A case report of haemolytic disease of the foetus and newborn caused by Alloantibody D and Jka in a Rhesus D negative Nigerian woman: Justification for the implementation of universal access to prophylaxis and evidenced-based best practices

1. Discussion

The clinical significance of a blood group system is dependent on that ability of antibodies of the blood group system to cause HDFN and Haemolytic transfusion reaction (HTR) [1]. The Rhesus blood group system is the second most clinically significant blood group system after the ABO. The most immunogenic of the Rhesus blood group antigens is the D antigen. Alloantibodies are antibodies produced in a patient as a result of exposure to foreign red cell antigen via transfusion, pregnancy or transplantation. Other common factors that facilitate alloantibody formation in the recipient include: the immune competence, the dose of the antigen the recipient is exposed to, the route of exposure and how immunogenic the foreign antigen is. Depending on the presence or absence of the Rhesus D antigen, an individual can be grouped as Rhesus D positive or negative respectively. A mother who is Rh D negative and married to a homozygous or heterozygous D positive husband has a 100 and 50% chance respectively of carrying a D positive baby. During such pregnancies as a result of feto maternal haemorrhage (FMH) following potentially sensitizing events during pregnancy [(Amniocentesis, chorionic villus biopsy and cordocentesis), APH, PV bleed during pregnancy, ECV, Abdo trauma (sharp/blunt, open/closed, ectopic pregnancy, evacuation of molar, pregnancy, IUFD, intrauterine intervention during pregnancy (transfusion, surgery, insertion of shunts and laser), miscarriages, TOP or during delivery (normal, instrument or C/S) and intraoperative cell salvage)]. As a result of the sensitizing event, some amounts of foetal blood can enter into the maternal circulation. The foreign foetal RhD-positive foetal cells can irreversibly sensitize the mother to produce alloantibody D. The risk of sensitization occurring is dependent on a number of factors; ABO blood group of the developing foetus (there is a higher risk if there is ABO compatibility between mum and baby), dose of foetal cells entering the maternal circulation, immune competence of the mother and type of pregnancy (risk is significantly higher in the first decreasing in the subsequent pregnancies). The antibody produced is immune IgG antibody that is a low molecular weight antibody and can potentially cross the placenta barrier. The first pregnancy is usually unaffected. However, the small molecular weight IgG immunoglobulin and can pass through the placenta in subsequent D positive pregnancy and destroy the foetal red cells with increased production of bilirubin a product of red cell breakdown resulting in HDFN. The baby can present with anaemia, hyperbilirubinaemia, hepatosplenomegaly, kernicterus and even death.

Women who are Rh D negative and are pregnant with Rh D positive babies run the risk of being sensitized following micro FMH that occur from potentially sensitizing events during pregnancy or during delivery. Evidenced-based recommendations to prevent HDFN include; performance of immunohaematological investigations in the antenatal and perinatal periods (alloantibody testing and identification, fetomaternal haemorrhage testing), the provision of universal access to anti-D immunoprophylaxis (IP) for Rh D negative and not-previously sensitized pregnant women to prevent HDFN (due to RhD incompatibility) [2, 3]. The anti-D alloantibody is the antibody most frequently responsible for HDFN [4]. Before the introduction of anti-D IP, HDFN secondary to anti-D immunisation affected 1% of neonates and it was the cause of death of one in every 2,200 babies born [5].

One of the evidence-based best practice in most developed countries to prevent Rhesus D negative women from being sensitized is the implementation of the Routine Antenatal Anti-D Prophylaxis (RAADP) program. This program is an alloimmunization prevention program where all Rh D negative women are universally offered prophylaxis during pregnancy (28 weeks gestation) and following the delivery of a Rhesus positive baby. The implementation of this program has significantly reduced the number of residual alloimmunization from 16% to less than 0.1% [6]. The justification for this implementation is based of scientific evidence that 92% of women who develop an alloantibody-D during pregnancy do so at or after 28 weeks gestation [7]. This implementation has significantly reduced the number of women who are sensitised and by extension the number of D HDFN. However, in countries like Nigeria where this policy has not been implemented, a significant 14% of unfortunate foetuses are born dead while 50% of those who are born alive suffer from brain injury [8].

