hemolytic disease of the newborn case presentation

Hemolytic Disease of the Newborn Clinical Presentation

• Author: Sameer Wagle, MBBS, MD; Chief Editor: Muhammad Aslam, MD  more...
• Sections Hemolytic Disease of the Newborn
• Pathophysiology
• Epidemiology
• Physical Examination
• Laboratory Studies
• Imaging Studies
• Approach Considerations
• Medical Care
• Complications
• Immunomodulators
• Colony-stimulating Factor
• Competitive heme oxygenase inhibitor
• Questions & Answers
• Media Gallery

Two usual patterns of Rh isoimmunization severity are noted. The disease may remain at the same degree of severity or may become progressively worst with each pregnancy. A history of hydropic birth increases the risk of fetal hydrops in the next pregnancy to 90%; the fetal hydrops occurs at about the same time or earlier in gestation in the subsequent pregnancy. Women at risk for alloimmunization should undergo an indirect Coombs test and antibody titers at their first prenatal visit. If results are positive, obtain a paternal blood type and genotype with serologic testing for other Rh antigens (C, c, E, e).

The paternal zygosity for the D allele is determined from race-specific gene frequency tables that take into account the serology results of Rh antigen expression, ethnicity, and number of previous Rh-positive children. [ 20 ] In the event of unclear ethnicity, quantitative polymerase chain reaction (PCR) of the RHD gene has been used to detect the heterozygous state. [ 21 ] Two such assays, one based on direct amplification of deletion and the other using RHD gene copy number with a reference gene, are available. Research revealed quantitative PCR to be highly accurate for detecting a paternal heterozygous state and now is preferred over serologic testing owing to the high frequency of interracial marriages. [ 22 ]

Obtaining serial maternal titers is suggested if the father is homozygous. If the father is heterozygous, determine fetal Rh genotype using PCR for the RHD gene on fetal cells obtained at amniocentesis [ 23 ] or on cell-free DNA in maternal circulation. [ 24 ] The sensitivity and specificity of PCR typing on amniotic fluid is 98.7% and 100%, respectively. However, obtaining maternal blood to rule out a maternal RHD pseudogene (in a Rh-positive fetus) and obtaining paternal blood to rule out RHD gene locus rearrangement (in a Rh-negative fetus) is important to improve the accuracy. [ 25 ] Determining fetal Rh genotype is also possible by performing cordocentesis, which is also called fetal blood sampling (FBS). FBS is associated with a more than 4-fold increase in perinatal loss compared with amniocentesis.

Indicators for severe hemolytic disease of the newborn include mothers who have had previous children with hemolytic disease, rising maternal antibody titers, rising amniotic fluid bilirubin concentration, and ultrasonographic evidence of fetal hydrops (eg, ascites , edema, pleural and pericardial effusions, worsening biophysical profile, decreasing hemoglobin [Hb] levels). The major advance in predicting the severity of hemolytic disease was the delta-OD 450 reported by Liley in 1961. [ 26 ] The serial values of deviation from baseline at 450 nm, the wavelength at which bilirubin absorbs light, are plotted on a Liley curve (see the image below) against the gestational weeks. The values above 65% on zone 2 indicate direct fetal monitoring by cordocentesis. Hematocrit (Hct) levels below 30% or a single value in zone 3 are indications for intrauterine transfusion.

The modification of Liley chart was developed by extrapolating the Liley curve [ 27 ] and is used to correct for gestations of less than 27 weeks because bilirubin levels normally peak at 23-25 weeks' gestation in unaffected fetuses (see the image below). [ 28 ]

Another curve was developed by Queenan for management of pregnancies before 27 weeks' gestation (see the image below). [ 29 ]

In a prospective evaluation, the Queenan curve predicted moderate anemia with a sensitivity of 83% and a specificity of 94%, whereas the sensitivity and specificity for severe anemia were 100% and 79%, respectively. [ 30 ] The delta-OD 450 value that plots out in the intrauterine death risk zone of Queenan curve indicates the need for FBS. A comparison of the curves found the Queenan curve to be superior to the Liley curve in overall sensitivity, specificity, and accuracy; when limited to less than 27 weeks' gestation, its sensitivity was higher by 10%, with both having a specificity of 40%. [ 31 ]

An infant born to an alloimmunized mother shows clinical signs based on the severity of the disease. The typical diagnostic findings are jaundice, pallor, hepatosplenomegaly, and fetal hydrops in severe cases. The jaundice typically manifests at birth or in the first 24 hours after birth with rapidly rising unconjugated bilirubin level. Occasionally, conjugated hyperbilirubinemia is present because of placental or hepatic dysfunction in those infants with severe hemolytic disease. Anemia is most often due to destruction of antibody-coated red blood cells by the reticuloendothelial system, and, in some infants, anemia is due to intravascular destruction. The suppression of erythropoiesis by intravascular transfusion (IVT) of adult Hb to an anemic fetus can also cause anemia. Extramedullary hematopoiesis can lead to hepatosplenomegaly, portal hypertension, and ascites.

Anemia is not the only cause of hydrops. Excessive hepatic extramedullary hematopoiesis causes portal and umbilical venous obstruction and diminished placental perfusion because of edema. Increased placental weight and edema of chorionic villi interfere with placental transport. Fetal hydrops results from fetal hypoxia, anemia, congestive cardiac failure, and hypoproteinemia secondary to hepatic dysfunction. Commonly, hydrops is not observed until the Hb level drops below approximately 4 g/dL (Hct < 15%). [ 8 ] Clinically significant jaundice occurs in as many as 20% of ABO-incompatible infants.

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• Hemolytic Disease of the Newborn. Liley curve. This graph illustrates an example of amniotic fluid spectrophotometric reading of 0.206, which when plotted at 35 weeks' gestation falls into zone 3, indicating severe hemolytic disease.
• Hemolytic Disease of the Newborn. Modified Liley curve for gestation of less than 24 weeks showing that bilirubin levels in amniotic fluid peak at 23-24 weeks' gestation.
• Hemolytic Disease of the Newborn. Queenan Curve: Modified Liley curve that shows delta-OD 450 values at 14-40 weeks' gestation.
• Hemolytic Disease of the Newborn. Slopes for peak systolic velocity in middle cerebral artery (MCA) for normal fetuses (dotted line), mildly anemic fetuses (thin line), and severely anemia fetuses (thick line).
• Hemolytic Disease of the Newborn. Management of first affected pregnancy.
• Hemolytic Disease of the Newborn. Management of pregnant women with previously affected fetus.
• Table. Comparison of Rh and ABO Incompatibility

Contributor Information and Disclosures

Sameer Wagle, MBBS, MD Consulting Staff, Division of Neonatology, Northwest Medical Center of Springdale and Willow Creek Women’s Hospital Sameer Wagle, MBBS, MD is a member of the following medical societies: American Academy of Pediatrics , American Medical Association Disclosure: Nothing to disclose.

Prashant G Deshpande, MD Attending Pediatrician, Department of Pediatrics, Christ Hospital Medical Center and Hope Children's Hospital; Assistant Clinical Professor of Pediatrics, Midwestern University Prashant G Deshpande, MD is a member of the following medical societies: American Academy of Pediatrics , American Medical Association , American Telemedicine Association Disclosure: Nothing to disclose.

Mary L Windle, PharmD Adjunct Associate Professor, University of Nebraska Medical Center College of Pharmacy; Editor-in-Chief, Medscape Drug Reference Disclosure: Nothing to disclose.

David A Clark, MD Professor and Martha Lepow Chairman of Pediatrics, Professor of Obstetrics and Gynecology, Albany Medical College; Director, Children's Hospital at Albany Medical Center David A Clark, MD is a member of the following medical societies: Alpha Omega Alpha , American Academy of Pediatrics , American College of Nutrition , American Forestry Association, American Pediatric Society , Capital District Pediatric Society, Christian Medical and Dental Associations , European Society for Paediatric Research , Eastern Society for Pediatric Research , Floyd W Denny Pediatric Alumni Society, Medical Society of the State of New York , New York Academy of Sciences , Society for Pediatric Research Disclosure: Nothing to disclose.

Muhammad Aslam, MD Professor of Pediatrics, University of California, Irvine, School of Medicine; Neonatologist, Division of Newborn Medicine, Department of Pediatrics, UC Irvine Medical Center Muhammad Aslam, MD is a member of the following medical societies: American Academy of Pediatrics Disclosure: Nothing to disclose.

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Meghan Delaney , Dana C. Matthews; Hemolytic disease of the fetus and newborn: managing the mother, fetus, and newborn. Hematology Am Soc Hematol Educ Program 2015; 2015 (1): 146–151. doi: https://doi.org/10.1182/asheducation-2015.1.146

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Hemolytic disease of the fetus and newborn (HDFN) affects 3/100 000 to 80/100 000 patients per year. It is due to maternal blood group antibodies that cause fetal red cell destruction and in some cases, marrow suppression. This process leads to fetal anemia, and in severe cases can progress to edema, ascites, heart failure, and death. Infants affected with HDFN can have hyperbilirubinemia in the acute phase and hyporegenerative anemia for weeks to months after birth. The diagnosis and management of pregnant women with HDFN is based on laboratory and radiographic monitoring. Fetuses with marked anemia may require intervention with intrauterine transfusion. HDFN due to RhD can be prevented by RhIg administration. Prevention for other causal blood group specificities is less studied.

