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Amniotic fluid studies in Rh-sensitized women
Amniotic fluid studies in Rh-sensitized women A preliminary report
CLAYTON T. BEECHAM, M.D. LYNDALL MOLTHAN, M.D. JOSEPH BOUTWELL, M.D. CHARLES W. ROHRBECK, M.D. Philadelphia, Pennsylvania
a fetus with hemolytic disease showed bilirubin in the liquor, causing a fairly consistent deviation in the curve. Walker, 4 following the work of Bevis, was able to forecast accurately the fetal condition in 70.3 per cent of his cases.
0 N E hardly need enumerate the difficulties and pitfalls attached to the prognosis for an infant of an Rh-sensitized woman. To assay potential danger to the fetus of a sensitized mother calls for a close look at the past newborn history, the titer levels, their behavior during pregnancy, and the husband's genotype. These factors along with clinical judgment have determined our course of action in the management of sensitized pregnant women. As everyone knows, these factors when considered individually may lead to errors in prognosis. Collectively they are more accurate but too often fall short of giving us the exact answer we want, i.e., when to deliver the baby. In our search for a solution we have turned to the liquor amnii as a likely source of information. Bevis 1 approached the same problem by the use of spectrophotometry to measure bilirubin, oxyhemoglobin, and methemalbumin in the fluid. After obtaining amniotic fluid by uterine paracentesis, he examined a spectral absorption curve, plotting optical density against wave length. Revis interpreted his results to indicate that
Studies and methods Eleven pregnant patients, 8 sensitized and 3 Rh-negative controls, were the basis for our study. We have investigated the Rh-D antibody content, levels of bilirubin (direct and total), iron, hemoglobin, and leucine aminopeptidase in amniotic fluid. Fluid was obtained by aminocentesis. In performing aminocentesis, fetal presentation and position were first determined by palpation. The depth to which the needle must penetrate to enter the amniotic sac was estimated. Conventional soap, ether, and alcohol skin preparation was employed as a preliminary to draping. The fetus was fixed by pressure from one hand while an infiltration of 1 per cent procaine solution into the layers of the abdominal wall was carried out. An 18 gauge spinal needle was then inserted into the amniotic sac and 20 c.c. of fluid withdrawn. A "bloody tap" rendered the specimen valueless for this study. Twenty cubic centimeters of venous blood was drawn at the same time for comparison with the amniotic fluid. Technique used for Rh-D antibody determination on aminotic fluid was basically
From the Departments of Obstetrics and Gynecology, Hematology, and Biochemistry, Temple University Medical Center. Presented at the Seventy-second Annual Meeting of the American Association of Obstetricians and Gynecologists, Hot Springs, Virginia, Se(lt. 7-9, 1961.
1053
1054
Beecham et al.
April 15, 1962
.\m.
J. Obst. & Gynec.
Table I. Clinical data I
No.I
Patient history Saline M. S. 5-1-2, white, 1 intra1:128 uterine death from hemolytic disease
L.A.P. 1:128
1:512
Negative Negative 1:32
0.23 U./ml.
:2
M. B. 4-2-0, white, 1 intrauterine death, 1 neonatal death, both hemolytic disease
0
1:64
1:512
Negative Negative 1: lfi
0.36
3.
C. B. 5-3-0, Negro, 1 intrauterine death, ? hemolytic disease
1: 1
1: 16
1:256
Negative Negative 1:4
0.55
4
L. S. 4-3-0, white, 1 hemolytic disease, 2 transfusions needed
1: 1
1:512
1:128
Negative Negative Negative
0. 76
5
R. B., 4-2-1, Negro, 1 hemolytic disease, 3 transfusions needed
1: 8
1:64
1:512
Negative Negative 1:4
5.0
6
T. C. 5-4-0, Negro, 1 jaundiced, 0 possibly transfused elsewhere
1:64
1:128
Negative Negative 1:8
1.1
7
A. B. 7-6-0, Negro, 1 kernicterus, died, pneumonia
0
1: 128
1:128
Negative Negative 1:2
0.44
8
F. P. 7-6, white, 1 neonatal death, ? jaundiced (elsewhere)
0
0
1: 16
Negative Negative Negative
0.85
Negative Negative Negative Negative Negative Negative
0.32
Controls N.D., negative
J.,
2
D.
