PRENATAL DIAGNOSIS, VOL. 3 , 4 1 4 6 (1983)

FETAL BLOOD GROUP STUDIES DURING

MANAGEMENT OF SEVERE ISOIMMUNIZATION MID-TRIMESTER PREGNANCY AND THE

IAN MACKENZIE,* CLAIRE M. GUEST? AND PETER I. BOWELLS Nufield Department of Obstetrics and Gynaecology and Oxford Regional Transfusion Centre,

John Radclire Hospital, Headington, Oxford, OX3 9D U, U.K.

SUMMARY Fetal blood samples collected at 14-22 weeks' gestation by fetoscopy have been analysed for 25 different red cell antigens and anti-A antibodies. The results obtained demonstrate that the technique can be used to determine fetal blood groups during mid-pregnancy and also to provide the opportunity to study the physiological and pathological developments of blood groups and their antibodies and to manage more rationally those pregnancies threatened by erythroblastosis fetalis.

KEY WORDS Blood grouping Mid-trimester Fetoscopy Rhesus isoimmunisation

INTRODUCTION

Fetoscopy offers many possibilities for the diagnosis of inherited disorders during early pregnancy (Kan et al., 1977; Mahoney et a[., 1977; Firschein et al., 1979; Cordesius et al., 1980). Pregnancies complicated by erythroblastosis fetalis resulting from incompatibility between fetal and maternal red cell antigens still occur despite the introduction of anti-D immunoglobulin prophylaxis (Clark and Whitfield, 1980). At present the management of severely affected cases depends upon extensive plasma exchange of the mother commencing in early pregnancy or intraperitoneal fetal transfusions in later pregnancy. In the event of the father of the pregnancy being heterozygous for the offending blood group gene, knowledge of the fetal blood group would make interpretation of high circulating antibody levels in maternal serum easier and management of the pregnancy more rational as suggested by Philip et al., 1978.

The present study was performed to decide how reliably fetal blood group antigens could be determined during mid-trimester pregnancy and is part of a larger work designed to evaluate the feasibility and suitability of fetoscopy in the management of haemolytic disease of the newborn (MacKenzie et al., 1982). In addition to the determi- nation of fetal blood groups, this work has provided an opportunity to study the development of red cell antigens and to monitor the passive transfer of maternal antibodies to the fetus.

* Clinical Reader. t Senior Medical Laboratory Scientific Officer, f Chief Medical Laboratory Scientific Officer.

0 197-38 5 1 /83/0 1004 1-06 $0 1 .OO 0 1983 by John Wiley & Sons, Ltd.

Received 21 May I982 Revised 28 September 1982

Accepted 2 October 1982

42 I. MACKENZIE. C. GUEST AND P. BOWELL

PATIENTS AND METHODS

Sixty-four patients admitted for therapeutic abortion within the terms of the Abortion Act 1967 consented to be studied: the gestational ages of the pregnancies ranged from 14 to 22 weeks, with 15 at 16 weeks’ gestation or less, 23 at 17-18 weeks’ gestation, 20 at 19-20 weeks’ gestation and six at 21-22 weeks. Samples of fetal blood were collected at fetoscopy using the technique previously described (MacKenzie and Maclean, 1980) immediately prior to the intra-amniotic administration of prosta- glandin E2 to induce abortion. In each instance, between 500 and 1500 pI of fetal blood was collected by aspiration from one of the umbilical cord vessels into sterile plastic 2 ml syringes and stored in plastic bottles for blood group analysis. A further 500 pl sample of fetal blood was examined using Shephard’s modification (Shephard et al., 1962) of the Kleihauer staining technique (Kleihauer et al., 1957). Red cell pheno- typing was performed using 25 antisera of differing specificities. These sera were used by the recommended techniques and were controlled in the accepted serological manner. An appreciation of antigen strength was determined from the score of the reaction. On occasions certain antisera not absorbed for anti A/B haemagglutinins, could not be used because the blood samples were group A and/or group B incom- patible. For 43 of the cases, matched samples of maternal blood were also collected and examined with the same 25 antisera, while for the remaining 21 cases, maternal ABO group and Rhesus factor only were determined. For all fetal and matched maternal samples, sera were tested at a dilution of 1/10 on the AutoAnalyzer for the presence of anti-A agglutinins (Bowell et af., 1980).

