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HISTOPATHOLOGIC TECHNIQUES > Tissue Processing > Flashcards

Tissue Processing Flashcards

1
Q

Steps in Tissue Processing

A

“Fat Danny Can Instantly Eat Tuna Sandwich So Much Lately”

“FDCIETS SMoL”
1. Fixation
(Decalcification)
2. Dehydration
3. Clearing/Dealcoholization
4. Impregnation/Infiltration
5. Embedding/Casting/Blocking
6. Trimming
7. Sectioning/Microtomy
8. Staining
9. Mounting
10. Labeling (slides)

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2
Q

1st and most critical step in tissue processing
Most important: stabilization of proteins

A

Fixation

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3
Q

Fixation aims to:

A

1’ aim: preserve cell (life-like)
2’ aim: harden & protect tissues

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4
Q

pH in fixation

A

6.0-8.0

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5
Q

Temperature in fixation

A

Room temp = Surgical specimen
0 to 4’C = EM and Histochem.

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6
Q

Microanatomical fixatives (8)

A

General microscopic study of tissues
a. 10% Formol saline
b. 10% NBF
c. Heidenhain’s SuSa
d. Formol sublimate (formol corrosive)
e. Zenker’s solution
f. Zenker-formol (Helly’s)
g. Bouin’s solution
h. Brasil’s solution

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7
Q

fixes specific parts of the cell

A

Cytological Fixatives

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8
Q

fixative that destroys mitochondria & golgi bodies (pH ≤4.6)

A

Nuclear fixatives: w/ glacial acetic acid

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9
Q

Types of cytological fixatives

A

1) Nuclear fixatives: w/ glacial acetic acid
2) Cytoplasmic fixatives: w/o glacial acetic acid
3) Histochemical fixatives

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10
Q

Enumerate nuclear fixatives

“BFNCH”

A

Bouin’s
Flemming’s w/ acetic acid
Newcomer’s
Carnoy’s
Heidenhain’s SuSa

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11
Q

Enumerate cytoplasmic fixatives

“HORFF”

A

Helly’s
Orth’s
Regaud’s
Flemming’s w/o acetic acid
Formalin w/ post chroming

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12
Q

Histochemical fixatives

“FANA”

A

10% Formol saline
Absolute alcohol
Newcomer’s fluid
Acetone

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13
Q

Enumerate aldehyde Fixatives

A

1) Formaldehyde
2) 10% Formol saline
3) 10% NBF
4) Formol-Corrosive
(formol sublimate)
5) Glutaraldehyde
6) Karnovsky’s paraformaldehyde-glutaraldehyde
7) Acrolein
8) Formol-calcium

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14
Q

Concentrated solutions should not be neutralized (explosion)
Stock solution: 37-40%
Working solution: 10% (no buffer: unstable)

A

Formaldehyde

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15
Q

Formalin pigments:

A

a. Paraformaldehyde
b. Acid formaldehyde hematin

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16
Q

White crystalline precipitates
- Due to prolonged standing
- Removed by: 10% METOH/filtration

A

Paraformaldehyde

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17
Q
  • Brown/black granular deposits that may obscure microscopic details
A

Acid formaldehyde hematin

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18
Q

Aldehyde fixative for CNS

A

10% Formol saline

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19
Q

Best general tissue fixative
Best fixative for tissue containing iron granules
w/ double phosphate buffer
1 mm/hr = rate of tissue penetration

A

10% NBF

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20
Q

w/ HgCl2

A

Formol-Corrosive
(formol sublimate)

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21
Q

EM

A

Glutaraldehyde

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22
Q

EM: electron histochemistry & electron immunocytochemistry

A

Karnovsky’s paraformaldehyde-glutaraldehyde

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23
Q

Mixture w/ formaldehyde/formaldehyde

A

Acrolein

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24
Q

Lipids (frozen section)

A

Formol-calcium

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25
Q

Tissue photography
For Trichrome stain (excellent)
Produce black granular deposits except SuSa

A

Mercuric Chloride

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26
Q

Enumerate mercuric chloride fixatives.

