Lecture 2

Question 1

Frozen Sections

Answer 1

Performed on stat samples delivered to the surgical pathology lab usually from the operating room
-intraoperative diagnosis
Advantage –speed
Disadvantage- slide quality

Question 2

Frozen Section Procedure

Answer 2

• Sample is frozen and a section is cut using a cryostat.
• The selected piece of tissue or sometimes the whole specimen if it is a small one is placed on a special holder which is placed inside the cryostat.
• A cryostat is an instrument that has a microtome inside a cabinet. It is kept at about ~ -20 degrees C.
• The tissue section inside the cryostat freezes quickly. –The technologist then cuts the tissue. A slide with the tissue section on it is prepared and stained.
• After this procedure has ended, the tissue sample is thawed. It is then fixed (placed in fixative) and processed in the routine manner

Question 3

Embedding

Answer 3

• performed at a machine called an embedding centre.
• piece of tissue is placed in a mold with liquid (molten) paraffin. The tissue is oriented in the mold so that the correct surface of the tissue will be cut during microtomy and placed on a microscope slide.
• labelled half of the original cassette is placed on top of the tissue.
• The paraffin cools and hardens so that the tissue is supported in a block of paraffin with the labelled cassette attached
• block of paraffin wax with the tissue in it is then removed from the embedding mold
• paraffin block supports the tissue and gives a suitable constituency for microtomy.

Question 4

microtome

Answer 4

the instrument used to accurately cut thin sections of tissue

Question 5

Microtomy/ Sectioning

Answer 5

Tissue sections are routinely cut at about 3 to 5 micrometers thick.

• trimming or rough cutting -tissue blocks are trimmed to remove excess wax and to expose a full cross-section of the tissue
• tissue block is then placed into the block holder of the microtome and cut or sectioned and a “ribbon” of tissue is then obtained
• placed on the surface of a water bath to remove any wrinkles
• slide labelled with surgical number and other patient identification, is then placed under the floating ribbon and used to pick up the tissue section
• surgical number on the slide corresponds to the surgical number on the block
• slides with the tissue sections on them are dried in an oven set at 60 to 65 degrees Celsius.

Question 6

routine stain

Answer 6

Hematoxylin and Eosin (H&E)

Question 7

Principle of the routine stain

Answer 7

hematoxylin-alum lake binds to the phosphate radicals of the DNA and RNA and this causes the nuclei to stain blue Eosin stains other tissue components (cytoplasm) varying degrees of pink/red.

Question 8

Staining Results
Nuclei -
Cytoplasm
Red Blood cells-
Muscle fibres

Answer 8

Nuclei - dark blue
Cytoplasm- pink
Red Blood cells- red
Muscle fibres- pink

Question 9

cryostat

Answer 9

instrument that has a microtome inside a cabinet. It is kept at about ~ -20 degrees C.

Question 10

Progressive staining:

Answer 10

The tissue is left in the dye solution until sufficient dye has been taken up and the desired intensity of colouring is attained.
A progressive H&E method is often used for rapid staining of frozen sections produced in the surgical lab for stat diagnosis.

Question 11

Regressive staining:

Answer 11

The tissue is over stained and excess dye is removed selectively until the desired intensity. The selective removal of this excess stain is called differentiation.

Regressive H&E method is most commonly used in most histology lab. This method takes longer than the progressive method. The acid alcohol solution is used as a differentiator.

Question 12

Xylene used first in routine staining - Regressive

Answer 12

to remove wax from the tissue section on the slide

If wax removal is not complete then there would be patchy staining- clear areas on the section that will be opaque and have no staining

Question 13

Absolute alcohol used 2nd - Regressive
100% ethanol

2 -3 changes

Answer 13

Xylene is miscible with alcohol. Dewaxing and then hydration with decreasing concentrations of alcohol are used to gradually “take the sections to water”.

The section is hydrated by transferring it to absolute alcohol
take it into 95% and then 70% and hydrate it
quick rinse with tap water

Question 14

Hematoxylin

Answer 14

Solution should be filtered before use to remove scum of over-oxidized dye at the surface

Question 15

Acid alcohol - because its the Regressive

Answer 15

Differentiates the Hematoxylin

In the regressive method the sections are overstained in Hematoxylin, then excess dye is removed by placing in acid alcohol solution,

The slide is washed quickly in tap water to stop differentiation

Question 16

Scotts Tap water or Ammonia water - Regressive

Answer 16

Alkaline solution used as a bluing agent
rinse with tap water
rinse with 1% eosin solution

Question 17

70% Alcohol and 95% Alcohol - Regressive

dehydrate

Answer 17

Dehydrating the section

70% ; 95% alcohol differentiate the eosin until the background has different shades of pink

Question 18

Xylene last time - Regressive

Answer 18

“Clears” section –raises the refractive index (R.I.)

