Alloantibodies in Pregnancy

Alloantibodies in pregnancy may be clinically significant. This depends on their ability to cause haemolytic disease of the fetus / newborn or haemolytic transfusion reactions.

• Maternal blood group should be checked at booking and 28 weeks to determine ABO/Rh status and screen for clinically significant red cell antibodies (e.g. anti-D, -c, -K)
  • If detected, further testing should be done to assess the likelihood of these causing HDFN
  • Antibody titres should also be measured once every four weeks until 28 weeks, then once every 2 weeks till delivery
    • Moderate risk of HDFN: anti-D levels 4-15 IU/ml, anti-c 7.5-20 IU/ml
    • Severe risk of HDFN: anti-D levels of >15 IU/ml, anti-c >20 IU/ml
  • If the mother has clinically-significant antibodies, then the father and fetus should be tested to ascertain the risk of the fetus carrying the relevant antigen
    • Free fetal DNA can be extracted from the maternal circulation from 16 weeks
• If the fetus is antigen positive and the mother has clinically-significant antibodies, then the pregnancy must be monitored closely. Middle cerebral artery peak systolic velocity is a non-invasive and sensitive way of detecting fetal anaemia.
• IgG antibodies can cross the placenta and cause haemolysis
• Incidence of clinically-significant antibodies in pregnancy: 0.3-1%
• Greatest risk with anti-D, anti-c and anti-K (and all other Kell system antigens, e.g. anti-k, -Kp^a, -Kp^b, -Js^a, -Js^b)

Antibody	Haemolytic Disease of Fetus/Newborn	Haemolytic Transfusion Reaction
D	Severe	Severe
c	Severe	Severe
K	Severe	Severe
c+E	Severe	Severe
E	Yes	Yes
C	Yes	Yes
e	Yes	Yes
Ce	Yes	Yes
Fya	Yes	Yes
Fyb	Yes	Yes
Jka	Yes	Yes
Jkb	No	Yes
S	Yes	Yes
s	Yes	Yes
U	Yes	Yes
M	Occasionally	Rarely, only if active at 37°C
N	Very rarely	Rarely, only if active at 37°C
H	Yes	Yes
k	Yes	Yes
Kpa	Yes	Yes

Basic Testing

• ABO grouping, Rh typing and antibody screen at booking and 28 weeks
  • For D-negative women, the 28 week sample should be taken before the administration of prophylactic anti-D, to reduce confusion between passive and immune anti-D.
• If antibody detected: identify antibody
• For anti-D and anti-c, quantify using a national standard preparation (UK), express in IU/ml
• For all other antibodies, quantify using titration and double dilutions against reagent red cells which are heterozygous for the antigen in question. All samples must be titrated in parallel with the previous sample, to reduce variation in methodology / interpretation.
  • UK guidelines recommend parallel titration of the NIBSC anti-D standard to reduce variability.
  • Antigen concentrations are expressed as the reciprocal of the highest dilution that gives a positive reaction.
  • Internal SOPs should specify what constitutes a positive reaction.
• All clinically-significant antibodies detected should be monitored 4-weekly till 28 weeks, then 2-weekly till delivery.

Detection of Anti-D in Pregnancy

• If anti-D is detected, especially if it was not detected in a previous sample, correlation with clinical history (especially anti-D administration) is essential to determine if it is a passive or immune anti-D.
  • There is no serological way to tell.
  • Passive anti-D concentrations rarely exceed 0.4 IU/ml (unless doses in excess of 1500 IU are given), and it has a half-life of 3 weeks.
  • Passive anti-D is detectable almost immediately after administration, reaching a peak within 3-7 days. It can be detected for up to 12 weeks or longer, depending on assay sensitivity.
  • Immune anti-D usually becomes detectable 4 weeks after exposure to a D-positive cell, reaching a peak concentration 6-8 weeks after exposure.
• Any anti-D detected at or before 28 weeks should be regarded as potentially-immune, and concentrations serially-monitored.
  • Although they should be monitored like immune anti-D, any woman with a titre of <0.4 IU/mL should still be offered anti-D prophylaxis, so as to avoid omitting anti-D prophylaxis for a non-sensitised woman.
• Increasing titres should prompt consideration of an immune anti-D and referral to a fetal medicine specialist once titres are >4 IU/mL.

