Tissue Processing Flashcards
Steps in Tissue Processing
“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)
1st and most critical step in tissue processing
Most important: stabilization of proteins
Fixation
Fixation aims to:
1’ aim: preserve cell (life-like)
2’ aim: harden & protect tissues
pH in fixation
6.0-8.0
Temperature in fixation
Room temp = Surgical specimen
0 to 4’C = EM and Histochem.
Microanatomical fixatives (8)
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
fixes specific parts of the cell
Cytological Fixatives
fixative that destroys mitochondria & golgi bodies (pH ≤4.6)
Nuclear fixatives: w/ glacial acetic acid
Types of cytological fixatives
1) Nuclear fixatives: w/ glacial acetic acid
2) Cytoplasmic fixatives: w/o glacial acetic acid
3) Histochemical fixatives
Enumerate nuclear fixatives
“BFNCH”
Bouin’s
Flemming’s w/ acetic acid
Newcomer’s
Carnoy’s
Heidenhain’s SuSa
Enumerate cytoplasmic fixatives
“HORFF”
Helly’s
Orth’s
Regaud’s
Flemming’s w/o acetic acid
Formalin w/ post chroming
Histochemical fixatives
“FANA”
10% Formol saline
Absolute alcohol
Newcomer’s fluid
Acetone
Enumerate aldehyde Fixatives
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
Concentrated solutions should not be neutralized (explosion)
Stock solution: 37-40%
Working solution: 10% (no buffer: unstable)
Formaldehyde
Formalin pigments:
a. Paraformaldehyde
b. Acid formaldehyde hematin
White crystalline precipitates
- Due to prolonged standing
- Removed by: 10% METOH/filtration
Paraformaldehyde
- Brown/black granular deposits that may obscure microscopic details
Acid formaldehyde hematin
Aldehyde fixative for CNS
10% Formol saline
Best general tissue fixative
Best fixative for tissue containing iron granules
w/ double phosphate buffer
1 mm/hr = rate of tissue penetration
10% NBF
w/ HgCl2
Formol-Corrosive
(formol sublimate)
EM
Glutaraldehyde
EM: electron histochemistry & electron immunocytochemistry
Karnovsky’s paraformaldehyde-glutaraldehyde
Mixture w/ formaldehyde/formaldehyde
Acrolein
Lipids (frozen section)
Formol-calcium
Tissue photography
For Trichrome stain (excellent)
Produce black granular deposits except SuSa
Mercuric Chloride
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
Su = sublimat (HgCl2)
Sa = saure (acid)
Heidenhain’s SuSa
Shrinks tissues
HgCl2
Swells tissues, counteracts HgCl2
G.HAc
Removal of mercuric deposits
H2O I2 H2O Sodium thiosulfate H2O
De-zenkerization
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)
Chromate pigments
Fine, yellow brown
Used in 4% aqueous solution of basic lead acetate
For acid MPS and mucin
Lead fixatives
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
Picric acid fixatives:
for embryos, Masson’s trichrome stain, glycogen
Bouin’s
Picric acid fixatives:
less messy than Bouin’s, glycogen (excellent)
Brasil’s alcoholic picroformol
Solidifies at 17’C
Fixes & precipitates nucleoproteins, chromosomes, & chromatin material
Most commonly combined w/ other fixatives
Glacial acetic acid
Disadvantage of alcoholic fixatives
Polarization (glycogen granules poles/ends of the cells)
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
Inhibits hematoxylin
Produce black precipitate crystals (osmium oxide)
For lipids
Osmium tetroxide
(Osmic acid)
Osmium tetroxide
(Osmic acid):
- permanently fixes fat, for nuclear structures (excellent)
- Fixative & decalcifying agent (chromic acid)
Flemming’s
Osmium tetroxide
(Osmic acid):
for mitochondria
Flemming’s w/o acetic acid
Precipitates proteins
Swelling effect counteract shrinkage by other fixatives
Weak decalcifying agent (softening effect)
Trichloroacetic acid
Recommended for H2O-diffusible enzymes (phosphatases, lipases)
Rabies
Acetone
Bacteriologic smears
Microwave: 45-55’C
Underheating: poor sectioning
Overheating (>65’C): vacuolation, overstained cytoplasm
Heat fixation
Placing an already fixed tissue in a 2nd fixative
2’ fixation
Primarily fixed tissue 2.5-3% K2CrO4 (mordant)
Post-chromatization
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
Glutaraldehyde
PtCl3
PtCl3 – formalin (Zamboni’s)
AuCl
Osmium tetroxide
10% NBF = acceptable but not recommended
EM fixatives
Stains (EM)
“PUL”
- PTA = 1st general stain
- Uranyl acetate = Best
- Lead
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
Factors that Affect Fixation of Tissues
Enhanced by:
a) Size & thickness
b) Agitation:
Automatic/mechanical tissue processing
c) Moderate heat:
37-56’C
Autopsy materials should be fixed ASAP.
If not possible, what should you do?
Mortuary refrigerator (4’C) or arterial embalming
Surgical specimens should be fixed ASAP.
If not, what should you do?
Refrigerate
If placed in NSS during operation, autolysis may occur (before/after) fixation?
before
If tissues are refrigerated, avoid slow freezing (ice crystal formation).
Repeated freezing & thawing causes what?
Repeated freezing & thawing destroy organelles, release enzymes…
Size of tissues
Not more than 5mm thick
*except lung edema: 1-2 cm thick
Ratio of fixative to tissue
20:1
*except osmium tetroxide (expensive) = ratio is 5-10:1
Ratio of fixative to tissue in prolonged fixation (ex. museum preparation)
50-100:1
Avoid drying of small tissue biopsies. To prevent this…
Place in a petri dish w/ moistened filter paper.
