Tissue Processing Flashcards
(129 cards)
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)
2
Q
1st and most critical step in tissue processing
Most important: stabilization of proteins
A
Fixation
3
Q
Fixation aims to:
A
1’ aim: preserve cell (life-like)
2’ aim: harden & protect tissues
4
Q
pH in fixation
A
6.0-8.0
5
Q
Temperature in fixation
A
Room temp = Surgical specimen
0 to 4’C = EM and Histochem.
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
7
Q
fixes specific parts of the cell
A
Cytological Fixatives
8
Q
fixative that destroys mitochondria & golgi bodies (pH ≤4.6)
A
Nuclear fixatives: w/ glacial acetic acid
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
10
Q
Enumerate nuclear fixatives
“BFNCH”
A
Bouin’s
Flemming’s w/ acetic acid
Newcomer’s
Carnoy’s
Heidenhain’s SuSa
11
Q
Enumerate cytoplasmic fixatives
“HORFF”
A
Helly’s
Orth’s
Regaud’s
Flemming’s w/o acetic acid
Formalin w/ post chroming
12
Q
Histochemical fixatives
“FANA”
A
10% Formol saline
Absolute alcohol
Newcomer’s fluid
Acetone
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
14
Q
Concentrated solutions should not be neutralized (explosion)
Stock solution: 37-40%
Working solution: 10% (no buffer: unstable)
A
Formaldehyde
15
Q
Formalin pigments:
A
a. Paraformaldehyde
b. Acid formaldehyde hematin
16
Q
White crystalline precipitates
- Due to prolonged standing
- Removed by: 10% METOH/filtration
A
Paraformaldehyde
17
Q
- Brown/black granular deposits that may obscure microscopic details
A
Acid formaldehyde hematin
18
Q
Aldehyde fixative for CNS
A
10% Formol saline
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
20
Q
w/ HgCl2
A
Formol-Corrosive
(formol sublimate)
21
Q
EM
A
Glutaraldehyde
22
Q
EM: electron histochemistry & electron immunocytochemistry
A
Karnovsky’s paraformaldehyde-glutaraldehyde
23
Q
Mixture w/ formaldehyde/formaldehyde
A
Acrolein
24
Q
Lipids (frozen section)
A
Formol-calcium
25
Tissue photography
For Trichrome stain (excellent)
Produce black granular deposits except SuSa
Mercuric Chloride
26
Enumerate mercuric chloride fixatives.
“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
27
Su = sublimat (HgCl2)
Sa = saure (acid)
Heidenhain’s SuSa
28
Shrinks tissues
HgCl2
29
Swells tissues, counteracts HgCl2
G.HAc
30
Removal of mercuric deposits
H2O I2 H2O Sodium thiosulfate H2O
De-zenkerization
31
Chromate fixatives
“ROCK”
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)
32
Chromate pigments
Fine, yellow brown
33
Used in 4% aqueous solution of basic lead acetate
For acid MPS and mucin
Lead fixatives
34
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)
Picric acid fixatives
35
Picric acid fixatives:
for embryos, Masson’s trichrome stain, glycogen
Bouin’s
36
Picric acid fixatives:
less messy than Bouin’s, glycogen (excellent)
Brasil’s alcoholic picroformol
37
Solidifies at 17’C
Fixes & precipitates nucleoproteins, chromosomes, & chromatin material
Most commonly combined w/ other fixatives
Glacial acetic acid
38
Disadvantage of alcoholic fixatives
Polarization (glycogen granules poles/ends of the cells)
39
Alcoholic fixatives:
"MEICAN"
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
40
Inhibits hematoxylin
Produce black precipitate crystals (osmium oxide)
For lipids
Osmium tetroxide
(Osmic acid)
41
Osmium tetroxide
(Osmic acid):
- permanently fixes fat, for nuclear structures (excellent)
- Fixative & decalcifying agent (chromic acid)
Flemming’s
42
Osmium tetroxide
(Osmic acid):
for mitochondria
Flemming’s w/o acetic acid
43
Precipitates proteins
Swelling effect counteract shrinkage by other fixatives
Weak decalcifying agent (softening effect)
Trichloroacetic acid
44
Recommended for H2O-diffusible enzymes (phosphatases, lipases)
Rabies
Acetone
45
Bacteriologic smears
Microwave: 45-55’C
Underheating: poor sectioning
Overheating (>65’C): vacuolation, overstained cytoplasm
Heat fixation
46
Placing an already fixed tissue in a 2nd fixative
2’ fixation
47
Primarily fixed tissue 2.5-3% K2CrO4 (mordant)
Post-chromatization
48
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
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
49
Glutaraldehyde
PtCl3
PtCl3 – formalin (Zamboni’s)
AuCl
Osmium tetroxide
10% NBF = acceptable but not recommended
EM fixatives
50
Stains (EM)
“PUL”
1. PTA = 1st general stain
2. Uranyl acetate = Best
3. Lead
51
Factors that Affect Fixation of Tissues
Retarded by:
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
52
Factors that Affect Fixation of Tissues
Enhanced by:
a) Size & thickness
b) Agitation:
Automatic/mechanical tissue processing
c) Moderate heat:
37-56’C
53
Autopsy materials should be fixed ASAP.