Following the research studies, fetal blood samples were obtained from two preg- nant Rhesus sensitized patients, both with a history of stillbirth due to erythroblas- tosis: in each case the father of the pregnancy was heterozygous for the Rhesus (D) factor. The procedure was carried out at 18 weeks’ and 22 weeks’ gestation respectively.

RESULTS

The results of the Kleihauer tests indicated that all the red blood cells in the fetal blood samples were fetal in origin. This finding was supported by the absence of a mixed field appearance in the blood grouping tests; there was no evidence of contrary homozygosity in the matched fetal/maternal samples. In the research study all the fetal samples tested were direct antiglobulin test negative.

The results of the blood grouping analyses on the fetal samples are summarized in Table 1. In the majority of instances the 25 blood group antigens examined were present both in normal strength and frequency. No positive reactions were observed with anti-Lea, anti-Leb, and anti-Sda sera. Weakened expression of A, e, Kpb, Jkb and Fyb antigens was encountered in a small number of cases. However, there was no correlation between reduced antigen strength and the gestational age of the fetuses examined.

There was a marked contrast both in strength and frequency of the PI antigen when fetal and maternal results were compared (see Table 2). The fetal samples showed an increased frequency of PI negative results (P c 0.001) as well as a tendency towards reduced antigen strength. All the maternal samples reacted in a comparable manner to other non-pregnant control cells tested in parallel with this study both in strength

FETAL BLOOD GROUP STUDIES 43

Table 1 . Nature and frequency of fetal blood group antigens exhibiting reduced antigen strength

Blood group antigens examined Antigens exhibiting weakened expression

Anti-sera used No. tested Nature No. weak and total positive?

Anti-A; B Anti-Cw; C; D; E; c; e Anti-M; N Anti-S; s Anti-PI Anti-Lua Anti-Lea; Leb Anti-K; k; Kpa; Kpb Anti-Fya; Fyb Anti-Jka; Jkb Anti-Sda

64 64 64

*56/45 *44 *38 *62/56 64

*50/46 *56/34 *51

A e None None P1 None Lea; Leb KP FYb Jkb Sda

3 of 25 1 of64

- See Table 2 -

All negative 2of64 1 of 35 1 of 22

All negative

* Certain antisera had not been absorbed for anti A/B agglutinins and could not be used for all

t The majority of antisera gave normal strength reactions and to normal frequencies. fetal samples.

Table 2. PI antigen in matched fetal and maternal blood

Sample Gestation Total PI reaction strength - (Wk) tested +++ + + + *

Maternal - 33 4 (12%) 10(30%) 7 (21%) 3 (9%) 9 (27%) 2(4%) 4(9%) 3 (7%) 35(79%) Fetal Total 44 -

1(11%) - 8 (89%) 14-16 9 17-19 22 - 2(9%) 3(14%) 1(5%) 16(73%)

- 2 (15%) 11 (85%) 20-22 13

- - - -

Table 3. Frequency and potency of anti-A in the sera of group 0 and group B fetuses of group 0 mothers

Gestation AutoAnalyser response A.O.D.

Total 0-0 .1 0-1-0.2 0.2-0.3 0.3-0.4 0.4-0.5 ~ 0 . 5 (Wk)

A.O.D. response 0-0.1 indicates absence of antibody. All maternal samples A.O.D. z 0.5.

and frequency of reaction with the exception of Lewis and Sda which are recognized to be influenced by pregnancy (Race and Sanger, 1975).

Table 3 lists the results of anti-A studies using the AutoAnalyzer technique in 23 cases when the mother was group 0 and fetuses were group 0 or B. All maternal samples gave A O.D. response in excess of 0.5 while in 12 (52 per cent) fetal samples no anti-A antibodies could be detected. The presence and potency of the anti-A antibody in fetal serum tended to appear more frequently and to a higher potency

44 I. MACKENZIE, C. GUEST AND P. BOWELL

with more advanced pregnancy. The sera of the three group 0 fetuses of group B mothers, however, did not exhibit anti-A activity. For seven group A fetuses carried by a group 0 mother there was no evidence of either free anti-A in the fetal serum or red cell sensitization and on nine occasions, when the fetus was either group 0 or B and the mother was group A, no anti-A could be found in the fetal serum.

