CORRESPONDENCE AND CORRECTIONS
Effect of Fixatives and Fixation
Times on Tissues
To the Editor:—Greer and colleagues
compared the effects of various fixatives
and fixation times on the polymerase
chain reaction-mediated amplification of
DNA obtained from paraffin-embedded
tissues.1 They reported better results in
tissues fixed in formaldehyde than those
fixed in coagulating fixatives. Their results
differ from those of previous workers who
reported that alcohol-based fixatives are
better than formaldehyde for the preservation of nucleic acids.2"4 Furthermore,
others have reported that the quality of
extractable DNA and RNA is inversely
proportional to the duration of formaldehyde fixation.2,5"7
It is noteworthy that Greer and colleagues found that the best amplification
was observed on samples "fixed" in formaldehyde for only 1 or 4 hours.
The disparity of Greer's findings with
those of previous researchers may be explained by critically considering the experimental design used by Greer and colleagues, in light of what is known about
the chemistry of formaldehyde fixation.
When formaldehyde is dissolved in water,
it quickly hydrates to methylene glycol, a
nonfixative, and only a small fraction
(0.1%) of the dissolved formaldehyde remains as free formaldehyde, available to
bind with aminoacids. As the scant available free aldehyde binds with tissue components, room is freed for additional
methylene glycol molecules to dehydrate
and release free formaldehyde, which in
turn binds to more tissue components,
and so on. This reaction, known to chemists as a "clock reaction," is slow and requires 24 to 48 hours to be complete.8
It follows therefore, that the tissues exposed to formaldehyde for 1 to 4 hours in
Greer's study were incompletely fixed by
the aldehyde. Fixation, rather, was largely
provided by the alcohol used after the brief
formalin exposure.
It is thus possible to reconcile the apparent difference between the results of
Greer and co-workers and those of previous workers. In their study Greer and
colleagues compared tissues exposed to
formaldehyde for periods of 1, 4, and 24
hours, with tissues fixed for the same period of time in other fixatives, including
alcohol-based fixatives. The best results
were obtained in tissues exposed to formalin for 1 to 4 hours and some deterioration was already evident at 24 hours.
Thus I believe that Greer's best results
were obtained in tissues essentially fixed
in alcohol, and thus are in agreement with
those of previous workers who compared
formaldehyde with alcohol fixation.2"4
Parenthetically, ethanol alone should have
been the appropriate control in the Greer
study.
Additional support for my interpretation is provided by Greer and her colleagues (note added in print), when she
states "preliminary studies suggest that
OmniFix (American Histology Reagent
Co., Stockton, CA) may be superior to
10% neutral buffered formalin for subsequent amplification of paraffin-embedded
tissues," because OmniFix does not contain aldehydes and is essentially an alcohol-based fixative.
In addition, it is important to emphasize that fixation times of 1 to 4 hours in
formaldehyde are unrealistic and practically never used in the diagnostic histology
laboratory. Furthermore, it is not uncommon to exceed the 24-hour fixation time
(weekends, large specimens, and so on).
Such prolonged exposures to formaldehyde are not only harmful to antigenic
sites9 but may also yield poorly preserved
DNA and RNA.2"7 In contrast, prolonged
immersion of tissues in ethanol followed
by paraffin embedding does not seem to
noticeably damage proteins, DNA, and
RNA.2-3,9 Thus, plain ethanol may be a
more appropriate fixative for DNA/RNA
amplification studies.
I agree with Greer and associates that
"a thorough review of the documentation
regarding the processing of each sample
when designing large retrospective studies" would be most useful. Unfortunately,
most pathology laboratories have not observed strict control over fixation time,
and few laboratories would be in a position to produce adequate documentation
of this nature. Thus, Greer and co-workers' recommendation that a pilot study to
evaluate the preservation of DNA on archival material be carried out before embarking on large retrospective studies is
sound and practical advice.
For prospective studies, however, cur144
rent evidence suggests that no single fixative is ideal for the preservation of a wide
range of molecules. It would then appear
prudent, until a hypothetical universal
fixative is developed, to fix tissues routinely in formaldehyde for periods not exceeding 24 hours, with an aliquot fixed in
a mild coagulating fixative such as absolute ethanol, or OmniFix. Even better, a
representative sample should be stored
frozen at —70°C. This approach, which is
routine in our laboratory, ensures the best
possible preservation of morphologic detail, antigens, and nucleic acids.
HECTOR BATTIFORA,
M.D.
City of Hope National Medical Center
Duarte, California
REFERENCES
1. Greer CE, Peterson SL, Kiviat NB, Manos
M. PCR amplification from paraffinembedded tissues. Effects of fixative and
fixation time. Am J Clin Pathol 1991 ;95:
117-124.
2. Ben-Ezra J, Johnson D, Rossi J, Wu A.
Effect of fixation on the amplification
of nucleic acids from paraffin embedded
material by the polymerase chain reaction. Lab Invest 1990;62:9(abstr).
3. Bramwell NH, Burns BF. The effects of
fixative type and fixation time on the
quantity and quality of extractable DNA
for hybridization studies on lymphoid
tissue. Exp Hematol 1988;16:730-732.
4. Jackson DP, Lewis FA, Taylor GR, Boylston AW, Quirke P. Tissue extraction of
DNA and RNA and analysis by the
polymerase chain reaction. J Clin Pathol
1990;43:499-504.
5. Barton Rogers B, Alpert LC, Hine EAS,
Buffone GJ. Analysis of DNA in fresh
andfixedtissue by the polymerase chain
reaction. Am J Pathol 1990; 136:541548.
6. Dubeau L, Chandler LA, Gralow JR, Nichols PW, Jones PA. Southern blot
analysis of DNA extracted from formalin-fixed pathology specimens. Cancer Res 1986;46:2964-2969.
7. Warford A, Pringle JH, Hay J, Henderson
SD, Lauder I. Southern blot analysis of
DNA extracted from formol-saline fixed
and paraffin embedded tissue. J Pathol
1988;154:313-320.
8. Fox CH, Johnson FB, Whiting J, Roller
PP. Formaldehydefixation.J Histochem
Cytochem 1985;33:845-853.
9. Battifora H, Kopinski M. The influence of
protease digestion and duration of fixation on the immunostaining of keratins. A comparison of formalin and
ethanolfixation.J Histochem Cytochem
1986;34:1095-1100.