Histologic Processing (Ch. 4) Flashcards Preview

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Flashcards in Histologic Processing (Ch. 4) Deck (42)
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1
Q

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

A

At least 1 part tissue to 10 parts formalin

2
Q

How far can formalin penetrate into tissue?

A

About 1cm

3
Q

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

A

It becomes hard and brittle

This can impede histologic sectioning and staining

4
Q

What are the five general characteristics of a good fixative?

A

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

5
Q

What are four drawbacks to fixation?

A

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

6
Q

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

A

Too much tissue in too little fixative

Tissue not received in formalin

7
Q

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

A

On a saline-moistened telfa pad

8
Q

How should tissue definitely NOT be received?

A

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

9
Q

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

A
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)
10
Q

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

A
4% formalin
Acetic zinc formalin (AZF)
Glutaraldehyde
100% ethanol
Bouin's solution
B5
11
Q

What are the disadvantages to using formalin for fixation?

A

Toxicity to the eyes, upper respiratory tract, and skin

Possible carcinogenesis

12
Q

What happens to microcalcifications in formalin?

A

They dissolve after 24 hours

13
Q

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

A

Acetic acid, formalin, and zinc chloride

Used for bone marrow cores and clots

14
Q

What is glutaraldehyde used for?

A

Electron microscopy sections

15
Q

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

A

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

16
Q

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

A

Picric acid, formalin, and acetic acid

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

17
Q

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

A

Mercuric chloride, sodium acetate, and formalin

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

18
Q

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

A

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

19
Q

What is in decalcification solution?

A

A strong acid (HCl) and a chelating agent

20
Q

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

A

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

21
Q

How should very small bone specimens be fixed and sectioned?

A

They can be immersed in decal then sectioned with a scalpel

22
Q

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

A

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

23
Q

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

A

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

24
Q

What special embedding instructions are often written on cassettes?

A
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)
25
Q

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

A
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)
26
Q

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

A

5 micron sections

27
Q

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

A

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

28
Q

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

A

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

29
Q

What color is hematoxylin? What color is eosin?

A
Hematoxylin = purple
Eosin = pink
30
Q

What solution is used to coverslip slides?

A

Permount

31
Q

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

A
Fat
Thick sectioning
Calcified tissue
Hair and other keratinous material
Foreign materal
"Floaters"
Unclear orientation instructions
Missing tissue
Missing block
Incorrectly labeled/wrong blocks
32
Q

Why does fat in tissue cause such a problem?

A

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

33
Q

How should keratinous material be dealt with before sectioning?

A

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

34
Q

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

A

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

35
Q

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

A
Nylon embedding/mesh bags
Microcassettes
Filter paper
Blue sponges
Agar
36
Q

When are the mesh bags used?

A

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

37
Q

For what specimens are the microcassettes used?

A

Liver needle core biopsies

38
Q

For what specimens is filter paper often used?

A

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

39
Q

For what specimens are blue sponges used?

A

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

40
Q

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

A

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

41
Q

What is agar used for?

A

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

42
Q

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

A

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