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Histotechnology > Safety, Fixation, Fixatives, Tissue Processing > Flashcards

Safety, Fixation, Fixatives, Tissue Processing Flashcards

1
Q

4 major hazard classes

A
  1. biological/infectious
  2. mechanical
  3. chemical
  4. physical
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2
Q

what inactivates TB?

A

moist heat 121°C for 15min

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3
Q

what is CJD?

A

prion disease

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4
Q

what are prions susceptible to? (4)

A
  1. autoclave 1hr at 132-134°C
  2. 5% sodium hypochlorite for 2hrs
  3. concentrated formic acid for 1hr
  4. 1N sodium hydroxide for 1hr
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5
Q

what do prions remain infectious in? (3)

A
  1. well fixed tissues
  2. paraffin blocks
  3. stained slides
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6
Q

recommended treatment for prion tissues:

A

48h-14days in NBF
conc. formic acid for 1h
fresh NBF for 48h

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7
Q

how is waste formalin and equipment from prion fixing treated? (3)

A
  1. waste formalin is diluted with 2N NaOH and let stand for 1h
  2. steel instruments and grossing station treated with 1N NaOH for 1h, then soap + water
  3. gloves/gowns/aprons autoclaved or incinerated
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8
Q

4 classifications of infectious waste:

A
  1. pathologic material specimens
  2. blood
  3. microbiological/cultures
  4. sharps
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9
Q

what is the PEL?

A

Permissible Exposure Limit: the maximum safe concentration of exposure, monitored per 8hr shift

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10
Q

what is the TWA?

A

Time Weighted Average: aka action level

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11
Q

what is the STEL?

A

Short Term Exposure Limit: highest possible TWA exposure for any 15 minute period during the work shift. should be measured during worst 15min period
Always > TWA

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12
Q

what is the CL?

A

Ceiling Limit: maximum permissible instantaneous exposure at any time

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13
Q

what is the TWA for formaldehyde?

A

0.75 ppm

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14
Q

what is the STEL for formaldehyde?

A

2.0 ppm

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15
Q

difference between combustibles and flammables:

A

combustibles have a higher FP
flammables vapours must be carefully controlled and has special storage conditions

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16
Q

what is an oxidizer?

A

can initiate or promote combustion in any other material

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17
Q

difference between toxic dose and toxic concentration low:

A

toxic dose is for any toxic method other than inhalation

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18
Q

what is lethal dose low?

A

lowest dose reported to have caused human death OR lowest single killing dose for animals

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19
Q

what is LD50?

A

calculated dose of a chemical expected to cause the death of 50% of an experimental animal population through any route EXCEPT inhalation

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20
Q

what is a class A fire?

A

ordinary combustibles (paper, wood)

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21
Q

what is a class B fire?

A

flammable liquids and gases, require oxygen to be blocked from the fuel

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22
Q

what is a class C fire?

A

electrical

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23
Q

what is a class D fire?

A

combustibles that are difficult to extinguish (metals like magnesium)

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24
Q

what are the colour categories of the National Fire Prevention Association?

A

Red = flammability
Yellow = instability
White = special (strong oxidizer, water reactive)
Blue = health

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25
Q

define histology:

A

the study of the structure of cells and tissues microscopically, demonstrating disease processes and the effects on microanatomy

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26
Q

define pathology:

A

study of diseased tissues and the processes/effects/causes of disease

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27
Q

8 tissue prep techniques:

A
  1. fixation
  2. accession
  3. grossing
  4. processing (dehydrate, clear, infiltrate)
  5. embedding
  6. paraffin sectioning
  7. frozen sectioning
  8. staining
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28
Q

define fixation:

A

stabilizing proteins and altering tissues to make them resistant to subsequent treatment

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29
Q

what is tissue grossing?

A

macroscopic review of received tissue specimens

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30
Q

5 parts of tissue grossing:

A
  1. all of specimen present
  2. 3D measurements
  3. overall appearance
  4. consistency
  5. abnormalities
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31
Q

5 steps of tissue processing:

A
  1. fixation
  2. dehydration
  3. clearing
  4. infiltration
  5. embedding
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32
Q

what is H&E staining and what does each stain show?

A

Hematoxylin (nucleus) and Eosin (cytoplasm)

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33
Q

what is the ischemic time of a tissue specimen?

A

the time at which blood flow was cut off

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34
Q

what is autolysis?

A

dissolving of cells by internal enzymatic action

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35
Q

what is desquamation?

A

sloughing off of epithelial cells from the basement membrane

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36
Q

what is putrefaction?

A

destruction of cellular components by external bacterial action

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37
Q

what does proper fixation maintain?

