FIXATION PAGES 40-60 BANCROFT Flashcards
most common fixative used in diagnostic
pathology
10% neutral buffered formalin
NBF
a bifunctional aldehyde
which probably combines with the same reactive
groups as formaldehyde
Glutaraldehyde
toxic solid which is
soluble in water as well as non-polar solvents. It can
react with hydrophilic and hydrophobic sites including
the side chains of proteins where it potentially
can cause cross-linking
Osmium tetroxide (OsO4)
Historically, this was favored for its ability
to enhance the staining properties of tissues, particularly with trichrome stains. However, it is now rarely used in the clinical laboratory due to the health and safety issues of mercury-containing fixatives and, the reduced reliance on ‘special stains’.
mercuric chloride
dissolves in water to produce an acidic solution of chromic acid with a pH of 0.85 and this is a powerful oxidizing agent which produces aldehyde from the 1, 2-diglycol residues of polysaccharides.
Chromium trioxide
are useful for specific tissues, e.g. alcoholic formalin for fixation of some fatty tissues,
such as breast, in which the preservation of the lipid is not important. Additionally, the fixation of gross specimens in alcoholic formalin may aid the identification of lymph nodes embedded in fat
Compound fixatives
may be fixed in NBF, usually for approximately 48 hours; to speed fixation one or two small windows can be cut into the globe, avoiding the retina and iris, after 24 hours.
Eyes
fixation of this takes 2-6 weeks
Brain
Clinical samples should be fixed in 10% NBF for a minimum of 6–8 hours, to a maximum of 72 hours and should be sliced at 5 mm intervals after appropriate gross inspection and margin designation
Breast
are typically fixed in NBF
Lung biopsies
fixed lungs can be cut within
2-6 hours,
many organisms, e.g. Mycobacterium tuberculosis and viruses may be present
lymphoreticular
system
Biopsies of these are fixed routinely in NBF.
Testis
received fresh and a portion is separated
for enzyme histochemistry. The tissue for routine
histological assessment is fixed in NBF and embedded so the fibers of the specimen are viewed in crosssection and longitudinally. After processing this is stained with H&E, a trichrome stain and Congo red if amyloid is suspected.
Muscle biopsies
biopsies should be subdivided into
three and each piece should contain an adequate number of glomeruli, e.g. 6-10 for light microscopy, 1-2 for electron microscopy and 3-6 for immunofluorescence. Each portion is then preserved depending upon the method to be used for subsequent analysis:
• NBF for routine histology.
• Buffered glutaraldehyde at pH 7.3 for ultrastructural analysis.
• Snap frozen in isopentane and liquid nitrogen for immunofluorescence examination.
Renal biopsies or Renal core biopsies
For routine histology this is is frequently used for initial fixation and for the
first station on tissue processors.
10% neutral buffered formalin
NBF
Tap water 900 ml
Formaldehyde (37%) 100 ml
Sodium phosphate, monobasic, monohydrate 4 g
Sodium phosphate, dibasic, anhydrous 6.5 g
Note
The pH should be 7.2–7.4. NBF purchased from
commercial companies may vary widely in its aldehyde content, and commercial companies may add material such as methanol (Fox et al. 1985) or other agents to stabilize NBF preparations.
10% Neutral buffered formalin (NBF)
Formaldehyde (37–40%) 10 ml
Tap water 90 ml
Sodium phosphate, monobasic 1.86 g
Sodium hydroxide 0.42 g
Note:
Deionized water can be used if tap water is hard and/
or contains solids. The pH should be 7.2–7.4. This
formula is reported to be better for ultrastructural
preservation than NBF.
