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mt > HISTOPATH TISSUE PREPARATION > Flashcards

HISTOPATH TISSUE PREPARATION Flashcards

1
Q

is a process whereby a
selected tissue specimen is immersed in a watch
glass containing isotonic salt solution, carefully
dissected or separated, and examined under the
microscope, either unstained by phase contrast or
bright field microscopy, or stained with differential dyes

A

teasing or dissociation

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2
Q

Small pieces of tissue not more than one mm. in
diameter are placed in a microscopic slide and
forcibly compressed with another slide or with a
cover glass

A

squash preparation (crushing)

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3
Q

Process of examining sections or sediments, whereby cellular materials are spread lightly over a slide by means of a wireloop or applicator or by making an apposition smear with another slide.

A

smear preparation

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4
Q

w/ an applicator stick or a platinum loop,
the material is rapidly and gently applied in a direct or zigzag line throughout the slide

A

streaking

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5
Q

kind of smear that should be avoided

A

too thin and too thick smear

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6
Q

a selected portion of the material is
transferred to a clean slide and gently spread into a moderately thick film by teasing the mucous strands apart with an applicator stick.

A

spreading

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7
Q

For fresh sputum ____

A

bronchial aspirates and thick
mucoid secretions

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8
Q

done by placing a drop of secretions
or sediment upon one slide and facing it to another clean slide

A

pull-apart

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9
Q

pull-apart is used for

A

Serous fluid, conc. Sputum, enzymatic lavage
samples from GIT and blood smears

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10
Q

the surface of a freshly cut piece of tissue is brought into contact and pressed on to the surface of a clean glass slide, allowing the cells to be transferred directly to the slide for examination by phase contrast microscopy or stained for light microscopic study

A

touch preparation (impression smear)

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11
Q

utilized when a rapid diagnosis of
the tissue is required

A

frozen section

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12
Q

frozen section is used for

A

For lipid, carbohydrates and nervous tissue elements
For rapid pathologic diagnosis during surgery
For diagnostic and research enzyme histochemistry
For silver stains (neuropathology) immunofluorescent and immunohistochemical staining

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13
Q

Commonly Used Methods of
Freezing

A
  1. Liquid nitrogen
  2. Isopentane cooled by liquid nitrogen
  3. Carbon dioxide gas
  4. Aerosol sprays
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14
Q
  • adequate for freezing small
    pieces of tissue except muscle
A

aerosol sprays

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15
Q

used for histochemistry and during operative procedures

A

Liquid nitrogen

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16
Q

disadvantage of liquid nitrogen

A

soft tissue is liable to crack due to the
rapid expansion of the ice w/in the tissue, producing ice crystals or freeze artifacts.

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17
Q

Overcools urgent biopsy blocks, causing damage to both..

A

block and blade if sectioning is done at -70 C or
below

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18
Q

Non-fatty unfixed tissues are sectioned well at
temperatures between

A

-10 C and -25 C

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19
Q

TISSUE PROCESSING

A
  1. Numbering
  2. Fixation
  3. Dehydration
  4. Clearing
  5. Infiltration
  6. Embedding
  7. Trimming
  8. Section-Cutting
  9. Staining
  10. Mounting
  11. Labeling
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20
Q

Identify properly all the specimens received without the need of writing the patient’s name to the accompanying specimen tag

A

Numbering

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21
Q

where is the details written in numbering

A

Entering the details of the specimen in alog book

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22
Q

how to do numbering

A

In numbering, the specimen number is preceded by either S (surgical), A (autopsy) or C (cytology). The year is also indicated. Example: S98-7677

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23
Q

After numbering the pathologist will describe the
_____ of the specimen. The MT will
write down the ____ at the back of the
request

A

gross description and description

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24
Q

Specimen size for processing:

A

3x2x0.5cm (length) and 3-5mmthick

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25
Q

the function of fixation

A

Preserve the tissue- stop all cellular activities

Prevent breakdown of cellular elements (prevents postmortem decomposition (autolysis) and prevents putrefaction)

Coagulate or precipitate protoplasmic substances

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26
Q

for conventional bright field light microscopy, we follow the following steps:

A

0.5 x 0.5 cm tissue is added to a small jar containing the fixative

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27
Q

adding tissue to a small jar containing fixative is to:

A

prevent autolysis
terminate cell metabolism
kill bacteria
harden tissue

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28
Q

fixative used is usually

A

Formalin 10% solution

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29
Q

tissues are fixed:

A

to preserve cells and tissues constituents
to prevent their degredation

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30
Q

with fixative’s volume 20x greater

A

Bouin’s Fixative / Formalin for 24 hrs

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31
Q

TWO basic mechanisms in fixation

A

additive fixation
Non- additive fixation

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32
Q

chemical constituent of the fixative is
taken in and becomes part of the tissue by forming cross-links or molecular complexes and giving stability to the protein

A

additive fixation

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33
Q

example of additive fixatives

A

Formalin, mercury and osmium tetroxide

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34
Q

fixation-fixing agent is not incorporated
into the tissue, but alters the tissue composition and stabilizes the tissue by removing the bound water attached to H bonds of certain groups w/in the CHON molecule.