Stomach, intestines
Packed w/ cotton soaked fixative or completely opened before being immersed in adequate fixing solution
Hollow organs
How do you prevent float on fixative when preparing air-filled lungs?
Cover w/ several layers of gauze to maintain it under surface.
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
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.
Can you use water on glycogen-containing tissues?
NO.
Do not use water.
Glycogen is water-soluble
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
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
Problem:
Removal of substances soluble in fixing agent
What is the cause?
Wrong choice of fixative
Problem:
Presence of artifact pigments on tissue sections
What is the cause?
Incomplete washing of fixative
Problem:
Tissues are soft & feather-like in consistency
What is the cause?
Incomplete fixation
Problem:
Loss/inactivation of enzymes needed for study
What is the cause?
Wrong choice of fixative
Problem:
Shrinkage & swelling of cells & tissue structure
What is the cause?
Overfixation
Problem:
Tissue blocks are brittle & hard
What is the cause?
Prolonged fixation
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
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
Pigment: Mercuric chloride pigment
Color: Black granules
This can be removed by?
Alcoholic iodine
Pigment: Chromate pigment
Color: Fine, yellow brown
This can be removed by?
Acid-alcohol
Pigment: Osmium tetroxide pigment
Color: Black precipitate crystals
This can be removed by?
Cold H2O
Intense eosinophilic staining at the center of the tissue (H & E)
Due to partial coagulation of partially fixed protein
Crush artifact
Ratio of decalcifying agent to tissue
20:1
At what temperature nuclear stain by Van Gieson’s stain be impaired?
37’C
At what temperature will tissue digestion occur (24-48 hrs)?
55’C
Optimum temperature and time required for decalcification
RT (18-30’C)
14-48 hrs
Enumerate categories of decalcifying agents.
1) Acids
2) Chelating agents (EDTA/versene)
3) Ion exchange resins
4) Elec. ionization (electrophoresis)
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
Yellow color has formed on tissue when using HNO3, what should you do?
neutralize w/ sodium thiosulfate
Both fixative & decalcifying agent
Best general decalcifying agent
For small pcs of bones & teeth
5% Formic acid
For small pcs of bones & teeth
For surface decalcification (HCl)
HCl (Von Ebner’s)
For EM, IHC, & enzyme staining
EDTA
Hastens decalcification by removing calcium ions from formic acid-containing decalcifying solutions
Ion exchange resins
Ca2+ are attracted to negative electrode (cathode)
Electrophoresis
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
most ideal, most sensitive, most reliable but very expensive method of measuring extent of decalcification
Removal/neutralization of acid from the tissues after decalcification
Lithium carbonate or sodium bicarbonate solution
Post-Decalcification
Enumerate tissue softeners (4)
4% phenol
Molliflex
2% HCl
1% HCl in 70% alcohol
This tissue softener, tissues might appear swollen & soapy.
Molliflex
Purpose of dehydration
To remove fixative & H2O
Dehydration:
Ascending grades of alcohol (Start: ____%)
Embryonic & animal tissues: __________
65%
30% ETOH
Ratio of dehydrating agent to tissue
10:1
Best dehydrating agent
Ethanol
Dehydrating agent for blood & tissue films
Methanol
Dehydrating agent for Plants & animals
Butanol
Ethanol + methanol
Denatured alcohol
is both a fixative & a dehydrating agent
Acetone
are both dehydrating & clearing agents
Dioxane (Diethylene dioxide)
Tetrahydrofuran (THF)
Dehydration w/ dioxane
Graupner’s method
Weiseberger’s method
Ethylene glycol monoethyl ether
Combustible and toxic
Cellosolve
- both dehydrating agent & indicator of H2O content of 100% ETOH
- (+) H2O = White Blue
Anhydrous CuSO4 (Last ETOH bath)
Additives to dehydrating agents
a. 4% phenol + 95% ETOH = softener
lec.mt 04 |Page | 19
agents
b. Anhydrous CuSO4 (Last ETOH bath)
Methods of determining incomplete dehydration
- Anhydrous CuSO4 method
- Xylene Milky
Most commonly used clearing agent
Xylene (Xylol)
Clearing time:
½ to 1 hr
In clearing,
Block size:
<5mm
Substitute for xylene/benzene
Clearing time: 1-2 hrs
Not carcinogenic
Toxic fumes
Toluene
Toxic to liver
For clearing tough tissues
Does not make tissue translucent but removes alcohol
Chloroform
For urgent biopsies
Minimum shrinkage
Aplastic anemia
Benzene
For double embedding techniques
Methyl salicylate
Methyl benzoate
For CNS, smooth muscles, skin
Cedarwood oil
For CNS, smooth muscles, skin
Cedarwood oil
Minimum shrinkage
Has tendency to become adulterated
Clove oil
Similar to chloroform but is cheaper
Disadvantage: similar to chloroform
CCl4
Clearing agent for delicate tissues, embryos and insects
Aniline oil
Clearing agent for delicate tissues, embryos and insects
Aniline oil
No dealcoholization but make the tissues clearer
Glycerin
Gum syrup
Other clearing agents
Citrus fruits oil
Trichloroethane & petrol
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
Ratio of infiltrating medium to tissue
25:1
Medium used in impregnation/infiltration
a) Paraffin wax
b) Celloidin (collodion)
c) Gelatin = H2O soluble, not a wax
d) Plastic = EM
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
