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Midtrimester amniotic fluid delta optical density at 450 nm in normal pregnancies
CURRENT INVESTIGATION
Midtrimester amniotic fluid delta optical density at 450 nm in normal pregnancies Uma Ananth, M.D., Steven L. Warsof, M.D., Jeanne M. Coulehan, R.N., Pamela H. Wolf, Dr.P.H., and John T. Queenan, M.D.
Was kington, D. C. Spectrophotometric analysis of amniotic fluid has become the standard for assessing the fetal condition in Rh-immunized pregnancies. Serial amniocentesis is usually started at 28 to 29 weeks of gestation unless the antibody titer or history indicates it should be done earlier. This study presents the values from 14 to 20 weeks in normal pregnancies, which will also assist in evaluating pathologically elevated values. (AM J OSSTET GVNECOL 1986;155:664-6.)
Key words: Delta optical density at 450 nm, amniotic fluid bilirubin Spectrophotometric analysis of amniotic fluid is a well-established laboratory test for the diagnosis and management of Rh-sensitized pregnancies. Since the classic work of Liley!. 2, this test has become the standard for assessing fetal condition in Rh-immunized pregnancies. Serial amniocenteses are usually begun at 28 to 29 weeks' gestation, unless the antibody titer or history indicates that the procedure should be done earlier.' The three-zone graph of Liley for predicting the severity of disease included values only as early as 27 weeks' gestation. Today, with sonographic guidance, intrauterine transfusions can be done considerably earlier in severely immunized pregnancies. But little information is available concerning normal values of delta optical density at 450 nm (~OD,,,,) in early pregnancy. This study presents the values from 14 to 20 weeks' gestation in normal pregnancies.
Material and methods Two hundred four amniotic fluid samples were obtained from normal pregnant women undergoing amniocentesis for advanced maternal age (35 to 39 years). All of the procedures were done by two investigators (S. L. W. and J. T. Q.). Only those women who were
From the Department of Obstetrics and Gynecology, Georgetown University School of Medicine. Reprint requests: Uma Ananth, M.D., Department of Obstetrics and Gynecology, Georgetown University School of Medicine, 3800 Reservoir Road, N.W., Washington, D. C. 20007.
664
0.16 0.14 0.12 '.'
..
0.10
:::
.:. ..·:S:· :.:.:.. •.•....
60.0. 450 nm 0.08
.:.
.~
16
17
18
-i-i-;-
..
0.04
:::=::-
'.'
::: .. .....
:~.:
0.06
'.'
.:.:.
.::-
0.02 14
15
19
20
Menstrual Weeks
Fig. 1. Distribution of amniotic fluid AOD 45o values for normal pregnancies between 14 and 20 menstrual weeks plotted against weeks of gestation (menstrual age).
either Rh-positive with no irregular antibodies or Rhnegative with no Rh or irregular antibodies were included in this study. Each sample of amniotic fluid was centrifuged immediately at 3000 rpm for 10 minutes. Then 3 ml aliquots of supernatant fluid were transferred to a quartz cuvette and analyzed in a Perkin Elmer 202 spectrophotometer with use of a visible light source. A continuous recording was obtained between the wavelengths 300 nm and 700 nm on linear-scale graph paper, with a slit of 25 and scan speed set at "fast." The deviation in optical density was plotted by drawing an arbitrary
Midtrimester amniotic fluid delta optical density 665
Volume 155 Number 3
2.0
0.40 0.30
1.0 0.90 0.80 0.70 0.60 0.50
0.20 0.18 0.16 0.14 0.12
0.40 0.30 0.20
0.10 0.09 0.08 0.07 0.06 0.05
• ---------------------"
0.04 0.03 L...--14'-----:-'15=---:-'16::---:-':17::---1~8:---1~9:----::' Weeks Gestation
Fig. 2. Ninety-five percent confidence limits for amniotic fluid ~OD45tl from 14 to 20 weeks' gestation.
