Hemolytic Anemia of Newborn (HDN)

Sample

1. The sample is serum from the mother.
2. Whole blood (RBC) from the fetus or newborn.

Indications for Coombs’ test

1. To diagnose hemolytic anemia of newborn (hemolytic disease of newborn = HDN).
2. To find out the presence of an Anti-D antibody in the mother’s serum.

Definition of Hemolytic anemia of newborn (HDN)

Hemolytic disease of the newborn (HDN) results from maternal alloantibodies that cross the placenta and enter the fetal circulation; the result is hemolysis.

Pathophysiology of hemolytic anemia of the newborn (HDN)

1. The red blood cells have various antigens on their surface, forming various blood groups. The Blood group ABO system and Rh-systems (D-antigen) are important.
2. Hemolytic disease of the newborn (HDN) results from fetal antigens, while the mother’s RBCs lack these antigens.
   1. Fetal antigens provoke the maternal antibodies formation that is IgG type. It happens when the fetal RBCs enter the maternal circulation during delivery (or pregnancy).
   2. These maternal antibodies (IgG) can cross the placenta and enter fetal circulation leading to hemolytic anemia by attacking the fetal RBCs.
3. If mother and fetus are ABO-incompatible, then sensitization can not occur due to maternal AB antibodies’ immediate destruction of the fetal RBCs.
4. An Rh-positive baby occurs in ∼10% of Rh-negative mothers in white females, 5% in black females, and 1% in Asian females.

Rh-incompatibility:

1. Hemolytic disease of the newborn due to Rh-incompatibility varies in severity from:
   1. Subclinical disease.
   2. Mild jaundice with anemia.
   3. Erythroblastosis fetalis is a dangerous condition.
   4. The main findings are jaundice and anemia.
   5. Reticulocytosis >6% accompanies the anemia.
   6. In severe cases, there are nucleated RBCs in the peripheral blood smear.
   7. Jaundice is due to mainly unconjugated (indirect) bilirubin.
      1. Jaundice is not present at birth; it appears after 24 hours.
      2. Physiologic jaundice may be confused with pathological jaundice. Physiologic jaundice has no anemia.
      3. Normal hemoglobin in newborns is 18 g/dL, and anemia is established when hemoglobin is <15 g/dL.
   8. Direct coombs’ testis positive.
2. In the Rh-system, the Rh antigen is present on some of the RBC, and those are called Rh-positive blood groups, while the RBC lacks Rh – antigens are called Rh-negative blood groups.
   1. The other common causes are Rh-c antigen and blood group Kell antigens.
3. There are a group of Rh antigens like Rh-C, Rh-D, Rh-E, and more.
4. Suppose the mother is Rh antigen (D-negative) and the baby is Rh antigen(D-positive), then because of the feto-maternal hemorrhage. In that case, the mother may be sensitized to Rh-antigen and develop Rh-antibody, mostly IgG type antibodies (anti-D antibodies).
   1. This sensitization occurs due to pregnancy, abortion, ectopic pregnancy, placental trauma, amniocentesis, blood transfusion with Rh-positive RBCs, or contaminated blood products like platelet concentrates.
   2. The most common is the fetomaternal mixing of fetal RBC Rh-positive cells.
   3. This mixing occurs after 16 weeks of the pregnancy or a single large dose of fetal RBCs mixing at the time of delivery.
   4. 10% to 13% of mothers risk sensitization by the Rh-positive fetal RBCs.
   5. Usually, the first child is not affected.
   6. First-born infants are affected 5% to 10% with HDN, either due to a previous pregnancy or abortion or high sensitivity of mother to Rh-antigens.
      

      HDN fetomaternal mixing of RBCs

5. Usually, mother and fetus are ABO compatible.
6. These maternal anti-D antibodies can cross the placental barrier, enter the fetal circulation, and lead to hemolytic disease of the newborn (HDN).
   

   Sensitization of the mother by Rh+ fetus

   

   Mechanism of the hemolytic disease of the newborn

Laboratory diagnosis of hemolytic anemia of newborn (HDN):

1. In the case of Rh-induced hemolytic anemia of newborn (HDN), direct Coomb’s test on cord blood is positive.
2. In the case of ABO-induced HDN on cord blood, coombs test is frequently but not always positive.
3. The direct Coombs test on infant blood is usually positive in Rh-induced HDN but is negative in ABO-induced HDN when done >24 hours after delivery.
4. Cord blood bilirubin is usually increased, and cord blood hemoglobin is decreased in severe HDN.

