Histologic Processing (Ch. 4)

Question 1

What is the best ratio of tissue to fixative for optimal fixation?

Answer 1

At least 1 part tissue to 10 parts formalin

Question 2

How far can formalin penetrate into tissue?

Answer 2

About 1cm

Question 3

What happens to tissue if it becomes overfixed? Why is this bad?

Answer 3

It becomes hard and brittle

This can impede histologic sectioning and staining

Question 4

What are the five general characteristics of a good fixative?

Answer 4

1) Preserves tissue to prevent autolysis and decomposition
2) Harden tissue to allow thin sectioning
3) Devitalizes or inactivates infectious agents
4) Stabilizes tissue components
5) Enhances avidity for dyes

Question 5

What are four drawbacks to fixation?

Answer 5

1) Alteration of protein structure
2) Loss of soluble tissue components such as lipid or glycogen
3) Tissue shrinkage
4) DNA and RNA degradation

Question 6

What are two fixation-related problems that are commonly encountered in the lab?

Answer 6

Too much tissue in too little fixative

Tissue not received in formalin

Question 7

How should specimens that are not received in formalin be received?

Answer 7

On a saline-moistened telfa pad

Question 8

How should tissue definitely NOT be received?

Answer 8

On a woven gauze pad - tissue can get stuck in the weave
Floating in saline - will macerate the tissue
Dry - tissue will desiccate and stick to the container

Question 9

What timing parameters must be recorded for breast cancer specimens? Why?

Answer 9

Ischemic time (time from removal from the body to placement in fixative) and length of fixation
To ensure proper fixation for preserving biomolecules for treatment assays (notably Her2/neu)

Question 10

What fixatives are currently or have historically been in use at Duke?

Answer 10

4% formalin
Acetic zinc formalin (AZF)
Glutaraldehyde
100% ethanol
Bouin's solution
B5

Question 11

What are the disadvantages to using formalin for fixation?

Answer 11

Toxicity to the eyes, upper respiratory tract, and skin

Possible carcinogenesis

Question 12

What happens to microcalcifications in formalin?

Answer 12

They dissolve after 24 hours

Question 13

What is AZF composed of? What is it used for?

Answer 13

Acetic acid, formalin, and zinc chloride

Used for bone marrow cores and clots

Question 14

What is glutaraldehyde used for?

Answer 14

Electron microscopy sections

Question 15

For what specimens is 100% ethanol used, and why?

Answer 15

Any specimens where gout is suspected, because uric acid crystals are soluble in aqueous fixatives like formalin

Question 16

What is in Bouin’s solution? What specimens is it used to fix?

Answer 16

Picric acid, formalin, and acetic acid

Used for testicular biopsies for infertility and lymph node dissections; good for sharp H&E staining

Question 17

What is in B5, and what specimens is it used to fix?

Answer 17

Mercuric chloride, sodium acetate, and formalin

Lymphoma workup tissues are placed in B5 because it preserves cytologic detail very well

Question 18

What are the solutions that are used to “hold” but not fix tissue?

Answer 18

Immunofixative (Michel’s/Zeus) - maintains tissue when formalin fixation is undesirable
RPMI (Roswell Park Memorial Institute) - holds tissue for flow cytometry, cytogenetics, other research protocols

Question 19

What is in decalcification solution?

Answer 19

A strong acid (HCl) and a chelating agent

Question 20

How should large bones like femoral heads be fixed and sectioned?

Answer 20

They should be fixed in formalin and sliced on the large band saw prior to decalcification

Question 21

How should very small bone specimens be fixed and sectioned?

Answer 21

They can be immersed in decal then sectioned with a scalpel

Question 22

How should very large specimens, like tumor amputations with bone and soft tissue, be fixed and sectioned?

Answer 22

They must be frozen solid in the -40 degree freezer, then sliced on the large band saw
Sections must be fixed overnight in formalin before being placed in decalcification solution

Question 23

What three things does the automated tissue processor do to specimens?

Answer 23

1) Dehydrates the tissue through graded alcohols
2) Clears the tissue using xylene
3) Infiltrates the tissue with paraffin, which stiffens it to allow thin sectioning

Question 24

What special embedding instructions are often written on cassettes?

Answer 24

On edge
On end
Lumen
Alopecia scalp punch 12 levels
GB (gallbladder)
Vas (vas deferens)
TA (temporal artery biopsies)
Punch
Punch bisect
Shave
Shave bisect
Number of tissue fragments (biospies)
Cornea
Tips (skin ellipses)

Question 25

What other special histological processing notes are often written on the side of a cassette?

Answer 25

Special stains ordered
Levels, serial or step sections
Special fixatives used
Presence of a microclip or other foreign hard object
Scant (if little tissue present)

Question 26

At what thickness is paraffin-embedded tissue sectioned on the microtome?

Answer 26

5 micron sections

Question 27

What must be done to tissue sections before they receive H&E staining? Why?

Answer 27

Tissue must be “run down to water” in baths of xylene, graded alcohols, and water
This is because H&E stains are aqueous

Question 28

Describe the staining steps in H&E staining, after the tissue is run down to water

Answer 28

1) Hematoxylin
2) Water wash
3) “Bluing” in ammonia water
4) Water wash
5) Eosin

Question 29

What color is hematoxylin? What color is eosin?

Answer 29

Hematoxylin = purple
Eosin = pink

Question 30

What solution is used to coverslip slides?

Answer 30

Permount

Question 31

What are 10 problems that can be encountered during tissue processing?

Answer 31

Fat
Thick sectioning
Calcified tissue
Hair and other keratinous material
Foreign materal
"Floaters"
Unclear orientation instructions
Missing tissue
Missing block
Incorrectly labeled/wrong blocks

Question 32

Why does fat in tissue cause such a problem?

Answer 32

It dehydrates very poorly unless the tissue section is very thin
The loose structure of fat makes it very difficult to cut thinly enough

Question 33

How should keratinous material be dealt with before sectioning?

Answer 33

It should be placed in a keratolytic depilatory (like Nair) until soften enough to section

Question 34

What’s a “floater?” How can you avoid them?

Answer 34

A little bit of tissue that shows up on a slide but shouldn’t be there because it came from a different block/case/patient/etc.
Floaters can be avoided by scrupulously cleaning instruments and work surfaces

Question 35

What materials are often used to prevent small specimens from getting lost during processing?

Answer 35

Nylon embedding/mesh bags
Microcassettes
Filter paper
Blue sponges
Agar

Question 36

When are the mesh bags used?

Answer 36

When there are multiple tiny tissue fragments that must be filtered and entirely submitted

Question 37

For what specimens are the microcassettes used?

Answer 37

Liver needle core biopsies

Question 38

For what specimens is filter paper often used?

Answer 38

Fragile specimens that may be damaged by mesh bags, like brain biopsies
Needle cores that are too small or fragile for microcassettes

Question 39

For what specimens are blue sponges used?

Answer 39

Prostate needle core biopsies
Small tissue specimens
Any tissue that must be kept oriented, like skin biopsies

Question 40

What is the major drawback to using mesh bags and blue sponges?

Answer 40

The pattern of the bag or sponge can imprint on fragile tissue, giving it a grid or “Swiss cheese” pattern

Question 41

What is agar used for?

Answer 41

To hold specimens in their proper orientation and to prevent small specimens from falling through slits in the cassette

Question 42

What are the drawbacks to using agar to hold tissue specimens?

Answer 42

It usually has a different consistency that the tissue it surrounds
It can be difficult to cut an adequate section
It must be kept heated during use
It has a tendency to deteriorate and not gel properly