- Email: [email protected]
Amniotic fluid tests for fetal maturity
Amniotic fluid tests for fetal maturity T.
A.
DORAN,
M.D.,
F.R.C.S.(C)
R.
J.
BENZIE,
M.B.,
CH.B.,
J.
L.
HARKINS,
M.D.,
M.
JONES
OWEN,
B.Sc.,
C.
J.
PORTER,
B.A.,
PH.D.
D.
W.
THOMPSON, LIEDGREN,
Toronto,
Ontario,
F.R.C.S.(C)
F.R.C.S.(C)
V.
S. I.
M.R.C.O.G.,
M.D.,
PH.D.
F.R.C.P.(C)
M.D. Canada
Eighteen biochemical parameters and one cytologic test (percentage of lipid-positive cells) in amniotic fluid were assessed for their value in establishing fetal maturity. Ten parameters showed a trend with gestation but the three best tests were percentage of lipid-positive cells, lecithin/sphingomyelin ratio (L/S), and creatinine. With the use of a graph containing the mean value of the three key parameters, an estimated period of gestation was produced which was 95 per cent accurate for any individual sample. With some minor deviations the three tests and the estimated period of gestation based on the three tests continued to be accurate in abnormal pregnancy states.
A c c u R A T E assessment Of fetal maturity is essential prior to elective induction, elective cesarean section, and premature termination of pregnancy indicated by such maternal conditions as diabetes, Rh isoimmunization, or hypertensive disorders. The purpose of this study was to determine which of the 18 biochemical parameters and one cytologic test (percentage of lipid-positive cells) performed on amniotic fluid could be used as tests for fetal maturity and whether
abnormal lidity. Materials
Revised
for
publication
Nouember
May
and
states altered
their
va-
methods
The sources of amniotic fluid and pregnancies studied were the same as shown in Tables I and II of our preceding artic1e.l Only patients of known gestation were included in the study. Where there was any discrepancy between the pediatric and obstetric data, the case was excluded. Samples contaminated by blood or meconium were also excluded. The tests performed on amniotic fluid were those described in detail in the preceding paper, viz., bilirubin, sodium, potassium, chloride, carbon dioxide, urea nitrogen, glucose, uric acid, total protein, albumin, lactic dehydrogenase (LDH) , hydroxybutyric dehydrogenase (HBDH) , creatinine kinase, alkaline phosphatase, acid phosphatase, creatinine, and osmolality. In addition cytology of the amniotic fluid (percentage of lipid-positive cells) was determined according to the method of Brosens and Gordon.*
From Toronto General Hospital. This study was carried out with the support of an Ontario Department of Health Grant, PR 279 by the Departments of Obstetrics and Gynaecology, Clinical Biochemistry and Pathology, Toronto General Hospital, University of Toronto, Canada. Received
pregnancy
30, 1973.
13, 1973.
Accepted December 17, 1973. Retrrint reauests: Dr. T. A. Doran. Amniotic &uid Study, Room 12, -’ 4 Urquhart Wing, Toronto General Hospital, 101 College St., Toronto, Ontario, Canada, M5G IL7. 829
830
Doran
July 15, 1974 Arm J. Obstet. Gynecol.
et al.
Fig. 1. Histogram of differential percentages positive cells, creatinine, L/S ratio) compared percentage of cases of 37 to 44 weeks; white 36% weeks.
Table I. Differential
percentages
of three key parameters (percentage of lipidto the LOC score. Shaded areas refer to the areas refer to the percentage of cases of 31 to
of 10 parameters
Parameter
Crit. value
Gest. trend
Lipid-pos. cells ( % ) Creatinine (mg./lOO ml.) L/S ratio Osmolality (mOsm./Kg.) Sodium (mEq./L.) Uric acid (mg./lOO ml.) Bilirubin (mg./lOO ml.) Potassium (mEq./L.) Urea diff. (mg./lOO ml.) Chloride (mEo./L.)
10 1.8 3/l 270 130 7.0 0.04 4 7 108
T 1 t 1 1 I‘ 1 ? I
31-36% Above 11 14 12 6 2; 10 17 26 2
The L/S ratio was estimated with a modification of Gluck’s original method.3 The amniotic fluid was centrifuged at 10,000 r.p.m. for 10 minutes and extraction was carried out. A 2 ml. quantity of supernatant was mixed with 2 ml. of methanol and shaken on a Vortex Mixer for 15 seconds; then 4 ml. of chloroform were added and shaken on a Vortex Mixer for 30 seconds. The resulting emulsion was separated by refrigeration and then centrifugation at 3,000 r.p.m. for 10 minutes. The chloroform layer was dried by warming to 40 degrees in a current of nitrogen and the residue was reconstituted in chloroform and spotted onto a thin-layer
in amniotic
wk. Below
Total
18 1.5 12 17 15 7 12 7 5 22
29 L’9 24 32 Iii 22 24 31 24
Per cent above 07 below 38 48 50 74 63 75 55 70 84 91
fluid
37+ Above 84 78 69 23 1.5 81 32 65 76 8
Total
Per cent above “7 below
Differential (%I
95 97 85 71 83 94 9’2 83 84 83
88 80 81 96 8i! 86 66 77 90 90
50 32 31 22 19 11 11 7 6 -1
wk. Below 11 19 16
62 68 13 60 18 8 75
plate (Silica Gel G) . Standard mixtures of lecithin and sphingomyelin were also spotted onto the plate. Chromatographic separation was achieved with the use of the solvent system chloroform : methanol : ammonium hydroxide : water (75 : 30: 4: 0.5 v/v). Lecithin and sphingomyelin were visualized by the method of Coch and Kessler4 by spraying the bismuth subnitrate solution and washing out the background color with 20 per cent acetic acid. The concentrations of lecithin and sphingomyelin present were estimated by visual comparison with the standards run on the sample plate. For various technical reasons not all of the 19 tests were performed
Volume Kumber
60:
1lY 6
Amniotic
fluid
tests
for
fetal
maturity
831
.
c-
.* _. . .
,--
c--
n
.
i--
.
