Fixation and Processing

Freida L. Carson

Fixation Rule #2 is that different fixatives produce their own

morphological patterns. That is an objective fact that
Fixation is the single most influential factor in the long
does not imply good or bad. Whether we like what we
sequence of steps between procurement of the specimen
see is a subjective matter predominantly based on our
and coverslipping the stained slide; nearly any other
individual training. Many chemicals act as fixatives in
step can be reversed to ameliorate a problem. Tissues can
that they denature macromolecules, but few produce
be reverse-processed and then reprocessed if a mistake
“acceptable” results because each creates its own unique
or breakdown occurs in tissue processing. Most stains
pattern of changes visible at the level of the light micro-
can be removed and reapplied to correct problems with
scope. We speak of “formaldehyde patterns” (implying
intensity or specificity. Bubbles under the coverslip can
“good”) versus “alcohol patterns” (“bad”) in describing
be removed simply by removing and resetting the cover-
how a specimen appears under the microscope. Some
slip. In sharp contrast to these examples, errors in fixa-
observers give high preference to mercuric fixatives over
tion are permanent. On the positive side, properly fixed
neutral buffered formalin (NBF) for lymphoid tissues,
tissue is nearly impervious to abuse during tissue pro-
and picric acid for gastric biopsies, because of the extra-
cessing and slide preparation. Understanding the role
sharp images they produce. Defining “good” fixation,
and mechanism of fixation is crucial to producing quali-
then, is difficult because of varying personal preferences.
ty slides and interpreting artifacts. Important aspects can
However, there are well-documented and accepted min-
be grouped under four rules.
imum staining criteria that specify well-defined nuclear
Rule #1 is that fixatives denature macromolecules; ie,
patterns, epithelial cell membranes, and cytoplasmic
fixation changes the shape of large molecules. This rule
staining exhibited by well-fixed tissues.1,2
is the basis for the varied functions of fixation and why
Rule #3 is that fixation is a chemical reaction that is
fixed specimens look the way they do under the micro-
not instantaneous. Its rate is dependent upon the chemi-
scope. Thus:
cal nature of the fixative solution and its temperature.
a. Fixation kills cells because denatured molecules can Closely correlated with this is Rule #4, which says that a
no longer engage in life-supporting chemical reac- fixative must be present for any reaction to occur. This
tions. self-evident notion is so frequently ignored that it war-
b. Fixation prevents autolysis because biological activity rants discussion. Raw specimens are not freely porous
of the specimen’s enzymes is destroyed as their shape objects. Fluids of any sort take time to diffuse into the
is altered. mass. If there are numerous intercellular channels, as in
c. Fixation prevents microbial attack because substrates lymph nodes, movement is faster than if cells are tightly
are no longer recognizable in their new conformation. adherent to one another, but penetration still is not
instantaneous. Most specimens present membrane barri-
d. Fixation firms the tissue (making it easier to gross and
ers that must be crossed each time the fluid moves into
to section) because denatured molecules form new
the next cell. Because membranes have fatty interiors,
intramolecular and intermolecular bonds.
aqueous fixatives penetrate poorly. Alcoholic versions of
e. Fixation changes the tissue’s receptivity to stains and common fixatives (alcoholic formalin, alcoholic zinc for-
histochemical procedures. In most cases the influence malin, and alcoholic glyoxal) are able to penetrate much
is positive because procedures have been optimized faster.
to work with fixed tissue, but prominent negative In most cases fixation increases permeability, but
examples exist (eg, masking of antigenic sites neces- some fixing agents (eg, mercuric salts) create such tight
sary for immunohistochemical staining).

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Histologic Preparations: Common Problems and Their Solutions

Table 1.1. Fixation/Processing Errors on the 2003-2004 HQIP H&E Challenges

% % % % % % % % %
Tissue Fixation Fixation Excessive Poor Cell Formalin or Nuclear Excessive Poor Section
H&E Delayed Incomplete Dehydration Processing Shrinkage Mercury Pigment Bubbling Decalcification Orientation

