https://orcid.org/0000-0002-7220-3064
Visual Abstract
Hemolytic disease of the fetus and newborn (HDFN) remains an important cause of perinatal morbidity and mortality. HDFN is caused by maternal alloimmunization to red blood cell (RBC) antigens. This article describes and highlights issues in the care of pregnant women with RBC alloimmunization. This includes monitoring for, and management of fetal anemia caused by maternal red cell alloantibodies, but also considerations for transfusion support for the woman in the event of major bleeding. Many aspects of care for women with RBC alloantibodies are not covered within specific guidelines, particularly with respect to best practice for antenatal management of women with prior significant obstetric morbidity or mortality due to HDFN, and we outline our approach in these cases. The use of noninvasive monitoring for fetal anemia through measurement of the middle cerebral artery peak systolic velocity has led to a paradigm shift in antenatal care for women with high-risk antibodies, and medical therapies hold promise for women with the most severe disease.
Introduction
Hemolytic disease of the fetus and newborn (HDFN) caused by maternal alloimmunization to red blood cell (RBC) antigens remains an important cause of perinatal morbidity and mortality. This is best recognized in the context of the RhD antigen; however, there are >50 RBC antigens that have the potential to cause HDFN.1-3
We describe and highlight issues in the care of pregnant women with RBC alloimmunization, including monitoring for and management of fetal anemia caused by maternal RBC alloantibodies, but also considerations for transfusion support for the patient in the event of major bleeding.
Pathophysiology
HDFN occurs when a maternal RBC antibody crosses the placenta and binds to fetal/neonatal RBCs or erythroid precursors that express the corresponding paternally-derived antigen, resulting in immune hemolysis. This can result in fetal/neonatal anemia, with a risk of serious consequences, including fetal hydrops, preterm birth, and perinatal loss, as well as neonatal morbidity, including anemia, hyperbilirubinemia, and potentially kernicterus.2-4 Hyperbilirubinemia is not a concern in the fetus given that fetal bilirubin crosses the placenta and is metabolized by the maternal liver.4
Formation of a maternal RBC alloantibody requires maternal exposure to the antigen. This may occur through various sensitizing events, both before and during pregnancy (Table 1). The likelihood of alloimmunization depends on variables such as the immunogenicity of the antigen, the volume of allogeneic blood to which the mother is exposed, and maternal immune function. After sensitization and development of an RBC alloantibody, further exposure to the paternally-derived antigen during a subsequent pregnancy will elicit a response that may vary in terms of intensity and timing of alloantibody production.2,4
Sensitizing events associated with the formation of maternal RBC alloantibodies
| Sensitizing event | Notes |
|---|---|
| Previous blood transfusion | Usually, RBC transfusion, but platelet transfusion is also possible due to the presence of small numbers of RBCs |
| Fetomaternal hemorrhage (FMH) | Transplacental bleeding may occur in miscarriage, antepartum hemorrhage, ectopic pregnancy, termination of pregnancy, fetal death in utero, delivery, abdominal trauma, and procedures, such as amniocentesis or external cephalic version FMH also occurs spontaneously with risk increased with advancing gestation, and highest at delivery |
| IV drug use | Sharing of needles is a reported cause of alloimmunization5 |
| None identified | Some alloantibodies occur naturally and may arise due to exposure from, for example, the gut microbiome (eg, anti-M) |
| Sensitizing event | Notes |
|---|---|
| Previous blood transfusion | Usually, RBC transfusion, but platelet transfusion is also possible due to the presence of small numbers of RBCs |
| Fetomaternal hemorrhage (FMH) | Transplacental bleeding may occur in miscarriage, antepartum hemorrhage, ectopic pregnancy, termination of pregnancy, fetal death in utero, delivery, abdominal trauma, and procedures, such as amniocentesis or external cephalic version FMH also occurs spontaneously with risk increased with advancing gestation, and highest at delivery |
| IV drug use | Sharing of needles is a reported cause of alloimmunization5 |
| None identified | Some alloantibodies occur naturally and may arise due to exposure from, for example, the gut microbiome (eg, anti-M) |
The presence of maternal RBC alloantibody does not automatically result in clinically significant HDFN. Unlike immunoglobulin G (IgG) antibodies, IgM isotype cannot cross the placenta. Additionally, once an antibody has crossed the placenta, HDFN cannot occur if the fetus/neonate does not express the associated antigen or expresses it only weakly. Even when the antibody is an IgG isotype and the cognate antigen is expressed on fetal red cells, HDFN does not always occur, and the severity may be variable. Additional factors influencing the impact of an antibody may include the degree of expression of antigen, avidity of antibody, and fucosylation of antibody as well as unknown factors that may limit the adverse effects of some antibodies.
