7 Clin Pathol 1997;50:193-196

ACP Broadsheet No 150 March 1997

Antenatal serological testing and prevention ofhaemolytic disease of the newborn

J K M Duguid

IntroductionRoutine antenatal serological screening hasbeen practised throughout the United King-dom and worldwide for about 30 years.' Origi-nally introduced to detect pregnancies at risk ofhaemolytic disease of the newborn (HDN) dueto Rh anti-D antibodies, its continued use hasled to an increasing realisation of the clinicalimportance of other red cell antibodies in thepathogenesis of HDN.Three factors are essential in the pathogen-

esis ofHDN: maternal red cell antibodies mustcross the placenta, the fetal red cells must pos-sess the appropriate red cell antigen againstwhich the antibodies are directed, and the anti-bodies must be capable of causing immunedestruction. The clinically relevant antibodiesare, therefore, virtually always IgG and reactiveat 37°C. About 2-5% ofwomen will have suchantibodies as a result of transfusion, pregnancy,or both; it is, therefore, important for the preg-nant patient, the fetus, and their clinicaladvisors that appropriate antenatal screeningtests are performed.

This Broadsheet has beenprepared by the authors at

the invitation of theAssociation of ClinicalPathologists who reserve

copyright. Further copies ofthis Broadsheet may beobtainedfrom thePublishing Manager,J7ournal of ClinicalPathology, BMvA House,Tavistock Square, LondonWClH 93R.

National BloodService, Mersey andNorth Wales, LiverpoolJ K M Duguid

Correspondence to:Dr Duguid, National BloodService, Mersey and NorthWales, West Derby St,Liverpool L7 8TW.

Accepted for publication6 November 1996

Aim of testingThere are three main reasons for routineantenatal serological testing:(1) to identify pregnancies at risk of HDN;(2) to identify RhD negative women for whom

anti-D immunoglobulin prophylaxis willbe required; and

(3) to ascertain maternal ABO group.

There is, however, no universal agreement on

the type of testing nor its optimal timing duringpregnancy.2 There are currently recommenda-tions that all women should be tested as early inpregnancy as possible, usually at 8 to 12 weeks'gestation.3 This initial testing must includeABO and RhD typing as well as a screening testto detect any irregular red cell antibodies. Fur-ther testing for irregular antibodies should beundertaken in all patients (irrespective of RhDtype) at a later stage in pregnancy. A suggestedtime for this to be undertaken is between 28and 36 weeks' gestation.3 Women who are

found to have red cell antibodies at initial test-ing may require more frequent testing through-out their pregnancy.

ABO typingInitial tests to establish maternal ABO type areconsidered important so that the mother'sABO group is recorded should she requiretransfusion support during her pregnancy. It isalso good practice to ascertain maternal ABOtype early, so that anomalous results can beinvestigated.There is no value in identifying group 0

mothers with high titres of anti-A or anti-B.Antenatal testing of these antibodies has beenshown to have no value in predicting theincidence ofABO HDN. The lytic potential ofmaternal IgG anti-A and anti-B is veryvariable, and frequently the IgG subclass whichpredominates in these group 0 patients is IgG2,a subclass which does not induce red celldestruction. A further reason for being unableto predict the incidence of ABO HDN is thevery variable density of A and B antigen onfetal red cells.4There is an increased incidence and an

increased severity of ABO HDN in certainpopulations: Arabs, Negroes, South-EastAsians, and Latin-Americans, but again there islittle predictive value from serological monitor-ing. However, it is essential that early postnataldiagnosis and management should be under-taken in children born to women from theseparticular ethnic groups.There are occasional patients with a history

of severe and recurrent ABO HDN associatedwith severe neonatal anaemia requiring ex-change transfusions. In this rare group ofpatients antenatal monitoring of anti-A andanti-B using an antibody dependent cell medi-ated cytotoxicity assay may be of use inpredicting ABO HDN.4Once a pregnant woman's ABO group has

been established by concordant results fromtwo samples obtained on different occasionsthere is no further need for ABO testing.

