TlIK AMKHU'AN .lOVRNAL OP CLINICAL PATHOLOGY
Vol. -11, No. 2, pp. 15S-102
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Co]>yriKlii yj JW4 by Tin* Williams & Wilkins G>.
Printed in U.S.A.
T H E T I M E OF FIXATION AND THE MITOTIC INDEX
JOSHUA L. EDWARDS, M.D., AND J. THOMAS DONALSON, B.S.
Department of Pathology, University of Florida College of Medicine, Gainesville, Florida
Available information about mitotic
activity, as a function of time, after somatic
death or removal of the tissue is not only
extremely scant, but also inconsistent.
Jolly3 observed that, upon removal of
tissues and placement in vitro, some cells
died while undergoing mitosis, others proceeded slowly through the process of division, and a few in interphase began
division. Mallory4 pointed out that although
mitotic figures are demonstrable in tissues
for as long as 24 hr. before fixation, they are
fewer than would be present in freshly fixed
tissue and that many of these are imperfectly preserved. Evans 2 attempted to
quantize this phenomenon by means of
placing fresh blocks of 3 different surgically
removed neoplasms at 37 C. room temperature, and refrigerated temperature, then
fixing samples of each at hourly intervals up
to 2-1 hr. He found that there was no
material variation in the number of mitotic
figures demonstrable under any of the 3
temperature conditions through 24 hr.
In sharp contrast, Bullough1 found that
the mitotic index of various tissues including
neoplasm left in situ after death, or removed
to non-nutrient mediums, decreased by a
factor of 6 to 20 times over a period of 5
hr. prior to fixation. Certain tissues with
numerous mitotic figures contained none
after 2 hr.
The experiment described in this paper
is the first of a series designed to determine
more precisely the effect of the time of fixing,
as well as other factors, on the incidence and
Received, May 31, 19G3; revision received,
August 27; accepted for publication November 5.
Dr. Edwards is Professor and Chairman of the
Department of Pathology, and Mr. Donalson is
a medical student.
This work was supported by Research Grants
#15GG and E-1542 from the National Institutes of
Health, I'nited States Public Health Service,
Bethesda, Maryland.
character of mitotic activity in removed
tissues.
MATERIALS A N D
METHODS
Two experiments were performed, as
described in the following paragraphs.
Experiment
1—regenerating liver. One
albino female Wistar rat, aged 11 weeks, was
partially hepatectomized with removal of
approximately 50 per cent of the liver. The
remaining liver was permitted to regenerate
for 38 hr., at which time mitotic activity is
intense. At this time, the rat was killed and
each of the 3 remaining liver lobes were
removed aseptically and cut into 6 pieces, 1
to 2 mm. in thickness. The 2 end pieces of
each lobe were immediately fixed hi 10 per
cent buffered formaldehyde. The unfixed
pieces were wrapped separately hi sterile
buffered saline solution moistened gauze, and
placed in a Petri dish at 4.5 C. Pieces from
each lobe were fixed at 4, 8, 12, and 24 hr.,
and 5 nui paraffin sections of these blocks
were stained with hematoxylin and eosin.
Experiment
2—Erhlich ascites tumor
(EAT) cells. A strain of Erhlich ascites
tumor cells was harvested aseptically from
ICR albino mice, and 1.8 X 106 of these
cells were injected into the peritoneal
cavity of 3 mice of the same strain. After 3
days, these mice were sacrificed and the
cells were aseptically recovered from the
peritoneal cavity and pooled. The cells were
washed 3 times and suspended in 10 parts of
saline solution, buffered at pH 7.4. This
suspension was placed in a refrigerator at
4.5 C. Samples were taken immediately and
at 2, 4, 6, 8, 12, and 24 hr., and fixed in
aceto-orcein. Wet preparations were made
and mitotic figures in 100 random fields
were counted, scoring all EAT cells as being
in the interphase, or one of the mitotic
phases. The average number of cells counted
per sample was 910. The mitotic index is
expressed in per cent of the total.
