Basic Histology and Methods

Question 1

Describe the steps involved in tissue processing

Answer 1

1. Selection of sample
2. Fixation: preserving the tissue in a living state
3. Dehydration of sample by placing it in increasingly concentrated solutions of alcohol
4. Clearing: removing alcohol using an organic solvent, often xylenes
5. Infiltration: placing tissue in a liquid wax so that the wax infiltrates the tissue
6. Embedding: Infiltrated tissue is placed in a liquid embedding media that allows the wax to solidify
7. Sectioning

Question 2

What are the different types of microscopy and relevant differences?

Question 3

Cytological significance of differential staining produced by Hematoxylin and eosin (H&E)

Answer 3

Hematoxylin is a basic dye that stains negatively charged substances (basophilic, acidic themselves).

Eosin is an acidic dye that stains positively charged structures (acidophilic, basic themselves)

Question 4

Cytological significance of differential staining produced by Periodic acid-Schiff (PAS):

Answer 4

PAS stains polysaccharides, so a positive stain indicates high concentration of carbohydrates in the sample.

Question 5

Cytological significance of differential staining produced by Trichrome:

Answer 5

Each trichrome is a stain that contains three different types of dye to highlight different aspects of the cell. For example, Masson’s TC stains collagen blue, muscle cells red, and cytoplasm of non-muscle cells pink/light red. Nuclei are black

Question 6

Cytological significance of differential staining produced by Elastin:

Answer 6

A positive elastin stain (dark blue or black) indicates presence of elastin fibers

Question 7

What is the function and significance of interpretation of immunohistochemistry?

Answer 7

Immunohistochemistry utilizes antibodies that are able to bind to the substance of interest. When the bound antibodies are illuminated and imaged, the location of the substance can be verified. Important in imaging of tumors because tumor cells often express distinct proteins

Question 8

What is the function and significance of interpretation of electron microscopy?

Answer 8

TEM (e pass through sample) allows visualization of organelles

SEM (e bounce off sample) allows visualization of 3d structures on the surface of the cell

Question 9

What is the function and significance of interpretation of fluorescent microscopy?

Question 10

What is histology and why does it matter?

Answer 10

Def: the study of tissues of the body and how these tissues are arranged to makeup organs. “Micro anatomy”

Important b/c: massively aids in understanding the cellular/microscopic constituents of the body

Question 11

Basic functions of ECM

Answer 11

Supports cell/tissue structure and contains fluid for transport between cells

Question 12

Tissue preparation and goals

Answer 12

Traditional def: slicing of tissues into thin (translucent) sections/slices.

Goal: preserve tissue structure and ability to differentiate between cells

Question 13

Methods of tissue firming preparation prior to slicing - list pros and cons of each (2)

Answer 13

Freezing - fast, but expansion of water causes artifacts

Embedding - replacing the water with a liquid that can be frozen without much expansion (most common)

Question 14

State the 8 steps of tissue prep for Light Microscopy (embedding)

Answer 14

1. Selection
2. Fixation
3. Dehydration
4. Dehydration
5. Clearing
6. Infiltration
7. Embedding
8. Sectioning

Question 15

Selection (tissue prep step)

Answer 15

Harvesting sample from macroscopic tissue

Question 16

Fixation (tissue prep step)

Answer 16

Preservation of tissue in “living state.” Treat small pieces of tissue with chemicals that preserve by cross-linking proteins and inactivating degradative enzymes

Question 17

Dehydration (tissue prep step)

Answer 17

Serial submersion in increasingly concentrated solutions of alcohol up to 100% to remove all water

Question 18

Clearing (tissue prep step)

Answer 18

Alcohol is removed in toluene/other agents in which both alcohol and paraffin are miscible (xylene the MOST common clearing agent).

Also removes fat from tissue

Question 19

Infiltration

Answer 19

Tissue is placed in liquid wax (embedding media) which INFLITRATES the tissue

Question 20

Embedding

Answer 20

The infiltrated tissue is placed in the liquid embedding media (paraffin) which is then allowed to solidify around the tissue in a mold that is designed to fit the microtome

Question 21

Sectioning

Answer 21

A microtome is used to cut thin slices (3-10 um). Slices are placed on glass slides for further processing and staining

Question 22

Why do you have to stain in histology?

Answer 22

Without stain, thinly sliced tissue is translucent with ~no contrast between cells

Question 23

What is the most common stain?

Answer 23

Hematoxylin and Eosin (H&E)

Question 24

Describe Hematoxylin and how it stains and common particles it stains

Answer 24

A basic dye that stains neg. charged molecules. Imparts a blue/purple dye.