A

proper relationship between cells and extracellular substances: connective fibers (collagen, reticulin, elastin)

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38
Q

5 major groups of fixatives;

A
  1. aldehydes
  2. alcohols
  3. mercurials
  4. oxidizing agents
  5. picrates
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39
Q

8 factors that affect fixation:

A
  1. temperature
  2. size
  3. time
  4. fixative choice
  5. penetration
  6. tissue storage
  7. pH
  8. osmolality
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40
Q

4 effects of temperature on fixation:

A
  1. increased speed of fixation BUT also increased speed of autolysis and putrefaction
  2. heat coagulates proteins
  3. cold/RT fixation is slower but autolysis and putrefaction are also slowed
  4. often the first step of processing is formalin at about 37°C
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41
Q

effects of size on fixation:

A
  1. thickness
  2. structural complexity
  3. density
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42
Q

what is the correct volume of tissue to fixative ratio?

A

1:20

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43
Q

how does time affect fixation?

A
  1. longer interval between ischemia and fixation = more autolysis and putrefaction
  2. fixatives usually penetrate ~ 1mm/hr
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44
Q

optimal pH for fixation:

A

6-8

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45
Q

what causes acidity in fixation?

A

hypoxia

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46
Q

what prevents acidity in fixation?

A

buffer

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47
Q

what is the result of acidity in formalin fixation?

A

formalin-heme pigment

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48
Q

what is commercial formalin buffered with?

A

phosphate

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49
Q

3 effects of fixation being too acid or alkaline:

A
  1. artefact
  2. damage of protein matrix
  3. damage of delicate structures by hydrolysis
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50
Q

buffers and their role in fixation: (3)

A
  1. indifferent salts
  2. maintain desired pH
  3. aid in penetration of fixative
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51
Q

effects of iso/hypotonic solutions on fixation:

A

swelling (water flows in)

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52
Q

effects of hypertonic solutions on fixation:

A

shrinkage (water flows out)

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53
Q

should fixatives be hypo or hypertonic? why?

A

slightly hypertonic to prevent swelling and cause only minimal shrinkage (done with buffering salts)

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54
Q

4 physical changes of fixation:

A
  1. hardening
  2. tissue protection
  3. stain enhancement
  4. visual differentiation
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55
Q

pros & cons of fixative hardening:

A

pro: tissues are stronger and easier to slice
con: over-hardening can make tissue brittle and shatter

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56
Q

what does fixation protect tissues from? (3)

A

future reagents:
1. organic solvents
2. heat
3. acids

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57
Q

what property of fixatives affects staining?

A

mordanting properties

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58
Q

how does fixation create visual differentiation?

A

changes the refractive index + allows cells to more easily stain

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59
Q

what is the RI of fresh tissue cut at 5 microns?

A

1.35

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60
Q

what is the RI of fixed tissue/borosilicate glass?

A

1.54

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61
Q

effects of fixation on proteins:

A

affects the side chain reactive groups, either degenerating or precipitating the protein

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62
Q

what are the most important protein side chain groups in fixation? (3)

A
  1. Amino (+ve)
  2. Sulphydryl (-ve)
  3. Carboxyl (-ve)
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63
Q

what is denaturation of protein?

A

irreversible alteration of the molecular structure: affects the secondary structure and causes collapse

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64
Q

how is the aqueous component of the cell changed by denaturation?

A

changes from SOL to GEL

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65
Q

what are the 2 basic elements of tissue?

A
  1. structural
  2. aqueous
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66
Q

key sources of error in poor fixation: (5)

A
  1. insufficient reagent
  2. poor penetration
  3. fixative not evenly exposed
  4. insufficient time
  5. too much time
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67
Q

how does insufficient reagent affect fixation?

A

incomplete immersion of the tissue in solution, poor fixation

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68
Q

what causes poor fixative penetration?

A

physical barriers like blood, mucous, fibrous capsules

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69
Q

what causes uneven fixation?

A

not all sides of tissue exposed (sitting on bottom of container, etc.)

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70
Q

how does time in fixative affect fixation?

A

too little = unfixed inside
too much = brittle and shrunken

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71
Q

what is the difference between tolerant and intolerant fixatives?

A

tissues can be left in tolerant fixatives almost indefinitely, tissues in intolerant fixatives must be closely monitored

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72
Q

9 kinds of fixative artefacts:

A
  1. protein precipitation
  2. autolysis/desquamation
  3. putrefaction
  4. diffusion/substance loss
  5. shrinkage
  6. distortion
  7. excessive hardening
  8. formalin pigments
    9.mercury pigments
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73
Q

3 effects of putrefaction on end result of tissue slide:

A
  1. ill-defined cell borders
  2. poor nuclear detail
  3. holes from gas bubbles
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74
Q

effect of diffusion/substance loss (fixation artefact):

A

glycogen and lipids are lost or diffused, glycogen tends to stream to one side of cell (polarization)

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75
Q

which structures show shrinkage the most? (3)

A
  1. muscle
  2. neurons
  3. glomeruli
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76
Q

best way to avoid distortion during fixation:

A

appropriate fixative

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77
Q

best way to prevent excessive hardening:

A

minimal exposure to intolerant fixatives (monitored exposure)

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78
Q

cause of formalin pigments:

A

pH is too low-acid degradation of hemoglobin

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79
Q

how to remove formalin pigments: (4)

A
  1. picric acid dissolved in ethanol then rinse in water or lithium carbonate to remove yellow colour
  2. soak tissue in 1% KOH dissolved in 80% ethanol
  3. soak tissue in 1% NaOH in 100% alcohol
  4. ammonium hydroxide in 70% alcohol
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80
Q

when do mercury pigments occur?

