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histopath Flashcards

1
Q

Squash
preparation /
Crushing

A

Process where small pieces of tissue not more than
1mm in diameter are placed in a microscopic slide and
forcibly compressed with another slide or with
coverglass .
Vital dyes are placed at the slide and coverslip junction
and absorbed through capillary action .

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2
Q

Frozen section

A
  • Normally utilized when a rapid diagnosis of the tissue
    in question is required , and especially recommended
    when lipids and nervous tissue elements are to be
    demonstrated .
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3
Q

Smearing

A

Useful in cytological examinations , particularly for
cancer diagnosis .

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4
Q

Fixation

A

Preservation

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5
Q

Decalcification

A

Calcium or lime salts are removed from the tissues
Optional process : for calcified tissues only such as
bones and teeth

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6
Q

Dehydration

A
  • Desiccation
    Removing intracellular and extracellular fluid / water
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7
Q

Clearing

A

De - alcoholization
Removing alcohol used in dehydration

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8
Q

Infiltration

A

Impregnation

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9
Q

Embedding

A

Casting or Blocking

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10
Q

Trimming

A

Removing excess wax from the tissue block
Optional process : not all tissue blocks have excess wax

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11
Q

Sectioning

A

Section cutting
- Cutting tissue blocks into uniformly thin slices

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12
Q

Staining

A

Dyeing

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13
Q

Labelling

A
  • Proper labelling
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14
Q

Smearing
Technique

A

Material

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15
Q

a . Streaking

A

Applicator stick
or platinum
loop .

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16
Q

b . Spreading
technique

A

Applicator stick
to tease the
mucous strands
to make a
moderately
thick film .

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17
Q

c . Pull - apart
technique

A

Slides facing
each other as a
drop of
secretion is
sandwiched in
between .

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18
Q

d . Touch
preparation
or
Impression
smear

A

One slide .

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19
Q

Autolysis

A

the destruction of the tissues ( breaking down of the
protein of the cell ) by enzymes which are produced by
the tissues and eventually liquefy it .
It is the first to occur among all post - mortem changes

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20
Q

Putrefaction or
Decomposition

A

the decomposition of organic matter under the influence
of microorganisms accompanied by the development of
disagreeable odors .

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21
Q

Degeneration

A
  • a retrogressive pathologic process in cells in which the
    cytoplasm undergoes deterioration while the nucleus is
    preserved .
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22
Q

Two Basic

A

Mechanisms in Fixation

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23
Q
  1. Additive
A

The chemical constituent of the
fixative is take in and becomes
part of the tissue through cross - link
formation or molecular complexes .

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24
Q
  1. Non
    additive
A

The fixing agent is not incorporated
into the tissue but alters the tissue
composition and stabilizes it
through water removal .
New cross - links are formed
preventing autolysis and bacterial
decomposition .

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25
Q

Main Factors Involved

A

in Fixation

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26
Q
  1. Hydrogen lon
    Concentration
A

pH : 6.0-8.0
Average : 7.0 ( neutral pH )

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27
Q
  1. Temperature
A

-
Traditional / usual : Room temperature ( 18-30 ° C )
Tissue processors : Autotechnicon ( 40-42 ° C )
Electron Microscopy and Histochemistry : 0-4 ° C
O Mast cells for EM : Room temperature
Nucleic acids fixation : Rapid at higher temperature
Formalin heated to 60 ° C → rapid fixation of very
urgent biopsy specimens
Formalin heated to 100 ° C → to fix tissues with
tuberculosis

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28
Q
  1. Thickness of
    sections
A

Small
O
Electron Microscopy : 1-2 mm²
Light Microscopy : 2 cm²
Thin
O Light Microscopy : ≤0.4 cm or as prescribed by
tissue processor manufacturer
Large solid tissue , such as uterus , should be
opened or sliced thinly
O Brain is usually suspended whole in 10 % Neutral
Buffered Formalin for 2-3 weeks .

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29
Q
  1. Osmolality
A

-
Best results are obtained using slightly hypertonic
solutions ( 400-450 mOsm )
Hypertonic solutions - Shrinkage =
Isotonic ( 340 mOsm ) / Hypotonic solutions
swelling and poor fixation
Added to Osmium tetroxide fixatives for EM : Sucrose
=

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30
Q
  1. Concentration
A

Formaldehyde : 10 %
Glutaraldehyde : 3 %
Glutaraldehyde for Immunoelectron microscopy :
0.25 %

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31
Q
  1. Duration
    fixation
    of
A

-
Most formalin fixatives : 24 hours ( washed out )
Buffered formalin : 2-6 hours up to 1 week
EM : 3 hours ( New books : 0-4 hours ; Average : 2 hours )
Prolonged fixation may cause shrinkage and
hardening of tissue

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32
Q

ROUTINE FORMALIN

A

FIXATIVES

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33
Q
  1. 10 % Formol - Saline
    Fixation time :
    24 hours at 35 ° C
    48 hours at 20-25 ° C
A

Traditionally , it is the most commonly used
fixative in pathology
Best fixative for central nervous tissues and
general post - mortem tissues for
histochemical examination .

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34
Q

2 . Calcium
formalin
Fixative )
acetate
( Lillies

A
  • It is used to preserve phospholipids
    Replaced formol - saline as the most
    commonly used fixative in pathology
    because :
    Simple to prepare
    Buffered to pH 7 by acetate
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35
Q

3 . 10 %
Buffered
Fixation time :
4-24 hours
Formalin / Phosphate
buffered Formalin
( pH 7 )
Neutral

A

-
Best general tissue fixative
Best Fixative for frozen sections
Recommended for surgical , post mortem
and research specimens .
Prevents precipitation of acid formalin
pigments .
Best fixative for iron - containing pigments
and elastic fibers .

