Processing

Question 1

Define: Dehydration

Answer 1

The removal of water from tissue.

Question 2

Define: Clearing

Answer 2

Process of replacing the alcohol (dehydrating agent) with a reagent that is miscible with paraffin - most leave the tissue transparent

Question 3

Define: Infiltration

Answer 3

Permeation, as in paraffin permeating (spreading or flowing throughout) tissue. AKA impregnation

Question 4

Define: Embedding

Question 5

Define: Universal solvent

Answer 5

Reagents that avoid the use of 2 solutions, performing both the dehydrating and clearing steps. They are miscible with water-based solutions and with paraffin.

Question 6

Define: Edge

Question 7

Define: Cross-section

Question 8

Define: Decalcification

Answer 8

The process of removing calcium from bone or tissue, thus allowing routine microtomy on paraffin embedded material

Question 9

Define: Ion exchange

Question 10

Define: Chelating agent

Answer 10

Organic compounds that form a ring compound by joining to metal ions (i.e. EDTA decalcifies by binding calcium ions)

Question 11

Define: Miscible

Answer 11

Capable of mixing or being mixed

Question 12

List the properties and chemical mechanism of Dehydrating agents.

Answer 12

Act to remove water in 2 ways - being hydrophilic and attracting water from the tissue, or dehydrating by repeated dilution of the aqueous tissue fluids

Question 13

List the properties and chemical mechanism of Ethyl alcohol.

Answer 13

○ Clear, colorless, moderately toxic, flammable liquid. Hydrophilic and is considered the best of the dehydrants
○ It should be used in a sequence of solutions that gradually increase in concentration - reducing some of the tissue shrinkage that occurs during the dehydration process (50-60% followed by 2 changes each of 95% and 100% EtOH)
If phosphate-buffered formaldehyde solutions were used for fixation, phosphate salts will precipitate in the tissues if the concentration of the initial alcohol solution is less than 70%
When used alone, it is moderately toxic and very flammable

Question 14

List the properties and chemical mechanism of Methyl alcohol (methanol).

Question 15

List the properties and chemical mechanism of Butyl alcohol (butanol).

Question 16

List the properties and chemical mechanism of Isopropyl alcohol (isopropanol).

Question 17

List the properties and chemical mechanism of Acetone.

Question 18

List the properties and chemical mechanism of Clearing agents (de-alcoholization).

Question 19

List the properties and chemical mechanism of Xylene.

Answer 19

Irritant, flammable,

Question 20

List the properties and chemical mechanism of Toluene.

Question 21

List the properties and chemical mechanism of Benzene.

Question 22

List the properties and chemical mechanism of Acetone.

Question 23

List the properties and chemical mechanism of Chloroform.

Question 24

List the properties and chemical mechanism of Limonene hydrocarbons.

Answer 24

Sensitizer, combustible

Question 25

Explain why a concentration of ethanol no higher than 65% should be used after fixation with phosphate-buffered formaldehyde.

Question 26

Identify 3 universal solvents.

Answer 26

Dioxane, Tertiary butanol, Tetrahydrofuran

Question 27

State the possible effects on hematoxylin & eosin stain if water is present in the clearing agent on the tissue processor.

Answer 27

The universal problem with uneven H&E staining and poor nuclear chromatin patterns is the result of incomplete clearing - often related to moisture in the clearing agent

Question 28

State the indication for using agar or gelatin for embedding.

Answer 28

Can be used to produce a single block of friable tissue or multiple fragments when cutting frozen sections.

Question 29

Explain the protocol for agar or gelatin embedding.

Answer 29

For gelatin embedding: 1. The tissue is washed overnight 2. Impregnated for 24 hrs with a 25% solution at 37C 3. Embedded in 25% gelatin and allowed to solidify, or harden, in the fridge 4. Further hardened by immersion in 5% formalin for 24hrs

Question 30

State the appropriate processing method or medium for Mohs surgery.

