Histologic Processing (Ch. 4) Flashcards
What is the best ratio of tissue to fixative for optimal fixation?
At least 1 part tissue to 10 parts formalin
How far can formalin penetrate into tissue?
About 1cm
What happens to tissue if it becomes overfixed? Why is this bad?
It becomes hard and brittle
This can impede histologic sectioning and staining
What are the five general characteristics of a good fixative?
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
What are four drawbacks to fixation?
1) Alteration of protein structure
2) Loss of soluble tissue components such as lipid or glycogen
3) Tissue shrinkage
4) DNA and RNA degradation
What are two fixation-related problems that are commonly encountered in the lab?
Too much tissue in too little fixative
Tissue not received in formalin
How should specimens that are not received in formalin be received?
On a saline-moistened telfa pad
How should tissue definitely NOT be received?
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
What timing parameters must be recorded for breast cancer specimens? Why?
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)
What fixatives are currently or have historically been in use at Duke?
4% formalin Acetic zinc formalin (AZF) Glutaraldehyde 100% ethanol Bouin's solution B5
What are the disadvantages to using formalin for fixation?
Toxicity to the eyes, upper respiratory tract, and skin
Possible carcinogenesis
What happens to microcalcifications in formalin?
They dissolve after 24 hours
What is AZF composed of? What is it used for?
Acetic acid, formalin, and zinc chloride
Used for bone marrow cores and clots
What is glutaraldehyde used for?
Electron microscopy sections
For what specimens is 100% ethanol used, and why?
Any specimens where gout is suspected, because uric acid crystals are soluble in aqueous fixatives like formalin
What is in Bouin’s solution? What specimens is it used to fix?
Picric acid, formalin, and acetic acid
Used for testicular biopsies for infertility and lymph node dissections; good for sharp H&E staining
What is in B5, and what specimens is it used to fix?
Mercuric chloride, sodium acetate, and formalin
Lymphoma workup tissues are placed in B5 because it preserves cytologic detail very well
What are the solutions that are used to “hold” but not fix tissue?
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
What is in decalcification solution?
A strong acid (HCl) and a chelating agent
How should large bones like femoral heads be fixed and sectioned?
They should be fixed in formalin and sliced on the large band saw prior to decalcification
How should very small bone specimens be fixed and sectioned?
They can be immersed in decal then sectioned with a scalpel
How should very large specimens, like tumor amputations with bone and soft tissue, be fixed and sectioned?
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
What three things does the automated tissue processor do to specimens?
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
What special embedding instructions are often written on cassettes?
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)
What other special histological processing notes are often written on the side of a cassette?
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)
At what thickness is paraffin-embedded tissue sectioned on the microtome?
5 micron sections
What must be done to tissue sections before they receive H&E staining? Why?
Tissue must be “run down to water” in baths of xylene, graded alcohols, and water
This is because H&E stains are aqueous
Describe the staining steps in H&E staining, after the tissue is run down to water
1) Hematoxylin
2) Water wash
3) “Bluing” in ammonia water
4) Water wash
5) Eosin
What color is hematoxylin? What color is eosin?
Hematoxylin = purple Eosin = pink
What solution is used to coverslip slides?
Permount
What are 10 problems that can be encountered during tissue processing?
Fat Thick sectioning Calcified tissue Hair and other keratinous material Foreign materal "Floaters" Unclear orientation instructions Missing tissue Missing block Incorrectly labeled/wrong blocks
Why does fat in tissue cause such a problem?
It dehydrates very poorly unless the tissue section is very thin
The loose structure of fat makes it very difficult to cut thinly enough
How should keratinous material be dealt with before sectioning?
It should be placed in a keratolytic depilatory (like Nair) until soften enough to section
What’s a “floater?” How can you avoid them?
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
What materials are often used to prevent small specimens from getting lost during processing?
Nylon embedding/mesh bags Microcassettes Filter paper Blue sponges Agar
When are the mesh bags used?
When there are multiple tiny tissue fragments that must be filtered and entirely submitted
For what specimens are the microcassettes used?
Liver needle core biopsies
For what specimens is filter paper often used?
Fragile specimens that may be damaged by mesh bags, like brain biopsies
Needle cores that are too small or fragile for microcassettes
For what specimens are blue sponges used?
Prostate needle core biopsies
Small tissue specimens
Any tissue that must be kept oriented, like skin biopsies
What is the major drawback to using mesh bags and blue sponges?
The pattern of the bag or sponge can imprint on fragile tissue, giving it a grid or “Swiss cheese” pattern
What is agar used for?
To hold specimens in their proper orientation and to prevent small specimens from falling through slits in the cassette
What are the drawbacks to using agar to hold tissue specimens?
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