A

“BOSCHZZ”
a. B5 = for BM biopsies
b. Ohlmacher’s
c. Schaudinn’s
d. Carnoy-Lebrun
e. Heidenhain’s SuSa = (-) black pigments
f. Zenker’s = recommended for trichrome staining
g. Zenker-formol (Helly’s) = pituitary gland, BM, & blood containing organs

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27
Q

Su = sublimat (HgCl2)
Sa = saure (acid)

A

Heidenhain’s SuSa

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28
Q

Shrinks tissues

A

HgCl2

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29
Q

Swells tissues, counteracts HgCl2

A

G.HAc

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30
Q

Removal of mercuric deposits
H2O I2 H2O Sodium thiosulfate H2O

A

De-zenkerization

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31
Q

Chromate fixatives
“ROCK”

A

a. Regaud’s (Moller’s) = chromatin, mitochondria, mitotic figures…
b. Orth’s = for Rickettsia, tissue necrosis
c. Chromic acid = preserves CHO
d. K2CrO4 = mitochondria (if acidified, fixes chromatin bodies & chromosomes but destroys mitochondria)

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32
Q

Chromate pigments

A

Fine, yellow brown

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33
Q

Used in 4% aqueous solution of basic lead acetate
For acid MPS and mucin

A

Lead fixatives

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34
Q

Highly explosive when dry
Excessive yellow staining of tissues
Picrates Protein Ppt. (H2O soluble) Add 70% ETOH Insoluble
Never wash in H2O before dehydration
For glycogen (excellent)

A

Picric acid fixatives

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35
Q

Picric acid fixatives:

for embryos, Masson’s trichrome stain, glycogen

A

Bouin’s

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36
Q

Picric acid fixatives:

less messy than Bouin’s, glycogen (excellent)

A

Brasil’s alcoholic picroformol

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37
Q

Solidifies at 17’C
Fixes & precipitates nucleoproteins, chromosomes, & chromatin material
Most commonly combined w/ other fixatives

A

Glacial acetic acid

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38
Q

Disadvantage of alcoholic fixatives

A

Polarization (glycogen granules poles/ends of the cells)

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39
Q

Alcoholic fixatives:
“MEICAN”

A

a. Methanol = BM & blood smears
b. Ethanol = preserves but does not fix glycogen (Disadv: polarization)
c. Isopropanol = for touch preparations
d. Carnoy’s = most rapid (1-3 hrs) | for chromosomes | Dx: rabies (acetone)
e. Alcoholic formalin (Gendre’s) = sputum
f. Newcomer’s = for MPS | nuclear & histochemical fixative

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40
Q

Inhibits hematoxylin
Produce black precipitate crystals (osmium oxide)
For lipids

A

Osmium tetroxide
(Osmic acid)

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41
Q

Osmium tetroxide
(Osmic acid):

  • permanently fixes fat, for nuclear structures (excellent)
  • Fixative & decalcifying agent (chromic acid)
A

Flemming’s

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42
Q

Osmium tetroxide
(Osmic acid):

for mitochondria

A

Flemming’s w/o acetic acid

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43
Q

Precipitates proteins
Swelling effect counteract shrinkage by other fixatives
Weak decalcifying agent (softening effect)

A

Trichloroacetic acid

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44
Q

Recommended for H2O-diffusible enzymes (phosphatases, lipases)
Rabies

A

Acetone

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45
Q

Bacteriologic smears
Microwave: 45-55’C
Underheating: poor sectioning
Overheating (>65’C): vacuolation, overstained cytoplasm

A

Heat fixation

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46
Q

Placing an already fixed tissue in a 2nd fixative

A

2’ fixation

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47
Q

Primarily fixed tissue 2.5-3% K2CrO4 (mordant)

A

Post-chromatization

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48
Q

Removing excess fixative
a. _________ = remove excess chromates, formalin, osmic acid (NOT Bouin’s)
b. _________= wash out excess picric acid (Bouin’s)
c. _________= remove excess mercuric fixatives

A

Washing out

Removing excess fixative
a. Tap H2O = remove excess chromates, formalin, osmic acid (NOT Bouin’s)
b. 50-70% alcohol = wash out excess picric acid (Bouin’s)
c. Alcoholic I2 = remove excess mercuric fixatives

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49
Q

Glutaraldehyde
PtCl3
PtCl3 – formalin (Zamboni’s)
AuCl
Osmium tetroxide
10% NBF = acceptable but not recommended

A

EM fixatives

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50
Q

Stains (EM)
“PUL”

A
  1. PTA = 1st general stain
  2. Uranyl acetate = Best
  3. Lead
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51
Q

Factors that Affect Fixation of Tissues
Retarded by:

A

a) Size & thickness:
size= fixation time

b) (+) Mucus:
Prevents complete penetration of fixative
Wash w/ NSS

c) (+) Fat:
Fatty tissues: cut in thin sections, fixed longer

d) (+) Blood:
Flush out w/ NSS fix

e) Cold temperature:
Inactivates enzymes

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52
Q

Factors that Affect Fixation of Tissues
Enhanced by:

A

a) Size & thickness

b) Agitation:
Automatic/mechanical tissue processing

c) Moderate heat:
37-56’C

53
Q

Autopsy materials should be fixed ASAP.
If not possible, what should you do?

A

Mortuary refrigerator (4’C) or arterial embalming

54
Q

Surgical specimens should be fixed ASAP.
If not, what should you do?

