Introduction

Hemolytic disease of the fetus and newborn (HDFN) is the

destruction of the red blood cells (RBCs) of a fetus and

neonate by antibodies produced by the mother. The mother

can be stimulated to form RBC antibodies naturally (ABO),

by previous pregnancy, or transfusion (RBC alloimmuniza-

tion). Before the advent of Rh immune globulin (RhIG),

about 95% of the cases of HDFN were caused by maternal

antibodies directed against the Rh antigen D (RhD). Theincidence of the disease caused by anti-D has
decreased since

1968 with the introduction of RhIG. Despite the decrease in

HDFN, RhD continues to remain an important cause of in-

compatibility, although its frequency has been equaled or

surpassed by other RBC antibody specificities.1,2

The initial diagnosis of maternal RBC alloimmunization

is serologic. After detection, the risk stratification and man-

agement of HDFN have been improved with advances in

technology. Ultrasonography, Doppler assessment of middle

cerebral artery peak systolic velocity, cordocentesis, fetal

DNA analysis from amniotic fluid or maternal plasma, and

intravascular intrauterine transfusion have greatly increased

the success of accurately diagnosing and adequately treating

this disease. This chapter will discuss the disease process,

diagnosis, therapy, and outcomes of HDFN.

Etiology

Although the clinical findings in the fetus and newborn were

noted as early as the 17th century, it was not until 1939 that

Levine and Stetson reported a transfusion reaction from

transfusing a husband’s blood to a postpartum woman. They

postulated that the mother had been immunized to the fa-

ther’s antigen through fetomaternal hemorrhage (FMH).

The antigen was later identified as RhD.

Maternal RBC alloimmunization can be caused by previ-

ous pregnancy or previous transfusion. Some authors suggest

significant impact of previous transfusion on development

of future HDFN.3 However, in a large international cohort of

infant-mother pairs that had a severe course of HDFN, most

maternal alloimmunization (83%) was due to previous preg-

nancy, while only 4% was due to previous transfusion and

14% was unable to be determined.4

HDFN is caused by the destruction of the fetal RBCs by

antibodies produced by the mother. Only antibodies of the

immunoglobulin G (IgG) class are actively transported

across the placenta via Fc receptors; other immunoglobulin

classes, such as IgA and IgM, are not.5 Most IgG antibodies

are directed against bacterial, fungal, and viral antigens, so

the transfer of IgG from the mother to the fetus is beneficial.

In the case of HDFN, the antibodies are directed against the

blood group antigens on the fetal RBCs that were inherited

from the father.

Pathogenesis

HDFN Caused by ABO

As the incidence of HDFN caused by RhD has declined, ABO

incompatibility has become the most common cause of

HDFN. Statistically, mother and infant are ABO-incompatible

in one in every five pregnancies. Maternal ABO antibodies

that are IgG can cross the placenta and attach to the ABO

antigens of the fetal RBCs. ABO antibodies are present in

the plasma of all individuals whose RBCs lack the corre-

sponding antigen (see Chapter 6 “The ABO Blood Group

System”). These antibodies, also called isohemagglutinins,

result from environmental stimulus in early life. Because

group O individuals are most likely to form high-titered

IgG anti-ABO antibodies, ABO HDFN is nearly always lim-

ited to A or B infants of group O mothers with potent anti-

A,B antibodies. Epidemiologically, clinically significant ABO

HDFN occurs most frequently in group O mothers who have

a group A infant in the white populations and group B

infants in the black population and appears to be more likely

when these antibody titers are high (≥512).6 ABO HDFN can

occur in the first pregnancy and in any, but not necessarily

all, subsequent pregnancies because it does not depend on

previous foreign RBC stimulation. Tetanus toxoid adminis-

tration and helminth parasite infection during pregnancy

have been linked to the production of high-titered IgG ABO

antibodies and severe HDFN.

The typically mild course of ABO HDFN is related to the

poor development of ABO antigens on fetal RBCs. ABO anti-

gens are not fully developed until after the first year of life.

Group A infant RBCs are serologically more similar to

A2 adult cells, with group A2 infant RBCs much weaker. The

weakened A antigen on fetal and neonatal RBCs is more

readily demonstrable with human than with monoclonal

anti-A reagents. As expected, group A2 infants are less likely

to have ABO HDFN.

The laboratory findings and clinical characteristics of the

infant affected by ABO HDFN differ from HDFN caused by

anti-D and other RBC alloantibodies (Table 20–1).

Microspherocytes and increased RBC fragility are charac-

teristic. Like other forms of HDFN, the severity of the disease

is independent of the presence of a positive DAT result or

demonstrable anti-A, anti-B, or anti-A,B in the eluate of the

infant’s RBCs. ABO HDFN causes a bilirubin peak at 1 to

3 days, which is later than with HDFN caused by other

antibody specificities. Phototherapy is usually sufficient for

slowly rising bilirubin levels. Rarely, when there are rapidly

increasing bilirubin levels, IVIG or exchange transfusion

with group O RBCs may be required. The serious conse-

quences seen with other causes of HDFN, such as stillbirth, hydrops fetalis, and kernicterus, are
extremely rare in ABO

induced HDFN.

