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Rhesus (Rh) incompatibility occurs when an Rh(D)-negative mother carries an Rh(D)-positive fetus and maternal exposure to fetal red blood cells triggers production of anti-D IgG antibodies. These antibodies can cross the placenta in subsequent pregnancies and destroy fetal red blood cells, causing Haemolytic Disease of the Fetus and Newborn (HDFN) — ranging from mild neonatal jaundice to life-threatening fetal anaemia, hydrops fetalis, and intrauterine death. The Rh(D) antigen is encoded by the RHD gene on chromosome 1. Approximately 15–17% of Caucasian women, 5–8% of Black African women, and 1–3% of Asian women are Rh(D) negative. Sensitising events (antepartum haemorrhage, amniocentesis, chorionic villus sampling, external cephalic version, abruption, miscarriage) can introduce fetal red cells into the maternal circulation, triggering sensitisation. The Kleihauer-Betke (KB) test — or equivalent flow cytometric methods — quantifies the volume of feto-maternal haemorrhage (FMH) in maternal blood, allowing calculation of the appropriate anti-D immunoglobulin dose. Routine antenatal anti-D prophylaxis (RAADP) at 28 weeks (and sometimes 34 weeks depending on local protocol) prevents sensitisation in the vast majority of Rh(D)-negative women. For sensitising events before 20 weeks, 500 IU anti-D is recommended; for events at or after 20 weeks, at least 1500 IU is given. If the KB test indicates FMH greater than 4 mL fetal cells (approximately 2 mL whole blood), additional anti-D doses are calculated: 500 IU per 4 mL of fetal red blood cells detected. Rh prophylaxis has reduced HDFN by over 95% in high-income countries.
Anti-D dose for FMH >4 mL fetal cells: Additional anti-D (IU) = (FMH volume in mL of fetal red cells / 4 mL) x 500 IU, rounded up to nearest 500 IU. Standard prophylaxis: 500 IU IM for events <20 weeks; 1500 IU IM for events ≥20 weeks and RAADP at 28 weeks
- 1Confirm maternal blood group and Rh(D) status on first antenatal booking bloods; check for pre-existing irregular red cell antibodies (indirect antiglobulin test, IAT) at booking and at 28 weeks.
- 2Identify the paternal (or fetal) Rh(D) status: if father is Rh(D) negative, no further prophylaxis required (unless paternity is uncertain). If father is Rh(D) positive or unknown, manage as per Rh(D) negative woman protocol.
- 3Administer Routine Antenatal Anti-D Prophylaxis (RAADP): 1500 IU anti-D IM at 28 weeks (UK NICE NG3 single-dose schedule) or 500 IU at 28 and 34 weeks (two-dose schedule, still used in some regions).
- 4For any sensitising event at any gestation: administer anti-D within 72 hours of the event; for events <20 weeks: minimum 250–500 IU; for events ≥20 weeks: 1500 IU.
- 5If significant FMH is suspected at or after 20 weeks (major haemorrhage, road traffic collision, domestic violence): perform Kleihauer-Betke test or flow cytometry on maternal blood to quantify FMH.
- 6Calculate FMH volume from KB test result: FMH (mL fetal red cells) = (% fetal cells / 100) × maternal blood volume (mL, typically assumed as 5000 mL for a term woman). If FMH >4 mL fetal red cells, additional anti-D is required.
- 7Administer additional anti-D: 500 IU per 4 mL fetal red cells detected, rounded up; repeat KB at 48 hours to confirm no further haemorrhage and check anti-D adequacy.
If delivery is Rh(D) positive baby: administer further 1500 IU anti-D postnatally within 72 hours
RAADP reduces maternal sensitisation risk from approximately 1.5–1.8% to below 0.2% per pregnancy. The 1500 IU dose at 28 weeks provides coverage for any undetected FMH during the third trimester.
Give 1500 IU immediately (standard for event ≥20 weeks) PLUS 2000 IU additional = 3500 IU total; repeat KB at 48 hours
KB showed 0.5% fetal cells in 5000 mL maternal blood = 25 mL total fetal blood. Dividing by 2 gives approximately 12.5 mL fetal red cells. Each 4 mL of fetal red cells requires 500 IU; 12.5/4 = 3.125, rounds to 4 × 500 = 2000 IU additional. This is added to the standard event dose of 1500 IU.
