Tissue Processing

Question 1

What are the three main steps of tissue processing after fixation?

Answer 1

Dehydration, Clearing, and Infiltration.

Question 2

What is the primary goal of tissue processing?

Answer 2

To make the tissue firm and rigid (harden it) so it can be cut into very thin sections for microscopy.

Question 3

What is an inevitable side effect of tissue processing?

Answer 3

Tissue shrinkage.

Question 4

How does the goal of “tissue processing” differ from “fixation”?

Answer 4

Fixation is done to preserve the tissue’s structure (prevent decay), while tissue processing is done to harden the tissue for sectioning.

Question 5

What is the purpose of the Dehydration step?

Answer 5

To remove all water from the tissue. This is done by passing the tissue through a graded series of alcohols (e.g., 60%, 70%, 95%, 100% alcohol).

Question 6

What is the purpose of the Clearing step?

Answer 6

To remove the dehydrating agent (alcohol) and make the tissue transparent, allowing it to be infiltrated with a solid support medium like paraffin wax.

Question 7

What is the purpose of the Infiltration step?

Answer 7

To permeate the tissue with a solid support medium (like paraffin wax), which gives the tissue the internal rigidity needed for sectioning.

Question 8

what is the general pattern of alcohol concentrations used in dehydration?

Answer 8

A graded series, starting from a lower concentration (e.g., 60%) and progressing to higher concentrations (70%, 95%, and finally 100% alcohol).

Question 9

What is the primary purpose of dehydration in tissue processing?

Answer 9

The primary purpose of dehydration is the controlled removal of free water (water not molecularly bound to the tissue) from specimens that have been fixed in aqueous reagents. This prepares the tissue for embedding in a non-aqueous medium (like paraffin wax).

Question 10

What are the two possible negative outcomes of improper dehydration, and how do they manifest in the tissue?

Answer 10

Excessive Dehydration: This removes molecularly bound water, resulting in tissue that is hard and brittle.

Incomplete Dehydration: Free water remains in the tissue, resulting in tissue that is soft and mushy.

Question 11

Briefly describe the two mechanisms by which dehydrants remove water from tissue.

Answer 11

Hydrophilic dehydrants: These reagents chemically attract and bind to the water, pulling it out of the tissue.

Diluting dehydrants: These reagents work by physically diluting the aqueous fluids present within the tissue.

Question 12

List the main categories and specific examples of reagents used for dehydration.

Answer 12

Alcohols:

A. Ethanol

B. Methanol

C. Isopropanol

D. Butanol

Acetone

Universal solvents: (e.g., dioxane, tertiary butanol, tetrahydrofuran)

Question 13

Why is ethanol considered “hydrophilic” and what are its general characteristics as a dehydrant?

Answer 13

Ethanol is hydrophilic, meaning it attracts water. It is a clear, colorless, and flammable liquid. It is considered reliable and fast-acting for dehydration. Ideally, denatured alcohol should be used.

Question 14

What important safety and legal precaution must be taken with ethanol?

Answer 14

Because ethanol is flammable, it should not be dumped down the drain. It is also a controlled substance by the government, so meticulous record-keeping of its use is mandatory.

Question 15

What is the correct technique for using ethanol to dehydrate tissue, and why is this necessary?

Answer 15

Ethanol must be used in ascending grades (e.g., from lower to higher concentration). This gradual increase in concentration helps to reduce tissue shrinkage by preventing a rapid osmotic shock to the cells.

Question 16

Why is it important to start dehydration with 60% ethanol after fixation with buffered formalin?

Answer 16

Starting at 60% ethanol is crucial after using phosphate-buffered formalin. If the tissue is placed directly into ethanol that is greater than 70%, the phosphate salts from the buffer will precipitate out within the tissue. This precipitation will cause problems during microtomy (section cutting).

Question 17

What is the potential consequence of exposing tissue to ethanol for too long?

Answer 17

Prolonged exposure to ethanol will cause excessive shrinkage and hardening of the tissue.

Question 18

What is the primary purpose of the clearing step in tissue processing?

Answer 18

The controlled removal of dehydrating agents (like alcohol) from tissues using multiple changes of a clearing agent.

Question 19

How do clearing agents make tissue transparent?

