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Changes in fetuses due to formalin preservation.

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CHANGES I N FETUSES DUE TO FORMALIN
PRESERVATION
ADOLF H. SCHULTZ
CARNEGIE
INSTITUTION
OF WASHINGTON,
DEPARTMENT
OF EMBRYOLOGY
Marked changes in size can frequently be noted in a fetus after it
has been in formalin for some time. These may vary in different parts
of the body, after different duration of preservation, in different specimens, and may consist in either shrinkage or an increase in size. Inasmuch as the age of a human fetus is generally computed upon the
sitting height, it is of importance to know how much this measurement
may be altered by the action of the fluid in which the specimen is
preserved. In an anthropological study of the fetal body such knowledge is of great assistance, and until the problem of possible changes
in the proportions of the body has been solved, any comparison between the fresh and preserved material is unsafe.
In this paper it is intended to determine in a preliminary way the
average changes produced by formalin in a few of the more important
measurements, and to detect, if possible, certain factors which influence the relative amount of change. Measurements were made of
the sitting height, head length and head breadth of 18 human fetuses
(ranging in age from the third to the ninth month) in the Carnegie
Collection of embryos, and of the sitting height of 48 fetal pigs in
various stages of development; the procedure being done first upon
the fresh specimens and repeated a t intervals after preservation in 10
per cent formalin (i. e., ten parts of 40 per cent formalin and 90 parts
of distilled water).
In Table I is given the sitting height of the human fetuses, the last
column showing the percentage of loss or gain after 36 weeks of preservation. On an average the sitting height decreases with the duration of preservation, although this may not be the rule in individual
instances. The decrease is most marked in the first week. The
final relative changes amount on an average to a loss of 2.54 per cent
1.8 to - 6.7 per cent.
and are somewhat variable, ranging from
+
35
AMER. JOUR. PHYS ANTHROP.,VOL. 11.. NO. 1
1
1
3
1
8
1
36
17.3
17.8
18.0
18.9
18.9
20.8
21.9
22.5
22.6
23.8
28.6
17.2
17.8
18.0
18.0
19.1
20.1
21.0
22.5
23.7
23.4
29.3
No.
mesh
17.3
17.8
18.2
18.2
19.1
20.4
20.9
22.6
23.5
23.3
128.8
17.2
17.8
18.0
18.2
19.1
20.4
20.8
22.8
23.3
23.1
28.7
+
-
+
17.2 -0.6
17.7 -0.6
17.8 - 1.1
18.1 - 4.2
1.1
19.1
20.2
2.9
20.7 - 5.5
22.5
0
23.0
1.8
23.0 - 3.4
128.2 - 1.4
1939
1924
2085
2063
2070
2049
2041
2089
1934
62
63
69
75
77
76
86
91
78
cm. cm. cm.
mm.
5.2 5.2 5.15 - 4.6 2066 22
9.85 9.85 9.7 - 2.5 1993 34
1973 45
2004 47
2082 59
2100 59
Change
i n Percentage
mm.
22
35
45
50
58
60
54
65
62
65
67
74
77
78
86
92
80
07
1
mm.
21
35
45
49.l
58
61
54
67
64
68
68
74
76
78
86
92
81
05
mm. mm.
21 20.1
34 35
46 46
49.5 49
58 58
61 60
54 54
66.5 67
64 62
68 66
70 69
74 74
77 77
78 78
87 87
92
92
80 82
08 106
3
--
-
-
Weeks In Formalin:
+ 0.8s
0
+
2.6
+ 1.2
+ 1.1
+ 5.1
0
- 1.3
- 6.8
Flnal
Change
in Percentage
+ 2.9
+ 2.2
+- 4.3
1.7
-+- 1.7
1.8
+ 01.5
+ 04.8
~
__
TABLEI1
HEADLENGTHS
OF HUMAN
FETUSES
______~______
Ave. 17.94 17.76'17.75 17.69117.53 - 2.54 Ave.1 65.( 65.4 65.7 66.01 65.i
I
I
-
1964
1939
1924
2085
2063
2070
2049
2041
2089
1934
1922
1-
1 1 1 1 1 1
Fresh
1 I
em. cm.
2066 5.4 5.2
1993 9.95 9.8
NO.
Weeks In Formalin:
Flnal
TABLEI
SITTINGHEIGHT
OF HUMAN
FETUSES
Fresh
3
16.:
30
37
37
51
51
45
50
51
51
55
52
61
59
64
71
59
87
-
18
31
36
44
50
50
44
50
51
54
55
55
59
61
66
71
60
91
mm.
17
31
37
43.
51
51 .E
44
51
53
60
57
56
58
61
69
72
60
91
mm.
16.t
30.1
37
42
52
52
43
52
54
56
59
56
62
64
71
73
63
93
--
mm.