Breast 2 14 1 9 0 2 9 NA 0

Skin 0 10 1 5 0 1 7 NA 2

Lymph Node 7 30 5 7 3 3 0 NA 0

Lung 7 11 2 3 2 6 5 NA 0

Uterus 11 19 0 3 1 1 27 NA 0

Colon 15 20 0 3 2 1 8 NA 2

Bone Marrow 5 16 1 11 2 1 3 8 0

Liver 8 12 5 6 1 0 8 NA 0

Average 7 17 2 8 1 2 8 NA 0.5

intermolecular bonds that diffusion may be impeded, Processing

and the fixative cannot penetrate all the way into the
If tissue is completely fixed, processing problems are less
specimen. While alcoholic solutions of aldehyde fixa-
likely to occur. However, today’s laboratory practices
tives do not seem to affect permeability adversely, plain
requiring ever faster turnaround times can result in
alcohol used for dehydration certainly does.
incompletely fixed specimens, so proper processing is
Rules #3 and #4 dictate that adequate time be given
imperative to minimize the overall detrimental effect.
for the fixation process (penetration + chemical reac-
The most common problems in processing are caused by
tion). Beyond that, no physical encumbrances should be
processing both biopsy and large tissue specimens
introduced during the handling of specimen. Squeezing
simultaneously on the same processing program. This
with forceps introduces localized artifacts because pene-
leads to overprocessing and excessive dehydration of
tration is hindered at the sites of tissue damage. More
the biopsy tissues and/or underprocessing and incom-
commonly, forcing oversized chunks of tissue into a cas-
plete dehydration of the larger specimens. These tissues
sette inhibits or prevents penetration by any fluid and
should be processed separately and on different sched-
may render processing an exercise in futility. The tissue
ules. As stated in the previous section under “Fixation,”
will remain unfixed, processing fluids will not dehy-
tissues must be cut as thin as possible, with the optimum
drate or clear, and the block will not section. If such a
thickness being no more than 3 mm, or about the thick-
disaster is then trimmed thinner and reprocessed, sec-
ness of a nickel. A problem that has been encountered
tions may be possible, but the tissue will be rotten. There
frequently within the last decade or so is one that leads
is no excuse for overly thick specimens.
to very poor staining of the nucleus. In the United States
this problem is referred to as smudginess or blue halo
effect, and in the United Kingdom as “nuclear melt-
down.” This is most often caused by incomplete dehy-
dration prior to clearing, but using too much heat on the
processor will also cause this same poor staining pattern.

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 Fixation and Processing

Heat should be used only for the paraffin, and that

should be at a temperature just above the melting point
of the paraffin. These problems will be discussed more
completely in the problem-solving section of this chap-
ter. The most common fixation and processing problems
on the 2003–2004 National Society for Histotech-
nology/College of American Pathologists (NSH/CAP)
HistoQIP (HQIP) program hematoxylin and eosin
(H&E) challenges are shown in Table 1.1. Percentages
have been rounded to the nearest whole percent. The
special stain challenges are not included because the
problems are not always as apparent on special stains.

What Should Be Seen in a Well-Fixed, Well-Processed

Specimen Stained With Hematoxylin and Eosin
Figure 1.1. A well-fixed and well-processed section of small intestine is demon-
• Nuclei should show a variety of chromatin patterns,
strated in this image. A variety of chromatin patterns can be seen in the nuclei,
with a crisp blue nuclear membrane. There should not
and the nuclear membranes are crisp and sharply defined. No cell shrinkage is
be any nuclear bubbling, smudginess, or fading.
noted.
• The cell cytoplasm should be well preserved and
should stain well with eosin.
• There should not be any artifactual spaces between
the individual cells.
• There should not be any cell shrinkage.
A well-fixed and well-processed section of small
intestine is demonstrated in Figure 1.1.3 The nuclei show
a variety of chromatin patterns, with crisp nuclear mem-
branes. There is no cell shrinkage; the cell membranes
are sharply defined, and the mucin droplets in the gob-
let cells and the brush border of the epithelium are also
well preserved. This tissue was fixed for 7 hours in zinc-
formalin.
Excellent fixation and processing of lymphoid tissue
is illustrated in Figure 1.2. There is excellent nuclear
detail in the germinal center, with eosinophilic nucleoli
Figure 1.2. Excellent fixation and processing is demonstrated on this section of
seen in several nuclei. Nuclei with a variety of chromatin
patterns can be seen, and no artifactual spaces are seen lymphoid tissue. The nuclear detail is excellent in the germinal center, with a vari-
around the lymphocytes. This tissue was received in ety of chromatin patterns seen. Eosinophilic nucleoli can be seen in several nuclei.
neutral buffered formalin early in the morning, sec-
tioned, put into cassettes, and fixed until 6:00 pm, when
it was put on the processor on an overnight schedule.
The first three stations on the processor are alcoholic for-
malin.
A number of the most common fixation and process-
ing problems are discussed and demonstrated in the fol-
lowing pages. It should be noted that in many instances
fixation and processing problems have multiple and var-
ied outcomes, so it is not always possible to correlate one
problem with only one solution.

