Immunohematology: LabCE Hemolytic Disease of the Fetus and Newborn

If possible, Rh immune globulin (RhIG) should be administered within what period of time following the delivery of an Rh positive or weak-D positive infant, or other Rh alloimmunizing event?

72 hours

Fetal monitoring

Used to assess the severity of HDFN - Doppler ultrasonography - Amniocentesis - Cordocentesis

Enlarged organs include

liver, eg, spleen and heart

Because RhIG contains IgG anti-D,

when given antepartum, it can cross the placenta and sensitize fetal D-positive red cells. Occasionally the fetus may be born with a weakly positive DAT, but significant hemolysis does not occur. For this reason some guidelines recommend that labs do NOT routinely perform DATs on infants whose mothers have received antepartum RhIG.

What dose of RhIG can suppress immunization of 30 mL of D-positive whole blood?

300 µg. One vial of 300µg RhIG can suppress immunization to a fetomaternal hemorrhage of approximately 30 mL of D+ whole blood

Donor RBC for exchange transfusion in cases of ABO HDFN must meet these criteria:

Group O Rh compatible with infant Crossmatch compatible with maternal serum* No more than 7 days old Reconstituted with AB FFP to obtain a prescribed hematocrit Cytomegalovirus (CMV) "safe" (leukoreduced and/or CMV seronegative) Negative for hemoglobin S to prevent blood from sickling under conditions of reduced oxygen concentration in the newborn Irradiated to prevent transfusion-associated graft-versus-host disease *Red Blood Cells are crossmatched with maternal plasma, although the infant's plasma can be used if a maternal blood specimen is unavailable.

Fetomaternal hemorrhage (FMH)

First screen for a large FMH and then quantitate the bleed is the screen is positive. Once the size of the FMH is determined, a formula is used to determine how much RhIG is needed. Recall that A standard vial of RhIG contains 1500 IU (300 µg) of IgG anti-D; 300 µg of RhIG can suppress immunization to approximately 30 mL of D-positive whole blood.

If available, DNA methods are typically used in these circumstances:

For women who type as weak D in serologic tests, to determine the Rh genotype of the mother to identify if she is partial D or weak D; For women who have made anti-D, to determine the Rh genotype of the father to see if fetal monitoring is needed; For women who have made anti-D, to determine the Rh type of the fetus if the father is heterozygous for RhD or unavailable for testing. Fetal blood typing can be done using fetal DNA from cells obtained by amniocentesis or by testing cell-free, fetal-derived DNA present in maternal plasma at 5 weeks gestation and later.

Hydrops fetalis

Gross edema occurs in severely affected infants, and often results in stillbirth or death soon after birth. Liver failure and hypoproteinemia also play a role in this syndrome.

Jaundice

Jaundice occurs after delivery as fetal bilirubin is cleared by the mother during pregnancy. Extravascular fetal red cell destruction by maternal antibody produces high bilirubin levels. The newborn, who is unable to produce adequate amounts of the liver enzyme glucuronyl transferase, is unable to conjugate the bilirubin into its water-soluble, excretable form

Which of the following tests are suitable for quantifying the size of fetomaternal hemorrhage (FMH)? Select all that apply.

Kleihauer-Betke test and Flow cytometry

Which of the following is generally considered equivalent to CMV seronegative RBC for use in an exchange transfusion to a newborn?

Leukoreduced RBC

RBC Transfusion

Many infants who have received IUTs also require simple RBC transfusions in the first few weeks of life to treat ongoing hemolysis caused by persistent maternal antibody in the newborn's circulation.

For which of these reasons would a molecular method be used to determine a pregnant woman's Rh type?

To differentiate between weak D or partial D

The pathophysiological events typically include

Anemia, jaundice, kernicterus, hydrops fetalis, enlarged organs Laboratory findings include a positive direct antiglobulin test (DAT), low hemoglobin, increased reticulocyte count, and increased nucleated red blood cells.

Which of the following blood group antibodies is generally NOT associated with HDFN?