. *la . w . . .. z-z-2
1
Fig. 2. Scatter diagram of percentage of lipid-positive on every one of the 252 samples but this was done in the vast majority of samples. Results Mean values of 17 parameters in amniotic fluid were reported in our first paper.* Of the parameters studied, the following showed either no trend or insufficient trends with gestation to be of clinical value in the assessment of fetal maturity: carbon dioxide, glucose, LDH, HBDH, creatine kinase, acid phosphatase, alkaline phosphatase, total protein, and albumin. The remaining 10 parameters did show a trend with gestation. We chose 37 weeks’ gestation as our level for maturity and excluded for purposes of analysis all cases less than 31 weeks’ gestation. For each parameter an arbitrary fixed level was chosen which tended to maximize the difference between the two gestational age groups (3 1 to 3S1/, weeks and 37 to 44 weeks). In the case of a parameter that increased with gestation (e.g., creatinine) we compared the percentage of samples above the fixed point at 31 to 36% weeks with the percentage of samples above the
.
: .
:
.
.
:
.
.
.:I
: .. .
. . -+.
. ... r-b
84
. .
%J
$.
.
+.
. L..
:
.... . ..
. . .
..
.. : 36
40
n
. 42
u
cells in amniotic fluid.
same point at 37 to 44 weeks. In the case of a parameter that fell with gestation the differential percentage applied to the percentage of samples below the fixed level in each gestational age group. We used this differential percentage as an index of the value of each parameter in the assessment of fetal maturity. The results of this analysis of our data are shown in Table I. The urea difference was obtained by subtracting the maternal serum urea from the amniotic fluid urea as described by Lind and Billewicz.’ In the case of amniotic fluid potassium, urea difference, and chloride there was a very small differential between the percentage of cases above our arbitrary critical level for each parameter in the 31 to 36vz week gestation group and the 37 to 44 week gestation group (7, 6, -1 per cent, respectively). For amniotic fluid osmolality, sodium, uric acid, and bilirubin there was a small difference between the two groups (31 to 36 weeks and 37 to 44 weeks), although there was a trend with gestation. The differential percentages were 22 for osmolality,
832
Doran
July 15, 1974 Am. J. O&et. Gynecol.
et al.
36 3.4 .32--
..
06-06 -12
I 14
:
i .*: rcr.p “.
.b . &<*&I:. . ..+p.4. . . ! 16
18
'&I
.
.
22
24
.
.
:
. 26
20
I 30
wmlrs-
Fig.
3. Scatter diagram of creatinine in amniotic fluid.
19 for sodium, 11 for uric acid, and 11 for bilirubin. Fig. 1 illustrates that three parameters--percentage of lipid-positive cells, creatinine, and L/S ratio-did show a relatively large differential percentage between the two gestational groups. Percentage of lipid-positive cells (Table I and Fig. 2), with the use of a critical level of 10 per cent at 37 weeks with a differential percentage of 50 between the two groups, was the best individual test for fetal maturity. There were still, however, 11/29 (38 per cent) false positives at 31 to 36f/2 weeks and 1 l/95 (12 per cent) false negatives at 37 to 44 weeks. The next best parameter was amniotic fluid creatinine (Table I and Fig. 3) with a differential percentage of 32 between the two gestational groups using the critical level of 1.8 mg. per 100 ml. at 37 weeks. There were 14/29 (48 per cent) false positives at 31 to 36vZ weeks and 19/92 (20 per cent) false negatives at 37 to 44 weeks. Of equal value to amniotic fluid creatinine in this context was the L/S ratio (Table I and Fig. 4) with a differential percentage of 31 between the two gestational groups, using a critical level of 3/l
at 37 weeks. There were 12/24 (50 per cent) false-positives and 16/85 (19 per cent) false negatives. We noted that the false-negative and the false-positive groups for each of the three parameters did not contain the same samples and so felt that an index incorporating the three tests might compensate for the deficiencies of any one test. Our initial attempts at a scheme incorporating the three tests utilized the same arbitrary critical values at 37 weeks and gave a score of 1 for each parameter that was at or above our critical level. This we called the LOC score (L/S ratio? orange-staining cells, and creatinine) There were 196 samples where all three tests were done. Of all samples taken at 37 weeks’ gestation or more, 70/81 (86 per cent) had a LOC score of 2 or more. If the LOC score was 2 or more the fetus was mature (37 weeks’ gestation or greater) in 70/79 (89 per cent) of cases. If the LOC score was 3 the fetus was mature in 93 per cent of cases (51/54). Comparison of the LOC score with the individual parameters (Fig. 1) showed, however, that at 37 weeks
Amniotic
fluid
tests
for
fetal
maturity
833
IO-
. . 69
76-
12
16
20
’ weeks
Fig. 4. Scatter
24
26
’
32
’
gestation
diagram of L/S ratio in amniotic fluid.
the score was no better than the best of the three leading tests (percentage of lipid-positive cells). When a critical LOC score of 2 was used, the differential percentage between the two gestational groups was 40 per cent with 9/24 (37 per cent) false positives and 11/81 (14 per cent) false negatives. The problem in utili&g the LOC score seemed to be that of the arbitrary critical level which automatically diminished the accuracy of the score. In an effort to eliminate this problem we then devised a scheme utilizing the mean values of the three key parameters. Fortunately, with minor deviations, the three mean values rose progressively during gestation (Fig. 5). By dropping a vertical line to the base line from the point on the graph where an individual value intersected the mean for that value, we arrived at the estimated period of gestation for that particular parameter (e.g., creatinine) . Repeating the same process for the other two parameters (e.g., L/S ratio and percentage of lipid-positive cells) and then averaging the three to the nearest half week gave us the mean estimated period of gestation (EPG) for any individual sample. Fig.
2.5 2.0
1.5
1.0
0.5
Fig. 5. Mean values of three leading AF parameters (percentage of lipid-positive cells, creatinine, L/S ratio).
6 illustrated our results, plotting estimated period of gestation against actual period of gestation. The coefficient of correlation was 0.95, indicating a high degree of accuracy for this method of assessing fetal maturity using the mean values of the three parameters. We also estimated the EPG using the individual parameters. Coefficients of correlation were 0.82 for L/S ratio, 0.89 for
834
Doran
July 15, 1974 Am. J. Obstet. Gynecol.
et al.
