MELS500 End of semester

Histology

The study of microscopic anatomy of tissue (cells) - morphology and its function

Histopathology

The study of structural and functional abnormalities that are expressed as diseases of organs and systems.

What is single piece workflow?

Working with one specimen at a time

Describe histological workflow

What is the technological workflow?

Accessioning, gross cutup verification, tissue processing, embedding, microtomy, primary and advanced staining, case assembly, pathologist review, archive

Name seven details you can find in a lab request form

Patient name, NHI, Date of birth and age, Specimen details, Theatre or clinic, Time/date taken, Name of requesting clinician, contact details of requesting clinician

Describe the four classifications of laboratory hazards

Chemical, Physical, Biological, Psychosocial

What happens when an anomaly is found during specimen reception?

Requesting clinician is contacted and they either visit/fax the ab to correct this error (including the request form) and the specimen is registered with its’ relevant anomaly code

What are the aims of fixation and how is this achieved?

To preserve cells and tissue to mimic the state of it as if it were in the body, stop enzyme breakdown (autolysis) and microbial activity (putrefaction).

Precipitation

Coagulation

Autolysis

The shutdown of oxygen supply and the breakup of living tissue

Putrefaction

The breakdown of tissue by bacteria

What are the 5 properties of a good fixative?

Safe to handle, readily available, cheap, stable, penetrates tissue well without causing damage

What affects fixation?

Temperature, concentration of fixative, duration, mode of fixation, use of buffer, use of vacuum and agitation, penetration rate

How can we speed up fixation?

Concentration, type of fixative, temperature, size and thickness of tissue, type of tissue (fatty or bloody)

What are some of the limitations of fixatives?

Slow procedure, limited use on hard tissue, section thickness, loss of tissue constituents (lipids and enzymes)

Artefacts

Pigments which are created when exposed to a certain fixative

List 3 different kinds of artefacts

Formalin, mercury, dichromate

How to artefacts effect tissues?

Osmotic effects (volume changes in tissue), diffusion of labile substances (glycogen), leaching of substances, chemical changes

What are the three classifications of fixatives?

Microanatomical, histochemical, cytological

Name three examples of simple (neat) fixatives

Picric acid, chromic acid, potassium dichromate

Name three examples of compound (combination) fixatives

Formaldehyde, Bouins, Mercury

What are the two buffer salts used for 10% NBF

Sodium hydrogen phosphate and sodium dihydrogen phosphate

Name the advantages and disadvantages of ethanol, methanol, and acetone as fixatives

ADVANTAGES

• Recommended for preservation of glycogen

• At low temp may preserve enzymes

DISADVANTAGES

• Acts by disrupting protein’s tertiary structure leading to protein coagulation

• Harden tissue

• Causes shrinkage

• Doesn’t fix lipids

How would you demonstrate fat in a tissue?

Decalcification

The removal for calcium salts (insoluble calcium salts are converted into soluble calcium salts by the action of the decalcifying agent so that the tissue becomes soft)

What is the process of decalcification?

Careful preliminary assesment, Proper fixation, Preperation of slices at a reasonable thickness for fixation and processing, choosing a suitable decalcifier (adequate volume, regular changing of this liquid), Determining an endpoint, processing using a suitable schedule

List the 3 criteria for decalcifying agents

Complete removal of calcium salts

Causes no damage to tissue

No adverse effect on the subsequent process

Minimal time

How is decalcification acheived?

Name one example of a strong and weak acid used

What does a chelating agent do?

Binds to calcium ion present in bone to carry out decalcification

What are the characteristics of ion exchange resins?

Faster decalcifying rate

Preserves cellular details

No need to changed solution

Resin can be re-used

What are the advantages and disadvantages of electrophoretic decalcification?

ADVANTAGES

• Shortened time for complete decalcification

• Better preservation of soft tissue

DISADVANTAGES

• Limited number of specimens processed at a time

• Not very practical

What four factors effect decalcification?

Concentration, temperature, agitation, fluid access

How do we know when decalcification is finished? What is the most ideal?

1. poking, prodding, bending (physical)

2. chemical tests (ammonium oxalate solution)

3. x-ray - most ideal (no damage)

Tissue processing

A procedure taking place between tissue fixation and embedding

What is the end goal of tissue processing?

Support the tissue before they can be sectioned for microscopy via infiltration and embedding

What is the most widely used infiltrating media used in histology?

What are the pre-requisites for tissue-processing?

Well-fixed, suitable size, proper placement in cassette (not squeezed in and wrapped in foam if fragile or minute)

What factors effect tissue processing?

Type of tissue (bone or fatty)

Reagent type

Agitation/fluid access

Temperature

Vacuum

What is the use of a vacuum in tissue processing?

Removes air bubbles, decreases boiling point of wax (viscosity), lowers boiling point of clearing agents (flammable)

What are the stages for tissue processing?

Fixation, dehydration, clearing, wax infiltration/impregnation

What agents are used for dehydration and what are their advantages and disadvantages?

What clearing agents can be used for tissue processing and what are their advantages and disadvantages?

Clearing agent - removes dehydrating agent

What considerations are there regarding clearing agents?

Must be miscible with dehydrating reagent and with infiltrating and embedding medium

What is the purpose of wax infiltration?

What should be considered for wax selection?

Stable at room temp, causes no tissue damage, safe to handle, readily available, used for all tissues

What will effect infiltration?

Size and type of tissue, clearing agent used, use of vacuum

Coagulant and Anticoagulant (fixatives)

Tolerant and anti-tolerant (fixatives)

Embedding

The technique of placing cells or tissue in a supporting medium so that thin sections can be cut using a microtome

What is the melting point of paraffin wax?

55º to 65º

What is the function of each embedding station?

Wax resovoir - holds wax

Forceps warmer

Cassette-holding tank

Mold bin

Wax dispenser

Cold plate - cool specimens

What is the procedure for embedding?

Open cassette

Check the tissue and lid

Select appropriately sized mould

Fill the mould a third-ways with paraffin

Place the tissue centrally in the mould ensuring the orientation is correct

Tamp the tissue down

Place the cassette onto the mould

Fill the cassette with wax to the top

Cold plate

Microtomy

The method of preparing thin sections using a microtome for microscopic study

What is the purpose of microtomy?

To enable the transparency of stained tissues and cells when viewing microscopically

How do you master microtomy?

Have a thorough knowledge of the equipment used and the type of investigation required and well -fixed good quality tissue

What is specimen tracking and why do we do it?

Identifies the patient information and any previous test carried out on the sample (esp. when additional tests are required)

Allows labs to know where samples are in the testing process

Why do we record anomalies and what are the repercussions of no recording them?

To establish a diagnosis and to allow the proper treatment for the patient. 

Why do we QC?