Transplacental haemorrhage and maternal iso-immunizationDaxa M Mehta, Snehlata C Gupte, HM Bhatia
Blood Group Reference Centre (I.C.M.R.), Seth G.S. Medical College, Parel, Bombay-400 012, India
Foetomaternal haemorrhage is one of the important factors influencing maternal iso-immunization. In the present report attempt is made to study various aspects of foetomaternal haemorrhage. While the overall incidence of foetal cell leak was observed in 21.43%, the incidence in the complicated deliveries ranged from. 27.94 to 85.72%. Incidence was not influenced by parity, however ABO compatible deliveries showed slight increase in the incidence (22.56%) as compared to ABO incompatible (18.75%) deliveries. ABO incompatible foetal cells were eliminated within 24 hours after delivery. Incidence of foetal cell leak was 8.28% in medical termination of pregnancy. Anth-Rh was produced in two out of nine women having foetal cell leak.
Mechanismm of Rh (D) immunization by transfer of foetal cells, was postulated by Levine et. al. (1941).  However the direct evidence for the transfer was presented by Javer and Reiss in 1952  Various techniques have been used to study foetal cell leak into maternal circulation. ,,,, Using either of these techniques, several foetal cell leak studies have been done during pregnancy and after delivery ,, In the present report various aspects of foetomaternal haemorrhage are presented.
Patients studied were drawn from the antenatal clinics and labour ward of the Cama and Albless Maternity Hospital, Bombay. Cases of medical termination of pregnancy were obtained from Nowrosjee Wadia Maternity Hospital.
Blood samples were collected in oxalated and plain bulbs. Among the cases with full term delivery, the first sample was collected within two hours of delivery and the subsequent samples were collected after six to twelve and twenty to twenty four hours after delivery.
All the details like age, past obstetric history, type of the delivery were obtained from the labour ward records. Attempt was also made to contact all the Rh (D) negative patients, 3 and 6 months after the delivery, for the detection of Rh (D) antibodies.
All serological methods used were the standard tube techniques outlined by Bhatia,  using locally prepared diagnostic reagents. Maternal blood samples were examined for ABO and Rh (D) groups, presence of Rh antibodies and detection and quantitation of foetal cells. Investigations on new born infants included ABO and Rh (D) groups and direct Coomb's test.
Detection of foetal cells:
One drop of blood was mixed with two drops of normal saline and one drop of this was taken on a clean slide and made into thin smear. Blood smears, thus prepared were fixed by keeping in 80% alcohol for 5 minutes and then rinsed with water and air dried. Slides were stained by Nierhaus and Betke (1968)  acid elution technique, using following reagents.
Solution A:- 0.75 gms haematoxylin
+100 ml of 95% alcohol.
Solution B: - 2.4 gms. Ferric Chloride
+ 2 ml of 25% HCI +
100 ml distilled water
Counter stain: 0.1% erythrocin in distilled water.
Five parts of solution A were mixed with one part of solution B. This mixture was used for six weeks with filtration every 4-5 days.
Fixed slides were kept in the mixture for exactly 20 seconds, rinsed in distilled water and counter stained for 2-3 minutes, in 0.1% erythrocin, washed with water and air dried. Foetal cells were recognised under microscope by their well defined distinct red colour, while the adult cells were light coloured ghost cells. Each slide was examined for 100,000 adult cells and the number of foetal cells encountered were counted. Amount of foetal blood in the maternal circulation was calculated on the following basis as obtained by the experimental artificial mixture of adult and cord cells.
300 Pregnant women were studied for the presence of foetal cells. Foetal cell leak was observed in 12 cases (4%). No foetal cells were detected in 129 women examined during I or II trimester, while 12 out of 171 women (7.01%) during third trimester showed the foetal cell leak.
[Table 1] gives the incidence of foetal cell leak among the women, at delivery in relation to their parity. Incidence of foetomaternal haemorrhage was 21.4 per cent among all the women. Incidence was slightly higher (26.82%) among primiparous women than in other parities (17.65 to 19.9%) though the difference was not statistically significant.
