Orth’s Fluid)
- Mercuric Chloride (Zenker’s Solution, Zenker-Formol Solution, Zenker Fluid, Lillie’s B-5 Fixative)
- Picric acid (Bouin’s Fluid, Gendre’s Solution,
