Hemolytic Disease of the Newborn (Causes and investigation)

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Hemolytic Disease of the Newborn MLTGEEKS

Haemolytic disease of the newborn abbreviated HDN is an immune haemolytic anaemia which causes newborn or infant to be born jaundiced and anaemic.

This condition is more likely caused by ABO incompatibility than Rhesus incompatibility in most tropical countries.This is because of the low probability or low number of Rh negative women in these countries.Other causes of HDN or Neonatal Jaundice are prematurity,sepsis and Glucose-6-Phosphate Dehydrogenase (G6PD) deficiency.

Causes of Haemolytic Disease of the Newborn

1.  HDN due to ABO Incompatibility

Hemolytic Disease of the Newborn due to ABO Incompatibility
Hemolytic Disease of the Newborn due to ABO Incompatibility

It can occur when a mother with blood group O has a high titre of  immunoglobulin G anti-A and anti-B in her serum becomes pregnant with an infant of  blood group A or B.Knowing that Immunoglobulin G(IgG) is the only antibody able to cross the placenta,IgG anti-A and anti-B crosses the placenta entering the fetal blood circulation which causes destruction of the fetal red cells due to immune response of foreign blood group antigens.

This situation is more severe with Blood group B infants than Blood Group A infants.When HDN due to ABO incompatibility is suspected,laboratory investigation is always required.

How to investigate HDN due to ABO incompatibility in the Laboratory

  • Do ABO cell grouping of the infant using wash cord cells in order to determine whether the infant is blood group A or B.
  • Check if there is maternal-infant blood group incompatibility by doing crossmatch as follows
  • Collect mother’s blood in a dry tube and centrifuge to obtain the serum
  • Now pipette 2 ml of the mother freshly obtained serum into a clean tube
  • Add 1 ml of 5% washed cells of the infant into the tube and mix.
  • Incubate the mixture at 37 °C for 15 minutes then centrifuge the mixture at slow speed (150 g (366 rpm) for 1 minute or 500 g (2113rpm) for 10 seconds)
  • Examine the tube for haemolysis or observe microscopically by making a wet mount of the solution
Also read  How to perform Direct and Indirect coombs test

If there is any lysis of  red cells,This indicates ABO HND (Incompatibility)

  • You can also perform a direct antiglobulin test known as DAT using the infant’s red cells to verify whether they have been coated with IgG antibodies from the mother as follows
  • Start by washing a 3% suspension of the infant’s red cell about four times in normal saline.
  • Decant the final supernatant fluid during the fourth washing
  • Now resuspend the packed red cells (sediment) and add 2 ml of Anti-Human Globulin(AHG) reagent and mix the solution
  • Slowly centrifuge the solution for 1 minute
  • Now  microscopically examine the tube for agglutination by transferring a small volume (1 or 2 drops) to a slide and observe using X10 to X40 objectives.
  • Positive DAT indicates antibody of the mother coated to infant’s red cells.
  • Another method of checking for HDN due to ABO incompatibility is by checking the haemoglobin and serum bilirubin level of the infant.This is done by gram staining the infant’s blood.

Confirm HDN by romanowski stain such as Giemsa or Field stain A and B will microscopically show marked spherocytosis,polychromasia which is due to reticulocytosis and nucleated red cells.

Notes that : HDN due to ABO incompatibility is rarely sufficiently severe to required an exchange of blood transfusion.In those situation which it is indicated,Blood Group O (Should be of the same rhesus group as the baby) be selected and crossmatched with mother’s serum.

Also HDN due to ABO incompatibility may occur in the first pregnancy ad may or may not affect subsequent pregnancies depending on the ABO group of the infant which is not the case with HDN due to Rhesus incompatibility.

Also Read :  Types of Blood transfusion

2. Haemolytic Disease of the Newborn due to Rhesus incompatibility

Hemolytic Disease of the Newborn due to Rhesus Incompatibility

Hemolytic Disease of the Newborn due to Rhesus Incompatibility

This is usually caused by immune anti-D and less commonly by other rhesus antibodies.