Explain the fetal and infant clinical findings associated with hemolytic disease of the fetus and newborn (HDFN)

Describe the approach to pregnancy management when a mother has red cell alloimmunization

Discuss the prevention strategies for HDFN

Hemolytic disease of the fetus and newborn (HDFN) is rare condition that occurs when maternal red blood cell (RBC) or blood group antibodies cross the placenta during pregnancy and cause fetal red cell destruction. The fetal physiological consequences of severe anemia in the fetus can also lead to edema, ascites, hydrops, heart failure, and death. In less severe cases, the in utero red cell incompatibility can persist postnatally with neonatal anemia due to hemolysis, along with hyperbilirubinemia and erythropoietic suppression.

There are an estimated 3/100 000 to 80/100 000 cases of HDFN per year in the United States. 1   The maternal blood group antibodies that cause HDFN can be naturally occurring ABO antibodies (isohemagglutinins), or develop after exposure to foreign RBC; the latter are called blood group alloantibodies. For HDFN to occur, the fetus must be antigen positive (paternally inherited) and the mother must be antigen negative. Several studies have investigated the prevalence of red cell sensitization. In a large series of 22 102 females in the US, 254 (1.15%) of the women were found to have a red cell alloantibodies, of whom 18% had more than one alloantibody. 2   In the Netherlands, the prevalence of red cell alloantibodies detected in the first trimester was 1.2%. 3  

The most common cause of blood group incompatibility results from the ABO blood group system, with incompatibility present in up to 20% of infants. 4   However, because anti-ABO antibodies are predominantly IgM class, most are not effectively transported across the placenta. In addition, the A and B antigens are not well developed on fetal red blood cells. Together, this results in a low rate of clinically severe HDFN due to ABO compatibility, although the incidence of more mild disease varies from 1:150 to 1:3000, depending on the parameters used for the case definition, such as bilirubin levels or neonatal anemia. 1   Because maternal ABO antibodies are present without previous sensitization, HDFN due to ABO antibodies can occur in the first pregnancy and has a recurrence rate up to 87%. 1   It is most commonly seen in group O mothers with group A infants (European ancestry) or group B infants (African ancestry).

The most clinically significant forms of HDFN are caused by maternal blood group alloantibodies are of IgG1 and IgG subclasses, which cause hemolysis more effectively than other IgG subclasses. IgG1 and IgG3 are transported across the placenta by the Fc receptor from the second trimester onward. 5   Once in the fetal circulation, the antibody binds antigen-positive fetal red cells that are then cleared by the fetal spleen. Free hemoglobin is metabolized into bilirubin that is conjugated by the maternal liver. As anemia worsens, fetal hematopoiesis increases, termed “erythroblastosis fetalis” and organs involved in red blood cell synthesis (liver, spleen) may enlarge. In the most severe cases, portal hypertension and reduced hepatic synthesis of albumin leads to low plasma oncotic pressure, edema and ascites. “Hydrops fetalis” refers to the state of widespread effusions and associated high-output cardiac failure and death. 6   A large population-based study in Sweden found that the presence of maternal red cell antibodies was significantly associated with adverse outcomes, with a 1.4-2.4 relative risk of preterm delivery and a 1.5-2.6 relative risk of stillbirth in mothers with red cell allosensitization as compared to those without. 7  

After delivery, the passive blood group antibody can continue to affect neonatal red cells causing ongoing anemia until the maternal antibody is no longer present, which can be weeks to months after birth. In early neonatal anemia, bilirubin from red cell destruction can rise quickly because the fetal liver's metabolic machinery is not well developed. Very high levels of unconjugated bilirubin can lead to bilirubin encephalopathy, which clinically presents acutely as lethargy, and can include neurological and muscular manifestations, such as hypotonia, hypertonia, a weak suck, seizures, and/or coma. Chronic and permanent effects of kernicterus, which is permanent neuronal damage from hyperbilirubinemia, includes cerebral palsy, auditory dysfunction, intellectual, or other handicaps. 6   Infants may also enter a hypoproliferative phase of anemia due to erythropoietic marrow suppression from maternal antibody, as well as intrauterine transfusion, and simple transfusion ( Table 1 ). 8   Low erythropoietin production by the infant may also be contributory to low hemoglobin levels. 9   Late hemolysis can continue to cause low blood counts and elevated bilirubin.

Neonatal manifestations of HDFN anemia by time of onset

Hb indicates hemoglobin; RBC, red blood cell; and IUT, intrauterine transfusion.

Adapted from Rath et al with permission. 8  

Maternal alloimmunization results from exposure to foreign red blood cells through previous or current pregnancy, previous transfusions, or organ transplant. During pregnancy, there is spontaneous mixing between fetal and maternal circulation (fetal–maternal hemorrhage; FMH). The mixing increases throughout the pregnancy; 3%, 12%, and 45% in trimesters I, II, and III, respectively, although the amounts of fetal blood in the maternal circulation is generally very small. Hemolytic disease of the fetus and newborn due to red cell alloantibodies rarely occurs in first pregnancies because the highest risk for FMH is later in the pregnancy, especially at delivery, and new alloantibodies are more likely to be formed after delivery. Any physical perturbation of a fetus or placenta in utero also increases the risk of FMH, such as trauma, abortion, ectopic pregnancy, amniocentesis, or multiple pregnancy. 10   Once exposed, the maternal immune system may or may not respond to foreign red cell antigens. 11   The immune response to red cell antigens is complex and not fully understood. It is clear that the RhD antigen is the most potent immunogen of all of the red cell antigens; 85% of RhD-negative individuals will sensitize (form anti-D) after challenge with a 200 mL transfusion of red cells, although more recent data suggests this is far lower. 12   Although as little as 0.1 to 1 mL of RhD-positive red cells can stimulate antibody production, the volumes of FMH are generally small, which contributes to relatively low alloimmunization rates in pregnancy. Before Rh(D) immunoprophylaxis was implemented in 1968, 16% of ABO compatible D-negative mothers with D-positive infants developed anti-D antibody. However, a much lower amount (≤2%) developed anti-D in mother/fetus pairs that were ABO incompatible because of the ABO antibody mediated clearance of fetal red cells from the maternal circulation. 13  

Although RhD remains the most prevalent cause for HDFN due to allosensitization, other red cell antigens are known to be commonly etiologic ( Table 2 ). Other antibodies that have been less commonly reported include E, k, Kp a , Kp b , Ku, Ge, M, Js a , Js b , Jk a , Fy a , Fy b , S, s, and U. 4   A Dutch case-controlled study utilizing a national database of 900 pregnant women with RBC sensitization to non-RhD red cell antibodies enumerated the risk factors for maternal allosensitization. Factors found to be associated with red cell allosensitization in the women were previous major surgery, red cell or platelet transfusion, multiparity, having had a previous male child, and operative removal of the placenta. 14  

Red blood cell antibody specificity in female population studies

Number of samples with antibody per 1000 samples.

Adapted from Giefman-Holtzman et al. 2  

All pregnant women should have testing performed, including a blood type (ABO, RhD) and antibody detection test (indirect antiglobulin test) that detects IgG antibodies. 15   For patients with red cell sensitization, the antibody specificity is determined and initial risk stratification occurs. Certain blood group antibodies such as anti-I, -P1, -Le a , and -Le b , may be ignored because the corresponding (cognate) antigens are incompletely developed at birth, the antibodies are typically not IgG, and clinical experience has established the rarity of their causing HDFN. 4   Women with red blood cell sensitization to clinically significant red cell antigens (such as D, E, c, K, etc) are transitioned into a pathway of more intensive diagnostic testing and monitoring. If paternity is assured, the paternal blood type is usually determined to predict fetal risk of inheriting the antigen that the maternal antibody is directed against. For most blood group systems, serological testing of the father's blood type is sufficient to predict homozygosity or heterozygosity of the antigen. For instance, anti-K and anti-k antisera can detect K and k antigens, respectively, and provide accurate prediction of the risk that the fetus has inherited the blood group antigen. For RhD, serological testing alone cannot predict the number of RhD genes that the father carries because there is no antithetical allele for the RhD gene. Thus, in the case of maternal anti-D sensitization, paternal genotyping to detect copy number of the RhD gene is recommended. 16   Fetuses may bear a 50% risk of antigen inheritance when testing identifies paternal heterozygosity for the antigen in question.. Direct fetal genotyping can determine fetal blood group expression in these settings and provide accurate prediction of fetal risk for HDFN in sensitized mothers. Red cell genotyping can be accomplished using fetal amniocyte genetic testing (obtained via amniocentesis or chorionic villus sampling), or using fetal DNA obtained from maternal serum. 17   The latter methodology has found broad appeal due to its noninvasive approach. 18  

For pregnancies at risk of HDFN due to maternal alloimmunization and possible fetal RBC expression of the cognate antigen, prenatal care by maternal–fetal medicine physicians is recommended. A detailed maternal history is useful to determine previous pregnancy outcomes, particularly for past stillbirths or hydropic fetal losses, and potential etiology of the offending red cell antibody. In addition, fetal ultrasound to determine gestational age and absence of ascites is indicated. 19  

The red cell antibody titer, or strength, helps with further stratification, although the relatively subjective nature of these assays should always be kept in mind. 20 , 21   Traditionally, serial antibody titers are used to detect ongoing sensitization, with arbitrary thresholds of increasing antibody strength used to indicate ongoing and increasing immune stimulation, presumably due to the presence of fetal red cell antigen. If there was a previously affected pregnancy, trending of the titer will not be a reliable measure of increasing sensitization. In addition, transfusion laboratories establish critical antibody titers at which the antibody strength has reached a level that may lead to significant fetal anemia (titers of 1:16-32 are commonly used). 4   However, because the Kell blood group antigens are present on early red cell precursors, a maternal anti-K of relatively low titer, such as 8, may lead to severe hypoproliferative anemia. 22   Using other techniques for antibody strength determination, such as flow cytometry, may be more precise than antibody titers. 23  

For pregnancies that have reached 16-24 weeks, or when a critical antibody titer is reached (depending on maternal history of previously affected pregnancies), fetal anemia is monitored using cerebral MCA Doppler velocity measurements every 2 weeks for risk stratification ( Figure 1 ). 19 , 24   Correlative studies support that the use of the noninvasive MCA Doppler technique as a surrogate measurement for assessing fetal anemia. 19   Doppler readings that are >1.5 multiples of the mean (MoM) are very sensitive, with a 12% false-positive rate, thus trending is important. Weekly fetal monitoring, such as ultrasound and fetal heart rate monitoring, is also often performed).