Anti-Lea

Passive versus immune

Unfortunately, there is no definitive test to determine if anti-D is passive or immune. Some labs perform a titration with the assumption that an anti-D titer greater than 4 likely represents active immunization. While a high titer can exclude passive anti-D, a low titer cannot.

Development of anti-D

- Some Rh-negative individuals produce anti-D after being exposed to a small volume of D-positive red cells (eg, 0.1 mL). - Some Rh-negative individuals must be exposed to a relatively large volume of D-positive cells to produce anti-D. - Some Rh-negative individuals will never produce anti-D, regardless of exposure.

A 300 µg dose of RhIG will be protective for up to what volume of fetomaternal hemorrhage of WHOLE BLOOD?

30 mL

If the mother has a clinically significant antibody, routine serologic tests done on the newborn include

ABO and Rh Direct antiglobulin test (DAT)

Red Blood Cell Units for exchange transfusion should meet the following criteria:

ABO and Rh compatible with the infant Crossmatch compatible with maternal serum Negative for antigens that correspond with maternal antibodies Less than 7 days old to minimize potassium and avoid decreased levels of 2,3 DPG Irradiated to avoid the risk of TA-GVHD Cytomegalovirus (CMV) "safe" (leukoreduced and/or CMV seronegative) Negative for HbS to avoid sickling in low O2 tension RBC units are normally crossmatched with maternal plasma, although the infant's plasma can be used if a maternal blood specimen is unavailable.

Intravenous transfusion (IVT)

Because there may be erratic and inconsistent absorption of intrauterine transfusions in severely hydropic fetuses, IVT was developed. IVT involves transfusing donor RBC directly into the umbilical vein.

Human Serum Albumin

Human serum albumin can also be transfused, either separately or as part of an exchange transfusion in place of FFP. Albumin binds unconjugated bilirubin, thus preventing its deposition in the fat-rich brain cells. Albumin must be used judiciously, because it can aggravate congestive heart failure.

Which of the following consequences of severe hemolytic disease of the fetus and newborn (HDFN) is most associated with neonatal death before or shortly after birth?

Hydrops fetalis

Intrauterine transfusion (IUT)

IUT is done when fetal monitoring indicates severe HDFN and the fetus is too premature for early delivery. IUT involves the intraperitoneal infusion of packed red cells. The success of the procedure depends on absorption of the red cells through the subdiaphragmatic lymphatic vessels of the fetus.

HDFN Due to Other Antibodies

Most common: anti-K anti-c anti-E anti-Fya (rarely) anti-Jka (rarely) anti-M,-N,-S,-s,-U (all rarely) Of these antibodies, anti-K, anti-c, and anti-E are more common causes. Anti-K is frequently associated with a severe form of HDFN due to the ability of the antibody to suppress fetal erythropoiesis in addition to causing hemolysis. Anti-c has also been known to cause severe HDFN.

Cordocentesis

Percutaneous umbilical blood cord sampling 18 weeks gestation Blood sample is removed from unbilical cord Confirmed by a rapid alkaline denaturation test Routine diagnostic tests (eg, blood group, DAT, reticulocyte count, platelet count, hemoglobin/hematocrit). used to deliver intravenous transfusions (IVTs).

When a screening test such as the rosette test indicates a significant FMH,

a quantitative test is required to determine the dose of RhIG required. Tests include -Kleihauer-Betke acid elution test (KB test); -Flow cytometry; -Other techniques that are less commonly used, eg, fluorescence microscopy and enzyme-linked antiglobulin test.

RhIG

has the ability to prevent immunization to D.

ABO HDFN

most common form of disease Most freuent with anti-A,B in group O mother who give birth to Group A infants Mild cases because: Fewer A and B antigens on fetal cells Poorly developed fetal A and B antigens Presence of A and B antigens on cells and tissues other than red cells. Typical symptoms of ABO HDFN include mild anemia and jaundice, usually appearing in the first 24 hours after birth. In rare cases the newborn can exhibit more severe symptoms of Rh HDFN; however, prenatal death is unlikely.