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36
Q

4 . Formol corrosive /
Fixation time :
3-24 hours
Formol - sublimate /
Formol - Mercuric
Chloride

A

-
Recommended for routine post - mortem
tissues .
It is excellent for silver reticulum methods .
It fixes lipids , especially neutral fats and
phospholipids .
Forms black deposits .
No frozen sections are made

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37
Q

Alcoholic
Gendre’s fixative
Formalin /

A
  • It contains ethyl alcohol saturated with picric
    acid .
    It enhances immunoperoxidase studies for
    EM if post - fixed with phenol formalin for 6
    hours or more .
    It fixes and dehydrates at the same time and
    fixes sputum since it coagulates mucus
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38
Q

SPECIAL FORMALIN
FIXATIVES :

A
  • Cajol’s formol ammonium bromide - good
    fixative for nervous tissue ( astrocytes ) .
    Fixatives for acid mucopolysaccharides .
    Baker’s formol calcium used for the
    preservation of lipids since most formalin
    fixatives are inert to lipids .
    -
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39
Q
  1. Chromic acid
A

Used in 1-2 % , used as a constituent of a
compound fixative .
It precipitates all proteins and adequately
preserves carbohdyrates .
Formaldehyde must be added to chrome
containing tissues before use to prevent
counteracting effects and consequent
decomposition of solution upon prolonged
standing .

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40
Q

2 . Regaud’s
Fluid /
Moeller’s fluid

A

Recommended for demonstration of Chromatin ,
Mitochondria , Mitotic figures , Golgi bodies ,
RBC’s and colloid - containing tissues

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41
Q

3 . Orth’s Fluid
Fixation time :
36-72 hours

A

Recommended for study of early degenerative
processes and tissue necrosis
Demonstrates Rickettsia and other bacteria

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42
Q

4 . Potassium
dichromate

A

Preserves mitochondria ( pH 4.5-5.2 )
Fixes lipids
Used in 3 % aqueous solution
It fixes but does not precipitate cytoplasmic
structures .

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43
Q

Mercuric Chloride

A

Fixatives

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44
Q
  1. Zenker’s fluid
    Fixation time :
    12-24 hours
A

Mercuric chloride + glacial acetic acid just before
its use .
To prevent turbidity and formation of dark
precipitates
Good general fixative for all kinds of tissue
Recommended for fixing small pieces of liver ,
spleen , connective tissue fibers and nuclei
May act as mordant to make certain special
staining possible
Contains glacial acetic acid which makes the
solution unstable

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45
Q

2 . Zenker
formol / Helly’s
Solution
Fixation time :
12-24 hours
“ Bloody Helly “

A

Mercuric chloride + 40 % formaldehyde just
before its use .
It is an excellent microanatomic fixative for
pituitary gland , bone marrow and blood
containing organs such as the liver and spleen .
Better nuclear fixations and staining than Zenker’s
It presevres cytoplasmic granules better than
Zenker’s
Disadvantage : Brown pigments are produced if
tissues are allowed to stay in the fixative for more
than 24 hours due to RBC lysis .
Remedy : immerse the tissue in alcoholic picric
acid or sodium hydroxide

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46
Q

3 . Heidenhain’s
Susa solution
Fixation time :
3-12 hours

A
  • Recommended for tumor biopsies especially of the
    skin
    It is an excellent Cytologic fixative
    Produces minimum shrinkage and hardening of
    tissues due to the counter - balance of the swelling
    effects of acid ( trichloroacetic acid ) and the
    shrinkage effect of a metal ( mercury ) .
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47
Q

4 . Schaudinn’s
Solution /
Sublimated
alcohol

A

A solution of mercuric
chloride , alcohol , and glacial acetic acid
Used on wet smears for cytologic examinations .
chloride , sodium

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48
Q

5 .
Fixation time :
1½ to 2 hours
( Rapid fixation )
B - 5 Fixative

A

Composed of mercuric chloride and sodium
acetate .
Commonly used for bone marrow biopsies .
Just prior to use , add 1 mL of 40 % formaldehyde
to 10 mL of B5

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49
Q

A. Bouin’s solution
Fixation time :
6-24 hours

A

-
Fixation of embryos and pituitary biopsies
It is an excellent fixative for preserving soft and
delicate structures ( endometrial curettings )
Yellow stain is useful in fragmentary biopsies
Preferred fixative for Masson’s trichrome
staining for collagen , elastic or connective tissue

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50
Q

B. Brasil’s Alcoholic
Picroformol Fixative

A

-
Best routine fixative for glycogen
It is better and less messy than Bouin’s solution

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51
Q
  1. Flemming’s Solution
    Fixation time :
    24-48 hours
A

The most commonly used Chrome - Osmium
acetic acid , recommended for nuclear
preparation of such sections .
Excellent for nuclear structures such as
chromosomes
Permanently fixes fats / lipids

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52
Q
  1. Flemming’s Solution
    without Acetic Acid
    Fixation time :
    24-48 hours
A

Recommended for cytoplasmic structures
such as mitochondria
The removal of acetic acid from the formula
serves to improve the cytoplasmic detail of the
cell .