Diagram of fetal middle cerebral artery Doppler velocimetry testing. Adapted from Moise 42   with permission.

When fetal anemia becomes moderate to severe as indicated by Doppler MoM measurements exceeding 1.5, invasive testing via cordocentesis is done to determine fetal hematocrit. If the fetus has not reached an acceptable gestational age for delivery, and the hematocrit level is <30%, intrauterine transfusion is usually indicated. Blood products for fetal transfusion should be ready at the start of the procedure for immediate use. Intrauterine transfusion (IUT) is performed by inserting a needle into the umbilical vein using ultrasound guidance and infusion of red cells at a predetermined hematocrit level. The selection of red cell products for intrauterine transfusion is typically Group O, Rh D-negative (or -positive, depending on maternal blood group antibody), leukocyte reduced, hemoglobin S-negative, CMV-safe (CMV seronegative or leukocyte reduced), irradiated, and antigen-negative for maternal red cell antibody/antibodies. Because authors have reported that there is a risk of additional maternal red cell antibody formation after IUT, some centers have adopted providing prospectively matched Rh C, c, E, e, and K-matched transfusions. 25   Despite this, there is evidence that women undergoing Rh- and K-matched IUTs still form additional alloantibodies [to Duffy (FY), Kidd (JK) and (MNS) S blood group antigens]. 26   Following one or more IUT procedures, the fetal circulation is comprised primarily of donor red cells, as fetal marrow production is suppressed and the remaining circulating red cells are destroyed. 27   The procedure of intrauterine transfusion carries a 1%-3% risk of fetal adverse events such as infection or rupture of membranes; procedure outcomes in the early second trimester are poor. 28-30  

Small patient series have explored using maternal treatment with plasma exchange and/or intravenous immunoglobulin (IVIG) to blunt the effect of the maternal antibody on the fetal red cells with some success. 31   The American Society for Apheresis considers plasma exchange in this setting a Category II (second line therapy), with a weak grade of evidence. 32   The ultimate decision for delivery is based on the treating physician's judgement, and it is standard to maintain pregnancy until the fetus has reached a safe gestational age. For severely affected fetuses, the risk of continued monitoring and intrauterine transfusion is balanced against this, and most suggest delivery at 37-38weeks, although earlier delivery may be warranted in severe HDFN. 24  

At birth, the connection to the maternal circulation is severed, and the risk of neonatal hyperbilirubinemia increases significantly because of the immature development of the metabolic pathway to break down bilirubin in the neonatal liver. Although most jaundice in newborns is benign, management of hyperbilirubinemia is critical in the neonatal period because of the risk for bilirubin-induced encephalopathy. 33   Affected infants may need phototherapy to oxidize unconjugated bilirubin to allow for urinary excretion. For patients with known HDFN, close observation of bilirubin levels and hemoglobin is warranted to determine whether neonatal exchange transfusion is needed to wash out bilirubin and maternal antibody, and/or if transfusions are indicated to support oxygen carrying capacity to the tissues. 8   Administration of IVIG to the newborn has been used to reduce the need for exchange transfusions and phototherapy, but it does not affect the need for top off transfusions, and a Cochrane review suggests that more study is needed to determine the best use of IVIG in this setting. 8   When levels of bilirubin reach critical levels, exchange transfusion is indicated ( Table 3 ). Blood product selection is similar to that of IUT, however, because infant whole blood is also being removed, the RBC unit is usually mixed with a plasma unit to create reconstituted whole blood. After a two-volume exchange transfusion, ∼90% of the red cells have been replaced and 50% of the bilirubin has been removed. After exchange transfusion, a platelet count should be performed to monitor for iatrogenic thrombocytopenia.

Guidelines for exchange transfusion in infants 35 or more weeks gestation

During birth hospitalization, exchange transfusion is recommended if the TSB rises to these levels despite intensive phototherapy. For readmitted infants, if the TSB level is above the exchange level, repeat TSB measurement every 2-3 hours and consider exchange if the TSB remains above the levels indicated after intensive phototherapy for 6 hours.

Adapted from the Clinical Guideline for Management of Hyperbilirubinemia. 33  

Hyporegenerative anemia can last for many weeks after birth ( Table 1 ). Infants must be carefully monitored for clinical signs of ongoing anemia, whichis most likely manifested by poor feeding, the most aerobic activity for neonates. They may also have increased sleep as anemia worsens. In ongoing anemia, the reticulocyte production from the fetal bone marrow may be decreased, and other cells lines, such as neutrophils can be affected. Weekly monitoring of reticulocytes and hematocrit will help to guide decision making about transfusion, and also provide reassurance when the marrow is recovering.

Prevention of HDFN can be divided into primary and secondary measures; there are no international standards, thus, nations differ on preventative measures including dosing and dosing schedules of RhIg and approach to transfusion. Primary prevention focuses on prevention of maternal alloimmunization in the first pregnancy. This is encompassed by the policy employed by some transfusion services, or blood banks, to provide red cell transfusions to females of childbearing potential that are more highly matched than standard transfusions to prevent transfusion-induced red cell sensitization. For instance, in some European countries, K-negative RBC units are provided to women younger than 45-50 years of age. Other nations provide additional matching for antigens, such as c and E. 34   In a retrospective review in Croatia, 48% of 214 pregnancies with maternal sensitization to E, K, or c had a history of transfusion, suggesting that matching for these antigens may be protective, however, the paternal antigen status was not reported. 35   A study in the Netherlands found that a proportion of maternal sensitization to non-RhD blood groups with clinically affected offspring was due to intrauterine transfusion itself. 26   Therefore, these measures may be effective in reducing the incidence of HDFN; however, further comparative studies are needed.

Secondary prevention of HDFN focuses on the RhD RBC antigen given that RhD immune globulin (RhIg) is available to prevent naïve RhD-negative immune systems from synthesizing anti-D antibody after exposure to small amounts of RhD antigen. The risk of an RhD-negative mother becoming allosensitized can be reduced to from 16% to <0.1% by the appropriate administration of RhIg. 13   At this time, there are no other pharmaceutical therapies that can prevent blood group sensitization for other blood group antigens. The transfusion service laboratory plays a critical role in guiding therapy with RhIg. For pregnant women who are RhD-negative, RhIg is typically administered at 2 time points over the course of the pregnancy. The first dose is provided at 28 weeks gestation, as recommended by the American College of Obstetricians and Gynecologists (ACOG) because the majority of allosensitization appears to occur after this time point, and the second after delivery of an RhD-positive infant. 36   The 28 week dose reduces the rate of allosensitization to RhD from 1.5% (antenatal administration only) to 0.1%. As noted above, any procedure or trauma that increases the rate of fetomaternal hemorrhage should elicit the administration of an additional RhIg dose. The antenatal RhIg dose is increased when the volume of fetomaternal hemorrhage is found to be ≥10 mL using the Kleihauer–Betke test. Although used for many years, the Kleihauer–Betke test has been found to be imprecise, and recent attention has focused on newer technologies, such as flow cytometry to provide more accurate quantification of the FMH volume 37  

As molecular testing advances throughout the field of medicine, so does the application of blood typing using molecular techniques. In certain patients, serological reagents do not accurately detect the RhD type. The most common genetic backgrounds that account for this serological typing problem are called weak D phenotypes. Recently, authors have encouraged the use of RhD genetic testing for patients with a weak D phenotype to provide accurate and actionable results for RhD blood typing and RhIg administration. 38 , 39   Further scientific study is needed to elucidate the clinical significance of different RhD genotypes in various ethnic backgrounds and the risk factors for RhIg failure. 40   Precise determination of fetal RhD typing has been widely accepted in Europe and improved the ability to guide RhIg therapy. 18  

In conclusion, HDFN is a multifaceted disease that has distinct technical considerations over several critical time periods of fetal and neonatal development. Advances in maternal–fetal medicine, such as the inventions of RhIg, IUT and noninvasive fetal genetic testing, have led to dramatic improvements in the outcomes of HDFN and prevention of maternal allosensitization. 41   Future advances in blood typing and noninvasive testing will continue to improve the care of mothers and their offspring affected by blood group incompatibility.

Meghan Delaney, Bloodworks NW, 921 Terry Ave, Seattle, WA 98104; Phone: 206-689-6500; e-mail: [email protected] .

Competing Interests

Conflict-of-interest disclosures: M.D. has consulted for Williams Kastner and has received honoraria from Grifols/Novartis and Bioarray/Immucor; and D.C.M. declares no competing financial interests.

Author notes

Off-label drug use: IVIG is included as a therapy for mothers with HDFN. This will be discussed in brief.

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Hemolytic Disease of the Newborn (HDN)

What is hemolytic disease of the newborn?

Hemolytic disease of the newborn (HDN) is a blood problem in newborn babies. It occurs when your baby's red blood cells break down at a fast rate. It’s also called erythroblastosis fetalis. 

• Hemolytic means breaking down of red blood cells.
• Erythroblastosis means making immature red blood cells.
• Fetalis means fetus.