Prenatal treatment

not routinely done because: - Titers of anti-A and anti-B do not correlate well with severity of disease; - The risks of fetal monitoring (eg, amniocentesis, cordocentesis) and fetal transfusion are greater than the risk of ABO HDFN since it is usually mild and subclinical. However, if a woman has a history of infants with moderate to severe ABO HDFN requiring treatment, she may be monitored so that the infant can be treated for possible HDFN as soon as possible.

Kleihauer-Betke Test (KB)

performed to quantitate the number of fetal cells present in the maternal circulation. The test is based on the principle that red cells containing fetal hemoglobin (HbF) are less susceptible to acid elution than cells containing adult hemoglobin (HbA). Limitations: Despite its widespread use, the KB test has significant limitations, including Low sensitivity Poor reproducibility.

Diagnosis of ABO HDFN is supported by these findings:

ABO incompatibility between mother and child, with mother typically group O Negative maternal antibody screen Weakly positive or negative cord DAT Newborn hyperbilirubinemia with jaundice occurring in the first 24 hours Increased spherocytes and reticulocytosis in the newborn Presence of IgG anti-A or anti-B in cord plasma/serum

Recommend tests for perinatal testing:

ABO, Rh*, and antibody screen at first prenatal visit; Test for weak D, if initial Rh typing appears to be D-negative (Optional -not mandated by blood safety standards). If weak-D testing is performed, antihuman globulin reagents that do not contain anti-complement activity would be recommended in order to avoid agglutination due to complement coating of maternal cells. D-negative females: Tested again (ABO, Rh, and antibody screen) at ~ 28 weeks gestation prior to administration of RhIg (depending on the country) and again at delivery. Antibody identification upon initial detection Titration of anti-D at initial detection; repeated at 18-20 weeks and periodically thereafter.

Dosage recommended by USA:

American College of Obstetricians and Gynecologists (2017): All Rh-negative women should receive at least one full dose of RhIG at 28 weeks gestation and another full dose after birth (assuming that the newborn is found to be D positive or weak-D positive).

If a mother has a clinically significant antibody, fetal blood for phenotyping can be obtained by several procedures, depending on gestational age and the antigen involved. These include

Amniotic fluid sampling* Chorionic villus sampling* Cell-free fetal DNA in maternal plasma* Percutaneous umbilical cord blood sampling (PUBS) / cordocentesis** *molecular genotyping**serologic tests As noted, typing the fetus is warranted when the father's blood type is unknown or the father tests as heterozygous positive.

For which of the following antibodies is the DAT most likely to be negative when testing a newborn for possible HDFN? Please select the single best answer anti-A anti-c anti-D anti-K anti-Fya

Anti-A The DAT is most likely to be negative in ABO HDFN. It's possible that the washing done as part of the DAT may break the bonds between anti-A (or anti-B) and the newborn's poorly developed A (or B) antigens.

If a pregnant woman is found to have an unexpected clinically significant antibody, routine antenatal serologic tests on the mother include

Antibody identification to detect clinically significant antibodies. Antigen typing: Once the antibody is identified, the mother is tested for the corresponding antigen, which she should lack. Antibody titration: Laboratories have different protocols. Depending on the antibody titer, titration may be performed at 2 or 4 week intervals after 18 weeks gestation.

Anemia

Cord Hb can be less than 10 g/dL (100 g/L) and as low as 3-5 g/dL (30-50 g/L).

Which procedure used to obtain a fetal blood sample to monitor severity of HDFN can also be used to deliver intravenous transfusions?

Cordocentesis

Diagnosis of ABO HDFN

DAT is unreliable. The infant's plasma is tested against group A1 (or B cells) and group O screen cells using the indirect antiglobulin test (IAT). A positive reaction with A1 or B cells, but not group O cells, would suffice to report a case of possible ABO HDFN. NOTE: Before ABO HDFN is considered as a possible cause of jaundice and anemia in the newborn, other causes should be considered, such as erythrocyte membrane defects or red cell enzyme deficiencies.