HDFN Caused by RBC Alloimmunization

There are several factors that affect maternal RBC alloimmu-

nization and severity of HDFN, including antigenic expo-

sure, maternal immune system factors, antibody specificity,

and influence of the ABO group.

Fetomaternal Hemorrhage

Previous pregnancy with FMH is the leading cause of maternal

alloimmunization. Transplacental hemorrhage of fetal RBCs

into the maternal circulation occurs in most women, but it is

usually a very small amount (0.5 mL in 93% of women).7 Large-

volume FMH of ≥30 mL occurs in only 3 of 1,000 women.

Interventions such as amniocentesis and chorionic villus

sampling and trauma to the abdomen can increase the risk of

FMH. At delivery, the incidence is more than 50%; this is the

time the placenta separates from the uterus, and fetal RBCs
can enter the maternal circulation (Fig. 20–1).

Maternal Factors

The ability of individuals to produce antibodies in response

to antigenic exposure varies, depending on complex genetic

factors that are not entirely defined.8 In Rh-negative individ-

uals who are transfused with 200 mL of RhD-positive RBCs,

approximately 85% respond and form anti-D.9 Nearly all of

the nonresponders will fail to produce anti-D even with

repeated exposure to RhD-positive blood. If RhIG is not

administered to an RhD-negative mother, the risk of immu-

nization is only about 16% after an RhD-positive pregnancy.

Immunoglobulin class and subclass of the maternal

antibody affects the severity of the HDFN. Of the im-

munoglobulin classes (i.e., IgG, IgM, IgA, IgE, and IgD),

only IgG is transported across the placenta. The active

transport of IgG begins in the second trimester and contin-

ues until birth. The IgG molecules are transported via the

Fc portion of the antibodies (see Chapter 3, “Fundamentals

of Immunology”). Of the four subclasses of IgG antibody,

IgG1 and IgG3 are more efficient in RBC intravascular he-

molysis than are IgG2 and IgG4.10 All subclasses of IgG are

transported across the placenta.

RBC Antibody Specificity

Of all the RBC antigens, RhD is the most antigenic. The

common antigens in the Rh system (C, E, and c) are also

potent immunogens and have been associated with moder-

ate to severe cases of HDFN (Table 20–2) Anti-E and anti-c

have caused severe HDFN that required intervention and

treatment.
Of the non–Rh system antibodies, anti-Kell is considered

the most clinically significant in its ability to cause HDFN.

Kell blood group antigens are present on immature erythroid

cells in the fetal bone marrow, so severe anemia occurs not

only by destruction of circulating RBCs but also by destruc-

tion of precursors.11 Because of the impact of anti-K on fetal

RBC precursors, the anti-K titer is less predictive of severe

fetal anemia, and thus, all pregnant women with anti-K RBC

antibodies should be followed closely for evidence of HDFNInfluence of ABO Group

When the mother is ABO-incompatible with the fetus (major

incompatibility), the incidence of detectable fetomaternal

hemorrhage is decreased. Investigators noted many years ago

that the incidence of D immunization is less in mothers with

major ABO incompatibility with the fetus. This apparent pro-

tection from RhD immunization is likely due to the clearing

and/or hemolysis of ABO-incompatible RhD-positive fetal

RBCs in the mother’s circulation before the RhD antigen can

be recognized by her immune system.

Hemolysis, Anemia, and Erythropoiesis

Once the mother is immunized to a RBC antigen, all sub-

sequent offspring who inherit the antigen will potentially

be affected. The maternal antibody crosses the placenta and

binds to the fetal antigen-positive cells (Fig. 20–1). Hemol-

ysis occurs when maternal IgG attaches to specific antigens

of the fetal RBCs. The antibody-coated cells are removed

from the circulation by the macrophages of the fetal spleen.

The rate of RBC destruction depends on antibody titer and

on the number of antigenic sites on the fetal RBCs. Destruc-

tion of fetal RBCs and the resulting anemia stimulate the

fetal bone marrow to produce RBCs at an accelerated

rate, even to the point that immature RBCs (erythroblasts)

are released into the circulation. The term erythroblastosis

fetalis was used to describe this finding. When the bone

marrow fails to produce enough RBCs to keep up with

the rate of RBC destruction, erythropoiesis outside the

bone marrow is increased in the hematopoietic tissues of

the fetal spleen and liver. These organs become enlarged

(hepatosplenomegaly), resulting in portal hypertension and

hepatocellular damage.