Carson’s modified Millonig’s phosphate
buffered formalin
Tap water 900 ml Formaldehyde (37%) 100 ml Calcium acetate 20 g Note This is a good fixative for preservation of lipids
Formal (10% formalin) calcium acetate
Tap water 900 ml
Formaldehyde (37%) 100 ml
Sodium chloride 9 g
Formal (10% formalin) saline
Tap water 900 ml
Formaldehyde (37%) 100 ml
Sodium chloride 9 g
Sodium phosphate, dibasic 12 g
Formal (10% formalin) buffered saline
Tap water 900 ml
Formaldehyde (37%) 100 ml
Sodium chloride 4.5 g
Zinc chloride or (zinc sulfate) 1.6 g (or 3.6 g)
Note
This is reported to be an excellent fixative for
immunohistochemistry
Formal (10% formalin) zinc, unbuffered
Distilled water 250 ml
Mercuric chloride 12.5 g
Potassium dichromate 6.3 g
Sodium sulfate 2.5 g
Note
Just before use, add 5 ml of glacial acetic acid to
95 ml of above solution. This is a good fixative for
bloody (congested) specimens and trichrome stains.
Zenker’s solution
Distilled water 250 ml
Mercuric chloride 12.5 g
Potassium dichromate 6.3 g
Sodium sulfate 2.5 g
Note
Just before use add 5 ml of 37% formaldehyde to
95 ml of above solution. It is excellent for bone
marrow extramedullary hematopoiesis and intercalated
discs.
Helly’s solution
Distilled water 50 ml
Mercuric chloride 3.5 g
Absolute ethanol 25 ml
Schaudinn’s solution
Absolute ethanol 32 ml Chloroform 6 ml Glacial acetic acid 2 ml Mercuric chloride 8 g Note This fixative penetrates rapidly.
Ohlmacher’s solution
Absolute ethanol 15 ml Chloroform 15 ml Glacial acetic acid 15 ml Mercuric chloride 8 g Note This fixative penetrates rapidly.
Carnoy-Lebrun solution
Mercuric chloride 12 g
Sodium acetate 2.5 g
Distilled water 200 ml
Note
Add 2 ml of formaldehyde (37%) to 20 ml of above
solution just before use. It is frequently used for
bone marrow, lymph nodes, spleen, and other hematopoietic
tissues.
B5 fixative
Potassium dichromate 2.5 g
Sodium sulfate 1 g
Distilled water 100 ml
Miller’s or Möller’s solution
Time of fixation (24 hours) is
critical for this type of fixatives. Tissue should be
washed after fixation and transferred to 70% ethanol.
Failure to wash the tissue after fixation may cause
pigments to be precipitated. Extensive shrinkage may
occur when tissues are processed to paraffin blocks.
Dichromate fixatives
Potassium dichromate 3 g
Distilled water 80 ml
Note
At time of use add 20 ml of formaldehyde (37%).
Möller’s or Regaud’s fluid
Potassium dichromate 2.5 g Sodium sulfate 1 g Distilled water 100 ml Note At time of use add 10 ml of formaldehyde (37%).
Orth’s solution
require a saturated aqueous
solution of picric acid. Aqueous picric acid 2.1%
will produce a saturated solution, and 5% picric acid
a saturated solution in absolute ethanol.
Picric acid fixatives
Saturated aqueous solution of picric acid 1500 ml
Formaldehyde (37%) 500 ml
Glacial acetic acid 100 ml
Note
Fixation time should not be more than 5 days.
Bouin’s solution
Distilled water 1000 ml Formaldehyde (37%) 100 ml Acetic acid 15 ml Picric acid 40 g Copper acetate 25 g Note A useful fixative for gastrointestinal biopsies and endocrine tissue. Specimens are washed before exposure to NBF.
Hollande’s solution
act to remove free and bound
water, causing a change to the tertiary structure of
proteins so that they precipitate, leaving the nucleic
acids relatively unchanged. Ultrastructure is also
destroyed due to the extraction of lipids and excessive
shrinking of tissue components may occur after
more than 3–4 hours of fixation. Each of the following
dehydrant fixatives can be modified by adding
other chemicals to produce specific effects.
• Absolute ethanol
• 95% ethanol
• 70-95% ethanol
• Methanol
• Acetone.
Dehydrant fixatives
useful for touch preparations and
smears, especially blood smears
Methanol
fixation should be short (1 hour) at 4°C on
small specimens. this also produces extensive shrinkage
and hardening, and results in microscopic distortion.
It is used for immunohistochemistry, enzyme
studies and in the detection of rabies.