Perinatal testing programs have two main purposes:

Detect the presence of any IgG antibody (at an early stage in pregnancy) that could cause HDFN in order to identify, monitor, and treat the infant as soon as possible. Determine which women are candidates for RhIg to prevent the production of anti-D.

Doppler Sonography

Detects & measures blood flood 18 weeks Measures the peak velocity of systolic blood flow in the fetal middle cerebral artery & is used to predict severity of fetal anemia. *Safer than amniocenteisis

If the newborn's DAT is positive, these procedures should be followed:

Elution of newborn's red cells to prepare an eluate containing the sensitizing antibody (unless assessed to be passive anti-D from RhIg*) Antibody identification using eluate Antibody in the eluate should correspond to at least one antibody found in the maternal serum. * The exception is a positive DAT in a newborn whose mother received RhIg antenatally and who has anti-D at delivery. If other maternal antibodies have been excluded, the positive DAT is assumed to be from RhIg and no elution is performed.

Factors that Affect production of Anti-D

Exposure to D+ red cells, volume of fetal bleed, ABO incompatibility between mother and fetus.

An Rh-negative pregnant female has produced anti-D and the physician has decided to use molecular typing to determine if the fetus is at risk. Is the following statement true or false? If molecular genotyping demonstrates that the father is homozygous for the RHD allele, molecular typing of the fetus is also indicated. True False

False This statement is false. If the father is homozygous for the RHD allele, all offspring will be Rh positive and do not need to be tested for D. The fetus will be at risk for HDFN and should be monitored.

ABO incompatibility between a D-negative mother and a D-positive fetus eliminates the possibility of HDFN due to anti-D. True False

False. This statement is false. ABO incompatibility between mother and fetus decreases the incidence of HDFN due to anti-D but does not eliminate it entirely.

Maternal antibody titer is a reliable indicator of fetal disease. True False

False. This statement is false. Maternal antibody titer is an unreliable indicator of fetal disease and is mainly done to determine if clinical fetal monitoring is warranted.

Blood for intrauterine transfusion (IUT) should meet the following criteria:

Group O negative or compatible with both maternal and fetal types if known.* Negative for the antigens that correspond to the maternal antibodies Crossmatch compatible with maternal serum. Irradiated to prevent transfusion-associated graft-versus-host disease (TA-GVHD) in the fetus. Cytomegalovirus (CMV) "safe" (leukoreduced and/or CMV seronegative). NOTE: Leukoreduced RBC units are generally considered equivalent to CMV seronegative RBC because CMV is carried intracellularly within leukocytes. Negative for HbS to prevent sickling in lower fetal O2 tensions. Optimally, the blood is less than 7 days old to reduce the risk from accumulated storage lesion products such as potassium and to avoid decreased levels of 2,3-DPG found in older stored blood, which reduces the ability of hemoglobin to deliver oxygen to the tissues. The hematocrit of the unit should generally be 75% - 85% to deliver maximum numbers of RBCs while minimizing the risk of volume overload in the fetus. *Some laboratories use red cells that are also K-negative since the K antigen is very immunogenic. This also applies to exchange transfusions.

Kernicterus

If indirect bilirubin levels reach approximately 20 mg/dL (340 mmol/L) the fat soluble unconjugated bilirubin deposits in the fat-rich brain cells, causing brain cell damage. Cerebral palsy, deafness, mental retardation, and other serious disorders can result.

Distinguishing between passive and immune anti-D is important clinically:

If passive anti-D is misinterpreted as active, RhIG prophylaxis may be omitted, leading to D sensitization. If active anti-D is misinterpreted as passive, appropriate antibody investigation may be curtailed putting the fetus at risk of developing HDFN. When this occurs, two main serologic questions need to be answered: Are the reactions due to passive anti-D from RhIG or due to active anti-D? Are there other antibodies that need to be excluded?