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53
Q

1 . Methyl Alcohol
100 %
Methanol )
( CH3OH /

A
  • Used in Wright’s stain as a diluent
    Excellent for fixing wet and dry smears , blood
    smears and bone marrow tissues
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54
Q

2 . Isopropyl Alcohol
95 % ( Isopropanol )

A
  • It is used for fixing touch preparations
    ( impression smears ) , although some are air
    dried and not fixed , for certain procedures
    such as Wright - Giemsa staining .
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55
Q

3 .
Fixation time :
18-24 hours
Ethyl Alcohol
( C2H5OH / Ethanol )

A

Is used in 70-100 % concentrations .
Lower concentrations ( 70-80 % ) will cause
RBC lysis and inadequate WBC preservation
It fixes blood , tissue films and smears
Used for histochemistry especially for enzyme
studies
Can be both used as a simple and compound
fixative .

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56
Q

4 .
Fixation time : 1-3 hrs .
Carnoy’s Fluid

A

-
The most rapid tissue fixative
Recommended for fixing chromosome , lymph
glands and urgent biopsies
It is used to fix brain tissue for rabies
diagnosis .
It preserves Nissl granules and very suitable
for Curettings ( small tissue fragments ) .

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57
Q

5 . Alcoholic
Formalin /
Gendre’s

A
  • Better preserves glycogen
    Capable of coagulating mucus and is used as
    a fixative for sputum cytology
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58
Q

6 . Newcomer’s Fluid
Fixation time :
12-18 hours @ 3 ° C

A
  • For fixing mucopolysaccharides and nuclear
    proteins
    It is both a nuclear and histochemical fixative .
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59
Q

Special Factors Affecting

A

Fixation

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60
Q
  1. Size and thickness
A

Larger and thicker tissues require more fixative
and fixation time

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61
Q
  1. Presence
    mucus
    of
A

Prevents complete penetration of fixative ,
hence , tissues that contain mucus are fixed
slowly and poorly
Excess mucus may be washed away with NSS .

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62
Q
  1. Presence of fats
A

Fatty tissues should be cut in thin sections and
should be fixed longer .

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63
Q
  1. Presence of blood
A

Tissues containing large amount of blood ( e.g.
Blood vessels and spleen ) should be flushed
out with saline ( arterial cannulization ) before
fixing .

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64
Q
  1. Cold temperature
A

Inactivates enzymes

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65
Q
  1. Hot temperature
A

Denatures enzymes

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66
Q
  1. Size and thickness
A

Smaller and thinner tissues require less fixative
and shorter fixation time

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67
Q
  1. Agitation
A
  • Fixation is accelerated when automatic or
    mechanical tissue processing is used .
    Autotechnicon
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68
Q
  1. Moderate heat
A

Accelerates fixation but hastens autolytic
changes and enzyme destruction

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69
Q

I. Acid Decalcifying

A

Agents

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70
Q
  1. Nitric acid
A

The most common and fastest decalcifying agent
used so far

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71
Q

Recommended
concentrations :

A

Very rapid decalcifying agent , producing minimal
distortion

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72
Q

5-10 %

A

Recommended for routine purposes

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73
Q

a . 10 % aqueous
Nitric Acid
Decalcification time :
12-24 hours

A

Recommended for urgent biopsies and for needle
and small biopsy specimen
Used for large or heavily mineralized cortical bone
specimen

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74
Q

b . Formol - Nitric
acid
Decalcification time :
1-3 days

A

For urgent biopsies with good nuclear staining .
Yellow color imparted by nitrous acid is removed
through neutralization with 5 % sodium sulfate and
running tap water for 12 hours

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75
Q

C. Perenyi’s fluid
Decalcification time :
2-7 days

A

-
It is recommended for routine purposes .
It decalcifies and softens tissues at the same time
Maceration is avoided due to the presence of
chromic acid and alcohol

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76
Q

d . Phloroglucin
Nitric Acid
Decalcification time :
12-24 hours

A

Most rapid decalcifying agent , recommended for
urgent works
Has poor nuclear staining
Yellow color formation

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77
Q
  1. Hydrochloric
    acid / Muriatic
    acid
A

Inferior compared to nitric acid ( slower action and
greater distortion of tissue )
Good nuclear staining
Recommended for surface decalcification of
tissue blocks if used in 1 % solution with 70 %
alcohol
Von Ebner’s fluid
Formula : 36 % Sat. Aq . NaCl , Conc . HCI , Distilled water

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78
Q
  1. Failure to arrest early cellular
    autolysis
A

Due to the failure to fix immediately by
which one first allowed the tissue to dry
before fixing or insufficient fixative

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79
Q
  1. Too brittle and too hard blocks
A

Due to prolonged fixation

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80
Q
  1. Soft and feather - like tissues
A

Due to incomplete fixation

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81
Q
  1. Removal of fixative soluble
    substances
A

Wrong choice of fixative

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82
Q
  1. Presence of artefact pigments on
    sections
A

Incomplete washing of fixative

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83
Q
  1. Shrinkage and swelling of cells in
    tissue blocks
A

Due to overfixation

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84
Q
  1. Enzyme inactivation and loss
A

Wrong choice of fixative

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85
Q

Incomplete fixation → Improper and

A

incomplete clearing and impregnation →

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86
Q
  1. Formic acid
A

Moderate - acting decalcifying agent
Recommended for routine decalcification of post
mortem research tissues

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87
Q

Decalcification time :
2-7 days

A

Disadvantage : Not suitable for urgent examination

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88
Q
  1. Trichloroacetic
    acid
A

-
Does not require washing out
Not recommended for urgent examinations ( very
slow acting )

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89
Q
  1. Sulfurous acid
A
  • Weakest decalcifying agent , suitable only for
    minute pieces of bone
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90
Q
  1. Chromic acid
A