A

always when a mercuric fixative is used

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81
Q

how to remove mercury pigments:

A

iodine followed by Na thiosulfate (hypo)

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82
Q

which fixatives react with nucleic acids?

A

none

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83
Q

which fixatives are preferred for nucleic acid?

A

Carnoy’s and acetic alcohol

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84
Q

at what temperatures will formaldehyde react with DNA and RNA?

A

RNA = 45°C
DNA = 65°C

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85
Q

what is believed to be the method of fixation of nucleic acids?

A

entrapment of the nucleic acids by the fixed nuclear protein

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86
Q

fixation agents will be either: (2)

A
  1. physical (heat/dessication)
  2. chemical
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87
Q

chemical fixation agents will be either: (2)

A
  1. solids in aqueous solution
  2. liquids (w/ or w/out H2O)
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88
Q

3 methods of fixation:

A
  1. heat
  2. freezing
  3. chemical
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89
Q

what do coagulating and non-coagulating fixatives have in common? (2)

A
  1. irreversible denaturation of proteins
  2. form a mesh of protein in a continuous phase
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90
Q

difference between coagulating and non-coagulating fixatives:

A

degree of harshness of protein denaturation

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91
Q

coagulant fixatives are _______ than non-coagulating fixatives

A

harsher

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92
Q

coagulant fixation is directed at:

A

proteins

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93
Q

how do coagulant fixatives affect proteins? (4)

A
  1. change the protein structure
  2. strengthen the linkage to prevent breaking
  3. very closed meshwork of denatured proteins
  4. meshwork allows for penetration of solutions
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94
Q

coagulant fixatives cause ______ shrinkage than non-coagulant fixatives

A

more

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95
Q

2 disadvantages of coagulant fixatives:

A
  1. may produce artefact
  2. may compromise cytological details
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96
Q

non-coagulant fixatives form _______________ with proteins. what is the effect?

A

additive compounds.
create a gel that prevents penetration of subsequent solutions

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97
Q

non-coagulant fixatives are _________ commonly used than coagulant

A

more

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98
Q

Compound fixatives are _____ commonly used than simple

A

more

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99
Q

most compound fixatives contain both:

A

coagulant and non-coagulant fixatives

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100
Q

how are larger specimens fixed?

A

perfusion: large volumes forcefully flooded into the tissue using a vacuum.

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101
Q

2 methods of causing irreversible protein denaturation:

A
  1. additive fixation
  2. non-additive fixation
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102
Q

how does additive fixation work? (2)

A
  1. binds proteins by acting as the link between them, joining to the reactive side groups
  2. alters the electrical charge on the molecule, thus altering the shape of the tertiary structure
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103
Q

all primary fixing agents are additive except:

A

ethanol (reacts like non-additive fixative)

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104
Q

how does non-additive fixation work? (2)

A
  1. precipitate/coagulate proteins without adding themselves to the structure of the tissue
  2. dehydration by dissociating bound H2O molecules from tissue protein groups
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105
Q

water loss always occurs in ________ fixation

A

non-additive

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106
Q

how do non-additive fixatives link proteins? (2)

A
  1. removing water brings reactive side groups together allowing linkage
  2. polymerization
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107
Q

non-additive fixation results in both:

A

shrinkage and hardening

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108
Q

in terms of composition, fixatives can be: (2)

A
  1. simple - one fixing chemical
  2. compound - more than one fixing chemical
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109
Q

3 categories of compound fixatives:

A
  1. micro-anatomical
  2. cytological
  3. histochemical
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110
Q

microanatomical fixatives: (3)

A
  1. routine
  2. target overall tissue
  3. retain anatomy
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111
Q

purpose of cytological fixatives: (2)

A
  1. used in instances where intracellular components need to be preserved
  2. target cellular components (nuclear and cytoplasmic)
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112
Q

histochemcial fixatives are used to:

A

target particular chemicals in tissues (eg enzymes and antigens)

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113
Q

8 common primary fixatives:

A
  1. formaldehyde
  2. potassium dichromate
  3. mercuric chloride
  4. ethanol
  5. picric acid
  6. acetic acid
  7. osmium tetroxide
  8. glutaraldehyde
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114
Q

formalin is produced by dissolving which gas into which solvent?

A

formaldehyde into water

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115
Q

what percent formaldehyde is undiluted formalin?

A

37-40%

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116
Q

what is 10% neutral buffered formalin?

A

4% solution of formaldehyde + phosphates to maintain neutral pH

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117
Q

formaldehyde is what type of fixative? (4)

A
  1. tolerant
  2. rapid
  3. non-coagulating
  4. additive
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118
Q

what is the main reaction site of formaldehyde?

A

amino group (NH2) found on side chain

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119
Q

what is the cross-linkage formed by formaldehyde and glutaraldehyde called?

A

methylene bridge

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120
Q

what other groups does formaldehyde react with?