The mean number was determined for
158
Feb. 1964
TIME OF FIXATION AND MITOTIC.INDEX
159
TABLE 1
VARIATION IN INCIDENCE OF M I T O S I S AND 5JTAC.ES' DURING INCUBATION
PRIOR TO F I X A T I O N OF BLOCKS ( R E G E N E R A T I N G
Hours after
Removal of
Tissue
Total Cells
Counted*
0
4
8
12
24
28,288
22,072
41,394
43,350
40,773
LIVER)
Mitotic
Index
SEt
Prophase
SEt
Metaphase
SEt
Anaphase
SEt
Telophase
SEt
per cent
per cent
per cent
per cent
per cent
per cent
per cent
per cent
per cent
per cent
1.37
1.28
0.S1
0.S3
0.33
0.750
O.COO
0.125
0.040
0.005
55. G
55.3
57.0
57.3
48.0
2.9
1.0
2.4
2.2
7.1
12.0
12.S
12.2
17.7
17.7
1.0
1.5
1.2
2.2
1.5
7.35
5.4
5.1
4.9
5.7
0.75
0.90
1.25
1.1
1.5
25.8
20.5
25.6
20.0
' 28.5
2.25
2.5
3.1
0.7
8.0
* E a c h value represents the cells counted in 4 sections, 2 from each of 2 different lobes. All other
values tabulated represent the mean of these 4 counts,
j Standard error of the mean of counts on 4 sections.
each section by means of counting 100
random fields in the section. "The total
number of hepatic parenchymal cells and
the number in mitosis were counted in at
least 800 randomly selected fields in each
section. The total number of hepatic
parenchymal cells was divided into the total
number of cells in mitosis to yield an index
of mitosis expressed in per cent of the
population of hepatic parenchymal cells.
The significance of these observations in
respect to the handling of surgical specimens
is discussed.
It seems reasonable to assume that the
conditions of our and Bullough's experi. ments are comparable in all respects but one,
i.e., temperature; and the slower rate of
decrease in the mitotic index observed by us
is the result of incubation at a lower temperature.
It may be safely assumed that temperature, availability of nutrients, mean duration of mitosis, and vulnerability of individual cells to autolysis are the major
factors determining the incidence of mitosis
in tissues after somatic death or removal of
tissue from the body.
KESULTS AND DISCUSSION
In Table 1 and Figure 1, it may be seen
that the incidence of mitosis in sections of
regenerating liver does not vary significantly
for the first 4 hr., after which it is decreased
to approximately 40 per cent at 8 hr., and
to slightly more than 75 per cent of the
original value at 24 hr.
TABLE 2
VARIATION IN INCIDENCE OF M I T O S I S AND S T A G E S
DURING INCUBATION PRIOR TO F I X A T I O N *
(EHBLICH ASCITES TUMOR C E L L S )
Hours
after
Removal
0
2
4
6
8
12
24
Mitotic
Index
Prophase
Metaphaset
Anaphasef
Telophaset
per cent
per cent
per cent
per cent
per ceil
9.05
5.35
3.29
1.31
0.64
0.34
0.11
6.79
4.55
3.29
1.31
0.64
0.23
0.11
1.6
0.8
0.20
0.23
0.47
0.11
* E a c h value represents per cent of approximately 1000 t o t a l cells counted.
t — Represents none counted in a total of 1000
cells.
The data in Table 2 indicate that, in
contrast, the incidence of mitosis in the
ascites tumor cells in suspension falls
precipitously beginning immediately, being
reduced by a factor of approximately 2 in 2
hr., and by a factor of 10 in 8 hr. The rate
of decrease is exponential during the first
8 hr. (Fig. 2).
The 2 populations are different also with
regard to the occurrence of the different
mitotic phases at different times, and thus
in relation to the progression of mitosis.
Analysis of Table 1 reveals that the
relative proportion of hepatic parenchymal
cells in various mitotic phases remains virtually the same throughout the period of 24
M l TOT I C
I N D E X(%)
loo.
MITOTIC
I N D E X(%)
HOURS
BE FOR
F i X AT I O N
FIG. 1 (upper). Per cent of hepatic parenchymal cells in mitosis in regenerating liver plotted in
relation to hours elapsing prior to fixation of the block of tissue.
FlG. 2 (lower). Per cent of Khrlich ascites colls in mitosis plotted in relation to hours elapsing
after placing in non-nutrient inediuin prior to fixation of the cells.