line from 375 nm to 525 nm and measuring the difference at 450 nm as described by Liley.' Twenty-four amniotic fluid samples demonstrating significant peaks at 412, 540, and 575 nm, and thus representing contamination with blood, were excluded from the study, since these peaks distort the AOD,,,, values. Therefore the following results were derived from 180 amniotic fluid samples, which were free of blood contamination. Results Fig. 1 shows the distribution of the AOD"o values plotted against the weeks since the last normal menstrual period. The lowest value is 0.0 I and the highest is 0.15. Regression analysis of these data reveals a slight but statistically significant upward trend (p = 0.03) in AOD"o as pregnancy advances. The 95% confidence limits for amniotic fluid AOD"" from 14 to 20 weeks' gestation in normal pregnancies are shown in Table I and Fig. 2. The black circles represent the upper 95% prediction levels and the open circles the lower ones. Seven patients underwent a second amniocentesis from 2 to 4 weeks later because of inadequate cell growth in culture. The amniotic fluid AOD.,o values are represented in Fig. 3. The second value was higher in four, lower in two, and the same in one.
Comment The AOD 4so is known to correlate well with the level of indirect bilirubin in the amniotic fluid. This study
Fig. 3. Serial amniotic fluid
~OD450
values in normal patients.
Table I. Ninety-five percent confidence limits for amniotic fluid AOD"" Week of gestation
Lower 95% prediction limit on J,OD 45V
Upper 95% prediction limit on J,OD 450
14 15 16 17 18 19 20
0.006 0.010 0.014 0.017 0.021 0.024 0.027
0.115 0.118 0.121 0.125 0.129 0.133 0.137
shows a wide range of normal values from 14 to 20 weeks' gestation, with a slight upward trend of values as pregnancy progressed. Berk and Sussman1 reported on amniotic fluid AOD 15o values in 54 women undergoing termination of pregnancy from 10 to 24 weeks' gestation and showed no trend with increasing gestation. Present results agree with those of Niswander,s who concluded from a study of 34 patients that the bilirubin content of amniotic fluid increases from 10 to 17 weeks' gestation, remains stable to 26 weeks, and thereafter decreases to term. But Niswander did not perform statistical analysis on his very small series to corroborate these trends. Later in pregnancy, from 22 to 26 weeks' gestation, the amniotic fluid bilirubin trend has been shown to be horizontal. 6 From approximately 25 to 26 weeks to 36 weeks the trend is downward. 1.2. 6 It is this well-documented downward trend that was the basis for Liley's three-zone graph. Over the years various attempts have been made to extend the slope of the Liley graph back earlier than 27 weeks' gestation. The data presented in
666 Ananth et al.
this study show that this is not logical because the normal values are much lower in early pregnancy. Furthermore, this practice could be dangerous because in early gestation fetal deterioration can occur in the presence of lower values. Since patients with very severe Rh disease are still being encountered, this information is extremely important in evaluating fetal condition and in determining whether an intrauterine transfusion is indicated during the second trimester of pregnancy. By determining the normal values for amniotic fluid dOD. 5o , pathologically elevated values can be evaluated better. REFERENCES I. Liley AW. Liquor amnii analysis in pregnancy complicated by rhesus sensitization. AM J OBSTET GYNECOL 1961; 82:1359.
September 1986 Am J Obstet Gynecol
2. Liley AW. Errors in assessment of hemolytic disease from amniotic fluid. AM J OBSTET GYNECOL 1963;86:485. 3. Queenan JT. Rh immunization. In: Queenan JT, Hobbins Je, eds. Protocols for high-risk pregnancies. Oradell, New Jersey: Medical Economics, 1982. 4. Berk H, Sussman L. Spectrophotometric analysis of amniotic fluid during early pregnancy. Obstet Gynecol 1970; 35: 170. 5. Niswander KR. Spectrophotometric analysis of amniotic fluid in early gestation. AM J OBSTET GYNECOL 1970; 108:1296. 6. Queenan JT. Amniotic fluid analysis. In: Modern management of the Rh problem. 2d ed. Hagerstown, Maryland:Harper & Row, 1977.
