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Fixation

Fixation
Of the many fixatives that have been proposed, 10% buffered formalin remains the best compromise
under most circumstances. It is inexpensive, the tissue can remain in it for prolonged periods without
deterioration, and it is compatible with most special stains, including immunohistochemical techniques,
[12,13,24] as long as the tissue is placed in fixative shortly (<30 min) after surgical removal, and
overfixation (>24–48 hours) is avoided.[34] ‘Pure’ formalin is a concentrated (40%) solution of the gas
formaldehyde in water. Thus a 10% formalin solution represents a 4% solution of the gas, which is 1.3
molar. If the final dilution is maintained in a range between 8% and 12%, no noticeable differences will
be noted. However, once the concentration of formalin drops below 5%, the quality of the preparation
will suffer. This may happen, unknowingly, in places where ‘pure formalin’ is adulterated by diluting it
with water. Rodriguez-Martinez et al.[26] have devised a simple-to-follow formula for checking the final
dilution of the fixative and correcting it if necessary by measuring the specific gravity of the fluid (Table
2.1). Contrary to popular belief, shrinkage of tissues is minimal in formalin fixation per se.[12,31] Any
shrinkage that occurs (and it may be considerable) is due to the contractile qualities of the specimen,
as supported by the observation that it tends to occur immediately after excision prior to fixation and
that is related to the amount of contractile tissue present.[17] The most obvious example is the external
muscle layer of the gastrointestinal tract. It has been calculated that segments of colorectum shrink by
57% of the in vivo length.[16] Much of this can be avoided by pinning down the specimen on a
corkboard prior to fixation.

Table 2.1 -- Formula for the preparation of 10% formalin on the basis of a solution of
formaldehyde of unknown concentration
DENSITY OF ‘PURE’ PERCENTAGE OF MILLILITERS OF MILLILITERS OF
FORMALIN FORMALDEHYDE FORMALIN WATER
NECESSARY TO PREPARE 10%
FORMALIN
1.090 40.00 10.00 90.00
1.086 39.00 10.25 89.75
1.083 38.00 10.56 89.44
1.080 37.00 10.84 89.16
1.075 35.15 11.37 88.63
1.070 33.30 12.00 88.00
1.065 31.45 12.70 87.30
1.060 29.60 13.35 86.65
1.055 27.75 14.40 85.60
1.050 25.90 15.44 84.56
1.045 24.05 16.62 83.38
1.040 22.20 18.00 82.00
1.035 20.35 19.61 80.39
1.030 18.50 21.65 78.35
1.025 14.80 27.00 73.00
1.020 12.95 30.92 69.08
1.015 11.10 36.10 63.90
1.012 9.25 43.24 56.74
1.010 7.40 54.00 46.00
1.0085 5.55 72.07 27.93
1.0065 4.00 100.00 0.00
Translated from Rodriguez-Martinez HA, Santos-Estrada L, Rosales MM, Cruz-Ortiz H. Formol o
formalina al diez por ciento? Patologia (Mexico) 1971, 9: 233–231.

Zenker fluid (which incorporates mercuric chloride) is an excellent fixative, one of the best that has ever
been devised for light microscopic work, but it is expensive, requires careful disposal of the mercury,
and necessitates meticulous attention to fixation times and washing procedures to remove the
precipitates of mercury. This fixative or sublimate sodium acetate formalin (‘B-5’) is often used for

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biopsies of the kidney, bone marrow, lymph node, and testicle.

Bouin fixative (which contains picric acid) has been especially recommended for testicular biopsies, but
Zenker fluid results in almost identical preparations. Bouin, Zenker, and B-5 are excellent fixatives for
routine work and for most immunohistochemical stains, but the preservation of nucleic acids is very
poor.[30]

Carnoy fixative is a mixture of ethanol, chloroform, and glacial acetic acid.[25] Thus at the same time
that it fixes the tissues, it dissolves most of the fat. This property has been found useful for the
identification of lymph nodes in radical resection specimens.