RhD typingEstablishing a mother's RhD type is anessential part of early pregnancy testing.Fifteen per cent of women will be RhDnegative and to try and prevent the develop-ment of anti-D antibodies during pregnancyand delivery they will require anti-D

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immunoglobulin prophylaxis. Despite the factthat anti-D immunoglobulin has been usedroutinely for more than 25 years, anti-Dantibodies are still the most common cause ofHDN, and their incidence is higher in theUnited Kingdom than in other Europeancountries.5 This continuing rate of anti-D sen-

sitisation is thought to result from a variety ofcauses including: failure to administer ad-equate doses of anti-D immunoglobulin whenpotentially sensitising antenatal episodesoccur; inadequate monitoring of routine post-natal anti-D immunoglobulin dose in the pres-ence of large feto-maternal haemorrhages; andprimary sensitisation occurring in late preg-nancy resulting from small undetected feto-maternal haemorrhages.6Once a mother's RhD type has been

established, assuming there are no irregular redcell antibodies present, both RhD negative andRhD positive mothers should be tested againlater in pregnancy, in case they have developedred cell antibodies capable of causing HDN.The exact timing of this testing is debated butbetween 28 and 36 weeks' gestation is usuallyrecommended.Each RhD negative mother needs to be noti-

fied of her RhD negative status, as does herattending clinician. This is to ensure that allconcerned in her antenatal management areaware of her RhD negative status and the needfor appropriate administration of anti-D im-munoglobulin to cover any sensitising epi-sodes. Local transfusion centres and pharma-ceutical companies responsible for anti-Dimmunoglobulin production produce bloodgroup cards and patient information leafletsexplaining the significance of being RhD nega-tive, and the occasions when anti-D immu-noglobulin needs to be administered.

Indications for antenatal anti-Dimmunoglobulin administrationTHERAPEUTICWomen who are RhD negative with no anti-Ddetectable in their serum should receive anti-Dimmunoglobulin whenever a sensitising epi-sode occurs (table 1). Anti-D immunoglobulin(250 IU) should be administered before 20weeks' gestation, and 500 IU at later stages ofpregnancy. A Kleihauer test should be per-formed within 72 hours of administration.Additional anti-D immunoglobulin can subse-quently be given as required to clear any fetalcells detected. Administration of anti-D immu-noglobulin should be repeated every six weeksif further sensitising episodes occur, or if anepisode such as a threatened abortion has beenon-going during that time.7

PROPHYLACTIC ROUTINE ANTENATAL

PROPHYLAXISRoutine antenatal prophylaxis is practised inCanada and the United States and at some

centres in the United Kingdom. The purposeof this is to reduce the incidence of thirdtrimester sensitisation to RhD by preventingsensitisation from feto-maternal haemorrhagethat occurs with increased frequency duringthis trimester.6 In the United Kingdom when

routine antenatal prophylaxis is undertaken,500 IU of anti-D immunoglobulin is adminis-tered at 28 weeks', and again at 32 weeks' ges-tation. In North America a single dose of1500 IU is administered at 28 weeks' gestation.

It is important to indicate clearly whenanti-D immunoglobulin has been adminis-tered, whether prophylactically or to cover aknown sensitising episode, as this passiveanti-D will be detected in any subsequent rou-tine antenatal serological tests. There is cur-rently no way of differentiating betweenpassively acquired and actively producedanti-D and, therefore, great care must be takenin interpreting the presence of anti-D in latepregnancy.

It is essential that all RhD negative womenwho deliver an RhD positive child receive thestandard United Kingdom dose of 500 IU ofanti-D immunoglobulin postdelivery. The onlyexceptions are women who are known to havehad anti-D antibodies throughout their preg-nancy, due to previous sensitisation. The pres-ence ofnon-D antibodies and prior administra-tion of antenatal anti-D immunoglobulin arenot contraindications to postnatal anti-Dimmunoglobulin administration. The standardpostdelivery dose of anti-D immunoglobulinvaries throughout the world. The dose used inthe United Kingdom was recommended by a

working party of the Medical Research Coun-cil following a trial of different doses of anti-Dimmunoglobulin.' It was realised that about0.7% of women would have a feto-maternalhaemorrhage of > 4 ml, and for these women adose of 500 IU anti-D immunoglobulin wouldbe insufficient. These women should be easilyidentified by the routine use of a Kleihauer testand additional anti-D immunoglobulin can beadministered. In North America a standarddose of anti-D immunoglobulin of 1500 IU isadministered and Kleihauer testing is only per-formed for certain "high risk" deliveries (mul-tiple gestation, placental abruptio, placentapraevia, and manual removal of the placenta).'Within the European Community the currentrecommendation is that a dose of 1500 IUanti-D immunoglobulin be used routinely butthere is no recommendation regarding Klei-hauer testing.'" The use of these larger doses ofanti-D immunoglobulin will prevent sensitisa-tion from feto-maternal haemorrhage up to15 ml; however, approximately 0.3% ofdeliver-ies are associated with feto-maternal haemor-rhage greater than this and, unless routineKleihauer testing is undertaken, sensitisationwill occur.