Used as a fixative and decalcifying agent
Caution : It is an environmental toxin , highly
corrosive to skin and mucous membrane and
carcinogenic

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91
Q
  1. Citric acid
    Citrate buffer
A

Does not produce cell or tissue distortion

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92
Q

3 Ways to Measure

A

the Extent of Decalcification

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93
Q
  1. Physical or
    Mechanical
    Method
A

By touching or bending the tissue with the fingers to
determine the consistency of tissues
By pricking the tissue with a fine needle / probe

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94
Q
  1. X - ray /
    Radiological
    method
A
  • Very expensive , although it is the most ideal , most
    sensitive and most reliable method
    Good but not always convenient
    Not recommended for mercuric chloride - fixed tissues
    ( radio - opacity will interfere with the plate interpretation )
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95
Q
  1. Chemical
    method /
    Calcium
    Oxalate test
A

-
1. Concentrated Ammonium Hydroxide
2. Saturated Aqueous Ammonium Oxalate
Detection of calcium in acid solutions by precipitation
of insoluble calcium hydroxide or calcium oxalate
Presence of cloudiness indicates the presence of Ca
( incomplete decalcification )
Simple , reliable and convenient method recommended
for routine purposes ( still favoured )
Decalcifying fluid is changed every 24-48 hrs
Solutions used :

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96
Q

a . Ethanol / Ethyl
alcohol

A
  • Undoubtedly the best dehydrating agent ( fast acting ,
    mixes with water and organic solvents and penetrates
    tissues easily )
    Has the advantage of not being poisonous and not
    very expensive
    Should be at least 99.7 % pure
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97
Q

b . Isopropanol /
Isopropyl
alcohol

A

Should be used if good - grade absolute ethyl alcohol is
not easily available

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98
Q

c . Methanol /
Methyl alcohol

A

Toxic dehydrating agent
For blood and tissue films and for smear preparations

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99
Q

d . Butanol / Butyl
alcohol

A

Utilized for plant and animal microtechniques .

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100
Q
  1. Cedarwood oil
    Clearing time :
    2-3 days
A

-
Clears both Paraffin and Celloidin sections
Recommended for CNS tissues and cytological
studies ( esp . Smooth muscles and skin )
Very expensive and it requires 2 changes in
clearing solution
Quality is not always uniform and good and is
extremely slow
It becomes milky on prolonged storage

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101
Q
  1. Aniline oil
A

Recommended for clearing embryos , insects
and very delicate specimens since it clears
70 % alcohol without excessive tissue shrinkage
and hardening

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102
Q
  1. Clove oil
A

-
It removes aniline dyes and dissolves Celloidin ;
Tissues become brittle
Its quality is not guaranteed due to its tendency
to be adulterated
Not suitable for routine purposes because it is
expensive

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103
Q
  1. CC14 ( Carbon
    Tetrachloride )
A

-
Properties are very similar to chloroform but it is
cheaper
Toxic on prolonged exposure

104
Q
  1. Tetrahydrofuran
A
  • Dehydrates and clears at the same time since it
    is miscible in both water and paraffin
105
Q
  1. Methyl
    benzoate /
    Methyl
    Salicylate
A

These are slow - acting clearing agents that can
be used when double embedding techniques
are required .

106
Q
  1. Acetone
    ( CH3COCH3 )
A

A cheap , rapid dehydrating agent utilized for
most urgent biopsies ( 1/2 to 2 hours )
Its use is limited only to small pieces of tissues
due to its extreme volatility and flammability

107
Q
  1. Dioxane /
    Diethylene
    Dioxide
A

Excellent dehydrating and clearing agent
miscible to water , melted paraffin , alcohol and
xylol / xylene
Expensive and toxic and extremely dangerous
( main disadvantage )

108
Q
  1. Cellosolve /
    Ethylene glycol
    monoethyl ether
A

Dehydrates rapidly and not harmful to tissues
Toxic by inhalation , skin contact and ingestion
( use propylene - based glycol esters )

109
Q
  1. Triethyl
    phosphate
A

Tissues can be transferred directly after fixation
& washing
Used to dehydrate sections and smears
following certain stains

110
Q
  1. Tetrahydofuran
A

Dehydrates and clears tissues since it is
miscible to water and paraffin
It is toxic if ingested or inhaled . Vapors cause
nausea , dizziness and headache .
It is an eye and skin irritant and prolonged
exposure ( up to 6 months ) may cause
conjunctival irritation .

111
Q

Commonly Used

A

Clearing Agents

112
Q

Clearing time :
½ to 2 hours ( usual )
15-30 Minutes
( urgent biopsies )
1. Xylene

A

Advantages :
An excellent and true clearing agent
1 .
2 .
3 .
4. Can be used with celloidin sections
Disadvantages :
1. It is highly flammable
The most rapid clearing agent
Cheap and does not extract out aniline dyes
It is miscible with absolute alcohol and paraffin
2. When dehydration is not complete , the xylene
becomes milky when the tissue or section is
added to it

113
Q
  1. Toluene
    Clearing time :
    1-2 hours
A

Substitute to xylene or benzene
It is miscible with absolute alcohol and paraffin
It is not carcinogenic but highly concentrated
emit fumes that are toxic upon prolonged
exposure
It acts slower than benzene and is expensive

114
Q
  1. Benzene
    Clearing time :
    15-60 minutes
A

-
Rapid acting , recommended for urgent biopsies
and routine purposes
It is miscible with absolute alcohol and paraffin
It is highly flammable
Carcinogenic or may damage bone marrow
( Aplastic anemia ; If antibiotic : Chloramphenicol )