negative
Negative Negative Negative Negative Negative Negative
0.37
3
B. D., negative
Negative Negative Negative Negative Negative Negative
0.9
-~---~-------
~--------------
the same as that for antibody determination on serum. No modifications were necessary. First, amniotic fluid was centrifuged at 2,000 to 3,000 rpm for 3 to 4 minutes and the supernatant removed for study. If the titer could not be carried out within 48 hours, the fluid was frozen at 2° F. Second, the fluid was titered against Group 0, Rh-D positive cells and Group 0, Rh-D negative cells (control) simultaneously. The patient's serum was also titered simultaneously against the same Rh-positive cells. Third, titrations were carried out by means of the 10 tube, twofold dilution technique, changing pipettes with each dilution. Titrations were carried out for the detection of saline-active antibody, albumin-active antibody, and indirect Coombs-active antibody.
Results were usually clear-cut and specific; however, an occasional amniotic fluid specimen would show slight nonspecific agglutination against the control cells. Bilirubin in amniotic fluid and serum was measured by a method developed by one of us (J. H. B.), as yet unpublished. Instead of methyl alcohol, as in the DucciWatson technique, acetamide is employed to develop the total bilirubin reaction. Iron in amniotic fluid and serum was measured by a modification of the method described by Ramsey. 2 Hemoglobin in amniotic fluid and serum was measured by determining the absorbance at 540 M.U. and 560 M.U. The difference was proportional to the hemoglobin content. Leucine aminopeptidase was measured by a slight modification of the Goldbarg and Rutenberg 3 method.
Volume 8.3 Number 8
Amniotic fluid studies in Rh-sensitized women
1055
Amniotic fluid Iron 19.1 mg. %
Hemoglobin 6.0 mg. %
l
Bilirubin
Newborn
0.21 mg.% direct 0.24 mg.% total
4-plus Coombs; Hgb. 8.1 Gm.; hematocrit 30; bilirubin 9.8 to 12.4 pre-exchange; 5 transfusions
17.6
5.96
0.07 direct 0.10 total
4-plus Coombs; Hgb. 11.0 Gm.; bilirubin 7 at birth; 1 transfusion
22.7
6.0
0 0
direct total
4-plus Coombs; Hgb. 15.1 Gm.; bilirubin 1.96 at birth, 4.8 at 10 hours, 20.3 at 72 hours; I exchange transfusion, 1 transfusion of packed cells
22.7
2.6
0.12 direct 0.08 total
4-plus Coombs; Hgb. 13 Gm.; bilirubin 2.1; one exchange transfusion
42.6
2.56
0 0
direct total
4-plus Coombs; Hgb. 16.5 Gm.; bilirubin 3.1; no exchange transfusion
26.8
10.2
0 0
direct total
Weakly positive Coombs; Hgb. 16.7 Gm.; bilirubin 1.2, no transfusion
17.9
7.7
0.09 direct 0.09 total
3-plus Coombs; Hgb. 16.1 Gm.; bilirubin 2.5; no transfusion
46.0
5.1
0.06 direct 0.09 total
Coombs negative; Hgb. 17.3 Gm.; hematocrit 53; bilirubin 2.7; no transfusion
24.2
9.4
0
direct and total
Coombs negative
29.2
3.4
1.2
direct and total
Coombs negative
"""
"O.:J
U.U'T
.
~~.:1
direct 0.018 total
One unit of leucine aminopeptidase activity is defined as that amount liberating one micromole of P-naphthylamide per hour per milliliter of the specimen analyzed.
Coon-1bs weakly positive
most part did. The indirect Coombs test was positive in 6 of the 8. Concentrations, where positive, were from 1:2 to 1:32. The highest concentration was in the case of the most senous1y arrecieu newoorn wnere Je:' exchange transfusions were needed. Further, the test was positive in two instances where the babies did not need transfusions and negative where another did. The amniotic fluid in none of the 3 controls reacted to Coombs (antiglobulin) test. The Coombs test was positive in serum of 7 infants of sensitized mothers and negative in 2 controls. One hal£4 of the newborns required exchange transfusions. Iron found in the liquor amnii of the sensitized women varied from 17.6 to 46 mg. per cent. Bilirubin when present was little more than a trace. It was also found .