As various special techniques have been incorporated into the diagnostic pathology armamentarium
and gained in popularity, attempts have been made to develop fixatives that were equally compatible
with routine handling and the performance of the techniques in question. When electron microscopy
was in vogue, a ‘universal fixative’ was proposed, made up of a mixture of 4% commercial
paraformaldehyde and 1% glutaraldehyde in a neutral buffer.[21] At the crest of the
immunohistochemistry wave, fixatives were introduced for the same purpose. With the current
excitement with molecular techniques, it is only natural that efforts are being made to develop fixatives
that would preserve as much as possible the amount and integrity of the nucleic acids present.[30,32,33]
One such proposal calls for 70% ethanol which – in contrast to formalin – is a noncross-linking agent
and brings very little chemical change to the DNA except for a reversible collapse.[15,30] Another
proposed fixative is methacarn, which is a Carnoy solution in which methanol is used in place of
ethanol.[29] While the search for the all-purpose fixative continues,[15] the most sensible approach is to
handle the tissue according to the recommendations for the particular technique being used. Naturally,
this implies that enough material is available for the purpose and that one has thought of it while the
tissue is still fresh. If these conditions have not been met in a particular case (a not uncommon
occurrence), one may console oneself by reflecting on the fact that formalin (a truly remarkable
substance) will still allow for most of these techniques to be carried out, however imperfectly. Regarding
DNA preservation, the best results are obtained with buffered (rather than acid) formalin at 4°C (rather
than at room temperature).[30]

Whenever formalin is used, the volume of fixative should be at least 10 times that of the tissue. The
container should have an opening large enough so that the tissue can be removed easily after it has
been hardened by the fixation. The fixative should surround the specimen on all sides. Large
specimens that float on a fixative should be covered by a thick layer of gauze. In cases of large, flat,
heavy specimens that rest on the bottom of the containers, the gauze should be placed between the
container bottom and the specimen.

The fixation can be carried out at room temperature or, in the case of large specimens, at 4°C (see
following discussion). Tissue should not be frozen once it has been placed in the fixative solution, for a
peculiar ice crystal distortion will result.[28] The freezing point of a 10% formalin solution is -3°C.

The speed of penetration of tissue by formalin is about 1 mm/h. However, tissue penetration is not
equivalent to fixation. It has been pointed out that formalin penetrates tissues rapidly as methylene
glycol but fixes slowly as carbonyl formaldehyde.[30] Therefore, a fixation time of several hours is
needed for most specimens.

An easy and inexpensive way of shortening the fixation time for routine specimens is by submerging
the specimen in a large beaker containing fixative kept at about 60°C and in continuous motor by the
action of a heater–rotor.

Fixation can also be achieved with microwaves, which are defined as electromagnetic waves with a
frequency between 300 MHz and 300 GHz. They can be used by themselves or in combination with
conventional chemical fixation. Microwaving tissue in formalin gives results somewhat inferior to those
obtained by first fixing tissues in formalin for a few hours at room temperature followed by microwave
irradiation for 1–2 minutes at 55°C.[30] The decreased fixation time achievable with microwaves is an
obvious advantage, but this is upset by the artifacts introduced, which include tissue shrinkage and
breakdown of red blood cells. However, these artifacts are very minor if the technique is carried out
carefully, so that no appreciable differences with routinely processed material will be evident.[27] As a
matter of fact, procedures have been described combining chemical fixation and microwave that result
in a 1 hour processing time, a section quality allegedly equivalent to that of routine processing, and
better preservation of RNA.[22,23]

It should also be taken into account that most laboratories use for this purpose household ovens, which
have obvious limitations in terms of reproducibility. Ovens specifically designed for histology use should
offer the standardization and calibration that these kitchen instruments sorely lack, and would

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presumably render the procedure even more satisfactory.

Parenthetically, microwaves are also used in the pathology laboratory for decalcification,[11] processing
for electron microscopy,[14,20] and immunohistochemical staining, including antigen retrieval.[10,18,19]

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