Blood Grouping and Red Cell Antibody Testing in Pregnancy (GTG 2014, bsh 2025)
Intro
15% of UK population Rh D-, therefore 60% of Rh D- mothers will carry a Rh D+ baby
1% of pregnant women are found to have a clinically significant antibody (most anti-D)
Group & Screen should be tested at booking and at 28 weeks in all pregnant women.
Effects of antibodies
Fetal and neonatal anaemia (hydrops)
Neonatal jaundice (kernicterus)
Perinatal loss (Anti-D, anti-K, anti-c)
Delays in timely provision of blood due to difficulty in performing screen
Sampling Principles
Patient consent
Hand-labeled at patient bedside
Lab Tests
ABO and D grouping
Test at booking to identify D-neg women who require anti-D prophylaxis. Also acts as useful confirmation of subsequent testing performed at a later date
Repeated at 28 weeks, before RAADP, to confirm ABO + D group and re-screen for antibodies
Red cell antibody screen
Test to detect clinically significant antibodies, ie. those that either may affect fetus/newborn, or complicate the provisional of blood components for the woman +/- fetus/newborn
Enzyme technique not required in antenal antibody screens as would not detect any additional clinically significant antibodies.
Antibody Identification
Once detected, further testing required to identify the specificity and concentration/strength of the antibody
Antibody concentration should then be monitored throughout pregnancy (see below)
Measuring Antibody Concentration
Used to guide initial referrals to fetal medicine units and then for ongoing monitoring/intervention
Antibody Quantification
Only available for Anti-D and Anti-c
Reported as IU per milliliter
Where possible test in parallel with previous stored samples to compare changes in concentration
Antibody Titration
Used to assess concentration of antibodies other than anti-D or anti-c
Doubling dilutions of plasma tested by IAT using heterozygous test cells (e.g. Kk)
If a rise in titre is detected, ideally re-test previous sample in parallel with current
Paternal Testing
If Ab detected, consider paternal testing to predict likelihood of HDN
Beware partner not being the biological father
Fetal Genotyping in alloimmunised pregnancies (cffDNA from 16+ weeks)
RHD, RHCE and KEL*01 genotypes
Avoids fetal sampling and pitfalls of partner testing
cffDNA is useful when either a, b or both are present with c:
a. Pregnant woman has a clinically significant antibody
b. Pregnant woman has a history of HDN
c. Father’s antigen status is unknown, or known to be corresponding antigen
cffDNA levels rise with gestation and so there is a corresponding fall in false negative rate with time
RHD and RHCE false negative rate at 16+ weeks is <1%.
KEL*01 testing should wait until 20+ weeks and repeat testing considered
Fetal Genotyping to guide anti-D prophylaxis in non-immunised RhD-neg women (cffDNA from 11 weeks)
High throughput screening for Rh D- mothers to D type the fetus
Recommended by NICE 2016. 99.9% sensitivity for excluding D positivity
False negative rate is approx. 0.1-0.3%
reporting laboratory investigations
Lab reports for antenal Group and Screen that identify an antibody must include:
The likely significance of the antibody detected:
For HDFN
And potential difficulty providing compatible blood components
The need to refer to fetal medicine unit (if applicable)
The timing of any further samples that are required
The need to give verbal and written information to the woman about the results
Red Cell Antibodies Detected in Pregnancy
Anti-D, Anti-K and Anti-c are the Ab most frequently implicated in HDN severe enough to warrant antenatal intervention.
Women with a history of HDN should be referred to fetal medicine specialist before 20 weeks, regardless of the antibodies detected.
Distinguishing between immune and passive Anti-D
Passive and immune anti-D cannot be separated serologically
Ab concentration should be quantified (IU/ml) by continuous flow analyser (CFA)
Passive anti-D
Concentration will fall with time
Concentration rarely exceeds 0.4 IU/ml
Detectable over 8 weeks after a 500 IU dose of Anti-D
Immune anti-D
Concentration will remain stable (or rise if there is re-stimulation)
First detectable 4 weeks after exposure to D+ cells
Procedure when anti-D is detected in pregnancy
Confirm whether anti-D was administered (not just issued)
And its indication, both asking patient and in written notes
Quantification of Anti-D (unless immediately prior to delivery, in which case monitor baby)
Monitor the antibody 4 weekly to 28 weeks, and 2 weekly thereafter until delivery (or until MCA Doppler has been instituted or anti-D is no longer detected) in any of the following situations:
Level ≥0.2 IU/ml
Anti-D detected at or before 28 week RAADP dose
There is no definite record of prior anti-D administration
Anti-D was present before first administration of anti-D
If Anti-D level is ≤0.4 IU/ml after up to 1500 IU of anti-D has been given, then prophylactic anti-D should continue to be offered in all appropriate situations, unless it is beyond doubt that this is immune anti-D
If doses >1500 IU have been given then passive anti-D can reach levels >0.4 IU/ml and so anti-D should continue to be offered.