A

sulfhydryl groups to form cross links with cysteine (amino acid)

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121
Q

which fixative causes the least amount of shrinkage?

A

formaldehyde

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122
Q

what are the only two fixatives more hardening than formaldehyde?

A

ethanol and acetone

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123
Q

which fixative allows for the most special staining techniques?

A

formaldehyde

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124
Q

how does formaldehyde fix glycogen?

A

indirectly: formaldehyde forms protein meshwork that traps glycogen inside

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125
Q

does formaldehyde cause streaming artefact?

A

yes

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126
Q

how does formaldehyde react with lipids?

A

preserves them but does not make them insoluble - lipids will gradually be lost with storage

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127
Q

does formaldehyde fix triglycerides?

A

no

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128
Q

is formaldehyde a reducing or oxidizing agent?

A

reducing

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129
Q

what happens if a reducer and oxidizer are combined in a compound fixative?

A

formation of precipitate and poor fixation

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130
Q

name 2 alkaline buffers that can be added to 10% formalin:

A
  1. sodium phosphate
  2. sodium borate
131
Q

what is formalin neutralized with? at what concentration?

A

borax at ~ 15mL/L

132
Q

3 disadvantages of formaldehyde:

A
  1. damage to mucous membranes/eyes
  2. carcinogen
  3. shrinkage
133
Q

when does potassium dichromate form additive compounds?

A

reacts with proteins

134
Q

which fixative’s major properties depend on pH?

A

potassium dichromate

135
Q

is potassium dichromate coagulating or non-coagulating?

A

depends on the pH:
< 3.4 = coagulating
> 3.8 = non-coagulating

136
Q

is potassium dichromate tolerant or intolerant?

A

depends on the pH:
< 3.4 = intolerant
> 3.8 = tolerant

137
Q

at what speed does potassium dichromate penetrate tissues?

A

1.33 mm/hr

138
Q

what protein groups does potassium dichromate have an affinity for?

A

COOH and OH

139
Q

what about potassium dichromate causes the enhanced acidophilic staining of tissues?

A

the disruption of salt linkages of proteins and the increase of reactive basic groups (-NH2)

140
Q

what happens when tissues are transferred directly from potassium dichromate to aqueous ethanol?

A

an insoluble precipitate, most likely chromic oxide

141
Q

how is chrome pigment prevented when fixing tissues in potassium dichromate?

A

all chromate salts must be removed from the tissue prior to exposure to alcohol - wash in running water 12-24h prior to alcohol

142
Q

what are the mordanting qualities of potassium dichromate?

A

it increases the affinity of tissue cytoplasm for acid dye

143
Q

2 unique disadvantages of potassium dichromate:

A
  1. insoluble lower oxide that must be washed to prevent a brown precipitate
  2. tissue is coloured yellow
144
Q

what is the main reason for using potassium dichromate as a fixative?

A

demonstrating adrenal gland disease called pheochromocytoma

145
Q

how does potassium dichromate react with carbohydrates?

A

oxidizes glycogen, not good for demonstrating glycogen

146
Q

what other fixatives are compatible with potassium dichromate? (3)

A
  1. picric acid
  2. mercuric chloride
  3. osmium tetroxide
147
Q

what are the additive/coagulation/tolerance properties of mercuric chloride?

A
  1. additive
  2. coagulating
  3. intolerant
148
Q

what 3 substances is mercuric chloride soluble in?

A
  1. water
  2. ethanol
  3. benzene
149
Q

is mercuric chloride a reducer or oxidizer?

A

neither

150
Q

what is the mode of action for mercuric chloride?

A

forms a bridge by chemically adding itself to the sulfhydryl, carboxyl, and amino groups

151
Q

mercuric chloride rate of penetration:

A

0.7-0.8mm/hr: this slows after 5 minutes due to extreme hardening

152
Q

what gives mercuric chloride its mordanting properties?

A

being a very powerful protein coagulant enhances the staining and makes tissues receptive to dye

153
Q

how does mercuric chloride react with carbohydrates?

A

no reaction, glycogen is not preserved in tissue fixed with mercuric chloride

154
Q

how does mercuric chloride react with lipids?

A

no reaction, does not destroy or preserve them, so they are destroyed by future exposure to organic solvents (like xylene)

155
Q

what does it mean that mercuric chloride is radio-opaque?

A

it does not allow x-rays to pass through, preventing the use of x-rays to determine end-point of decalcifcation

156
Q

why is mercuric chloride never used alone?

A

too harsh - its extreme intolerance needs to be counteracted by a gentler fixative

157
Q

what is mercuric chloride used for aside from its mordanting properties?

A

somewhat useful for fixing some mucopolysaccharides, like mucin

158
Q

what 2 substances is it important that ethanol is miscible with?

A
  1. water
  2. xylene
159
Q

what are the additive/coagulation/tolerance properties of ethanol?

A
  1. non-additive
  2. coagulating
  3. intolerant
160
Q

what type of fixative is ethanol classed as?

A

cytological

161
Q

what makes ethanol intolerant?