Examples of how women may still produce anti-D some 40+ years after the implementation of RhIg prophylaxis:

Immunization to D occurred before the administration of RhIG, eg, before 28 weeks gestation*; Immunization to D occurred after the administration of RhIG at 28 weeks and before delivery because an antenatal fetomaternal hemorrhage (FMH) occurred that was too large for residual passive anti-D to give protection; Female was already immunized from a prior pregnancy but anti-D was too weak to be detected in antibody screen tests prior to RhIG administration; RhIG dosage was insufficient to clear a larger fetal bleed at delivery (eg, FMH screen was not done or a false negative occurred); Incorrect calculation of RhIG dosage; RhIG administered too late, ie, well after 72 hours of delivery; Antenatal RhIG not given, eg, mother had no, or limited, access to prenatal care, or did not seek it, and an FMH occurred during pregnancy; Failure of a physician to carry out prenatal blood testing; RhIG is not given due to laboratory clerical or technical error in Rh typing the mother or child; RhIG is not given in cases such as abortions, ectopic pregnancies, and trauma (eg, car accidents).

Postnatal treatment: Exchange transfusion

In exchange transfusions, up to 85-90% of the infant's blood can be exchanged with donor blood by a process of removing 5-20 mL of blood at a time and injecting an equivalent amount until the exchange is complete. An exchange transfusion accomplishes the following: - Removes bilirubin and thus helps prevent kernicterus - Removes sensitized red cells that have not been broken down yet - Removes circulating maternal antibody - Provides antigen-negative red cells that will not be destroyed by the maternal antibody, thus will survive and provide oxygen to the tissues.

Phototherapy helps to prevent which symptom of HDFN? Anemia Jaundice Enlarged spleen Hydrops

Jaundice

Weak D

Multiple weak D variants exist. Red cells have fewer D antigens/red cell (quantitative difference) and only minor variations in D antigen proteins. Some, but not all, weak D phenotypes are detected by today's Rh typing sera and may be classified as Rh positive or Rh negative by routine testing but will be positive when a weak D test (IAT with anti-D) is done. An extreme form of weak D is the Del phenotype, in which the D antigen is so weakly expressed that it may be demonstrated only by adsorption and elution of anti-D. Weak D individuals do NOT produce anti-D and can be considered to be Rh positive for transfusion and RhIg purposes

Calculating RhIG dosage

Number of vials of 300 µg RhIG = Volume of fetal bleed/30 mL Volume of fetal bleed: % fetal cells x maternal blood volume Maternal blood volume: 70 mL/kg x weight (kg) (assume 5,000 mL if maternal information is unknown)

Amniocentesis

Obtain amniotic fluid for diagnostic test 28 weeks & repeated to provide serial estimates of the amount of bilirubin in amniotic fluid. The process measures the difference in optical density at a wavelength of 450 nm between the observed density and an extrapolated baseline (if no excess bilirubin was being produced). Serial values are plotted on charts (eg, Queenan chart or the extended Liley chart), which categorize HDFN into 3 zones of severity.

Partial D

Partial D variants have altered Rh(D) proteins that differ sufficiently from normal D antigens (qualitative difference) to allow anti-D production. Partial D red cells may react with some but not all anti-D typing reagents. There are many categories of partial D antigens (e.g., DIIIa, DVI, DAR), each with a unique genetic basis. Some persons with partial D have weakly expressed D epitopes and are designated "partial weak D." In practice, partial D and weak partial D can be considered similarly, i.e., ideally they should be transfused with Rh negative RBC and are candidates to receive perinatal Rh immune globulin depending on the policy in their location.

A Rh positive individual has produced an anti-D antibody. Which D variant possesses the ability to stimulate this production of anti-D? (Choose all that apply.) Weak D Partial D Partial weak D None of the listed D variants can produce anti-D

Partial D, Partial weak D Both partial D and partial weak D can produce anti-D to the D epitopes that they lack.

RhIG recommended for

Partial D, Partial weak D, D negatuve

Phototherapy

Phototherapy is used to treat jaundice in preterm infants without HDFN and in infants with mild HDFN. Intensive phototherapy has also been used to treat moderate and severe HDFN and decrease the need for exchange transfusion. The newborn is placed under a "blue light," which chemically alters the bilirubin in the surface capillaries to a harmless substance.