The use ofKleihauer testing in theprevention ofHDNKleihauer testing is a means of detecting fetalred cells in the maternal circulation. Fetal redcells are resistant to acid lysis because of the

presence of fetal haemoglobin. Maternal bloodsubjected to acid lysis and then counterstainedresults in fetal cells being preferentially identi-fied. A quantitative estimate of the size of anyfeto-maternal haemorrhage can then bemade."

Table 1 Antenatal episodeswhich may cause RhDsensitisation

AbortionSpontaneous > 12 weeks'

gestationTherapeutic, any gestation

Chorionic villous samplingAmniocentesisExternal cephalic versionAntepartum haemorrhageAbdominal trauma

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Antenatal serological testing and prevention of haemolytic disease of the newborn

The routine postnatal dose of 500 IU ofanti-D immunoglobulin is sufficient to preventsensitisation following fetal haemorrhage of upto 4 ml. If Kleihauer testing indicates a largerbleed then additional anti-D immunoglobulinis required. Whenever anti-D immunoglobulinis administered, postnatally or antenatally, tocover a potentially sensitising episode, aKleihauer test must be performed. If additionalanti-D immunoglobulin has to be adminis-tered, repeat Kleihauer testing should beundertaken every 48 hours until fetal red cellshave cleared.7 The main disadvantage of theKleihauer test is that it does not differentiatebetween RhD positive and RhD negative fetalred cells. It is also a relatively imprecise test thatrequires considerable technical skill for correctinterpretation. 2 There is also considerableinterlaboratory variation in the way a Kleihauertest is performed and in how results areinterpreted and reported, and more impor-tantly whether specific comments are maderegarding the need for additional anti-Dimmunoglobulin when a large feto-maternalhaemorrhage has occurred.'" Staff need to beaware of the problems of interpretation whenpatients with hereditary persistence of fetalhaemoglobin are tested. Its main use is as ascreening test to identify patients who have afeto-maternal haemorrhage of > 4 mf, withflow cytometric measurement of RhD positivecells within the maternal circulation alsoperformed, if available. On the basis of thisresult the appropriate dose of anti-D immu-noglobulin can be recommended and itsefficacy assessed by monitoring clearance offetal red cells. An additional advantage of theuse offlow cytometry is that, because it specifi-cally measures RhD positive cells, it is moreuseful for antenatal patients in whom the fetalRhD status is unknown.

Antibody screeningAll women must have a red cell antibody screenperformed during pregnancy. This is ideallyundertaken early in pregnancy so that antibod-ies can be investigated thoroughly and appro-priate clinical action taken at an early stage. Asignificant number (2-5%) ofwomen will havered cell antibodies and there is an increasingincidence of non-RhD antibodies in thesepatients. There are two principal reasons forthe detection and identification of red cell anti-bodies in pregnant women: prevention ofHDN, and identification of potential transfu-sion problems.Both problems are equally relevant to RhD

negative and RhD positive women and, there-fore, timing and nature of testing applies to allantenatal patients. Antibody screening shouldbe undertaken using an indirect antiglobulintest (IAT) and a red cell panel conforming tocurrent United Kingdom guidelines,'4 andtesting should be undertaken at both an earlystage of pregnancy, and at 28 to 36 weeks' ges-tation for all women. More frequent testing willbe required in women in whom clinicallysignificant antibodies are detected. When anantibody is detected, the clinician responsiblefor the woman's antenatal care must be alerted

to its likely significance, both with regard toHDN and transfusion problems. Clear recom-mendations for further testing, and, whereneeded, referral to a specialist centre must bemade. Management of pregnancies in whichred cell antibodies are detected varies depend-ing on the clinical significance of the antibodydetected.Antibody screening tests using enzyme

treated red cells are not required for routineantenatal screening. Their use is associatedwith an increased antibody detection rate, butmany of these reactions are false positives.Antibodies are also detected which are notassociated with HDN because they are notIgG, or because they react only in the presenceof enzyme modified red cells. Laboratories thathave access to samples taken only at an earlystage of pregnancy for their RhD positivepatients will often use this technique in thehope that its increased sensitivity will lead toearly detection of antibodies that will developclinical significance during pregnancy. Earlydetection of these antibodies enables the clini-cian to be alerted to the need for further sam-pling later in pregnancy for these patients.

