ABO incompatibility

ABO incompatibility is the most common cause of hemolytic disease of the newborn (HDN) which occurs in 15 to 25% of pregnancies ¹. ABO incompatibility happens when a mother’s blood type is type-O becomes pregnant with a fetus with a different blood type (type A, B, or AB) ². The mother’s immune system may react and make antibodies against her baby’s red blood cells.

Blood is made up of red blood cells, white blood cells and platelets in a liquid called plasma. Your blood group is identified by antibodies and antigens in the blood.

Antibodies are proteins found in plasma. They’re part of your body’s natural defences. They recognize foreign substances, such as germs, and alert your immune system, which destroys them.

Antigens are protein molecules found on the surface of red blood cells.

The type-O mother’s serum contains naturally occurring anti-A and anti-B, which tend to be of the immunoglobulin G (IgG) class and can therefore cross the placenta and hemolyse fetal red blood cells (breakdown of red blood cells). Hemolysis associated with ABO incompatibility exclusively occurs in type-O mothers with fetuses who have type-A or type-B blood, although it has rarely been documented in type-A mothers with type-B infants with a high titer of anti-B immunoglobulin G (IgG).

In mothers with blood type-A or type-B, naturally occurring antibodies are of the immunoglobulin M (IgM) class and do not cross the placenta, whereas 1% of type-O mothers have a high titer of the antibodies of immunoglobulin G (IgG) class against both A and B. They cross the placenta and cause hemolysis in fetus.

The direct antiglobulin test (direct Coombs test) determines whether your red blood cells (RBCs) circulating in the bloodstream are covered with antibodies. The antibodies that are attached to the surface of the red blood cells are responsible for their destruction.

Hemolysis due to anti‐A is more common (1 in 150 births) than hemolysis due to anti-B. Affected newborns will usually be direct anti‐globulin test (DAT) positive, but, in contrast with the clinical picture with anti‐Rh antibodies, both anti‐A and anti‐B hemolytic disease of the newborn usually result predominantly in hyperbilirubinemia without significant neonatal anemia. This is mainly because of the relatively few group A or B antigenic sites on neonatal red blood cells, allowing the antibody‐coated cells to remain in the circulation for a longer period than in Rh-D disease (Rh incompatibility) ³. As a reflection of this, the blood film in ABO hemolytic disease characteristically shows very large numbers of spherocytes, with little or no increase in nucleated red blood cells, whereas in RhD hemolytic disease of the newborn there are few spherocytes and large numbers of circulating nucleated red blood cells.

However, hemolysis due to anti-B IgG can be severe and can lead to exchange transfusion. Because A and B antigens are widely expressed in various tissues besides red blood cells, only a small portion of antibodies crossing the placenta are available to bind to fetal red blood cells. Recent analysis of IgG subclass in ABO incompatible direct Coombs positive neonates showed IgG2 was predominent antibody which is poorly transferred across placenta and less efficient in causing hemolysis while IgG1 was noted in 22% of neonates and as a group had similar rate of hemolysis and severity of hyperbilirubinemia ⁴.

In addition, fetal RBCs appear to have less surface expression of A or B antigen, resulting in few reactive sites; hence the low incidence of significant hemolysis in affected neonates. This results in hyperbilirubinemia as a predominant manifestation of incompatibility (rather than anemia), and peripheral blood film frequently reveals a large number of spherocytes and few erythroblasts, unlike what is seen in Rh incompatibility (erythroblastosis fetalis), in which blood film reveals a large number of nucleated RBCs and few spherocytes ⁵.

Hemolytic disease of the newborn due to ABO incompatibility is usually less severe than Rh incompatibility (Rhesus disease). One reason is that fetal red blood cells express less of the ABO blood group antigens compared with adult levels. In addition, in contrast to the Rh antigens, the ABO blood group antigens are expressed by a variety of fetal (and adult) tissues, reducing the chances of anti-A and anti-B binding their target antigens on the fetal red blood cells.

ABO incompatibility consequences and treatment are similar to Rhesus disease or Rh incompatibility.

