Blood Grouping and Red Cell Antibody Testing in Pregnancy (BSH 2015, GTG 2014)

  

Intro

15% of UK population Rh D-, therefore 60% of Rh D- mothers will carry a Rh D+ baby

G&S should be tested at booking and at 28 weeks in all pregnant women.

 

1% of pregnant women are found to have a clinically significant antibody (most anti-D)

 

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

  • Patient consent

  • Hand-labeled at patient bedside

 

Lab Tests

 

ABO and D grouping

Fetal D Group by cffDNA from 11+2 weeks

  • High throughput screening for Rh D- mothers to D type the fetus

  • Recommended by NICE 2016. 99.9% sensitivity for excluding D positivity

Red cell antibody screen (no need to use enzyme technique in the screen)

Antibody Identification

  • Once detected, antibody concentration should be monitored throughout pregnancy

Antibody Quantification

  • Only available for Anti-D and Anti-c

  • Reported as IU per milliliter

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 by 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

 

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 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 od 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 cause 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 HDN

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

These patients are at higher risk of developing immune anti-D

 

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

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 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

KEL*01 status of fetus using cffDNA if father is heterozygous or unknown.

 

Pregnant women with other red cell antibodies

Only IgG antibodies cross placenta, and red cell antibodies with 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

But many others have been reported

In the absence of a history of HDN, the risk is much lower than for D, c or Kell

So frequency of monitoring assessed on basis of history

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

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