A

causes extreme shrinkage

162
Q

is ethanol a reducer or oxidizer?

A

reducer

163
Q

how does ethanol react with carbohydrates?

A

best fixative for preserving glycogen: ethanol directly precipitates glycogen to be demonstrated by glycogen stains

164
Q

does ethanol cause glycogen streaming?

A

yes

165
Q

how does ethanol react with lipids?

A

its close relation to H2O means it competes for hydrogen bonds, replacing H2O molecules in the tissues

166
Q

which fixative causes the worst tissue shrinkage?

A

ethanol

167
Q

what is the only primary fixative that hardens more than ethanol?

A

acetone

168
Q

3 useful aspects of ethanol:

A
  1. preservation of uric acid crystals that would dissolve in an aqueous solution
  2. antigens remain intact
  3. enzymes remain intact
169
Q

what is the chemical name for picric acid?

A

trinitrophenol (TNP)

170
Q

what is the standard concentration for picric acid?

A

saturated aqueous

171
Q

what are the additive/coagulation/tolerance properties of picric acid?

A
  1. additive
  2. coagulating
  3. intolerant
172
Q

why is picric acid not suitable for staining DNA/RNA?

A

strong coagulant of nucleoprotein but leaves DNA/RNA soluble, hydrolyzing it

173
Q

what are the hardening properties of picric acid?

A

it does not harden tissue

174
Q

is picric acid an oxidizer or reducer?

A

neither

175
Q

what is the main protein reaction with picric acid?

A

the negative charged ionized radical of picric acid reacts with the positive charged amine group on protein chains

176
Q

why does picric acid create the need for washing?

A

picric acid combines with the amine groups and forms protein-picrate complexes

177
Q

why shouldn’t tissues be exposed to water after picric acid? what should they go into instead?

A
  1. a small number of protein-picrate complexes are water soluble and would be lost
  2. alcohol to prevent the loss of those picrates
178
Q

how does picric acid react with carbohydrates and lipids?

A

does not fix either one, but indirectly fixes glycogen the way formaldehyde does

179
Q

what is picric acid used for aside from fixation?

A

dye

180
Q

why must picric acid be kept in water and not allowed to dry?

A

EXPLOSION!!!

181
Q

what other fixative is picric acid likely to be found in a compound with? why?

A

acetic acid, because it counteracts the shrinkage caused by picric acid

182
Q

what are the additive/coagulation/tolerance properties of acetic acid? (5)

A
  1. additive
  2. non-coagulating (cytoplasm)
    2.5 coagulating (nuclei)
  3. tolerant (cytoplasm)
    3.5 intolerant (nuclei)
183
Q

why is acetic acid frequently added to compound fixatives? (2)

A
  1. ability to fix nuclei
  2. counteracts shrinkage
184
Q

what is the mode of action of acetic acid? (2)

A
  1. the acetate ion reacts with the amine group of the protein
  2. tissue structural fibres are joined by the amino-carboxyl bonds - acetic acid breaks those bonds
185
Q

why does acetic acid cause swelling? what substance in particular is affected?

A
  1. acetic acid exposes hydrophilic groups that attract water between the structural fibres
  2. collagen
186
Q

why is acetic acid a poor preserver of RBCs?

A

lyses them

187
Q

what is the main function of acetic acid as a fixative? why?

A
  1. preservation of nucleoproteins
  2. yields distinct chromatin pattern + precipitates dNA
188
Q

what are the hardening and penetrating properties of acetic acid?

A
  1. leaves tissues soft
  2. penetrates very rapidly
189
Q

3 unique disadvantages of acetic acid:

A
  1. destroys mitochondria and golgi apparatus
  2. lyses RBCs
  3. PEL = 10ppm
190
Q

how does acetic acid interact with carbohydrates and lipids?

A

no reaction with either one, neither is preserved/demonstrable

191
Q

why is osmium tetroxide an infrequently used fixative?

A

interferes with subsequent staining

192
Q

what is the primary use of osmium tetroxide as a fixative?

A

specimens for electron microscopy

193
Q

what are the additive/coagulation properties of osmium tetroxide?

A
  1. additive
  2. non-coagulating
194
Q

is osmium tetroxide an oxidizer or reducer?

A

oxidizer obviously

195
Q

what happens if tissues go from osmium tetroxide into alcohol?

A

going from oxidizer to reducer will create a brown precipitate

196
Q

what must be done to tissues fixed in osmium tetroxide prior to alcohol exposure?

A

washed in a buffer solution to remove residual OT

197
Q

how does osmium tetroxide interact with proteins?

A

OT reacts with the hydrogen groups on adjacent protein chains

198
Q

how does osmium tetroxide interact with carbohydrates?

A

no reaction - after prolonged exposure though, glycogen will appear as black granules

199
Q

how does osmium tetroxide interact with lipids? (4)

A
  1. OT has a high affinity for lipids
  2. OT will chemically combine with lipids and make them insoluble
  3. OT will oxidize lipids
  4. OT will become osmium dioxide and appear black
200
Q

3 unique disadvantages of osmium tetroxide:

A
  1. more expensive than gold
  2. deteriorates into osmium dioxide in sunlight
  3. after fixation tissues only have affinity for basic dyes
201
Q

what are the dual purposes of osmium tetroxide?

