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Foundations of Medicine I > Basic Histology and Methods > Flashcards

Basic Histology and Methods Flashcards

1
Q

Describe the steps involved in tissue processing

A
  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
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2
Q

What are the different types of microscopy and relevant differences?

A
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3
Q

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

A

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)

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4
Q

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

A

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

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5
Q

Cytological significance of differential staining produced by Trichrome:

A

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

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6
Q

Cytological significance of differential staining produced by Elastin:

A

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

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7
Q

What is the function and significance of interpretation of immunohistochemistry?

A

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

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8
Q

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

A

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

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9
Q

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

A
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10
Q

What is histology and why does it matter?

A

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

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11
Q

Basic functions of ECM

A

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

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12
Q

Tissue preparation and goals

A

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

Goal: preserve tissue structure and ability to differentiate between cells

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13
Q

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

A

Freezing - fast, but expansion of water causes artifacts

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

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14
Q

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

A
  1. Selection
  2. Fixation
  3. Dehydration
  4. Dehydration
  5. Clearing
  6. Infiltration
  7. Embedding
  8. Sectioning
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15
Q

Selection (tissue prep step)

A

Harvesting sample from macroscopic tissue

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16
Q

Fixation (tissue prep step)

A

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

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17
Q

Dehydration (tissue prep step)

A

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

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18
Q

Clearing (tissue prep step)

A

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

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19
Q

Infiltration

A

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

20
Q

Embedding

A

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

21
Q

Sectioning

A

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

22
Q

Why do you have to stain in histology?

A

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

23
Q

What is the most common stain?

A

Hematoxylin and Eosin (H&E)

24
Q

Describe Hematoxylin and how it stains and common particles it stains

A

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

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

25
Q

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

A

Basophilic (or said to have basophilia)

26
Q

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

A

Acidic dye that stains positively charged molecules red/pink.

Commonly stains: cytoplasm and collagen

27
Q

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

A

Eosinophilc or Acidophilic (or said to have acidophilia)

28
Q

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

A

Basophilic molec are ACIDIC

Acidophilic molec are BASIC

29
Q

Describe PAS stain and commonly stained substances

A

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

30
Q

What does a PAS-positive stain indicate?

A

There are large amounts of carbohydrates in the tissue sample

31
Q

Describe Trichrome Stain

A

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

32
Q

Masson’s Trichrome - Colors and Meanings

A

Blue/green: collagen

Red: muscle cells

Pink/light red: cytoplasm of non-muscle cells

Black: nuclei

33
Q
  1. What does it mean when an elastin (VVG) stain shows dark blue/black?
  2. What is a common use of an elastin stain?
A
  1. There are elastic fibers present
  2. Commonly used to highlight the structure of large arteries
34
Q

Define immunohistochemistry and its mechanism

A

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

35
Q

Define the “Brown Wave” and what indicates a positive stain

A

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

36
Q

Basis of Light Microscopy and various types

A

Interaction of light and tissue (different interactions for different types of tissue)

Types: bright-field, fluorescent, confocal

37
Q

Bright Field Microscopy: state basis and pros and cons

A

Light passing through specimen.

Pros: cheap, fast, a lot of option for tissue processing

Cons: relatively limited magnification resolution

38
Q

Fluorescent Microscopy: state basis and pros and cons

A

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

39
Q

Confocal microscopy: state basis and pros and cons

A

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

40
Q

How does electron microscopy work?

How does it compare to light microscopy?

A

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

41
Q

Describe the two types of electron micropscopy

A
  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
42
Q

Which method of electron microscopy allows visualization of organelles?

A

TEM: transmission electron microscopy

Draw out a cell shown by TEM!

43
Q

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

A

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.

44
Q

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

Additional tips in reading

A

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

45
Q

Significance of multiple factors in reading a histological image

3/4 rule

A

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

46
Q

Can you rely on color from one image to another when reading histo images? Why?

What is a good alternative characteristic to look at?

A

NO! Because stain quality varies so much between institutions

Good alternative: texture!

Foundations of Medicine I (3 decks)

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