115
Q
  1. Chloroform
    Clearing time :
    6-24 hours
A

-
It is the best of the traditional clearing agents for
routine use
Gives the widest latitude
It is recommended for tough ( skin , fibroid and
decalcified tissues ) and large tissue specimens
Also best for nervous tissue , lymph nodes ,
granulation tissue , and fetal and other delicate ,
highly cellular specimens

116
Q

3 Ways of Paraffin

A

Wax Impregnation ( and Embedding )

117
Q
  1. Manual
    processing
A

Requires at least 4 changes of wax with 15 minutes ‘
interval
Total : 1 hour

118
Q
  1. Automatic
    Processing
A

Makes use of an automatic tissue processing machine
( e.g. Autotechnicon ) which fixes , dehydrates , clears
and infiltrates tissues , thereby decreasing the time
and labor needed during the processing of tissues ,
resulting in a more rapid diagnosis with less
technicality
Autotechnicon / Elliot - Bench Type Tissue Processor :
Fixation , Dehydration , Clearing , Infiltration

119
Q
  1. Vacuum
    Embedding
A

Impregnation under negative atmospheric pressure
Recommended for urgent biopsies and delicate
tissues such as lung , brain , connective tissues ,
decalcified bones , eyes spleen and CNS .
Reduces 25-75 % of impregnation time
Most rapid

120
Q

Several Types

A

of Blocking - out Molds

121
Q
  1. Leukhart’s
    embedding
    mold
A

Consist of 2 L - shaped pieces of heavy brass or metal , a
base being formed by a piece of 1/8 inch thick copper
brass , about 3X2 inches square , or a piece of plate glass .

122
Q
  1. Compound
    embedding
    mold
A

Made up of a series of interlocking plates resting on a flat
metal base , forming several compartments .

123
Q
  1. Plastic
    embedding
    rings with
    base mold
A

Consist of a special stainless steel base mold fitted with a
plastic embedding ring which later serves as the block
holder during cutting .
Example : Tissue Tek

124
Q
  1. Disposable
    Embedding
    Molds
A

a . Peel - aways
o available in 3 different sizes , peeled off one at a time
b . Plastic Ice Trays
o Used in ordinary refrigerators recommended for busy
routine laboratories
c . Paper boat
o Cheap and easy to make
o Used for embedding celloidin and paraffin blocks

125
Q

a . Wet Celloidin Method

A

Recommended for bones , teeth , large
brain sections and whole organs
Tissues must be cut wet ( both the knife &
tissue block are kept moist with 70 %
alcohol while cutting )

126
Q

b . Dry Celloidin Method
* Gilson’s mixture -equal
parts of chloroform and
cedarwood oil

A
  • Preferred for processing of whole eye
    sections
    Material embedded with the dry method
    can be cut without alcohol due to the
    presence of the cedarwood oil in the block
127
Q

Reagent to Tissue Ratio

A

Step

128
Q

20 : 1

A

Fixation

129
Q

10 : 1

A

Dehydration

130
Q

25 : 1

A

Infiltration

131
Q
  1. SLIDING
    MICROTOME
    1789 : Adams
A

a . Base - Sledge Microtome
b . Standard Sliding Microtome
For cutting celloidin embedded sections
Favored in laboratories where very hard tissue
or blocks are sectioned
Most dangerous because of its movable
exposed knife
The block remains stationary while the knife is
moved backward and forward

132
Q

2 . ROTARY
MICROTOME
1885-1886 : Minot

A

For cutting paraffin embedded sections
Most popular and most common type used for
routine and research studies

133
Q
  1. ROCKING
    MICROTOME
    AKA : Cambridge
    1881 : Paldwell &
    Trefall
A

The simplest
For cutting serial sections of large blocks of
paraffin embedded tissues

134
Q

4 . FREEZING
MICROTOME
1848 : Queckett

A

For cutting unembedded frozen sections
Replaced in most laboratories by the cryostat ,
which is easier to operate ( faster and gives
thinner sections ) .

135
Q
  1. ULTRATHIN
    MICROTOME
A

For cutting specimens into extremely thin
slices ( 0.5u ) for electron microscopy work

136
Q
  1. Carborundum hone
A

Available in a wide variety of fineness
Used only for badly nicked knives

137
Q
  1. Arkansas stone
A

A stone of medium fineness

138
Q
  1. Yellow Belgian / Belgium
A
  • The finest ( best result )
139
Q
  1. Belgian Black Vein
A

-
Oils used :
a .
b .
Also called oil stones , because oil is
commonly used as a lubricant
C.
d .
e .
light machine oil
Liquid paraffin oil ( mineral oil )
Vegetable oil
Xylene - tends to evaporate too rapidly
Natural oil - become messy

140
Q
  1. PLANE CONCAVE
    ( 25mm in length )
    Frok A
    Planoconcave
A

One side of the knife is flat / straight ( Celloidin
tissues ) while the other is concave ( Paraffin
tissues )
Base - sledge / Rocking / Rotary microtome

141
Q
  1. BICONCAVE
    ( 120mm in length )
    Profile B
    Biconcave
A

With both sides concave ( Paraffin sections )
Rotary microtome

142
Q
  1. PLANE WEDGE
    ( 100 mm in length )
    Paris . C
    Wilge shaped
A

Have both sides straight ( frozen sections and
extremely hard and tough specimens )
Base - sledge / Sliding microtome