Infants with hemolytic disease of the newborn may be treated with:

• Feeding often and receiving extra fluids.
• Light therapy (phototherapy) using special blue lights to convert bilirubin into a form which is easier for the baby’s body to get rid of.
• Antibodies (intravenous immunoglobulin, or IVIG) to help protect the baby’s red cells from being destroyed.
• Medicines to raise blood pressure if it drops too low.
• In severe cases, an exchange transfusion may need to be performed. This involves removing a large amount of the baby’s blood, and thus the extra bilirubin and antibodies. Fresh donor blood is infused.
• Simple transfusion (without exchange). This may need to be repeated after the baby goes home from the hospital.

Management of ABO hemolytic disease of the newborn is usually successful with phototherapy alone provided by modern equipment. However, close monitoring of the affected neonate is essential, and exchange transfusion is occasionally required. This is particularly the case in ABO hemolytic disease of the newborn due to anti‐B IgG where racial differences in disease severity exist, severe cases being prevalent in mothers and neonates of black African origin. In such cases, severe anemia as well as hyperbilirubinemia ⁶ can occur and rarely antenatal hydrops fetalis has been described ⁷.

Blood groups

Blood types are based on the markers (specific carbohydrates or proteins) or antigens on the surface of red blood cells. Two major antigens or surface identifiers on human red blood cells are the A and B antigens. Another important surface antigen is called Rh (Rhesus). Blood typing detects the presence or absence of these antigens to determine a person’s ABO blood group and Rh type.

There are 4 main blood groups (types of blood): A, B, AB and O. Your blood group is determined by the genes you inherit from your parents.

Each group can be either RhD positive or RhD negative, which means in total there are 8 blood groups.

The ABO system is regarded as the most important blood-group system in transfusion medicine because of severe hemolytic transfusion reactions and, to a lesser degree, hemolytic disease of the newborn.

ABO grouping is a test performed to determine an individual’s blood type. It is based on the premise that individuals have antigens on their red blood cells (RBCs) that correspond to the four main blood groups: A, B, O, and AB. Antibodies (isohemagglutinins) in an individual’s plasma are directed against blood group antigens that their own red blood cells lack (see Table 1, below). These antibodies (isohemagglutinins) form early in life. ABO antigens are expressed on red blood cells, platelets, and endothelial cells and are present in body fluids.

ABO testing is performed to prevent an adverse transfusion reaction that could be caused by ABO incompatibility between the blood of a patient (recipient) and that of a donor.

The ABO system

There are 4 main blood groups defined by the ABO system:

• blood group A – has A antigens on the red blood cells with anti-B antibodies in the plasma
• blood group B – has B antigens with anti-A antibodies in the plasma
• blood group O – has no antigens, but both anti-A and anti-B antibodies in the plasma
• blood group AB – has both A and B antigens, but no antibodies

Blood group O is the most common blood group. Almost half of the US population (48%) has blood group O.

Receiving blood from the wrong ABO group can be life threatening. For example, if someone with group B blood is given group A blood, their anti-A antibodies will attack the group A cells.

This is why group A blood must never be given to someone who has group B blood and vice versa.

As group O red blood cells do not have any A or B antigens, it can safely be given to any other group.

Table 1. ABO Genotyping

Blood group	Antigens present on red blood cells	Antibody present in serum	Genotype
A	A antigen	Anti-B	AA or AO
B	B antigen	Anti-A	BB or BO
AB	A antigen B antigen	None	AB
O	None	Anti-A, anti-B, anti-A,B	OO

Table 2. ABO Phenotype frequencies among different ethnic groups

Race	O	A	B	AB
White	44%	43%	9%	4%
Black	49%	27%	20%	4%
Asian	43%	27%	25%	5%

[Source ⁸ ]

The Rh system

Red blood cells sometimes have another antigen, a protein known as the RhD antigen. If this is present, your blood group is RhD positive. If it’s absent, your blood group is RhD negative.

This means you can be 1 of 8 blood groups:

• A RhD positive (A+)
• A RhD negative (A-)
• B RhD positive (B+)
• B RhD negative (B-)
• O RhD positive (O+)
• O RhD negative (O-)
• AB RhD positive (AB+)
• AB RhD negative (AB-)

About 85% of the US population is RhD positive (36% of the population has O+, the most common type).

In most cases, O RhD negative blood (O-) can safely be given to anyone. It’s often used in medical emergencies when the blood type is not immediately known.

It’s safe for most recipients because it does not have any A, B or RhD antigens on the surface of the cells, and is compatible with every other ABO and RhD blood group.