RhIG is indicated in these circumstances:

Pregnancy termination through delivery or abortion Antepartum fetomaternal hemorrhage (FMH), suspected or proven as a result of placenta previa, amniocentesis, chorionic villus sampling, cordocentesis, other obstetrical manipulative procedures (eg, external version or abdominal trauma such as may occur in a car accident or fall) Vaginal bleeding during pregnancy Loss, or threatened loss, of fetus at any gestational stage Ectopic pregnancy For non-routine indications, depending on the level of medical care the pregnant woman receives or the awareness level of the risks involved, there is increased potential for RhIG to be administered inappropriately or not at all.

In the first pregnancy,

Rh-positive fetal red cells enter the maternal circulation during the pregnancy and/or at delivery. The mother has a 1o immune response in which mainly IgM antibody is produced, with lower levels of IgG anti-D produced. Thus the first infant is rarely affected because: - Larger fetal bleeds occur at delivery and these are more likely to cause antibody production than smaller antenatal bleeds. - Antibody is produced slowly and is mostly IgM.

Several screening methods are available to detect FMHs that require additional RhIG, including

Rosette method Commercial fetal bleed screening tests Gel agglutination fetal cell screening technique Note: The rosette test may be falsely positive if the mother is weak-D positive and may be falsely negative if the baby is weak-D positive. The rosette method, commonly used by many laboratories, will be briefly reviewed.

Rosette Test

Screen test for FMH that detects fetal D+ red cells in maternal Rh negative blood. Note: The rosette test may be falsely positive if the mother is weak-D positive and may be falsely negative if the baby is weak-D positive. In such cases, a Kleihauer-Betke test or flow cytometry should be done.

Dosage recommended by Canada:

Society of Obstetricians and Gynaecologists of Canada (2010): Antepartum RhIg dose of 300 µg (1500 IU)at 28 weeks (alternatively, 2 doses of 100-120 µg, one at 28 weeks and one at 34 weeks). After delivery of a D-positive infant, another 300 µg (alternatively, 120 µg IM or IV).

RhIG Policies differ between countries and even within countries.

Some labs do not test pregnant women for weak D and rely on routine D typing to determine Rh status. Some labs perform weak D tests on pregnant women who appear to be Rh negative and, if weak D, do NOT inject with RhIG. Some labs perform weak D tests on pregnant women who appear to be Rh negative and, if weak D, inject with RhIG based on the possibility that they may be partial D and capable of forming anti-D.

A mother's serologic results are shown above. Her newborn types as group A Rh positive with a (1+) positive direct antiglobulin test (DAT). Which of the following investigative tests would be most useful to resolve the cause of the positive DAT and should be done FIRST?

Test newborn's plasma against group A1 red cells and group O antibody screen cells by IAT.

The following protocols are examples only and assume:

That the patient received RhIG (this needs to be confirmed) That the antibody screen is positive (2+ or less) Antibody reacts only in the IAT phase and only with D+ screen cells. In other words, the following protocols assume that the antibody looks like a relatively weak IgG anti-D consistent with RhIG administration.

Like all diagnostic methods, DNA typing has limitations and is not 100% sensitive and specific. For example:

The blood group's molecular basis may be unknown; Not all alleles in ethnic populations are known; Rare mutations in the RHD and other genes may not be detected; Silencing changes (switching off of a gene) may affect antigen expression; Fetal typing using amniotic fluid may give false-negative results because of maternal cell contamination.

Which of the following must be true in order for a fetus to be at risk for hemolytic disease of the fetus and newborn (HDFN)?

The fetus must inherit a paternal red blood cell (RBC) antigen that is not present on maternal RBCs.

At 28 weeks gestation, a group A Rh-negative female had a negative antibody screen and was injected with Rh immune globulin (RhIG). Anti-D is detected in the mother's serum at delivery. A panel is performed that excludes the presence of other antibodies. The most likely conclusion would be passive anti-D due to RhIG administration.

