FIXATION PAGES 40-60 BANCROFT

Question 1

most common fixative used in diagnostic

pathology

Answer 1

10% neutral buffered formalin

NBF

Question 2

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

Answer 2

Glutaraldehyde

Question 3

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

Answer 3

Osmium tetroxide (OsO4)

Question 4

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’.

Answer 4

mercuric chloride

Question 5

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.

Answer 5

Chromium trioxide

Question 6

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

Answer 6

Compound fixatives

Question 7

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.

Answer 7

Eyes

Question 8

fixation of this takes 2-6 weeks

Answer 8

Brain

Question 9

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

Answer 9

Breast

Question 10

are typically fixed in NBF

Answer 10

Lung biopsies

Question 11

fixed lungs can be cut within

Answer 11

2-6 hours,

Question 12

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

Answer 12

lymphoreticular

system

Question 13

Biopsies of these are fixed routinely in NBF.

Answer 13

Testis

Question 14

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.

Answer 14

Muscle biopsies

Question 15

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.

Answer 15

Renal biopsies or Renal core biopsies

Question 16

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

Answer 16

10% neutral buffered formalin

NBF

Question 17

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.

Answer 17

10% Neutral buffered formalin (NBF)

Question 18

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.

Answer 18

Carson’s modified Millonig’s phosphate

buffered formalin

Question 19

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

Answer 19

Formal (10% formalin) calcium acetate

Question 20

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

Answer 20

Formal (10% formalin) saline

Question 21

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

Answer 21

Formal (10% formalin) buffered saline

Question 22

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

Answer 22

Formal (10% formalin) zinc, unbuffered

Question 23

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.

Answer 23

Zenker’s solution

Question 24

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.

Answer 24

Helly’s solution

Question 25

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

Answer 25

Schaudinn’s solution

Question 26

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

Answer 26

Ohlmacher’s solution

Question 27

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

Answer 27

Carnoy-Lebrun solution

Question 28

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.

Answer 28

B5 fixative

Question 29

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

Answer 29

Miller’s or Möller’s solution

Question 30

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.

Answer 30

Dichromate fixatives

Question 31

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

Answer 31

Möller’s or Regaud’s fluid

Question 32

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

Answer 32

Orth’s solution

Question 33

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.

Answer 33

Picric acid fixatives

Question 34

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.

Answer 34

Bouin’s solution

Question 35

``` 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. ```

Answer 35

Hollande’s solution

Question 36

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.

Answer 36

Dehydrant fixatives

Question 37

useful for touch preparations and | smears, especially blood smears

Answer 37

Methanol

Question 38

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.

Answer 38

Acetone

Question 39

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.

Answer 39

Clarke’s solution

Question 40

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.

Answer 40

Carnoy’s fixative

Question 41

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.

Answer 41

Methacarn’s fixative

Question 42

Compound fixatives with both dehydrant and crosslinking | actions include alcohol-formalin mixtures

Answer 42

Dehydrant cross-linking fixatives

Question 43

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:

Answer 43

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

Question 44

Ethanol (95%) 895 ml | Formaldehyde (37%) 105 ml

Answer 44

Alcoholic formalin

Question 45

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.

Answer 45

Alcohol-formalin-acetic acid fixative

Question 46

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

Answer 46

Alcoholic Bouin’s (Gendre’s solution)

Question 47

``` 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): ```

Answer 47

Gendre’s solution

Question 48

``` 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 ```

Answer 48

Equivalent to aged alcoholic Bouin’s

Question 49

``` 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). ```

Answer 49

Alternative alcoholic form of Bouin’s solution

Question 50

``` 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. ```

Answer 50

Rossman’s solution

Question 51

``` 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). ```

Answer 51

Davidson’s fixative for eyes

Question 52

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.

Answer 52

Fixation for fatty tissue

Question 53

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.

Answer 53

Zinc-Tris fixative