A

non-additive fixation

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35
Q

non-additive fixatives example

A

alcohol fixatives

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36
Q

FACTORS INVOLVED IN FIXATION

A

pH
Temperature
Thickness of section
Osmolality
Concentration
Duration of Fixation

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37
Q

the pH in fixation

A

6-8

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38
Q

temperature in fixation

A

Surgical spe - room temp.
tissue processors- 40 deg celsius
electron microscopy & histochem - 0-4 deg celsius

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39
Q

Formalin at 60 deg celsius is for

A

rapid fixation of urgent biopsy

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40
Q

Formalin 100 deg celsius is for

A

TB

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41
Q

Thickness of section for EM

A

1-2 mm^2

42
Q

Thickness of section for LM

A

2cm^2 or not more than 0.4 cm

43
Q

Thickness of section for Brain

A

10% buffered formalin for 2-3 weeks

44
Q

osmolality for slightly hypertonic solution

A

around 400-450mOsm

45
Q

osmolality of isotonic solutions

A

340 mOsm

46
Q

concentration in fixation

A

Formaldehyde- 10%
Glutaraldehyde- 3%
Glutaraldehyde - 0.25% immunoelectron microscopy

47
Q

duration of fixation for buffered formalin

A

2-6 hours

48
Q

duration of fixation for EM

A

fixation 3 hrs and then placed in a buffer

49
Q

PRACTICAL CONSIDERATION OF FIXATIVES

A

SPEED
PENETRATION
VOLUME
DURATION

50
Q

formalin penetration

A

1mm/hr

51
Q

volume of fixatives

A

10-25 times that of the tissue

52
Q

the maximum effectiveness for volume of fixatives

A

20x the tissue volume

53
Q

duration for fixatives

A

uterus & intestinal tract take longer time than small or loosely textured such as biopsies and scrapings

54
Q

Characteristics of good fixatives

A

Cheap
Stable
Safe to handle
Kill cell quickly with minimal distortion
Inhibit bacterial decomposition and autolysis
Minimum shrinkage
Penetrate rapidly and permit application of stains
Isotonic, insoluble to hypotonic

55
Q

FIXATIVE TYPES

A

Simple
Compound

56
Q

what are the simple fixatives

A

Aldehydes
Metallic Fixatives
Lead Fixatives

57
Q

what are the Aldehyde Fixatives

A

Formaldehyde, Glutaraldehyde

58
Q

what are the Metallic Fixatives

A

Mercuric Chloride, Chromate (potassium dichromate, chromic acid)

59
Q

what are the Lead Fixatives

A

Picric, Acetic, Acetone, Alcohol, Osmic Acid

60
Q

2 or more fixatives

A

Compound Fixative

61
Q

Fixative according to action

A

Microanatomical Fixatives and Cytologic Fixatives

62
Q

permit the general microscopic study of tissue structures without altering the structural pattern and normal intercellular relationship of the tissues in question.

A

Microanatomical Fixatives

63
Q

types of Microanatomical Fixatives

A
  1. 10% formal saline
  2. 10% neutral buffered formalin
  3. Heidenhain ‘s Susa
  4. Formal sublimate (formal corrosive)
  5. Zenker ‘s solution
  6. Zenker-formal (Kelly ‘s solution)
  7. Bouin’s solution
  8. Brasil’s solution
64
Q

Are those (fixatives) that preserve specific parts and particular microscopic elements of the cell itself

A

Cytologic Fixatives

65
Q

the types of cytologic fixatives

A
  1. Nuclear
  2. Cytoplasmic
  3. Histochemical
66
Q

Preserves nuclear structures

A

Nuclear Fixatives

67
Q

Fluids in Nuclear Fixatives

A

Flemming’s Fluid
Carnoy’s Fluid
Bouin’s Fluid
Newcomer’s Fluid
Heidenheins Susa

68
Q

are those that preserve cytoplasmic structures in particular

A

Cytoplasmic Fixatives

69
Q

Fluids in Cytoplasmic Fixatives

A

Flemming’s fluid without acetic acid
Kelly’s fluid
Formalin with “post-chroming”
Regaud ‘s fluid (Muller ‘s fluid)
Orth ‘s fluid

70
Q

are those that preserve the chemical constituents of cells and tissues.