Severe anemia and hypoproteinemia caused by decreased

hepatic production of plasma proteins leads to the develop-

ment of high-output cardiac failure with generalized edema,

effusions, and ascites, a condition known as hydrops fetalis.

In severe cases, hydrops fetalis can develop by 18 to 20 weeks’

gestation. In the past, hydrops fetalis was almost uniformly

fatal; today, most fetuses with this condition can be treated

successfully, although many suffer permanent consequences.

In a large cohort study of children treated with intrauterine

transfusion (IUT, below), those with severe hydrops were

the most likely to have severe long-term neurological

impairment.12

The process of RBC destruction continues after birth as

long as maternal antibody persists in the newborn infant’s

circulation. The rate of RBC destruction after birth de-

creases because no additional maternal antibody is entering

the infant’s circulation through the placenta. However, IgG

is distributed both extravascularly and intravascularly and

has a half-life of 25 days, so antibody binding and hemol-

ysis of RBCs can continue for several days to weeks after

delivery. There are three different phases of anemia caused

by HDFN: early (within 7 days of birth) due to antibody-

mediated hemolysis; late hemolytic anemia (2 weeks or

more after birth) due to continued hemolysis, the expand-

ing intravascular compartment, and natural decline of

hemoglobin levels; and late hyporegenerative anemia due

to marrow suppression as a result of transfusions and IUT,

antibody destruction of RBC precursors, and deficiency of

erythropoietin (Table 20–3).13

Bilirubin

RBC destruction releases hemoglobin, which is metabolized

to bilirubin in different metabolic stages. Indirect bilirubin is

unconjugated and does not dissolve in water. It travels

through the bloodstream to the liver to be conjugated and

rendered water soluble (direct bilirubin) and is excreted

through the gastrointestinal tract. The fetal liver is not

able to metabolize the indirect bilirubin. During pregnancy,

the indirect bilirubin made by the fetus is transported across

the placenta and conjugated by the maternal liver and safely

excreted. After birth, the immature infant liver cannot yet

metabolize bilirubin efficiently, and this leads to the accu-

mulation of unconjugated bilirubin and neonatal jaundice.

When a newborn infant is affected by HDFN, the levels of

indirect bilirubin are higher due to the pathological RBC

destruction. With moderate to severe hemolysis of HDFN, the unconjugated, or indirect, bilirubin can
reach levels toxic

to the infant’s brain (generally, more than 18 to 20 mg/dL),

and if left untreated can cause kernicterus or permanent

damage to the brain.

Diagnosis

HDFN Caused by ABO

Many investigators have tried to use the immunoglobulin

class and titer of maternal ABO antibodies to predict ABO

HDFN. These tests are laborious and at best demonstrate the

presence of IgG maternal antibody, but they do not correlate

well with the extent of fetal RBC destruction. Consequently,

detection of ABO HDFN is best done after birth.

Postnatal Diagnosis

No single serologic test is diagnostic for ABO HDFN. When

a newborn develops jaundice within 12 to 48 hours after

birth, various causes of jaundice need to be investigated. The

DAT on the cord or neonatal RBCs is the most important

diagnostic test. In all cases (100%) of ABO HDFN requiring

transfusion therapy and in 90% of those with jaundice, the

DAT result has been positive.14 On the other hand, the DAT

result can be positive even in the absence of signs and symp-

toms of clinical anemia in the newborn infant. However, these

infants may have compensated anemia or the RBCs are not

being destroyed by the reticuloendothelial system.

Collecting cord blood samples on all delivered infants is

highly recommended. The sample should be collected by

venipuncture to avoid contamination with maternal blood

and Wharton’s jelly (the material surrounding the blood

vessels) and should be anticoagulated for storage. If the

neonatal infant develops jaundice, ABO, RhD, and DAT

testing can be carried out and the results can be assessed.

When the DAT result is negative but the infant is jaundiced,

other causes of jaundice should be investigated. In the rare

cases in which ABO incompatibility can be the only cause of

neonatal jaundice, but the DAT result is negative, the eluate

of the cord RBCs may reveal ABO antibodies. The eluate can

also be helpful when the mother’s blood specimen is not

available.

HDFN Caused by RBC Alloimmunization

The diagnosis of HDFN due to previous RBC exposure and

alloimmunization requires close cooperation among the

pregnant patient, her health-care provider, her partner, and

the personnel of the clinical laboratory performing the test-

ing (Fig. 20–2).15 The recommended practice is to perform

the type and antibody detection test at the first prenatal visit,

preferably during the first trimester.16 At that time, a mater-

nal history must be taken to understand if there is a history

of HDFN, the previous pregnancy outcomes, and whether

there is a history of prior transfusions. Previous severe dis-

ease and poor outcome predict similar findings in the current

pregnancy. Although consecutive antibody titers are useful

in assessing the extent of intrauterine fetal anemia during

the first affected pregnancy, antibody titers are less predictive

in subsequent pregnancies.