Rho(D) immune globulin (RhIG) is a medication that can be given to non-sensitised Rhesus D negative pregnant women to prevent Rh D sensitization. The medication is often administered intramuscularly or intravenously and has a half-life of 12 weeks. Rho(D) immune globulin used for prophylaxis is a human derived fractionated plasma product produced from blood donors who have high levels of anti-D Ig either due to previous sensitization or intentional immunization of Rh D negative men to produced immune D. Implementation of universal access to prophylactic anti-D given during antenatal and post-partum period following the delivery of a Rh D positive baby can help prevent primary Rh D immunization and risk of HDFN in subsequent pregnancies [9]. It is recommended that routine antenatal anti-D prophylaxis (RAADP) is offered to all non-sensitised pregnant women who are Rh D negative to reduce the risk of sensitization and by extension D-related HDFN [10]. The World Health Organization (WHO) recommends that antenatal prophylaxis with anti-D immunoglobulin should be given to non-sensitised Rh-negative pregnant women at 28 and 34 weeks of gestation to prevent Rh D alloimmunization. It is estimated that single dose of anti-D can cost around US$ 50 (equivalent of N18,000 Nigerian naira) (500 IU) to US$ 87 (equivalent of N31,320 Nigerian naira) (1500 IU), depending on the brand and local taxes. Table 1 show a number of potentially sensitising events during pregnancy requiring evidenced-based administration of anti-D prophylaxis.

Although the introduction of post-partum IP in RhD negative pregnant women has drastically reduced the incidence of cases of HDFN [11], HDFN due to anti-D continues to occur in 0.4 of every 1,000 births particularly in settings like Nigeria where universal access to IP is unavailable [12] and red blood cell alloimmunisation still remains the most common cause of foetal anaemia [13]. There are various reasons for the continued occurrence of this disease; the possible development of anti-D immunisation during a pregnancy as a result of an occult foetal-maternal haemorrhage (FMH), usually after the 28 th week of gestation, which affects about 1% of RhD negative mothers of a RhD positive foetus [14]; lack of administration of IP; ineffective IP because the amount administered was not sufficient for the volume of the FMH; possible errors in the typing of the pregnant woman and possible errors in 1 of 20 the transfusion treatment of females of childbearing potential (transfusion of red blood cell concentrates with mismatched RhD antigen). The fundamental cause of HDFN is the reaction between class IgG maternal antibodies and antigens on foetal red blood cells, leading to the destruction of these cells, mainly in the spleen. The immune response depends on the entity of the FMH, the number of immunising events and the capacity of the woman’s response. ABO incompatibility between mother and foetus partially protects against immunisation. In the natural history of HDFN, without any kind of intervention, in 50% of cases the foetus has only mild signs of the disease and recovers without any treatment; in 25% of cases the foetus develops haemolysis and kernicterus, if not treated adequately at birth; and in the remaining 20–25% of cases, HDFN due to anti-D may present in its most severe form (hydrops foetalis and death) before the 34 th week of gestation [15].

There are several challenges associated with the management of Rhesus negative pregnancies and pregnancies associated with clinically significant alloantibodies in Nigeria. These include the non-implementation of policy on routine antenatal anti-D prophylaxis (RAADP) and poor management of Rhesus negative women that require termination of pregnancy (TOP).

RAADP or routine antenatal anti-D prophylaxis is a recommended treatment option for all Rhesus D negative pregnant women who are not known to be sensitized previously to the RhD antigen. Routine antenatal anti-D prophylaxis (RAADP) is a program under which non-sensitised Rhesus D negative pregnant women are universally offered anti-D prophylaxis at 28–34 weeks’ gestation with the aim of preventing sensitization and by extension anti-D HDFN in subsequent pregnancy [89]. The aim of the RAADP programme is the reduction in the incidence of anti-D related HDFN; improve the survival of the children delivered by Rhesus D negative women, reduce the incidence of disability and health-related quality of life of children and mortality in children delivered by Rhesus D negative women. These women run the risk of developing anti-D and its associated HDFN if mothers are not offered this life saving prophylaxis. The RAADP program is based on offering either two standard doses (500 IU) at 28- and 34-weeks’ gestation or a single dose of at least 1500 IU at 28 weeks gestation followed by a further dose of at least 1500 IU within 72 h of delivery of a Rh D positive baby. The dose offered post-delivery is dependent on the result of feto maternal haemorrhage testing result (flow cytometry of Kleihauer testing). Both methods quantify in millilitres the amount of foetal red cells that has entered maternal circulation to facilitate the administration of adequate dose of prophylactic anti-D. As a general rule 125 IU of anti-D is administered to clear 1 ml of foetal red cells from maternal circulation. The introduction of anti-D prophylaxis using Rhesus D immunoglobulin (given intramuscular or intravenous administration) has led to a significant fall in the number of women becoming sensitised and by extension reduced the incidence and severity of this condition. The anti D immunoglobulin used as prophylaxis is prepared from plasma of male Rhesus D negative donors who have high levels of plasma anti-D due to deliberate or intentional immunisation with D positive red cells. Anti D is also administered to Rhesus D negative women following potentially sensitizing events during pregnancy. The anti-D facilitates the clearance of foetal red cells from the maternal circulation to prevents active immunisation, thus preventing the production of alloantibody D. Prior to the availability and widespread use of anti-D prophylaxis for Rhesus negative pregnant women, the incidence of Rh D sensitization among Rhesus D negative women following two deliveries of D positive and ABO-compatible, infants was approximately 16% and haemolytic disease of the foetus and newborn (HDFN) due to immune anti-D was a significant cause of morbidity and mortality [16]. There are several reasons why women in Nigeria continues to be sensitized and carry babies that suffer and die from HDFN.