ABO incompatibility signs and symptoms

ABO incompatibility can destroy the newborn baby’s blood cells very quickly, which can cause symptoms such as:

• Edema (swelling under the surface of the skin)
• Newborn jaundice which occurs sooner and is more severe than normal

Signs of ABO incompatibility include:

• Anemia or low blood count
• Enlarged liver or spleen
• Hydrops (fluid throughout the body’s tissues, including in the spaces containing the lungs, heart, and abdominal organs), which can lead to heart failure or respiratory failure from too much fluid.

ABO incompatibility complications

Acute bilirubin encephalopathy from the buildup of bilirubin in an infant’s brain may manifest as hypotonia or poor suck reflex, which then progresses to irritability and hypertonia with retrocollis and opisthotonos. Long term consequences of chronic bilirubin encephalopathy may lead to cerebral palsy, auditory dysfunction, paralysis of upward gaze, and permanent intellectual dysfunction ⁹. Thus, early recognition and treatment are imperative to prevent the detrimental progression of hemolytic disease of the newborn.

ABO incompatibility diagnosis

Hemolytic disease of the fetus and newborn should be considered in the differential diagnosis of newborns with jaundice or hyperbilirubinemia and certainly in the case of neonatal anemia. Diagnosis of hemolytic disease of the newborn can be made by identifying the presence of maternal red blood cell antibodies (agglutination in an indirect antibody test) and/or a positive direct antibody test (DAT) in the infant’s serum ². If a pregnant woman is identified to have alloimmunization, the first step in further evaluation is to determine the paternal red blood cell antigen status. If positive, the next step is to identify the fetal blood type, typically done through amniocentesis ¹⁰.

Which tests are done depends on the type of blood group incompatibility and the severity of symptoms, but may include:

• Complete blood count and immature red blood cell (reticulocyte) count
• Bilirubin level
• Blood typing

According to the American Academy of Pediatrics, “if a mother has not had prenatal blood grouping or is Rh-negative, a direct antibody test (Coombs’ test), blood type, and an Rh (D) type on the infant’s (cord) blood are strongly recommended” ⁹.

ABO incompatibility treatment

If hemolytic disease of the fetus and newborn is identified or suspected in utero, a consult to maternal-fetal medicine should be placed as early as possible in the pregnancy. Affected pregnancies can be managed by monitoring antibody titers, and fetal middle cerebral artery velocities, intrauterine transfusions, and possibly early delivery as infants with severe anemia may not tolerate term labor well ¹⁰.

Hemolytic disease of the newborn is managed by treating hyperbilirubinemia with phototherapy and exchange transfusions if needed. Routine universal screening with transcutaneous bilirubin often occurs at 24 hours of life, but screening should be conducted as soon as hyperbilirubinemia is suspected. An elevated transcutaneous bilirubin should always be verified with a serum total bilirubin. The hour-specific Bhutani nomogram is then used to risk stratify the amount of bilirubin in the infant’s blood ⁹. This nomogram provides a recommended threshold for starting phototherapy versus early transfusions depending on the infant’s risk level.

Phototherapy was introduced in the 1970s and has become the mainstay of hyperbilirubinemia management in newborns. Photo isomerization causes the transformation of bilirubin into a water-soluble isomer that can then be excreted by the kidneys and stool without the need for processing in the liver. The main determinants of phototherapy efficacy are the wavelength of light used, the intensity of that light, the total light dose (time exposed and surface area exposed), and the threshold at which phototherapy is initiated. The American Academy of Pediatrics recommends the use of intensive phototherapy in hemolytic disease of the newborn. Optimal light used for phototherapy has a wavelength of 460-490 nm. The light should be at a close distance (about 20cm above the infant), and double phototherapy has proven to be more efficacious than single. There is limited data on the efficacy of continuous versus intermittent phototherapy for infants >2000g ¹¹. During the use of phototherapy, mothers should be encouraged to breastfeed their infants at timely intervals despite needing to remove them from phototherapy to do so.