What causes HDN in a newborn?

All people have a blood type (A, B, AB, or O). Everyone also has an Rh factor (positive or negative). There can be a problem if a mother and baby have a different blood type and Rh factor.

HDN happens most often when an Rh negative mother has a baby with an Rh positive father. If the baby's Rh factor is positive, like his or her father's, this can be an issue if the baby's red blood cells cross to the Rh negative mother.

This often happens at birth when the placenta breaks away. But it may also happen any time the mother’s and baby's blood cells mix. This can occur during a miscarriage or fall. It may also happen during a prenatal test. These can include amniocentesis or chorionic villus sampling. These tests use a needle to take a sample of tissue. They may cause bleeding.

The Rh negative mother’s immune system sees the baby's Rh positive red blood cells as foreign. Your immune system responds by making antibodies to fight and destroy these foreign cells. Your immune system stores these antibodies in case these foreign cells come back again. This can happen in a future pregnancy. You are now Rh sensitized.

Rh sensitization normally isn’t a problem with a first pregnancy. Most problems occur in future pregnancies with another Rh positive baby. During that pregnancy, the mother's antibodies cross the placenta to fight the Rh positive cells in the baby's body. As the antibodies destroy the cells, the baby gets sick. This is called erythroblastosis fetalis during pregnancy. Once the baby is born, it’s called HDN.

Which children are at risk for HDN?

The following can raise your risk for having a baby with HDN:

• You’re Rh negative and have an Rh positive baby but haven’t received treatment.
• You’re Rh negative and have been sensitized. This can happen in a past pregnancy with an Rh positive baby. Or it can happen because of an injury or test in this pregnancy with an Rh positive baby. 

HDN is about 3 times more common in Caucasian babies than in African-American babies.

What are the symptoms of HDN in a newborn?

Symptoms can occur a bit differently in each pregnancy and child.

During pregnancy, you won't notice any symptoms. But your healthcare provider may see the following during a prenatal test:

• A yellow coloring of amniotic fluid. This color may be because of bilirubin. This is a substance that forms as blood cells break down.
• Your baby may have a big liver, spleen, or heart. There may also be extra fluid in his or her stomach, lungs, or scalp. These are signs of hydrops fetalis. This condition causes severe swelling (edema).

After birth, symptoms in your baby may include:

• Pale-looking skin. This is from having too few red blood cells (anemia).
• Yellow coloring of your baby’s umbilical cord, skin, and the whites of his or her eyes (jaundice). Your baby may not look yellow right after birth. But jaundice can come on quickly. It often starts within 24 to 36 hours.  
• Your newborn may have a big liver and spleen.
• A newborn with hydrops fetalis may have severe swelling of their entire body. They may also be very pale and have trouble breathing.

How is HDN diagnosed in a newborn?

HDN can cause symptoms similar to those caused by other conditions. To make a diagnosis, your child’s healthcare provider will look for blood types that cannot work together. Sometimes, this diagnosis is made during pregnancy. It will be based on results from the following tests:

• Blood test. Testing is done to look for for Rh positive antibodies in your blood.
• Ultrasound. This test can show enlarged organs or fluid buildup in your baby.
• Amniocentesis. This test is done to check the amount of bilirubin in the amniotic fluid. In this test, a needle is put into your abdominal and uterine wall. It goes through to the amniotic sac. The needle takes a sample of amniotic fluid.
• Percutaneous umbilical cord blood sampling. This test is also called fetal blood sampling. In this test, a blood sample is taken from your baby’s umbilical cord. Your child’s healthcare provider will check this blood for antibodies, bilirubin, and anemia. This is done to check if your baby needs an intrauterine blood transfusion.

The following tests are used to diagnose HDN after your baby is born:

• Testing of your baby's umbilical cord. This can show your baby’s blood group, Rh factor, red blood cell count, and antibodies.
• Testing of the baby's blood for bilirubin levels.

How is HDN treated in a newborn?

Treatment will depend on your child’s symptoms, age, and general health. It will also depend on how severe the condition is.

During pregnancy, treatment for HDN may include the following.

A healthcare provider will check your baby’s blood flow with an ultrasound.

Intrauterine blood transfusion

This test puts red blood cells into your baby's circulation. In this test, a needle is placed through your uterus. It goes into your baby’s abdominal cavity to a vein in the umbilical cord. Your baby may need sedative medicine to keep him or her from moving. You may need to have more than 1 transfusion.

Early delivery

If your baby gets certain complications, he or she may need to be born early. Your healthcare provider may induce labor may once your baby has mature lungs. This can keep HDN from getting worse.  

After birth, treatment may include the following.

Blood transfusions

This may be done if your baby has severe anemia.

Intravenous fluids

This may be done if your baby has low blood pressure.

Phototherapy

In this test, your baby is put under a special light. This helps your baby get rid of extra bilirubin.

Help with breathing

Your baby may need oxygen, a substance in the lungs that helps keep the tiny air sacs open (surfactant), or a mechanical breathing machine to breathe better.

Exchange transfusion

This test removes your baby’s blood that has a high bilirubin level. It replaces it with fresh blood that has a normal bilirubin level. This raises your baby’s red blood cell count. It also lowers his or her bilirubin level. In this test, your baby will alternate giving and getting small amounts of blood. This will be done through a vein or artery. Your baby may need to have this procedure again if his or her bilirubin levels stay high.

Intravenous immunoglobulin (IVIG)

IVIG is a solution made from blood plasma. It contains antibodies to help the baby's immune system. IVIG reduces your baby’s breakdown of red blood cells. It may also lower his or her bilirubin levels.  

What are possible complications of HDN in a newborn?

When your antibodies attack your baby’s red blood cells, they are broken down and destroyed (hemolysis).

When your baby’s red blood cells break down, bilirubin is formed. It’s hard for babies to get rid of bilirubin. It can build up in their blood, tissues, and fluids. This is called hyperbilirubinemia. Bilirubin makes a baby’s skin, eyes, and other tissues to turn yellow. This is called jaundice.

When red blood cells breakdown, this makes your baby anemic. Anemia is dangerous. In anemia, your baby’s blood makes more red blood cells very quickly. This happens in the bone marrow, liver, and spleen. This causes these organs to get bigger. The new red blood cells are often immature and can’t do the work of mature red blood cells.

Complications of HDN can be mild or severe.

During pregnancy, your baby may have the following:

• Mild anemia, hyperbilirubinemia, and jaundice. The placenta gets rid of some bilirubin. But it can’t remove all of it.
• Severe anemia. This can cause your baby’s liver and spleen to get too big. This can also affect other organs.
• Hydrops fetalis. This happens when your baby's organs aren’t able to handle the anemia. Your baby’s heart will start to fail. This will cause large amounts of fluid buildup in your baby's tissues and organs. Babies with this condition are at risk for being stillborn.

After birth, your baby may have the following:

• Severe hyperbilirubinemia and jaundice. Your baby’s liver can’t handle the large amount of bilirubin. This causes your baby’s liver to grow too big. He or she will still have anemia.
• Kernicterus. This is the most severe form of hyperbilirubinemia. It’s because of the buildup of bilirubin in your baby’s brain. This can cause seizures, brain damage, and deafness. It can even cause death.

What can I do to prevent hemolytic disease of the newborn?

HDN can be prevented. Almost all women will have a blood test to learn their blood type early in pregnancy.

If you’re Rh negative and have not been sensitized, you’ll get a medicine called Rh immunoglobulin (RhoGAM). This medicine can stop your antibodies from reacting to your baby’s Rh positive cells. Many women get RhoGAM around week 28 of pregnancy.

If your baby is Rh positive, you’ll get a second dose of medicine within 72 hours of giving birth. If your baby is Rh negative, you won’t need a second dose

Key points about hemolytic disease of the newborn

• HDN occurs when your baby's red blood cells break down at a fast rate.
• HDN happens when an Rh negative mother has a baby with an Rh positive father.
• If the Rh negative mother has been sensitized to Rh positive blood, her immune system will make antibodies to attack her baby.
• When the antibodies enter the baby's bloodstream, they will attack the red blood cells. This causes them to break down. This can cause problems.
• This condition can be prevented. Women who are Rh negative and haven’t been sensitized can receive medicine. This medicine can stop your antibodies from reacting to your baby’s Rh positive cells.

Tips to help you get the most from a visit to your child’s healthcare provider:

• Know the reason for the visit and what you want to happen.
• Before your visit, write down questions you want answered.
• At the visit, write down the name of a new diagnosis, and any new medicines, treatments, or tests. Also write down any new instructions your provider gives you for your child.
• Know why a new medicine or treatment is prescribed and how it will help your child. Also know what the side effects are.
• Ask if your child’s condition can be treated in other ways.
• Know why a test or procedure is recommended and what the results could mean.
• Know what to expect if your child does not take the medicine or have the test or procedure.
• If your child has a follow-up appointment, write down the date, time, and purpose for that visit.
• Know how you can contact your child’s provider after office hours. This is important if your child becomes ill and you have questions or need advice.

Related Links

• Neonatology Care
• Pediatric Hematology
• Bass Center for Childhood Cancer and Blood Diseases
• Hemolytic Anemia in Children
• Vitamin K Deficiency Bleeding in the Newborn

Related Topics

High-Risk Newborn Blood Disorders

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Hemolytic Disease of the Newborn

What is hemolytic disease of the newborn (hdn).

Hemolytic disease of the newborn is also called erythroblastosis fetalis. This condition occurs when there is an incompatibility between the blood types of the mother and baby.