Case 1
A gravida 2 para 1 woman referred at 13 weeks of gestation has a positive antibody screen. Her blood group was A RhD-positive with anti-c detected. Her first pregnancy was uncomplicated, with forceps delivery after spontaneous labor at 39 weeks of gestation. Postpartum hemorrhage of 1500 mL occurred and she received 2 units of RBC. Her antibody screen was negative throughout the first pregnancy and her infant was healthy, without anemia or jaundice.
Screening for maternal RBC antibodies
Antenatal guidelines recommend determining maternal blood group (ABO and RhD) and antibody status in the first trimester. Provided the antibody screen is negative at that time, some guidelines recommend repeat testing at 28 weeks of gestation; however, this may not be cost-effective.6-8 The sample for the blood group and antibody screen at 28 weeks should be collected before giving prophylactic RhD immunoglobulin (RhD Ig) in RhD-negative women.
The rationale for these tests includes: (1) identification of maternal RBC antibodies associated with HDFN; (2) identification of RhD-negative women, who should receive RhD Ig provided they are not already alloimmunized to RhD; and (3) to enable appropriate transfusion support to the mother should it be required, including identification of maternal RBC antibodies and rare blood groups, which may complicate selection of compatible blood for transfusion.6
If an antibody is identified, the next step is obtaining a clinical history, including prior transfusion, pregnancy, or other sensitizing events, and identifying any previous pregnancies affected by HDFN. If the identified antibody is anti-D, then obtaining a history of recent prophylactic RhD Ig administration and liaising with the transfusion laboratory for further advice is important to determine whether the detected antibody is passive or due to alloimmunization.
The prevalence of maternal RBC alloantibodies has been estimated in various studies. An Australian study reported a positive RBC-antibody screen in 0.73% of samples from 66 354 women presenting for routine antenatal care (2011-2013).9 This rate is comparable with the data from other settings, including the United Kingdom, the Netherlands, Israel, and the United States.10-13 Higher alloimmunization rates have been reported in other settings,14,15 demonstrating that the prevalence and specificity of antibodies may vary due to population gene frequencies of RBC antigens, local transfusion practices (and therefore the risk of alloimmunization), blood banking resources and techniques, as well as other factors such as the presence and effectiveness of RhD Ig immunoprophylaxis programs. Prevention of maternal alloimmunization to RhD is the rationale for the administration of RhD Ig to D-negative women during their antenatal care, for sensitizing events, at the beginning of the third trimester, and delivery. In some countries, RhD Ig prophylaxis is targeted to women who are known to be carrying an RhD-positive fetus by offering noninvasive prenatal testing (NIPT) using cell-free fetal DNA.
Immunoprophylaxis programs do not exist for other Rh and non-Rh antigens, and an important aspect of alloimmunization prevention more broadly is sound transfusion practice, which adheres to the principles of patient blood management. This includes optimization of hemoglobin (Hb) to avoid or minimize transfusion where possible and the appropriate selection of red cells when transfusion is required. Australia, Canada, the Netherlands, and other jurisdictions have adopted a K–matched transfusion policy for women with childbearing potential. This practice can decrease the likelihood of K-sensitization, a desirable outcome, given the potential severity of anti-K HDFN.16 Women with chronic hematological disorders requiring transfusion support, such as sickle cell disease, are ideally managed with red cell genotyping and extended red cell phenotype matching, where possible to reduce the likelihood of alloimmunization, primarily for their own care but also to minimize risks of HDFN in future pregnancies. More widespread phenotype matching to prevent alloimmunization impacting pregnancy is not practiced in Australia, and its effectiveness remains uncertain.17
Antibody specificity
Once a maternal RBC alloantibody is identified, further laboratory and clinical workup is required to assess the risk to the fetus of developing HDFN. Antibody specificity is crucial in determining HDFN risk. An overview of RBC alloantibodies and the associated blood groups most associated with HDFN is included in Table 2, with more extensive lists found elsewhere.4,6
RBC antigen groups and risk for HDFN
| RBC antigen group | Specific RBC antigen | Potential severity of HDFN |
|---|---|---|
| ABO ABO incompatibility is actually the most common cause of HDFN, affecting up to 1%-4% of pregnancies4, However, it only rarely causes clinically significant or recognized disease Most anti-A and anti-B antibodies are naturally occurring, IgM antibodies. A proportion can be IgG isotype (particularly in Group O individuals) and therefore can cross the placenta ABO antigens are poorly expressed in the fetus/neonate.4 A group A or B fetus/neonate with a group O mother is the most likely scenario in which ABO HDFN may occur4 Resultant disease is typically mild, with expectant management of the neonate in most cases; if treatment is required, phototherapy alone is usually sufficient | A, B | Mild-moderate, rarely severe |