/’ ,.L.. *. . .-A*.. / , .
.
..L/ :/’
:
.
:. .. . .:.
16 16
20
24
sctusl
28
32
36
40
44
gestation
Fig. 6. Regression graph between estimated period of gestation (based on three means) and actual period of gestation.
3. l
IUGR
-i
OlUGR
+ Hypertension /-
I
I 12
Fig. 7. AF retardation.
20 weeks
28
36
in
intrauterine
1 44
growth
expected range throughout gestation with the exception of creatinine, where two out of the 18 values (toxemia and hypertension with superimposed toxemia) were above the test group range. The EPG continued to be a valid test in hypertensive disorders.
test group range. The EPG continued to be of value in Rh isoimmunization. The severity of the Rh isoimmunization process did not affect the distribution of values with the exception of amnoitic fluid creatinine in hydrops fetalis (four cases) where values were within the test group range but below the mean. Comment
creatinine, and 0.92 for percentage of lipidpositive cells, confirming the increased accuracy of the three-mean method. The next question to be answered was whether these tests continued to be of value in abnormal pregnancy states. We then analyzed our data for each of the abnormal pregnancy states studied, recognizing that the number of individual samples in each group was small.
Intrauterine growth retardation (7 patients, 13 samples). All three parameterscreatinine, lipid-positive
Hypertensive disorders ( 13 patients, 19 samples). All parameters were within the
Rh isoimmunization (18 patients, 35 samples). All three parameters were within the
gestation
creatinine
group range (mean plus or minus two standard deviations). Fig. 7 illustrates amniotic fluid creatinine in these cases of intrauterine growth retardation. The EPG according to the three tests also continued within the test group range, although in this small group of cases nine of 13 observations were below the regression line. Diabetes (10 patients, 13 samples). Creatinine and L/S ratio were within the espetted range for the test group in diabetes. When the diabetic samples were tested for percentage of lipid-positive cells, lo/12 results were within the test group range but below the mean. The EPG continued to be accurate in diabetes.
L/S
ratio, cells-were
and
percentage within the
of test
Accurate estimation of fetal maturity is necessary where termination of pregnancy before term is indicated and is essential prior to elective induction or elective cesarean section. Currently available methods of fetal maturity estimation are all imperfect. Because of these deficiencies the biochemical estimation of amniotic fluid constituents has received ,great attention in recent years and various attempts have been made to correlate amniotic fluid tests with fetal maturity. Mandelbaum and Evans” found that the disappearance of bilirubin as measured by spectrophotometric analysis was “the most
Volume Number
119 6
reliable criterion of functional fetal maturity.” Droegemueller and associates,? however, were unable to find a significant correlation between amniotic fluid bilirubin and gestational age. Jools and Jones8 agreed with Mandelbaum and Evans and stated that a disappearance of the bilirubin peak without a subsequent rise was a fairly constant finding after the thirty-sixth week. White and associates” and Thiede,‘” however, found that the O.D. difference at 450 mp was less than 0.01 in a significant percentage of samples before 36 weeks. Finally, Parmley and Miller’l did find a decrease in the O.D. of amniotic fluid at 450 mp, but were disappointed by the spread of their data. We share their disappointment and conclude that in our hands bilirubin estimation in amniotic fluid is an unreliable guide to fetal maturity due to the spread of our data, with a differential percentage of only 11 between the two key gestational groups. The fall in amniotic fluid osmolality with increasing gestation has been demonstrated by several workers.lZ-l” Miles and Pearson14 found that a level of 250 Osm. per kilogram or less was suggestive of fetal maturity and suspected that lower levels indicated fetal compromise. Mattison, ” however, maintained that levels of amniotic fluid osmolality higher than normal indicated fetal jeopardy. Our data indicate that although amniotic fluid osmolality does decrease with increasing gestation, the decline is gradual with an insufficient differential percentage between the two gestational groups (11 per cent). We did not find any deviation from the normal trend in osmolality in abnormal pregnancy states. Harrison’” found 79 per cent of amniotic fluid uric acid levels above 8.5 mg. per 100 ml. at 38 weeks and no values below this level. Our finding of a differential percentage of only 11 per cent between the two key gestational groups (critical level 7.0 mg. per 100 ml.) indicates to us that this test is of very limited value in fetal maturity assessment. The L/S ratio first reported by Gluck and associates” and now by others,17-19 has been utilized as a test of fetal pulmonary maturity.
Amniotic
fluid
tests
for
fetal
maturity
835
Its predictive accuracy has been confirmed by Donald and associates.” It was not our intention in setting up this study to analyze fetal pulmonary maturity and we had only two cases of respiratory distress syndrome in the series; however, in both cases the L/S ratios were less than 2. Our results, therefore, would certainly confirm the value of this test in assessing fetal pulmonary maturity. It is our opinion, however, based on our own results (Table I, Fig. 4) that the test is also of value in the assessment of fetal maturity per se. Bryson and associates’” reported that the L/S ratio did not correlate with maturity; however, on analyzing their data, it appears to us that there is in fact a good trend with gestation in their results. In assessing the L/S ratio with respect to abnormal pregnancy states, we found that it continued to be of value in these situations. In diabetes we found that the expected surge in L/S ratio did occur at 35 weeks, as opposed to the findings of Whitfield and Sproule,2” who found that the surge did not take place consistently. Gluck and Kulovitrh?’ have stated that the L/S ratio is accelerated in Class D, E, and F diabetes, decelerated in Class A, B, and C diabetes. Analysis of our small number of cases, which were all Class A, B, and C, failed to reveal any significant deceleration. Donald and associates” found that even with a L/S ratio greater than 2 infants of diabetic mothers showed a higher risk of respiratory distress syndrome. One might conclude that the L/S ratio must be studied further in diabetic patients and that, until more is known, L/S ratios in diabetics should be interpreted with caution. We did note a slight trend toward acceleration of the L/S ratio in hypertensive disorders, but all values were within the test group range. This trend, we believe, should be explored further. Since Kittrichz2 first investigated the use of Nile Blue stain in identifying fetal cells in amniotic fluid, numerous papers have attested to its value in estimating fetal maturity. 22-24 Anderson and Griffith? found that the presence of orange-stained cells indicated a gestation of 36 weeks. We have, however, noted the presence of orange-
836
Doran
et al.