ABO, RhD, and Antibody Screen

The prenatal specimen must be typed for ABO and RhD. The

antibody detection method, or indirect antihuman globulin

test (IAT), must be able to detect clinically significant IgG

alloantibodies that are reactive at 37°C and in the anti -

globulin phase. At least two separate reagent screening RBCs

that express all of the common blood group antigens (prefer-

ably homozygous) should be used. For tube testing, an

antibody-enhancing medium such as polyethylene glycol

(PEG) or low ionic strength solution (LISS) can increase

sensitivity of the assay.

Prenatal patients may produce clinically insignificant anti -

bodies, such as anti-Lea or anti-Leb. Therefore, immediatespin and room temperature incubation phases
can be omitted,

and anti-IgG rather than polyspecific antiglobulin reagent is

used. These steps reduce detection of IgM antibodies, which

cannot cross the placenta. Other antibody screening methods,

such as solid phase or gel column, may be used.

If the antibody screen is nonreactive, repeat testing is rec-

ommended before RhIG therapy in RhD-negative prenatal

patients and in the third trimester if the patient has been

transfused or has a history of unexpected antibodies.17

Antibody Identification

If the antibody screen is reactive, the antibody identity must

be determined. Follow-up testing will depend on the anti-

body specificity. Cold reactive IgM antibodies such as anti-I,

anti-IH, anti-Lea, anti-Leb, and anti-P1 can be ignored. Lewis

system antibodies are rather common in pregnant women

but have not been reported to cause HDFN. Antibodies such

as anti-M and anti-N can be IgM or IgG or a combination of

both. Both anti-M and anti-N can cause mild to moderate

HDFN, although rarely. To establish the immunoglobulin

class, the serum can be treated with a sulfhydryl reagent,

such as dithiothreitol or 2-mercaptoethanol, and then

retested with appropriate controls. The J-chain of IgM anti-

bodies will be destroyed by this treatment; IgG antibodies

will remain reactive.

Many Rh-negative pregnant women have weakly reactive

anti-D, particularly during the third trimester. Most of these

women have received RhIG, either after an event with

increased risk of fetomaternal hemorrhage or at 28 weeks’

gestation (antenatal). The passively administered anti-D will

be weakly reactive in testing and will remain demonstrable

for 2 months or longer. This must be distinguished from

active immunization. A titer higher than 4 almost always

indicates active immunization; with a titer under 4, active

immunization cannot be ruled out, but it is less likely. If

encountering a sample from a pregnant patient, using PEG

appears to provide the least number of false positives com-

pared to gel cards and solid phase.18 Communication with

the patient’s provider to obtain a history of RhIG administra-

tion is an important confirmatory step as serological methods

alone are not able to determine the derivation of the antibody.

If the antibody specificity is determined to be clinically

significant and the antibody is IgG, further testing is re-

quired. Other than anti-D, the most common and most sig-

nificant antibodies are anti-K, anti-E, anti-c, anti-C, and

anti-Fya (Table 20–2).

Antibody Titers

The relative concentration of all antibodies capable of cross-

ing the placenta and causing HDFN is determined by anti-

body titration. The patient serum or plasma is serially

diluted and tested against appropriate RBCs to determine the

highest dilution at which a reaction occurs.19 The method

must include the indirect antiglobulin phase using anti-IgG

reagent. The result is expressed as either the reciprocal of

the titration endpoint or as a titer score.

The titration must be performed exactly the same way

each time the patient’s serum is tested. The recommended

method is the saline antiglobulin tube test, with 60-minute

incubation at 37°C and the use of anti-IgG reagent, although

other methods have also been proposed.19,20 The RBCs used

for each titration should have the same genotype (preferably

homozygous for the antigen of interest), approximately the

same storage time, and the same concentration. The first

serum or plasma specimen should be frozen and run in par-

allel with later specimens to increase accuracy since the titer

test results are difficult to reproduce.21 Only a difference of

greater than 2 dilutions or a score change of more than 10 is

considered a significant change in titer.

The method chosen is critical for the appropriate clinical

correlation. Methods using enhancing media or gel columns

result in higher titers, as shown by comparative proficiency

testing. Therefore, the critical titer level for these other meth-

ods must be determined by the individual laboratory by re-

viewing the outcome of several pregnancies complicated by

HDFN, if this method is to be used.