| Rh RhD is highly immunogenic4 Anti-D HDFN was the most common form of severe HDFN before immunoprophylaxis programs The Rh system includes many antigens other than D; >40 other serologically defined antigens have been described18 From the point of view of HDFN, the most clinically significant of these are c and E; followed by C and e Routine RhD Ig immunoprophylaxis does not prevent alloimmunization against other Rh antigens | D, c∗, C∗, E∗, e∗ G | Severe Mild, rarely moderate Moderate, rarely severe Mild Mild, rarely severe |
| Kell Large blood group system, with antigens including K, k, Kp(a), Kp(b), Ko, Js(a), Js(b), and many other rare antigens The most antigenic and clinically important of these from the point of view of HDFN is K Anti-K antibodies are the most common RBC alloantibodies after ABO and Rh antibodies, responsible for a relatively large portion of non-Rh HDFN4, K is expressed on early RBC precursors during erythroid development. Anti-K can cause severe, hyporegenerative anemia, with a lower reticulocyte and erythroblast count seen in anti-K HDFN than in anti-D HDFN. This results in the fetus/neonate being less able to compensate for anemia.4 Anti-K HDFN can cause severe disease as early as 18 to 20 weeks of gestation. In K-sensitized pregnancies with a K-positive fetus, ∼50% will require an IUT, and perinatal death reported in ∼3%.19 | K, k, Ku, Jsb Kpb Kpa, Jsa | Severe Moderate Mild |
| Duffy Duffy antigens Fy(a) and Fy(b) are inherited in a codominant fashion, giving rise to the following phenotypes: Fy(a+b+), Fy(a−b−), Fy(a+b−), or Fy(a−b+). Several other antigens also comprise the Duffy blood group system. Anti-Fya is a relatively common RBC alloantibody. Although is it not a common cause of HDFN, it is possible and has been reported to cause mild to moderate disease. Anti-Fyb is not typically associated with HDFN, although rare case reports of anti-Fyb HDFN do exist4,6 | Fya Fyb Fy3, Fy4, Fy5, Fy6 | Moderate Mild to severe None |
| Kidd Defined by 2 antigens, Jka and Jkb The null phenotype, Jk(a−b−), is rare (1% of Polynesians, a higher proportion of Filipinos, also reported in Subcontinent and African ethnicities) Kidd antibodies can cause clinically significant hemolytic transfusion reactions but are rarely associated with clinically significant HDFN | Jka, Jkb Jk(3-) | Mild, rarely severe Mild |
| MNS Large blood group system with >30 antigens Anti-M and anti-N antibodies can be naturally occurring in a small proportion of people Anti-M is usually an IgM antibody, hence cannot cross the placenta. A small proportion may be IgG, with the potential for HDFN, which is rare but can be severe.4,6 Severe anti-M HDFN appears to be more common in people of East Asian ethnicity. Similarly, anti-N antibodies are typically IgM and are very uncommon causes of HDFN Antibodies to S, s, and U are more likely to be IgG, and, although uncommon, can possibly cause severe HDFN Within the MNS blood group, there is a subsystem of hybrid glycophorins (“Miltenberger subsystem”). These hybrid glycophorins are difficult to detect serologically but can be clinically significant. For example, the hybrid glycophorin family GP.Mur (also called Mi.III) has a population frequency of ∼7%-15% or even higher in South East Asia20-22 and antibodies against these antigens have been reported to cause significant HDFN, including cases of hydrops fetalis.23 Molecular methods have been developed to detect these antigens. | M N S, s, U | Generally mild, rarely severe None to mild Mild to severe |
| RBC antigen group | Specific RBC antigen | Potential severity of HDFN |
|---|---|---|
| ABO ABO incompatibility is actually the most common cause of HDFN, affecting up to 1%-4% of pregnancies4, However, it only rarely causes clinically significant or recognized disease Most anti-A and anti-B antibodies are naturally occurring, IgM antibodies. A proportion can be IgG isotype (particularly in Group O individuals) and therefore can cross the placenta ABO antigens are poorly expressed in the fetus/neonate.4 A group A or B fetus/neonate with a group O mother is the most likely scenario in which ABO HDFN may occur4 Resultant disease is typically mild, with expectant management of the neonate in most cases; if treatment is required, phototherapy alone is usually sufficient | A, B | Mild-moderate, rarely severe |
| Rh RhD is highly immunogenic4 Anti-D HDFN was the most common form of severe HDFN before immunoprophylaxis programs The Rh system includes many antigens other than D; >40 other serologically defined antigens have been described18 From the point of view of HDFN, the most clinically significant of these are c and E; followed by C and e Routine RhD Ig immunoprophylaxis does not prevent alloimmunization against other Rh antigens | D, c∗, C∗, E∗, e∗ G | Severe Mild, rarely moderate Moderate, rarely severe Mild Mild, rarely severe |