stained cells at earlier gestation---even on occasions prior to 20 weeks. Brosens and Gordon? have pointed out that false negatives are likely to accur before 36 weeks and false positives do not. In our hands false negatives may occur after 37 Meeks and false positives before then. In our opinion, percentage of lipid-positive cells is the best single test for estimation of fetal maturity. It does, however, have the disadvantage of being subject to individual interpretation by the examiner. We found, as did Bishop and CorsonZ-’ that when fat cells were more than 20 per cent, that a11 cases studied were 36 weeks
July 15, 1974 Am. J. Obstet. Gynecol.
seems to us to be a valid one. This is particularly true if this process improves the over-all accuracy by correcting for the deficiencies of any individual test. This concept was first introduced by Thiede,‘” utilizing lipid-staining cells, creatinine, and bilirubin. In 49 samples he had no false positives and five false negatives (20 per cent) in predicting maturity (37 weeks). Jools and ones8 utilized creatinine, percentage of J lipid-positive cells, and bilirubin in 91 samples of amniotic fluid. Gestation was predicted as a range only and their method of . arriving at the predicted gestation was not clearly described. However, a “very satisfaccalculation was obtained. Lind and tory” Billewicz” described three different scoring systems using cytology, creatinine, and urea difference. The percentage accuracy was only 72, 70, and 60 per cent. Our method, utilizing EPG based on three parameters-percentage of lipid-positive cells? L/S ratio, and creatinine-seems to be more accurate than those previously described, with an accuracy of 95 per cent. Its predictive accuracy does not vary with different periods of gestation and it can be utilized for any sample from 16 weeks to past term. Reporting fetal maturity as estimated period of gestation is more meaningful to the clinician than a number or index. With some deviations the three key parameters and the EPG continue to be of value in abnormal pregnancy states. We intend to investigate further the trends in the three parameters and in the EPG in abnormal pregnancy states, but have sufficient confidence in the tests that we now report to the clinician the values of the three tests plus the EPG based on the three means. We feel that this gives the physician an important and accurate tool for assessment both of fetal maturity and fetal pulmonary maturity. We also feel very strongly that he should incorporate the results of these tests with other clinical, radiologic, ultrasonographic, and laboratory data (estriols, etc.) before making a decision for or against the delivery in any individual pregnancy.
Volume Number
119 6
REFERENCES
Benzie, R. J., Doran, T. A., Harkins, J. L., Jones Owen, V. M., and Porter, C. J.: AM. J. OBSTET. GYNECOL. 119: 798, 1974. 2. Brosens, I., and Gordon, J.: J. Obstet. Gvnaecol. Br. Commonw. 72: 343. 1965. 3. Gluck, L., Kulovich, M. V., Borer, R. C., Jr., Brenner, P. H., Anderson, G. G., and Spellacy, W. N.: AM. J. OBSTET. GYNECOL. 109: 440, 1971. 4. Coch, E. H., and Kessler, G.: Clin. Chem. 18: 490. 1972. 5. Lind, T., and Billewicz, W. Z.: Br. J. Hosp. Med. 5: 681. 1971. 6. Mandelbauxn, B., and Evans, T. N.: AM. J. OBSTET. GYNECOL. 104: 365, 1965. 7. Droegemueller, W., Jackson, C., Makowski, E. L., and Battaglia, F. C.: AM. J. OBSTET. GYNECOL. 104: 424, 1969. 8. Jools, N. D., and Jones, W. R.: Minn. Med. 54: 403. 1971. 9. White, ‘C. A., Doorenbos, D. E., and Bradbury, J. T.: AM. I. OBSTET. GYNECOL. 104: 655,1969. ” 10. Thiede, H.: Discussion, in AM. J. OBSTET. GYNECOL. 104: 668, 1969. 11. Parmley, T., and Miller, E.: AM. J. OBSTET. GYNECOL. 105: 354, 1969. 12. Gillibrand, P. N.: J. Obstet. Gynaecol. Br. Commonw. 76: 898, 1969. 13. Turnbull, A. C.: Devel. Med. Child. Neurol. 12: 375, 1970. 14. Miles, P. A., and Pearson, J. W.: Obstet. Gynecol. 34: 701, 1969.
Amniotic
15.
1.
16. 17. 18.
19.
20. 21. 22. 23. 24. 25.
26. 27. 28.
fluid
tests
for
fetal
maturity
837
Mattison, D. R.: Obstet. Gynecol. 36: 420, 1970. Harrison, R. F.: J. Obstet. Gynaecol. Br. Commonw. 79: 708, 1972. Lemons, J. A., and Jaffe, R. B.: AM. J. OBSTET. GYNECOL. 45: 233, 1973. Donald, I. R., Freeman, R. K., Goebelsmann, U. Chan, W. H., and Nakamura, R. M.: AM. J. OBSTET. GYNECOL. 115: 547, 1973. Bryson, J, J., Gabert, H. A., and Stenchever, M. A.: AM. J. OBSTET. GYNECOL. 114: 208, 1972. Whitfield, C. R., and Sproule, W. B.: Br. Med. J. 2: 85, 1972. Gluck, L., and Kulovich, M. V.: AM. J. OBSTET. GYNECOL. 115: 539, 1973. Kittrich, M.: Geburtshilfe Frauenheilkd. 23: 156, 1963. Bishop, E. H., and Corson, S.: AM. J. OBSTET. GYNECOL. 104: 654, 1968. Huisjes, H. J., and Arendzen, J. D.: Obstet. Gynecol. 35: 725, 1970. Anderson, A. B. M., and Griffiths, A. D.: J. Obstet. Gynaecol. Br. Commonw. 75: 300, 1968. Doran, T. A., Bjerre, S., and Porter, C. J.: AM. J. OBSTET. GYNECOL. 106: 325, 1970. Pitkin, R. M., and Zwirek, S. J.: AM. J. OBSTET. GYNECOL. 98: 1135, 1967. Moore, W. M. O., Murphy, P. J., and Davis, J. A.: AM. J. OBSTET. GYNECOL. 100: 908, 1971.