For the recommended method, 16 is considered the crit-

ical titer. If the initial titer is 16 or higher, a second titer

should be done at about 18 to 20 weeks’ gestation. A titer

reproducibly and repeatedly at ≥32 is an indication for color

Doppler imaging to assess middle cerebral artery peak sys-

tolic velocity (MCA-PSV) after 16 weeks’ gestation.22 When

the titer is less than 32, it should be repeated at 4-week in-

tervals, beginning at 16 to 20 weeks’ gestation and then every

2 to 4 weeks during the third trimester. Antibody titer alone

cannot predict severity of HDFN. In some sensitized women,

the antibody titer can remain moderately high throughout

pregnancy while the fetus is becoming more severely af-

fected. Similarly, a previously sensitized woman can have

consistently high antibody titer, whether pregnant or not,

and whether the fetus is RhD-positive or RhD-negative. In

others, the titer can rise rapidly, which portends increasing

severity of HDFN. However, antibody titers consistently

below the laboratory’s critical titer throughout the pregnancy

reliably predict an unaffected or mild-to-moderately affected

fetus, with the exception of anti-K with a K-positive fetus.

Titration studies at time of delivery are not recommended

because they provide no clinically useful information.

Paternal Phenotype and Genotype

A specimen of the father’s blood should be obtained and

tested for the presence and zygosity (predicted copy number

of the gene) of the corresponding blood group antigen to

predict fetal risk of being affected by HDFN. Fathers that are

homozygous for the blood group gene have a 100% chance

of passing this gene to their offspring; heterozygous fathers

have a 50% chance. The mother should be counseled in pri-

vate as to the paternity of the fetus to ensure accurate pater-

nal phenotyping and genotyping.

If the mother has anti-D and the father is RhD-positive,

the paternal genotype determined by DNA methods is the

only way to definitively determine how many copies of

the RHD gene an RhD-positive person carries. Thus, RHD

zygosity genotype testing is the recommended test for

RhD-positive fathers when the mother has anti-D antibody

(Fig. 20–2). In cases of maternal antibody specificity other than anti-D,

paternal red blood cell phenotype testing of the father can

predict the chance of the fetus inheriting the implicated

blood group antigen. For example, only 9% of the population

is positive for the Kell antigen.

Fetal DNA Testing

Risk stratification of the fetus can be directly carried out by

obtaining fetal cells through amniocentesis or chorionic vil-

lous sampling (CVS) as early as 10 to 12 weeks’ gestation.

The fetal cells are grown in tissue culture, and then DNA is

extracted to determine if the fetus has genes coding for the

blood group genes, including RHD and others (c, e, C, E, K,

Fya, Fyb, Jka, Jkb, M). To avoid an invasive procedure, fetal

DNA can be isolated from maternal plasma from a peripheral

blood sample to determine RHD and KEL genotype.23,24 The

cell-free DNA (cfDNA) method has advanced fetal risk strat-

ification for mothers with RBC alloimmunization. In some

countries cfDNA methods are also used when the mother is

RhD-negative to determine if the fetus is predicted to be

RhD-positive and therefore if RhIG should be adminis-

tered.25 This may be a cost-effective approach.26

Management of the Fetus

During pregnancy, women with known RBC alloimmuniza-

tion and/or a history of HDFN are closely monitored using

antibody titers as described above. The fetus is also closely

monitored throughout the pregnancy to check for fetal well-

being and fetal anemia and to determine when is the best

time to deliver.

Fetal Ultrasound

At about 16 to 20 weeks’ gestation, the clinical diagnosis of

fetal anemia can be made using an ultrasound technique

called fetal middle cerebral artery peak systolic velocity

(MCA-PSV) (Fig. 20–3).15 The measurement is based on the

reduced blood viscosity at lower hematocrits and resulting

in faster velocity of the blood. Readings are typically done

every 2 weeks to track the degree of fetal anemia; those

that are greater than 1.5 multiples of the mean (MoM) are

sensitive enough to predict significant fetal anemia in which

intervention may be needed.15,27

Invasive Monitoring—Cordocentesis

and Amniocentesis

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 hemat-

ocrit.15 Using high-resolution ultrasound with color

Doppler enhancement of blood flow, the umbilical vein is

visualized at the level of the cord insertion into the placenta.

A spinal needle is inserted into the umbilical vein, and a

sample of the fetal blood is obtained. If the fetus has not

reached an acceptable gestational age for delivery, and the

hematocrit level is less than 30%, intrauterine transfusion

is usually indicated. During the procedure, an RBC blood

product to be used for intrauterine transfusion (IUT) should

be ready in case it is needed (see below). The fetal blood

sample can also be tested for bilirubin, ABO, Rh, DAT, and

antigen phenotype and genotype.

For risk stratification of fetal anemia, amniocentesis to

monitor amniotic fluid bilirubin levels has been replaced

with MCA-PSV.28 In the past, the concentration of bilirubin

pigment in the amniotic fluid was used to estimate the extent

of fetal hemolysis. The amniotic fluid is tested by a spec-

trophotometric scan optical density (∆OD) at 450 nm (the

absorbance of bilirubin). The measurement is plotted on a

graph (Liley Curve Graph) according to gestational age. An

increasing or unchanging ∆OD 450 nm as pregnancy pro-

ceeds predicts worsening of the hemolysis. High values

indicate severe and often life-threatening hemolysis (fetal he-

moglobin less than 8 g/dL) and require urgent intervention.