A

Refrigerate

55
Q

If placed in NSS during operation, autolysis may occur (before/after) fixation?

A

before

56
Q

If tissues are refrigerated, avoid slow freezing (ice crystal formation).
Repeated freezing & thawing causes what?

A

Repeated freezing & thawing destroy organelles, release enzymes…

57
Q

Size of tissues

A

Not more than 5mm thick

*except lung edema: 1-2 cm thick

58
Q

Ratio of fixative to tissue

A

20:1

*except osmium tetroxide (expensive) = ratio is 5-10:1

59
Q

Ratio of fixative to tissue in prolonged fixation (ex. museum preparation)

A

50-100:1

60
Q

Avoid drying of small tissue biopsies. To prevent this…

A

Place in a petri dish w/ moistened filter paper.

60
Q

Stomach, intestines
Packed w/ cotton soaked fixative or completely opened before being immersed in adequate fixing solution

A

Hollow organs

61
Q

How do you prevent float on fixative when preparing air-filled lungs?

A

Cover w/ several layers of gauze to maintain it under surface.

62
Q

Human brains:

Suspended by a cord tied under the Circle of Willis to prevent flattening
Avoid Ringer’s lactate for washing out of blood intravascular perfusion
Fixation time: __________

A

2 weeks

63
Q

This organ/specimen, is not dissected before fixation to avoid tissue collapse & wrinkling (escape of vitreous humor).
When do you inject formol-alcohol?

A

Inject formol-alcohol before immersing the organ in the fixative.

64
Q

Can you use water on glycogen-containing tissues?

A

NO.

Do not use water.
Glycogen is water-soluble

65
Q

Cervix, uterus, fibroids, hyperkeratotic skin, fingernails
Wash in running water overnight immerse in 4% aqueous phenol for 1-3 days (Lendrum’s method)

A

Hard tissues

66
Q

Difficulties Encountered because of Improper Fixation:

Problem:
Failure to arrest early cell autolysis. What could be the cause?

A

Failure to fix immediately (tissue was allowed to dry before fixing)
Insufficient fixative

67
Q

Problem:
Removal of substances soluble in fixing agent

What is the cause?

A

Wrong choice of fixative

68
Q

Problem:
Presence of artifact pigments on tissue sections

What is the cause?

A

Incomplete washing of fixative

69
Q

Problem:
Tissues are soft & feather-like in consistency

What is the cause?

A

Incomplete fixation

70
Q

Problem:
Loss/inactivation of enzymes needed for study

What is the cause?

A

Wrong choice of fixative

71
Q

Problem:
Shrinkage & swelling of cells & tissue structure

What is the cause?

A

Overfixation

72
Q

Problem:
Tissue blocks are brittle & hard

What is the cause?

A

Prolonged fixation

73
Q

An ________________ tissue may lead to improper & incomplete clearing & impregnation, and may later prove to be a hindrance to normal sectioning & staining of specimen

A

incompletely fixed

74
Q

Pigment:
Acid formaldehyde hematin
Color: Brown/black granules

This can be removed by?

A

“SAKaL”
a. Saturated picric acid
b. Alcoholic KOH
c. Kardasewitsch method
d. Lillie’s method

75
Q

Pigment: Mercuric chloride pigment
Color: Black granules

This can be removed by?

A

Alcoholic iodine

76
Q

Pigment: Chromate pigment
Color: Fine, yellow brown

This can be removed by?

A

Acid-alcohol

77
Q

Pigment: Osmium tetroxide pigment
Color: Black precipitate crystals

This can be removed by?

A

Cold H2O

78
Q

Intense eosinophilic staining at the center of the tissue (H & E)
Due to partial coagulation of partially fixed protein

A

Crush artifact

79
Q

Ratio of decalcifying agent to tissue

A

20:1

80
Q

At what temperature nuclear stain by Van Gieson’s stain be impaired?

A

37’C

81
Q

At what temperature will tissue digestion occur (24-48 hrs)?

A

55’C

82
Q

Optimum temperature and time required for decalcification

A

RT (18-30’C)

14-48 hrs

83
Q

Enumerate categories of decalcifying agents.

A

1) Acids
2) Chelating agents (EDTA/versene)
3) Ion exchange resins
4) Elec. ionization (electrophoresis)

84
Q

HNO3
Most common
a. _______ = tissue softener & decalcifying agent
b. ______________ = most rapid
- Disadvantage: ______ color on tissue

A

Most common
a. Perenyi’s = tissue softener & decalcifying agent
b. Phloroglucin-HNO3 = most rapid
- Disadvantage: Yellow color on tissue

85
Q

Yellow color has formed on tissue when using HNO3, what should you do?