For the two patients undergoing diagnostic fetoscopy and blood sampling, the fetus in one was shown to be Rhesus negative and the pregnancy continued to term while the other was shown to be Rhesus positive, direct antiglobulin positive and severely affected by erythroblastosis fetalis: this latter fetus was already noted to be grossly hydropic at examination and died three days later, the diagnosis confirmed at postmortem.

DISCUSSION

We have confirmed the findings of Philip et al. (1978) that red cell phenotyping of samples of fetal blood obtained at fetoscopy can be reliably performed in the second trimester. By aspirating blood from the umbilical cord under direct vision using our method (MacKenzie and Maclean, 1980) or that proposed by Rodeck and Campbell (1 978) the difficulties of sample contamination experienced by Philip and his colleagues have been largely circumvented.

The results obtained indicate that the majority of red cell antigens examined are fully formed early in the second trimester of pregnancy. This work largely supports the findings of Toivanen and Hirvonen (1973) with the possible exception of the strength of reaction with the anti-Lua antisera, for the three Lua positive fetal blood samples in our study reacted in a comparable manner to maternal and other adult control cells.

On eight occasions the fetal expression of the blood group antigens was weaker than maternal and non-pregnant adult controls; there was no indication that there was any direct association between antigen strength and gestational age. On three occasions the blood group antigen concerned was A. It is possible that this is attribu- table to the known weakened expression of the A2 sub-type as demonstrated by Constantoulakis et al. (1973). On these occasions, when the blood group antigen was absent or very poorly developed (Lea, Leb, Sda and PI), there would be little danger of haemolytic disease of the newborn: furthermore, antibodies to these factors are usually IgM in nature and therefore unable to cross the placenta.

Ikin et al. (1961) have previously reported that the PI antigen, although less frequent in fetal than adult samples, is more commonly present at an early gestation with increased strength of agglutination, especially in fetuses with a crown-rump length of 10 cm or less-equivalent to a gestational age up to 16 weeks. In the present study there was no evidence of a similar trend, but the 20 per cent frequency of PI positive fetuses compared well with Ikin’s figures.

Using the sensitive AutoAnalyzer technique, no group 0 or B fetuses of group A mothers had anti-A antibodies in their sera, indicating that the fetus is incapable of producing the antibody at the gestations studied. The passive transfer of maternal anti-A antibodies was observed on 11 of the 23 occasions (48 per cent) from group 0 mothers to group 0 or B fetuses. The presence and strength of anti-A antibodies in the fetus tended to increase with gestational age. The absence of anti-A in the sera of three group 0 fetuses of group B mothers is possibly further evidence that the pre-

FETAL BLOOD GROUP STUDIES 45

dominant anti-A immunoglobulin of group B maternal sera is IgM in nature, unlike group 0 maternal sera where IgG anti-A is commonly found (Rawson and Abelson, 1960). Interesting also is the observation that anti-A antibodies present in Group 0 mothers were not found in the sera of group A fetuses.

Clearly, blood sampling using the fetoscope has application within the management of haemolytic disease of the newborn. In cases where incompatibility between mother and fetus are thought to exist, examination of fetal blood samples can now be con- firmatory. However, because of the risk of inducing abortion (Nathan et al., 1979) or transplacental haemorrhage resulting from fetoscopy, careful selection of cases is essential. The technique would be of benefit in cases where the father of the pregnancy is known to be heterozygous for the offending blood group antigen in the following situations: (i) patients with a history of severe erythroblastosis fetalis due to blood group incompatibility including D, E, E and Kell and (ii) patients with a high maternal serum anti-D level in excess of 20 iu/rnl-l existing during the first twenty weeks of pregnancy (Bowell et al,, 1982). The knowledge that the fetus is blood group compatible allows for a considerable saving of time, effort and expense by obviating the need for further diagnostic tests and exacting methods of treatment.

ACKNOWLEDGEMENTS

Acknowledgement is made to Dr Douglas Maclean, Dr Stephen Lloyd-Evans and Sister Anna Fry for assistance in collecting the fetal blood samples, Dr Fred Bolton and staff for performing the Kleihauer ananlyses and Dr Harold Gunson, formerly Director of the Oxford Regional Transfusion Centre, for advice in planning this study. The fetoscopic equipment was purchased with a grant awarded by the Oxford Hospital Services Development Trust.

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46 I. MACKENZIE, C. GUEST AND P. BOWELL

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