100
Feb. 1964
TIME OF FIXATION AND MITOTIC INDEX
101
hr. after removal from the body, even though that (2) after this period, the mitotic index
the absolute number of cells in a specific will progressively decline, as a result of the
phase was greatly reduced. In sharp con- reduced rate at which cells enter the mitotic
trast, mitotic phases observed in the cycle, and their continued egress from the
population of Erhlieh ascites tumor cells mitotic cycle'. Thus, in evaluating the
were limited to the prophase after 4 hr. significance of the abundance or dearth of
(Table 2).
mitoses in sections of tissue, the period of
Even in thin blocks of tissue, most cells time elapsing between removal of the
remain in direct contact with tissue fluid specimen and its fixation i s a factor of conhaving some, if minimal, nutrient value. siderable importance.
Under the conditions of this experiment,
The observations and conclusions are. in
many cells in the sections of regenerating full accord with Bullough's demonstration
liver enter, proceed through, and leave the that an extraneous source of energy is
division cycle at the same progressively essential for a cell to enter the mitotic cycle,
decreasing rate throughout 24 hr. after although cells that have begun such division
removal of the tissue. This must be the can complete the mitotic cycle without an
situation, inasmuch as the ratio of cells in extraneous source of energy.
different phases of mitosis remains the same,
even though the mitotic index steadily
SUMMARY •
decreases. If cells in mitosis at any or all
The mitotic index of suspensions of
stages died before completing the division
cycle, the mitotic index would tend to Ehrlich Ascites Tumor Cells in non-nutrient
remain stationary. Inasmuch as the mitotic mediums decreased exponentially by a factor
index is reduced by a factor of 4 in 24 hr., of 10 during 8 hr., whereas in blocks of
one may assume that at least 75 per cent regenerating liver it decreased by a factor of
of the cells that enter mitosis under these only 1.7 during 8 hr., and 4 in 24 hr. Proporconditions complete the mitotic cycle during tions of cells in different mitotic phases
remained constant throughout 24 hr. in
this period.
On the other hand, in the case of the blocks of regenerating liver, whereas in
suspensions of Ehrlich ascites tumor cells, Ehrlich ascites tumor (EAT) cells mitotic
where each cell is in contact with a non- figures are limited to the prophase after 4
nutrient medium from the start, few or no hr.
These observations support the belief that
cells enter division, although it is clear that
cells
having entered the prophase complete
all in any stage of mitosis at the time of
removal will continue to proceed through the cycle independent of outside sources of
the mitotic cycle for a period of at least 12 energy, that extraneous energy is essential
hr. The absence of cells in the later stages of to entering the prophase, and that tissue
mitosis after 4 hr. is ascribed to the rela- fluids present in blocks of fresh tissue protively short duration of these stages. vide nutrients adequate for maintenance of
Inasmuch as no evidence of lysis of cells was mitotic activity at the original level for
found hi the suspensions, the continued approximately 4 hr. prior to fixation.
decrease in mitotic index from 8 to 12 hr.
SUMMARIO IN INTERLINGUA
indicates that cells in the prophase must be
completing the cycle.
Le observationes supporta conclusioncs
On the basis of these observations, it may que es plenmeute de accordo con le demonalso be assumed that (1) surgical specimens stration de Bullough que un fonte extranee
(except for small biopsy specimens), if kept de energia es essential pro que un ccllula
moist at refrigerator temperatures, will not entra in un eyclo mitotic, sed cellulas que
change significantly in the incidence of ha comenciate lor division potc compler le
mitosis for the first 4 hr. after removal; but cyclo mitotic sin fonte extranee de energia.
162
EDWAKDS AM) DONALSON
REFERENCES
1. BULLOUGH, \V. S.: Mitotic activity in the tissues of dead mice, and in tissues kept in
physiological salt, solutions. Exper. Cell
Res., 1: 410-420, l'J50.
2. EVANS, N.: Mitotic figures in malignant
tumors as affected by time before fixation of
Vol. Jfl
tissues. Arch. Path, and Lab. Med., 1:
894-898, 1920.
3. JOLI.Y, M. J.: Sur la duroc de la vie et de la
multiplication des cellules animales en dehors
de l'organisme. Coinpt. rend. Soc. biol.,
55: 12(iG-120S, 1903.
4. MAU.OHY, F. B.: Pathological Technique. Philadelphia. W. B. Saunders, 193S, 3S0 pp.