Midtrimester amniotic fluid delta optical density at 450 nm in normal pregnancies Uma Ananth, M.D., Steven L. Warsof, M.D., Jeanne M. Coulehan, R.N., Pamela H. Wolf, Dr.P.H., and John T. Queenan, M.D.
Was kington, D. C. Spectrophotometric analysis of amniotic fluid has become the standard for assessing the fetal condition in Rh-immunized pregnancies. Serial amniocentesis is usually started at 28 to 29 weeks of gestation unless the antibody titer or history indicates it should be done earlier. This study presents the values from 14 to 20 weeks in normal pregnancies, which will also assist in evaluating pathologically elevated values. (AM J OSSTET GVNECOL 1986;155:664-6.)
Key words: Delta optical density at 450 nm, amniotic fluid bilirubin Spectrophotometric analysis of amniotic fluid is a well-established laboratory test for the diagnosis and management of Rh-sensitized pregnancies. Since the classic work of Liley!. 2, this test has become the standard for assessing fetal condition in Rh-immunized pregnancies. Serial amniocenteses are usually begun at 28 to 29 weeks' gestation, unless the antibody titer or history indicates that the procedure should be done earlier.' The three-zone graph of Liley for predicting the severity of disease included values only as early as 27 weeks' gestation. Today, with sonographic guidance, intrauterine transfusions can be done considerably earlier in severely immunized pregnancies. But little information is available concerning normal values of delta optical density at 450 nm (~OD,,,,) in early pregnancy. This study presents the values from 14 to 20 weeks' gestation in normal pregnancies.
Material and methods Two hundred four amniotic fluid samples were obtained from normal pregnant women undergoing amniocentesis for advanced maternal age (35 to 39 years). All of the procedures were done by two investigators (S. L. W. and J. T. Q.). Only those women who were
From the Department of Obstetrics and Gynecology, Georgetown University School of Medicine. Reprint requests: Uma Ananth, M.D., Department of Obstetrics and Gynecology, Georgetown University School of Medicine, 3800 Reservoir Road, N.W., Washington, D. C. 20007.
664
0.16 0.14 0.12 '.'
..
0.10
:::
.:. ..·:S:· :.:.:.. •.•....
60.0. 450 nm 0.08
.:.
.~
16
17
18
-i-i-;-
..
0.04
:::=::-
'.'
::: .. .....
:~.:
0.06
'.'
.:.:.
.::-
0.02 14
15
19
20
Menstrual Weeks
Fig. 1. Distribution of amniotic fluid AOD 45o values for normal pregnancies between 14 and 20 menstrual weeks plotted against weeks of gestation (menstrual age).
either Rh-positive with no irregular antibodies or Rhnegative with no Rh or irregular antibodies were included in this study. Each sample of amniotic fluid was centrifuged immediately at 3000 rpm for 10 minutes. Then 3 ml aliquots of supernatant fluid were transferred to a quartz cuvette and analyzed in a Perkin Elmer 202 spectrophotometer with use of a visible light source. A continuous recording was obtained between the wavelengths 300 nm and 700 nm on linear-scale graph paper, with a slit of 25 and scan speed set at "fast." The deviation in optical density was plotted by drawing an arbitrary
Midtrimester amniotic fluid delta optical density 665
Volume 155 Number 3
2.0
0.40 0.30
1.0 0.90 0.80 0.70 0.60 0.50
0.20 0.18 0.16 0.14 0.12
0.40 0.30 0.20
0.10 0.09 0.08 0.07 0.06 0.05
• ---------------------"
0.04 0.03 L...--14'-----:-'15=---:-'16::---:-':17::---1~8:---1~9:----::' Weeks Gestation
Fig. 2. Ninety-five percent confidence limits for amniotic fluid ~OD45tl from 14 to 20 weeks' gestation.