Question 19

Advantages of the automated stainer

Answer 19

• Some automatic stainers have a heated station to dry slides before staining
• Speed – can handle large volumes of slides at a time
• Some can perform different staining programs at the same time
• Gives more consistent staining
• Reduces exposure to solvents and other staining reagents
• Frees up technologist/technician time to do other tasks

Question 20

Quality control Requirements for Staining

Answer 20

• Daily solution/reagent changes and filtering of stain solutions (e.g. hematoxylin and eosin). This solution changes must recorded in a log
• Run stain control (H&;E control) which must be checked microscopically by a technologist. An H&E control must be run daily and anytime solutions on the stainer are changed.
• Record any equipment problems that occur with the autostainer on a designated log. Contact service representative for repairs if required
• Yearly preventative maintenance must be performed on the instrument, This may be provided by the vendor or another outside contractor with expertise on the equipment

Question 21

Coverslipping

Answer 21

-stained slide is covered with a thin glass or plastic called a coverslip
kept for 20 years

Purpose of Coverslipping:
To improve microscopic detail
To protect the tissue section from damage
To preserve the stain

Question 22

Resinous Mounting Media - permount

Answer 22

This type of mounting media is used to mount routine slides in the histology lab.
o These are resins and may be either natural or synthetic
o They have a refractive index which is close to that of glass (1.52)
o These dissolve in xylene so stained slides being mounted must be dehydrated and then cleared (in xylene) before Coverslipping.
o This is considered to be permanent mounting media
o Coverslips can be removed by soaking the slides in xylene
-tape can be used for coverslipping

Question 23

Aqueous Mounting Media - Glycerol or gelatin based mountants

Answer 23

o Slides are mounted from water or an aqueous solution
o Used for certain stains where dehydrating solutions such as alcohol and acetone cannot be used
o Slides mounted with aqueous mounting media are not permanent and if the slide has to be kept for a long period then the edges of the cover glass (coverslip) needs to be sealed.
not as permenant as resinous media

Question 24

Manual method of Coverslipping

Answer 24

• a drop of mounting media is applied to the slide then a coverslips is placed on top of the mounting media.
• A drop of mounting media is placed on the coverslip and then the slide with the tissue section on top of this.
  o Care must be taken to prevent air bubbles
  they must be labelled with a final label

Question 25

Advantages of the automated coverslipper

Answer 25

o Speed–some can coverslip up to 500 slides per hour o Reduces exposure to solvents and other staining reagents o Frees up technologist/technician time to do other tasks

Question 26

Maintenance and quality control of automated coverslipper

Answer 26

Automated coverslippers need daily maintenance such as: o Coverslips and mounting media have to be replenished o Surfaces and slide racks have to be kept clean o Solutions have to be maintained

Question 27

Quality in the Histology Lab

Answer 27

o All Medical labs in Ontario licensed by the Ontario Ministry of Health and Long-Term Care must be accredited by IQMH ( Institute for Quality Management in Healthcare) o Standard Operating Procedures (SOP’s) must be available for staff in the lab to follow and must be reviewed annually by the lab director and by staff performing the procedures. o All temperature dependent equipment such as water baths, ovens and refrigerators used in the lab must have their temperature taken every day of use and recorded in a log. o Staining Control slides must be run daily and checked microscopically and results must be recorded in a log. o Equipment in the lab such as the tissue processor, automated stainers must have annual preventative maintenance and records must be kept on file for reference o Daily/Weekly Maintenance of equipment must be performed according to procedures and must be recorded in the appropriate logs. r. o Incidents such as labelling errors or any other errors that could affect the specimen diagnosis must be recorded on a lab incident report form. o Staff training and competency testing records must be kept in their files

Question 28

Main types of hematoxylin

Answer 28

Harris Hx Gill III Erlich’s

Question 29

Harris Alum Hematoxylin

Answer 29

Hematoxylin Absolute alcohol-is used to dissolve the Hematoxylin precipitates Ammonium or potassium alum - – mordant to link the dye to the tissue Distilled water Mercuric oxide - oxidizing agent Glacial acetic acid Hematoxylin must be filtered daily before use

Question 30

Eosin is used as either as

Answer 30

aqueous solution or alcoholic solution

Question 31

Acid-Alcohol

Answer 31

1% Hydrochloric acid in 70% ethanol can remove an entire stain if needed

Question 32

Differentiation

Answer 32

The section/slide is quickly dipped in acid- alcohol . | It is then rinsed in water to stop the action of the acid-alcohol

Question 33

Bluing Agents

Answer 33

Alkaline solution if too alkaline sections of tissue from the slide will be removed ``` Examples: Warm tap water ( may not be alkaline enough) Saturated solution of lithium carbonate Tris buffer ph 7.6 Ammonia water Scott’s tap water substitute ```

Question 34

Tissue Processing- Summary

Answer 34

``` Accessioning Fixation Decalcification (if required) Grossing Dehydration Clearing Infiltration Embedding Microtomy Staining Microscopic examination by pathologist ```

Question 35

Romanowsky stains

Answer 35

``` Wright's stain Jenner's or May-Grunwald Leishman’s stain Giemsa stain Diff –Quik ```