Results
Aminocentesis was relatively easy to perform between the thirtieth and the thirtyfourth week of gestation. Clear fluid was obtained in eleven of thirteen attempts. The procedure was more difficult after the thirtysixth week. We successfully obtained fluid during the last month of pregnancy in only one patient of the three attempted in this stage of pregnancy. There were no apparent complications resulting from aminocentesis. The amniotic fluid of the 8 sensitized women failed to react to saline or albuminsuspended cells, although their sera for the
•
1
1¥
'
'I
1
,
1056
.'\p!il J'J, 1~6~ \m. ]. Obst. & Gynec.
Beecham et al.
in 2 of the controls. Hemoglobin varied from 2.6 to 10.2 mg. per cent and leucine aminopeptidase activity 5.0 to 0.23 unit per milliliter. Four of the 8 sensitized patients were delivered between 36 and 37Yz weeks of gestation. In these, all factors indicated that the babies were affected and all 4 did need transfusions. The other 4 sensitized patients had equivocal histories of hemolytic disease in the newborn, heterozygous husbands, and/or titers that either did not change or dropped during pregnancy. The latter group were allowed to go to term and the newborn infants (except in one case) did show evidence of mild hemolytic disease. Nonf' required transfusions. The entire group of 8 babies left the hospital in good condition. Oxytocin infusion with rupture of the membranes at an appropriate time successfully induced labor in 2 of 4 patients. Cesarean section was employed in 2 failed inductions. The 3 Rh-negative controls had no antibodies in either their sera or liquor amnii. The fluid revealed leucine aminopeptidase 0.32 to 0.9 units per milliliter, iron 22.9 to 29.2 mg. per cent and hemolysis 3.4 to 9.4 mg. per cent. All infants in the control group were free of hemolytic disease.
no conclusions beyond the point that the enzyme is present in amniotic fluid. Normally, a 24 hour urine specimen contains 0.06 to 0.2 mg. of iron. Our figures of 17.9 to 49 mg. per cent in amniotic fluid covered a wide range. If we assume these values represent the total fetal excretory iron up to the time of aminocentesis, we are failing to take into consideration the amount of fluid ingested by the growing fetus. Correlating the iron level with other data and the status of the newborn failed to yield significant prognostic information. One would assume that hemoglobin levels would be significantly higher in fluid surrounding- an affected baby. Such was not the case, for the controls had more hemoglobin than the sensitized group. In the light of Bevis' work and the nature of hemolytic disease in the newborn, we expected bilirubin determinations to offer prognostic information. This was not borne out by chemical analysis and, further, we cannot explain the high bilirubin values in 2 of the controls. The Coombs test was positive in the amniotic fluid of 6 patients. However, the inconsistencies made it unreliable as an index of hemolytic disease in the newborn. Conclusions
Comment
There have been few studies on the constituents of amniotic fluid. Thus, we undertook this preliminary study with a certain degree of optimism. Leucine aminopeptidase determinations, it was thought, might offer information of prognostic value since hematologic diseases where a hemolytic component is known to exist result in elevated leucine aminopeptidase activity in both serum and urine. Normal 24 hour urine contains 50 to 350 units (mean 190 units) of leucine aminopeptidase activity. The values obtained in these determinations allow us
Determinations of antibody titers, leucine aminopeptidase activity, iron, hemolysis, and bilirubin levels in amniotic fluid have not assisted us in predicting the severity of hemolytic disease in the newborn. Addendum. This paper was submitted for publication before a significant contribution was brought to our attention. E. V. Mackay, writing in the new Australian and New Zealand Journal of Obstetrics and Gynaecology 1: 78, 1961, does not share our pessimism. Using spectrophotometric methods in a large series ( 223 patients) Mackay feels that his studies are an aid in prognosticating affected infants.