| Kell Large blood group system, with antigens including K, k, Kp(a), Kp(b), Ko, Js(a), Js(b), and many other rare antigens The most antigenic and clinically important of these from the point of view of HDFN is K Anti-K antibodies are the most common RBC alloantibodies after ABO and Rh antibodies, responsible for a relatively large portion of non-Rh HDFN4, K is expressed on early RBC precursors during erythroid development. Anti-K can cause severe, hyporegenerative anemia, with a lower reticulocyte and erythroblast count seen in anti-K HDFN than in anti-D HDFN. This results in the fetus/neonate being less able to compensate for anemia.4 Anti-K HDFN can cause severe disease as early as 18 to 20 weeks of gestation. In K-sensitized pregnancies with a K-positive fetus, ∼50% will require an IUT, and perinatal death reported in ∼3%.19 | K, k, Ku, Jsb Kpb Kpa, Jsa | Severe Moderate Mild |
| Duffy Duffy antigens Fy(a) and Fy(b) are inherited in a codominant fashion, giving rise to the following phenotypes: Fy(a+b+), Fy(a−b−), Fy(a+b−), or Fy(a−b+). Several other antigens also comprise the Duffy blood group system. Anti-Fya is a relatively common RBC alloantibody. Although is it not a common cause of HDFN, it is possible and has been reported to cause mild to moderate disease. Anti-Fyb is not typically associated with HDFN, although rare case reports of anti-Fyb HDFN do exist4,6 | Fya Fyb Fy3, Fy4, Fy5, Fy6 | Moderate Mild to severe None |
| Kidd Defined by 2 antigens, Jka and Jkb The null phenotype, Jk(a−b−), is rare (1% of Polynesians, a higher proportion of Filipinos, also reported in Subcontinent and African ethnicities) Kidd antibodies can cause clinically significant hemolytic transfusion reactions but are rarely associated with clinically significant HDFN | Jka, Jkb Jk(3-) | Mild, rarely severe Mild |
| MNS Large blood group system with >30 antigens Anti-M and anti-N antibodies can be naturally occurring in a small proportion of people Anti-M is usually an IgM antibody, hence cannot cross the placenta. A small proportion may be IgG, with the potential for HDFN, which is rare but can be severe.4,6 Severe anti-M HDFN appears to be more common in people of East Asian ethnicity. Similarly, anti-N antibodies are typically IgM and are very uncommon causes of HDFN Antibodies to S, s, and U are more likely to be IgG, and, although uncommon, can possibly cause severe HDFN Within the MNS blood group, there is a subsystem of hybrid glycophorins (“Miltenberger subsystem”). These hybrid glycophorins are difficult to detect serologically but can be clinically significant. For example, the hybrid glycophorin family GP.Mur (also called Mi.III) has a population frequency of ∼7%-15% or even higher in South East Asia20-22 and antibodies against these antigens have been reported to cause significant HDFN, including cases of hydrops fetalis.23 Molecular methods have been developed to detect these antigens. | M N S, s, U | Generally mild, rarely severe None to mild Mild to severe |
This table is not exhaustive and does not cover all known antigens for each antigen group.
C/c and E/e are inherited in a codominant manner.
Overall, the most important antibodies from the point of view of antibody frequency and potential for causing HDFN are anti-D, anti-c, and anti-K, followed by anti-C and anti-E. Some antibodies are recognized not to cause HDFN and therefore, require no further surveillance or intervention throughout pregnancy. These include anti-I, anti-P1, anti-Lea, and anti-Leb.3,14 Liaison between clinical teams and transfusion laboratory staff is important once a maternal alloantibody has been identified. The transfusion laboratory staff will be able to provide input on the potential significance and recommended ongoing monitoring of any antibody identified.
Antibody titer/quantification
If a clinically significant RBC alloantibody is identified, serial monitoring of the strength of the antibody is performed to identify which pregnancies require referral and more intensive surveillance, as there is some correlation between antibody strength and the risk of fetal anemia in most cases (an important exception being anti-K, as discussed below).4,6,24
The antibody strength can be assessed by titration or quantification. Titration involves serial twofold dilutions of patient plasma and testing for antibody activity. The reciprocal of the highest dilution of plasma that gives a score of 5 reaction is referred to as the titer end point. There is variability in the titration results between laboratories due to inherent variation in assay methodology, as well as biological variation in reagent red cells. Serial antibody monitoring should ideally be performed in the same laboratory.4,6 Quantitation methods provide results in IU/mL against an international standard and are comparable between laboratories. In the past, laboratory testing included IgG subclass analysis and other assays used in an attempt to predict the severity of disease.25 These assays are now largely restricted to research.