True

The ability to produce anti-D varies significantly among Rh negative individuals.

True

Tiny volumes of D+ red cells (eg, as little as 0.1 mL) can stimulate production of anti-D in some individuals.

True

When given during pregnancy, RhIG may cross the placenta and sensitize fetal D-positive RBCs. True False

True

Ectopic pregnancy is an indication for administering RhIG to an Rh negative woman. True False

True This statement is true. RhIG is indicated in any circumstance in which D-positive fetal red cells may enter the circulation of an Rh-negative female, including ectopic pregnancy.

A rosette test may be FALSELY POSITIVE if the mother is weak-D positive. True or flase

True.

All of the following criteria for donor RBC to be used for an exchange transfusion relate to both ABO HDFN and HDFN due to anti-D: Less than or equal to 7 days old (or fresher) Reconstituted with AB FFP CMV negative Negative for hemoglobin S Irradiated True False

True.

Kernicterus due to high levels of unconjugated bilirubin can cause brain damage in newborns suffering from severe HDFN. True False

True. This statement is true. Kernicterus causes brain damage when fat soluble, unconjugated bilirubin deposits in the fat rich brain cells. Kernicterus is not a risk until indirect bilirubin levels reach approximately 20 mg/dL (340 mmol/L).

Postnatal treatment

Usually consists of phototherapy in which the newborn is placed under a "blue light" that chemically alter bilirubin in the surface capillaries. In more severe cases, exchange transfusion may be preformed.

Hemolytic Disease of the Fetus and Newborn (HDFN)

a set of pathophysiological consequences that occur before and/or after birth resulting from the destruction of fetal red blood cells by maternal antibody that reacts with an antigen or antigens inherited from the father.

anti-Lea is not generally associated with HDFN for two reasons

anti-Lea is most often IgM and therefore it does not cross the placenta. In addition, the Lewis system antigens are not well developed at birth.

The 50 µg dose is used

for Rh-negative women whose pregnancy terminates in the first 12 weeks of pregnancy

In the second pregnancy,

if the fetus is again D-positive, when fetal cells enter the mother, they cause a 2o immune response in which higher levels of IgG anti-D are produced. Depending on the antibody titer, the second child may suffer mild to severe HDN.

After 12 weeks a full dose (300 µg)

is administered when there is a similar risk of fetomaternal hemorrhage (FMH). A full dose of RhIG protects against a bleed of 30 mL of Rh-positive fetal whole blood (15 mL packed cells).

Those with partial D, but not usually weak D,

may make anti-D and should be considered D negative for transfusion and as RhIg candidates. Currently, serologic reagents cannot distinguish the two D variants, but RHD genotyping can.

Secondary (2°)/anamnestic immune response

occurs following subsequent exposures When exposure to D occurs in subsequent pregnancies, the lag phase is short (3-7 days) due to the presence of memory B cells that quickly differentiate into antibody-secreting plasma cells. An increase in anti-D is usually detectable within days. The amount of anti-D rises to a higher level. Anti-D titers tend to remain higher for longer but eventually decline. The main type of anti-D produced is IgG (although small amounts of IgM may be produced).

Primary (1°) immune response

the response that occurs following the first exposure to a foreign antigen. Following the first exposure to the D antigen, a lag phase occurs in which no anti-D is produced, but activated B cells differentiate into plasma cells. The lag phase can be as short as several days, but often is longer. Depending on the antibody detection method, it often takes 5-15 weeks before anti-D is detectable in serologic tests. The amount of anti-D produced is relatively low. Anti-D titers decline fairly rapidly and may become undetectable. The first anti-D produced is mainly IgM (although small amounts of IgG are usually also produced).

If a third or fourth pregnancy results in D-positive infants,

these infants (by also bleeding into the mother) cause the production of even higher titers of IgG anti-D and offspring will be more severely affected, perhaps dying in utero or soon after birth.

Flow cytometry

used to quantitate the number of fetal D+ red cells present in the maternal circulation (also the amount of HbF).