REFERENCES
1. Bevis, D. C. A.: J. Obst. & Gynaec. Brit. Emp. 63: 68, 1956. 2. Ramsey, W. M.: Clin. chem. acta 2: 214, 1957.
3. Goldbarg, J. A., and Rutenberg, A. M.: Cancer 11: 283, 1958. I. Walker, A. H. C.: Brit. M. J. 2: 376, 1957.
Volume 83 Number 8
Discussion DR. H. CLOsE HESSELTINE, Chicago, Illinois. Dr. Beecham and his colleagues deserve our commendation, because too frequently people are inclined to minimize negative findings. 1'-Jeg-
ative findings are just as much a part of our education as are positive ones. One may have objections to the method used to obtain amniotic fluid. My colleagues and I have had some experience in this direction when determinations were made on the transfer of antibiotics to the fetus and amniotic fluid from the mother and also transfer from the amniotic sac back to the mother. The insertion of the needle may present a possible risk of damage to the fetus. Conceivably, vital structures could bt" inadvertently or unintentionally injured. However, by selecting the triangle formed by the arms, thighs, and ventral aspect of the fetus, it seems that there should be less likelihood of injury to the fetus.
The prospect of contamination of the amniotic cavity is inescapable. It is unlikely with proper care that a lethal complication will occur. Even so, some years back, a mother and
Amniotic fluid studies m Rh-sensitized women
1057
her unborn child died from infection. Another complication is the laceration of a blood vessel on the placental surface or of partial separation of the placenta. Thus, the fetus might face exsanguination or suffocation. The a\\rareness of these risks encourages greater caution. With sufficient reduction of harm, moral justification increases for educational inquiry. It seems that these gentlemen have satisfied scientific curiosity and documented the study, and it is assumed that this study is probably completed. DR. CARL P. HuBER, Indianapolis, Indiana. would simply like to rrcord the fact that Dr. Stander in our department has been doing similar studies during the past year and has found no correlation with the state of the infant. DR. WILLIS E. BRowN, Little Rock, Arkansas. One of our residents read of the work being done abroad on this problcn1. He vvas stimulated to study, in a manner somewhat similar to that of Dr. Beecham and his co-workers, a small number of cases. Likewise, he found no significant prognostic information.
CLAYTON T. BEECHAM, M.D. LYNDALL MOLTHAN, M.D. JOSEPH BOUTWELL, M.D. CHARLES W. ROHRBECK, M.D. Philadelphia, Pennsylvania
a fetus with hemolytic disease showed bilirubin in the liquor, causing a fairly consistent deviation in the curve. Walker, 4 following the work of Bevis, was able to forecast accurately the fetal condition in 70.3 per cent of his cases.
0 N E hardly need enumerate the difficulties and pitfalls attached to the prognosis for an infant of an Rh-sensitized woman. To assay potential danger to the fetus of a sensitized mother calls for a close look at the past newborn history, the titer levels, their behavior during pregnancy, and the husband's genotype. These factors along with clinical judgment have determined our course of action in the management of sensitized pregnant women. As everyone knows, these factors when considered individually may lead to errors in prognosis. Collectively they are more accurate but too often fall short of giving us the exact answer we want, i.e., when to deliver the baby. In our search for a solution we have turned to the liquor amnii as a likely source of information. Bevis 1 approached the same problem by the use of spectrophotometry to measure bilirubin, oxyhemoglobin, and methemalbumin in the fluid. After obtaining amniotic fluid by uterine paracentesis, he examined a spectral absorption curve, plotting optical density against wave length. Revis interpreted his results to indicate that
Studies and methods Eleven pregnant patients, 8 sensitized and 3 Rh-negative controls, were the basis for our study. We have investigated the Rh-D antibody content, levels of bilirubin (direct and total), iron, hemoglobin, and leucine aminopeptidase in amniotic fluid. Fluid was obtained by aminocentesis. In performing aminocentesis, fetal presentation and position were first determined by palpation. The depth to which the needle must penetrate to enter the amniotic sac was estimated. Conventional soap, ether, and alcohol skin preparation was employed as a preliminary to draping. The fetus was fixed by pressure from one hand while an infiltration of 1 per cent procaine solution into the layers of the abdominal wall was carried out. An 18 gauge spinal needle was then inserted into the amniotic sac and 20 c.c. of fluid withdrawn. A "bloody tap" rendered the specimen valueless for this study. Twenty cubic centimeters of venous blood was drawn at the same time for comparison with the amniotic fluid. Technique used for Rh-D antibody determination on aminotic fluid was basically
From the Departments of Obstetrics and Gynecology, Hematology, and Biochemistry, Temple University Medical Center. Presented at the Seventy-second Annual Meeting of the American Association of Obstetricians and Gynecologists, Hot Springs, Virginia, Se(lt. 7-9, 1961.