A

Histochemical Fixatives

71
Q

fluids in Histochemical Fixatives

A

Formal Saline 10%
Absolute Ethyl Alcohol
Acetone
Newcomer’s Fluid

72
Q

Bouin’s Fluid composition

A

Picric Acid - 75 mL
Formalin - 25mL
Glacial Acetic Acid - 5mL

73
Q
  • it has rapid and even penetration
  • fixed tissue gives brilliant staining with trichome methods
  • used to demonstrate glycogen
  • good for GIT biopsies
A

Bouin’s Fluid

74
Q

Involves thermal coagulation of tissue proteins for rapid diagnosis

A

Heat Fixation

75
Q

Heat Fixation is used for..

A

For frozen tissue sections
For bacteriological smears
For nuclear and cytoplasmic detail

76
Q

Heat Fixation destroys

A

RBC

77
Q

Heat Fixation dissolves

A

starch and glycogen

78
Q

Is the process of placing an already fixed tissue in a second fixative

A

secondary fixation

79
Q

secondary fixation Is the process of placing an already fixed tissue in a second fixative in order to:

A

Facilitate & improve demonstration of particular subs.
Make special staining techniques possible
Ensure further and complete hardening and preservation of tissues
Done before dehydration and on deparaffinized sections before staining (10% formalin/10% formol saline as primary fixative)

80
Q

Is a Form of secondary fixation whereby a primarily fixed is placed in aqueous solution of 2.5-3% potassium dichromate for 24 hours to act as mordant for better staining effects and in cytologic preservation of tissues

A

POST-CHROMATIZATION

81
Q

Removing excess fixative from the tissue after fixation

A

Washing Out

82
Q

remove excess chromates, formalin, osmic acid

A

Tap Water

83
Q

wash excess amount of picric acid

A

50 to 70% Alcohol

84
Q

remove excess mercuric fixatives

A

Alcoholic Iodine

85
Q

Factors Affecting Fixation

A

Size and thickness
Presence of mucus
Presence of fat
Presence of blood-
Cold Temp

86
Q

larger tissue requires more fixatives and longer fixation time

A

Size and thickness

87
Q

maybe washed with saline solution

A

presence of mucus

88
Q

cut in thin section and fixed longer

A

presence of fat

89
Q

flushed out with saline

A

presence of blood

90
Q

inactivates enzymes

A

cold temp

91
Q

FIXATION IS ENHANCED BY

A

Size and thickness of tissues
agitation
Moderate heat (37-56 C) accelerates fixation but hastens autolytic changes and enzyme destruction

92
Q

Principles and precautions in handling and fixation of Specimen

A

Fix Autopsy ASAP
Fix surgical specimen ASAP
Proper labeling and Identification
Tissues size not more than 5mm thick except lung edema (1-2cm thick)
Volume 20x except for osmium tetroxide 5-10x
For prolong fixation vol not less than 50-100x
Hollow organs (stomach) shld be completely opened before fixing
Lungs may be covered with several layers of gauze
Human brain (2 weeks) may be suspended by acord tied under circle of willis to prevent flattening
Water should not be used for glycogen-containing tissue

93
Q

Works as a physical agent similar in mechanism to vacuum, oven and agitation to increase the movement of molecules and accelerate fixation.

A

MICROWAVE TECHNIQUE

94
Q

MICROWAVE TECHNIQUE used to..

A

Used to accelerate staining, decalcification, immunohistochemistry and electron microscopy

95
Q

the tissue in microwave technique..

A

Tissue is heated right thru the block in a very short time.

96
Q

For antibodies demonstration
Formalin-fixed and paraffin embedded sections may be used
Prepared as Cryostat section and fixation limited to a few seconds in absolute methanol or acetone.

A

IMMUNOFLUORESCENCE AND IMMUNOPEROXIDASE TECHNIQUES

97
Q

preserve the maximum enzyme activity at its localization

A

ENZYME HISTOCHEMISTRY

98
Q

Fixed in 4% formaldehyde or formol saline

A

ENZYME HISTOCHEMISTRY

99
Q

Fresh frozen cryostat sections may be fixed in..

A

acetone or formaldehyde and washed in distilled water prior to enzyme staining

100
Q

primary fiaxatives in EM

A

Osmium tetroxide, glutaraldehyde and paraformaldehyde

101
Q

Osmium tetroxide, glutaraldehyde and paraformaldehyde is performed at

A

4C

102
Q

For electron histochemistry and electron immunocytochemistry ____ is useful

A

karnovsky’s paraformaldehyde-glutaraldehyde is useful

mt (54 decks)

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