A

neutralize w/ sodium thiosulfate

86
Q

Both fixative & decalcifying agent
Best general decalcifying agent
For small pcs of bones & teeth

A

5% Formic acid

87
Q

For small pcs of bones & teeth
For surface decalcification (HCl)

A

HCl (Von Ebner’s)

88
Q

For EM, IHC, & enzyme staining

A

EDTA

89
Q

Hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions

A

Ion exchange resins

90
Q

Ca2+ are attracted to negative electrode (cathode)

A

Electrophoresis

91
Q

How do you measure extent of decalcification?

A

1) Physical method

2) Chemical method = CaOx test (routine) | Turbidity = (+) Ca2+

3) X-ray
- X-ray paper = Kodak X-omat or Faxitron

92
Q

most ideal, most sensitive, most reliable but very expensive method of measuring extent of decalcification

A
93
Q

Removal/neutralization of acid from the tissues after decalcification
Lithium carbonate or sodium bicarbonate solution

A

Post-Decalcification

94
Q

Enumerate tissue softeners (4)

A

4% phenol

Molliflex

2% HCl

1% HCl in 70% alcohol

95
Q

This tissue softener, tissues might appear swollen & soapy.

A

Molliflex

96
Q

Purpose of dehydration

A

To remove fixative & H2O

97
Q

Dehydration:

Ascending grades of alcohol (Start: ____%)
Embryonic & animal tissues: __________

A

65%

30% ETOH

98
Q

Ratio of dehydrating agent to tissue

A

10:1

99
Q

Best dehydrating agent

A

Ethanol

100
Q

Dehydrating agent for blood & tissue films

A

Methanol

101
Q

Dehydrating agent for Plants & animals

A

Butanol

102
Q

Ethanol + methanol

A

Denatured alcohol

103
Q

is both a fixative & a dehydrating agent

A

Acetone

104
Q

are both dehydrating & clearing agents

A

Dioxane (Diethylene dioxide)
Tetrahydrofuran (THF)

105
Q

Dehydration w/ dioxane

A

Graupner’s method
Weiseberger’s method

106
Q

Ethylene glycol monoethyl ether
Combustible and toxic

A

Cellosolve

107
Q
  • both dehydrating agent & indicator of H2O content of 100% ETOH
  • (+) H2O = White Blue
A

Anhydrous CuSO4 (Last ETOH bath)

108
Q

Additives to dehydrating agents

A

a. 4% phenol + 95% ETOH = softener
lec.mt 04 |Page | 19
agents
b. Anhydrous CuSO4 (Last ETOH bath)

109
Q

Methods of determining incomplete dehydration

A
  1. Anhydrous CuSO4 method
  2. Xylene Milky
110
Q

Most commonly used clearing agent

A

Xylene (Xylol)

111
Q

Clearing time:

A

½ to 1 hr

112
Q

In clearing,

Block size:

A

<5mm

113
Q

Substitute for xylene/benzene
Clearing time: 1-2 hrs
Not carcinogenic
Toxic fumes

A

Toluene

114
Q

Toxic to liver
For clearing tough tissues
Does not make tissue translucent but removes alcohol

A

Chloroform

115
Q

For urgent biopsies
Minimum shrinkage
Aplastic anemia

A

Benzene

116
Q

For double embedding techniques

A

Methyl salicylate
Methyl benzoate

117
Q

For CNS, smooth muscles, skin

A

Cedarwood oil

118
Q

For CNS, smooth muscles, skin

A

Cedarwood oil

119
Q

Minimum shrinkage
Has tendency to become adulterated

A

Clove oil

120
Q

Similar to chloroform but is cheaper
Disadvantage: similar to chloroform

A

CCl4

121
Q

Clearing agent for delicate tissues, embryos and insects

A

Aniline oil

122
Q

Clearing agent for delicate tissues, embryos and insects

A

Aniline oil

123
Q

No dealcoholization but make the tissues clearer

A

Glycerin
Gum syrup

124
Q

Other clearing agents

A

Citrus fruits oil
Trichloroethane & petrol

125
Q

Enumerate clearing agents.

A

1) Xylene
2) Toluene
3) Chloroform
4) Benzene
5) Methyl salicylate
6) Methyl benzoate
7) Cedarwood oil
8) Clove oil
9) CCl4
10) Aniline oil
11) Glycerin, Gum syrup

126
Q

Ratio of infiltrating medium to tissue

A

25:1

127
Q

Medium used in impregnation/infiltration

A

a) Paraffin wax
b) Celloidin (collodion)
c) Gelatin = H2O soluble, not a wax
d) Plastic = EM

128
Q

Introduced by Bütschlii
Not recommended for fatty tissues
Low MP = paraffin is soft
High MP = paraffin is hard
Manual: At least 4 changes of wax at 15mins interval

A

Paraffin

HISTOPATHOLOGIC TECHNIQUES (8 decks)

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