143
Q
  1. PARAFFIN SECTION
    ( 4-6 μ thick )
A
  • For paraffin embedded tissue blocks
    May be cut by rotary and rocking microtome
144
Q
  1. CELLOIDIN SECTION
    ( 10-15μ thick )
A

-
For celloidin embedded tissues
Usually used by means of the sliding
microtome

145
Q
  1. FROZEN SECTION
A

May be cut from tissues that have been
fixed and frozen with CO2 ( cold knife
procedure ) or for fresh or fixed tissues
frozen with the cryostat ( cold microtome )

146
Q

Special Processing

A

Techniques

147
Q
  1. Freeze
    Drying
A

-
Preserving tissues by rapid freezing ( Quenching ) and
removing water ( Dessication ) by a physical process
from the still frozen tissue block without the use of any
chemical fixative .
Tissue size : 2 mm thick
Complete processing time : 24-48 hours
Advantages :
1. Produces minimum shrinkage
2. Allows tissues to be processed in a fresh state
3. Less displacement

148
Q

2 . Freeze
Substitution

A

-
Similar to freeze - drying
Difference : Tissue is fixed in Rossman’s fluid or in
1 % acetone and dehydrated in absolute alcohol
Rossman’s fluid : Satrurated Picric acid and
Formaldehyde

149
Q

a . Hematoxylin

A

The most important and the most commonly used for
routine histologic studies

150
Q

b . Orcein

A

A vegetable dye extracted from lichens which are
normally colorless , but when treated with ammonia and
expose to air , produce blue or violet colors
Mainly used for staining Elastic fibers

151
Q

c . Cochineal

A

An old histologic dye extracted from the female Cochineal
Bug ( Coccus cacti )

152
Q

d . Saffron

A

A plant with orange stigmas yielding a dye

153
Q
  1. Crystal Violet
A
  • A nuclear or chromatin stain used for staining
    amyloid in frozen sections and platelets
    Gentian Violet Mixture of crystal violet , methyl
    violet and Dextrin
154
Q
  1. Giemsa
A

Used for staining blood to differentiate
leukocytes

155
Q
  1. Gold Sublimate
A

Used for metallic impregnation , made up of gold
chloride and mercuric chloride

156
Q
  1. lodine
    a . Gram’s
    b . Lugol’s
A
  • The oldest of all stains
    Stains amyloid , cellulose , starch , carotenes and
    glycogen
157
Q
  1. Janus Green B
A

Used for demonstrating mitochondria ( intravital
staining )

158
Q
  1. Malachite Green
A

Used as a contrast stain for staining ascaris
eggs and erythrocytes
Used as bacterial spore stain

159
Q
  1. Methyl Green
A
  • Stains chromatin green in the presence of an
    acid
160
Q
  1. Methylene Blue
A

Plasma cells , Fresh sputum for malignant cells ,
evaluation and differentiation of bacterial
organisms , Diphtheria diagnosis and Nervous
tissue vital staining

161
Q
  1. Loeffler’s
    Polychrome
    Methylene Blue
A
  • Used in frozen sections for rapid diagnosis
162
Q
  1. Methylene Violet
A
  • Coloring nuclei of leukocytes reddish - purple in
    the presence of methylene blue
163
Q
  1. Neutral Red
A
  • For observing cell granules and vacuoles of
    phagocytic cell
164
Q
  1. Night Blue
A

Substitute for carbol fuchsin in acid - fast staining

165
Q
  1. Orcein
A
  • An excellent stain for Elastic fibers ( Taenzer
    Unna Orcein Method ) ; Demonstrates the finest
    and most delicate fibers in skin ( Dermatological
    studies )
166
Q
  1. Osmium
    Tetroxide
A
  • Used as a fixative ; Used to stain fats
167
Q
  1. Prussian Blue
A

Normally utilized for the manufacture of paints ;
Used as microanatomical contrast stain ; For
demonstration for the circulatory system by
injection ( intravital staining )

168
Q
  1. Rhodamine B
A

Used with osmic acid to fix and stain blood and
glandular tissues

169
Q
  1. Silver Nitrate
A
  • Used in identification of Spirochetes , reticulum
    and other fiber stains
170
Q
  1. Toluidine Blue
A
  • Recommended for staining of Nissl granules or
    chromophilic bodies ; Nuclear stain for fixed
    tissues ; Used as a substitute for thionine in fresh
    tissue sections
171
Q
  1. Victoria blue
A
  • For demonstration of neuroglia in frozen
    sections
172
Q
  1. Cytoplasm & proteins in edema fluid
A

Pale pink

173
Q

2 . Decalcified bone
Osteoid
Collagen

A

Pink

174
Q

3 . Muscle fiber

A

Deep pink

175
Q

4 . RBC
Eosinophil granules
Keratin

A

Bright orange to Red

176
Q

5 . Calcium & Calcified bone

A

Purplish blue

177
Q

6 . Plasma cells
Osteoblast
Basophilic cytoplasm

A

Purplish pink

178
Q

7 . Karyosome

A

Dark blue

179
Q

8 . Nucleus

A

Blue to Blue - Black

180
Q

9 . Cartilage

A

Light blue to Dark Blue

181
Q
  1. Van Gieson’s
    ( Acid fuchsin picric
    acid )
A

Mixture of picric acid and acid fuchsin for the
demonstration of Connective tissues , mucin
and Elastic tissue

182
Q
  1. Acridine Orange
A

A basic acridine fluorochrome which permits
discrimination between dead and living cells ,
giving GREEN fluorescence for DNA and a RED
fluorescence for RNA