Acetone
Absolute ethanol 60 ml
Glacial acetic acid 20 ml
Note
This solution produces good general histological results
for H&E stains. It has the advantage of preserving
nucleic acids whilst lipids are extracted. A short
fixation is recommended and tissues are transferred
to 95% ethanol following fixation.
Clarke’s solution
Acetic acid 10 ml
Absolute ethanol 60 ml
Chloroform 30 ml
Note
Carnoy’s fixative is useful for RNA stains, e.g. methyl
green pyronine and for glycogen preservation. It shrinks
and hardens tissues, hemolyzing red blood cells. It may
destroy the staining of acid-fast bacilli. It is useful in
cytology to clear heavily blood-stained specimens.
Carnoy’s fixative
Acetic acid 10 ml
100% methanol 60 ml
Chloroform 30 ml
Note
Causes less hardening and less shrinkage than
Carnoy’s, but with the same pattern of staining.
Methacarn’s fixative
Compound fixatives with both dehydrant and crosslinking
actions include alcohol-formalin mixtures
Dehydrant cross-linking fixatives
Absolute ethanol 650 ml
Distilled water 250 ml
Formaldehyde (37%) 100 ml
Note
Carson recommended this formula because it was
noted that the concentration of ethanol should
be less than 70% to prevent the precipitation of
phosphates in 10% NBF saturated tissues. For initial
fixation the following formulas can be used:
post-fixation (e.g. after 10% NBF)
Ethanol (95%) 895 ml
Formaldehyde (37%) 105 ml
Alcoholic formalin
Ethanol (95%) 85 ml
Formaldehyde (37%) 10 ml
Glacial acetic acid 5 ml
Note
Methanol may be substituted for ethanol with care.
Similarly, various mixtures of ethanol, acetic acid and
formalin may be used.
Alcohol-formalin-acetic acid fixative
This fixative is similar to Bouin’s except it is less
aqueous and there is better carbohydrate retention,
particularly glycogen. Fixation should be
between 4 hours and overnight, followed by washing
in 70% ethanol and then several washes in 95%
ethanol. This is the one alcoholic fixative which
improves with aging
Alcoholic Bouin’s (Gendre’s solution)
95% ethanol saturated with picric acid (5 g per 100 ml) 800 ml Formaldehyde (37%) 150 ml Glacial acetic acid 50 ml Note To increase the effectiveness of alcoholic Bouin’s, if there is no time for aging, the following formula has been recommended (Gregory, 1980):
Gendre’s solution
Picric acid 0.5 g Formaldehyde (37%) 15 ml 95% ethanol 25 ml Glacial acetic acid 5 ml Ethyl acetate 25 ml Tap water 30 ml
Equivalent to aged alcoholic Bouin’s
Bouin’s solution (page 58) 75 ml 95% ethanol 25 ml Note This solution is excellent for lymph nodes (24 hours) and for fatty tissue (48 hours).
Alternative alcoholic form of Bouin’s solution
Tap water 10 ml Formaldehyde (37%) 10 ml Absolute ethanol 80 ml Lead nitrate 8 g Note Fix for 24 hours at room temperature. This is a good fixative for connective tissue mucins and umbilical cord.
Rossman’s solution
95% ethyl alcohol 300 ml Formaldehyde (37%) 200 ml Glacial acetic acid 100 ml Distilled water 300 ml Note This fixative gives good nuclear details and may also be used to fix hematopoietic organs as well as rodent testes (fix for 24 hours).
Davidson’s fixative for eyes
Bouin’s solution (page 58) 75 ml
95% ethanol 25 ml
Note
May require up to 48 hours for good sections of
lipomas or well-differentiated liposarcomas.
Fixation for fatty tissue
0.1M Tris buffer, pH 7.4 1000 ml
Calcium acetate 0.5 g
Zinc acetate 5.0 g
Zinc chloride 5.0 g
Notes
Mix to dissolve. The final pH will be approximately
6.8. Do not readjust the pH, as this will cause the
zinc to come out of solution. Fix for 24–48 hours.
This fixative is useful for the preservation of fixationsensitive
antigens in paraffin-embedded sections
(Beckstead, 1994). It is a recommended fixative for
the detection of CD4 and CD8 on murine tissue.
Zinc-Tris fixative
