W7L1 - Introduction to Microscopy

Question 1

Specimen Collection - Reporting Location and Types of Lesions

Answer 1

Description of anatomical sites 
- e.g. head of pancreas
ID by placement of a suture or staple
ID by radiological imaging
Number of lesions and distance between lesions

Question 2

Specimen Collection - Prior/Current Patient Treatment

Answer 2

Radiotherapy/chemotherapy can result in changes that may mimic malignancy
Certain cancers can be difficult to find on gross examination following recent treatment, but may be visible microscopically
Drug therapies/immunotherapy can alter histologic appearance of tissues
Drugs can also make patients more susceptible to infections by lowering the immune system

Question 3

Urgent Specimens

Answer 3

Those from critically ill patients, are often given priority over routine specimens, to aid clinical management
Urgent samples are usually required for surgical margins during resection

Question 4

What to do to prevent autolysis from occurring with recently collected sample?

Answer 4

Usually commences immediately after surgical removal of tissues
Can be prevented by storing specimens in -20◦C
Extended delays prior to adequate fixation will affect the diagnostic quality of tissues and may reduce immunoreactivity for some biomarkers
All tissue or objects removed from a patient should be considered hazardous and must be transported safely in a rigid leakproof container
- most are double sealed in an additional zip locked bag to prevent loss or contamination of specimen

Question 5

Orientation of Specimen

Answer 5

Sutures of variable length, or number can be used as anatomical landmarks
Two sutures at right angles are required to ID the remaining 4 margins
Stitches can also be used
Commonly, the long sutures are used to represent lateral margins and short sutures represent superior margins

Question 6

Inking

Answer 6

Small bx for non neoplastic disease (i.e. hyperplastic polyps in colons or diverticulitis) are not required to be inked
Small bx for neoplastic lesions should be inked in their entirety before processing

Question 7

Dissection/Prosection

Answer 7

Specimens need to be completely dissected or serially sectioned prior to reporting
Most small biopsies are not dissected further
Most large organs/hollow structures are described, photographed, weighed and opened for further investigation

Question 8

Margins

Answer 8

The margins are observed on all resections to document the presence or absence of tumour or viability of the resection margin
Two types of margins are used:
- en face margins
- perpendicular to resection
Type of margin used during sectioning must be documented

Question 9

En Face Margins - Advantages and Disadvantages

Answer 9

Advantages:
- 10-100 times more surface area is examined than when sectioned in a perpendicular plane
- entire anatomical structure can be evaluated
Disadvantages:
- the exact distance of the tumour from the margin cannot be measured

Question 10

Perpendicular Margins - Advantages and Disadvantages

Answer 10

Advantages:
- the exact distance of the tumour from the margin is determined
- recommended when a small rim of uninvolved tissue would be considered a negative
margin
- most pathologists are familiar with this type of margin
Disadvantage:
- minimal tissue in the margin is sampled in large resections

Question 11

Selection of Tissue for Microscopy

Answer 11

All lesions;
If multiple similar lesions are present, tissue between the lesions is submitted to determine if this is the same neoplasm, different or interconnected
Lesional tissue placed in special fixative
Lymph nodes - most important of all tumour resection

Question 12

Oversampling and Undersampling

Answer 12

Oversampling - wasteful of resources and unnecessarily increasings expenses
Undersampling - important dx or prognostic information may be lost, leading to suboptimal pathology and incorrect patient management

Question 13

Aims of Fixation

Answer 13

Fixation aims to;

• preserve tissue - by preventing autolysis by cellular enzymes and further decomposition by bacteria/molds
• harden tissue - to allow sectioning
• inactivate further infectious organisms
• stabilise tissue components - proteins, biomarker, antigenic sites
• enhance avidity for dyes

Question 14

Undesirable Effects during Fixation - Alteration of Protein Structure

Answer 14

May result in cross linked proteins or change the tertiary structure resulting in loss of antigenic targets
May cause solubility of tissue components such as lipids and carbohydrates (e.g. glycogen may be lost)
May result in artificial shrinkage of tissue resulting in incorrect tumour sizes
DNA and RNA degradation, especially in those fixatives that contain picric acid, Mol Pathology can not be performed on these cases

Question 15

Undesirable Effects during Fixation - Volume of Fixative

Answer 15

An adeq amount is considered to be 15 to 20 times the volume of the tissue
If a sample is received in saline, the saline should be discarded prior to fixation
Fixative contaminated with blood or other fluids will be diluted and therefore the tissue will not fix well

Question 16

Undesirable Effects during Fixation - Fixatives Penetrate Slowly (0.1 cm/hour)

Answer 16

There may be anatomical barriers to prevent fixation
Large specimens should be thinly sectioned
Gauge pads can be helpful in smaller biopsies
Large resections like colectomy, can be pinned out and immersed upside down in a large container of fixative

Question 17

Undesirable Effects during Fixation - Time and Temperature

Answer 17

Time:
- usually 6 to 8 hrs is required for adequate fixation in formalin
- other fixatives may penetrate more rapidly or slowly depending on the type used
Temperature
- increasing the temperature increases the rate of fixation but will increase the rate of autolysis

Question 18

Formalin Fixative

Answer 18

Used primarily for the routine fixation of specimens
Advantages
- standard fixative in most path labs
- fixes most tissue well and compatible with most histologic stains
Disadvantage
- causes cross links of proteins even in small needle bx
- over-fixation can destroy immunoreactive sites
- can dissolve calcification in breast within a 24hr period
It is toxic
- effects eyes, URT
- can cause dermal irritation
- can cause pulmonary edema and haemorrhage

Question 19

Non Formalin Fixatives

Answer 19

Alcohol based
Indications for other fixatives include those tissues which may require molecular protocols
Advantages:
- most of these types of fixatives are not hazardous and can be disposed of down the drain
Disadvantages
- time of fixation is critical and under or over fixation may lead to suboptimal results

Question 20

Bouin’s Solution

Answer 20

Consists of picric acid (hazardous solution), formaldehyde and acetic acid
Usually used for small biopsies
Advantages:
- result is sharp H&E staining
- fixation can facilitate finding small lymph nodes because the nodes will
remain white and the fat is stained yellow
- prolonged fixation can be used to decalcify tissue
Disadvantages:
- tissues can become quite brittle and fixation >18hrs
should be avoided (transfer tissue to alcohol to avoid this)
- RBCs will be lysed, iron and calcium deposits will be dissolved
- IHC studies may be less sensitive
- picric acid can lead to degradation of DNA for PCR and molecular analysis

Question 21

Zenker’s Fixation

Answer 21

Contains potassium dichromate, mercuric chloride and acetic acid
Often used in bone marrow bx, requires 8-12 hrs for decalcification and optimum preservation
Advantages
- rapid fixation with excellent histological detail
- slowly decalcifies tissue
- preferred from bloody specimens so RBCs will be lysed
Disadvantages
- poor penetration
- fixation > 24hrs may result in the tissue becoming brittle – transfer to formalin
- RBCs lysed and iron dissolved
- may demonstrate mercury pigment
- poor antigen preservation for IHC

Question 22

Glutaraldehyde

Answer 22

Contains glutaraldehyde, cacodylate buffer
Used for preservation of tissue for EM
Advantages
- excellent preservation of ultrastructural cellular detail
Disadvantages
- slow and poor penetration
- tissues must be very small for rapid fixation
- fixative must be stored at -4◦C
- can result in false PAS +ve staining reactions