183
Q
  1. Acridine Red 3B
A

For calcium salts and phosphatase activity

184
Q
  1. Alcian Blue
A
  • Stain acid mucopolysaccharides ; More specific
    for connective tissue and epithelial mucin
185
Q
  1. Aniline blue
A
  • A cytoplasmic stain used for counterstaining of
    Epithelial sections
186
Q
  1. Basic Fuchsin
    a . Carbol fuchsin
    b . Coleman’s feulgen
    c . Schiff’s
    d . Mallory’s fuchsin
    e . Aldehyde fuchsin
    ( Gomori’s )
A
  • A plasma stain utilized also for deep staining of
    acid fast organisms , mitochondria and
    differentiation of smooth muscles with the use of
    picric acid
187
Q
  1. Benzidine
A

Used for staining haemoglobin

188
Q
  1. Bismarck brown
A

Use for staining diphtheria ; Used as a contrast
stain for Gram’s technique , acid fast and
Papanicolau method .

189
Q
  1. Carmine
A

-
Used as a chromatin stain for fresh materials in
smear preparations
Best Carmine Combined with aluminium
chloride to stain glycogen
=

190
Q
  1. Mayer’s
    Carmalum solution
A
  • A mordanted dye acting as a basic dye and
    staining acid substrances
191
Q
  1. Celestine Blue
A

Recommended for routine staining of fixed
sections

192
Q
  1. Congo Red
A

Best known as indicator ; May be utilized as a
stain for axis cylinders in embryos ; Used for
staining elastic tissues , amyloid and myelin
( Krajian’s method )

193
Q

RED

A

Yellow

194
Q

Eosin Y
Eosin B
Phloxine B
Rose Bengal

A

Picric acid
Orange G

195
Q

Red

A

Blue

196
Q

Neutral red

A

Methylene blue

197
Q

Safranin

A

Toluidine Blue

198
Q

Carmine

A

Celestine Blue

199
Q

LYSOCHROMES

A

( Oil Soluble Dyes )

200
Q
  1. Sudan Black
    B ( SBB )
A

Greatest affinity for phospholipids or neutral fats
( triglycerides )
A more sensitive coloring agent than other
lysochromes
Should be discarded if the brownish black color
appeared .
Demonstrates lipids that are resistant to paraffin
embedding

201
Q
  1. Sudan IV or
    Sharlach R
A

Recommended for neutral fats ( triglycerides )
Do not color phospholipids and fine lipid droplets

202
Q
  1. Sudan III
A

-
First Sudan dye introduced into Histochemistry
Fat soluble
A good stain for the CNS

203
Q

a . Water

A

Low refractive index ; good only for temporary
mounting
-

204
Q

b . Glycerin ( RI : 1.47 )

A

Also used as preservative , has high index of
refraction
-

205
Q

c . Farrant’s Medium
( Gum syrup ) ( RI : 1.43 )

A

Does not solidify upon storage -

206
Q

d . Apathy’s Medium

A

Used for Methylene Blue - stained nerve
preparations and as general purpose aqueous
mountant

207
Q

e . Brun’s Fluid

A
  • Recommended for mounting frozen sections from
    water or paraffin sections which require
    dehydration and clearing
208
Q

a . Canada Balsam
( RI : 1.524 )

A
  • Natural resin extracted from the Canadian Tree ,
    Abus balsamea
  • Recommended for whole mounts and for thick
    sections because it does not shrink much
    Miscible with xylene and is quite expensive
209
Q

b . DPX ( RI : 1.532 )

A
  • Recommended for small tissue sections
210
Q

c . XAM ( RI : 1.52 )

A

Synthetic resin mixture in xylene in pale yellow or
colorless solution

211
Q

d . Clarite ( RI : 1.544 )

A
  • Synthetic resin which is soluble in xylene ( usually
    diluted to 60 % with xylene )
212
Q
  1. Mayer’s Egg Albumin
A

Most commonly used because it is very
easy to make and relatively inexpensive
Formula : Egg white ( 50 mL )
Glycerin ( 50 mL )
Thymol crystals ( 100 mg )

213
Q
  1. Dried Albumin
A

Formula : Dried albumin ( 5 g )
Sodium chloride ( 5 g )
Distilled water ( 100 mL )
Thymol crystals

214
Q
  1. 1 % Gelatin
A

Formula : Gelatin ( 1 g )
Distilled water ( 100 mL )
Glycerol ( 15 mL )
Phenol crystals ( 2 g )

215
Q
  1. Gelatin
A

Formula : 1 % Gelatin ( 5 mL )
2 % Formaldehyde ( 5 mL )

216
Q
  1. Starch Paste
A

Formula : Powdered starch ( 1 g )
Distilled water ( 30 mL = 10 mL
cold , 20 mL boiling )
Hydrochloric acid ( 2 drops )
Thymol crystals

217
Q
  1. Plasma
A

Readily available from outdated blood
stored in blood banks , dispensed into
sterile tubes of 0.5 mL each .

218
Q
  1. Poly - L - Lysine
A

0.1 % aqueous detergent solution which
is further diluted 1:10 with distilled water
prior to use ( Final dilution : 0.01 % ) .
Used as a section adhesive in
immunohistochemistry .