Common stain-ees: Nucleus, RNA-rich portions of cytoplasm, cartilage matrix

Question 25

What is the term used to describe molecules that are stained by basic dyes?

Answer 25

Basophilic (or said to have basophilia)

Question 26

Describe Eosin, how it stains, and common particles it stains

Answer 26

Acidic dye that stains positively charged molecules red/pink. Commonly stains: cytoplasm and collagen

Question 27

What is the term that is used to describe molecules that are stained by acidic dyes?

Answer 27

Eosinophilc or Acidophilic (or said to have acidophilia)

Question 28

Basophilic molecules themselves are ______, whereas acidophilic molecules are ______!

Answer 28

Basophilic molec are ACIDIC Acidophilic molec are BASIC

Question 29

Describe PAS stain and commonly stained substances

Answer 29

Periodic-acid Schiff stain - strongly stains polysaccharides and other carb-rich tissue deep purple or magenta Common stain-ees: mucus, basal laminae, glycogen rich tissue (tissue), thyroid colloid, some pathogenic organisms

Question 30

What does a PAS-positive stain indicate?

Answer 30

There are large amounts of carbohydrates in the tissue sample

Question 31

Describe Trichrome Stain

Answer 31

Several types; each type contains three stains that indicate different aspects of the tissue.

Question 32

Masson's Trichrome - Colors and Meanings

Answer 32

Blue/green: collagen Red: muscle cells Pink/light red: cytoplasm of non-muscle cells Black: nuclei

Question 33

1. What does it mean when an elastin (VVG) stain shows dark blue/black? 2. What is a common use of an elastin stain?

Answer 33

1. There are elastic fibers present 2. Commonly used to highlight the structure of large arteries

Question 34

Define immunohistochemistry and its mechanism

Answer 34

A method to visualize specific molecules in a tissue sample Mechanism: antibodies bind to a molecule of interest; the antibody is then labeled in some way to allow visualization

Question 35

Define the "Brown Wave" and what indicates a positive stain

Answer 35

Common method of immunohistochemistry used that uses a brown chromagen to visualize the bound antibodies Positive stain: brown stain matches the expected localization of the protein

Question 36

Basis of Light Microscopy and various types

Answer 36

Interaction of light and tissue (different interactions for different types of tissue) Types: bright-field, fluorescent, confocal

Question 37

Bright Field Microscopy: state basis and pros and cons

Answer 37

Light passing through specimen. Pros: cheap, fast, a lot of option for tissue processing Cons: relatively limited magnification resolution

Question 38

Fluorescent Microscopy: state basis and pros and cons

Answer 38

Uses fluorescent compounds either naturally occurring in tissue or introduced as a stain Pros: higher contrast, multiplex staining Cons: slower and more expensive than bright-field, needs to work in dark

Question 39

Confocal microscopy: state basis and pros and cons

Answer 39

Small point of high-intensity light is delivered through a pinhole aperture in front of the image detector, then scans the whole slide Pros: reduces light scatter and increase contrast/resolution, greater localization, 3D image Cons: slow and expensive

Question 40

How does electron microscopy work? How does it compare to light microscopy?

Answer 40

A beam of electrons interacts with tissue ad emits wavelengths that are picked up by a detector. Better resolution that light microscopy because wavelength of electron beam is shorter than light. Although, EM is not widely utilized because of high cost and time expense

Question 41

Describe the two types of electron micropscopy

Answer 41

1. TEM=Transmission electron microscopy: electron beam passes through specimen; view organelles (ultrastructure) 2. SEM=Scanning electron microscopy: electron beam bounces off the specimen: surface details are shown in high res with 3D appearance

Question 42

Which method of electron microscopy allows visualization of organelles?

Answer 42

TEM: transmission electron microscopy Draw out a cell shown by TEM!

Question 43

Cellularity and specific tricks (skill in reading histo. image)

Answer 43

Counting how many cells there are. Look for nuclei: Remember that cells usually only have 1 nucleus each, and most non-cancerous cells do not overlap each others' boundaries.

Question 44

What is the significance of cell size when reading a histological image? Additional tips in reading

Answer 44

Size helps determine cell type. Must find a good reference in the image! RBCs are a good reference for size if you can find them

Question 45

Significance of multiple factors in reading a histological image 3/4 rule

Answer 45

SO IMPORTANT to integrate findings regarding multiple criteria because of the high variability among cell anatomy between different parts of the body and different individuals. 3/4 rule: if 3 out of 4 criteria math for a specific cell type/organ, that one missing criterion is most likely over-ruled

Question 46

Can you rely on color from one image to another when reading histo images? Why? What is a good alternative characteristic to look at?

Answer 46

NO! Because stain quality varies so much between institutions Good alternative: texture!