ANTIBODIES OF CLINICAL SIGNIFICANCEThe antibodies most often implicated in caus-ing severe to moderate HDN are anti-D, -c,and -Kell, but other antibodies are also recog-nised as a cause of HDN (table 2).There arenumerous reports of IAT reacting antibodiesbeing implicated in HDN of moderate severity,and all women found to have an IAT reactingred cell antibody in early pregnancy should beregarded as requiring further investigation.The majority will merely require repeat testinglater in pregnancy (28 to 36 weeks); after deliv-ery the baby should be tested for HDN andhis/her red cells typed for the implicatedantigen. Women with a previous history ofchildren with HDN in association with thesame red cell antibody should be managedmore actively in line with the managementoutlined below for anti-D, -c, -Kell.Women with anti-D, -c, -Kell antibodies

(and others with a previous clinical history ofHDN) require regular testing to monitor thelevel of the antibody, and to detect thedevelopment of additional antibodies. Testingshould be at least monthly until 28 weeks' ges-tation, then every two weeks. Testing of anti-Dand anti-c involves a quantitative measure-ment; levels of anti-D > 4 IU/ml and of anti-c> 10 IU/ml are associated with a moderate riskofHDN, and these patients should be referredearly to a specialist centre for fetal ultrasoundassessment and possible fetal blood sampling.Assessment of anti-Kell (and other HDN-associated antibodies (table 2) and antibodiespreviously implicated in HDN for a particularpatient) is less definitive, titration studiesusually being undertaken, but no clear cutassociation between titre and HDN has beenestablished."' Laboratory assessment of thesepatients should be carried out at a specialistcentre where additional techniques, such asfunctional assays that measure cytotoxic lysisor phagocytosis, or a combination of these, are

Table 2 Antibodiesassociated with HDN

Blood group system Antigen

Rhesus DcCE

Kell

eCe

Kk

Duffy FyaKidd Jk'

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used. Functional antibody assays, in particularthe monocyte mediated antibody dependentcell cytotoxicity assay, provide a means ofassessing functional activity of the specificmaternal antibody present in any pregnancy,and provide a more reliable means of predict-ing HDN severity than either quantification ortitre.16 Fetal blood typing from amniocyteDNA for D, C, c, Rh antigens, and Kell andDuffy a (Fy a) antigens is also available throughthe International Blood Group ReferenceLaboratory in Bristol. As for patients withanti-D antibodies, early referral to a specialistcentre for fetal assessment is important forthese patients.

Paternal blood testing for any red cellantigen to which the mother has antibodies canbe undertaken. However, it must be realisedthat the paternity of the fetus is oftenuncertain, and clinical decisions should not bemade solely on the basis of paternal phenotyp-ing.

ANTIBODIES UNLIKELY TO CAUSE HDNWomen who are found to have antibodies notusually associated with HDN and who have noprevious history of HDN, and women with noantibodies on initial testing should be testedagain at 28 to 36 weeks' gestation to ensure noadditional antibodies have developed. If theantibody is likely to cause problems with provi-sion of blood to cover obstetric emergencies,the clinician in charge of the patient must beinformed as well as the local transfusionlaboratory, and efforts made to ensure thatblood of a suitable phenotype can be acquiredat short notice. Patients with antibodies tocommon antigens may be considered for anautologous blood donation programme. Atdelivery, any baby born to a mother with IATreacting antibodies must be assessed forevidence of HDN.

RhD negative mothers with non-DantibodiesIt is essential that all RhD negative mothers whodo not have preformed anti-D receive appropri-ate doses of anti-D immunoglobulin both ante-natally and at delivery. This particularly appliesto RhD negative mothers with other antibodieswho have already been proven 'good' respond-ers to red cell antigenic stimulus.

ConclusionsThe routine testing of a pregnant woman's RhDtype forms an essential component of antenatalmanagement. The appropriate administration ofanti-D immunoglobulin to RhD negativewomen is an important part of optimal obstetricmanagement, and involves close cooperationand informed decision making from manyhealth care professionals. The role of the labora-tory in RhD typing and Kleihauer testing isessential in delivering a successful RhD immu-noprophylaxis programme.