• "Hemolytic" means breaking down of red blood cells
• "Erythroblastosis" refers to making of immature red blood cells
• "Fetalis" refers to fetus

What causes hemolytic disease of the newborn (HDN)?

HDN most frequently occurs when an Rh negative mother has a baby with an Rh positive father. When the baby's Rh factor is positive, like the father's, problems can develop if the baby's red blood cells cross to the Rh negative mother. This usually happens at delivery when the placenta detaches. However, it may also happen anytime blood cells of the two circulations mix, such as during a miscarriage or abortion, with a fall, or during an invasive prenatal testing procedure (such as an amniocentesis or chorionic villus sampling).

The mother's immune system sees the baby's Rh positive red blood cells as "foreign." Just as when bacteria invade the body, the immune system responds by developing antibodies to fight and destroy these foreign cells. The mother's immune system then keeps the antibodies in case the foreign cells appear again, even in a future pregnancy. The mother is now "Rh sensitized."

In a first pregnancy, Rh sensitization is not likely. Usually, it only becomes a problem in a future pregnancy with another Rh positive baby. During that pregnancy, the mother's antibodies cross the placenta to fight the Rh positive cells in the baby's body. As the antibodies destroy the red blood cells, the baby can become sick. This is called erythroblastosis fetalis during pregnancy. In the newborn, the condition is called hemolytic disease of the newborn.

Who is affected by hemolytic disease of the newborn?

Babies affected by HDN are usually in a mother's second or higher pregnancy, after she has become sensitized with a first baby. HDN due to Rh incompatibility is about three times more likely in Caucasian babies than African-American babies.

Why is hemolytic disease of the newborn a concern?

When the mother's antibodies attack the red blood cells, they are broken down and destroyed (hemolysis). This makes the baby anemic. Anemia is dangerous because it limits the ability of the blood to carry oxygen to the baby's organs and tissues. As a result:

• The baby's body responds to the hemolysis by trying to make more red blood cells very quickly in the bone marrow and the liver and spleen. This causes these organs to get bigger. The new red blood cells, called erythroblasts, are often immature and are not able to do the work of mature red blood cells.
• As the red blood cells break down, a substance called bilirubin is formed. Babies are not easily able to get rid of the bilirubin and it can build up in the blood and other tissues and fluids of the baby's body. This is called hyperbilirubinemia. Because bilirubin has a pigment or coloring, it causes a yellowing of the baby's skin and tissues. This is called jaundice.

Complications of hemolytic disease of the newborn can range from mild to severe. The following are some of the problems that can result:

During pregnancy:

• Mild anemia, hyperbilirubinemia, and jaundice. The placenta helps rid some of the bilirubin, but not all.
• Severe anemia with enlargement of the liver and spleen. When these organs and the bone marrow cannot compensate for the fast destruction of red blood cells, severe anemia results and other organs are affected.
• Hydrops fetalis. This occurs as the baby's organs are unable to handle the anemia. The heart begins to fail and large amounts of fluid build up in the baby's tissues and organs. A fetus with hydrops is at great risk of being stillborn.

After birth:

• Severe hyperbilirubinemia and jaundice. The baby's liver is unable to handle the large amount of bilirubin that results from red blood cell breakdown. The baby's liver is enlarged and anemia continues.
• Kernicterus. Kernicterus is the most severe form of hyperbilirubinemia and results from the buildup of bilirubin in the brain. This can cause seizures, brain damage, deafness, and death.

What are the symptoms of hemolytic disease of the newborn?

The following are the most common symptoms of hemolytic disease of the newborn. However, each baby may experience symptoms differently. During pregnancy symptoms may include:

• With amniocentesis, the amniotic fluid may have a yellow coloring and contain bilirubin.
• Ultrasound of the fetus shows enlarged liver, spleen, or heart and fluid buildup in the fetus's abdomen, around the lungs, or in the scalp.

After birth, symptoms may include:

• A pale coloring may be evident, due to anemia.
• Jaundice, or yellow coloring of amniotic fluid, umbilical cord, skin, and eyes may be present. The baby may not look yellow immediately after birth, but jaundice can develop quickly, usually within 24 to 36 hours.
• The newborn may have an enlarged liver and spleen.
• Babies with hydrops fetalis have severe edema (swelling) of the entire body and are extremely pale. They often have difficulty breathing.

How is hemolytic disease of the newborn diagnosed?

Because anemia, hyperbilirubinemia, and hydrops fetalis can occur with other diseases and conditions, the accurate diagnosis of HDN depends on determining if there is a blood group or blood type incompatibility. Sometimes, the diagnosis can be made during pregnancy based on information from the following tests:

• Testing for the presence of Rh positive antibodies in the mother's blood
• Ultrasound - to detect organ enlargement or fluid buildup in the fetus. Ultrasound is a diagnostic imaging technique which uses high-frequency sound waves and a computer to create images of blood vessels, tissues, and organs. Ultrasound is used to view internal organs as they function, and to assess blood flow through various vessels.
• Amniocentesis - to measure the amount of bilirubin in the amniotic fluid. Amniocentesis is a test performed to determine chromosomal and genetic disorders and certain birth defects. The test involves inserting a needle through the abdominal and uterine wall into the amniotic sac to retrieve a sample of amniotic fluid.
• Sampling of some of the blood from the fetal umbilical cord during pregnancy to check for antibodies, bilirubin, and anemia in the fetus.

Once a baby is born, diagnostic tests for HDN may include the following:

• Testing of the baby's umbilical cord blood for blood group, Rh factor, red blood cell count, and antibodies
• Testing of the baby's blood for bilirubin levels

Treatment for hemolytic disease of the newborn

Once HDN is diagnosed, treatment may be needed. Specific treatment for hemolytic disease of the newborn will be determined by your baby's doctor based on:

• Your baby's gestational age, overall health, and medical history
• Extent of the disease
• Your baby's tolerance for specific medications, procedures, or therapies
• Expectations for the course of the disease
• Your opinion or preference

During pregnancy, treatment for HDN may include:

• Intrauterine blood transfusion of red blood cells into the baby's circulation. This is done by placing a needle through the mother's uterus and into the abdominal cavity of the fetus or directly into the vein in the umbilical cord. It may be necessary to give a sedative medication to keep the baby from moving. Intrauterine transfusions may need to be repeated.
• Early delivery if the fetus develops complications. If the fetus has mature lungs, labor and delivery may be induced to prevent worsening of HDN.

After birth, treatment may include:

• Blood transfusions (for severe anemia)
• Intravenous fluids (for low blood pressure)
• Help for respiratory distress using oxygen, surfactant,  or a mechanical breathing machine
• Exchange transfusion to replace the baby's damaged blood with fresh blood. The exchange transfusion helps increase the red blood cell count and lower the levels of bilirubin. An exchange transfusion is done by alternating giving and withdrawing blood in small amounts through a vein or artery. Exchange transfusions may need to be repeated if the bilirubin levels remain high.
• Intravenous immunoglobin(IVIG). IVIG is a solution made from blood plasma that contains antibodies to help the baby's immune system. IVIG may help reduce the breakdown of red blood cells and lower bilirubin levels.  

Prevention of hemolytic disease of the newborn

Fortunately, HDN is a very preventable disease. Because of the advances in prenatal care, nearly all women with Rh negative blood are identified in early pregnancy by blood testing. If a mother is Rh negative and has not been sensitized, she is usually given a drug called Rh immunoglobulin (RhIg), also known as RhoGAM. This is a specially developed blood product that can prevent an Rh negative mother's antibodies from being able to react to Rh positive cells. Many women are given RhoGAM around the 28th week of pregnancy. After the baby is born, a woman should receive a second dose of the drug within 72 hours, if her baby is Rh positive. If her baby is Rh negative, she does not need another dose.

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• Case report
• Open access
• Published: 20 February 2012

Hemolytic disease of the fetus and newborn caused by anti-D and anti-S alloantibodies: a case report

• Rabeya Yousuf 1 ,
• Suria Abdul Aziz 1 ,
• Nurasyikin Yusof 1 &
• Chooi-Fun Leong 1  

Journal of Medical Case Reports volume  6 , Article number:  71 ( 2012 ) Cite this article

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Introduction

Hemolytic disease of the fetus and newborn is most commonly caused by anti-D alloantibody. It is usually seen in Rhesus D (RhD)-negative mothers that have been previously sensitized. We report here a case of hemolytic disease of the fetus and newborn in a newborn baby caused by anti-D and anti-S alloantibodies, born to a mother who was RhD negative, but with no previous serological evidence of RhD alloimmunization.

Case presentation

A one-day-old Chinese baby boy was born to a mother who was group A RhD negative. The baby was jaundiced with hyperbilirubinemia, but with no evidence of infection. His blood group was group A RhD positive, his direct Coombs' test result was positive and red cell elution studies demonstrated the presence of anti-D and anti-S alloantibodies. Investigations performed on the maternal blood during the 22 weeks of gestation showed the presence of anti-S antibodies only. Repeat investigations performed post-natally showed the presence of similar antibodies as in the newborn and an anti-D titer of 1:32 (0.25 IU/mL), which was significant. A diagnosis of hemolytic disease of the fetus and newborn secondary to anti-D and anti-S was made. The baby was treated with phototherapy and close monitoring. He was discharged well after five days of phototherapy.

Conclusions

This case illustrates the possibility of an anamnestic response of allo-anti-D from previous sensitization in a RhD-negative mother, or the development of anti-D in mid-trimester. Thus, it highlights the importance of thorough antenatal ABO, RhD blood grouping and antibody screening, and if necessary, antibody identification and regular monitoring of antibody screening and antibody levels for prevention or early detection of hemolytic disease of the fetus and newborn, especially in cases of mothers with clinically significant red cell alloantibody.