A

stain + dye

202
Q

what is unique about glutaraldehyde at RT?

A

oily liquid

203
Q

what is the primary use of glutaraldehyde as a fixative? why?

A

electron microscopy because it preserves ultra structure

204
Q

which fixative is also an effective antiseptic against AIDS, and used as an equipment sterilizer and embalming fluid?

A

glutaraldehyde

205
Q

what are the additive/coagulation/tolerance properties of glutaraldehyde?

A
  1. additive
  2. non-coagulating
  3. tolerant
206
Q

why can’t glutaraldehyde be combined with an oxidizer?

A

it’s a reducer

207
Q

what is the mode of action of glutaraldehyde?

A

reacts like formaldehyde, but faster due to its 2 aldehyde groups

208
Q

how does glutaraldehyde interact with carbohydrates?

A

like formaldehyde - indirect fixation

209
Q

how does glutaraldehyde interact with lipids?

A

no reaction, lipids will not be preserved

210
Q

what is a unique disadvantage of glutaraldehyde?

A

unstable, must be stored in fridge in small 2-4% vials to retard oxidation

211
Q

how long should tissues be fixed in glutaraldehyde? what should they be transferred to after?

A
  1. 2 hrs
  2. buffer solution
212
Q

which fixative is a rarely used and less toxic aldehyde than glutar and form?

A

glyoxal

213
Q

9 main compound fixatives:

A
  1. NBF
  2. formal saline
  3. formal calcium
  4. buffered formal sucrose
  5. formal alcohol
  6. Zenker’s
  7. Bouin’s
  8. B - 5
  9. Zinc-containing
214
Q

why are compound fixatives used? (2)

A
  1. to gain the advantages of multiple primary fixatives
  2. to counteract the downsides
215
Q

why are salts/buffers added to compound fixatives? (3)

A
  1. maintain osmotic pressure (hypo/iso/hyper-tonic)
  2. aid in penetration of fixatives into tissues
  3. maintain desired pH
216
Q

why are salts termed “indifferent”

A

take no part in protein precipitation

217
Q

what are the two diluents used in compound fixatives?

A

water or alcohol

218
Q

3 subcategories of compound fixatives:

A
  1. microanatomical
  2. cytological
  3. histochemical
219
Q

when is a microanatomical fixative used?

A

to preserve architecture

220
Q

when is a cytological fixative used?

A

to preserve individual cells

221
Q

when is a histochemical fixative used? (3)

A

to preserve a specific substance like cell membrane, antigens, and enzymes

222
Q

what does nbf contain? (3)

A
  1. formaldehyde
  2. phosphate buffer
  3. water
223
Q

what does formal saline contain? (3)

A
  1. formaldehyde
  2. sodium chloride
  3. water
224
Q

what does formal calcium contain?

A

10% formaldehyde in 1.0% calcium chloride

225
Q

what is formal calcium used for? (2)

A
  1. demonstrating phospholipids in lung & brain tissue
  2. recommended for lipid histochemistry
226
Q

what does buffered formal sucrose contain? (3)

A
  1. 10% formaldehyde
  2. 7.5% sucrose
  3. phosphate buffer (pH7.4)
227
Q

what is buffered formal sucrose used to demonstrate? (5)

A
  1. phospholipids
  2. fine structures
  3. some enzymes
  4. mitochondria
  5. endoplasmic reticulum
228
Q

what is in Zenker’s stock solution? (5)

A
  1. mercuric chloride
  2. potassium dichromate
  3. sodium sulphate
  4. distilled water
  5. glacial acetic acid
229
Q

what is Zenker’s stock solution combined with to make Zenker’s acetic?

A

acetic acid

230
Q

what is added to Zenker’s stock solution to form Helly’s? what is another name for this new solution?

A
  1. formaldehyde
  2. Zenker’s formal
231
Q

why does zenker’s stock solution require substances to be added right before use?

A

the solution will turn muddy brown about 24hrs after the addition of acetic acid

232
Q

what is zenker’s good for fixing? (4)

A
  1. liver
  2. spleen
  3. nuclei
  4. connective tissue
233
Q

what is the fixative of choice for blood-forming tissue? what are the blood-forming tissues?

A
  1. Helly’s
  2. bone marrow, spleen
234
Q

what is in bouin’s fixative? (3)

A
  1. picric acid
  2. formalin
  3. glacial acetic acid
235
Q

what is the dual purpose of acetic acid in Bouin’s?

A
  1. best fixative for giving good nuclear detail
  2. counteracts the shrinkage caused by picric acid
236
Q

which fixatives give brilliant trichrome stains? (2)

A
  1. Bouin’s
  2. Zenker’s
237
Q

which fixative is recommended for fixing endocrine tissue and to distinguish collagen from muscle?

A

bouin’s

238
Q

which fixative distorts/dissolves cytoplasmic organelles?