There are recognized antibody titer/concentration thresholds (“critical threshold”) that predict sufficient risk of fetal anemia that the patient should be promptly referred to a specialist fetal medicine unit for further monitoring and appropriate subsequent management (Table 3).26 Women with a known history of a pregnancy affected by HDFN, particularly if intervention such as intrauterine transfusion (IUT) was required, should be promptly referred to a fetal medicine unit, ideally before 18 to 20 weeks of gestation, regardless of the current antibody screening result or titer.6,24
Critical RBC alloantibody titers/thresholds
| RBC alloantibody | Critical thresholds (saline IAT) | Additional notes |
|---|---|---|
| Anti-D† | Titer 32∗ Concentration ≥4 IU/mL | |
| Anti-c† | Titer 32∗ Concentration ≥7.5 IU/mL | Concurrent anti-E can result in more severe fetal anemia due to anti-c antibodies; referral at lower antibody levels recommended (eg, at detection of both antibodies) |
| Anti-K† | No critical threshold; refer for specialist management at detection of antibody, regardless of titer | Potential for early and severe HDFN with anti-K, and less robust correlation between antibody titer and risk of fetal anemia |
| Other clinically significant RBC alloantibodies‡ | Titer 32∗ |
| RBC alloantibody | Critical thresholds (saline IAT) | Additional notes |
|---|---|---|
| Anti-D† | Titer 32∗ Concentration ≥4 IU/mL | |
| Anti-c† | Titer 32∗ Concentration ≥7.5 IU/mL | Concurrent anti-E can result in more severe fetal anemia due to anti-c antibodies; referral at lower antibody levels recommended (eg, at detection of both antibodies) |
| Anti-K† | No critical threshold; refer for specialist management at detection of antibody, regardless of titer | Potential for early and severe HDFN with anti-K, and less robust correlation between antibody titer and risk of fetal anemia |
| Other clinically significant RBC alloantibodies‡ | Titer 32∗ |
Titer cutoffs may vary between laboratories particularly related to testing methodology such as column agglutination or solid phase techniques. Cutoffs should be locally established.
For anti-D, anti-c, and anti-K, guidelines recommend obtaining serial titers or quantifications monthly until 28 weeks, and fortnightly thereafter until delivery. There is minimal benefit for ongoing monitoring of antibody titers once active surveillance for fetal anemia is commenced.
For other clinically significant antibodies, recommendations are to perform titer/quantification at detection, and then again at 28 weeks.
Predicting fetal antigen expression
Once the alloantibody has crossed the placenta, it can only cause fetal/neonatal anemia if the fetus expresses its cognate antigen. If the fetus has not inherited or does not express this antigen, hemolysis cannot occur. Therefore, predicting fetal RBC antigen expression is an important step in the risk assessment of HDFN.
Blood group systems are inherited from both parents: if the father is homozygous for a particular antigen, then all his offspring will inherit the antigen; if he is heterozygous, then there is a 50% chance that the fetus will inherit the antigen. Determining the paternal RBC antigen phenotype and/or genotype can be an important tool for predicting the likelihood of fetal RBC antigen expression and therefore, the potential risk of HDFN. However, this comes with the caveat of assuming paternity and paternal availability.
Fetal red cell antigen expression can now be predicted using NIPT and molecular typing of cell-free fetal DNA, which circulates in maternal plasma. By ∼10 to 12 weeks of gestation, these levels are high enough to permit testing on a maternal plasma sample. This noninvasive method carries no risks to the fetus and avoids the issue of nonpaternity. RBC antigens that are most commonly genotyped in this manner include D, C, c, E, e, and K. Access to this technology depends on the local laboratory capabilities and funding. Where available, it is a powerful tool for the management of alloimmunized pregnancies. If a maternal RBC alloantibody is present, but the fetus is predicted to be negative for the corresponding antigen by NIPT, then there is no concern for HDFN and surveillance for fetal anemia is not required. The reader is referred elsewhere for further information on the NIPT.27,28
Managing mild to moderate HDFN
Case 1 continued
A red cell phenotype performed on her partner demonstrates that he is heterozygous for the c antigen, conferring a 50% likelihood that the fetus will be c antigen-positive, with NIPT confirming this. The antibody titer remained at 8 in the second trimester, rising to 16 and then to 32 by 30 weeks of gestation. Ultrasound monitoring for features of fetal anemia and measurement of the middle cerebral artery (MCA) peak systolic velocity (PSV) were commenced. These measurements remained normal throughout the pregnancy. Labor was induced at 38 weeks of gestation, and she delivered a healthy fetus with a positive cord blood direct antiglobulin test (DAT). Initial Hb and bilirubin were within normal range. The infant was observed for jaundice with noninvasive bilirubinometry, required 12 hours of phototherapy on day 2 and was discharged with stable Hb and serially decreasing bilirubin measurements.
This case highlights a typical course for a woman with a clinically significant RBC alloantibody who requires antenatal monitoring but without evidence of significant antenatal HDFN. In such cases, consideration must be given to the appropriate timing of delivery. Doppler measurements for fetal anemia become less reliable toward the end of the third trimester; hence, for patients with clinically significant RBC alloantibody at critical thresholds or above, we would typically recommend delivery at ∼37 to 38 weeks of gestation, although these decisions must be individualized.
At delivery, a cord blood sample should be collected for the neonatal blood group and antigen typing, DAT, Hb, and bilirubin.4,6,24 If DAT is positive, elution may be performed to determine the antibody specificity. Red cell antigen typing using monoclonal antisera for the relevant clinically significant antigens to which the woman is alloimmunized can also assist in determining which infants require closer clinical surveillance, with management including regular clinical monitoring and assessment of neurobehavioral state and presence of jaundice, as well as the formal measurement of Hb and bilirubin.
