Histotechnique

Question 1

Cytology

Answer 1

the study of individual cells

Question 2

Goal of histopathology

Answer 2

Preserve tissues in a life-like manner

–> abnormalities can be identified microscopically

Question 3

Simplified workflow

Answer 3

obtain –> fix –> slice –> stain

Question 4

Reason for fixative solution

Answer 4

tissues and cells die/degrade immediately after removal from body
fresh tissues must be transported immediately
placed in fixative solution for transportation

Question 5

Sources of samples (2)

Answer 5

Surgical (biopsy with clean margins)

Post-mortem (autopsy)

Question 6

Grossing

Answer 6

Macroscopic evaluation of specimen

Question 7

Parameters (5)

Answer 7

Size
Texture
Number/Proportion
Markings
Locations

Question 8

Fixation

Answer 8

Preservation go cells and tissues
Life-like as possible
STABILIZE PROTEIN so it is RESISTANT TO FURTHER CHANGES

Question 9

Function of Fixatives (4)

Answer 9

1. Prevents putrefaction (rotting) and autolysis (breakdown)
2. Maintain proper relationships between cells and extracellular substances
3. Bring out differences in refractive indexes and increase the visibility of/contrast between different tissue elements
4. Secondary functions: enhance staining, limiting osmotic effects, prevent desiccation

Question 10

Autolysis

Answer 10

Self-destruction after cells death
Caused by Intracellular enzymes
Temperature (warm=more damage)
Specialized cells - more rapid, worse damage
Nuclear changes: pyknosis –> karyorrhexis –> karyolysis
*condensed chromatin –> loose
Cytoplasm: granular and swollen

Question 11

Putrefaction

Answer 11

decomposition by microorganisms

Question 12

Modes of Action (fixing) (3)

Answer 12

Render - inactivate enzymes by stabilizing proteins
Kill - kill bacteria and molds (toxic)
Make - make tissues more receptive to dyes

Question 13

Impact of Fixing on Tissues (5)

Answer 13

1. Change in size (smaller)
2. Change in texture (brittle, hard)
3. Lost material (dissolve lipids)
4. Chemical alterations (charges and properties may change)
5. Fixation artefacts (deposits on and around tissues - affect microscopic imagine)

Question 14

Volume ratio of Fixative/Tissue

Answer 14

20/1

Question 15

T/F: dense tissue is faster to fix

Answer 15

FASLE - dense tissues required more time than porous tissues

Question 16

Fixative Classification (4)

Answer 16

1. Chemical action on proteins
2. Effect on microscopic appearance of the tissues
3. Number of fixing reagents in fixative solution
4. Amount of time tissues can remain in fixative

Question 17

Chemical action on Proteins:

Answer 17

Coagulant - tertiary structure, organelles, mesh
Non-coagulant - cross-linkages (impermeable), insoluble gel (hard to stain)

Additive - combines with protein
Non-additive - changes protein nature, structural configuration or activity

Question 18

Microscopic appearance of tissue

Answer 18

1. Microanatomical fixative - preserve microarchitecture
2. Cytological fixative - preserve intracellular structures/inclusions (electron micro)
3. Histochemical fixative - minimal changes

Question 19

simple vs compound fixing sequence

Answer 19

Simple - 1 fixative

Compound - more than 1 fixative in solution

Question 20

Tolerant vs intolerant fixing

Answer 20

Tolerant - time is flexible

Intolerant - timing is critical

Question 21

Simple Fixatives (8)

Answer 21

1. Formaldehyde and Formalin based fixatives
2. Glutaraldehyde
3. Osmium Tetroxide
4. Potassium Dichromate
5. Mercuric Chloride
6. Picric Acid
7. Ethanol
8. Acetic Acid

Question 22

Universal Fixative

Answer 22

Formaldehyde

Question 23

Formaldehyde

Question 24

Formalin-based fixatives

Question 25

Glutaraldehyde

Question 26

Osmium Tetroxide

Question 27

Potassium Dichromate

Question 28

Mercric Chloride

Question 29

Picric Acid

Question 30

Ethanol

Question 31

Acetic Acid

Question 32

Compound Fixative Formula

Answer 32

1. 1 or more coagulating agent 2. May contain a non-coagulating agent 3. always in solution 4.

Question 33

B-Plus Fixative

Question 34

Bouin's Fixative

Question 35

Secondary Fixation

Question 36

Formalin Pigment

Question 37

How do you remove formalin pigment?

Question 38

Mercuty Pigment

Question 39

Chrome Pigment

Question 40

Goal of Tissue Processing

Answer 40

- remove all water | - replace with an inert, stable material

Question 41

3 steps to remove water

Answer 41

1. Dehydration 2. Clearing 3. Infliltration

Question 42

Incomplete dehydration

Answer 42

mushy & soft tissue

Question 43

Prolonged dehydration

Answer 43

brittle & hard tissue

Question 44

Dehydration

Answer 44

slow and gentle | increasing concentrations of alcohol (70%-100%)

Question 45

Clearing

Answer 45

Remove dehydrating agent, replace with material miscible with infiltrating agent Xylene TOXIC agents Improper: greasy feel and odor

Question 46

Infiltration

Answer 46

``` Removal of clearing agent, replaced with intent, stable supportive material INTERNAL SUPPORT Long step (vacuum to speed up) Paraffin wax (with or without additives) ```

Question 47

Processing Maintenance

Answer 47

Replace all single solutions with new "dirtiest" repeat is replaced and shifted to end position Occurs after every run

Question 48

Processing Outcomes

Answer 48

Tissue shrinkage Brittle Lipids dissolved (tightly bound myelin stays)

Question 49

Microwave processing

Answer 49

FAST finicky 1 sample at a time