Faculty of Allied Medical Science Blood Banking (MLBB 201)

HEMOLYTIC DISEASE OF THE NEWBORN Prof. Dr Nadia A. Sadek Prof. in Hematology, Head of Blood Bank, MRI

Learning outcome This lecture will enable the students to:- Recognize HDN, its presentation and complications Know the laboratory tests for its diagnosis and management.

Hemolytic Disease of the Newborn It is a condition in which the life span of the fetal/neonatal red cells is shortened due to maternal alloantibodies against red cell antigens acquired from the father.

Maternal IgG cross the placenta and bind to the corresponding antigen on fetal red cells which are then removed by the fetal phagocytic system. The most common antibodies are related to the Rh and ABO system. The D antigen is highly immunogenic followed by c, anti K.

Lewis and P1 antibodies occur frequently in pregnancy, but being of IgM type they do not cross the placenta. In addition, the Lewis antibodies are not fully developed at birth. All women who had previous pregnancies or blood transfusion may be immunized against foreign red cell antigens.

Women without such a history can also be immunized either because the antibodies are naturally occurring or because a spontaneous abortion has passed unrecognized.

Blood samples from pregnant females are tested early for alloantibodies at 12-16 weeks, at the booking visit and at 28-32 weeks for a rising anti-D titre. In case of anti-K, severe HDN can occur even at low titres.

Anti-A,B may occur in group O mothers delivering an A or B infants, but the disease is mild and does not need exchange transfusion. The difference between HDN due to ABO and Rh incompatibilities includes:-

Low incidence of need to exchange transfusion in ABO versus Rh antibodies. ABO is found in the first and the . subsequent pregnancies. In Rh the first pregnancy may be unaffected. ABO antigens are expressed by a variety of fetal and adult tissues reducing the chances of anti-A binding on the fetal RCs.

The risk of sensitization to the RhD antigen is reduced if the fetus is ABO incompatible. This is because any fetal cells that leak into the maternal circulation are rapidly destroyed by potent anti-A and anti-B thus reducing the likelihood of maternal exposure to the D antigen.

In the first sensitization the maternal anti D is IgM and does not cross the placenta, while in subsequent pregnancy, it is of IgG type and crosses the placenta to destroy the fetal red cells.

Coombs test in HDN Direct Coombs:- A sample of fetal RBCs is washed to remove any unbound Ig. The reagent agglutinates any fetal RBCs to which maternal antibodies are already bound.

Indirect test:- To prevent HDN. It is carried out on mother’s serum to find anti-D antibodies. The serum is incubated with Rh D-positive cells, washed to remove excess, reagent is added. If the mother is sensititized agglutination occurs.

Clinical features HDN may manifest as a mild hemolytic anaemia with slight jaundice on the second or third days of life and mild anaemia. More severely affected infants have severe hyperbilirubinaemia (icterus gravis neonatorum).

If left untreated, bilirubin reaches the basal ganglia (kernicterus) and the condition may be fatal or leads to mental retardation and spasticity. In the most severe cases, profound anaemia occurs in utero and intrauterine fetal death may occur.

The fetus presents with severe anaemia, oedema and ascites The fetus presents with severe anaemia, oedema and ascites. The placenta is bulky, swollen and friable, a condition known as hydrops fetalis which necessitates ultrasound-guided intravascular transfusions.

In hydrops fetalis extravascular hemolysis stimulates extramedullary haematopoiesis in the liver with distortion of the hepatic circulation, portal hypertension, impaired albumin production resulting in oedema, pleural and pericardial effusions. The severe anemia leads to heart failure and tissue hypoxia, and fluid extravasation to the extravascular space.

Blood picture The fetal blood film shows polychromasia and increased numbers of nucleated red cells (normoblasts). The direct antiglobulin test (DAT) on the infant’s red cells is positive except in some ABO antibodies. In ABO incompatibility the infant’s blood film shows spherocytosis (not seen in Rh HDN).

Rh hemolytic disease of the newborn Anti-D accounts for 90% of the cases. Maternal antibodies are usually undetected before 28 weeks. The most objective means of quantitating anti-D levels is by automated analyser, flow cytometry or manually.

Following delivery, all RhD-negative women unsensitized for RhD should be given prophylactic anti-D immunoglobulin if the infant is RhD positive. The antibody level in maternal blood is affected by:-

- IgG subclass - Rate of rise of antibody - Past history of immunization - Presence of blocking antibodies. Level below 1iu/ml (0.2ug/ml) require no action, a level of 10iu/ml (20ug/ml) necessitates amniocentesis.

Antenatal assessment of HD Ultrasound-guided fetal blood sampling from 18-20 weeks of gestation. Bile pigments measurements in the amniotic fluid from week 28 onwards. Fetal haemoglobin deficit necessitates intravascular transfusion of O-negative blood, CMV-negative and irradiated blood. Rh genotyping of amniotic cells by DNA.

Intrauterine transfusions Injection of antigen-negative donor red cells in infant in utero either into the peritoneal cavity, or directly into fetal umbilical vessels . In the latter, ascites should not be present otherwise absorption of the red cells into the fetal circulation will be slow.

Management Plasma exchange during pregnancy in cases of high antibody titres in the mother. Premature delivery after 34 weeks. Exchange transfusion to the newborn. The compatibility test should be performed to the mother and newborn against mother serum.

ABO HDN The A and B antigens are not fully developed in the infant. In A and B subjects the anti-B and anti-A are IgM and do not reach the fetus. ABO HDN occurs in group O mothers possessing IgG anti-A, anti-B

HDN due to other antibodies Anti-c and anti-K are due to previous maternal blood transfusions. Anti-K may cause severe fetal anaemia necessitating intrauterine or exchange transfusion.

Prevention of HDN - Routine administration of 500iu of anti-D to all Rh-negative mothers within 72 hours of delivery of an Rh-positive infant. - Detection and quantitation of fetal red cells in maternal blood sample taken within 1 hour after delivery by Kleinhauer acid-elution test.

All females in childbearing period requiring blood transfusion should receive c-negative, K-negative blood to prevent sensitization.

Case presentation Accurate Rh testing can be difficult if the red cells are heavily coated with IgG anti-D antibodies; a phenomenon called “BLOCKED D” Rh D negative report was obtained in a newborn male baby with severe HDN and features of kernicterus. He was born to a second gravida B Rh negative mother.

The baby was grouped as B Rh D negative by direct grouping, but after elution, D Ag was detected and he was phenotyped as CcDe. Direct Coombs was strongly positive after 3 exchange transfusions.

The baby had free antibodies apart from the red cell bound The baby had free antibodies apart from the red cell bound. The mother had IgM and IgG anti D as well as anti C and IgG anti-A.

Prevention of HDN - Determine the Rh status of the mother - Indirect Coombs on the mother’s serum to detect sensitization. - Give anti D at 28 weeks (time of starting fetal D antigen expression) - Give another dose at 34 weeks (risk of fetomaternal haemorrhage) - A final dose after the baby is born

If the father is homozygous for D allele D/D, the fetus will be D-positive. If he is D/d, there is 50/50 chance that the fetus is D-positive. Monitor the pregnancy by ultrasound A rising anti-D titre denotes active hemolysis.

Treatment If fetal anemia is severe, blood transfusion in utero. After delivery, exchange transfusion with O- negative blood.

Study Question How to diagnose HDN?

Assignments Flow Cytometry سيد احمد كمال