MTAP (Gross Exam- Fixation) Flashcards
1
Q
A properly completed histopathology requisition form contain:
A
- patient’s name
- age
- sex
- relevant clinical data
- surgical findings
- nature of operation
- name of tissue submitted
2
Q
The specimen placed on the cutting board in an anatomic position and the following information must be recorded:
Quick recall ^_^
A
- Types of specimen
- Structure include
- Dimensions (length and width)
- Weight
- Shape
- Color
- Consistency
- Surgical margin
3
Q
In recording the suture margin of the specimen what must be included?
A
- Sutures
- Surgical inking
4
Q
These should be measured by aggregate pieces in volume
A
Endometrial and prostatic tissue
5
Q
Measurements are usually given in _________ unless the specimen is very small in which ______ can be used
A
centimeters; millimeters
6
Q
Most specimens from solid tissues are cut in the form of pieces measuring ___________ on the slides and _______ in thickness
A
10-15 mm; 2-3 mm
7
Q
Discrete areas of calcification or ossication should be taken out and should be decalcified in?
A
3% nitric acid
8
Q
Small fragments of tissue must be wrapped in?
A
thin paper
9
Q
Ovaries filtrated with hard tissues have white portions which are _____ and discolorations that could be _______
A
tumors; cancers
10
Q
Decalcification in nitric acid takes about
A
24 hrs to 3 days
11
Q
Its aim is to have good output in biopsy
A
Fixation
12
Q
As soon as cells or tissues are removed from the body they begin to die and undergo?
A
post mortem changes (decomposition)
13
Q
A self-destructive process where cells release autolytic enzymes, leading to the breakdown of cell components.
A
Autolysis
14
Q
Action from cells
A
Autolysis
15
Q
Occurs when bacteria invade the tissue and cause decomposition; unlike autolysis, it is driven by bacterial action
A
Putrefaction
16
Q
Action from bacteria
A
Putrefaction
17
Q
To stop the decomposition what fixative is commonly used?
A
10% buffered formalin
18
Q
List the main aims of fixation
A quick recall lang hehehe ^_^
A
- 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).
18
Q
Most commonly used fixative because it penetrates tissues effectively, prevents autolysis/putrefaction, and is relatively inexpensive compared to other fixatives.
A
10% buffered formalin
18
Q
Fixative to specimen ratio
A
20:1 (10:1 sa other books daw)
19
Q
What factors are crucial for obtaining satisfactory fixation results? CUP
A
- Correct choice of fixative
- Use of fresh tissue
- Proper penetration of the fixative
19
Q
What is a key limitation regarding the penetration of fixatives?
A
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).
20
Q
These are cut into slices as necessary but not thicker than 5 mm
A
Solid organ
21
Q
It is fatty and not fully miscible in formaldehyde and require to be cut twice before soaking in 10% buffered formalin
A
Breast tissue
21
It is either opened or filled with fixative or pack lightly with wool soaked in fixative.
Hollow organ
21
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
21
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
21
Most of these act by denaturing or precipitating proteins which then form a sponge or meshwork, tending to hold the other constituents
fixatives
22
Methods of fixation:
- Heat fixation
- Perfusion fixation
- Immersion fixation (most common)
- Vapor method
- Phase partition method
*last 4 (PIVP) are quite expensive*
22
Method of fixation requires special equipment to deliver the fixative.
Perfusion fixation
22
Method of fixation that uses the vapors of the fixative to fix the tissue
Vapor method
22
Method of fixation often used in microbiology
Heat fixation
22
Method of fixation where the tissue is submerged in the fixative
Immersion fixation
22
What is the simplest form of physical fixation?
Heat fixation
23
It refers to using an elevated temperature to fix tissue or cells.
Heat fixation
23
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)
24
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
25
Factors that speed up fixation: AIA
1. agitation in fixations
2. Increase temperature
3. add in osmolality or osmolarity of the solution to penetrate easily
26
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
26
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
26
Utilizes organic or non-organic solutions to maintain adequate morphological preservation.
Chemical fixation
26
Chemical fixatives can be considered as members of three major categories:
3Cs
● Coagulant
● Cross-linking
● Compound fixatives
27
It is prepared by mixing 40% Formaldehyde gas in 100 w/v of distilled water.
Formalin
27
- It forms cross-links between amino acids of proteins, thereby making them insoluble.
- It fixes 4 mm thick tissue in 8 hours.
Formalin
27
Made up of two formaldehyde residues, linked by a three-carbon chain.
Glutaraldehyde
28
Causes rapid and irreversible changes, fixes quickly, and is well suited for electron microscopy
Glutaraldehyde
29
This is satisfactory for electron microscopy
A standard fixative (2% buffered glutaraldehyde) followed by secondary fixation in osmium tetroxide
30
Glutaraldehyde in fixing small tissue fragment and neddle biopsies:
2.5% solution fixed in 2-4 hours at room temperature
31
Glutaraldehyde in fixing larger tissues less than 4mm thick:
4% solution fixed in 6-8 hours up to 24 hours
32
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
33
Causes the complete denaturation of protein and is used in electron microscopy both as a fixative and a heavy metal stain.
Osmium Tetroxide
34
A secondary fixative by reacting with lipids.
Osmium Tetroxide
35
Penetrates slower than glutaraldehyde (**0.5 mm/hr**) and the tissue takes on a progressively blackened appearance
Osmium Tetroxide
36
Most common chrome-osmium acetic acid, for nuclear preparation of such sections.
Flemming's Solution
36
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(?).