Answer 19

They have a high index of refraction, which allows light to pass through the tissue more easily.

Question 20

What are the two key chemical properties a clearing agent must have?

Answer 20

It must be miscible with both the dehydration agent (e.g., alcohol) and the infiltration medium (e.g., paraffin wax).

Question 21

What is the chain reaction of errors caused by inadequate dehydration?

Answer 21

Inadequate dehydration → Inadequate clearing → Inadequate infiltration.

Question 22

What is the consequence of inadequate clearing on tissue texture?

Answer 22

The tissues will become soft and mushy.

Question 23

What is the consequence of prolonged exposure to a clearing agent on tissue texture?

Answer 23

The tissues will become hard and brittle.

Question 24

What is the most widely used clearing agent?

Answer 24

Xylene

Question 25

What is the visual sign of adequate clearing when using xylene?

Answer 25

The tissues become transparent.

Question 26

Prolonged exposure to xylene is especially damaging to which types of tissues?

Answer 26

Fibrous, muscular, CNS (central nervous system), and cartilaginous tissues.

Question 27

Xylene displaces alcohol rapidly and is miscible with what two substances?

Answer 27

Alcohol and paraffin.

Question 28

What happens to xylene if it becomes contaminated with water?

Answer 28

It turns cloudy. This indicates the reagent should be changed as soon as possible.

Question 29

List three important safety and handling precautions for xylene.

Answer 29

It is flammable, a neurotoxin, and a defatting agent. It also cannot be poured down the sink.

Question 30

What is the purpose of the infiltration step in tissue processing?

Answer 30

The controlled removal of clearing agents and their replacement with an infiltrating (supporting) medium. This holds the cells and intercellular structures in their proper relationships so that thin sections can be cut.

Question 31

How can incomplete infiltration be detected?

Answer 31

It is manifested on the H&E-stained section.

Question 32

What process can greatly enhance infiltration?

Answer 32

Vacuum.

Question 33

What is the most popular infiltrating medium?

Answer 33

Paraffin.

Question 34

What is paraffin wax primarily made of?

Answer 34

Inert hydrocarbons with additives.

Question 35

List five common additives to paraffin wax and their purposes.

Answer 35

Beeswax: Reduces crystal size (and increases stickiness and adhesion). Rubber: Reduces brittleness (increases stickiness and allows for good ribboning). Other waxes: Produce smooth texture and smaller crystal size. Plastic: Increases hardness and support.

Question 36

How does the melting point (MP) of paraffin correlate with its hardness and sectioning properties?

Answer 36

Higher MP: Paraffin becomes harder, providing more support for hard tissues and allowing for thinner sections, but ribboning is difficult. Lower MP: Paraffin becomes softer, providing less support, making thin sections difficult to obtain, but ribboning is easier.

Question 37

What is the commonly used melting point range for paraffin?

Answer 37

55°C to 58°C.

Question 38

What type of paraffin is used for IHC staining and why?

Answer 38

Low melting point paraffin is used to preserve antigens.

Question 39

What is "crystallization" in the context of paraffin embedding?

Answer 39

The formation of crystals in the wax surrounding the tissue as it solidifies.

Question 40

What are the two main factors affecting paraffin infiltration?

Answer 40

Time and Temperature.

Question 41

What is the effect of prolonged exposure of tissue to paraffin?

Answer 41

It causes shrinkage and hardening.

Question 42

What is the ideal temperature range for a paraffin bath?

Answer 42

2°C to 4°C above the melting point of the wax.

Question 43

What is the consequence of using overheated paraffin?

Answer 43

It causes over-hardening of tissues.

Question 44

What types of tissue specimens should be processed separately to avoid over-processing?

Answer 44

Biopsies with large tissues or fatty tissues should not be processed on the same cycle as smaller, more delicate biopsies.

Question 45

What are the results of over-processed biopsy tissues?

Answer 45

They become hard and friable, making them difficult to section and leading to microtomy and staining artifacts.

Question 46

List six key points for quality control of paraffin tissue processing.

Answer 46

Use adequate volume of reagents. Rotate or change reagents regularly following a routine schedule. Check alcohols for contamination using hydrometers. Record the daily temperature and adjust if necessary. Use three changes of paraffin. Ensure the last paraffin bath is the cleanest.