Answer 30

○ Excise the lesion with minimal uninvolved tissue, this is notched for specimen orientation and inked to indicate specific margins ○ Specimens must be embedded with the epidermis down on the chuck, leaving the deep margin exposed for initial sectioning - This technique of specimen orientation allows the surgeon to simultaneously evaluate both the deep and lateral margins of the lesion ○ Sections are collected as soon as tissue is visible, levels are repeated at defined intervals until a full face is obtained ○ Sections are stained with H&E

Question 31

State the appropriate processing method or medium for Enzyme histochemistry.

Question 32

State the appropriate processing method or medium for Undecalcified bone.

Question 33

State the appropriate processing method or medium for Fat demonstration.

Question 34

Relate the melting point of paraffin to hardness, section thickness, and ribboning quality.

Answer 34

○As the melting point increases, the paraffin contains more polymers and becomes harder, providing better support for the tissue and allowing for thinner sections to be obtain but ribboning becomes harder ○ As the melting point decreases, the wax becomes softer and provides less support for hard tissues making thinner sections more difficult to obtain but ribboning is easier ○ Paraffin hardness is related to the plastic point (the lowest temperature at which permanent deformation can occur without fracture) and is a few degrees below the melting point

Question 35

Identify the problem encountered if the infiltration paraffin overheats.

Answer 35

Tissue overhardens

Question 36

Identify the problem encountered if the embedding paraffin overheats.

Answer 36

May change the sectioning quality, but the tissue will not be affected because the tissue is not immersed in this paraffin for a long period at the higher temperature

Question 37

Identify the problem encountered if dehydration is inadequate.

Question 38

Identify the problem encountered if dehydration is excessive.

Question 39

Identify the problem encountered if clearing is inadequate.

Question 40

Identify the problem encountered if clearing is excessive.

Question 41

Describe how an increase or decrease in the time that the specimen remains in each solution will affect the quality of processing.

Question 42

Outline paraffin processing schedules.

Question 43

Describe the microwave processing procedure.

Question 44

Identify the differences between the microwave procedure and the procedure used on an enclosed processor.

Question 45

Identify 2 means of decreasing processing time when using paraffin.

Question 46

Identify at least 3 paraffin processing errors and describe the method of correction for each error.

Question 47

Identify at least 2 quality control procedures used in processing tissues for paraffin embedding.

Question 48

Describe the method for validation of a new processing program.

Question 49

State 3 reasons for reprocessing tissue.

Question 50

Describe a method for gently reprocessing tissue.

Question 51

State 3 histological reagents that may be recycled and quality control activities for each.

Question 52

Relate rapid chilling of paraffin to crystal size.

Question 53

State why paraffin wax of small crystalline structure is important.

Question 54

Describe the proper orientation when embedding skin.

Question 55

Describe the proper orientation when embedding tubular structures.

Question 56

Describe the proper orientation when embedding structures with a wall (gallbladder).

Question 57

Describe the proper orientation when embedding bone.

Question 58

State at least 2 ways that specimen orientation can be indication during gross dissection to ensure proper embedding.

Question 59

Briefly describe simple acid decalcification.

Question 60

Briefly describe decalcification using ion exchange resin.

Question 61

Briefly describe electrolytic decalcification.

Question 62

Briefly describe chelation decalcification.

Question 63

Describe the methods for determining the endpoint of decalcification.

Question 64

Identify methods for increasing the rate of the decalcification reaction.

Question 65

State the effects of overdecalcification and undercalcification on calcified tissue sections.

Question 66

State the effects of decalcification on fixed and unfixed tissues.

Question 67

Describe a method of handling paraffin tissue when areas of calcification are discovered during microtomy.

Question 68

State 1 reason for examining undecalcified bone sections.

Question 69

Identify 3 methods of freezing tissue in preparation for cutting frozen sections.

Question 70

Which procedure of freezing tissue should be used for skeletal muscle enzyme studies?

Question 71

Identify 2 problems with frozen sectioning.

Question 72

Identify a problem with using eosin as colorant in dehydrating solutions.

Question 73

Describe and explain the embedding technique used during Mohs surgery.