Significance of Coombs’ test for hemolytic anemia of newborn (HDN):

1. Coombs’ test is used to detect antibodies in Rh-negative mothers or newborns.
   1. Mother has free antibodies in the serum.
   2. Fetal/newborn has coated RBCs by the antibodies.
2. One can monitor the presence of the antibody during pregnancy.
   1. Mother titer of more than 1:16 at 8th month indicates the presence of Anti-D antibodies in the mother. These can cross the placental barrier and enter fetal circulation.
      1. The fetus can develop hemolytic disease.
3. An indirect Coombs’ test is done to find a free anti-D antibody in the serum on the maternal blood.
   

   Coombs’ indirect test

4. A direct Coombs’ test is done to find RBC-coated antibodies in the fetus or newborn.
   

   Coombs’ direct test

Prenatal screening:

1. Must do blood group ABO and Rh at the first prenatal visit during the pregnancy.
2. Advise indirect coombs’ test for mother for ABO or different antigens.
3. Rh-negative mothers should be given anti-D immunoglobulin (Rhogam) at the end of the second trimester and again within 72 hours of the delivery in case of Rh-positive babies. This treatment will decrease the prevalence of HDN.
4. Monitor anti-D titer on mother serum to find the sensitization when the titer is >1:8.
   1. If the titer is ≥1:32, advise serial estimation of the amniotic fluid bilirubin (indirect) level every 2 to 3 weeks to prevent the risk to the fetus.
5. Lecithin/sphingomyelin ratio estimation gives the idea about lung maturity.
6. Amniocentesis is more reliable than the anti-D titer to determine the severity of the HDN.
   1. DNA analysis of amniotic fluid by PCR can determine the D-antigen status of the fetus.
7. At birth, determine the cord blood (baby blood):
   1. Hemoglobin.
   2. Bilirubin level.
   3. Direct Coombs’ test.

Postnatal workup and follow-up:

1. Check indirect serum bilirubin of the infant because it rises rapidly. There may be an increase of 0.3 to 1.0 mg/dl/hours. It may reach 30 mg/dL in untreated babies in 3 to 5 days; at this level, the baby may die.
2. It is increased urobilinogen in urine and feces. This increase will be parallel to blood serum bilirubin.
3. Direct Coombs’ test is positive on cord blood RBCs in the case of Rh, Kell, Kidd, and Duffy antibodies.
   1. It is weakly positive in anti-A antibodies.
4. Anemia is not evident at birth, but it becomes maximum by the 3rd or 4th day.
5. Blood examination of the baby:
   1. MCHC is normal, MCV and MCH are increased.
   2. Peripheral blood smears show an increased number of nucleated RBCs in the first 2 days. Their number will decrease and may be absent on the 3rd or 4th day.
   3. It shows anisocytosis, polychromatophilia, and increased macrocytes.
   4. There is reticulocytosis >6% and in some cases may reach 30% to 40%.
   5. WBC count is increased.
   6. The platelets count is mostly normal.
6. This episode will be over in 3 to 6 weeks when these babies are treated, and there are no more maternal antibodies.

Prognosis:

• Its (HDN) prevalence is markedly decreased due to in-time administration of Rh-globulin (Rhogam) after abortion or delivery.

Treatment of hemolytic anemia of newborn (HDN):

1. The exchange of blood transfusion can save infants with HDN.
2. Indications/criteria for blood exchange transfusion:
   1. Infants serum bilirubin level >20 mg/dL or in premature or severely ill infants with a bilirubin level of 15 mg/dL.
   2. Cord blood indirect bilirubin level >3 mg/dL.
   3. Cord blood hemoglobin <13 g/dL, and there are references in favor of 8 to 14 g/dL.
   4. Maternal Rh-antibody titer of 1:64 or greater. If bilirubin does not rise, then this is not the indication.
3. Criteria for the blood exchange transfusion:
   

   Lab parameters	No treatment is needed, only follow-up	Exchange transfusion needed
   Cord hemoglobin	>14 g/dL	<12 g/dL
   Cord indirect bilirubin	<4 mg/dL	>5 mg/dL
   Rh-antibody titer mother	<1:64	>1:64
   Infant serum bilirubin	<18 mg/dL	18 to 20 mg/dL 20 mg/dL in first 24 hours Premature baby 15 mg/dL is indication
   Infant hemoglobin	>12 g/dL	<12 g/dL and decreasing

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