A.
DORAN,
M.D.,
F.R.C.S.(C)
R.
J.
BENZIE,
M.B.,
CH.B.,
J.
L.
HARKINS,
M.D.,
M.
JONES
OWEN,
B.Sc.,
C.
J.
PORTER,
B.A.,
PH.D.
D.
W.
THOMPSON, LIEDGREN,
Toronto,
Ontario,
F.R.C.S.(C)
F.R.C.S.(C)
V.
S. I.
M.R.C.O.G.,
M.D.,
PH.D.
F.R.C.P.(C)
M.D. Canada
Eighteen biochemical parameters and one cytologic test (percentage of lipid-positive cells) in amniotic fluid were assessed for their value in establishing fetal maturity. Ten parameters showed a trend with gestation but the three best tests were percentage of lipid-positive cells, lecithin/sphingomyelin ratio (L/S), and creatinine. With the use of a graph containing the mean value of the three key parameters, an estimated period of gestation was produced which was 95 per cent accurate for any individual sample. With some minor deviations the three tests and the estimated period of gestation based on the three tests continued to be accurate in abnormal pregnancy states.
A c c u R A T E assessment Of fetal maturity is essential prior to elective induction, elective cesarean section, and premature termination of pregnancy indicated by such maternal conditions as diabetes, Rh isoimmunization, or hypertensive disorders. The purpose of this study was to determine which of the 18 biochemical parameters and one cytologic test (percentage of lipid-positive cells) performed on amniotic fluid could be used as tests for fetal maturity and whether
abnormal lidity. Materials
Revised
for
publication
Nouember
May
and
states altered
their
va-
methods
The sources of amniotic fluid and pregnancies studied were the same as shown in Tables I and II of our preceding artic1e.l Only patients of known gestation were included in the study. Where there was any discrepancy between the pediatric and obstetric data, the case was excluded. Samples contaminated by blood or meconium were also excluded. The tests performed on amniotic fluid were those described in detail in the preceding paper, viz., bilirubin, sodium, potassium, chloride, carbon dioxide, urea nitrogen, glucose, uric acid, total protein, albumin, lactic dehydrogenase (LDH) , hydroxybutyric dehydrogenase (HBDH) , creatinine kinase, alkaline phosphatase, acid phosphatase, creatinine, and osmolality. In addition cytology of the amniotic fluid (percentage of lipid-positive cells) was determined according to the method of Brosens and Gordon.*
From Toronto General Hospital. This study was carried out with the support of an Ontario Department of Health Grant, PR 279 by the Departments of Obstetrics and Gynaecology, Clinical Biochemistry and Pathology, Toronto General Hospital, University of Toronto, Canada. Received
pregnancy
30, 1973.
13, 1973.
Accepted December 17, 1973. Retrrint reauests: Dr. T. A. Doran. Amniotic &uid Study, Room 12, -’ 4 Urquhart Wing, Toronto General Hospital, 101 College St., Toronto, Ontario, Canada, M5G IL7. 829
830
Doran
July 15, 1974 Arm J. Obstet. Gynecol.
et al.
Fig. 1. Histogram of differential percentages positive cells, creatinine, L/S ratio) compared percentage of cases of 37 to 44 weeks; white 36% weeks.
Table I. Differential
percentages
of three key parameters (percentage of lipidto the LOC score. Shaded areas refer to the areas refer to the percentage of cases of 31 to
of 10 parameters
Parameter
Crit. value
Gest. trend
Lipid-pos. cells ( % ) Creatinine (mg./lOO ml.) L/S ratio Osmolality (mOsm./Kg.) Sodium (mEq./L.) Uric acid (mg./lOO ml.) Bilirubin (mg./lOO ml.) Potassium (mEq./L.) Urea diff. (mg./lOO ml.) Chloride (mEo./L.)
10 1.8 3/l 270 130 7.0 0.04 4 7 108
T 1 t 1 1 I‘ 1 ? I
31-36% Above 11 14 12 6 2; 10 17 26 2
The L/S ratio was estimated with a modification of Gluck’s original method.3 The amniotic fluid was centrifuged at 10,000 r.p.m. for 10 minutes and extraction was carried out. A 2 ml. quantity of supernatant was mixed with 2 ml. of methanol and shaken on a Vortex Mixer for 15 seconds; then 4 ml. of chloroform were added and shaken on a Vortex Mixer for 30 seconds. The resulting emulsion was separated by refrigeration and then centrifugation at 3,000 r.p.m. for 10 minutes. The chloroform layer was dried by warming to 40 degrees in a current of nitrogen and the residue was reconstituted in chloroform and spotted onto a thin-layer
in amniotic
wk. Below
Total
18 1.5 12 17 15 7 12 7 5 22
29 L’9 24 32 Iii 22 24 31 24
Per cent above 07 below 38 48 50 74 63 75 55 70 84 91
fluid
37+ Above 84 78 69 23 1.5 81 32 65 76 8
Total
Per cent above “7 below
Differential (%I
95 97 85 71 83 94 9’2 83 84 83
88 80 81 96 8i! 86 66 77 90 90
50 32 31 22 19 11 11 7 6 -1
wk. Below 11 19 16
62 68 13 60 18 8 75
plate (Silica Gel G) . Standard mixtures of lecithin and sphingomyelin were also spotted onto the plate. Chromatographic separation was achieved with the use of the solvent system chloroform : methanol : ammonium hydroxide : water (75 : 30: 4: 0.5 v/v). Lecithin and sphingomyelin were visualized by the method of Coch and Kessler4 by spraying the bismuth subnitrate solution and washing out the background color with 20 per cent acetic acid. The concentrations of lecithin and sphingomyelin present were estimated by visual comparison with the standards run on the sample plate. For various technical reasons not all of the 19 tests were performed
Volume Kumber
60:
1lY 6
Amniotic
fluid
tests
for
fetal
maturity
831
.
c-
.* _. . .
,--
c--
n
.
i--
.