17
30.5
38
43
51
52
44
52
54
59
58.5
56
62
63
71
71
60
92
+
9.8
7.3
7.7
1.6
8.5
10.9
f 2.8
t 6.8
f 6.9
f
f
f
f
f
+ 001.7
4- 13.5
++ 2.0
2.0
- 4.4
+f 4.0
5.9
Flnal
Change
in Percentage
Ave. 51.5 52.f 53.5 54.1 54.4 f 4.83
-
-__
2066
1993
1973
2004
2082
2100
1928
1964
1939
1924
2085
2063
2070
2049
2041
2089
1934
1922
1
8
36
--
Weeks in Formalin:
-
-mm.
mm.
No.
TABLEI11
HEADBREADTHS
OF HUMAN
FETUSE
CHANGES IN FETUSES DUE TO FORMALIN PRESERVATION
37
The absolute size of the specimen seems to have no noticeable influence upon the relative change in the sitting height. It was thought
that the condition of the specimen may be largely responsible for the
amount of change; this, however, was not confirmed by the findings,
as the changes were as marked in specimens in excellent condition as
in those which showed slight maceration.
Table I1 is a compilation of the head lengths of the same series taken
in the fresh state and after preservation. On an average there is a
slight increase in this measurement during preservation, and this
reaches its maximum after the eighth week. I n many individual
cases, however, the changes were rather irregular. The increase in
the head length after 36 weeks of preservation amounts on an average
to 0.88 per cent. Again, this relative change is found to show a con5.1 per cent.
siderable variability, its extremes being - 6.8 and
The absolute size and the condition of the specimen have no apparent
influence upon the relative amount of change in the head length.
In Table I11 is given the head breadth of each human fetus in the
fresh state and a t the different stages of preservation. As in the preceding tables, the last column represents the percentage of final
change. On an average the head breadth increases during preservation, and this is most marked in the first three weeks. The average
increase after 36 weeks in formalin amounts to 4.83 per cent, which is
five and a half times as great as the average relative increase of the
head length. In only one case was there a decrease after preservation, and this amounted to 4.4 per cent. The greatest increase was
13.5 per cent. Like in the preceding measurements, the relative changes
are rather variable, and appear to be independent of the absolute size
or the condition of the specimen.
The much greater average change in the breadth of the head as
compared to its length involves as a necessary consequence a change
in the cephalic index from that noted in the fresh specimen. The
changes in this index after 36 weeks of preservation resulted in an
average gain of 3.93 per cent, the extremes of these relative changes
lying at - 2.7 and
9.7 per cent. In only 17 per cent of the fetuses
was there a slight decrease in the cephalic index due to the preservative; in all the remaining specimens there was a more or less marked
increase. As would be expected from the above, the cephalic index
changes as much in the larger fetuses as in the smaller ones. It may
be stated in this connection that the combination of a decrease in the
sitting height and an increase in the length and breadth of the head
+
+
38
ADOLF H . SCHULTZ
results in the head showing a relative to the body greater size after
preservation.
The pig fetuses were all obtained in an absolutely fresh state, and
were still warm when measured. Only a portion of each litter was
put in formalin immediately, the others being kept either in distilled
water for 3 days or in air for 15 hours before fixation. This was done
in order to determine to what extent, if any, the changes may be influenced by the fact that the fetus was dead some time before abortion,
or had not been fixed immediately following abortion. It may be
mentioned that the human, as well as the pig fetuses, were kept in
large jars with plenty of formalin, so that a loss in strength of the solut,ion through absorption of water from the specimens was negligible.
In the fresh fetuses the sitting height (from the most cranial point
on the forehead to the most caudal point beneath the tail) ranged from
64 to 269 mm., as in the human series, a wide variety of sizes being
represented. The fetuses that were kept in water changed their sitting height very little during this time, and showed either a slight
shrinkage or an increase in length of not more than 1 per cent. The
loss in sitting height after 36 weeks in formalin was practically as
great in these specimens as in those from corresponding litters that
were fixed when fresh, and on an average closely approaches the average relative loss of the whole series. The sitting height of the fetuses
that had been kept in air for 15 hours before fixation diminished in
that time from 2 to 5 per cent. These specimens lose less in their
sitting height during preservation than those fixed when fresh, but
when this decrease is added to that resulting from exposure to the air
the final loss when compared with the sitting height of the fresh specimens is quite as great. These facts tend to prove the conclusion previously drawn from a study of the human fetuses; i. e., that a fetus
in good condition will undergo as much change in formalin as will one
in poor condition, no matter whether the latter may be due to death
in utero or to delay in fixation.
