BIOM5713: Research Methods and Adv. Biomedical Techniques HDFN | DMU

Investigation of Haemolytic Disease of the Foetus and Newborn (HDFN).

Mild to severe anaemia in the foetus or anaemia and/or jaundice in the newborn caused by reaction of maternal antibodies with foetal antigens of paternal origin.

Learning Outcome of BIOM5713:

• Relate the pathophysiology of HDFN with clinical symptoms and laboratory tests.
• Compare the impact of blood group system on the severity of HDFN.
• Review current approaches to the prevention of HDFN.

BIOM5713 Overview

• Pathophysiology.
• Predicting and determining severity.
• Antenatal and post natal procedures.
• Prevention, treatment.
• Foeto-maternal haemorrhage.
• Other causes besides anti-D.

First pregnancy

Subsequent pregnancy

Pathophysiology

Increased foetal red cell destruction

Predicting and determining severity of HDFNB.

Amniotic fluid bilirubin levels

Amniotic fluid analysis

Bilirubin (Amniotic Fluid)

• A level of 0.28 OD to 0.46 OD at 28 to 31 weeks is considered low. No abnormal red blood cell breakdown or very mild anemia.
• A level of 0.47 OD to 0.90 OD - moderate red cell disease. - blood transfusion in the womb. recommend an early delivery if pregnancy has reached 32 or 33 weeks.
• A level of 0.91 OD to 1.0 OD means your baby is at risk for severe anemia from haemolysis. -blood transfusion in the womb. - early delivery at 32 to 33 weeks recommnded.
• A level of less than 0.04 OD toward the end of your pregnancy means baby is reaching maturity and is not in danger of haemoytic anemia.

Foetal DNA in amniotic fluid or maternal plasma

• PCR on DNA amplifies regions of the RhCcEe and RhD genes.
• Identifies at risk fetus in first trimester.
• Avoids unnecessary invasive fetal blood sampling.

Post delivery treatment If jaundice is severe

• Phototherapy: blue light 420-475nm range.
• Failure to respond - Exchange transfusion.
• Donor blood ABO compatible with both mother and baby, Rh(D) compatible with mother.
• Donor must lack the antigen which corresponds with antibody identified in mother.

Donor blood crossmatched using mother’s serum/plasma.

Blood for exchange Transfusion

• CMV negative.
• Irradiated.
• HbS negative.
• <5 days old.
• High Hct (semi-packed).

Blood for neonatal Transfusion

Prevention of Rh HDFNB due to anti-D

• All Rh D Negative women of childbearing age should be given Anti-D injection between 28 and 30 weeks of pregnancy or within 72 hours of a sensitising event (usually occurs at delivery)
• 125 IU of IM anti-D suppresses sensitisation by 1mL of D positive packed red cells.

Foeto-maternal Haemorrhage (FMH)

Can occur any time during the pregnancy:

• invasive prenatal diagnosis e.g. amniocentesis, chorionic villus sampling
• ante-partum haemorrhage
• ectopic pregnancy
• external version of the foetus
• abdominal injury
• iud
• Stillbirth, abortion

Estimation of FMH

• Red cells containing Hbf resist acid elution more than red cells containing Hba.
• On staining and counterstaining foetal cells appear as darkly-stained red cells with a background of palely-stained ghost red cells.

Estimation of FMH

Kleihäuer and Betke Test

Estimation of FMH

• Some reticulocytes resist acid elution.
• Rare hereditary persistence of foetal haemoglobin (HPFH) and other genetic disorders e.g. thalassaemia, have high levels of HbF.
• Method is subjective and imprecise.

Estimation of FMH

• Fluorescent markers attached to HbF containing RBC.
• Mother’s blood treated with anti-D
• Fluorescein labelled anti-IgG added.
• Detects down to 1 D+ve cell in 1000 RBCs.
• Flowcytometry

Estimation of FMH

• Monoclonal anti-D directly conjugated with fluorescein.
• Murine monoclonal fluorescein labelled anti-HbF.
• Using sensitive fluorescein anti-HbF all adults have some +ve cells (up to4.4%),

Other causes of HDFN

Anti-c

• Found in 0.7 per 1000 pregnancies.
• Frequency of HDFN lower than anti-D.
• 45% immunised by transfusion.
• Causes 65% of HDFN cases not due to anti-D.
• Found in 1 per 1000 pregnancies.
• Most immunised by transfusion.
• Moderate to severe anaemia.
• Poor corelation between severity and antibody titre.
• Poor corelation between severity of anaemia and jaundice.
• Retics, erythroblasts, bilirubin lower than matched cases of anti-D HDFN.
• K antigen present on early erythroblasts.
• D expressed much later.
• Anti-K causes immune destruction of early erythroid precursors.

Most common cause of HDNB

ABO HDFN

• Mild, short lived bilirubinaemia 12-48hrs post delivery.
• DAT wk +ve in 25-30%
• Phototherapy usually adequate.
• Severe cases very rare.