1053
1054
Beecham et al.
April 15, 1962
.\m.
J. Obst. & Gynec.
Table I. Clinical data I
No.I
Patient history Saline M. S. 5-1-2, white, 1 intra1:128 uterine death from hemolytic disease
L.A.P. 1:128
1:512
Negative Negative 1:32
0.23 U./ml.
:2
M. B. 4-2-0, white, 1 intrauterine death, 1 neonatal death, both hemolytic disease
0
1:64
1:512
Negative Negative 1: lfi
0.36
3.
C. B. 5-3-0, Negro, 1 intrauterine death, ? hemolytic disease
1: 1
1: 16
1:256
Negative Negative 1:4
0.55
4
L. S. 4-3-0, white, 1 hemolytic disease, 2 transfusions needed
1: 1
1:512
1:128
Negative Negative Negative
0. 76
5
R. B., 4-2-1, Negro, 1 hemolytic disease, 3 transfusions needed
1: 8
1:64
1:512
Negative Negative 1:4
5.0
6
T. C. 5-4-0, Negro, 1 jaundiced, 0 possibly transfused elsewhere
1:64
1:128
Negative Negative 1:8
1.1
7
A. B. 7-6-0, Negro, 1 kernicterus, died, pneumonia
0
1: 128
1:128
Negative Negative 1:2
0.44
8
F. P. 7-6, white, 1 neonatal death, ? jaundiced (elsewhere)
0
0
1: 16
Negative Negative Negative
0.85
Negative Negative Negative Negative Negative Negative
0.32
Controls N.D., negative
J.,
2
D.
negative
Negative Negative Negative Negative Negative Negative
0.37
3
B. D., negative
Negative Negative Negative Negative Negative Negative
0.9
-~---~-------
~--------------
the same as that for antibody determination on serum. No modifications were necessary. First, amniotic fluid was centrifuged at 2,000 to 3,000 rpm for 3 to 4 minutes and the supernatant removed for study. If the titer could not be carried out within 48 hours, the fluid was frozen at 2° F. Second, the fluid was titered against Group 0, Rh-D positive cells and Group 0, Rh-D negative cells (control) simultaneously. The patient's serum was also titered simultaneously against the same Rh-positive cells. Third, titrations were carried out by means of the 10 tube, twofold dilution technique, changing pipettes with each dilution. Titrations were carried out for the detection of saline-active antibody, albumin-active antibody, and indirect Coombs-active antibody.
Results were usually clear-cut and specific; however, an occasional amniotic fluid specimen would show slight nonspecific agglutination against the control cells. Bilirubin in amniotic fluid and serum was measured by a method developed by one of us (J. H. B.), as yet unpublished. Instead of methyl alcohol, as in the DucciWatson technique, acetamide is employed to develop the total bilirubin reaction. Iron in amniotic fluid and serum was measured by a modification of the method described by Ramsey. 2 Hemoglobin in amniotic fluid and serum was measured by determining the absorbance at 540 M.U. and 560 M.U. The difference was proportional to the hemoglobin content. Leucine aminopeptidase was measured by a slight modification of the Goldbarg and Rutenberg 3 method.