In Nigeria, alloantibody screen, identification and titration/quantification are not included in the antenatal testing menu for pregnant women like it obtains in the developed world. Maternal antibody screening is undertaken to detect the presence of clinically significant antibodies which has the potential to cause HDFN and affect the foetus and/or newborn. Approximately 1% of pregnant women are found to have clinically significant red cell antibodies [17, 18, 19]. The detection of clinically significant antibodies among antenatal women plays an important role in preventing HDFN. A previous study among antenatal multiparous women in Jammu region of India indicated that the commonly observed alloantibodies were anti-D, anti-E, anti-C and anti-K [20]. Once an antibody has been detected, it is usually identified by carrying out a panel test. If the antibody specificity is one that has the potential to cause HDFN, the titre/quantification must be carried out periodically (every 4 weeks from booking at 16 weeks to 28 weeks and every 2 weeks from 28 weeks until delivery). Antibody quantification (anti-D, and anti-c) and titration (Anti-e, c, E, C+ e, G, CW, Fya, Fyb, K, k, Kpa, Kpb, Jka, Jkb, Jsa, Jsb, M, S, and s) is performed on the maternal sample to support the prediction and management of HDFN. All clinically significant maternal antibodies detected during antenatal booking or follow up visit must be identified by carrying out a panel to enable us confirm the specificity, determine the potential of the antibody to cause HDFN and to enable the determination of the titre/quantification. The trend in the titre/quantification is used to monitor and to determine the severity of the disease. Evidenced- based best practice in developed countries requires the monitoring of women with red cell antibodies of specificities such as anti-D which is known to cause significant HDFN every 4 weeks from booking until 28 weeks and every 2 weeks there after till delivery. Obstetricians and neonatologist can use this information in conjunction with middle cerebral artery Doppler assessment to determine the extent to which the developing infant is affected inutero, the presence of foetal anaemia, decision to carry out intrauterine transfusion as well as decision on whether to deliver the baby earlier to allow for a better and less risky physical management rather than ultrasound-guided inutero management [21]. Foetal middle cerebral arterial (MCA) Doppler assessment is an important part of assessing foetal cardiovascular distress, foetal anaemia or foetal hypoxia. Colour Doppler ultrasonography a non-invasive technique currently being used to assess cerebral artery blood flow [22]. Increased cerebral artery blood flow is an indicator of foetal anaemia [22]. Diagnosis of foetal anaemia used to be carried out using procedures such as amniocentesis or cordocentesis, with subsequent analysis of amniotic fluid and foetal blood sampling obtained by ultrasonography guided procedure. These procedures are invasive and significantly risky with potential attendant negative effect to the foetus. A rising antibody level or titre is an indication that the developing foetus is significantly affected by HDFN. The antibody titre/quantification often rises as a result of re-exposure of the maternal immune system to red cell antigen from the foetus following the initial sensitizing events that occur during pregnancy. Every re-exposure to the initial offending antigen from the foetus to the mother’s immune system often becomes booster doses and results in an increased anamnestic alloantibody production. The innocent and vulnerable Nigerian woman discussed in this case study was Rhesus D negative. She was not offered prophylaxis following the first miscarriage she had. This miscarriage may have led to her sensitization and development of alloantibody D. The anti-D may have led to HDFN in the first baby that was delivered severely jaundiced and needed management post-delivery for HDN. The woman has gone through significant trauma of a further stillbirth and five subsequent miscarriages. Women who experience a late-stage miscarriage or stillbirth may develop post-traumatic stress disorder, anxiety and depression. There is need to improve the diagnosis and treatment of psychological disorders following miscarriage or still birth in these unfortunate women.