36
Chromic and osmic acid, for **cytoplasmic structures**, particularly the ***mitochondria***
Flemming's Solution without Acetic Acid
36
Flemming's Solution without Acetic Acid fixation time:
24-48 hours
37
Why is acetic acid removed in Flemming's Solution without Acetic Acid?
To improve the cytoplasmic detail of the cell.
37
Used in a **3% aqueous solution**, it fixes but does not precipitate cytoplasmic structures. It also **preserves lipids and mitochondria**.
Potassium Dichromate
37
Potassium dichromate 2.5gm 100 ml, Sodium sulfate (optional) 1 gm.
➔ Fixation time: 36-72 hours.
Orth's Fluid
37
3% Potassium dichromate - 80 ml.
➔ Fixation time: 12-48 hours
Regaud’s (Muller’s) Fluid
37
Most common metallic fixative, frequently used in saturated aqueous solutions of 5-7%.
Mercuric Chloride
37
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
37
Widely used as a secondary fixative reacting with a number of amino acid residues.
Mercuric Chloride
37
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)
37
Staining of the **nucleus, cytoplasm, and connective tissue** are good with _________
Helly’s fluid
37
Fixation time of Lillie's B-5 Fixative
4-8 hours
37
By adding __________ to Zenker’s solutions, the beneficial effects of both fixatives are combined, minimizing their disadvantages.
formalin
37
Formula:
● Mercuric chloride- 5 gm
● Potassium dichromate- 2.5 gm
● Distilled water- 100 ml
➔ Fixation time: 4-24 hours.
Zenker-Formol (Helly’s) Solution
37
Rapidly penetrate tissues and permit excellent staining of **nuclei and connective tissues**.
Zenker's Fluid (Mercuric Chloride Fixative)
37
Lillie's B-5 Fixative working solution (prepare immediately before use):
● B-5 stock solution - 20 ml
● 40% formaldehyde - 2 ml
37
Fixed tissue should be washed overnight in running tap water before processing.
Disadvantage: Causes hardening of tissues.
Zenker's Fluid (Mercuric Chloride Fixative)
37
These are used to preserve the anatomy of the tissue.
Microanatomical Fixatives
37
Used to fix intracellular structures.
Cytological Fixatives
37
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
37
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)
37
Lillie's B-5 Stock solution:
● Mercuric chloride - 12 g
● Sodium acetate anhydrous - 2.5 g
● Distilled water - 200 ml
37
Explosive hazard in dry form.
Picric Acid
37
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)
37
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
37
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
37
Formula:
● Formaldehyde (37-40%) - 10 ml
● Distilled water - 90 ml
● Mix well.
1-10% Formal Saline
37
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)
38
Causes even fixation and very little shrinkage due to its isotonicity.
1-10% Formal Saline
38
Disadvantage of 10% Neutral Buffered Formalin (NBF)
● Laborious and time-consuming.
● A lot of residue and artifacts seen during microscopic examination.
38
This is NOT used to fix Pap smear
Formalin
38
Used for Pap smear and FNAB
Alcohol fixative
38
Formula:
● Absolute alcohol - 60 ml
● Chloroform - 30 ml
● Glacial acetic acid - 10 ml
➔ Fixation Time: 1-3 hours
Carnoy's Fluid
39
When using this fixative, the fixed tissue should be processed immediately or transferred to 80% alcohol.
Carnoy's Fluid
40
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
41
Formula:
● Ethanol (absolute) - 75 ml
● Acetic acid glacial - 25 ml
➔ Fixation time: 3 - 4 hours
Clarke's fluid
42
Formula:
● Chromium trioxide - 1 g
● Potassium dichromate - 3 g
● Osmium tetroxide - 1 g
● Distilled water - 250 ml
Champy's fluid
42
Formula:
● Potassium dichromate 3% - 80 ml
➔ Fixation time: 12-48 hours
Regaud’s (Muller’s) Fluid
42
Used to demonstrate the chemical constituents of the cell.
Histochemical Fixatives
43
These are the reagents used for histopathology and are monitored by the government:
● Benzene
● Chloroform
● Some stains
● Acetone
43
Include formaldehyde (formalin) and glutaraldehyde. Good for immunohistochemistry techniques.
Aldehydes
43
Penetrates tissue well but is relatively slow.
Formalin
43
Aldehydes standard solution
10% neutral buffered formalin.
44
Fix tissue by an unknown mechanism. Contain mercuric chloride and include well-known fixatives like Zenker’s.
Mercurials
45
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
45
Penetrate relatively poorly and cause some tissue hardness but are fast and give excellent nuclear detail
Mercurials
46
These histochemical fixatives are used for fatty tissue
Mercurials and aldehydes
46
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
46
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)
46
Compound Fixative include:
1. Microanatomical Fixative (1-10% Formol Saline, 10% Neutral Buffered Formalin)
2. Cytological Fixatives (Alcohol Fixative and Nuclear Fixative which under this kay ang Carnoy's Fluid, Clarke's Fluid, Newcomer's Fluid, Flemming's Solution)
3. Cytoplasmic Fixative (Champy's Fluid and Regaud's Fluid)
4. Histochemical Fixatives
46
Simple fixatives include:
1. Formalin
2. Glutaraldehyde
3. Osmium Tetroxide (Flemming's Solution, Flemming's Solution without Acetic Acid)
4. Potassium Dichromate (Regaud Fluid, Orth's Fluid)
5. Mercuric Chloride (Zenker's Solution, Zenker-Formol Solution, Zenker Fluid, Lillie's B-5 Fixative)
6. Picric acid (Bouin's Fluid, Gendre's Solution,