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Histologic Preparations: Common Problems and Their Solutions

Figure 1.3. Some of the nuclei are very faded and have almost totally disap- Figure 1.6. Except for the crypts, the epithelial layer has totally disappeared in
peared in this section of intestine, while others are very pyknotic. This is a mani- this very autolysed section of small intestine. This is typical of delayed fixation of
festation of early autolysis or delayed fixation.3 autopsy tissues, and sections of this type should not be used as control tissue.

Problems Encountered With Fixation and Processing

PROBLEM: Fixation Delayed

APPEARANCE: Nuclei may show a loss of chromatin,
blue halo, fading, or complete disappearance (Figures
1.3 and 1.4). There may be cell shrinkage, disruption of
the cytoplasm, and artifactual spaces around cells
(Figure 1.5). If the delay is prolonged, some cells may
completely disappear, such as the epithelial cells in intes-
tinal specimens obtained at autopsy (Figure 1.6).
CAUSES:
• Specimens are obtained long after the blood supply
has been compromised (eg, autopsy).
Figure 1.4. The effects of mild autolysis can be seen in this section of kidney.
• The specimen is not opened so that fixative can come
No nuclei remain in some of the tubules.
in contact with all surfaces (eg, uterus, small intestine,
colon).
• The specimen is not thinly cut so that fixatives can
penetrate more easily (eg, spleen, breast, organ resec-
tions, large tumors).
• Inadequate volume of fixative relative to the amount
of tissue (20:1 minimum).
SOLUTIONS:
• Place specimens in fixative as soon as possible after
the blood supply has been interrupted.
• Open specimens wherever possible. Gastrointestinal
specimens should be opened, pinned to a cork or
paraffin wax board, and placed in fixative. Uterus
specimens should also be opened and placed in fixa-
tive. Lungs can be inflated with fixative by gravity
Figure 1.5. A section of central nervous system tissue demonstrates the results
flow.
of delayed fixation, with marked disruption of the normal morphology.
• Slice specimens, such as spleen, breast, kidney, any
organ resection, or large tumor, into thin slices and
place in fixative.

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 Fixation and Processing

Figure 1.7. A section of an incompletely fixed lymph node shows slight disin- Figure 1.8. A section of fallopian tube demonstrates “smudgy” or “muddy”
tegration in the water bath due to incomplete fixation. This node is completely nuclei that result from incomplete fixation. The nuclear chromatin patterns would
surrounded by fat and was not bisected; thus the fixative had a difficult time be much more apparent after a longer period of fixation.
penetrating.

• Bisect lymph nodes when appropriate and place in

fixative.
• Place formalin container for holding cassettes on stir
plates, and provide agitation to enhance the fixation
and penetration process.
• Ensure that the volume of fixative is 15 to 20 times
that of the tissue.
• Sort cassettes by specimen thickness and size for
appropriate processing schedule.

PROBLEM: Fixation Incomplete

APPEARANCE: Nuclei may be muddy, or smudgy.
Tissue components can separate easily on the flotation Figure 1.9. A section of spleen demonstrating the results of incomplete fixation.
bath during microtomy. Tissue morphology is not well There is a large crack that occurred during flotation on the water bath due to the
maintained (Figures 1.7, 1.8, and 1.9). incomplete fixation. The white pulp also shows the poor cell adhesion due to the
CAUSES: inadequate fixation. This problem will not occur with well-fixed tissue.
• Tissue sections not allowed enough time in fixative.
COMMENTS: Well-grossed sections of routine tissue
• Inadequate amount of fixative relative to tissue vol- should be fixed at least 8 to 12 hours to ensure at least
ume. adequate fixation; however, various authorities have
• Sections grossed too thick for good penetration. stated that anywhere from 48 hours to 1 week is neces-
• Formalin solution is depleted. sary for complete fixation. In a carefully controlled
study, Dapson3 found that artifact-free specimens could
SOLUTIONS: be produced with neutral buffered formalin fixation
• Ensure that enough time is allowed for good fixation only if they were fixed a minimum of 30 to 40 hours, and
(see “Comments”). that profound artifacts were apparent after 7 hours of
• Ensure that the fixative volume is 15 to 20 times the fixative exposure. The use of alcoholic formalin on the
tissue volume. tissue processor will help this somewhat, but enough
• Ensure that the grossed sections are thin, preferably exposure time is still needed between the tissue and
no more than 3 mm thick. the fixative solution. Excellent sections can be obtained
after only 7 hours fixation in zinc formalin (see Figure
• Change formalin solutions frequently throughout the 1.1).3 Thin sections are critical in all instances for good
process to prevent depletion. fixation.