Evidenced- based management of HDFN in pregnant women with alloantibodies requires that facilities for the effective management of the foetus are available (alloantibody titration and quantification, foetal genotype determination; non-inversive determination of anaemia in the foetus using doppler ultrasound to measure the velocity of blood through the cerebral artery; intrauterine transfusion to manage foetal anaemia inutero, facilities to test and provide donor unit that meets the requirements for blood intended for intrauterine transfusion). Foetal blood group genotype of a developing foetus can also be determined for Rh C, c, E and Kell (K) using cffDNA from maternal plasma [24]. All these evidenced based best practices are not available in Nigeria. The net result is that there is increase in the preventable death of neonates and foetuses. Many European Union countries have suggested the mass application of foetal genotyping for all foetuses carried by D-negative women. This advocacy is based on clear benefit of conserving anti-D stocks and prevention of unnecessary administration of this human-derived blood product that has associated risk [25, 26]. In Denmark and the Netherlands this evidenced-based best practice of foetal Rh D testing for all non-sensitised D negative pregnant women have been introduced [27]. Also, non-invasive foetal blood group genotyping (DNA) testing of maternal plasma to determine the antigen status of the developing foetus carried by an already alloimmunised pregnant women like in this case study is vital as it provides useful information as to whether a foetus is carrying the group specific antigen to which the maternal alloantibody is specific and to determine the risk of haemolytic disease. This will help prevent the need of extensive laboratory (quantification of antibody every 4 weeks till 28 weeks’ gestation and every 2 weeks from 28 weeks to delivery) and clinical monitoring in antigen-negative cases.

One of the common causes of death in severely affected HDFN cases is foetal anaemia. In utero the diagnosis of foetal anaemia used to be made by carrying out ultrasound guided foetal blood sampling for analysis for foetal haemoglobin and haematocrit or amniocentesis-an invasive ultrasound guided procedure to obtain amniotic fluid. This procedure has a significant 2% risk of foetal loss associated with entering the amniotic sac and obtaining amniotic fluid. The amniotic fluid analysis for product of haemoglobin breakdown can be used to determine how severely affected a developing foetus is. However, non-invasive diagnosis of foetal anaemia can now be made using non-invasive Doppler ultrasound technology by measuring the middle cerebral artery peak systolic velocities (MCA PSV). The foetal anaemia can be managed with intrauterine blood transfusions. Intrauterine transfusion is associated with a number of risks; foetal bradycardia, foetal death, cord haematoma, haemorrhage from the puncture site, miscarriage, preterm labour and vessel spasm. The blood used for such transfusions must meet certain requirements; gamma irradiated red cells to prevent transfusion-associated graft-versus-host disease, haemoglobin S negative, CMV negative and significantly high PCV (0.70–0.85) [28], unit must be C-, D-, E- and K-, leucocyte depleted to less than 1 × 10 6 leucocytes per unit, less than 5 days old, free from clinically significant irregular blood group antibodies and negative for high-titre anti-A and anti-B haemolysin. If the mother has other alloantibodies apart from D, it is vital that suitable blood donor units negative for the specific antigens to which the maternal alloantibodies are specific are selected. The Nigerian government does not seem to be implementing these evidenced- best practices nor offering the best possible obstetric and neonatal care to her citizens [29, 30, 31, 32, 33]. This has often resulted in the preventable death of a significant number of severely affected foetuses and neonates.

These cases of preventable death of Nigerian children is a humanitarian emergency that require urgent attention and good will by all well-meaning Nigerians and people of good will across to globe. This case study is associated with 1 foetal demise and 5 miscarriages as a result of lack of political will by the Nigerian government to implement best practices aimed at prevention and management of HDFN. This case study is a clear picture of the state of obstetric management particularly of pregnant women with a clinically relevant alloantibody. Pregnant women in Nigeria are not as fortunate like their counterparts in the West because of the absence of a fit for purpose obstetric care. The Nigerian government must come to the realization that there are economic benefits associated with the implementation of universal access to prophylactic anti-D for non-previously sensitized Rhesus D negative women. The cost of antenatal prophylaxis for two 500 IU doses could be as much as US$ 100 (equivalent of N36,000 Nigerian Naira) per woman. Although providing anti-D prophylaxis universally to all Rh D negative non-sensitised pregnant women is not cheap policy, however the cost benefit analysis of preventing sensitization, HDFN and its related physical disabilities, mental retardation and death of affected neonates supports investing in the implementation of this policy by any responsible government who believes that every life count. Routine antenatal anti-D prophylaxis provides a cost-effective intervention for preventing HDFN in non-sensitised Rh D-negative pregnant women [34]. It is suggested that a programme of routine prophylaxis would be cost saving if HDFN were eradicated by its implementation [30]. It does make economic sense for the Nigerian government to rise to their responsibilities by proving universally anti-D prophylaxis for all non-sensitised Rhesus D negative women. She can also significantly reduce the cost of implementing RAADP by investing on facilities for the non-invasive detection of foetal genotype. This implementation will identify Rhesus D negative women who are carrying a Rhesus negative foetus in whom the anti-D prophylaxis is not clinically indicated. The spared anti-D can be used for women in whom the prophylaxis is indicated.