. *la . w . . .. z-z-2
1
Fig. 2. Scatter diagram of percentage of lipid-positive on every one of the 252 samples but this was done in the vast majority of samples. Results Mean values of 17 parameters in amniotic fluid were reported in our first paper.* Of the parameters studied, the following showed either no trend or insufficient trends with gestation to be of clinical value in the assessment of fetal maturity: carbon dioxide, glucose, LDH, HBDH, creatine kinase, acid phosphatase, alkaline phosphatase, total protein, and albumin. The remaining 10 parameters did show a trend with gestation. We chose 37 weeks’ gestation as our level for maturity and excluded for purposes of analysis all cases less than 31 weeks’ gestation. For each parameter an arbitrary fixed level was chosen which tended to maximize the difference between the two gestational age groups (3 1 to 3S1/, weeks and 37 to 44 weeks). In the case of a parameter that increased with gestation (e.g., creatinine) we compared the percentage of samples above the fixed point at 31 to 36% weeks with the percentage of samples above the
.
: .
:
.
.
:
.
.
.:I
: .. .
. . -+.
. ... r-b
84
. .
%J
$.
.
+.
. L..
:
.... . ..
. . .
..
.. : 36
40
n
. 42
u
cells in amniotic fluid.
same point at 37 to 44 weeks. In the case of a parameter that fell with gestation the differential percentage applied to the percentage of samples below the fixed level in each gestational age group. We used this differential percentage as an index of the value of each parameter in the assessment of fetal maturity. The results of this analysis of our data are shown in Table I. The urea difference was obtained by subtracting the maternal serum urea from the amniotic fluid urea as described by Lind and Billewicz.’ In the case of amniotic fluid potassium, urea difference, and chloride there was a very small differential between the percentage of cases above our arbitrary critical level for each parameter in the 31 to 36vz week gestation group and the 37 to 44 week gestation group (7, 6, -1 per cent, respectively). For amniotic fluid osmolality, sodium, uric acid, and bilirubin there was a small difference between the two groups (31 to 36 weeks and 37 to 44 weeks), although there was a trend with gestation. The differential percentages were 22 for osmolality,
832
Doran
July 15, 1974 Am. J. O&et. Gynecol.
et al.
36 3.4 .32--
..
06-06 -12
I 14
:
i .*: rcr.p “.
.b . &<*&I:. . ..+p.4. . . ! 16
18
'&I
.
.
22
24
.
.
:
. 26
20
I 30
wmlrs-
Fig.
3. Scatter diagram of creatinine in amniotic fluid.
19 for sodium, 11 for uric acid, and 11 for bilirubin. Fig. 1 illustrates that three parameters--percentage of lipid-positive cells, creatinine, and L/S ratio-did show a relatively large differential percentage between the two gestational groups. Percentage of lipid-positive cells (Table I and Fig. 2), with the use of a critical level of 10 per cent at 37 weeks with a differential percentage of 50 between the two groups, was the best individual test for fetal maturity. There were still, however, 11/29 (38 per cent) false positives at 31 to 36f/2 weeks and 1 l/95 (12 per cent) false negatives at 37 to 44 weeks. The next best parameter was amniotic fluid creatinine (Table I and Fig. 3) with a differential percentage of 32 between the two gestational groups using the critical level of 1.8 mg. per 100 ml. at 37 weeks. There were 14/29 (48 per cent) false positives at 31 to 36vZ weeks and 19/92 (20 per cent) false negatives at 37 to 44 weeks. Of equal value to amniotic fluid creatinine in this context was the L/S ratio (Table I and Fig. 4) with a differential percentage of 31 between the two gestational groups, using a critical level of 3/l
at 37 weeks. There were 12/24 (50 per cent) false-positives and 16/85 (19 per cent) false negatives. We noted that the false-negative and the false-positive groups for each of the three parameters did not contain the same samples and so felt that an index incorporating the three tests might compensate for the deficiencies of any one test. Our initial attempts at a scheme incorporating the three tests utilized the same arbitrary critical values at 37 weeks and gave a score of 1 for each parameter that was at or above our critical level. This we called the LOC score (L/S ratio? orange-staining cells, and creatinine) There were 196 samples where all three tests were done. Of all samples taken at 37 weeks’ gestation or more, 70/81 (86 per cent) had a LOC score of 2 or more. If the LOC score was 2 or more the fetus was mature (37 weeks’ gestation or greater) in 70/79 (89 per cent) of cases. If the LOC score was 3 the fetus was mature in 93 per cent of cases (51/54). Comparison of the LOC score with the individual parameters (Fig. 1) showed, however, that at 37 weeks
Amniotic
fluid
tests
for
fetal
maturity
833
IO-
. . 69
76-
12
16
20
’ weeks
Fig. 4. Scatter
24
26
’
32
’
gestation
diagram of L/S ratio in amniotic fluid.
the score was no better than the best of the three leading tests (percentage of lipid-positive cells). When a critical LOC score of 2 was used, the differential percentage between the two gestational groups was 40 per cent with 9/24 (37 per cent) false positives and 11/81 (14 per cent) false negatives. The problem in utili&g the LOC score seemed to be that of the arbitrary critical level which automatically diminished the accuracy of the score. In an effort to eliminate this problem we then devised a scheme utilizing the mean values of the three key parameters. Fortunately, with minor deviations, the three mean values rose progressively during gestation (Fig. 5). By dropping a vertical line to the base line from the point on the graph where an individual value intersected the mean for that value, we arrived at the estimated period of gestation for that particular parameter (e.g., creatinine) . Repeating the same process for the other two parameters (e.g., L/S ratio and percentage of lipid-positive cells) and then averaging the three to the nearest half week gave us the mean estimated period of gestation (EPG) for any individual sample. Fig.
2.5 2.0
1.5
1.0
0.5
Fig. 5. Mean values of three leading AF parameters (percentage of lipid-positive cells, creatinine, L/S ratio).
6 illustrated our results, plotting estimated period of gestation against actual period of gestation. The coefficient of correlation was 0.95, indicating a high degree of accuracy for this method of assessing fetal maturity using the mean values of the three parameters. We also estimated the EPG using the individual parameters. Coefficients of correlation were 0.82 for L/S ratio, 0.89 for
834
Doran
July 15, 1974 Am. J. Obstet. Gynecol.
et al.