Also read  Blood Types,Rhesus and ABO Blood Group Test (Beth-Vincent and Simonin blood group test)

Rhesus HDN occurs particularly when a Rhesus Negative mother with circulating Ig G anti-D antibody becomes pregnant with a rhesus positive infant(The circulating Anti-D antibodies result from a previous rhesus incompatibility pregnancy).

The Ig G anti-D cross the placenta into the fetal circulation which destroys the fetal red cells.This can cause the infant to be born severely anaemic and jaundiced and also the severity of this disease increases with each Rhesus positive pregnancy.

Those infant born with Rhesus HDN are usually more severely affected s compare to those born with ABO HDN.

Maternal Ig G anti-D is mostly detected by laboratory especially during routine antenatal visits and which ease the monitoring of the strength of the antibody.

How to investigate HDN due to Rhesus incompatibility in the Laboratory

  • Determine the ABO and Rhesus blood of  the mother and infant knowing that there can be no Rhesus incompatibility caused by anti-D antibody unless the mother is rhesus negative and the infant rhesus positive.

Sometimes when checking the infant’s blood group,they may not appear rhesus positive  especially when antigen D receptors on the baby’s cell have been coated with maternal anti-D

  • Do a Direct Antiglobulin test (DAT) on the infant’s cord cells .If this is a Rhesus HDN,The DAT will be positive
  • Also measure the infant’s haemoglobin and serum bilirubin.Hemoglobin is usually below 140 g/l while serum unconjugated bilirubin may rise to over 340 µmol/l.Such levels of unconjugated bilirubin can cause irreversible brain damage terme kernicterus
  • Stain the infant blood with a romanowski stain such as field stain A and B or Giemsa and examine for the features of HDN such as spherocytosis,which is usually less marked than in ABO HDN,other features such as polychromasia (reticulocytosis) and many nucleated red cells.
  • Also test the mother’s serum for anti-D antibody

Exchange Blood Transfusion due to HDN

Exchange blood transfusion
Exchange blood transfusion

In situation where the haemoglobin level of an infant with Rhesus HDN continues to fall while the unconjugated bilirubin rise to dangerous levels,Exchange blood transfusion may be required and the laboratory is responsible for the preparation an exchange blood transfusion.

Also read  ABO and Rhesus Blood Grouping principles and Result interpretation

An exchange blood transfusion will help the anaemia,lower the level of bilirubin and also remove sensitized red cells  from the infant’s circulation.

Blood used during an exchange transfusion must be negative for the antigen against which the maternal antibody is reactive taking for example

  • if the anti-D,ABO compatibility Rhesus negative blood must be used and the mother’s serum must be used in carrying out the crossmatch.
  • In the case where the mother’s serum is incompatible with the infant’s ABO group,Blood group O should be used.
  • If the mother and infant has the same blood group,the blood of choice should be that to which the baby and the mother belong.Let’s say if they all belongs to blood group O hence the blood group of choice will be O

This is summarized below

Mother’s GroupInfant’s GroupABO Blood group to use
OA,B or OGroup O only
AA or ABO or BGroup A or OGroup O only
BB or ABO or AGroup B or OGroup O only
ABABABGroup A or OGroup B or OGroup A, B, AB or O

In order to carry out an exchange transfusion,fresh blood not more than 3 days old should be used

Also Read :  Types of Blood transfusion


  • Monica Cheesbrough Part 2,p 376-377

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About the Author: Arthur Westmann

DEFFE ARTHUR (AMOEBAMANN) is the founder and author of MLTGEEKS and MLTEXPO.He’s from Cameroon and is currently a Final year State Medical Laboratory Technician (MLT MA). Beyond lab works, he’s a passionate internet user with a keen interest in web design and blogging. Furthermore He likes traveling, hanging around with friends and social networking to do in his spare time.

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