After 28 weeks, if all of the following apply, no further antibody testing is required:
Anti-D was not detectable in sample at 28 weeks prior to RAADP
And There is a written record of anti-D administration in the previous 8 weeks
And Level of detected anti-D is now <0.2 IU/ml
Pregnant women with immune Anti-D
Anti-D is most frequent cause of serious HDFN
Test anti-D level monthly to 28 weeks and every 2 weeks thereafter until delivery
An increase in concentration >50% is a significant increase
Prev history of HDN + Concentration —> predicted outcome:
<4 IU/ml – HDN unlikely
4-15 IU/ml – moderate risk, refer to fetal medicine unit
>15 IU/ml – high risk, refer to fetal medicine unit
MCA Doppler to detect fetal anaemia may be used at the fetal medicine unit
Fetuses at risk of HDN should be delivered at 37 weeks to minimize exposure to maternal blood group antibodies
D status of fetus should be determined using cffDNA grouping (from 11+2 weeks)
Pregnant women with apparent anti-C+D, possible anti-G
A proportion of antibodies apparently typing as anti-C+D (often with high anti-C titre) are actually found to be anti-G or anti-C+G —> all such cases should be referred to reference lab for confirmation
These patients are at higher risk of developing immune anti-D and should be offered RAADP + post-delivery Anti-D prophylaxis
Pregnant women with immune anti-c
May cause delayed anaemia in the baby
Test anti-c level monthly to 28 weeks and every 2 weeks thereafter until delivery
Prev history of HDN + Concentration —> predicted outcome:
<7.5 IU/ml – HDN unlikely
7.5-20 IU/ml – moderate risk, refer to fetal medicine unit
>20 IU/ml – high risk, refer to fetal medicine unit
(Note: Co-existence of an Anti-E increases severity of fetal anemia. Refer at lower levels.)
MCA Doppler to detect fetal anaemia may be used at the fetal medicine unit
Fetuses at risk of HDN should be delivered at 37 weeks to minimize exposure to maternal blood group antibodies
c status of fetus should be determined using cffDNA genotyping from 16 weeks
Pregnant women with immune anti-K or other Kell antibodies
Other Kell antigens (k, Kpa, Kpb, Jsa, Jsb) should be treated in the same way as anti-K
Majority of historical cases of anti-K in pregnant women are consequence of previous K+ transfusion
—> Female <50 y.o. who are K- or unknown should receive K- blood
HDN due to anti-K is characterized by low Hb with normal amniotic/cord bilirubin levels
Test anti-K level monthly to 28 weeks and every 2 weeks thereafter until delivery, unless father is confirmed to be negative for the relevant antigen.
Prev history of HDN + Concentration —> predicted outcome:
Refer to fetal medicine unit once anti-K detected. Anaemia can occur at low titres
Titre 1:32 or greater –-> HDN more likely
MCA Doppler to detect fetal anaemia may be used at the fetal medicine unit
Fetuses at risk of HDN should be delivered at 37 weeks to minimize exposure to maternal blood group antibodies
KEL*01 status of fetus using cffDNA if father is heterozygous or unknown (9% of general pop. are K+)
Pregnant women with other red cell antibodies
Only IgG antibodies cross placenta, and red cell antibodies w/ a significant IgG component are detectable by IAT.
Other involved antibodies include:
Anti-C, -e, -E, -Fya, -Jka
Anti-M in Japanese, anti-Ge3 in Hispanic, anti-Mur / anti-Dia in Chinese
But many others have been reported
The risk of significant HDFN is much lower than for D, c or Kell
—> So frequency of monitoring assessed on basis of maternal history and in the absence of previous HDFN then women with a antibody titre <32 may not require intensive monitoring. Discuss with fetal medicine unit.
A titre of >32 would suggest increased risk of HDN
Action at time of Birth
D typing of cord samples from D-negative women with no immune anti-D
Maternal and cord blood sample for D grouping if baby group not already known
Post-delivery anti-D as required
DAT on cord samples
Not required routinely for D+ babies born to D- mothers with no red cell antibodies (Anti-D Ig can cross the placenta —> 3-6% of D+ cord samples found to also have a positive DAT)
DAT, Hb and bilirubin should be tested on cord blood of babies born to women with IAT-reactive red cell antibodies and the baby observed for jaundice.
If DAT+ and baby shows signs of HDN then a red cell eluate may help confirm the red cell antibody specificity.
IgG ABO antibodies can cause severe HDN so in cases of baby having major ABO mismatch with mother, the eluate should also be tested with A1 and B cells.
Babies who received IUT with D- cells as prevention of HDN may type as D- for several months until their own D+ cells are released into circulation.
Perform FMH at delivery on D- women with no immune anti-D to inform anti-D requirements