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Hemolytic disease of the fetus and newborn (HDFN) is characterized by the presence of IgG antibodies in the maternal circulation, directed against a paternally derived antigen present in the fetal/neonatal red cells that cause hemolysis in the fetus by crossing the placenta and sensitizing red cells for destruction by the macrophages in the fetal spleen [ 1 ]. It was first described in 1609 by a French midwife [ 2 ] but established in 1939 by Levine and Stetson. They reported a transfusion reaction from transfusing the husband's blood to a postpartum woman who had been immunized through a feto-maternal hemorrhage [ 3 ]. The serological tests for the diagnosis of HDFN includes a positive direct Coombs' test (DCT) on the baby's red blood cells and the presence of an IgG red cell alloantibody in both cord blood eluate and maternal sera. The presence of the corresponding antigen on cord cells confirms the diagnosis of HDFN [ 4 , 5 ]. The most severe HDFN is caused by IgG antibodies directed against D, c or K antigens on the fetal red cells, but any IgG antibodies can cause HDFN [ 6 ]. Anti-S has been documented as a rare cause of HDFN [ 7 ].

In this study, we report a case of HDFN caused by anti-D and anti-S in a para 3+1 mother who had no anti-D antibodies detected during the first trimester of pregnancy. The presence of allo-anti-D in the newborn and the mother herself postpartum may suggest an anamnestic response from previous sensitization or the development of anti-D during early trimesters of pregnancy. It also highlights the importance of regular monitoring of antibody screening in pregnant women, especially Rhesus D (RhD)-negative mothers, in view of the high immunogenicity of the RhD antigen.

A full-term, Chinese baby boy was born to a 30-year-old woman at 38 weeks of gestation. The baby weighed 2.8 kg with an Apgar score of 9/10. The baby was noted to have mild jaundice with normal vital signs on day one of life; there was no evidence to suggest other causes of neonatal jaundice such as intrauterine infections and his glucose-6-phosphate dehydrogenase screen was normal. Laboratory investigations showed that his total bilirubin was 198 μmol/L and hemoglobin was 19 g/dL. The baby's blood group was A RhD positive with a red cell phenotype of ccDEe (R2r) and SS. The result of a DCT was positive and red cell elution studies of the baby's blood identified the presence of anti-D and anti-S antibodies.

The mother was para 3+1. Her first pregnancy was aborted five years ago and unfortunately no investigation was performed to find out the cause of abortion. Subsequent pregnancies were uneventful with no history of HDFN in the last three years. She denied any previous history of blood transfusion. Her transfusion record at our center showed that the mother developed anti-S antibodies during her second pregnancy three years ago. An antenatal antibody screening test performed at 22 weeks identified only allo-anti-S; no anti-D was detected. She was given RhD Ig prophylaxis at 28 weeks of pregnancy. Her other laboratory investigation results showed that she was grouped as A RhD negative with red cell phenotype ccdee (rr), and homozygous ss. At postpartum, the result of her DCT was negative, but the antibody screening test performed using the indirect Coombs' test method and antibody investigations showed the presence of anti-D and anti-S, and the anti-D titer was 1:32 (0.25 IU/mL).

In view of the presence of allo-anti-D and allo-anti-S in the postpartum maternal blood, supported by the presence of similar alloantibodies in the baby's red cell eluates and clinical presentation of hemolytic anemia, a diagnosis of hemolytic disease of the fetus and newborn secondary to anti-D and anti-S was made. The baby was immediately started on a course of conventional phototherapy with a single tungsten halogen bulb. His serum bilirubin levels subsequently reduced to normal levels over a few days. On the sixth day of life, the baby was discharged well with no complications.

This case illustrates an uncommon case of HDFN caused by anti-D and anti-S antibodies, which were identified from the red cell eluate of the baby as well as the mother's serum post-natally. In this case, there was no anti-D detected at 22 weeks of gestation and there was no subsequent follow-up at our center. However, at postpartum, when the newborn developed jaundice an investigation for HDFN demonstrated that there were both anti-D and anti-S. The possible explanation for the anti-D at postpartum could be due to (a) RhD Ig prophylaxis given at the early third trimester of pregnancy. A previous case report by Hensley et al. [ 8 ] showed that the maternal antibody screen becomes normal at 37 weeks of pregnancy in a mother who was given an RhD Ig prophylaxis at 28 weeks of pregnancy. The maternal serum sample was taken 40 minutes after RhD Ig immunization and showed an anti-D titer of 1:8, which subsequently peaked at a titer of 1:32 at 24 hours and remained at that level for about two weeks and then leveled off at 1:16 from week three through to week nine. At term, about 37 weeks of gestation, the maternal antibody screens reverted to normal [ 8 ]. Besides that, it is thought that the administration of RhD Ig during pregnancy can cause a positive antibody screening in the mother but the anti-D titer rarely reaches above 1:4 [ 9 ]. In our case study, the mother claimed that she was only given RhD Ig at 28 weeks of gestation and her anti-D titer was 1:32 (0.25 IU/mL). The high anti-D titer of 1:32 in our case study is most probably due to RhD alloimmunization from exposure of the mother to RhD positive fetal red blood cells later in gestation and unlikely to be due to the administration of RhD Ig at 28 weeks.

Another possible explanation could be (b) an anamnestic response to anti-D. The mother could have had previous exposure to the RhD antigen during her previous abortion or pregnancies, and these anti-D antibodies were not initially detectable in her plasma but subsequent exposure to the RhD antigen from this pregnancy at a later point provoked a rapid and robust production of anti-D antibodies. The titer detected was also significant, as it is described in the literature that anti-D titers of ≥1:32 are significant and can lead to HDFN [ 3 , 9 ]. Unfortunately, we were unable to differentiate between the two concentrations without the regular monitoring of antibody screening and identification and quantification of the antibody titer in our patient.

Anti-S antibody is an IgG antibody developed following red cell alloimmunization. It is reactive at 37°C and best detected by the indirect antiglobulin test. A literature search revealed that anti-S is a rare cause of HDFN and usually presents as a mild form of jaundice [ 7 ]. However, there are a few reports of severe and fatal HDFN due to this antibody. The first case of severe HDFN due to anti-S was described as early as 1952 where a baby died secondary to kernicterus at 60 hours of life [ 10 ]. Griffith [ 11 ] and Mayne et al. [ 12 ] also identified two other cases of severe HDFN due to anti-S. Fortunately, in our patient, despite the presence of anti-D and anti-S on the baby's red cells, the severity of HDFN was relatively mild and the baby's condition improved with phototherapy.

In this report of an uncommon case of HDFN due to anti-D and anti-S antibodies, the detection of anti-D in postpartum serum could be explained by the anamnestic effect of previous alloimmunization or the sensitization after 22 weeks of gestation in the mother's current pregnancy. This case report highlights the importance of regular antenatal follow-up and monitoring of red cell alloantibody development and antibody titers, especially in a mother who is RhD negative. This is because the red blood cell alloantibody may lead to HDFN of variable severity, and early detection in utero may allow early intervention and thus minimize morbidity at birth.

Written informed consent was obtained from the patient's legal guardian for publication of this case report and any accompanying images. A copy of the written consent is available for review by the Editor-in-Chief of this journal.

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Rabeya Yousuf, Suria Abdul Aziz, Nurasyikin Yusof & Chooi-Fun Leong

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RY obtained the case history and consent from our patient's mother. SAA and NY analyzed and interpreted our patient's laboratory investigation results and assisted with the literature review. RY and LCF played a major role in the literature review and writing the manuscript. LCF edited the manuscript. All authors read and approved the final manuscript.

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Yousuf, R., Abdul Aziz, S., Yusof, N. et al. Hemolytic disease of the fetus and newborn caused by anti-D and anti-S alloantibodies: a case report. J Med Case Reports 6 , 71 (2012). https://doi.org/10.1186/1752-1947-6-71

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DOI : https://doi.org/10.1186/1752-1947-6-71

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Hemolytic disease of the fetus and newborn caused by anti-D and anti-S alloantibodies: a case report

Affiliation.

• 1 Blood Bank Unit, Department of Pathology, Universiti Kebangsaan Malaysia Medical Centre, Jalan Yaakob Latif, Cheras, 56000, Kuala Lumpur, Malaysia. [email protected].
• PMID: 22348809
• PMCID: PMC3299637
• DOI: 10.1186/1752-1947-6-71

Introduction: Hemolytic disease of the fetus and newborn is most commonly caused by anti-D alloantibody. It is usually seen in Rhesus D (RhD)-negative mothers that have been previously sensitized. We report here a case of hemolytic disease of the fetus and newborn in a newborn baby caused by anti-D and anti-S alloantibodies, born to a mother who was RhD negative, but with no previous serological evidence of RhD alloimmunization.

Case presentation: A one-day-old Chinese baby boy was born to a mother who was group A RhD negative. The baby was jaundiced with hyperbilirubinemia, but with no evidence of infection. His blood group was group A RhD positive, his direct Coombs' test result was positive and red cell elution studies demonstrated the presence of anti-D and anti-S alloantibodies. Investigations performed on the maternal blood during the 22 weeks of gestation showed the presence of anti-S antibodies only. Repeat investigations performed post-natally showed the presence of similar antibodies as in the newborn and an anti-D titer of 1:32 (0.25 IU/mL), which was significant. A diagnosis of hemolytic disease of the fetus and newborn secondary to anti-D and anti-S was made. The baby was treated with phototherapy and close monitoring. He was discharged well after five days of phototherapy.