A

bouin’s

239
Q

which fixative should not be used for kidney tissue?

A

bouin’s

240
Q

what is in B - 5 fixative? (4)

A
  1. mercuric chloride
  2. sodium acetate
  3. distilled water
  4. formalin
241
Q

which fixative requires tissue storage in 70% alcohol post-fixation?

A

B - 5

242
Q

which fixative is good for lymphoreticular and hematopoietic tissues?

A

B - 5

243
Q

is B - 5 suitable for IHC studies?

A

yes

244
Q

what is in Z - 5 fixative? (3)

A
  1. zinc sulphate
  2. formalin (unbuffered)
  3. ethanol
245
Q

why is Z -5 slowly taking the place of B - 5? (3)

A
  1. less distortion
  2. tolerant
  3. no mercury
246
Q

5 cytological fixatives:

A
  1. carnoy’s
  2. zenker’s
  3. helly’s
  4. bouin’s
  5. ether/alcohol
247
Q

what is in carnoy’s fixative? (3)

A
  1. absolute alcohol
  2. chloroform
  3. glacial acetic acid
248
Q

why is carnoy’s fixative only good for small/STAT specimens?

A

fixes rapidly for the first 2mm and then stops, allowing 2mm tissues to be fixed in 15 minutes

249
Q

which fixative (cytological) fixes and preserves nissl substances and glycogen very well?

A

carnoy’s

250
Q

which cytological fixative is good for smears and tissue imprints?

A

95% ether/alcohol

251
Q

what are histochemical fixatives used for?

A

chemical constituents of cells, like enzymes and antigens

252
Q

what are secondary fixatives?

A

fixation in a second fixative to enhance preservation and for a mordanting effect

253
Q

what do secondary fixatives usually contain?

A

mercury or picric acid

254
Q

what is dehydration?

A

removal of water not bound to tissue components, but contained within the protein network

255
Q

why is dehydration necessary?

A

paraffin cannot enter the tissue if water is present

256
Q

how is diffusion involved in dehydration?

A

inward flow of dehydrating agent, outward flow of water

257
Q

why must water be removed gradually during the dehydration process?

A

rapid removal of water will rupture cells

258
Q

what types of tissues will require starting at a lower concentration of alcohol when dehydrating?

A

friable tissue like brain, blood clots

259
Q

what issue with dehydration yields soft, mushy blocks?

A

incomplete dehydration prevents clearing agent from acting properly

260
Q

how can you tell if dehydration is complete just from looking at the alcohol solution?

A

if the solution is cloudy = water remains
if the solution is clear = dehydration complete

261
Q

2 areas where dehydrating procedures are used:

A
  1. prepping of tissue for embedding
  2. prepping of stained tissue for mounting
262
Q

how is water removed during the dehydration process?

A

hydrophilic reagents attract water from the tissue

263
Q

3 common dehydrating agents:

A
  1. ethanol
  2. isopropyl alcohol
  3. acetone
264
Q

what is the best routine dehydrant?

A

ethanol

265
Q

how does xylene indicate if water is present?

A

milky

266
Q

which dehydrant is a good substitute for ethanol?

A

isopropyl alcohol

267
Q

which dehydrant cannot be used with celloidin?

A

isopropyl alcohol

268
Q

can isopropyl alcohol be absolute?

A

no, will always contain approx. 1% water

269
Q

why is acetone not the best dehydrant?

A

excessive hardening and shrinkage

270
Q

3 less common dehydrants:

A
  1. butanol
  2. dioxane
  3. ethylene glycol monoethyl ether
271
Q

which dehydrant is miscible with water, alcohol, xylene, and paraffin?

A

dioxane

272
Q

what are the 3 universal solvents?

A
  1. butanol
  2. dioxane
  3. tetrahyrdofuran
273
Q

stain interference occurs when tissues remain in dehydrant lower than what %, and higher than what %?

A

70, 80

274
Q

what is clearing?

A

de-alcoholization

275
Q

2 times when clearing is done:

A
  1. in processing, to remove the alcohol from the tissue
  2. in staining, to remove wax from the cut sections
276
Q

why is it called clearing?

A

the chemicals used have a high index of refraction and render tissue transparent

277
Q

what is the result of inadequate clearing?

A

inadequate infiltration, mushy tissue

278
Q

what is the only clearant with no effect on the RI?

A

chloroform

279
Q

why is excessive clearing bad?

A

tissue hardens, and further protein denaturation occurs, making microtomy harder

280
Q

what should the reagent to tissue ratio be for clearing?

A

50-100x the tissue volume

281
Q

2 consequences of improper clearing:

A
  1. moth eaten appearance
  2. sections crumble and tear out of the block
282
Q

7 clearing agents:

A
  1. xylene
  2. toluene tetrachloride
  3. benzene substitutes
  4. cedarwood oil
  5. chloroform
  6. carbon
  7. xylene substitutes
283
Q

health-related disadvantage of xylene:

A

in high concentrations it becomes a narcotic and habit-forming, affects the brain (memory loss)

284
Q

which clearant is the best of the aromatic hydrocarbons?