Volume 8.3 Number 8
Amniotic fluid studies in Rh-sensitized women
1055
Amniotic fluid Iron 19.1 mg. %
Hemoglobin 6.0 mg. %
l
Bilirubin
Newborn
0.21 mg.% direct 0.24 mg.% total
4-plus Coombs; Hgb. 8.1 Gm.; hematocrit 30; bilirubin 9.8 to 12.4 pre-exchange; 5 transfusions
17.6
5.96
0.07 direct 0.10 total
4-plus Coombs; Hgb. 11.0 Gm.; bilirubin 7 at birth; 1 transfusion
22.7
6.0
0 0
direct total
4-plus Coombs; Hgb. 15.1 Gm.; bilirubin 1.96 at birth, 4.8 at 10 hours, 20.3 at 72 hours; I exchange transfusion, 1 transfusion of packed cells
22.7
2.6
0.12 direct 0.08 total
4-plus Coombs; Hgb. 13 Gm.; bilirubin 2.1; one exchange transfusion
42.6
2.56
0 0
direct total
4-plus Coombs; Hgb. 16.5 Gm.; bilirubin 3.1; no exchange transfusion
26.8
10.2
0 0
direct total
Weakly positive Coombs; Hgb. 16.7 Gm.; bilirubin 1.2, no transfusion
17.9
7.7
0.09 direct 0.09 total
3-plus Coombs; Hgb. 16.1 Gm.; bilirubin 2.5; no transfusion
46.0
5.1
0.06 direct 0.09 total
Coombs negative; Hgb. 17.3 Gm.; hematocrit 53; bilirubin 2.7; no transfusion
24.2
9.4
0
direct and total
Coombs negative
29.2
3.4
1.2
direct and total
Coombs negative
"""
"O.:J
U.U'T
.
~~.:1
direct 0.018 total
One unit of leucine aminopeptidase activity is defined as that amount liberating one micromole of P-naphthylamide per hour per milliliter of the specimen analyzed.
Coon-1bs weakly positive
most part did. The indirect Coombs test was positive in 6 of the 8. Concentrations, where positive, were from 1:2 to 1:32. The highest concentration was in the case of the most senous1y arrecieu newoorn wnere Je:' exchange transfusions were needed. Further, the test was positive in two instances where the babies did not need transfusions and negative where another did. The amniotic fluid in none of the 3 controls reacted to Coombs (antiglobulin) test. The Coombs test was positive in serum of 7 infants of sensitized mothers and negative in 2 controls. One hal£4 of the newborns required exchange transfusions. Iron found in the liquor amnii of the sensitized women varied from 17.6 to 46 mg. per cent. Bilirubin when present was little more than a trace. It was also found .
Results
Aminocentesis was relatively easy to perform between the thirtieth and the thirtyfourth week of gestation. Clear fluid was obtained in eleven of thirteen attempts. The procedure was more difficult after the thirtysixth week. We successfully obtained fluid during the last month of pregnancy in only one patient of the three attempted in this stage of pregnancy. There were no apparent complications resulting from aminocentesis. The amniotic fluid of the 8 sensitized women failed to react to saline or albuminsuspended cells, although their sera for the
•
1
1¥
'
'I
1
,
1056
.'\p!il J'J, 1~6~ \m. ]. Obst. & Gynec.
Beecham et al.
in 2 of the controls. Hemoglobin varied from 2.6 to 10.2 mg. per cent and leucine aminopeptidase activity 5.0 to 0.23 unit per milliliter. Four of the 8 sensitized patients were delivered between 36 and 37Yz weeks of gestation. In these, all factors indicated that the babies were affected and all 4 did need transfusions. The other 4 sensitized patients had equivocal histories of hemolytic disease in the newborn, heterozygous husbands, and/or titers that either did not change or dropped during pregnancy. The latter group were allowed to go to term and the newborn infants (except in one case) did show evidence of mild hemolytic disease. Nonf' required transfusions. The entire group of 8 babies left the hospital in good condition. Oxytocin infusion with rupture of the membranes at an appropriate time successfully induced labor in 2 of 4 patients. Cesarean section was employed in 2 failed inductions. The 3 Rh-negative controls had no antibodies in either their sera or liquor amnii. The fluid revealed leucine aminopeptidase 0.32 to 0.9 units per milliliter, iron 22.9 to 29.2 mg. per cent and hemolysis 3.4 to 9.4 mg. per cent. All infants in the control group were free of hemolytic disease.