If a Clinically-Significant Antibody is Detected

• Quantify the antibody.
• It is useful to know if the fetus expresses the corresponding antigen to predict the risk of HDFN.
• Anti-D:
  • <4 IU/mL: HDFN unlikely, continue monitoring
  • 4-15 IU/mL: moderate risk, refer to fetal medicine specialist
  • >15 IU/mL: high risk, refer to fetal medicine specialist
• Anti-c:
  • <7.5 IU/mL: HDFN unlikely, continue monitoring
  • 7.5-20 IU/mL: moderate risk, refer to fetal medicine specialist
  • >20 IU/mL: high risk, refer to fetal medicine specialist
• Options:
  • Phenotype /genotype the father. If he is homozygous for the antigen, the fetus is at definite risk of HDFN. If he is heterozygous, fetal genotyping will be necessary.
  • Non-invasive fetal genotyping using fetal free DNA in the maternal circulating. This can be used to predict the RHD, RHCE and KEL*01 (K) genotype of the fetus, with a 1% false negative rate. Rh genotyping can be performed from 16 weeks, K from 20 weeks. However, due to variations in individual lab protocols, specific recommendations for when to test should be followed to preserve sensitivity. Non-invasive genotyping is currently not available for other red cell antigens.
  • It is reasonable to proceed straight to fetal genotyping to avoid issues of non-paternity.
  • Invasive testing (chorionic villious sampling / amniocentesis). The risks of this (miscarriage, further / worsening alloimmunisation) must be weighed against the benefits. Such an option may be reasonable if the patient has a history of significant HDFN previous pregnancies, a baby with unexplained severe neonatal jaundice / anaemia requiring transfusions, or if ultrasound monitoring detects signs of anaemia and intrauterine transfusion is being considered.
• If the fetus expresses the corresponding antigen, then it is at risk of HDFN, and must be referred to a fetal medicine specialist. Twins should be monitored individually.
• Fetuses of women with anti-D > 4IU/mL, anti-c > 7.5IU/mL, anti-K of any titre or all other clinically-significant antibodies with a titre of 32 or more are at risk of HDFN.
• Such fetuses should be monitored with weekly (or biweekly, if the antibody is anything other than anti-D, anti-c or anti-K) with peak systolic flow velocities of the middle cerebral artery (MCA PSV), measured by Doppler ultrasound.
• Any fetus with a MCA PSV > 1.5 multiples of the mean is at risk of significant fetal anaemia, and intrauterine transfusions should be considered.
• All babies at risk of HDFN should be delivered at 37 weeks to reduce the risk of exposure to maternal antibodies.

Intrauterine Transfusions

• Must be carried out in a centre with expertise in IUT
• Red cell preparations should be group O (or ABO-matched to the fetus and mother). Other requirements:
• Antigen-negative for the corresponding maternal antibody
• IAT cross-match compatible with maternal plasma
• Leukocyte-reduced, irradiated (irradiation within 1 day of transfusion)
• In citrate phosphate dextrose (CPD) anticoagulant
• CMV-negative
• HbS negative 
• Kell negative
• Haematocrit 0.70-0.85
• Less than 5 days old (reduce potassium load)

Neonatal Transfusion Requirements

• Red cell preparations should be group O (ABO compatible with both mother and neonate). Other requirements:
• Antigen-negative for the corresponding maternal antibody
• IAT cross-match compatible 
• Leukocyte-reduced
• Ideally irradiated (exchange transfusion or prior IUT only), unless irradiation would delay treatment unacceptably
• In citrate phosphate dextrose (CPD) anticoagulant
• CMV-negative
• HbS negative
• Haematocrit 0.50-0.60
• Less than 5 days old (reduce potassium load, exchange transfusion only)

Maternal Transfusion Requirements

• ABO-matched (group O if ABO matched unavailable)
• Rh negative if the woman is Rh negative
• K negative
• CMV negative