Managing potential severe HDFN
Case 2
A gravida 2 para 1 woman is found to have anti-K antibodies (titer, 16) during routine screening in the first trimester. Her obstetric history is of a forceps birth of a healthy male infant at term; she had never received a blood transfusion. No RBC antibodies were identified in the pregnancy, and the baby had no demonstrated features of HDFN. The woman’s partner is found to be heterozygous for the K antigen, and NIPT predicts the fetus to be K-positive. By 16 weeks of gestation, the titer is 32, and Doppler monitoring of fetal MCA PSV is commenced. The titer continues to rise, as does the MCA PSV; at 28 weeks, the latter is consistently measuring at 1.8 multiples of the median (MoM), and the fetal circulation appears hyperdynamic, with tricuspid regurgitation evident. Considering these features of fetal anemia, fetal blood sampling (FBS) is performed under ultrasound guidance, confirming fetal anemia with a Hb of 68 g/L. An IUT of RBCs is performed, and the posttransfusion Hb is 130 g/L. Further transfusions are required at 31 and 35 weeks, with delivery of a healthy infant occurring at 38 weeks, thus avoiding complications associated with prematurity. Neonatal care involves top-up transfusions for persisting hyporegenerative anemia associated with Kell alloimmunization.
The discovery that pulsed wave Doppler interrogation of the fetal MCA permits the assessment of anemia by determining the PSV of blood flow in that vessel represents a paradigm shift in the management of women with RBC alloimmunization.29 Hitherto, assessment required amniocentesis to determine the optical density of bile pigments in amniotic fluid at 450 nm (by definition only useful in anemia caused by hemolysis) or cordocentesis for direct measurement of fetal Hb and hematocrit. MCA PSV Doppler monitoring avoids the risks associated with invasive procedures. It remains a screening test, and the established threshold of 1.5, MoM for gestation, has a 15% false-positive rate in fetuses known to be at risk of anemia (ie, those with a high pretest probability).30 A key advantage of MCA Doppler assessment is that it can indicate anemia before the development of late signs, such as hydrops fetalis or significant cardiac dysfunction, which normally only becomes apparent when anemia becomes severe. Current guidelines recommend starting MCA Doppler monitoring from the second trimester onwards once the “critical titer” has been reached. Critical titers have been best established for RhD, in which a titer >16 is used to determine when to initiate fetal surveillance. Similar cutoffs are used for most non-D antibodies and although they generally cause less severe disease, such cutoffs are adequately sensitive in predicting those that would benefit from Doppler monitoring. Because anti-K in contrast, can cause severe anemia at low titers, a threshold of 4 to 8 is used.24
A fetal MCA PSV that is consistently >1.5 MoM (when the fetus is quiescent) is predictive of moderate-to-severe fetal anemia and is generally considered an indication for FBS at gestations <35 weeks, with delivery being indicated thereafter. FBS should only be performed when IUT can be performed at the same time and anemia should be confirmed. FBS/IUT is performed under ultrasound guidance and generally involves fetal paralysis through intramuscular injection of vecuronium or equivalent, followed by needling the umbilical vein at its placental or fetal insertion (or less commonly, a free loop) or the hepatic vein. At very early gestation, intravascular needle placement may not be possible; therefore, intraperitoneal transfusion of blood can be attempted after inserting the needle into the fetal abdomen.31
The fetal Hb, in conjunction with the gestation and donor unit Hb/hematocrit, will determine the volume of blood to be transfused with various calculators available in commonly used ultrasound reporting software such as ViewPoint (GE HealthCare, Chicago, IL), and online at the Fetal Medicine Foundation (https://fetalmedicine.org/research/assess/anemia, accessed 8 March 2024). Transfusion of a severely anemic fetus may need to be performed on 2 occasions to avoid volume overload. Optimal transfusion strategy/technique that minimizes procedural risk and optimizes outcomes has not been addressed in any randomized studies. Some use intravascular transfusion alone, whereas others use a combination of intravascular and intraperitoneal. Preparation for an IUT involves close collaboration between interventional sonologist, hematologist, and blood bank scientist. The requirements for the red cells for IUT are listed in Table 4.
Specifications for red cells for IUT
| Specification |
|---|
| 5 d old or less∗ |
| ABO compatible with mother and fetus† |
| Negative for the antigen which the maternal alloantibody is directed against, and cross-match compatible with maternal plasma |
| Antigen matched to the extended maternal red cell phenotype‡ |
| Cytomegalovirus (CMV) seronegative§ |
| K negative |
| Irradiated and used within 24 hours of irradiation§ |
| Hyperconcentrated|| |
| Specification |
|---|
| 5 d old or less∗ |
| ABO compatible with mother and fetus† |
| Negative for the antigen which the maternal alloantibody is directed against, and cross-match compatible with maternal plasma |
| Antigen matched to the extended maternal red cell phenotype‡ |
| Cytomegalovirus (CMV) seronegative§ |
| K negative |
| Irradiated and used within 24 hours of irradiation§ |
| Hyperconcentrated|| |
IUT is administered as a rapid, large-volume transfusion. The use of fresh RBC reduces the risk associated with high supernatant K+ in older red cell units.
