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.