If not possible, what should you do?
Mortuary refrigerator (4’C) or arterial embalming
54
Surgical specimens should be fixed ASAP.
If not, what should you do?
Refrigerate
55
If placed in NSS during operation, autolysis may occur (before/after) fixation?
before
56
If tissues are refrigerated, avoid slow freezing (ice crystal formation).
Repeated freezing & thawing causes what?
Repeated freezing & thawing destroy organelles, release enzymes…
57
Size of tissues
Not more than 5mm thick
*except lung edema: 1-2 cm thick
58
Ratio of fixative to tissue
20:1
*except osmium tetroxide (expensive) = ratio is 5-10:1
59
Ratio of fixative to tissue in prolonged fixation (ex. museum preparation)
50-100:1
60
Avoid drying of small tissue biopsies. To prevent this...
Place in a petri dish w/ moistened filter paper.
60
Stomach, intestines
Packed w/ cotton soaked fixative or completely opened before being immersed in adequate fixing solution
Hollow organs
61
How do you prevent float on fixative when preparing air-filled lungs?
Cover w/ several layers of gauze to maintain it under surface.
62
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: __________
2 weeks
63
This organ/specimen, is not dissected before fixation to avoid tissue collapse & wrinkling (escape of vitreous humor).
When do you inject formol-alcohol?
Inject formol-alcohol before immersing the organ in the fixative.
64
Can you use water on glycogen-containing tissues?
NO.
Do not use water.
Glycogen is water-soluble
65
Cervix, uterus, fibroids, hyperkeratotic skin, fingernails
Wash in running water overnight immerse in 4% aqueous phenol for 1-3 days (Lendrum’s method)
Hard tissues
66
Difficulties Encountered because of Improper Fixation:
Problem:
Failure to arrest early cell autolysis. What could be the cause?
Failure to fix immediately (tissue was allowed to dry before fixing)
Insufficient fixative
67
Problem:
Removal of substances soluble in fixing agent
What is the cause?
Wrong choice of fixative
68
Problem:
Presence of artifact pigments on tissue sections
What is the cause?
Incomplete washing of fixative
69
Problem:
Tissues are soft & feather-like in consistency
What is the cause?
Incomplete fixation
70
Problem:
Loss/inactivation of enzymes needed for study
What is the cause?
Wrong choice of fixative
71
Problem:
Shrinkage & swelling of cells & tissue structure
What is the cause?
Overfixation
72
Problem:
Tissue blocks are brittle & hard
What is the cause?
Prolonged fixation
73
An ________________ tissue may lead to improper & incomplete clearing & impregnation, and may later prove to be a hindrance to normal sectioning & staining of specimen
incompletely fixed
74
Pigment:
Acid formaldehyde hematin
Color: Brown/black granules
This can be removed by?
“SAKaL”
a. Saturated picric acid
b. Alcoholic KOH
c. Kardasewitsch method
d. Lillie’s method
75
Pigment: Mercuric chloride pigment
Color: Black granules
This can be removed by?
Alcoholic iodine
76
Pigment: Chromate pigment
Color: Fine, yellow brown
This can be removed by?
Acid-alcohol
77
Pigment: Osmium tetroxide pigment
Color: Black precipitate crystals
This can be removed by?
Cold H2O
78
Intense eosinophilic staining at the center of the tissue (H & E)
Due to partial coagulation of partially fixed protein
Crush artifact
79
Ratio of decalcifying agent to tissue
20:1
80
At what temperature nuclear stain by Van Gieson’s stain be impaired?
37’C
81
At what temperature will tissue digestion occur (24-48 hrs)?