The average relative loss in sitting height of the pig fetuses after
36 hours in formalin is 5.67 per cent. This is more than twice as high
as in the human specimens. These relative changes are also rather
variable, the extremes being 0 and - 10.5 per cent; i . e., the sitting
height of a fresh fetus may either not alter at all or may decrease as
much as one tenth during preservation. Not one of the pig fetuses
showed an increase in the sitting height after being in formalin. As
was found in the human fetuses, that the absolute size does not notice-
CHANGES IN FETUSES DUE TO FORMALIN PRESERVATION
39
ably influence the relative change in the sitting height, no correlation
between the sitting height and its relative change during preservation
could be detected, the larger fetuses changing relatively quite as much
as the smaller ones. It is not impossible that embryos and very small
fetuses, which are not included in this study, may show a somewhat
different behavior in formalin. The averages of the sitting height of
the pig fetuses were 132.6 mm. in fresh condition, 126.8 mm. after
1 day in formalin, 126.6 mm. after 1 week, 125.2 n m . after 8 weeks,
and 124.7 mm. after 36 weeks. From these figures it can be seen that
the greatest and most rapid reduction in length occurs during the first
clay of preservation.
The weight of the pig fetuses, in the fresh state and at intervals
during preservation, was also taken, and here the influence exerted
by the condition of the specimen upon the relative change in formalin
was clearly manifested. The fetuses that were placed in formalin
immediately after they were measured and weighed, still warm, increased in weight during the 36 weeks of preservation on an average
of 6.51 per cent. The variability of the relative changes, however, is
17.7 per cent, without showing
considerable, ranging from - 12.7 to
any correlation with the absolute weight of the specimen. A decrease
in weight in a specimen preserved in a fresh state is exceptional, occurring in only two instances. The average weight of all these specimens was before preservation 139.3 grams, after the f i s t day in formalin 144 grams, after l week 157.3 grams, after 8 weeks 156.9 grams,
and after 36 weeks 151.7 grams. These figures show that the increase
in weight reaches its maximum at the end of the first week, after
which the weight decreases slowly. HrdliEka in a careful study of
the influence of preservatives upon the brain (Brains and brain preservatives, Proc. U. Nat. Mus., 1906, xxx, 245), found the same sharp
initial rise and subsequent gradual decline of weight due to the formalin. According to him the brain also shows marked individual variation of change in weight during preservation, as was noted in the
fetuses.
The pig fetuses that were kept in distilled water for 3 days prior to
fixation showed considerable variability in their weight changes during this time, as well as in formalin. The weight of some increased
during the 3 days in water, while that of others, even from the same
litter, decreased. In ail of this series the weight was less after the
first day in formalin than it was after 3 days in water. After one week
in formalin the weight, without exception, increased again. In the
+
s.
40
ADOLF H. SCHULTZ
subsequent weeks, however, it either increased or decreased, the extremes being respectively
4.2 and - 10.3 per cent of the weight of
the fresh specimen. On an average the relative change consisted in a
loss of 0.36 per cent.
The fetuses kept in air for 15 hours before being fixed lost during
this period from 2 t o 8 per cent of their weight, but became heavier
again after 1 day in formalin. The final changes after 15 hours in air
and 36 weeks in formalin amount on an average to a loss of 1.9 per
cent. These relative changes are rather variable in the individual
cases, and may consist in even a gain in weight. The loss during preservation in the fetuses kept in water or in air before fixation contrasts
with the gain of 6.5 per cent in those preserved while in a perfectly
fresh state.
In summarizing briefly the study of the changes in the fetal body
produced by a solution of 10 per cent formalin, the following results
may be enumerated :
The sitting height decreases during 9 months of preservation on an
average of 2.5 per cent in human, and 5.7 per cent in pig fetuses.
The head length of the human fetus increases during preservation
on an average of 0.9 per cent.
The head breadth of the human fetus increases on an average of
4.8 per cent.
The greatest and most rapid change in these measurements occurs
a t the beginning of preservation.
The cephalic index increases in human fetuses during 9 months of
preservation on an average of 3.9 per cent.
The absolute and relative size of the head of a human fetus increases
during preservation.
The absolute size and the conditionof the fetus have no apparent
influence upon the relative amount of change in the above measurements.
The weight of fetuses increases if they are preserved immediately
after death, but decreases if the specimen is in poor condition when
placed in formalin. The weight of the former will increase most
within the first week of preservation, and will drop again slowly
later on.
The individual variation of change in all the measurements taken,
and also in the weight, is quite marked.
The demonstration of the possibility of changes in proportions and
relative sizes of different parts of the fetal body under the infl-ience
+
CHANGES IN FETUSES DUE TO FORMALIN PRESERVATION
41
of preservatives invites further study in this direction, in order to
enable us to reduce the anthropometric findings on preserved fetuses
to conditions in the fresh material. The disadvantage incident upon
these changes is not attributable alone to 10 per cent formalin; very
probably it is even more pronounced in solutions of greater strength,
as well as in alcohol; it appears, in fact, to be associated with all
known preservative fluids.
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