Generally group O.
Where possible, matched to the extended maternal phenotype to reduce the risk of additional alloimmunization.
The fetus is considered a vulnerable recipient at risk of CMV and transfusion–associated graft-versus-host disease
Some centers recommend a concentration to a hematocrit of 0.8. This may present an advantage of reduced volume transfused in a fetus with hydrops, however as no clear evidence is available demonstrating the superiority of hyperconcentrated red cells, compared with packed red cells, IUT should not be delayed if this is not available.
FBS and IUT are not benign interventions: fetal loss occurs in 0.6% of procedures and, at viable gestations, an emergency cesarean section is required in 0.4% of cases.32 As a consequence, these procedures are reserved for circumstances where there is reasonable clinical suspicion of moderate-to-severe anemia.
MCA PSV monitoring becomes less sensitive after each successive IUT, as transfused adult RBCs behave differently from fetal RBCs when assessed by Doppler velocimetry.30 Subsequent transfusions may be scheduled based on the projected decline in fetal Hb, which largely reflects the natural degradation of transfused adult RBCs (not hemolysis) and fetal growth. Frequent ultrasounds to observe other features of anemia remain an important safeguard, as empiric predictions of declining fetal Hb levels are estimates at best.
The care of neonates born after 1 or more IUTs is a specialized domain.33 If the delivery is by emergency cesarean section immediately after a transfusion, the baby will likely still be paralyzed from muscle relaxant and will need invasive respiratory support. After several transfusions, the neonatal blood group reflects that of the donor blood rather than the neonate’s pretransfusion blood type, and the neonate may exhibit transfusion-related suppression of erythropoiesis, placing the baby at risk of delayed anemia requiring top-up transfusions, occasionally up to the age of 4 months. Darbopoetin alfa has been shown to reduce transfusion requirements in delayed anemia. Infants born with significant anemia and hyperbilirubinemia may require an exchange transfusion, a highly specialized procedure that is less commonly required in contemporary practice and thus should be conducted by tertiary units with a concentration of experience.
Managing subsequent pregnancies at high risk for severe HDFN
Case 3
A gravida 3 para 2 woman presents for prepregnancy counseling. She has a high titer alloantibody to RhD. Her partner was genotyped and is known to be D homozygous. Anti-D was detected antenatally during her second pregnancy in which MCA PSV surveillance was commenced from 20 weeks of gestation. During the pregnancy, 3 IUTs were performed for fetal anemia, with an initial fetal Hb of 36 g/L at 28 weeks. She delivered at 35 weeks of gestation and the infant is achieving normal milestones at age 4 years. In her third pregnancy, evidence of fetal hydrops was present at 16 weeks of gestation and fetal death occurred shortly thereafter.
This case raises the issue of management of an at-risk pregnancy with previous significant morbidity and mortality due to HDFN. Due to immune memory, alloimmunized women tend to mount earlier and more robust immune responses when exposed to the same antigen in future pregnancies; therefore, HDFN tends to present earlier and be more severe with each affected pregnancy. Although there are no specific guidelines available to direct therapy in such cases, various options exist. This includes strategies to avoid having a fetus that carries the corresponding antigen, including assisted reproductive techniques using a known antigen-negative (in this case, RhD-negative) sperm donor, or, if the partner is heterozygous for the relevant antigen, pursuing in vitro fertilization with preimplantation genetic diagnosis to select only antigen-negative embryos. These options are obviously costly and resource-intensive.
Various antenatal medical therapies have been used to try to mitigate severe early-onset fetal anemia due to HDFN and enable pregnancy to reach a gestation at which IUT can safely be used. Administration of high-dose IV immunoglobulin (IVIg) to the mother is one such option. Although there is no randomized controlled trial evidence for IVIg in HDFN, several case series indicate a beneficial effect in delaying the onset of fetal anemia and mitigating the severity of subsequent disease course.31,34-36 Importantly, IVIg will not be of benefit once significant fetal anemia has already been established. IVIg is usually well-tolerated at 1 g/kg per week.37 It is our practice to offer IVIg, where there is a likelihood of severe fetal anemia before the third trimester.