no conclusions beyond the point that the enzyme is present in amniotic fluid. Normally, a 24 hour urine specimen contains 0.06 to 0.2 mg. of iron. Our figures of 17.9 to 49 mg. per cent in amniotic fluid covered a wide range. If we assume these values represent the total fetal excretory iron up to the time of aminocentesis, we are failing to take into consideration the amount of fluid ingested by the growing fetus. Correlating the iron level with other data and the status of the newborn failed to yield significant prognostic information. One would assume that hemoglobin levels would be significantly higher in fluid surrounding- an affected baby. Such was not the case, for the controls had more hemoglobin than the sensitized group. In the light of Bevis' work and the nature of hemolytic disease in the newborn, we expected bilirubin determinations to offer prognostic information. This was not borne out by chemical analysis and, further, we cannot explain the high bilirubin values in 2 of the controls. The Coombs test was positive in the amniotic fluid of 6 patients. However, the inconsistencies made it unreliable as an index of hemolytic disease in the newborn. Conclusions
Comment
There have been few studies on the constituents of amniotic fluid. Thus, we undertook this preliminary study with a certain degree of optimism. Leucine aminopeptidase determinations, it was thought, might offer information of prognostic value since hematologic diseases where a hemolytic component is known to exist result in elevated leucine aminopeptidase activity in both serum and urine. Normal 24 hour urine contains 50 to 350 units (mean 190 units) of leucine aminopeptidase activity. The values obtained in these determinations allow us
Determinations of antibody titers, leucine aminopeptidase activity, iron, hemolysis, and bilirubin levels in amniotic fluid have not assisted us in predicting the severity of hemolytic disease in the newborn. Addendum. This paper was submitted for publication before a significant contribution was brought to our attention. E. V. Mackay, writing in the new Australian and New Zealand Journal of Obstetrics and Gynaecology 1: 78, 1961, does not share our pessimism. Using spectrophotometric methods in a large series ( 223 patients) Mackay feels that his studies are an aid in prognosticating affected infants.
REFERENCES
1. Bevis, D. C. A.: J. Obst. & Gynaec. Brit. Emp. 63: 68, 1956. 2. Ramsey, W. M.: Clin. chem. acta 2: 214, 1957.
3. Goldbarg, J. A., and Rutenberg, A. M.: Cancer 11: 283, 1958. I. Walker, A. H. C.: Brit. M. J. 2: 376, 1957.
Volume 83 Number 8
Discussion DR. H. CLOsE HESSELTINE, Chicago, Illinois. Dr. Beecham and his colleagues deserve our commendation, because too frequently people are inclined to minimize negative findings. 1'-Jeg-
ative findings are just as much a part of our education as are positive ones. One may have objections to the method used to obtain amniotic fluid. My colleagues and I have had some experience in this direction when determinations were made on the transfer of antibiotics to the fetus and amniotic fluid from the mother and also transfer from the amniotic sac back to the mother. The insertion of the needle may present a possible risk of damage to the fetus. Conceivably, vital structures could bt" inadvertently or unintentionally injured. However, by selecting the triangle formed by the arms, thighs, and ventral aspect of the fetus, it seems that there should be less likelihood of injury to the fetus.
The prospect of contamination of the amniotic cavity is inescapable. It is unlikely with proper care that a lethal complication will occur. Even so, some years back, a mother and
Amniotic fluid studies m Rh-sensitized women
1057
her unborn child died from infection. Another complication is the laceration of a blood vessel on the placental surface or of partial separation of the placenta. Thus, the fetus might face exsanguination or suffocation. The a\\rareness of these risks encourages greater caution. With sufficient reduction of harm, moral justification increases for educational inquiry. It seems that these gentlemen have satisfied scientific curiosity and documented the study, and it is assumed that this study is probably completed. DR. CARL P. HuBER, Indianapolis, Indiana. would simply like to rrcord the fact that Dr. Stander in our department has been doing similar studies during the past year and has found no correlation with the state of the infant. DR. WILLIS E. BRowN, Little Rock, Arkansas. One of our residents read of the work being done abroad on this problcn1. He vvas stimulated to study, in a manner somewhat similar to that of Dr. Beecham and his co-workers, a small number of cases. Likewise, he found no significant prognostic information.