NICE NG3: anti-D for all sensitising events including miscarriage if Rh(D) negative; KB not required below 20 weeks
Although spontaneous miscarriage at 9 weeks rarely causes sensitisation (fetal red cell antigens are not fully expressed this early), anti-D prophylaxis is given for surgical management (ERPC) as a precaution. KB testing is not necessary before 20 weeks.
If KB >4 mL fetal cells: additional anti-D as per calculation; repeat KB at 48 hours
Postnatal anti-D is given regardless of RAADP having been given in the third trimester, as delivery is the most common sensitising event. The 72-hour window is critical — effectiveness decreases rapidly beyond this window.
Routine antenatal anti-D prophylaxis (RAADP) administration to all Rh(D)-negative pregnant women at 28 weeks., where accurate rh incompatibility analysis through the Rh Incompatibility supports evidence-based decision-making and quantitative rigor in professional workflows
Emergency anti-D administration after sensitising events (APH, miscarriage, trauma, invasive procedures)., where accurate rh incompatibility analysis through the Rh Incompatibility supports evidence-based decision-making and quantitative rigor in professional workflows across diverse organizational contexts and analytical requirements
Kleihauer-Betke testing to calculate additional anti-D dosing after significant feto-maternal haemorrhage., where accurate rh incompatibility analysis through the Rh Incompatibility supports evidence-based decision-making and quantitative rigor in professional workflows across diverse organizational contexts and analytical requirements
Postnatal anti-D administration after confirmed delivery of an Rh(D)-positive infant., where accurate rh incompatibility analysis through the Rh Incompatibility supports evidence-based decision-making and quantitative rigor in professional workflows across diverse organizational contexts and analytical requirements
Non-invasive fetal RHD genotyping to target anti-D prophylaxis only to pregnancies where the fetus is Rh(D) positive.
Already sensitised Rh(D)-negative women
Once sensitisation has occurred (anti-D detectable in maternal serum), further anti-D prophylaxis is of no benefit and is not given. Management focuses on serial anti-D titre monitoring, fetal MCA Doppler assessment for anaemia (MCA-PSV >1.5 MoM threshold), and intrauterine transfusion if severe anaemia is detected. These women require specialist fetal medicine centre care.
Weak D and partial D phenotypes
Some individuals have reduced expression of the D antigen ('weak D') or partial D antigen expression. Laboratory determination of weak D vs. partial D is important: weak D individuals usually do not need prophylaxis (they can be managed as Rh(D) positive), while partial D individuals can produce anti-D and should be treated as Rh(D) negative. Molecular RHD typing is increasingly used to clarify ambiguous serological results.
Rh(D)-negative women with Rh(D)-negative partners
If paternity is certain and the partner is confirmed Rh(D) negative (homozygous), all fetuses will be Rh(D) negative and anti-D prophylaxis is not required. However, paternity certainty must be established with sensitivity and care. Non-invasive fetal RHD testing from maternal plasma is an increasingly available and more robust alternative.
Anti-D administration in rhesus-negative multiply-sensitised women
Women with multiple irregular antibodies (including non-D antibodies such as anti-c, anti-K) require complex management. Red cell phenotyping guides transfusion selection; fetal genotyping from cell-free DNA or amniotic cells identifies the fetal antigen status and guides the need for and intensity of monitoring.
| Situation | Anti-D Dose | KB Test Required |
|---|---|---|
| Sensitising event <20 weeks (no surgery) | 250 IU IM | No |
| Sensitising event <20 weeks (surgical) | 500 IU IM | No |
| Sensitising event ≥20 weeks | 1500 IU IM | Yes — quantify FMH |
| RAADP at 28 weeks (single-dose schedule) | 1500 IU IM | No |
| Postnatal (Rh(D)+ baby) | 1500 IU IM within 72 hours | Yes — check FMH |
| FMH >4 mL fetal red cells | 500 IU per 4 mL FMH additional | Repeat KB at 48 hours |
What is the Kleihauer-Betke test?
The Kleihauer-Betke (KB) test is an acid elution test that exploits the fact that fetal haemoglobin (HbF) is resistant to acid while adult haemoglobin (HbA) is eluted. A maternal blood smear is treated with acid, then stained: fetal red cells appear pink (HbF retained) while maternal cells appear as ghost cells. The percentage of fetal cells is counted and used to calculate the volume of FMH. Flow cytometry (anti-HbF immunofluorescence) is more accurate and is increasingly preferred.
What happens if anti-D is not given?