219
Q
  1. 3 - APES ( 3
    aminopropyltriethoxysilane )
A
  • Very useful in cytology particularly for
    cytospin preparations of proteinaceous
    or bloody material .
220
Q
  1. Keratin
A

Highly sensitive marker for epithelial cells
( Epithelial tumors )

221
Q

a . Cytokeratin 7
( CK7 )

A

Carcinomas of the lung , breast , uterus and
ovaries ( serous tumors )

222
Q

b . Cytokeratin 20
( CK20 )

A

Carcinomas of colon and stomach

223
Q

c . CK 7 and CK20

A
  • Transitional carcinomas of the bladder and
    mucinous ovarian tumor
224
Q
  1. Epithelial membrane
    antigen ( EMA )
A

-
Determining site tumor ,
Breast , lung and kidney adenocarcinomas

225
Q

3 . Carcinoembryonic
membrane antigen
( CEA )

A

Differentiating
mesothelioma
GIT , pancreas , lung , breast , ovary , uterus
and cervix
Thyroid , lung and neuroendocrine tumors
adenocarcinoma and

226
Q
  1. Thyroid
    transcription factor
    1 ( TTF - 1 )
A
  • Distinguishing lung adenocarcinoma from
    mesotheliomas
227
Q
  1. Prostate specific
    antigen ( PSA )
A
  • Prostatic
    salivary gland tumor .
    carcinoma ; pancreatic and
228
Q

1 . Actin

A

Muscle differentiation ( smooth , skeletal and
cardiac muscle )

229
Q

2 . ntin

A

Melanomas and Schwannomas

230
Q

3 . Desmin

A
  • Myogenic tumors
    rhabdomyosarcoma )
    ( leiomyoma and
231
Q

4 . Glial fibrillary acidic
protein ( GFAP )

A
  • Astrocytoma
232
Q
  1. Neurofilament ( NF )
A
  • Neuronal or neuroendocrine differentiation
233
Q

6 . S100 protein

A
  • Calcium - binding protein that is expressed in
    CNS glial cells , Schwann cells ,
    melanocytes , chondrocytes , skeletal and
    cardiac muscle and myoepithelial cells
234
Q
  1. Neuron - specific
    enolase ( NSE )
A

Neural or neuroendocrine differentiation

235
Q
  1. Chromogranin
A

Neuroendocrine differentiation

236
Q
  1. Synaptophysin
A

Associated with presynaptic vesicles of neurons ;
identified in normal neurons and neuroendocrine
cells .

237
Q
  1. Human chorionic gonadotropin
    ( HCG )
A

Choriocarcinoma

238
Q
  1. Alpha - fetoprotein ( AFP )
A
  • Endodermal sinus tumors
    ( yolk sac differentiation ) ;
    Hepatocellular carcinomas
239
Q
  1. Placenta - like alkaline phosphatase
    ( PLAP )
A

Germ tumors
( Germinomas )
cell

240
Q
  1. Muscle - specific actin and myosin ;
    Myo - D1 , myoglobin and myogenin
A

Myogenic tumors ( Skeletal
muscle )

241
Q

2 . CD68 or FAM 56 ; alpha - 1
antiptrypsin and alph - 1
antichymotrypsin

A

Fibrohistiolytic tumors

242
Q

3 . Endothelial markers : Factor VII
related antigen , CD31 and Ulex
Europaeus ( UEA )

A

Vascular
( Angiosarcoma )
tumors

243
Q

4 . $ 100 protein
Melanosome ( HMB - 45 )
Melan - A ( MART - 1 )

A

Melanomas

244
Q
  1. Best : Leukocyte Common Antigen
    ( LCA ) or CD45
A

Lymphomas

245
Q

SPECIMENS

A

FIXATIVES

246
Q

Cervical / vaginal smears

A

95 % Ethanol and Ether
95 % Ethanol

247
Q

Sputum and bronchopulmonary
specimens

A

95 % Ethyl alcohol / Cough directly on a
container with Brasil’s fixative

248
Q

Pleural and peritoneal fluids

A

Brasil’s fixative

249
Q

Gastric specimens

A

95 % Ethanol

250
Q

CELLS FOUND IN VAGINAL

A

SMEARS

251
Q
  1. Mature Superficial
    cells
A

Exhibits true acidophilia ( under estrogen
influence )
It has darken and shrunken nuclei ( pyknotic )
One must differentiate true acidophilia from
pseudoacidophilia which occurs in the
following conditions :
1. Infection
2 .
3 .
4 .
Chemicals
Smear drying before fixation
Vaginal prolapse and drying

252
Q
  1. Intermediate cells
A

-
Medium - sized
Basophilic Cytoplasm with vacuoles
Elongated or polyhedral cells .
It may look like a boat at times and it is called
as Navicular cells
O Found in the latter half of menstrual
cycle , during pregnancy and menopause
O Under progesterone control

253
Q
  1. Pregnancy cells
A

Oval , round or boat - shaped
Nucleus is eccentric
Has basophilic and translucent cytoplasm
Double - walled boundary with a deep blue
stain at the cytoplasmic periphery

254
Q
  1. Parabasal Cells
    Parabasal cells
A
  • Round to oval in shape
    Smaller than Intermediate cells
    Normally found from 2 weeks of age to
    puberty , during abortions , after menopause
    and after childbirth .
255
Q
  1. Endometrial cells
A

Similar to parabasal cells in appearance
Occurs in groups of 3 or more
Slightly cylindrical with a less basophilic
cytoplasm
Found 1-4 days after menstruation

256
Q
  1. Endocervical
    glandular cells
A
  • Honeycomb in appearance
    With pale blue / gray cytoplasm that is finely
    vacuolated
    The nuclei have finely granular chromatin
257
Q
  1. Basal cells
    Basal cells
A

-
Oval , small and round - shaped
With very large nuclei more than half of the
cell in size
Found before puberty and after menopause
Its cytoplasm is strongly basophilic

histopath (2 decks)

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