Currently, amniocentesis may be used for obtaining fetal

amniocytes for DNA testing, as described above.

Intrauterine Transfusion

Intervention in the form of intrauterine transfusion becomes

necessary when one or more of the following conditions

exists:

• MCA-PSV indicates anemia (>1.5 MoM).

• Fetal hydrops is noted on ultrasound examination.

• Cordocentesis blood sample has hemoglobin level less

than 10 g/dL.

• Amniotic fluid ∆OD 450 nm results are high and/or

increasing.

Intrauterine transfusion is performed by accessing the

fetal umbilical vein (cordocentesis) and injecting donorRBCs directly into the vein.29 The goal of
intrauterine trans-

fusion is to maintain fetal hemoglobin above 10 g/dL. Once

intrauterine transfusion is initiated, the procedure is typi-

cally repeated every 2 to 4 weeks until delivery to suppress

fetal hematopoiesis. The initial intrauterine transfusion is

rarely performed after 36 weeks’ gestation.

The selection of red blood cell products for intrauterine

transfusion is typically group O, RhD-negative (or RhD-

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 blood cell antibody/antibodies.

The RBC unit is irradiated to prevent transfusion-associated

graft-versus-host disease. The hematocrit level of the RBCs

should be greater than 70% because of the small volume

transfused and the need to correct severe anemia.

Cordocentesis, intrauterine transfusion, and amniocentesis

have several risks, including infection, premature labor, and

trauma to the placenta, which may cause increased antibody

titers because of antigenic challenge to the mother through

fetomaternal hemorrhage. To protect the mother from addi-

tional sensitization due to the exposure to donor RBCs through

the procedure, some authors tried to prevent alloimmunization

by using Rh C, c, E, e, and K maternally antigen-matched RBCs

for IUT, but found that the risk of additional maternal RBC

antibodies remained.30 High-level antigen matching (Duffy,

Kidd, Ss) of mothers with HDFN does seem to reduce the risk

of further alloimmunization, but the RBCs become increasingly

difficult to find and the clinical impact is not clear.31 Intrauter-

ine transfusion alone carries a 1% to 3% chance of adverse fetal

events such as premature rupture of membranes.32 When done

in the early second trimester, the outcomes are poor.33,34

Despite the risks of IUT, children older than 2 years who were

treated with this procedure while in utero had a relatively

low rate (4.8%) of neurocognitive impairment (cerebral palsy,

severe developmental delay, bilateral deafness, blindness) so

long as they were not severely hydropic.12

Management of the Infant

When birth occurs, the connection from the fetal to maternal

circulation is severed, and the risk of hyperbilirubinemia

increases because the fetal metabolic pathway to metabolize

bilirubin is immature. Although many babies are affected with

neonatal jaundice, those with HDFN-induced hemolysis are at

greater risk of the bilirubin reaching very high levels and thus

for bilirubin-induced encephalopathy. Blood product transfu-

sion can be used to treat anemia and hyperbilirubinemia.

Cord Blood Testing

Serologic testing of the cord blood sample drawn at the time

of birth is used to confirm HDFN and prepare for possible

transfusion.

ABO Grouping

ABO antigens are not fully developed in newborn infants;

their RBCs may show weaker reactions with anti-A and

anti-B antisera than for older children and adults. In addi-

tion, infants do not have their own isohemagglutinins but

may have those of the mother, so reverse grouping cannot

be used to confirm the ABO group.

RhD Typing

Rarely, the infant’s RBCs can be heavily antibody-bound with

maternal anti-D, causing a false-negative Rh type, or what

has been called blocked Rh.35 An eluate from these RBCs

will reveal anti-D, and typing of the eluted RBCs will show

reaction with anti-D.

Direct Antiglobulin Test

The most important serologic test for diagnosing HDFN

is the DAT with anti-IgG reagent. A positive test result

indicates that there is antibody coating the infant’s RBCs;

however, the strength of the reaction does not correlate well

with the severity of the HDFN. A positive test result may be

found in infants without clinical or other laboratory evidence

of hemolysis (e.g., mother received RhIG).

Elution

The routine preparation of an eluate of all infants with a

positive DAT result is unnecessary. Elution in cases of known

HDFN and postnatal ABO incompatibility is not needed, be-

cause eluate results do not change therapy. The preparation

of an eluate may be helpful when the cause of HDFN is in

question or suspected. As noted above, the resolution of a

case of blocked RhD typing requires an eluate.

