Accelerated clotting time (ACT) : a simple test to assess foetal maturity.
Several methods have been described in the literature to assess the foetal maturity (M). The older established tests like L/S ratio, serum creatinine, cytological examination and spectrophotometric analysis of the amniotic fluid (AF) are still not ideal. We describe below a new test to assess the foetal maturity by measuring "Accelerated Clotting Time (ACT)" which has been found to be a simple, rapid and economic method. We have tried to evaluate its predictability by comparing the results with those of two other tests viz. (a) Nile blue sulphate stained fat cell measurement and (b) OD400 measurement both of which have been routinely used in our department for the assessment of foetal maturity and also with gestational age determination by paediatric assessment at delivery and occurrence of respiratory distress syndrome in the newborn.
The tests were performed on the amniotic fluid of  patients admitted in the Department of Obstetrics and Gynaecology, K.E.M. Hospital. Thirty samples were from patients admitted in the labour ward in active labour,  were from cases who had come for M.T.P. during the second trimester between the 16th and 20eth week of gestation, six samples were from the amniotic fluid stored over a period of ½-1 year at 3°C of patients with doubtful maturity and whose ODD400 and nile blue sulphate results were known.
In  cases the amniotic fluid was blood stained and in  cases, meconium stained. All the  tests carried out in our study were performed within two hours of collection of the amniotic fluid and the results were correlated with the gestation o£ the baby at delivery and also with the presence or absence of the respiratory distress syndrome.
(a) Accelerated clotting time, ,  is thought to be a possible measure of maturity of coagulation systems at the uteroplacental sites. 1.5 ml of patient's blood was taken quickly from the cubital vein and added to  ml of freshly drawn amniotic fluid at 37°C in a 10 ml plastic test tube. The time taken for the blood: amniotic fluid mixture to clot was measured in seconds. Clotting time of less than 110, seconds usually indicated a mature fetus of more than  weeks' gestation. ACT of more than 110 seconds indicated a fetus of less than  weeks' gestation with 95-120 seconds being the transitional zone.
(b) ODD at 400 mµ, , : Fresh amniotic fluid was centrifuged at 3200 RPM (2000 g) for  minutes. Absorbance of the supernatent fluid was measured by a Spectronic  spectrophotometer at 400 nm. In the absence of blood or meconium staining, the ODD400 of more than 0. usually indicated a mature fetus and less than 0.28 an immature fetus.
In all M.T.P. cases, the fluid was collected by transabdominal amniocentesis as also in  cases of patients in labour. In the rest of the cases, the amniotic fluid was collected by the transvaginal route at the time of artificial rupture of the membranes.
(c) Nile blue sulphate test5: One drop of the amniotic fluid was mixed directly on a slide with  drop of 0.1% aqueous nile blue sulphate solution. A cover slip was applied and the preparations was examined under the 45X of the microscope.
In each preparation, the number of orange cells as a proportion of the stained cells was counted and the results were interpreted as shown in [Table 1].
Graph  demonstrates the clotting time in seconds in normal pregnant women in various weeks of pregnancy. The dotted line at 110 seconds separates the amniotic fluid into two broad groups of mature and immature foetuses. Samples of mature fetuses show clotting times of less than 110 seconds while those of immature foetuses show clotting times of more than 110 seconds. The second line intersects at approximately 34 weeks indicating that the maturity is probably reached at that period of gestation.
Graph  compares the clotting time measured in seconds with the maturity of the amniotic fluid as obtained by the nile blue sulphate test. The vertical line intersects the horizontal line at 20% fat cells which is the criteria for maturity by the nile blue sulphate test. When the results of the two tests correlated with each other, they lay in the upper left and the lower right quadrants, while when they did not, they fell in the other two quadrants.
[Table 2] shows the relationship between the accelerated clotting time and the nile blue sulphate test as well as the OD400 in the  cases examined.
Meyer3 first studied the syndrome of amniotic fluid embolism. Steiner and Lushbaugh7 demonstrated the presence of disseminated microemboli containing squamae in the lungs of patients who died of obstetric shock in late pregnancy. Infusion of immature amniotic fluid did not produce the same result. It is apparent that only cloudy amniotic fluid rich in squamae and vernix will promote the formation of thromboemboli through its thromboplastic or clot accelerating activity.
Thromboplastins are non-specific phospholipid protein complexes liberated from damaged cells. Their release initiates the extrinsic or tissue coagulation pathway promoting the conversion of prothrombin to thrombin and finally fibrinogen to fibrin. As term approaches, the amniotic fluid contains increasing amounts of both free phospholipid and thromboplastins in the form of increasing numbers of desquamating and degenerated fetal cells; and hence the clot accelerating activity.
In our study of  cases, 11 cases were for MTP. In  of these cases the clotting time was greater than 130, seconds. In one case, the clotting time was only  seconds. Significantly, this fluid contained. a lot of cell debris of unknown origin. In  cases, the amniotic fluid was stored for periods varying between  months to  year and here the clotting time increased beyond 150 seconds irrespective of whether the fetus was mature or not. Hence we concluded that stored amniotic fluid cannot be used for maturity studies by this method and these cases were not included in our calculations.
Where the clotting time was less than 110 seconds babies were usually mature and did not develop respiratory distress syndrome (RDS) as was seen in  cases in our study. With clotting time of more than 110 seconds the chances of RDS increased rapidly. Four cases which died due to prematurity had clotting times varying between 130 and 160 seconds and birth weights between 1.0 and 1.8 kg. 95-120 seconds was considered the transitional zone between prematurity and maturity. In  amniotic fluid samples with clotting times between 95-110 seconds, babies birth weight varied between 1.9 and 2.1 kg. None of these babies developed RDS. In  cases the clotting time was  and  seconds and the babies had birth weights of 2-2.050 kg; however, both these cases were full term as calculated from their last menstrual period and were cases of pre-eclampsia with intra-uterine growth retardation. None of these babies developed respiratory distress syndrome.
When the accelerated clotting time was compared with maturity as determined by fat cells, there were  cases where the two results did not correlate. In all  cases the nile blue sulphate test was found to have given a false negative result. In 4 cases the amniotic fluid was meconium stained, in  cases blood stained and in  cases the cause of the discrepancy was not detectable.
On comparing the accelerated clotting times with OD400,  cases were found to have OD400 of more than 0.28 and ALIT less than 110 seconds indicating a mature fatus by both parameters. Of these,  newborns were mature with no signs of RDS while one was an MTP case.
OD400 was less than 0.28 and ACT more than 110 seconds in  cases. Of these, five babies were premature of which  died and one was from the amniotic fluid stored for a prolonged period of time.
OD400 of more than 0.28 and ACT more than 110 seconds was seen in  cases. Of these,  were the cases of MTP (OD400 more than 0.28 due to increased bilirubin content at earlier gestations),  were from stored amniotic fluid and two fluids were meconium and one blood stained. The foetuses associated with meconium and blood staining were fully mature.
In Hastwell's study of 244 cases, the false positive rate was 1.3% and the false negative rate was 11%. In our study of accelerated clotting times, the false positive rate was 2.12% and the false negative rate was nil. As compared to that, nile blue sulphate test had a false negative rate of 23.4% especially in the presence of blood and meconium while OD400 was also not reliable under the same conditions. Accelerated clotting time is adversely affected by storage and may also be affected by blood though the latter was not evident in our study.
We thank the Dean, K.E.M. Hospital, Bombay, for permitting us to report the hospital data.