Conclusions: This case illustrates the possibility of an anamnestic response of allo-anti-D from previous sensitization in a RhD-negative mother, or the development of anti-D in mid-trimester. Thus, it highlights the importance of thorough antenatal ABO, RhD blood grouping and antibody screening, and if necessary, antibody identification and regular monitoring of antibody screening and antibody levels for prevention or early detection of hemolytic disease of the fetus and newborn, especially in cases of mothers with clinically significant red cell alloantibody.

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Severe Cholestasis in Neonates with Hemolytic Disease of the Fetus and Newborn—A Case Report

Agnieszka drozdowska-szymczak.

1 Department of Neonatology and Neonatal Intensive Care, Institute of Mother and Child, Kasprzaka 17a, 01-211 Warsaw, Poland

Julia Proczka

Natalia mazanowska.

2 Department of Obstetrics and Gynecology, Institute of Mother and Child, Kasprzaka 17a, 01-211 Warsaw, Poland

Artur Ludwin

3 Department of Obstetrics and Gynecology, Medical University of Warsaw, Pl. Starynkiewicza 1/3, 02-015 Warsaw, Poland

Paweł Krajewski

Associated data.

Data presented in this study are available in Table 1 , Table 2 and Table 3 and Figure 1 and Figure 2 .

Hemolytic disease of the fetus and newborn (HDFN) may cause severe cholestasis with direct bilirubin concentrations reaching up to 50 times the upper limit of normal. This case report describes twins whose highest direct bilirubin concentrations were 32.2 mg/dL and 50.2 mg/dL, with no significant signs of hepatic impairment. The index pregnancy was complicated by Rhesus factor immunization with anti-D antibodies present in maternal serum, which caused fetal anemia requiring intrauterine blood transfusions. Complementary tests demonstrated Rhesus D alloimmunization as the sole cause of cholestasis. To the best of our knowledge, this is the first study to describe such elevated direct bilirubin concentrations caused by HDFN.

1. Introduction

Hemolytic disease of the fetus and newborn (HDFN) is an immune-mediated disorder caused by the mother’s antibodies directed against fetal red blood cells (RBCs). Alloimmunization can be caused by any Rhesus antigen (D, d, C, c, E, e) or other antigens absent in maternal RBCs but inherited from the father. Fetal blood cells may migrate into maternal circulation and stimulate the production of immunoglobulin G (IgG) that can cross the placenta. Maternal IgG antibodies attach to fetal RBCs, causing hemolysis leading to anemia and increased bilirubin production.

Bilirubin is an end-product of heme degradation, primarily deriving from hemoglobin. Serum transport of poorly water-soluble bilirubin requires binding with albumin as a carrier. This indirect (unconjugated) bilirubin can cross the blood–brain barrier (BBB) and exert a neurotoxic effect.

Unconjugated bilirubin is transported to the liver, where it is modified into a water-soluble compound—direct (conjugated with glucuronic acid) bilirubin. This form cannot cross the BBB and does not affect the brain. Direct bilirubin is actively excreted into the bile and to the small intestine, where it is transformed into bile pigments (stercobilin) by bacterial enzymes.

Hyperbilirubinemia is not a concern when considering fetal life, owing to placental clearance. After birth, the neonatal liver cannot metabolize bilirubin due to the immaturity of its enzyme activity. This results in unconjugated hyperbilirubinemia, which may require treatment to prevent kernicterus and encephalopathy.

During alloimmunized pregnancies, intrauterine blood transfusions (IUTs) may be necessary to treat fetal anemia. To assess the need for intrauterine therapy, the middle cerebral artery peak systolic velocity (MCA PSV) values, expressed as multiples of the median (MoM), are used, with a value of MoM MCA PSV higher than 1.5 MoM regarded as an indication for cordocentesis and intrauterine transfusion [ 1 ].

IUT is often a life-saving procedure; however, its negative consequences should also be noted. Procedure-related complications may include fetal demise, acute fetal distress, volume overload with heart failure, chorioamnionitis, preterm rupture of membranes, or preterm labor. Suppression of fetal erythropoiesis, minimal risk for anaphylactic reactions, and transmission of viral diseases have also been observed [ 2 ].

Although HDFN is a rare cause of cholestasis, elevated direct bilirubin concentration is common [ 3 , 4 ]. It is caused mainly by iron overload of the liver, and is more frequent in neonates requiring IUTs, especially neonates experiencing Rhesus D alloimmunization [ 4 ].

The term cholestasis describes an interruption of bile flow or excretory hepatocyte dysfunction. Consequently, we have observed elevations of direct bilirubin, bile acids, and other bile component levels. The diagnosis is based on a direct bilirubin concentration exceeding 1 mg/dL (17.1 µmol/L) regardless of the total serum bilirubin (TSB) concentration [ 5 ]. Symptoms of cholestasis include jaundice, hepatomegaly, pale stools, and rarely, green discoloration of the skin [ 6 ]. Increased concentrations of conjugated bilirubin, gamma-glutamyl transpeptidase (GGT), transaminases, and alkaline phosphatase (ALP) are also often observed in patients with cholestasis. ALP alone should not be utilized as a diagnostic parameter because elevated levels of bone-specific alkaline phosphatase (BAP) are typical during the neonatal period [ 6 ].

2. Case Presentation

We present the case of male monochorionic diamniotic (MCDA) twins born at 34 weeks of gestation (para 5, gravida 5) via emergency cesarean section in the first stage of labor because of the transverse lie of the first twin. The index pregnancy was complicated by red cell alloimmunization with an anti-D antibody titer > 1:2000 and an anti-C antibody titer of 1:64, diet-controlled gestational diabetes mellitus, and hypertension. A single course of steroids (betamethasone) was given antenatally. The presence of fetal anemia required two IUTs for both fetuses, at 31 and 33 weeks of gestation. Before the first IUT, hemoglobin concentration levels of 9.1 g/dL in twin A and 8.9 g/dL in twin B were observed. Further details can be found in Table 1 .

Red blood cell parameters before and after intrauterine transfusions.

IUT—intrauterine transfusion; Hgb—hemoglobin concentration; Hct—hematocrit; RBC—red blood cell count.

Physical examination of the patients revealed subcutaneous edema, hepatosplenomegaly, and green skin discoloration. Twin A, weighing 3100 g, was delivered non-vigorous, with Apgar scores of 1, 4, 6, and 6 at 1, 3, 5, and 10 min after birth, respectively, an arterial cord blood pH of 7.07, and a base excess (BE) of −10.1 mmol/L. Twin B, weighing 3200 g, was delivered non-vigorous, with Apgar scores of 2, 4, 6, and 7 at 1, 3, 5, and 10 min after birth, respectively, an arterial cord blood pH of 7.11, and a BE of −9.0 mmol/L.

Severe hyperbilirubinemia was observed in both neonates due to HDFN. The patients’ serious condition was considered as a contraindication for exchange transfusions on the first day of life. The newborns were treated with intensive phototherapy for four days and intravenous immunoglobulin (IVIG) infusion.

During the first weeks of hospitalization, we observed increasing bilirubin levels in both twins, with direct bilirubin making up most of their TSBs since their fourth day of life. Consequently, an exchange transfusion was not performed. In the following days, a gradual decrease in TSB concentrations was noted, and the twins were discharged with bilirubin levels vaguely exceeding the upper limit of normal. Their bilirubin values are presented in Figure 1 and Figure 2 . Other treatment details can be found in Table 2 .

Bilirubin concentrations of twin A.

Bilirubin concentrations of twin B.

Treatment details.

1 At 31 and 33 weeks of gestation. 2 In the 1st, 2nd, and 12th days of life. 3 In the 1st and 20th days of life. 4 In the 1st day of life. 5 Length of stay was defined as the time from birth until discharge from hospital to home. Both twins were treated in three hospitals.

Laboratory tests revealed severe cholestasis in both patients, with elevated GGT and transaminases. Their most extreme laboratory results are presented in Table 3 .

Initial and extreme laboratory results during hospitalization.

* The highest ferritin concentrations were obtained on the 57th day of life. Previous ferritin concentrations might have been higher, but due to laboratory limitations, they were labeled as above the detection range of the test (>1650 ng/mL).

During hospitalization, laboratory value improvement was observed for both patients. The twins did not present coagulation disorders, and there was no need for albumin administration. Due to the suboptimal 25-hydroxyvitamin D status of twin A, high-dose vitamin D supplementation was administered, and twin B maintained adequate vitamin D levels during regular-dose supplementation.

Both patients temporarily passed pale stools, yet normal stool color was observed at discharge. Symptomatic treatments with ursodeoxycholic acid (UDCA), fat-soluble vitamins (A, D, E, K), docosahexaenoic acid (DHA), and formulas with medium chain triglycerides (MCT) were used.

Complementary tests were performed to rule out any causes of cholestasis other than HDFN. Serum alpha-1 antitrypsin levels were deemed normal. Thyroid stimulating hormone (TSH) levels and the activity of galactose-1-phosphate uridyl transferase (GALT) in RBCs were within normal range. TANDEM MS, GC–MS, and cystic fibrosis screening tests showed no abnormalities. No signs of Alagille syndrome were observed. Cortisol levels were within normal range. PCR-based panels for viruses were negative, and congenital cytomegalovirus infection and congenital toxoplasmosis were excluded.

Both twins required blood transfusions due to anemia. Twin A received three transfusions, while twin B received two transfusions.

Echocardiography on the first day of life revealed persistent pulmonary hypertension of the newborn (PPHN) and increased chamber volumes in both twins, with normal cardiac contractibility during vasopressor administration. In ensuing examinations, gradual normalization was observed.