Phototherapy implementation guidelines were addressed in clinical practice guidelines published by the American Academy of Pediatrics ¹². The recommendations are as follows:

• The guidelines are based on total serum bilirubin levels and the direct fraction should not be subtracted from the total unless it is more than 50% of the total serum bilirubin level.
• Intensive phototherapy should be started for babies with hemolytic disease. This implies the use of irradiance in the 430-490 nm band of more than 30 µW/cm²/nm delivered to as much of the infant’s surface area as possible. This can be accomplished using special blue fluorescent tubes that are labeled F20T12/BB or TL52/20W and positioning them 10-15 cm above the infant. When fluorescent tubes are used, they should be brought as close to the infant as possible to increase irradiance. However, when halogen spotlights are used, the distance above the infant should be as per the manufacturer’s instructions because spotlights can cause burns. Phototherapy lights emit minimal ultraviolet (UV) radiation that does not cause erythema and is completely absorbed by the acrylic Plexiglas covering of the tubes.
• Irradiance should be measured using radiometers recommended by the manufacturers of phototherapy systems at multiple sites on the infant’s body surface illuminated by the phototherapy lamp and the measurements averaged.
• The infant should be in the bassinet, and the sides should be lined with white cloth or aluminum foil to expose more surface area. The exposed surface area is increased by the use of 1-2 fiberoptic pads that should be placed under the infant or by the use of BiliBed or Bili-Bassinet, which provides phototherapy from above and below. The diaper should be removed if bilirubin is approaching exchange levels.
• The serum bilirubin declines by 0.5-1 mg/dL in the first 4-8 hours on intensive phototherapy and should be measured in 2-3 hours to document the effectiveness.
• If the serum bilirubin level continues to rise despite intensive phototherapy or is within 2-3 mg/dL of exchange level, administer intravenous immunoglobulin (IVIG) at 0.5-1 g/kg over 2 hours and repeat every 12 hours if needed.
• High-dose IVIG 1 g/kg given early in high-risk neonates with rapid rise of bilirubin level (>0.5 mg/kg/h) and worsening anemia (hemoglobin [Hb] < 2 g/dL) despite intensive phototherapy, is be able to eliminate the need for exchange transfusion and to reduce duration of phototherapy. The number needed to treat is 6 ¹².

Phototherapy is indicated in the term infant with hemolytic disease of the newborn immediately after birth due to Rh disease and due to ABO incompatibility as follows ¹³:

• Unborn (cord blood): Total serum bilirubin level of more than 3.5 mg/dL
• Age less than 12 hours: Total serum bilirubin level of more than 10 mg/dL
• Age less than 18 hours: Total serum bilirubin level of more than 12 mg/dL
• Age less than 24 hours: Total serum bilirubin level of more than 14 mg/dL
• Age 2-3 days: Total serum bilirubin level of more than 15 mg/dL
• Immediately after birth in all preterms who weigh less than 2500 g

An exchange transfusion may be needed for severely anemic newborns, which involves replacing infant red blood cells with antigen-negative red blood cells, thereby preventing further hemolysis. 5mL/kg aliquots are removed and replaced over several minutes for a total of 25-50mL/kg exchange of red blood cells. The process is time consuming and labor intensive but remains the ultimate treatment to prevent kernicterus. The process involves the placement of a catheter via the umbilical vein into the inferior vena cava and removal and replacement of 5- to 10-mL aliquots of blood sequentially, until about twice the volume of the neonate’s circulating blood volume is reached (ie, double-volume exchange). Exchange transfusions are recommended by the American Academy of Pediatrics if total bilirubin levels remain above the transfusion threshold despite intensive phototherapy or if signs of bilirubin encephalopathy are present ¹¹. If an exchange transfusion is being considered, an albumin level should be measured. Albumin of 3.0 g/dL or less is considered an independent risk factor for hyperbilirubinemia and lowers the phototherapy threshold. Without sufficient albumin to bind bilirubin, the amount of free, unconjugated bilirubin increases, thereby increasing the risk for kernicterus ¹¹.

This process removes approximately 70-90% of fetal red blood cells. The amount of bilirubin removed directly varies with the pretransfusion bilirubin level and amount of blood exchanged. Because most of the bilirubin is in the extravascular space, only about 25% of the total bilirubin is removed by an exchange transfusion. A rapid rebound of serum bilirubin level is common after equilibration and frequently requires additional exchange transfusions. However, continued hemolysis and anemia in spite of multiple exchange transfusions and negative direct antiglobulin test (DAT), should raise the possibility of absorption of IgG anti-D acquired from maternal breast milk leading to hyporegenerative anemia caused by ongoing hemolysis of erythroid precursor and marrow supression ¹⁴.