Findings in [Table 2] suggests that the incidence of foetal cell leak was higher in complicated deliveries than in normal deliveries. Highest incidence of 85.7 per cent was observed in twins deliveries.
While the overall incidence of foetal cell leak was 21.3%, it was 20.73% in ABO compatible and 18.75 per cent in ABO incompatible deliveries [Table 3]. [Table 3] also gives the follow up studies of those women during 24 hours. Findings suggest that though the incidence of foetal cell leak is 20.3% among ABO compatible and 18.75 per cent among incompatible, when the samples were collected within 2 hours after delivery the incidence of foetal cell leak at 12 and 24 hours was 20.73 and 13.42 in ABO compatible and 5.1 and 1.7% among ABO incompatible deliveries.
Incidence and amount of foetal haemorrhage among Rh positive and Rh negative women was not significantly different. The data showed that 49 out of 222 Rh (D) negative (22.0%) and 98 out of 466 Rh positive (21.00%) women had foetal cell leak. Amount of foetal bleed was also similar among Rh positive and Rh negative mothers. In 60% of the cases having foetal leak, the amount of foetal cell leak was less than 0.15 ml. 22% had a leak up to 1.0 ml., 6% had upto 2.5 ml and only one case had more than 5 ml.
Thirty three cases of Rh (D) negative women were available for follow up to detect Rh antibodies. Among these 33 women, 9 had foetal cell leak. Anti-Rh was detected in 2 out of 9 Rh (D) negative women having foetal cell leak. In both these cases the amount of foetal leak was 0.5 and 1.0 ml. respectively, while the remaining 7 cases had foetal cell leak less than 0.5 ml.
Foetomaternal haemorrhage in medical termination of pregnancy
193 cases of medical termination were studied for foetal cell leak. Findings given in [Table 4] indicate that. 8.29 percent of the medical terminations had foetal cell leak. Incidence was higher (14.06%) in those where intra-amniotic hypertonic saline injections were given for terminating the pregnancy. In all the sixteen cases the amount of foetal cell leak was less than 0.1 ml.
Haemolytic disease of the newborn due to Rh (D) immunization is more common in Western countries than in India, since the incidence of Rh (D) negatives is 15% in Caucasians. In India the incidence of Rh (D) HDN has been reported as 1:30 Rh (D) negative women or 1:600 randam pregnancies.  One of the factors stimulating Rh immunization is foeto-maternal haemorrhage which may occur either during pregnancy or at the time of delivery. Woodrow and Finn  reported an incidence of 12.6% during pregnancy with increasing incidence from the first to the third trimester. In the present study the foetal cells were not detected in women during the first and second trimester. Present data, like those of other investigators , gives significantly higher incidence of foetomaternal haemorrhage in complicated deliveries in comparison to normal deliveries. Incidence was particularly high (85.7%) in twins delivery. Such significantly high incidence among women with complicated deliveries may suggest this as one of the factors influencing Rh (D) immunization. Among the MTP patients, intraamniotic saline procedure involved more risk of foetal cell leak (14.06%). The role of ABO compatibility is also reflected in the present data since the foetal cell leak was very much similar among ABO compatible and incompatible deliveries. ABO incompatible foetal cells were eliminated within 24 hours. These finding support the observation of higher risk of Rh immunization in Rh negative women with ABO compatible pregnancies.
The present data does not give any correlation of foetal cell leak with parity. Zipursky et al  observed that primary sensitisation requires 0.5 ml. or more blood and sensitisation may take place after a booster dose of 0.1 to 0.2 ml. of blood. In the present study, out of 9 Rh (D) negative women showing foetal cell leak two had foetal cell leak of 0.5 and 1 ml respectively after delivery. Both of these women developed Rh antibodies during their subsequent pregnancy suggesting primary response due to delivery. Remaining seven cases who did not develop anti-Rh had foetal cell leak less than 0.5 ml.
Authors are thankful to the Superintendent of Cama and Albless Hospital, Bombay and Dean of the Nowrosjee Wadia Maternity Hospital for the facilities provided.
[Table 1], [Table 2], [Table 3], [Table 4]