The second alloantibody identified in the Nigerian woman discussed in this case study was anti-Jka. This alloantibody is clinically significant (can cause HDFN and HTR). Jka antigens have been identified to have the potential to cause maternal alloimmunization and subsequent haemolytic disease of the foetus or newborn (HDFN). This antibody was first discovered in 1951 in the serum of a pregnant patient called Mrs. Kidd [35]. The Jka antigen was present on the RBCs of her foetus, and the maternal antibodies targeted against it caused a fatal haemolytic disease in her newborn child. Majority of people are Jka negative; 23% Caucasians, 8% Blacks, 27% Asians are Jk(a-b+) and thus are at risk for developing anti-Jka [36]. During pregnancy, foetal Kidd antigens are capable of causing alloimmunization of the mother [37, 38]. Kidd antibodies (anti-Jka and anti-Jkb) are only rarely responsible for severe HDN [39]. Anti-Jka has been implicated in at least one severe case of HDN, but most cases of HDN caused by the anti-Kidd antibodies are mild in nature [40]. A previous report described HDN in a Korean neonate with severe anaemia, early-onset hyperbilirubinaemia, low haemoglobin and spherocytosis due to anti-Jk(a) antibodies [41]. Previous report [40] indicated that 1 of 20 pregnancies associated with anti-Jka, using maternal antibody titer and MCA PSV were effective in detecting severe HDFN resulting from to anti-Jka. Anti-Jka has been reported to cause severe and fatal haemolytic transfusion reactions but is more commonly associated with less severe DHTRs [42].

This case study is a clear indication of the poor states of HDFN prevention and suboptimal management of pregnant women particularly those with a clinically relevant alloantibody in Nigeria. We recommend that the Nigerian government should fulfil her obligations under goal 3.8 of the Universal Health Coverage initiative and the Sustainable Development goal 3.b which emphasizes the need for access to safe, effective, quality and affordable essential medicines for all by implementing universal access to anti-D prophylaxis for previously unsensitized Rhesus D negative pregnant women. Anti-D prophylaxis should be available for non-sensitized Rhesus D negative women following potentially sensitizing events such as amniocentesis, cordocentesis, antepartum haemorrhage, ectopic pregnancy, vaginal bleeding during pregnancy, external cephalic version, abdominal trauma, intrauterine foetal death and stillbirth, in utero therapeutic interventions, miscarriage, and therapeutic termination of pregnancy. All Rhesus D negative women having a termination of pregnancy (abortion) must be tested for Rhesus D group and those found to be Rhesus D negative must be offered prophylaxis with 72 hours of carrying out the procedure. Routine foetal genotyping using non-invasive testing of maternal sample to identify Rhesus negative pregnant women who are carrying Rhesus negative infants for whom prophylaxis is not indicated should be implement to prevent the administration of anti-D for women in whom it is not clinically indicated. There is need to introduce alloantibody testing, identification and titration/quantification into the antenatal management menu for pregnant women in Nigeria. Facilities for intrauterine transfusion of infants who become anaemic in utero as a result of HDFN and require a red cell transfusion should be provided in teaching hospitals and federal medical centers across the country. Facilities to enable Medical Laboratory Scientists effectively test blood donors to facilitate the selection of donor red cells that meet the requirements for intrauterine, exchange and top up transfusion should be provided. Also, facilities for fetomaternal haemorrhage (FMH) testing to facilitate the administration of optimal amount of anti-D prophylaxis following a sensitizing event aimed at preventing sensitization and subsequent HDFN should be provided. There is the need to optimize the knowledge of Medical Laboratory Scientists, Obstetricians, Neonatologist, Midwives, Pharmacist and Traditional Birth Attendants in Nigeria on anti-D prophylaxis, HDFN prevention strategies and management of women with clinically significant antibodies.