/’ ,.L.. *. . .-A*.. / , .
.
..L/ :/’
:
.
:. .. . .:.
16 16
20
24
sctusl
28
32
36
40
44
gestation
Fig. 6. Regression graph between estimated period of gestation (based on three means) and actual period of gestation.
3. l
IUGR
-i
OlUGR
+ Hypertension /-
I
I 12
Fig. 7. AF retardation.
20 weeks
28
36
in
intrauterine
1 44
growth
expected range throughout gestation with the exception of creatinine, where two out of the 18 values (toxemia and hypertension with superimposed toxemia) were above the test group range. The EPG continued to be a valid test in hypertensive disorders.
test group range. The EPG continued to be of value in Rh isoimmunization. The severity of the Rh isoimmunization process did not affect the distribution of values with the exception of amnoitic fluid creatinine in hydrops fetalis (four cases) where values were within the test group range but below the mean. Comment
creatinine, and 0.92 for percentage of lipidpositive cells, confirming the increased accuracy of the three-mean method. The next question to be answered was whether these tests continued to be of value in abnormal pregnancy states. We then analyzed our data for each of the abnormal pregnancy states studied, recognizing that the number of individual samples in each group was small.
Intrauterine growth retardation (7 patients, 13 samples). All three parameterscreatinine, lipid-positive
Hypertensive disorders ( 13 patients, 19 samples). All parameters were within the
Rh isoimmunization (18 patients, 35 samples). All three parameters were within the
gestation
creatinine
group range (mean plus or minus two standard deviations). Fig. 7 illustrates amniotic fluid creatinine in these cases of intrauterine growth retardation. The EPG according to the three tests also continued within the test group range, although in this small group of cases nine of 13 observations were below the regression line. Diabetes (10 patients, 13 samples). Creatinine and L/S ratio were within the espetted range for the test group in diabetes. When the diabetic samples were tested for percentage of lipid-positive cells, lo/12 results were within the test group range but below the mean. The EPG continued to be accurate in diabetes.
L/S
ratio, cells-were
and
percentage within the
of test
Accurate estimation of fetal maturity is necessary where termination of pregnancy before term is indicated and is essential prior to elective induction or elective cesarean section. Currently available methods of fetal maturity estimation are all imperfect. Because of these deficiencies the biochemical estimation of amniotic fluid constituents has received ,great attention in recent years and various attempts have been made to correlate amniotic fluid tests with fetal maturity. Mandelbaum and Evans” found that the disappearance of bilirubin as measured by spectrophotometric analysis was “the most
Volume Number
119 6
reliable criterion of functional fetal maturity.” Droegemueller and associates,? however, were unable to find a significant correlation between amniotic fluid bilirubin and gestational age. Jools and Jones8 agreed with Mandelbaum and Evans and stated that a disappearance of the bilirubin peak without a subsequent rise was a fairly constant finding after the thirty-sixth week. White and associates” and Thiede,‘” however, found that the O.D. difference at 450 mp was less than 0.01 in a significant percentage of samples before 36 weeks. Finally, Parmley and Miller’l did find a decrease in the O.D. of amniotic fluid at 450 mp, but were disappointed by the spread of their data. We share their disappointment and conclude that in our hands bilirubin estimation in amniotic fluid is an unreliable guide to fetal maturity due to the spread of our data, with a differential percentage of only 11 between the two key gestational groups. The fall in amniotic fluid osmolality with increasing gestation has been demonstrated by several workers.lZ-l” Miles and Pearson14 found that a level of 250 Osm. per kilogram or less was suggestive of fetal maturity and suspected that lower levels indicated fetal compromise. Mattison, ” however, maintained that levels of amniotic fluid osmolality higher than normal indicated fetal jeopardy. Our data indicate that although amniotic fluid osmolality does decrease with increasing gestation, the decline is gradual with an insufficient differential percentage between the two gestational groups (11 per cent). We did not find any deviation from the normal trend in osmolality in abnormal pregnancy states. Harrison’” found 79 per cent of amniotic fluid uric acid levels above 8.5 mg. per 100 ml. at 38 weeks and no values below this level. Our finding of a differential percentage of only 11 per cent between the two key gestational groups (critical level 7.0 mg. per 100 ml.) indicates to us that this test is of very limited value in fetal maturity assessment. The L/S ratio first reported by Gluck and associates” and now by others,17-19 has been utilized as a test of fetal pulmonary maturity.
Amniotic
fluid
tests
for
fetal
maturity
835
Its predictive accuracy has been confirmed by Donald and associates.” It was not our intention in setting up this study to analyze fetal pulmonary maturity and we had only two cases of respiratory distress syndrome in the series; however, in both cases the L/S ratios were less than 2. Our results, therefore, would certainly confirm the value of this test in assessing fetal pulmonary maturity. It is our opinion, however, based on our own results (Table I, Fig. 4) that the test is also of value in the assessment of fetal maturity per se. Bryson and associates’” reported that the L/S ratio did not correlate with maturity; however, on analyzing their data, it appears to us that there is in fact a good trend with gestation in their results. In assessing the L/S ratio with respect to abnormal pregnancy states, we found that it continued to be of value in these situations. In diabetes we found that the expected surge in L/S ratio did occur at 35 weeks, as opposed to the findings of Whitfield and Sproule,2” who found that the surge did not take place consistently. Gluck and Kulovitrh?’ have stated that the L/S ratio is accelerated in Class D, E, and F diabetes, decelerated in Class A, B, and C diabetes. Analysis of our small number of cases, which were all Class A, B, and C, failed to reveal any significant deceleration. Donald and associates” found that even with a L/S ratio greater than 2 infants of diabetic mothers showed a higher risk of respiratory distress syndrome. One might conclude that the L/S ratio must be studied further in diabetic patients and that, until more is known, L/S ratios in diabetics should be interpreted with caution. We did note a slight trend toward acceleration of the L/S ratio in hypertensive disorders, but all values were within the test group range. This trend, we believe, should be explored further. Since Kittrichz2 first investigated the use of Nile Blue stain in identifying fetal cells in amniotic fluid, numerous papers have attested to its value in estimating fetal maturity. 22-24 Anderson and Griffith? found that the presence of orange-stained cells indicated a gestation of 36 weeks. We have, however, noted the presence of orange-
836
Doran
et al.