Exchange Transfusion

For patients with known HDFN, close observation of bilirubin

levels and hemoglobin is warranted to determine if neonatal

exchange transfusion is needed to remove bilirubin and

maternal antibody.13,36 When levels of bilirubin reach critical

levels (which depends on the infant’s gestational age), ex-

change transfusion is indicated.36 Exchange transfusion is

the use of whole blood or equivalent to replace the neonate’s

circulating blood and simultaneously remove maternal anti-

bodies and bilirubin. Exchange transfusion is rarely required

because of advances in phototherapy and the use of IVIG.

Blood product selection is similar to that of IUT; group O,

RhD-negative (or RhD-positive, depending on maternal

blood group antibody), leukocyte reduced, hemoglobin S

negative, CMV-safe (CMV seronegative or leukocyte re-

duced), irradiated, and antigen-negative for maternal red

blood cell antibody/antibodies. However, because infant

whole blood is also being removed, the RBC unit is usually

mixed with a plasma unit to create reconstituted whole

blood. Usually, RBCs units less than 7 to 10 days from

collection from the donor are selected to reduce the risk of

hyperkalemia. However, special circumstances, such as the

need for units of the mother’s blood when high-incidence

antibodies are involved, have shown that older blood units

can be safe and effective for the newborn when infused

slowly. After a two-volume exchange transfusion, approxi-

mately 90% of the red blood cells have been replaced and50% of the bilirubin has been removed. After
the procedure,

a platelet count should be performed to monitor for iatro-

genic thrombocytopenia.

Simple Transfusions

The infant may receive small-volume or “top-off” RBC trans-

fusions to correct anemia anytime from after birth to many

weeks later. The hemoglobin or hematocrit level at which a

newborn needs an RBC transfusion has been the subject of

a number of studies, and is typically set by the neonatology

physician group. Infants must be carefully monitored for

clinical signs of ongoing anemia, which can be clinically sus-

pected when the infant has poor feeding or increased sleep.13

When transfused, the RBCs selected have the same attributes

as noted with IUT and exchange transfusion. Many hospitals

will keep 1 unit dedicated to an infant with HDFN and draw

small aliquots from the parent RBC unit over time to de-

crease donor exposure over multiple transfusion episodes.

Phototherapy

Phototherapy at 460 to 490 nm is used to metabolize the

unconjugated bilirubin to isomers that are less lipophilic,

less toxic to the brain, and able to be excreted through

urine.37 Relatively high doses are given by using two banks

of lights to surround the infant’s body. For infants with mild

to moderate hemolysis or history of intrauterine transfusion,

phototherapy is generally sufficient to adequately conjugate

the bilirubin and lessen the need for transfusion.

Intravenous Immune Globulin

Intravenous immune globulin (IVIG) is used to treat hyper-

bilirubinemia of the newborn caused by HDFN. The IVIG

competes with the mother’s antibodies for the Fc receptors

on the macrophages in the infant’s spleen, reducing the

amount of hemolysis. IVIG appears to reduce the need for

exchange transfusions and phototherapy, but it does not

affect the need for top-off transfusions.13

Prevention

Prevention of HDFN can be divided into primary and second-

ary measures. Because there are no international standards,

nations differ on preventative measures for HDFN, including

dosing and dosing schedules of RhIG and the approach to

RBC transfusion.

Selection of RBCs for Females

One of the tenets of transfusion medicine is to preserve RhD-

negative RBCs in the blood bank inventory for use for

women of childbearing potential. This is entirely in place to

reduce the risk of RhD sensitization and of HDFN affecting

future pregnancies.

Furthermore, in some countries, minor blood group anti-

gens are matched (or provided as negative) for women of

childbearing potential. For instance, a number of countries

use K-negative RBC units for women younger than 45 to

50 years of age. Other nations provide additional matching

for antigens such as c and E.38 In a large international review

of women with infants with severe HDFN, a transfusion pol-

icy of prospective antigen matching for RBC transfusion did

not provide protection from HDFN. This appears to be

driven by the fact that 83% of maternal sensitization that

went on to cause severe HDFN was due to previous preg-

nancy and not transfusion itself.4

Rh Immune Globulin

The risk of an RhD-negative mother becoming allosensitized

can be reduced to from 16% to less than 0.1% by the appro-

priate administration of RhIG.39 At this time, there are no

other pharmaceutical therapies that can prevent blood group

sensitization for other blood group antigens. The mechanism

of action of RhIG is uncertain. Evidence indicates it inter-

feres with B-cell priming to make anti-D, although other

modes of action may occur.40

Because of the known risk of Rh immunization during

pregnancy, RhIG should be given to RhD-negative mothers.

The first dose is provided at 28 weeks’ gestation, as recom-

mended by the American College of Obstetricians and

Gynecologists (ACOG), since the majority of allosensitiza-

tion appears to occur after this time. A second dose is given

after delivery of an RhD-positive infant.41 Based on experi-

ments conducted many years ago, it is recommended to give

RhIG within 72 hours after delivery. Even if more than

72 hours have elapsed, RhIG should still be given, as it may

be effective and is not contraindicated.