Plasma exchange, which removes antibodies from the maternal circulation, has also been used in women with a history of previous severe HDFN. Maternal risks relate to vascular access (infection, catheter-related thrombosis), hemodynamic shifts, and instability, which also may affect placental perfusion, electrolyte disturbance, and depletion of other plasma proteins, such as coagulation factors and other immunoglobulins. There is also a described phenomenon of antibody rebound, in which there is an increase in antibody levels after plasma exchange, which is thought to be due to immune stimulation or other immunomodulatory effects. Concomitant use of IVIg may mitigate this effect.38
Immunosuppressive or immunomodulatory therapies to reduce antibody production have also been described, although there is little robust evidence of efficacy. Capraru et al39 described the successful use of rituximab, along with plasma exchange with immunoadsorption and IVIg in 2 high-risk patients with anti-K HDFN.
Emerging therapies
Nipocalimab (M281) is an experimental monoclonal antibody therapy that has promising preclinical and early-phase clinical trial evidence.40,41 It binds with high specificity to the neonatal Fc receptor (FcRn), responsible for IgG transport across the placenta, and therefore blocks the transplacental passage of antibodies, including RBC alloantibodies. The phase 2 Unity study of nipocalimab enrolled 13 anti-D or anti-K alloimmunized pregnant women with singleton pregnancies, who were at high risk of early onset HDFN, with a prior obstetric history of severe fetal anemia, fetal hydrops, or stillbirth at or before 24 weeks of gestation.42 The participants received once-weekly IV infusions of nipocalimab, with no major maternal or fetal safety issues reported. The trial reported favorable results, with 54% of patients in this high-risk cohort achieving the primary end point (live birth at or after 32 weeks of gestation, without IUT throughout the entire pregnancy). The US Food and Drug Administration granted a breakthrough therapy designation for nipocalimab in February 2024. The phase 3 Azalea trial is currently underway, and we await outcome data with interest.
Case 4
A primigravida of Southeast Asian ethnicity with a monochorionic diamniotic twin pregnancy is referred with an unidentified antibody on routine testing at 28 weeks of gestation. She has no history of blood transfusions or sensitizing events. Her blood group is O RhD-positive, with no antibodies detected in her first-trimester screen. Further testing identified an anti-Jk3 antibody and her red cell phenotype is Jk(a−b−).
The Jk(a−b−) phenotype, (Jk-null phenotype), is an uncommon phenotype of the Kidd RBC antigen system, which is rare in most populations, with reported incidences of <1%. Once sensitized to other Kidd antigens (usually via transfusion or pregnancy), Jk(a−b−) individuals form an antibody called anti-Jk3. Anti-Jk3 has been associated with hemolytic transfusion reactions, as well as with case reports of mild HDFN.
This case highlights that apart from potential HDFN risk, the identification of a maternal RBC alloantibody may have important implications for transfusion support for the mother herself. The presence of the antibody can complicate pretransfusion testing in the laboratory and delay the provision of appropriate blood products. This is particularly true if the antibody specificity is for a high-frequency RBC antigen, in which case, it may be difficult to source fully compatible RBC units for transfusion at all. High-frequency antigens are those which >90% (and typically >99%) of the population express. The lack of a high-frequency antigen is, by definition, a rare phenotype and sometimes exclusive to certain ethnicities. Antibodies to high-frequency antigens can be difficult for general transfusion laboratories to identify and samples are typically sent to reference laboratories for further workup. Examples of antibodies against high-frequency antigens include anti-Jk3 (as in this case), anti-k, anti-Kpb, anti-Jsb, and anti-U among many others.
For women with antibodies to high-frequency antigens, we perform a risk assessment for bleeding at delivery and ensure that Hb and iron stores are optimized during the antenatal period with oral or IV iron, as required. We use our local transfusion medicine service to identify compatible units for transfusion and may type family members to determine if they are compatible and eligible to make a directed donation. Minimization of blood loss at birth is important with experienced staff ensuring optimal care, including active management of the third stage, consideration of tranexamic acid, and prompt escalation of care in the event of bleeding. Autologous blood collection is usually considered safe during pregnancy and may be considered where antigen-negative blood is in short supply among the donors. Consideration of future blood donation after pregnancy for frozen storage or to contribute to rare red cell inventories is recommended.
Conclusion
Appropriate transfusion practices and the success of RhD immunoprophylaxis have reduced the prevalence of pregnancies impacted by HDFN. Notwithstanding, screening for these antibodies remains a routine part of antenatal care. Management when alloantibodies occur requires a multidisciplinary approach with maternal-fetal medicine, neonatology, ultrasound, transfusion medicine, and hematology expertise. Advances in outcomes are largely the result of improvements in monitoring, improvements in the technical aspects of IUT, and care of preterm infants. Recently reported experimental agents preventing the passage of harmful alloantibodies across the placenta hold promise for dramatically changing outcomes in women with the most severe disease.
Authorship
Contribution: H.F.S., A.P., and S.C.K. contributed equally to writing and editing the manuscript.
Conflict-of-interest disclosure: The authors declare no competing financial interests.
Correspondence: Helen Frances Savoia, The Royal Children's Hospital, 50 Flemington Rd, Parkville, VIC 3052, Australia; email: helen.savoia@rch.org.au.
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