Routine screening of pregnant women foratypical red cell antibodies is also an essentialpart of antenatal care. The decline in the inci-dence of anti-D antibodies has led to adecreased incidence ofHDN but the cases that

occur are associated with a greater variety ofantibodies. These require careful laboratoryinvestigation both for their identification andassessment of clinical significance. Few hospi-tals have wide experience in handling the vari-ety of cases which may occur and it is,therefore, important that, not only is there veryclose cooperation between laboratory staff,haematologists, obstetricians, and midwiveswithin a single hospital setting but also withreference laboratories and centres capable ofperforming fetal transfusions. Modern labora-tory techniques, employing cellular assays andamniocyte DNA fetal blood typing, coupledwith skilled ultrasound assessment of early fetalhydrops, enables obstetricians to help theirpatients to make informed decisions beforeinvasive procedures such as fetal blood sam-pling are undertaken.A multidisciplinary approach involving care-

ful clinical assessment, evaluation of previoushistory, laboratory testing, and, where neces-sary, non-invasive and invasive assessment offetal wellbeing, sometimes coupled with intra-uterine transfusion, is the only way ofprovidingeffective management of pregnancies compli-cated by red cell antibodies. Maternal and fetalwellbeing must be assured in these cases andcareful informed consideration of the likelysignificance of any red cell antibodies detectedfor both HDN and transfusion support willhelp to ensure the optimal outcome for themother and her child.

1 Mollison PL, Engelfriet CP, Contreras M. Blood transfusionin clinical medicine. 9th ed. Oxford: Blackwell ScientificPublications, 1993:543-91

2 International Forum. Laboratory Procedures for the Predic-tion of the Severity of Haemolytic Disease of the Newborn.Vox Sang 1995;69:61-9.

3 British Committee for Standards in Haematology BloodTransfusion Task Force. Guidelines for blood grouping andred cell antibody testing during pregnancy. TransfusionMedicine 1996;6:71-4.

4 Brouwers HAA, Overbeeke MAM, van Ertbruggen I,Schaasberg W, Alsbach GPL, van der Heiden C, et al. Whatis the best predictor of the severity of ABO-haemolytic dis-ease of the newborn? Lancet 1988;ii:641-4.

5 Letsky EA, De Silva M. Preventing Rh immunisation. BM31994;309:213-4.

6 Tovey LAD, Townley A, Stevenson BJ, Tavener J. The York-shire antenatal anti-D immunoglobulin trial in primigravi-dae. Lancet 1983;ii:244-6.

7 National Blood Transfusion Service ImmunoglobulinWorking Party. Recommendations for the use of anti-Dimmunoglobulin. Prescriber's Journal 199 1;31: 135-45.

8 Medical Research Council. Report of a working party on theuse of anti-D immunoglobulin for the prevention of isoim-munization of Rh-negative women during pregnancy. BM31974;ii:75-81.

9 American College of Obstetricians and Gynecologists Tech-nical Bulletin No. 79: Prevention of Rho (D) isoimmunisa-tion. Washington DC: American College of Obstetriciansand Gynecologists, 1984.

10 Committee for Proprietary Medicinal Products. Note forguidance: core summary of product characteristics foranti-D immunoglobulin. Brussels: Commission of Euro-pean Communities, 1992. (111(3463/92-EN).)

11 Mollison PL, Engelfreit CP, Contreras M. Blood transfusionin clinical medicine. 9th ed. Oxford: Blackwell Scientific,1993:798-800.

12 Polesky HF, Sebring ES. Evaluation of methods fordetection and quantitation of fetal cells and their effect onRh IgG usage. Am J Clin Pathol 1981;76(Suppl 1):525-9.

13 Duguid JKM, Bromilow I. Value of Kleihauer testing afteradministration of anti-D immunoglobulin. BM3 1994;309:240.

14 Guidelines for the Blood Transfusion Service. London:HMSO, 1992:157-62.

15 van Dijk BA, Dooren MC, Overbeeke MAM. Red cell anti-bodies in pregnancy: there is no "critical titre". TransfusionMedicine 1995;5:199-202.

16 Hadley AG, Kumpel BM, Leader KA, Poole GD, Fraser ID.Correlation of serological, quantitative and cell-mediatedfunctional assays of maternal alloantibodies with the sever-ity of haemolytic disease of the newborn. Br J Haematol1991;77:221-8.

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