Figure 20–4 outlines a decision tree for the indications

and dose of postpartum RhIG administration. The mother

should be D-negative, and the infant should be D-positive

or D-variant. If the type of the infant is unknown (e.g., if the

infant is stillborn), RhIG should also be administered. Anti-

body titers are not recommended because the amount of

circulating RhIG does not correlate with effectiveness of the

immune suppression or with the amount of fetomaternal

hemorrhage. The half-life of IgG is about 25 days, so only

about 10% of the antenatal dose will be present at 40 weeks’

gestation. It is essential that the anti-D from antenatal RhIG

present at delivery not be interpreted erroneously as active

rather than passive immunization.

Dose and Administration

The regular-dose vial of RhIG in the United States contains

sufficient anti-D to protect against 15 mL of packed RBCs or

30 mL of whole blood. This is equal to 300 µg of the World

Health Organization (WHO) reference material. In the United

Kingdom, the regular-dose vial contains about 100 µg, which

appears to be adequate for postpartum prophylaxis. The mi-

crodose (United States) can be used for abortions and ectopic

pregnancies before the 12th week of gestation. The total fetal

blood volume is estimated to be less than 5 mL at 12 weeks.

Intravenous preparations (IV) of RhIG are approved for use

in the United States. These products also contain 300 µg in

each vial and can be administered either intramuscularly orintravenously. The RhIG must be injected
according to the

product label. The IV product can also be given intramuscu-

larly. The intramuscular form must be given intramuscularly

only. IV injections of intramuscular preparations can cause se-

vere anaphylactic reactions because of the anticomplementary

activity of these products. RhIG also contains IgA and may be

contraindicated in patients with anti-IgA and IgA deficiency

who have had anaphylactic reactions to blood products.

Massive fetomaternal hemorrhages of more than 30 mL

of whole blood occur in less than 1% of deliveries.7 These

massive hemorrhages can lead to immunization if adequate

RhIG is not administered. A maternal sample should be ob-

tained within 1 hour of delivery and screened using a test

such as the rosette technique for massive fetomaternal hem-

orrhage. If positive, quantitation of the hemorrhage must be

done by Kleihauer-Betke or by flow cytometry assays.

In the Kleihauer-Betke test, a maternal blood smear is

treated with acid and then stained with counterstain. Fetal

cells contain fetal hemoglobin (Hgb F), which is resistant to

acid and will remain pink. The maternal cells will appear as

436 PART III Transfusion Practices

RhD negative RhD positive

Rhlg at 28 weeks gestation or

when procedure or trauma occurs

FMH screen
test (Rosette)

Quantitative FMH

assessment

Cord blood

sample

RhD

negative

RhD positive

or weak D

Negative Positive

Maternal postpartum

sample

Birth of infant

Anti-D present: Rhlg not

indicated if anti-D not due to

previous Rhlg

One vial Rhlg

(300 ug)

Calculate Rhlg

dose

Maternal Sample

During First Trimester

Figure 20–4. Decision tree for the indications and dose of RhIG as determined

by laboratory test results. If the cord or neonatal blood specimen is unavailable,

assume the fetus is D-positive.

ghosts. After 2,000 cells are counted, the percentage of fetal

cells is determined, and the volume of fetal hemorrhage is

calculated using this formula:

Number of fetal cells × Maternal blood volume = Number of maternal cells

Volume of fetomaternal hemorrhage

Because one 300-µg dose covers 30 mL of whole blood,

the calculated volume of fetomaternal hemorrhage is then

divided by 30 to determine the number of required vials of

RhIG. Because the Kleihauer-Betke is an estimate, one vial

is added to the calculated answer.16 When needed, the addi-

tional vials of RhIG should be administered within 72 hours

of delivery or as soon as possible. Although it has been a key

test as described for many years, the Kleihauer-Betke test is

imprecise, and recent attention has focused on newer tech-

nologies, such as flow cytometry, to provide more accurate

quantification of the FMH volume.42

Maternal Weak D

As molecular testing advances throughout the field of med-

icine, so does the application of blood typing using molecu-

lar techniques. In certain patients, serologic reagents do not

accurately detect the RhD type. The most common genetic

backgrounds that account for this serologic typing problem

are called weak D phenotypes. Recently, authors have en-

couraged 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.43,44 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.45

Other Considerations

RhIG is of no benefit once a person has been actively immu-

nized and has formed anti-D. However, care must be taken

to distinguish women who have been passively immunized

by antenatal administration of RhIG from those who have

been actively immunized by exposure to Rh-positive RBCs.

RhIG is not indicated for the mother if the infant is found

to be D-negative. The blood type of fetuses in abortions, still-

births, and ectopic pregnancies usually cannot be deter-

mined; therefore, RhIG should be administered in these

circumstances.