line from 375 nm to 525 nm and measuring the difference at 450 nm as described by Liley.' Twenty-four amniotic fluid samples demonstrating significant peaks at 412, 540, and 575 nm, and thus representing contamination with blood, were excluded from the study, since these peaks distort the AOD,,,, values. Therefore the following results were derived from 180 amniotic fluid samples, which were free of blood contamination. Results Fig. 1 shows the distribution of the AOD"o values plotted against the weeks since the last normal menstrual period. The lowest value is 0.0 I and the highest is 0.15. Regression analysis of these data reveals a slight but statistically significant upward trend (p = 0.03) in AOD"o as pregnancy advances. The 95% confidence limits for amniotic fluid AOD"" from 14 to 20 weeks' gestation in normal pregnancies are shown in Table I and Fig. 2. The black circles represent the upper 95% prediction levels and the open circles the lower ones. Seven patients underwent a second amniocentesis from 2 to 4 weeks later because of inadequate cell growth in culture. The amniotic fluid AOD.,o values are represented in Fig. 3. The second value was higher in four, lower in two, and the same in one.
Comment The AOD 4so is known to correlate well with the level of indirect bilirubin in the amniotic fluid. This study
Fig. 3. Serial amniotic fluid
~OD450
values in normal patients.
Table I. Ninety-five percent confidence limits for amniotic fluid AOD"" Week of gestation
Lower 95% prediction limit on J,OD 45V
Upper 95% prediction limit on J,OD 450
14 15 16 17 18 19 20
0.006 0.010 0.014 0.017 0.021 0.024 0.027
0.115 0.118 0.121 0.125 0.129 0.133 0.137
shows a wide range of normal values from 14 to 20 weeks' gestation, with a slight upward trend of values as pregnancy progressed. Berk and Sussman1 reported on amniotic fluid AOD 15o values in 54 women undergoing termination of pregnancy from 10 to 24 weeks' gestation and showed no trend with increasing gestation. Present results agree with those of Niswander,s who concluded from a study of 34 patients that the bilirubin content of amniotic fluid increases from 10 to 17 weeks' gestation, remains stable to 26 weeks, and thereafter decreases to term. But Niswander did not perform statistical analysis on his very small series to corroborate these trends. Later in pregnancy, from 22 to 26 weeks' gestation, the amniotic fluid bilirubin trend has been shown to be horizontal. 6 From approximately 25 to 26 weeks to 36 weeks the trend is downward. 1.2. 6 It is this well-documented downward trend that was the basis for Liley's three-zone graph. Over the years various attempts have been made to extend the slope of the Liley graph back earlier than 27 weeks' gestation. The data presented in
666 Ananth et al.
this study show that this is not logical because the normal values are much lower in early pregnancy. Furthermore, this practice could be dangerous because in early gestation fetal deterioration can occur in the presence of lower values. Since patients with very severe Rh disease are still being encountered, this information is extremely important in evaluating fetal condition and in determining whether an intrauterine transfusion is indicated during the second trimester of pregnancy. By determining the normal values for amniotic fluid dOD. 5o , pathologically elevated values can be evaluated better. REFERENCES I. Liley AW. Liquor amnii analysis in pregnancy complicated by rhesus sensitization. AM J OBSTET GYNECOL 1961; 82:1359.
September 1986 Am J Obstet Gynecol
2. Liley AW. Errors in assessment of hemolytic disease from amniotic fluid. AM J OBSTET GYNECOL 1963;86:485. 3. Queenan JT. Rh immunization. In: Queenan JT, Hobbins Je, eds. Protocols for high-risk pregnancies. Oradell, New Jersey: Medical Economics, 1982. 4. Berk H, Sussman L. Spectrophotometric analysis of amniotic fluid during early pregnancy. Obstet Gynecol 1970; 35: 170. 5. Niswander KR. Spectrophotometric analysis of amniotic fluid in early gestation. AM J OBSTET GYNECOL 1970; 108:1296. 6. Queenan JT. Amniotic fluid analysis. In: Modern management of the Rh problem. 2d ed. Hagerstown, Maryland:Harper & Row, 1977.