stained cells at earlier gestation---even on occasions prior to 20 weeks. Brosens and Gordon? have pointed out that false negatives are likely to accur before 36 weeks and false positives do not. In our hands false negatives may occur after 37 Meeks and false positives before then. In our opinion, percentage of lipid-positive cells is the best single test for estimation of fetal maturity. It does, however, have the disadvantage of being subject to individual interpretation by the examiner. We found, as did Bishop and CorsonZ-’ that when fat cells were more than 20 per cent, that a11 cases studied were 36 weeks
July 15, 1974 Am. J. Obstet. Gynecol.
seems to us to be a valid one. This is particularly true if this process improves the over-all accuracy by correcting for the deficiencies of any individual test. This concept was first introduced by Thiede,‘” utilizing lipid-staining cells, creatinine, and bilirubin. In 49 samples he had no false positives and five false negatives (20 per cent) in predicting maturity (37 weeks). Jools and ones8 utilized creatinine, percentage of J lipid-positive cells, and bilirubin in 91 samples of amniotic fluid. Gestation was predicted as a range only and their method of . arriving at the predicted gestation was not clearly described. However, a “very satisfaccalculation was obtained. Lind and tory” Billewicz” described three different scoring systems using cytology, creatinine, and urea difference. The percentage accuracy was only 72, 70, and 60 per cent. Our method, utilizing EPG based on three parameters-percentage of lipid-positive cells? L/S ratio, and creatinine-seems to be more accurate than those previously described, with an accuracy of 95 per cent. Its predictive accuracy does not vary with different periods of gestation and it can be utilized for any sample from 16 weeks to past term. Reporting fetal maturity as estimated period of gestation is more meaningful to the clinician than a number or index. With some deviations the three key parameters and the EPG continue to be of value in abnormal pregnancy states. We intend to investigate further the trends in the three parameters and in the EPG in abnormal pregnancy states, but have sufficient confidence in the tests that we now report to the clinician the values of the three tests plus the EPG based on the three means. We feel that this gives the physician an important and accurate tool for assessment both of fetal maturity and fetal pulmonary maturity. We also feel very strongly that he should incorporate the results of these tests with other clinical, radiologic, ultrasonographic, and laboratory data (estriols, etc.) before making a decision for or against the delivery in any individual pregnancy.
Volume Number
119 6
REFERENCES
Benzie, R. J., Doran, T. A., Harkins, J. L., Jones Owen, V. M., and Porter, C. J.: AM. J. OBSTET. GYNECOL. 119: 798, 1974. 2. Brosens, I., and Gordon, J.: J. Obstet. Gvnaecol. Br. Commonw. 72: 343. 1965. 3. Gluck, L., Kulovich, M. V., Borer, R. C., Jr., Brenner, P. H., Anderson, G. G., and Spellacy, W. N.: AM. J. OBSTET. GYNECOL. 109: 440, 1971. 4. Coch, E. H., and Kessler, G.: Clin. Chem. 18: 490. 1972. 5. Lind, T., and Billewicz, W. Z.: Br. J. Hosp. Med. 5: 681. 1971. 6. Mandelbauxn, B., and Evans, T. N.: AM. J. OBSTET. GYNECOL. 104: 365, 1965. 7. Droegemueller, W., Jackson, C., Makowski, E. L., and Battaglia, F. C.: AM. J. OBSTET. GYNECOL. 104: 424, 1969. 8. Jools, N. D., and Jones, W. R.: Minn. Med. 54: 403. 1971. 9. White, ‘C. A., Doorenbos, D. E., and Bradbury, J. T.: AM. I. OBSTET. GYNECOL. 104: 655,1969. ” 10. Thiede, H.: Discussion, in AM. J. OBSTET. GYNECOL. 104: 668, 1969. 11. Parmley, T., and Miller, E.: AM. J. OBSTET. GYNECOL. 105: 354, 1969. 12. Gillibrand, P. N.: J. Obstet. Gynaecol. Br. Commonw. 76: 898, 1969. 13. Turnbull, A. C.: Devel. Med. Child. Neurol. 12: 375, 1970. 14. Miles, P. A., and Pearson, J. W.: Obstet. Gynecol. 34: 701, 1969.
Amniotic
15.
1.
16. 17. 18.
19.
20. 21. 22. 23. 24. 25.
26. 27. 28.
fluid
tests
for
fetal
maturity
837
Mattison, D. R.: Obstet. Gynecol. 36: 420, 1970. Harrison, R. F.: J. Obstet. Gynaecol. Br. Commonw. 79: 708, 1972. Lemons, J. A., and Jaffe, R. B.: AM. J. OBSTET. GYNECOL. 45: 233, 1973. Donald, I. R., Freeman, R. K., Goebelsmann, U. Chan, W. H., and Nakamura, R. M.: AM. J. OBSTET. GYNECOL. 115: 547, 1973. Bryson, J, J., Gabert, H. A., and Stenchever, M. A.: AM. J. OBSTET. GYNECOL. 114: 208, 1972. Whitfield, C. R., and Sproule, W. B.: Br. Med. J. 2: 85, 1972. Gluck, L., and Kulovich, M. V.: AM. J. OBSTET. GYNECOL. 115: 539, 1973. Kittrich, M.: Geburtshilfe Frauenheilkd. 23: 156, 1963. Bishop, E. H., and Corson, S.: AM. J. OBSTET. GYNECOL. 104: 654, 1968. Huisjes, H. J., and Arendzen, J. D.: Obstet. Gynecol. 35: 725, 1970. Anderson, A. B. M., and Griffiths, A. D.: J. Obstet. Gynaecol. Br. Commonw. 75: 300, 1968. Doran, T. A., Bjerre, S., and Porter, C. J.: AM. J. OBSTET. GYNECOL. 106: 325, 1970. Pitkin, R. M., and Zwirek, S. J.: AM. J. OBSTET. GYNECOL. 98: 1135, 1967. Moore, W. M. O., Murphy, P. J., and Davis, J. A.: AM. J. OBSTET. GYNECOL. 100: 908, 1971.