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HSTCYT > FIXATION PAGES 40-60 BANCROFT > Flashcards

FIXATION PAGES 40-60 BANCROFT Flashcards

1
Q

most common fixative used in diagnostic

pathology

A

10% neutral buffered formalin

NBF

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2
Q

a bifunctional aldehyde
which probably combines with the same reactive
groups as formaldehyde

A

Glutaraldehyde

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3
Q

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

A

Osmium tetroxide (OsO4)

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4
Q

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’.

A

mercuric chloride

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5
Q

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.

A

Chromium trioxide

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6
Q

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

A

Compound fixatives

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7
Q

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.

A

Eyes

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8
Q

fixation of this takes 2-6 weeks

A

Brain

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9
Q

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

A

Breast

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10
Q

are typically fixed in NBF

A

Lung biopsies

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11
Q

fixed lungs can be cut within

A

2-6 hours,

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12
Q

many organisms, e.g. Mycobacterium tuberculosis and viruses may be present

A

lymphoreticular

system

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13
Q

Biopsies of these are fixed routinely in NBF.

A

Testis

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14
Q

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.

A

Muscle biopsies

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15
Q

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.

A

Renal biopsies or Renal core biopsies

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16
Q

For routine histology this is is frequently used for initial fixation and for the
first station on tissue processors.

A

10% neutral buffered formalin

NBF

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17
Q

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.

A

10% Neutral buffered formalin (NBF)

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18
Q

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.

A

Carson’s modified Millonig’s phosphate

buffered formalin

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19
Q 
Tap water 900 ml
Formaldehyde (37%) 100 ml
Calcium acetate 20 g
Note
This is a good fixative for preservation of lipids
A

Formal (10% formalin) calcium acetate

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20
Q

Tap water 900 ml
Formaldehyde (37%) 100 ml
Sodium chloride 9 g

A

Formal (10% formalin) saline

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21
Q

Tap water 900 ml
Formaldehyde (37%) 100 ml
Sodium chloride 9 g
Sodium phosphate, dibasic 12 g

A

Formal (10% formalin) buffered saline

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22
Q

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

A

Formal (10% formalin) zinc, unbuffered

23
Q

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.

A

Zenker’s solution

24
Q

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.

A

Helly’s solution

25
Q

Distilled water 50 ml
Mercuric chloride 3.5 g
Absolute ethanol 25 ml

A

Schaudinn’s solution

26
Q 
Absolute ethanol 32 ml
Chloroform 6 ml
Glacial acetic acid 2 ml
Mercuric chloride 8 g
Note
This fixative penetrates rapidly.
A

Ohlmacher’s solution

27
Q 
Absolute ethanol 15 ml
Chloroform 15 ml
Glacial acetic acid 15 ml
Mercuric chloride 8 g
Note
This fixative penetrates rapidly.
A

Carnoy-Lebrun solution

28
Q

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.

A

B5 fixative

29
Q

Potassium dichromate 2.5 g
Sodium sulfate 1 g
Distilled water 100 ml

A

Miller’s or Möller’s solution

30
Q

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.

A

Dichromate fixatives

31
Q

Potassium dichromate 3 g
Distilled water 80 ml
Note
At time of use add 20 ml of formaldehyde (37%).

A

Möller’s or Regaud’s fluid

32
Q 
Potassium dichromate 2.5 g
Sodium sulfate 1 g
Distilled water 100 ml
Note
At time of use add 10 ml of formaldehyde (37%).
A

Orth’s solution

33
Q

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.

A

Picric acid fixatives

34
Q

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.

A

Bouin’s solution

35
Q 
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.
A

Hollande’s solution

36
Q

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.

A

Dehydrant fixatives

37
Q

useful for touch preparations and

smears, especially blood smears

A

Methanol

38
Q

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.

A

Acetone

39
Q

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.

A

Clarke’s solution

40
Q

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.

A

Carnoy’s fixative

41
Q

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.

A

Methacarn’s fixative

42
Q

Compound fixatives with both dehydrant and crosslinking

actions include alcohol-formalin mixtures

A

Dehydrant cross-linking fixatives

43
Q

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:

A

post-fixation (e.g. after 10% NBF)

44
Q

Ethanol (95%) 895 ml

Formaldehyde (37%) 105 ml

A

Alcoholic formalin

45
Q

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.

A

Alcohol-formalin-acetic acid fixative

46
Q

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

A

Alcoholic Bouin’s (Gendre’s solution)

47
Q 
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):
A

Gendre’s solution

48
Q 
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
A

Equivalent to aged alcoholic Bouin’s

49
Q 
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).
A

Alternative alcoholic form of Bouin’s solution

50
Q 
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.
A

Rossman’s solution

51
Q 
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).
A

Davidson’s fixative for eyes

52
Q

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.

A

Fixation for fatty tissue

53
Q

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.

A

Zinc-Tris fixative

HSTCYT (6 decks)

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