Both patients required mechanical ventilation, nitrous oxide due to PPHN, and surfactant therapy due to respiratory distress on the first day of life. Catecholamines were administered due to circulatory failure. In both twins we also observed hypoglycemia on the first day of life, thrombocytopenia in the first week of life, and necrotizing enterocolitis (NEC). Parenteral nutrition with fish oil-based lipid emulsions was used during hospitalization.

Both twins required antibiotic therapy. Potentially ototoxic ampicillin and gentamicin were used until the exclusion of early-onset sepsis (EOS) in the first four days of life. During their hospital stay, each twin also received gentamicin and vancomycin, as NEC treatment (twin A), and during late-onset sepsis before an antibiogram could be obtained (twin B).

Abdominal ultrasounds showed hepatosplenomegaly in both twins, with no abnormalities of the gall bladder or bile ducts. Cranial ultrasounds revealed abnormal hemodynamic parameters in cerebral vessels (reverse flow in the middle cerebral artery), increased periventricular echogenicity, and hyperechogenicity in the left thalamus of both patients. Signs of grade 1 intraventricular hemorrhage (IVH) were also noted: bilaterally in twin A, and on the right side in twin B. Both boys underwent brain magnetic resonance imaging (MRI), which revealed no significant abnormalities. Ophthalmic consultations were obtained multiple times for both twins; stage 2 retinopathy of prematurity was diagnosed, with no indication for treatment. Additionally, yellow discoloration of the retina was present.

Currently, the twins are 3.5 years old and do not require medication. They receive physical therapy and orofacial myofunctional therapy. There are no signs of cerebral palsy or neurodevelopmental impairment. Neurological examinations show no significant abnormalities. Although both patients receive audiological care due to suspected partial hearing loss, they do not require hearing aids.

3. Discussion

The severity of HDFN differs between mild anemia with hyperbilirubinemia and intrauterine fetal demise as a result of fetal hydrops. Anemia requiring multiple transfusions and hyperbilirubinemia with jaundice are often observed. Other postnatal complications include thrombocytopenia, iron overload, cholestasis, bilirubin-induced neurologic dysfunction, and kernicterus [ 3 ]. Long-term consequences of severe HDFN may consist of altered cardiovascular development, cerebral palsy, severe developmental delay, bilateral deafness, and blindness [ 7 ].

In the described case, both twins required two IUTs due to fetal anemia. These procedures were performed with no complications. No signs of hydrops fetalis were observed. During the neonatal period, both patients needed blood transfusions due to anemia. Twin A received three transfusions, while twin B received two transfusions

Hyperbilirubinemia caused by HDFN may require phototherapy or exchange transfusions [ 8 , 9 , 10 , 11 , 12 ]. The latter is associated with an increased risk of thrombocytopenia, hypocalcemia, hypernatremia, and leucopenia [ 13 ]. Serious complications can include cardiac arrest, NEC, sepsis, and neonatal death [ 8 ]. In some neonatal centers, hyperbilirubinemia in HDFN may also be treated with IVIG [ 3 , 9 , 11 ].

In the described case, phototherapy and IVIG were used to treat both twins. Exchange transfusion was contraindicated on the first day of life due to poor birth conditions, and was not necessary in the following days.

HDFN has been linked to cholestasis, as conjugated hyperbilirubinemia has been observed in 7–13% of neonates with HDFN. Recently, diagnostic criteria for cholestasis changed according to new guidelines (conjugated bilirubin concentration above 1 mg/dL instead of the previous standard), which might influence the prevalence of cholestasis diagnoses [ 3 , 4 , 5 , 9 , 14 ]. Hemolysis increases the concentration of free iron, which is stored mainly in the liver. In neonates with HDFN, ferritin levels are significantly above the normal range [ 15 , 16 , 17 ]. In both patients, ferritin concentrations exceeded the detection range of the test, reaching over 1650 ng/mL. On their 56th day of life, the twins were transferred to a multi-specialist hospital to continue differential diagnosis of cholestasis, where ferritin concentrations exceeding 5000 ng/mL were obtained from both patients. Iron overload may lead to liver dysfunction and cholestasis with coagulopathy, and severe cases may require chelation therapy [ 4 , 16 , 18 , 19 , 20 , 21 , 22 ].

The risk of cholestasis is higher in children who received IUTs and who have HDFN due to Rhesus D alloimmunization [ 3 , 4 ]. Higher risk is also observed in children with low birth weight, anemia, and high TSB and ferritin levels in the first days of life. Liver dysfunction may also be caused by hypoxia due to anemia and extramedullary hematopoiesis in the liver [ 4 ].

Our patients suffered from HDFN due to anti-D antibodies treated with IUTs, and hepatomegaly was observed. According to the literature, direct bilirubin concentrations of patients with HDFN can reach over 30 times the upper limit of normal [ 23 ]. In their first days of life, both twins developed severe cholestasis, with direct bilirubin exceeding 50 mg/dL in twin B. Similar cases of cholestasis since birth in patients with HDFN can be found in the literature. However, in no other case was such a high level (>50 mg/dL) of direct bilirubin described. In 1997, Grobler et al. reported the case of a term infant with HDFN complicated with kernicterus who had a maximal TSB concentration of 45.2 mg/dL, and a direct bilirubin of 31.6 mg/dL [ 23 ]. A 2022 review identified one patient with kernicterus due to hyperbilirubinemia caused by ABO hemolytic disease of the newborn. Their maximal TSB concentration reached 61 mg/dL, with a direct bilirubin of 27.7 mg/dL [ 24 ]. The author previously published a case report of a neonate with severe cholestasis and coagulation disorders in the course of hemolytic disease of the newborn who required chelation therapy, and who had a maximal direct bilirubin concentration of 33.14 mg/dL and a maximal ferritin concentration exceeding 33,000 ng/mL [ 22 ].

To our knowledge, the studies mentioned above identified the highest bilirubin levels reported in the available literature.

The causes of cholestasis can be classified into two categories: extrahepatic, in which obstruction of biliary ducts occurs, and intrahepatic, in which dysfunction of hepatocytes or hypoplasia of intrahepatic biliary ducts is observed. Differential diagnoses must include biliary atresia, abdominal tumor, cholelithiasis, pancreaticobiliary anomalies, alpha-1 antitrypsin deficiency, galactosemia, bacterial infection, sepsis, listeriosis, viral infection (including TORCH), cystic fibrosis, Alagille syndrome, limy bile syndrome, long-term parenteral nutrition, toxic-induced and drug-induced liver injury, Caroli disease, and many others [ 6 , 25 , 26 ]. Necessary diagnostic imaging tests include abdominal ultrasound with evaluation of the bile ducts, hepatobiliary scintigraphy, liver biopsy, and cholangiography [ 6 , 25 ]. Laboratory tests that should be performed include tests for alpha-1 antitrypsin, the activity of GALT in RBCs, TSH, thyroid hormones, cortisol, electrolytes, and acid-base parameters, as well as an infection workup.

In our case, impairment of bile flow caused by HDFN and iron accumulation was deemed the most probable cause of cholestasis. Abnormal direct bilirubin levels had already been detected by the first day of life. The concentration of direct bilirubin could also have been influenced by other factors, e.g., NEC, parenteral nutrition, the severe condition of the children caused by HDFN, and the treatments used, such as antibiotic therapy.

Intrahepatic cholestasis requires causal treatment and diet therapy with partial replacement of long-chain triglycerides (LCT) for MCT, fat-soluble vitamin supplementation, and UDCA treatment [ 6 , 27 ].

Both patients received UDCA, vitamin supplementation, DHA with fish oil in parenteral nutrition, and high-MCT formula. No severe consequences of cholestasis were observed—the only symptoms present included pale stools and hepatosplenomegaly. Laboratory tests revealed low levels of vitamin D3, protein, and albumin, with normal values in the coagulation profile.

According to the literature, bilirubin concentration in infants with HDFN normalizes within one week to three months. Laboratory results should be monitored until normalization [ 4 ]. In both patients, direct bilirubin slowly returned to the normal range. Currently, both twins are developing normally, require no medication, and receive outpatient care at a gastroenterology and hepatology clinic. The described patients do not present any signs of cerebral palsy or kernicterus.

4. Conclusions

This case report shows that HDFN may cause severe cholestasis in newborns. Patients with risk factors (such as an MCDA pregnancy, multiple IUTs, and poor birth condition) and extremal conjugated hyperbilirubinemia may be treated successfully with no severe consequential disability. It should be noted that long-term multidisciplinary care is necessary for these patients. Further investigation in this field is required to improve these outcomes.

This study, however, was subject to several limitations. First, a systematic search including additional databases may provide additional information on the subject. Also, some data from the patients’ medical histories might be missing due to multicenter management. Furthermore, the highest ferritin concentrations of the patients were unknown due to laboratory limitations.

Funding Statement

This research received no external funding.

Author Contributions

Conceptualization, A.D.-S. and P.K.; methodology, A.D.-S.; software, A.D.-S. and J.P.; validation, A.L. and N.M.; formal analysis, J.P.; investigation, A.D.-S. and N.M.; resources, A.D.-S., A.L. and N.M.; data curation, A.D.-S., A.L. and N.M.; writing—original draft preparation, A.D.-S. and J.P.; writing—review and editing, A.D.-S., A.L., P.K. and N.M.; visualization, J.P.; supervision, N.M. and P.K.; project administration, A.D.-S. and N.M.; funding acquisition, P.K. All authors have read and agreed to the published version of the manuscript.

Institutional Review Board Statement

Ethical review and approval were waived for this case report because the parents provided written consent for the treatment.

Informed Consent Statement

Informed consent was obtained from the parents of the patients.

Data Availability Statement

Conflicts of interest.

The authors declare no conflicts of interest.

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