The indications for exchange transfusion are controversial, except for the fact that severe anemia and the presence of a rapidly worsening jaundice despite optimal phototherapy in the first 12 hours of life indicate the need for exchange transfusion. In addition, the presence of conditions that increase the risk of bilirubin encephalopathy lowers the threshold of safe bilirubin levels.

Guidelines for exchange transfusion in neonates with hemolytic disease of the newborn are as follows ¹⁵:

• Total serum bilirubin level of more than 20 mg/dL: Weight more than 2500 g (healthy)
• Total serum bilirubin level of more than 18 mg/dL: Weight more than 2500 g (septic)
• Total serum bilirubin level of more than 17 mg/dL: Weight 2000-2499 g
• Total serum bilirubin level of more than 15 mg/dL: Weight 1500-1999 g
• Total serum bilirubin level of more than 13 mg/dL: Weight 1250-1499
• Total serum bilirubin level of 9-12 mg/dL: Weight less than 1250

The following are indications for exchange transfusion ¹⁶:

• Severe anemia (Hb < 10 g/dL)
• Cord bilirubin above 4 mg/dL.
• Rate of bilirubin rises more than 0.5 mg/dL despite intensive phototherapy
• Severe hyperbilirubinemia ¹²
• Serum bilirubin-to-albumin ratio exceeding levels that are considered safe

Exchange transfusion should be considered in newborns born at more than 38 weeks’ gestation with a bilirubin-to-albumin ratio of 7.2 and in newborns born at 35-37 weeks’ gestation with a bilirubin-to-albumin ratio of 6.8. Exchange transfusion is not free of risk, with the estimated morbidity rate at 5% and the mortality rate as high as 0.5%. Apnea, bradycardia, cyanosis, vasospasm, and hypothermia with metabolic abnormalities (eg, hypoglycemia, hypocalcemia) are the most common adverse effects.

Anemic infants may require blood transfusions with ABO-matched packed red blood cells. If immediate transfusion is thought to be needed, O-type, Rh-negative blood that has been leukodepleted and irradiated should be available at delivery ¹¹.

Other treatment modalities have been considered, but are still controversial. Intravenous immunoglobulin (IVIG) in the infant may block Fc receptors on macrophages, thereby decreasing the breakdown of antibody-coated red blood cells. IVIG is recommended by the American Academy of Pediatrics if total serum bilirubin continues to rise despite intensive phototherapy or is within 2-3 mg/dL of the exchange transfusion level ¹¹. Intravenous immunoglobulin (IVIG) has been shown to reduce the need for exchange transfusion in hemolytic disease of the newborn due to Rh or ABO incompatibility. The number needed to treat to prevent one exchange transfusion was noted to be 2.7 and was estimated to be 10, if all the infants with strongly positive direct Coombs test were to receive the medication ¹⁷. In addition, it also reduced the duration of hospital stay and phototherapy ¹⁷. Although it was very effective as a single dose, multiple doses were more effective in stopping the ongoing hemolysis and reducing the incidence of late anemia.

A randomized, controlled trial by Smits-Wintjens et al ¹⁸, however, failed to show the benefit of prophylactic single-dose IVIG at 0.75 g/kg within 4 hours of life in severely sensitized neonates with prior IUT due to Rh alloimmunization. Although IVIG has been proven to be safe, a retrospective review reported almost 30-times increased risk of necrotizing enterocolitis (NEC) in late preterm and term infants ¹⁹.

Administration of IVIG to mothers prior to delivery has not been shown to be efficacious and is not currently recommended. Other agents such as albumin, phenobarbital, metalloporphyrins, zinc, clofibrate, and prebiotics have been studied as possible treatment options for hyperbilirubinemia, but none are currently recommended ¹¹. In a recent randomized control trial of 70 infants with Rh-alloimmunization, delayed cord clamping was shown to improve anemia without increasing the incidence of adverse events. Delayed cord clamping had no significant impact however, on the need for exchange transfusion or duration of phototherapy ²⁰.

ABO incompatibility prognosis

The severity of this condition can vary. Some babies have no symptoms. In other cases, problems such as hydrops can cause the baby to die before, or shortly after, birth. Severe hemolytic disease of the newborn may be treated before birth by intrauterine blood transfusions.

The overall prognosis of hemolytic disease of the newborn is good if identified and treated promptly. While permanent neurologic dysfunction may result from delays in care, this is now a rare occurrence with the advancements in monitoring as well as prophylaxis against hemolytic disease of the newborn.

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