A

toluene

285
Q

5 advantages of cedarwood oil as a clearing agent:

A
  1. eco-friendly and safe
  2. gentle/tolerant
  3. can clear from 90% alcohol
  4. makes tissue transparent
  5. does not affect aniline dyes
286
Q

4 disadvantages of cedarwood oil as a clearant:

A
  1. slow penetrating
  2. difficult to remove from wax
  3. any remaining oil will make cutting difficult
  4. very expensive
287
Q

which clearant is ideal for hard and delicate tissues?

A

chloroform

288
Q

what is the conflict associated with chloroform?

A

it acts on tissues very desirably, but cannot be used in automated tissue processors because it is volatile and will melt plastic

289
Q

which clearants are the most similar?

A

chloroform and carbon tetrachloride

290
Q

what are the xylene alternatives?

A
  1. new class of clearants
  2. alkanes
  3. low in reactivity and toxicity
291
Q

what are terpenes?

A
  1. xylene substitute
  2. primary constituent of essential oils
  3. must be removed using xylene
292
Q

essential oils used in histology: (4)

A
  1. cloves
  2. origanum
  3. sandalwood
  4. cedarwood
293
Q

what are limonenes?

A

xylene substitute (less toxic), cause more contamination of paraffin

294
Q

what is infiltration/impregnation?

A

replacement of clearing agent with wax

295
Q

what is the purpose of infiltration?

A

maintains tissue structure, allowing for cutting of thin sections

296
Q

what is the pressure used for vacuum infiltration?

A

300-500mmHg

297
Q

how does the melting point of paraffin affect sectioning?

A

higher MP = thinner sections
lower MP = easier ribbons

298
Q

what is the plastic point?

A

lowest temp at which permanent deformation can occur without fracture

299
Q

additives used in paraffin wax: (4)

A
  1. plastic polymers
  2. asphalt
  3. rubber
  4. beeswax
300
Q

what is paraffin artefact?

A

shrinkage that occurs when tissue is attached to the glass

301
Q

which fixative causes more paraffin artefact? which one causes less?

A
  1. formalin
  2. bouin’s/zenker’s
302
Q

what is celloidin?

A

nitrocellulose specialty embedding compound used for CNS tissues

303
Q

what is double embedding?

A

using more than one substance to impregnate the tissue

304
Q

what are plastic/resin embedding/infiltration agents used for? (2)

A
  1. hard tissues like bone that need more support
  2. tissues that need extremely thin sections
305
Q

what are some major disadvantages of plastic/resins for infiltration/embedding?

A

require glass or diamond knives

306
Q

what is the main purpose of agar/gelatin for embedding/infiltration?

A

friable tissue

307
Q

what is 30% sucrose used for?

A

preparing frozen sections from unprocessed, formalin-fixed tissues

308
Q

requirements for a good automated processor: (7)

A
  1. LIS connectivity
  2. closed system
  3. easy access to retort chamber
  4. heat and vacuum
  5. constant agitation
  6. alarm system
  7. filter/ventilation for noxious fumes
309
Q

what is the risk of starting dehydration at 70% alcohol?

A

formation of precipitates from zinc or phosphate buffered formalin (increased pH)

310
Q

what is sponge artefact?

A

occurs when small tissues are placed between dry sponges prior to processing

311
Q

how to prevent sponge artefact?

A

soak sponges in fixative prior to processing

312
Q

what is processing by microwave?

A

increases TAT and eliminates xylene as a clearing agent

313
Q

what is microwave processing used for?

A

biopsy tissues

314
Q

what is the most critical step of embedding?

A

tissue orientation

315
Q

what is tissue inking?

A

indication of how to orient tissue when embedding

316
Q

why is it important for embedded blocks to be cooled rapidly?

A

smaller paraffin crystals provide better support for sectioning

317
Q

which primary fixatives are NOT additive?

A
  1. ethanol
318
Q

which primary fixatives are coagulating? (5**)

A
  1. potassium dichromate (IF pH < 3.8)
  2. mercuric chloride
  3. ethanol
  4. picric acid
  5. acetic acid (nuclei only)
319
Q

which fixatives are INtolerant? (7**)

A
  1. potassium dichromate (IF pH < 3.8)
  2. mercuric chloride
  3. ethanol
  4. picric acid
  5. bouin’s
  6. B - 5
  7. acetic acid (nuclei only)
320
Q

which primary fixatives ARE coagulating AND additive? (4**)

A
  1. potassium dichromate (IF pH<3.8)
  2. mercuric chloride
  3. picric acid
  4. acetic acid (nuclei only)
321
Q

which primary fixatives are reducers? (3)

A
  1. formaldehyde
  2. glutaraldehyde
  3. ethanol
322
Q

which primary fixatives are oxidizing? (2)

A
  1. potassium dichromate
  2. osmium tetroxide
323
Q

which primary fixatives are neither oxidizers nor reducers? (3)

A
  1. mercuric chloride
  2. picric acid
  3. acetic acid

Histotechnology (2 decks)

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