55’C
82
Optimum temperature and time required for decalcification
RT (18-30’C)
14-48 hrs
83
Enumerate categories of decalcifying agents.
1) Acids
2) Chelating agents (EDTA/versene)
3) Ion exchange resins
4) Elec. ionization (electrophoresis)
84
HNO3
Most common
a. _______ = tissue softener & decalcifying agent
b. ______________ = most rapid
- Disadvantage: ______ color on tissue
Most common
a. Perenyi’s = tissue softener & decalcifying agent
b. Phloroglucin-HNO3 = most rapid
- Disadvantage: Yellow color on tissue
85
Yellow color has formed on tissue when using HNO3, what should you do?
neutralize w/ sodium thiosulfate
86
Both fixative & decalcifying agent
Best general decalcifying agent
For small pcs of bones & teeth
5% Formic acid
87
For small pcs of bones & teeth
For surface decalcification (HCl)
HCl (Von Ebner’s)
88
For EM, IHC, & enzyme staining
EDTA
89
Hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions
Ion exchange resins
90
Ca2+ are attracted to negative electrode (cathode)
Electrophoresis
91
How do you measure extent of decalcification?
1) Physical method
2) Chemical method = CaOx test (routine) | Turbidity = (+) Ca2+
3) X-ray
- X-ray paper = Kodak X-omat or Faxitron
92
most ideal, most sensitive, most reliable but very expensive method of measuring extent of decalcification
93
Removal/neutralization of acid from the tissues after decalcification
Lithium carbonate or sodium bicarbonate solution
Post-Decalcification
94
Enumerate tissue softeners (4)
4% phenol
Molliflex
2% HCl
1% HCl in 70% alcohol
95
This tissue softener, tissues might appear swollen & soapy.
Molliflex
96
Purpose of dehydration
To remove fixative & H2O
97
Dehydration:
Ascending grades of alcohol (Start: ____%)
Embryonic & animal tissues: __________
65%
30% ETOH
98
Ratio of dehydrating agent to tissue
10:1
99
Best dehydrating agent
Ethanol
100
Dehydrating agent for blood & tissue films
Methanol
101
Dehydrating agent for Plants & animals
Butanol
102
Ethanol + methanol
Denatured alcohol
103
is both a fixative & a dehydrating agent
Acetone
104
are both dehydrating & clearing agents
Dioxane (Diethylene dioxide)
Tetrahydrofuran (THF)
105
Dehydration w/ dioxane
Graupner’s method
Weiseberger’s method
106
Ethylene glycol monoethyl ether
Combustible and toxic
Cellosolve
107
- both dehydrating agent & indicator of H2O content of 100% ETOH
- (+) H2O = White Blue
Anhydrous CuSO4 (Last ETOH bath)
108
Additives to dehydrating agents
a. 4% phenol + 95% ETOH = softener
lec.mt 04 |Page | 19
agents
b. Anhydrous CuSO4 (Last ETOH bath)
109
Methods of determining incomplete dehydration
1. Anhydrous CuSO4 method
2. Xylene Milky
110
Most commonly used clearing agent
Xylene (Xylol)
111
Clearing time:
½ to 1 hr
112
In clearing,
Block size:
<5mm
113
Substitute for xylene/benzene
Clearing time: 1-2 hrs
Not carcinogenic
Toxic fumes
Toluene
114
Toxic to liver
For clearing tough tissues
Does not make tissue translucent but removes alcohol
Chloroform
115
For urgent biopsies
Minimum shrinkage
Aplastic anemia
Benzene
116
For double embedding techniques
Methyl salicylate
Methyl benzoate
117
For CNS, smooth muscles, skin
Cedarwood oil
118
For CNS, smooth muscles, skin
Cedarwood oil
119
Minimum shrinkage
Has tendency to become adulterated
Clove oil
120
Similar to chloroform but is cheaper
Disadvantage: similar to chloroform
CCl4
121
Clearing agent for delicate tissues, embryos and insects
Aniline oil
122
Clearing agent for delicate tissues, embryos and insects
Aniline oil
123
No dealcoholization but make the tissues clearer
Glycerin
Gum syrup
124
Other clearing agents
Citrus fruits oil
Trichloroethane & petrol
125
Enumerate clearing agents.
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
Ratio of infiltrating medium to tissue
25:1
127
Medium used in impregnation/infiltration
a) Paraffin wax
b) Celloidin (collodion)
c) Gelatin = H2O soluble, not a wax
d) Plastic = EM
128
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
Paraffin