Without anti-D prophylaxis, an Rh(D)-negative woman exposed to Rh(D)-positive fetal red cells may form anti-D IgG antibodies (sensitisation). In a subsequent Rh(D)-positive pregnancy, these antibodies cross the placenta and destroy fetal red cells, causing HDFN — progressive fetal anaemia, hydrops fetalis, fetal heart failure, and potentially intrauterine death. Before anti-D prophylaxis, HDFN was a major cause of perinatal mortality globally.
What is feto-maternal haemorrhage (FMH)?
FMH is the transfer of fetal red blood cells into the maternal circulation. Small volumes (<1 mL) occur in the vast majority of pregnancies at delivery (micro-haemorrhage), but significant FMH (>4 mL fetal red cells) is uncommon without a specific cause. Sensitising events associated with significant FMH include abdominal trauma, antepartum haemorrhage, placental abruption, external cephalic version, invasive prenatal procedures, and delivery itself.
Does anti-D affect breastfeeding?
No. Anti-D immunoglobulin is safe to use in breastfeeding women and is not excreted in significant quantities in breast milk. It should not be withheld on grounds of breastfeeding. If administered before delivery, the half-life of anti-D is approximately 3 weeks, so it may still be detectable in the baby's blood at birth via cord blood serology, which can cause a weakly positive DAT (direct antiglobulin test) — a clinically insignificant finding.
What is non-invasive prenatal testing (NIPT) of fetal RHD?
Cell-free fetal DNA from maternal plasma can be tested to determine the fetal RHD genotype non-invasively from a simple maternal blood draw. This is now offered in some countries (including the UK NHS) at approximately 11–16 weeks. If the fetus is Rh(D) negative (15–20% of cases), anti-D prophylaxis throughout pregnancy can be avoided, reducing unnecessary treatment and anti-D immunoglobulin demand.
Can a sensitised woman still have a healthy baby?
Yes. With modern management including serial fetal MCA Doppler monitoring (MCA-PSV >1.5 MoM is the threshold for investigating severe anaemia), intrauterine transfusion (IUT) for severely anaemic fetuses, and timely delivery, the survival rate for fetuses with HDFN is over 90% in specialist centres. IUT has transformed the prognosis for severely affected pregnancies.
What other red cell antibodies (besides anti-D) cause HDFN?
While anti-D remains the most clinically significant antibody, other irregular antibodies can cause HDFN: anti-c (Rh system) and anti-K (Kell system) are the next most common causes of severe HDFN. Anti-E, anti-C, anti-Jka, and anti-Fyb can cause mild-moderate HDFN. The antenatal red cell antibody screen (indirect antiglobulin test, IAT) detects all clinically significant irregular antibodies.
Is anti-D given if the pregnancy has ended in early miscarriage without surgical management?
For spontaneous miscarriage (complete or incomplete) before 12 weeks without surgical intervention, anti-D is not routinely recommended in the UK (per NICE NG3 2021 update) if there is no heavy bleeding and no surgical management. Anti-D is still recommended for any surgical or medical management (ERPC, mifepristone/misoprostol), threatened miscarriage above 12 weeks, ectopic pregnancy, and termination of pregnancy at any gestation.
Consiglio Pro
When performing the KB test after a sensitising event, always record the time of sampling relative to the event, and repeat the KB at 48 hours if the initial result shows significant FMH. Occasionally a second wave of FMH occurs after initial treatment, and a repeat KB confirms adequacy of anti-D coverage.
Lo sapevi?
Rhesus (Rh) blood group nomenclature came from a series of early experiments in the 1940s where Karl Landsteiner and Alexander Wiener immunised rabbits and guinea pigs with red blood cells from rhesus monkeys (Macaca mulatta). The antibodies produced agglutinated approximately 85% of human red blood cell samples — designating the carriers as Rh-positive. It was only later discovered that the antibody raised in animals against rhesus monkey cells was not identical to the human anti-D antibody, but the name 'Rhesus' had already become embedded in medical terminology.
Riferimenti
- ›NICE — Ectopic Pregnancy and Miscarriage (NG126) — Anti-D guidance
- ›RCOG Green-top Guideline No. 22 — The Use of Anti-D Immunoglobulin for Rhesus D Prophylaxis
- ›NICE — Routine Antenatal Anti-D Prophylaxis for Women who are Rhesus D Negative (TA156)
- ›Qureshi H et al — BCSH guideline for the use of anti-D immunoglobulin for the prevention of haemolytic disease of the fetus and newborn — Transfusion Medicine 2014